(Geoffrey Michael Gadd, Sima Sariaslani) PDF

VOLUME NINETY FOUR
ADVANCES IN
APPLIED MICROBIOLOGY
VOLUME NINETY FOUR
ADVANCES IN
APPLIED MICROBIOLOGY
Edited by
SIMA SARIASLANI
Wilmington, Delaware, USA
GEOFFREY MICHAEL GADD

Dundee, Scotland, UK
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ISBN: 978-0-12-804803-0
ISSN: 0065-2164
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CONTRIBUTORS
Varenyam Achal
School of Ecological and Environmental Sciences, East China Normal University,
Shanghai, China
A. Fox
Department of Life Sciences, University of Limerick, Limerick, Ireland
Geoffrey Michael Gadd
Geomicrobiology Group, School of Life Sciences, University of Dundee, Dundee,
Scotland, UK; Xinjiang Key Laboratory of Environmental Pollution and Bioremediation,
Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi,
China
M. It€avaara
VTT Technical Research Centre of Finland Ltd, Espoo, Finland
Deepika Kumari
Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Xinjiang
Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, China
Qianwei Li
Geomicrobiology Group, School of Life Sciences, University of Dundee, Dundee,
Scotland, UK
K. Marjamaa
Xiangliang Pan
Xin-Yi Qian
T. Ruskeeniemi
GTK, Geological Survey of Finland, Espoo, Finland
H. Salavirta
A. Schmalenberger
vii j
CHAPTER ONE
Geomicrobiology and
Metagenomics of Terrestrial
Deep Subsurface Microbiomes
avaara*, 1, H. Salavirta*, K. Marjamaa* and T. Ruskeeniemix
M. It€
*VTT Technical Research Centre of Finland Ltd, Espoo, Finland
x
GTK, Geological Survey of Finland, Espoo, Finland
1
Corresponding author: E-mail: Merja.Itavaara@vtt.fi
Contents
1. IntroductiondDeep Crustal Life 3
1.1 Microbial Life at the Surface and Deep Subsurface Habitats 3
1.2 The Lithosphere as a Host of Life 4
1.3 Physical and Geochemical Constraints on Deep Subsurface Life 6
1.4 Geological Carbon Sources for Deep Subsurface Life 8
1.5 Biogeochemistry 10
1.5.1 Sources of Energy in the Lithosphere 10
1.5.2 Deep Carbon Cycling 15
1.5.3 Microbial Activity 18
2. Exploring the Diversity of Terrestrial Deep Subsurface Microbiomes 20
2.1 Bacteria 20
2.2 Archaea 21
2.3 Eukaryotes 22
2.4 Viruses 22
3. Sampling of the Deep Biosphere 23
3.1 Groundwater Sampling 24
3.2 Processing and Maintenance of Samples for Microbiological Research 28
4. Terrestrial Deep Subsurface Microbiomes: Metagenomics 30
4.1 Metagenomics and Metatranscriptomics as Tools for Exploring the 30
Deep Subsurface Life
4.2 Amplicon Sequencing 31
4.2.1 Biodiversity 31
4.2.2 Gene Markers of Functional Diversity 32
4.3 Metagenome and Metatranscriptome Sequencing and Data Analysis 33
4.3.1 Metagenomics 33
Advances in Applied Microbiology, Volume 94

© 2016 Elsevier Inc.
j
ISSN 0065-2164
http://dx.doi.org/10.1016/bs.aambs.2015.12.001 All rights reserved. 1
2 M. It€avaara et al.
4.3.2 Metatranscriptomics 36
4.3.3 Data Analysis 37
4.4 Single-Cell Isolation and Sequencing 39
5. A Case Study: Metagenomics of the Outokumpu Deep Borehole 39
5.1 Metagenomics of Deep Subsurface Life in the Outokumpu Deep Borehole, 41
Finland
5.2 Data Analysis of Borehole Water and Fracture Zone Metagenomes 42
5.2.1 Quantity and Quality of Metagenomic Sequences 42
5.2.2 Metagenome Assembly 44
5.2.3 Gene Prediction and Annotation 45
5.3 Species Distribution in Borehole Water and in Fracture Zone Samples 46
5.4 Representativeness of the Assemblies and Insights into the Microbial 53
Communities in the Outokumpu Bedrock
5.5 Summary 60
6. Conclusions 61
References 63
Abstract
Fractures in the deep subsurface of Earth’s crust are inhabited by diverse microbial
communities that participate in biogeochemical cycles of the Earth. Life on Earth,
which arose c. 3.5e4.0 billion years ago, reaches down at least 5 km in the crust.
Deep mines, caves, and boreholes have provided scientists with opportunities to
sample deep subsurface microbiomes and to obtain information on the species diver-
sity and functions. A wide variety of bacteria, archaea, eukaryotes, and viruses are now
known to reside in the crust, but their functions are still largely unknown. The crust at
different depths has varying geological composition and hosts endemic microbiomes
accordingly. The diversity is driven by geological formations and gases evolving from
deeper depths. Cooperation among different species is still mostly unexplored, but
viruses are known to restrict density of bacterial and archaeal populations. Due to
the complex growth requirements of the deep subsurface microbiomes, the new
knowledge about their diversity and functions is mostly obtained by molecular
methods, eg, meta‘omics’. Geomicrobiology is a multidisciplinary research area
combining disciplines from geology, mineralogy, geochemistry, and microbiology.
Geomicrobiology is concerned with the interaction of microorganisms and geolog-
ical processes. At the surface of mineralogical or rock surfaces, geomicrobial pro-
cesses occur mainly under aerobic conditions. In the deep subsurface, however,
the environmental conditions are reducing and anaerobic. The present chapter de-
scribes the world of microbiomes in deep terrestrial geological environments as
well as metagenomic and metatranscriptomic methods suitable for studies of these
enigmatic communities.
Deepterrameta 3
1. INTRODUCTIONdDEEP CRUSTAL LIFE

1.1 Microbial Life at the Surface and Deep Subsurface
Habitats
Deep crustal subsurfaces have been explored for decades in search of
new energy reservoirs such as oil and methane hydrate deposits. The
discovery of deep subsurface microorganisms may be related to drilling for
oil reservoirs where microorganisms have caused problems such as
biocorrosion and plugging of oil drilling casings due to formation of a bio-
film by microbes that utilize hydrocarbons as a source of energy and nutri-
ents. Sulfate-reducing bacteria in particular are specializing in hydrocarbon
degradation under anaerobic conditions and can produce corrosive sulfides
(Youssef, Elsahed, & McInerney, 2009).
The role of microbes and their connection to inner Earth’s geochemical
processes have been recognized and are studied in several scientific drilling
projects. Deep carbon reservoirs and fluxes releasing gases from the mantle
via the crust to the atmosphere are known to affect the microbial carbon and
energy economy. Deep carbon cycling, accordingly, is a major process that
has been studied by the global scientific network Deep Carbon Observatory
(http://www.deepcarbon.net) connected to the Ocean Drilling (http://
www.iodp.org/) and the Continental Scientific Drilling programs (http://
www.icdp-online.org). Biogeochemical processes and biodiversity of deep
subsurface life are now actively studied, and characterization of microbial
ecosystems has revealed that a considerable part of deep subsurface life is still
new to science.
Terrestrial deep subsurface life has been discovered across the globe
(Chivian et al., 2008; Dong et al., 1999; Fredrickson & Balkwill, 2006;
It€avaara, Nyyss€onen, Bomberg, et al., 2011; J€agevall, Rabe, & Pedersen,
2011; Kato & Takai, 2000; Miettinen, Bomberg, et al., 2015; Miettinen,
Kiet€av€ainen, et al., 2015). There are estimates that as much as 50% of the
planet’s total biomass exists in the fluid-filled pores and fractures of
continental sedimentary and crystalline rocks as well as the oceanic floor
(Horsfield & Kieft, 2007; Pedersen, 2000; Whitman, Coleman, & Wiebe,
1998). The microorganisms in the terrestrial deep subsurface can live under
variable, often extreme, environmental conditions and have adapted to sur-
vive high pressure, drought, extreme temperatures, high salinity, and varying
pH. Many of these organisms are autotrophic and survive with scarce carbon
and energy resources arising from watererock interactions and from gases
forming in the deep interior of the Earth’s crust (Abe, 2007; Bartlett, 2002;
Horsfield et al., 2007; Lauro, Chastain, Blankenship, Yayanos, & Bartlett,
2007).
The abundance of microbial biomass at the surface of the Earth is c.
3.91 108 to 5.69 109 cells/g soil (Bressan et al., 2015; Marteinsson
et al., 2015; Torsvik, 2002). Generally, deep continental fracture waters
contain c. 103e105 microbial cells per milliliter, and typically, the number
of cells decreases as a function of depth (It€avaara, Nyyss€ onen, Bomberg,
et al., 2011; J€agevall et al., 2011; Nyyss€
onen et al., 2014; Purkamo et al.,
2013). Furthermore, the density of microbes appears to be dependent on
crustal thickness and temperature as well as availability of electron acceptors
(Hallbeck & Pedersen, 2008; Miettinen, Kiet€av€ainen, et al., 2015; Pedersen,
2010).
At the surface of the Earth, degraders of organic matter transform organic
carbon to several compounds and metabolites, which sustain new biomass
production in the environment. Photosynthetic microorganisms and plants
utilize solar energy for carbon fixation, which is then consumed by hetero-
trophic organisms. Oxygen is abundantly available at the surface as a terminal
electron acceptor in respiration and enables fast biodegradation of organic
compounds and production of carbon dioxide as a result of mineralization.
In deeper geological environments, however, both organic carbon and
oxygen are depleted rapidly, and organic compounds are biodegraded by
several microbiological transformation processes generating carbon dioxide
and methane as end products. Under anaerobic conditions, microbiological
processes are linked to reduction of nitrate, nitrite, iron (III), manganese
(IV), sulfate, or carbon dioxide which are acting as electron acceptors
(Amend & Teske, 2005; Boettger, Lin, Cowen, Hentscher, & Amend,
2013; Osburn, LaRowe, Momper, & Amend, 2014). The elements required
for construction of organic molecules include carbon, hydrogen, nitrogen,
oxygen, phosphorus, and sulfur, which are all cycled by microorganisms.
These cycling processes extend from the surface to the depth several kilome-
ters within the Earth’s crust.
1.2 The Lithosphere as a Host of Life

The Earth’s radius is c. 6300 km, and the outermost layer is a solid crust,
which “floats” on the more viscous mantle. The crust and the upper part
of the mantle are collectively referred to as the lithosphere. It is thought
that the iron-rich Earth’s interior is composed of a liquid outer core and a
solid inner core. The crust is subdivided into the oceanic crust
Deepterrameta 5
(which underlies ocean basins) and the continental crust. Crust thickness
varies from 5 to 70 km: the average thickness of the continental crust is
45 km, whereas the crust under oceans is only 8 km thick on average.
According to the present knowledge, microbial ecosystems are found as
deep as 3e5 km from the surface of the continental crust if environmental
conditions are suitable (Hallbeck & Pedersen, 2008). The environmental
factors that support or limit life include availability of electron acceptors
and donors, prevailing hydrogeochemical conditions, temperature, and
pressure (Pedersen, 2012, 2000).
There are important chemical and mineralogical differences between the
oceanic and continental types of crust. The ocean basin is composed of dense
iron- and magnesium-rich silicate rocks (mafic composition) such as basalt.
The continental crust is less dense and is predominantly composed of so-
diumepotassiumealuminum silicate rocks (felsic composition), for
example, granite. The lithosphere is broken up into seven or eight major
plates and many minor ones, which move relative to one another. This
concept of continental drift forms the basis of the theory describing large-
scale motion in the lithosphere (Hess, 1962; Wegener, 1912). The oceanic
crust is formed at midocean ridges and spreads outward, thus forcing the
plates to move. Where the plates meet, they either move past each other
(eg, San Andreas Fault in California) or collide, or one will move under
the other to form a subduction zone (eg, east of Japan). Typically, the dense
ocean floor moves under the continent and drags along huge amounts of
seawater and sediments. Volcanic activity, earthquakes, and mountain build-
ing occur along the plate boundaries. The continental crust has formed in
the geological past as a result of volcanism and accretion through tectonic
processes. Later, weathering and erosion of the continental areas on the scale
of kilometers with subsequent deposition of sediments and sedimentary
rocks have together resulted in the geological complexity of lithologies, lith-
ogeochemistry, bedrock structures, and the range of ages observed today.
According to the present understanding, the distribution of continents has
varied in the past. They have formed supercontinents or smaller clusters and
have again broken apart. The last reconstructed supercontinent was Pangea,
which included all the present continents. It is believed to have been formed
w300 million years ago (mya) and break apart 175 mya (Condie, 1989).
Evidence of the existence of this supercontinent comes from the continuation
of geological units and from similar fossil findings, eg, across the Atlantic
Ocean (Benton, 2004). The breakup included several stages; for example,
North America and Europe separated 60e50 mya. After breaking apart,
the continents have drifted relative to each other and to the equator. During
Paleozoic, Fennoscandia was located in the Southern Hemisphere, at or
close to the equator, and only w350 mya started to drift toward the present
latitude (Torsvik, 2002). The plate tectonics must have also affected the
biogeography of deep microbiomes. This notion may explain why similar
microbial species that were identified deep in the Fennoscandian Shield can
also be found at distant sites, such as South Africa (Chivian et al., 2008;
It€avaara, Nyyss€onen, Bomberg, et al., 2011; It€avaara, Nyyss€ onen, Kapanen,
et al., 2011). Perhaps these species separated a long time ago due to geological
changes, and due to slow metabolic activity and evolution, they have adapted
to remote prevailing geological conditions.
1.3 Physical and Geochemical Constraints

on Deep Subsurface Life
Although deep subsurface life is known to tolerate extreme environmental
conditions, the increasing temperature and pressure in the bedrock are likely
to control the diversity of life. The temperature limit is estimated to be c.
121 C (Kashefi & Lovley, 2003). Nonetheless, Takai et al. (2008) reported
that a hyperthermophilic methanogen (Methanopyrus kandleri) can multiply
and generate isotopically labeled methane during high-pressure cultivation
at 122 C. This finding suggests that the known temperature limit for life
may increase in the future when more knowledge is obtained.
High temperatures can damage all cellular macromolecules (eg, nucleic
acids, proteins, and lipids); the rate of depurination of DNA (hydrolysis of
purine bases) and racemization of proteins and their amino acids increase
exponentially with temperature (Lever et al., 2015). The hyperthermophilic
microorganisms have adaptations both at the molecular and organelle levels
that protect the cells from heat-induced damage (eg, reviewed by Kashefi,
2012).
In areas far away from tectonic plate boundaries and active volcanism,
the continental average temperature gradient is w25 C/km. On the other
hand, the upper part of the bedrock tends to adopt the prevailing annual
average surface temperature. For example, in Scandinavia, as an impact of
the Quaternary ice ages, a low-temperature anomaly can be observed
down to the depth of several 100 m (Cermak, Kukkonen, & Safanda,
1993; Kukkonen & Safanda, 1996). In the 2.5-km-deep Outokumpu
research drill hole in Finland, 40 C is reached only at the 2.5-km depth,
corresponding to w16 C/km (Kukkonen, 2011). Microbiology of the
Outokumpu borehole is presented later in this chapter as a case study.
Deepterrameta 7
The hydrostatic pressure in the upper part of the bedrock increases with
depth. A simplistic approximation suitable for the majority of practical
applications is that the hydrostatic pressure at shallow depths of the bedrock
increases by 1 MPa per 100 m (10 bar per 100 m). Locally there may be
deviations from this rule of thumb in relation to topography, geometry of
the hydraulic zones, and density differences in the water column. Microbes
have been reported to be active at pressures up to 80 MPa (Kato et al., 1998;
Kato & Takai, 2000). The present knowledge about physiological adapta-
tions of microorganisms to high pressure has been gained mainly from
ocean-drilling projects, where deep sea microorganisms have been cultured
in pressure bioreactors at 10e130 MPa (Lauro & Bartlett, 2008; Lauro et al.,
2007; Martin, Bartlett, & Roberts, 2002). The effects of high-pressure phys-
ics and chemistry on key cellular macromolecules, eg, proteins, nucleic acids,
and lipids and their interactions, have been reviewed elsewhere (Meersman
et al., 2013). High pressure denatures proteins (typically at 400e800 MPa)
and interferes with crucial proteineDNA interactions at 70e130 MPa;
the latter figure is close to the upper pressure limit for growth of most
microbes (Merrin, Kumar, & Libchaber, 2011). Maintenance of the physi-
ologically active fluidlike state of membrane phospholipids and the transition
of different phospholipids to the gel-like phase are affected by temperaturee
pressure conditions (Meersman et al., 2013). Alterations in the membrane
lipid composition are some of the adaptation mechanisms of microorganisms
living under high-pressure conditions (DeLong & Yayanos, 1985).
Water is essential for life. In the deep bedrock, water enables transport of
nutrients and gases that support microbial life. Water is present in all geolog-
ical materials, even in hot magmas (Sparks et al., 1997). In the bedrock,
water is present in the rock matrix and/or in water-conducting fractures.
Matrix water occupies pore spaces between mineral grains and is generally
in a more stagnant phase than water in fractures. This is because pore spaces
are small and weakly connected as compared to rough planar fractures,
which cut geological units on the scale of centimeters to hundreds of meters
or more. The tendency to form interconnected fracture networks further
promotes the conductive flow as a response to the hydraulic gradient.
Geological evolution, age of a rock formation, and its deformation his-
tory determine how water can move through the rock. Major sets of frac-
tures have formed deep in the crust at high pressure and temperature as a
result of large-scale tectonic processes. Cooling fluids precipitate minerals
into the fractures and tend to seal them; these processes prevent the flow.
In contrast, later, bedrock movements at lower temperatures may reopen
these sealed fractures, thus providing routes for water flow. During billions
of years, erosional processes have removed kilometers of rock from the sur-
face, and at high latitudes, numerous glaciations with extensive ice sheets
have strained the upper part of the bedrock. Probably, this unloading has
caused “loosening” of the bedrock structures and opening of fractures close
to the surface. Drill core studies and hydraulic testing of boreholes in Finland
and Sweden have shown that the water-conducting fracturing in the meta-
morphic and plutonic bedrock is more common and interconnected within
the upper 150e300 m (Fig. 1), whereas at deeper levels, water conductivity
diminishes significantly and is concentrated in discrete zones (Follin et al.,
2014; It€avaara, Nyyss€ onen, Bomberg, et al., 2011; It€avaara, Nyyss€ onen,
€
Kapanen, et al., 2011; Ohberg, 2006).
Sedimentary rocks are characterized by significantly greater pore
volumes, up to tens of percentage points, as compared to plutonic rocks
(eg, granites) or metamorphosed, crystalline rocks (eg, gneisses or schists),
which typically have porosity well below 1%. In addition, in sedimentary
rocks, precipitation of minerals can seal pore spaces and reduce the water
content.
As noted above, pore water and fracture water often coexist. The differ-
ences in their physical environments, particularly in crystalline rocks, often
result in a chemical imbalance. This phenomenon is due to the mechanism
whereby the water phases may exchange. The fracture network allows for a
quick response to changing hydraulic conditions, and the fracture water
readily mixes with the introduced water, but diffusion is the principal pro-
cess of movement of solutes from one pore to another. In dynamic hydro-
geological systems (eg, during a glacial cycle or in relation to rapid changes in
the sea level), the chemical differences may be considerable and can persist
for a long time. In principle, the geochemical stability in the rock matrix
can be considered attractive for microbes. Climatological or other changes
occurring at the surface are affecting these factors more slowly or may
even be buffered at deeper levels, but the other side of the coin is the limited
availability of nutrients. The interaction of microbes with their geological
environment (biogeochemistry), especially with carbon and energy sources,
is discussed in the next sections (Sections 1.4 and 1.5).
1.4 Geological Carbon Sources for Deep Subsurface Life

Carbon sources in the bedrock include volatile compounds derived from
solidifying magma as well as organic and inorganic solid phases. Organic
solids are varieties of coal, petroleum, and natural gas which are the main
Deepterrameta 9
Figure 1 A conceptual view of the hydrogeological and hydrogeochemical conditions

in the upper part of the crystalline bedrock and electron acceptors in microbiological
processes. (Panel 1) The upper 300e500 m is characterized by a connected fracture
network, which allows for recharging of meteoric waters and groundwater flow along
the network. Already at a small depth, oxic conditions are transformed to anoxic.
Typically, the groundwaters are fresh or brackish, but the salinity increases with depth.
(Panel 2) Fracture frequency decreases in the deeper bedrock, and the hydraulic zones
tend to become more isolated; these changes decrease the groundwater flow rate.
Salinity continues to increase and brines appear. (Panel 3) Regional deformation zones
divide the bedrock into large blocks. These zones result from ancient bedrock
movements, which are tens to hundreds of meters wide, kilometers or tens of
kilometers long, and are believed to extend deeply into the crust. Due to the long
geological history, the lateral and vertical hydrogeological properties within these
zones are variable and difficult to predict.
source of mineral carbon. Inorganic solid phases are predominantly graphite

(reduced carbon) and carbonates (oxidized carbon) present in various lithol-
ogies as primary or secondary components (Sephton & Hazen, 2013). Thick
carbonate rock units are common in sedimentary and metamorphic terrains.
Extensive graphite-bearing black shale/schist formations have been
described all over the world, and there is evidence of a biogenic origin of
their carbon content (Klein et al., 2015; Loukola-Ruskeeniemi, 1999;
Young, Loukola-Ruskeeniemi, & Pratt, 2013).
In addition, methane is present in the upper crust in variable quantities as
a dissolved phase in groundwater moving toward the surface via fractures in
the bedrock (Kiet€av€ainen et al., 2013; Kiet€av€ainen & Purkamo, 2015;
Stotler et al., 2010). In the deep subsurfaces, the origin of this methane
can be either biotic or abiotic. Abiotic formation of methane has been
reviewed recently (Etiope & Sherwood Lollar, 2013; McCollom, 2013),
and the whole terrestrial methane cycling is reviewed by Kiet€av€ainen and
Purkamo (2015).
Huge amounts of methane are tied up in methane clathrates, also known
as gas hydrates and methane ice found in marine sediments and in sedimen-
tary rocks in permafrost regions (Buffett, 2000; Kvenvolden, 1995; Sephton
& Hazen, 2013). A methane clathrate is a solid ice-type compound, where
methane is trapped in a water cage. The compound is stable in a relatively
narrow range of pressure and temperature limiting the occurrence to the up-
per lithosphere: the minimum depth is w300 m and maximum w2000 m
depending on the local geothermal gradient (eg, Guggenheim and van
Groos, 2003). At greater depths, the temperature increases, and gas becomes
the stable phase. The origin of the methane clathrate is a subject of debate
(eg, Abrajano et al., 1988; Fu, Sherwood Lollar, Horita, Lacrampe-
Couloume, & Seyfried, 2007; Schrenk, Brazelton, & Lang, 2013).
1.5 Biogeochemistry
1.5.1 Sources of Energy in the Lithosphere
The deep subsurface microbes have developed diverse mechanisms for
procurement of energy (Boettger et al., 2013; Lever et al., 2015; Wright,
Grasby, Williamson, Spear, & Templeton, 2011). The use of different
energy sources is dependent on their availability, which is affected by
geological constrains, depth, and the presence of organic and inorganic
compounds and gases in the lithosphere.
Microbes can use both inorganic (eg, hydrogen, minerals, or abiotic
carbon compounds) and organic energy sources (originating from
Deepterrameta 11
degradation of living organisms). Organic carbon sources can provide both

energy and carbon. A more detailed description of carbon cycling in deep
terrestrial subsurfaces is given in Section 1.5.2.
The subsurface geological layers may receive dissolved organic carbon
flow from the surface where the sun is the primary energy source. In addi-
tion, dead microbial biomass that formed as a consequence of the normal
cycle of life provides organic carbon to the microbes, thereby also support-
ing the growth of heterotrophic microorganisms. The hydrocarbons (eg,
methane and longer-chain hydrocarbons) that are present in deep Earth
crust reservoirs can also feed microbial life.
Geochemical energy in the form of hydrogen and minerals is generally
considered the primary source of energy for deep subsurface microorgan-
isms (Ahonen et al., 2011; Chivian et al., 2008; Fredrickson & Balkwill,
2006; Fredrickson et al., 1997; Gebert, Knoblauch, Gadd, Pfeiffer, & Dilly,
2011; Lin et al., 2014; Mayhew, Ellison, McCollom, Trainor, &
Templeton, 2013; McCollom & Amend, 2005; Meyer-Dombard et al.,
2014; Pedersen, 2010, 2000, 2012). Hydrogen can be generated by abiotic
and biotic reactions. Abiotic global hydrogen production in the oceanic
crust as a result of watererock interactions is estimated to be c.
1011 moles per year and continental hydrogen production was considered
negligible (Bach & Edwards, 2003; Canfield, Rosing, & Bjerrum, 2006).
Sherwood Lollar, Onstott, Lacrampe-Couloume, and Ballentine (2014)
recently reviewed global hydrogen production on the basis of new pub-
lished data pertaining to both the oceanic and continental lithosphere.
The calculations included watererock interactions and radiolysis of water
caused by the radioactive decay of uranium (U), thorium (Th), and potas-
sium (K) in the Precambrian ultramafic and mafic rocks. According to the
revised calculations, the upper limit of hydrogen production in ultramafic
and mafic rocks is 0.78e1.8 1011 mol per year, and the lower limit is
0.2e0.4 1011 mol per year: as much as in marine ecosystems. The con-
tinental crustal surface area represents c. 30% of the globe and is therefore
contributing considerably to global hydrogen production.
Hydrogen production by abiotic reactions during watererock interac-
tions is mediated at least by the following processes: (1) radiolysis of water,
(2) hydration of iron silicate minerals, and in particular (3) hydration of ul-
tramafic rocks (serpentinization) in the oceanic crust at the plate boundaries.
In addition, hydrogen is reported to form as an intermediate compound of
the organic carbon biodegradation processes, where volatile fatty acids and
hydrogen are typical intermediate compounds (Mayhew et al., 2013).
The following are the reactions producing abiotic hydrogen:

1. Radiolysis of water due to the natural radioactivity (U, Th, and K) of
the rock:
H2 O / OH þ H
2. Hydration of iron silicate minerals (Sherwood Lollar et al., 2014):
3FeOðin silicatesÞ þ H2 O / Fe3 O4 ðmagnetiteÞ þ H2 ðaqÞ
3. Serpentinization of olivine in ultramafic rock (Abrajano et al., 1990;

Brazelton, Morrill, Szponar, & Schrenk, 2013):
3Fe2 SiO4 ðFe olivineÞ þ 2H2 O / 3SiO2 þ 2Fe3 O4 ðmagnetiteÞ

þ 2H2 ðaqÞ
2Mg2 SiO4 ðMg olivineÞ þ 3H2 O / Mg3 Si2 O5 ðOHÞ4 ðserpentineÞ

þ MgðOHÞ2 ðbruciteÞ
ðFe; MgÞ2 SiO4 ðolivineÞ þ H2 O þ CO2 / Mg3 Si2 O5 ðOHÞ4

ðserpentineÞ þ Fe3 O4 ðmagnetiteÞ þ CH4
Hydrogen formation due to radiolysis of water can be calculated from the

concentration of radioactive compounds (M€ uller, 2015; Ringelberg, 1997).
The serpentinization process involves both Fe and Mg end members
present in a solid solution in the olivine mineral (Mayhew et al., 2013). As
a result, a complex mixture of secondary minerals is formed, including one
or more minerals of the serpentine group (antigorite, lizardite, or
chrysotile), magnetite, or brucite (group 3 reactions). This mixture forms a
rock called serpentinite. Hydrogen is specifically formed during serpentiniza-
tion of iron-olivine (fayalite), which is hydrated owing to the oxidation of
reduced iron present in the mineral. Thus, iron seems to be the key compo-
nent that catalyzes abiotic formation of hydrogen (Amend, McCollom,
Hentscher, & Bach, 2011; Mayhew, Lau, McCollom, Webb, & Templeton,
2011; Mayhew, Webb, & Templeton, 2011; Wright, Williamson, Grasby,
Spear, & Templeton, 2013). Serpentinization yields alkaline fluids
(pH 10) with unique chemical properties (ie, Ca-Mg-OH- waters) and
hydrogen gas, which may also result in abiogenic production of hydrocarbons
by chemosynthetic metabolism (ie, FischereTropsch-type synthesis)
(Abrajano et al., 1990; Mayhew et al., 2013)
Deepterrameta 13
Serpentinization is generally reported to occur at elevated temperatures

(50e600 C) (Brazelton, Nelson, & Schrenk, 2012; Evans, 2004; Evans,
Johannes, & Oterdoom, 1976; Mayhew, Lau, et al., 2011; Mayhew,
Webb, et al., 2011; Szponar et al., 2013). In laboratory experiments,
Mayhew, Lau, et al. (2011), Mayhew, Webb, et al. (2011) studied iron oxida-
tion in peridotite rock, pyroxene, olivine, and magnetite minerals with anoxic
fluids at 55 and 100 C and monitored hydrogen gas production. Studies on
the changes in the speciation of iron involving synchrotron-based micro-X-
ray fluorescence and X-ray absorption near-edge structure spectroscopy
showed that serpentinization may also occur at a lower temperature than
expected earlier, via different, still unknown processes (Kelley et al., 2005;
Mayhew, Lau, et al., 2011; Mayhew, Webb, et al., 2011; Sanchez-Murillo
et al., 2014). If this notion is confirmed, then serpentinization have occurred
in environments at various temperatures in the Earth’s lithosphere now and in
the past (Brazelton et al., 2013, 2012; Meyer-Dombard et al., 2014; Schrenk
et al., 2013). Understanding serpentinization of ultramafic rocks means link-
ing life sciences with geology; thus, serpentinization may be a fundamental
process in evolution and in the startup of prebiotic and biotic environments.
Fig. 2 shows abiotic and biotic sources of hydrogen and their connection to
microbiological processes and carbon cycling.
In general, utilization of chemical energy in biological systems involves
transfer of electrons in oxidationereduction (redox) reactions from a pri-
mary electron donor, which gives up its energy, to a terminal electron
acceptor having the most negative electron potential in the system (Amend
et al., 2011; Konhauser, 2007; Shock & Holland, 2004). Many geomicrobes
are chemolithotrophs owing to their ability to utilize geochemical energy
by oxidizing inorganic compounds such as hydrogen, iron, manganese,
sulfur, and nitrogen (Hoehler, 2004; Morita, Mitsialis, Koike, Liu, &
Kourembanas, 1997). Energy in the deep Earth’s crust can also be gained
from organic compounds; for example, fermenting microorganisms were
found in the black schist layer of the Outokumpu formation at the depth
of 1000e1300 m (Nyyss€ onen et al., 2014).
Several geomicrobiological books describe in detail the redox processes
connected to geological environments (Barton, Mandl, & Loy, 2010;
Ehrlich, 2002; Konhauser, 2007; Rosling, Finlay, & Gadd, 2009). The redox
reactions are active in variable environments from aerobic surface conditions
to anaerobic deep subsurface conditions, to increasingly reducing conditions
where molecular oxygen is depleted, and then preferentially nitrate and nitrite
are reduced to molecular nitrogen. Oxygen depletion happens already at very
small depths below the surface. If organic carbon is present, oxygen consump-
tion and a change of the redox state to an anaerobic one is rapid. Several other
terminal electron acceptors are involved after nitrate and nitrite are depleted.
Manganese, ferric iron, sulfate, and carbon dioxide are then used as terminal
electron acceptors in respiration (Mayer & M€ uller, 2014) Fig.1.
So far, energy mechanisms of deep-sea ecosystems have received more
attention than did terrestrial deep subsurface ecosystems (Amend et al., 2011;
Amend & Teske, 2005; M€ uller, 2015). Different thermodynamic models
Figure 2 Hydrogen and carbon cycling in the deep terrestrial subsurface.

Deepterrameta 15
have been used to quantify the energetic mechanisms of microbial metabolism

in hydrothermal vents. Up to 150 hydrothermal vents are estimated to be pre-
sent along the 60,000-km-long mid-ocean ridge system (McCollom & Shock,
1997; Shock & Holland, 2004). According to energy calculations for these eco-
systems, most energy is provided by aerobic respiration at a low temperature
(less than 40 C) and anaerobic respiration linked to sulfate reduction and
methanogenesis at elevated temperatures (McCollom & Shock, 1997; Shock
& Holland, 2004).
1.5.2 Deep Carbon Cycling

Understanding the sources of carbon, whether derived from the surface,
crust, or mantle is essential for elucidation of the microbial biogeochemistry
of the deep subsurface. The quantity of dissolved organic carbon originating
from decaying organisms is very low and is present in various forms, from
simple amino acids to complex high-molecular weight compounds
(Neff & Asner, 2001). Total organic carbon content, which is w0.4e
20.0 mol/kg in the terrestrial deep subsurface, correlates well with the abun-
dance of biomass, meaning that the organic carbon is tied up in the microbial
biomass (Lipp, Morono, Inagaki, & Hinrichs, 2008). The whole ecosystem
may potentially be controlled by viruses, which can lyse microorganisms in
deep subsurfaces (Eydal, J€agevall, Hermansson, & Pedersen, 2009; Labonté
et al., 2015; Roux et al., 2014). The lysed biomass can be again degraded by
deep subsurface chemoorganotrophic heterotrophic microorganisms to car-
bon dioxide and methane. The low amount of dissolved organic carbon in
the deep subsurface implies that decaying organic compounds are effectively
consumed by the microbial populations already at small depths.
Hydrocarbons (methane and longer-chain hydrocarbons) are probably
the most abundant carbon sources in the deep terrestrial biosphere. It was
suggested that the hydrocarbons in the Earth’s crust may have been formed
via both biogenic (eg, oil) and abiotic routes (Sephton & Hazen, 2013).
Evidence of abiotic synthesis of hydrocarbons has been demonstrated theo-
retically and experimentally (McCollom, 2013). According to the present
knowledge, the Earth crust hydrocarbons can evolve via two major path-
ways: from the breakup of organic matter or via organic synthesis of small
carbon- and hydrogen-containing molecules and further polymerization
of these molecules into more complex ones. Abiotic formation of methane
has been reviewed recently (Etiope & Sherwood Lollar, 2013; McCollom,
2013), and the whole methane cycling was reviewed by Kiet€av€ainen and
Purkamo (2015).
The deep earth gas hypothesis postulates that abiogenic methane that is
released from the mantle migrates to the crust, where it acts as an energy
source for microorganisms constituting a deep microbial ecosystem: the
deep hot biosphere (Lloyd et al., 2013; Sephton & Hazen, 2013). Stable
isotope studies have confirmed that abiotic formation of methane is the
dominant process in deep terrestrial subsurfaces in the Fennoscandian Shield
and in Canada, as opposed to biological formation via methanogenesis
(Kiet€av€ainen & Purkamo, 2015). Abiotic formation is expected to occur
via organic FischereTropsch-type or Sabatier-type reactions and can also
result in abiotic formation of other hydrocarbons. The primary production
in the deep biosphere is believed to rely on organic molecules synthetized
abiotically from carbon dioxide and hydrogen in geochemical processes
(Amend & Teske, 2005; Schrenk et al., 2013). Biological formation of
methane in the deep biosphere is due to methanogenic microorganisms,
which are all included in the phylogenetically diverse group Euryarchaeota
in the domain Archaea.
1.5.2.1 Microbes Involved in Carbon Cycling

Methane-cycling microbes are essential members of deep subsurface micro-
biomes. Seven methanogenic, ie, methane-producing archaeal orders are
known to exist, among which Methanopyrales, Methanococcales, Metha-
nomicrobiales, Methanocellales, and Methanoplasmatales all use hydrogen
and carbon dioxide as an energy source and carbon source, respectively.
Methanosarcina spp. are more versatile in their carbon utilization than the
other methanogens and can utilize diverse organic compounds (Borrel
et al., 2013; Liu & Whitman, 2008; Paul, Nonoh, Mikulski, & Brune,
2012). Methanoplasmatales have been recently found to utilize methanol,
methylamines, and hydrogen (Borrel et al., 2013; Paul et al., 2012). Metha-
nogens are present in the most nutrient-depleted deep anaerobic environ-
ments where all electron acceptors, except CO2, have been depleted
(Kiet€av€ainen & Purkamo, 2015).
The mcrA gene (a methyl-coenzyme M reductase subunit) is often used
as a marker for detection and characterization of methanogens in the
environment. This gene is involved in the methane-metabolic pathway
and production of methane (Bomberg, Lamminm€aki, & It€avaara, 2015;
Bomberg, Nyyss€ onen, Pitk€anen, Lehtinen, & It€avaara, 2015; Kiet€av€ainen
& Purkamo, 2015; Miettinen, Bomberg, et al., 2015; Rajala et al., 2015).
In addition, heterodisulfide reductases are enzymes needed for the final
step in methanogenesis and can be either membrane bound or cytoplasmic.
Deepterrameta 17
According to Liu and Whitman (2008), hydrogenotrophic methanogens are

more abundant than acetoclastic or methylotrophic methanogens.
Methanotrophs are methane-cycling microbes consuming methane to
gain energy and carbon. In the near-surface oxic/anoxic interface of the
lithosphere, aerobic methanotrophs are involved in oxidation of methane
to carbon dioxide, thus reducing methane emissions into the atmosphere
(Smith, Schiater, Mohamund, & Agrawal, 2007). Although a tremendous
amount of methane is available in the Earth’s crust, only a few species can
oxidize it anaerobically (Hanson & Hanson, 1996; Haroon et al., 2013;
Knittel & Boetius, 2009; Nyyss€ onen et al., 2012). The biochemical
mechanisms of anaerobic methane oxidation are still unclear. Anaerobic
methanotrophs are mostly Archaea except for Methylomirabilis oxyfera,
which is a bacterium (Haroon et al., 2013). Electron acceptors in
oxygen-depleted environments include nitrite, nitrate, iron, manganese,
and sulfate (Hanson & Hanson, 1996; Kiet€av€ainen & Purkamo, 2015;
Knittel & Boetius, 2009). Recently, multiple studies were published on
anaerobic methanotrophic (ANME) archaea, which live in syntrophic con-
sortia with sulfate reducers and are abundant in sulfateemethane transition
zones in deep terrestrial groundwater (Bomberg, Nyyss€ onen, et al., 2015;
Brazelton, Schrenk, Kelley, & Baross, 2006; Mayer & M€ uller, 2014;
Nyyss€ onen et al., 2012). Milucka et al. (2012) recently showed that no syn-
trophic growth between sulphate-reducing bacteria and ANME is needed
but ANME can carry out in addition to anaerobic methane oxidation also
dissimilatory sulphate reduction to form disulphide and other zero-valent
sulphur compounds.
Deep subseafloor sediments are typical habitats for ANME archaea
(Knittel, Losekann, Boetius, Kort, & Amann, 2005; Lever et al., 2013). In
terrestrial crustal subsurfaces, ANME archaea were recently detected in
boreholes of the Fennoscandian Shield in Olkiluoto, Finland (Bomberg,
Lamminm€aki, et al., 2015; Bomberg, Nyyss€ onen, et al., 2015; Miettinen,
Bomberg, et al., 2015; Miettinen, Kiet€av€ainen, et al., 2015; Nyyss€ onen
et al., 2012). In these processes, sulfates generally serve as terminal electron
acceptors during anaerobic methane oxidation, which may result in sulfide
formation (Knittel & Boetius, 2009). Sulfides may cause problems for long-
term storage of spent nuclear fuel, which is planned in Olkiluoto (It€avaara,
Vehkom€aki, & Nousiainen, 2008). Sulfate reducers Deltaproteobacteria,
Desulfosarcina, and Desulfococcus are often associated with anaerobic methane
oxidizers from groups ANME-1 and ANME-2 (Knittel & Boetius, 2009).
In addition, ANME-2d archaea have been demonstrated to grow
syntrophically with ammonia oxidizers in an anaerobic environment

(Haroon et al., 2013). ANME-3 archaea are typically associated with Desul-
fobulbus-type sulfate reducers (Knittel & Boetius, 2009). Methylomirabilis oxy-
fera, the only bacterial representative of all known anaerobic methane
oxidizers, reduces nitrite while simultaneously producing oxygen, which
is then used in aerobic oxidation of methane (Ettwig et al., 2010). Other
electron acceptors such as Nitrate, Nitrite, Iron, Manganase and Sulfate in
oxide minerals can also be used as terminal electron acceptors for anaerobic
methane oxidation (Beal, House, & Orphan, 2009).
Terminal electron acceptors in anaerobic methane oxidation are
Nitrate : 5CH4 þ 8NO3 þ 8Hþ / 5CO2 þ 4N2 þ 14H2 O
Nitrite : 3CH4 þ 8NO2 þ 8Hþ / 3CO2 þ 4N2 þ 10H2 O
Iron : CH4 þ 8FeðOHÞ3 þ 15Hþ / HCO3 þ 8Fe2þ þ 21H2 O
Manganese : CH4 þ 4MnO2 þ 7Hþ / HCO3 þ 4Mn2þ þ 5H2 O
Sulfate : CH4 þ SO4 2 / HS þ HCO3 þ H2 O
1.5.3 Microbial Activity

How active are the deep subsurface microorganisms? What is the activity
level of dormant microorganisms or microorganisms that have access to
only the minimal maintenance energy? What activity level is needed for
reproduction and for a specific environmental function?
Because of the scarcity of energy sources, microbial metabolic activity in
deep subsurface habitats is very slow. Based upon geochemical models, some
research groups studying the subsurface biosphere have deduced the average
cellular doubling time: hundreds to thousands of years (Lever et al., 2015;
Onstott et al., 2014). Racemization is a paleobiological method for estima-
tion of age of a specimen. All living organisms use amino acids of the “L”
configuration, but after death, the configuration of the amino acids is no
longer maintained, and the ratio of D-to L-amino acids moves from a value
near 0 toward an equilibrium value near 1.0. Thus, the ratio of D-to L-amino
acids allows researchers to estimate the time when a specimen died
(Lever et al., 2015). This method has also been used to estimate the in
situ average cellular protein turnover or doubling time of metabolically
active microorganisms as a function of abundance of cellular amino acids,
Deepterrameta 19
the D/L ratio of cellular aspartic acid, and the in vivo racemization rate of
aspartic acid. Application of this method to planktonic microbial commu-
nities collected in deep fractures in South Africa yielded maximal cellular
turnover time of amino acids: 89 years for the depth of 1 km at 27 C,
and 1e2 years for the depth 3 km at 54 C. These values of turnover time
are much smaller than previous estimates based upon geochemical
arguments (Onstott et al., 2014). The aspartic acid racemization rate at
higher temperatures yields cellular protein doubling time that is consistent
with the survival period of hyperthermophilic strains and predicts that at
85 C, cells must replace proteins every 2 days to maintain enzymatic activ-
ity. Such a high maintenance requirement may be the principal limit on the
abundance of living microorganisms in the deep hot subsurface biosphere as
well as a potential limit on their activity. The measurement of the D/L ratio
of aspartic acid in biological samples may become an effective analytical tool
for deep continental- and oceanic-crust settings where geochemical models
of carbon turnover time are poorly constrained.
Metabolic conversions take place within individual cells, but in complex
ecosystems, metabolic processes also occur at the population level in syntro-
phy of several organisms. The transfer of electrons through the biosphere can
result from a variety of biogeochemical cycles. Understanding how physics
and chemistry constrain life is central to understanding the metabolism. The
origin and evolutionary modularity of metabolism have been discussed else-
where (Braakman & Smith, 2013). Those authors stated that more diverse
chemistry and greater evolutionary dynamics exist in core metabolic path-
ways because of the higher mass flux.
Microbes tend to get attached to the fracture surfaces to interact with the
rock. Nonetheless, most studies have been focused on planktonic cells of
borehole water because of difficulties with direct sampling of rock surfaces.
Pedersen (2013) studied attachment of microbes and their activation in the
flow cell circulating systems in Onkalo, Finland. Similar in situ cultivation
methods for subsurface microorganisms have been used at a depth of
1474 m in Evander Gold Mines in South Africa in order to study methano-
genic, Fe3þ-, and sulfate-reducing microorganisms (Silver et al., 2010). This
kind of in situ growth systems allow the microbiomes to benefit from
specific electron donors and acceptors in natural ecosystems (at ambient
pressure and temperature), where gases, salts, and minerals are present. After
sampling, researchers can study their diversity and functions by molecular
methods. These methods have expanded our understanding of the diversity
of species and their functions in relation to the environment.
2. EXPLORING THE DIVERSITY OF TERRESTRIAL DEEP

SUBSURFACE MICROBIOMES
Only a few deep terrestrial sites have been thoroughly studied, and
as such, the biogeography of the deep terrestrial biosphere remains largely
unknown. Species distribution in deep terrestrial sites in Olkiluoto,
Finland (Bomberg, Lamminm€aki, et al., 2015; Miettinen, Kiet€av€ainen,
et al., 2015; Nyyss€ onen et al., 2012), and in South African gold mines
(Takai, Moser, DeFlaun, Onstott, & Fredrickson, 2001) has been found
to vary greatly. In general, sampling depth does not appear to predict the
species composition of the deep subsurface microbial communities.
Instead, biogeochemical parameters, such as pH and availability of specific
electron acceptors and donors, appear to have more influence on the
composition of these microbial communities. The deep terrestrial
biosphere contains unexpectedly rich biodiversity and includes representa-
tives of all three domains of life, ie, archaea, bacteria, and eukaryotes as well
as viruses.
2.1 Bacteria
Proteobacteria constitute the largest and phenotypically most diverse divi-
sion among prokaryotes (Gupta, 2000) and represent nearly a half of the
partial and complete prokaryotic genomes hosted at the National Center
for Biotechnology Information (NCBI; ftp://ftp.ncbi.nlm.nih.gov/
genomes/GENOME_REPORTS/prokaryotes.txt). Thus, it is not sur-
prising that Proteobacteria are also abundant in the deep terrestrial
biosphere. Proteobacteria are considered a dominant microbial clade of
the shallow layers of the Fennoscandian Shield, where their functional roles
are connected to such processes as nitrogen fixation and oxidation of iron,
sulfur, and methane. At greater depths, however, under more reducing
environmental conditions, other bacterial taxa belonging to such phyla as
Firmicutes, Tenericutes, and Actinobacteria are more prevalent (It€avaara,
Nyyss€ onen, Bomberg, et al., 2011; Nyyss€ onen et al., 2014; Purkamo
et al., 2013; Purkamo, Bomberg, Kiet€av€ainen, et al., 2015; Purkamo,
Bomberg, Nyyss€ onen, et al., 2015; Sohlberg et al., 2015). Similar findings
have been reported out of South Africa (Magnabosco et al., 2014).
Notably, the first known single-species ecosystem was reported at the
depth of 2.8 km in a South African gold mine (Chivian et al., 2008). In
this ecosystem, domain-crossing horizontal gene transfer of crucial genes
(such as those related to nitrogen fixation) from archaea to bacteria appears
Deepterrameta 21
to have enabled the lone survival of the firmicute Candidatus Desulforudis

audaxviator (Chivian et al., 2008).
Notably, Brown et al. (2015) recently isolated numerous novel bacterial
phyla that they named candidate phyla radiation (CPR) bacteria from
groundwater. CPR bacteria have previously evaded detection mostly
because of their ultrasmall cell size and insertions in their 16S ribosomal
RNA (rRNA) genes. Brown et al. (2015) estimated that CPR bacteria,
which are mainly obligate fermenters that mediate carbon and hydrogen
cycles, comprise more than 15% of the bacterial domain. The prevalence
of CPR bacteria in the deep terrestrial biosphere is unknown.
2.2 Archaea
Archaea are similar to bacteria in morphology and size, but their membranes
differ from those of bacteria and eukaryotes. Many archaea are extremophilic
microorganisms that can be found in extreme environments such as hot
springs and halophilic, alkaline, or acid environments and in the deep
biosphere. Archaea can utilize a variety of electron acceptors and donors
and play an important role in global biochemical cycles. For example,
Euryarchaeotal ANME microbes, which are close phylogenetic relatives of
methane-producing archaea, participate in anaerobic oxidation of methane
with various electron acceptors including nitrate, manganese, and iron
(Boetius et al., 2000; Haroon et al., 2013; Knittel & Boetius, 2009; Beal
et al., 2009). In Finland, a 16S rRNA-based survey of the deep biosphere
of Olkiluoto (a geological disposal site for nuclear waste) revealed that
archaea make a big contribution to the total microbial community sizes,
particularly in proximity to a sulfate- and methane-rich water-mixing zone
(the so-called sulfateemethane transition zone). Euryarchaea was found to
be the dominant archaeal phylum at 19 sampled boreholes and depths, except
for a single sample, where Crenarchaeota were the most abundant archaeal
phylum (Miettinen, Bomberg, et al., 2015; Miettinen, Kiet€av€ainen, et al.,
2015). Euryarchaea are mainly represented by Methanobacteria, Methano-
microbia, and Methanoplasmatales Thermoplasmata (Dridi, Fardeau,
Ollivier, Raoult, & Drancourt, 2012; Poulsen et al., 2013). Likewise, diverse
archaeal communities have been detected in South African gold mines. In
fact, multiple archaeal groups were first detected in South African gold mines
and are named accordingly, eg, SAGMEG (South African Gold Mine Eur-
yarchaeotic Group) and SAGMEG (South African Gold Mine Crenarch-
aeotic Group). In South Africa, archaea are most abundant at depths of
2.7 km (17.7% of a total community), 870 m (26.1%), and 1.8 km
(74.4%), with the latter sample water being characterized by a particularly

high concentration of sulfate and nitrate (Takai et al., 2001). Recently,
members of the Euryarchaeotal SM1 lineage, which can form near-single-
species biofilms in the subsurface, were found to have a novel reductive
acetyl-coenzyme A (CoA) pathway (Probst et al., 2014).
2.3 Eukaryotes
Fungi are best known as degraders of organic matter (saprotrophs), symbiotic
partners of plant species, and anaerobic fermenters (eg, yeast). Rock-
inhabiting fungi are known to oxidize or reduce minerals and can grow
on the surface of a rock or in small pores and cavities within rocks (Burford,
Kierans, & Gadd, 2003; Gadd, 2010). The role and prevalence of fungi in
the deep terrestrial biosphere are still largely unexplored. Nonetheless,
they have been found to be a part of the subseafloor ecological environments
(Nagano & Nagahama, 2012). Recently, it was recognized that diverse and
active fungal communities exist in the crystalline bedrock of the Fennoscan-
dian Shield (Sohlberg et al., 2015). In Olkiluoto boreholes, fungi belonging
mainly to Ascomycota but also Basidiomycota and Chytridiomycota were
detected at all sampled depths. These fungal taxa were mostly the same as
those that have also been detected in deep-sea environments, eg, Sordario-
mycetes, Eurotiomycetes, Dothideomycetes, Microbotryomycetes, and
Tremellomycetes (Sohlberg et al., 2015). The functional role of fungi in
deep subsurfaces has yet to be studied.
As expected, the majority of the deep terrestrial biosphere eukaryotes are
unicellular organisms such as diatoms. Nonetheless, Metazoa and Viridiplan-
tae rRNA sequences have also been detected in anoxic marine subsurface
samples (Orsi, Biddle, & Edgcomb, 2013). Another unexpected finding is
the detection of microscopic asexually reproducing bacteria-grazing Nema-
toda, Halicephalobus mephisto, in 3000- to 12,000-year-old palaeometeoric
fracture water from the depth 0.9e3.6 km in a South African gold mine
(Borgonie et al., 2011).
2.4 Viruses
The deep subsurface virosphere was recently reviewed elsewhere (Anderson,
Brazelton, & Baross, 2013). Viruses are estimated to kill 10e20% of oceanic
planktonic biomass every day (Evans & Brussaard, 2012; Suttle, 2007). These
infections have a major impact on composition of the oceanic microbial
community and its evolution and global geochemical cycles (Jover, Effler,
Buchan, Wilhelm, & Weitz, 2014). A similar influence can be expected
Deepterrameta 23
from the deep-biosphere viruses with a potential impact on carbon cycling,

for example, via lysis of microbial cells. Members of ancient order Caudo-
virales (bacteriophages), which represents the most abundant viral group,
have been detected in deep-subseafloor culture collections (Engelhardt,
Sahlberg, Cypionka, & Engelen, 2011), and the presence of viral particles
in subsurface sediments has been confirmed by electron and epifluorescence
microscopy (Engelhardt, Kallmeyer, Cypionka, & Engelen, 2014). Lytic
Podoviridae from deep granitic groundwater have been characterized by
Eydal et al. (2009), and viral proteins of Caudovirales have been detected
in terrestrial deep subsurface metagenomic studies (Nyyss€
onen et al., 2014).
3. SAMPLING OF THE DEEP BIOSPHERE

If direct studies of the deeper parts of the Earth are to be conducted,
there must be adequate means of observation and collection of samples.
Underground mines and deep drilling sites provide such opportunities. At
present, the deepest borehole is 12,262 m deep and was drilled in the
Kola Peninsula, Russia (Butler, 1994), whereas the deepest mines reach
the depth of 4 km in South Africa (Borgonie et al., 2011; Davidson et al.,
2011; Magnabosco et al., 2014).
Our globe is under intensive drilling: both industrial and scientific drilling
projects are undertaken at the ocean bottom and on continents (Ahonen
et al., 2011; Inagaki & Orphan, 2014; Kieft et al., 2015). Industrial drilling
projects are aimed at exploitation of oil and gas reserves or metallic ores.
Most of the exploratory boreholes for mineral deposits are less than 1 km
deep, whereas in the oil industry, it is routine to drill down to (add) 2e
3km. Because of the commercial objectives, the scientific aims are often
not fully appreciated, technically or geologically. On the other hand, the ma-
jority of groundwater-related research projects worldwide have utilized exist-
ing boreholes and drill cores to benefit from the preexisting huge investments,
often in good cooperation with the private sector. Despite the compromises,
which may have been necessary to accept, this approach has been successful
and has helped to implement numerous research projects.
On the other hand, scientific drilling is focused on finding answers to
specific questions. In this case, the site, drilling methods, and all other pro-
tocols can be optimized to serve the scientific objective as effectively as
possible. Nuclear waste disposal programs have promoted basic hydrosphere
research in various geological and climatic environments to support their
data needs for long-term safety assessments. For example, Posiva in Finland,
SKB in Sweden, and NWMO in Canada have conducted extensive research
programs in their homelands and in Arctic areas (Claesson Liljedahl, et al.,
2015; Harper et al., 2011; Hobbs, Frape, Shouakar-Stash, & Kennell,
2011; Nilsson et al., 2011; Pitk€anen, Luukkonen, & Partamies, 2003; Stotler
et al., 2011; Stotler, Frape, Ruskeeniemi, Pitk€anen, & Blowes, 2012). Major
scientific deep-drilling activities are coordinated by the Integrated Ocean
Drilling Program (IODP http://www.iodp.org) and the International Con-
tinental Scientific Drilling Program (ICDP http://www.icdp-online.org).
They also promote collaboration of multidisciplinary science communities.
To date, ICDP has supported more than 30 drilling projects investigating
plate boundaries, volcanoes, impact structures, and other topics. The work
with deep subsurface boreholes and especially with superdeep boreholes
requires specific downhole research equipment and groundwater sampling,
which tend to increase the budget.
Deep biosphere studies have become an increasingly important part of
drilling objectives. Even though vast data on deep subsurface microbiomes
have already been generated, the knowledge about microbial functions is still
lacking. Most studies have been focused on identification of biodiversity of
deep subsurface microbiomes. Novel biodiversity and metabolic processes,
biogeochemical cycling, and the impact of human activities on these ecosys-
tems are still poorly understood. In the future, more microbial observatories
must be constructed to monitor online geobiological processes that are linked
to the development of early life. The CROMO facility in California, where
the role of serpentinization is studied in a borehole drilled in 2011 (Cardace
et al., 2013), may serve as a guide for the construction of such monitoring
systems. Particularly, prebiotic organic compounds that tentatively formed
during serpentinization will be monitored (Cardace et al., 2013). At the
time of this writing, there have been some discussions regarding submission
of a proposal to ICDP to drill the first deep borehole specifically designed
for microbial research (http://deep-biosphere.icdp-online.org/).
3.1 Groundwater Sampling

Microbes, hydrogeochemistry, dissolved gases, and the bedrock are coupled
to one another, and any biosphere research should include research on the
other topics above because only comprehension of the whole system can
serve as a sound basis for profound interpretations. This broad approach
has been adopted in many projects, because often, the research is initially
focused on groundwater and only later is extended to microbes.
Deepterrameta 25
First, it is beneficial to proceed in this order because these studies share

the strict requirements for the drilling activities as compared to standard
exploration drilling. For example, the use of potentially disturbing chemicals
is banned. Second, groundwater research provides the necessary geochem-
ical background information. Third, irrespective of how cautiously the
drilling is done, it temporarily disturbs the natural conditions at some depths.
One of the main issues is the necessity to use drilling water. This means that a
large volume of surface water is pumped into depth during drilling and some
of this water intrudes into the bedrock and is mixed with the formation
groundwater.
The backflow of foreign water into the hole is naturally a challenge for
the hydrogeochemical and microbe sampling. Quality assurance is a neces-
sary step before sampling for more sophisticated and expensive studies.
There are geochemical and isotopic geochemical tools (d18O, d2H, and
tritium assays) to evaluate the level of contamination, but the microbial
representativeness is more difficult to determine.
A standard procedure for groundwater research drilling is to characterize
the chemistry of the drilling water, to mix inert tracers with the drilling wa-
ter and to keep track of the water consumption (SKB, 2010). Once the
tracer concentration is known both in the drilling water and in the sample,
it is possible to monitor the cleaning of the drill hole and to determine the
correct composition for the groundwater. Experience has shown that it may
take years before the drilling-water contamination is reduced to an accept-
able level, unless active cleaning pumping is conducted.
Sampling equipment and other downhole instruments have been origi-
nally designed for groundwater or geological studies without consideration
of the asepticity requirements of microbiology. There is an obvious risk that
the instruments transfer microorganisms from the surface down to the drill
hole. Although it has been estimated that microorganisms from the aerobic
surface environment cannot survive harsh chemolithotrophic high-pressure
conditions in the deep subsurface for several years, the contamination in the
form of organic compounds and horizontal gene transfer from the surface
cannot be ruled out.
In practice, the purity requirements are difficult to meet during drilling
and sampling in field conditions. Microbiologists must often put up with
continued pumping and successive samplings in the hope that the represen-
tativeness of the samples will increase with time. Nonetheless, there are
different sampling methods, and for some of them, the purity/aseptic con-
cerns are easier to manage than for others. In the Finnish nuclear waste
disposal program, the nuclear waste management company, Posiva has used
and developed a number of sampling and monitoring protocols (Pitk€anen
€
et al., 2007; Ohberg, 2006).
The most common sampling methods in hydrogeochemical research are
(1) direct pumping of groundwater, (2) tube sampling, (3) packer sampling,
and (4) pressurized sampling devices/retrievers.
1. Direct pumping of water from drill holes is a low-cost method and is
technically easy to conduct. Nevertheless, there are major concerns
related to representativeness of the samples. In undisturbed drill holes,
water is often at a stagnant stage and in contact with the fresh rock
face of the hole and with surface-derived organic matter as well as the
rock powder deposited at the bottom. Thus, the chemical and microbial
composition of the water may be abnormal, and such water should not
be sampled without cleaning pumping. Any deeper drill hole is likely to
intersect with several hydraulic zones, which may produce different types
of water. As the open-hole water column is agitated with pumping, the
waters from different sources are mixed in unknown proportions and
form “artificial” water. From the microbiological point of view, the
other concerns in open-hole sampling are related to the possible distur-
bance of the anoxiceoxic boundary and the drop of ambient pressure.
Technically speaking, microbiological sampling involving submersible
pumps is relatively easy due to the limited amount of equipment to be
cleaned.
2. Tube sampling was developed for sampling of a continuous water profile
from an open drill hole (Nurmi & Kukkonen, 1986). The sampler is
compiled from sections of polyamide tubes, which are connected with
shut-off valves, and the string is equipped with a back-pressure valve
and a weight at the bottom. The length of the sampler is fixed to match
the depth of the drill hole, and the length of the subsections is generally
25e100 m. A tube sampler is slowly lowered into the drill hole down to
the end of the hole and then is lifted up. Valves are closed as soon as they
reach the surface. The depth of each section is known, and the captured
water sample remains isolated from the atmosphere and pressurized until
a valve is opened. Tube sampling is a low-tech and low-cost method to
get the first impression about the layering and characteristics of the
groundwater.
From the microbiological perspective, the tube samplers are a challenge. A
considerable source of contamination is related to the tubing and valves.
Basso et al. (2005) studied the effect of chlorination treatments for
Deepterrameta 27
disinfection of the tubing and found that attached microorganisms indeed

pose a considerable contamination problem. Therefore, the microbiolog-
ical sampling campaigns should preferably utilize factory-new tubing
(It€avaara, Nyyss€ onen, Bomberg, et al., 2011; It€avaara, Nyyss€ onen,
Kapanen, et al., 2011). Ideally, the tubing and valves should be autoclaved
prior to the compilation of the string, and all components should be
handled only with sterile gloves.
3. Pumping of water samples from sections isolated by rubber discs or inflat-
able packers is a significant improvement and solves the quality problems
related to open-hole conditions. The general idea is to isolate the section
of interest from the rest of the drill hole and thus to prevent the mixing
along the hole and reduce the duration of cleaning pumping. Due to the
limited volume of the section, the cleaning pumping is effective, and the
samples are known to represent bedrock water.
Water is brought up from the sections through thin plastic or steel
tubing by means of narrow pumps installed in the extended upper parts
€
of the tubing (Ohberg, 2006). In other solutions, the water can be
driven up by gas applied through a special valve system (Freifeld,
2005). The arriving water can be collected into bottles, or if pressurized
conditions and isolation from the atmosphere are preferred, directly
into specific containers.
Anaerobic conditions and the ambient hydraulic pressure at the sampling
depth can be maintained during sampling with advanced packer sam-
plers, and the source-related uncertainties are minimized. The challenges
are again associated with cleaning of the numerous components of the
sampler, especially the tubing.
4. The forth approach for groundwater sampling is retriever-type sam-
plers. In principle, a sample container is lowered down to the sampling
level, water is allowed to fill the container, and then surface-operated
valves are closed, and the sealed sample container is brought up to
the surface. The sample is under in situ pressure and remains isolated
from the atmosphere. The sampling operation is conducted between
packers. In some types of equipment, the packers and the container
are in the same unit, whereas in other instruments, the container moves
inside a casing and is docked to a selected sampling port for sample
collection.
In the early 1990s, a PAVE-type sampler (pressurized water sampling
device) was developed by Posiva www.posiva.fi specifically for gas and
microbe sampling (Ruotsalainen, Alhonm€aki-Aalonen, Aalto, Helenius,
& Sellge, 1996). The wire line system has one or two inflatable packers,
one or more sterilized and vacuumed 250-ml pressure containers, a rub-
ber membrane pump, and some valves. Continued through-flow is
maintained for several hours prior to the sampling.
Retriever-type samplers have an advantage: sample water does not
flow through the extended length of the tubing. Thus, it is easier to
sterilize all the components that are in immediate contact with the
sampled water. From the microbiological point of view, this type of
samplers are currently the most promising option for collection of un-
contaminated samples.
3.2 Processing and Maintenance of Samples for

