Professional Documents
Culture Documents
PETROLEUM BIOTECHNOLOGY
Developments and Perspectives
Vol. 151
PETROLEUM BIOTECHNOLOGY
Developments and Perspectives
Edited by
Rafael Vazquez-Duhalt
Institute of Biotechnology
National University of Mexico
Morelos, Mexico
and
Rodolfo Quintero-Ramirez
Mexican Petroleum Institute
Colonia San Bartolo
Atephehuacan, Mexico
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PREFACE
Without a doubt, historians will describe 20th and 21st centuries as the oil-based society. One
hundred years ago oil exploitation began, first as a source of energy and later to include oil as
a source of raw material. In addition to the 1 trillion barrels that have already been harvested,
recent estimations shows that about 3 trillion barrels of oil remain to be recovered worldwide,
half from proven reserves and half from undeveloped or undiscovered sources. Oil production
is expected to peak sometime between 2010 and 2020, and then fall inexorably until the end
of this century. After the production peak, the more expensive fuel sources will come into
production. These include hard-to-extract oil deposits, tarry sands, and Synfuels from coal
that requires alternative or complementary to conventional oil refining technologies.
Our society has an inexorable challenge: to increase the production of goods and
services for people, using new process technology that should be energetically efficient and
environmental friendly. This also will be the case for the petroleum industry. Improvements
in conventional oil refining processes such as cracking, hydrogenation. isomerization,
alkylation. polymerization, and hydrodesulfurization, certainly will occur. Nevertheless, nonconventional biotechnological processes could be implemented. In contrast to the available
processes, biological processing may offer less severe process conditions and higher
selectivity for specific reactions. Biochemical processes are expected to be low demand
energy processes and certainly environmentally compatible.
The primary target of the petroleum industry is to enhance and maintain a continuous
oil production. Preconceived ideas and misconceptions about biotechnology continue to limit
the applications of biological processes in the chemical industry. Nevertheless, there are
biotechnological processes that have been demonstrated to be industrially successful and that
are shown to be sufficiently stable, productive and economic for commercial applications.
Even if wastewater treatment and soil bioremediation are common biotechnological
applications in the oil industry, petroleum biotechnology is still in its infancy. Doubtless,
though, biotechnology will play an increasingly important role in future industrial processes.
In this book, experts from 11 countries critically discuss the developments and perspectives of
biotechnological processes for the petroleum industry.
An integrated approach into the possibility of using petroleum biotechnology
throughout the value chain of an oil company is presented. The authors discuss the evaluation
of biotechnology as a general toolbox for solving some of the technology problems of today
and future possibilities to implement new refinery processes. Petroleum refining could be
enhanced by biochemical reactions in which the specificity exceeds by far these of chemical
reactions. The selective removal of sulfur, nitrogen, and metals from petroleum by
biochemical reactions performed by microorganisms and/or enzymes is discussed. Increasing
supply of heavy crude oils and bitumens has increased the interest in the conversion of the
high-molecular weight fractions of these materials into refined fuels and petrochemicals. This
upgrading has typically been accomplished either with high-temperature and expensive
processes thermal conversion (cracking or coking) or by catalytic hydroconversion. In
contrast to the available processes, biological processing may offer less severe process
conditions and higher selectivity to specific reactions. Enzymatic transformations of
asphaltenes in non- conventional media, and biological upgrading to improve the quality of
certain crude oils and liquid fuels could be envisaged, using biocatalysts to decrease
aromaticity and sensitize aromatic heterocycles to subsequent heteroatom removal.
Bioprocessing would complement conventional refining technologies and result in improved
fuel quality at lower capital and operating costs and with reduced environmental impact.
vi
vii
The powerful tools of molecular biochemistry can be used to improve the enzyme
stability and efficiency. These techniques may be applied to the particular needs of the
petroleum industry. In addition, the enzymes isolated from extremophilic microorganisms are
extremely thermostable and generally resistant to non-conventional conditions such as organic
solvents and extreme pH. Thus, many enzymes and enzymatic proteins are still to be
discovered.
Rafael Vazquez-Duhalt
The only way to discover the limits of the possible is to go beyond them into the impossible.
(Arthur C. Clarke).
ix
Table of Contents
Preface
List of Contributors
Chapter 1
Use of Petroleum Biotechnology throughout the value chain of an oil company:
An integrated approach.
H.Kr. Kotlar, O.G. Brakstad, S. Markussen and A. Winnberg
Statoil ASA. Trondheim, Norway
v
xiii
Chapter 2
Petroleum biorefining: the selective removal of sulfur, nitrogen, and metals
J.J. Kilbane II" and S. Le Borgneb
a
Gas Technology Institute, Illinois U.S.A.
b
Instituto Mexicano del Petroleo, Mexico
29
Chapter 3
Enzymatic catalysis on petroleum products
M. Ayala" and R. Vazquez-Duhaltb
a
lnstituto Mexicano del Petroleo. Mexico
b
Instituto de Biotecnologia, UNAM, Mexico
67
Chapter 4
Prospects for biological upgrading of heavy oils and asphaltenes
K.M. Kirkwood, J.M. Foght, and M.R. Gray
University of Alberta, Canada
I 13
Chapter 5
Whole-cell bio-processing of aromatic compounds in crude oil and fuels
J.M. Foght
University of Alberta, Canada
145
Chapter 6
Biocatalysis by methane monooxygenase and its implications for the petroleum
industry
T.J. Smith" and H. Dalton 3
a
University of Warwick, United Kingdom
Sheffield Hal lam University, United Kingdom
177
Chapter 7
Biocorrosion
H.A. Videla" and L.K. Herrerah
a
University of La Plata, Argentina
University of Antioquia, Colombia,
193
Chapter 8
Molecular tools in microbial corrosion
X. Zhu and J.J. Kilbane II
Gas Technology Institute, Illinois U.SA.
219
Chapter 9
Potential applications of bioemulsifiers in the oil industry
H. Bach" and D.L. Gutnick1'
b
Tel-Aviv University, Tel-Aviv, 69978, Israel
a
Taro Pharmaceuticals New York, U.S.A.
233
Chapter 10
Anaerobic hydrocarbon biodegradation and the prospects for microbial
enhanced energy production
J.M. Suflita", I.A. Davidova\ L.M. Gieg", M. Nanny" and R.C. Prince1'
"University of Oklahoma, U.S.A.
b
ExxonMobil Research and Engineering Co., U.S.A.
283
Chapter 11
Using nitrate to control microbially-produced hydrogen sulfide in oil field waters
R.E. Eckford and P.M. Fedorak
University of Alberta, Edmonton, Canada
307
Chapter 12
Regulation of toluene catabolic pathways and toluene efflux pump expression
in bacteria of the genus Pseudomonas
J.L. Ramos, E. Duque, M.T. Gallegos, A. Segura and S. Marques
Estacion Experimental del Zaidin, CSIC, Granada, Spain
341
Chapter 13
Bacterial hydrocarbon biosynthesis revisited
B. Valderrama
Instituto de Biotecnologia, UNAM. Mexico
373
Chapter 14
The microbial diversity of deep subsurface oil reservoirs
N.-K. Birkeland
University of Bergen, Norway
385
xi
Chapter 15
Biotechnological approach for development of microbial enhanced oil recovery
technique
K. Fujiwara11, Y. Sugai1', N. Yazawa1', K. Ohno\ C.X. Hong" and H. Enomoto1
a
Chugai Technos Co. Ltd., Japan
Akita University, Japan
c
Japan National Oil Corporation, Japan
PetroChina Company Limited, China
c
Tohoku University, Japan
405
Chapter 16
Phytoremediation of hydrocarbon-contaminated soils: principles and applications
R. Kamath, J. A. Rentz, J. L. Schnoor and P. J. J. Alvarez
University of Iowa, U.S.A.
447
Chapter 17
Biological treatment of polluted air emissions
S. Revah* and R. Auria"
a
Universidad Autonoma Metropolitana-lztapalapa, Mexico.
b
Universite de Provence, France
479
Chapter 18
Bioremediation of marine oil spills
R. C. Prince and J. R. Clark
ExxonMobil Research & Engineering Co.
495
Chapter 19
Biotreatment of water pollutants from the petroleum industry
E. Razo-Flores, P. Olguin-Lora, S. Alcantara and M. Morales-Ibarria
Institute Mexicano del Petroleo, Mexico
513
xiii
List of Contributors
S. Alcantara
Institute Mexicano del Petroleo
Eje Central Lazaro Cardenas 152. C.P. 07730, Mexico D.F.
P. J. J. Alvarez
Department of Civil and Environmental Engineering, Seamans Center
University of Iowa, Iowa City, Iowa, U.S.A. - 52242
R. Auria
Laboratoire 1RD de Microbiologie, Universite de Provence
CESB/ESIL, Case 925, 163 Avenue de Luminy 13288, Marseille Cedex 9 France
M. Ayala
Institute Mexicano del Petroleo.
Eje Central Lazaro Cardenas 152, San Bartolo Atepehuacan 07730 Mexico DF, Mexico
H. Bach
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University
Tel-Aviv, 69978, Israel
N.-K. Birkeland
Department of Biology, University of Bergen, Box 7800, N-5020 Bergen, Norway
O.G. Brakstad
Sintef Materials and Chemistry, Trondheim, Norway
.1. R. Clark
ExxonMobil Research & Engineering Co.
Annandale, NJ 08801
H. Dalton
Department of Biological Sciences, University of Warwick
Coventry CV4 7AL, United Kingdom
I.A. Davidova
Institute for Energy and the Environment and Department of Botany and Microbiology,
University of Oklahoma, Norman, OK 73019, USA.
E. Duque
Estacion Experimental del Zaidin. CS1C
C / Profesor Albareda 1, 18008 Granada, Spain
xiv
R.E. Eckford
Department of Biological Sciences, University of Alberta
Edmonton, Alberta, Canada T6G 2E9
H. Enomoto
Department of Geoscience and Technology, Graduate School of Environmental Studies,
Tohoku University, Aramaki, Aoba-ku, Sendai 980-0845, Japan
P.M. Fedorak
Department of Biological Sciences, University of Alberta
Edmonton, Alberta, Canada T6G 2E9
J. M. Foght
Department of Biological Sciences, University of Alberta
Edmonton, Alberta Canada T6G 2E9
K. Fujiwara
Chugai Technos Co. Ltd.
9-20 Yokogawa-Shinmachi Nisi-ku Hiroshima City 733-0013, Japan
M.T. Gallegos
Estacion Experimental del Zaidin, CSIC
C / Profesor Albareda 1, 18008 Granada, Spain
L.M. Gieg
Institute for Energy and the Environment and Department of Botany and Microbiology,
University of Oklahoma, Norman, OK 73019, USA.
M.R. Gray
Department of Chemical and Materials Engineering, University of Alberta
Edmonton, Alberta, Canada T6G 2G6
D.L. Gutnick
Present address, Biotechnology Research Laboratories. Taro Pharmaceuticals U.S.A.,
3 Skyline Drive, Hawthorne, New York, 10532, U.S.A.
L.K. Herrerab
Faculty of Engineering, University of Antioquia, Medellin, Colombia
C.X. Hong
PetroChina Company Limited, Jilin Oilfield Company
Jilin province, China
R. Kamath
Department of Civil and Environmental Engineering, Seamans Center
University of Iowa, Iowa City, Iowa, U.S.A. - 52242
XV
J.J. Kilbanell
Gas Technology Institute, 1700 S. Mt. Prospect Rd.. Des Plaines 1L 60018
K.M. Kirk wood
Department of Chemical and Materials Engineering, University of Alberta
Edmonton, Alberta, Canada T6G 2G6
H.Kr. Kotlar
Statoil ASA, R & D Center, Postuttak, N-7005 Trondheim, Norway
S. Le Borgne 1
Institute Mexicano del Petroleo, Eje Central Lazaro Cardenas 152, Col. San Bartolo
Atepehuacan, 07730 Mexico D.F., Mexico
S. Markussen
Department of Marine Environmental Technology, Trondheim, Norway
S. Marques
Estacion Experimental del Zaidin, CSIC
C / Profesor Albareda 1, 18008 Granada, Spain
M. Morales-lbarria
Instituto Mexicano del Petroleo
Eje Central Lazaro Cardenas 152, C.P. 07730, Mexico D.F.
M. Nanny
Institute for Energy and School of Civil Engineering and Environmental Science,
University of Oklahoma, Norman, OK 73019, USA.
K. Ohno
Technology Research Center, Japan National Oil Corporation
1-2-2 Hamada, Mihama-ku, Chiba 261-0025, Japan
P. Olguin-Lora
Instituto Mexicano del Petroleo
Eje Central Lazaro Cardenas 152, C.P. 07730, Mexico D.F.
R. C. Prince
ExxonMobil Research & Engineering Co.
Annandale. NJ 08801
J.L. Ramos
Estacion Experimental del Zaidin, CSIC
C / Profesor Albareda 1, 18008 Granada, Spain
xvi
E. Razo-Flores,
Institute Potosino de Investigation Cienti'fica y Tecnologica
Camino a la Presa San Jose 2055,. C.P. 78216, San Luis Potosi, SLP, Mexico.
.1. A. Rentz
Department of Civil and Environmental Engineering, Seamans Center
University of Iowa, Iowa City, Iowa. U.S.A. - 52242
S. Revah
Department of Process Engineering, Universidad Autonoma Metropolitana-Iztapalapa
(UAM-I). Apdo. Postal 55-534, 09340 Mexico D.F., Mexico
J. L. Schnoor
Department of Civil and Environmental Engineering, Seamans Center
University of Iowa, Iowa City, Iowa, U.S.A. - 52242
A. Segura
Estacion Experimental del Zaidin, CSIC
C / Profesor Albareda 1, 18008 Granada, Spain
T.J. Smith
Biomedical Research Centre, Sheffield Hallam University
Howard Street, Sheffield SI 1WB, United Kingdom
.I.M. Suflita
Institute for Energy and the Environment and Department of Botany and Microbiology,
University of Oklahoma, Norman. OK 73019, USA.
Y. Sugai
Akita University Venture Business Laboratory
1-1 Tegatagakuen-cho Akita City ,010-8502, Japan
B. Valderrama
Departamento de Ingenieria Celular y Biocatalisis, Universidad Nacional Autonoma de
Mexico. AP 510-3. Cuernavaca, Morelos, 62250, Mexico.
R. Vazquez-Duhalt
Instituto de Biotecnologia, UNAM.
Apartado Postal 510-3 Cuernavaca, Morelos 62250 Mexico
H.A. Videla
Department of Chemistry. College of Pure Sciences, IN1FTA, University of
La Plata, Argentina
A. Winnberg
Department of Biotechnology, N7465 Trondheim, Norway
xvii
N. Yazawa
Technology Research Center, Japan National Oil Corporation
1-2-2 Hamada. Mihama-ku, Chiba 261-0025, Japan
X. Zhu
Gas Technology Institute, 1700 S. Mt. Prospect Rd., Des Plaines 1L 60018
Chapter 1
other microbes [9, 15]. Thus, one would expect to find genetic markers of
microbial activities both during exploration, drilling and production.
Statoil has filed a patent application for utilization of DNA technologies
as a tool for identification and characterization of hydrocarbon sources during
drilling or sampling from sea floor seep zones. Drill cuttings from exploration
wells, sediments from sea floor seep zones or other specimens could be analyzed
with a selection of specific DNA probes/markers. These specific DNA probes
are taken from microbes found to be linked to different oil producing fields in
the North Sea and other sources. The energy sources for these organisms will be
constituents of the oil, gas or others, specific for the reservoir zones and
conditions of the particular field
This genetic tool may give valuable information on possible migration
routes of the hydrocarbon from the source rock. Specific recognition patterns
might also be used in monitoring different reservoir zones during production,
and further indicate the individual contribution of the particular zone to the
overall production. Possibly, sweep efficiency pattern could be calculated.
Detection of DNA from drill cuttings, sediments, or core samples during
explorative drilling may result in defined species pattern, resulting in indications
of potential hydrocarbon bearing zones (Fig. 2).
the two reservoirs showed dominance of small rods, single or in short chains,
and sheathed rods (Thermotogales like). Pure isolates were obtained from only
one of the reservoirs, reservoir A. Even though the enrichments from the other
reservoir, reservoir B, showed a variety of organisms, it was not possible to
obtain any pure isolates from these. The 16S rDNA clones from these
enrichments aligned to Thermosipho japonicus, Bradyrhizobium and
Aquabacterium. 16S rDNA clones from isolates from reservoir A, showed
dominance of Archaeobglobus fulgidus, Methanococcus thermolithotrophicus,
Thermococcus sibiricus and Thermosipho japonicus. Several of the sequences
abundant in the cultures were not found in the clone library from the cultureindependent approach (2.1.1). This is in accordance with other studies [9], and
suggests that several of the predominant members of the enrichment cultures
(e.g. Thermosipho) are not the predominant member of the reservoir
communities, but show fast-growing characteristics in several of the culture
media. Other cultures included a-, P-, s- and y-Proteobacteria Sphingomonas,
Stenotrophomonas, Halomonas meridiana, and Geospirillum, and the Grampositive bacterium Thermoanaerobacter ethanolicus.
Fig. 3. DGGE analysis of PCR-amplified 16S rDNA sequences from two North Sea oil
reservoirs, reservoir A (1, 2) and reservoir B (3, 4, 5). Only sample 2 contained fluids with
seawater penetration.
Thermophilic species of Thermotogales, Archaeoglobus, Thermoanaerobacter, Methanococcus and Thermococcus have been reported from hightemperature oil reservoirs [6-9, 14]. Several of these microbes are typical sulfurutilizers, being active in desulphurization of crude oil. These microbes may be
the predominant sources for H2S generation rather than typical sulphatereducing bacteria, and interestingly several of them were enriched in culture
media designed for SRB.
2.1.3. Detection of specific microbes
Monitoring of microbes in the oil reservoir has traditionally been
accomplished by culture methods, e.g. MPN methods for quantification of
viable sulphate-reducing bacteria (SRB), as recommended by the American
Petroleum Institute [24]. Some commercial techniques have also been
introduced, for instance a commercialized immunoassay for semi-quantification
of the SRB-specific enzyme APS reductase [25]. Monitoring may also include
molecular biology methods. Currently, two RNA-based methods are
investigated, fluorescence in-situ hybridization (FISH) and nucleic acid
sequence-based amplification (NASBA). By using RNA detection mainly the
metabolic active cells are assessed. The FISH methods include fluorescencelabeled DNA probes for the targeting of specific microbes. An example is given
in Fig. 4 where bacteria, archaea, Archaeoglobus, Arcobacter and Erythobacter
are enumerated in production fluids from two reservoirs. These methods may be
further refined for offshore analysis by using field equipment, e.g. the Microcyte
fluorescence cell counter. NASBA is an isothermic alternative to PCR [26].
Real-time miniaturized lab-on-a-chips systems are currently under development
with the NASBA technology as basis [27].
2.1.4. Characterization ofmicrobial dynamics by microarrays
Nucleic acid microarrays have recently been introduced for phylogenetic
identification in microbial ecology. Basically, microarrays consist of series of
specific DNA probes (grabber probes) that are printed on glass slides. Sample
nucleic acids are extracted and labeled (e.g. by fluorescence) and incubated on
the slides, followed by recording. Labeled detector probes may be used for
detection as alternatives or supplements to labeled target DNA [28]. The
microarrays are made quantitative by employing reference DNA to normalize
variations in spot size and hybridization (29). The methods provide a powerful
tool for parallel detection of 16S rRNA genes [30-31] and may be particularly
useful for environmental studies of phylogenetically diverse groups. Although
most arrays are based on the PCR amplification of target genes prior to array
hybridization, systems have also been described where direct profiling of
extracted rRNA from environmental samples have been used [32]. Printed slides
may be brought offshore and target genes quantified directly on the platforms by
portable devices. Arrays have also been established for the assessment of
functional gene diversities and distribution, for instance with genes from the
nitrogen cycling [33-34]. For offshore conditions the sulphur and nitrogen
cycles may be addressed during curing of biological souring by nitrate injection.
3. BIOREACTOR: POTENTIAL USE OF BIOCATALYSTS IN CRUDE
OIL UP-GRADING AND REFINING
Until recently, research within oil biotechnology mainly focused on biodegradation and bioremediation in connection with clean up after oil spills, and
less on the application of microbial systems in industrial processes. However,
the interest in the latter has been growing the last years, addressing problems
like asphaltenes, high sulfur content, the poor transportability of heavy crudes
due to high viscosity, the presence of heavy metals and polyaromatic/
heterocyclic compounds (see chapters 2, 3, 4 and 5).
The aim of our activity is to use biotechnological processes in up-grading of
"problem" oils/heavy oil and refinery fractions. The overall scope is to define
microbial/biotechnological technologies along the crude oil value chain that will
give the potential highest cost-benefits, competing with or being superior to
existing methods, or even better, provide solutions where no acceptable methods
exist. In the current program there has been focused on:
Fig. 4. FISH enumeration of the total concentrations of cells (DAPI), bacteria (EUB338),
archaea (ARCH915), Arcoglobus (ARGLO605) and thermotogales (THERSI672) in produced
fluids from two North Sea reservoirs, Reservoir A and Resevoir B wl and w2.
10
11
12
13
Fig. 5. Schematic outline of the procedure for making blocked mutants with an inactive
enzyme by gene disruption.
14
Fig. 6. Bioconversion of light gas oil by the specially designed Sphingomonas spp. N2.
15
16
Fig. 8. Bio-reactor for conversion of PAH's in a real feedstock from crude oil
17
Study of pure enzyme vs. whole cell based biocatalysts. In future investigations
this will include "the aromatic ring opening dioxygenase system". The
Sphingomonas yanoikuyae N2 will be used as a model system for comparing
enzyme and whole cell biocatalysts. In many instances it is an advantage to use
pure enzyme systems instead of whole cells as biocatalysts (see chapter 3).
Enzyme reactions are specific and easy to control, they can be carried out in
non-aquatic environments, and enzymes, as other chemical catalysts, will not
consume carbon i.e. the carbon content in the fuel will be preserved. The
opening of the aromatic ring (e.g. naphthalene, Fig. 9) is a four step enzymatic
process starting with a dioxygenase reaction, then a dehydrogenation followed
by a second dioxygenase reaction and finally an isomerization. The first
oxygenation requires NADH, but the formed NAD+ is recycled to NADH in the
dehydrogenation reaction. The challenge is to develop a system where this
multistep enzyme reaction could proceed efficiently in a cell free system.
Fig. 9: Metabolic pathway of naphthalene showing the enzymes involved See reference [57],
18
3.2.2. Bioreactors
Bioconversion of refinery fractions may take place using growing or resting
cells, "dead" cells, or immobilized cells or enzymes as biocatalysts. Aromatic
ring-opening involves a multistep metabolic pathway. Multistep enzymatic
reactions often require co-factors and/or reducing power (NAD (P) H) that has
to be regenerated or supplied for the enzymatic reaction to take place. Thus,
whole cells, rather than pure enzymes, are often required. The biocatalysts are
usually contained in the aqueous phase and the reaction take place either in this
phase or at the interface between the aqueous and the organic/oil phase. The
components in the refinery fraction that are being up-graded usually show low
water solubility, while the converted products usually are more soluble in the
aqueous phase than in the organic/oil phase. Mass transfer of substrates and
products between the water and oil phase is a major challenge. To achieve
adequate mass transfer, reactors capable of generating a large interface between
oil and water should be chosen. Various types of bioreactors have been
employed by others [58], including stirred tank reactors, airlift reactors,
emulsion phase contactors reactor and fluidized bed reactors. The current
investigation has used stirred tank reactors run in batch, fed-batch and
continuous mode with free growing or resting cells. However, immobilized cells
and enzymes are included in the next phase of studies.
19
20
in productivity occur in the well. The preventive actions are to avoid the onset of
these predicted situations.
With the advance in drilling and completion, increasing number of complex
and expensive wells are being installed, e.g. multilateral, multi-zones, sidetrack
and horizontal. The infrastructures that are in place, such as flow lines and
platforms, also enable the targeting and drainage of the additional reserves found
near the exiting fields. Very often these additional oil and/or gas are produced
via tieback and satellite facilities. Successful treatments of stimulation, scale
squeeze and tubing deposit removal in these wells can no longer rely on the
traditional method of bullheading. Special tools such as coil tubing and
inflatable plug will be needed to place the chemicals accurately down-hole.
Intervention in these wells will be prohibitory expensive due to tools hire,
personnel and extended period of deferred oil production (tools run). It is
important to realize that for certain type of completion, well re-entry is almost
impossible despite accepting the financial penalty. There is clearly a need to
develop an intervention free system for these wells that allow the flow of oil
unhindered and preferably with the chemicals pre-delivered down-hole.
Fig. 11. OPEX profile in developments of new technology for mining bitumen. The curve
shows the measured cost until 1998, then the further projection. The bars in 99, 00 and 01 are
the actual cost. (Maurice B. Dusseault, personal communication).
21
22
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29
Chapter 2
Gas Technology Institute, 1700 S. Mt. Prospect Rd., Des Plaines, Illinois 60018
Instituto Mexicano del Petroleo, Eje Central Lazaro Cardenas 152, Col. San
Bartolo Atepehuacan, 07730 Mexico D.F., Mexico
1. INTRODUCTION
The quality of petroleum is progressively deteriorating as the highest quality
petroleum deposits are preferentially produced. Consequently the concern about
the concentrations of compounds/contaminants such as sulfur, nitrogen, and
metals in petroleum will intensify. These contaminants not only contribute to
environmental pollution resulting from the combustion of petroleum, but also
interfere with the processing of petroleum by poisoning catalysts and
contributing to corrosion. The selective removal of contaminants from
petroleum while retaining the fuel energetic value is a difficult technical
challenge. New processes are needed and bioprocesses are an option. Existing
thermo-chemical processes, such as hydrodesulfurization, can efficiently remove
much of the sulfur from petroleum but the selective removal of sulfur from
compounds such as dibenzothiophene, the removal of organically bound
nitrogen, and the removal of metals cannot be efficiently accomplished using
currently available technologies. The specificity of biochemical reactions far
exceeds that of chemical reactions. The selective removal of sulfur, nitrogen,
and metals from petroleum by biochemical reactions performed by
microorganisms and/or enzymes has been demonstrated. However, further
research is needed before biorefining technology can be commercialized. This
chapter reviews the status of biorefining and discusses topics requiring further
research.
30
31
32
available crude oils. However, the bottom line is that light crude oil is more
readily recovered, and more readily processed/refined, than heavy oil.
Consequently, deposits of light, low heteroatom content oil are preferentially
brought into full production while known deposits of heavy/high heteroatom
content petroleum are produced at less than full capacity or may even be idle.
Moreover, as a deposit of light oil is harvested, the lighter fractions are
preferentially removed such that after primary or secondary production mainly
heavy oil remains. Because of this irreversible trend, the time when available
crude oil is predominantly or exclusively heavy with a high heteroatom and
heavy metals content is not far off.
The chief concern is for the sulfur content of petroleum, but the nitrogen
and metal content of petroleum is also of concern due to environmental,
processing and corrosion concerns [23]. In North America, over 3 trillion barrels
of known petroleum reserves are largely untapped or underutilized because of
their high sulfur content and viscosity [22]. It is well known in petroleum
chemistry that sulfur and heavy metals are preferentially associated with the
higher molecular weight fractions of oil [4, 7]. So, not only is light oil easier to
produce because of its physical properties, but it also contains significantly less
undesirable impurities in comparison with heavy oils. Sulfur, nitrogen and heavy
metal impurities are of great environmental concern since they originate acid
rain as a consequence of sulfur and nitrogen oxides emissions from the
combustion of petroleum derived fuels, and potential health effects due to high
concentrations of heavy metals on combustion ashes [2, 10, 24, 25]. Some sulfur
and nitrogen heterocycles are suspected carcinogens [8, 12] and sulfur
compounds in oil have been implicated in the corrosion of pipelines and refinery
equipment [7, 24, 26]. Heavy metals content, mainly nickel and vanadium,
contributes to the poisoning of catalysts used in hydrodesulfurization or in
catalytic cracking [5, 20, 23]. In addition to catalysts poisoning by heavy metals,
sulfur and nitrogen in heterocyclic compounds are capable of poisoning catalysts
by causing electronic modifications in Pd, Pt, Ni, and Ru compounds. The
poisoning of catalysts exasperates the problems associated with the processing
of heavy oils and residuum by interfering with the methods employed to reduce
the heteroatom content and molecular weight, i.e. hydro treatment and cracking.
The quantity of heavy oils to be processed is increasing not only due to
the depletion of light oils but also to the increasing demand for cleaner
transportation fuels and other low molecular weight products, so the importance
of technologies capable of dealing with heavy oils and residuum has increased
[11, 27]. This increased demand for lower molecular weight petroleum products
seems incompatible with the use of heavy oils as primary feedstocks because of
their metals, nitrogen and sulfur content that increases the production of coke
and gas and accelerates catalyst deactivation [6]. But at the same time, the need
to obtain greater quantities of gasoline, diesel and aviation fuels from each
33
34
products. Of all of the chemical forms of sulfur in crude oil, the most recalcitrant
to hydrodesulfurization is the thiophenic sulfur in thiophene and
dibenzothiophene derivatives. Because of the abundance of alkylated
dibenzothiophenes in crude oil and the recalcitrance of these compounds to
hydrodesulfurization, there is a high level of interest in technologies that can
effectively desulfurize dibenzothiophenes [2].
Researchers have been examining the possibility of biodesulfurization of
petroleum or other fossil fuels for over 4 decades [32]. Presently, there is no
commercial operation for biodesulfurization of fossil fuels, however several
economic studies indicate a favorable prospect of developing such a technology
[10, 24, 25, 27, 33]. Numerous microorganisms have been described in the
literature that are capable of utilizing dibenzothiophene (DBT) as sole source of
carbon, energy and sulfur. However, the complete degradation of organosulfur
compounds is not beneficial for upgrading crude oils and derived fuels. The
selective cleavage of carbon-sulfur bonds in DBT and derivatives is preferred,
this way sulfur is selectively removed and the calorific value of the treated fuel
remains intact.
The first microorganism that was shown to be capable of selectively
cleaving carbon-sulfur bonds in crude oil, coal, and a wide range of model
compounds, resulting in the selective removal of sulfur and the retention of
carbon and calorific value, is Rhodococcus erythropolis IGTS8 (ATCC 53968)
[34]. Subsequently, numerous other bacteria capable of selectively cleaving
carbon-sulfur bonds in DBT were isolated and characterized. The biochemical
pathway used by these aerobic microorganisms to desulfurize DBT compounds
has been termed the 4S pathway due to the progressive oxidation of sulfur that
occurs through 4 steps [2, 35]. The selective removal of sulfur from DBT and
from crude oil by anaerobic bacteria has also been reported. Sulfate-reducing
bacteria such as Desulfovibrio desulfuricans have been shown to metabolize
DBT to H2S and biphenyl [36]. The desulfurization of oil under anaerobic
conditions avoids costs associated with aeration, and has the advantage of
liberating sulfur as a gas. However, an anaerobic biodesulfurization process has
not been developed due to low reaction rates, safety and cost concerns, and the
lack of identification of specific enzymes and genes responsible for anaerobic
desulfurization. Consequently, aerobic biodesulfurization has been the focus of
the majority of research [2, 32].
2.1. Substrate range of desulfurization
A desulfurization competent moderate thermophile was recently isolated,
Mycobacterium phlei GTIS10, that metabolizes DBT by the same pathway as R.
erythropolis IGTS8 [37]. A comparison of the capabilities of these two
microorganisms that have optimum growth temperatures of 30C and 50C
35
36
Table 1.
Range of organosulfur substrates used as sole source of sulfur for growth
byMpezGTIS10
37
Fig. 1. The 4S metabolic pathway for DBT desulfurization. DszC is the DBT
monooxygenase, DszA the DBT sulfone monooxygenase, DszB the HPBSi desulfmase and
DszD is a flavin reductase. I, DBT; II, DBT sulfoxide; III, DBT sulfone; IV,
hydroxyphenylbenzenesulfmate; V, 2-hydroxybiphenyl.
38
Table 2.
Concentrations of metabolites of dibenzothiophene produced by M. phlei GTIS10 and R.
erythropolis IGTS8
1
= All concentrations of metabolites are expressed as |j.g/ml of the ethyl acetate extract (1 ml
total) derived from each culture grown with 1,440 (ig DBT as the sole sulfur source.
Dihydroxybiphenyl #1 and #2 have identical molecular formulas, but could not be assigned
specific molecular structures based on data available.
39
40
41
42
conjugal transfer of plasmids containing the dsz genes or the transposition of dsz
genes is sparse, the distribution of dsz genes in bacterial cultures strongly
support the hypothesis that these genes are commonly subjected to horizontal
transfer in nature. Indeed the DNA sequences of dsz genes from numerous
bacterial cultures isolated in geographically distinct locations have been found to
be nearly identical. Various Rhodococcus [68], Mycobacterium [37], Gordona
[59], Corynebacterium
[69], Arthrobacter
[70], Enterobacter [39],
Stenotrophomonas [39], Klebsiella [39], Bacillus [71], and Nocardia [72]
species have been isolated that possess dsz gene sequences that are identical or
highly homologous to the DNA sequence of the dsz gene of R. erythropolis
IGTS8. However, some variation in the sequences of the dsz genes has been
observed. The dsz genes of the moderate thermophile Paenibacillus sp. A 11-2
and Nocardia asteroides are only 52-65% and 89% homologous to R.
erythropolis IGTS8, respectively [61, 73]. Moreover, PCR amplification of dsz
genes from soil samples revealed relatively few variations in dsz gene
sequences, with the majority of variations found in dszA, and even then
homology to the R. erythropolis IGTS8 dszA sequence was 95% or more [66].
It is interesting to note that while several bacterial genera apparently
participate in horizontal transfer of dsz genes in nature, and laboratory studies
demonstrate that dsz genes and enzymes function well in Pseudomonas and E.
coli strains, a naturally occurring desulfurization-competent Pseudomonas sp. is
rarely encountered [74] and a desulfurization competent E. coli isolate has never
been reported [2]. The reasons for the restricted range of distribution of dsz
genes in nature are currently unknown, but one factor may be the ability of
bacterial species to withstand exposure to substrates such as petroleum.
Laboratory studies indicated that Pseudomonas sp. containing dsz genes could
efficiently metabolize DBT in aqueous culture or DBT added in a solvent such
as hexadecane. However, the ability of these same Pseudomonas cultures to
metabolize DBT in diesel oil or other petroleum product is much reduced [75,
76]. Naturally occurring desulfurization-competent bacterial cultures are almost
exclusively gram positive or gram variable and it may be that the cell
wall/membrane structure of gram negative bacterial species is less able to
tolerate exposure to petroleum compounds and solvents. There are many
thousands of gram positive and gram variable bacterial species, yet the observed
occurrence of dsz genes in naturally occurring desulfurization-competent
bacterial isolates is restricted to only a few species. Clearly, more remains to be
learned about the role dsz genes play in microbial ecology and the functioning of
desulfurization enzymes in different bacterial hosts [77].
Other topics regarding biodesulfurization that are not well understood are
the access of desulfurization enzymes to insoluble and high molecular weight
substrates, and the mechanism by which the sulfur liberated from organosulfur
substrates by the desulfurization enzymes is subsequently incorporated into
43
biomass. When R. erythropolis IGTS8 was first isolated, it was obtained from an
enrichment culture growing in a defined mineral salts medium devoid of
inorganic sulfur [78, 79]. All essential nutrients were present in abundance with
the exception of sulfur, which was supplied in the form of coal or DBT creating
an environment where any bacterial species that could utilize organically bound
sulfur had a strong selective advantage. The mixed culture that grew with DBT
as the sole source of sulfur was streaked onto a variety of agar plates allowing
pure cultures to be obtained from each of the types of colonies present. Then
each pure culture was tested individually to determine if it could utilize DBT as
a sole source of sulfur for growth.
It soon became clear that none of the pure cultures most readily isolated
from the desulfurization-competent mixed culture were capable of utilizing DBT
as a sole sulfur source. Perseverance in investigating this desulfurizationcompetent mixed culture eventually led to the isolation of a relatively slow
growing pure culture that was demonstrated to utilize DBT as a sole source of
sulfur, and this culture was subsequently identified as R. erythropolis IGTS8
[34]. R. erythropolis IGTS8 was present at low abundance in the original
desulfurization-competent mixed culture and even when pure cultures of R.
erythropolis IGTS8 and a desulfurization-deficient, but faster growing, bacterial
culture such as Enterobacter cloacae, were combined in various ratios and used
to inoculate growth experiments in sulfur-limited media where DBT was the
sole source of sulfur, R. erythropolis IGTS8 invariably emerged as the least
abundant species in the resulting culture [34].
These results cannot be explained if DBT is taken up into the cytoplasm
of/?, erythropolis IGTS8 and only then is DBT converted to 2-HBP and sulfite,
unless it is also hypothesized that sulfite is then excreted. While intracellular
metabolism of DBT by R. erythropolis IGTS8 is stated to occur [46] there is no
evidence for DBT transport/uptake in desulfurization competent Rhodococcus
cultures [25], nor is there evidence for mass transfer limitations in DBT
metabolism [25, 80]. If sulfur is liberated intracellularly within R. erythropolis
IGTS8 and sulfur is the growth limiting nutrient, it seems unlikely that sulfite
would be excreted extracellularly unless intracellular oxidation of sulfite to
sulfate were not possible. Sulfate has been demonstrated to be the form of
inorganic sulfur that is utilized by R. erythropolis IGTS8 [81] while sulfite has
been demonstrated to be the form of sulfur obtained as a product of the
desulfurization of DBT [43]. Further research into sulfite and sulfate metabolism
by desulfurization competent cultures is warranted.
One hypothesis that is consistent with the observation that faster growing
desulfurization-deficient bacterial species can dominate mixed cultures when R.
erythropolis IGTS8 is the only desulfurization competent culture present, is that
desulfurization of DBT occurs in association with the external surface of R.
erythropolis IGTS8 cells. The Dsz proteins are known to have membrane-
44
spanning domains [47, 82] so that the desulfurization pathway may function in
association with the cell membrane such that extracellular substrates and
intracellular cofactors can both be accessed. A further consideration regarding
the localization of the Dsz enzymes is the size of some of the substrates that can
be metabolized. Solid coal particles and high molecular weight coal derived
polymers can be effectively desulfurized [83-85], yet there has never been a
report documenting the intracellular uptake of substrates such as coal by any
bacterial species. Moreover, the size of coal particles vastly exceeds the size of
bacterial cells in experiments where biodesulfurization has been demonstrated to
remove 72% of organic sulfur without otherwise altering the composition of the
coal [85]. There is no evidence whatsoever that desulfurization enzymes are
excreted from R. erythropolis IGTS8 cells, but the size of substrates metabolized
and the ability of other bacterial species to successfully compete for sulfur
liberated from organosulfur substrates by R. erythropolis IGTS8 make it likely
that desulfurization does not occur intracellularly, but in association with the
external surface of cells.
A consequence of the fact that desulfurization-deficient bacterial species
can successfully compete for sulfur liberated from organosulfur substrates by R.
erythropolis IGTS8 is that a selective pressure favoring the evolution of a high
specific activity for desulfurization enzymes is created. In a mixed culture
environment where sulfur is the growth limiting nutrient and R. erythropolis
IGTS8 is the only desulfurization-competent culture, this bacterium must
liberate many times more sulfur than it needs to meet its own nutritional
requirements because competition from other bacteria leaves only a fraction of
the utilizable sulfur actually available for use by R. erythropolis IGTS8 [34]. If
this dynamic typified the natural environment for R. erythropolis IGTS8 and
other desulfurization competent cultures it would be reasonable to expect that a
high level of desulfurization activity would have evolved in such cultures.
However, that is not the case and even when grown as pure cultures, all
naturally occurring desulfurization competent cultures have levels/activities of
desulfurization enzymes that are growth limiting rather than capable of
supplying sulfur in excess of the needs of the culture [2]. This further illustrates
that we have much to learn about the role of Dsz enzymes in nature and the
characterization of the microenvironment occupied in nature by R. erythropolis
IGTS8 and other desulfurization-competent bacteria. Nevertheless, it is worth
considering that enrichment cultures and directed evolution experiments
designed to obtain cultures with higher levels of desulfurization activity may
benefit from the intentional use of mixed cultures.
2.5. Influence of the bacterial host on biodesulfurization
M. phlei GTIS10 appears to be highly similar to R. erythropolis IGTS8 as
regards to biodesulfurization capability except that the maximum growth
45
46
identical desulfurization genes are unknown. However, it is clear that the host
contributes to the functioning of the desulfurization pathway in yet
uncharacterized ways so that the manipulation of the dsz (or tds) genes alone
may be insufficient to yield bacterial cultures with substantially higher
desulfurization activity, such as would be required for a commercial
biodesulfurization process.
2.6 Desulfurization activity of various cultures
The maximum specific desulfurization activity for M. phlei GTIS10 was
1.1 0.07 umole 2-HBP/min/g DCW [37]. The maximum specific
desulfurization activity for R. erythropolis IGTS8 observed at the Gas
Technology Institute (1.2 0.08 umole 2-HBP/min/g DCW) is higher than
previous studies where other researchers reported specific desulfurization
activity values ranging from approximately 0.6 to 5.8 umole 2-HBP/min/g
DCW [46, 57]. The reason why our culture of R. erythropolis IGTS8 showed
higher desulfurization specific activity than previously reported may be due to
the continuous culturing (> 10 years) of this bacteria in our laboratory under
conditions where DBT, or other organosulfur compounds, served as sole sulfur
source for growth. The highest desulfurization specific activity reported for the
thermophilic Paenibacillus sp. strain A l l - 2 was approximately 0.08 umole 2HBP/min/g DCW [57]. Bacterial cultures containing cloned desulfurization
genes from Paenibacillus (tdsABC) were reported to have a maximum
desulfurization specific activity of 0.16 umole 2-HBP/min/g DCW [61], while
strains containing cloned Rhodococcus desulfurization genes (dszABC) were
reported to have a maximum desulfurization specific activity of 4.7 umole 2HBP/min/g DCW [52, 58]. The moderate thermophile Mycobacterium phlei
WU-F1 was described as having greater desulfurization activity than
Paenibacillus sp. strain Al 1-2, but no specific activity data was reported [53].
2.7. Genetic modifications to increase desulfurization activity
Largely due to the interest in biodesulfurization and the high percentage
of desulfurization competent bacteria that are Rhodococcus species as well as
other potential applications of this genus, there has been a lot of research on the
genetics of Rhodococci [88, 89]. Multiple cloning and shuttle vectors are
available and genetic manipulation of Rhodococcus can now be conveniently
and reliably performed [90-92]. However, one area of genetic research that has
received comparatively little attention is gene expression in Rhodococcus.
Overexpression of genes in Rhodococcus has been reported [89, 90, 92, 93];
however, an array of gene expression vectors and a knowledge of the consensus
sequences of transcriptional promoters in Rhodococcus is lacking.
47
Fig. 2. Resting cells of M. phlei GTIS10 exhibit specific desulfurization activity at higher
temperatures than resting cells of R. etythropolis IGTS8. The amount of 2-HBP produced by
the conversion of DBT by each culture after incubation for 24 hours at various temperatures
was quantified by HPLC analysis. Rate of change in 2-HBP concentration was calculated
from the linear portion of the curve, generally the first 4 hours of the incubation. The specific
desulfurization activity values recorded are averages of three replicate samples from three
separate experiments for a total of nine data points. Standard deviation was less than 10 %. O,
M. phlei GTIS10; , R. erythropolis IGTS8.
48
The highest rate of 2-HBP formation was obtained without the cloned
FMN oxidoreductase, perhaps because the accumulation of intermediates
inhibited the Dsz enzymes. The maximum amount of 2-HBP produced was 0.2
mM regardless of the amount of DBT added or the incubation time, which
suggests that inhibition by 2-HBP is also a key factor limiting biodesulfurization
efficiency in E. coli, and probably in other bacterial hosts as well [52]. Oshiro et
al. [94] screened 80 bacterial and 20 yeast cell extracts to find flavin reductases
with the best ability to support DszC and DszA activity. A flavin oxidoreductase
from Paenibacillus polymyxa was found to be the best allowing 3.5 to 5-fold
better activity of DszC and DszA as compared with the Rhodococcus flavin
oxidoreductase.
Rhodococcus strains containing increased copies of dszABC genes on
plasmids or integrated into the chromosome have resulted in higher DBT
conversion rates, but also in the accumulation of pathway intermediates as
DBTSO and DBTSO2 [58, 96]. When the copy number of the dszD gene was
increased in Rhodococcus erythropolis KA 2-5-1 cultures containing their
natural complement of dszABCD genes, then DBTSO and DBTSO2
accumulation occurred. However, if the copy number of all of the dszABCD
genes was increased then the accumulation of intermediates was avoided, but
only when the correct balance between dsz genes was achieved [58]. Derivatives
of R. erythropolis KA 2-5-1 originally had a specific desulfurization activity of
0.05 mmol/g DCW/hr while derivative cultures that, in addition to their natural
complement of dszABCD genes, contained a plasmid with one copy of the
dszABC genes had a specific desulfurization activity of 0.14 mmol/g DCW/hr;
0.19 mmol/g DCW/hr with dszABCD genes on a plasmid, and 0.28 mmol/g
DCW/hr with two copies of dszABC genes and one dszD gene on a plasmid.
Derivative cultures that contained additional copies of the dszABC operon or the
dszD gene did not yield cultures with higher enzymatic activity. Similar results
were obtained for the expression of various combinations of dsz and tds genes in
Rhodococcus, E. coli, and Pseudomonas hosts [96, 97], demonstrating that in
order to obtain bacterial cultures with the highest possible desulfurization
activities it is necessary to obtain the proper ratio of desulfurization enzymes
and cofactors.
The results of experiments in which different copy numbers,
combinations of dsz/tds genes and promoters were used also revealed that a limit
in desulfurization activity is reached that can not be overcome by increasing the
amount of the Dsz/Tds enzymes in cells [24, 76, 93, 95, 98]. There are other
factors affecting desulfurization activity and/or the intrinsic properties of the
Dsz/Tds enzymes needs to be improved if higher desulfurization activity is to be
achieved. A way of improving the intrinsic properties of enzymes is directed
evolution.
49
50
51
yet been tested in pilot scale experiments so the costs and efficiency of such a
process are not yet known.
Energy BioSystems Corporation (EBC) conducted a comprehensive
evaluation, particularly as regards crude oil and fractions, of the
biodesulfurization technology originally developed by the Institute of Gas
Technology (IGT) [now known as the Gas Technology Institute (GTI)] under a
program funded by the U.S. Department of Energy. Encouraged by their
experimental results and feedback from the petroleum industry, EBC licensed
the technology, and assembled a team of executives, engineers, and scientists
from the petroleum industry, committed to the commercialization of
biodesulfurization technology.
The development of bioprocesses for biodesulfurization of petroleum
have been almost exclusively focused on the use of biocatalysts that are
derivatives of, or related to, R. erythropolis IGTS8, and diesel has been the
target for the development of the first biodesulfurization processes [24, 25].
EBC was the first organization to seriously attempt the development of a
commercial biodesulfurization process. They chose diesel fuel desulfurization as
the target for initial process development efforts because environmental
regulations mandating a reduction of the maximum permissible concentration of
sulfur in diesel to 50 ppm had been proposed and existing refinery processes
were not able to efficiently and economically meet this requirement [25]. The
most abundant organosulfur compounds in diesel includes DBT and its
derivatives which are recalcitrant to traditional hydrodesulfurization but are
good substrates for biodesulfurization [2].
The application of any technology, chemical or biochemical, to the
treatment of petroleum requires a highly efficient process as the resulting
products are low priced commodities [105]. Moreover, the volume of petroleum
processed, even at a small refinery, dwarfs the scale of bioprocesses typically
used in the pharmaceutical and biotechnology industries. To address these
process concerns, EBC claimed to have achieved a 200-fold improvement in the
specific activity of the R. erythropolis IGTS8 biocatalyst using a combination of
medium improvement, reaction conditions and genetic engineering [24].
Moreover, process engineering research increased the volumetric reaction rate
(oil/water ratio), biocatalyst life and solved separations issues. Specific details
about EBC's biodesulfurization process and the results achieved were not
published, but a desulfurization rate of 20 umole DBT/min/g DCW was stated
as a target for a commercially successful process [25]. The literature contains a
large amount of information regarding the use of genetic engineering to achieve
higher desulfurization rates as previously discussed in this chapter. It has been
shown that R. erythropolis IGTS8 biocatalysts are capable of functioning at 9to-1 oil-to-water ratios [106], and maximum cell yields in fed batch fermentation
52
53
The key reasons that EBC did not succeed in developing a commercially
viable biodesulfurization process included changes in the environmental
regulations and improvements in hydrodesulfurization technologies. When EBC
began process engineering efforts to develop a commercial process for the
biodesulfurization of diesel, the environmental regulations specified a maximum
total sulfur content of 50 ppm and the existing hydrodesulfurization processes
could not efficiently achieve that goal. However, while EBC was involved in the
challenging task of implementing the first bioprocess in the petroleum industry
(other than waste remediation), stricter environmental regulations were proposed
decreasing the maximum permissible sulfur content in diesel to 10 to 15 ppm.
Additionally, during this same time frame, improvements were made in
hydrodesulfurization technology that allowed these lower sulfur levels to be
reached [109].
Integrating a biodesulfurization process into a refinery is the only way to
treat a product such as diesel, but this requires a substantial modification of
current operations in a refinery and requires that the biodesulfurization process
operate at the same speed and reliability as other refinery processes so as not to
disrupt normal refining operations. It is very challenging for any new technology
to be embraced by a conservative industry such as the petroleum industry so that
employing biodesulfurization as a component of refinery operations met with
understandable opposition. However, alternative ways of implementing a
biodesulfurization process exist (see chapter 4)[104].
54
Fig. 4. Overview of the Energy Biosystems Corporation process for the simultaneous
biodesulfurization diesel oil and the production of a sulfinate/surfactant byproduct.
55
56
57
3. BIODENITROGENATION OF PETROLEUM
The removal of organically bound nitrogen from crude oil, without the loss of
significant calorific value, requires the selective cleavage of carbon-nitrogen
bonds. The selective cleavage of carbon-sulfur bonds in crude oil using
biocatalysts has been demonstrated [2, 24, 113] and it may be possible to
selectively cleave carbon-nitrogen bonds using biocatalysts developed from
microorganisms capable of metabolizing compounds such as quinoline and
carbazole [114]. The cleavage of carbon-nitrogen bonds resulting in the
conversion of quinoline to 8-hydroxycoumarin and ammonia has been
demonstrated [115], and the genes that encode the enzymes participating in the
quinoline degradation pathway have been identified and sequenced [116]. The
removal of nitrogen from crude oil by a quinoline degrading culture,
Pseudomonas ayucida IGTN9m, has also been demonstrated [115]. However,
the abundance of quinoline relative to other organonitrogen compounds in crude
oil is low and existing quinoline degradation enzymes have a narrow substrate
range. Consequently, even though removal of 68% of quinoline from crude oil
was demonstrated the total nitrogen content was reduced by only 5%. An
appropriate topic for future research is the development of cultures that express
higher levels of quinoline degrading enzymes that have wider substrate ranges,
but it is also important to develop biocatalysts that can remove nitrogen from
other compounds typically found in petroleum such as carbazole.
Fig. 6. Carbazole Degradation Pathways. The top pathway illustrates the existing carbazole
degradation pathway that results in overall degradation, whereas the bottom pathway
illustrates a potential pathway for the selective removal of nitrogen from carbazole that could
be developed using metabolic engineering.
58
59
60
61
62
63
64
65
[106] S. Patel, J. J. Kilbane, and D. A. Webster, J. Chem. Technol. Biotechnol. 69 (1997) 100.
[107]O. Yoshikawa, Y. Ishii, K-I. Koizumi, T. Ohshiro, Y. Izumi, and K. Marahashi, J.
Biosci. Bioeng. 94 (2002) 447.
[108]L. Linguist, and M. Pacheco, Oil & Gas Journal (1999), pp. 45-48.
[109] S. T. Oyama, and Y-K. Lee, American Chemical Society, Fuel Chemistry Division 48
(2003), 173-174.
[110]S. Reeson, Energy World 235 (1996) 9.
[111]M. S. Lin, T. Premuzic, J. H. Yablon, and W. M. Zhou, Appl. Biochem. Biotechnol. 5758(1996)659.
[112]E. T. Premuzic, M. S. Lin, M. Bohenek, and W. M. Zhou, Enegy & Fuels 13 (1999)
297.
[113] J. J. Kilbane II, Final Report, Energy BioSystems Project No. 40308-02 (1992).
[114]M. J. Benedik, P. R. Gibbs, R. R. Riddle, and R. C. Wilson, Trends in Biotechnology 16
(1998)390.
[115] J. J. Kilbane, R. Ranganathan, K. J. Kayser, L. Cleveland, C. Ribiero and M. M.
Linhares, Appl. Environ. Microbiol. 66 (2000) 688.
[116]U. Frerichs-Deeken, B. Goldenstedt, R. Gahl-Janben, R. Kappl, J. Huttermann, and S.
Fretzner, European J. Biochem. 270 (2003) 1567.
[117]S. Mitra-Kirtley, O. C. Mullins, J. van Elp, S. J. George, J. Chen, and S. P. Cramer, J.
Am. Chem. Soc. 115 (1993) 252.
[118] J. J. Kilbane II, A. Daram, J. Abbasian, and K. J. Kayser, Biochem. Biophys. Res.
Comm. 297 (2002) 242.