Microbiological Research
The required volumes and further processing of microbiological samples
depend on the aims of research. The cell density in the sample is routinely
determined by visualization of the cells with a fluorescent dye such as
4,6-diamidino-2-phenylindole (DAPI, Sigma) followed by counting
under an epifluorescence microscope or by flow cytometry (It€avaara
et al., 2008; It€avaara, Nyyss€ onen, Bomberg, et al., 2011; It€avaara,
Nyyss€ onen, Kapanen, et al., 2011; Rajala et al., 2015). Fixation of the
samples with paraformaldehyde prolongs the interval that can be allowed
between sampling and cell counting. The fixation also enables more
advanced studies such as detection of an individual gene or transcripts at
the cellular level, eg, using fluorescence in situ hybridization (Breuker,
K€ oweker, Blazejak, & Schippers, 2011). A viability assay with the LIVE/
DEADÒ BacLightÔ Bacterial Viability Kit (Thermo Fisher) has been
used for discrimination of active microorganisms from the rest of the pop-
ulation (eg, Nyyss€ onen et al., 2014).
Deep microbiome biomass for molecular biological research is typically
collected by filtration, preferably anaerobically. The amount of water needed
to obtain a sufficient amount of biomass can vary from one to hundreds of
liters (Chivian et al., 2008; It€avaara, Nyyss€
onen, Bomberg, et al., 2011; Silver
et al., 2012), depending on the objective (see Sections 4 and 5). Filtration can
be performed directly from the sampling tubes by allowing the pressure
generated in situ to push the water through a connected SterivexÔ filter
(Millipore) (It€avaara, Nyyss€onen, Bomberg, et al., 2011; It€avaara, Nyyss€
onen,
Kapanen, et al., 2011) Fig. 3. Alternatively, the water sample can be brought
to an anaerobic hood or glove box where filtration is performed by means
Deepterrameta 29
Figure 3 Field sampling for molecular microbiological analysis based on filtration of

microbial biomass during the sampling campaign in the Outokumpu deep borehole,
Finland. At the back Outokumpu deep borehole (2.5km).
of regular filtration systems, eg, polyethersulfone filters (Bomberg,

Lamminm€aki, et al., 2015; Bomberg, Nyyss€ onen, et al., 2015; Rajala et al.,
2015). The collected biomass should be immediately frozen on dry ice or
in liquid nitrogen to prevent changes in structure and activity of the microbial
community. Chemical preservation of the cells, eg, using commercial prod-
ucts such as RNALaterÒ (Thermo Fisher Scientific) has also been used (Lau
et al., 2014). Microbiomes in the bedrock pores and fracture surfaces have
not been well studied (Mason et al., 2010). This research may be possible
immediately after drilling activities when a fresh core is available. Just as the
water samples, rock samples should be kept frozen, and the sample for micro-
biological studies should be taken from the inner part of the core because of
contamination at the surface due to the drilling.
4. TERRESTRIAL DEEP SUBSURFACE MICROBIOMES:

METAGENOMICS
4.1 Metagenomics and Metatranscriptomics as Tools
for Exploring the Deep Subsurface Life
The vast majority of microbes are unculturable; thus, the traditional
culture-based methods are inadequate for community level analysis. Meta-
genomics represents molecular methods designed to analyze the biodiversity
and metabolic functions of microbial communities (reviewed by Vieites,
Guazzaroni, Beloqui, Golyshin, & Ferrer, 2009). The protocols rely on
direct isolation of genetic material (DNA), eg, from a soil or water sample.
Metagenomic DNA can be analyzed by various methods to obtain informa-
tion on the microbial-species diversity, metabolic pathways, and individual
enzyme genes. Metagenomics can be further expanded to metatranscrip-
tomics, where an RNA pool, which represents the set of expressed, ie, active
genes in a sample population, is isolated and analyzed (eg, reviewed by
Moran et al., 2013). Metatranscriptomics can give some clues about the
microbial processes that are active in the sample at the moment of isolation
(Johansson et al., 2013).
Good-quality, uncontaminated DNA or RNA is necessary for successful
metagenomics and metatranscriptomics. Important quality metrics include
concentration, purity, and integrity of the nucleic acids. Cell numbers in
terrestrial deep subsurface microbiomes are typically low (eg, see Section 5)
and can make the isolation of sufficient amounts of good-quality DNA or
RNA challenging. Sensitivity of traditional ultraviolet spectrophotometry
or agarose gel methods for assessment of the quantity and quality of nucleic
acid samples is often insufficient, and more advanced methods (eg, Qubit
and Agilent Analyzer) are more suitable. Nevertheless, metagenomics has
been successfully used for exploring the microbes and their functions in
the deep biosphere (Bomberg, Lamminm€aki, et al., 2015; Bomberg,
Nyyss€ onen, et al., 2015; Brazelton et al., 2012; Eiler et al., 2014; It€avaara,
Nyyss€ onen, Bomberg, et al., 2011; It€avaara, Nyyss€ onen, Kapanen, et al.,
2011; Lau et al., 2014; Nyyss€ onen et al., 2014; Orsi et al., 2013; Purkamo
et al., 2013; Rajala et al., 2015; Wang, Xia, Dong, & Dong, 2005). The pro-
tocols used for isolation of DNA or RNA from the deep-biosphere samples
include both traditional methods with organic solvent extraction steps and
commercial kits (eg, PowerWater (MOBIO) and NucleoSpin
(MachereyeNagel)) (It€avaara, Nyyss€ onen, Bomberg, et al., 2011; It€avaara,
Nyyss€ onen, Kapanen, et al., 2011; Lau et al., 2014; Nyyss€ onen et al.,
Deepterrameta 31
2014; Purkamo et al., 2013; Purkamo, Bomberg, Kiet€av€ainen, et al., 2015;

Purkamo, Bomberg, Nyyss€ onen, et al., 2015; Rajala et al., 2015).
4.2 Amplicon Sequencing

At present, the most commonly used metagenomic method that is used to
explore the species diversity and functions of the microbiomes in deep
terrestrial environments is amplicon sequencing. The amount of DNA or
RNA required for this type of approach is often low (<1 ng) as compared
to the direct metagenome or metatranscriptome sequencing (typically,
20e100 ng of DNA or RNA is needed). Due to amplification of genes
by PCR, the method is prone to biases related to primer specificity and
coverage, and as such, cannot be considered fully quantitative, as reviewed
by Sanschagrin and Yergeau (2014). This approach, however, provides valu-
able insights into the composition and life of microbial communities in the
deep bedrock (see Section 2).
4.2.1 Biodiversity
Woese et al. pioneered molecular biodiversity studies and classified cellular
life into three domains: archaea, bacteria, and eukaryotes (Woese, 1987;
Woese, Kandler, & Wheelis, 1990). Since then, rapid advancements in
molecular and computational methods have enabled the research into whole
microbial communities and thereby revolutionized the field of microbial
ecology. Prior to these approaches, microbial ecology was largely limited
to cultivation-based studies: they were quite inefficient because less than
1% of microbial species can be cultivated in the laboratory.
First sequencing-based microbial ecological studies largely relied on
cloning and 16S rRNA PCR with denaturing gradient gel electrophoresis
(DGGE) (Muyzer, de Waal, & Uitterlinden, 1993). PCR-DGGE was
used to separate the DNA fragments in a gel, which were then cut out
and purified for sequencing. This method has been widely used until the
advancement of high-throughput sequencing technologies in the early
2000s. Roche’s 454 pyrosequencing was then the most widely used
sequencing method particularly for amplicon sequencing but has now
been largely replaced by Illumina HiSeq and MiSeq (Illumina Inc.). Other
current sequencing technologies include the RS line of products from
PacBio, which output relatively long DNA sequence reads (up to 60 kbp)
as well as the ion line of products from Thermo Fisher Scientific, which
primarily compete with Illumina’s MiSeq platform. Up-and-coming
sequencing technologies include Oxford Nanopore’s MinIon: a device that

is not much larger than a matchbox.
Biodiversity of bacteria and archaea in the deep terrestrial biosphere has
been most commonly studied by amplicon sequencing of the small subunit
rRNA (16S rRNA) (Anderson, Sogin, & Baross, 2015; Bomberg,
Lamminm€aki, et al., 2015; Bomberg, Nyyss€ onen, et al., 2015; Chivian
et al., 2008; It€avaara, Nyyss€
onen, Bomberg, et al., 2011) (also see Sections
2.1 and 2.2). Generally, this approach involves amplification and sequencing
of a fragment of the 16S rRNA gene, with length varying from 100 to
400 bp depending on the sequencing technology. The 16S rRNA gene
contains both conserved and variable regions, which are useful for identifi-
cation and phylogenetic analysis of prokaryotes. Fungal biodiversity in the
deep terrestrial subsurface has been studied by sequencing the internal tran-
scribed spacer (ITS) region between the genes of small and large subunits of
rRNA, which is the recommended barcode sequence for identification of
fungi (Schoch et al., 2012; Sohlberg et al., 2015) (also see Section 2.3).
Biodiversity of a given sample can be identified from the sequence data.
When molecular protocols are used for studies on unculturable microbes,
the biodiversity measures are calculated on the basis of operational taxo-
nomic units (OTUs), eg, interpreted from 16S rRNA and ITS sequence
sets (Sogin et al., 2006). The OTUs are sequence groups or clusters that share
a defined level of sequence similarity (eg, 85%, 90%, or more; commonly,
97% for 16S rRNA amplicons) and are presumed to originate from the
same species. The diversity can be described, for example, by distribution
of the sequences within the OTUs (evenness) and by the amount of different
OTUs in the sample (richness). Rarefaction analysis (an average OTU count
when a sample is resampled multiple times) can be used to determine how
well diversity of the sample was captured. Various biodiversity indexes can
be calculated in order to compare the diversity among samples. For example,
the Shannon index (H0 ) imparts high values to samples where the number of
OTUs is high and/or OTUs are evenly distributed (Sogin et al., 2006).
Abiotic factors affecting the microbial community structure and their
connection to biodiversity are analyzed by means of a multivariate statistical
metric called b-diversity (Griffiths et al., 2011). b-Diversity is linked to
biogeography of microorganisms (Martiny et al., 2006).
4.2.2 Gene Markers of Functional Diversity

Gene-encoding enzymes that catalyze key reactions in certain biochemical
pathways can be used to study functions of microbial populations. The
Deepterrameta 33
presence of these genes or their transcripts in DNA or RNA samples is

detected by conserved sequence motifs. Functional gene markers have
been used for monitoring of carbon, nitrogen, and sulfur cycling in deep
subsurface microbial communities by PCR, quantitative PCR, and ampli-
con and metagenome sequencing (Bomberg, Lamminm€aki, et al., 2015;
Bomberg, Nyyss€ onen, et al., 2015; Katsuyama et al., 2013; Lau et al.,
2014; Purkamo, Bomberg, Kiet€av€ainen, et al., 2015; Rajala et al., 2015;
Yoshioka et al., 2010).
Functional gene markers can be used to assess the presence, quantity, and
diversity of microbes with specific functions as well as active metabolic
processes when RNA pools are used as a starting material. As an example,
Rajala et al. (2015) utilized gene markers and quantitative PCR to study in-
duction gene expression in microbial communities of fracture fluids from
the Outokumpu deep borehole. Carbon sources, namely, methane and
methanol, were fed to the microbes with and without sulfate as an electron
acceptor; subsequently, DNA and RNA were isolated from the samples, and
RNA was converted to complementary DNA (cDNA). The gene expres-
sion was monitored by quantifying genes (DNA samples) and transcripts
(cDNA samples) representing marker genes pmoA, dsrB, and narG (Table 1).
The results showed that expression of these marker genes in native fraction
fluid was minimal, but addition of carbon sources (especially methane) and
particularly the presence of sulfate activated gene expression related to
methane oxidation (pmoA) and nitrate (narG) and sulfite reduction (dsrB).
Thus, it was possible to show that although microbes in a deep subsurface
microbiome may be in a dormant state, their metabolism is rapidly activated
when suitable sources of carbon and energy become available.
4.3 Metagenome and Metatranscriptome Sequencing and

Data Analysis
4.3.1 Metagenomics
Direct sequencing of total microbial metagenomes is becoming more
common due to the advances in sequencing technologies (higher
throughput, smaller sample requirements, and decreasing prices), as
reviewed by Reuter, Spacek, and Snyder (2015). For example, c. 20e
50 ng of DNA can be sufficient for successful sequencing on the Illumina
HiSeq platform, one of the most popular sequencing technologies. No prior
knowledge about the genetic material of the community under study is
needed. This is an important feature for deep microbiome research because
many microbes in such communities may be previously unknown.
34
Table 1 Examples of marker genes utilized in deep biosphere research
Process Gene Enzyme Enzyme class Description References
Carbon mcrA Methyl coenzyme EC 2.8.4.1 Final step in Yoshioka et al. (2010),
cycling M reductase methanogenesis Katsuyama et al. (2013),
Lau et al. (2014),
Bomberg, Lamminm€aki,
et al. (2015), Bomberg,
Nyyss€ onen, et al. (2015),
Rajala et al. (2015), ,
Purkamo, Bomberg,
Nyyss€ onen, et al. (2015),
Purkamo, Bomberg,
Kiet€av€ainen, et al. (2015)
pmoA, Methane EC 1.14.18.3 Oxidation of methane Rajala et al. (2015),,
mmo monooxygenase to methanol in Purkamo, Bomberg,
M. It€avaara et al.
methanotrophy Nyyss€ onen, et al. (2015),
Purkamo, Bomberg,
Kiet€av€ainen, et al. (2015),
Lau et al. (2014)
Deepterrameta
Nitrogen amoA Ammonia EC 1.14.99.39 Oxidation of ammonia Lau et al. (2014), Kutvonen
cycling monooxygenase to hydroxylamine in et al. (2015)
nitrification
nifH Nitrogenase EC 1.18.6.1 Reduction of nitrogen Lau et al. (2014)
to ammonia in
nitrogen fixation
nirK, nirS Nitrite reductase EC 1.7.2.1 Reduction of nitrite to Katsuyama et al. (2013), Lau
nitric oxide in et al. (2014)
anaerobic denitrification
(respiration)
napA, narG Dissimilatory nitrate EC 1.7.99.4 Reduction of nitrate to Lau et al. (2014), Kutvonen
reductase nitrite in anaerobic et al. (2015), Rajala et al.
denitrification (2015)
(respiration)
Sulfur cycling dsrB Dissimilatory sulfite EC 1.8.99.3 Reduction of sulfite to It€avaara, Nyyss€ onen,
reductase sulfide in anaerobic Bomberg, et al. (2011),
respiration It€avaara, Nyyss€
onen,
Kapanen, et al. (2011),
Purkamo et al. (2013),
Bomberg, Lamminm€aki,
et al. (2015), Bomberg,
Nyyss€ onen, et al. (2015)
Rajala et al. (2015)
35
Whole-metagenome amplification with PCR or isothermal amplification

by means of commercially available kits have also been successfully used
to obtain enough material for metagenome sequencing (Duhaime, Deng,
Poulos, & Sullivan, 2012; Lau et al., 2014; Nyyss€ onen et al., 2014; Orsi
et al., 2013). On the other hand, the amplification may alter the
metagenome composition because of uneven replication of different
DNA molecules (de Bourcy et al., 2014; Dupinay et al., 2012). This effect
may be reduced by several parallel amplifications and by pooling the result-
ing amplified products (Nyyss€
onen et al., 2014). A case study on the micro-
bial communities in Outokumpu deep borehole ground waters by
metagenome sequencing is described later in this chapter.
4.3.2 Metatranscriptomics
Active metabolic pathways of prokaryotes and fungi have been studied
by sequencing of metatranscriptomes from deep-sea drilling campaigns
(Baker et al., 2013; Orsi, Biddle, Edgcomb, 2013; Orsi, Richards, &
Santoro, 2015). Rapid freezing and possibly additional chemical preserva-
tion of a sample are desirable for metatranscriptomic analysis because of
rapid changes in microbial gene expression and the short half-life of
messenger RNA (mRNA), which represents the expressed genes. The
mRNA is converted to cDNA for sequencing. In an isolated RNA sam-
ple, the proportion of mRNA is typically low (1e5%) as compared to the
rRNA fraction. Consequently, enrichment of mRNA in a sample is often
needed in order to clear mRNA sequence data from rRNA sequences,
especially in the case of deep terrestrial-microbiome samples where low
gene expression levels can be expected. In the case of eukaryotes,
mRNA can be converted to cDNA by means of the typical polyA tails,
but in the case of prokaryotes, alternative methods are needed. These
typically rely on removal of rRNA prior to the conversion of mRNA
to cDNA. He et al. (2010) compared rRNA-specific exonuclease treat-
ment and rRNA hybridization capture-based protocols for removal of
rRNA from samples of artificial microbial communities. The least bias
was observed in the case of a hybridization-based system. Giannoukos
et al. (2012) compared several commercial kits in terms of rRNA removal
from isolated strains and mixed cultures; they found that the hybridiza-
tion-based RiboZero kit yielded the best results. The efficiency of the
hybridization method may be improved by designing sample-specific
hybridization probes if rRNA sequences are already known for the sample
in question (Stewart, Ottesen, & DeLong, 2010).
Deepterrameta 37
4.3.3 Data Analysis

Many steps in the analysis of metagenomic and metatranscriptomic sequence
data sets require substantial computing power and thus are best performed
on a high-performance computing cluster, where the task can be split
among tens to thousands of central processing units. The analysis of metage-
nomes and metatranscriptomes begins with raw sequence data (reads). The
read length depends on the sequencing technology and can be 50, 100, or
150 bp of DNA or cDNA. Quality control (QC) is the first bioinformatic
step in virtually all metagenomics studies.
QC of the sequence reads includes removal of sequencing adapters, of
low-quality reads and bases, and of possible contaminants. The FastQC soft-
ware, which generates numerous informative graphs concerning sequence
content and quality, is perhaps the most popular tool for the assessment of
quality of raw data from next-generation sequencing (Andrews, 2015).
One convenient solution is Trim Galore (Krueger, 2015), which is a
wrapper tool built around Cutadapt (Martin, 2011) and FastQC and can
identify and remove sequencing adapters as well as low-quality read seg-
ments automatically. Contamination with human DNA can be detected
and removed manually by aligning the sequence reads against the human
genome with software such as Bowtie 2 (Langmead & Salzberg, 2012),
BWA (Li & Durbin, 2009), and BBMap. Nonetheless, convenient wrapper
tools, such as DeconSeq (Schmieder & Edwards, 2011), which make use of
these aligners and automatically remove the contamination, are also avail-
able. In a typical metagenomic bioinformatics workflow, the short sequence
fragments (reads) are assembled, ie, merged into longer contiguous
sequences (contigs) after QC. The majority of the state-of-the-art metage-
nome assemblers are based on the de Bruijn graph approach (Pevzner, Tang,
& Waterman, 2001) and include IDBA-UD (Peng, Leung, Yiu, & Chin,
2012) and MetaVelvel-SL (Afiahayati, Sato, Sakakibara, 2015). Quality of
the metagenome assemblies is usually quantified by means of the N50 statis-
tic, which describes the length of the assembled contigs. The N50 value is
the length of the shortest contig, which together with all contigs longer
than itself makes up a half of the sum of the length of all contigs. Assembly
of genome-length contigs from environmental DNA samples is mostly
impossible. Nonetheless, postassembly methods such as binning can some-
times reveal near-complete microbial genomes in metagenomic assemblies
(eg, Brown et al., 2015). The binning algorithms generally cluster contigs
that meet a length threshold of c. 1000 bp into groups based on 4-mer
frequencies and/or depth of sequencing coverage and ultimately either
rely on the human eyeebrain system for cluster identification (Laczny et al.,
2015) or are fully automated (Wu, Tang, Tringe, Simmons, & Singer, 2014).
The assembled metagenomes and metatranscriptomes can be analyzed in
different ways depending on research purposes. Typically, protein-coding
genes as well as rRNA genes and transfer RNAs are predicted from contig
data by specialized algorithms, which often take into account the fragmented
nature of the sequence data. Commonly used protein prediction algorithms
include Prodigal (Hyatt et al., 2010) and Glimmer-MG (Kelley, Liu,
Delcher, Pop, & Salzberg, 2012), whereas rRNAs can be predicted by
means of such applications as meta_rna (Huang, Gilna, & Li, 2009) and
SSU-ALIGN (Nawrocki, 2009), and transfer RNAs by means of tRNAs-
can-SE (Lowe & Eddy, 1997).
The protein-coding sequences and ribosomal genes are then compared
to known sequences for functional and taxonomical annotation (Fig. 4).
For example, the popular bioinformatic tool BLAST (Altschul, Gish, Miller,
Myers, & Lipman, 1990) can be used in BLASTP mode (protein versus pro-
tein search) to query the predicted proteins against a database such as nr
Figure 4 A bioinformatics workflow in metagenomics. The light gray background indi-

cates that the step is mainly based on high-throughput DNA sequencing (DNA-Seq),
whereas the dark gray background indicates that the step involves both DNA-Seq
and RNA-Seq.
Deepterrameta 39
(a nonredundant protein database, NCBI) for annotation of function and

taxonomy simultaneously. More detailed and trustworthy information con-
cerning the function can be obtained by comparison with curated databases
such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa
& Goto, 2000; Kanehisa et al., 2014) and MetaCyc (Caspi et al., 2014).
Furthermore, even more information, such as domain structure of proteins,
can be acquired with such software as InterProScan (Zdobnov & Apweiler,
2001). In addition, more remote homologs may be detected with such tools
as HHpred (S€ oding, Biegert, & Lupas, 2005). The (annotated) protein-
coding sequences can then be used for analysis of metabolic pathways in
microbial communities (see a case study in Section 5).
4.4 Single-Cell Isolation and Sequencing

The data from metagenome sequencing may be complex, and single-cell
isolation and sequencing have been developed to characterize isolated
members of a community (Stepanauskas, 2012; Wasmund et al., 2014).
The single-cell analysis requires extra clean laboratory conditions to
prevent contamination during isolation and biochemical protocols (Woyke
et al., 2011).
Fluorescence-activated cell sorting (FACS) equipment is used to separate
cells from the rest of the community: specific probes or activation methods
can be used to isolate the cells of interest (Garcia et al., 2013; Martinez-
Garcia et al., 2012; Woyke et al., 2011). Single-cell isolation and sequencing
have been used in the research on virusehost interactions (Roux et al.,
2014; Yoon et al., 2011) and in biotechnology (Ishii, Tago, & Senoo,
2010) as well as to study community level dynamics of natural environments
(Yoon et al., 2011).
5. A CASE STUDY: METAGENOMICS OF THE

OUTOKUMPU DEEP BOREHOLE
Outokumpu scientific deep borehole in Finland is probably the most
actively studied borehole in the Fennoscandian shield and represents an
excellent platform for deep subsurface geobiological research and method
development. Since its drilling in 2004e05, extensive research efforts
have been devoted to characterization of the fluids, geology, geophysics,
geochemistry, and gases of the borehole, and a more comprehensive picture
of the Outokumpu deep biosphere has started to emerge (Kiet€av€ainen et al.,
2013; Kiet€av€ainen, Ahonen, Kukkonen, Niedermann, & Wiersberg, 2014;
Kukkonen, 2011). The Outokumpu borehole is 2.5 km deep and contains

five fracture zones that discharge ancient saline ocean water and gases into
the borehole (Ahonen et al., 2011). The Palaeoproterozoic Outokumpu for-
mation, intersected at the depth of 1300e1500 m, is w2 billion years old,
dating back to the period when atmospheric oxygen concentration increased
rapidly (S€antti, Kontinen, Sorjonen-Ward, Johanson, & Pakkanen, 2006).
The formation was originally a sequence of sea bottom sediments and
oceanic lithosphere (Outokumpu ophiolite). Organic material originating
from the ancient deposits now forms graphite- and sulfide-rich layers (“black
schist”) in the Outokumpu metasedimentary rocks and in the contact zones
of the ophiolite-derived ultramafic rocks (Loukola-Ruskeeniemi, 2011,
1999, 1991). Traces of hydrocarbons have been found in the organic
graphitic matter of the Outokumpu core sample (Taran, Onoshko, &
Mikhailov, 2011).
Deep groundwater in the Outokumpu area and in the borehole is saline,
and in terms of residence time, is up to tens of millions of years old
(Kiet€av€ainen et al., 2014). No downward flow of fresh surface water,
groundwater, or contamination from drilling fluid has been observed in
the borehole. The latter was controlled by marking the drilling fluid with
sodium fluorescein. Stable-isotope analyses of the water (d18O and d2H)
did not detect mixing with meteoric water. Consequently, the level of
contamination in the borehole from the surface is negligible. The main
ions in the borehole water are calcium, potassium, sodium, and chloride,
and they constitute up to 1.2% of total dissolved solids. Major gases are
methane, nitrogen, and helium. Traces of longer-chain hydrocarbons have
also been identified. One particularly important source of gas-bearing saline
fluid is an open fracture zone at the depth of 967 m (Kiet€av€ainen et al., 2013).
In addition to extensive geophysical and geochemical research
(Kukkonen, 2011), studies on deep subsurface life have also been conduct-
ed in the Outokumpu borehole (Fig. 5). Microbiological sampling cam-
paigns were performed during the summers of 2007e12 (It€avaara,
Nyyss€ onen, Kapanen, et al., 2011). Despite the low cell density in deep
groundwater (c. 103e105/mL), development of sampling and molecular
biological methods has enabled community-level microbiological research
into the Outokumpu bedrock microbial communities and yielded new in-
sights into the life in the deep subsurface (It€avaara, Nyyss€onen, Bomberg,
et al., 2011; Nyyss€ onen et al. 2014; Purkamo et al., 2013; Purkamo,
Bomberg, Kiet€av€ainen, et al., 2015; Purkamo, Bomberg, Nyyss€ onen,
et al., 2015; Rajala et al. 2015).
Deepterrameta 41
(A) (B)
(C) (D)
Figure 5 Scanning electron micrographs of microorganisms from the Outokumpu

deep borehole sampled from the 1000-m depth.
Major findings about the microbiology of the Outokumpu deep bore-

hole have been published in several articles (It€avaara, Nyyss€
onen, Bomberg,
et al., 2011; It€avaara, Nyyss€
onen, Kapanen, et al., 2011; Nyyss€ onen et al.,
2014; Purkamo, Bomberg, Kiet€av€ainen, et al., 2015; Purkamo, Bomberg,
Nyyss€ onen, et al., 2015; Purkamo et al., 2013; Rajala et al., 2015). The
key technologies and findings are summarized in the following sections.
5.1 Metagenomics of Deep Subsurface Life in the

Outokumpu Deep Borehole, Finland
The first samples (for microbiological research) from the Outokumpu bore-
hole were collected from the water column (at 100-m intervals) by the tube
sampling technique (Ahonen et al., 2011; It€avaara, Nyyss€onen, Bomberg,
et al., 2011; It€avaara, Nyyss€onen, Kapanen, et al., 2011). Sampling
techniques have been described in detail in Section 3.1. Sample volumes
of c. 500 mL were sufficient for isolation of DNA for studies of the species
and functional diversity of bacterial and archaeal communities by means
of 16S rRNA DGGE (It€avaara, Nyyss€ onen, Bomberg, et al., 2011), 16S
rRNA 454 amplicon sequencing (Nyyss€ onen et al., 2014), and 454 ampli-
con sequencing of marker genes related to carbon, nitrogen, and sulfate
cycling (Rajala et al., 2015; Purkamo, Bomberg, Kiet€av€ainen, et al.,
2015). Furthermore, whole-metagenome DNA samples from the depths
of 600, 1500, and 2300 m were processed by isothermal amplification and
analyzed by 454-shotgun sequencing (Nyyss€ onen et al., 2014).
Water that was pumped from fractures by using packer sampling tech-
nique as described above is known to represent in situ deep groundwater
better than the water in the open borehole can. By the same token, the mi-
crobial communities in fracture zones are considered more representative of
the native communities in the bedrock; thus, they were selected as the next
objects of research. Water samples were collected from three fracture zones
(at 500, 967, and 2260 m) using packer sampling approaches described in
Section 3.1 and by Purkamo et al. (2013). The first sample volumes ranged
from 1.5 to 3.0 L and allowed for isolation of DNA and RNA for 16S
rRNA DGGE and for marker gene analysis by quantitative PCR (Purkamo,
Bomberg, Kiet€av€ainen, et al., 2015; Purkamo, Bomberg, Nyyss€ onen, et al.,
2015). Bigger samples, 5e10 L, were subsequently collected for recovery of
DNA for sequencing on an Illumina HiSeq100 in order to achieve better
coverage of microbial metagenomes. Nonetheless, whole-metagenome
amplification was still necessary to obtain enough DNA for preparation of
the sequencing library (Nyyss€ onen et al., 2014).
In this case study, we summarize these results; assemble the 454-
sequenced metagenomes from Nyyss€ onen et al. (2014); introduce three
additional Illumina HiSeq-sequenced shotgun metagenomes from fracture
zones at the depths of 500, 967, and 2260 m; and attempt to present a
more consistent picture of the Outokumpu deep biosphere.
5.2 Data Analysis of Borehole Water and Fracture Zone

Metagenomes
5.2.1 Quantity and Quality of Metagenomic Sequences
The sizes of sequenced data sets obtained from a borehole water column
(depths 500e600, 1400e1500, and 2200e2300 m) and fracture zones
(depths 400e500, 867e967, and 2160e2260 m) were considerably
different. The total size of the dereplicated-sequence samples from the bore-
hole water ranged from 108 to 144 million bp, whereas the size range of the
fracture zone sequence samples was 7.8e24.3 billion bp (Table 2). In part,
these numbers reflect the difference between the productivity levels of the
Deepterrameta
Table 2 Basic statistics on the metagenomic samples in the Outokumpu deep borehole
Sample Length Length Predicted 16S 16S 18S
(m) Sequencing (clean reads) (assembly) N50 proteins archaea bacteria eukaryota
600 454 107,939,831 8,022,306 721 17,545 0 7 0

2 454 143,983,116 8,202,050 621 19,723 0 7 0
2300 454 130,453,592 8,630,083 1084 17,761 2 14 0
500 Illumina 7,806,660 413 94,607 542 6372 136,360 3 95 0
967 Illumina 24,303 119 600 230,996,094 4162 333,953 3 146 1
2260 Illumina 11,139 803 325 46,324 877 3447 66,549 3 30 2
43
two sequencing platforms: although 454-sequencing typically produces

longer reads than Illumina HiSeq does, the amount of sequence data
obtained from Illumina HiSeq is orders of greater magnitude.
Contamination with human nucleic acids in the samples was very low.
The contamination rate in the borehole water column samples was only
0.0002% in the samples collected from the depths 600 and 1500 m, and
0.0007% in a sample from the depth 2300 m (DeconSeq analysis; Schmieder
& Edwards, 2011). Similarly, the contamination rates in the fracture zone
samples from depths of 500 and 967 m were only 0.001% and 0.005%,
respectively. On the other hand, the sample from the fracture zone at the
2260-m deep showed a much higher rate of contamination with human
nucleic acids (1.298%). In addition, a nearly full-length eukaryotic 18S
rRNA, which was almost identical to that of Pinus luchuensis (Okinawa
pine), was detected in the assembly from the depth 2260 m (see below).
This result indicated that this sample also included contamination from sour-
ces other than humans.
5.2.2 Metagenome Assembly

The 454 reads from borehole water samples and Illumina HiSeq reads from
the fracture zones were assembled in the IDBA-UD software (Peng et al.,
2012). Default settings were used for the assembly, except that the k-mer
length was incremented by 10 in every assembly iteration. IDBA-UD was
originally designed for paired-end reads, where DNA fragments are
sequenced from both ends. In this study, however, only the fracture zone
samples were sequenced in this fashion. IDBA-UD can also process
single-end reads when the final scaffolding step is omitted (joining of contigs
on the basis of read pair linkage information). The N50 values, which reflect
the length of the assembled contigs, were significantly greater for the
HiSeq-sequenced samples (Table 2). Likewise, the assembly sizes of the
454-sequenced samples were relatively smalldfrom 8 to 8.6 million bpd
which amount to a few complete prokaryote genomes, whereas the assem-
bly sizes of the HiSeq-sequenced samples ranged from 46.3 to 231 million
bp (Table 2).
The sequencing depth, ie, how many times each nucleotide in the sam-
ple was sequenced, was first estimated on the basis of the order of magnitude
of reduction in total sequence length from reads to the assemblies. This anal-
ysis revealed c. 15-fold sequencing depth for the 454-sequenced samples
and 82- to 239-fold sequencing depth for HiSeq-sequenced samples. To
assess the actual sequencing depth of the contigs, the reads were mapped
Deepterrameta 45
to the assemblies in the BWA software (Li & Durbin, 2009). Indicating
possible redundancy in the sequencing, the contigs with the greatest
coverage were all short and not much longer than a typical read of the
sequencing technologies used. This result was expected in the case of the
HiSeq-sequenced samples because the reads were not dereplicated prior
to the assembly. In contrast, the 454-sequenced assemblies were generated
from dereplicated reads; therefore, the redundancy was unexpected. One
possible explanation for the redundancy is that the dereplication of the reads
was carried out at a very high sequence identity threshold, eg, 100%. The
454-sequencing has a mean base call error rate of c. 1% (Gilles et al.,
2011); consequently, a typical read includes w3 wrong base calls on average;
thus, generally, a more relaxed similarity threshold should be considered for
454 data dereplication.
5.2.3 Gene Prediction and Annotation

Protein-coding genes were predicted in the contigs by means of Prodigal
(Hyatt et al., 2010), which has a metagenome mode and can optionally
output convenient file formats for downstream bioinformatics work. The
predicted protein counts of the 454-sequenced assemblies were in the range
17,545e19,723: approximately the size of the human proteome. As
expected from the assembly sizes, the predicted protein counts of the
HiSeq-sequenced assemblies far exceeded the protein counts of the 454
assemblies (Table 2). Small subunit rRNA genes (16S rRNA) were
predicted in the SSU-ALIGN software (Nawrocki, 2009). The relative
abundance levels of the detected archaeal and bacterial 16S rRNAs indicated
that bacteria most likely dominated all the sampled communities (Table 2).
On the other hand, in agreement with the results of Nyyss€ onen et al. (2014),
according to the 16S rRNA ratios, archaea appeared to be more abundant in
the deep subsurface samples, ie, those coming from the fracture zone and
water column at depths 2260 and 2300 m, respectively.
For taxonomic and functional annotations, the predicted proteins were
queried in BLASTP (Altschul et al., 1990) against the nr protein database
of the NCBI and against the KEGG protein database (Kanehisa & Goto,
2000; Kanehisa et al., 2014).
Taxonomy was assigned on the basis of the BLASTP queries against the
nr database by the last common ancestor (LCA) method implemented in the
Blast2lca software (https://github.com/emepyc/Blast2lca). In a general
BLAST search, the search sequence is compared with the database
sequences, and similar sequences are returned with a corresponding bit
score, e-value, and similarity and identity percentage values, which describe
the significance of the sequence similarity between the search sequence and
a database hit. In the Blast2lca method, the taxonomy of a given protein is
calculated from a subset of the hits that are within 0.9 bit score of the best
hit. Accordingly, the taxonomic level of the annotations varies from protein
to protein, depending on how taxonomically similar the annotated
sequences found in the database are. The threshold e-value for significant
BLASTP hits against KEGG was 1e-6. The more stringent e-value of
1e-12 and minimum sequence identity of 50% were applied to the hits
considered for LCA annotations (hereafter referred to as species).
In order to estimate how comprehensively the predicted proteomes
represented the sampled microbial communities, UNIX tools awk, cut,
grep, shuf, and sort were utilized to generate accumulation data from the
species annotations and from the KEGG annotations. In this approach, an
increasing number of annotations was subsampled from the total data in
order to produce accumulation curves, which were visualized by means of
the ggplot2 package (Wickham, 2009) of the R software (R Development
Core Team, 2008). Because nearly identical DNA sequences (such as those
from closely related bacterial strains) merge into one sequence during
sequence assembly, the species annotation counts were normalized prior
to subsampling on the basis of the sequencing depth of the open reading
frame regions.
5.3 Species Distribution in Borehole Water and in Fracture

Zone Samples
According to the taxonomic assignments, bacteria were the most abundant
group at all sampled depths both in borehole water and in fracture zone sam-
ples (Table 3). The relative abundance of archaea was mostly low (c. 0.1%) in
the samples. One exception was the borehole water sample from the depth
Table 3 Relative abundance levels of viruses and the domains of life in the protein
space of assemblies from the Outokumpu deep borehole
Sample (m) Sequencing Archaea Bacteria Eukaryota Viruses Unknown
600 454 0.1% 92.4% 0.0% 0.1% 7.3%

1500 454 0.1% 87.6% 0.0% 0.2% 12.1%
2300 454 15.6% 72.0% 0.0% 0.0% 12.4%
500 Illumina 0.0% 93.0% 0.1% 0.0% 6.9%
967 Illumina 0.1% 90.3% 0.0% 0.0% 9.5%
2260 Illumina 7.2% 86.5% 0.0% 0.0% 6.3%
Deepterrameta 47
2300 m (454-sequenced) and a fracture zone sample from the depth 2260 m
(HiSeq-sequenced), where archaea were found to represent nearly 16% and
7% of the community, respectively. The difference between the archaeal
abundance levels in the samples in the vicinity of the 2300-m depth could
be explained by temporal variation. Purkamo et al. (2013) observed tempo-
ral changes in the microbial communities of the Olkiluoto borehole
although these changes were assumed to be related to the disturbances asso-
ciated with water pumping. Alternatively, the different sampling sources
(water column vs fracture water) as well as the relatively small size of samples
(particularly the 454-sequenced sample) may explain the discrepancy.
Eukaryotes and viruses were present in all assemblies at very low abundance
(generally less than 0.1%). Overall, these domain level results were in
agreement with the results of Nyyss€ onen et al. (2014), particularly in the
sense that archaea appeared to be relatively abundant only in proximity to
the 2300-m depth, where temperature (c. 40 C), pressure (c. 230 bar),
and salinity (Ca/Na 2.31) are the highest.
Nyyss€onen et al. (2014) obtained higher-resolution taxonomic profiles
from 16S rRNA amplicons and from 16S rRNA fragments that were
detected in the metagenomic 454-reads from the depths 600, 1,500, and
2300 m. The most abundant bacterial families were detected by both
methods although at different relative abundance levels. For example, on
the basis of amplicons, Comamonadaceae was either the most abundant
or the second most abundant bacterial family at all studied depths. In
contrast, according to the 16S rRNA metagenomic fragments, Comamona-
daceae were relatively abundant only at the 2300-m depth. Both methods of
Nyyss€ onen et al. (2014) indicated that Acholeplasmataceae was the most
abundant bacterial family at the 600-m depth, whereas Peptococcaceae
and Clostridiales were among the most abundant bacterial taxa throughout
the depth profile. As for archaea, Nyyss€onen et al. (2014) inferred from the
amplicon data that the genus Methanobacterium of the Methanobacteria-
ceae family was the most abundant archaeal taxon at all depths except
1100 m, where Methanolobus of the Methanosarcinaceae family was the
dominant archaeal genus. Judging by the amplicon data, SAGMEG-1
(South Africa Goldmine Euryarchaeotic Group 1) was the second most
abundant archaeal taxon at the 600-m depth, whereas Methanolobus was
the second most abundant archaeal group at the depth of 1500 m; the
archaeal fraction corresponding to the 2300-m depth consisted almost
exclusively of Methanobacterium species. In terms of metagenomic reads,
Nyyss€ onen et al. (2014) detected archaeal 16S rRNA fragments only in
the sample from the depth 2300 m. These reads were associated with
Methanobacterium and unclassified Methanobacteriaceae (Nyyss€ onen et al.,
2014).
When abundance of the bacterial taxa was interpreted in the protein
space of the 454-sequence assemblies (this study), Comamonadaceae were
not among the dominant groups. Instead, the most abundant classified bac-
terial families were Acholeplasmataceae, Peptococcaceae, and Clostridia-
ceae. Acholeplasmataceae was the most abundant in the sample of the
600-m depth, whereas Peptococcaceae and Clostridiaceae were the most
abundant taxa in the two deeper samples. In addition, unclassified Firmicutes
and unclassified bacteria represented a significant fraction of all these pro-
teomes. In the two deeper samples, unknown bacteria were also relatively
abundant. Taxa that represented less than 5% of the samples at the family
level (hereafter referred to as rare taxa) were abundant and represented
36.8, 40.7, and 35.2% of the proteomes from the depths 600, 1,500, and
2300 m, respectively, as shown in Fig. 6.
Methanosarcinaceae was the most abundant archaeal family in pro-
teomes from the depths 600 and 1500 m. In addition, Methanomassiliicoc-
caceae, unclassified Euryarchaeota, and rare archaeal taxa were abundant in
the proteome from the depth 600 m, whereas Methanoperedenaceae
(ANME-2d), Methanococcaceae, Methanobacteriaceae, and rare archaeal
taxa were abundant in the proteome from the 1500-m depth. The sample
from the 2300-m depth was the most homogeneous in terms of archaeal
families because Methanobacteriaceae represented over 82% of the archaeal
proteome.
Purkamo et al. (2013) studied taxa distribution in three fracture zones of
the Outokumpu deep borehole at the depths of 500, 967, and 2260 m by
16S PCR-DGGE. According to their study, Alphaproteobacteria, Betapro-
teobacteria, and Mollicutes are the dominant bacterial taxa in the fracture
zone at 500 m. The same taxa were also abundant in the protein space of
the Illumina HiSeq-sequenced assembly from the depth 500 m. According
to the assembly, Alphaproteobacteria at the 500-m depth belongs to an
unclassified family, whereas Betaproteobacteria were mainly Comamonada-
ceae, and Mollicutes were Acholeplasmataceae. In addition, unclassified
Gammaproteobacteria, unclassified and unknown bacteria, and rare bacterial
taxa were abundant at this depth.
According to Purkamo et al. (2013), the archaeal community in the
fracture at the depth 500 m was relatively homogeneous and consisted of
Methanobacteriaceae and to a lesser degree Methanosarcinaceae. In contrast,
Deepterrameta
Figure 6 The relative abundance levels of bacterial families in the Outokumpu deep borehole (2.5-km deep). The numbers are based on
coverage-normalized abundance values of taxonomic annotations, which were derived from the protein space of the corresponding meta-
49
genomic assemblies. Taxa that were present in less than 5% abundance at the family level are grouped into the “other” category.
protein space of the HiSeq-sequenced assembly suggested that the archaeal

community of the fracture zone at the 500-m depth was relatively diverse
and included (in addition to Methanobacteriaceae and Methanosarcinaceae)
Methanoperedenaceae, Methermicoccaceae, Methanomassiliicoccaceae,
unclassified Euryarchaeota, unknown archaea, and rare archaeal taxa (Fig. 7).
Purkamo et al. (2013) detected the most diverse bacterial and archaeal
communities in the Outokumpu deep borehole in the fracture zone at
the 967-m depth. Their results showed that at this depth, the community
includes Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria,
Mollicutes, Peptococcaceae (Clostridiales), Firmicutes, and Flavobacteria
(Bacteroidetes). Among these taxa, only Mollicutes (Acholeplasmataceae)
and Peptococcaceae were abundant when the protein space of the assembly
from the 967-m depth was inspected. Other abundant bacterial taxa in the
proteome from the 967-m depth were potentially nitrogen-fixing Prolixi-
bacteraceae, unclassified Bacteroidetes, unclassified bacteria, and rare bacte-
rial taxa. Purkamo et al. (2013) determined that SAGMEG and
Methanobacteriaceae dominate the archaeal community of the fracture
zone at the depth of 967 m. Judging by the proteome from this depth,
the archaeal fraction also includes Thermococcaceae, Methanomassiliicocca-
ceae, Methanoperedenaceae as well as unclassified Euryarchaeota, unclassi-
fied archaea, unknown archaea, and rare archaeal taxa (Fig. 7).
According to Purkamo et al. (2013), the bacterial community of the
fracture zone at the 2260-m depth consists of Actinobacteria, Firmicutes, and
Alphaproteobacteria. In the corresponding HiSeq-sequenced assembly, Pepto-
coccaceae (Firmicutes) represent nearly 60% of the bacterial protein space.
Other abundant bacterial taxa in this proteome were Gammaproteobacteria
(Legionellaceae and unclassified bacteria) and rare bacterial taxa (Fig. 6).
According to the results of Purkamo et al. (2013), at the family level, the
archaeal community of the fracture zone at the depth of 2260 m is relatively
similar to the archaeal community of the fracture zone at the 500-m depth.
On the other hand, the proteomes analyzed here suggest that the archaeal
community of the fracture zone at the depth 2260 m is very similar to the
archaeal community inferred from the borehole water retrieved by
Nyyss€ onen et al. (2014) from the depth of 2300 m (Fig. 7).
Altogether, only 132 proteins got a viral classification; therefore, the
relative abundance levels of viruses in the samples from the Outokumpu
deep borehole (as shown here) represent ambiguous estimates. Because viral
particles are generally very small, it is unlikely that the sample collection by
our protocols and by the methods of Nyyss€ onen et al. (2014) has captured
Deepterrameta
Figure 7 The relative abundance levels of archaeal families in the Outokumpu deep borehole. The numbers are based on coverage-normal-
ized abundance values of taxonomic annotations, which were derived from the protein space of the corresponding metagenomic assem-
blies. Taxa that were present in less than 5% abundance at the family level are grouped into the “other” category.
51
them, ie, the viral particles must have passed through the 0.22-mm filters.
Furthermore, detection of some viruses, such as those with RNA genomes
and no DNA intermediates in the life cycle (ie, Groups III, IV, and V in the
Baltimore classification system) is not possible with the approaches used. As
expected, the genes that were classified as viral in this study come mainly
from bacteriophages that have integrated into prokaryotic genomes as
prophages and from horizontally transferred genes. With the exception of
the samples from the greatest depths, proteins belonging to unclassified
viruses and the three Caudovirales familiesdMyoviridae, Podoviridae, and
Siphoviridaedwere relatively evenly represented at all sampled depths.
Other viral families, eg, Microviridae, Iridoviridae, and Mimiviridae, were
detected more sporadically (Fig. 8). In the case of the two latter groups,
the presence of suitable eukaryotic hosts should also be expected.
Proteins with eukaryotic classification were likewise rare (n ¼ 260), and
major groups rarely occurred at more than one depth. The detected abun-
dant groups included fungi, worms (Nematoda and Annelida), Oomycetes,
Figure 8 The relative abundance levels of viral families in the Outokumpu deep bore-
hole. The numbers are based on coverage-normalized abundance values of LCA anno-
tations, which were derived from the protein space of the corresponding metagenomic
assemblies.
Deepterrameta 53
and Alveolates as well as putative contaminants in the sample from the depth
of 2260 m, including Streptophyta and Hominidae. The inclusion of
coverage normalization in the relative-abundance calculations affected the
eukaryotic-abundance results significantly (data not shown). For example,
on the basis of just one high-coverage protein, Perkinsus marinus (Perkinsidae)
represented >91% of the eukaryotic protein space in the fracture zone
sample from the 500-m depth. Nonetheless, when abundance was based
on hit counts alone, the most abundant eukaryotes were a Rhizophagus
irregularis-related Glomeromycete (24%), Plasmodium yoelii-like Apicom-
plexan (12%), and Elaeis guineensis-like palm tree (12%). These results
highlight the difficulties with taxonomic assignment, in particular, in rela-
tion to partial multidomain eukaryotic proteins. More stringent BLASTP
cut-offs especially in terms of coverage percentage of the subject sequence
should certainly decrease the number of false hits, but at the same time, a
larger fraction of the protein space would be left without taxonomic
annotations.
5.4 Representativeness of the Assemblies and Insights into

the Microbial Communities in the Outokumpu Bedrock
The species accumulation curves shown in Fig. 9 indicate that the fracture
zones at depths 500 and 967 m are more species-rich than the fracture
zone at 2260 m. Nevertheless, the total unique species counts differed vastly
depending on the signal threshold of a species’ presence. For example, there
were w4000 unique species in the proteome at the depth 967 m when a
single hit was enough to signal a species’ presence. In contrast, when the
signal threshold was 100 hits, the unique species count of the proteome at
the depth 967 m approached 200. Regardless of the signal threshold, the
unique-species counts were not saturated, ie, deeper sequencing of the sam-
ples would have uncovered more species.
The one-hit threshold of a species’ presence (top of the shaded areas in
Fig. 9) exaggerates the total species counts significantly. This is because there
is no reason to expect that the proteomes of two closely related prokaryotic
species (on the basis of 16S rRNA similarity) would evolve similarly, ie, we
cannot expect that all their genes would represent the best matches to each
other. In addition, gene content even between very closely related bacterial
strains can vary enormously, especially due to horizontal gene transfer
(Gogarten & Townsend, 2005), which might be frequent in the deep
biosphere (Labonté et al., 2015). For example, when we queried the prote-
ome of Methylobacterium extorquens AM1 (NCBI accession #: NC_012808.1)
Figure 9 Species accumulation curves based on the protein space of metagenomic

assemblies from the Outokumpu deep borehole. The vertices of the shaded areas
represent the number of species when each unique LCA hit increases the count. The
bottoms of the shaded areas represent the number of species when 100 hits to a given
LCA are required to increase the count. The lines represent the number of species when
10 hits to a given LCA are required to increase the count. The dashed line intersects the
species/LCA count at 200. The proteomes from the depths 600, 1500, and 2300 m are
stacked in the lower left corner.
against the nr database and excluded self-hits, the best hits were distributed
among 15 species (LCAs). The presence of only this one M. extorquens strain
in the Outokumpu samples could have potentially increased the species
count by 15.
As shown in Fig. 9 and Table 2, the proteomes of the 454-sequenced
samples were small in comparison with the proteomes that were predicted
from the Illumina-sequenced samples. Nyyss€ onen et al. (2014) estimated
on the basis of 16S rRNA amplicons at 3% distance (which is a commonly
used threshold for species) that there are c. 150e200 prokaryotic species at
each sampled depth of the Outokumpu borehole. In Fig. 9, the accumula-
tion curves never break the threshold count of 200 species when 100 hits are
required to signal the presence of a species. There are currently 4250
completely sequenced prokaryotic genomes in the NCBI database, which
Deepterrameta 55
encode on average 3233 proteins. If the estimate of 200 species is valid for
the Outokumpu samples, then the ideal sequencing would have revealed
c. 650,000 proteins per sample. In other words, the smaller metagenomic as-
semblies presented here (in particular the 454-sequenced ones) may cover as
little as 1e2% of the sampled microbial communities. In contrast, the
HiSeq-sequenced samples, particularly the assembly from the 967-m depth,
clearly captured more representative slices of the Outokumpu deep
biosphere.
To test how well the metabolic capabilities of the microbial communities
in the Outokumpu deep borehole were represented by the assemblies, the
predicted proteins were also queried against the KEGG database for func-
tional annotations. According to pathway accumulation curves, the fracture
zone at the depth of 967 m was more metabolically diverse than the samples
at other depths regardless of the level of subsampling (Fig. 10). The fracture
zone microbial communities from depths 500 and 2260 m appeared to
encode a relatively similar number of pathways, whereas the results from
the 454-sequenced samples were inconclusive due to the small sample
size. These findings indicate that the functioning of the microbial commu-
nity in the fracture zone at the depth 967 m, where water is particularly rich
in methane, is more diverse than the functioning in the other studied
samples. Purkamo et al. (2013) found the highest microbial diversity in
this fracture zone; therefore, the results support each other.
Nyyss€onen et al. (2014) interpreted metagenomic reads and concluded
that the microbial communities of the Outokumpu borehole water column
at depths 600, 1500, and 2300 m assimilate carbon in various ways, including
Calvin, incomplete reductive tricarboxylic acid (TCA), reductive acetyl-
CoA, and 3-hydroxypropionate cycles. Nonetheless, the genes that are
associated with these cycles largely overlap. Consequently, the presence of
one cycle can simultaneously signal possible presence of other cycles. We
decided to find out what kind of KEGG modules for carbon fixation and
for nitrogen, methane, and sulfur metabolism were present in the microbial
communities of the Outokumpu deep borehole. To this end, we assessed
their presence in the assembled metagenomes by means of the module
reconstruction tool on the KEGG website. Only complete modules and
those with a maximum of one missing block were considered. The Calvin
cycle was detected in all the HiSeq-sequenced samples but in none of the
454-sequenced samples; this finding likely means that the Calvin cycle is
present at all sampled depths but could not be detected as a complete or
near-complete metabolic pathway in the very sparse 454-sequenced samples
Figure 10 Accumulation curves of Kyoto Encyclopedia of Genes and Genomes (KEGG)

pathways on the basis of metagenomic assemblies in the protein space of the
Outokumpu deep borehole. The vertices of the shaded areas represent the number
of pathways when each unique pathway hit increases the count. The bottoms of the
shaded areas represent the number of pathways when 100 hits to a given pathway
are required to increase the count. The lines represent the number of pathways
when 10 hits to a given pathway are required to increase the count. The dashed line
intersects the LCA count at 200. The proteomes at depths 600, 1500, and 2300 m are
stacked in the lower left corner.
(Table 3). The same observation is generally applicable to reductive TCA,

reductive acetyl-CoA, and incomplete reductive TCA cycles. As for nitro-
gen metabolism, a metabolic pathway for nitrogen fixation (nitro-
gen / ammonia) and a pathway for dissimilatory nitrate reduction were
likely to be present at all depths of the borehole, whereas the metabolic
pathways for assimilatory nitrate reduction and denitrification (nitrate /
nitrogen) were likely to be present at depths 500, 600, and 967 m, but a
pathway for nitrification (ammonia / nitrite) only at the 967-m depth.
The metabolic pathway for carbon dioxide-, acetate-, or methanol-to-
methane methanogenesis was likely to be present only in the two samples
from the greatest depths, ie, 2260 and 2300 m. However, a near-complete
module for methylamine-, dimethylamine-, or trimethylamine-to-methane
Deepterrameta 57
methanogenesis was also identified at the 967-m depth; this result points to
fermentation, eg, degradation of organic compounds to methane via several
microbial processes. As for the sulfur metabolism, complete modules for
assimilatory and dissimilatory sulfate reduction were detected in all the
HiSeq-sequenced samples but in none of the 454-sequenced samples
(Table 4). Sulfate reducers have been found earlier by marker gene analysis
(dsrB) at all the tested depths of the borehole (It€avaara, Nyyss€
onen, Kapanen,
et al., 2011; Nyyss€ onen et al. 2014).
The three more representative assemblies, ie, the HiSeq-sequenced sam-
ples from fracture zones at depths 500, 967, and 2260 m, encoded a total of
544 complete or partial KEGG modules, of which 62.5% were shared
among all three proteomes (Fig. 11). For the most part, the shared modules,
which were associated with such pathways as carbon, nitrogen, and purine
metabolism; biosynthesis of amino acids; ABC transporters; the phospho-
transferase system; DNA repair mechanisms; and two-component regulato-
ry systems, could be expected to be present in essentially any microbial
community. On the other hand, less universal modules, such as those asso-
ciated with sulfur and methane metabolism, were also detected in the pro-
teomes of all three Outokumpu fracture zones. Sulfate reducers have also
been previously detected at the depth of 1500 m and above in the borehole
water column (It€avaara, Nyyss€ onen, Bomberg, et al., 2011). Some of the
modules that were unique to the fracture zone proteome at the 2260-m
depth were clearly a result of contamination, eg, photosystems I and II.
The majority of the other modules unique to this proteome were associated
with antibiotic biosynthesis and resistance and may be related to life in the
deep subsurface. Likewise, many of the modules that were unique to the
fracture zone proteome at the depth 500 m were associated with antibiotic
resistance and biosynthesis of antibiotics (particularly cationic antimicrobial
peptides). Other modules unique to the proteome at the 500-m depth
included thiamine, vitamin B12, hemophore/metalloprotease, taurine,
and magnesium transport systems and modules associated with the degrada-
tion of aromatic compounds such as benzoate and terephthalate. The prote-
ome at the depth 967 m included the greatest number of modules that were
unique to a specific sample. Due to the most successful sampling, however,
this proteome was over twice the size of the proteome at the depth 500 m
and approximately fivefold larger than the proteome at the 2260-m depth.
Consequently, many modules were likely to be unique to this sample by
chance. As with the other samples, some of the unique modules of the pro-
teome at 967 m were associated with antibiotic resistance and biosynthesis.
Table 4 Distribution of complete or near-complete carbon, nitrogen, methane, and sulfur metabolism-associated KEGG modules
58
detected in metagenomic assemblies from the Outokumpu deep borehole.
500 m 600 m 967 m 1500 m 2260 m 2300 m
Carbon fixation
M00165 reductive pentose phosphate cycle Complete 1 missing Complete
(Calvin cycle)
M00166 reductive pentose phosphate cycle, Complete 1 missing 1 missing Complete 1 missing
ribulose-5P ¼> glyceraldehyde-3P
M00167 reductive pentose phosphate cycle, Complete 1 missing Complete 1 missing Complete 1 missing
glyceraldehyde-3P ¼> ribulose-5P
M00168 CAM (Crassulacean acid metabolism), dark Complete 1 missing Complete Complete Complete Complete
M00169 CAM (Crassulacean acid metabolism), light Complete 1 missing Complete Complete Complete Complete
M00172 C4-dicarboxylic acid cycle, NADPdmalic 1 missing 1 missing 1 missing 1 missing 1 missing
enzyme type
M00173 reductive citrate cycle (ArnoneBuchanan 1 missing 1 missing 1 missing 1 missing
cycle)
M00377 reductive acetyl-CoA pathway (Wood Complete 1 missing Complete 1 missing Complete
eLjungdahl pathway)
M00579 phosphate acetyltransferase-acetate kinase Complete 1 missing Complete Complete Complete 1 missing
pathway, acetyl-CoA ¼> acetate
M00620 incomplete reductive citrate cycle, 1 missing Complete Complete Complete
acetyl-CoA ¼> oxoglutarate
Nitrogen metabolism
M00175 nitrogen fixation, nitrogen ¼> ammonia Complete Complete 1 missing Complete Complete
M. It€avaara et al.
M00531 assimilatory nitrate reduction, 1 missing 1 missing
nitrate ¼> ammonia
M00530 dissimilatory nitrate reduction, Complete 1 missing 1 missing Complete
nitrate ¼> ammonia
Deepterrameta
M00529 denitrification, nitrate ¼> nitrogen Complete 1 missing
M00528 nitrification, ammonia ¼> nitrite 1 missing
[PATH:map00910] (1) (1 block missing)
Methane metabolism
M00567 methanogenesis, CO2 ¼> methane Complete
M00357 methanogenesis, acetate ¼> methane Complete
M00356 methanogenesis, methanol ¼> methane 1 missing Complete 1 missing
M00563 methanogenesis, methylamine/ 1 missing 1 missing
dimethylamine/trimethylamine ¼> methane
M00358 coenzyme M biosynthesis 1 missing Complete Complete
M00608 2-oxocarboxylic acid chain extension, Complete 1 missing
2-oxoglutarate ¼> 2-oxoadipate ¼> 2-
oxopimelate ¼> 2-oxosuberate
M00346 formaldehyde assimilation, serine pathway 1 missing
M00345 formaldehyde assimilation, ribulose Complete 1 missing Complete 1 missing Complete Complete
monophosphate pathway
M00344 formaldehyde assimilation, xylulose 1 missing 1 missing
monophosphate pathway
M00378 F420 biosynthesis 1 missing Complete 1 missing
M00422 acetyl-CoA pathway, CO2 ¼> acetyl-CoA Complete 1 missing Complete Complete
Sulfur metabolism
M00176 assimilatory sulfate reduction, sulfate ¼> H2S Complete Complete Complete
M00596 dissimilatory sulfate reduction, sulfate ¼> H2S Complete Complete Complete
M00595 thiosulfate oxidation by SOX complex, Complete 1 missing
thiosulfate ¼> sulfate
“1 missing” means that one gene or “block” necessary for the functioning of the said module was not detected.
59
Figure 11 A Venn diagram of complete and/or partial Kyoto Encyclopedia of Genes

and Genomes modules encoded by the proteomes from fracture zone samples at
depths 500, 967, and 2260 m in the Outokumpu deep borehole. The diagram was
generated with Venny 2.0. Bardou, P., Mariette, J., Escudié, F., Djemiel, C., & Klopp, C. (2014).
jvenn: an interactive Venn diagram viewer. BMC Bioinformatics, 15, 293. http://dx.doi.org/
10.1186/1471-2105-15-293.
Numerous two-component systems, such as nitrate respiration associated