[119]H. Habe, Y. Ashikawa, Y. Saiki, T. Yoshida, H. Nojiri, and T. Omori, FEMS Microbiol.
Lett. 211(2002), 43.
[120]K. Kirimura, H. Nakagawa, K. Tsuji, K. Matsuda, R. Kurane, and S. Usami, Biosci.
Biotechnol. Biochem. 63 (1999) 1563.
[121]H. Nojiri, J. W. Nam, M. Kosaka, K. I. Morii, T. Takemura, K. Furihata, H. Yamane,
and T. Omori, J. Bacteriol. 181 (1999) 3105.
[122]N. Oichiyama, T. Omori, and T. Kodama, Biosci. Biotech. Biochem. 57 (1993) 455.
[123] J. Schneider, R. J. Grosser, K. Jayasimhulu, W. Xue, B. Kinkle, and D. Warshawsky,
Can. J. Microbiol. 46 (2000) 269.
[124]J. M. Shepherd, and G. Lloyd-Jones, Biochem. Biophys. Res. Comm. 247 (1998) 129.
[125]R. R. Riddle, P. R. Gibbs, R. C. Wilson, and M. J. Benedik, J. Ind. Microbiol.
Biotechnol. 30 (2003) 6.
[126]S. I. Sato, J. W. Nam, K. Kasuga, H. Nojiri, H. Yamane, and T. Omori, J. Bacteriol. 179
(1997)4850.
67
Chapter 3
Instituto Mexicano del Petroleo. Eje Central Lazaro Cardenas 152, San Bartolo
Atepehuacan 07730 Mexico DF, Mexico
b
1. INTRODUCTION
The contemporary society is highly dependent on oil supply for energy,
transportation, food production, and in general, industrial production. A century
ago the oil exploitation began, first as a source of energy and now as a source
both of energy and raw material. Thus, history will describe our time as the oil
based society. Nature took 500 millions years to accumulate the world's oil;
nevertheless, the world's petroleum could be consumed in two centuries [1]. The
inexorable production peak is estimated to occur sometime between 2010 and
2020 and then the oil resources will be drastically reduced at the end of this
century (Fig. 1) [2]. When the world's oil reserves become scarce, the more
expensive fuel sources as hard-to-extract oil deposits, tarry sands, and synfuels
from coal will be brought to the front of production.
68
69
of product per liter per hour have been achieved. Biocatalytic reactions that have
been successfully applied in the industry, at large scale, include: production of
high fructose corn syrup, fatty acids and triglyceride oils, aspartame, acrylamide,
antibiotic precursors, amino acids, S-2-chloropropionic acid, polylactic acid and
cyclodextrins [8].
At first glance, enzymes seem to be more expensive than chemical
catalyst. Enzyme prices ranges from $ 100 per kilogram, as for crude
preparations of amylase, to $ 100,000 per kilogram, as for lactic dehydrogenase.
However, the key cost to consider in biocatalysis should be not the cost of the
enzyme itself, but rather the cost-contribution of the final product. This cost
contribution could be as low as $ 0.10 per kilogram as in the case of aspartase in
the L-aspartic acid production. When compared with the cost of other catalyst,
especially those with similar selectivity, the prices of enzymes are not very
different (Table 1).
Still the industrial use of enzymatic catalysts is limited by their instability
under harsh conditions, which are usually found in large-scale processes.
Nevertheless, chemical and genetic modifications of enzymes to improve both
activity and stability, together with solvent engineering and new catalytic
activities from extremophiles microorganisms will provide better biocatalysts
for the specific needs of the petroleum industry. Non-conventional uses of
enzymatic transformation are still in their infancy. Non-aqueous systems, high
temperatures and hydrophobic substrates are the three main characteristics of oil
industry that represent the most important challenges for the enzymatic catalysis
to be applied in the petroleum refining industry. The success of biocatalysis in
the petroleum industry depends on the development of biocatalysts able to
perform transformations of oil products in non-aqueous systems and stable
under the conditions usually found in the refineries.
Table 1
70
71
72
73
need an aqueous phase to accomplish sulfur removal, while enzymes are able to
function in media containing very low water content. Thermodynamic water
activity (aw) influences both enzyme activity and stability, as water acts as a
lubricant altering the flexibility of enzyme molecules. Protein mobility and
therefore protein unfolding is restrained in a low water content medium.
However, a certain amount of protein-bound water is essential to allow enough
molecules flexibility to execute catalysis [40]. Thus it is possible to optimize
enzyme performance in hydrophobic media by controlling aw [10, 41-42]. In
addition, it has been shown that in certain organic media enzymes are active and
more thermostable than in aqueous media [12, 13, 43], and it is possible to
perform enzymatic transformations at temperatures higher than 100C.
Although sulfur elimination might not be achieved by a single enzymatic
step, the enzyme-mediated transformation of sulfur-containing compounds may
facilitate its removal. An enzymatic procedure to reduce the sulfur content from
straight-run diesel has been described [44]. A fungal chloroperoxidase from
Caldariomyces fumago was able to oxidize the sulfur-containing fraction of
untreated diesel containing 1.6% sulfur, in the presence of low concentrations of
hydrogen peroxide.
Figure 2 shows gas chromatograms with both Flame Ionization (FID,
general) and Flame Photometric (FPD, sulfur selective) detectors. The
distribution of compounds in straight-run diesel fuel before and after oxidation
with chloroperoxidase, are shown in panel a and b, respectively. The oxidation
is clearly detected by the increase of boiling point (retention time) of these
compounds on the gas chromatogram monitored by the sulfur selective detector
(FPD). The higher boiling point of the oxidized compounds allowed its removal
by a distillation step. Microdistillation of both chloroperoxidase-oxidized and
untreated diesel fuels monitored by FID and FPD (Fig. 3) shows that the
hydrocarbon distillation profile changes slightly after enzymatic treatment. In
contrast, the sulfur selective detector (FPD) shows a significant change of the
distillation profile, in which most of organosulfur compounds were effectively
oxidized and their boiling points increased after enzymatic treatment.
Table 2
Process characteristics of enzymatic and metabolic desulfurization.
Enzymatic desulfurization
Activity in low water systems
Activity at temperature higher than 100C
Activity in toxic systems
Activity only on organosulfur compounds
Life-time depending on molecule stability
Metabolic desulfurization
Activity in aqueous phase
Inactivation at high temperature
Sensitive to toxics
Needs carbon source
Self-producing catalytic system
74
Fig. 2. Gas chromatograms of straight ran diesel fuel before (a) and after (b) chloroperoxidase
treatment [44].
75
Table 3
Sulfur content of straight-run diesel fuel after enzymatic oxidation with chloroperoxidase from Caldariomyces fumago followed by a distillation to 325C as final
distillation point.
Distillation
TPH (%)
Sulfur (%)
Destillate
83
1.27
Residue
17
3.21
TPH. Total petroleum hydrocarbons.
Enzymatic -H distillation
TPH (%)
Sulfur(%)
71
0.27
29
5.51
76
77
Table 4
Sulfur-containing substrates of chloroperoxidase.
78
79
activity (see chapters 1 and 4). Some reports on oil biodegradation claim the
degradation of asphaltenic fraction by mixed bacteria [63, 64]. However, none
of these reports described the analytical results of extractable materials
recovered from appropriate sterile controls. On the other hand, although
microorganisms have been found associated with bitumens containing high
amounts of asphaltenes [65], the asphaltenic fraction did not support bacterial
growth and no changes in asphaltene content could be found after bioconversion
of heavy oils and asphaltenes [66, 67]. Because the asphaltene content was
usually determined gravimetrically after n-alkane precipitation, the reported
changes could be attributed to the disruption of the asphaltenic matrix by the
production of surfactants during bacterial growth, liberating trapped
hydrocarbons. Therefore, most of the asphaltene losses during microbial activity
could be considered to be abiotic losses [68].
Nevertheless, a clear experimental evidence that enzymes are able to
modify asphaltene molecules has been reported [69]. Chloroperoxidase from the
fungus Caldariomyces fumago was able to transform petroporphyrins and
asphaltenes, and this modification was significantly higher in systems containing
organic solvent than in aqueous systems [69, 70]. Asphaltenes and
petroporphyrins are highly hydrophobic materials, thus mass transfer limitations
are expected in aqueous reactions. The biocatalytic oxidation of a
petroporphyrin rich-fraction of asphaltenes in the ternary solvent system and in
the presence of hydrogen peroxide was performed. Chloroperoxidase catalyzed
reaction produced notable spectral changes in the petroporphyrin rich-fraction of
asphaltenes (Fig. 5). The destruction of petroporphyrins by chloroperoxidase in
the presence of hydrogen peroxide leads to the removal of Ni and V from
asphaltene molecules, as in the case of synthetic nickel and vanadium
porphyrins (Table 5).
On the other hand, a doubly modified cytochrome c (PEG-Cyt-Met) was
able to catalyze the oxidation of a petroporphyrin rich-fraction of asphaltenes in
the ternary solvent system and in the presence of 100 mM of tert-butyl
hydroperoxide [71]. As chloroperoxidase, the PEG-Cyt-Met catalyzed reaction
produced spectral changes in the petroporphyrin rich-fraction of asphaltenes
(Fig. 5). The oxidative porphyrin ring disruption entails the simultaneously
release of metal. The biocatalytic process with PEG-Cyt-Met removed 95% of
the vanadium and 74 % of the nickel (Table 5). The destruction of the
petroporphyrin molecules is conformed by the Soret band loss and metal
removal.
80
81
Table 5
Nickel and Vanadium removal from petroporphyrin rich fractions of asphaltenes by
chloroperoxidase-mediated reaction.
Heavy metal
Nickel
Vanadium
Chloroperoxidase [69]
Chloroperoxidase [70]
20%
19%
57%
52%
Chemically modified
cytochrome c [71]
74%
95%
82
the spectra from both untreated and treated samples showed an important 5.29
ppm signal, probably due to protons from non aromatic (C = C) double bonds,
which are not detectable in whole asphaltenes fractions. The main differences of
enzyme treated samples when compared with the untreated fraction appeared in
the saturated hydrocarbon region: a quartet placed on 2.28 ppm, a triplet placed
on 2.49 ppm, and a singlet placed on 3.63 ppm. These signals seem to be
originated from the hydrocarbon chains of polar compounds, may be from
oxygenated compounds as 13C spectrum shows (see below). The singlet shift can
be attributed to ether or alcohol groups. The ester-amide signal (4.3-4.36 ppm)
was very important in the oxidized sample, while was minor in the control
petroporphyrins.
The 13C NMR analysis showed the presence of 58.78 ppm and 46.11 ppm
shifts in the control, which are attributed to the C-N bond (Fig. 9). These signals
disappeared in the oxidized petroporphyrins. Signals between 10 ppm and 60
ppm are usually assigned to the hydrocarbon chains. The NMR spectrum from
oxidized petroporphyrins showed a more intense terminal methyl (-CH3) signal
than in the untreated sample. The (-CH2-) / (-CH3) intensities ratio was lower in
the treated asphaltenes fraction than in the untreated ones, which could be
attributed to the presence of shorter alkyl chains or more branched chains. Thus,
this lower ratio could be the consequence of molecule cracking. The aromatic
region of the spectra (110 ppm to 160 ppm) showed significant differences. The
control showed a signal-hill between 133 ppm and 146 ppm, which include the
carbon atoms corresponding to heteroatom moieties (S, N, O), aromatic carbons
bonded to alkyl moieties, and aromatic carbons bonded to other aromatic
carbon. This signal-hill disappeared in the oxidized fraction, suggesting a loss of
heteroatoms or alkyl derivatives in the aromatic molecules. A reduction in the
number of substituted aromatic carbons and an increase of the number of
aromatic carbons bonded to hydrogen are observed.
The enzymatic treatment of asphaltenes is an interesting alternative for the
removal of heavy metals in order to reduce catalyst poisoning in hydrotreatment
and cracking processes. On the other hand, enzymatic cracking of asphaltenes
molecules should not be excluded. The enormous amount of energetic resource
found as asphaltenes-rich deposits justify the exploration of alternative
upgrading technologies.
83
Fig. 6. FTIR spectra of untreated and biocatalytically treated porphyrin-rich fractions from
asphaltenes. FTER was performed using the film-spreading technique [71].
Fig. 7. Absorbance contours from gel permeation chromatography (GPC) of untreated and
biocatalytically treated porphyrin-rich fractions from asphaltenes [71].
84
85
86
Fig. 10. The influence of ionization potential of PAHs on the specific activity of lignin
peroxidase oxidation [82].
87
Table 6
Products from in vitro oxidation of polycyclic aromatic hydrocarbons by lignin
peroxidase from Phanerochaete chrysosporium.
PAH
Anthracene
Acenaphthene
1 -Methylanthracene
2-Methylanthracene
9-Methylanthracene
Pyrene
Benzo(a)pyrene
Products
9,10- Anthraquinone
1-Acenaphthenol
1 - Acenaphthenone
1 -Methyl-9,10-anthraquinone
2-Methyl-9,10-anthraquinone
9-Methyl-9,10-anthraquinone
9-Methyl-10-anthranone
9,10-Anthraquinone
1,8-Pyrenedione
1,6-Pyrenedione
1,6-Benzo(a)pyrenedione
3,6-Benzo(a)pyrenedione
6,12-Benzo(a)pyrenedione
Ref.
83,84
83
83
83
83
83
83
83
83,84
82
87
87
87
88
89
Table 7
In vitro PAHs oxidation with manganese peroxidase in lipid
peroxidation reactions [96].
.
,
Aromatic compound
r
Fluorene
Benz(a)antrhracene
Benzo(a)pyrene
Anthracene
Dibenz(a,c)anthracene
Benzo(e)pyrene
Diphenylmethane
Benzo(c)phenanthrene
Benzo(b)fluoranthene
Fluoranthene
Phenanthrene
Oxidation rate
,
...
(nmol/h)
3.10
1.08
0.96
0.93
0.60
0.31
0.30
0.21
0.19
0.14
0.06
Table 8
Oxidation of aromatic compounds by versatil peroxidase at pH 4.0 in the absence
ofMn(II)[113].
Specific activity (min"1)
PAH
Ionization potential (eV)a
9-Methylanthracene
7.25
52 (2.7) b
C
1 -Methylanthracene
NA
22.4(1.7)
2-Methylanthracene
7.37
12.4 (1.1)
Anthracene
7.41
2.5 (+0.01)
Benzo(a)pyrene
7.41
0.32 (0.04)
Pyrene
7.42
0.008 (0.01)
Benzo(e)pyrene
7.43
NRd
Chrysene
7.60
NR
Carbazol
7.68
2.4 (0.05)
1 -Methylphenanthrene
7.70
NR
Acenaphthene
7.76
NR
Phenanthrene
7.91
NR
Dibenzothiophene
7.93
NR
Fluoranthene
7.95
NR
Naphthalene
8.15
NR
a
Photoelectron spectroscopy values (http://webbook.nist.gov)
b
Values in parentheses are standard deviations.
C
NA, not available
d
NR, no reaction
90
Table 9
Kinetic constants of purified cytochrome P448
cerevisiea for oxidation of benzo(a)pyrene [115, 116].
System
Reconstituted with NADPH
Cumene hydroperoxide
Hvdrosen oeroxide in situ
kcat (min"1)
33
125
200
from
Saccharomyces
KM ( U M )
0.017
0.022
0.034
91
Fig. 11. Engineering cytochrome P450 BM-3 for oxidation of polycyclic aromatis hydrocarbons
[127].
92
Table 10
Specific activity of yeast cytochrome c on aromatic compounds [135].
Aromatic compound
Dibenzothiophene
Anthracene
Pyrene
Benzothiophene
Carbazole
Acenaphthene
Chrysene
Fluoranthrene
Fluorene
Phenanthrene
Triphenylene
NR, no reaction detected.
Product
Dibenzothiophene sulfoxide
9,10-Anthraquinone
1,8-Pyrenodione
Benzothiophene sulfoxide
Unknown
(0.1)
(0.1)
(0.3)
(0.2)
(0.1)
NR
NR
NR
NR
NR
NR
93
Table 11
Kinetic constants of wild-type and variants of yeast iso-1cytochrome c for pyrene oxidation [135].
kcat
Variant
Ala86;ThrlO2
Phe67;ThrlO2
Ala72;ThrlO2
Ala52;ThrlO2
Ala73;ThrlO2
Ala87;ThrlO2
Phe82;CyslO2(WT)
Ala79;ThrlO2
(s'1)
0.17
0.10
0.13
0.18
0.28
0.39
0.31
3.28
K M ,app
kcat/KM,app
(mM)
4.0
3.3
4.0
4.7
7.5
3.9
9.7
101.8
(s"1 M"1)
33
32
33
39
38
99
32
32
Table 12
Oxidation of polycyclic aromatic hydrocarbon by unmodified- and methylated
poly(ethylene)glycol-modified-cytochrome c [137].
Aromatic compound
7,12-Dimethylbenzanthracene
1,2:3,4-Dibenzanthracene
Azulene
3-Methylcholanthrene
7-Methylbenzo(a)pyrene
1,2:5,6-Dibenz anthracene
Triphenylene
Dibenzothiophene
Anthracene
Thianthrene
Pyrene
Fluoranthene
Acenaphthene
Benzo(a)pyrene
Fluorene
Phenanthrene
Chrysene
9,10-Dimethylanthracene
Naphthalene
Biphenyl
NR. No reaction detected
94
95
Product
9,10-Anthraquinone
9,10-Anthraquinone
Unknown
1,8-Pyrenodione
Dibenzothiophene sulfoxide
9-Fluorenone
96
Table 14
Specific activity of chloroperoxidase from Caldariomyces
fumago against aromatic compounds.
Aromatic compound
9-Methylanthracene
Azulene
Anthracene
2-methylanthracene
7,12-Dimethylbenzanthracene
Benzo[a]pyrene
7-Methylbenzo [ajpyrene
Acenaphthene
Pyrene
Benzo[ghi]perylene
Perylene
Biphenylene
Phenanthrene
Fluoranthene
Fluorene
Triphenylene
Naphtalene
Biphenyl
Dibenzofuran
Anthrone
NR: no reaction detected.
97
reported for other peroxidases. Lignin peroxidase is able to oxidize PAH's and
form quinones up to a PAH's IP of 8.0 eV [85] and manganese peroxidase from
P. chrysosporium shows a threshold value for PAH's substrates of 8.1 eV [98].
4.8. Laccase
Laccases (EC 1.10.3.2) are copper-containing enzymes widespread in
white rot fungi which catalyze the oxidation of a variety of aromatic phenols and
anilines, reducing oxygen to water. Their characteristics have been
comprehensively reviewed [148, 149]. While the substrate range for laccase is
normally limited to phenolic substrates, it can be extended to nonphenolic
compounds with the addition of mediating substrates such as ABTS and HBT
[150-155]. In vitro oxidation of PAH's has been demonstrated by purified fungal
laccases [156-160]. The rate of oxidation of several PAH's has been shown to be
enhanced by the addition of the cooxidant ABTS [158-161]. Purified laccase of
C. gallica transformed 7 of 10 PAHs examined in the presence of ABTS (Table
15). Benzo[a]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene,
biphenylene, acenaphthene, and phenathrene were oxidized by laccase [160].
The synthetic or natural mediating substances acts as free-radical mediators.
These mediators are sbstrates for laccase and tranformed into free radicals by
one electron subtraction, and then they diffuse and oxidize the aromatic
compound prducing, as peroxidases, mainly quinones. Unlike peroxidases, no
clear relationship between the substrate ionization potential and first-order rate
constant could be detected.
Fig. 12. Influence of the PAH ionization potential on the chloroperoxidase activity.
98
Table 15
First rate constants of reactions of C. Gallica laccase with polycyclic aromatic
hydrocarbons [160].
PAH
9-Methylanthracene
Benzo[a]pyrene
Acenaphthene
Anthracene
2-Methylanthracene
Biphenylene
Phenanthrene
Pyrene
Fluoranthene
Azulene
NR, no reaction detected.
NEO, nonenzymatic oxidation.
Rate constant
(h"1)
240
83
10
5.2
4.9
3.8
0.8
NR
NR
NEO
Ionization potential
(eV)
7.23
7.12
7.7
7.55
7.42
7.58
8.03
7.72
7.76
7.43
Fig. 13. Effects of the free-radical mediators HBT and ABTS on the anthracene oxidation by
purified laccase from C. gallica [160].
99
Ethane
Propane
Usual
Feedstock
Methane
Hydrocarbons
Methanol
Ethylene
Methanol
Ethylene
Ethylene
Propylene
Propylene
Propylene
Conventional Process
Product
Reforming
Steam cracking
Oxidation/dehydrogenation
Oxychlorination
Carbonylation
Oxidation
Oxidation
Ammoxidation
Oxidation
Chlorhydrination, epoxidation
Methanol
Ethylene
Formaldehyde
Vinyl chloride
Acetic acid
Acetaldehyde
Ethylene oxide
Acrylonitrile
Acrylic acid
Propylene oxide
100
Paraffinic substrate
Active site
p-Methane monooxygenase
(1.14.13.25)
s-Methane monooxygenase
(1.14.13.25)
Alkane hydroxylase
(1.14.15.3)
P-450 monooxygenase
(1.14.14.1)
Methane, Ci to C5 linear
alkanes
Methane, C5 to C7 linear and
branched alkanes
C5 to C24 alkanes
Ref.
169-171
172
101
Fig. 14. Steps involved in the oxidation reaction catalyzed by alkane hydroxylase and
methane monooxygenase (A) and cytochrome P450 (B).
102
Table 20
Relative activities of alkane hydroxylase for the oxidation of
medium-chain alkanes [177].
Substrate
Major product
R elative
n-Hexane
n-Heptane
n-Octane
n-Nonane
n-Decane
n-Undecane
n-Dodecane
None
n-Heptanol
n-Octanol
n-Nonanol
n-Decanol
n-Undecanol
None
0
0. 58
1
0.83
0. 16
< 0.05
0
103
Table 21
Alkane hydroxylation by different enzymatic systems [172]
Enzymatic system
Substrate
Hexane
Hexane
Octane
Methane
3800
182
210
222
104
several opportunities for some sectors of the petroleum industry, such as deep
desulfurization and denitrogenation, and asphaltene upgrading.
The powerful tools of molecular biochemistry can be used to improve
the enzyme stability and efficiency. These techniques may be applied to the
particular needs of the petroleum industry. Protein engineering is generally
understood as the use of site-directed or random mutagenesis to alter the
properties of a protein or enzyme. In addition, the enzymes isolated from
extremophilic microorganisms are extremely thermostable and generally
resistant to non conventional conditions such as organic solvents and extreme
pH. Thus, many enzymes and enzymatic proteins are still to be discovered. In
addition, over the past two decades people have seen many examples of the
improvement of biocatalysts by chemical and genetic techniques.
Still, there is not any enzymatic process ready to by applied in the
petroleum refining industry, and three main research fields may be suggested
to obtain an enzyme catalysts to be used in the petroleum industry:
1) The search of new enzymatic activities upon petroleum products,
specially from extreme environments. New microorganisms are currently
discovered from extreme environments such as thermal vents in the ocean
deep and fossilized salt rocks. The enzymes isolated from extremophilic
microorganisms are extremely thermostable and generally resistant to organic
solvents and extreme pH. Enzymes from these microorganisms working in
non-aqueous systems at temperatures higher as 200C (operating conditions
found in refineries) could be expected. Moreover at high temperatures, the
hydrocarbons bioavailability and solubility is increased. All these unknown
organisms are a potential source of new enzyme forms with different
physicochemical properties: the potential source of biocatalytic activity for
the oil industry could be there.
2) The improvement of the enzymatic activities in very low water
systems, in order to increase the transformation rates using petroleum
fractions without further addition of water. Since petroleum is a hydrophobic
material, it is suitable to speculate that new enzymatic processes for the oil
industry should be carried out in non-aqueous systems. The use of reaction
mixtures containing organic solvents reduces mass transfer limitations,
promoting the establishment of productive interactions between the enzyme
and the hydrophobic substrates (oil-derived compounds). In addition, a
biocatalyst placed in a non-aqueous medium shows interesting properties,
such as improved thermostability, higher substrate accessibility, adjustable
selectivity, and high storage stability. The study of the relationship between
the solvent properties and the enzyme activity seems to be essential to
understand and to improve the biocatalytic processes.
105
Table 22
Improvement of cytochrome P450 BM-3 for the catalysis of small alkanes oxidation [179]
Enzyme mutant
Mutant 139-3
Substrate
Propane
Octane
Mutant J
Propane
Octane
Propane
Mutant 9- 10A
Octane
Mutant 9-10A-A82L Propane
Octane
Major product
n-propanol
2-octanol
n-propanol
2-octanol
n-propanol
2-octanol
n-propanol
4-octanol
106
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113
Chapter 4
1. INTRODUCTION
Increasing supply of heavy crude oils and bitumens, mainly from Canada,
Mexico and Venezuela, has increased the interest in transportation and
conversion of the high-molecular weight fractions of these materials into refined
fuels and petrochemicals. The high viscosity of these crudes requires addition of
a solvent in order to allow pipelining over a significant distance. The cost of
suitable solvents, such as naphtha or natural gas condensate, has led to study of
new methods to reduce the viscosity of heavy crudes. Once they enter a refinery,
processing of heavy crudes and bitumens requires conversion of the vacuum
residue components, including the asphaltenes, into distillable oils. This
upgrading has typically been accomplished with either thermal conversion
(cracking or coking) or by catalytic hydroconversion. Thermal processing can
range from mild cracking, to reduce viscosity, to severe cracking with attendant
formation of coke. These high-temperature processes require expensive
investment in process equipment and supporting infrastructure for supply of
hydrogen and treatment of hydrogen sulfide in cracked off-gases.
In contrast to the available processes, biological processing may offer less
severe process conditions and higher selectivity to specific reactions. This
chapter reviews the characteristics of the molecules in the vacuum residue
fraction of crude oils, and examines the prospects for using biological processes
to improve the value of these materials.
114
2. MOLECULES OF INTEREST
Heavy crude oils pose new upgrading challenges, in addition to the upgrading
needs common to lighter crudes. These problems are related to two types of high
molecular weight molecules present in these oils: waxes and asphaltenes. Waxes
are long-chain paraffinic molecules, or alkanes, which typically cause
operational problems if longer than 40 carbon atoms [3]. Asphaltenes, on the
other hand, are not classified by structure, but are defined as a solubility class,
including material that is soluble in toluene but not in -pentane (or alternatively
-heptane). There are two different views on the molecular structure of
asphaltenic material. The first represents asphaltenes as having a single large
condensed polycyclic aromatic core, with aliphatic chains attached on the
periphery (Fig. la) [1, 4-6]. This type of structure, however, does not account
for all of the physical and chemical properties of asphaltenes. The second
Fig. 1. Representative models of asphaltene molecules showing either (a) a single large
condensed polycyclic aromatic core [1] or (b) multiple smaller polycyclic aromatic cores with
aliphatic bridges [2].