NarQ-NarP, were also present. In addition, many of the unique modules
of the proteome at the depth 967 m were associated with eukaryotic
ATPases, DNA and RNA polymerases, and other housekeeping protein
complexes such as minichromosome maintenance and replication factor C
(RF-C).
5.5 Summary
Composition of each microbial community in the water column samples
from depths 600, 1500, and 2300 m in the Outokumpu deep borehole
was assessed by three methods: analyses of 16S rRNA amplicons, of 16S
rRNA fragments from metagenomic reads, and of relative abundance of
proteins in the assembled metagenomes. All three methods yielded rather
different results on composition of each community. Nonetheless, our
most consistent finding is the relatively high abundance of archaea in the
sample at the 2300-m depth. This result was obtained by interpreting the
metagenomic reads and metagenomic assembly. It€avaara, Nyyss€ onen,
Bomberg, et al. (2011) found that Comamonadaceae dominate the upper
Deepterrameta 61
part of the Outokumpu borehole, and using 16S rRNA amplicons,

Nyyss€ onen et al. (2014) found that this bacterial family is among the most
abundant bacteria at essentially every depth of the Outokumpu borehole
water column. According to the metagenomic assemblies, however,
Comamonadaceae members are relatively abundant only in the fracture
zone at the 500-m depth. Likewise, discrepancies were noted between
the results of Purkamo et al. (2013) and the HiSeq-sequenced metagenomes
that were analyzed here, especially in terms of the composition of archaeal
communities. Multiple factors are likely to contribute to the observed differ-
ences. These factors include, eg, conflicting taxonomies, PCR bias, variable
numbers of 16S rRNA copies, differences in throughput (tens of sequences
in 16S PCR-DGGE vs thousands of 16S rRNA 454 amplicons versus up to
hundreds of thousands of proteins in HiSeq-sequenced assemblies) as well as
the target of quantification (the relative number of 16S rRNA fragments
vs the relative number of proteins). A particularly interesting explanation
of the discrepancy in the abundance of Comamonadaceae in the Outo-
kumpu deep borehole may be that the genes encoding the proteins analyzed
here are hosted due to horizontal gene transfer largely among Comamona-
daceae genomes. More comprehensive metagenomes and/or single-cell
sequenced genomes would be needed to assess the validity of this hypothesis.
Overall, the metagenomic assemblies present a relatively consistent pic-
ture of the microbial communities of the Outokumpu deep borehole. To be
precise, the upper three samples (depths 500, 600, and 967 m) have relatively
similar community composition, as do the two samples from the greatest
depths (2260 and 2300 m). The community composition of the sample
from the 1500-m depth is somewhere between the two extremes. These
results are largely in agreement with the 16S rRNA-based dendrograms
of Nyyss€ onen et al. (2014) and indicate that the microbial communities in
the borehole water column and in fracture zones do not differ significantly.
6. CONCLUSIONS
Exploration of deep mines and boreholes and their fracture fluids has
expanded our knowledge about the deep subsurface life existing at the depth
of kilometers in the continental Earth’s crust.
Terrestrial deep subsurface microorganisms are thought to live mainly in
the fractures of the bedrock, where ancient fluids and gases are found
(Kiet€av€ainen et al., 2013; Purkamo, Bomberg, Kiet€av€ainen, et al., 2015;
Purkamo, Bomberg, Nyyss€ onen, et al., 2015; Sohlberg et al., 2015). Biodi-
versity in deep subsurfaces seems to vary considerably among various sites
presumably as a result of differences in environmental conditions including
temperature, pH, and availability of energy and carbon sources. The contin-
uous flow of hydrogen that is formed during rockewater interactions is
thought to be globally important for provision of energy to deep subsurface
life (Libert, Bildstein, Esnault, Jullien, & Sellier, 2011; Sherwood Lollar
et al., 2014). The information on biodiversity is still patchy, and global
biogeography of microbial species has yet to be fully characterized. It is still
poorly understood how global geological transformations and the separation
of continents affected the biogeography of deep subsurface microbiomes.
Further research is needed regarding the evolutionary perspective on the
early life forms, their separation among the continents, and how this separa-
tion has affected the species diversity and functions.
In the continental crust, all three main domains of life are represented:
bacteria, archaea, and eukaryotes as well as viruses. Generally, bacteria and
especially Proteobacteria tend to be among the major taxa at small depths
of the continental crust, whereas Firmicutes become more common at
greater depths. Many of the bacteria are involved in iron and sulfur oxida-
tion of rock, and these processes may increase the concentration of sulfates in
deep groundwater. Carbon dioxide-fixing and hydrogen-utilizing microor-
ganisms are also often found in the same locations (Purkamo, Bomberg,
Kiet€av€ainen, et al., 2015; Purkamo, Bomberg, Nyyss€ onen, et al., 2015;
Schrenk et al., 2013). Methanogenic archea generally constitute a small
part of the microbial communities but have a considerable impact. Sulfates
formed by microbes serve as terminal electron acceptors in other
biogeochemical processes, eg, during methanotrophic sulfide formation in
anaerobic methane oxidation. Methanogenic, sulfate-reducing, and metha-
notrophic microbial communities have been the major focus of deep subsur-
face studies in recent years. These biogeochemical processes are connected to
one another, and the microbial species involved benefit from one another’s
activities by providing or using electron donors and acceptors.
The knowledge about microbial processes in the deep subsurface can be
utilized for risk evaluations in various practical applications, eg, in nuclear
waste disposal, oil, and mining industries.
The increasing amount of sequence data on deep subsurface microbiomes
may be useful for development of novel biocatalysts for biotechnological ap-
plications. The knowledge about the constraints and adaptations of microbial
life in the deep subsurface is even used in astrobiology and space research.
Deepterrameta 63
REFERENCES
Abe, F. (2007). Exploration of the effects of high hydrostatic pressure on microbial growth,
physiology and survival: perspectives from piezophysiology. Bioscience Biotechnology and
Biochemistry, 71, 2347e2357. http://dx.doi.org/10.1271/bbb.70015.
Abrajano, T. A., Sturchio, N. C., Bohlke, J. K., Lyon, G. L., Poreda, R. J., & Stevens, C. M.
(1988). Methane-hydrogen gas seeps, Zambales ophiolite, Philippines: deep or shallow
origin? Chemical Geology, 71, 211e222. http://dx.doi.org/10.1016/0009-2541(88)90116-7.
Abrajano, T. A., Sturchio, N. C., Kennedy, B. M., Lyon, G. L., Muehlenbachs, K., &
Bholke, J. (1990). Geochemistry of reduced gas related to serpentinization of the Zam-
bales ophiolites, Philippines. Applied Geochemistry, 5, 625e630.
Afiahayati, Sato, K., & Sakakibara, Y. (2015). MetaVelvet-SL: an extension of the velvet
assembler to a de novo metagenomic assembler utilizing supervised learning. DNA
Research, 22, 69e77.
Ahonen, L., Kiet€av€ainen, R., Kortelainen, N., Kukkonen, I. T., Pullinen, A.,
€
Toppi, T. … Oster, M. (2011). Hydrogeological characteristics of the outokumpu deep
drill hole. Special Paper e Geological Survey of Finland, 2011, 151e168.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local align-
ment search tool. Journal of Molecular Biology, 215, 403e410.
Amend, J. P., McCollom, T. M., Hentscher, M., & Bach, W. (2011). Catabolic and anabolic
energy for chemolithoautotrophs in deep-sea hydrothermal systems hosted in different
rock types. Geochimica et Cosmochimica Acta, 75, 5736e5748. http://dx.doi.org/
10.1016/j.gca.2011.07.041.
Amend, J. P., & Teske, A. (2005). Expanding frontiers in deep subsurface microbiology.
Palaeogeography Palaeoclimatology Palaeoecology, 219, 131e155. http://dx.doi.org/
10.1016/j.palaeo.2004.10.018.
Anderson, R. E., Brazelton, W. J., & Baross, J. A. (2013). The deep viriosphere: assessing the
viral impact on microbial community dynamics in the deep subsurface. Reviews in Miner-
alogy and Geochemistry, 75, 649e675. http://dx.doi.org/10.2138/rmg.2013.75.20.
Anderson, R. E., Sogin, M. L., & Baross, J. A. (2015). Biogeography and ecology of the rare
and abundant microbial lineages in deep-sea hydrothermal vents. FEMS Microbiology
Ecology, 91, 1e11. http://dx.doi.org/10.1093/femsec/fiu016.
Andrews, S., (2015). http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. (WWW
Document).
Bach, W., & Edwards, K. J. (2003). Iron and sulfide oxidation within the basaltic ocean crust:
implications for chemolithoautotrophic microbial biomass production. Geochimica et Cos-
mochimica Acta, 67, 3871e3887. http://dx.doi.org/10.1016/S0016-7037(03)00304-1.
Baker, B. J., Sheik, C. S., Taylor, C. A., Jain, S., Bhasi, A., Cavalcoli, J. D., & Dick, G. J.
(2013). Community transcriptomic assembly reveals microbes that contribute to deep-
sea carbon and nitrogen cycling. ISME Journal, 7, 1962e1973. http://dx.doi.org/
10.1038/ismej.2013.85.
Bardou, P., Mariette, J., Escudié, F., Djemiel, C., & Klopp, C. (2014). jvenn: an interactive
Venn diagram viewer. BMC Bioinformatics, 15, 293. http://dx.doi.org/10.1186/1471-
2105-15-293.
Bartlett, D. H. (2002). Pressure effects on in vivo microbial processes. Biochimica et Biophysica
Acta (BBA) e Protein Structure and Molecular Enzymology, 1595, 367e381. http://
dx.doi.org/10.1016/S0167-4838(01)00357-0.
Barton, L. L., Mandl, M., & Loy, A. (2010). Geomicrobiology: Molecular and environmental
perspective. Netherlands, Dordrecht: Springer. http://dx.doi.org/10.1007/978-90-481-
9204-5.
Basso, O., Lascourreges, J.-F., Jarry, M., Magot, M., Lascourreges, J. F.,
Jarry, M. … Kukkonen, I. T. (2005). The effect of cleaning and disinfecting the sampling
well on the microbial communities of deep subsurface water samples. Environmental

Microbiology, 7, 13e21. http://dx.doi.org/10.1111/j.1462-2920.2004.00660.x.
Beal, E. J., House, C. H., & Orphan, V. J. (2009). Manganese- and iron-dependent marine
methane oxidation. Science, 325, 184e187. http://dx.doi.org/10.1126/science.1169984.
Benton, M. J. (2004). Vertebrate palaeontology (3rd ed.). Oxford: Wiley-Blackwel Publishing.
Boetius, A., Ravenschlag, K., Schubert, C. J., Rickert, D., Widdel, F.,
Gieseke, A. … Pfannkuche, O. (2000). A marine microbial consortium apparently medi-
ating anaerobic oxidation of methane. Nature, 407, 623e626.
Boettger, J., Lin, H. T., Cowen, J. P., Hentscher, M., & Amend, J. P. (2013). Energy yields
from chemolithotrophic metabolisms in igneous basement of the Juan de Fuca ridge
flank system. Chemical Geology, 337e338, 11e19. http://dx.doi.org/10.1016/
j.chemgeo.2012.10.053.
Bomberg, M., Lamminm€aki, T., & It€avaara, M. (2015). Estimation of microbial metabolism
and co-occurrence patterns in fracture groundwaters of deep crystalline bedrock at Olki-
luoto, Finland. Biogeosciences Discuss, 12, 13819e13857. http://dx.doi.org/10.5194/
bgd-12-13819-2015.
Bomberg, M., Nyyss€ onen, M., Pitk€anen, P., Lehtinen, A., & It€avaara, M. (2015). Active
microbial communities inhabit sulphate-methane interphase in deep bedrock fracture
fluids in olkiluoto, Finland. BioMed Research International, 2015.
Borgonie, G., García-Moyano, A., Litthauer, D., Bert, W., Bester, A., van
Heerden, E. … Onstott, T. C. (2011). Nematoda from the terrestrial deep subsurface
of South Africa. Nature, 474, 79e82. http://dx.doi.org/10.1038/nature09974.
Borrel, G., O’Toole, P. W., Harris, H. M. B., Peyret, P., Brugere, J.-F., & Gribaldo, S.
(2013). Phylogenomic data support a seventh order of methylotrophic methanogens
and provide insights into the evolution of methanogenesis. Genome Biology and Evolution,
5, 1769e1780. http://dx.doi.org/10.1093/gbe/evt128.
de Bourcy, C. F. A., De Vlaminck, I., Kanbar, J. N., Wang, J., Gawad, C., & Quake, S. R.
(2014). A quantitative comparison of single-cell whole genome amplification methods.
PLoS One, 9, e105585. http://dx.doi.org/10.1371/journal.pone.0105585.
Braakman, R., & Smith, E. (2013). The compositional and evolutionary logic of metabolism.
Physical Biology, 10, 011001. http://dx.doi.org/10.1088/1478-3975/10/1/011001.
Brazelton, W. J., Morrill, P. L., Szponar, N., & Schrenk, M. O. (2013). Bacterial commu-
nities associated with subsurface geochemical processes in continental serpentinite
springs. Applied and Environmental Microbiology, 79, 3906e3916. http://dx.doi.org/
10.1128/AEM.00330-13.
Brazelton, W. J., Nelson, B., & Schrenk, M. O. (2012). Metagenomic evidence for H(2)
oxidation and H(2) production by serpentinite-hosted subsurface microbial
communities. Frontiers in Microbiology, 2, 268. http://dx.doi.org/10.3389/
fmicb.2011.00268.
Brazelton, W. J., Schrenk, M. O., Kelley, D. S., & Baross, J. A. (2006). Methane- and sulfur-
metabolizing microbial communities dominate the Lost City hydrothermal field
ecosystem. Applied and Environmental Microbiology, 72, 6257e6270. http://dx.doi.org/
10.1128/AEM.00574-06.
Bressan, M., Trinsoutrot Gattin, I., Desaire, S., Castel, L., Gangneux, C., & Laval, K. (2015).
A rapid flow cytometry method to assess bacterial abundance in agricultural soil. Applied
Soil Ecology, 88, 60e68. http://dx.doi.org/10.1016/j.apsoil.2014.12.007.
Breuker, A., K€ oweker, G., Blazejak, A., & Schippers, A. (2011). The deep biosphere in
terrestrial sediments in the Chesapeake Bay area, Virginia, USA. Frontiers in Microbiology,
2. http://dx.doi.org/10.3389/fmicb.2011.00156.
Brown, C. T., Hug, L. A., Thomas, B. C., Sharon, I., Castelle, C. J.,
Singh, A. … Banfield, J. F. (2015). Unusual biology across a group comprising more
than 15% of domain bacteria. Nature, 523, 208e211.
Deepterrameta 65
Buffett, B. A. (2000). Clathrate hydrates. Annual Review of Earth and Planetary Sciences, 28,
477e507. http://dx.doi.org/10.1146/annurev.earth.28.1.477.
Burford, E. P., Kierans, M., & Gadd, G. M. (2003). Geomycology: fungi in mineral substrata.
Mycologist, 17, 98e107. http://dx.doi.org/10.1017/S0269-915X(03)00311-2.
Butler, R. (1994). Drilling deep holes in the continents. Geology Today, 10, 32e35. http://
dx.doi.org/10.1111/j.1365-2451.1994.tb00856.x.
Canfield, D. E., Rosing, M. T., & Bjerrum, C. (2006). Early anaerobic metabolisms. Philo-
sophical Transactions of the Royal Society B: Biological Sciences, 361, 1819e1836. http://
dx.doi.org/10.1098/rstb.2006.1906.
Cardace, D., Hoehler, T., McCollom, T., Schrenk, M., Carnevale, D., Kubo, M., &
Twing, K. (2013). Establishment of the coast range ophiolite microbial observatory
(CROMO): drilling objectives and preliminary outcomes. Scientific Drilling, 16, 45e
55. http://dx.doi.org/10.5194/sd-16-45-2013.
Caspi, R., Altman, T., Billington, R., Dreher, K., Foerster, H.,
Fulcher, C. A. … Karp, P. D. (2014). The MetaCyc database of metabolic pathways
and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids
Research, 42, D459eD471.
Cermak, V., Kukkonen, I. T., & Safanda, J. (1993). Temperature logs in deep wells? a useful
tool for past climatic reconstruction. Terra Nova, 5, 134e143. http://dx.doi.org/
10.1111/j.1365-3121.1993.tb00238.x.
Chivian, D., Brodie, E. L., Alm, E. J., Culley, D. E., Dehal, P. S.,
DeSantis, T. Z. … Onstott, T. C. (2008). Environmental genomics reveals a single-
species ecosystem deep within earth. Science, 322, 275e278. http://dx.doi.org/
10.1126/science.1155495.
Claesson Liljedahl, L., Lehtinen, A., Harper, J., N€aslund, J.-O., Selroos, J.-O.,
Pitk€anen, P. … Vidstrand, P. (2015). The Greenland analogue project: Final report. Swedish
Nucl. Fuel Waste Manag. Co. Rep. TR-14e13 (in press).
Condie, K. C. (1989). Plate tectonics and crustal evolution (3rd ed.). Oxford: Pergamon Press.
Davidson, M. M., Silver, B. J., Onstott, T. C., Moser, D. P., Gihring, T. M.,
Pratt, L. M. … Ralston, C. (2011). Capture of planktonic microbial diversity in fractures
by long-term monitoring of flowing boreholes, evander basin, South Africa. Geomicrobi-
ology Journal, 28, 275e300. http://dx.doi.org/10.1080/01490451.2010.499928.
DeLong, E. F., & Yayanos, A. A. (1985). Adaptation of the membrane lipids of a deep-sea
bacterium to changes in hydrostatic pressure. Science, 228, 1101e1103.
Dong, H., Onstott, T. C., DeFlaun, M. F., Fuller, M. E., Gillespie, K. M., &
Fredrickson, J. K. (1999). Development of radiographic and microscopic techniques
for the characterization of bacterial transport in intact sediment cores from Oyster,
Virginia. Journal of Microbiological Methods, 37, 139e154. http://dx.doi.org/10.1016/
S0167-7012(99)00056-1.
Dridi, B., Fardeau, M.-L., Ollivier, B., Raoult, D., & Drancourt, M. (2012).
Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon
isolated from human faeces. International Journal of Systematic and Evolutionary Microbiology,
62, 1902e1907.
Duhaime, M. B., Deng, L., Poulos, B. T., & Sullivan, M. B. (2012). Towards quantitative
metagenomics of wild viruses and other ultra-low concentration DNA samples: a
rigorous assessment and optimization of the linker amplification method.
Environmental Microbiology, 14, 2526e2537. http://dx.doi.org/10.1111/j.1462-
2920.2012.02791.x.
Dupinay, T., Nguyen, A., Croze, S., Barbet, F., Rey, C., Mavingui, P. … Legras-Lachuer, C.
(2012). Next-generation sequencing of ultra-low copy samples : from clinical FFPE
samples to single-cell sequencing. Current Topics in Virology, 10, 63e83.
Ehrlich, H. L. (2002). Geomicrobiology. Marcel Dekker, Inc.
Eiler, A., Zaremba-Niedzwiedzka, K., Martínez-García, M., McMahon, K. D.,

Stepanauskas, R., Andersson, S. G. E., & Bertilsson, S. (2014). Productivity and salinity
structuring of the microplankton revealed by comparative freshwater metagenomics.
Environmental Microbiology, 16, 2682e2698. http://dx.doi.org/10.1111/1462-
2920.12301.
Engelhardt, T., Sahlberg, M., Cypionka, H., & Engelen, B. (2011). Induction of prophages
from deep-subseafloor bacteria. Environmental Microbiology Reports, 3, 459e465.
Engelhardt, T., Kallmeyer, J., Cypionka, H., & Engelen, B. (2014). High virus-to-cell ratios
indicate ongoing production of viruses in deep subsurface sediments. ISME Journal, 8,
1503e1509.
Etiope, G., & Sherwood Lollar, B. (2013). Abiotic methane on Earth. Reviews of Geophysics,
51, 276e299. http://dx.doi.org/10.1002/rog.20011.
Ettwig, K. F., Butler, M. K., Le Paslier, D., Pelletier, E., Mangenot, S.,
Kuypers, M. M. M. … Strous, M. (2010). Nitrite-driven anaerobic methane oxidation
by oxygenic bacteria. Nature, 464, 543e548. http://dx.doi.org/10.1038/nature08883.
Evans, B. W. (2004). The serpentinite multisystem revisited: chrysotile is metastable. Interna-
tional Geology Review, 46, 479e506. http://dx.doi.org/10.2747/0020-6814.46.6.479.
Evans, B. W., Johannes, W., & Oterdoom, H. (1976). Stability of crysotile and antigorite in
the serpentine multisystem. Schweizerische Mineralogische und Petrographische Mitteilungen,
56, 79e93.
Evans, C., & Brussaard, C. P. D. (2012). Regional variation in lytic and lysogenic viral
infection in the southern ocean and its contribution to biogeochemical cycling. Applied
and Environmental Microbiology, 78, 6741e6748. http://dx.doi.org/10.1128/
AEM.01388-12.
Eydal, H. S. C., J€agevall, S., Hermansson, M., & Pedersen, K. (2009). Bacteriophage lytic to
Desulfovibrio aespoeensis isolated from deep groundwater. ISME Journal, 3, 1139e1147.
http://dx.doi.org/10.1038/ismej.2009.66.
Follin, S., Hartley, L., Rhén, I., Jackson, P., Joyce, S., Roberts, D., & Swift, B. (2014). A
methodology to constrain the parameters of a hydrogeological discrete fracture network
model for sparsely fractured crystalline rock, exemplified by data from the proposed
high-level nuclear waste repository site at Forsmark, Sweden. Hydrogeology Journal, 22,
313e331. http://dx.doi.org/10.1007/s10040-013-1080-2.
Fredrickson, J. K., & Balkwill, D. L. (2006). Geomicrobial processes and biodiversity in the
deep terrestrial subsurface. Geomicrobiology Journal, 23, 345e356. http://dx.doi.org/
10.1080/01490450600875571.
Fredrickson, J. K., McKinley, J. P., Bjornstad, B. N., Long, P. E., Ringelberg, D. B.,
White, D. C. … Onstott, T. C. (1997). Porelsize constraints on the activity and survival
of subsurface bacteria in a late cretaceous shale-sandstone sequence, northwestern
New Mexico. Geomicrobiology Journal, 14, 183e202. http://dx.doi.org/10.1080/
01490459709378043.
Freifeld, B. M. (2005). The U-tube: a novel system for acquiring borehole fluid samples from
a deep geologic CO2 sequestration experiment. Journal of Geophysical Research, 110,
B10203. http://dx.doi.org/10.1029/2005JB003735.
Fu, Q., Sherwood Lollar, B., Horita, J., Lacrampe-Couloume, G., & Seyfried, W. E. J.
(2007). Abiotic formation of hydrocarbons under hydrothermal conditions: constraints
from chemical and isotope data. Geochimica et Cosmochimica Acta, 71, 1982e1998.
Gadd, G. M. (2010). Metals, minerals and microbes: geomicrobiology and bioremediation.
Microbiology, 156, 609e643. http://dx.doi.org/10.1099/mic.0.037143-0.
Garcia, S. L., McMahon, K. D., Martinez-Garcia, M., Srivastava, A., Sczyrba, A.,
Stepanauskas, R. … Warnecke, F. (2013). Metabolic potential of a single cell belonging
to one of the most abundant lineages in freshwater bacterioplankton. ISME Journal, 7,
137e147. http://dx.doi.org/10.1038/ismej.2012.86.
Deepterrameta 67
Gebert, J., Knoblauch, C., Gadd, G., Pfeiffer, E.-M., & Dilly, O. (2011). Sustainability of
geochemical cycling. Journal of Geochemical Exploration, 110, viieviii. http://
dx.doi.org/10.1016/j.gexplo.2011.04.008.
Giannoukos, G., Ciulla, D. M., Huang, K., Haas, B. J., Izard, J., Levin, J. Z. … Gnirke, A.
(2012). Efficient and robust RNA-seq process for cultured bacteria and complex com-
munity transcriptomes. Genome Biology, 13, R23. http://dx.doi.org/10.1186/gb-
2012-13-3-r23.
Gilles, A., Meglécz, E., Pech, N., Ferreira, S., Malausa, T., & Martin, J.-F. (2011). Accuracy
and quality assessment of 454 GS-FLX Titanium pyrosequencing. BMC Genomics, 12,
245. http://dx.doi.org/10.1186/1471-2164-12-245.
Gogarten, J. P., & Townsend, J. P. (2005). Horizontal gene transfer, genome innovation and
evolution. Nature Reviews Microbiology, 3, 679e687. http://dx.doi.org/10.1038/
nrmicro1204.
Griffiths, R. I., Thomson, B. C., James, P., Bell, T., Bailey, M., & Whiteley, A. S. (2011).
The bacterial biogeography of British soils. Environmental Microbiology, 13, 1642e1654.
http://dx.doi.org/10.1111/j.1462-2920.2011.02480.x.
Guggenheim, S., & Koster van Groos, A. F. (2003). New gas-hydrate phase: synthesis and
stability of clayemethane hydrate intercalate. Geology, 31, 653. http://dx.doi.org/
10.1130/0091-7613(2003)031<0653:NGPSAS>2.0.CO;2.
Gupta, R. S. (2000). The phylogeny of proteobacteria: relationships to other eubacterial
phyla and eukaryotes. FEMS Microbiology Reviews, 24, 367e402.
Hallbeck, L., & Pedersen, K. (2008). Characterization of microbial processes in deep aquifers
of the Fennoscandian shield. Applied Geochemistry, 23, 1796e1819. http://dx.doi.org/
10.1016/j.apgeochem.2008.02.012.
Hanson, R. S., & Hanson, T. E. (1996). Methanotrophic bacteria. Microbiological Reviews, 60,
439e471.
Haroon, M. F., Hu, S., Shi, Y., Imelfort, M., Keller, J., Hugenholtz, P. … Tyson, G. W.
(2013). Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal
lineage. Nature, 500, 567e570.
Harper, J., Hubbard, A., Ruskeeniemi, T., Claesson Liljedahl, L., Lehtinen, A.,
Booth, A. … V, A. D. (2011). The Greenland analogue project yearly report 2010 (p. 162).
Stockholm: Swedish Nucl. Fuel Waste Manag. Co. Rep. SKB R-11-23, SKB: R-11-
23/Posiva: WR 2012e16.
He, S., Wurtzel, O., Singh, K., Froula, J. L., Yilmaz, S., Tringe, S. G. … Hugenholtz, P.
(2010). Validation of two ribosomal RNA removal methods for microbial
metatranscriptomics. Nature Methods, 7, 807e812. http://dx.doi.org/10.1038/
nmeth.1507.
Hess, H. H. (1962). History of ocean basins. In A. E. J. Engel, H. L. James, & B. F. Leonard
(Eds.), Petrologic studies: A volume in honor of a. F. Buddington (pp. 599e620). Buddington.
Boulder, CO: Geological Society of America.
Hobbs, M. Y., Frape, S. K., Shouakar-Stash, O., & Kennell, L. R. (2011). Regional hydrogeo-
chemistry e Southern Ontario. Toronto, Canada: NWMO. Rep. NWMO DGR-TR-
2011-12 157.
Hoehler, T. M. (2004). Biological energy requirements as quantitative boundary conditions
for life in the subsurface. Geobiology, 2, 205e215. http://dx.doi.org/10.1111/j.1472-
4677.2004.00033.x.
Horsfield, B., & Kieft, T. L. (2007). Continental scientific drilling, continental scientific drilling: A
decade of progress, and challenges for the future. Berlin Heidelberg: Springer. http://
dx.doi.org/10.1007/978-3-540-68778-8.
Horsfield, J. A., Anagnostou, S. H., Hu, J. K.-H., Cho, K. H. Y., Geisler, R.,
Lieschke, G. … Crosier, P. S. (2007). Cohesin-dependent regulation of Runx genes.
Development, 134, 2639e2649. http://dx.doi.org/10.1242/dev.002485.
Huang, Y., Gilna, P., & Li, W. (2009). Identification of ribosomal RNA genes in metage-
nomic fragments. Bioinformatics, 25, 1338e1340.
Hyatt, D., Chen, G.-L., Locascio, P. F., Land, M. L., Larimer, F. W., & Hauser, L. J. (2010).
Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC
Bioinformatics, 11, 119.
Inagaki, F., & Orphan, V. (2014). Earth and life processes discovered from subseafloor environments
e A decade of science achieved by the integrated ocean drilling program (IODP), developments in
Marine geology, developments in Marine geology. Elsevier. http://dx.doi.org/10.1016/B978-
0-444-62617-2.00002-5.
Ishii, S., Tago, K., & Senoo, K. (2010). Single-cell analysis and isolation for microbiology and
biotechnology: methods and applications. Applied Microbiology and Biotechnology, 86,
1281e1292. http://dx.doi.org/10.1007/s00253-010-2524-4.
It€avaara, M., Nyyss€ onen, M., Bomberg, M., Kapanen, A., Nousiainen, A.,
Ahonen, L. … Kukkonen, I. T. (2011). Microbiological sampling and analysis of the
Outokumpu deep drill hole biosphere in 2007e2009. Special Paper e Geological Survey
of Finland, 2011, 199e206.
It€avaara, M., Nyyss€ onen, M., Kapanen, A., Nousiainen, A., Ahonen, L., & Kukkonen, I.
(2011). Characterization of bacterial diversity to a depth of 1500 m in the Outokumpu
deep borehole, Fennoscandian shield. FEMS Microbiology Ecology, 77, 295e309. http://
dx.doi.org/10.1111/j.1574-6941.2011.01111.x.
It€avaara, M., Vehkom€aki, M.-L., & Nousiainen, A. (2008). Sulphate-reducing bacteria in
ground water samples from olkiluoto e Analyzed by quantitative PCR. Posiva Work. Rep.
2008-2.
J€agevall, S., Rabe, L., & Pedersen, K. (2011). Abundance and diversity of biofilms in
€ o hard rock laboratory. Sweden Microbial Ecology,
natural and artificial aquifers of the Asp€
61, 410e422. http://dx.doi.org/10.1007/s00248-010-9761-z.
Johansson, H., Dhaygude, K., Lindstr€ om, S., Helanter€a, H., Sundstr€ om, L., & Trontti, K.
(2013). A metatranscriptomic approach to the identification of microbiota associated
with the ant Formica exsecta. PLoS One, 8, e79777. http://dx.doi.org/10.1371/
journal.pone.0079777.
Jover, L. F., Effler, T. C., Buchan, A., Wilhelm, S. W., & Weitz, J. S. (2014). The elemental
composition of virus particles: implications for marine biogeochemical cycles. Nature
Reviews Microbiology, 12, 519e528. http://dx.doi.org/10.1038/nrmicro3289.
Kanehisa, M., & Goto, S. (2000). KEGG: kyoto encyclopedia of genes and genomes. Nucleic
Acids Research, 28, 27e30.
Kanehisa, M., Goto, S., Sato, Y., Kawashima, M., Furumichi, M., & Tanabe, M. (2014).
Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids
Research, 42, D199eD205.
Kashefi, K. (2012). Hyperthermophiles: metabolic diversity and biotechnological applications.
In R. P. Anitori (Ed.), Extremophiles: Microbiology and biotechnology (pp. 183e231). Norfolk,
UK: Caister Academic Press.
Kashefi, K., & Lovley, D. R. (2003). Extending the upper temperature limit for life. Science,
301, 934. http://dx.doi.org/10.1126/science.1086823.
Kato, C., Li, L., Nogi, Y., Nakamura, Y., Tamaoka, J., & Horikoshi, K. (1998). Extremely
barophilic bacteria isolated from the Mariana trench, challenger deep, at a depth of
11,000 meters. Applied and Environmental Microbiology, 64, 1510e1513.
Kato, C., & Takai, K. (2000). Microbial diversity of deep-sea extremophilesePiezophiles,
Hyperthermophiles, and subsurface microorganisms. Biological Sciences in Space ¼ Uchu
seibutsu kagaku, 14, 341e352.
Katsuyama, C., Nashimoto, H., Nagaosa, K., Ishibashi, T., Furuta, K.,
Kinoshita, T. … Kato, K. (2013). Occurrence and potential activity of denitrifiers and
methanogens in groundwater at 140 m depth in Pliocene diatomaceous mudstone of
Deepterrameta 69
northern Japan. FEMS Microbiology Ecology, 86, 532e543. http://dx.doi.org/10.1111/

1574-6941.12179.
Kelley, D. R., Liu, B., Delcher, A. L., Pop, M., & Salzberg, S. L. (2012). Gene prediction
with glimmer for metagenomic sequences augmented by classification and clustering.
Nucleic Acids Research, 40, e9.
Kelley, D. S., Karson, J. A., Fr€ uh-Green, G. L., Yoerger, D. R., Shank, T. M.,
Butterfield, D. A. … Sylva, S. P. (2005). A serpentinite-hosted ecosystem: the Lost
City hydrothermal field. Science, 307, 1428e1434. http://dx.doi.org/10.1126/
science.1102556.
Kieft, T. L., Onstott, T. C., Ahonen, L., Aloisi, V., Colwell, F. S.,
Engelen, B. … Wilkins, M. J. (2015). Workshop to develop deep-life continental
scientific drilling projects. Scientific Drilling, 19, 43e53. http://dx.doi.org/10.5194/
sd-19-43-2015.
Kiet€av€ainen, R., Ahonen, L., Kukkonen, I. T., Hendriksson, N., Nyyss€ onen, M., &
It€avaara, M. (2013). Characterisation and isotopic evolution of saline waters of the
Outokumpu deep drill hole, Finland e implications for water origin and deep terrestrial
biosphere. Applied Geochemistry, 32, 37e51. http://dx.doi.org/10.1016/
j.apgeochem.2012.10.013.
Kiet€av€ainen, R., Ahonen, L., Kukkonen, I. T., Niedermann, S., & Wiersberg, T. (2014).
Noble gas residence times of saline waters within crystalline bedrock, Outokumpu
deep drill hole, Finland. Geochimica et Cosmochimica Acta, 145, 159e174. http://
dx.doi.org/10.1016/j.gca.2014.09.012.
Kiet€av€ainen, R., & Purkamo, L. (2015). The origin, source, and cycling of methane in deep
crystalline rock biosphere. Frontiers in Microbiology, 6, 725. http://dx.doi.org/10.3389/
fmicb.2015.00725.
Klein, F., Humphris, S. E., Guo, W., Schubotz, F., Schwarzenbach, E. M., & Orsi, W. D.
(2015). Fluid mixing and the deep biosphere of a fossil Lost City-type hydrothermal
system at the Iberia Margin. Proceedings of the National Academy of Sciences of the United
States of America, 112, 12036e12041. http://dx.doi.org/10.1073/pnas.1504674112.
Knittel, K., & Boetius, A. (2009). Anaerobic oxidation of methane: progress with an
unknown process. Annual Review of Microbiology, 63, 311e334. http://dx.doi.org/
10.1146/annurev.micro.61.080706.093130.
Knittel, K., Losekann, T., Boetius, A., Kort, R., & Amann, R. (2005). Diversity and distri-
bution of methanotrophic archaea at cold seeps. Applied and Environmental Microbiology,
71, 467e479. http://dx.doi.org/10.1128/AEM.71.1.467-479.2005.
Konhauser, K. (2007). Introduction to geomicrobiology. Blackwell Publishing.
Krueger, F.., 2015. http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
(WWW Document).
Kukkonen, I. (2011). Outokumpu deep drilling project 2003e2010. In Geological survey of
Finland. Special Paper 51, p. 255.
Kukkonen, I. T., & Safanda, J. (1996). Palaeoclimate and structure: the most important
factors controlling subsurface temperatures in crystalline rocks. A case history from Out-
okumpu, eastern Finland. Geophysical Journal International, 126, 101e112. http://
dx.doi.org/10.1111/j.1365-246X.1996.tb05270.x.
Kutvonen, H., Rajala, P., Carpén, L., & Bomberg, M. (2015). Nitrate and ammonia as ni-
trogen sources for deep subsurface microorganisms. Frontiers in Microbiology. http://
dx.doi.org/10.3389/fmicb.2015.01079.
Kvenvolden, K. A. (1995). A review of the geochemistry of methane in natural gas hydrate.
Organic Geochemistry, 23, 997e1008. http://dx.doi.org/10.1016/0146-6380(96)
00002-2.
Labonté, J. M., Field, E. K., Lau, M., Chivian, D., Van Heerden, E.,
Wommack, K. E. … Stepanauskas, R. (2015). Single cell genomics indicates horizontal
gene transfer and viral infections in a deep subsurface Firmicutes population. Frontiers in
Microbiology, 6, 349. http://dx.doi.org/10.3389/fmicb.2015.00349.
Laczny, C. C., Sternal, T., Plugaru, V., Gawron, P., Atashpendar, A.,
Margossian, H. H. … Wilmes, P. (2015). VizBin e an application for reference-
independent visualization and human-augmented binning of metagenomic data.
Microbiome, 3, 1.
Langmead, B., & Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nature
Methods, 9, 357e359.
Lau, M. C. Y., Cameron, C., Magnabosco, C., Brown, C. T., Schilkey, F.,
Grim, S. … Onstott, T. C. (2014). Phylogeny and phylogeography of functional genes
shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial
evolutionary relationships. Frontiers in Microbiology, 5, 531. http://dx.doi.org/10.3389/
fmicb.2014.00531.
Lauro, F. M., & Bartlett, D. H. (2008). Prokaryotic lifestyles in deep sea habitats. Extremo-
philes, 12, 15e25. http://dx.doi.org/10.1007/s00792-006-0059-5.
Lauro, F. M., Chastain, R. A., Blankenship, L. E., Yayanos, A. A., & Bartlett, D. H. (2007). The
unique 16S rRNA genes of piezophiles reflect both phylogeny and adaptation. Applied and
Environmental Microbiology, 73, 838e845. http://dx.doi.org/10.1128/AEM.01726-06.
Lever, M. A., Rogers, K. L., Lloyd, K. G., Overmann, J., Schink, B.,
Thauer, R. K. … Jorgensen, B. B. (2015). Life under extreme energy limitation: a syn-
thesis of laboratory- and field-based investigations. FEMS Microbiology Reviews, 1e41.
http://dx.doi.org/10.1093/femsre/fuv020.
Lever, M. A., Rouxel, O., Alt, J. C., Shimizu, N., Ono, S., Coggon, R. M. … Teske, A.
(2013). Evidence for microbial carbon and sulfur cycling in deeply buried ridge flank
basalt. Science, 339, 1305e1308. http://dx.doi.org/10.1126/science.1229240.
Li, H., & Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler
transform. Bioinformatics, 25, 1754e1760.
Libert, M., Bildstein, O., Esnault, L., Jullien, M., & Sellier, R. (2011). Molecular hydrogen:
an abundant energy source for bacterial activity in nuclear waste repositories. Physics and
Chemistry of the Earth, Parts A/B/C, 36, 1616e1623. http://dx.doi.org/10.1016/
j.pce.2011.10.010.
Lin, H.-T., Cowen, J. P., Olson, E. J., Lilley, M. D., Jungbluth, S. P., Wilson, S. T., &
Rappé, M. S. (2014). Dissolved hydrogen and methane in the oceanic basaltic
biosphere. Earth and Planetary Science Letters, 405, 62e73. http://dx.doi.org/10.1016/
j.epsl.2014.07.037.
Lipp, J. S., Morono, Y., Inagaki, F., & Hinrichs, K.-U. (2008). Significant contribution of
archaea to extant biomass in marine subsurface sediments. Nature, 454, 991e994.
http://dx.doi.org/10.1038/nature07174.
Liu, Y., & Whitman, W. B. (2008). Metabolic, phylogenetic, and ecological diversity of the
methanogenic archaea. Annals of the New York Academy of Sciences, 1125, 171e189.
http://dx.doi.org/10.1196/annals.1419.019.
Lloyd, K. G., Schreiber, L., Petersen, D. G., Kjeldsen, K. U., Lever, M. A.,
Steen, A. D. … Jørgensen, B. B. (2013). Predominant archaea in marine sediments degrade
detrital proteins. Nature, 496, 215e218. http://dx.doi.org/10.1038/nature12033.
Loukola-Ruskeeniemi, K. (1991). Geochemical evidence for the hydrothermal origin of
sulphur, base metals and gold in proterozoic metamorphosed black shales, Kainuu and
Outokumpu areas, Finland. Mineralium Deposita, 26. http://dx.doi.org/10.1007/
BF00195262.
Loukola-Ruskeeniemi, K. (1999). Origin of black shales and the serpentinite-associated
Cu-Zn-Co ores at Outokumpu, Finland. Economic Geology, 94, 1007e1028. http://
dx.doi.org/10.2113/gsecongeo.94.7.1007.
Deepterrameta 71
Loukola-Ruskeeniemi, K. (2011). Graphite and sulphide-bearing schists in the Outokumpu

R2500 drill core: comparison with the Ni-Cu-Co-Zn-Mn-rich black schists at Talvi-
vaara, Finland. In Outokumpu deep drilling project 2003-2010 (pp. 229e252). Geological
Survey of Finland. Special Paper 51.
Lowe, T. M., & Eddy, S. R. (1997). tRNAscan-SE: a program for improved detection of
transfer RNA genes in genomic sequence. Nucleic Acids Research, 25, 955e964.
Magnabosco, C., Tekere, M., Lau, M. C. Y., Linage, B., Kuloyo, O.,
Erasmus, M. … Onstott, T. C. (2014). Comparisons of the composition and biogeo-
graphic distribution of the bacterial communities occupying South African thermal
springs with those inhabiting deep subsurface fracture water. Frontiers in Microbiology,
5, 679. http://dx.doi.org/10.3389/fmicb.2014.00679.
Martin, D. D., Bartlett, D. H., & Roberts, M. F. (2002). Solute accumulation in the deep-sea
bacterium photobacterium profundum. Extremophiles, 6, 507e514. http://dx.doi.org/
10.1007/s00792-002-0288-1.
Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing
reads. EMBnet.journal, 17, 10.
Martinez-Garcia, M., Brazel, D., Poulton, N. J., Swan, B. K., Gomez, M. L.,
Masland, D. … Stepanauskas, R. (2012). Unveiling in situ interactions between marine
protists and bacteria through single cell sequencing. ISME Journal, 6, 703e707.
Marteinsson, V., Klonowski, A., Reynisson, E., Vannier, P., Sigurdsson, B. D., &

Olafsson, M. (2015). Microbial colonization in diverse surface soil types in surtsey and
diversity analysis of its subsurface microbiota. Biogeosciences, 12, 1191e1203. http://
dx.doi.org/10.5194/bg-12-1191-2015.
Martiny, J. B. H., Bohannan, B. J. M., Brown, J. H., Colwell, R. K., Fuhrman, J. A.,
Green, J. L. … Staley, J. T. (2006). Microbial biogeography: putting microorganisms
on the map. Nature Reviews Microbiology, 4, 102e112. http://dx.doi.org/10.1038/
nrmicro1341.
Mason, O. U., Nakagawa, T., Rosner, M., Van Nostrand, J. D., Zhou, J.,
Maruyama, A. … Giovannoni, S. J. (2010). First investigation of the microbiology of
the deepest layer of ocean crust. PLoS One, 5, e15399. http://dx.doi.org/10.1371/
journal.pone.0015399.
Mayer, F., & M€ uller, V. (2014). Adaptations of anaerobic archaea to life under extreme en-
ergy limitation. FEMS Microbiology Reviews, 38, 449e472. http://dx.doi.org/10.1111/
1574-6976.12043.
Mayhew, L. E., Lau, G. E., McCollom, T. M., Webb, S., & Templeton, A. S. (2011). The
effect of methanogenesis on the geochemistry of low temperature watereFe0ebasalt
reactions. Applied Geochemistry, 26, S318. http://dx.doi.org/10.1016/j.apgeochem.
2011.03.074.
Mayhew, L. E., Webb, S. M., & Templeton, A. S. (2011). Microscale imaging and identifi-
cation of Fe speciation and distribution during fluid-mineral reactions under highly
reducing conditions. Environmental Science & Technology, 45, 4468e4474. http://
dx.doi.org/10.1021/es104292n.
Mayhew, L. E., Ellison, E. T., McCollom, T. M., Trainor, T. P., & Templeton, A. S.
(2013). Hydrogen generation from low-temperature watererock reactions. Nature
Geoscience, 6, 478e484. http://dx.doi.org/10.1038/ngeo1825.
McCollom, T. M. (2013). Laboratory Simulations of abiotic hydrocarbon formation in
Earth’s deep subsurface. Reviews in Mineralogy & Geochemistry, 75, 467e494. http://
dx.doi.org/10.2138/rmg.2013.75.15.
McCollom, T. M., & Amend, J. P. (2005). A thermodynamic assessment of energy require-
ments for biomass synthesis by chemolithoautotrophic micro-organisms in oxic and
anoxic environments. Geobiology, 3, 135e144. http://dx.doi.org/10.1111/j.1472-

4669.2005.00045.x.
McCollom, T. M., & Shock, E. L. (1997). Geochemical constraints on chemolithoautotro-
phic metabolism by microorganisms in seafloor hydrothermal systems. Geochimica et
Cosmochimica Acta, 61, 4375e4391.
Meersman, F., Daniel, I., Bartlett, D. H., Winter, R., Hazael, R., & McMillan, P. F. (2013).
High-pressure biochemistry and biophysics. Reviews in Mineralogy & Geochemistry, 75,
607e648. http://dx.doi.org/10.2138/rmg.2013.75.19.
Merrin, J., Kumar, P., & Libchaber, A. (2011). Effects of pressure and temperature on the
binding of RecA protein to single-stranded DNA. Proceedings of the National Academy
of Sciences of the United States of America, 108, 19913e19918. http://dx.doi.org/
10.1073/pnas.1112646108.
Meyer-Dombard, D. R., Woycheese, K. M., Yargıçoglu, E. N., Cardace, D., Shock, E. L.,
G€ uleçal-Pektas, Y., & Temel, M. (2014). High pH microbial ecosystems in a newly
discovered, ephemeral, serpentinizing fluid seep at Yanartaş (Chimera), Turkey. Frontiers
in Microbiology, 5, 723. http://dx.doi.org/10.3389/fmicb.2014.00723.
Miettinen, H., Bomberg, M., Nyyss€ onen, M., Salavirta, H., Sohlberg, E., Vikman, M., &
It€avaara, M. (2015). The diversity of microbial communities in olkiluoto bedrock groundwaters
2009e2013.
Miettinen, H., Kiet€av€ainen, R., Sohlberg, E., Numminen, M., Ahonen, L., & It€avaara, M.
(2015). Microbiome composition and geochemical characteristics of deep subsurface
high-pressure environment, Pyh€asalmi mine Finland. Frontiers in Microbiology, 6.
http://dx.doi.org/10.3389/fmicb.2015.01203.
Milucka, J., Ferdelman, T., Polerecky, L., Frazke, D., Wegener, G.,
Schmid, M. … Kuypers, M. (2012). Zero-valent sulphur is a key intermediate in marine
methane oxidation. Nature, 491, 541e546. http://dx.doi.org/10.1038/nature11656.
Moran, M. A., Satinsky, B., Gifford, S. M., Luo, H., Rivers, A., Chan, L.-K. …
Hopkinson, B. M. (2013). Sizing up metatranscriptomics. ISME Journal, 7, 237e243.
Morita, T., Mitsialis, S. A., Koike, H., Liu, Y., & Kourembanas, S. (1997). Carbon monoxide
controls the proliferation of hypoxic vascular smooth muscle cells. Journal of Biological
Chemistry, 272, 32804e32809.
M€
uller, V. (2015). Microbial life at the thermodynamic limit: how much energy is required
to sustain life? Environmental Microbiology Reports, 7, 31e32. http://dx.doi.org/10.1111/
1758-2229.12232.
Muyzer, G., de Waal, E. C., & Uitterlinden, A. G. (1993). Profiling of complex microbial
populations by denaturing gradient gel electrophoresis analysis of polymerase chain reac-
tion-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology, 59,
695e700, 0099-2240/93/030695-06$02.00/0.
Nagano, Y., & Nagahama, T. (2012). Fungal diversity in deep-sea extreme environments.
Fungal Ecology, 5, 463e471. http://dx.doi.org/10.1016/j.funeco.2012.01.004.
Nawrocki, E. P. (2009). Structural RNA homology search and alignment using covariance models.
Washington University in Saint Louis.
Neff, J. C., & Asner, G. P. (2001). Dissolved organic carbon in terrestrial ecosystems: synthesis
and a model. Ecosystems, 4, 29e48. http://dx.doi.org/10.1007/s100210000058.
Nilsson, A., Ab, G., Tullborg, E., Ab, T., Smellie, J., Ab, C. … Auqué, L. F. (2011). SKB
R-11-06 SFR site investigation bedrock hydrogeochemistry.
Nurmi, P. A., & Kukkonen, I. T. (1986). A new technique for sampling water and gas from
deep drill holes. Canadian Journal of Earth Sciences, 23, 1450e1454. http://dx.doi.org/
10.1139/e86-138.
Nyyss€ onen, M., Bomberg, M., Kapanen, A., Nousiainen, A., Pitk€anen, P., & It€avaara, M.
(2012). Methanogenic and sulphate-reducing microbial communities in deep
Deepterrameta 73
groundwater of crystalline rock fractures in olkiluoto, Finland. Geomicrobiology Journal,

29, 863e878. http://dx.doi.org/10.1080/01490451.2011.635759.
Nyyss€ onen, M., Hultman, J., Ahonen, L., Kukkonen, I., Paulin, L., Laine, P. … Auvinen, P.
(2014). Taxonomically and functionally diverse microbial communities in deep crystal-
line rocks of the Fennoscandian shield. ISME Journal, 8, 126e138. http://dx.doi.org/
10.1038/ismej.2013.125.
€
Ohberg, A. (2006). Investigation equipment and methods used by posiva. POSIVA Working report
2006-81. Olkiluoto.
Onstott, T. C., Magnabosco, C., Aubrey, A. D., Burton, A. S., Dworkin, J. P.,
Elsila, J. E. … Bada, J. L. (2014). Does aspartic acid racemization constrain the depth limit
of the subsurface biosphere? Geobiology, 12, 1e19. http://dx.doi.org/10.1111/
gbi.12069.
Orsi, W., Biddle, J. F., & Edgcomb, V. (2013). Deep sequencing of subseafloor eukaryotic
rRNA reveals active fungi across marine subsurface provinces. PLoS One, 8, e56335.
Orsi, W. D., Richards, T. A., & Santoro, A. E. (2015). Cellular maintenance processes
that potentially underpin the survival of subseafloor fungi over geological
timescales. Estuarine Coastal and Shelf Science, 1e9. http://dx.doi.org/10.1016/
j.ecss.2015.04.009.
Osburn, M. R., LaRowe, D. E., Momper, L. M., & Amend, J. P. (2014). Chemolithotrophy
in the continental deep subsurface: Sanford Underground Research Facility (SURF),
USA. Frontiers in Microbiology, 5, 610. http://dx.doi.org/10.3389/fmicb.2014.00610.
Paul, K., Nonoh, J. O., Mikulski, L., & Brune, A. (2012). ‘Methanoplasmatales,’ thermoplas-
matales-related archaea in termite guts and other environments, are the seventh order of
methanogens. Applied and Environmental Microbiology, 78, 8245e8253.
Pedersen, K. (2000). Exploration of deep intraterrestrial microbial life: current perspectives.
FEMS Microbiology Letters, 185, 9e16. http://dx.doi.org/10.1016/S0378-1097(00)
00061-6.
Pedersen, K. (2010). The deep biosphere. GFF, 132, 93e94. http://dx.doi.org/10.1080/
11035891003692942.
Pedersen, K. (2012). Subterranean microbial populations metabolize hydrogen and acetate
under in situ conditions in granitic groundwater at 450 m depth in the Asp€ € o hard
rock laboratory, Sweden. FEMS Microbiology Ecology, 81, 217e229. http://dx.doi.org/
10.1111/j.1574-6941.2012.01370.x.
Pedersen, K., Bomberg, M., & It€avaara, M. (2012). Summary report microbiology of olkiluoto and
onkalo groundwater. POSIVA 2012-42. Olkiluoto.
Pedersen, K. (2013). Metabolic activity of subterranean microbial communities in deep
granitic groundwater supplemented with methane and H(2). ISME J, 7, 839e849.
Peng, Y., Leung, H. C. M., Yiu, S. M., & Chin, F. Y. L. (2012). IDBA-UD: a de novo
assembler for single-cell and metagenomic sequencing data with highly uneven depth.
Bioinformatics, 28, 1420e1428.
Pevzner, P. A., Tang, H., & Waterman, M. S. (2001). An Eulerian path approach to DNA
fragment assembly. Proceedings of the National Academy of Sciences of the United States of
America, 98, 9748e9753.
Pitk€anen, P., Ahokas, H., Partamies, S., Yl€a-Mella, M., Snellman, M., & Hell€a, P. (2007).
Quality review of hydrochemical baseline data from the Olkiluoto site. POSIVA 2007-05. Posiva
Oy, Olkiluoto.
Pitk€anen, P., Luukkonen, A., & Partamies, S. (2003). Hydrochemical interpretation of baseline
groundwater conditions at the Olkiluoto site. POSIVA 2003-07. Olkiluoto.
Poulsen, M., Schwab, C., Borg Jensen, B., Engberg, R. M., Spang, A., Canibe, N. … Urich, T.
(2013). Methylotrophic methanogenic thermoplasmata implicated in reduced methane
emissions from bovine rumen. Nature Communications, 4, 1428.
Probst, A. J., Weinmaier, T., Raymann, K., Perras, A., Emerson, J. B., Rattei, T. … Moissl-
Eichinger, C. (2014). Biology of a widespread uncultivated archaeon that contributes to
carbon fixation in the subsurface. Nature Communications, 5, 5497.
Purkamo, L., Bomberg, M., Kiet€av€ainen, R., Salavirta, H., Nyyss€ onen, M., Nuppunen-
Puputti, M. … It€avaara, M. (2015). The keystone species of Precambrian deep bedrock
biosphere belong to Burkholdeariales and Clostridiales. Biogeosciences Discuss, 12, 18103e
18150, 2015 www.biogeosciences-discuss.net/12/18103/2015/doi:10.5194/bgd-
12-18103-2015.
Purkamo, L., Bomberg, M., Nyyss€ onen, M., Kukkonen, I., Ahonen, L., & It€avaara, M.
(2015). Heterotrophic communities supplied by ancient organic carbon predominate
in deep fennoscandian bedrock fluids. Microbial Ecology, 69, 319e332. http://
dx.doi.org/10.1007/s00248-014-0490-6.
Purkamo, L., Bomberg, M., Nyyss€ onen, M., Kukkonen, I., Ahonen, L., Kiet€av€ainen, R., &
It€avaara, M. (2013). Dissecting the deep biosphere: retrieving authentic microbial com-
munities from packer-isolated deep crystalline bedrock fracture zones. FEMS Microbiology
Ecology, 85, 324e337. http://dx.doi.org/10.1111/1574-6941.12126.
R Team Development Core. (2008). R: A language and environment for statistical computing
[WWW Document]. R Found. Stat. Comput. URL http://www.r-project.org.
Rajala, P., Bomberg, M., Kiet€av€ainen, R., Kukkonen, I., Ahonen, L., Nyyss€ onen, M., &
It€avaara, M. (2015). Rapid reactivation of deep subsurface microbes in the presence of
C-1 compounds. Microorganisms, 3, 17e33. http://dx.doi.org/10.3390/
microorganisms3010017.
Reuter, J. A., Spacek, D. V., & Snyder, M. P. (2015). High-throughput sequencing
technologies. Molecular Cell, 58, 586e597. http://dx.doi.org/10.1016/j.molcel.2015.
05.004.
Ringelberg, D. (1997). Biomass, bioactivity and biodiversity: microbial ecology of the deep
subsurface: analysis of ester-linked phospholipid fatty acids. FEMS Microbiology Reviews,
20, 371e377. http://dx.doi.org/10.1016/S0168-6445(97)00019-3.
Rosling, A., Finlay, R. D., & Gadd, G. M. (2009). Geomycology. Fungal Biology Reviews, 23,
91e93. http://dx.doi.org/10.1016/j.fbr.2010.03.005.
Roux, S., Hawley, A. K., Torres Beltran, M., Scofield, M., Schwientek, P.,
Stepanauskas, R. … Sullivan, M. B. (2014). Ecology and evolution of viruses infecting
uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics. eLife, 3,
e03125. http://dx.doi.org/10.7554/eLife.03125.
Ruotsalainen, P., Alhonm€aki-Aalonen, S., Aalto, E., Helenius, J., & Sellge, R. (Eds.). (1996).
Development of the pressurized groudwater sampling equipment PAVE. Posiva Work. Rep.
PATU-96e82.
Sanchez-Murillo, R., Gazel, E., Schwarzenbach, E. M., Crespo-Medina, M., Schrenk, M. O.,
Boll, J., & Gill, B. C. (2014). Geochemical evidence for active tropical serpentinization in
the Santa Elena Ophiolite, Costa Rica: an analog of a humid early Earth? Geochemistry,
Geophysics, Geosystems, 15, 1783e1800. http://dx.doi.org/10.1002/2013GC005213.
Sanschagrin, S., & Yergeau, E. (2014). Next-generation sequencing of 16S ribosomal rna
gene amplicons. Journal of Visualized Experiments. http://dx.doi.org/10.3791/51709.
S€antti, J., Kontinen, A., Sorjonen-Ward, P., Johanson, B., & Pakkanen, L. (2006). Metamor-
phism and chromite in serpetinized and carbonate-silica-altered peridotites of the
Paleoproterozoic Outokumpu-Jormua ophiolite belt, eastern Finland. International
Geology Review, 48, 494e546.
Schmieder, R., & Edwards, R. (2011). Fast identification and removal of sequence contam-
ination from genomic and metagenomic datasets. PLoS One, 6, e17288.
Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L.,
Levesque, C. A. … Schindel, D. (2012). Nuclear ribosomal internal transcribed spacer
(ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National
Deepterrameta 75
Academy of Sciences of the United States of America, 109, 6241e6246. http://dx.doi.org/

10.1073/pnas.1117018109.
Schrenk, M. O., Brazelton, W. J., & Lang, S. Q. (2013). Serpentinization, carbon, and deep
life. Reviews in Mineralogy and Geochmistry, 75, 575e606. http://dx.doi.org/10.2138/
rmg.2013.75.18.
Sephton, M. A., & Hazen, R. M. (2013). On the origins of deep hydrocarbons. Reviews in
Mineralogy, 75, 449e465. http://dx.doi.org/10.2138/rmg.2013.75.14.
Sherwood Lollar, B. S., Onstott, T. C., Lacrampe-Couloume, G., & Ballentine, C. J. (2014).
The contribution of the Precambrian continental lithosphere to global H2 production.
Nature, 516, 379e382. http://dx.doi.org/10.1038/nature14017.
Shock, E. L., & Holland, M. (2004). Geochemical energy sources that support the subsur-
face biosphere. In W. S. D. Wilcock, E. F. DeLong, D. S. Kelley, J. A. Baross, &
S. Craig (Eds.), The subseafloor biosphere at mid-ocean ridges. Geophysics monograph series
(pp. 153e165). Washington, DC: AGU.
Silver, B. J., Onstott, T. C., Rose, G., Lin, L.-H., Ralston, C., Sherwood-
Lollar, B. … McCuddy, S. (2010). In situ cultivation of subsurface microorganisms in a
deep mafic Sill: Implications for SLiMEs. Geomicrobiology Journal, 27, 329e348. http://
dx.doi.org/10.1080/01490451003705183.
Silver, B. J., Raymond, R., Sigman, D. M., Prokopeko, M., Sherwood Lollar, B., Lacrampe-
Couloume, G. … Onstott, T. C. (2012). The origin of NO3 and N2 in deep subsurface
fracture water of South Africa. Chemical Geology, 294-295, 51e62. http://dx.doi.org/
10.1016/j.chemgeo.2011.11.017.
SKB. (2010). The Greenland analogue project. Yearly report 2009s. Swedish Nucl. Fuel Waste
Manag. Co. Rep. SKB R-10e59 116.
Smith, C., Schiater, P., Mohamund, Y., & Agrawal, A. (2007). Using a constructed wetland
to treat groundwater contaminated with chlorinated ethenes. In Battelle press e 9th
international in situ and on-site bioremediation symposium 2007.
S€
oding, J., Biegert, A., & Lupas, A. N. (2005). The HHpred interactive server for
protein homology detection and structure prediction. Nucleic Acids Research, 33,
W244eW248.
Sogin, M. L., Morrison, H. G., Huber, J. A., Welch, D. M., Huse, S. M.,
Neal, P. R. … Herndl, G. J. (2006). Microbial diversity in the deep sea and the under-
explored ‘rare biosphere’. Proceedings of the National Academy of Sciences of the United States
of America, 103, 12115e12120. http://dx.doi.org/10.1073/pnas.0605127103.
Sohlberg, E., Bomberg, M., Miettinen, H., Nyyss€ onen, M., Salavirta, H., Vikman, M., &
It€avaara, M. (2015). Revealing the unexplored fungal communities in deep groundwater
of crystalline bedrock fracture zones in Olkiluoto, Finland. Frontiers in Microbiology, 6,
573. http://dx.doi.org/10.3389/fmicb.2015.00573.
Sparks, R. S. J., Bursik, M. I., Carey, S. N., Gilbert, J. S., Glaze, L. S., Sigursson, H., &
Woods, A. W. (1997). Volcanic plumes. Geological Magazine, 135. http://dx.doi.org/
10.1017/S0016756897278258. S0016756897278258.
Stepanauskas, R. (2012). Single cell genomics: an individual look at microbes. Current
Opinion in Microbiology, 15, 613e620. http://dx.doi.org/10.1016/j.mib.2012.09.001.
Stewart, F. J., Ottesen, E. A., & DeLong, E. F. (2010). Development and quantitative ana-
lyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics. ISME
Journal, 4, 896e907. http://dx.doi.org/10.1038/ismej.2010.18.
Stotler, R. L., Frape, S. K., Ahonen, L., Clark, I., Greene, S., Hobbs, M. … Tarasov, L.
(2010). Origin and stability of a permafrost methane hydrate occurrence in the Canadian
shield. Earth and Planetary Science Letters, 296, 384e394. http://dx.doi.org/10.1016/
j.epsl.2010.05.024.
Stotler, R. L., Frape, S. K., Freifeld, B. M., Holden, B., Onstott, T. C., Ruskeeniemi, T., &
Chan, E. (2011). Hydrogeology, chemical and microbial activity measurement through
deep permafrost. Ground Water, 49, 348e364. http://dx.doi.org/10.1111/j.1745-

6584.2010.00724.x.
Stotler, R. L., Frape, S. K., Ruskeeniemi, T., Pitk€anen, P., & Blowes, D. W. (2012). The
interglacial-glacial cycle and geochemical evolution of Canadian and Fennoscandian
shield groundwaters. Geochimica et Cosmochimica Acta, 76, 45e67. http://dx.doi.org/
10.1016/j.gca.2011.10.006.
Suttle, C. A. (2007). Marine viruses d major players in the global ecosystem. Nature Reviews
Microbiology, 5, 801e812. http://dx.doi.org/10.1038/nrmicro1750.
Szponar, N., Brazelton, W. J., Schrenk, M. O., Bower, D. M., Steele, A., & Morrill, P. L.
(2013). Geochemistry of a continental site of serpentinization, the Tablelands Ophiolite,
Gros Morne National Park: a Mars analogue. Icarus, 224, 286e296. http://dx.doi.org/
10.1016/j.icarus.2012.07.004.
Takai, K., Moser, D. P., DeFlaun, M., Onstott, T. C., & Fredrickson, J. K. (2001). Archaeal
diversity in waters from deep South African gold mines. Applied and Environmental Micro-
biology, 67, 5750e5760.
Takai, K., Nakamura, K., Toki, T., Tsunogai, U., Miyazaki, M., Miyazaki, J. … Horikoshi, K.
(2008). Cell proliferation at 122 C and isotopically heavy CH4 production by a hyperther-
mophilic methanogen under high-pressure cultivation. Proceedings of the National Academy of
Sciences of the United States of America, 105, 10949e10954. http://dx.doi.org/10.1073/
pnas.0712334105.
Taran, L. N., Onoshko, M. P., & Mikhailov, N. D. (2011). Structure and composition of
organic matter and isotope geochemistry of the palaeoproterozoic graphite and
sulphide-rich metasedimentary rocks from the Outokumpu Deep Drill Hole, eastern
Finland. Special Paper e Geological Survey of Finland, 2011, 219e228.
Torsvik, V. (2002). Prokaryotic diversityemagnitude, dynamics, and controlling factors.
Science, 296, 1064e1066. http://dx.doi.org/10.1126/science.1071698.
Vieites, J. M., Guazzaroni, M.-E., Beloqui, A., Golyshin, P. N., & Ferrer, M. (2009). Meta-
genomics approaches in systems microbiology. FEMS Microbiology Reviews, 33, 236e
255. http://dx.doi.org/10.1111/j.1574-6976.2008.00152.x.
Wang, Y. Y., Xia, Y., Dong, H. H., & Dong, X. X. (2005). 16S rDNA analysis of bacterial
community in Donghai terrestrial deep subsurface: microbiology research of the Chinese
continent scientific drilling. Acta Petrologica Sinica, 21, 533e539.
Wasmund, K., Schreiber, L., Lloyd, K. G., Petersen, D. G., Schramm, A.,
Stepanauskas, R. … Adrian, L. (2014). Genome sequencing of a single cell of the widely
distributed marine subsurface Dehalococcoidia, phylum Chloroflexi. ISME Journal, 8,
383e397. http://dx.doi.org/10.1038/ismej.2013.143.
Wegener, A. L. (1912). Die Entestehung der kontinente (The drift of continents). Geologische
Rundschau, 3, 276e292.
Whitman, W. B., Coleman, D. C., & Wiebe, W. J. (1998). Prokaryotes: the unseen majority.
Proceedings of the National Academy of Sciences of the United States of America, 95, 6578e6583.
Wickham, H. (2009). ggplot2: Elegant graphics for data analysis. New York, New York, NY:
Springer. http://dx.doi.org/10.1007/978-0-387-98141-3.
Woese, C. R. (1987). Bacterial evolution. Microbiological Reviews, 51, 221e271.
Woese, C. R., Kandler, O., & Wheelis, M. (1990). Towards a natural system of organisms:
proposal for the domains archaea, bacteria and eucarya. Proceedings of the National Academy
of Sciences of the United States of America, 87, 44576e44579.
Woyke, T., Sczyrba, A., Lee, J., Rinke, C., Tighe, D., Clingenpeel, S. … Cheng, J.-F.
(2011). Decontamination of MDA reagents for single cell whole genome
amplification. PLoS One, 6, e26161. http://dx.doi.org/10.1371/journal.pone.0026161.
Wright, K. E., Grasby, S. E., Williamson, C., Spear, J., & Templeton, A. S. (2011).
Bioenergetics of microbial sulfur-redox reactions in a glacial environment. Applied
Geochemistry, 26, S323. http://dx.doi.org/10.1016/j.apgeochem.2011.03.076.
Deepterrameta 77
Wright, K. E., Williamson, C., Grasby, S. E., Spear, J. R., & Templeton, A. S. (2013).
Metagenomic evidence for sulfur lithotrophy by Epsilonproteobacteria as the major
energy source for primary productivity in a sub-aerial arctic glacial deposit, Borup Fiord
Pass. Frontiers in Microbiology, 4, 63. http://dx.doi.org/10.3389/fmicb.2013.00063.
Wu, Y.-W., Tang, Y.-H., Tringe, S. G., Simmons, B. A., & Singer, S. W. (2014). MaxBin:
an automated binning method to recover individual genomes from metagenomes using
an expectation-maximization algorithm. Microbiome, 2, 26.
Yoon, H. S., Price, D. C., Stepanauskas, R., Rajah, V. D., Sieracki, M. E.,
Wilson, W. H. … Bhattacharya, D. (2011). Single-cell genomics reveals organismal
interactions in uncultivated marine protists. Science, 332, 714e717. http://dx.doi.org/
10.1126/science.1203163.
Yoshioka, H., Maruyama, A., Nakamura, T., Higashi, Y., Fuse, H., Sakata, S., &
Bartlett, D. H. (2010). Activities and distribution of methanogenic and methane-
oxidizing microbes in marine sediments from the Cascadia Margin. Geobiology, 8,
223e233. http://dx.doi.org/10.1111/j.1472-4669.2009.00231.x.
Young, S. A., Loukola-Ruskeeniemi, K., & Pratt, L. M. (2013). Reactions of hydrothermal
solutions with organic matter in Paleoproterozoic black shales at Talvivaara, Finland:
evidence from multiple sulfur isotopes. Earth and Planetary Science Letters, 367, 1e14.
http://dx.doi.org/10.1016/j.epsl.2013.02.004.
Youssef, N., Elsahed, M. S., & McInerney, M. J. (2009). Advances in applied microbiology.
Academic Press.
Zdobnov, E. M., & Apweiler, R. (2001). InterProScanean integration platform for the
signature-recognition methods in InterPro. Bioinformatics, 17, 847e848.
CHAPTER TWO
Microbially-induced Carbonate
Precipitation for Immobilization
of Toxic Metals
Deepika Kumari*, Xin-Yi Qian*, Xiangliang Pan*, 1,
Varenyam Achalx, Qianwei Li{ and Geoffrey Michael Gadd*, {
*Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology
and Geography, Chinese Academy of Sciences, Urumqi, China
x
School of Ecological and Environmental Sciences, East China Normal University, Shanghai, China
{
Geomicrobiology Group, School of Life Sciences, University of Dundee, Dundee, Scotland, UK
1
Corresponding author: E-mail: panxl@ms.xjb.ac.cn
Contents
1. Introduction 80
2. Urease 82
3. Biomineralization 83
3.1 Microbially-induced Calcium Carbonate Precipitation 84
4. Bioprecipitation of Metal(loid)s by Bacterial-induced Carbonate Precipitation 87
4.1 Arsenic 87
4.2 Cadmium 89
4.3 Chromium 91
4.4 Copper 93
4.5 Lead 93
4.6 Radionuclide Bioprecipitation by Urease-producing Bacteria 94
5. Bioprecipitation of Metals by Fungal-induced Carbonate Precipitation 97
6. Conclusions 100
Acknowledgments 101
References 101
Abstract
Rapid urbanization and industrialization resulting from growing populations contribute
to environmental pollution by toxic metals and radionuclides which pose a threat to
the environment and to human health. To combat this threat, it is important to develop
remediation technologies based on natural processes that are sustainable. In recent
years, a biomineralization process involving ureolytic microorganisms that leads to

j
ISSN 0065-2164
80 Deepika Kumari et al.
calcium carbonate precipitation has been found to be effective in immobilizing toxic

metal pollutants. The advantage of using ureolytic organisms for bioremediating metal
pollution in soil is their ability to immobilize toxic metals efficiently by precipitation or
coprecipitation, independent of metal valence state and toxicity and the redox poten-
tial. This review summarizes current understanding of the ability of ureolytic microor-
ganisms for carbonate biomineralization and applications of this process for toxic
metal bioremediation. Microbial metal carbonate precipitation may also be relevant
to detoxification of contaminated process streams and effluents as well as the produc-
tion of novel carbonate biominerals and biorecovery of metals and radionuclides that
form insoluble carbonates.
1. INTRODUCTION
With rapid urbanization and increasing populations, increasing indus-
trial development is inevitable despite awareness of possible adverse effects
on human health and the environment. Various industrial wastes, such as
those from mining and metal refining, fuel and energy production including
atomic energy, iron and steel production, aerospace industries, and many
others, contain toxic metals which are directly or indirectly discharged
into the environment causing pollution (Bishop, 2002). Metals are regarded
as the main soil contaminants in many countries (Guimaraes et al., 2010).
Important pollutants include toxic metal(loid)s, such as Cu, Cr, Cd, Hg,
Sb, Pb, As, Co, Zn, and Sn, and radionuclides such as Sr, U, Th, Am,
and Ra (Singh, Gautam, Mishra, & Gupta, 2011; Wuana & Okieimen,
2011).
The contamination of soil with toxic metals affects human health
directly or indirectly in addition to causing great economic losses (Zin-
jarde, Apte, Mohite, & Ravi Kumar, 2014). The behaviour of metals in
soil always makes them challenging substances to decontaminate as they
may form complexes with naturally occurring substances, bind to soil com-
ponents, and precipitate as insoluble mineral forms. All soils naturally
contain trace levels of metals; however, when this level exceeds tolerable
concentrations, it results in pollution. In soils, metals may dissolve in the
soil solution, occupy exchange sites or be adsorbed on inorganic soil con-
stituents, associate with insoluble soil organic matter or precipitate as pure
or mixed solids (Shuman, 1991) as well as be accumulated by the biota
(Gadd, 2010).
Microbially-Induced Metal Carbonate Precipitation 81
Conventional methods for the treatment of contaminant metals in soil

include physico-chemical methods that suffer from high costs associated
with energy and chemical consumption in addition to possible emission
of secondary pollutants (Krishna & Philip, 2005). Phytoremediation
methods are also highly popular and have been used for in situ remediation
of heavy metals. However, this also has limitations because of the depen-
dence on plant growth conditions such as climate, geology, altitude,
and temperature (Achal, Pan, Zhang, & Fu, 2012a). Phytoremediation
may also be a long-term method to clean the soils because of the low
amounts of metals that can be accumulated by plants before toxic symp-
toms result.
There have been various reports of bacterial decontamination of metal-
polluted soils. Fundamental processes that enable bioremediation include
changes in pH and/or redox reactions, increases or decreases in solubility
by means of complexation or precipitation, and adsorption or uptake of
pollutants (Smith, Alleman, & Copley-Graves, 1994). Different oxidation
states of many metal(loid)s are of differing mobility and toxicity meaning
that variations in soil redox potential may affect microbial redox transfor-
mations and result in failure to stabilize a metal in contaminated soil (Achal
et al., 2012a).
When a problem associated with a bioremediation method exists, it may
be solved with an advanced or unexplored approach. Biotechnology applied
to the remediation of metal pollution has been a topic of great interest for
many years. Various enzymic systems have been used effectively for the
remediation of different organic pollutants (Nessner Kavamura & Esposito,
2010), including those from bacteria and fungi (Ruggaber & Talley, 2006).
Most of the degradative enzymes involved in organic bioremediation
are mono- or dioxygenases, oxidoreductases, dehalogenases, cytochrome
P450 monooxygenases, enzymes involved in lignin degradation, and phos-
photriesterases (Pieper, Martins dos Santos, & Golyshin, 2004; Rao, Scelza,
Scotti, & Gianfreda, 2010). However, there are many enzymes which are
less studied. Microbial urease, a type of hydrolase, is one such enzyme which
has been demonstrated to have an effective role in the immobilization of
various metals as insoluble carbonates. This article therefore reviews the
properties and applications of urease for toxic metal immobilization and dis-
cusses future prospects for the use of ureolytic microorganisms in bioreme-
diation and metal biorecovery.
2. UREASE
Urease (or urea amidohydrolase) was discovered around 150 years ago.
The first ureolytic microorganism, Micrococcus ureae, was isolated from urine
in 1864 by van Tieghem. However, Musculus obtained the first ureolytic
enzyme in 1874 from putrid urine, and as proposed by Miquel in 1890, it
was named urease (see Mobley & Hausinger, 1989; Mobley, Island, &
Hausinger, 1995; Krajewska, 2009). Initially, the ureolytic enzyme was
considered to be a potent virulence factor in pathogenic bacteria such as
Helicobacter pylori, Proteus mirabilis, Campylobacter pyloridis, and Staphylococcus
saprophyticus. However, it was subsequently found that urease is produced
by many taxonomically diverse bacterial species, including normal non-
pathogenic microbiota from terrestrial and aquatic habitats (Dunn, Campbell,
Perez-Perez, & Blaser, 1990; Gatermann & Marre, 1989; Graham et al.,
1987; Jones & Mobley, 1988). Mobley and Hausinger (1989) have high-
lighted the significance of urease as a virulence factor in animal pathogenesis,
its role in ruminant metabolism and in environmental transformations of
urea-based compounds. Furthermore, Mobley et al. (1995) reviewed
numerous urease gene clusters for which the entire nucleotide sequence
was known in addition to exploring mechanisms by which urease gene
expression is regulated in different bacterial species.
Urease belongs to the hydrolase class and superfamily of amidohydrolases
and phosphotriesterases with EC number 3.5.1.5. Urease hydrolyzes urea to
yield ammonia and carbamate, which is unstable, and spontaneously forms
carbonic acid and ammonia upon further hydrolysis. Urease activity is
widely found among prokaryotes, as well as in eukaryotes including fungi
and plants (Blakeley & Zerner, 1984; Li, Csetenyi, & Gadd, 2014). To
date, the widest analytical application of urease has been for the quantifica-
tion of urea in blood and urine (Francis, Lewis, & Lim, 2002). Recently,
there has been a growing demand for urease in applications in other
areas such as food production (Krajewska, 2009).
Urease plays an essential role in the nitrogen metabolism of terrestrial and
aquatic microorganisms. Ureolytic activity minimizes crop damage during
urea fertilization of agricultural soil and solves the problem of fixed nitrogen
availability (Mobley & Hausinger, 1989). Such urease activity is attributable
to a variety of soil microbes. Lloyd and Sheaffe (1973) reported that 17e30%
of the cultivable bacterial population from soil produced urease. This urease
activity in soil is known to be extracellular and is stabilized by association of
the urease with certain soil components (Mulvaney & Bremner, 1981).
Urease levels in different soils vary. The cellular content of urease among
microbes also varies, suggesting different regulatory mechanisms of urease
production. Urease production in many microbes may be tightly regulated
in conjunction with the nitrogen regulatory system, which is controlled by a
complex cascade that ultimately triggers ribonucleic acid polymerase synthe-
sis, recognizing specific promoters of nitrogen-regulated gene products
(Mobley & Hausinger, 1989). In some microbial species urease production
is dependent on the presence of urea, which acts as an enzyme inducer,
while other microbial species produce urease constitutively. It has also
been demonstrated that urea can significantly increase soil respiration but
may not influence soil urease activity (Margesin, Zimmerbauer, & Schinner,
2000). In brief, urease production has been demonstrated to be constitutive,
or inducible and repressible (Mobley & Hausinger, 1989). Most earlier
studies on environmental urease have been confined to its significance in
soil chemistry and agricultural practice. More recent studies have shown
that urease-producing microbes show considerable potential for mediating
metal bioprecipitation through the formation of insoluble metal carbonates
(Achal, Pan, & Zhang, 2011; Fujita, Ferris, Lawson, Colwell, & Smith,
2000; Fujita, Redden, et al., 2004; Fujita, Taylor, et al., 2008; Li, Cheng,
& Guo, 2013; Li et al., 2014; Li, Csetenyi, Paton, & Gadd, 2015).
3. BIOMINERALIZATION
Biomineralization is the process by which organisms form minerals
(Ben Omar, Arias, & Gonzalez-Mu~ noz, 1997; Gadd, 2010; Lowenstam &
Weiner, 1989). The process of biomineralization can be categorized into
biologically induced mineralization (BIM) and biologically controlled
mineralization (BCM) (Bazylinski, 2001; Benzerara et al., 2011; Fouke,
2011; Gadd, 2010; Li et al., 2014; Northup & Lavoie, 2001; Phillips
et al., 2013; Rhee, Hiller, & Gadd, 2015). BCM depends on the cellular ac-
tivities of the biomineralizing organism (eg, coccolithophores, diatoms, and
magnetic bacteria) which directly influence the nucleation, growth, and
morphology of the produced biominerals and control the final biomineral
locations (Bazylinski, 2001; Mukkamala, Anson, & Powell, 2006; Gadd,
2010). In the context of BIM, the organism modifies its local microenviron-
ment to create appropriate physicochemical conditions for the precipitation
of minerals (Gadd, 2010; Gadd et al., 2014; Gadd, Rhee, Stephenson, &
Wei, 2012; Li et al., 2014, 2015). Most microbial biomineralization pro-
cesses therefore usually refer to BIM (Burford, Hillier, & Gadd, 2006;
Gadd, 2010; Li et al., 2014; Rhee et al., 2015; Uroz, Calvaruso, Turpault,
& Frey-Klett, 2009).
Calcium carbonate is a major biomineralization product (Berman et al.,
1990; Lakshminarayanan, Kini, & Valiyaveettil, 2002; Perito & Mastromei,
2011) and calcite (CaCO3) precipitation is a common microbially-mediated
phenomenon in the biosphere (Ehrlich, 1998; Castanier, Levrel, & Perthuisot,
1999). Carbonates, especially calcite (CaCO3) and dolomite (CaMg(CO3)2),
are often found as limestones on the Earth’s surface (Ehrlich & Newman,
2009). Moreover, 13% of the total land surface of the Earth is occupied by
the near-surface calcretes and dolocretes in the soil environment, and they
are important carbon reservoirs in the Earth’s lithosphere (Ehrlich &
Newman, 2009; Goudie, 1996). A significant proportion of such carbonate
minerals at the Earth’s surface is of biogenic origin, and many microorganisms,
including bacteria and fungi, can deposit calcium carbonate extracellularly
(Barua, Suzuki, Pham, & Inatomi, 2012; Burford et al., 2006; Goudie,
1996; Li et al., 2014, 2015; Navarathna, Harris, Roberts, & Nickerson,
2010; Verrecchia, 2000; Verrecchia, Dumont, & Rolko, 1990; Yamanaka,
1999). Calcium carbonate precipitation by bacteria is generally regarded
to be inducible, and the type of mineral produced is largely dependent
on environmental conditions (Ben Omar et al., 1997; Brennan, Lowenstein,
& Horita, 2004; Rivadeneyra, Delgado, del Moral, Ferrer, & Ramos-
Cormenzana, 1994). Bacteria involved in the nitrogen cycle are important
organisms for calcium carbonate precipitation in various environments
through the production of urease which mediates the precipitation of
CaCO3, a process known as microbially-induced calcium carbonate precipi-
tation (MICP) (Achal, 2015).
3.1 Microbially-induced Calcium Carbonate Precipitation

MICP by urease-producing bacteria involves a series of biochemical reac-
tions. Apart from urease, the process requires calcium ions at a concentration
that permits precipitation of carbonate, while nucleation sites with a strong
affinity for cations enable the accumulation of calcium ions on cell walls.
In MICP, urease hydrolyzes urea into ammonia and carbamate
(Eq. [1]) which on subsequent hydrolysis releases ammonia and carbonic
acid (Eq. [2]). These products equilibrate in water to form bicarbonate
and ammonium and hydroxyl ions (Eqs. [3] and [4]), resulting in an increase
in pH that ultimately shifts the bicarbonate equilibrium to form carbonate

ions (Eq. [5]). Metabolic CO2 from respiration further contributes to an in-
crease in the level of dissolved inorganic carbon in the microenvironment to
enhance the precipitation of calcium carbonate (Hammes & Verstraete,
2002). The conditions of high pH favour the formation of CO3 2 from
HCO3 (Knoll, 2003). The increased carbonate concentration therefore
leads to CaCO3 precipitation around cells, and in media, in the presence
of calcium ions (Eqs. [6] and [7]).
COðNH2 Þ2 þ H2 O/NH2 COOH þ NH3 [1]
NH2 COOH þ H2 O/NH3 þ H2 CO3 [2]
H2 CO3 4HCO3 þ Hþ [3]
2NH3 þ 2H2 O42NH4 þ þ 2OH [4]
HCO3 þ Hþ þ 2NH4 þ þ 2OH 4CO3 2 þ 2NH4 þ þ 2H2 O [5]
Ca2þ þ Cell/Cell Ca2þ [6]
Cell Ca2þ þ CO3 2 /Cell CaCO3 [7]

MICP has been shown to have potential as a remediation strategy for
toxic metals, since toxic metals can also be precipitated as insoluble carbon-
ates (Achal et al., 2011; Fujita et al., 2000, 2004, 2008; Li et al., 2013).
Furthermore, carbonates can be highly effective in further absorbing toxic
metals (Plassard, Winiarski, & Petit-Ramel, 2000; Sipos, Németh, Mohai,
& D odony, 2005).
Urease-based MICP has been applied to enhance the durability of
building structures by improving strength, reducing water permeation
and corrosion (Achal, Mukherjee, Goyal, & Reddy, 2012; De Muynck,
Belie, & Verstraete, 2010; Phillips et al., 2013) and for cementation of
cracks and fissures (Ramachandran, Ramakrishnan, & Bang; 2001; Van
Tittelboom, De Belie, De Muynck, & Verstraete, 2010). It has also been
used as a “bio-grout” for ground permeability control and reinforcement
(Whiffin, Van Paassen, & Harkes, 2007; DeJong, Mortensen, Martinez,
& Nelson, 2010; Akiyama & Kawasaki, 2012), and the restoration of
historical monuments (Tiano, Biagiotti, & Mastromei, 1999). Urease-
producing organisms have also been proposed for novel applications in
the bioremediation of toxic metals and radionuclides through the formation
of insoluble metal-containing carbonates (Table 1). A diagram showing

how urease mediates metal carbonate bioprecipitation is shown in Fig. 1.
Toxic metals may also precipitate on the Ca mineral surface as discrete com-
pounds or form mixed solid solutions, eg, CdnCan-1CO3 (Papadopoulos &
Table 1 Some examples of the application of ureolytic bacteria for immobilization

of metal(loid)s by MICP
Bioremediation
Bacterium Metal(loid) efficiency Reference
Sporosarcina As 96% removal from Achal et al. (2012a)

ginsengisoli aqueous media
(ic ¼ 10 mg/L)
96% removal from
exchangeable soil
fraction
(ic ¼ 500 mg/kg)
Exiguobacterium Cd 84% removal from Kumari et al. (2014)
undae aqueous media
90% in exchangeable
soil fraction
Lysinibacillus Cd 99.95% removal from Kang et al. (2014)
sphaericus aqueous media
(ic ¼ 2 g/L)
Terrabacter Cd 99% removal from Li et al. (2013)
tumescens aqueous media
(ic ¼ 2 g/L)
Kocuria flava Cu 96% removal from Achal et al. (2011)
exchangeable soil
fraction
(ic ¼ 340 mg/kg)
Bacillus sp. Cr(VI) >68% removal from Cr Achal et al. (2013)
slag
Enterobacter Pb 68% removal from Kang et al. (2015)
cloacae aqueous media
(ic ¼ 7.2 mg/L)
Sporosarcina Pb 99% removal from Li et al. (2013)
koreensis aqueous media
(ic ¼ 2 g/L)
Halomonas sp. Sr 86% removal from Achal et al. (2012b)
quartz sand
(ic ¼ 100 mg/kg)
Sporosarcina sp. Zn 99% removal from Li et al. (2013)
aqueous media
(ic ¼ 2 g/L)
ic, initial concentration of metal(loid).
Figure 1 Diagram of precipitation of metal carbonates by urease-producing microor-

ganisms. M2þ represents a divalent metal cation. Adapted from Li, Q., Csetenyi, L., &
Gadd, G.M. (2014). Biomineralization of metal carbonates by Neurospora crassa. Envi-
ronmental Science and Technology, 48, 14409e14416.
Rowell, 1988). The following sections discuss bioprecipitation of those

metals where MICP has been successfully applied using urease-producing
bacteria and fungi.
4. BIOPRECIPITATION OF METAL(LOID)S BY
BACTERIAL-INDUCED CARBONATE PRECIPITATION
4.1 Arsenic
Arsenic, a crystalline metalloid, is highly toxic to all forms of life. The
permissible limit of arsenic in soil is 24 mg/kg (TCEQ, 2009). The major
sources of arsenic in soil are natural weathering from bedrock, atmospheric
deposition, agricultural materials, and the coal industry. Arsenic is highly
dangerous to human health as it can cause skin cancer, melanosis, and kera-
tosis, as well as other physiological disorders (Singh, Singh, Parihar, Singh, &
Prasad, 2015). Removal of arsenic from contaminated soil is therefore very
important and a great challenge using bioremediation methods. Arsenic
exists in four oxidation states (O, -III, III, and V) with arsenate [As(V)] and
arsenite [As(III)] as predominant forms in contaminated environments. Due
to the toxicity of arsenic, microorganisms possess mechanisms to resist its haz-
ardous effects, mainly by active efflux, extracellular precipitation, chelation,
or intracellular sequestration (Kruger, Bertin, Heipieper, & Arsene-Ploetze,
2013). Bioremediation may employ redox transformations of As via As(V)

reduction and As(III) oxidation which can be carried out by a wide variety
of As(V)-reducing and As(III)-oxidizing bacteria including Chrysiogenes arsen-
atis, Sulfurospirillum barnesii, Bacillus arsenicoselenatis, Desulfitobacterium hafniense,
and Thiomonas arsenivorans (see Yamamura & Amachi, 2014). While removing
As from contaminated soil using Bacillus selenatarsenatis SF-1, Yamamura,
Watanabe, Kanzaki, Soda, and Ike (2008) successfully reported mobilization
of As into the aqueous phase from contaminated soil through reduction of
solid-phase As(V) and Fe(III); however, a maximum of 56% removal occured
from soil containing 250 mg/kg As. Biovolatilization has also been used for
As remediation, and this resulted in about 2.2e4.5% of arsenic removal
from soil after a 30-day incubation using Sphingomonas desiccabilis and Bacillus
idriensis (Liu, Zhang, Chen, & Sun, 2011). There was a significant rate of bio-
volatilization of As(V) and As(III) from culture medium by Staphylococcus sp.
(Srivastava et al., 2012).
Arsenic in soils is most commonly associated with its primary minerals
derived from bedrock, secondary minerals (primarily Fe oxy/hydroxides;
sulfides) formed in the course of mineral weathering, and As adsorbed to
mineral surfaces. Association of As with calcium minerals is well known
(Chang & Jackson, 1957). The precipitation of Ca arsenates was shown in
highly acidic waste pile leachates after association with carbonate subsurface
layers ( Juillot et al., 1999). Furthermore, significant adsorption of As on car-
bonate mineral phases has been reported (Goldberg & Glaubig, 1988;
Roman-Ross, Cuello, Turrillas, Fernandez-Martínez, & Charlet, 2006). It
was demonstrated that arsenate may substitute for CO3 2 in calcite from
travertine, suggesting the possibility of As immobilization through carbonate
precipitation (Di Benedetto et al., 2006).
In order to improve the efficiency of As removal, Achal et al. (2012a)
used Sporosarcina ginsengisoli CR5 for remediation of As(III) in contami-
nated soil. This ureolytic bacterium significantly reduced the As concentra-
tion in the exchangeable fraction of soil to 0.88 mg/kg in a soil
supplemented with 500 mg/kg As(III). It was proposed that calcite produc-
tion by the bacterium-facilitated precipitation of a strong arsenicecalcite
complex leading to reduced As mobility. Such an immobilization process
may enable metal(loid)s to be transformed in situ into insoluble and chem-
ically inert forms and are applicable to removing metals from aqueous
solution (Gadd, 2004; 2010). Analysis of the mineralogical products in
MICP-treated As-contaminated soil samples showed that various minerals
such as gwihabaite, calcite, vaterite, and aragonite were formed along with
Figure 2 XRD spectra conforming biomineralization products in soil induced by Spor-

osarcina ginsengisoli CR5. C, calcite; A, aragonite; C-As, calcite-arsenite precipitate;
V, vaterite; G, gwihabaite; Q, quartz; H, halite. Adapted with permission from Achal, V.,
Pan, X., Zhang, D., & Fu, Q.L. (2012a). Biomineralization based remediation of As(III)
contaminated soil by Sporosarcina ginsengisoli. Journal of Hazardous Materials, 201e
202, 178e184.
As(III)ecalcite co-precipitation products (see Fig. 2). Such co-precipitation

is unaffected by the oxidation state of arsenic which confirms the efficiency
of calcite as an effective scavenger of a variety of metals (Alexandratos,
Elzinga, & Reeder, 2007; Rouff, Elzinga, & Reeder, 2004). Urease-
producing bacteria have therefore been shown to be effective for immobi-
lization of high amounts of arsenic and are therefore potential candidates
for application in arsenic contaminated sites.
4.2 Cadmium
Cadmium (Cd) is a non-essential heavy metal, naturally present in soils and
enriched by anthropogenic and agricultural activities. It occurs typically in
the range of 0.1 and 1.0 mg/kg. Cd-contaminated soils pose a threat to
human health through consumption of cereals or other crops grown in
such soil (Smolders & Mertens, 2013). Cd can form complexes with various
anions, such as Cl, SO4 2 , CO3 2 , and PO4 3 (Makino et al., 2006), and
this property makes it a suitable candidate for immobilization by MICP.
Though various methods of Cd bioremediation from soil have been sug-

gested, immobilization of Cd is generally recognized as the most practical
technology as it does not affect agricultural activity (Wang, Sun, Jiang,
Mao, & Zhang, 2014). Cadmium sorption has been studied in calcareous
soil which implicated the efficiency of calcium carbonate in Cd removal
(O’Connor, O’Connor, & Cline, 1984). Waste oyster shells containing
high amounts of CaCO3 were used to stabilize Cd-contaminated soils
(Ok, Lim, & Moon, 2011). Addition of calcium chloride was reported as
the most appropriate soil-washing treatment for Cd-contaminated soil,
and this resulted in 55% Cd removal from the exchangeable soil fraction
(Makino et al., 2006). However, the major drawback of such an approach
was that this was not sufficient to remove the high remaining amount of
Cd. There is scope therefore to enhance Cd bioremediation using urease-
producing organisms that would lead to further Cd immobilization.
Terrabacter tumescens, a urease-producing bacterium, was reported to
effectively remove more than 90% Cd within 72 h when 2 g/L CdCl2
was present in laboratory media (Li et al., 2013). The Cd in solution was
assumed to precipitate as cadmium carbonate (CdCO3). Li et al. (2013)
also found effective immobilization of other metals, such as Ni, Cu, Pb,
Co and Zn using T. tumescens, which were precipitated as NiCO3,
CuCO3, PbCO3, CoCO3, and ZnCO3. These biominerals exhibited
different morphologies and were rhombohedral, needle-like, or spherical
in shape, and of size 10e50 mm (see Fig. 3).
Lysinibacillus sphaericus CH-5 has been demonstrated to precipitate Cd
based on ureolytic activity (Kang, Han, Shin, Oh, & So, 2014). This bacte-
rium was isolated from an abandoned mine site and showed high urease
activity (2.41 mmol/min) and produced 10 mg/mL calcite in broth contain-
ing beef extract, peptone and urea. Urease production was also evident in a
consolidated sand column using L. sphaericus that resulted in improved
mechanical properties. Urease production (1.72 mmol/min) after 48 h in
the presence of 2 g/L Cd resulted in 99.95% Cd removal (Kang et al.,
2014). The precipitated Cd appeared mostly as spherical forms with a diam-
eter of 10e40 mm, while X-ray diffraction (XRD) revealed calcite peaks
along with otavite showing clear precipitation of Cd as carbonate.
Kumari, Pan, Lee, and Achal (2014) reported MICP for Cd immobiliza-
tion from soil at low temperature. Exiguobacterium undae YR10 was added to
soil artificially contaminated with 100 mg CdSO4/kg soil, in the form of a
bacterial culture grown in nutrient broth containing urea and calcium chlo-
ride. The experiments were terminated after 2 weeks and thereafter the
(A) (B) (C)
(D) (E) (F)
Figure 3 Environmental scanning electron microscopy of (A) Ni-containing minerals

precipitated by Terrabacter tumescens, (B) Cu- (C) Pb-containing minerals precipitated
by bacterial isolate UR47, (D) Co- (E) Zn-containing minerals precipitated by bacterial
isolate UR31, and (F) Cd-containing minerals precipitated by T. tumescens. Scale bars:
(a, b, c, e, f) ¼ 10 mm, (d) ¼ 40 mm. Adapted with permission from Li, M., Cheng, X., &
Guo, H. (2013). Heavy metal removal by biomineralization of urease producing bacteria
isolated from soil. International Biodeterioration and Biodegradation, 76, 81e85.
soluble-exchangeable soil fraction contained 0.87 mg Cd/kg soil at 25 C,

and 1.2 mg Cd/kg soil at 10 C in the same fraction. The carbonate fraction
of the soil had a significantly higher Cd concentration, suggesting that most
of the Cd was either converted to CdCO3 or coprecipitated with calcite.
Although CdCO3 is sparely soluble in the soil solution, it may combine
with CaCO3 and remain immobilized (Kumari et al., 2014). Li et al.
(2014) have reported calcium and cadmium carbonate biomineralization
by the ureolytic fungus Neurospora crassa. The Cd precipitates were identified
as pure otavite (CdCO3). This suggested an important role for ureolytic
microbes in providing a means of metal biorecovery as well as bioremedia-
tion (Li et al., 2014).
4.3 Chromium
Chromium (Cr) is often considered to be a “local-source” contaminant and
presumed not to constitute a widespread environmental problem (Samborska,
Stepniewska, & Stepniewski, 2004). However, its toxic effects cannot be
ignored. It contaminates soils from metallurgy operations, electroplating,
production of paints and pigments, tanning, wood preservation, chromium

chemical production, and pulp and paper production. Cr exists primarily in
two different oxidation states as Cr(III) and Cr(VI), of which Cr(III) is
non-toxic and exhibits limited environmental disruption, while Cr(VI) is
highly mobile, soluble, and toxic with strong oxidizing properties (Zhang
& Li, 2011). The disposal of Cr-containing wastes over large areas has led
to extensive contamination of soil in many parts of the world. The sites
around such dumping zones are highly prone to further contamination due
to leaching and seepage of Cr(VI) into the groundwater (Zayed & Terry,
2003). In view of the seriousness of Cr(VI) pollution, efforts have been
made based on a bioconsolidation approach involving urease-producing bac-
teria for the treatment of Cr-contaminated soils and slags.
Co-precipitation of Cr(VI), in which chromate incorporates into mineral
structures, has been considered as an alternative means of limiting the
mobility of chromate although few studies address the interaction of Cr
with calcium carbonate minerals (Tang, Elzinga, Jae Lee, & Reeder,
2007). In one study, urease-producing bacteria were used to produce calcite
and consequently entrap chromate. A calcifying ureolytic Bacillus sp. CS8
was used to consolidate Cr slag in the form of bricks of size
18 9.5 3.5 cm (Achal, Pan, Lee, Kumari, & Zhang, 2013). The bio-
consolidation resulted in a significant decrease in Cr(VI) in the exchangeable
fraction that was 95% lower than the control. At the same time, the
increased carbonate-bound Cr(VI) suggested preferential incorporation
into the calcite during crystal growth (Tang et al., 2007). MICP was also
tested to confirm its efficency in preventing metal leaching in soil column
experiments. Bacillus sp. CS8 reduced the flow rate from a Cr slag column
by reducing permeability due to a calcium carbonate layer being precipitated
by the bacteria (Achal et al., 2013).
In another study, soils artificially contaminated with 100 mg/kg Cr(VI)
were treated with ureolytic B. cereus YR5 which resulted in a significant
decrease (92%) of Cr(VI) in the exchangeable fraction of the polluted soil
and increased the carbonate-bound Cr(VI) fraction (Kumari et al., 2014).
One report suggested the presence of urea enhanced Cr(VI) removal effi-
ciency during electrochemical remediation of Cr(VI) in chromium slag.
The Cr(VI) in the calcium carbonate structure showed resistance to gaseous
reductants or solution-phase extractants (Hua, Deng, Thornton, Yang, &
Amonette, 2007; Thornton & Amonette, 1999) implying long-term stabil-
ity of Cr(VI) incorporation in the calcium carbonate and prevention of
Cr(VI) release.
4.4 Copper
Copper is a common soil contaminant (Santorufo, Van Gestel, & Maisto,
2012). Anthropogenic activities (such as application of sewage sludge,
mine slags, industrial wastewaters, fungicides, and fertilizers) can lead to
the elevation of copper to toxic levels in agricultural soils (Anjum et al.,
2015; Hu, Liang, Liu, Lei, & Yu, 2014; Wang, Hua, & Ma, 2012). Soluble
and exchangeable metals such as copper are often considered as being the
most potentially toxic in soil (Yang, Liu, Zheng, & Feng, 2006; Hu et al.,
2014), and copper remediation from this soil fraction is therefore highly
desired.
The versatility of Kocuria flava CR1 with a high tolerance to copper and
urease-producing ability has been documented for effective treatment of
copper in contaminated soil (Achal et al., 2011). This bacterium produced
a very high amount of urease (472 U/ml) in nutrient brotheurea media,
establishing MICP for copper immobilization. Copper removal was 95%
from a solution containing 500 mg/L CuSO4$5H2O. The resulting
precipitates were evaluated by Fourier transform infrared spectroscopy and
identified as calcium carbonate and aragonite (Vagenas, Gatsouli, &
Kontoyannis, 2003). MICP using ureolytic bacteria was also effective in
copper-contaminated soil and 98% copper was immobilized from soil
containing 340 mg/kg copper (Achal et al., 2011). Only 3.5 mg Cu/kg
soil remained in the exchangeable fraction after treatment compared to
67 mg Cu/kg in untreated soil. Copper was also immobilized as CuCO3
by the ureolytic bacterium T. tumescens (Li et al., 2013).
4.5 Lead
Lead (Pb) is a toxic metal that may pollute soil or water due to emission from
automobiles, waste irrigation, pesticide application, mining and smelting,
and ultimately may pose a health risk (Gworek, 1992; Li, Shi, Shao, &
Shao, 2009). Lead is also the most distinctive heavy metal contaminant of
urban soils. Once it accumulates inside humans, it can cause neurodegener-
ative damage, DNA damage, apoptosis, cancer, and various disabilities in
children (Gworek, 1992; Li et al., 2009).
Urease-based MICP has been shown to be highly effective in lead
immobilization. A urease-producing K. flava CR1 that grew well in nutrient
media supplemented with 50 mM Pb was able to remove 80% Pb from the
soluble-exchangeable fraction of contaminated soil (Achal, Pan, Zhang, &
Fu, 2012b). The bioremediation efficiency of MICP was confirmed in terms
of the distribution coefficient (gi) of each Pb fraction, indicating a significant

increase in gi of carbonate-bound Pb, while at the same time the gi of
soluble-exchangeable Pb was reduced greatly. It was concluded that Pb
immobilization by such a mechanism could be of considerable relevance
because of its stability in a variety of geologic environments (Achal et al.,
2012b). Another efficient urease producer, Sporosarcina koreensis (UR47),
was reported to remove 99% lead from a solution containing 2 g/L PbCl2
through MICP (Li et al., 2013).
A lead-resistant Bacillus sp. KK1 isolated from Pb-contaminated mine tail-
ings effectively biomineralized mobile Pb (Govarthanan et al., 2013). The
lead mineral products were lead sulfide (PbS) and lead silicon oxide (PbSiO3)
(see Fig. 4A). Bacillus sp. KK1 was used to treat lead-contaminated mine tail-
ings containing Pb 1050 mg/kg, and this resulted in a 26% decrease in the
exchangeable Pb fraction in the bioaugmented tailings (Govarthanan et al.,
2013). At the same time, the carbonate Pb fraction increased by 38% due
to bacterially mediated precipitation of Pb (see Fig. 4B). XRD spectra
showed differences in PbO and Pb(OH)2 in bioaugmented mine tailings
when compared with the control, indicating that MICP could effectively
scavenge different species of Pb (Govarthanan et al., 2013).
Urease-producing Sphingobacterium sp., Enterobacter cloacae, and L. sphaericus
which showed a high Pb tolerance were isolated from soils at abandoned
metal mine sites (Kang et al., 2015). These bacteria showed the presence of
ureC genes which were amplified using UreC-F and UreC-R primers
(Gresham, Sheridan, Watwood, Fujita, & Colwell, 2007). A high removal
rate (68%) of Pb was observed within 48 h based on MICP resulting in
lead carbonate precipitates of diameter w5 mm. The MICP process also
resulted in a significant increase in enzyme activities (phosphatase 37%, dehy-
drogenase 14%, and urease 334%) in the treated mine tailings (Govarthanan
et al., 2013). Increased urease and dehydrogenase activity in Pb-contaminated
soils after adding ureolytic bacteria has also been reported by others (Achal
et al., 2012b).
4.6 Radionuclide Bioprecipitation by Urease-producing

Bacteria
Radioactive contamination has been a serious problem since the develop-
ment of nuclear technology. Significant amounts of radionuclides are dis-
charged by industrial activities allied to nuclear power generation, nuclear
weapons, and accidental release (Pollmann, Raff, Merroun, Fahmy, &
Selenska-Pobell, 2006). Soils contaminated with radionuclides, such as
6000
(A) PbSiO3 KK1
PbS
KK1 + Pb(NO3)2
PbS
5000
4000
3000
2000
Intensity
1000
(B) C Bioremediated soil
50000 Control soil
40000
30000
20000
PbO
C
10000 Pb(OH)2
A C
A C
10 20 30 40 50 60
2-Theta degree
Figure 4 X-ray diffractograms of (A) Bacillus sp. KK1 before and after incubation with
lead nitrate, (B) mine soil samples before and after bioaugmentation. C, calcite; A,
aragonite. Adapted with permission from Govarthanan, M., Lee, K.J., Cho, M., Kim, J.S.,
Kamala-Kannan, S., & Oh, B.T. (2013). Significance of autochthonous Bacillus sp. KK1 on
biomineralization of lead in mine tailings. Chemosphere, 90, 2267e2272.
137
Cs, 235U, and 90Sr, pose a long-term radiation hazard to human health
through exposure via the food chain and other pathways. They pose serious
health impacts on humans and cause neurological disorders, infertility, birth
defects, and various types of cancer (Najem & Voyce, 1990; Mossman, 2003;
Das, 2012).
The concept of biomineralization in radionuclide bioremediation was
introduced several years ago. Radionuclides can be immobilized through
interactions between microbially produced sulfide (Lebranz et al., 2000;
White, Sharman, & Gadd, 1998) and phosphate (Boswell, Dick, &
Macaskie, 1999; Jeong & Macaskie, 1999; Macaskie, Empson, Cheetham,
Grey, & Skarnulis, 1992), or through bacterial iron oxidation (Banfield,

Welch, Zhang, Ebert, & Penn, 2000) in the general process of biomineral-
ization (Martinez et al., 2007). Uranium phosphate precipitation has been
facilitated by diverse bacterial genera including Arthrobacter, Bacillus, Rahnella,
Deinococcus, Escherichia, and Pseudomonas (Appukuttan, Rao, & Apte, 2006;
Basnakova, Stephens, Thaller, Rossolini, & Macaskie, 1998; Powers et al.,
2002). It has also been shown that Bacillus subtilis can immobilize U through
the formation of uranyl-hydroxide, uranyl-carbonate, and calcium-uranyl-
carbonate species with functional groups present on cell surfaces (Fowle,
Fein, & Martin, 2000; Gorman-Lewis, Elias, & Fein, 2005). Pseudomonas
aeruginosa, an indigenous bacterial isolate from uranium mine waste, could
sequester soluble uranium in mineral form, the bioaccumulated uranium
being sequestered as crystalline needle-shaped U phosphate compounds
within the cell envelope, identified as UO2(PO3)2, (UO2)3(PO4)2$H2O,
and U2O(PO4)2 (Choudhary & Sar, 2011).
Biomineralization of radionuclides has been further investigated using
urease-producing bacteria. The remediation of 90Sr from the Snake River
Plain Aquifer, which underlies the Idaho National Engineering and Envi-
ronmental Laboratory (INEEL), USA, was evaluated based on a ureolyti-
cally driven calcite precipitation approach (Fujita et al., 2004). 90Sr is a
significant aquifer and vadose zone contaminant at the INEEL, as well as
at a number of DOE (Department of Energy) facilities across the USA (Riley
& Zachara, 1992). Native ureolytic microbes were used to remediate 90Sr
contamination at the Hanford 100-N area in Washington where ureolytic
activities of microbes were confirmed by UreC amplification (Fujita, Taylor,
Wendt, Reed, & Smith, 2010). Quantitative assays detected up to 2 104
putative ureC gene copies per mL in water and up to 9 105 copies per g in
sediment. Further analyses indicated that the Sr was incorporated into calcite
ensuring the relative stability of 90Sr (Fujita et al., 2010).
In another study, a possible role of ureolytic Halomonas sp. was reported
for the remediation of strontium (Sr) in aquifer sand (Achal, Pan, & Zhang,
2012). The overall reactions involved in the bioremediation process
included urease producing NH4 þ and HCO3 , desorption of Ca2þ and/
or Sr2þ from solid surfaces by NH4 þ and HCO3 promoted precipitation
of CaCO3 and co-precipitation of 90Sr (Wu et al., 2011). The hydrolysis
of urea produces bicarbonate and ammonium, where bicarbonate partici-
pates directly in calcite precipitation, and ammonium can exchange for
sorbed strontium, calcium, and other metals, resulting in their enhanced
susceptibility to recapture via carbonate mineral formation (Fujita et al.,

2010). Some possible chemical reactions can be summarized as follows
(Achal et al., 2012) (Eqs. [8] and [9]):
1. Urease-mediated reaction producing NH4 þ and HCO3
H2 NðCOÞNH2 þ Hþ þ 2H2 O/2NH4 þ þ HCO3 [8]
2. Precipitation of calcite and coprecipitation of Sr, promoted by HCO-3
90
x90Sr2þ þ ð1 xÞCa2þ þ 2HCO3 4Cað1 xÞ90 Srx CO3

[9]
þ H2 O þ CO2
5. BIOPRECIPITATION OF METALS BY FUNGAL-

INDUCED CARBONATE PRECIPITATION
Fungi are ubiquitous chemoorganotrophic (heterotrophic) organisms,
and their importance as animal and plant symbionts and pathogens, and
spoilage organisms of natural and manufactured materials is profound
(Gadd, 2008). Metals, metalloids, metal radionuclides, organometals, and
organometalloids, and their compounds, interact with fungi in various
ways depending on the chemical speciation, organism, and environmental
factors (Gadd, 1993, 1999, 2007; Gadd et al., 2012). Both metabolism-
independent and -dependent fungal activities can result in the precipitation
of secondary organic and inorganic minerals (eg, oxalates, oxides, phos-
phates, and carbonates). Fungi can act as effective biosorbents for a variety
of metals including U, Th, Pb, Cu, Zn, Cd, and Ni, and can also affect speci-
ation and mobility of metals and radionuclides through mineral dissolution
and bioprecipitation (Gadd, 1993, 2007, 2009, 2010). The key factors that
can influence the nucleation, growth, and deposition of biominerals on
and around fungal biomass include pH and cell wall composition as well
as excretion of various organic and inorganic metabolites (Gadd, 2010).
The precipitation of carbonates, phosphates, and hydroxides can increase
soil aggregation and cations such as Si4þ, Fe3þ, Al3þ and Ca2þ (that may
be released through mineral dissolution mechanisms) may act as bonding
agents for soil particles. Hyphae can also enmesh soil particles (Bronick &
Lal, 2005). Apart from the biomineral examples that follow, several other
carbonate minerals precipitated by fungi have been recorded (Table 2).
One mechanism commonly associated with the biomineralization of
CaCO3 is based on urea degradation, as in bacteria, which leads to the
Table 2 Fungal species reported for the biomineralization of various metal carbonates
Fungal species Carbonate minerals Reference
Acremonium strictum Calcite (CaCO3) Li and Gadd (unpublished)

Cephalosporium sp. CaCO3 Gadd and Raven (2010)
Cephalotrichum (syn Calcite (CaCO3) Burford et al. (2006)
Doratomyces) sp.
Fusarium oxysporum Calcite (CaCO3) Ahmad, Rautaray, and
Sastry (2004)
Morchella sp. Calcite (CaCO3) Masaphy, Zabari, Pastrana,
and Dultz (2009)
Myrothecium Calcite (CaCO3), vaterite Li et al. (2015)
gramineum ((CaxSr1-x)CO3), strontianite
(SrCO3)
Neurospora crassa Calcite (CaCO3), otavite Li et al. (2014)
(CdCO3)
Neurospora crassa Strontianite (SrCO3), CoCO3, Li and Gadd (unpublished)
nickel carbonate, La2(CO3)$
8H2O
Paecilomyces javanicus Hydrocerussite Rhee et al. (2015)
(Pb3(CO3)2(OH)2),
plumbonacrite
(Pb10(CO3)6O(OH)6)), lead
hydroxycarbonate
Penicillium CaCO3 Gadd and Raven (2010)
corylophilum
Penicillium Hydromagnesite Burford, Kierans, and Gadd
simplicissimum (Mg5(CO3)4(OH)2.4$H2O) (2003)
Pestalotiopsis sp. Calcite (CaCO3), strontianite Li et al. (2015)
(SrCO3), olekminskite
(Sr(Sr, Ca)(CO3)2), (Ca,Sr)
CO3, vaterite (CaCO3)
Serpula himantioides Calcite (CaCO3) Burford et al. (2006)
Trichothecium sp. Calcite (CaCO3) Ahmad et al. (2004)
Verrucaria spp. CaCO3 Easton (1997)
Verticillium sp. CaCO3, BaCO3 Rautaray, Ahmad, and
Sastry (2004)
release of carbonate which is then precipitated by available Ca (Burbank,

Weaver, Green, Williams, & Crawford, 2011; Whiffin et al., 2007).
Li et al. (2014) used urea-hydrolyzing N. crassa grown in a urea- and
calcium-rich medium in order to produce ammonium (NH4 þ ) and
dissolved carbonate which together with increasing medium pH, resulted
in calcite bioprecipitation (Eqs. [10] and [11]).
Fungal
COðNH2 Þ2 ðaqÞ þ 2H2 OðaqÞ / 2NHþ
4 ðaqÞ þ CO3 ðaqÞ
2
[10]
urease
CO3 2 ðaqÞ þ Ca2þ ðaqÞ/CaCO3 ðsÞ [11]