115
116
117
118
Fig. 2. Examples of hydrogenation reactions in the biodegradation of (a) TNT [26, 27] and (b)
naphthalene [28, 29].
119
120
121
under nitrate-reducing conditions [45, 46] and w-dodecane [39] under sulfatereducing conditions. The addition of fumarate to alkanes does not occur at a
terminal methyl C-H, but rather at either a C2 or C3 subterminal methylene C-H
[39, 46], producing a branched dicarboxylic acid (Fig. 3b), which is degraded
through fatty acid metabolism. As with the aerobic pathway described above,
anaerobic alkane degradation therefore proceeds from the terminus of the
molecule.
A subterminal attack on long-chain -alkanes may occur in some aerobic
bacterial cultures. A mutant Rhodococcus strain, designated KSM-B-3M,
accumulated c-unsaturated metabolites of -hexadecane, 1-chlorohexadecane,
and heptadecanonitrile, which were not growth substrates [47]. (The first two
compounds did support growth of the wild-type strain.) In all three compounds
the unsaturation was at position 9. Unsaturated products were also detected for
1-hexadecanol, 1,2-epoxyhexadecane, hexadecyl benzene, and hexadecyl
chloroformate. The mutant had likely lost the ability to cleave the alkane chain
at the unsaturated bond, resulting in the inability to grow on these substrates
[47].
The degradation of phytanyl octadecyl ether by a mixed soil culture and by
Rhodococcus ruber (DSMZ 7512) also showed evidence of an initial
subterminal dehydrogenation [48]. Degradation occurred initially on the linear
side chain, and initial degradation products were the phytanyl ethers of C2 to C8
primary alcohols. The corresponding carboxylic acids appeared next as the
alcohols disappeared from the cultures. The final products were the phytanyl
ethers of acetic acid and propanoic acid. One other metabolite was observed,
phytanyl octadec-9-enyl ether. The formation of unsaturated products was
proposed to be analogous to the dehydrogenation of Cig fatty acids in the cell
membrane, which also occurs at position 9. The alternate degradation pathway
proposed starts with the observed internal dehydrogenation, followed by a
hypothesized olefinic oxidation to a secondary alcohol, oxidation to a ketone,
Baeyer-Villiger oxidation to an ester, ester cleavage, and P-oxidation [48].
A subterminal attack of this type has not been shown for a diterminally
substituted alkyl chain, but a similar pathway has been shown as the mechanism
of degradation for both small and large cyclic alkanes. Both cyclohexane
(reviewed in Ref. [49]) and cyclododecane [40] are oxidized via an alcohol to a
cyclic ketone. The ketones are oxidized to lactones by Baeyer-Villiger
monooxygenases, followed by ester cleavage to an co-hydroxycarboxylic acid
(Fig. 3c) and oxidation to a dicarboxylic acid [40] that can be degraded through
central metabolic pathways. The Baeyer-Villiger monooxygenases appear to
have fairly narrow substrate specificities. Cyclododecanone monooxygenase
from R. ruber strain CD4 could also oxidize cyclopentadecanone, but not
cyclohexanone or cyclooctanone [40]. In growth assays, R. ruber strain SCI,
isolated on cyclododecanone, could also grow on Ci5, C13, C n , and C10 cyclic
122
ketones, but not on C8, C7, or C6 cyclic ketones [50]. Conversely, cyclohexanone
monooxygenases are known to favour shorter chain cyclic ketones. For example,
two enzymes from Brevibacterium sp. strain HCU could oxidize C4-C7 cyclic
ketones, but not C 8 -C| 2 compounds [51].
Molecular weight reduction in the residue fraction of heavy oils requires
cleavage of alkyl bridges, where both ends of the carbon chain are blocked by
attachment to aromatic groups. The more common aerobic and anaerobic
bacterial alkane-degradation pathways are not appropriate for molecular weight
reduction in crude oil, because they only activate the free end of the molecule to
create a fatty acid for central metabolic pathways. More relevant research has
been done with long-chain -alkanes and cycloalkanes. This work shows that an
alkyl chain can be cleaved through bacterial attack in the absence of a terminal
methyl group. This reaction is more directly analogous to the alkyl bridges
found in high molecular weight crude oil components, and is a promising
avenue for further work.
3.3. Deposition control
Both asphaltenes and waxes may cause deposition problems in the
reservoir, pipelines, and storage and processing equipment. Asphaltenes deposit
due to an increase in the aliphatic content of the oil, while waxes crystallize and
precipitate due to a drop in temperature (e.g. from the reservoir to the surface,
[52]) or an increase in aromaticity of the bulk oil. Changes in solvency occur
due to dilution or to blending of different oils [19]. Both types of compounds
may co-precipitate, through entrapment of one type in a deposit of the other.
Generally, a wax content greater than 2% by weight is found to lead to wax
deposition problems [3]. Deposition prevention is accomplished through
chemical treatment to maintain the molecules in solution, as well as through
temperature and flow control. Waxes are a valuable feedstock for refinery
operations, so prevention of wax deposition is important to preserve the value of
the oil as well as to avoid operational problems. Existing deposits are removed
through circulation of hot water, hot oil, solvents, and surfactants, or through
"pigging" of transfer lines [53]. In the case of asphaltenes, treatments include
addition of aromatic streams to dissolve the deposits, or the addition of
dispersants to prevent flocculation of the asphaltenes into particles that
subsequently deposit on surfaces.
Although biological treatments for deposition control are commercially
available, little scientific literature is available in this area [54, 55]. Three modes
of biological activity are conceivably relevant to deposition control: production
of metabolites (from carbon sources other than the oil) which improve the
solubility of either waxes or asphaltenes, biotransformation of waxes and
asphaltenes to more soluble products (through molecular weight reduction or
functionalization), and biodegradation to remove the problematic compounds
123
either from the oil or from existing deposits. The ability of bacteria to degrade
solid alkanes is limited by mass transfer rates. For instance, liposome
encapsulation was required to achieve biodegradation of hexatriacontane (n-C^)
by a Pseudomonas isolate which did not grow on the crystalline compound [38].
The usefulness of biological treatments for removal of deposits may therefore be
limited to the production of solubilizing agents rather than direct transformation
or degradation of the crystallized molecules. Isolated bacteria and consortia
from paraffin deposits, hydrocarbon contaminated soils and waters, and brine
have been shown to produce biosurfactants, as well as to degrade hydrocarbons
from samples of paraffin deposits and paraffinic oils [56]. In a flow system, a
consortium of these bacteria decreased the paraffin content of a heavy oil. The
treated oil also had a lower freezing point and a decreased low temperature
viscosity, but the effect of these changes on deposition in the flow system was
not reported [56]. To the extent that microorganisms adsorb wax or asphaltic
material, then bacteria could serve to disperse the deposits and prevent
deposition on surfaces, however, no systematic research has been conducted in
this area.
3.4. Emulsion behaviour and de-emulsiflcation
Water-in-oil (W/O) and oil-in-water (O/W) emulsions occur throughout oil
production, transportation, and processing. The water may be from the
formation or may be added through water or steam injection to improve oil
recovery, or addition of wash water in desalting operations. Emulsions may be
produced incidentally through handling or deliberately to improve flow
properties for enhanced oil recovery and transportation [57]. Desirable
emulsions produced for pipeline transportation are O/W emulsions, usually
containing around 30% aqueous phase [58]. Undesirable O/W emulsions are
typically found in waste waters from the oil industry. Although deemulsification does recover some oil, treatment is generally driven by
environmental concerns rather than economic incentive. On the other hand,
resolution of W/O emulsions improves the quality of the oil, and is therefore
economically driven [53]. Problems associated with water in oil include
corrosion, scale formation, sludge accumulation in storage tanks, altered
viscosity and flow properties, and reduced distillation efficiency [53].
Regardless of the source, emulsions must be resolved at some point before
refining. This is accomplished through heating, settling, centrifugation,
filtration, electrical dehydration, and chemical treatment. The pipeline
specification includes both solids and water, and is typically a maximum of
0.5% bottom solids and water (BS&W) [58].
Emulsions are formed from two immiscible phases through mixing to
produce a fine dispersion of droplets of one phase in the other, where the
interface between the two phases is stabilized by emulsifying components. The
124
energy added through mixing is essential, since the emulsified state is not
usually thermodynamically stable. Emulsifying agents associate at the interface
of the two phases and impart kinetic stability to the emulsion, either through
reduction of interfacial tension (chemical stabilization), or by providing a barrier
to coalescence (physical stabilization). Resolution of emulsions, or deemulsification, proceeds via two steps: flocculation or aggregation of droplets,
and coalescence of droplets to form a continuous second phase. De-emulsifiers
may promote one or both of these phenomena [58].
Crude oil emulsions are complex, and vary from location to location. The
emulsifying agents may be amphiphilic molecules from the oil, especially the
resin fraction, including naphthenic acids. Many crude oil emulsions are
stabilized by fine solids, including clays, scale, or wax crystals [59], or bacteria
themselves [60], which present a barrier to droplet coalescence. Asphaltenes are
especially important in heavy crude oil emulsions. After association with the
interface, asphaltenes agglomerate to form a skin, which prevents coalescence of
droplets. Resins are also believed to play a part in stabilizing this skin [58].
Complex emulsion structures, such as water-in-oil-in-water emulsions, have also
been observed [61]. De-emulsification in the oil industry is challenging due to
the variety of possible emulsion properties, and treatments are currently tailored
to each site and adapted over time [59].
Biological de-emulsification has been studied using a variety of
microorganisms. Whole bacterial cells have received the most research [62-69],
but Streptomyces spores [70], bacterial metabolites [71], and yeast cells [64]
have also been studied. The organisms and emulsion systems used are
summarized in Table 1. The majority of studies have examined model,
chemically stabilized emulsions consisting of water, hydrocarbon, and a
commercial surfactant. This research has allowed some assessment of the mode
of action. De-emulsification ability appears to be associated with the surface of
the bacterial cells. Depending on their hydrophobicity, cells may aggregate at
the oil-water interface, promoting flocculation and coalescence of droplets [72].
Differences in hydrophobicity may account for changes in effectiveness of
microbes in different growth phases, as well as for differing abilities to resolve
O/W or W/O emulsions. In general, it appears that more hydrophilic cells are
required to treat W/O emulsions, while relatively more hydrophobic cells are
able to resolve O/W emulsions [62, 65, 66, 69, 70].
The ability of bacterial cells to de-emulsify both model and oilfield O/W
and W/O emulsions has been demonstrated, but the potential for treating the true
spectrum of real crude oil emulsions has not been rigorously tested. As with
chemical treatments, no single biological treatment will likely be effective for all
chemically stabilized crude oil emulsions. Biological products may still be a
valuable complement to existing chemical technologies.
125
Table 1
Biological systems shown to de-emulsify oil-water emulsions
Organism
Emulsion system
Comments
Nocardia amarae
strain LL-Se6
(ATCC 27808)
Refs.
O/W emulsions:
Alkanes / water
Kerosene / water
Oilfield emulsions
W/O emulsions:
Water / kerosene
Oilfield emulsions
Corvnebacterium
petrophilum
(ATCC 21404)
W/O emulsions:
Oilfield emulsions
[64, 691
Micrococcus sp.
O/W emulsions:
Kerosene / water
W/O emulsions:
Water / kerosene
[631
Mixed aerobic
bacterial culture
W/O emulsions:
Water / kerosene
Oilfield emulsions
Streptomvces sp.
strain AA8321
O/W emulsions:
Kerosene / water
Alkanes / water
Diesel / water
Gasoline / water
Paraffin oil / water
Soybean oil / water
[701
Bacillus subtilis
W/O emulsions:
Water / crude oil
[711
Torulopsis
bombicola
(ATCC 22214)
W/O emulsions:
Oilfield emulsions
[641
126
127
Fig. 4. Representative naphthenic acids structures (based on Ref. [74]). (R - alkyl group; m
number of carbons in the side chain excluding the carboxyl group)
128
129
Fig. 5. Viscosity data for whole oils, residuals, and distillates [88], oil sand bitumens and
topped crudes [84], oil fractions [85], synthetic crude oils [86], and crude oils and natural
bitumens [20] showing correlation to (a) average molecular weight and (b) asphaltene content.
(Analysis temperatures are indicated in the legends)
130
Table 2
Selected organic sulfur compounds successfully used for enrichment of
microorganisms able to use the compounds as sole sulfur source under sulfurlimited conditions
Compound
Procedures used
Refs.
Ametryne
prometryne
(herbicides)
Naphthalenesulfonic
acids
Benzenesulfonic acids
(detergents)
Endosulfan
(an insecticide)
131
132
Table 3
Selected dibenzothiophene desulfurizine bacteria and alternate sulfur sources
133
was also hypothesized, but not directly observed. JVH1 was shown to use a
variety of compounds with aliphatic carbon-sulfur bonds as sulfur sources
(including dialkyl sulfides, thiacycloalkanes, and aryl-terminated sulfides), but
not thiophenic compounds. This selective ability to cleave compounds with
aliphatic carbon-sulfur bonds is extremely interesting for research into
biological viscosity reduction in heavy crude oils.
Fig. 6. (a) Structure of phytanyl octadecyl sulfide. (b) Metabolites produced by Rhodococcus
erythropolis ATCC 13260 and proposed reactions in the degradation of phytanyl octadecyl
sulfide [15].
134
Fig. 7. Proposed pathway of PFPS metabolism in Rhodococcus sp. strain JVH1 [104].
Compounds in brackets were not directly observed. (PFPP-OH - 3-pentafluorophenylpropanl-ol; PFPP-acid - 3-pentafluorophenylpropanoic acid).
135
136
Fig. 6. Dimethyl sulfide biodegradation pathways, (a) aerobic marine thiobacilli and
hyphomicrobia [106]. (b) Thiobacillus sp. strain ASN-1 [107]. (c) Rhodococcus sp. strain
SY1 [95]. (X -cobalamin carrier of methyltransferase)
137
138
Fig. 7. Metabolites formed in the biodegradation of the sulfur mustard analogues (a)
thiodiglycol [111] and (b) 2-chloroethyl ethyl sulfide [114]. Products in brackets were not
directly observed.
For the sulfur-mustard analogue TDG, both sulfur oxidation and terminal
carbon oxidation were observed in a bacterium using the compound as a carbon
source. These reactions were apparently independent and mutually exclusive
with the sulfoxide produced accumulating as a dead-end metabolite. Carbonsulfur bond cleavage was assumed to occur subsequent to terminal carbon
oxidation, but only in the absence of sulfur oxidation. Some fungal strains were
also able to degrade sulfur mustard analogues, although metabolites were not
identified. As with DMS, 2-chloroethyl ethyl sulfide was subject to sulfurspecific degradation by a DBT-desulfurizing strain, demonstrating again that the
desulfurization enzymes may have a sufficiently broad substrate specificity to
allow attack on both thiophenes and aliphatic sulfides.
5. CONCLUSIONS
Known interactions between microbes and the high molecular weight
components of crude oils include oxidation of aliphatic and aromatic carbon
groups, oxidation of naphthenic acids, and oxidation and desulfurization of
aromatic and aliphatic sulfur groups. Hydrogenation and dehydrogenation
reactions have been demonstrated only on lower-molecular weight components.
All of these reactions are of potential interest for upgrading heavy crude oils and
bitumens, but a major barrier is the transport of reactants to the active site of
reaction, particularly for intracellular enzymes in bacteria. Although membranes
may give significant barriers for bioprocessing of heavy hydrocarbons, the
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145
Chapter 5
146
147
Fig. 1. Classical "upper pathway" for phenanthrene ring cleavage (adapted from Refs. [7, 8])
showing stepwise oxidation and metabolites from the "aromatic activation" [6] and BioARC
[9] processes. Theoretical products after hydrogenation are indicated. Compounds: I,
phenanthrene; II, cw-3,4-dihydroxy-3,4-dihydrophenanthrene; III, 3,4-dihydroxyphenanthrene; IV, 2-hydroxy-2#-benzo[h]chromene-2-carboxylic acid; V, fra.s-4-(l-hydroxynaph-2-yl)-2-oxobut-3-enoic acid; VI, l-hydroxy-2-naphthoaldehyde. Enzymatic steps: A;
aromatic dioxygenase; B, dihydrodiol dehydrogenase; C, extradiol dioxygenase; D,
isomerase; E, hydratase-aldolase.
148
149
Fig. 2. Classical angular attack on carbazole showing main pathway (adapted from Ref. [11])
and some minor products [12]. Compounds: I, carbazole; II, postulated intermediate; III, 2'aminobiphenyl-2,3-diol; IV, 2-hydroxy-6-oxo-6-(2'aminophenyl)-hexa-2,4-dienoic acid; V,
2-hydroxypenta-2,4-dienoic acid; VI, anthranilic acid. Other metabolites selected from Ref.
150
[12].
151
152
emulsions and ultimately the separation of the two liquid phases and the
biocatalyst. In fact, many of the same technical problems previously reported
with bench-scale and pilot-scale biodesulfurization processes will likely be
encountered with aromatic ring oxidation. Several types of reactors may be
suitable for petroleum biocatalysis (reviewed in Ref. [26]), with the choice
depending on process economics and product recovery considerations. Two
reactor types are described here: conventional stirred tank bioreactors and
electro-spray reactors.
2.1.1. Two-liquid-phase stirred tank bioreactors
The efficient mixing essential for bio-processing can be achieved in
conventional "two-liquid-phase" stirred tank bioreactors, such as those
described by Villemur and Daugulis and their co-workers [27, 28, 29]. Whereas
other applications of two-liquid-phase bioreactors may require the addition of a
non-degradable, water-immiscible, biocompatible carrier phase to dissolve the
substrate [27], with petroleum the feedstock itself is the solvent for the aromatic
substrates and there should be no need for addition of carrier phases. However,
for toxic feedstocks, it may be necessary to consider diluting the feed with a
non-toxic (e.g. aliphatic) carrier. In this case, it is even more important to ensure
that the biocatalyst cannot oxidize or grow on non-target components of the
feedstock or diluent. Both batch-fed and continuous loop bioreactors may be
suitable for bio-processing, but the former are easier to set up and manipulate.
Oxygen required for biocatalysis is provided by aeration of the two liquid
phases, and therefore may be mass transfer-limited.
Although chemical and microbial surfactants and emulsifiers can increase
the efficiency of droplet formation and stabilize those droplets during
biocatalysis, Marcoux et al. [28] reported that addition of surfactants to a twoliquid-phase bioreactor did not enhance conversion of high molecular weight
PAH, and rhamnolipids had an inhibitory effect. Another consideration
associated with use of surfactants is whether they will interfere with subsequent
separation of the liquid phases or removal of the biocatalyst cells.
To reduce operational costs associated with water handling, separation,
and disposal, ideally the volume ratios of oihwater should be maximized.
However, this can lead to additional problems of emulsification (e.g., water-inoil emulsions), hindering post-treatment separation of the phases. Therefore,
optimum volume ratios likely would have to be determined experimentally for
specific feedstocks and biocatalysts, and emulsions may have to be broken by
centrifugation or by hydrocyclone, such as the patented process for separating
three-phase systems (oil/water/biocatalyst) [30].
153
154
155
For the BioARC process, and possibly also for bio-denitrogenation, problems of
separating and recovering the polar products from the aqueous phase must be
addressed, as well as their treatment after recovery (e.g. chemical hydrogenation
or further bio-processing to produce specialty chemicals).
3. ATTRIBUTES OF AROMATIC BIO-PROCESSING BIOCATALYSTS
Aromatic ring cleavage, as exploited in the BioARC process [17, 19], and
hydroxylation, as used in the patented "aromatic activation" process [6], require
multiple, sequential enzymatic steps and various co-factors such as NADH and
FMNH2. Whole cells are the most readily applicable biocatalysts to encompass
these requirements, rather than treatment with a series of pure enzymes.
Fortunately, there is a relatively long history of research into biodegradation of
aromatic hydrocarbons and heterocycles for purposes of bioremediation, which
can be tapped for relevant information. Bio-processing of oil can be seen as a
logical extension of natural biodegradation capabilities. The key difference is
that biodegradation aims to achieve complete oxidation of the substrate
(mineralization to CO2), whereas bio-processing should oxidize or cleave the
aromatics without carbon loss. This generally requires truncation or
modification of existing catabolic pathways through genetic manipulation
(Section 4).
Generally desirable attributes of aromatic bio-processing biocatalysts
include:
1.
2.
3.
4.
5.
6.
7.
156
suspension;
Tolerance towards any toxic effects of the feedstock or biocatalytic
products;
9. Non-pathogenicity of the biocatalyst for safe handling and disposal;
10. The ability to scale up active biomass production to commercial scale
operations.
8.
157
and peroxidases (e.g., [47, 48]), and may be suitable for the "aromatic
activation" process. However, fungi that accumulate quinones or transdihydrodiol-PAHs without ring cleavage would not be suitable for the BioARC
process. This chapter does not consider the fungal enzymes, which are reviewed
in Chapter 3.
Table 1
Bacteria with broad aromatic substrate ranges for oxidizing di- and tri-cyclic aromatic
hydrocarbons and heterocycles. This table is not a comprehensive list, but rather an
indicator of the diversity of genera with potential for aromatic bio-processing.
Genus, species and strain names
Gram negative
Alcaligenes denitrificans WW1
Burkholderia sp. RP007
Comamonas testosteroni
Cycloclasticus pugetii
Cycloclasticus sp. A5
Neptunomonas naphthovorans NAG-2N-113
Pseudomonads
Pseudomonas putida G7 (NAH7)
Pseudomonas fluorescens LP6a
Pseudomonas resinovorans CA10
Ralstoniasp. RJGII.123
Sphingomonads
Sphingomonas paucimobilis EPA505
Sphingobium yanoikuyae B8/36
Sphingomonas sp. ANT 17
Novosphingobium aromaticivorans F199
Xanthomonas ampellina
Substrates*
References
N, MeN, P, A
N,P,A
[49]
[50]
[51]
[52]
[53]
[54]
N,P
N,P, A
N, P, F, D
N, MeN, P, Ac
N,P,A
N, MeN, P, A, F, D
C, Df
C
reviewed in [7]
[9, 55]
[56]
[57]
N, P, A
N, P, A, Ac, D, C
N, MeN, P, F, D
MeN, P, Ac, F, D,
[58, 59]
[6]
[60]
[61,62]
[63]
Gram positive
Bacillus sp.
N,P, A
[64]
P
Microbacterium esteraromaticus B21
[65]
Mycobacterium sp. PYR-1
P,A
[66]
Mycobacterium gilvum Bl
N, P
[65]
Nocardioides sp. KP7
P
[67]
P
Porphyrobacter sp. B51
[65]
Rhodococcus sp. NCMB12038
N, Ie, Io
[68]
F, Df
[69]
Terrabacter (Staphylococcus auriculans) sp.
DBF63
* N, naphthalene; Me-N, methylnaphthalenes; P, phenanthrene; A, anthracene; Ac,
acenaphthene; F, fluorene; D, dibenzothiophene; C, carbazole; Df, dibenzofuran; Ie,
indene; Io, indole.
158
Suitable new candidate biocatalysts surely exist and could be enriched and
isolated from suitable environments where natural selection processes favor
desirable catabolic phenotypes. Using conventional microbial methods,
enrichment can be facilitated by judicious application of additional selective
pressures in the laboratory, with subsequent genetic manipulation to remove
undesirable properties or add novel phenotypes. To identify a new potential
biocatalyst it may be adequate to screen the mixed community for the desired
genetic complement by DNA:DNA hybridization or PCR, followed by chemical
confirmation of catabolism. However, isolation of truly novel biocatalysts (i.e.,
those with little homology to known aromatic catabolism genes; Section 3.2)
requires classical microbiological selection and biochemical screening
techniques. Notably, Kilbane et al. [70] found that simple chemostat and shakeflask enrichments of novel bacteria capable of selectively removing the N
heteroatom from quinoline were unsuccessful, and that repeated rounds of
enrichment and mutagenesis were required to select a strain with the desired
catabolic activity.
3.2. Examples of genetic systems for aromatic oxidation
Because of the wide variety of potential bacterial biocatalysts, there is
also a diverse array of genetic systems that encode and regulate aromatic
hydroxylation and ring cleavage activity. In general, the aromatic catabolic
genes are regulated as one or more operons that may be encoded on very large
plasmids (e.g. Novosphingobium (formerly Sphingomonas) aromaticivorans
F199 plasmid pNLl, 184 kb [62]), medium-sized plasmids (e.g., Pseudomonas
putida NCIB 9816-4 plasmid pDTGl, 84 kb [71]) or on the chromosome (e.g.,
Mycobacterium sp. PYR-1 [72] and Sphingomonas sp. ANT 17 [60]). Many of
these bacteria use degradative pathways analogous to that shown in Fig. 1, but
there are increasing numbers of reports of new pathways, such as anthracene
degradation by Mycobacterium sp. LB501T [73]. Unfortunately, detailed genetic
characterization of most of these novel pathways remains to be addressed and
the bulk of the literature deals with descriptions of "re-discovered" classical
PAH catabolic genes.
3.2.1. Gene organization and homology
The genes for PAH oxidation are commonly organized into "upper" and
"lower" pathway operons (Fig. 3), which may be separated by many kilobases
[74]. Using the archetypal example of the naphthalene degradation genes
encoded on plasmid NAH7, the upper pathway {nah operon) comprises
oxidation of naphthalene to salicylate, and the lower pathway {sal operon)
encodes oxidation of salicylate to central metabolites (reviewed in Ref. [71];
Fig. 3). It is the upper pathway genes that are important for the aromatic bioprocessing described here.
159
Functional analogues of the nah genes have been described, including the
ndo [75], dox [76] and pah [77] genes in Pseudomonas spp., phb genes in
Sphingomonaspaucimobilis EPA505 [5S],phn genes in Burkholderia sp. RP007
[50], phd genes in Comamonas testosteroni strains [51] and Nocardioides sp.
KP7 [67], nag genes in Ralstonia {Pseudomonas) sp. U2 [78], and nid genes in
Mycobacterium sp. PYR-1 [72]. Some of these operons are genetically
homologous to the nah genes (nah-\ike), whereas others are unconventional
(non-nah-like) in that their sequences and (or) gene arrangements differ from the
classical nah genes (see comprehensive review in Ref. [7]). For example, some
dox and ndo genes are virtually identical [76], whereas the phdA gene is only
60% homologous to other nah dioxygenase genes [67] and the phn genes not
only show little homology with archetypal ndo genes but are also organized
differently within the operon [50]. In the case of the sphingomonads (reviewed
in Ref. [59]), Sphingobium (Sphingomonas) yanoikuyae Bl and N.
aromaticivorans F199 harbour aromatic catabolism genes that are only distantly
related to the well-studied pseudomonad PAH catabolic genes but appear to be
conserved among other sphingomonads. Lack of cross-hybridization and
sequence similarity among non-homologous PAH-degrading organisms [79, 80]
may hinder molecular screens to identify new, unconventional candidate
biocatalysts in the environment.