It was shown that more than 90% of supplied calcium (at a concentration
of 50 mM) could be precipitated as calcite by the fungus (Li et al., 2014).
When incubated in urea-containing medium modified with different con-
centrations of CaCl2 and SrCl2, various other minerals were deposited in the
medium and around the biomass (see Fig. 5), and these were identified as
calcite and strontianite (SrCO3) (unpublished data). Furthermore, cracks
involving hyphae were observed on the surface of some of the crystals
(see Fig. 5A) which indicated that hyphae may act as nucleation sites for
some of the calcite precipitation observed. Compared to the simpler bacte-
rial cell form, the fungal filamentous growth habit could provide more
(A) (B)
(C) (D)
Figure 5 Scanning electron microscopy of mineral deposition by Neurospora crassa

grown in different media. (A, B) AP1 media amended with 40 mM urea and 50 mM
CaCl2, (B) is a higher magnification image of the area indicated by the square in (A),
scale bars: a ¼ 10 mm, b ¼ 1 mm; (C) AP1 media amended with 40 mM urea, 25 mM
CaCl2, and 25 mM SrCl2, scale bar ¼ 10 mm; (D) AP1 media amended with 40 mM
urea and 50 mM SrCl2, scale bar ¼ 10 mm. All samples were incubated for 12 days at
25 C in the dark. Typical images are shown from many similar examples (Li and Gadd,
unpublished data).
framework support and stability for the precipitation of calcite or other

biominerals. Such performance of a urease-positive fungus in urea-
supplemented media suggests a promising method for calcite synthesis as
well as other metal-containing carbonates. For example, 50% of supplied
CdCl2 (at a concentration of 0.5 M) was precipitated as pure otavite
(CdCO3) by the culture supernatant obtained after growth of N. crassa in
urea-supplemented medium (Li et al., 2014). Urease-positive fungi (Pestalo-
tiopsis sp. and Myrothecium gramineum) isolated from calcareous soil were also
found to precipitate CaCO3 and SrCO3 as well as olekminskite
(Sr(Sr,Ca)(CO3)2) and Sr-containing vaterite ((CaxSr1-x)CO3) (Li, Csetenyi,
Paton, & Gadd, 2015). The soil fungus Paecilomyces javanicus was found to
mediate the transformation of metallic lead into lead secondary minerals:
plumbonacrite (Pb10(CO3)6O(OH)6), hydrocerussite (Pb3(CO3)2(OH)2),
and a new lead hydroxycarbonate (Rhee et al., 2015). The roles of fungi
in the environmental fate of toxic metals is of considerable interest although
biologically induced calcium carbonate precipitation has received little
attention as a potential remediating strategy for contaminated environments
or for element biorecovery (Achal, Mukherjee, et al., 2012; Achal et al.,
2012a; Pan, 2009). Many free-living fungi are capable of urea degradation
(Li et al., 2014, 2015). Most ammonia fungi as well as ectomycorrhizal fungi
also show strong abilities of urea degradation (Barua et al., 2012; Yamanaka,
1999). Ammonia fungi are an abundant group of soil fungi which flourish
when additional nitrogenous substances are present, such as urea, the
degradation of which leads to soil alkalinization to pH 9e10 (Navarathna
et al., 2010).
6. CONCLUSIONS
One of the primary objectives of bioremediation of contaminated soil
is to reduce the bioavailability of metals. The urease-driven MICP process
may offer a promising option for immobilizing heavy metals. Since urea-
hydrolyzing microorganisms show the ability to precipitate Ca as CaCO3,
this means they can also be applied to other toxic metals to form other metal
carbonates. During the precipitation of calcite, toxic metal ions may be
incorporated into the CaCO3 by substituting for Ca2þ or may also copreci-
pitate within the CaCO3 lattice structure. Although the total toxic metal
concentration in soil remains unchanged during MICP, a significant major-
ity of the contaminant may be removed from the soluble-exchangeable
fraction to the carbonate-bound fraction. Microbial metal carbonate precip-

itation is also relevant to detoxification of contaminated process streams and
effluents, as well as the synthesis of novel metal carbonates and biorecovery
of metals and radionuclides that form insoluble carbonates.
ACKNOWLEDGMENTS
The work was supported by National Natural Science Foundation of China
(Nos. U1403181, U1503281, 41450110430, 41450110458). G. M. Gadd gratefully
acknowledges an award under the 1000 Talents Plan with the Xinjiang Institute of Ecology
and Geography, Chinese Academy of Sciences, Urumqi, China. We also acknowledge finan-
cial support from the China Scholarship Council through a PhD scholarship to Qianwei Li
(No. 201206120066).
REFERENCES
Achal, V. (2015). Production of bacteria for structural concrete. In F. Pacheco Torgal,
J. A. Labrincha, M. V. Diamanti, C. P. Yu, & H. K. Lee (Eds.), Biotechnologies and
biomimetics for civil engineering (pp. 309e324). Dordrecht: Springer.
Achal, V., Mukherjee, A., Goyal, S., & Reddy, M. S. (2012). Corrosion prevention of
reinforced concrete with microbial calcite precipitation. ACI Materials Journal, 109,
157e164.
Achal, V., Pan, X., Lee, D. J., Kumari, D., & Zhang, D. (2013). Remediation of Cr(VI) from
chromium slag by biocementation. Chemosphere, 93, 1352e1358.
Achal, V., Pan, X., & Zhang, D. (2011). Remediation of copper contaminated soil by Kocuria
flava CR1, based on microbially induced calcite precipitation. Ecological Engineering, 37,
1601e1605.
Achal, V., Pan, X., & Zhang, D. (2012). Bioremediation of strontium (Sr) contaminated
aquifer quartz sand based on calcite precipitation induced by Sr resistant Halomonas sp.
Chemosphere, 89, 764e766.
Achal, V., Pan, X., Zhang, D., & Fu, Q. L. (2012a). Biomineralization based remediation
of As(III) contaminated soil by Sporosarcina ginsengisoli. Journal of Hazardous Materials,
201e202, 178e184.
Achal, V., Pan, X., Zhang, D., & Fu, Q. L. (2012b). Bioremediation of Pb-contaminated soil
based on microbially induced calcite precipitation. Journal of Microbiology and Biotech-
nology, 22, 244e247.
Ahmad, A., Rautaray, D., & Sastry, M. (2004). Biogenic calcium carbonate: calcite crystals of
variable morphology by the reaction of aqueous Ca2þ ions with fungi. Advanced Func-
tional Materials, 14, 1075e1080.
Akiyama, M., & Kawasaki, S. (2012). Novel grout material comprised of calcium phosphate
compounds: in vitro evaluation of crystal precipitation and strength reinforcement.
Engineering Geology, 125, 119e128.
Alexandratos, V. G., Elzinga, E. J., & Reeder, R. J. (2007). Arsenate uptake by calcite:
macroscopic and spectroscopic characterization of adsorption and incorporation
mechanisms. Geochimica et Cosmochimica Acta, 71, 4172e4187.
Anjum, N. A., Singh, H. P., Khan, M. I. R., Masood, A., Per, T. S., Negi, A. … Ahmad, I.
(2015). Too much is bad e an appraisal of phytotoxicity of elevated plant-beneficial
heavy metal ions. Environmental Science and Pollution Research, 22, 3361e3382.
Appukuttan, D., Rao, A. S., & Apte, S. K. (2006). Engineering of Deinococcus radiodurans R1
for bioprecipitation of uranium from dilute nuclear waste. Applied and Environmental
Microbiology, 72, 7873e7878.
Banfield, J. F., Welch, S. A., Zhang, H. Z., Ebert, T. T., & Penn, R. L. (2000). Aggregation-
based crystal growth and microstructure development in natural iron oxyhydroxide
biomineralization products. Science, 289, 751e754.
Barua, B. S., Suzuki, A., Pham, H. N., & Inatomi, S. (2012). Adaptation of ammonia fungi to
urea enrichment environment. Journal of Agricultural Science and Technology, 8, 173e189.
Basnakova, G., Stephens, E. R., Thaller, M. C., Rossolini, G. M., & Macaskie, L. E. (1998).
The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel
from aqueous flows. Applied Microbiology and Biotechnology, 50, 266e272.
Bazylinski, D. A. (2001). Bacterial mineralization. In K. H. J. Buschow, R. Cahn,
M. Flemings, B. Ilschner, E. Kramer, S. Mahajan, & P. Veyssiere (Eds.), Encyclopedia of
materials: Science and technology (pp. 441e448). Amsterdam: Elsevier.
Ben Omar, N., Arias, J. M., & Gonzalez-Mu~ noz, M. T. (1997). Extracellular bacterial miner-
alization within the context of geomicrobiology. Microbiologia, 12, 161e172.
Benzerara, K., Miot, J., Morin, G., Ona-Nguema, G., Skouri-Panet, F., & Férard, C. (2011).
Significance, mechanisms and environmental implications of microbial
biomineralization. Comptes Rendus Geoscience, 343, 160e167.
Berman, A., Addadi, L., Kvick, A., Leiserowitz, L., Nelson, M., & Weiner, S. (1990). Inter-
calation of sea urchin proteins in calcite: study of a crystalline composite material. Science,
250, 664e667.
Bishop, P. L. (2002). Pollution prevention: Fundamentals and practice. Beijing: Tsinghua
University Press.
Blakeley, R. L., & Zerner, B. (1984). Jack bean urease: the first nickel enzyme. Journal of
Molecular Catalysis, 23, 263e292.
Boswell, C. D., Dick, R. E., & Macaskie, L. E. (1999). The effect of heavy metals and other
environmental conditions on the anaerobic phosphate metabolism of Acinetobacter
johnsonii. Microbiology, 145, 1711e1717.
Brennan, S. T., Lowenstein, T. K., & Horita, J. (2004). Seawater chemistry and the advent of
biocalcification. Geology, 32, 473e476.
Bronick, C. J., & Lal, R. (2005). Soil structure and management: a review. Geoderma, 124,
3e22.
Burbank, M. B., Weaver, T. J., Green, T. L., Williams, B. C., & Crawford, R. L. (2011).
Precipitation of calcite by indigenous microorganisms to strengthen liquefiable soils.
Geomicrobiology Journal, 28, 301e312.
Burford, E. P., Hillier, S., & Gadd, G. M. (2006). Biomineralization of fungal hyphae with
calcite (CaCO3) and calcium oxalate mono- and dihydrate in carboniferous limestone
microcosms. Geomicrobiology Journal, 23, 599e611.
Burford, E. P., Kierans, M., & Gadd, G. M. (2003). Geomycology: fungal growth in mineral
substrata. Mycologist, 17, 98e107.
Castanier, S., Levrel, G. L. M., & Perthuisot, J. P. (1999). Ca-carbonates precipitation and
limestone genesis-the microbiogeologist point of view. Sedimentary Geology, 126, 9e23.
Chang, S. C., & Jackson, M. L. (1957). Fractionation of soil phosphorus. Soil Science, 84,
133e144.
Choudhary, S., & Sar, P. (2011). Uranium biomineralization by a metal resistant Pseudomonas
aeruginosa strain isolated from contaminated mine waste. Journal of Hazardous Materials,
186, 336e343.
Das, N. (2012). Remediation of radionuclide pollutants through biosorption e an overview.
Clean-Soil, Air, Water, 40, 16e23.
De Muynck, W., Belie, N., & Verstraete, W. (2010). Microbial carbonate precipitation in
construction materials: a review. Ecological Engineering, 36, 118e136.
DeJong, J. T., Mortensen, M. B., Martinez, B. C., & Nelson, D. C. (2010). Biomediated soil
improvement. Ecological Engineering, 36, 197e210.
Di Benedetto, F., Costagliola, P., Benvenuti, M., Lattanzi, P., Romanelli, M., & Tanelli, G.
(2006). Arsenic incorporation in natural calcite lattice: evidence from electron spin echo
spectroscopy. Earth and Planetary Science Letters, 246, 458e465.
Dunn, B. E., Campbell, G. P., Perez-Perez, G. I., & Blaser, M. J. (1990). Purification and
characterization of urease from Helicobacter pylori. Journal of Biological Chemistry, 265,
9464e9469.
Easton, R. M. (1997). Lichen-rock-mineral interactions: an overview. In J. M. McIntosh, &
L. A. Groat (Eds.), Biological-mineralogical interactions (pp. 209e239). Ottawa: Mineralog-
ical Association of Canada.
Ehrlich, H. L. (1998). Geomicrobiology: its significance for geology. Earth-Science Reviews,
45, 45e60.
Ehrlich, H. L., & Newman, D. K. (2009). Geomicrobiology (5th ed.). Boca Raton, FL, USA:
CRC Press/Taylor and Francis Group.
Fouke, B. W. (2011). Hot-spring systems geobiology: abiotic and biotic influences on trav-
ertine formation at Mammoth Hot Springs, Yellowstone National Park, USA. Sedimen-
tology, 58, 170e219.
Fowle, D. A., Fein, J. B., & Martin, A. M. (2000). Experimental study of uranyl adsorption
onto Bacillus subtilis. Environmental Science and Technology, 34, 3737e3741.
Francis, P. S., Lewis, S. W., & Lim, K. F. (2002). Analytical methodology for the determi-
nation of urea: current practice and future trends. TrAC Trends in Analytical Chemistry,
21, 389e400.
Fujita, Y., Ferris, F. G., Lawson, R. D., Colwell, F. S., & Smith, R. W. (2000). Calcium car-
bonate precipitation by ureolytic subsurface bacteria. Geomicrobiology Journal, 17, 305e318.
Fujita, Y., Redden, G. D., Ingram, J. C., Cortez, M. M., Ferris, F. G., & Smith, S. W. (2004).
Strontium incorporation into calcite generated by bacterial ureolysis. Geochimica et
Cosmochimica Acta, 68, 3261e3270.
Fujita, Y., Taylor, J., Gresham, T., Delwiche, M., Colwell, F. S., McLing, T. L. …
Smith, R. W. (2008). Stimulation of microbial urea hydrolysis in groundwater to
enhance calcite precipitation. Environmental Science and Technology, 42, 3025e3032.
Fujita, Y., Taylor, J., Wendt, L., Reed, D., & Smith, R. (2010). Evaluating the potential of
native ureolytic microbes to remediate Sr contaminated environment. Environmental
Science and Technology, 44, 7652e7658.
Gadd, G. M. (1993). Interactions of fungi with toxic metals. New Phytologist, 124, 25e60.
Gadd, G. M. (1999). Fungal production of citric and oxalic acid: importance in metal speci-
ation, physiology and biogeochemical processes. Advanced in Microbial Physiology, 41,
47e92.
Gadd, G. M. (2004). Microbial influence on metal mobility and application for
bioremediation. Geoderma, 122, 109e119.
Gadd, G. M. (2007). Geomycology: biogeochemical transformations of rocks, minerals,
metals and radionuclides by fungi, bioweathering and bioremediation. Mycological
Gadd, G. M. (2008). Fungi and their role in the biosphere. In S. E. Jorgensen, & B. Fath
(Eds.), Encyclopedia of ecology (pp. 1709e10177). Amsterdam: Elsevier.
Gadd, G. M. (2009). Biosorption: critical review of scientific rationale, environmental impor-
tance and significance for pollution treatment. Journal of Chemical Technology and Biotech-
nology, 84, 13e28.
Gadd, G. M. (2010). Metals, minerals and microbes: geomicrobiology and bioremediation.
Gadd, G. M., Bahri-Esfahani, J., Li, Q., Rhee, Y. J., Wei, Z., Fomina, M. … Liang, X.
(2014). Oxalate production by fungi: significance in geomycology, biodeterioration
and bioremediation. Fungal Biology Reviews, 28, 36e55.
Gadd, G. M., & Raven, J. A. (2010). Geomicrobiology of eukaryotic microorganisms.

Geomicrobiology Journal, 27, 491e519.
Gadd, G. M., Rhee, Y. J., Stephenson, K., & Wei, Z. (2012). Geomycology: metals,
actinides and biominerals. Environmental Microbiology Reports, 4, 270e296.
Gatermann, S., & Marre, R. (1989). Cloning and expression of Staphylococcus saprophyticus
urease gene sequences in Staphylococcus carnosus and contribution of the enzyme to
virulence. Infection and Immunity, 57, 2998e3002.
Goldberg, S., & Glaubig, R. (1988). Anion sorption on a calcareous, montmorillonitic soil e
arsenic. Soil Science Society of America Journal, 52, 1297e1300.
Gorman-Lewis, D., Elias, P. E., & Fein, J. B. (2005). Adsorption of aqueous uranyl com-
plexes onto Bacillus subtilis cells. Environmental Science and Technology, 39, 4906e4913.
Goudie, A. S. (1996). Organic agency in calcrete development. Journal of Arid Environments,
32, 103e110.
Govarthanan, M., Lee, K. J., Cho, M., Kim, J. S., Kamala-Kannan, S., & Oh, B. T. (2013).
Significance of autochthonous Bacillus sp. KK1 on biomineralization of lead in mine
tailings. Chemosphere, 90, 2267e2272.
Graham, D. Y., Klein, P. D., Evans, D. J., Alpert, L. C., Opekun, A. R., & Boutton, T. W.
(1987). Campylobacter pyloridis detected by the 13C-urea test. Lancet, 1174e1177.
Gresham, T. L. T., Sheridan, P. P., Watwood, M. E., Fujita, Y., & Colwell, F. S. (2007).
Design and validation of ureC-based primers for groundwater detection of urea-
hydrolyzing bacteria. Geomicrobiology Journal, 24, 353e364.
Guimaraes, B. C. M., Arends, J. B. A., van der Ha, D., de Wiele, T. V., Boon, N., &
Verstraete, W. (2010). Microbial services and their management: recent progresses in
soil bioremediation technology. Applied Soil Ecology, 46, 157e167.
Gworek, B. (1992). Lead inactivation in soils by zeolites. Plant and Soil, 143, 71e74.
Hammes, F., & Verstraete, W. (2002). Key roles of pH and calcium metabolism in microbial
carbonate precipitation. Reviews in Environmental Science and Biotechnology, 1, 3e7.
Hu, B., Liang, D., Liu, J., Lei, L., & Yu, D. (2014). Transformation of heavy metal fractions
on soil urease and nitrate reductase activities in copper and selenium cocontaminated soil.
Ecotoxicology and Environmental Safety, 110, 41e48.
Hua, B., Deng, B., Thornton, E. C., Yang, J., & Amonette, J. E. (2007). Incorporation of
chromate into calcium carbonate structure during coprecipitation. Water Air and Soil
Pollution, 179, 381e390.
Jeong, B. C., & Macaskie, L. E. (1999). Production of two phosphatases by a Citrobacter sp.
grown in batch and continuous culture. Enzyme and Microbial Technology, 24, 218e224.
Jones, B. D., & Mobley, H. L. T. (1988). Proteus mirabilis urease: genetic organization, regu-
lation, and expression of structural genes. Journal of Bacteriology, 170, 3342e3348.
Juillot, F., Ildefonse, P., Morin, G., Calas, G., de Kersabiec, A. M., & Benedetti, M. (1999).
Remobilization of arsenic from buried wastes at an industrial site: mineralogical and
geochemical control. Applied Geochemistry, 14, 1031e1048.
Kang, C. H., Han, S. H., Shin, Y. J., Oh, S. J., & So, J. S. (2014). Bioremediation of Cd by
microbially induced calcite precipitation. Applied Biochemistry and Biotechnology, 172,
1929e1937.
Kang, C. H., Oh, S. J., Shin, Y. J., Han, S.-H., Nam, I.-H., & So, J.-S. (2015). Bioremedi-
ation of lead by ureolytic bacteria isolated from soil at abandoned metal mines in South
Korea. Ecological Engineering, 74, 402e407.
Knoll, A. H. (2003). Biomineralization and evolutionary history. Reviews in Mineralogy and
Geochemistry, 54, 329e356.
Krajewska, B. (2009). Ureases. II. Properties and their customizing by enzyme immobiliza-
tions: a review. Journal of Molecular Catalysis B: Enzymatic, 59, 22e40.
Krishna, K. R., & Philip, L. (2005). Bioremediation of Cr(VI) in contaminated soils. Journal of
Hazardous Materials, 121, 109e117.
Kruger, M. C., Bertin, P. N., Heipieper, H. J., & Arsene-Ploetze, F. (2013). Bacterial meta-
bolism of environmental arsenic-mechanisms and biotechnological applications. Applied
Microbiology and Biotechnology, 97, 3827e3841.
Kumari, D., Pan, X., Lee, D. J., & Achal, V. (2014). Immobilization of cadmium in soil by
microbially induced carbonate precipitation with Exiguobacterium undae at low
temperature. International Biodeterioration and Biodegradation, 94, 98e102.
Lakshminarayanan, R., Kini, R. M., & Valiyaveettil, S. (2002). Investigation of the role of
ansocalcin in the biomineralization in goose eggshell matrix. Proceedings of the National
Academy of Sciences of the United States of America, 99, 5155e5158.
Lebranz, M., Druschel, G. K., Thomsen-Ebert, T., Gilbert, B., Welch, S. A.,
Kemner, K. M. … Banfield, J. F. (2000). Formation of sphalerite (ZnS) deposits in natural
biofilms of sulfate-reducing bacteria. Science, 290, 1744e1747.
Li, M., Cheng, X., & Guo, H. (2013). Heavy metal removal by biomineralization of urease
producing bacteria isolated from soil. International Biodeterioration and Biodegradation, 76,
81e85.
Li, Q., Csetenyi, L., & Gadd, G. M. (2014). Biomineralization of metal carbonates by Neuros-
pora crassa. Environmental Science and Technology, 48, 14409e14416.
Li, Q., Csetenyi, L., Paton, G. I., & Gadd, G. M. (2015). CaCO3 and SrCO3 bioprecipitation
by fungi isolated from calcareous soil. Environmental Microbiology, 17, 3082e3097.
Li, H., Shi, W. Y., Shao, H. B., & Shao, M. A. (2009). The remediation of the lead-polluted
garden soil by natural zeolite. Journal of Hazardous Materials, 169, 1106e1111.
Liu, S., Zhang, F., Chen, J., & Sun, G. (2011). Arsenic removal from contaminated soil via
biovolatilization by genetically engineered bacteria under laboratory conditions. Journal of
Environmental Science-China, 23, 1544e1549.
Lloyd, A. B., & Sheaffe, M. J. (1973). Urease activity in soils. Plant Soil, 39, 71e80.
Lowenstam, H. A., & Weiner, S. (1989). On biomineralization. Oxford: Oxford University
Press.
Macaskie, L. E., Empson, R. M., Cheetham, A. K., Grey, C. P., & Skarnulis, A. J. (1992).
Uranium bioaccumulation by a Citrobacter sp. as a result of enzymatically-mediated
growth of polycrystalline HUO2PO4. Science, 257, 782e784.
Makino, T., Sugahara, K., Sakurai, Y., Takano, H., Kamiya, T., Sasaki, K. … Sekiya, N.
(2006). Remediation of cadmium contamination in paddy soils by washing with chem-
icals: selection of washing chemicals. Environmental Pollution, 144, 2e10.
Margesin, R., Zimmerbauer, A., & Schinner, F. (2000). Monitoring of bioremediation by
soil biological activities. Chemosphere, 40, 339e346.
Martinez, R. J., Beazley, M. J., Taillefert, M., Arakaki, A. K., Skolnick, J., & Sobecky, P. A.
(2007). Aerobic uranium(VI) bioprecipitation by metal resistant bacteria. Environmental
Masaphy, S., Zabari, L., Pastrana, J., & Dultz, S. (2009). Role of fungal mycelium in the
formation of carbonate concentrations in growing media e an investigation by SEM
and synchrotron-based X-ray tomographic microsocopy. Geomicrobiology Journal, 26,
442e450.
Mobley, H. L. T., & Hausinger, R. P. (1989). Microbial ureases: significance, regulation and
molecular characterization. Microbiological Reviews, 53, 85e108.
Mobley, H. L. T., Island, M. D., & Hausinger, R. P. (1995). Molecular biology of microbial
ureases. Microbiological Reviews, 59, 451e480.
Mossman, K. I. (2003). Restructuring nuclear regulations. Environmental Health Perspectives,
111, 13e17.
Mukkamala, S. B., Anson, C. E., & Powell, A. K. (2006). Modelling calcium carbonate bio-
mineralisation processes. Journal of Inorganic Biochemistry, 100, 1128e1138.
Mulvaney, R. L., & Bremner, J. M. (1981). Control of urea transformations in soils. Soil
Biochemistry, 5, 153.
Najem, G. R., & Voyce, L. K. (1990). Health effects of a thorium waste disposal site. American
Journal of Public Health, 80, 478e480.
Navarathna, D. H., Harris, S. D., Roberts, D. D., & Nickerson, K. W. (2010). Evolutionary
aspects of urea utilization by fungi. FEMS Yeast Research, 10, 209e213.
Nessner Kavamura, V., & Esposito, E. (2010). Biotechnological strategies applied to the
decontamination of soils polluted with heavy metals. Biotechnology Advances, 28, 61e69.
Northup, D. E., & Lavoie, K. H. (2001). Geomicrobiology of caves: a review. Geomicrobiology
Journal, 18, 199e222.
O’Connor, G. A., O’Connor, C., & Cline, G. R. (1984). Sorption of cadmium by calcar-
eous soils: influence of solution composition. Soil Science Society of America Journal, 48,
1244e1247.
Ok, Y. S., Lim, J. E., & Moon, D. H. (2011). Stabilization of Pb and Cd contaminated soils
and soil quality improvements using waste oyster shells. Environmental Geochemistry and
Health, 33, 83e91.
Pan, X. (2009). Micrologically induced carbonate precipitation as a promising way to in situ
immobilize heavy metals in groundwater and sediment. Research Journal of Chemistry and
Environment, 13, 3e4.
Papadopoulos, P., & Rowell, D. L. (1988). The reactions of cadmium with calcium carbon-
ate surfaces. Journal of Soil Science, 39, 23e36.
Perito, B., & Mastromei, G. (2011). Molecular basis of bacterial calcium carbonate
precipitation. In W. E. G. Muller (Ed.), Molecular biomineralization, aquatic organisms form-
ing extraordinary materials (pp. 113e140). Berlin: Springer.
Phillips, A. J., Gerlach, R., Lauchnor, E., Mitchell, A. C., Cunningham, A. B., & Spangler, L.
(2013). Engineered applications of ureolytic biomineralization: a review. Biofouling, 29,
715e733.
Pieper, D. H., Martins dos Santos, V. A., & Golyshin, P. N. (2004). Genomic and mecha-
nistic insight into the biodegradation of organic pollutants. Current Opinion in Biotech-
nology, 15, 215e224.
Plassard, F., Winiarski, T., & Petit-Ramel, M. (2000). Retention and distribution of three
heavy metals in a carbonated soil: comparison between batch and unsaturated column
studies. Journal of Contaminant Hydrology, 42, 99e111.
Pollmann, K., Raff, J., Merroun, M., Fahmy, K., & Selenska-Pobell, S. (2006). Metal binding
by bacteria from uranium mining waste piles and its technological applications. Biotech-
nology Advances, 24, 58e68.
Powers, L. G., Mills, H. J., Palumbo, A. V., Zhang, C., Delaney, K., & Sobecky, P. A.
(2002). Introduction of a plasmid-encoded phoA gene for constitutive overproduction
of alkaline phosphatase in three subsurface Pseudomonas isolates. FEMS Microbiology Ecol-
ogy, 41, 115e123.
Ramachandran, S. K., Ramakrishnan, V., & Bang, S. S. (2001). Remediation of concrete
using microorganisms. ACI Materials Journal, 98, 3e9.
Rao, M. A., Scelza, R., Scotti, R., & Gianfreda, L. (2010). Role of enzymes in the remedi-
ation of polluted environments. Journal of Soil Science and Nutrition, 10, 333e353.
Rautaray, D., Ahmad, A., & Sastry, M. (2004). Biological synthesis of metal carbonate min-
erals using fungi and actinomycetes. Journal of Materials Chemistry, 14, 2333e2340.
Rhee, Y. J., Hiller, S., & Gadd, G. M. (2015). A new lead hydroxycarbonate produced dur-
ing transformation of lead metal by the soil fungus Paecilomyces javanicus. Geomicrobiology
Journal, 33, 1e11.
Riley, R. G., & Zachara, J. M. (1992). Chemical contaminants on DOE lands and selection of
contaminant mixtures for subsurface science research (p. 77). Washington, DC: Office of Energy
Research, Subsurface Science Program, U.S. Department of Energy.
Rivadeneyra, M. A., Delgado, R., del Moral, A., Ferrer, M. R., & Ramos-Cormenzana, A.
(1994). Precipitation of calcium carbonate by Vibrio spp. from an inland saltern. FEMS
Microbiology Ecology, 13, 197e204.
Roman-Ross, G., Cuello, G. J., Turrillas, X., Fernandez-Martínez, A., & Charlet, L. (2006).
Arsenite sorption and co-precipitation with calcite. Chemical Geology, 233, 328e336.
Rouff, A. A., Elzinga, E. J., & Reeder, R. J. (2004). X-ray absorption spectroscopic evidence
for the formation of Pb (II) inner-sphere adsorption complexes and precipitates at the
calciteewater interface. Environmental Science and Technology, 38, 1700e1707.
Ruggaber, T. P., & Talley, J. W. (2006). Enhancing bioremediation with enzymatic pro-
cesses: a review. Practice Periodical of Hazardous, Toxic, and Radioactive Waste Management,
10, 73e85.
Samborska, A., Stepniewska, Z., & Stepniewski, W. (2014). Influence of different oxidation
states of chromium (VI, III) on soil urease activity. Geoderma, 122, 317e322.
Santorufo, L., Van Gestel, C. A. M., & Maisto, G. (2012). Ecotoxicological assessment of
metal-polluted urban soils using bioassays with three soil invertebrates. Chemosphere,
88, 418e424.
Shuman, L. M. (1991). Chemical forms of micronutrients in soils. In J. J. Mortvedt, F. R. Cox,
L. M. Shuman, & R. M. Welch (Eds.), Micronutrients in Agriculture (pp. 113e144).
Madison,WI: Soil Science Society of America.
Singh, R., Gautam, N., Mishra, A., & Gupta, R. (2011). Heavy metals and living systems: an
overview. Indian Journal of Pharmacology, 43, 246e253.
Singh, R., Singh, S., Parihar, P., Singh, V. P., & Prasad, S. M. (2015). Arsenic contamination,
consequences and remediation techniques: a review. Ecotoxicology and Environmental
Safety, 112, 247e270.
Sipos, P., Németh, T., Mohai, I., & D odony, I. (2005). Effect of soil composition on
adsorption of lead as reflected by a study on a natural forest soil profile. Geoderma,
124, 363e374.
Smith, L. A., Alleman, B. C., & Copley-Graves, L. (1994). Biological treatment options.
In J. L. Means, & R. E. Hinchee (Eds.), Emerging technology for bioremediation of metals
(pp. 1e12). New York: Lewis Publishers.
Smolders, E., & Mertens, J. (2013). Cadmium. In B. J. Alloway (Ed.), Environmental pollution:
vol. 22. Heavy metals in soils: Trace metals and metalloids in soils. Netherlands: Springer.
Srivastava, S., Verma, P. C., Singh, A., Mishra, M., Singh, N., Sharma, N., & Singh, N. (2012).
Isolation and characterization of Staphylococcus sp. strain NBRIEAG-8 from arsenic
contaminated site of West Bengal. Applied Microbiology and Biotechnology, 95, 1275e1291.
Tang, Y., Elzinga, E. J., Jae Lee, Y., & Reeder, R. J. (2007). Coprecipitation of chromate
with calcite: batch experiments and X-ray absorption spectroscopy. Geochimica et Cosmo-
chimica Acta, 71, 1480e1493.
TCEQ (Texas Commission on Environmental Quality), Tier 1 Protective Concentration
Levels, 2009, http://www.tceq.texas.gov/remediation/trrp/trrppcls.html.
Thornton, E. C., & Amonette, J. E. (1999). Hydrogen sulfide gas treatment of Cr(VI)-
contaminated sediment samples from a plating-waste disposal site e implications for
in-situ remediation. Environmental Science and Technology, 33, 4096e4101.
Tiano, P., Biagiotti, L., & Mastromei, G. (1999). Bacterial bio-mediated calcite precipitation
for monumental stones conservation: methods of evaluation. Journal of Microbiological
Methods, 36, 139e145.
Uroz, S., Calvaruso, C., Turpault, M. P., & Frey-Klett, P. (2009). Mineral weathering by
bacteria: ecology, actors and mechanisms. Trends in Microbiology, 17, 378e387.
Vagenas, N. V., Gatsouli, A., & Kontoyannis, C. G. (2003). Quantitative analysis of synthetic
calcium carbonate polymorphs using FT-IR spectroscopy. Talanta, 59, 831e834.
Van Tittelboom, K., De Belie, N., De Muynck, W., & Verstraete, W. (2010). Use of bacteria
to repair cracks in concrete. Cement and Concrete Research, 40, 157e166.
Verrecchia, E. P. (2000). Fungi and sediments. In R. E. Riding, & S. M. Awramik (Eds.),
Microbial sediments (pp. 69e75). Berlin: Springer-Verlag.
Verrecchia, E. P., Dumont, J. L., & Rolko, K.,E. (1990). Do fungi building limestones exist
in semi-arid regions? Naturwissenschaften, 77, 584e586.
Wang, X., Hua, L., & Ma, Y. (2012). A biotic ligand model predicting acute copper toxicity
for barley (Hordeum vulgare): Influence of calcium, magnesium, sodium, potassium and
pH. Chemosphere, 89, 89e95.
Wang, T., Sun, H., Jiang, C., Mao, H., & Zhang, Y. (2014). Immobilization of Cd in soil and
changes of soil microbial community by bioaugmentation of UV-mutated Bacillus subtilis
38 assisted by biostimulation. European Journal of Soil Biology, 65, 62e69.
Whiffin, V. S., Van Paassen, L. A., & Harkes, M. P. (2007). Microbial carbonate precipitation
as a soil improvement technique. Geomicrobiology Journal, 24, 417e423.
White, C., Sharman, A. K., & Gadd, G. M. (1998). An integrated microbial process for the
bioremediation of soil contaminated with toxic metals. Nature Biotechnology, 16, 572e575.
Wu, Y., Ajo-Franklin, J. B., Spycher, N., Hubbard, S. S., Zhang, G., Williams, K. H. …
Smith, R. (2011). Geophysical monitoring and reactive transport modeling of
ureolytically-driven calcium carbonate precipitation. Geochemical Transactions, 12, 7.
Wuana, R. A., & Okieimen, F. E. (2011). Heavy metals in contaminated soils: a review of
sources, chemistry, risks and best available strategies for remediation. ISRN Ecology,
2011, 1e20.
Yamamura, S., & Amachi, S. (2014). Microbiology of inorganic arsenic: from metabolism to
bioremediation. Journal of Bioscience and Bioengineering, 118, 1e9.
Yamamura, S., Watanabe, M., Kanzaki, M., Soda, S., & Ike, M. (2008). Removal of arsenic
from contaminated soils by microbial reduction of arsenate and quinone. Environmental
Science and Technology, 42, 6154e6159.
Yamanaka, T. (1999). Utilization of inorganic and organic nitrogen in pure cultures by sap-
rotrophic and ectomycorrhizal fungi producing sporophores on urea-treated forest floor.
Mycological Research, 103, 811e816.
Yang, Z., Liu, S., Zheng, D., & Feng, S. (2006). Effects of cadmium, zinc, and lead on soil
enzyme activities. Journal of Environmental Science, 18, 1135e1141.
Zayed, A. M., & Terry, N. (2003). Chromium in the environment: factors affecting biolog-
ical remediation. Plant and Soil, 249, 139e156.
Zhang, K., & Li, F. (2011). Isolation and characterization of a chromium-resistant bacterium
Serratia sp. Cr-10 from a chromate-contaminated site. Applied Microbiology and Biotech-
nology, 90, 1163e1169.
Zinjarde, S., Apte, M., Mohite, P., & Ravi Kumar, A. (2014). Yarrowia lipolytica and pollut-
ants: interactions and applications. Biotechnology Advances, 32, 920e925.
CHAPTER THREE
Bacterial Mobilization of Nutrients

From Biochar-Amended Soils
A. Schmalenberger1 and A. Fox
1
Corresponding author: E-mail: achim.schmalenberger@ul.ie
Contents
1. Introduction 110
2. Biochar and the Soil Microbiota 112
3. Biochar as a Source of Nutrients 116
4. Biochar and the Bacterial Cycling of Nitrogen 117
4.1 Nitrification 118
4.2 Denitrification 126
4.2.1 Laboratory Studies 127
4.2.2 Field Studies 128
4.3 Fixation of Atmospheric Di-Nitrogen 129
5. Biochar and the Bacterial Cycling of Phosphorus 132
5.1 Mobilization of Inorganically Bound Phosphorus 133
5.2 Mobilization of Ester-Bound Phosphorus 135
5.3 Carbon-Bonded Phosphorus 138
6. Biochar and the Bacterial Cycling of Sulfur 139
6.1 Mobilization of Ester-Bound Sulfur 140
6.2 Mobilization of Sulfonate-Bound Sulfur 141
7. Biochar and Bacterial Cycling of Other Nutrients 143
8. Conclusions and Outlook 144
Acknowledgments 145
References 146
Abstract
Soil amendments with biochar to improve soil fertility and increase soil carbon stocks
have received some high-level attention. Physical and chemical analyses of amended
soils and biochars from various feedstocks are reported, alongside some evaluations
of plant growth promotion capabilities. Fewer studies investigated the soil microbiota
and their potential to increase cycling and mobilization of nutrients in biochar-
amended soils. This review is discussing the latest findings in the bacterial contribution
to cycling and mobilizing nitrogen, phosphorus, and sulfur in biochar-amended soils
and potential contributions to plant growth promotion. Depending on feedstock,
j
ISSN 0065-2164
110 A. Schmalenberger and A. Fox
pyrolysis, soil type, and plant cover, changes in the bacterial community structure were
observed for a majority of the studies using amplicon sequencing or genetic finger-
printing methods. Prokaryotic nitrification largely depends on the availability of ammo-
nium and can vary considerably under soil biochar amendment. However,
denitrification to di-nitrogen and in particular, nitrous oxide reductase activity is
commonly enhanced, resulting in reduced nitrous oxide emissions. Likewise, bacterial
fixation of di-nitrogen appears to be regularly enhanced. A paucity of studies suggests
that bacterial mobilization of phosphorus and sulfur is enhanced as well. However,
most studies only tested for extracellular sulfatase and phosphatase activity. Further
research is needed to reveal details of the bacterial nutrient mobilizing capabilities
and this is in particular the case for the mobilization of phosphorus and sulfur.
1. INTRODUCTION
Biochars are products of organic material which were subjected to a
heat treatment under restricted oxygen supply, commonly at temperatures
below 700 C (Lehmann & Joseph, 2009); however, temperatures of
800 C have also been used (Al-Wabel, Al-Omran, El-Naggar, Nadeem, &
Usman, 2013; Klose & Wiest, 1999). Biochar is vaguely separated from
charcoal that is usually produced under similar conditions albeit commonly
at higher temperatures and less stringent oxygen restrictions during pyrolysis
(Antal & Grønli, 2003) with the aim to produce a solid fuel. Furthermore,
the level of organic carbon in biochar tends to be higher than usually found
in charcoal (Lehmann & Joseph, 2009). The term biochar is relatively
new and found its entry in peer-reviewed research papers in 2000
(Karaosmanoglu, Işigig€ur-Erg€
udenler, & Sever, 2000) and subsequently
replaced terms like charcoal when the product was aimed to be added to
soils for benefits that include carbon sequestration and plant growth.
This resulted in a more widely accepted description of biochar by the
International Biochar Initiative ( Joseph, Peacocke, Lehmann, & Munroe,
2009). As such, the historic addition of charcoal to the tropical soils of the
Amazon region as an agricultural amendment (Glaser, Balashov, Haumaier,
Guggenberger, & Zech, 2000; Glaser, Haumaier, Guggenberger, & Zech,
2001) could be considered as the first widespread application of what we
would now call biochar. Initial investigations into the systematic burial of
charcoal/biochar to soil have been reported since 1980 (Devonald, 1982;
Iswaran, Jauhri, & Sen, 1980; Kishimoto & Sugiura, 1985). Interestingly,
the effect of charcoal on root nodule formation and Rhizobium has been
reported 60 years ago (Turner, 1955), while the effect of charcoal on seed
Bacterial Mobilization 111
germination was investigated a century ago (Retan, 1915). In recent years,

the number of publications addressing biochar in soils (in agricultural, bio-
logical, and environmental science subjects) has increased considerably.
Since 2011 the annual biochar publication rate (www.scopus.com)
exceeded 120 publications and so far increased year on year to over 400 pub-
lications in 2014. The bulk of these publications were associated with the
United States, China, and member states of the European Union (exceeding
300 publications each).
Incorporation of biochar into soils has been practised over the last few
years partially with the aim to replicate a sustained improvement in soil
fertility as observed in the Amazon region (Glaser et al., 2001) as well as
to sequester carbon into the soil (Lehmann, Gaunt, & Rondon, 2006).
More recently, the potential benefits of biochar application to reduce or
limit soil erosion and to improve soil restoration have been highlighted as
well (Lal, 2009). The physical and chemical attributes of biochar have
been highlighted to be mostly responsible for these potential beneficial ap-
plications in soils (Jeffery, Verheijen, Van Der Velde, & Bastos, 2011). Addi-
tion of biochar to soils will have an effect on soil physical properties
including the surface area, porosity, and bulk density since biochar’s physical
properties diverge substantially from many soil types and these physical
changes will affect for instance the soil’s response to water retention
(Downie, Crosky, & Munroe, 2009; Sohi, Krull, Lopez-Capel, & Bol,
2010). During pyrolysis, a rudimentary structure of the raw material will
be retained (Downie et al., 2009) in most reactors at low to medium tem-
peratures and thus the primary structure on a microscopic level will be an
important determinative in the physical properties of the biochar. Fig. 1
shows the obtained cellular structure for biochar produced at 600 C from
Miscanthus giganteus grass. Pore sizes of biochar buried in soil is of importance
as it will allow colonization by soil microbes that are small enough to enter
the spaces available while at the same time will not allow grazing predators of
larger sizes to follow (Warnock, Lehmann, Kuyper, & Rillig, 2007).
Chemical studies identified a negative surface charge on the investigated
charcoal/biochar that resulted in enhanced sorption ability (Liang et al.,
2006; Mohan, Pittman, & Steele, 2006). Depending on the raw material
and the type of pyrolysis, biochar deposition to soils can lead to a pH
neutralization effect, increased electro conductivity, and cation exchange
capacities (Atkinson, Fitzgerald, & Hipps, 2010; Fox, Kwapinski, Griffiths, &
Schmalenberger, 2014; Liang et al., 2006). Although biochar is regarded to
as recalcitrant with lower mineralization rates than most other forms of
Figure 1 SEM image of biochar particle (non-incubated; feedstock: Miscanthus

giganteus) scanned at 5 kV.
organic carbon (Lehmann et al., 2006), it may also serve as a source of

nutrients including phosphorus (P), potassium (K), sulfur (S), magnesium
(Mg), calcium (Ca), and other micronutrients, depending on the elemental
composition of the raw materials and the pyrolysis conditions (Amonette &
Joseph, 2009). The availability of these nutrients depends on whether, they
are present in an inorganic form in the feedstock and then concentrated dur-
ing pyrolysis due to the loss of carbon and oxygen in the pyrolysis, or are
organically bound to the carbon fraction in the biochar (Amonette & Joseph,
2009). This has a substantial impact on the immediate availability of nutri-
ents when added to the soil, as organically bound nutrients will only be
released slowly via biological mineralization and thus the immediate value
of biochar burial to plants can vary considerably (Yin Chan & Xu, 2009).
An indirect nutrient value of biochar can be attributed to its potential ability
to retain and absorb nutrients from soil solution (Yin Chan & Xu, 2009) that
would be otherwise lost into the ground water, and this has been extensively
demonstrated in the Terra Preta (Glaser et al., 2001).
2. BIOCHAR AND THE SOIL MICROBIOTA

During pyrolysis, pore spaces are increased more than a 1000-fold and
the resulting biochar pores are likely to provide a favorable habitat for
microbiota in which microbes such as mycorrhizal hyphae are protected
from being grazed by predators (see also Fig. 1), while at the same time
benefiting from access to nutrient and energy sources (Saito, 1990; Thies &
Rillig, 2009; Warnock et al., 2007). Many of the pores that represent the
remnants of the cell walls from the feedstock are too small for predators
such as mites, collembola, and nematodes to gain access to the hyphae
and bacteria which colonize the char. The diameter of prokaryotes such as
bacteria and eukaryotic hyphae are commonly in the single digit micrometer
as opposed to the commonly larger protists (Thies & Rillig, 2009; Warnock
et al., 2007). Research into the microbial communities associated with bio-
char is somewhat lacking behind other studies of biochar research as
expressed by Thies and Rillig (2009) and also by Lehmann et al. (2011).
However, the number of studies analyzing the effect of biochar on the
soil microbiota has increased over recent years alongside a general increased
activity around biochar research.
Studies undertaken in the Terra Preta in the Amazonian region have
shown that the historical addition of black carbon (which we would now
label as biochar) has a strong influence on the soil microbial community
structure. Cultivation-independent analysis of 16S rRNA gene fragments
revealed that soils from the Terra Preta had a 25% higher species richness
when compared to adjacent undisturbed forest soil (Kim, Sparovek, Longo,
DeMelo, & Crowley, 2007). A cultivation-based study of the same Amazo-
nian black earths in close proximity to the nutrient-poor ferrisols showed
that the Terra Preta had higher abundances of cultivable bacteria of up to
two orders of magnitude per gram soil present (O’Neill et al., 2009). Phylo-
genetic analysis of bacterial isolates also identified a higher level of diversity
compared to the adjacent ferrisols. More recently, the employment of culti-
vation-independent fingerprinting methods and next generation sequencing
confirmed that bacterial communities of the Amazonian black earths are
distinctive to the bacterial communities from the adjacent native soils
(Grossman et al., 2010; Navarrete, Cannavan, Taketani, & Tsai, 2010;
Taketani et al., 2013). At the same time, a number of microbiological studies
investigated the effect of biochar burial in various soils.
The addition of biochars made from oak to soils with and without a
historical record of burning revealed that soil type and source of pyrogenic
carbon influenced the microbial community composition with specific taxa
more abundant, reducing the overall bacterial diversity in the treatments
with low-temperature biochar but increasing diversity with high-
temperature biochar (Khodadad, Zimmerman, Green, Uthandi, & Foster,
2011). The authors concluded that biochars induce taxon-specific shifts in
microbial biomass and diversity. It can be assumed that low-temperature
biochar pyrolyzed at 250 C contain reasonable amounts of labile carbon that

is utilized preferentially by certain microbes, thus apparently reducing the
overall bacterial diversity. Anderson et al. (2011) hypothesized that biochar
addition to soil will undoubtedly shift the microbial community structure
and function by simply changing the physiochemical properties of the soil
and introducing labile C compounds. Terminal restriction fragment length
polymorphism (T-RFLP) coupled with 454 pyrosequencing revealed
statistically significant changes over time. Increases in bacterial family
abundances upon biochar amendment that exceeded 5% included the
Bradyrhizobiaceae (8%), Hyphomicrobiaceae (14%), Streptosporangineae (6%),
and Thermomonosporaceae (8%), while reduction of the families Streptomyceta-
ceae (11%) and Micromonosporaceae (7%) were recorded (Anderson et al.,
2011). Similar shifts in the bacterial community structure upon biochar
soil amendment were also observed in the rhizosphere of peppers that
included a near fivefold increase of the genus Flavobacterium as opposed to
previous reported changes in the bulk soil (Kolton et al., 2011), suggesting
that biochar amendment is influencing the rhizosphere.
Since 2012, a number of studies investigated the effect of biochar onto
the abundance and diversity of soil bacteria of which most reported a clear
community shift and/or increase in abundance (Chen et al., 2013, 2015; Fox
et al., 2014; Han, Meng, Zhang, & Chen, 2013; L. He et al., 2014; L. Hu,
Cao, & Zhang, 2014; Sun et al., 2012; Thuy Thu et al., 2014; Yoo & Kang,
2012), while others have identified little or no change (Anderson, Hamonts,
Clough, & Condron, 2014; Nelissen et al., 2015; Rutigliano et al., 2014).
Fox et al. (2014) identified a significant plant growth promotion effect
arising from Miscanthus-based biochar soil amendment in Irish soil with
ryegrass as host plant that also resulted in a significant shift in the bacterial
community structure. Shifts of the bacterial rhizosphere communities
were made visible via PCR and denaturing gradient gel electrophoresis
(DGGE) (Fox et al., 2014). Likewise, similar shifts were observed when
tomato plants (same plant growth-promoting effect) were used instead of
ryegrass (Fig. 2). On other occasions, biochar was explored as a carrier for
bacterial inoculation (Hale, Luth, Kenney, & Crowley, 2014). Furthermore,
biochar was also applied to mitigate pollutants in soils in conjunction with
microbial activity (Chen, Yuan, & Qian, 2012; Liu, Chen, Sun, Shen, &
Shang, 2015; Meynet et al., 2014).
The earliest studies on the effects of biochar on fungi were reported on
ectomycorrhiza in forests with charcoal depositions (Harvey, Jurgensen, &
Larsen, 1976) and ericoid mycorrhiza formation upon charcoal addition
1.0
B1 2
B1 3 Shoot weights
B1 5
pH
B1 4
B1 6 B2 4
B1 8
B2 2
B2 8
B1 1
Sulfonate utilizing
bacteria
Tri-calcium-phosphate
utilizing bacteria
Heterotrophic
bacteria
B2 5 B2 3 B2 1
B2 7
C1
C4
C6 C2 B2 6
C7 C3
C5 C8
-0.6
-0.6 1.0
Figure 2 Canonical correspondence analysis of the effect of the environmental
variables of soil pH, shoot weight, sulfonate utilizing, tri-calcium phosphate mobilizing,
and heterotrophic bacteria values on the 16S rRNA gene-based bacterial communities
in the Solanum lycopersicum (var. “Tiny Tim”) rhizospheres of the control (C1eC8),
biochar 1% (w/w; B1 1eB1 8) and biochar 2% (w/w; B2 1eB2 8) treatment (pot exper-
imental setup as in Fox et al., 2014). All presented environmental variables significantly
shifted the bacterial 16S community structure upon biochar amendment. Significant
differences between biochar 1%, 2% and control were confirmed via permutation
testing (Monte Carlo; P 0.05).
(Duclos & Fortin, 1983). Later, the effect of charcoal on the germination of
spores from the arbuscular mycorrhizal (AM) fungus Glomus was reported
(Gunasekaran, Sundaresan, Raja, & Lakshmanan, 1987). In 1990, increased
AM infections of soybean roots upon charcoal addition to soils were
revealed in a field study (Saito, 1990). These initial reports were followed
by further studies of charcoal/biochar amendments and their effect on
mycorrhizal fungi that were conceptually summarized by Warnock and
colleagues. They found evidence in the literature of (1) indirect effects on
mycorrhizae via other soil microbes, (2) effects on signaling dynamics
between plant and fungus, and (3) protection from grazing (Warnock
et al., 2007). Since then, further evidence was reported of the positive effect
of biochar on mycorrhization of plant hosts in the field (Solaiman,

Blackwell, Abbott, & Storer, 2010). Furthermore, monoxenic cultures of
root organs and AM fungi allowed identifying a transfer of labeled P from
biochar to the plant host through the AM hyphae of Rhizophagus (Hammer
et al., 2014). General fungal community analysis was conducted in biochar-
amended soils as well and reports are now published that show a similar
pattern to the observed changes in bacterial community structures (L. Hu
et al., 2014; Liang et al., 2014; Sun et al., 2012). However, shifts from a
fungal dominated microbial community towards a bacterial one were also
documented (Chen et al., 2013) as well as a lack of a fungal response to
biochar amendment (Nelissen et al., 2015).
Biochar deposition to soil may not only affect prokaryotes and fungi but
also microscopic animals such as the nematodes. Indeed, recent investiga-
tions into nematode diversity and abundance found that biochar soil amend-
ment can change nematode diversity, increase the abundance of bacteria
feeding nematodes, and reduce the abundance of plant-parasitic nematodes
(Fox et al., 2014; Rahman, Whitelaw-Weckert, & Orchard, 2014; Zhang
et al., 2013).
3. BIOCHAR AS A SOURCE OF NUTRIENTS

Biochar is generally regarded as relatively inert when compared to
their feedstocks and the carbon of biochars tends to be present in the soils
for hundreds to thousands of years, depending on the feedstock and type
of pyrolysis (Thies & Rillig, 2009). Short-term carbon usage by soil
microbes is more likely to happen when residues of bio oils are accessible.
However, these bio oils may also inhibit microbial activities and plant
growth as observed by Kwapinski and colleagues (oral communication).
Increased bacterial abundance in biochar-amended soils as described above
does not automatically increase microbial respiration as found out by Thies
and Rillig (2009), where increases in biochar deposition resulted in reduced
CO2 emissions. The reason for the lower respiration rate can have multiple
causes, ranging from higher metabolic efficiency ranges to absorbance of
CO2 to the biochar. Short- to medium-term changes on the biochar surface
are more likely to occur than general alteration of buried biochar. Not only
carbon may be lost but most likely also essential macro- and micronutrients
as identified on biochar surfaces using SEM-EDS (Amonette & Joseph,
2009). Besides the obvious presence of carbon, other elements such as
N, P, K, S, and Mg may be present in the biochar and on its surfaces that

originated from the used feedstocks, provided that the nutrients have not
volatilized during pyrolysis. A collection of elemental analyses from different
biochars was presented by Yin Chan and Xu (Yin Chan & Xu, 2009) where
N content varied from 1.8 to 56.4 g/kg and P from 2.7 to 480 g/kg. From
these findings it is clear that nutrient content is dependent on the individual
biochar produced and is not principally transferrable.
In many cases, however, the C/N ratio in biochars is very high with a
mean of 67, suggesting that net N immobilization will be likely in the
amended soil with the risk of N limitation in plant growth (Yin Chan &
Xu, 2009). Higher temperatures during pyrolysis result in higher losses of
nitrogen as demonstrated by Bagreev, Bandosz, and Locke (2001), where
temperature increases from 400 to 800 C resulted in a loss of N by over
50%. However, difference in the feedstock may result in substantial varia-
tions of N loss over a temperature gradient (Lang, Jensen, & Jensen,
2005). The total amount of P in biochar appears to be increasing with higher
pyrolysis temperature (Yin Chan & Xu, 2009), suggesting that little or no P
will be lost to the gas phase during pyrolysis up to 800 C. The fate of S dur-
ing pyrolysis appears to be connected to the feedstock and applied temper-
ature. While Lang et al. (2005) identified losses of around 50% at
temperatures above 400 C, Di Blasi, Signorelli, Di Russo, and Rea
(1999) found no evidence of S loss from wheat straw and wood chips
even at 850 C.
Even when total amounts of nutrients are established, their molecular
form may determine which types of organisms have access to the nutrient
as it is believed, for instance, that N and S are commonly bound to
organic carbon, thus microbial mineralization may be needed before these
nutrients are accessible to plants (Kertesz, Fellows, & Schmalenberger,
2007; Schimel & Bennett, 2004). The various nutrients that are absorbed
or chemically bound to the biochar-C could be mobilized and mineralized
by a range of soil microbes.
4. BIOCHAR AND THE BACTERIAL CYCLING

OF NITROGEN
Nitrogen is the nutrient that plants need in high amounts but cannot
access directly from the atmosphere as they can with carbon. Plants are
largely dependent on the uptake of nitrate and ammonium from the soil
solution, or in the case of leguminous plants from symbiotic bacteria. As a
result, nitrogen can be commonly limiting to plant growth (Vitousek &

Howarth, 1991). Nitrogen exists in soils in various organic forms where it
forms part of nucleic acids (part of the sugars of the nucleosides), is essential
in amino acids to establish the peptide bond to build peptides and proteins
and can be found in considerable amounts in amino sugars that are used to
build cell wall structures such as fungal chitins and the bacterial peptido-
glycan. The dominant inorganic forms of nitrogen in soils are the oxidized
form of inorganic nitrogen, nitrate, and the reduced inorganic form, ammo-
nium; alongside further intermediate forms (ammonia, nitrous oxide, nitric
oxide, nitrite, nitrogen dioxide, and di-nitrogen) (Robertson & Groffman,
2015).
The nitrogen cycle can be principally divided into three sections: (1) the
elemental cycle, (2) the anabolic process, and (3) the catabolic process.
Transformations in the elemental cycle are conducted by prokaryotic
microbes. They are responsible for the reduction and oxidation of the inor-
ganic forms of nitrogen, can reduce it to atmospheric nitrogen as di-nitrogen
or the greenhouse gas nitrous oxide (Robertson & Groffman, 2015).
Furthermore, microbes are largely responsible for the mineralization of
organically bound nitrogen (ammonification) which forms part of the cata-
bolic process. The assimilation of nitrate and ammonium for the buildup of
organically bound forms (anabolism) can be carried out by prokaryotes,
fungi, and plants alike employing enzymes such as the glutamate
dehydrogenase (GDH) and the glutamine synthetase-glutamate synthase
(GS-GOGAT) (Schwartz, Misri, & Fock, 1991). Whether the soil environ-
ment is experiencing a net mineralization of organic nitrogen or a net
immobilization into organically bound nitrogen is determined by the carbon
to nitrogen ratio. Organic carbon with low levels of nitrogen (degraded by
saprophytic microbes) results in an additional demand for nitrogen from the
inorganic pool, while organic carbon with high nitrogen content will result
in net mineralization as the degrading microbes do not need all of the
released nitrogen for the incorporation into their own biomass (Tammeorg,
Brandstaka, Simojoki, & Helenius, 2013).
4.1 Nitrification
Nitrification is the process of oxidizing ammonium (ammonia at the enzyme
level) to nitrate via nitrite. Ammonium oxidation to nitrite via NH2OH as
an intermediate step is catalyzed by the ammonia monooxygenase (Amo)
that can be found among bacteria and archaea (Robertson & Groffman,
2015). This is an aerobic process. A by-product of this oxidation is nitrous
oxide (N2O). Under oxygen-limiting conditions the amount of nitrous

oxide formation can increase considerably as the formed nitrite is then
used as a substitute electron acceptor ( Jia et al., 2013). Thus, the use of
biochar to increase soil aeration could help to reduce formation of nitrous
oxide gas. In the second part of the nitrification reaction, nitrite can be
further oxidized to nitrate with the nitrite oxidoreductase (Nor) by nitrify-
ing bacteria. These oxidation steps allow gaining energy from an inorganic
source and the prokaryotes capable of this oxidation are commonly chemo-
lithotrophic. When this energy is utilized to fix carbon dioxide from the
atmosphere, these prokaryotes are then classified as chemolithoautotrophs;
however, heterotrophic nitrification is also possible and can be carried out
by bacteria and fungi (Robertson & Groffman, 2015). Its benefit is not clear
as the heterotrophic oxidation is not directly coupled with a gain of energy.
Nitrification is generally believed to be higher in soils when the pH is neutral
and not acidic, although a few acidic nitrifiers have been also reported
(De Boer & Kowalchuk, 2001). Since nitrification is a reaction that is also
reducing the environmental pH (Neina & Dowuona, 2013), biochar that
has a neutralizing capacity may therefore depress the inhibition of nitrifica-
tion. Furthermore, in an acidic soil, pH neutral microsites could serve as a
home for nitrifying prokaryotes including biochar particles and their imme-
diately surrounded soils.
Increases in nitrification through biochar or charcoal addition to soils
were reported for temperate and boreal forests, where the control nitrifica-
tion was below the detection limit (Ball, MacKenzie, DeLuca, & Holben,
2010; Berglund, DeLuca, & Zackrisson, 2004; DeLuca, MacKenzie,
Gundale, & Holben, 2006). More recently, increases in nitrification rates
were also reported in arable soils (Case et al., 2015; F. He, Liang, Wu,
Rong, & Liu, 2014; Y.L. Hu, Wu, Zeng, & Chang, 2014; Nelissen,
R€ utting, Huygens, Ruysschaert, & Boeckx, 2014; Nelissen et al., 2012;
Pereira et al., 2015; Prommer et al., 2014; Ulyett, Sakrabani, Kibblewhite, &
Hann, 2014; Xu et al., 2014; Zhao, Wang, & Xing, 2014) while others
have found changes in the bacterial and archaeal diversity of ammonia
oxidizers and bacterial nitrite oxidizers but not necessarily a net increase in
nitrification (Anderson et al., 2014; Ball et al., 2010; Harter et al., 2014;
Liu et al., 2014; Song, Zhang, Ma, Chang, & Gong, 2014). However, a
substantial number of publications have found no evidence of biochar
having an effect on nitrification (Castaldi et al., 2011; Cheng, Cai, Chang,
Wang, & Zhang, 2012; Martin, Clarke, Othman, Ramsden, & West,
2015; Sun et al., 2014; Yanardag, Zornoza, Cano, Yanardag, & Mermut,
2014; Yao, Campbell, & Qiao, 2011) or found a reduction in net nitrifica-
tion (Ali, Hoque, & Kim, 2013; Anderson et al., 2011; Lentz, Ippolito, &
Spokas, 2014; Schulz & Glaser, 2012; Yang, Cao, Gao, Zhao, & Li, 2015;
Zhu et al., 2015). Thies and colleagues have compiled an extensive table
of biochar experiments related to nitrogen cycling research that also includes
an overview of outcomes in nitrification for comparison (Table 13.3, Thies,
Rillig, & Graber, 2015). The fundamental differences in these reports, pub-
lished since 2010, can be explained by the diversity of feedstock utilized, py-
rolysis regime and temperatures employed, and types of soils used for the
deposition. Furthermore, a diverse range of experiments were established
with soils in incubators without plants, microcosms, pots with and without
plants, and field trials. Therefore, it is not entirely surprising that apparently
entirely different results were obtained. A few general trends can be estab-
lished across these findings though. Soils with low levels of ammonium to
begin with are unlikely to experience an increase in prokaryotic nitrification
upon biochar amendment due to a lack of substrate. This, however, can
change substantially when soils are also fertilized with ammonium in addi-
tion to biochar amendment. If the biochars applied have a high potential
of sorption of ammonium, then nitrification rates may be reduced at least
short term. Likewise, if nitrate is increasingly adsorbed, then net nitrification
rates may appear to be lower than they actually are. Plants that grow signif-
icantly better upon soil biochar amendment may take up larger amounts of
nitrate and ammonium, thus influencing the amount of inorganic nitrogen
in the soil and affecting the calculation of nitrification rates.
Molecular tools are capable of investigating nitrifying prokaryotes in
more detail. As a result, the importance of ammonia-oxidizing archaea
(AOA) over ammonia-oxidizing bacteria (AOB) in soils was discovered
by quantifying archaeal and bacterial amoA genes (Leininger et al.,
2006). Variations in bacterial and archaeal amoA gene abundance were
linked to soil types, soil pH, and soil fertilization (He et al., 2007; Leininger
et al., 2006). A low soil pH appeared to be favoring archaeal ammonia
oxidation, while bacterial activity increased at a neutral pH (Nicol, Lei-
ninger, Schleper, & Prosser, 2008). In some cases, bacterial ammonia
oxidation was found to be dominating agricultural soils despite high abun-
dances of archaeal ammonia oxidizers, which seemed to be less active
though (Di et al., 2009; Jia & Conrad, 2009). Research in amoA abundance
and expression by bacteria or archaea is less well researched in biochar-
amended soils with reports now appearing in the public domain. Table 1
provides an overview of soil biochar research studies that investigated
Table 1 Experimental conditions and methods used to study bacterial cycling/mobilization of nutrients under biochar amendment
Biochar
Bacterial Mobilization
application Soil type or
Microbial activity Type of study rates Feedstock type Pyrolysis ( C) description References
Nitrification, ammonia- Wildfire woodland n.s. Wood Wildfire North Idaho Ball et al. (2010)
oxidizing bacterial
abundance (amoA)
increased
Changes in nirK, nirS, nosZ Field trial, ryegrass 15, 30 kg/ha þ Wood 450 Silt loam Anderson et al. (2014)
gene abundance pasture urine
Increased nosZ copy numbers Soil microcosms 2, 10% (w/w) Green waste 700 Loamy sand Harter et al. (2014)
and gene expression; amoA
and nifH copy numbers
varied over time
Reduced amoA gene copy Soil microcosms 1, 2, 4, and 8% Rice straw; bovine 300 and 500 Pujiang, Shanghai Liu et al. (2014)
numbers, nirS copy (w/w) manure
numbers varied
Bacterial and archaeal amoA Soil microcosms/pots 5, 10, 20% (w/w) Cotton stalks 650 Coastal sandy loam, Song et al. (2014)
copy numbers and diversity pH 8
vary with biochar
concentrations
Increased nosZ, amoA in Soil microcosm þ labile 1% Poultry litter; 550 Vertosol, ferrosol, Van Zwieten et al. (2014)
Tenosol with biochar, N2O C þ wetting/drying wheat chaff; calcarosol, tenosol
emission reduced oil mallee
Reduced N2O emission Manure composting 3% (w/w) Bamboo 600 Pig manure, sawdust, Wang et al. (2013)
correlated with abundance wood chips
of nosZ, nirK, nirS
Reduced N2O emissions; Microcosms 2% (w/w) 9 different types 500 15 agricultural soils Cayuela et al. (2013)
biochar proposed as (leaves, woody,
electron shuttle manure)
Increased amoA, nirS, nirK, Pot experiment, no plant 1, 2, 10% (w/w) Switchgrass 350 (steam Portneuf (from Ducey et al. (2013)
nosZ, nifH, 16S gene copy cover activated 800) Idaho)
numbers
121
(Continued)
Table 1 Experimental conditions and methods used to study bacterial cycling/mobilization of nutrients under biochar amendmentdcont'd
122
Biochar
Most probable number of Spinach in glasshouse 12.5, 25 t/ha Corncob 350e550 n.s. Han et al. (2013)
ammonifying bacteria plots
increased, denitrifier
abundances varied
Increased denitrification rate; Pot experiment with 5% Rice straw 500 Acrisol Xu et al. (2014)
16S community shifts; nosZ plant cover
and archaeal amoA
expression increased
Abundance of nosZ unchanged Field trial, N fertilizer 18e69 t/ha Maize silage, wood 210 (hydrochar), Haplic cambisol Dicke et al. (2015)
added chip 600, 850,
digestate added
Denitrification, plant growth Field trial with 25, 50 t/ha Wood 450 Eutric cambisol Jones et al. (2012)
and microbial abundance wheat þ fertilizer
increased
Biological nitrogen fixation Pot trial with fertilizer 3, 6, 9% (w/w) Wood 350 Haplustox Rondon et al. (2007)
increased and common bean
Plant growth and biological Pots/mesocosms with 1, 10, 50, 120 t/ha Grass 400 Nature reserve, Mia et al. (2014)
nitrogen fixation increased grass/clover cover, Netherlands
at 10 t/ha fertilizer
A. Schmalenberger and A. Fox