In some bacteria the catabolic gene organization is considerably more
complicated than the a/z-like operons. For example, the chromosomal genes for
aromatic catabolism in several sphingomonads are disjointed and redundant
[59], possibly resulting from repeated lateral gene transfers [7]. Aromatic
catabolism in S. yanoikuyae Bl involves six operons with convoluted pathways
for mono- and polycyclic aromatic catabolism [83]. Some catabolic operons are
flanked by insertion elements or associated with transposons [56, 84], forming
potentially mobile "catabolic cassettes". This could account for their apparent
lateral transfer and occurrence in diverse bacteria, but also has implications for
the genetic stability of some candidate biocatalysts. Repeated insertion into large
plasmids or the chromosome may also account for the presence of multiple
isofunctional enzymes; such redundancy may complicate efforts to engineer
mutants blocked at specific enzymatic steps (Section 4).
Much less has been published about Gram positive PAH-degrading
bacteria, such as Mycobacterium sp. PYR-1 which has both dioxygenase and
monooxygenase activity against PAH and an unusual gene order in the catabolic
operon [72]. The aromatic dioxygenase genes in this strain cluster
phylogenetically with other Gram positive dioxygenase genes, including
Rhodococcus and Nocardioides spp. [72], rather than the well-studied Gram
negative nah genes.
160
Fig. 3. A. Generalized operon organization for classical nah-Mke genes, adapted from Refs. [7,
71].The arrows indicate the direction of transcription, and shading indicates genes for related
enzymes. Aabcd encode the initial dioxygenase; B the dehydrogenase, C the extradiol
dioxygenase, D the isomerase, E the hydratase-aldolase, F the salicylaldehyde dehydrogenase.
Q is uncharacterized. "R" is the divergently transcribed regulatory gene described for P.
putida NAH7 [81]. G, H, I, N, L, J, and K comprise the lower pathway operon for salicylate
degradation, which may be separated from the upper operon by several kilobases. B. Gene
organization of the Pseudomonas sp. LD-2 carbazole catabolic operon, adapted from Ref.
[82]. Genes for related enzyme subunits are indicated by shading, car A genes encode the
multi-subunit terminal oxygenase, carB the weto-cleavage enzyme, and carC the hydrolase.
161
162
163
unexpected, as these non-toxic compounds are carbon sources for the organism.
Why the organism would develop such an efflux system is unknown; it is
possible that the hydrocarbons are gratuitous substrates rather than primary
substrates for the pump. The effect of efflux on aromatic biocatalysis has not yet
been investigated in this organism or any other to date.
3.4. Enzyme specificity
Biocatalysts for aromatic bio-processing must be capable of attacking the
diverse aromatic substrates present in feedstocks, including alkyl- and
heterocyclic homologues. Two general modes of attack occur: oxidation by a
limited number of enzymes with broad substrate range (e.g. P. fluorescens LP6a
[55]), or by suites of enzymes, each with a narrow substrate range but with
overlapping, complementary functions (e.g. S. yanoikuyae Bl [83]).
The details of naphthalene degradation have been known for some time,
whereas the genetic basis for catabolism of phenanthrene was initially unknown.
Menn et al. [98] were the first to show that phenanthrene and anthracene were
also substrates of the naphthalene oxidation pathway, confirming earlier
speculation about broad specificity degradation pathways for aromatic
hydrocarbons and heterocycles (e.g., Ref. [99]). Evidence of broad substrate
specificity has been reported with increasing frequency, with the most
comprehensive inventory having been described for the naphthalene
dioxygenase (NDO) of Pseudomonas sp. NCIB9816 [100]. This multi-subunit
enzyme produces m-dihydrodiols from a wide array of aromatic hydrocarbons
and heterocycles as well as monohydroxylating, desaturating, dealkylating or
sulfoxidizing various aromatic substrates. Depending upon the biocatalytic
approach being used, the substrate ranges of the other enzymes in the pathway
may also be important but they are poorly characterized [8]. Unfortunately, the
substrate ranges of the unconventional dioxygenases have not yet been fully
explored.
Some strains demonstrate a highly restrictive substrate range. For
example, Rhodococcus sp. SI oxidizes only anthracene and 2-methylanthracene
[101] and Nocardioides sp. KP7 grows on phenanthrene but not naphthalene
[67]. In contrast, other bacteria exhibit a broad substrate range (Table 1)
including alkyl and heteroatom substitutions. Interestingly, the substrate range
can vary even between similar catabolic genes: the homologous nah upper
pathway genes encoded on plasmids found in P. putida PpG7 (NAH7) and
NCIB9816 have differing abilities to oxidize 2- and 3-ring PAHs [102].
The ability to attack alkyl homologues of aromatic substrates varies with
the degree and position of substitution. Some bacteria attack only the
unsubstituted aromatic ring whereas others may also indiscriminately oxidize
the alkyl group or the heteroatom [100], generating alcohols, ketones,
sulfoxides, sulfones, etc. These undesirable enzymatic activities may be suitable
164
165
166
167
168
derivatives (steps A and B, Fig. 1). The second step is chemical dehydration at
high temperature (200 - 600C) in the presence of CO and absence of O2.
Notably, the chemical hydrogenation or hydrogenolysis treatment described in
the patent does not require prior separation of the aqueous and organic phases
(although the effect of the presence of biocatalyst cells is not addressed). This
treatment theoretically achieves ring cleavage and (or) removal of aromatic N
and S heteroatoms, yielding product streams with lower molecular weight and
decreased heteroatom content. Depending on the reaction conditions, chemical
hydrogenolysis of dihydroxybiphenyls yields phenols, alkyl-phenols,
monohydroxybiphenyls, methylated dihydroxybiphenyls, cyclohexyl- and
cyclopentylbenzenes, alkylbenzenes and dihydronaphthalenes, among other
products. However, no specific examples of heteroatom removal were described
in the patent, nor the efficiency of conversion given for the mixed substrates
present in authentic feedstocks.
Several bacterial strains were suggested to be suitable biocatalysts for the
process, including mutant strains of P. putida F blocked either at the
dehydrogenation step (Step B, Fig. 1) so that cw-dihydrodiols accumulate, or at
the catechol dioxygenase step (Step C, Fig. 1) so that 1,2-dihydroxy compounds
accumulate. Alternatively, dioxygenase genes from strains of Pseudomonas
spp., C. testosteroni or S. yanoikuyae are proposed as sources of genes for
creating recombinant biocatalysts by cloning.
5.3. Bio-denitrogenation
Several species of nitrogen heterocycles are found in petroleum, including
the predominant "non-basic" carbazoles, pyrroles, and indoles, and the "basic"
quinolines and pyridines. Two general modes of attack on these nitrogen
heterocycles are recognized (reviewed in Ref. [2]). Quinolines are sequentially
mono-oxygenated to yield hydroxyquinolines, with subsequent N removal and
further oxidation. Carbazole, however, undergoes an unusual "angular
dioxygenation" at the 1,9 position [112, 113], generating 2'-aminobiphenyl-2,3diol, followed by a ring-opened weto-cleavage metabolite and finally anthranilic
acid (Fig. 2), with side-reactions generating additional minor products [12, 57].
The extensive metabolism required to remove the nitrogen heteroatom from
these metabolites would also remove carbon, thus reducing fuel value unless the
metabolites could be recovered for separate processing.
One approach to circumvent this carbon loss would be to block the
angular attack pathway after ring cleavage but before carbon loss, to generate an
aromatic metabolite with a free amine group (Compounds III or IV, Fig. 2), then
hydrogenating this "sensitized" molecule under mild conditions to specifically
remove the nitrogen. Genes encoding the angular attack enzymes have been
cloned, sequenced, and characterized [56, 114, 115], facilitating future genetic
manipulation by analogy with other aromatic-oxidizing systems, and as
169
described in Section 4.1, Riddle et al. [82] deleted genes from the carbazole
operon to achieve metabolite accumulation.
170
171
172
quality at lower capital and operating costs and with reduced environmental
impact. However, the reality awaits further research.
Acknowledgments.
The author acknowledges the financial support of ChevronTexaco (USA),
the National Centre for Upgrading Technology (Canada) and NSERC (Canada)
in developing the BioARC process, and R. Shong, H. Dettman, M. Gray, and K.
Kirkwood for helpful suggestions on the manuscript.
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Chapter 6
1. INTRODUCTION
Methane monooxygenases (MMOs) are unique among known catalytic systems
in their ability to convert methane to methanol under ambient conditions using
dioxygen as the oxidant.
CH4 + O2 + 2H+ + 2e" -> CH3OH + H2O
Thus, MMOs activate the kinetically inert O2 molecule to react at 30-45 C, 1
atmosphere pressure with the unreactive hydrocarbon methane (C-H bond
energy = 1 1 9 kcal mol"1) to produce methanol with stoichiometric yield and
high turnover (up to 6.0 s"1 [1]). In addition to this important transformation,
they also catalyse the oxygenation of a diversity of adventitious substrates,
including numerous petroleum constituents and by-products, which have led to
intense interest in their exploitation for biocatalyis and bioremediation. In this
chapter, we review the biological distribution of MMOs, their structural and
catalytic properties and then discuss their applications and potential for
transformation of petroleum-related products. Lastly, we discuss how MMOs
may inspire the synthesis of biomimetic catalysts to facilitate methane to
methanol conversion and other commercially important transformations.
178
179
although in many cases almost indistinguishable results have been obtained with
the Ms. trichosporium system.
3.1. sMMO structure
sMMO comprises three components (Fig. 1): (1) a hydroxylase with an
(a|3y)2 structure in which the a, |3 and y subunits have masses of 61, 45 and 20
kDa, respectively; (2) a 39-kDa NAD(P)H-dependent reductase; (3) a third
component known as protein B or the coupling/gating protein, which comprises
a single polypeptide of 16 kDa [8]. The a subunits of the hydroxylase each
contain a u-(hydr)oxo-bridged binuclear iron site [10-12] that is coordinated by
four glutamate and three histidine sidechains and is the site of O2 activation. Xray crystallography of the hydroxylases from Me. capsulatus (PDB accession
codes 1MM0, 1MTY) and Ms. trichosporium (1MHY, 1MHZ) has shown that
the hydroxylase is a predominantly a-helical structure in which the binuclear
iron centres reside within the a subunits, in a hydrophobic pocket that is almost
certainly critical in binding substrates [13-15]. Indeed, energy minimisation
calculations have suggested that the most favourable binding site for methane
and other small substrates lies inside this pocket, within 3 A of the binuclear
iron centre (Fig. 2) [16].
The reductase component, which is required for the supply electrons from
NADH or NADPH, contains flavin adenine dinucleotide (FAD) and Fe2S2
prosthetic groups. Protein B has no prosthetic groups. NMR structural analysis
(PDB accession codes 1CKV [17] and 2MOB [18]) has shown that it has a core
ot/p structure with highly mobile regions at the N- and C-termini.
Fig. 1. Components of sMMO. The individual polypeptides of the enzyme are named
according to the genes that encode them [9].
180
Fig. 2. Active site structure of sMMO based on crystallographic data in Ref. [14]. The iron
atoms are shown as large grey spheres and the residues that ligate them as a grey ball-andstick representation. The residues forming the hydrophobic active site pocket [ 16] are shown
in white and two solvent molecules that bridge the dinuclear iron site in black.
181
182
Fig. 3. Principal intermediates during the sMMO catalytic cycle. References are given in the text.
183
sMMO; they are probably oxidised to highly reactive ketenes which then attack
catalytically essential groups on the enzyme [33].
4. PARTICULATE METHANE MONOOXYGENASE
4.1. Structure and mechanism of pMMO
Although pMMO appears to be responsible for the bulk of methane
oxidation catalysed by methanotrophic bacteria in the environment, its
instability during purification procedures has meant that a consensus about its
composition and catalytic properties has been slow to emerge. pMMO is well
established as a copper-containing enzyme. The intracytoplasmic membranes of
methanotrophic bacteria that contain it are produced only during high-copper
growth [5] and, in methanotrophs that can express either pMMO or sMMO,
pMMO is induced in response to high copper-to-biomass ratio in the culture [6].
In addition, Cu2+ ions stimulate pMMO activity in vitro and the copper content
of methanotroph membranes has been observed to be directly proportional to the
pMMO activity [36].
One of the chief problems with purification of pMMO has been
solubilising the enzyme without permanent loss of activity. In 1989, it was
observed in our laboratory that solubilisation of pMMO by dodecyl-P-Dmaltoside was reversible, because activity could be restored by adding
phospholipids back to the solubilised enzyme [37]. Dodecyl-(3-D-maltoside
appears to be the most effective detergent tested to date for solubilisation of
pMMO and to the authors' knowledge has formed the basis for all recent studies
of purified pMMO. Shiemke and coworkers [38] observed that pMMO activity
could be detected in solubilised pMMO if duroquinol or decyl plastoquinol were
used as electron donor in lieu of NADH, suggesting that the direct electron
donor into pMMO is a quinol.
Protocols for purification of pMMO typically comprise preparation of the
membrane fraction, washing with buffer containing up to 1 M monovalent salt
and then one or more chromatographic separations, usually including at least
one anion exchange column [39-43]. Some workers have found anaerobic
conditions favourable to retention of activity [43], whilst others have found the
presence of oxygen tolerable [40] or beneficial [41]. Active preparations of
pMMO have generally contained three polypeptides, of about 49, 27 and 22 kDa
[37], among which the 27-kDa subunit is most likely to contain the active centre
because it becomes labelled by the inhibitor acetylene [33, 39]. The 49, 27 and
22-kDa components are encoded by the genes pmoA, B and C, respectively,
which are multicopy genes [44, 45] induced in response to high copper-tobiomass ratio [46, 47].
184
Table 1
Principal oxidation reactions catalysed by sMMO
Substrate
Major detected product(s); relative
molar proportions of multiple
products are shown in parentheses.
Alkanes
Methane
Methanol
Ethane
Ethanol
Propane
Propan-1-ol (39); propan-2-ol (61)
Butane
Butan-1-ol (54); butan-2-ol (46)
Pentane
Pentan-1-ol (28); pentan-2-ol (72)
Hexane
Hexan-1-ol (63); hexan-2-ol (37)
Heptane
Heptan-1-ol (22); heptan-2-ol (78)
Octane
Octan-1-ol (9); octan-2-ol (91)
2-Methylpropane
2-Methylpropan-2-ol (70); 2methylpropan-1-ol (30)
2-Methylpropane
2-Methylpropan-2-ol (70); 2methylpropan-1-ol (30)
2,3-Dimethylpentane 3,4-Dimethylpentan-2-ol
Alkenes
Ethene
Propene
But-1-ene
czs-But-2-ene
Zrarc.s-But-2-ene
Epoxyethane
Epoxypropane
1,2-Epoxybutane
cw-2,3-Epoxybutane (47); cis-2buten-1-ol (53)
?ra5-2,3-Epoxybutane (27); trans-2buten-1-ol (73)
Specific activity
(nmol of product
min~ mg" )
Reference
(type of
assay) 2
84
68
69
77
73
40
27
9
33
32 (SE)
32 (SE)
32 (SE)
32 (SE)
32 (SE)
32 (SE)
32 (SE)
32 (SE)
26 (PP)
33
26 (PP)
20
26 (PP)
148
83
49
33
32
32
32
26
39
26 (PP)
25
34 (SE)
26 (PP)
(SE)
(SE)
(SE)
(PP)
26 (PP)
3
1.3
26 (PP)
26 (PP)
0.5
26 (PP)
748
682
31 (PP)
31 (PP)
185
Substrate
l,l-Dichloroethene J
Trifluoroethene
Chlorotrifluoroetheylene 3
Tribromoethylene 3
Monoaromatics
Benzene
Toluene
Ethylbenzene 3
Styrene 3
Pyridine
Diaromatics
Naphthalene
Biphenyl 3
2-Hydroxybiphenyl 3
2-Methylbiphenyl 3
2-Chlorobiphenyl 3
2-Bromobiphenyl 3
2-Iodobiphenyl 3
Specific activity
(nmol of product
mirf'mg" 1 ) 1
648
Reference
(type of
assay) 2
31 (PP)
79
31 (PP)
17
31 (PP)
31 (PP)
Phenol
Benzyl alcohol; 4-cresol.
1-Phenylethanol (30); 4hydroxyethylbenzene (70)
Styrene oxide
Pyridine N-oxide
74
53
18.7
34 (SE)
32 (SE)
34 (SE)
82
29
34 (SE)
32 (SE)
1-Naphthol, 2-naphthol
2-Hydroxybiphenyl (9); 3hydroxybiphenyl (1); 4hydroxybiphenyl (90)
Dihydroxybiphenyls
Ring (56) and sidechain (44)
hydroxylated products
Hydroxychlorobiphenyls
Hydroxybromobiphenyls (41); 2hydroxybiphenyl (59)
Hydroxyiodobiphenyls (18); 2hydroxybiphenyl (82)
7 (W)
35 (W)
35 (W)
35 (W)
35 (W)
35 (W)
35 (W)
84
82
35
66
45
64
246
32
32
32
32
32
32
32
(SE)
(SE)
(SE)
(SE)
(SE)
(SE)
(SE)
Others
Diethyl ether
Ethanol (47); acetaldehyde (53)
32 (SE)
Carbon monoxide
Carbon dioxide
61
32 (SE)
Specific activities are as reported in the original publications. Caution is advised when
comparing values from different studies, since various protein preparation and assay
procedures were used by the different authors.
Type of enzyme preparation used for each assay: PP, purified protein; SE, soluble extract;
W, whole cells.
3
sMMO of Ms. trichosDorium OB3b: other entries refer to Me. capsulatus (Bath).
186
187
mechanism of C-H bond breakage rather than one involving radical or cation
intermediates [51].
4.2 Substrate profile of pMMO
The substrate profile of pMMO (Table 2) is considerably narrower than that of
sMMO. It includes methane and linear short-chain hydrocarbons but excludes
aromatics (benzene, ethyl benzene and styrene), the alicyclic hydrocarbon
cyclohexane [34] and the branched aliphatic 2-methylpropane [52], all of which
are substrates of sMMO. Thus it appears that access to the active site of pMMO
is sterically more restricted than in the soluble enzyme. Consistent with this,
acetylene is a potent suicide substrate of both soluble and particulate enzymes
[33], whereas the bulkier phenylacetylene is much more effective against the
sMMO than pMMO [53].
5. METHANE MONOOXYGENASE IN BIOCATALYIS
As indicated above MMO-containing bacteria are able to catalyse the oxidation
of alkenes to epoxides. Since there is no direct, commercially viable, chemical
route to epoxide formation from alkenes attempts have been made to exploit the
MMO-catalysed reaction on a large scale. In a comprehensive study to produce
epoxypropane from propene we have shown how a two stage system using Me.
capsulatus (Bath) can be used to give high yields of product in a continuous
operation [55]. This methanotroph was chosen because it grows readily at 45C
and at this temperature the epoxypropane is released into the gas phase. The
product could then be readily condensed from the gas phase at a lower
temperature. A major problem that had to be overcome was the inherent in vivo
toxicity of the epoxides to the cells. In fact epoxypropane was shown to be an
inhibitor of MMO and so caused an inactivation of the cells if grown in a single
stage operation [56]. The problem was solved by using a two stage bioreactor
system in which the first stage was used to catalyse the oxidation of propene to
the epoxide and a second reactor was fed with cells from the catalytic reactor to
reactivate inactivated cells. In the first reactor methanol was used as the electron
donor for the whole cell reaction which had to be fed as a growth-limiting
substrate to minimise its interference in MMO catalysis (methanol is a substrate
for sMMO [see Table 1] but it has a much lower affinity for the enzyme than
propene). In the second stage reactivator, methane was supplied as the growth
substrate along with all nutrients necessary to allow resynthesis of inactivated
cells. Critical to the success of the system were the operating parameters that
included the ratio of the size of the reactors such that a continuous operation was
possible that permitted active cells to be fed back to the first stage to maintain
high catalytic productivity. The continuous recycling of cells from the reactor
vessel to the reactivator (at an aspect ratio of 1:10) gave rates of 20-30 g L"1
188
day"1 over a three week period. Similar experiments with Methylocystis parvum
(OBBP) gave rates of up to 90 g L"' day"1 over a one week period. In a feasibility
study [55] using cells at 30 g L"1 and a production rate of 250 g L"1 day"1 the total
cost of epoxypropane production was estimated at $1.26 kg"1. As such it was not
competitive with the commercial oxirane process which values the product at
around this price; no account in the biological; process was taken for storage,
transport or profit which need to be added to this cost. Consequently, at the
present time this system has not been commercialised although patents for the
process have been filed worldwide.
In principle such an operation could be adapted to produce any of the
sMMO products listed in Table 1 although the separation and purification of the
product will dictate the precise mode of operation. The process has been
evaluated with other substrates including 1-butene and ethane [57] to produce
epoxybutane and ethanal respectively.
pMMO shows moderate stereoselectivity with some reactions (up to 80 %
enantiomeric excess of the R-enantiomer of pentan-2-ol product formed from
pentane) [52] and so may be suitable for eventual development as an
enantioselective catalyst. Interestingly, whilst neither pMMO nor sMMO shows
a high stereoselectivity in epoxide-generating reactions (enantiomeric excesses
for epoxypropane generation by the two enzymes are 18.5 % S [52] and 21 % R
[58], respectively), they show opposite enantioselectivity and so may be suitable
for future genetic development into a pair of enantiocomplementary biocatalysts.
Table 2
Principal oxidation reactions catalysed by pMMO
Reference
Substrate
Product(s); relative molar proportions of multiple
products are shown in parentheses.
Alkanes
Methanol
Methane
Ethane
52
Ethanol
Propane
52
Propan-2-ol (ca. 100); propan-1-ol (trace)
Butan-2-ol (95); butan-1-ol (5)
52
Butane
Pentane
Pentan-2-ol (95); pentan-1-ol (5)
52
Alkenes
52
Propene
Epoxypropane
52
But-1-ene
1,2-Epoxybutane (58); but-3-en-2-ol (42)
1,3-Butadiene
1,2-Epoxybut-3-ene
52
s-But-2-ene
2,3-cw-Epoxybutane
52
fr-ans-But-2-ene
2,3-/ra^-Epoxybutane
52
Chlorinated aliphatics
Trichloroethene'
Carbon dioxide
54
sMMO of Ms. trichosporium OB3b; other entries refer to Me. capsulatus (Bath).
189
190
7. FUTURE PROSPECTS
The unusual reactivity and broad substrate profiles of MMOs suggest many
possible applications in synthetic chemistry and bioremediation for the enzymes
and biomimetics based on them. Our recent development of a system for sitedirected mutagenesis of the soluble enzyme [64] opens the way for fine-tuning
of the catalytic versatility of sMMO for more precise and profitable
biotransformations than are possible with the wild-type enzyme. Coupled with
the sequencing of the Me. capsulatus genome, which is currently being
undertaken by the University of Bergen and The Institute for Genomic Research,
genetic technology may shortly enable metabolic engineering of novel pathways
incorporating engineered methane monooxygenases for the synthesis of valuable
Pharmaceuticals and other products using methane and other inexpensive
starting materials.
Acknowledgements
We gratefully acknowledge research funding from the Biotechnology and Biological
Sciences Research Council (UK), British Gas (UK), British Petroleum (UK), Idemitsu (Japan)
and the Gas Research Institute (GRI) (Chicago, IL).
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193
Chapter 7
Biocorrosion
H.A. Videlaa and L.K. Herrerab
department of Chemistry. College of Pure Sciences, INIFTA, University of
La Plata, Argentina
b
1. INTRODUCTION
Microorganisms influence corrosion by changing the electrochemical
conditions at the metal/solution interface. These changes may have different
effects, ranging from the induction of localized corrosion, through a change in
the rate of general corrosion, to corrosion inhibition [1]. Any biological effect
that either facilitates or impedes one of the anodic or cathodic reactions of the
corrosion process, or permanently separates anodic and cathodic sites, will
increase corrosion. For instance, stimulation of the anodic reaction by acidic
metabolites or the cathodic reaction by microbial production of a cathodic
reactant like hydrogen sulfide, the breakdown of protective films or the increase
in conductivity of the liquid environment will enhance corrosion.
Although the electrochemical nature of corrosion remains valid for
microbial corrosion, the participation of the microorganisms in the process
induces several unique features, mainly the modification of the metal/solution
interface by biofilm formation. Biofilms affect the interaction between metal
surfaces and the environment, not only in biodeterioration processes like
corrosion, but also in several biotechnological processes applied to materials
recovery and handling [2]. Thus, the key to the alteration of conditions at a
metal surface, and hence the enhancement or inhibition of corrosion is the
formation of a biofilm [3]. This can be considered as a gel containing 95% or
more water, made of a matrix of exopolysaccharidic substances (EPS) in which
microbial cells, and inorganic detritus are suspended [4].
Biofilm formation is the result of an accumulation process -not
necessarily uniform in time or space [5]- that starts immediately after metal
immersion in the aqueous environment. A thin film (approximately 20-80 nm
194
thick), due to the deposition of inorganic ions and high relative molecular mass
organic compounds, is formed in a first stage. This initial film is able to alter the
electrostatic charges and wettability of the metal surface facilitating its further
colonization by bacteria. In a short time, (minutes or hours according to the
aqueous environment where the metal is immersed), microbial growth and EPS
production results in the development of a biofilm. This biofilm is a dynamic
system and the different transport processes and chemical reactions occurring at
the biofouled interface will take place from now on, through the biofilm
thickness [6] (Fig. 1).
Microbial colonization of metal surfaces drastically changes the classical
concept of the electrical interface commonly used in inorganic corrosion.
Important changes in the type and concentration of ions, pH values and
oxidation-reduction potential are induced by the biofilm, altering the passive or
active behavior of the metallic substratum and its corrosion products, as well as
the electrochemical parameters used for assessing corrosion rates [7].
Simultaneously with the biological changes that lead to biofilm
accumulation, a sequence of inorganic changes takes place at the metal surface
immediately after its immersion in an aggressive aqueous medium. This
sequence involves the process of metal dissolution and corrosion product
formation.
195
Both biological and inorganic processes occur within the same time
period, but following opposite directions at the metal/solution interface.
Whereas corrosion and corrosion product accumulation occur from the metal
surface towards the solution, biofilm formation is the result of accumulation
processes directed from the bulk towards the metal surface (Fig. 2) [8]).
Thus, a very active interaction between the corrosion product layers and
the biofilms can be expected. The consequent corrosion behavior of the metal
will vary according to the degree of this reciprocal interaction and a concept of a
new biologically conditioned interface must be kept in mind [9]. The approach
for a sound interpretation of any microbial corrosion case must then be
interdisciplinary, and include a thorough process analysis combined with well
defined microbiological and electrochemical methodologies.
Biocorrosion has been focusing increasing attention from different
research areas in the last two decades as an answer to the demand of a wide
variety of industries. Fortunately an increasing intellectual and technical crossfertilization of ideas between researchers from different disciplines like
microbiology, electrochemistry and materials science has allowed a considerable
improvement in the understanding of biocorrosion to be reached.
2. SRB INDUCED CORROSION OF STEEL
Biocorrosion of carbon steel in anaerobic environments involving the
presence of SRB have been the focus of most biocorrosion research. Starting
196
with the cathodic depolarization theory (CDT) of von Wolzogen Kuhr & Van
der Vlugt [10] (Fig. 3), a copious list of papers and reviews on the anaerobic
corrosion of iron has been published [11-13]. Bacterial biofilms may develop
anaerobic regions, even in aerobic bulk water environments [2], thus allowing
SRB a very favorable environment for growth. The final result of these
processes within biofilms is to produce a wide variety of sites on the metal
surface that are markedly different from neighboring sites from a
physicochemical standpoint, thus facilitating the initiation of localized corrosion
processes.
Fig. 3. Sequence of reactions of the Cathodic Depolarization Theory. The three elements of
biocorrosion (metal/solution/microorganisms) are involved in different reactions of the whole
process.
197
198
199
Fig. 5. AFM image of a SRB biofilm on AISI 316 SS (from Ref. [21]).
200
Fig. 6. XPS spectra of a biogenic sulfide film (top) and of an abiotic sulfide film (bottom)
(from Ref. [23]).
201
Fig. 7. Hydrogen permeation through 50D steel, cathodically protected or unprotected and
coated or uncoated, exposed to open seawater and embedded in marine mud. 1 = Grit blasted
plus cathodic protection. 2 = CTE coating plus cathodic protection. 3 = anti-fouling paint. 4 =
grit basted only. 5 = As received (with mill scale, uncoated) (from Ref. [29]).
202
bacteria and the metal). Indeed, the local environment surrounding the metal
surface is very different from that without bacteria, even if the same levels of
sulfide are detectable.
These areas are particularly important, as pipelines and the bottoms of the
legs of offshore platforms can be buried in marine muds where anaerobic
conditions are predominant and SRB activity is intense (Fig. 8). Moreover, sour
environments, such as those frequently found in oil production activities, are
particularly aggressive due to high levels of hydrogen available at the metal
surface or in a crack, as a consequence of sulfide poisoning of the recombination
reaction at the cathode [31]. In such habitats hydrogen effects can be altered by
the presence of organic molecules on the metal surface and the existence of a
biofilm with its EPS matrix. This feature can explain the differences between
general embrittlement effects (as measured by hydrogen flux) and crak tip
effects (as measured by crack growth). Embrittlement results in the general
lowering of strength of the material causing it to fail in a catastrophic way at a
lower load as it has been reported in high sulfide biological environments more
than in low sulfide abiotic environments, even though the rest of the crack
growth curve is very similar (Fig. 9) [28].
EPS and other organic molecules related to biofilms hinder dissolution
and dissociation reactions and adsorption processes in the crack. Even under low
frequency cyclic loading the crack tip opens and closes rapidly. Thus, any effect
of the environment must occur fast and organic material dragged into the crack
could have a blocking impact.
The results referred here serve to illustrate the complex nature of the
interactions between SRB biofilm and the steel. In many cases, bacterial
metabolism within the biofilm generates sulfides, and consequently, this is the
main cause of the corrosiveness of the environment. However, microbial
metabolic activity is also responsible for the release of EPS which may have a
blocking effect on hydrogen entry into the metal.
3. BIOCORROSION OF ALUMINUM ALLOYS IN FUEL/WATER
SYSTEMS
Microbial contamination of hydrocarbon fuels is the main cause of serious
problems concerning the quality of maintenance of the product, as well as the
corrosion of metals used during the processes of extraction, production,
distribution, and storage of the fuel.
203
Fig. 8. Composite diagram of an offshore structure showing the main sites of biodeterioration
problems: 1. marine fouling; 2. drill cuttings around legs; 3. oil storage and transport; 4.
water-filled legs; 5. production system; 6. seawater injection system; 7. downhole pipework;
8. reservoir problems (from Ref. [31]).
204
Fig. 9. Crack growth rates of a RQT 501 steel in biologically active H2S seawater
environments; solid line: crack growth rate in seawater; dotted line: crack growth rate in 520
ppm H2S in abiotic natural seawater (from Ref. [28]).
205
206
Fig. 10. Simplified scheme of the initiation of pitting attack on aluminum alloys in fuel/water
systems (from Ref. [45]).
207
208
209
210
Table 1
Biocides used in industrial water systems. Properties and usual concentrations.
Chlorine: effective against bacteria and algae; oxidizing; pH dependent; concentration range:
0.1-0.2 mg/1 (continuous treatment)
Chlorine dioxide: effective against bacteria, in a lesser extent against fungi and algae;
oxidizing; not dependent on the pH; concentration range: 0.1-1.0 mg/1
Bromine: effective against bacteria and algae; oxidizing; wide pH range; concentration
range: 0.05-0.1 mg/1
Ozone: effective against bacteria and biofilms; oxidizing; pH dependent; concentration
range: 0.2-0.5 mg/1
Methylene-bis-thiocyanate: effective against bacteria; non-oxidizing; hydrolyses at pH
higher than 8.0; concentration range: 1.5-8.0 mg/1
Isothiazolones: effective against bacteria, algae and biofilms; non-oxidizing; not dependent
on the pH; concentration range: 0.9-10 mg/1
QUATS: effective against bacteria and algae; non-oxidizing; surface activity; concentration
range: 8-35 mg/1
Glutaraldehyde: effective against bacteria, algae, fungi and biofilms; non-oxidizing; wide
pH range; concentration range: 10-70 mg/1
THPS (tetra kis-hydroximethil phosphonium): effective against bacteria, algae and fungi;
low environmental toxicity; specific action againts BRS.
211
212
sampler device.
213
214
atomic force microscope (AFM) permit biofilm observation in real time and
without intoducing distortion of the samples. There is an increasing number of
references using these innovative technologies in recent biocorrosion literature
[61-63].
A combination of CSL and microelectrode techniques allowed correlation
of oxygen concentration profiles with biofilm structure [64]. CSL facilitates the
visualization of biofilm structures by eliminating the interference arising from
out of focus objects [65]. Observations performed under flow conditions and
using physiologically active biofilms, provided information to construct a new
conceptual model of biofilm structure.
On the corrosion side, new electrochemical test methods for the study of
localized corrosion phenomena in biocorrosion analysis and monitoring have
been reported [66]. As an example, an electrochemical sensor for monitoring
biofilms on metallic surfaces in real time has been recently presented [67]. The
system provides an immediate indication of the condition of biological activity
on probe surfaces and it is a powerful tool to optimize biocide treatment (Fig.
12).
10. CONCLUDING REMARKS
Biocorrosion is rarely linked to a single mechanism or to a single species of
microorganisms.
Biofilms mediate the interaction between metal surfaces and the liquid
environment. This interaction leads to an important modification of the
metal/solution interface drastically changing the types and concentrations of
ions, pH and oxygen levels.
Biofilm/corrosion product layers interactions at the metal/solution
interface condition the electrochemical behavior of metal surfaces in biological
media.
Acknowledgement
H.A. Videla acknowledges the financial support of the Agencia de Promocion
Cientifica y Tecnologica of Argentina through the project PICT/99 6782 on
Biodeterioration of materials.
215
Fig. 12. Scheme of an electrochemical sensor for monitoring biofilms (from Ref. [67])
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219
Chapter 8
220
the medicine, food, and cosmetic industries has largely replaced microbial
growth tests. These modern biotechnology methods are only beginning to be
employed in the gas and oil industry for problems related to microbiologically
influenced corrosion but it is likely that genetic techniques will be the methods
of choice for monitoring MIC in the future. Initial efforts to introduce the use of
genetic techniques for monitoring MIC or other environmental samples have
involved a type of DNA hybridization test called reverse sample genome
probing (RSGP). There are other hybridization-based genetic techniques
including whole cell in situ fluorescent hybridization, DNA amplification
followed by hybridization (dot-blot hybridization) or gel electrophoresis
(denaturing gradient gel electrophoresis) [17-21] that could also be used to
examine MIC samples, but this has not yet occurred. Another type of genetic
test method that could be used to investigate MIC samples is based on DNA
amplification using the polymerase chain reaction (PCR). PCR-based
approaches include quantitative competitive PCR (cPCR), quantitative real-time
PCR (q-PCR), and reverse transcriptase PCR (RT-PCR) [22-25]. This chapter
summarizes the status of genetic tests to monitor MIC and discusses possible
applications of genetic monitoring techniques for the future.
2. DNA HYBRIDIZATION TECHNIQUES TO MONITOR
MICROBIOLOGICALLY INFLUENCED CORROSION
One of the most commonly used genetic approaches to the monitoring of
bacteria associated with corrosion is reverse sample genome probing (RSGP)
[26-29]. This technique is based upon the hybridization of whole-community
DNA obtained from environmental samples to the DNA of various characterized
bacterial species (reference cultures) that are spotted on the master membrane.
Hybridization experiments take advantage of the fact that DNA is normally
double stranded so that if DNA is denatured (the strands are separated)
complementary strands/DNA sequences will come back together. The additive
nature of the hydrogen bonds formed by complementary nucleic acid bases
provides a strong thermodynamic force favoring the formation of double
stranded DNA by complementary DNA sequences. In RSGP various pure
cultures, most frequently sulfate reducing bacteria (SRB), are isolated from
environments of interest and then grown in the laboratory. About thirty cultures
of SRB have been cultivated from gas and oil production operations so that the
DNA of these cultures can be used in RSGP [30]. RSGP technique was also
used to identify and quantify bacteria communities capable of degrading various
aromatic hydrocarbons in contaminated soil [31-34]. Chromosomal DNA is
purified from each species of bacterial culture that was cultivated in the
laboratory. The DNA is then denatured and added as discrete drops to specific
locations on a membrane, typically a nitrocellulose membrane. The DNA is then
221
chemically /thermally bound to the membrane, which then serves as the master
grid in a subsequent hybridization experiment. To perform RSGP an
environmental sample of interest, such as biomass obtained from a gas and oil
production pipelines, is processed to obtain DNA from the mixture of bacterial
species present. This mixed DNA sample is subjected to a biochemical labeling
process that results in the addition of fluorescent or radioactive compounds to
the DNA mixture [32]. When the labeled DNA mixture is denatured and added
to the master grid complementary DNA strands form double stranded DNA, a
washing step then removes any DNA not bound to the membrane. The amount
of DNA in the mixture that corresponds to each species of bacteria present on
the master grid is determined by quantifying the amount of fluorescence or
radioactivity associated with each spot/location on the master grid. The quantity
of signals in each spot on the master grid reflects the abundance of each
reference organism in the environmental sample. This technique allows the
quantification of many different microorganisms simultaneously.
It can be readily appreciated that there are several drawbacks to RSGP.
The technique requires specialized training and equipment and involves multiple
steps so that several days are typically needed to obtain results. However, a
more important limitation is that the only species of bacteria quantified by
RSGP are those whose genomic DNA is spotted onto the master grid. Since it is
generally accepted that less than 1% of bacterial species in nature can be
cultivated in the lab [35, 36], only a small subset of bacterial species are
available with which to prepare master grids. In microbial corrosion research,
RSGP has been applied almost exclusively to the quantification of SRB. While
SRB are unquestionably capable of causing metal corrosion it is also
unquestionably true that several other types of bacterial groups such as acid
producing bacteria, iron respiring bacteria, denitrifying bacteria, sulfur oxidizing
bacteria, and methanogenic bacteria also can cause metal corrosion (see chapter
7). It would be extremely cumbersome to quantify all of these bacterial types
using RSGP, but it may be possible to develop such tests in the future using
microarray technology. Genetic methods can indeed provide more accurate data
more quickly than microbial growth tests, but methods more convenient than
RSGP are needed. The genetic method of choice for monitoring food and
cosmetic products for microbial contamination, for the detection and
identification of infectious microorganisms in medicine and bio-warfare
monitoring is quantitative real-time PCR. Quantitative PCR has recently been
adapted for use in monitoring microbial populations in gas and oil pipelines, and
may become the most convenient and reliable method to monitor MIC in the
future.
222
223
found for sulfur [39]. Moreover, many sulfate reducing bacteria can also utilize
nitrate [38-40].
Other results obtained from characterizing the microbial populations
within gas pipeline samples were that methanogens were frequently present in
pipeline and biofilm samples, and that sulfate reducers were often present at
lower levels than indicated by microbial growth tests. The presence of
methanogens in gas pipelines was unexpected, but it is significant because any
microbial process, such as methanogenesis, that consumes hydrogen is capable
of accelerating corrosion by cathodic depolarization (a process which pulls the
cathodic reduction of protons by removal of the product and thereby accelerates
anodic metal dissolution) [8, 41, 42].
These results highlight the fact that the composition of microbial
communities in gas pipelines has not been thoroughly investigated and prior to
the genetic studies described here the entirety of our knowledge about
microorganisms in gas pipelines was restricted to information gathered by
growing bacteria in various media under laboratory conditions. There are many
types of bacteria, and testing for all types of bacteria using growth experiments
would be very tedious, and in fact it has never been done. We only have data
concerning those types of bacteria that have been tested for, so our view of the
microbial ecology of gas pipelines is biased indeed. A commonly held belief
regarding microbial corrosion is that SRB are the most important contributors to
corrosion. Since traditional microbial growth tests most frequently test for SRB,
and very few other types of microorganisms, it is not surprising that this belief is
widely held. However, our genetic tests of gas pipeline samples were not biased
in looking for any particular type of microorganism, and SRB were not among
the most abundant microorganisms in any of the pipeline samples characterized.
However, when these same pipeline samples were used to inoculate bacterial
growth tests using SRB medium, SRB were invariably found. The differences in
the number of particular types of bacteria detected by microbial growth tests and
by genetic tests were investigated further, and it was found that the species of
SRB that grew in laboratory media inoculated with gas pipeline samples were
not the same species of SRB detected when the gas pipeline samples were
examined directly. In other words, by growing bacteria in a particular medium
an artificial environment is created where nutrient concentrations, and other
factors we have yet to fully appreciate, differ significantly from the conditions
that are present in gas pipelines. Thus, the composition of microbial
communities detected in laboratory growth experiments differs profoundly from
the composition of microbial communities actually present in a gas pipeline.
224
Microbial type
Methanogen
Cultures
Methanoarcina
Iron-reducing
Shewamlla
Sulfate reducing
Desulfovibrio
Beta-Proteobacteria
Acidovorax
Denitrifier
biack
++
+++
black
+++
++
+++
+ ++
++
+++
++
+++
++-).
++
+++
++
+++
225
226
227
spiked into gas pipeline samples. This is best seen by inspecting the ratio
between the actually detected gene copy number from the spiked PCR reaction
and the calculated copy number per reaction (copy number detected from unspiked reaction, plus the copies spiked into the reaction). If all of the DNA
added in spiked samples was accurately quantified the ratio of detected and
calculated would be 1; values above 1 means an overestimate of the actual
concentration, and values below 1 means an underestimate of spiked gene
copies. The average ratios of the detected and calculated were 1.02 0.09, 0.69
0.1, 1.2 0.15, 0.87 0.09, and 1.22 0.16 for bacteria, archaea, SRB,
denitrifiers, and methanogens, respectively, which are considered very accurate
for the analysis of complex environmental samples [24].
The data in Table 3 illustrate that genetic tests employing quantitative
PCR techniques provide accurate and reliable data concerning the quantity of
various types of bacteria that may be present in gas pipeline samples.
5.2. Applying the genetic testing methods to quantify various groups of
microorganisms present in pipeline samples
Seven pipeline samples were collected from various natural gas
companies at various geographical locations. The biomass was centrifuged down
and used for DNA extraction using FastDNA SPIN Kit for Soil (Qbiogene,
Carlsbad, CA). The extracted DNA was further purified by phenol/chloroform
extraction to remove inhibitory substances commonly present in this type of
samples. The spiking experiment showed that the extra purification step was
sufficient to eliminate inhibitory effect of PCR amplification (Table 3). The
purified genomic DNA was then amplified and quantified using quantitative
real-time PCR with DNA standards of corresponding target genes. The results
were summarized in Table 4. The results confirmed our previous observation
[18] using DGGE analysis of 16S rRNA gene sequences that denitrifying
bacteria and methanogens are two types of organisms which were commonly
present in relatively high abundance in gas pipeline samples. Denitrifying
bacteria were detected in all seven samples, and the concentration in the sample
was as high as 7.95 x lO^/ml; methanogens were detected in five out of seven
samples, and the concentration was as high as 3.7 x 10^/ml; SRB were detected
in six samples, but at lower concentration in most of samples. The lower archaea
concentration detected using 16S rRNA gene than methanogens based on the
detection of mcrA gene is due to archaea primers, which will exclude many
methanogens, especially Methanococcus species [52-54].
Table 2
Real-time PCR quantification of gas pipeline samples grown in various microbial growth media
Growth
Sample ID Medium
1
3
6
8
9
2
4
5
MET
MET
MET
MET
MET
MET
MET
MET
8
9
4
2
DNB
DNB
DNB
DNB
7.84E+09
3.80E+10
1.06E+08
7.28E+05
ND = not detected
MET = methanogenic bacteria
DNB = denitrifying bacteria
2.35E+05
1.55E+06
ND
4.26E+07
4.38E+09
8.50E+05
ND
ND
0.00
0.00
ND
ND
ND
ND
ND
0.54
11.53
0.80
ND
229
Table 3
Accuracy of real-time PCR quantification of bacteria, archaea, SRB, denitrifiers, and
methanogens in natural gas pipeline samples
Bacteria detected (copy/rnx)
Bacteria calculated (copy/rnx)
w/ spiking
w/o spiking + spiked copies
1
7.32E+08
7.97E+08
2
1.04E+08
1.16E+08
3
4.56E+06
4.54E+06
1.05E+08
4
1.14E+08
5
6.73E+O5
6.40E+05
8
1.05E+O9
9.76E+08
9
2.35E+09
2.61E+09
Spiking: 5.36E+05 copies of 16S rRNA genes of'/'seudomonas aeruginosa PAO-1 per reaction.
Ratio
det:cal
0.92
1.12
1.01
1.08
0.95
0.93
1.11
Ratio
det:cal
0.78
0.63
0.61
0.83
0.56
0.75
0.64
Ratio
det:cal
1.22
1.24
1.38
1.24
1.31
0.96
1.05
Ratio
det:cal
0.76
0.94
0.85
0.91
1.02
0.84
0.78
Ratio
det:cal
1.29
1.27
1.10
1.22
1.15
1.56
1.20
Sample ID
Sample ID
Sample ID
Sample ID
Sample ID
230
Table 4
Quantification of bacteria, archaea, SRB, denitrifiers, and methanogens in natural
gas pipeline samples (/mL)
Sample ID
Bacteria
Archaea
SRB
Denitrifier Methanogen
1
1.59E+03
7.95E+06
1.18E+04
1.99E+08
8.12E+03
2
2.58E+07
1.49E+05
4.05E+04
2.18E+05
1.98E+03
3
1.00E+06
5.93E+01
5.29E+01
4.00E+03
ND
4
2.75E+07
1.75E+03
9.51E+O3
1.02E+05
4.14E+03
5
3.42E+04
1.96E+02
ND
4.67E+01
ND
8
2.62E+08
6.97E+06
6.75E+05
1.64E+05
3.70E+07
9
5.88E+08
6.33E+05
6.48E+06
3.25E+06
4.11E+04
ND = not detected
6. CONCLUSIONS
Quantifying various types of bacteria that may be present in gas and oil
production operation samples is a difficult challenge. Traditional tests employ
microbial growth media of various types that are intended to foster the growth of
particular types of microorganisms. Unfortunately, microbial growth media are
not uniquely selective and microorganisms other than the intended type often
grow in the test medium. In some cases, even though significant growth occurs
the concentration of the target population of bacteria is below detection limits.
Thus, if the results of growth in microbial test media are used to determine the
quantity of various types of bacteria present in gas and oil production operation
samples the results obtained can be very misleading. Traditional microbial
growth tests require weeks of incubation, are not accurate, and do not provide
information about what microbial species were actually present in the
environment. An improved means of quantifying various types of bacteria
present in gas pipeline samples is using genetic techniques, such as RSGP and qPCR. Other industries such as medicine, food, and cosmetics share with gas and
oil industry the need to detect, identify, and quantify microorganisms. These
other industries have largely abandoned microbial growth tests in favor of
genetic methods. Hybridization test methods such as RSGP are not typically
used in characterizing environmental samples, but future improvements in
microarray technology may change this situation. q-PCR has been adopted by
other industries as the method of choice for rapid quantification of
microorganisms, but this technique has not yet been in the gas and oil industry.
In this chapter data was presented that demonstrated that q-PCR can obtain data
within a few hours that specifically and accurately quantifies bacterial types
present in gas pipeline samples. The gas pipeline samples are analyzed directly
without any cultivation or other manipulation in the laboratory that would alter
the composition of the microbial community. Therefore, q-PCR provides data
231
concerning the actual microbial community present in the gas pipeline. GTI has
developed an accurate and reliable method to quantify bacterial types present in
gas pipeline samples and is now offering this service to the industry.
REFERENCES
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[2]
[3]
[4]
[5]
[6]
[7]
[8]
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[10]
[II]
[12]
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[14]
[15]
[16]
[17]
232
[48] G. Braker, A. Fesefeldt, and K.-P. Witzel, Appl. Environ. Microbiol. 64 (1998) 3769.
[49] G. Braker, H. L. Ayala-del-Rio, A. H. Devol, A. Fesefeldt, J. M. Tiedje, Appl. Environ.
Microbiol. 67(2001)1893.
[50] P. E. Luton, J. M. Wayne, R. J. Sharp, P. W. Riley, Microbiology 148 (2002) 3521.
[51] T. Lueders, M. W. Fnedrich, Appl. Environ. Microbiol. 69 (2003) 320.
[52] A.-L. Reysenbach, K. Longnecker, and J. Kirshtein, Appl. Environ. Microbiol. 66
(2000), 3798-3806.
[53] K. Takai, andK. Honkoshi, Appl. Environ. Microbiol. 66 (2000), 5066-5072.
[54] M. T. Suzuki, L. T. Taylor, and E. F. DeLong, Appl. Environ. Microbiol. 66 (2000)
4605.
233
Chapter 9
234
While chemical surfactants are both inexpensive and efficient, they may
have very negative effects on the environment. Increasing awareness on the part
of the consumer, coupled with the potential for legislation governing excessive
use of such chemicals offers new opportunities for biotechnological alternatives.
The potential advantages of such products include, 1. biodegradability resulting
in lower levels of pollution, 2. selectivity and specificity towards hydrocarbon
substrates, 3. potential for using recombinant DNA technology to engineer
changes in surfactant structure and function, 4. compatibility with chemical
products leading to novel formulations, 5. natural products may have unique
characteristics which cannot be produced by simple chemical synthesis. One
such product, which is used in the oil industry is xanthan gum, a microbial
polymeric exopolysaccharide with unique, sheer thinning rheological properties
[5]. In this Chapter we will focus primarily on microbial biosurfactants as
bioemulsifiers and their potential applications in the petroleum industry.
2. LOW MOLECULAR WEIGHT BIOSURFACTANTS
As is the case with chemical surfactants of non-biological origin, biosurfactants
exhibit a wide variety of surface activities ranging from reduction of surface
tension, formation of water-in-oil and oil-in-water emulsions, adsorption to and
coating of surfaces, surface wetting, flocculation of solids, foaming and
defoaming at air/liquid interfaces and emulsion breakage. Moreover, many of
these compounds can exhibit more than a single activity. Since there is a very
wide range of structures and producing organisms it is often useful to consider
the lower molecular mass biosurfactants as a group distinct from the polymeric
bioemulsifiers [6-9]. In this section we will refer to the biosurfactants as low
molecular mass detergents whose primary activity is to lower interfacial tension,
while bioemulsifiers are generally higher molecular mass polymers.
Biosurfactants generally are smaller and exhibit molecular masses in the range
of a few thousands of Daltons. As detergents, their primary surface activity is to
lower interfacial tension at the air/water interface. As is the case with chemical
surfactants, biosurfactant formulations may include biosurfactants in
combination with chemical or biological co-surfactants, solvents, biocides and
other ingredients. Their production can be the result of denovo synthesis [10], or
in some cases the result of biotransformations resulting from transacylation or
other post synthetic processing [11]. From the point of view of large-scale
production, microbial systems have been considered as the major sources of
biosurfactants largely owing to the availability of fermentation technology for
their production. Moreover, in certain cases, it is possible to manipulate the
biosynthetic pathways and their regulation using modern recombinant DNA
technology [12]. Since, in recent years a number of original papers and excellent
235
reviews dealing with low molecular weight biosurfactants have been published
[7,9,13-16], the subject will be treated only briefly in this section.
2.1. Structure-function features of low molecular weight biosurfactants
2.1.1. Chemical characterization of biosurfactants
As illustrated in Table 1A and IB, biosurfactants exhibit a wide variety of
structures including fatty acids and alcohols, phospholipids, lipopeptides, which
can be either cyclic or linear, and glycolipids consisting of disaccharides linked
via esters or ethers to fatty acids. While most of these compounds are
extracellular, an interesting exception has been reported, in which the cells
themselves exhibit emulsifying activity [13].
Lipopeptides. In the lipopeptides, produced by several species of Bacilli
and other organisms, the hydrophilic moiety is represented either by a cyclic
oligopeptide, as in surfactin [17] or a 2, 4-diaminobutyric acid intercalated
cyclic oligopolypeptide, as in polymyxin [18]. Another class of biodetergents
was isolated from a variety of microorganisms belonging to the genera
Pseudomonas. They share a common cyclic oligopeptide structure represented
by charged amino acids such as serine, aspartic acid and glutamine, while noncharged amino acids as leucine, and isoleucine are present as well [19].
Interestingly, amino acids which are normally not found in proteins, such as Lnorvaline, L-norleucine, L-allylglycine, etc. have been found in some
lipopeptide biodetergents strongly suggesting that NRPS's (Non-Ribosomal
Protein Synthases) play a role in the generation of these amphipathic peptides
[20]. The amino acid number that constitutes the oligopeptide varies among the
species, but they share the common lactonized structure formed as a result of an
ester bond. For example, serrawetin W2 contains 5 amino acids, surfactin is a
heptapeptide; WLIP, viscosinamide, massetolides and viscosin contain 9 amino
acids, while tensin, hodersin, pholipeptin, lokisin, arthrofactin and amphisin
contain 11 amino acids (Table IB). In all of these compounds, a hydrophobic 3hydroxydecanoyl moiety serves as the lipophilic component [19].
Glyco- and flavolipids. Certain microorganisms produce biodetergents
consisting disaccharides as the hydrophilic moieties and fatty acids linked via
ester or ether linkages. In the case Pseudomonas aeruginosa the sugar residue is
rhamnose to form a rhamnolipid [21], while Arthrobacter parqffineus produces
a trehalose lipid [22]. Similarly, the yeast Torulopsis produces a biodetergent
consisting of two molecules of glucose. Interestingly, here the acyl moiety is
introduced by biotransformation of an exogenously added source of fatty acid
[31]. Another fungal glycolipid contains sophorose as the hydrophilic moiety.