Root nodulation reduced, Pot trial 25, 50, 25 þ 25, Wood 450 Eutric cambisol Quilliam et al. (2013)
nodule weight and di- 50 þ 50 t/ha
nitrogenase activity/nodule
increased
Plant growth promotion, Pot trial with modified 15 t/ha Wood, shrub, 350, 550 Humic acrisol G€
uere~
na et al. (2015)
higher biological nitrogen biochar and common maize stover
fixation, mycorrhization, P bean and cob, rice
uptake hulls
Altered nifH diversity Pot experiment 1, 3% (w/w) Miscanthus 600 Mollic, histic, Corkery (2014)
stagnosol
Bacterial Mobilization
Plant growth promotion, Pot experiment 1, 3% (w/w) Miscanthus 600 Mollic, histic, Fox et al. (2014)
higher TCP, phytate, stagnosol
phosphonate, sulfonate
utilizers, nematodes;
changed phnJ, asfA diversity
Community shifts infer Field and pot trial with 15, 30 t/ha; Wood n.s. Templeton silt-loam Anderson et al. (2011)
changes in N cycle and ryegrass 10% (w/w) soil
phosphate-solubilizing
bacteria
Phosphatase increased Compost experiment 2% (v/v) Wood 400e600 Poultry manure with Jindo et al. (2012)
organic wastes
Bacterial and fungal Vineyard with biochar, 8 t/ha Hard 750 Haplic regosol Mackie et al. (2015)
abundances, phosphatase, compost wood þ wood
sulfatase unchanged chips
Fungal and bacterial Field trial (paddy rice) 20, 40 t/ha Wheat straw 350, 550 Hydragric anthrosol Chen et al. (2013)
communities changed,
alkaline phosphatase
increased, acid phosphatase
unchanged
Neutral phosphatase, fungal Cu-contaminated field 10, 20, 40 t/ha Wheat straw 450 Ferric-accumulic Cui et al. (2013)
abundance increased trial, fertilized (rice/ stagnic anthrosols
wheat)
Plant growth, alkaline and Pot experiment, maize 0.1, 0.3, 0.5, Water hyacinth 300 Ustorthents Masto, Kumar, et al. (2013)
acidic phosphatase 1, 2% (w/w)
increased
Acid phosphatase reduced/ Field trial with fly ash n.s. Sage n.s. Sandy loam Masto, Ansari, et al. (2013)
alkaline phosphatase and maize
increased
Alkaline phosphatase increased Field trial, fertilizer þ 4.5, 9 t/ha Crushed corncob 360 Fluvic cambisols Du et al. (2014)
maize
Alkaline phosphatase and Apple orchard 10 t/ha Wood 500 Haplic calcisol Ventura et al. (2014)
arylsulfatase increased
123
(Continued)
124
Table 1 Experimental conditions and methods used to study bacterial cycling/mobilization of nutrients under biochar amendmentdcont'd
Biochar
Barley biochar: phosphatase, Microcosm, no plant 2% (w/w) Barely stover; 320; 600e800 Pasture and rice Yoo and Kang (2012)
bacteria, archaea increased, cover swine manure paddy utisols
fungi varied; N2O
emissions varied
Acid phosphatase decreased, Microcosms, no plant 0.5, 1.5% (w/w) Pig manure 400 Clay and silt loam Jin et al. (2015)
alkaline phosphatase cover soils
increased
Arylsulfatase unchanged Cups, no plant cover 1, 5% (w/w) Corn stover, wood 850 Frigid entic Chintala et al. (2014)
chips, hapludolls; frigid
switchgrass pachic hapludolls
Acid phosphatase and Pot experiment, no plant 4, 8% Sewage sludge 600 Umbrisol Paz-Ferreiro et al. (2012)
arylsulfatase unchanged cover
A. Schmalenberger and A. Fox

Arylsulfatase increased Pot experiment, no plant 3% w/w Poultry litter 400 Haplic acrisol Paz-Ferreiro et al. (2015)
cover
n.s. ¼ not specified.

bacterial or archaeal nitrification alongside other bacterial N, P, and S

cycling/mobilization studies.
A statistically nonsignificant increase in archaeal amoA over bacterial copy
numbers was reported by Harter et al. (2014) when green waste biochar and
a fertilizer was added to soils without plants. Song et al. (2014) established a
plant-free biochar soil amendment with up to 20% biochar addition. They
found over a 12-week incubation period that gene copy numbers of amoA
for AOA and AOB varied greatly with AOA copies responding within
1 week of the establishment of the incubations, while AOB responded
only after 4 weeks into the incubation. At various time points controls con-
tained highest copy numbers, whereas at other times 5% biochar addition
was found to have highest numbers. Potential ammonia oxidation (PAO)
rates appeared to be correlated to AOA amoA copy numbers while PAO
was highest in the 15% biochar amendment in the first 4 weeks (Song
et al., 2014). Diversities of amoA have changed with biochar addition
with a reported increase of the Shannon index for AOB, while changes in
AOA were ambiguous (Song et al., 2014). The effect of soil wetting cycles
in the absence or presence of biochar on the amoA abundance revealed that
AOA increased in abundance by the fifth wetting cycle, whereas AOB
abundance was largely unaffected by the wetting/drying events regardless
of the soil tested (Van Zwieten et al., 2014). In a different study of biochar
amendment, plant growth was, albeit nonsignificantly, increased for one
biochar type, while at the same time abundance of AOB was reduced
(Liu et al., 2014). However, other biochar amendments by Liu et al.
(2014) showed a similar reduction in AOB, while plant growth was reduced
over the controls (not significant).
In summary, to date, very little information is available on AOB and
AOA activity in relation to biochar and this is particularly the case in
connection to plant growth as no study was able to show changes in
AOA or AOB abundance when the deposited biochar was able to signifi-
cantly increase plant growth. Therefore, a substantial knowledge gap still
exists when it comes to the application of biochar in agriculture and rates
and mechanisms of nitrification. Likewise, gene expression of amoA in the
presence and absence of biochar is to date not investigated and thus it is
unclear under which conditions AOA or AOB will be mainly responsible
for the nitrification process. Since our knowledge of nitrification under bio-
char soil amendment is quite limited, we also have very limited information
on whether biochar addition to soils could reduce nitric oxide emissions
during nitrification. A working hypothesis would be that highly porous
biochar amendment would increase soil aeration and therefore minimize

nitrous oxide production during oxidation of ammonium. The use of nitri-
fication inhibitors in agriculture has been investigated on several occasions
with the aim to reduce nitrous oxide emission either during nitrification
or subsequently during the denitrification process. Interestingly, the use of
dicyandiamide (DCD) in combination with various biochars in a soil incu-
bation experiment reduced the efficacy of DCD, possibly due to sorption of
nitrification-inhibiting components from the soil and DCD directly (Shi,
Zhang, & Zhao, 2015).
4.2 Denitrification
Besides the assimilation of nitrate as a source of nitrogen, the oxidized form
of inorganic nitrogen can be utilized by soil prokaryotes as an alternative
electron acceptor, when either oxygen is not available or the microbes are
not capable of utilizing oxygen as a terminal electron acceptor. The reduc-
tion of nitrogen follows several enzymatic steps starting with the nitrate
reductase (Nar), followed by the nitrite reductase (Nir), nitric oxide reduc-
tase (Nor), and finally the nitrous oxide reductase (Nos), reducing nitrate via
nitrite, nitric oxide, and nitrous oxide to di-nitrogen gas (Knowles, 1982).
Nitrous oxide as the last intermediate in this reaction is also known to be
released into the environment as a greenhouse gas. Furthermore, nitrate
can also be reduced to ammonium via nitrite by the pathway called
dissimilatory nitrate reduction to ammonium (DNRA) (Tiedje, Sexstone,
Parkin, & Revsbech, 1984), which can be responsible for a substantial
proportion of nitrate reduction in temperate soils (R€ utting, Boeckx,
M€ uller, & Klemedtsson, 2011). However, to date the influence of biochar
addition to soil on the DNRA is largely unknown. Indirect evidence via
fingerprinting and next generation sequencing of SSU rRNA gene
fragments suggests that biochar soil amendment has the potential to increase
DNRA though (Anderson et al., 2011). In contrast, the dissimilatory reduc-
tion of nitrate to di-nitrogen gas has received considerably more attention,
largely because of the potential loss of nitrogen from soils and the potential
emission of nitrous oxide. Unlike the presented results from the nitrification
process, the majority of research publications available find a reduction in
nitrous oxide emissions in soils amended with biochar (Thies et al., 2015).
This can have several causes, ranging from lower amounts of nitrate that
may be available due to reduced nitrification rates or sorption effects of
the biochar, a more efficient nitrous oxide reductase activity, to a shift of
dissimilatory nitrate reduction accomplished via DNRA.
4.2.1 Laboratory Studies

Yanai, Toyota, and Okazaki (2007) found that charcoal made from house-
hold waste was able to reduce N2O emissions over a 120-h incubation
period at up to 78% of water-filled pore space. Interestingly, further
increases in soil wetting resulted in a significant increase in nitrous oxide
emissions. The alkalinity of the biochar was proposed to be the main driver
that increased soil pH and with it higher microbial nitrous oxide reductase
activity, resulting in reduced emissions of N2O (Yanai, Hatano, Okazaki, &
Toyota, 2008). In contrast to these findings, Van Zwieten et al. (2010) found
that even under 100% water-filled porosity, biochars made from green
waste, poultry litter, paper-mill waste, and bio-solids were able to reduce
nitrous oxide emissions from an acidic ferrosol fertilized with urine. In a
different experiment, however, soil incubation experiments with pasture
soil, urine, and biochar showed that addition of biochar to soil with urine
can increase nitrous oxide fluxes within the first 30 days of incubation
(Ciough et al., 2010). Addition of pig slurry to soil columns with biochar
increased N2O emission, but biochar application alone showed no increase
in nitrous oxide emission over the control (Troy, Lawlor, O’Flynn, &
Healy, 2013). Laboratory-based incubation of soils from Colorado and
Minnesota with oak-based biochar showed a reduction in nitrous oxide
emissions but increases in methane emissions were recorded (Zheng,
Stewart, & Cotrufo, 2012). Addition of switchgrass biochar to soil in an in-
cubation experiment recorded increased abundances of genes nirK, nirS, and
nosZ (qPCR) with increasing concentrations of biochar, suggesting higher
denitrification rates to di-nitrogen under biochar amendment (Ducey,
Ippolito, Cantrell, Novak, & Lentz, 2013). Likewise, higher abundances
of cultivable denitrifying bacteria were found in the rhizosphere of spinach
when biochar was added to the soil (Han et al., 2013). Reductions in N2O
emission in compost were identified, while gene abundance in the compost
showed that biochar addition increased nosZ but not nirK, suggesting a
more efficient nitrous oxide reduction with biochar amendment (Wang
et al., 2013).
The above-mentioned studies are limited in the number of biochars and
soils tested under identical experimental conditions. Cayuela et al.
(2013) tested 14 different soils and 9 different biochars. There, 13 out of
14 soils with biochar amendment showed reductions in nitrous oxide emis-
sions. Comparisons of the biochar revealed that all biochars were capable of
reducing N2O emissions with the exception of two biochars with high N
content. Even acidified biochar deposition resulted in lower nitrous oxide
emissions. Only after further addition of nitrate and glucose to the soil, the
beneficial effect of biochar was largely eliminated (Cayuela et al., 2013). The
authors concluded that pyrolysis at 500 C minimizes microbial inhibitory
effects and sorption of nitrate to the char. Also, the soil alkalization had at
least partially an indirect effect on the denitrification that was largely attrib-
uted to the change in microbial activity. There, the biochar may act as an
electron shuttle supporting the dissimilatory reduction of inorganic nitrogen
and thus reducing the loss of N2O in the microbiological process (Cayuela
et al., 2013). Further lab-based incubation experiments are in agreement
with the often observed reductions in N2O emissions (Chintala et al.,
2015; Harter et al., 2014; Martin et al., 2015; Nelissen, Saha, Ruysschaert, &
Boeckx, 2014; Rizhiya, Buchkina, Mukhina, Belinets, & Balashov, 2015).
Reductions in nitrous oxide release may be linked to higher levels of
nosZ gene expression, thus denitrification is followed through to the forma-
tion of di-nitrogen gas instead of the release of N2O (Harter et al., 2014; Xu
et al., 2014). This was also detected in plant pot experiments (Xu et al.,
2014) where in addition archaeal amoA gene expression was observed
despite the reduction in N2O emission. Nevertheless, this effect was not uni-
versally observed as other studies were not able to find significant reductions
in N2O emission (Xiang, Liu, Ding, Yuan, & Lin, 2015) or higher abun-
dances of nosZ (Dicke et al., 2015).
4.2.2 Field Studies

In an Australian intensively managed subtropical pasture, biochar (cattle
feedlot, pyrolyzed at 550 C) soil amendment had no significant impact on
nitrous oxide emissions, but episodes of high rainfall contributed to short-
term increases in greenhouse gas emissions (Scheer, Grace, Rowlings,
Kimber, & van Zwieten, 2011). In a field experiment with wheat in Italy
over two growing seasons, contrasting findings could be identified as well
(Castaldi et al., 2011), where biochar soil amendment (3 and 6 kg/m2 of
wood-based commercial biochar) reduced nitrous oxide emissions but this
effect was reversed upon urine application. Furthermore, spatial variations
appeared to play an important role, resulting often in nonsignificant biochar
effects. While plant growth was recorded in this experiment, no evidence of
plant growth promotion through biochar application was reported (Castaldi
et al., 2011). Similar findings were reported by Kammann, Ratering,
Eckhard, and M€ uller (2012), where biochar and hydrochar application in
a field increased ryegrass growth and significantly reduced nitrous oxide
emissions, but again this effect was reversed upon nitrogen fertilization.
In a 3-year field trial in Wales, grassland growth promotion due to biochar

deposition at 50 kg/ha was reported in the third year, and in the same year
higher denitrification enzyme activity was found (both significant) ( Jones,
Rousk, Edwards-Jones, DeLuca, & Murphy, 2012). By the third year,
most of the pH neutralization effect by the wood chip-based commercial
biochar was lost. However, by then the microbial community has shifted to-
wards a bacterial dominance ( Jones et al., 2012). While further short-term
benefits such as reduced nitrous oxide emissions without reductions in inor-
ganic nitrogen availabilities in the field were reported (Case et al., 2015),
there is to date a shortfall in long-term investigations.
4.3 Fixation of Atmospheric Di-Nitrogen

Early advancements in di-nitrogen fixation research were most notably
accomplished in the 19th century with the discovery and isolation of di-ni-
trogen-fixing bacteria from root legumes by M. Beijerinck. This initiated
the microbiological research into the biological fixation of di-nitrogen gas
that is limited to members of the prokaryotic kingdoms of the bacteria
and the archaea (Postgate, 1982). Fixation of di-nitrogen can be accom-
plished by free-living prokaryotes such as Azospirillum that may be associated
to plant hosts due to their activities in the rhizosphere, with the potential to
enhance plant growth (Baldani, Alvarez, Baldani, & D€ obereiner, 1986;
Okon, 1985). Bacteria such as Rhizobium are known to be able to establish
a symbiosis with their plant host and fix large amounts of di-nitrogen gas in-
side root nodules of their plant host as bacteroids, where all other nutrients
are provided by the host (Van Rhijn & Vanderleyden, 1995). The nitroge-
nase enzyme complex, responsible for reduction of di-nitrogen to ammonia,
is formed as a tetramer unit (Postgate, 1982) with an MoFe protein
(di-nitrogenase) and an Fe protein (nitrogenase reductase) with the latter
being a homodimer encoded by nifH (Fuhrmann & Hennecke, 1984). Since
the sequence of nifH is well conserved, it has been successfully used as a
molecular marker to study nitrogenase diversity and phylogeny (Zehr,
Jenkins, Short, & Steward, 2003).
Due to a partial loss of nitrogen to the atmosphere during pyrolysis, bio-
char tends to have lower concentrations of nitrogen than the corresponding
feedstocks (Yin Chan & Xu, 2009). Therefore, di-nitrogen-fixing (diazotro-
phic) bacteria colonizing biochar in soils may be able to provide additional
nitrogen resources. Despite the accumulation of thousands of research arti-
cles and book chapters dealing with biological (di-)nitrogen fixation (BNF),
research in BNF under soil biochar amendment is rather rare. First
discoveries were made with the use of charcoal as a carrier for diazotrophic
bacteria as an alternative to peat (Pramanik & Iswaran, 1973), and
subsequently charcoal was used in field trials to inoculate plant hosts with
Rhizobium (Beck, 1991; Kavimandan, 1986; Muniruzzaman & Khan,
1992). Later, the application of charcoal and diazotrophic bacteria to pro-
mote plant growth was introduced (Ogawa, 1998). Gehring found evidence
of high BNF activity in the Amazon secondary forests after slash and burn of
the primary forests (Gehring, 2003). Abundant root nodulation was found in
the Amazonian Black Earths as opposed to low nodulation in adjacent fer-
risols (Sylvester-Bradley, DeOliveira, DePodestaFilho, & St. John, 1980).
Rondon, Lehmann, Ramírez, and Hurtado (2007) established pot experi-
ments with Columbian soil and added eucalyptus-based biochar to grow
common beans. Via 15N stable isotope dilution, they found increased levels
of formerly atmospheric nitrogen in the plant with biochar soil amendment
that ultimately resulted in significantly improved plant growth (Rondon
et al., 2007). Unlike other studies, Rondon and colleagues found no signif-
icant changes in the mycorrhiza of the common bean (George, Wagner,
K€ucke, & Rillig, 2012; Robertson, Michael Rutherford, L opez-Gutiérrez, &
Massicotte, 2012).
Soil incubations with biochar revealed that biochar-amended soils con-
tained more copies of nifH than the control soils (Ducey et al., 2013; Harter
et al., 2014). Quilliam, DeLuca, and Jones (2013) found that in a field exper-
iment in Wales with clover, root nodules did not increase in abundance with
soil biochar (wood based) amendment but interestingly, nitrogenase activity
in the nodules of the biochar amendment was indeed higher than in the
controls. Likewise, Mia et al. (2014) found increased rates of BNF in a
pot experiment with clover, when grass-based biochar was introduced to
the soil. A variety of biochars from different feedstocks were applied in a
pot experiment with common beans by G€ uere~na et al. (2015), that resulted
in higher root nodulation rates and elevated levels of nitrogen in the plant
biomass fixed from the atmosphere. From the findings above mentioned,
it can be concluded that the selection of a biochar, well suited for the soil
type and plant host, will promote BNF either in root nodules or by associ-
ated free-living bacteria.
However, to date very little is known about the diversity of such diazo-
trophic bacteria that may colonize biochar and rhizosphere under plant
growth promoting conditions. In a recent study, the plant growth promoting
effect of a Miscanthus grass-based biochar was tested in pot experiments with
ryegrass, tomato, and spring barley (Fox, 2013; Fox et al., 2014). While these
studies were primarily investigating the mobilization of sulfur and phos-

phorus, diversity of nifH was also investigated in rhizosphere soil of spring
barley (Corkery, 2014). There, clone libraries from a biochar free control,
1% and 2% biochar addition (w/w) were conducted from nifH amplicons
and then screened via restriction fragment length polymorphism (Poly,
Ranjard, Nazaret, Gourbiere, & Monrozier, 2001). The resulting dominating
operational taxonomical units (OTUs) were translated into protein sequences
(NifH) and aligned with selected sequences from pure cultures obtained from
the Zehr Laboratory nifH database for arb (Heller, Tripp, Turk-Kubo, &
Zehr, 2014; Ludwig et al., 2004). A randomized axelerated maximum likeli-
hood tree (RAxML) was calculated (Stamatakis, 2006) using a base sequence
filter to exclude the truncated sections obtained from the spring barley pot
experiment as can be seen in Fig. 3. Sequences associated to NifH from pro-
teobacteria Xanthobacter (OTU7), Rhizobium (OTU4), Dechlorosoma (OTU3),
Sideroxydans (OTU14), Pelobacter (OTU10 and 21), Desulfovibrio (OTU8), and
Anaeromyxobacter (OTU15) were not preferentially found in any of the three
clone libraries (control, 1% biochar, 2% biochar). In contrast, NifH associated
to Azoarcus sp. BH72 (OTU33) was only found in the control. NifH exclu-
sively found in biochar amendments were associated to Geobacter (OTU19, 1,
and 5) and Pelobacter (OTU 6 and 16). Further sequence comparison of these
OTUs using protein BLAST confirmed the close association to the Geobac-
teraceae with 99% sequence identity to NifH sequenced from various
temperate forest soils (Berthrong et al., 2014). Their potential role in bio-
char-amended rhizosphere soil may well be based on their ability to fix nitro-
gen, utilize a wide range of carbon sources other than the rhizosphere
exudates and their ability to use an extracellular electron transfer (Reguera
et al., 2005). It may be speculated that these bacteria use biochar particles as
electron acceptors as they use artificial electrodes (Bond & Lovley, 2003),
similar to the proposed use of biochar as an electron shuttle for denitrification
(Cayuela et al., 2013). OTU33, found exclusively in the controls, has a 100%
protein sequence identity to NifH sequences found in forest soils (Berthrong
et al., 2014; same sites that identified highly similar sequences to Geobacter,
above). However, its closest cultivate relative (>95% sequence identity) is
Azoarcus sp. BH72, which is an endophytic proteobacterium isolated from
Kallar grass roots (Pakistan) where it exerts nitrogenase activity (Hurek &
Reinhold-Hurek, 2003; Hurek, Reinhold-Hurek, Van Montagu, & Kellen-
berger, 1994). Further research is needed to find out how nitrogen-fixing
bacteria may promote plant growth the most in biochar-amended soils and
who the key players are.
Figure 3 Randomized axelerated maximum likelihood tree (RAxML), calculated using

NifH protein sequences and from pure cultures from Heller et al. (2014) as a scaffold
to add dominating operational taxonomical units (OTUs) (OTUs with only one clone
each were not sequenced); a base sequence filter was used to exclude truncated sec-
tions of the cloned nifH amplicons; sequences for this study were obtained from spring
barley (var. SY Taberna) pot experiment with 1 and 2% biochar amendment (B1, B2) and
biochar free control (C) (pot experimental setup as in Fox et al., 2014); sequences from
this study (GenBank accession KT599445-KT599462) are highlighted in bold with OTU
identifiers (left), followed by a clone identifier in brackets; added table shows the num-
ber of clones obtained per OTU selected for sequencing.

OF PHOSPHORUS
Concentration of orthophosphate in soils is often below 0.01 mg/L
and despite of this, it is the primary source of P for plants (Randriamanantsoa
et al., 2013). The vast majority of soil P is bound organically or inorganically
(Metcleff & Wanner, 1991) with the organic fraction classified into
the phosphate esters, phosphonate, and phosphoric acid anhydrides
(Turner, Cade-Menun, Condron, & Newman, 2005). It is estimated that
only 5e30% of the 15 million tonnes of P fertilizer applied to agricultural

soils are assimilated by plants, with the remainder leached to the ground
water or immobilized by physiochemical reactions and microbial activities
(Trolove, Hedley, Kirk, Bolan, & Loganathan, 2003; Wang, Camps-
Arbestain, Hedley, & Bishop, 2012). As outlined above, pyrolysis is not
believed to release P from the feedstock at temperatures up to 800 C and
thus may be retained in the biochar in inorganic or organic form. As carbon
gets partially volatilized, parts of the organically bound feedstock P may be
turned into inorganic salts of P, while the remainder stays organically bound
(DeLuca, Gundale, MacKenzie, & Jones, 2015). Phytate in manure and
plant-based feedstocks may be converted to inorganic P (Uchimiya &
Hiradate, 2014) and consequently, this extractable phosphate could enter
the soil solution. However, increases in pyrolysis temperature to produce
wood-based chars reduced the amount of extractable phosphate (Gundale &
DeLuca, 2006) and reduced leaching of P in soil columns filled with loamy
soil from Iowa (Laird, Fleming, Wang, Horton, & Karlen, 2010). Likewise,
soil microcosms with biochar and ryegrass also found a reduction in P leach-
ing even though availability of P increased (Borchard et al., 2012). Soil bio-
char amendment can also influence soil P availability due to changes in soil
pH, alterations of enzyme efficiencies, formation of organo-mineral com-
plexes that may increase P solubility, and through induced changes in the
soil microbial community (DeLuca et al., 2015).
5.1 Mobilization of Inorganically Bound Phosphorus

In alkaline soils, inorganic P is principally bound to calcium, while in acidic
soils, insoluble aluminum and iron phosphates are formed (Shen et al.,
2011). Many microbes including the bacterial genera Pseudomonas, Bacillus,
and Rhizobium are able to solubilize particulate inorganic P (Gyaneshwar,
Kumar, Parekh, & Poole, 2002), as seen for a tri-calcium phosphate
(TCP) solubilizing Pseudomonas strain in Fig. 4. This is achieved via exuda-
tion of an organic acid, such as citrate, gluconate, or oxalate, which lowers
the pH of the surrounding soil (Alikhani, Saleh-Rastin, & Antoun, 2006).
Gluconate is thought to be the most common organic acid exuded for inor-
ganic phosphate solubilization in gram-negative bacteria including Pseudo-
monas (Babu-Khan et al., 1995) and Burkholderia (Lin, Huang, Shen, &
Young, 2006). While charcoal has been used as a carrier to inoculate soils
with phosphate-solubilizing bacteria (Gaind & Gaur, 1990; Packialakshmi &
Aliya Riswana, 2014), there is limited knowledge on how biochar depo-
sition affects bacteria that can mobilize inorganically bound P. This is
Figure 4 Growth of a soil isolate associated to Pseudomonas linii on a tri-calcium phos-

phate agar plate (experimental procedure as described in Fox et al., 2014) with visible
zones of clearance around the colonies.
probably due to the ability of breaking the Ca-, Al-, or Fe-P bond with the
exudation of organic acids as described above, which is not genetically
encoded in a specific bacterial pathway as this is the case for the cleaving
of P from an organic molecule.
Increased abundances of phosphate-solubilizing bacteria upon biochar
amendment were indicated indirectly by sequence comparison, where
higher abundances of Burkholderiales and Microbacteriaceae in biochar-
amended soils suggested a potential higher ability of phosphate solubilization
(Anderson et al., 2011, Table 1). Bacteria, isolated from the rhizosphere of
maize from wood chip biochar-amended soil were found to be capable of
TCP solubilization and were associated to Microbacterium, Rahnella,
Enterobacter, Acidovorax, and Pseudomonas (Fox, Cullen, Kwapinski, &
Schmalenberger, 2012). These findings represent an interesting overlap to
the sequence-based analysis by Anderson et al. (2011). More specifically,
significantly increased abundances of TCP solubilizing bacteria were identi-
fied in a pot experiment when biochar amendment resulted in a significant
growth promotion of ryegrass (Fox et al., 2014). These experiments were
successfully repeated with tomato, where 1 and 2% biochar amendment
(A) (B) 100000

B
1000000
B
MPN of phytate ulizing bacteria g-1

Cfu of TCP solubilizing bacteria g-1
C
10000
rhizosphere soil
100000
rhizosphere soil
B
10000 1000
A
1000 100
Control Biochar 1% Biochar 2% Control Biochar 1% Biochar 2%
(C) (D)
100000
100000
B B
MPN of phosphonate ulizing bacteria g-1
B
MPN of sulfonate ulizing bacteria g-1
10000 10000
rhizosphere soil
rhizosphere soil
A
A
1000 1000
100
100 Control Biochar 1% Biochar 2%
Control Biochar 1% Biochar 2%
Figure 5 (A) Colony forming units (cfu) of tri-calcium phosphate (TCP) solubilizing bac-
teria identified to create a zone of clearance around its colony as seen in Figure 4; (B)
Most probable number (MPN) of phytate utilizing bacteria; (C) MPN of phosphonoace-
tate utilizing bacteria; (D) MPN of toluenesulfonate utilizing bacteria (as sole source of P
and S, respectively); from a tomato (var. “Tiny Tim”) pot experiment with Miscanthus-
based biochar (control ¼ biochar free; 1 and 2% biochar w/w; experimental procedure
as described in Fox et al., 2014). Error bars indicate standard error of the mean. Different
letters indicate significant differences (P 0.05, KruskaleWallis, manual posthoc test).
resulted in a significant increase in TCP solubilizing bacteria (Fig. 5A) that

reached nearly two orders of magnitude. This effect was also significantly
(P < 0.05) correlated with shifting bacterial communities as indicated in
Fig. 2 and confirmed by permutation tests (Monte Carlo; 9999 repeats).
5.2 Mobilization of Ester-Bound Phosphorus

Many microbes mobilize organically bound P with phosphatases, hydrolyz-
ing phosphate-ester bonds (Eivazi & Tabatabai, 1977). Of the phosphate
esters, the monoesters are thought to be dominating the organo-P pool in

soils with the diesters contributing up to 10%, for instance as nucleic acids
(Turner et al., 2005). Phosphatase activity has been used as an indicator of
soil activities in the past ( Joner & Jakobsen, 1995; Tabatabai & Bremner,
1969) that also potentially includes the activity of plant roots ( Juma & Taba-
tabai, 1988) and fungal hyphae ( Juma & Tabatabai, 1988; Tarafdar, Bareja,
& Panwar, 2003). Phosphatases can be subdivided in phosphomono-, phos-
phodi-, and phosphotriesterases that are hydrolyzing one to three ester
bonds of the phosphor group. Alternatively, phosphatases can be described
as acid and alkaline phosphomonoesterases, phosphoprotein phosphatases,
phytases, and nucleotidases (Nannipieri, Giagnoni, Landi, & Renella, 2011).
Unfortunately, phosphate ester hydrolysis is not restricted to a single class
of enzymes. Among bacteria and fungi, a multitude of enzyme classes exist,
which are capable of phosphate ester hydrolysis. Just for the hydrolysis of phy-
tate alone, four different types of microbial phytases can be found that include
the histidine acid phosphatases, b-propeller phytases, purple acid phytases, and
the cysteine phytases/protein tyrosine phosphatases (Lim, Yeung, Cheng, &
Hill, 2007). Phytate (inositol phosphate) is the most common phosphate ester
in many soils as it is largely produced by plants for instance in seeds as a storage
of phosphorus (Turner, Paphazy, Haygarth, & McKelvie, 2002). Most
recently, primers were developed to target the bacterial b-propeller phytases
for molecular studies of phytase diversity in soil and rhizosphere (Sanguin,
Wilson, & Kertesz, 2015). Bacterial nonspecific acid phosphatases can be
divided into classes A, B, and C and share some similarities with eukaryotic
acid phosphatases including the ones from plants (Gandhi & Chandra,
2012). In contrast, alkaline phosphatases appear to be restricted to bacteria,
fungi and members of the fauna and specific primers for the bacterial alkaline
phosphatase are now available to generate DGGE fingerprints (Sakurai,
Wasaki, Tomizawa, Shinano, & Osaki, 2008; Tarafdar & Claassen, 1988).
Luo, Benner, Long, and Hu (2009) investigated marine metagenomic data-
bases to study three prokaryotic alkaline phosphatase gene families PhoA,
PhoD, and PhoX. More recently, the diversity of phoD has been analyzed
in pastures using next generation sequencing (Tan et al., 2013).
While there has been progress made in our understanding about how
microbes can hydrolyze phosphate esters, very little is known about this
function in biochar-amended soils directly. The bulk of research in phytase
and phosphatase activity has been carried out through measurement of enzy-
matic activities in biochar-amended soils. A majority of studies found
evidence of increased phosphatase activity upon biochar amendment
(Table 1). This was reported either as neutral phosphatase, alkaline, or acid
phosphatase activity in combination with compost ( Jindo et al., 2012),
paddy soil (Chen et al., 2013; Cui et al., 2013; Yang, Yan, & Ding,
2013), or various other field studies (Du et al., 2014; Ventura et al., 2014)
and laboratory (Yoo & Kang, 2012) or pot (Masto, Kumar, et al., 2013)
experiments. No changes in (acid) phosphatase activity were found in
Cu-contaminated soil (Mackie, Marhan, Ditterich, Schmidt, & Kandeler,
2015) and a pot experiment with sewage sludge biochar (Paz-Ferreiro,
Gasco, Gutiérrez, & Méndez, 2012). A pot experiment with biochar and
cucumber showed reduced phosphatase activity (Zou et al., 2015). Interest-
ingly, 31P NMR showed that feedstock phytate can be transformed to inor-
ganic P during pyrolysis (Uchimiya & Hiradate, 2014), thus microbial
phosphatase activity would be reduced in environments with high levels
of inorganic P. Likewise, alkaline and acidic phosphatase activity can also
change differently. Chen and colleagues studied shifts of bacterial and fungal
communities upon biochar addition to a paddy rice field, where no signif-
icant changes in the acidic phosphatase activity was measured, but alkaline
phosphatase activity was significantly increased. At the same time, an in-
crease in bacterial abundance alongside a decrease of fungal abundance
was determined as well as community shifts of both bacteria and fungi
(Chen et al., 2013). These changes in alkaline phosphatase activity could
be a result of higher bacterial abundance of some particular phylogenetic
groups as indicated by T-RFLP analysis that included the families Burkhol-
deriaceae and Anaerolineaceae and the Nitrospira class. Decreases in acid
phosphatase activity were also reported alongside the increases in alkaline
phosphatase activity with biochar of high inorganic P levels made from
manure ( Jin et al., 2015) and a field trial with biochar and fly ash (Masto,
Ansari, George, Selvi, & Ram, 2013).
Abundances of bacteria that utilize phytate as sole source of phosphorus
were significantly increased in pot experiments with ryegrass (Fox et al.,
2014) and tomato (Fig. 5B) under biochar amendment. However, increased
abundances in the tomato rhizosphere were less pronounced than for the
TCP-utilizing bacteria and shifts in the bacterial community structure as
observed for the tomato rhizosphere community were not significantly
affected by the increase in phytate-utilizing bacteria (Fig. 2). To date, abun-
dances of bacterial phosphatase genes and gene expression are not yet deter-
mined under a biochar soil amendment scenario, thus further research is
needed to study the bacterial ability to utilize phosphate esters directly under
the influences of biochar.
5.3 Carbon-Bonded Phosphorus

Beyond the world of phosphate esters that often dominate the organo-phos-
phorus pool in soil, there are other organically bound forms of phosphorus
that are directly bound to carbon. One carbon-bonded form belongs to the
phosphonates (Metcleff & Wanner, 1991). Many gram-negative bacteria
seem to be capable of using phosphonates as a source of P under limiting
conditions (Kamat, Williams, & Raushel, 2011). The CeP phosphonate
bond is extremely stable, often resisting denaturation through heat, chemical
hydrolysis or photolysis, but many bacteria are capable of cleaving the CeP
bond (Ternan, McGrath, McMullan, & Quinn, 1998). Phosphonolipids are
commonly found in lower organisms. These and other type phosphonates
such as in glycoproteins, glycolipids, some antibiotics and man-made herbi-
cides such as glyphosate are degraded by many soil bacteria (Ternan et al.,
1998) and some soil-borne fungi (Krzysko-qupicka & Orlik, 1997). Several
metabolic microbial pathways are described in the literature for phosphonate
degradation including the C-P lyase, the phosphonatase, the phosphonoace-
tate hydrolase, and the phosphonopyruvate hydrolase (Kononova &
Nesmeyanova, 2002; Ternan et al., 1998). For both the phosphonatase
and the C-P lyase pathway sequence alignments of phnX and phnG-M,
respectively, have identified phylogenetic relationships among the utilizing
bacteria of these pathways and identified evidence of horizontal gene transfer
(Huang, Su, & Xu, 2005). The use of these genes as markers for investigating
bacterial phosphonate diversity and abundance is scarce so far, with phnJ
receiving some initial attention (Fox et al., 2014; Karl, 2007). The phyloge-
netic diversity of PhnJ under a biochar soil amendment scenario with
ryegrass revealed Rhizobiales, Rhodospirillales, and Burkholderiales to be
the dominating orders that appeared to be in possession of the C-P lyase
pathway (Fox et al., 2014). Abundance of bacteria capable of utilizing phos-
phonoacetic acid as sole source of P increased significantly in pot experi-
ments with ryegrass (Fox et al., 2014) and tomato (Fig. 5C) under biochar
amendment. As for the phytate-utilizing bacterial abundance above, shifts
in the bacterial community structure of the tomato rhizosphere were not
significantly affected by the increase in phosphonate utilizing bacteria
(Fig. 2). Again, further research is needed to study the bacterial ability to uti-
lize phosphonates directly under the influences of biochar, as current studies
are more commonly focusing on the degradation of xenobiotic phospho-
nates (Panas, Ternan, Dooley, & McMullan, 2006) or phosphonates in
the marine environment (Villarreal-Chiu, Quinn, & McGrath, 2012).

OF SULFUR
Sulfur (S) is an essential macronutrient required for growth of plants,
microbes, and the fauna alike. A significant amount of S was released into the
atmosphere since the industrial revolution due to the burning of fossil fuels.
Indeed, only a few decades ago, plants would receive the majority of their S
from atmospheric depositions in the industrialized regions of the world (De
Kok et al., 1997; McGrath & Zhao, 1995). Over the last 20e30 years, a
reduction in atmospheric S levels through cleaner fuels as well as stricter
controls on fossil fuel combustion has resulted in greatly reduced
atmospheric S inputs to soils and plants (Fowler, Smith, Muller, Hayman, &
Vincent, 2005). As a result, S limitations during plant growth can now be
regularly observed across Europe and other industrialized countries (Blair,
2002; McGrath, Zhao, & Blake-Kalff, 2003). S present in soil is approxi-
mately 95% organically bound, largely in the forms of sulfate esters and
sulfonates (Autry & Fitzgerald, 1990; Kertesz & Mirleau, 2004) that are
not directly available to plants, which rely upon soil microbes for mobiliza-
tion of S (Kertesz et al., 2007). Many bacteria and fungi in soil are capable of
hydrolyzing sulfate ester bonds to release S ready for plant uptake (Klose,
Moore, & Tabatabai, 1999). The capacity to cleave the ester bond with a
sulfatase has been used as a measure of soil health in the past and is thought
to be influenced by various factors including soil temperature, moisture con-
tent, vegetative cover, and crop rotation (Tabatabai & Bremner, 1970). In
contrast, a bacterial multicomponent monooxygenase enzyme complex is
needed to mobilize S from a large variety of sulfonates, the dominating
organically bound S source (organo-S) in many soils (Kertesz & Mirleau,
2004; Vermeij, Wietek, Kahnert, W€ uest, & Kertesz, 1999). Mineralization
of organo-S may not always be carried out by microbes to access S, but
to utilize the organic molecule as a carbon or energy source, using alternative
pathways (Cook, Laue, & Junker, 1998), or in some cases as for the genus
Rhodococcus, sulfonates can serve both purposes (Schmalenberger, Hodge,
Hawkesford, & Kertesz, 2009). For sulfonate utilization, however, the abun-
dances of bacteria capable of utilizing toluenesulfonate as a source of S was
several orders of magnitude higher than the bacteria that were capable of
utilizing it as a source of carbon (Schmalenberger et al., 2008).
Pyrolysis may or may not result in a loss of S to the atmosphere (Di Blasi
et al., 1999; Lang et al., 2005) and this may well depend on the pyrolysis
conditions as well as the feedstock used. Consequently, biochar soil
deposition may result in an increased S pool that is available to the soil

microbes. However, improved soil conditions may also lead to a more active
mobilization of S from soil directly. Cheah, Malone, and Feik (2014) studied
S K-edge XANES spectra of various biochars and found that inorganic S in
biochar is increasingly transformed to organo-S with increase of the pyrolysis
temperature. Thus, mobilization of such newly formed organo-S would be
again dependent on microbial activity for mobilization. Apart from the
mobilization of S for uptake and incorporation into biomass (assimilation),
sulfur can also be reduced dissimilatorily in order to use sulfate or elemental
sulfur as an electron acceptor in the form of anaerobic respiration (Canfield &
Raiswell, 1999). This prokaryotic form of respiration takes place in soils,
when oxygen and other energetically more favorable electron acceptors
are exhausted (Postgate, 1984). As the amount of sulfate in biochar may
be reduced to levels below that of the original feedstock, use of biochar-
derived sulfate as an electron acceptor may be less common. Nevertheless,
as outlined before, biochar may actually also serve as an electron shuttle as
described in the role of biochar in the dissmilatory reduction of nitrate
(Cayuela et al., 2013). Consequently, biochar deposition to anaerobic soils
may have an impact on dissimilatory sulfate reduction rates after all, but
this needs further attention in the future as there is currently no scientific
evidence of it in the public domain.
6.1 Mobilization of Ester-Bound Sulfur

Mineralization of sulfate esters is facilitated by sulfatases of the esterase class
(Deng & Tabatabai, 1997). Arylsulfatases split the OeS bond of aromatic
(aryl) sulfate esters, while alkylsulfatases hydrolyze the CeO bond of
aliphatic sulfate esters (Kertesz, 1999). Both of these reactions release
inorganic S into the environment and are common in the rhizosphere where
there is an elevated demand for sulfate to be taken up by plant roots (Kertesz
et al., 2007; Kertesz & Mirleau, 2004). In Pseudomonas aeruginosa,
arylsulfatase activity is repressed in the presence of sulfate and induced during
S starvation, while in a Streptomyces strain, sulfatase activity was also detected
independently of the substrate presence (Cregut, Piutti, Slezack-
Deschaumes, & Benizri, 2013; Hummerjohann, Laudenbach, Rétey,
Leisinger, & Kertesz, 2000). Sulfateeester hydrolysis capability has been
reported for a range of bacterial genera including Pseudomonas, Klebsiella,
Salmonella, Enterobacter, Serratia, and Comamonas (Hummerjohann et al.,
2000). Sulfateeester hydrolysis is not exclusively carried out by bacteria as
for instance, sulfatase activity has been reported for filamentous fungi as
well (Marzluf, 1997), but this ability was never closely examined in relation
to utilizing sulfate esters from soils (Kertesz et al., 2007). As for the utilization
of P, most of the current knowledge gathered from biochar deposition and
utilization of sulfate esters comes from the measurements of the extracellular
sulfatase activity (Table 1), using nitrophenolsulfate as a substrate (Tabatabai &
Bremner, 1970). In a pot experiment with biochar obtained from sewage
sludge, sulfatase activity was slightly increased over the control treatments
but significance was not achieved (Paz-Ferreiro et al., 2012). No significant
changes in sulfatase activity were also reported for another laboratory-based
incubation experiment using swine manure and barley stover-based biochars
(Yoo & Kang, 2012). Likewise, Chintala et al. (2014) measured sulfatase
activity in two soil types under amendment with biochars from corn stover,
wood chips, and switchgrass in a laboratory experiment and found no
significant changes. In contrast, in a pot experiment without plant cover
with poultry litter-based biochar, substantially higher sulfatase activity was
identified (Paz-Ferreiro, Fu, Méndez, & Gasc o, 2015). An increase in sulfa-
tase activity was also reported from an apple tree orchard under wood-based
biochar amendment (Ventura et al., 2014).
Enzymatic assays as reported, above all, share the limitation that they
only measure extracellular activity, while intracellular bacterial enzymatic
activity will be overlooked (Chintala et al., 2014). Furthermore, nitrophe-
nylsulfate could be potentially absorbed to the biochar surface and thus alter
the outcome of the assay (Fox et al., 2014). The addition of biochar directly
to the assay may be needed as a control to exclude such a potential bias. To
date, very little research has been conducted on a molecular level to study
diversity or expression of sulfatase genes among microbes. Houlden and
Kertesz (oral communication) have recently developed a set of primers
targeting the diversity of the arylsulfatase gene atsA. However, no such
applications have been reported for studies of biochar soil amendments,
thus there is a need for future studies in this particular direction. This is
particularly the case for studies looking directly into plant growth promo-
tion, as to date most of the few studies that have looked into sulfate-ester
utilization in biochar-amended soils were soil incubation studies without
vegetation covers, conducted in the laboratory.
6.2 Mobilization of Sulfonate-Bound Sulfur

Aliphatic and aromatic sulfonates represent the most abundant forms of
organo-S in many soils (Autry & Fitzgerald, 1990; Zhao, Lehmann,
Solomon, Fox, & McGrath, 2006). The ability to mobilize S from aliphatic
C2-sulfonates appears to be widespread among soil bacteria (King & Quinn,

1997). However, aromatic sulfonates may be of greater importance for S
nutrition, since the ability to mobilize arylsulfonates was directly linked to
growth promotion of tomato and Arabidopsis (Kertesz et al., 2007; Kertesz &
Mirleau, 2004). Release of inorganic S from aryl- and alkylsulfonates is best
studied in Pseudomonas putida S-313, where it is catalyzed by a FMNH2-
dependent monooxygenase enzyme complex, encoded in the ssu gene
cluster (Kertesz, 1999). For aromatic desulfonation, the asf gene cluster is
additionally required. A LysR-type regulator activates the system when sul-
fate is limited (Vermeij et al., 1999). Mutagenesis of asfA in P. putida S-313
resulted in the loss of the ability to specifically utilize arylsulfonates as source
of S and with it lost the ability to promote the growth of tomato plants
(Kertesz & Mirleau, 2004). So far, the delicate nature of the monooxygenase
enzyme complex has prevented the establishment of an enzymatic assay.
Instead, the use of genetic markers as well as cultivation-dependent studies
progressed the state of the art. Various recent studies on the bacterial phylog-
eny of arylsulfonate-mobilizing bacteria have expanded the diversity of aryl-
sulfonate utilizing soil, rhizosphere, and mycorrhizosphere bacteria to the
Beta-Proteobacteria: Burkholderia, Acidovorax, Cupriavidus, Hydrogenophaga,
Polaromonas, and Variovorax; Gamma-Proteobacteria: Stenotrophomonas; and
Actinobacteria: Rhodococcus and Williamsia (Gahan & Schmalenberger,
2014, 2015).
Abundances of cultivable arylsulfonate utilizing bacteria under biochar
soil amendment have been studied so far in the rhizospheres of maize,
ryegrass, spring barley (Fox, 2013; Fox et al., 2012, 2014), and tomato
(Fig. 5D). From the rhizosphere of maize under biochar amendment, an
unprecedented diversity of isolates capable of growing with toluenesulfonate
as the sole source (desulfonating) of S was retrieved. These included isolates
associated to the Actinobacteria: Arthrobacter, Microbacterium, Rhodococcus;
Alpha-Proteobacteria: Agrobacterium, Bosea, Rhizobium; Beta-Proteobacteria:
Acidovorax, Duganella, Herminiimonas, Hydrogenophaga, Polaromonas; and
Gamma-Proteobacteria: Enterobacter, Erwinia, Pseudomonas, Pseudoxanthomo-
nas, Rahnella, Stenotrophomonas, and Yersinia (Fox et al., 2012). In the ryegrass
rhizosphere under biochar amendment, desulfonating isolates identified
were associated to Acidovorax, Arthrobacter, Pseudomonas, and Stenotrophomo-
nas. With the exception of the maize rhizosphere, where no significant plant
growth promotion effect was identified upon biochar amendment, sig-
nificantly higher abundances of bacteria were identified (alongside plant
growth promotion), capable of utilizing toluenesulfonate as sole source of
S. Significant correlations were made between these abundances and shifts in

the bacterial community structure of the pot experiments with ryegrass (Fox
et al., 2014) and tomato (Fig. 2).
Clone libraries of molecular marker asfA for arylsulfonate desulfurization
from the ryegrass experiment revealed a broad phylogenetic diversity of aryl-
sulfonate desulfurizing bacteria in biochar-amended soils (Fox et al., 2014).
The dominating clone from the biochar treatments was not closely related
to any identified bacterial genus but most likely belonged to the Beta-
Proteobacteria (Fox et al., 2014). Further research is needed to establish
the role of sulfonate mobilization in biochar-amended soils more accurately
and to get a clearer picture of the importance of this process, especially under
field conditions. This will have a significant impact on fertilization regimes
alongside biochar deposition in the future or management where inorganic
fertilizer application is not desired such as organic farming.
7. BIOCHAR AND BACTERIAL CYCLING OF OTHER

NUTRIENTS
Further nutrients are needed for plant and soil microbiota growth
albeit commonly in smaller quantities than N, P, and S. Plants do require
adequate amounts of potassium (K), magnesium (Mg), calcium (Ca), iron
(Fe), and further metals as trace elements (Fageria, Baligar, & Jones, 2010).
Unlike P and S, the amount of K, Mg, Ca, and most other metals in the
earth’s crust are much higher than in the plant biomass (Brantley et al.,
2011). Furthermore, they are not major constituents of organic material
and are primarily needed for osmotic and ion balance roles, enzyme confor-
mation and catalysis, and as structural chelates (Fageria et al., 2010). Concen-
trations of K, Mg, and Ca in biochar are dependent on the feedstock,
pyrolysis type, and temperature. Due to high levels of volatilization of other
elements to the atmosphere, concentrations of K, Mg, and Ca appear to
increase with pyrolysis temperature (Ippolito, Spokas, Novak, Lentz, &
Cantrell, 2015) and may be subsequently solubilized by soil bacteria. Indeed,
bacteria such as Bacillus were reported to improve K uptake by plants when
mineral forms of K were added to soil (Sheng, 2005). The mechanism of K
release is believed to be related to bacterial weathering activity, where
organic acid and/or proton release is responsible for the nutrient release as
this is also the case for Mg and Ca (Uroz et al., 2011). The latter nutrient
may be actually more abundant in silicate rocks and particularly in limestone
than actually desired by the microbiota and the plants. Luckily, some of the
calcium is immobilized through secondary mineral formation in the form of

calcium oxalate, for instance via ectomycorrhizal exudation of oxalate as the
main mineral/rock weathering agent (Schmalenberger et al., 2015). As out-
lined above, P and S are much lower in abundance in the earth’s rocks (crust)
than Mg, K, and Ca and thus a hypothesis would be that most of the release
of Ca, K, and Mg could be related to mineral weathering of bacteria and
fungi in order to access primarily mineral P (eg, apatite). This may also be
the case for some biochars, where acid release by microbes such as bacteria
is related to the mobilization of P and other cations are released as a by-
product such as Ca in the case of apatite. Indeed, bacterial TCP solubiliza-
tion was enhanced in biochar-amended soils (Fox et al., 2014, Fig. 5A).
Further investigations are needed in this area to provide evidence for or
against this hypothesis.
8. CONCLUSIONS AND OUTLOOK