Recently a new product was discovered during a screen for surfactant producers
from soil samples in the Southwestern desert of the U.S. [24]. The new
surfactant, produced by a Flavobacteria contains a hydrophilic group consisting
236
of citric acid and two cadaverine molecules. The hydrophobic moiety contains
two acyl groups of from 6-10 carbons.
237
238
239
240
241
242
243
244
245
246
247
and/or capacity for emulsification need not be directly related to the natural role
of the surface-active molecule. A standard measure for surface activity is the
reduction of surface tension. This method may vary somewhat from instrument
to instrument and is somewhat cumbersome since frequently measurements
must be made at several concentrations. Generally, a good candidate for a
surfactant lowers the surface tension from 70mN/m to below 30mN/m. An
additional screen based on surface activity was to search for organisms, which
produced materials which lyse eukaryotic cells [50-53]. The problem with this
approach was that it also enriched for microbial pathogens. Moreover, it was
shown to have the weakest correlation with other modes of screening and to
yield the highest number of false positives [14]. Another useful and effective
screen involves examining the capacity of a drop of culture broth from a
putative surfactant producer to collapse an aqueous droplet formed on a
hydrophobic surface [54-55]. The surfactant in this case increases the contact
angle and droplet collapse can be estimated as a function of surfactant
concentration and related to standard materials. The droplet collapse method is
rapid and yields relatively low numbers of false positives [14]. The oil spreading
technique involves placing a droplet of a surfactant containing solution on a
surface coated with a liquid hydrocarbon. The surface activity causes oil
spreading leaving a clear zone at the point of application the diameter of which
is a qualitative measure of surface activity [56]. Recently the three screening
methods, lysis of blood agar, droplet collapse and oil spreading were compared
with surface tension measurements for 205 natural isolates. The oil spreading
technique appeared to give the highest correlation with the surface tension
lowering, although there was strong negative correlation between clear zone
diameter and droplet collapse suggesting that the two procedures measured
similar activities and could be correlated well with surface tension
measurements. It was suggested that an effective protocol for screening natural
isolates is to use the droplet collapse method and subsequently employ the oil
spreading technique for more quantitative preliminary evaluations [14].
The wide variability in structures (see Table 1A and IB) and the
production and secretion of bio surfactants by organisms, which do not grow on
hydrocarbons indicates that there is probably no general biological role for all
biosurfactants [57]. Moreover, unless the search is for a closely related analog
whose synthesis may be catalyzed by similar gene products, it is unlikely that
modern methods in molecular ecology will be productive in identifying new
organisms or products. Recently, natural isolates from arid soil samples from the
Southwestern U.S. were screened for their ability to produce extracellular
materials in the culture broth, which lowered surface tension at the air/water
interface [12], According to this screen over two percent of the isolates
produced surfactants when grown on a rich medium. Interestingly, biosurfactant
producers were found in both uncontaminated soils as well as in soils showing
248
249
acid side chain to provide the hydrophobic tail. Homologous of this complex are
found in Bacilli, which produce cyclic lipopeptides such as surfactin.
3. BIOEMULSIFIERS
Bioemulsifiers are another group of biosurfactant. Many surface-active agents
exhibit multiple surface activities such as reduction of surface tension and
reduction of interfacial tension. However, this is not always the case.
Bioemulsifiers may be poor detergents, while the ability to lower interfacial
tension may not be sufficient to stabilize water in oil or oil in water emulsions
[62-64]. Bioemulsifiers are microbial products, which form and stabilize water
in oil, or oil in water emulsions. Their discovery was based initially on the
observation that growth of microorganisms on various oils is often accompanied
by the emulsification of the hydrophobic carbon source in the aqueous growth
medium [8-9,49, 65]. Although some low molecular weight biosurfactants are
also bioemulsifiers, here we will focus primarily on the polymeric
bioemulsifiers since their higher viscosity enhances their stabilization properties
[66-69]. Moreover, the polymeric bioemulsifiers exhibit a preference for the
oil/water interface and do not form micelles which enables them to be used at
lower concentrations (i.e. higher ratios of oil: emulsifier).
3.1. Protein-Polysaccharide Interactions
As shown in Table 2 most of the isolated and characterized bioemulsifiers
show a composition where a protein fraction is part of a complex, which
contains a polysaccharide backbone, however the role of the nature of the
protein-polysaccharide interaction at the oil/water interface remains to be
clarified. What follows below is a brief consideration of the interactions of
proteins and polysaccharides at interfaces. This very general treatment may help
to understand some of the complex interactions governing bioemulsifier
function.
3.1.1. Proteins at oil-water interfaces
Proteins often act as surface-active agents e.g. p*-lactoglobulin [83] and
elastin [84], In order to exhibit this surface activity they should be able to be
transported from the bulk solution and to concentrate at the interface of the
system. Subsequently, they should be able to penetrate into the surface layer.
Each stage is accompanied by a specific energy barrier, which, once overcome,
results in a successive lowering of interfacial energy [85]. This property of the
protein stems from its flexibility, which in turn is conferred by the peptide bonds
[86]. At the interface the protein may unfold to varying extents, reorient,
rearrange and spread to form a continuous cohesive film [87-89].
250
Table 2
Composition of biosurfactants
Compound
Organism
MW*
Composition
C+ P FA*
Reference
Exopolysaccharides
Acinetohacter
>3xlO5
calcoaceticus MM5
Bacillus sp. IAF343
Bacillus cereus IAF 346
Halomonas euhhalina H28
9xl0 5
Acinetobacter
radioresistens K53
Acinetobacter
51400
calcoaceticus A2
Acinetobacter venetianus
lxlO6
RAG-1
Candida lypolytica ATCC 27600
8662
Alasan
Biodispersan
Emulsan
Liposan
15
20
[70]
44
44
35
2
2
4
20
[71]
[71]
[72]
[73]
70
30
[74]
70
15
83
17
12
[75]
[76]
Lipids
Rhodococcus sp. Q15
Saccharomyces uvarum
14-18
18
[77]
[78]
15
10
7
[79]
[19]
[56\
[34]
[80]
[80]
[38]
Lipopeptides
Amphisin
Arthrofactin
Bacitracin
Hodersin
Lokisin
Lychesin A
Serrawettin
Surfactin
Tensin
Viscosin
Viscosinamide
Glycolipids
Pentasaccharide
Rhamnolipids
Sophorose
1035
1395
1354
1600
1409
1355
1030
10
10
12-17
732
923
8
12
[42]
[43]
1410
10
[45]
1126
1126
10
10
[46]
[47]
Nocardia
corynebacteroides SMI
Pseudomonas strains
750
18-20
800
10
1084
1371
[27]
[27]
[28]
22
[81]
[23]
Other biosurfactants
Aerobactin
Mannoprotein
+
Saccharomyces cerevisiae
44
17
[82]
251
252
253
254
255
256
Table 3A
Production and surface activity of bioemulsifiers
Organism
C-source
Compound
Exopolysaccharides
Yield*
glucose
0.6
tetradecanc
0.06
ST
Reference
[157]
\70]
sucrose
0.5-1.2
53
sucrose
0.5-1.2
28
crude oil
Alasan
ethanol
2.2
[73]
Biodispersan
Acinetobacter calcoaceticus A2
ethanol
[74]
Emulsan
ethanol
15
[158\
Liposan
hexadecane
glucose, acetate
36
[77]
29
[159\
Saccharomyces uvarum
hexadecane,
kerosene
n-dccane
20
[78]
glucose
25-34
0.35
\7l]
[71]
[72]
[76]
Lipids
Lipopeptides
30
[160]
glucose
27
[80]
L-broth
24
[56]
27
[34]
glucose
27
[80]
glucose
27
[S01
28
[38]
28
[42]
27
[43]
glucose
27
ISO]
glucose
26.5
[19\
glucose
27
[80]
Cory'nebacterium lepus
kerosene
Amphisin
Arthrofactin
Bacitracin
Bacillus liqueniformis
Hodersin
Pseudomonas sp.
Lokisin
Lychesin A
glucose
Scrrawettin
glucose
Surfactin
glucose
Tcnsin
Viscosin
Pseudomonas viscosa
Viscosinamide
ing/I.
Surface Tension in mN/m.
[79]
0.16
3-4
257
258
Yield'
32
8
0.1
26
[24]
2.8
26
[27]
29
[167]
70
31
[168]
43
[31]
Flavolipid
Flavobacterium sp MTN11
Mihagol L
Mihagol S
glucose
Pentasaccharide
Nocardia corynebacteroides
nCi4_ 15
DSM 43215
Reference
C-source
ST
[25]
SMI
Rhamnolipids
Pseudomonasputida 21BN
Sophorose
hexadecane,
glucose
glucose,
safflower oil
glucose
glucose
[SI]
Others biosurfactants
Aerobactin
Mannoprotein
glucose
glucose
1
8"
Synthetic surfactants
Cetyl Triethyl Ammonium Bromide (CTAB)
Linear alkylbenzene sulfonate
Sodium dodecyl sulphate
Tween 20
30
47
37
30
Water
72
ing/1.
Surface Tension in mN/m.
[23]
[82]
259
Table 4
Enhancement of apoemulsan activity on different hydrophobic substrates by recombinant
esterase
Hydrophobic substrate
Anthracene
Crude oil
Dicyclohexane
Diesel oil
Eicosane
Fluoranthene
Heptadecane
Immersion oil
2-Methyl Naphthalene
Mineral oil
Octadecane
Petroleum refinery sludge
Pyrene
Soya oil
Squalene
Tetracosane
966
4.3
8.9
5.7
1800
593
28
10.4
1984
6.6
2250
2
420
1260
600
506
260
261
spite of the fact that A. venetianus RAG-1 is thus far the only natural isolate
which has been shown to produce emulsan [180]. According to convention, the
specific genes for emulsan biosynthesis were termed wee; the first letter
signifies that the gene is a biosynthetic gene from a polysaccharide biosynthetic
cluster, the second land third letters identifying the specific product (emulsan,
exopolysaccharide). In accordance with convention, some gene products exhibit
similar functions in all organisms, and thus are allowed to retain their original
names. Figure 3 summarizes a hypothetical biosynthetic pathway for
apoemulsan, with, the rightward operon encoding proteins involved in precursor
synthesis and activation, aminoglycosyl transferases for assembling the
trisaccharide subunit on the inner side of the cytoplasmic membrane, a
polymerase, decorating enzymes for the acylation of the aminosugars, a
translocase which moves the polymer from the cytoplasmic face of the
membrane to the outer or periplasmie face, and enzymes involved in subsequent
translocation of the polymer through a specific channel or porin to the outer
surface of the cell [180].
Regulation and the production of viscoemulsan. Located in the
intercistronic region are two putative d promoters. The leftward operon
consists of three repeating frames wza, wzb and wzc, which encode a porin, a
protein tyrosine phosphate phosphatase and a protein tyrosine kinase,
respectively [180-181]. Knockout mutants in any of these genes resulted in
defects in emulsan production. Both Wzc and Wzb proteins of RAG-1 were
cloned and over-expressed in E. coli. The Wzc Ptk was shown to be an
autophosphyorylase in which a tyrosine (s) in the C-terminal portion of the
protein is phosphorylated and subsequently dephosphorylated by the
phosphatase [182]. Similarly, the phosphotyrosine of Wzc from RAG-1 was
shown to be dephosphorylated by Wzb [181]. According to other reports, the
phosphorylated form of Wzc is expected to negatively regulate polymer export
through the porin Wza. Elevated levels of extracellular biopolymer production
would then be initiated with the activity of Wzb, the phosphatase, which
removes the phosphates, permiting the enhanced export. Consistent with the
hypothesis was the finding that knockout mutants in the phosphatase were also
emulsan deficient. However, the results did not explain why knockouts in Wzc
would be emulsan deficient as well. Apparently there is a requirement for the
Wzc protein even in its non-phosphorylated state. The Wzc protein contains a
series of five tyrosine residues in close proximity to each other at the C
terminus. When these tyrosines were deleted, the resulting protein was made but
could be phosphorylated and surprisingly, a high molecular mass
polysaccharide, termed viscoemulsan, was produced [181]. This product appears
to contain the same constituents as emulsan, but is not active as an emulsifier
(Nakar, In preparation). The introduction of a wild-type allele of wzc gave rise
to the production of a wild-type allele of emulsan suggesting that the protein
262
tyrosine kinase may act to control the size of the exported polymer. It is also of
interest that the Wzc protein is required for viscoemulsan production even
though it cannot be phosphorylated [181] suggesting that there is an additional
role for the protein. A model to describe the role of phosphorylation and
dephosphorylation is shown in Fig. 4 [181]. According to this model Wzc, Wzb,
Wza proteins and others interact in a multienzyme complex to control the export
of the exopolysaccharide. The process is initiated by dephosphorylation of the
protein relaxing the control on the porin diameter and enabling larger amounts
of polymer to be translocated to the external surface of the cell. Under
conditions of rapid growth and high ATP, all of the tyrosine residues are
phosphorylated and polymer production is low. In fact, emulsan production does
not take place in rich media, although its biosynthesis has been shown to occur.
The polymer can be detected immunologically. The manipulation of the export
process coupled with the modifications of the biosynthetic genes offers new
approaches to the generation of new and novel products and is currently in
progress.
3.3.4. Engineering novel derivatives of emulsan
The production of new viscous derivatives such as viscoemulsan
represents one approach to engineering new biopolymers. Kaplan and coworkers have used nutritional modification to modify fatty acid composition and
surface active properties of the resulting derivatives of emulsan [161-164].
Moreover, as described above, the surface activity of apoemulsan-containing
formulations can be enhanced by the addition of a particular cell surface
enzyme, the cell surface esterase of RAG-1 [139].
263
Fig. 3. The wee cluster for the biosynthesis of emulsan. The scale of the cluster size is in
kilobases. The black arrows represent putative orf sequences. White arrows represent partially
sequenced orf s. Putative promoter sites are indicated with thin black arrows. The names of
the genes are shown below the corresponding orf s. Orf s labeled solely with capital letters
are putative pathway specific genes encoding Wee A-K respectively.
264
for an esterase from another member of the genus Acinetobacter, the strain A.
calcoaceticus BD4 [188] and its miniencapsulated derivative BD413. While this
enzyme shows strong sequence and structural homology to the RAG-1 enzyme,
it did not display any emulsification enhancement when added to apoemulsan
[139].
Specificity towards hydrocarbons. As shown in Table 4 the recombinant
esterase protein enhances emulsification of apoemulsan towards a variety of
pure and crude hydrophobic substrates. EEP activity was observed with mutants
of the esterase defective in catalytic activity, suggesting a role for the protein
other than as an enzyme.
Esterase exhibits EEP activity towards other polysaccharides.
Surprisingly, the interaction of the recombinant RAG-1 esterase with the water
soluble, rhamnose-containing exopolysaccharide from A. calcoaceticus BD4 led
to the formation of a new bioemulsifier complex. In sharp contrast, the esterase
from BD4 did not enhance emulsifying activity of apoemulsan towards
hydrophobic substrates [140]. Remarkably, the recombinant esterase from RAG1 exhibited EEP activity with over 25 different natural biopolymers, none of
which exhibited any emulsifying activity in the absence of the protein. In these
cases, the enhancement was not dependent on catalytic activity of the
recombinant protein (Bach and Gutnick, in preparation). The results point to a
new approach to generation of amphipathic emulsifiers, which is no longer
dependent on fermentation to produce the polymer emulsifier. Among the
inexpensive materials, which can be converted into bioemulsifiers using this
unique formulation with the RAG-1 esterase are cellulose, dextran, starch,
xanthan, alginic acid, and a variety of plant and bacterial polysaccharides
including the inactive viscoemulsan described above (Table 5). The mode of
action of the EEP remains to be elucidated although evidence is discussed below
demonstrating that there is a unique motif in the RAG-1 esterase, which is
missing from other homologues.
Mapping the EEP domain. Initial observations showed that limited
proteolysis of the recombinant esterase yielded a fragment of about 10 kDa,
which retained the ability to enhance emulsification of hydrophobic substrates
such as hexadecane. Accordingly, a series of site directed mutants were
generated and over-expressed to produce different fragments of the esterase.
Since the fragments were rapidly degraded even in strains of E. coli lacking Clp
or Lon proteases, fragments were prepared which were fused in frame to the Cterminus of the maltose binding protein [189]. The various constructs are shown
in Fig. 5. The each over-expressed fusion was tested with apoemulsan using the
model hydrophobic substrate, hexadecane as a substrate for emulsification.
Virtually all the enhancing activity was localized to the C-terminal third of the
esterase. It was of interest that the maltose binding protein itself exhibited no
EEP activity. Moreover the fusion protein containing the active polypeptide was
265
no less active than the intact enzyme. Removal of the terminal 15 amino acids
from the C-terminus completely abolished the EEP activity. Sequence analysis
showed that this 15 amino acid C-terminal peptide is unique to the RAG-1
esterase and probably accounts for the unique characteristics of this protein.
However, as shown in Table 2, many organisms produce emulsifiers consisting
of protein/polysaccharide complexes [7, 9, 190]. In most cases the protein
requirement has yet to be clarified and it is possible that there are other proteins
or peptides, which exhibit unique EEP activity. Regardless, EEP technology
offers a new approach to bioemulsifier production and paves the way for new
families of inexpensive, non-toxic, amphiphiles.
Fig. 4. Hypothetical model for the role of protein tyrosine kinase (Wzc) and protein tyrosine
phosphatase (Wzb) in emulsan export. 1. Dephosphorylated Wzc allows for polymerization
and translocation of emulsan. 2. Phosphorylation of Wzc halts the process, thereby
determining the size of the exported polymer. 3. Emulsan release and beginning of a new
round of polymerization, translocation and release. Wza-translocation channel; Wzb-protein
tyrosine phosphatase; Wzc-Protein tyrsoine kinase; Wzx-polymerase; Wzy-translocase.
266
Table 5
Enhancement of the emulsifying activity of different polysaccharides by recombinant
esterase in the presence of hexadecane
_ ,
,
,
Polysacchande
Agarose
Alginic acid
Apoemulsan
BD-4 exopolysaccharide
Carrageenan
Cellobiose
Cellulose
Chitin
Colamc acid
Dextran
Emulsan
Ficoll 400
Gum Arabic
Pectin
Polyvinyl Pyrrolydone
Potato starch
Pullulan
Stewartan
Xanthan
Xylan
Emulsifying activity
,..,
,
, , .
.
(U/mg polysacchande/mg esterase)
963
496
5430
3396
3345
626
766
540
2050
583
6752
263
1895
1830
1950
544
3400
1196
2720
1854
267
Fig. 4. Generated esterase constructs fused in frame to the C-terminus of the maltose binding
protein.
268
269
approval from the regulatory agencies, are offset by the high prices and
profitability of the product, the cost of applications in the oil industry must be
kept relatively competitive with products of the chemical industry. This is a
particularly difficult constraint considering that production of many of the
biotechnological products may involve large-scale fermentation processes,
which exert a considerable impact on the cost of the product, particularly if
extensive downstream processing is required. Several approaches may be used
to enhance the cost effectiveness of biosurfactants.
4.1.1. Searching for the "world beater"
Occasionally, the search for natural products yields a compound with
unique properties unlike others either natural or developed by the chemical
industry. Such products are termed "world beaters" because their properties are
unique and unmatched by products of chemical synthesis. Antibiotics represent
a classical example of natural materials of major chemotherapeutic importance
whose activities are unmatched by chemical synthesis, although they are
generally modified by various chemical transformations [ 197]. Another example
of a microbial product with unique properties is the exopolysaccharide product
of the plant pathogen Xanthomonas campestris, xanthan, is a major polymer
whose sheer thinning properties and high reduced viscosity make it a major
industrial product in foods as a thickening agent, in drilling muds, and as a
gelling agent for use in oil field fracturing programs [5]. Moreover, xanthan
viscosity is exploited in oil field flooding during enhanced oil recovery, since
the extraordinary high viscosity enables it to actually "push" the released oil out
of the well. In most cases, however, the natural biosurfactant exhibits
characteristics, which are promising but not necessarily unique.
4.1.2. Cutting the cost of production
The cost of production of biosurfactants is frequently a function of the cost
of fermentation and subsequent downstream processing. This is particularly true
for production of products in which the carbon source must be a hydrocarbon,
which, is often the case with low molecular weight glycolipids [9, 16, 53, 198199]. A key approach in this system involves enhancing the product yield by
upgrading and optimizing the fermentation [21]. In the latter case the
fermentation of rhamnolipids has been upgraded such that lOOg/liter was
produced from 160g of soybean oil as a carbon source, a remarkable conversion
of substrate to product. In addition, production on various industrial waste
products has also lowered the cost of production [200-202]. Assuming that the
product biosurfactant is sufficiently active, this presents a way of upgrading the
waste material by producing a product of higher added value. This approach
may be particularly advantageous in the oil industry, since the crude product
270
need not be purified to any significant extent and may not require expensive
downstream processing [203].
Enhanced productivity can in principle be obtained by transferring
biosynthetic genes into an organism such E. coli K-12 which is easy to grow and
which utilizes a "friendlier" source of carbon and energy [204-205].
At least in pilot scale, most biosurfactants are produced in batch
fermentations. Cooper and co-workers developed a semi-continuous approach to
producing biosurfactants via self-recycling system [206]. Similarly, emulsan
was produced in a similar protocol in which the product was allowed to grow
and accumulate in early stationary phase followed by the removal of 90% of the
cells and emulsan, which was harvested downstream. The fermentor was filled
with fresh media and the culture again entered exponential and early stationary
growth, the major portion of the emulsan recovered and the cycles repeated in
the same fermentor for several semi-continuous production runs. The cost of
production is thus significantly reduced (Cooper, D., Personal communication).
Another way to cut the cost of production is to upgrade the producing
strain in order to enhance overall productivity [8, 199], This approach has been
used in the case of the emulsan producing strain A. venetianus RAG-1 [166].
The positive selection for emulsan overproducers was based on the fact that the
emulsan polyanion binds the toxic cation cetyl-trimethylammonium bromide
(CTAB). Among the mutants of RAG-1 resistant to CTAB, were those such as
strain A. venetianus CTR49, which overproduce the extracellular polyanion and
are thus significantly more resistant to the CTAB than the parent. In the
laboratory, mutants of this variety produced up to twice as much emulsan per
gram of ethanol carbon source than the wild-type.
4.1.3. Upgrading the product
Metabolic engineering of new products. Another approach to generating a
viable technology employing biosurfactants is to employ physiology,
formulation and/or recombinant DNA technology to generate modified products
with improved properties. One such approach involved preparing emulsan from
RAG-1 cells grown in the presence of various fatty acids [161-164] in order to
modify the nature of the acyl groups present in the side chains of the
bioemulsifier. We have carried out similar experiments and have found that the
emulsan produced is significantly more active towards hydrophobic substrates
such as hexadecane alone. Of course, the enhanced efficacy still needs to be
weighed against the increased cost of the fermentation due to the inclusion of
fatty acids in the media.
Formulation packages.
Emulsifiers and surfactants are generally
incorporated into surfactant packages, which include a mixture of surfactants
designed to lower interfacial tension between water and oil phases. Also, in
cleaning applications, the formulations may also contain a biopolymer to
271
stabilize the emulsion [207-209] and prevent coalescence of the phases, and they
can also contain a compatible solvent. The solvent need not be a water based
solvent, but it should be able to dissolve both the low molecular weight
surfactants as well as the biopolymer. Materials such as pine oil, liquid
terpenoids, dimethyl sulfoxide, and various light crude oils have all been
included in various surfactant packages [210]. In addition to solubilizing all of
the components into a pumpable mixture, the solvent addition has also been
shown to enhance the cleaning of oil contaminated tanks by removing the last
remnants of sludge and other flammable materials from the walls of the
container rendering the tank not only clean, but also gas-free. The choice of
suitable components for various surfactant packages must also take into account
other potential components, which must be included. For example, if routine
cleaning operations include rinses with anticorrosive materials, the formulation
package must be designed on the basis of compatibility with such components.
Similarly, emulsion based fuels may need to be formulated together with
materials which lower sulfur emissions. Specially designed surfactant
formulations may also require compatibility with a variety of materials including
flame retardants, biocides etc.
4.1.4. EEP technology
In section 3.3.2 the ability of a recombinant cell surface esterase from
RAG-1 to enhance the emulsification of apoemulsan towards a variety of
hydrophobic substrates was described [139]. The remarkable feature of this
system was the finding that the RAG-1 esterase and several of its derivatives
were able to interact with a host of polysaccharides to generate a series of
amphipathic complexes, which exhibited strong emulsifying activity [140]. This
surprising activity, has paved the way for the generation of a whole suite of
bioemulsifiers, in which the polymeric component need not be produced as a
fermentation product. In fact, it may be possible to upgrade waste materials such
as crude celluloses, starch, pectins, etc. to bioemulsifier when combined with a
specific peptide derived from the esterase. This peptide can be produced as an
over-expressed protein fusion following cloning in a suitable vector [Bach and
Gutnick, in preparation], or it can be generated via proteolysis of the esterase
itself. The feasibility of employing EEP technology for emulsification in oil
industry applications will become clearer once larger scale field trials are
conducted and evaluated.
4.2. Bioemulsification, cleaning and sludge recovery
4.2.1. Tank clean ing
Oil storage containers accumulate enormous quantities of sludges and
bottom sediments. Previous work from this and other laboratories have
272
273
somewhat higher than with a regular hydrocarbon fuel since the surface area is
larger. What is even more interesting is that the quality of the burn was
indistinguishable from that of a light high quality fuel oil suggesting that
emulsion based fuels can be a viable alternative for some applications [207210,213].
Interestingly, these larger scale burn experiments were performed with a
biosurfactant containing formulation. However, at Petroferm U.S.A. specialty
chemical formulations were developed some of which did not contain the
emulsan, but was composed of chemical surfactants, a solvent and other
components. Emulsion based fuels have become more and more popular,
because they permit the efficient combustion of various hydrocarbons which are
normally difficult to burn. Arguably, the best studied system is the waterbitumen emulsion system, termed Oriemulsion which has been commercialized
and is currently exported from Venezuela throughout the world. The emulsion is
chemical based, and resembles the initial Petroferm formulations.