Recent investigations have revealed new details about the role of
bacteria in nutrient mobilization in biochar-amended soils.
Studies targeting the role of bacteria in biochar-amended soils over the
last 3e4 years have created a baseline for our understanding, what the
role of bacteria can be under soil biochar amendment. Depending on soil
type, climate, and biochar used, nitrification can be enhanced or inhibited.
In most cases, release of nitrous oxide gas is reduced due to higher bacterial
nitrous oxide reductase activity. BNF can be enhanced in biochar-amended
soils and with it can change the diversity of the di-nitrogen-fixing bacterial
population. Microbial alkaline phosphatase is often enhanced upon biochar
soil amendment resulting in putative higher phosphorus availability for
plants and microbes. This effect is also likely the case for the mobilization
of particulate inorganic phosphate and carbon-bonded P. Bacterial mobili-
zation of S from esters and sulfonates is also likely to be enhanced as revealed
through arylsulfatase measurements, cultivation, and genetic marker analysis.
Particularly, further research is needed in BNF, P, and S mobilization in
order to gain a more complete picture. This is of critical importance should
biochar become a major soil amendment to establish a more sustainable from
of agriculture.
Nowadays, a range of cutting-edge techniques are at our disposal to
answer some of these remaining open questions. The use of (stable) isotopes
could be a significant step to better our understanding, whether biochar
serves as a major source of P and S or if indeed biochar is primarily

improving the bacterial mobilization of bonded P and S from the soil.
Techniques to identify the S species in biochar material includes
synchrotron-based S K-edge XANES. Previous studies were able to separate
the potential feedstock S sources in wood as sulfides, thiols, sulfonates, and
sulfate esters (Schmalenberger, Wolfgang, Ojeda, & Noll, 2011). S K-edge
XANES spectra of corn stover and oak biochar showed that the majority of
S in biochar can be associated to reduced thiols (2472 eV) and oxidized S, eg,
sulfate ester (2482 eV) and intermediate S sulfonates (2480 eV) (Cheah et al.,
2014); the latter was only obvious in one of the oak biochar spectra though.
Likewise, 31P NMR has been used to identify P species in biochar samples
(Uchimiya & Hiradate, 2014).
The application of massive parallel sequencing or next generation
sequencing over recent years has created a wealth of information on bacterial
diversities in environments including ones under biochar amendment
(Anderson et al., 2011; Xu et al., 2014). However, 16S amplicon sequencing
is providing only indirect information on bacterial functions in a best-case
scenario. Thus, further advancements are needed in this sector to reveal
processes and bacterial functions in these environments. Quantification of
gene abundances and expression related to the nitrogen cycle (Table 1)
have been conducted already as indicated in the sections above and this needs
to be investigated further across other microbial functions. Meta-
transcriptomics and meta-proteomics work, possibly linked to stable isotope
tracing is another promising line of research to progress our knowledge.
Reductions in costs of whole genome sequencing will create a renewed desire
to isolate bacteria again in order to gain in-depth insight into the metabolic
capabilities of bacteria in biochar-amended soils. Finally, soil bacteria are
not fulfilling functions in soils, rhizospheres, and on biochar particles in isola-
tion. Thus, the study of saprophytic and mycorrhizal fungal hyphae is essential
as well, in particular when some of the key functional bacteria may be located
on the hyphoplane of mycorrhizal hyphae as observed recently for sulfonate-
utilizing bacteria (Gahan & Schmalenberger, 2015).
ACKNOWLEDGMENTS
This research was co-funded by FP7 People (CIG No. 293429) and the Department of Life
Sciences. We are grateful to Maire Corkery and Ruth Cullen for their contributions as proj-
ect students to this study, Witold Kwapinski for his support in the biochar manufacture, Paula
Olsthoorn for her support in the SEM capture, and JJ Leahy for critically reading the
manuscript.
REFERENCES
Al-Wabel, M. I., Al-Omran, A., El-Naggar, A. H., Nadeem, M., & Usman, A. R. A. (2013).
Pyrolysis temperature induced changes in characteristics and chemical composition of
biochar produced from conocarpus wastes. Bioresource Technology, 131, 374e379.
Ali, M. A., Hoque, M. A., & Kim, P. J. (2013). Mitigating global warming potentials of
methane and nitrous oxide gases from rice paddies under different irrigation regimes.
Ambio, 42, 357e368.
Alikhani, H. A., Saleh-Rastin, N., & Antoun, H. (2006). Phosphate solubilisation activity of
rhizobia native to Iranian soils. Plant and Soil, 287, 35e41.
Amonette, J. E., & Joseph, S. (2009). Characteristics of biochar: micro-chemical properties.
In J. Lehmann, & S. Joseph (Eds.), Biochar for environmental management: Science and tech-
nology (pp. 33e52). London, UK: Earthscan.
Anderson, C. R., Condron, L. M., Clough, T. J., Fiers, M., Stewart, A., Hill, R. A., et al.
(2011). Biochar induced soil microbial community change: implications
for biogeochemical cycling of carbon, nitrogen and phosphorus. Pedobiologia, 54,
309e320.
Anderson, C. R., Hamonts, K., Clough, T. J., & Condron, L. M. (2014). Biochar does not
affect soil N-transformations or microbial community structure under ruminant urine
patches but does alter relative proportions of nitrogen cycling bacteria. Agriculture Ecosys-
tems and Environment, 191, 63e72.
Antal, M. J., & Grønli, M. (2003). The art, science, and technology of charcoal production.
Industrial and Engineering Chemistry Research, 42, 1619e1640.
Atkinson, C. J., Fitzgerald, J. D., & Hipps, N. A. (2010). Potential mechanisms for achieving
agricultural benefits from biochar application to temperate soils: a review. Plant and Soil,
337, 1e18.
Autry, A. R., & Fitzgerald, J. W. (1990). Sulfonate S e a major form of forest soil organic
sulfur. Biology and Fertility of Soils, 10, 50e56.
Babu-Khan, S., Tiong Chia, Y., Martin, W. L., Duron, M. R., Rogers, R. D., &
Goldstein, A. H. (1995). Cloning of a mineral phosphate-solubilizing gene from Pseudo-
monas cepacia. Applied and Environmental Microbiology, 61, 972e978.
Bagreev, A., Bandosz, T. J., & Locke, D. C. (2001). Pore structure and surface chemistry
of adsorbents obtained by pyrolysis of sewage sludge-derived fertilizer. Carbon, 39,
1971e1979.
Baldani, V. L. D., Alvarez, M. A. D. B., Baldani, J. I., & D€ obereiner, J. (1986). Establishment
of inoculated Azospirillum spp. in the rhizosphere and in roots of field grown wheat and
sorghum. Plant and Soil, 90, 35e46.
Ball, P. N., MacKenzie, M. D., DeLuca, T. H., & Holben, W. E. (2010). Wildfire and char-
coal enhance nitrification and ammonium-oxidizing bacterial abundance in dry montane
forest soils. Journal of Environmental Quality, 39, 1243e1253.
Beck, D. P. (1991). Suitability of charcoal-amended mineral soil as carrier for Rhizobium
inoculants. Soil Biology and Biochemistry, 23, 41e44.
Berglund, L. M., DeLuca, T. H., & Zackrisson, O. (2004). Activated carbon amendments
to soil alters nitrification rates in Scots pine forests. Soil Biology and Biochemistry, 36,
2067e2073.
Berthrong, S. T., Yeager, C. M., Gallegos-Graves, L., Steven, B., Eichorst, S. A.,
Jackson, R. B., et al. (2014). Nitrogen fertilization has a stronger effect on soil
nitrogen-fixing bacterial communities than elevated atmospheric CO2. Applied and
Environmental Microbiology, 80, 3103e3112.
Blair, G. J. (2002). Sulphur fertilisers: A global perspective. International Fertiliser Society.
Bond, D. R., & Lovley, D. R. (2003). Electricity production by Geobacter sulfurreducens
attached to electrodes. Applied and Environmental Microbiology, 69, 1548e1555.
Borchard, N., Wolf, A., Laabs, V., Aeckersberg, R., Scherer, H. W., Moeller, A., et al.
(2012). Physical activation of biochar and its meaning for soil fertility and nutrient leach-
ing e a greenhouse experiment. Soil Use and Management, 28, 177e184.
Brantley, S. L., Megonigal, J. P., Scatena, F. N., Balogh-Brunstad, Z., Barnes, R. T.,
Bruns, M. A., et al. (2011). Twelve testable hypotheses on the geobiology of
weathering. Geobiology, 9, 140e165.
Canfield, D. E., & Raiswell, R. (1999). The evolution of the sulfur cycle. American Journal of
Science, 299, 697e723.
Case, S. D. C., McNamara, N. P., Reay, D. S., Stott, A. W., Grant, H. K., & Whitaker, J.
(2015). Biochar suppresses N2O emissions while maintaining N availability in a sandy
loam soil. Soil Biology and Biochemistry, 81, 178e185.
Castaldi, S., Riondino, M., Baronti, S., Esposito, F. R., Marzaioli, R., Rutigliano, F. A., et al.
(2011). Impact of biochar application to a Mediterranean wheat crop on soil microbial
activity and greenhouse gas fluxes. Chemosphere, 85, 1464e1471.
Cayuela, M. L., Sanchez-Monedero, M. A., Roig, A., Hanley, K., Enders, A., & Lehmann, J.
(2013). Biochar and denitrification in soils: when, how much and why does biochar
reduce N2O emissions? Scientific Reports, 3, 1732.
Cheah, S., Malone, S. C., & Feik, C. J. (2014). Speciation of sulfur in biochar produced from
pyrolysis and gasification of oak and corn stover. Environmental Science and Technology, 48,
8474e8480.
Chen, B., Yuan, M., & Qian, L. (2012). Enhanced bioremediation of PAH-contaminated
soil by immobilized bacteria with plant residue and biochar as carriers. Journal of Soils
and Sediments, 12, 1350e1359.
Chen, J., Liu, X., Li, L., Zheng, J., Qu, J., Zheng, J., et al. (2015). Consistent increase in
abundance and diversity but variable change in community composition of bacteria in
topsoil of rice paddy under short term biochar treatment across three sites from South
China. Applied Soil Ecology, 91, 68e79.
Chen, J., Liu, X., Zheng, J., Zhang, B., Lu, H., Chi, Z., et al. (2013). Biochar soil amend-
ment increased bacterial but decreased fungal gene abundance with shifts in community
structure in a slightly acid rice paddy from Southwest China. Applied Soil Ecology, 71,
33e44.
Cheng, Y., Cai, Z., Chang, S. X., Wang, J., & Zhang, J. (2012). Wheat straw and its biochar
have contrasting effects on inorganic N retention and N2O production in a cultivated
Black Chernozem. Biology and Fertility of Soils, 48, 941e946.
Chintala, R., Owen, R. K., Schumacher, T. E., Spokas, K. A., McDonald, L. M., Kumar, S.,
et al. (2015). Denitrification kinetics in biomass- and biochar-amended soils of different
landscape positions. Environmental Science and Pollution Research, 22, 5152e5163.
Chintala, R., Schumacher, T. E., Kumar, S., Malo, D. D., Rice, J. A., Bleakley, B., et al.
(2014). Molecular characterization of biochars and their influence on microbiological
properties of soil. Journal of Hazardous Materials, 279, 244e256.
Ciough, T. J., Bertram, J. E., Ray, J. L., Condron, L. M., O’Callaghan, M., Sherlock, R. R.,
et al. (2010). Unweathered wood biochar impact on nitrous oxide emissions from a
bovine-urine-amended pasture soil. Soil Science Society of America Journal, 74, 852e860.
Cook, A. M., Laue, H., & Junker, F. (1998). Microbial desulfonation. FEMS Microbiology
Reviews, 22, 399e419.
Corkery, M. (2014). Cloning and analysis of the nifH gene, associated with plant growth promotion of
spring barley, Hordeum vulgare, in a biochar incubation experiment. Limerick: BSc, Life
Sciences, University of Limerick.
Cregut, M., Piutti, S., Slezack-Deschaumes, S., & Benizri, E. (2013). Compartmentalization
and regulation of arylsulfatase activities in Streptomyces sp., Microbacterium sp. and Rhodo-
coccus sp. soil isolates in response to inorganic sulfate limitation. Microbiological Research,
168, 12e21.
Cui, L., Yan, J., Yang, Y., Li, L., Quan, G., Ding, C., et al. (2013). Influence of biochar on
microbial activities of heavy metals contaminated paddy fields. BioResources, 8, 5536e5548.
De Boer, W., & Kowalchuk, G. A. (2001). Nitrification in acid soils: Micro-organisms and
mechanisms. Soil Biology and Biochemistry, 33, 853e866.
De Kok, L. J., Elisabeth, C., Stuiver, E., Rubinigg, M., Westerman, S., & Grill, D. (1997).
Impact of atmospheric sulfur deposition on sulfur metabolism in plants: H2S as sulfur
source for sulfur deprived Brassica oleracea L. Botanica Acta, 110, 411e419.
DeLuca, T. H., Gundale, M. J., MacKenzie, D. M., & Jones, D. L. (2015). Biochar effects on
soil nutrient transformation. In J. Lehmann, & S. Joseph (Eds.), Biochar for environmental
management: Science, technology and implementation (pp. 421e454). London, UK:
Earthscan.
DeLuca, T. H., MacKenzie, M. D., Gundale, M. J., & Holben, W. E. (2006). Wildfire-
produced charcoal directly influences nitrogen cycling in Ponderosa pine forests. Soil Sci-
ence Society of America Journal, 70, 448e453.
Deng, S., & Tabatabai, M. (1997). Effect of tillage and residue management on enzyme
activities in soils: III. Phosphatases and arylsulfatase. Biology and Fertility of Soils, 24,
141e146.
Devonald, V. G. (1982). The effect of wood charcoal on the growth and nodulation of gar-
den peas in pot culture. Plant and Soil, 66, 125e127.
Di, H. J., Cameron, K. C., Shen, J. P., Winefield, C. S., Ocallaghan, M., Bowatte, S., et al.
(2009). Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils.
Nature Geoscience, 2, 621e624.
Di Blasi, C., Signorelli, G., Di Russo, C., & Rea, G. (1999). Product distribution from
pyrolysis of wood and agricultural residues. Industrial and Engineering Chemistry Research,
38, 2216e2224.
Dicke, C., Andert, J., Ammon, C., Kern, J., Meyer-Aurich, A., & Kaupenjohann, M. (2015).
Effects of different biochars and digestate on N2O fluxes under field conditions. Science of
the Total Environment, 524e525, 310e318.
Downie, A., Crosky, A., & Munroe, P. (2009). Physical properties of biochar. In J. Lehmann,
& S. Joseph (Eds.), Biochar for environmental management: Science, technology and implementa-
tion (pp. 13e32). London, UK: Earthscan.
Du, Z., Wang, Y., Huang, J., Lu, N., Liu, X., Lou, Y., et al. (2014). Consecutive biochar
application alters soil enzyme activities in the winter wheat-growing season. Soil Science,
179, 75e83.
Ducey, T. F., Ippolito, J. A., Cantrell, K. B., Novak, J. M., & Lentz, R. D. (2013). Addition
of activated switchgrass biochar to an aridic subsoil increases microbial nitrogen cycling
gene abundances. Applied Soil Ecology, 65, 65e72.
Duclos, J. L., & Fortin, J. A. (1983). Effect of glucose and active charcoal on in-vitro synthesis
of ericoid mycorrhiza with Vaccinium spp. New Phytologist, 94, 95e102.
Eivazi, F., & Tabatabai, M. A. (1977). Phosphatases in soils. Soil Biology and Biochemistry, 9,
167e172.
Fageria, N. K., Baligar, V. C., & Jones, C. A. (2010). Growth and mineral nutrition of field crops.
Boca Raton, FL: CRC Press.
Fowler, D., Smith, R., Muller, J., Hayman, G., & Vincent, K. (2005). Changes in the atmo-
spheric deposition of acidifying compounds in the UK between 1986 and 2001. Environ-
mental Pollution, 137, 15e25.
Fox, A. (2013). Microbial mobilization of sulphur and phosphorus in biochar amended soils. Limerick:
MSc, Life Scienes, University of Limerick.
Fox, A., Cullen, R., Kwapinski, W., & Schmalenberger, A. (2012). Bacterial mobilization of
sulfur and phosphorous in biochar amended soils (pp. 549e550). Copenhagen: ISME
Proceedings.
Fox, A., Kwapinski, W., Griffiths, B. S., & Schmalenberger, A. (2014). The role of sulfur and
phosphorus mobilizing bacteria in biochar induced growth promotion of Lolium perenne.
FEMS Microbiology Ecology, 90, 78e91.
Fuhrmann, M., & Hennecke, H. (1984). Rhizobium japonicum nitrogenase Fe protein gene
(nifH). Journal of Bacteriology, 158, 1005e1011.
Gahan, J., & Schmalenberger, A. (2014). The role of bacteria and mycorrhiza in plant sulfur
supply. Frontiers in Plant Science, 5, 723.
Gahan, J., & Schmalenberger, A. (2015). Arbuscular mycorrhizal hyphae in grassland select
for a diverse and abundant hyphospheric bacterial community involved in sulfonate
desulfurization. Applied Soil Ecology, 89, 113e121.
Gaind, S., & Gaur, A. C. (1990). Shelf life of phosphate-solubilizing inoculants as influenced
by type of carrier, high temperature, and low moisture. Canadian Journal of Microbiology,
36, 846e849.
Gandhi, N. U., & Chandra, S. B. (2012). A comparative analysis of three classes of bacterial
non-specific acid phosphatases and archaela phosphoesterases: evolutionary perspective.
Acta Informatica Medica, 20, 167e173.
Gehring, C. (2003). The role of biological nitrogen fixation in secondary and primary forests in Central
Amazonia. Goettingen, Germany: Cuvillier Verlag.
George, C., Wagner, M., K€ ucke, M., & Rillig, M. C. (2012). Divergent consequences of
hydrochar in the plant-soil system: arbuscular mycorrhiza, nodulation, plant growth
and soil aggregation effects. Applied Soil Ecology, 59, 68e72.
Glaser, B., Balashov, E., Haumaier, L., Guggenberger, G., & Zech, W. (2000). Black carbon
in density fractions of anthropogenic soils of the Brazilian Amazon region. Organic
Geochemistry, 31, 669e678.
Glaser, B., Haumaier, L., Guggenberger, G., & Zech, W. (2001). The ‘Terra Preta’ phenom-
enon: a model for sustainable agriculture in the humid tropics. Naturwissenschaften, 88,
37e41.
Grossman, J. M., O’Neill, B. E., Tsai, S. M., Liang, B., Neves, E., Lehmann, J., et al. (2010).
Amazonian anthrosols support similar microbial communities that differ distinctly from
those extant in adjacent, unmodified soils of the same mineralogy. Microbial Ecology,
60, 192e205.
G€
uere~ na, D. T., Lehmann, J., Thies, J. E., Enders, A., Karanja, N., & Neufeldt, H. (2015).
Partitioning the contributions of biochar properties to enhanced biological nitrogen
fixation in common bean (Phaseolus vulgaris). Biology and Fertility of Soils, 51, 479e491.
Gunasekaran, P., Sundaresan, P., Raja, N. U., & Lakshmanan, M. (1987). Effect of pH, tem-
perature and nutrients on the germination of a vesicular-arbuscular mycorrhizal fungus,
Glomus fasciculatum in vitro. Proceedings: Plant Sciences, 97, 231e234.
Gundale, M. J., & DeLuca, T. H. (2006). Temperature and source material influence ecolog-
ical attributes of ponderosa pine and Douglas-fir charcoal. Forest Ecology and Management,
231, 86e93.
Gyaneshwar, P., Kumar, G. N., Parekh, L. J., & Poole, P. S. (2002). Role of soil microor-
ganisms in improving P nutrition of plants. Plant and Soil, 245, 83e93.
Hale, L., Luth, M., Kenney, R., & Crowley, D. (2014). Evaluation of pinewood biochar as a
carrier of bacterial strain Enterobacter cloacae UW5 for soil inoculation. Applied Soil Ecology,
84, 192e199.
Hammer, E. C., Balogh-Brunstad, Z., Jakobsen, I., Olsson, P. A., Stipp, S. L. S., &
Rillig, M. C. (2014). A mycorrhizal fungus grows on biochar and captures phosphorus
from its surfaces. Soil Biology and Biochemistry, 77, 252e260.
Han, G., Meng, J., Zhang, W., & Chen, W. (2013). Effect of biochar on microorganisms quan-
tity and soil physicochemical property in rhizosphere of spinach (Spinacia oleracea L.).
Applied Mechanics and Materials, 295e298, 210e219.
Harter, J., Krause, H. M., Schuettler, S., Ruser, R., Fromme, M., Scholten, T., et al. (2014).
Linking N2O emissions from biochar-amended soil to the structure and function of the
N-cycling microbial community. ISME Journal, 8, 660e674.
Harvey, A. E., Jurgensen, M. F., & Larsen, M. J. (1976). Comparative distribution of ecto-
mycorrhizae in a mature Douglas-fir/Larch forest soil in western Montana. Forest Science,
22, 350e358.
He, F., Liang, Y., Wu, A., Rong, X., & Liu, Q. (2014). Effect of biochar on nitrification from
vegetable-planting acid soil. Huanjing Kexue Xuebao/Acta Scientiae Circumstantiae, 34,
2376e2383.
He, J. Z., Shen, J. P., Zhang, L. M., Zhu, Y. G., Zheng, Y. M., Xu, M. G., et al. (2007).
Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria
and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertiliza-
tion practices. Environmental Microbiology, 9, 2364e2374.
He, L., Yang, H., Zhong, Z., Gong, P., Liu, Y., L€ u, H., et al. (2014). PCR-DGGE analysis of
soil bacterium community diversity in farmland influenced by biochar. Shengtai Xuebao/
Acta Ecologica Sinica, 34, 4288e4294.
Heller, P., Tripp, H. J., Turk-Kubo, K., & Zehr, J. P. (2014). ARBitrator: a software pipeline
for on-demand retrieval of auto-curated nifH sequences from GenBank. Bioinformatics,
30, 2883e2890.
Hu, L., Cao, L., & Zhang, R. (2014). Bacterial and fungal taxon changes in soil microbial
community composition induced by short-term biochar amendment in red oxidized
loam soil. World Journal of Microbiology and Biotechnology, 30, 1085e1092.
Hu, Y. L., Wu, F. P., Zeng, D. H., & Chang, S. X. (2014). Wheat straw and its biochar had
contrasting effects on soil C and N cycling two growing seasons after addition to a Black
Chernozemic soil planted to barley. Biology and Fertility of Soils, 50, 1291e1299.
Huang, J. L., Su, Z. C., & Xu, Y. (2005). The evolution of microbial phosphonate degrada-
tive pathways. Journal of Molecular Evolution, 61, 682e690.
Hummerjohann, J., Laudenbach, S., Rétey, J., Leisinger, T., & Kertesz, M. A. (2000). The
sulfur-regulated arylsulfatase gene cluster of Pseudomonas aeruginosa, a new member of the
cys regulon. Journal of Bacteriology, 182, 2055e2058.
Hurek, T., & Reinhold-Hurek, B. (2003). Azoarcus sp. strain BH72 as a model for nitrogen-
fixing grass endophytes. Journal of Biotechnology, 106, 169e178.
Hurek, T., Reinhold-Hurek, B., Van Montagu, M., & Kellenberger, E. (1994). Root colo-
nization and systemic spreading of Azoarcus sp. strain BH72 in grasses. Journal of Bacteri-
ology, 176, 1913e1923.
Ippolito, J. A., Spokas, K. A., Novak, J. M., Lentz, R. D., & Cantrell, K. B. (2015). Biochar
elemental composition and factors influencing nutrient retention. In J. Lehmann, &
S. Joseph (Eds.), Biochar for environmental management: Science technology and implementation
(pp. 139e163). London, UK: Earthscan.
Iswaran, V., Jauhri, K. S., & Sen, A. (1980). Effect of charcoal, coal and peat on the yield of
moong, soybean and pea. Soil Biology and Biochemistry, 12, 191e192.
Jeffery, S., Verheijen, F. G. A., Van Der Velde, M., & Bastos, A. C. (2011). A quantitative
review of the effects of biochar application to soils on crop productivity using meta-
analysis. Agriculture Ecosystems and Environment, 144, 175e187.
Jia, W., Liang, S., Zhang, J., Ngo, H. H., Guo, W., Yan, Y., et al. (2013). Nitrous oxide
emission in low-oxygen simultaneous nitrification and denitrification process: sources
and mechanisms. Bioresource Technology, 136, 444e451.
Jia, Z., & Conrad, R. (2009). Bacteria rather than Archaea dominate microbial ammonia
oxidation in an agricultural soil. Environmental Microbiology, 11, 1658e1671.
Jin, Y., Liang, X., He, M., Liu, Y., Tian, G., & Shi, J. (2015). Manure biochar influence upon
soil properties, phosphorus distribution and phosphatase activities: a microcosm incuba-
tion study. Chemosphere. http://dx.doi.org/10.1016/j.chemosphere.2015.07.015.
Jindo, K., Suto, K., Matsumoto, K., García, C., Sonoki, T., & Sanchez-Monedero, M. A.
(2012). Chemical and biochemical characterisation of biochar-blended composts
prepared from poultry manure. Bioresource Technology, 110, 396e404.
Joner, E. J., & Jakobsen, I. (1995). Growth and extracellular phosphatase activity of arbuscular
mycorrhizal hyphae as influenced by soil organic matter. Soil Biology and Biochemistry, 27,
1153e1159.
Jones, D. L., Rousk, J., Edwards-Jones, G., DeLuca, T. H., & Murphy, D. V. (2012).
Biochar-mediated changes in soil quality and plant growth in a three year field trial.
Soil Biology and Biochemistry, 45, 113e124.
Joseph, S., Peacocke, C., Lehmann, J., & Munroe, P. (2009). Developing a biochar classifi-
cation and test methods. In J. Lehmann, & S. Joseph (Eds.), Biochar for environmental man-
agement: Science, technology and implementation (pp. 107e126). London, UK: Earthscan.
Juma, N. G., & Tabatabai, M. A. (1988). Phosphatase activity in corn and soybean roots:
conditions for assay and effects of metals. Plant and Soil, 107, 39e47.
Kamat, S. S., Williams, H. J., & Raushel, F. M. (2011). Intermediates in the transformation of
phosphonates to phosphate by bacteria. Nature, 480, 570e573.
Kammann, C., Ratering, S., Eckhard, C., & M€ uller, C. (2012). Biochar and hydrochar effects
on greenhouse gas (carbon dioxide, nitrous oxide, and methane) fluxes from soils. Journal
of Environmental Quality, 41, 1052e1066.
Karaosmanoglu, F., Işigig€
ur-Erg€ udenler, A., & Sever, A. (2000). Biochar from the straw-stalk
of rapeseed plant. Energy and Fuels, 14, 336e339.
Karl, H. (2007). Charakterisierung von mikrobiellen, C-P-Lyasen kodierenden Genen in zwei
unterschiedlichen Ackerb€oden. M€unchen: Wissenschaftszentrum Weihenstephan f€ ur Ern€ah-
rung, Landnutzung und Umwelt, PhD. Technische Universit€at M€ unchen.
Kavimandan, S. K. (1986). Influence of Rhizobial inoculation on yield of wheat (Triticum
aestivum L.). Plant and Soil, 95, 297e300.
Kertesz, M. A. (1999). Riding the sulfur cycle e metabolism of sulfonates and sulfate esters in
gram-negative bacteria. FEMS Microbiology Reviews, 24, 135e175.
Kertesz, M. A., Fellows, E., & Schmalenberger, A. (2007). Rhizobacteria and plant sulfur
supply. Advances in Applied Microbiology, 62, 235e268.
Kertesz, M. A., & Mirleau, P. (2004). The role of soil microbes in plant sulfur nutrition.
Journal of Experimental Botany, 55, 1939e1945.
Khodadad, C. L. M., Zimmerman, A. R., Green, S. J., Uthandi, S., & Foster, J. S. (2011).
Taxa-specific changes in soil microbial community composition induced by pyrogenic
carbon amendments. Soil Biology and Biochemistry, 43, 385e392.
Kim, J.-S., Sparovek, G., Longo, R. M., De Melo, W. J., & Crowley, D. (2007). Bacterial
diversity of terra preta and pristine forest soil from the Western Amazon. Soil Biology
and Biochemistry, 39, 684e690.
King, J., & Quinn, J. (1997). The utilization of organosulphonates by soil and freshwater
bacteria. Letters in Applied Microbiology, 24, 474e478.
Kishimoto, S., & Sugiura, G. (1985). Charcoal as a soil conditioner. International Achieve
Future, 5, 12e23.
Klose, S., Moore, J. M., & Tabatabai, M. A. (1999). Arylsulfatase activity of microbial biomass
in soils as affected by cropping systems. Biology and Fertility of Soils, 29, 46e54.
Klose, W., & Wiest, W. (1999). Experiments and mathematical modeling of maize pyrolysis
in a rotary kiln. Fuel, 78, 65e72.
Knowles, R. (1982). Denitrification. Microbiological Reviews, 46, 43e70.
Kolton, M., Harel, Y. M., Pasternak, Z., Graber, E. R., Elad, Y., & Cytryn, E. (2011). Impact of
biochar application to soil on the root-associated bacterial community structure of fully
developed greenhouse pepper plants. Applied and Environmental Microbiology, 77, 4924e4930.
Kononova, S. V., & Nesmeyanova, M. A. (2002). Phosphonates and their degradation by
microorganisms. Biochemistry-Moscow, 67, 184e195.
Krzysko-qupicka, T., & Orlik, A. (1997). Use of glyphosate as the sole source of phosphorus
or carbon for the selection of soil-borne fungal strains capable to degrade this herbicide.
Chemosphere, 34, 2601e2605.
Laird, D., Fleming, P., Wang, B., Horton, R., & Karlen, D. (2010). Biochar impact on
nutrient leaching from a Midwestern agricultural soil. Geoderma, 158, 436e442.
Lal, R. (2009). Soils and food sufficiency. A review. Agronomy for Sustainable Development, 29,
113e133.
Lang, T., Jensen, A. D., & Jensen, P. A. (2005). Retention of organic elements during solid
fuel pyrolysis with emphasis on the peculiar behavior of nitrogen. Energy and Fuels, 19,
1631e1643.
Lehmann, J., Gaunt, J., & Rondon, M. (2006). Bio-char sequestration in terrestrial ecosys-
temsda review. Mitigation and Adaptation Strategies for Global Change, 11, 395e419.
Lehmann, J., & Joseph, S. (2009). Biochar for environmental management: an introduction.
In J. Lehmann, & S. Joseph (Eds.), Biochar for environmental management: Science, technology
and implementation (pp. 1e12). London, UK: Earthscan.
Lehmann, J., Rillig, M. C., Thies, J., Masiello, C. A., Hockaday, W. C., & Crowley, D.
(2011). Biochar effects on soil biota e a review. Soil Biology and Biochemistry, 43,
1812e1836.
Leininger, S., Urich, T., Schloter, M., Schwark, L., Qi, J., Nicol, G. W., et al. (2006).
Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature, 442,
806e809.
Lentz, R. D., Ippolito, J. A., & Spokas, K. A. (2014). Biochar and manure effects on net ni-
trogen mineralization and greenhouse gas emissions from calcareous soil under corn. Soil
Science Society of America Journal, 78, 1641e1655.
Liang, B., Lehmann, J., Solomon, D., Kinyangi, J., Grossman, J., O’Neill, B., et al. (2006).
Black carbon increases cation exchange capacity in soils. Soil Science Society American Jour-
nal, 70, 1719e1730.
Liang, C., Zhu, X., Fu, S., Méndez, A., Gasc o, G., & Paz-Ferreiro, J. (2014). Biochar alters
the resistance and resilience to drought in a tropical soil. Environmental Research Letters, 9,
064013.
Lim, B. L., Yeung, P., Cheng, C., & Hill, J. E. (2007). Distribution and diversity of phytate-
mineralizing bacteria. ISME Journal, 1, 321e330.
Lin, T. F., Huang, H. I., Shen, F. T., & Young, C. C. (2006). The protons of gluconic acid
are the major factor responsible for the dissolution of tricalcium phosphate by Burkholde-
ria cepacia CC-Al74. Bioresource Technology, 97, 957e960.
Liu, L., Chen, P., Sun, M., Shen, G., & Shang, G. (2015). Effect of biochar amendment on
PAH dissipation and indigenous degradation bacteria in contaminated soil. Journal of Soils
and Sediments, 15, 313e322.
Liu, L., Shen, G., Sun, M., Cao, X., Shang, G., & Chen, P. (2014). Effect of biochar on
nitrous oxide emission and its potential mechanisms. Journal of the Air and Waste Manage-
ment Association, 64, 894e902.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, et al. (2004).
ARB: a software environment for sequence data. Nucleic Acids Research, 32, 1363e1371.
Luo, H., Benner, R., Long, R. A., & Hu, J. (2009). Subcellular localization of marine
bacterial alkaline phosphatases. Proceedings of the National Academy of Sciences, 106,
21219e21223.
Mackie, K. A., Marhan, S., Ditterich, F., Schmidt, H. P., & Kandeler, E. (2015). The effects
of biochar and compost amendments on copper immobilization and soil microorganisms
in a temperate vineyard. Agriculture, Ecosystems and Environment, 201, 58e69.
Martin, S. L., Clarke, M. L., Othman, M., Ramsden, S. J., & West, H. M. (2015). Biochar-
mediated reductions in greenhouse gas emissions from soil amended with anaerobic
digestates. Biomass and Bioenergy, 79, 39e49.
Marzluf, G. A. (1997). Molecular genetics of sulfur assimilation in filamentous fungi and

yeast. Annual Reviews in Microbiology, 51, 73e96.
Masto, R. E., Ansari, M. A., George, J., Selvi, V. A., & Ram, L. C. (2013). Co-application of
biochar and lignite fly ash on soil nutrients and biological parameters at different crop
growth stages of Zea mays. Ecological Engineering, 58, 314e322.
Masto, R. E., Kumar, S., Rout, T. K., Sarkar, P., George, J., & Ram, L. C. (2013). Biochar
from water hyacinth (Eichornia crassipes) and its impact on soil biological activity. Catena,
111, 64e71.
McGrath, S. P., & Zhao, F. J. (1995). A risk assessment of sulfur deficiency in cereals using soil
and atmospheric deposition data. Soil Use and Management, 11, 110e114.
McGrath, S. P., Zhao, F., & Blake-Kalff, M. (2003). History and outlook for sulphur fertil-
izers in Europe. Fertilizers Fertilization, 2, 5e27.
Metcleff, W. W., & Wanner, B. L. (1991). Involvement of the Escherichia coli phn (psiD) gene
cluster in assimilation of phosphorus in the form of phosphonates, phosphite, Pi esters,
and Pi. Journal of Bacteriology, 173, 587e600.
Meynet, P., Moliterni, E., Davenport, R. J., Sloan, W. T., Camacho, J. V., & Werner, D.
(2014). Predicting the effects of biochar on volatile petroleum hydrocarbon biodegrada-
tion and emanation from soil: a bacterial community finger-print analysis inferred
modelling approach. Soil Biology and Biochemistry, 68, 20e30.
Mia, S., van Groenigen, J. W., van de Voorde, T. F. J., Oram, N. J., Bezemer, T. M.,
Mommer, L., et al. (2014). Biochar application rate affects biological nitrogen fixation
in red clover conditional on potassium availability. Agriculture, Ecosystems and Environ-
ment, 191, 83e91.
Mohan, D., Pittman, C. U., & Steele, P. H. (2006). Pyrolysis of wood/biomass for bio-oil: a
critical review. Energy Fuels, 20, 848e889.
Muniruzzaman, S., & Khan, S. I. (1992). Suitability of some local agro-industrial wastes as
carrier materials for Rhizobium sp. infecting Sesbania bispinosa. World Journal of Microbiology
and Biotechnology, 8, 329e330.
Nannipieri, P., Giagnoni, L., Landi, L., & Renella, G. (2011). Role of phosphatase enzymes
in soil. In E. B€ unemann, A. Oberson, & E. Frossard (Eds.), Phosphorus in action: Biological
processes in soil phosphorus cycling. New York, NY: Springer.
Navarrete, A. A., Cannavan, F. S., Taketani, R. G., & Tsai, S. M. (2010). A molecular survey
of the diversity of microbial communities in different Amazonian agricultural model
systems. Diversity, 2, 787e809.
Neina, D., & Dowuona, G. N. N. (2013). Short-term effects of human urine fertiliser and
wood ash on soil pH and electrical conductivity. Journal of Agriculture and Rural Develop-
ment in the Tropics and Subtropics, 114, 89e100.
Nelissen, V., R€ utting, T., Huygens, D., Ruysschaert, G., & Boeckx, P. (2014). Temporal
evolution of biochar’s impact on soil nitrogen processes e a 15N tracing study. GCB Bio-
energy, 7, 635e645.
Nelissen, V., R€ utting, T., Huygens, D., Staelens, J., Ruysschaert, G., & Boeckx, P. (2012).
Maize biochars accelerate short-term soil nitrogen dynamics in a loamy sand soil. Soil
Biology and Biochemistry, 55, 20e27.
Nelissen, V., Ruysschaert, G., Manka’Abusi, D., D’Hose, T., De Beuf, K., Al-Barri, B., et al.
(2015). Impact of a woody biochar on properties of a sandy loam soil and spring barley
during a two-year field experiment. European Journal of Agronomy, 62, 65e78.
Nelissen, V., Saha, B. K., Ruysschaert, G., & Boeckx, P. (2014). Effect of different
biochar and fertilizer types on N2O and NO emissions. Soil Biology and Biochemistry,
70, 244e255.
Nicol, G. W., Leininger, S., Schleper, C., & Prosser, J. I. (2008). The influence of soil pH on
the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and
bacteria. Environmental Microbiology, 10, 2966e2978.
Ogawa, M. (1998). Utilization of symbiotic microorganisms and charcoal for desert greening.
Green Age, 14, 5e11.
Okon, Y. (1985). Azospirillum as a potential inoculant for agriculture. Trends in Biotechnology,
3, 223e228.
O’Neill, B., Grossman, J., Tsai, M. T., Gomes, J. E., Lehmann, J., Peterson, J., et al. (2009).
Bacterial community composition in Brazilian Anthrosols and adjacent soils characterized
using culturing and molecular identification. Microbial Ecology, 58, 23e35.
Packialakshmi, N., & Aliya Riswana, T. (2014). Screening and production of phosphate
solubilising bacterial inoculants using different carrier. Research Journal of Pharmaceutical,
Biological and Chemical Sciences, 5, 1762e1772.
Panas, P., Ternan, N. G., Dooley, J. S. G., & McMullan, G. (2006). Detection of phospho-
noacetate degradation and phnA genes in soil bacteria from distinct geographical origins
suggest its possible biogenic origin. Environmental Microbiology, 8, 939e945.
Paz-Ferreiro, J., Fu, S., Méndez, A., & Gasc o, G. (2015). Biochar modifies the thermody-
namic parameters of soil enzyme activity in a tropical soil. Journal of Soils and Sediments,
15, 578e583.
Paz-Ferreiro, J., Gasc o, G., Gutiérrez, B., & Méndez, A. (2012). Soil biochemical activities
and the geometric mean of enzyme activities after application of sewage sludge and
sewage sludge biochar to soil. Biology and Fertility of Soils, 48, 511e517.
Pereira, E. I. P., Suddick, E. C., Mansour, I., Mukome, F. N. D., Parikh, S. J., Scow, K., et al.
(2015). Biochar alters nitrogen transformations but has minimal effects on nitrous oxide
emissions in an organically managed lettuce mesocosm. Biology and Fertility of Soils, 51(5),
573e582.
Poly, F., Ranjard, L., Nazaret, S., Gourbiere, F., & Monrozier, L. J. (2001). Comparison of
nifH gene pools in soils and soil microenvironments with contrasting properties. Applied
and Environmental Microbiology, 67, 2255e2262.
Postgate, J. R. (1982). Biological nitrogen fixation: fundamentals. Philosophical Transactions of
the Royal Society of London. Series B, Biological Sciences, 296, 375e385.
Postgate, J. R. (1984). The sulfate reducing bacteria. Cambridge, UK: University Press.
Pramanik, M., & Iswaran, V. (1973). Survival of Rhizobium japonicum in various carriers. Zentral-
blatt f€ur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene. Zweite Naturwissen-
schaftliche Abteilung: Allgemeine, Landwirtschaftliche und Technische Mikrobiologie, 128, 232e239.
Prommer, J., Wanek, W., Hofhansl, F., Trojan, D., Offre, P., Urich, T., et al. (2014). Bio-
char decelerates soil organic nitrogen cycling but stimulates soil nitrification in a
temperate arable field trial. PLoS One, 9, 86388.
Quilliam, R. S., DeLuca, T. H., & Jones, D. L. (2013). Biochar application reduces nodula-
tion but increases nitrogenase activity in clover. Plant and Soil, 366, 83e92.
Rahman, L., Whitelaw-Weckert, M. A., & Orchard, B. (2014). Impact of organic soil
amendments, including poultry-litter biochar, on nematodes in a Riverina, New South
Wales, vineyard. Soil Research, 52, 604e619.
Randriamanantsoa, L., Morel, C., Rabeharisoa, R. L., Douzet, J. M., Jansa, J., & Frossard, E.
(2013). Can the isotopic exchange kinetic method be used in soils with a very low water
extractable phosphate content and a high sorbing capacity for phosphate ions? Geoderma,
200e201, 120e129.
Reguera, G., McCarthy, K. D., Mehta, T., Nicoll, J. S., Tuominen, M. T., & Lovley, D. R.
(2005). Extracellular electron transfer via microbial nanowires. Nature, 435, 1098e1101.
Retan, G. A. (1915). Charcoal as a means of solving some nursery problems. Forestry Quar-
terly, 13, 25e30.
Rizhiya, E. Y., Buchkina, N. P., Mukhina, I. M., Belinets, A. S., & Balashov, E. V. (2015).
Effect of biochar on the properties of loamy sand Spodosol soil samples with different
fertility levels: a laboratory experiment. Eurasian Soil Science, 48, 192e200.
Robertson, G. P., & Groffman, P. M. (2015). Nitrogen transformations. In E. A. Paul (Ed.),

Soil microbiology, ecology and biochemistry (pp. 421e446). London, UK: Academic Press.
Robertson, S. J., Michael Rutherford, P., L opez-Gutiérrez, J. C., & Massicotte, H. B. (2012).
Biochar enhances seedling growth and alters root symbioses and properties of sub-boreal
forest soils. Canadian Journal of Soil Science, 92, 329e340.
Rondon, M. A., Lehmann, J., Ramírez, J., & Hurtado, M. (2007). Biological nitrogen
fixation by common beans (Phaseolus vulgaris L.) increases with bio-char additions. Biology
and Fertility of Soils, 43, 699e708.
Rutigliano, F. A., Romano, M., Marzaioli, R., Baglivo, I., Baronti, S., Miglietta, F., et al.
(2014). Effect of biochar addition on soil microbial community in a wheat crop. European
Journal of Soil Biology, 60, 9e15.
R€utting, T., Boeckx, P., M€ uller, C., & Klemedtsson, L. (2011). Assessment of the importance
of dissimilatory nitrate reduction to ammonium for the terrestrial nitrogen cycle.
Biogeosciences, 8, 1779e1791.
Saito, M. (1990). Charcoal as a micro habitat for VA mycorrhizal fungi, and its practical
application. Agriculture Ecosystem and Environment, 29, 341e344.
Sakurai, M., Wasaki, J., Tomizawa, Y., Shinano, T., & Osaki, M. (2008). Analysis of bacterial
communities on alkaline phosphatase genes in soil supplied with organic matter. Soil
Science and Plant Nutrition, 54, 62e71.
Sanguin, H., Wilson, N. L., & Kertesz, M. A. (2015). Assessment of functional diversity and
structure of phytate-hydrolysing bacterial community in Lolium perenne rhizosphere.
Plant and Soil. http://dx.doi.org/10.1007/s11104-015-2512-7.
Scheer, C., Grace, P. R., Rowlings, D. W., Kimber, S., & van Zwieten, L. (2011). Effect of
biochar amendment on the soil-atmosphere exchange of greenhouse gases from an
intensive subtropical pasture in northern New South Wales, Australia. Plant and Soil,
345, 47e58.
Schimel, J. P., & Bennett, J. (2004). Nitrogen mineralization: challenges of a changing
paradigm. Ecology, 85, 591e602.
Schmalenberger, A., Duran, A. L., Bray, A. W., Bridge, J., Bonneville, S., Benning, L. G.,
et al. (2015). Oxalate secretion by ectomycorrhizal Paxillus involutus is mineral-specific
and controls calcium weathering from minerals. Scientific Reports, 5, 12187.
Schmalenberger, A., Hodge, S., Bryant, A., Hawkesford, M. J., Singh, B. K., &
Kertesz, M. A. (2008). The role of Variovorax and other Comamonadaceae in sulfur
transformations by microbial wheat rhizosphere communities exposed to different sulfur
fertilization regimes. Environmental Microbiology, 10, 1486e1500.
Schmalenberger, A., Hodge, S., Hawkesford, M. J., & Kertesz, M. A. (2009). Sulfonate
desulfurization in Rhodococcus from wheat rhizosphere communities. FEMS Microbiology
Ecology, 67, 140e150.
Schmalenberger, A., Wolfgang, P., Ojeda, J. J., & Noll, M. (2011). Characterization of
main sulfur source of wood-degrading basidiomycetes by S K-edge X-ray absorption
near edge spectroscopy (XANES). International Biodeterioration and Biodegradation, 65,
1215e1223.
Schulz, H., & Glaser, B. (2012). Effects of biochar compared to organic and inorganic fertil-
izers on soil quality and plant growth in a greenhouse experiment. Journal of Plant Nutri-
tion and Soil Science, 175, 410e422.
Schwartz, T., Misri, B. K., & Fock, H. P. (1991). The involvement of glutamate
dehydrogenase and glutamine synthetase/glutamate synthase in ammonia assimilation
by the basidiomycete fungus Stropharia semiglobata. Journal of General Microbiology, 137,
2253e2258.
Shen, J., Yuan, L., Zhang, J., Li, H., Bai, Z., Chen, X., et al. (2011). Phosphorous dynamics:
from soil to plant. Plant Physiology, 156, 997e1005.
Sheng, X. F. (2005). Growth promotion and increased potassium uptake of cotton and rape
by a potassium releasing strain of Bacillus edaphicus. Soil Biology and Biochemistry, 37,
1918e1922.
Shi, Y., Zhang, L., & Zhao, M. (2015). Effect of biochar application on the efficacy of the
nitrification inhibitor dicyandiamide in soils. BioResources, 10, 1330e1345.
Sohi, S. P., Krull, E., Lopez-Capel, E., & Bol, R. (2010). A review of biochar and its use and
function in soil. In L. S. Donald (Ed.), Advances in Agronomy (Vol. 105, pp. 47e82).
Academic Press.
Solaiman, Z. M., Blackwell, P., Abbott, L. K., & Storer, P. (2010). Direct and residual effect
of biochar application on mycorrhizal root colonisation, growth and nutrition of wheat.
Australian Journal of Soil Research, 48, 546e554.
Song, Y., Zhang, X., Ma, B., Chang, S. X., & Gong, J. (2014). Biochar addition affected the
dynamics of ammonia oxidizers and nitrification in microcosms of a coastal alkaline soil.
Biology and Fertility of Soils, 50, 321e332.
Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses
with thousands of taxa and mixed models. Bioinformatics, 22, 2688e2690.
Sun, D., Jun, M., Zhang, W., Guan, X., Huang, Y., Lan, Y., et al. (2012). Implication of
temporal dynamics of microbial abundance and nutrients to soil fertility under biochar
application e field experiments conducted in a brown soil cultivated with soybean, north
China. Advanced Materials Research, 518e523, 384e394.
Sun, Z., Bruun, E. W., Arthur, E., de Jonge, L. W., Moldrup, P., Hauggaard-Nielsen, H.,
et al. (2014). Effect of biochar on aerobic processes, enzyme activity, and crop yields
in two sandy loam soils. Biology and Fertility of Soils, 50, 1087e1097.
Sylvester-Bradley, R., De Oliveira, L. A., De PodestaFilho, J. A., & St. John, T. V. (1980).
Nodulation of legumes, nitrogenase activity of roots and occurrence of nitrogen-
fixing Azospirillum ssp. in representative soils of central Amazonia. Agro-Ecosystems,
6, 249e266.
Tabatabai, M. A., & Bremner, J. M. (1969). Use of p-nitrophenyl phosphate for assay of soil
phosphatase activity. Soil Biology and Biochemistry, 1, 301e307.
Tabatabai, M. A., & Bremner, J. M. (1970). Arylsulfatase activity of soils. Proceedings e Soil
Science Society of America, 34, 225e229.
Taketani, R. G., Lima, A. B., Jesus, E. D. C., Teixeira, W. G., Tiedje, J. M., & Tsai, S. M.
(2013). Bacterial community composition of anthropogenic biochar and Amazonian
anthrosols assessed by 16S rRNA gene 454 pyrosequencing. Antonie Van Leeuwenhoek In-
ternational Journal of General and Molecular Microbiology, 104, 233e242.
Tammeorg, P., Brandstaka, T., Simojoki, A., & Helenius, J. (2013). Nitrogen mineralisation
dynamics of meat bone meal and cattle manure as affected by the application of softwood
chip biochar in soil. Earth and Environmental Science Transactions of the Royal Society of Edin-
burgh, 103, 19e30.
Tan, H., Barret, M., Mooij, M., Rice, O., Morrissey, J., Dobson, A., et al. (2013). Long-
term phosphorus fertilisation increased the diversity of the total bacterial community
and the phoD phosphorus mineraliser group in pasture soils. Biology and Fertility of Soils,
49, 661e672.
Tarafdar, J. C., Bareja, M., & Panwar, J. (2003). Efficiency of some phosphatase producing
soil-fungi. Indian Journal of Microbiology, 43, 27e32.
Tarafdar, J. C., & Claassen, N. (1988). Organic phosphorus compounds as a phosphorus
source for higher plants through the activity of phosphatases produced by plant roots
and microorganisms. Biology and Fertility of Soils, 5, 308e312.
Ternan, N. G., Mc Grath, J. W., McMullan, G., & Quinn, J. P. (1998). Organophospho-
nates: occurrence, synthesis and biodegradation by microorganisms. World Journal of
Microbiolology and Biotechnology, 14, 635e647.
Thies, J. E., & Rillig, M. C. (2009). Characteristics of biochar: biological properties. In

J. Lehmann, & S. Joseph (Eds.), Biochar for environmental management: Science, technology
Thies, J. E., Rillig, M. C., & Graber, E. R. (2015). Biochar effects on the abundance, activity
and diversity of the soil biota. In J. Lehmann, & S. Joseph (Eds.), Biochar for environmental
management: Science, technology and implementation (pp. 327e389). London, UK: Earthscan.
Thuy Thu, D., Bouvier, C., Bettarel, Y., Bouvier, T., Henry-des-Tureaux, T., Janeau, J. L.,
et al. (2014). Influence of buffalo manure, compost, vermicompost and biochar amend-
ments on bacterial and viral communities in soil and adjacent aquatic systems. Applied Soil
Ecology, 73, 78e86.
Tiedje, J. M., Sexstone, A. J., Parkin, T. B., & Revsbech, N. P. (1984). Anaerobic processes
in soil. Plant and Soil, 76, 197e212.
Trolove, S. N., Hedley, M. J., Kirk, G. J. D., Bolan, N. S., & Loganathan, P. (2003). Progress
in selected areas of rhizosphere research on P acquisition. Australian Journal of Soil Research,
41, 471e499.
Troy, S. M., Lawlor, P. G., O’Flynn, C. J., & Healy, M. G. (2013). Impact of biochar
addition to soil on greenhouse gas emissions following pig manure application. Soil
Biology and Biochemistry, 60, 173e181.
Turner, B. L., Cade-Menun, B. J., Condron, L. M., & Newman, S. (2005). Extraction of soil
organic phosphorus. Talanta, 66, 294e306.
Turner, B. L., Paphazy, M. J., Haygarth, P. M., & McKelvie, I. D. (2002). Inositol phosphates
in the environment. Philosophical Transactions of the Royal Society B: Biological Sciences, 357,
449e469.
Turner, E. R. (1955). The effect of certain adsorbents on the nodulation of clover plants.
Annals of Botany, 19, 149e160.
Uchimiya, M., & Hiradate, S. (2014). Pyrolysis temperature-dependent changes in dissolved
phosphorus speciation of plant and manure biochars. Journal of Agricultural and Food Chem-
istry, 62, 1802e1809.
Ulyett, J., Sakrabani, R., Kibblewhite, M., & Hann, M. (2014). Impact of biochar addition
on water retention, nitrification and carbon dioxide evolution from two sandy loam soils.
European Journal of Soil Science, 65, 96e104.
Uroz, S., Oger, P., Lepleux, C., Collignon, C., Frey-Klett, P., & Turpault, M. P. (2011).
Bacterial weathering and its contribution to nutrient cycling in temperate forest
ecosystems. Research in Microbiology, 162, 821e831.
Van Rhijn, P., & Vanderleyden, J. (1995). The rhizobium-plant symbiosis. Microbiological Re-
views, 59, 124e142.
Van Zwieten, L., Kimber, S., Morris, S., Downie, A., Berger, E., Rust, J., et al. (2010).
Influence of biochars on flux of N2O and CO2 from Ferrosol. Australian Journal of Soil
Van Zwieten, L., Singh, B. P., Kimber, S. W. L., Murphy, D. V., Macdonald, L. M., Rust, J.,
et al. (2014). An incubation study investigating the mechanisms that impact N2O flux from
soil following biochar application. Agriculture, Ecosystems and Environment, 191, 53e62.
Ventura, M., Zhang, C., Baldi, E., Fornasier, F., Sorrenti, G., Panzacchi, P., et al. (2014).
Effect of biochar addition on soil respiration partitioning and root dynamics in an apple
orchard. European Journal of Soil Science, 65, 186e195.
Vermeij, P., Wietek, C., Kahnert, A., W€ uest, T., & Kertesz, M. A. (1999). Genetic organi-
zation of sulfur-controlled aryl desulfonation in Pseudomonas putida S-313. Molecular
Villarreal-Chiu, J. F., Quinn, J. P., & McGrath, J. W. (2012). The genes and enzymes of
phosphonate metabolism by bacteria, and their distribution in the marine
environment. Frontiers in Microbiology, 3, 19.
Vitousek, P., & Howarth, R. (1991). Nitrogen limitation on land and in the sea: how can it
occur? Biogeochemistry, 13, 87e115.
Wang, C., Lu, H., Dong, D., Deng, H., Strong, P. J., Wang, H., et al. (2013). Insight into the
effects of biochar on manure composting: evidence supporting the relationship between
N2O emission and denitrifying community. Environmental Science and Technology, 47,
7341e7349.
Wang, T., Camps-Arbestain, M., Hedley, M., & Bishop, P. (2012). Predicting phosphorus
bioavailability from high-ash biochars. Plant and Soil, 357, 173e187.
Warnock, D. D., Lehmann, J., Kuyper, T. W., & Rillig, M. C. (2007). Mycorrhizal responses
to biochar in soil e concepts and mechanisms. Plant and Soil, 300, 9e20.
Xiang, J., Liu, D., Ding, W., Yuan, J., & Lin, Y. (2015). Effects of biochar on nitrous oxide
and nitric oxide emissions from paddy field during the wheat growth season. Journal of
Cleaner Production, 104, 52e58.
Xu, H. J., Wang, X. H., Li, H., Yao, H. Y., Su, J. Q., & Zhu, Y. G. (2014). Biochar impacts
soil microbial community composition and nitrogen cycling in an acidic soil planted with
rape. Environmental Science and Technology, 48, 9391e9399.
Yanai, Y., Hatano, R., Okazaki, M., & Toyota, K. (2008). Analysis of the C2H2 inhibition-
based N2O production curve to characterize the N2O-reducing activity of denitrifying
communities in soil. Geoderma, 146, 269e276.
Yanai, Y., Toyota, K., & Okazaki, M. (2007). Effects of charcoal addition on N2O emissions
from soil resulting from rewetting air-dried soil in short-term laboratory experiments.
Soil Science and Plant Nutrition, 53, 181e188.
Yanardag, I., Zornoza, R., Cano, A. F., Yanardag, A. B., & Mermut, A. R. (2014). Evalu-
ation of carbon and nitrogen dynamics in different soil types amended with pig slurry, pig
manure and its biochar by chemical and thermogravimetric analysis. Biology and Fertility of
Soils, 51, 183e196.
Yang, F., Cao, X., Gao, B., Zhao, L., & Li, F. (2015). Short-term effects of rice straw biochar
on sorption, emission, and transformation of soil NHþ 4 -N. Environmental Science and Pollu-
tion Research, 22, 9184e9192.
Yang, Y., Yan, J., & Ding, C. (2013). Effects of biochar amendment on the dynamics of
enzyme activities from a paddy soil polluted by heavy metals. Advanced Materials Research,
610e613, 2129e2133.
Yao, H., Campbell, C. D., & Qiao, X. (2011). Soil pH controls nitrification and carbon sub-
strate utilization more than urea or charcoal in some highly acidic soils. Biology and
Fertility of Soils, 47, 515e522.
Yin Chan, K., & Xu, Z. (2009). Biochar: nutrient properties and their enhancement. In
J. Lehmann, & S. Joseph (Eds.), Biochar for environmental management: Science, technology
Yoo, G., & Kang, H. (2012). Effects of biochar addition on greenhouse gas emissions and
microbial responses in a short-term laboratory experiment. Journal of Environmental Qual-
ity, 41, 1193e1202.
Zehr, J. P., Jenkins, B. D., Short, S. M., & Steward, G. F. (2003). Nitrogenase gene diversity
and microbial community structure: a cross-system comparison. Environmental Microbi-
ology, 5, 539e554.
Zhang, X. K., Li, Q., Liang, W. J., Zhang, M., Bao, X. L., & Xie, Z. B. (2013). Soil nem-
atode response to biochar addition in a Chinese wheat field. Pedosphere, 23, 98e103.
Zhao, F. J., Lehmann, J., Solomon, D., Fox, M. A., & McGrath, S. P. (2006). Sulphur speci-
ation and turnover in soils: evidence from sulphur K-edge XANES spectroscopy and
isotope dilution studies. Soil Biology and Biochemistry, 38, 1000e1007.
Zhao, X., Wang, S., & Xing, G. (2014). Nitrification, acidification, and nitrogen leaching
from subtropical cropland soils as affected by rice straw-based biochar: laboratory incu-
bation and column leaching studies. Journal of Soils and Sediments, 14, 471e482.
Zheng, J., Stewart, C. E., & Cotrufo, M. F. (2012). Biochar and nitrogen fertilizer alters soil
nitrogen dynamics and greenhouse gas fluxes from two temperate soils. Journal of
Environmental Quality, 41, 1361e1370.
Zhu, J., Wei, X., Zhu, P., Yu, H., Shu, L., Xu, Z., et al. (2015). Biochar addition inhibiting
nitrification of reclaimed soils in coal-mining subsidence area. Nongye Gongcheng Xuebao/
Transactions of the Chinese Society of Agricultural Engineering, 31, 264e271.
Zou, C. J., Zhang, Y. Y., Zhang, Y. M., Guo, X. O., Li, M. J., & Li, T. L. (2015). Regulation
of biochar on matrix enzyme activities and microorganisms around cucumber roots
under continuous cropping. Chinese Journal of Applied Ecology, 26, 1772e1778.
INDEX
Note: Page numbers followed by “f ” indicate figures and “t” indicate tables.
A B
Abiotic formation, 16–17 Bacillus, 143–144
Abundant root nodulation, 129–130 Bacteria, 21, 81–82, 84
Acholeplasmataceae, 48 Bacterial community structure, 113–114,
Aliphatic sulfonates, 141–142 137
Alkylsulfatases, 140–141 Bacterial cycling
Alphaproteobacteria, 48 of nitrogen, 117–132
AM. See Arbuscular mycorrhizal of other nutrients, 143–144
(AM) of phosphorus, 132–138
Ammonia monooxygenase (Amo), of sulfur, 139–143
118–119 Bacterial nonspecific acid phosphatases,
Ammonia-oxidizing archaea (AOA), 136
120–125 Bacterial-induced carbonate precipitation,
Ammonia-oxidizing bacteria (AOB), bioprecipitation of metal(loid)s by,
120–125 87–97
Amo. See Ammonia monooxygenase BCM. See Biologically controlled
(Amo) mineralization (BCM)
amoA genes, 120–126 Betaproteobacteria, 48
Amplicon sequencing, 31 BIM. See Biologically induced
biodiversity, 31–32 mineralization (BIM)
gene markers of functional diversity, Biochars, 110–111, 112f
32–33 and bacterial cycling
Anabolic process, 118 of nitrogen, 117–131
Anaerobic methanotrophic (ANME), of other nutrients, 143–144
17–18 of phosphorus, 132–138
ANME. See Anaerobic methanotrophic of sulfur, 139–143
(ANME) canonical correspondence analysis, 115f
AOA. See Ammonia-oxidizing archaea chemical studies, 111–112
(AOA) physical and chemical attributes, 111
AOB. See Ammonia-oxidizing bacteria soil microbiota, 112–116
(AOB) as source of nutrients, 116–117
Arbuscular mycorrhizal (AM), 114–116 Biogeochemistry
Archaea, 22 deep carbon cycling, 15–19
Aromatic sulfonates, 141–142 field sampling for molecular
Arsenic, 87–89 microbiological analysis, 15f
Arylsulfatases, 140–141 hydrogeological and hydrogeochemical
asf gene cluster, 141–143 conditions, 12f
Atmospheric di-nitrogen fixation, microbial activity, 19–20
129–132 sources of energy in lithosphere, 9–15
Azoarcus sp. BH72, 130–131 Biological (di-)nitrogen fixation (BNF),
Azospirillum, 129 129–130
161 j
162 Index
Biologically controlled mineralization D

(BCM), 83–84 DAPI. See 4,6-Diamidino-2-phenylindole
Biologically induced mineralization (BIM), (DAPI)
83–84 DCD. See Dicyandiamide (DCD)
Biomineralization, 83–87 Deep biosphere research, marker genes in,
MICP, 84–87 34t–35t
precipitation of metal carbonates, 87f Deep biosphere sampling, 24
of radionuclides, 96 groundwater sampling, 25–28
ureolytic bacteria application, 86t processing and maintenance of samples for
Bioprecipitation of metal(loid)s microbiological research, 29
by bacterial-induced carbonate Deep carbon cycling, 3, 15–16
precipitation, 87–97 hydrocarbons, 16
arsenic, 87–89 microbes in, 17–19
Cd, 89–91 Deep carbon reservoirs, 3
Copper, 93 Deep crustal life. See also Deep subsurface
Cr, 92–93 life; Terrestrial deep subsurface
Pb, 93–94 microbiomes
radionuclide bioprecipitation by biogeochemistry
urease-producing bacteria, deep carbon cycling, 15–19
95–97 field sampling for molecular
by fungal induced carbonate precipitation, microbiological analysis, 15f
97–100 hydrogeological and
Bioremediation, 81 hydrogeochemical conditions, 12f
BNF. See Biological (di-)nitrogen fixation microbial activity, 19–20
(BNF) sources of energy in lithosphere, 9–15
deep subsurface habitats, 3–4
C lithosphere as host of life, 4–6
Cadmium (Cd), 89–91 microbial life at surface, 3–4
Calcite (CaCO3), 84 Deep subseafloor sediments, 18–19
Calcium carbonate, 84 Deep subsurface life. See also Deep crustal
Candidate phyla radiation (CPR), 21 life
Carbon sources, 33 geological carbon sources for, 8–9
for deep subsurface life, 8–9 metagenomics, 41–42
Carbon-bonded phosphorus, 138 physical and geochemical constraints on,
Catabolic process, 118 6–8
cDNA. See complementary DNA Deep subsurface microbes, 9
(cDNA) Deep terrestrial subsurface, hydrogen and
Chromium (Cr), 91–92 carbon cycling in, 14f
Clostridiaceae, 48 Deep-sea ecosystems, 14–15
CoA. See Coenzyme A (CoA) Denaturing gradient gel electrophoresis
Coenzyme A (CoA), 22 (DGGE), 31–32, 114
Comamonadaceae, 47–48 Denitrification, 126–129. See also
complementary DNA (cDNA), 33 Nitrification
Conventional methods, 81 field studies, 128–129
Copper, 93 laboratory studies, 127–128
CPR. See Candidate phyla radiation DGGE. See Denaturing gradient gel
(CPR) electrophoresis (DGGE)
Index 163
4,6-Diamidino-2-phenylindole (DAPI), by abiotic reactions, 10

29 formation, 11
Diazotrophic bacteria, 130–131 Hyperthermophilic microorganisms, 6
Dicyandiamide (DCD), 125–126
Direct pumping of water, 26–27 I
Dissimilatory nitrate reduction to ICDP. See International Continental
ammonium (DNRA), 126 Scientific Drilling Program (ICDP)
Dolomite (CaMg(CO3)2), 84 Idaho National Engineering and
Environmental Laboratory
E (INEEL), 96
Elemental cycle, 118 Inorganic solid phases, 8–9
Enzymatic assays, 141 Inorganically bound phosphorus
Enzymic systems, 81–82 mobilization, 133–135
Ester-bound Inositol phosphate. See Phytate
phosphorus mobilization, 135–137 Integrated Ocean Drilling Program
sulfur mobilization, 140–141 (IODP), 24–25
Eukaryotes, 22–23 Internal transcribed spacer (ITS), 32
Euryarchaea, 22 International Continental Scientific
Exiguobacterium undae YR10, 91 Drilling Program (ICDP), 24–25
F K
FastQC software, 37–38 Kyoto Encyclopedia of Genes and
Field studies, 128–129 Genomes (KEGG), 38–39
Filtration, 29 accumulation curves of, 56f
Fluorescence-activated cell sorting carbon, nitrogen, methane, and sulfur
(FACS), 40 metabolism-associated KEGG
Functional gene markers, 33 modules, 58t–59t
Fungal induced carbonate precipitation, Venn diagram, 60f
bioprecipitation of metal(loid)s by,
97–100 L
Fungi, 22–23, 81–82, 84 Laboratory studies, 127–128
Last common ancestor method (LCA
G method), 45–46
Gas hydrates. See Methane clathrates Lead (Pb), 93–94
Gene prediction and annotation, Lithosphere as host of life, 4–6
45–46 Lysinibacillus sphaericus CH-5, 90
Gene-encoding enzymes, 32–33
Geomicrobes, 13 M
Glutamate dehydrogenase (GDH), 118 mcrA gene, 17
Glutamine synthetase-glutamate synthase messenger RNA (mRNA), 36
(GS-GOGAT), 118 Metagenome assembly, 44–45
Groundwater sampling, 25–28 Metagenomics, 30–31
amplicon sequencing, 31–33
H bioinformatics workflow in, 38f
Heterotrophic nitrification, 118–119 data analysis, 37–39
Hydrocarbons, 16 marker genes in deep biosphere research,
Hydrogen, 10 34t–35t
164 Index
Metagenomics (Continued ) NCBI. See National Center for

metagenomics, 33–36 Biotechnology Information
metatranscriptomics, 30–31 (NCBI)
metatranscriptomics, 36 nifH, 129–131
Outokumpu deep borehole, 40–61 Nir. See Nitrite reductase (Nir)
single-cell isolation and sequencing, Nitrate reductase (Nar), 126
39–40 Nitrification, 118–126. See also
Metatranscriptomics, 30–31 Denitrification
Methane, 9 Nitrite oxidoreductase (Nor), 118–119,
clathrates, 9 126
methane-cycling microbes, 17 Nitrite reductase (Nir), 126
Methane ice. See Methane clathrates Nitrogen bacterial cycling, 117–131.
Methanobacteriaceae, 48–50 See also Phosphorus bacterial
Methanogen (Methanopyrus kandleri), 6 cycling; Sulfur bacterial cycling
Methanoplasmatales, 17 atmospheric di-nitrogen fixation,
Methanopyrus kandleri. See Methanogen 129–131
(Methanopyrus kandleri) denitrification, 126–129
Methanosarcina spp., 17 experimental conditions and methods,
Methanosarcinaceae, 48 121t–124t
Methanotrophs, 17–18 nitrification, 118–126
Methylomirabilis oxyfera (M. oxyfera), sections, 118
17–18 Nitrogen regulatory system, 82–83
MICP. See Microbially induced calcium Nitrous oxide (N2O), 118–119
carbonate precipitation (MICP) Nitrous oxide reductase (Nos), 126
Microbes, 9–10 Nor. See Nitrite oxidoreductase (Nor)
in carbon cycling, 17–19 Nucleic acids, 117–118
in deep carbon cycling, 17–19
Microbial activity, 19–20 O
Microbial phytases, 136 Operational taxonomical units (OTUs),
Microbial urease, 81–82 32, 130–131
Microbially induced calcium carbonate Organic carbon sources, 9–10
precipitation (MICP), 84–87 Organic solids, 8–9
Microbiological research, processing and OTUs. See Operational taxonomical units
maintenance of samples for, 29 (OTUs)
Micrococcus ureae (M. ureae), 82 Outokumpu deep borehole
million years ago (mya), 5–6 metagenomics, 40. See also
Miscanthus grass-based biochar, Terrestrial deep subsurface
130–131 microbiomes
Molecular tools, 120–125 assemblies and insights into microbial
Mollicutes, 48 communities, 53–60
mRNA. See messenger RNA (mRNA) data analysis of borehole water and
mya. See million years ago (mya) fracture zone metagenomes
gene prediction and annotation, 45–46
N metagenome assembly, 44–45
Nar. See Nitrate reductase (Nar) quantity and quality of metagenomic
National Center for Biotechnology sequences, 42–44
Information (NCBI), 21 in fracture zone samples, 46–53
Index 165
KEGG Q
accumulation curves of, 56f Quality assurance, 26
carbon, nitrogen, methane, and sulfur Quality control (QC), 37
metabolism-associated KEGG
modules, 58t–59t
R
Venn diagram, 60f
Racemization, 19–20
metagenomic samples in, 43t
Radionuclide bioprecipitation by
metagenomics of deep subsurface life,
urease-producing bacteria,
41–42
95–97
relative abundance levels
Randomized axelerated maximum
of archaeal families in, 51f
likelihood tree (RAxML),
of bacterial families in, 49f
130–131, 132f
of viral families in, 52f
Rarefaction analysis, 32
species distribution in borehole water,
redox reactions. See Oxidation-reduction
46–53
reactions (redox reactions)
Oxidation-reduction reactions (redox
Retriever-type samplers, 28
reactions), 13
Rhizobium, 110–111, 129
Oxygen, 4
Ribosomal genes, 38–39
ribosomal RNA (rRNA), 21
P
Paecilomyces javanicus (P. javanicus), 100
PAO. See Potential ammonia oxidation S
(PAO) Sequencing-based microbial ecological
PAVE-type sampler, 28 studies, 31–32
Peptococcaceae, 48 Serpentinite, 11
Phosphatases, 135–136 Serpentinization process, 11, 13
Phosphorus bacterial cycling, 132–138. Single-cell isolation and sequencing,
See also Nitrogen bacterial cycling; 39–40
Sulfur bacterial cycling Soil
carbon-bonded phosphorus, 138 incubations, 130
ester-bound phosphorus mobilization, microbiota, 112–116
135–137 South African Gold Mine Crenarchaeotic
growth of soil isolate, 134f Group (SAGMEG), 22
inorganically bound phosphorus Strontium (Sr), 97
90
mobilization, 133–135 Sr, 96
Photosynthetic microorganisms and plants, Sulfate–ester hydrolysis, 140–141
4 Sulfate–methane transition zone, 22
Phytate, 136 Sulfonate-bound sulfur mobilization,
Phytoremediation methods, 81 141–143
Potassium (K), 10 Sulfur bacterial cycling, 139–143. See also
Potential ammonia oxidation (PAO), 125 Nitrogen bacterial cycling;
Protein-coding Phosphorus bacterial cycling
genes, 45 ester-bound sulfur mobilization,
sequences, 38–39 140–141
Proteobacteria, 21 sulfonate-bound sulfur mobilization,
Pyrolysis, 139–140 141–143
166 Index
T Thorium (Th), 10
Terminal restriction fragment length Toxic metals, 80, 85
polymorphism (T-RFLP), 113–114 Tri-calcium phosphate (TCP), 133–135,
Terra Preta (T. Preta), 113 135f
Terrabacter tumescens (T. tumescens), 90 Tricarboxylic acid (TCA), 55–57
Terrestrial deep subsurface life, 3–4 Tube sampling, 27
Terrestrial deep subsurface microbiomes.
See also Outokumpu deep U
borehole metagenomics Uranium (U), 10
exploring diversity, 20–21 phosphate precipitation, 95–96
archaea, 22 Urea amidohydrolase. See Urease
bacteria, 21 Urease, 82–83
eukaryotes, 22–23 Urease-based MICP, 85–87
viruses, 23 Ureolytic enzyme, 82
metagenomics, 30–31, 33–36
amplicon sequencing, 31–33
data analysis, 37–39 V
marker genes in deep biosphere Viruses, 23
research, 34t–35t
metatranscriptomics, 30–31, 36 W
single-cell isolation and sequencing, Water, 7, 28
39–40 samples, 42
CONTENTS OF PREVIOUS VOLUMES
VOLUME 40
Manipulations of Catabolic Genes for the
Microbial Cellulases: Protein Degradation and Detoxification of
Architecture, Molecular Properties, Xenobiotics
and Biosynthesis Rup Lal, Sukanya Lal, P. S. Dhanaraj,
Ajay Singh and Kiyoshi Hayashi and D. M. Saxena
Factors Inhibiting and Stimulating Bacterial Aqueous Two-Phase Extraction for
Growth in Milk: An Historical Downstream Processing of Enzymes/
Perspective Proteins
D. K. O’Toole K. S. M. S. Raghava Rao, N. K. Rastogi,
M. K. Gowthaman, and N. G. Karanth
Challenges in Commercial Biotechnology.
Part I. Product, Process, and Market Biotechnological Potentials of Anoxygenic
Discovery Phototrophic Bacteria. Part I. Production
Ales Prokop of Single Cell Protein, Vitamins,
Ubiquinones, Hormones, and Enzymes
Challenges in Commercial Biotechnology.
and Use in Waste Treatment
Part II. Product, Process, and Market
Ch. Sasikala and Ch. V. Ramana
Development
Ales Prokop Biotechnological Potentials of Anoxygenic
Phototrophic Bacteria. Part II. Bio-
Effects of Genetically Engineered
polyesters, Biopesticide, Biofuel, and
Microorganisms on Microbial
Biofertilizer
Populations and Processes in Natural
Ch. Sasikala and Ch. V. Ramana
Habitats
Jack D. Doyle, Guenther Stotzky, Index
Gwendolyn McClung, and
Charles W. Hendricks
VOLUME 42
Detection, Isolation, and Stability of
Megaplasmid-Encoded Chloroaromatic The Insecticidal Proteins of Bacillus
Herbicide-Degrading Genes within thuringiensis
Pseudomonas Species P. Ananda Kumar, R. P. Sharma,
Douglas J. Cork and Amjad Khalil and V. S. Malik
Index Microbiological Production of Lactic
Acid
John H. Litchfield
VOLUME 41 Biodegradable Polyesters
Ch. Sasikala
Microbial Oxidation of Unsaturated Fatty
The Utility of Strains of Morphological
Acids
Group II Bacillus
Ching T. Hou
Samuel Singer
Improving Productivity of Heterologous
Phytase
Proteins in Recombinant Saccharomyces
Rudy J. Wodzinski and A. H. J. Ullah
cerevisiae Fermentations
Amit Vasavada Index
167 j
168 Contents of Previous Volumes
VOLUME 43 Thermal Processing of Foods, A Retro-

spective, Part I: Uncertainties in Thermal
Production of Acetic Acid by Clostridium Processing and Statistical Analysis
thermoaceticum M. N. Ramesh, S. G. Prapulla,
Munir Cheryan, Sarad Parekh, Minish Shah, M. A. Kumar, and M. Mahadevaiah
and Kusuma Witjitra
Thermal Processing of Foods, A
Contact Lenses, Disinfectants, and Retrospective, Part II: On-Line Methods
Acanthamoeba Keratitis for Ensuring Commercial Sterility
Donald G. Ahearn and Manal M. Gabriel M. N. Ramesh, M. A. Kumar,
Marine Microorganisms as a Source of New S. G. Prapulla, and M. Mahadevaiah
Natural Products
Index
V. S. Bernan, M. Greenstein,
and W. M. Maiese
Stereoselective Biotransformations in VOLUME 45
Synthesis of Some Pharmaceutical
One Gene to Whole Pathway:
Intermediates
The Role of Norsolorinic Acid in
Ramesh N. Patel
Aflatoxin Research
Microbial Xylanolytic Enzyme System: J. W. Bennett, P.-K. Chang, and
Properties and Applications D. Bhatnagar
Pratima Bajpai
Formation of Flavor Compounds in Cheese
Oleaginous Microorganisms: An Assessment P. F. Fox and J. M. Wallace
of the Potential
The Role of Microorganisms in Soy Sauce
Jacek Leman
Production
Index Desmond K. O’Toole
Gene Transfer Among Bacteria in Natural
VOLUME 44 Environments
Xiaoming Yin and G. Stotzky
Biologically Active Fungal Metabolites
Cedric Pearce Breathing Manganese and Iron: Solid-State
Respiration
Old and New Synthetic Capacities of
Kenneth H. Nealson and Brenda Little
Baker’s Yeast
P. D’Arrigo,G. Pedrocchi-Fantoni, Enzymatic Deinking
and S. Servi Pratima Bajpai
Investigation of the Carbon- and Sulfur- Microbial Production of Docosahexaenoic
Oxidizing Capabilities of Microorganisms Acid (DHA, C22:6)
by Active-Site Modeling Ajay Singh and Owen P. Word
Herbert L. Holland Index
Microbial Synthesis of D-Ribose: Metabolic
Deregulation and Fermentation Process
VOLUME 46
P. de Wulf and E. J. Vandamme
Production and Application of Tannin Acyl Cumulative Subject Index
Hydrolase: State of the Art
P. K. Lekha and B. K. Lonsane
VOLUME 47
Ethanol Production from Agricultural
Biomass Substrates Seeing Red: The Story of Prodigiosin
Rodney J. Bothast and Badal C. Saha J. W. Bennett and Ronald Bentley
Contents of Previous Volumes 169
Microbial/Enzymatic Synthesis of Chiral The Role of Microorganisms in Ecological

Drug Intermediates Risk Assessment of Hydrophobic
Ramesh N. Patel Organic Contaminants in Soils
Recent Developments in the C. J. A. MacLeod, A. W. J. Morriss,
Molecular Genetics of the Erythromycin- and K. T. Semple
Producing Organism Saccharopolyspora The Development of Fungi: A New
erythraea Concept Introduced By Anton de Bary
Thomas J. Vanden Boom Gerhart Drews
Bioactive Products from Streptomyces Bartolomeo Gosio, 1863–1944: An
Vladisalv Behal Appreciation
Advances in Phytase Research Ronald Bentley
Edward J. Mullaney, Catherine B. Daly, Index
and Abdul H. J. Ullah
Biotransformation of Unsaturated Fatty
Acids of industrial Products VOLUME 49
Ching T. Hou
Ethanol and Thermotolerance in the Biodegredation of Explosives
Bioconversion of Xylose by Yeasts Susan J. Rosser, Amrik Basran,
Thomas W. Jeffries and Yong-Su Jin Emmal R. Travis, Christopher E. French,
and Neil C. Bruce
Microbial Degradation of the Pesticide
Lindane (g-Hexachlorocyclohexane) Biodiversity of Acidophilic Prokaryotes
Brajesh Kumar Singh, Ramesh Chander Kevin B. Hallberg and D. Barrie Johnson
Kuhad, Ajay Singh, K. K. Tripathi, Laboratory Birproduction of Paralytic
and P. K. Ghosh Shellfish Toxins in Dinoflagellates
Microbial Production of Oligosaccharides: Dennis P. H. Hsieh, Dazhi Wang,
A Review and Garry H. Chang
S. G. Prapulla, V. Subhaprada, Metal Toxicity in Yeasts and the Role of
and N. G. Karanth Oxidative Stress
S. V. Avery
Index
Foodbourne Microbial Pathogens and the
Food Research Institute
M. Ellin Doyle and Michael W. Pariza
VOLUME 48 Alexander Flemin and the Discovery of
Penicillin
Biodegredation of Nitro-Substituted J. W. Bennett and King-Thom Chung
Explosives by White-Rot Fungi:
Index
A Mechanistic Approach
Benoit Van Aken and Spiros N. Agathos
Microbial Degredation of Pollutants in Pulp VOLUME 50
Mill Effluents
Pratima Bajpai Paleobiology of the Archean
Bioremediation Technologies for Sherry L. Cady
Metal-Containing Wastewaters A Comparative Genomics Approach for
Using Metabolically Active Studying Ancestral Proteins and
Microorganisms Evolution
Thomas Pumpel and Kishorel M. Paknikar Ping Liang and Monica Riley
Chromosome Packaging by Archaeal The Development of the Penicillin

Histones Production Process in Delft, The
Kathleen Sandman and John N. Reeve Netherlands, During World War II
DNA Recombination and Repair in the Under Nazi Occupation
Archaea Marlene Burns and Piet W. M. van Dijck
Erica M. Seitz, Cynthia A. Haseltine, Genomics for Applied Microbiology
and Stephen C. Kowalczykowski William C. Nierman and Karen E. Nelson
Basal and Regulated Transcription in Index
Archaea
J€org Soppa
Protein Folding and Molecular Chaperones
in Archaea VOLUME 52
Michel R. Leroux
Soil-Based Gene Discovery: A New
Archaeal Proteasomes: Proteolytic
Technology to Accelerate and Broaden
Nanocompartments of the Cell
Biocatalytic Applications
Julie A. Maupin-Furlow,
Kevin A. Gray, Toby H. Richardson,
Steven J. Kaczowka, Mark S. Ou,
Dan E. Robertson, Paul E. Swanson,
and Heather L. Wilson
and Mani V. Subramanian
Archaeal Catabolite Repression: A Gene
The Potential of Site-Specific
Regulatory Paradigm
Recombinases as Novel Reporters in
Elisabetta Bini and Paul Blum
Whole-Cell Biosensors of Pollution
Index Paul Hinde, Jane Meadows, Jon Saunders,
and Clive Edwards
Microbial Phosphate Removal and Poly-
phosphate Production from Wastewaters
VOLUME 51
John W. McGrath and John P. Quinn
The Biochemistry and Molecular Biology of Biosurfactants: Evolution and Diversity in
Lipid Accumulation in Oleaginous Bacteria
Microorganisms Raina M. Maier
Colin Ratledge and James P. Wynn Comparative Biology of Mesophilic and
Bioethanol Technology: Developments and Thermophilic Nitrile Hydratases
Perspectives Don A. Cowan, Rory A. Cameron,
Owen P. Ward and Ajay Singh and Tsepo L. Tsekoa
Progress of Aspergillus oryzae From Enzyme Adaptation to Gene
Genomics Regulation
Masayuki Machida William C. Summers
Transmission Genetics of Microbotryum Acid Resistance in Escherichia coli Hope
violaceum(Ustilago violacea): T. Richard and John W. Foster
A Case History Iron Chelation in Chemotherapy
E. D. Garber and M. Ruddat Eugene D. Weinberg
Molecular Biology of the Koji Molds Angular Leaf Spot: A Disease Caused by the
Katsuhiko Kitamoto Fungus Phaeoisariopsis griseola (Sacc.)
Noninvasive Methods for the Investigation Ferraris on Phaseolus vulgaris L.
of Organisms at Low Oxygen Levels Sebastian Stenglein, L. Daniel Ploper,
David Lloyd Oscar Vizgarra, and Pedro Balatti
The Fungal Genetics Stock Center: From Tissue Infection and Site-Specific Gene
Molds to Molecules Expression in Candida albicans
Kevin McCluskey Chantal Fradin and Bernard Hube
Adaptation by Phase Variation in LuxS and Autoinducer-2: Their
Pathogenic Bacteria Contribution to Quorum Sensing and
Laurence Sala€un, Lori A. S. Snyder, Metabolism in Bacteria
and Nigel J. Saunders Klaus Winzer, Kim R. Hardie,
What Is an Antibiotic? Revisited and Paul Williams
Ronald Bentley and J. W. Bennett Microbiological Contributions to the Search
An Alternative View of the Early History of of Extraterrestrial Life
Microbiology Brendlyn D. Faison
Milton Wainwright Index
The Delft School of Microbiology, from the
Nineteenth to the Twenty-first Century
Lesley A. Robertson VOLUME 54
Index Metarhizium spp.: Cosmopolitan Insect-
Pathogenic Fungi – Mycological Aspects
Donald W. Roberts and Raymond
VOLUME 53 J. St. Leger
Biodegradation of Organic Pollutants in the Molecular Biology of the Burkholderia
Rhizosphere cepacia Complex
Liz J. Shaw and Richard G. Burns Jimmy S. H. Tsang
Anaerobic Dehalogenation of Organohalide Non-Culturable Bacteria in Complex
Contaminants in the Marine Commensal Populations
Environment William G. Wade
Max M. H€aggblom, Young-Boem Ahn, l Red-Mediated Genetic Manipulation of
Donna E. Fennell, Lee J. Kerkhof, Antibiotic-Producing Streptomyces
and Sung-Keun Rhee Bertolt Gust, Govind Chandra, Dagmara
Biotechnological Application of Jakimowicz, Tian Yuqing, Celia J. Bruton,
Metal-Reducing Microorganisms and Keith F. Chater
Jonathan R. Lloyd, Derek R. Lovley, Colicins and Microcins: The Next
and Lynne E. Macaskie Generation Antimicrobials
Determinants of Freeze Tolerance in Osnat Gillor, Benjamin C. Kirkup,
Microorganisms, Physiological Impor- and Margaret A. Riley
tance, and Biotechnological Applications Mannose-Binding Quinone Glycoside,
An Tanghe, Patrick Van Dijck, MBQ: Potential Utility and Action
and Johan M. Thevelein Mechanism
Fungal Osmotolerance Yasuhiro Igarashi and Toshikazu Oki
P. Hooley, D. A. Fincham, Protozoan Grazing of Freshwater Biofilms
M. P. Whitehead, and N. J. W. Clipson Jacqueline Dawn Parry
Mycotoxin Research in South Africa Metals in Yeast Fermentation Processes
M. F. Dutton Graeme M. Walker
Electrophoretic Karyotype Analysis in Fungi Interactions between Lactobacilli and
J. Beadle, M. Wright, L. McNeely, Antibiotic-Associated Diarrhea
and J. W. Bennett Paul Naaber and Marika Mikelsaar
Bacterial Diversity in the Human Gut Toxic Mold Syndrome

Sandra MacFarlane and Michael B. Levy and Jordan N. Fink
George T. MacFarlane Fungal Hypersensitivity: Pathophysiology,
Interpreting the Host-Pathogen Dialogue Diagnosis, Therapy
Through Microarrays Vincent A. Marinkovich
Brian K. Coombes, Philip R. Hardwidge, Indoor Molds and Asthma in Adults
and B. Brett Finlay Maritta S. Jaakkola and Jouni J. K. Jaakkola
The Inactivation of Microbes by Sunlight: Role of Molds and Mycotoxins in Being
Solar Disinfection as a Water Treatment Sick in Buildings: Neurobehavioral and
Process Pulmonary Impairment
Robert H. Reed Kaye H. Kilburn
Index The Diagnosis of Cognitive Impairment
Associated with Exposure to Mold
Wayne A. Gordon and Joshua B. Cantor
VOLUME 55 Mold and Mycotoxins: Effects on the
Fungi and the Indoor Environment: Their Neurological and Immune Systems in
Impact on Human Health Humans
J. D. Cooley, W. C. Wong, C. A. Jumper, Andrew W. Campbell, Jack D. Thrasher,
and D. C. Straus Michael R. Gray, and Aristo Vojdani
Fungal Contamination as a Major Identification, Remediation, and
Contributor to Sick Building Syndrome Monitoring Processes Used in a
De-Wei LI and Chin S. Yang Mold-Contaminated High School
S. C. Wilson, W. H. Holder,
Indoor Moulds and Their Associations with
K. V. Easterwood, G. D. Hubbard,
Air Distribution Systems
R. F. Johnson, J. D. Cooley,
Donald G. Ahearn, Daniel L. Price,
and D. C. Straus
Robert Simmons, Judith Noble-Wang,
and Sidney A. Crow, Jr. The Microbial Status and Remediation of
Contents in Mold-Contaminated
Microbial Cell Wall Agents and Sick
Structures
Building Syndrome
Stephen C. Wilson and Robert C. Layton
Ragnar Rylander
Specific Detection of Fungi Associated With
The Role of Stachybotrys in the Phenom-
SBS When Using Quantitative
enon Known as Sick Building Syndrome
Polymerase Chain Reaction
Eeva-Liisa Hintikka
Patricia Cruz and Linda D. Stetzenbach
Moisture-Problem Buildings with Molds
Causing Work-Related Diseases Index
Kari Reijula
Possible Role of Fungal Hemolysins in Sick VOLUME 56
Building Syndrome Potential and Opportunities for Use of
Stephen J. Vesper and Mary Jo Vesper Recombinant Lactic Acid Bacteria in
The Roles of Penicillium and Aspergillus in Human Health
Sick Building Syndrome (SBS) Sean Hanniffy, Ursula Wiedermann, Andreas
Christopher J. Schwab and David C. Straus Repa, Annick Mercenier, Catherine Daniel,
Pulmonary Effects of Stachybotrys Jean Fioramonti, Helena Tlaskolova, Hana
chartarum in Animal Studies Kozakova, Hans Israelsen, Søren Madsen,
Iwona Yike and Dorr G. Dearborn Astrid Vrang, Pascal Hols, Jean Delcour,
Peter Bron, Michiel Kleerebezem, and Jerry VOLUME 57

Wells
Microbial Transformations of Mercury:
Novel Aspects of Signaling in Streptomyces
Potentials, Challenges, and Achievements
Development
in Controlling Mercury Toxicity in the
Gilles P. van Wezel and Erik Vijgenboom
Environment
Polysaccharide Breakdown by Anaerobic Tamar Barkay and Irene Wagner-D€obler
Microorganisms Inhabiting the
Interactions Between Nematodes and
Mammalian Gut
Microorganisms: Bridging Ecological and
Harry J. Flint
Molecular Approaches
Lincosamides: Chemical Structure, Keith G. Davies
Biosynthesis, Mechanism of Action,
Biofilm Development in Bacteria
Resistance, and Applications
Katharine Kierek-Pearson and Ece Karatan
Jaroslav Spízek, Jitka Novotna,

and Tomas Rezanka Microbial Biogeochemistry of Uranium
Mill Tailings
Ribosome Engineering and Secondary
Edward R. Landa
Metabolite Production
Kozo Ochi, Susumu Okamoto, Yuzuru Yeast Modulation of Wine Flavor
Tozawa, Takashi Inaoka, Takeshi Hosaka, Jan H. Swiegers and Isak S. Pretorius
Jun Xu, and Kazuhiko Kurosawa Moving Toward a Systems Biology
Developments in Microbial Methods for the Approach to the Study of Fungal
Treatment of Dye Effluents Pathogenesis in the Rice Blast Fungus
R. C. Kuhad, N. Sood, K. K. Tripathi, Magnaporthe grisea
A. Singh, and O. P. Ward Claire Veneault-Fourrey
and Nicholas J. Talbot
Extracellular Glycosyl Hydrolases from
Clostridia The Biotrophic Stages of Oomycete–Plant
Wolfgang H. Schwarz, Vladimir V. Zverlov, Interactions
and Hubert Bahl Laura J. Grenville-Briggs and Pieter van West
Kernel Knowledge: Smut of Corn Contribution of Nanosized Bacteria to the
María D. García-Pedrajas and Scott E. Gold Total Biomass and Activity of a Soil
Microbial Community
Bacterial ACC Deaminase and the
Nicolai S. Panikov
Alleviation of Plant Stress
Bernard R. Glick Index
Uses of Trichoderma spp. to Alleviate or
Remediate Soil and Water Pollution VOLUME 58
G. E. Harman, M. Lorito, and J. M. Lynch
Physiology and Biotechnology of
Bacteriophage Defense Systems and
Aspergillus
Strategies for Lactic Acid Bacteria
O. P. Ward, W. M. Qin, J.
Joseph M. Sturino and Todd R.
Dhanjoon, J. Ye, and A. Singh
Klaenhammer
Conjugative Gene Transfer in the
Current Issues in Genetic Toxicology
Gastrointestinal Environment
Testing for Microbiologists
Tine Rask Licht and Andrea Wilcks
Kristien Mortelmans and Doppalapudi S.
Rupa Force Measurements Between a Bacterium
and Another Surface In Situ
Index Ruchirej Yongsunthon and Steven K. Lower
Actinomycetes and Lignin Degradation The Role of Helen Purdy Beale in the Early
Ralph Kirby Development of Plant Serology and
An ABC Guide to the Bacterial Toxin Virology
Complexes Karen-Beth G. Scholthof
Richard ffrench-Constant and Nicholas and Paul D. Peterson
Waterfield Index
Engineering Antibodies for Biosensor
Technologies
Sarah Goodchild, Tracey Love, VOLUME 60
Neal Hopkins, and Carl Mayers Microbial Biocatalytic Processes and Their
Molecular Characterization of Development
Ochratoxin A Biosynthesis and John M. Woodley
Producing Fungi Occurrence and Biocatalytic Potential of
J. O’Callaghan and A. D. W. Dobson Carbohydrate Oxidases
Index Erik W. van Hellemond, Nicole G. H.
Leferink, Dominic P. H. M. Heuts,
Marco W. Fraaije, and Willem J. H.
van Berkel
VOLUME 59 Microbial Interactions with Humic
Substances
Biodegradation by Members of the Genus
J. Ian Van Trump, Yvonne Sun,
Rhodococcus: Biochemistry, Physiology,
and John D. Coates
and Genetic Adaptation
Michael J. Larkin, Leonid A. Kulakov, Significance of Microbial Interactions in the
and Christopher C. R. Allen Mycorrhizosphere
Gary D. Bending, Thomas J. Aspray,
Genomes as Resources for Biocatalysis
and John M. Whipps
Jon D. Stewart
Escherich and Escherichia
Process and Catalyst Design Objectives for
Herbert C. Friedmann
Specific Redox Biocatalysis
Daniel Meyer, Bruno B€uhler, Index
and Andreas Schmid
The Biosynthesis of Polyketide Metabolites VOLUME 61
by Dinoflagellates
Kathleen S. Rein and Richard V. Snyder Unusual Two-Component Signal Trans-
Biological Halogenation has Moved far duction Pathways in the Actinobacteria
Beyond Haloperoxidases Matthew I. Hutchings
Karl-Heinz van Pée, Changjiang Dong, Acyl-HSL Signal Decay: Intrinsic to
Silvana Flecks, Jim Naismith, Bacterial Cell–Cell Communications
Eugenio P. Patallo, and Tobias Wage Ya-Juan Wang, Jean Jing Huang,
Phage for Rapid Detection and Control of and Jared Renton Leadbetter
Bacterial Pathogens in Food Microbial Exoenzyme Production in Food
Catherine E. D. Rees and Christine E. R. Peggy G. Braun
Dodd Biogenetic Diversity of Cyanobacterial
Gastrointestinal Microflora: Probiotics Metabolites
S. Kolida, D. M. Saulnier, and G. R. Ryan M. Van Wagoner, Allison K.
Gibson Drummond, and Jeffrey L. C. Wright
Pathways to Discovering New Microbial Rhizobacteria and Plant Sulfur Supply

Metabolism for Functional Genomics and Michael A. Kertesz, Emma Fellows,
Biotechnology and Achim Schmalenberger
Lawrence P. Wackett Antibiotics and Resistance Genes:
Biocatalysis by Dehalogenating Influencing the Microbial Ecosystem in
Enzymes the Gut
Dick B. Janssen Katarzyna A. Kazimierczak
Lipases from Extremophiles and Potential and Karen P. Scott
for Industrial Applications Index
Moh’d Salameh and Juergen Wiegel
In Situ Bioremediation VOLUME 63
Kirsten S. Jørgensen
Bacterial Cycling of Methyl Halides A Ferment of Fermentations: Reflections on
Hendrik Sch€afer, Laurence G. Miller, the Production of Commodity Chemicals
Ronald S. Oremland, Using Microorganisms
and J. Colin Murrell Ronald Bentley and Joan W. Bennett
Submerged Culture Fermentation of
Index “Higher Fungi”: The Macrofungi
Mariana L. Fazenda, Robert Seviour,
Brian McNeil, and Linda M. Harvey
VOLUME 62
Bioprocessing Using Novel Cell Culture
Anaerobic Biodegradation of Methyl Systems
tert-Butyl Ether (MTBE) and Related Sarad Parekh, Venkatesh Srinivasan,
Fuel Oxygenates and Michael Horn
Max M. H€aggblom, Laura K. G. Youngster, Nanotechnology in the Detection and
Piyapawn Somsamak, and Hans H. Richnow Control of Microorganisms
Controlled Biomineralization by and Pengju G. Luo and Fred J. Stutzenberger
Applications of Magnetotactic Bacteria Metabolic Aspects of Aerobic Obligate
Dennis A. Bazylinski and Sabrina Sch€ubbe Methanotrophy
The Distribution and Diversity of Yuri A. Trotsenko and John Colin Murrell
Euryarchaeota in Termite Guts Bacterial Efflux Transport in Biotechnology
Kevin J. Purdy Tina K. Van Dyk
Understanding Microbially Active Antibiotic Resistance in the Environment,
Biogeochemical Environments with Particular Reference to MRSA
Deirdre Gleeson, Frank McDermott, William Gaze, Colette O’Neill,
and Nicholas Clipson Elizabeth Wellington, and Peter Hawkey
The Scale-Up of Microbial Batch and Host Defense Peptides in the Oral Cavity
Fed-Batch Fermentation Processes Deirdre A. Devine and Celine Cosseau
Christopher J. Hewitt and Alvin W. Neinow
Index
Production of Recombinant Proteins in
Bacillus subtilis
VOLUME 64
Wolfgang Schumann
Quorum Sensing: Fact, Fiction, and Diversity of Microbial Toluene Degradation
Everything in Between Pathways
Yevgeniy Turovskiy, Dimitri Kashtanov, R. E. Parales, J. V. Parales,
Boris Paskhover, and Michael L. Chikindas D. A. Pelletier, and J. L. Ditty
Microbial Endocrinology: Experimental Conor P. O’ Byrne and Kimon A. G.

Design Issues in the Study of Interking- Karatzas
dom Signalling in Infectious Disease Protein Secretion and Membrane Insertion
Primrose P. E. Freestone and Mark Lyte Systems in Bacteria and Eukaryotic
Molecular Genetics of Selenate Reduction Organelles
by Enterobacter cloacae SLD1a-1 Milton H. Saier, Chin Hong Ma,
Nathan Yee and Donald Y. Kobayashi Loren Rodgers, Dorjee G. Tamang,
Metagenomics of Dental Biofilms and Ming Ren Yen
Peter Mullany, Stephanie Hunter, Metabolic Behavior of Bacterial Biological
and Elaine Allan Control Agents in Soil and Plant
Biosensors for Ligand Detection Rhizospheres
Alison K. East, Tim H. Mauchline, Cynthia A. Pielach, Daniel P. Roberts,
and Philip S. Poole and Donald Y. Kobayashi
Islands Shaping Thought in Microbial Copper Homeostasis in Bacteria
Ecology Deenah Osman and Jennifer S. Cavet
Christopher J. van der Gast Pathogen Surveillance Through Monitoring
Human Pathogens and the Phyllosphere of Sewer Systems
John M. Whipps, Paul Hand, Ryan G. Sinclair, Christopher Y. Choi,
David A. C. Pink, and Gary D. Bending Mark R. Riley, and Charles P. Gerba
Microbial Retention on Open Food Index
Contact Surfaces and Implications for
Food Contamination
Joanna Verran, Paul Airey, Adele Packer,
and Kathryn A. Whitehead VOLUME 66
Index
Multiple Effector Mechanisms Induced by
Recombinant Listeria monocytogenes
VOLUME 65 Anticancer Immunotherapeutics
Anu Wallecha, Kyla Driscoll Carroll,
Capsular Polysaccharides in Escherichia coli Paulo Cesar Maciag, Sandra Rivera,
David Corbett and Ian S. Roberts Vafa Shahabi, and Yvonne Paterson
Microbial PAH Degradation Diagnosis of Clinically Relevant Fungi in
Evelyn Doyle, Lorraine Muckian, Medicine and Veterinary Sciences
Anne Marie Hickey, and Nicholas Clipson Olivier Sparagano and Sam Foggett
Acid Stress Responses in Listeria Diversity in Bacterial Chemotactic
monocytogenes Responses and Niche Adaptation
Sheila Ryan, Colin Hill, and Lance D. Miller, Matthew H. Russell,
Cormac G. M. Gahan and Gladys Alexandre
Global Regulators of Transcription in Cutinases: Properties and Industrial
Escherichia coli: Mechanisms of Action Applications
and Methods for Study Tatiana Fontes Pio and Gabriela Alves
David C. Grainger and Stephen J. W. Busby Macedo
The Role of Sigma B (sB) in the Stress Microbial Deterioration of Stone
Adaptations of Listeria monocytogenes: MonumentsdAn Updated Overview
Overlaps Between Stress Adaptation and Stefanie Scheerer, Otto Ortega-Morales,
Virulence and Christine Gaylarde
Microbial Processes in Oil Fields: Culprits, Biotechnological Applications of

Problems, and Opportunities Recombinant Microbial Prolidases
Noha Youssef, Mostafa S. Elshahed, Casey M. Theriot, Sherry R. Tove,
and Michael J. McInerney and Amy M. Grunden
Index The Capsule of the Fungal Pathogen
Cryptococcus neoformans
Oscar Zaragoza, Marcio L. Rodrigues,
VOLUME 67 Magdia De Jesus, Susana Frases,
Ekaterina Dadachova,
Phage Evolution and Ecology and Arturo Casadevall
Stephen T. Abedon Baculovirus Interactions In Vitro and
Nucleoid-Associated Proteins and Bacterial In Vivo
Physiology Xiao-Wen Cheng and
Charles J. Dorman Dwight E. Lynn
Biodegradation of Pharmaceutical and Posttranscriptional Gene Regulation in
Personal Care Products Kaposi’s Sarcoma-Associated
Jeanne Kagle, Abigail W. Porter, Herpesvirus
Robert W. Murdoch, Giomar Rivera-Cancel, Nicholas K. Conrad
and Anthony G. Hay
Index
Bioremediation of Cyanotoxins
Christine Edwards and Linda A. Lawton
Virulence in Cryptococcus Species VOLUME 69
Hansong Ma and Robin C. May
Variation in Form and Function: The
Molecular Networks in the Fungal
Helix-Turn-Helix Regulators of the
Pathogen Candida albicans
GntR Superfamily
Rebecca A. Hall, Fabien Cottier, Paul A. Hoskisson and
and Fritz A. M€uhlschlegel
Sébastien Rigali
Temperature Sensors of Eubacteria
Biogenesis of the Cell Wall and Other
Wolfgang Schumann Glycoconjugates of Mycobacterium
Deciphering Bacterial Flagellar Gene tuberculosis
Regulatory Networks in the Genomic Devinder Kaur, Marcelo E. Guerin,
Era Henrieta Skovierova, Patrick J. Brennan,
Todd G. Smith and Timothy R. Hoover and Mary Jackson
Genetic Tools to Study Gene Expression Antimicrobial Properties of
During Bacterial Pathogen Infection Hydroxyxanthenes
Ansel Hsiao and Jun Zhu Joy G. Waite and Ahmed E. Yousef
Index In Vitro Biofilm Models: An Overview
Andrew J. McBain
Zones of Inhibition? The Transfer of
VOLUME 68 Information Relating to Penicillin in
Europe during World War II
Bacterial L-Forms
Gilbert Shama
E. J. Allan, C. Hoischen, and J. Gumpert
The Genomes of Lager Yeasts
Biochemistry, Physiology and Biotech-
Ursula Bond
nology of Sulfate-Reducing Bacteria
Larry L. Barton and Guy D. Fauque Index
VOLUME 70 Cell Immobilization for Production of

Lactic Acid: Biofilms Do It Naturally
Thermostable Enzymes as Biocatalysts in the Suzanne F. Dagher, Alicia L. Ragout,
Biofuel Industry Faustino Si~neriz, and José M. Bruno-Barcena
Carl J. Yeoman, Yejun Han, Dylan Dodd,
Microbial Fingerprinting using Matrix-
Charles M. Schroeder, Roderick I. Mackie,
Assisted Laser Desorption Ionization
and Isaac K. O. Cann
Time-Of-Flight Mass Spectrometry
Production of Biofuels from Synthesis Gas (MALDI-TOF MS): Applications and
Using Microbial Catalysts Challenges
Oscar Tirado-Acevedo, Mari S. Chinn, R. Giebel, C. Worden, S. M. Rust,
and Amy M. Grunden G. T. Kleinheinz, M. Robbins,
Microbial Naphthenic Acid Degradation and T. R. Sandrin
Corinne Whitby
Index
Surface and Adhesion Properties of
Lactobacilli
G. Deepika and D. Charalampopoulos VOLUME 72
Shining Light on the Microbial World: The
Application of Raman Evolution of the Probiotic Concept: From
Microspectroscopy Conception to Validation and
Wei E. Huang, Mengqiu Li, Acceptance in Medical Science
Roger M. Jarvis, Royston Goodacre, Walter J. Dobrogosz, Trent J. Peacock,
and Steven A. Banwart and Hosni M. Hassan
Detection of Invasive Aspergillosis Prokaryotic and Eukaryotic Diversity of the
Christopher R. Thornton Human Gut
Julian R. Marchesi
Bacteriophage Host Range and Bacterial
Resistance Oxalate-Degrading Bacteria of the Human
Paul Hyman and Stephen T. Abedon Gut as Probiotics in the Management of
Kidney Stone Disease
Index Valerie R. Abratt and Sharon J. Reid
Morphology and Rheology in Filamentous
Cultivations
VOLUME 71 T. Wucherpfennig, K. A. Kiep, H. Driouch,
C. Wittmann, and R. Krull
Influence of Escherichia coli Shiga Toxin on
the Mammalian Central Nervous System Methanogenic Degradation of Petroleum
Fumiko Obata Hydrocarbons in Subsurface Environ-
ments: Remediation, Heavy Oil Forma-
Natural Products for Type II Diabetes
tion, and Energy Recovery
Treatment
N. D. Gray, A. Sherry, C. Hubert,
Amruta Bedekar, Karan Shah,
J. Dolfing, and I. M. Head
and Mattheos Koffas
Experimental Models Used to Study Index
Human Tuberculosis
Ronan O’Toole
VOLUME 73
Biosynthesis of Peptide Signals in
Gram-Positive Bacteria Heterologous Protein Secretion by Bacillus
Matthew Thoendel and Alexander R. Species: From the Cradle to the Grave
Horswill Susanne Pohl and Colin R. Harwood
Function of Protein Phosphatase-1, Glc7, in Mucosal Biofilm Communities in the

Saccharomyces cerevisiae Human Intestinal Tract
John F. Cannon Sandra Macfarlane, Bahram Bahrami,
Milliliter-Scale Stirred Tank Reactors for and George T. Macfarlane
the Cultivation of Microorganisms Index
Ralf Hortsch and Dirk Weuster-Botz
Type I Interferon Modulates the Battle of VOLUME 76
Host Immune System Against Viruses
The Regulation of Secondary Metabolism
Young-Jin Seo and Bumsuk Hahm
and Mutualism in the Insect Pathogenic
Index Bacterium Photorhabdus luminescens
Susan A. Joyce, Lea Lango,
VOLUME 74 and David J. Clarke
Assessing the Relevance of Light for Fungi:
Bacterial Strategies for Growth on Aromatic
Implications and Insights into the
Compounds
Network of Signal Transmission
Kevin W. George and Anthony G. Hay
Monika Schmoll
Recent Advances in Hantavirus Molecular
Detection and Quantification of Microbial
Biology and Disease
Cells in Subsurface Sediments
Islam T. M. Hussein, Abdul Haseeb,
Jens Kallmeyer
Absarul Haque, and Mohammad A. Mir
Antigenic Variation and the Genetics and Index
Epigenetics of the PfEMP1 Erythrocyte
Surface Antigens in Plasmodium VOLUME 77
falciparum Malaria
David E. Arnot and Anja T. R. Jensen Phage Therapy Pharmacology: Calculating
Phage Dosing
Biological Warfare of the Spiny Plant: Stephen Abedon
Introducing Pathogenic Microorganisms
into Herbivore’s Tissues From Rio Tinto to Mars: The Terrestrial
Malka Halpern, Avivit Waissler, Adi Dror, and Extraterrestrial Ecology of
and Simcha Lev-Yadun Acidophiles
R. Amils, E. Gonzalez-Toril, A. Aguilera,
Index N. Rodríguez, D. Fernandez-Remolar,
F. Gomez, A. García-Moyano, M. Malki,
VOLUME 75 M. Oggerin, I. Sanchez-Andrea,
and J. L. Sanz
Myxobacterial Vesicles: Death at a Distance?
Fungal Adaptation to Extremely High Salt
David E. Whitworth
Concentrations
Diversity, Structure, and Size of N2O- Cene Gostinc ar, Metka Lenassi,
Producing Microbial Communities in Nina Gunde-Cimerman, and Ana Plemenitas
SoilsdWhat Matters for Their
Resistance of Yeasts to Weak Organic Acid
Functioning?
Food Preservatives
Gesche Braker and Ralf Conrad
Peter W. Piper
Solar-Driven Hydrogen Production in
Silver Nanoparticles: A Microbial
Green Algae
Perspective
Steven J. Burgess, Bojan Tamburic,
M. J. Sweet and I. Singleton
Fessehaye Zemichael, Klaus Hellgardt,
and Peter J. Nixon Index
VOLUME 78 Pleiomorphism in Mycobacterium

Leif A. Kirsebom, Santanu Dasgupta,
Phage Therapy Pharmacology: Phage and Br€annvall M. Fredrik Pettersson
Cocktails
Review: Metal-Based Nanoparticles; Size,
Benjamin K. Chan and Stephen T. Abedon
Function, and Areas for Advancement in
Utility of Greater Wax Moth Larva (Galleria Applied Microbiology
mellonella) for Evaluating the Toxicity Michael J. Sweet, Ashley Chesser,
and Efficacy of New Antimicrobial and Ian Singleton
Agents
Andrew P. Desbois and Peter J. Coote Index
Bacteriophages and Nanostructured
Materials VOLUME 81
Paul Hyman Heterologous Gene Expression in
Microbial Communities Associated with Filamentous Fungi
House Dust Xiaoyun Su, George Schmitz, Meiling
Helena Rintala, Miia Pitk€aranta, Zhang, Roderick I. Mackie,
and Martin T €aubel and Isaac K. O. Cann
Serpula lacrymans, Wood and Buildings Staphylococcal Biofilms: Quest for the
S. C. Watkinson and D. C. Eastwood Magic Bullet
Index Jamie L. Brooks and Kimberly K. Jefferson
Climate Change and Defense against
VOLUME 79 Pathogens in Plants
Adrian C. Newton, Lesley Torrance,
The Molecular Basis of pH Sensing, Nicola Holden, Ian K. Toth, David E. L.
Signaling, and Homeostasis in Fungi Cooke, Vivian Blok, and Eleanor M. Gilroy
Elaine Bignell Advances in the In-Field Detection of
Barriers to Horizontal Gene Transfer in Microorganisms in Ice
Campylobacter jejuni Megan J. Barnett, David A. Pearce,
Susan P. Gardner and Jonathan W. Olson and David C. Cullen
Innate Immunity to Intracellular Pathogens: Microsatellites for Microbiologists
Lessons Learned from Legionella Michael J. Sweet, Lucinda A. Scriven,
pneumophila and Ian Singleton
Sunny Shin Modern Advances against Plague
Culture Collections Petra C.F. Oyston and E. Diane Williamson
David Smith Salmonella Enteritidis in Shell Eggs:
Index Evolving Concerns and Innovative
Control Measures
VOLUME 80 Jennifer J. Perry and Ahmed E. Yousef
Index
The Bacterial Etiology of Preterm Birth
Kimberly K. Jefferson
VOLUME 82
The Future of Taxonomy
Amanda Lousie Jones Insights into Lignin Degradation and its
Mathematics Make Microbes Beautiful, Potential Industrial Applications
Beneficial, and Bountiful Ahmed M. Abdel-Hamid, Jose O. Solbiati,
John R. Jungck and Isaac K. O. Cann
Bacterial Volatiles and Diagnosis of Carbon-Rich Wastes as Feedstocks for

Respiratory Infections Biodegradable Polymer (Poly-
James E. Graham hydroxyalkanoate) Production Using
Polymicrobial Multi-functional Approach Bacteria
for Enhancement of Crop Productivity Jasmina Nikodinovic-Runic, Maciej Guzik,
Chilekampalli A. Reddy and Ramu S. Shane T. Kenny, Ramesh Babu,
Saravanan Alan Werker, and Kevin E. O Connor
Recombinant Production of Spider Silk Index
Proteins
Aniela Heidebrecht and Thomas Scheibel
Mechanisms of Immune Evasion in
VOLUME 85
Leishmaniasis Yeast Petites and Small Colony Variants: For
Gaurav Gupta, Steve Oghumu, and Abhay Everything There Is a Season
R. Satoskar Martin Day
Index Fungal Spores for Dispersion in Space and
Time
Timon T. Wyatt, Han A. B. W €osten,
VOLUME 83 and Jan Dijksterhuis
Regulation of Bacterial Pathogenesis
Screening and Expression of Genes from
by Intestinal Short-Chain
Metagenomes
Fatty Acids
Benedikt Leis, Angel Angelov, and Wolfgang
Yvonne Sun and Mary X. D. O’Riordan
Liebl
Chromera velia: The Missing Link in the
The Escherichia coli Nucleoid in Stationary
Evolution of Parasitism
Phase
Kate Weatherby and Dee Carter
Anne S. Meyer and David C. Grainger
Living with Stress: A Lesson from the Index
Enteric Pathogen Salmonella enterica
Sebastian Runkel, Hannah C. Wells, and
VOLUME 86
Gary Rowley
Chitin and Glucan, the Yin and Yang of the Pseudomonas aeruginosa Biofilms:
Fungal Cell Wall, Implications for Mechanisms of Immune Evasion
Antifungal Drug Discovery and Therapy Maria Alhede, Thomas Bjarnsholt,
Carol A. Munro Michael Givskov, and Morten Alhede
Index Insights into the Biology of Borrelia burg-
dorferi Gained Through the Application
of Molecular Genetics
VOLUME 84 Ashley M. Groshong and Jon S. Blevins
Shiga Toxin-Producing Escherichia coli
Sensing and Adapting to Anaerobic James L. Smith, Pina M. Fratamico,
Conditions by Staphylococcus aureus and Nereus W. Gunther IV
Jeffrey W. Hall and Yinduo Ji Modern Taxonomy of Biotechnologically
The Clinical Importance of Fungal Biofilms Important Aspergillus and Penicillium
Gordon Ramage and Craig Williams Species
The Natural History of Yeast Prions Jos Houbraken, Ronald P. de Vries,
Mick F. Tuite and Robert A. Samson
Upstream Regulation of Mycotoxin Benzoyl-CoA, a Universal Biomarker for

Biosynthesis Anaerobic Degradation of Aromatic
Fahad Alkhayyat and Jae-Hyuk Yu Compounds
Abigail W. Porter and Lily Y. Young
Index
Index
VOLUME 87 VOLUME 89
The Tools for Virulence of Cryptococcus Morphogenesis of Streptomyces in
neoformans Submerged Cultures
Carolina Coelho, Anamelia Lorenzetti Bocca, Dino van Dissel, Dennis Claessen,
and Arturo Casadevall and Gilles P. van Wezel
Community Interactions of Oral Interactions Between Arbuscular
Streptococci Mycorrhizal Fungi and Organic Material
Nicholas S. Jakubovics, Sufian A. Yassin, Substrates
and Alexander H. Rickard Angela Hodge
Bioprospecting in the Genomic Age Transcription Regulation in the Third
Michael A. Hicks and Kristala L.J. Prather Domain
Environmental and Animal-Associated Elizabeth A. Karr
Enterococci Bacteria–Phage Interactions in Natural
Christopher Staley, Gary M. Dunny, Environments
and Michael J. Sadowsky Samuel L. Díaz-Mu~noz and Britt Koskella
An Introduction to Nitric Oxide Sensing The Interactions of Bacteria with Fungi in
and Response in Bacteria Soil: Emerging Concepts
Andrew M. Stern and Jun Zhu Irshad Ul Haq, Miaozhi Zhang, Pu Yang,
Index and Jan Dirk van Elsas
Production of Specialized Metabolites by
Streptomyces coelicolor A3(2)
VOLUME 88 Geertje van Keulen and Paul J. Dyson
Synthetic Polyester-Hydrolyzing Enzymes
The Genetic Basis of the Symbiosis Between From Thermophilic Actinomycetes
Photorhabdus and Its Invertebrate Hosts Ren Wei, Thorsten Oeser, and Wolfgang
David J. Clarke Zimmermann
Regulation of Plant Biomass Utilization in
Index
Aspergillus
Joanna E. Kowalczyk, Isabelle Benoit,
and Ronald P. de Vries VOLUME 90
Threonine Aldolases Sugar Catabolism in Aspergillus and Other
Sarah E. Franz and Jon D. Stewart Fungi Related to the Utilization of Plant
Carbohydrate-Binding Modules of Fungal Biomass
Cellulases: Occurrence in Nature, Func- Claire Khosravi, Tiziano Benocci,
tion, and Relevance in Industrial Biomass Evy Battaglia, Isabelle Benoit, and
Conversion Ronald P. de Vries
Aniko Varnai, Miia R. M€akel€a, The Evolution of Fungicide Resistance
Demi T. Djajadi, Jenni Rahikainen, John A. Lucas, Nichola J. Hawkins, and
Annele Hatakka, and Liisa Viikari Bart A. Fraaije
Genetic Control of Asexual Development The Escherichia coli Acid Stress Response and
in Aspergillus fumigatus Its Significance for Pathogenesis
Fahad Alkhayyat, Sun Chang Kim, and Daniela De Biase and Peter A. Lund
Jae-Hyuk Yu Challenges for the Production of Bioethanol
Escherichia coli ST131: The Quintessential from Biomass Using Recombinant Yeasts
Example of an International Multi- William Kricka, James Fitzpatrick, and
resistant High-Risk Clone Ursula Bond
Amy J. Mathers, Gisele Peirano, and Modulation of Bacterial Proliferation as
Johann D.D. Pitout a Survival Strategy
Colonization Factors of Enterotoxigenic Kristina Heinrich, David J. Leslie, and
Escherichia coli Kristina Jonas
T.P. Vipin Madhavan and Harry Sakellaris
Index
Index
VOLUME 91 VOLUME 93
Microbiota Regulation of the Mammalian Toward Modeling the Resistance and
Gut–Brain Axis Resilience of “Below-ground” Fungal
Aurelijus Burokas, Rachel D. Moloney, Communities: A Mechanistic and
Timothy G. Dinan, and John F. Cryan Trait-Based Approach
Aromatic Metabolism of Filamentous Fungi Ruth E. Falconer, Wilfred Otten, and Nia A.
in Relation to the Presence of Aromatic White
Compounds in Plant Biomass The Importance of the Microbial N Cycle
Miia R. M€akel€a, Mila Marinovic , in Soil for Crop Plant Nutrition
Paula Nousiainen, April J.M. Liwanag, Penny R. Hirsch and Tim H. Mauchline
Isabelle Benoit, Jussi Sipil€a, Annele Hatakka, Polyhydroxyalkanoates: Much More than
Ronald P. de Vries, and Kristiina S. Hildén Biodegradable Plastics
Candida Survival Strategies Nancy I. Lopez, M. Julia Pettinari, Pablo I.
Melanie Polke, Bernhard Hube, and Nikel, and Beatriz S. Méndez
Ilse D. Jacobsen Catabolism of Phenol and Its Derivatives in
Tailoring Specialized Metabolite Bacteria: Genes, Their Regulation, and
Production in Streptomyces Use in the Biodegradation of Toxic
Jana K. Hiltner, Iain S. Hunter, and Pollutants
Paul A. Hoskisson Jan Nesvera, Lenka Rucka, and Miroslav
Patek
Index
Index
VOLUME 92
The Genus Geobacillus and Their
Biotechnological Potential
Ali H. Hussein, Beata K. Lisowska, and
David J. Leak

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VOLUME NINETY FOUR

GEOFFREY MICHAEL GADD

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

First edition 2016

Copyright Ó 2016 Elsevier Inc. All rights reserved.

For information on all Academic Press publications

Advances in Applied Microbiology, Volume 94

1. INTRODUCTIONdDEEP CRUSTAL LIFE

1.2 The Lithosphere as a Host of Life

1.3 Physical and Geochemical Constraints

1.4 Geological Carbon Sources for Deep Subsurface Life

Figure 1 A conceptual view of the hydrogeological and hydrogeochemical conditions

source of mineral carbon. Inorganic solid phases are predominantly graphite

degradation of living organisms). Organic carbon sources can provide both

The following are the reactions producing abiotic hydrogen:

3. Serpentinization of olivine in ultramaﬁc rock (Abrajano et al., 1990;

3Fe2 SiO4 ðFe olivineÞ þ 2H2 O / 3SiO2 þ 2Fe3 O4 ðmagnetiteÞ

2Mg2 SiO4 ðMg olivineÞ þ 3H2 O / Mg3 Si2 O5 ðOHÞ4 ðserpentineÞ

ðFe; MgÞ2 SiO4 ðolivineÞ þ H2 O þ CO2 / Mg3 Si2 O5 ðOHÞ4

Hydrogen formation due to radiolysis of water can be calculated from the

Serpentinization is generally reported to occur at elevated temperatures

Figure 2 Hydrogen and carbon cycling in the deep terrestrial subsurface.

have been used to quantify the energetic mechanisms of microbial metabolism

1.5.2 Deep Carbon Cycling

1.5.2.1 Microbes Involved in Carbon Cycling

According to Liu and Whitman (2008), hydrogenotrophic methanogens are

syntrophically with ammonia oxidizers in an anaerobic environment

Nitrite : 3CH4 þ 8NO2 þ 8Hþ / 3CO2 þ 4N2 þ 10H2 O

Iron : CH4 þ 8FeðOHÞ3 þ 15Hþ / HCO3 þ 8Fe2þ þ 21H2 O

Manganese : CH4 þ 4MnO2 þ 7Hþ / HCO3 þ 4Mn2þ þ 5H2 O

Sulfate : CH4 þ SO4 2 / HS þ HCO3 þ H2 O

1.5.3 Microbial Activity

2. EXPLORING THE DIVERSITY OF TERRESTRIAL DEEP

to have enabled the lone survival of the ﬁrmicute Candidatus Desulforudis

(74.4%), with the latter sample water being characterized by a particularly

from the deep-biosphere viruses with a potential impact on carbon cycling,

3. SAMPLING OF THE DEEP BIOSPHERE

3.1 Groundwater Sampling

First, it is beneﬁcial to proceed in this order because these studies share

disinfection of the tubing and found that attached microorganisms indeed

3.2 Processing and Maintenance of Samples for

Figure 3 Field sampling for molecular microbiological analysis based on ﬁltration of

of regular ﬁltration systems, eg, polyethersulfone ﬁlters (Bomberg,

4. TERRESTRIAL DEEP SUBSURFACE MICROBIOMES:

2014; Purkamo et al., 2013; Purkamo, Bomberg, Kiet€av€ainen, et al., 2015;

4.2 Amplicon Sequencing

sequencing technologies include Oxford Nanopore’s MinIon: a device that

4.2.2 Gene Markers of Functional Diversity

presence of these genes or their transcripts in DNA or RNA samples is

4.3 Metagenome and Metatranscriptome Sequencing and

Whole-metagenome ampliﬁcation with PCR or isothermal ampliﬁcation

4.3.3 Data Analysis

Figure 4 A bioinformatics workﬂow in metagenomics. The light gray background indi-

(a nonredundant protein database, NCBI) for annotation of function and

4.4 Single-Cell Isolation and Sequencing

5. A CASE STUDY: METAGENOMICS OF THE

Kukkonen, 2011). The Outokumpu borehole is 2.5 km deep and contains

Figure 5 Scanning electron micrographs of microorganisms from the Outokumpu

Major ﬁndings about the microbiology of the Outokumpu deep bore-

5.1 Metagenomics of Deep Subsurface Life in the

5.2 Data Analysis of Borehole Water and Fracture Zone

600 454 107,939,831 8,022,306 721 17,545 0 7 0

two sequencing platforms: although 454-sequencing typically produces