4.3. Viscosity reduction and oil transportation
As discussed above, the stability of emulsan-based oil-in-water emulsions
results from the coating of the oil droplets with emulsan in an oriented
conformation; hydrophobic moieties coming into contact with the oil surface,
and the hydrophilic components oriented towards the aqueous phase. This
results in a homogeneous suspension in which the viscosity of the oil component
is significantly reduced at room temperature. The extent of the viscosity
reduction is a function of the water composition of the bulk phase, which for an
emulsanosol can be as high as 30%. Under these conditions the viscosity of a
high viscosity oil from the Orinoco basin in Venezuela, (Boscan crude) was
reduced from >50,000 Cp to about 85 Cp in the form of an emulsanosol
generated with an emulsan based surfactant package [207-210, 213] at a ratio of
1 part surfactant to 500 parts oil. About fifty barrels of this emulsion was
transported through an experimental pipeline of 1.25 inches for 96 h during
which the mixture was subjected to over 500 pump transits. There was no effect
on the low viscosity, although the shear forces on the emulsion might have been
expected to produce an inversion from an oil-in-water to a water-in-oil
emulsion. Moreover, even after the system was shut down and the emulsion
allowed to stand undisturbed for 48 h, there was no breakage or inversion of the
emulsion and the low viscosity was maintained through the pipeline for an
additional 48 h. The results support the use of surfactant packages to generate
oil-in-water emulsions for pipelining highly viscous oils. In fact, this is the basis
of the Orimulsion technology.
274
5. CONCLUDING REMARKS
There is little doubt that biosurfactants and bioemulsifiers exhibit characteristics
and activities, which are applicable in the oil industry. As noted, in some cases
product efficacy has been tested in large scale and successful trials have been
recorded. However, the impact of such materials on the oil industry is likely to
be far less than in other industrial sectors, where the price of the final product is
high relative to the costs of production. Ongoing efforts to isolate new
biosurfactants, genetically modify existing ones, enhance productivity of the
producing organism and otherwise lower the cost of production, and formulate
new and more effective biosurfactant packages should yield a host of products
and applications in the future.
Moreover, for some applications such as enhanced oil recovery or
bioremediation, the biotechnology associated with in-situ biosurfactant
production accompanying the growth of microorganisms, discussed elsewhere in
this book, can be a useful and economically competitive strategy. Finally, the
incorporation of biosurfactants in novel formulations suitable for sludge
liquification and viscosity reduction leading to economically feasible techniques
for waste recovery and recycling.
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283
Chapter 10
1. INTRODUCTION
Hydrocarbon-based energy underpins the economic, social, and political fabric
of the world and demand for oil is expected to grow unabated for the foreseeable
future. It is forecast that global oil consumption will increase annually by an
average of more than 4 x 107 barrels per day to eventually reach 4.3 x 1010
barrels per year by 2020 [1]. This is projected to be about a 58% increase over
current usage by 2025 [2]. The U.S. Geological Survey recently predicted that
about 3 trillion barrels of oil remain to be recovered worldwide (with 1 trillion
barrels already harvested), half from proven reserves and half from undeveloped
or undiscovered sources [3]. However, as proven reserves get exploited and
develop into mature fields, secondary and tertiary recovery technologies will
increasingly be relied upon to obtain the remaining residual oil. This is
particularly true for the U.S. and other oil-importing countries that tend to rely
more heavily on mature, domestic energy sources. With demand far surpassing
energy production in the U.S., there is heightened interest in diversifying energy
sources, tapping unconventional energy supplies and the development of new
technology to more fully exploit domestic reserves.
Although oil is expected to remain the dominant energy fuel in the next
20 years, the use of natural gas as a substantial energy source has risen
significantly in the past 10 years [4]. In fact, natural gas is projected to be the
fastest growing primary energy source and an increasingly important alternative
to oil [2]. Natural gas, consisting mainly of methane (>95%) but also with small
amounts of other short-chain hydrocarbons (C2 to C4), can be harvested from
284
large gas fields, sometimes associated with oil reservoirs, or be obtained from
unconventional sources such as shale, coalbeds, or tight sands. The use of
natural gas is becoming increasingly popular due to its abundance across the
globe. Further, the lower price of natural gas relative to oil makes it an attractive
energy source [5]. Natural gas is also a cleaner energy source than oil or coal,
and thus can help reduce greenhouse gas emissions [6]. Natural gas combustion
produces only about 56% and 71% of the CO2 associated with the equivalent
amount of energy produced from coal or oil, respectively [Energy Information
Administration (1999). Natural Gas 1998: Issues and Trends (http://www.eia.
doe.gov/oil_gas/natural_gas/analysis_publications/natural_gas_1998_issues_and
_trends/it98.html). Moreover, methane use results in less NOX, SO2, and
particulates per equivalent amount of energy generated, relative to other sources.
Despite the increasing use of natural gas and its attendant environmental
advantages, world reliance on oil is unlikely to wane in the near future given the
existing energy infrastructure and the aforementioned dependence of many
societies on this energy form. However, there is a biotechnological link between
oil and natural gas that is the product of the relatively recent recognition that
many hydrocarbons are susceptible to anaerobic biodegradation and can be
converted to methane and carbon dioxide [7-9]. Unlike the well-documented
patterns of aerobic oil biodegradation [10], anaerobic hydrocarbon metabolism
was essentially dismissed as ecologically insignificant for many years. This
view has been completely altered in recent years with the growing appreciation
for the metabolism of hydrocarbons coupled with the consumption of electron
acceptors other than oxygen.
Not surprisingly then, the majority of world oil reserves are believed to be
biodegraded to at least some degree, but it was generally accepted that aerobic
oxidation processes were largely responsible for such alterations [11, 12].
Recent evaluations of many petroliferous formations have convincingly argued
that it is actually anaerobic processes that predominate in oil and gas reservoirs,
sometimes leading to the production of biogenic methane [12-14]. Geological
evidence has suggested that such methanogenic processes occur very slowly
over millennia, and are most important in reservoirs shallower than 4 km and at
temperatures of less than 80C [12, 15, 16]. Microbial decay of oils in deep
subsurface reservoirs can clearly reduce oil quality, and a better understanding
of the microbial principles behind such decay will be important to help
distinguish between degraded, low-value oils and untouched, high-value oils
[11]. However, if methanogenesis continues to be identified as an important
process in deep reservoirs worldwide, the recovery of methane gas as an
alternate form of energy from otherwise unrecoverable or biodegraded sources
might have far-reaching economic and environmental implications.
The purpose of this chapter is to review evidence for anaerobic
hydrocarbon biodegradation and to provide an overview of some of the more
285
286
287
288
289
only initiate alkane degradation via fumarate addition, but most probably share
the entire degradation pathway.
Polycyclic aromatic hydrocarbons (PAHs) are also susceptible to
anaerobic decay. Naphthalene can be completely mineralized by pure cultures of
sulfate-reducing and denitrifying bacteria [87, 88]. Enrichments from coal-tar
contaminated sediments and garden soil were reported to mineralize [14C]naphthalene with soluble Fe(III) and insoluble FeOOH, although not more than
15% of added radioactive substrate was recovered as 14CO2 [89]. Anaerobic
degradation of phenanthrene was also demonstrated in sediments [22, 90] and by
a sulfate-reducing enrichment culture [91]. Studies with marine sediments also
indicated the loss of 2 to 5-ringed PAHs under anaerobic conditions, with the
smaller PAHs degrading more rapidly than the heavier molecular weight
counterparts [90]. It has been shown that unsubstituted PAHs, such as
naphthalene and phenanthrene, are initially attacked by carboxylation to form 2naphthoic acid and phenanthrenecarboxylic acid, respectively [91, 92]. The
carbon in both cases arises from inorganic CO2. 2-Methylnaphthalene is
converted to 2-naphthoic acid following the anaerobic oxidation of the methyl
group [93]. A mechanism for the activation of 2-methylnaphthalene is the
addition of fumarate to the methyl group [92, 94]. The product of this reaction,
naphthyl-2-methyl-succinic acid, is subsequently oxidized to 2-naphthoic acid
which further decomposes by ring reduction reactions to form the fully saturated
decalin-2-carboxylic acid prior to ring cleavage and ultimate mineralization [91,
92, 95].
Alicyclic hydrocarbons can comprise a substantial fraction (often up to
~12% wt/wt) of the organic molecules in petroleum mixtures. Despite this
quantitative importance, little is known about the metabolic fate of this class of
materials. Recently, a study of the anaerobic metabolism of a model alicyclic
hydrocarbon, ethylcyclopentane, revealed that it too was initially activated by
fumarate addition to form ethylcyclopentylsuccinic acid [25]. Wilkes et al. [84]
recently observed that when the denitrifying strain HxNl was incubated with
crude oil, a series of C4 to C8 -alkanes as well as cyclic alkanes, were activated
to their corresponding alkylsuccinates and methyl-branched fatty acids. Further,
cyclopentane, cyclohexane, and their methyl-and ethyl substituted congeners
were rapidly consumed in live incubations of sulfate-amended anoxic sediment
enrichments from a gas condensate-contaminated aquifer [26]. Though alicyclic
biodegradation was more extensive under sulfate-reducing conditions, there was
biodegradation of simpler alicyclic compounds under methanogenic conditions.
In parallel methanogenic incubations, 90% of cyclopentene and methylcyclopentene was lost in 100 days [26]. Thus, this class of materials is also
susceptible to methanogenic biodegradation.
290
291
(1)
292
the relatively higher bond dissociation energies, fumarate addition to the alkyl
side chain of an alkylated alicyclic hydrocarbon is therefore unexpected.
Table 1
Bond Dissociation Energies (AH298) at 298 K for various hydrocarbons in the reaction
RH -> R* + H* Bolded hydrogen atom represents abstracted hydrogen.
zl//2(kcal mol"1)
reference
[98]
[99]
[99]
[99]
[99]
[100]
[100]
[100]
[100]
95.6 +/- 1
93.7
93.7
[101]
[102]
[102]
112.9+/-0.5
89.8 +/- 0.6
85.4+/- 1.5
87.5
[103]
[104]
[105]
[106]
86.7
83.5
98.7
[107]
[107]
[107]
112.2+/-1.3
111.9+/-1.4
[108]
[108]
[105]
[107]
Alkanes
CH3-H (methane)
CH3CH2-H (ethane)
(CH3)2CH-H (propane)
CH3CH2CH2CH3 (H-butane)
(CH3)3C-H (wo-butane)
fl-CjHu-H (-pentane)
CH3CH2(CH2)2CH3 (-pentane)
-C6H]3-H (-hexane)
CH3CH2(CH2)3CH3 (-hexane)
Alicvclic Alkanes
CP-H (cyclopentane)
CPH(CH3) (methylcyclopentane)
CPH(CH2CH3) (ethylcyclopentane)
Alkyl Aromatics
C6H5-H (benzene)
C6H5CH2-H (toluene)
C6H5CH2 CH3 (ethylbenzene)
C6H5CH2 CH2CH3 (n-propylbenzene)
Y-C6H5CH(CH3)2
(zso-propylbenzene - substituted)
Y = 2,5 dimethyl
Y = 4-;-butyl
C6H5C(CH3)2CH2-H(?-butylbenzene)
Naphthalene-H
(Ci position)
(C2 position)
Naphthalene-CH2-H
(CH3 at Ci position)
(CH3 at C2 position)
293
294
295
yet been identified in oils, but they may be so polar that they primarily partition
to the aqueous phase.
296
297
298
299
to produce methane gas that could help decrease the viscosity of oil and aid in
further recovery. What if such an inoculation procedure resulted in at least some
fraction of the available energy being recovered as usable methane gas? Such
speculative technology is quite far from being addressed or realized, especially
from an economic point of view, but initial laboratory experimentation on this
topic has been promising (Fig. 1). Samples (10 g) taken from a field in Nowata,
OK that had undergone secondary oil recovery procedures (water flooding) were
used to test the importance of a methane-producing oil-degrading inoculum
enriched from a gas-condensate contaminated aquifer [9]. When residual oil
core samples were ground or broken into small portions, the oil-degrading
inoculum was effective in stimulating methanogenesis relative to a variety of
controls. The latter included a heat-inactivated preparation, an oil-unamended
control, and production water from the same field that received the inoculum (
Fig. 1).
Interestingly, the rate of methanogenesis was much greater with the
residual oil core samples than that observed when a standard oil or even when
the formation (Nowata) crude alone served as a substrate for the inoculum.
While the reasons for this result are under investigation, it is clear that such
inocula may play a potential role for the enhanced recovery of methane from oil
trapped in mature reservoirs.
Fig. 1. Methane production from residual oil in core samples inoculated with a methanogenic
bacterial enrichment capable of anaerobic hydrocarbon metabolism. Symbols: Oil unamended
control (); Nowata crude oil (); Production water (X); An artificially weathered Alaska
north slope oil standard (A); Crushed core (o); Pebbled core (). Heat inactivated and
uninoculated controls are not depicted, but were uniformly negative.
300
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307
Chapter 11
1. INTRODUCTION
The presence of hydrogen sulfide (H2S) in oil fields can be the result of abiotic
or biotic processes. In the later case, sulfate-reducing bacteria (SRB) are the
culprits that produce this nocuous gas, leading to "souring" that is defined as the
process whereby petroleum reservoirs experience an increase in the production
of H2S during the economic production life of the field [1]. The increase in H2S
content leads to a decrease in the economic value of the gas and oil, as well as
operational problems associated with the H2S.
This microbial process in wastewaters and oil field waters can be
controlled by another group of microbes, known as nitrate-reducing bacteria
(NRB). Their metabolic activities stop sulfate reduction by SRB, and in many
cases the NRB can actually consume sulfide, thus decreasing H2S concentration
in the waters. Jenneman et al. [2] have referred to these sulfide-consuming
bacteria as "sulfide bioscavengers". Hitzman and Sperl [3] used the term
"biocompetitive exclusion" to describe the microbial process in which NRB use
volatile fatty acids and out-complete SRB to prevent or decrease sulfide
production, and enhance oil recovery.
This chapter will review (a) H2S in the petroleum industry, (b) the
metabolism of SRB leading to sulfide production, (c) the occurrence, types and
activities of NRB that might be found in oil field waters, (d) some laboratory
studies that have elucidated the mechanisms by which NRB control sulfide
produced by SRB, (e) some oil field experiences with nitrate injection to control
sulfide in wastewaters, surface waters and oil field waters, and (f) some of the
U.S. patents that apply to this microbial process.
308
Although nitrite, rather than nitrate, addition has been studied, this chapter
focuses solely on the use of nitrate to control sulfide in oil field waters. This is a
proven biotechnology that is under-utilized by the petroleum industry.
2. H2S AND THE PETROLEUM INDUSTRY
2.1. Formation of H2S
Kerogen is the organic source material from which petroleum is formed
and released [4-5]. The formation of petroleum occurs in the deeper subsurfaces
as burial continues and temperature and pressure increase [5]. First oil, then gas
is expelled from kerogen as the maturation process continues. Significant oil
generation occurs between 60 and 120C, and significant gas generation occurs
between 120 and 2v25C [5]. During the maturation process, H2S is also
released.
Machel [6] wrote, "The association of dissolved sulfate and hydrocarbons
are thermodynamically unstable in virtually all diagenetic environments. Hence,
redox-reactions occur, whereby sulfate is reduced by hydrocarbons either
bacterially (bacterial sulfate reduction) or inorganically (thermochemical sulfate
reduction)." Temperature is the major factor determining which process occurs.
The microbiological process is common at temperatures for 0 to 60 or 80C,
whereas, the thermochemical process occurs at temperatures greater that 100 to
140C [6]. Because temperature increases with burial depth, H2S found at
shallow depths is usually the result of bacterial sulfate reduction whereas, H2S
found at greater depths is the result of thermochemical sulfate reduction [7].
However, there are shallow pools that contain higher than expected
concentrations of thermochemically generated sulfide [8]. These are believed to
be the result of thermochemical sulfate reduction occurring downdip and
migrating upward to a shallow reservoir [8].
At the time of discovery, the H2S concentration in an oil field depends
upon its maturation history and/or the migration of H2S into the oil field.
However, during oil recovery from some oil fields, an increase in H2S
concentration (souring) can occur as a result of pressurizing the formation by
injecting water into the reservoir. This process, know as waterfiooding, is
discussed in section 3. Three well-documented examples of oil field souring are
given in the following paragraphs.
Cochrane et al. [9] describe the souring of the Ninian field in the North
Sea. This field was discovered in 1974, and after several years of operation,
injection of sea water was used to maintain the production rate. This was
followed by an increase in sulfide production attributed to bacterial sulfate
reduction. The reservoir temperature was initially between 100 to 120C, but in
the areas adjacent to the injection well bores, the temperature was cooled to as
low as 40C, which was conducive to bacterial sulfate reduction.
309
Frazer and Boiling [10] described the souring of the Kuparuk River field
on the North Slope of Alaska. The field was initially sweet, but after injection of
Beaufort Sea water, detectable levels of H2S began to appear at the producing
wells. The connate water contained essentially no sulfate. However, the sulfate
in the sea water stimulated bacterial sulfate reduction in the reservoir that had a
temperature of about 70C.
The Skjold oil field in the North Sea soured upon the onset of
waterflooding [11]. Oil and gas production began from this field in 1982 and sea
water injection began in April 1985. In September 1985, the first recorded H2S
production was measured to be 1.8 ppm in the gas phase. In 2002, the
concentrations varied from 10 to 1000 ppm [11]. In late 1999, this field
produced 1150 kg H2S d"1.
These examples clearly demonstrate that waterflooding can stimulate
bacterial sulfate reduction, leading to souring. Although these examples refer to
offshore oil fields, souring also occurs in land-based oil fields using
waterflooding [12-15]. As a result of the bacterial production of toxic H2S, the
value of the oil decreases as the oil field sours.
2.2. H2S toxicity and properties
H2S is a very dangerous gas, even though it occurs in nature. Its
characteristic rotten egg smell is generally obvious at 0.13 ppm by volume and
quite noticeable at 4.6 ppm [16]. Unfortunately the smell sense becomes quickly
fatigued and can fail to warn of higher concentrations. Collapse, coma and death
from respiratory failure may occur within a few seconds after one or two
inspirations of the undiluted H2S [17]. The U.S. Occupational Safety and Health
Administration has established the acceptable ceiling concentration of 20 ppm
(by volume) for H2S with an acceptable maximum peak above the acceptable
ceiling concentration of 50 ppm for an 8-h shift [16].
The specific gravity of H2S is 1.19; therefore it will collect in low places
and accumulate under poorly ventilated conditions [18]. H2S is soluble in water
and oil. It is a weak acid existing in aqueous solutions as H2S, HS~, or S~ (pKa
values of 7.04 and 11.96). Aqueous solutions of H2S absorb O2 leading to the
formation of elemental sulfur [17].
2.3. Detrimental effects of H2S
Besides its toxicity, H2S is a nuisance in the petroleum industry because it
contaminates gas and stored oil, it corrodes iron in the absence of air (anaerobic
corrosion), and it precipitates as amorphous ferrous sulfide (FeS), plugging and
diminishing the injectivity of water injection wells [18]. In addition, fluids with
water and H2S, may cause sulfide stress cracking of susceptible metals. This is
affected by metal composition, pH, H2S concentration, total pressure, total
tensile stress, temperature and time [19].
310
Fig. 1. Iron metal corrosion mediated by SRB in a biofilm. The process is caused by the
consumption of H2 causing cathodic depolarization. Adapted from Ref. [18].
311
Removal of dissolved gases (O2, H2S and CO2) from drilling and
produced fluids is necessary to minimize corrosion damage. H2S in oil base
drilling fluid is removed by gas separators and vacuum degassers, and then
neutralized. Controlling corrosion in H2S-containing environments requires
proper selection of materials, including the use of low-hardness steels,
application of inhibitors and complete exclusion and removal of O2 from water
used in petroleum production [21]. Clearly, the presence of H2S greatly
increases the cost of exploration for oil and natural gas, and the cost of
production and storage of petroleum.
Plugging (or biofouling) of injection wells is also caused by SRB. The
sulfide they produce, precipitates soluble iron in the injection or formation water
forming colloidal FeS [23]. This colloidal material becomes associated with
bacterial cells and oil, forming a gummy mass that can clog reservoirs and plug
injection wells. The activities of SRB can also produce calcite (CaCO3) that can
add to the plugging problem.
3. OIL RECOVERY AND WATERFLOODING
Under primary oil recovery, typically less than 30% of the original oil is
produced, so that improved or enhanced methods are used to recover some of
the remaining oil [24]. These processes, known as secondary and tertiary
recovery methods, include the addition of energy into the reservoir and are
accomplished by injecting some type of fluid through injection wells. This is
referred to as enhanced oil recovery and involves water injection, gas injection,
steam injection, combustion, miscible fluid displacement and polymer injection
[24]. In this paper, only water injection or waterflooding will be discussed.
Waterflooding involves pumping water into the reservoir to stimulate
production. The injected water provides pressure to force the oil out of the rock
and to sweep it toward producing wells as shown in Fig. 2. Waterflooding has
been attempted in almost every type of reservoir, with its greatest success in
relatively homogenous reservoirs having sufficient permeability to allow water
injection at a reasonable rate [24]. Up to 60% of the oil can be recovered with
waterflooding [5]. Water handling can become a major operational procedure.
For example, in some western Canadian oil fields, the proportion of water in the
oil-water emulsion brought to the surface can be 95% by volume [15]. That is,
the volume of water handled is 19 times greater than the volume of oil produced.
Water used as injection water can be of three types: formation water, sea
water or fresh water. Formation water is subsurface brackish or brine water
produced from a petroleum or non-petroleum producing formation. Sea water
may also include water from a salty (non-potable) lake. Fresh water, containing
312
less than 2000 ppm dissolved solids, is primarily water that can be made potable
by flocculation, filtration and chlorination [25].
Because oil field reservoir rocks are porous, they are susceptible to plugging by
solids suspended in or precipitated from an injection fluid [26]. This makes
water quality testing necessary to determine parameters such as: amount and
composition of suspended solids, clay sensitivities, presence of bacteria,
compatibility of two or more waters, and compatibility of the injection solution
with reservoir rock. An example of incompatible waters occurs when sulfate
scales, such as barium sulfate, calcium sulfate or strontium sulfate are formed by
mixing waters containing sulfate with waters containing barium, calcium or
strontium ions [26]. As well, the gases O2, H2S and CO2 found in injection
waters and implicated in corrosion [25-26], must be monitored. Water quality
testing, should be continued after the enhanced oil recovery operation hasstarted,
to ensure that the system is maintained at optimum conditions [25]. Water
treatment methods are outlined by Rose et al. [27].
Fig. 2. A simple waterflooding operation. Oil, gas and water are collected from the production
wells and the produced water is separated from the oil and gas. The produced water is
combined with source water and injected into the oil-bearing rock to pressurize the formation
and sweep the oil to the producing wells.
313
(1)
314
(2)
As late as the 1970's, only a few genera of SRB were recognized, and
these were known to use only a few growth substrates, most notably lactate,
pyruvate or H2. Now it is apparent that SRB are capable of using various
compounds for electron donors.
Based on their metabolic capabilities, heterotrophic SRB fall into two
groups: those that cannot oxidize acetate, and those that carry out complete
oxidation of acetate to C0 2 [36]. Reaction (3) illustrates the overall reaction of
lactate-utilizing SRB that cannot oxidize acetate. One mol of acetate
accumulates for each mol of lactate that is consumed.
2CH3CHOHCOO" + S04 = + 2H+ -> 2CH3COO" + 2H2O + 2CO2 + H2S
G' = -77 kJ (mol lactate)"1
(3)
The complete oxidation of acetate is given by reaction (4), showing that less
energy is available per mol of acetate than per mol of lactate (reaction 3).
CH3COO" + SOzf + 3H+ - 2CO2 + H2S + 2H2O
G' = -41 kJ (mol acetate)"1
(4)
315
316
317
318
reduce nitrate to ammonium [53-56]. In the presence of nitrate, some SRB will
preferentially use nitrate, and some will use both concomitantly [54].
Thiobacillus denitrificans is listed as one of the chemolithotrophs in
Fig. 4. In general, this species is not tolerant to high sulfide concentrations, but
Sublette and Woolsey [57] enriched Thiobacillus denitrificans strain F that
initially tolerated up to 1.75 mM sulfide, and later up to 2.5 mM sulfide [58].
This strain has been used in studies to demonstrate its ability to reduce H2S
concentrations in porous rock cores [59-60] and in sour produced waters
[58,61].
Gevertz et al. [62] described two novel bacterial isolates that are obligate
chemolithotrophs, using nitrate as a terminal electron acceptor, and sulfide as an
energy source. Both grow under anaerobic conditions. One isolate is a denitrifier
that closely resembles Thiomicrospria denitrificans, and it has been called
Thiomicrospria strain CVO (Fig. 4). The other isolate was called Arcobacter
strain FWKO B, and it reduces nitrate to nitrite.
Fig. 3. Examples of some heterotrophic bacteria that could be stimulated by the presence of
nitrate in anaerobic environments that contain suitable organic substrates.
319
Fig. 4. Examples of some chemolithotrophic bacteria that could be stimulated by the presence
of nitrate in anaerobic environments. See text for details.
Injection of nitrate into an oil field might also stimulate the activity of
bacteria similar to P. pantotrophus [63] (formerly Paracoccus denitrificans [64]
and Thiosphaera pantotropha strain GB17 [65]). This bacterium was isolated
from a denitrifying effluent treatment system. It is a facultative anaerobe and
facultative autotroph (Fig. 4) that uses nitrate as an electron acceptor. It grows
autotrophically with sulfide as an electron donor, or heterotrophically with a
variety of organic compounds (including acetate which is commonly found in
produced waters [66-67]) as electron donors [65]. We are not aware of any
research that has detected facultative chemolithotrophs in oil field waters.
The bacteria shown in Fig. 4 all have the capability of oxidizing sulfide
while reducing nitrate. These are referred to as nitrate-reducing, sulfideoxidizing bacteria (NR-SOB). Greene et al. [68] compared the sulfide tolerance
of four species of NR-SOB. In their liquid medium, sulfide was oxidized by
Thiobacillus denitrificans strain F at concentrations less than 0.5 mM, by
Thiomicrospira denitrificans and Arcobacter sp. strain FWKO B at up to 3 mM,
and by Thiomicrospira strain CVO at up to 15 mM.
320
Although only a few NR-SOB have been identified in oil field waters,
Loka Bharathi et al. [69] isolated over 100 strains of anaerobic colorless NRSOB from sea water and a sulfide-rich creek. Their data showed that different
isolates oxidized sulfide at different rates. For example, one isolate oxidized all
of the sulfide in the medium within 9 days, whereas another isolate oxidized
only 2.9% of the sulfide in the same time. Thus, it is likely that different NRSOB in the produced water from oil fields would oxidize sulfide at different
rates.
5.2. NRB in oil field waters
The presence of NRB in oil field waters has not be studied extensively.
This group of microorganisms was not even mentioned in a review entitled
"Microbiology of petroleum reservoirs" [46]. Several investigations have
enumerated NRB in oil field waters using most probable number (MPN)
methods with different media formulations. Some of the results are summarized
in Table 1, in chronological order. One of the first enumeration studies [70] used
molasses or sucrose as electron donors in the media to count heterotrophic NRB
in samples taken as near the wellheads as possible. Very low numbers ( 4 L"1)
were found in these samples.
Most of the other media formulations preferentially, but not exclusively,
cultured autotrophs. For example, the medium used by Davidova et al. [14]
(Table 1) contained only inorganic compounds except for yeast extract, with
thiosulfate serving as the electron donor. This would preferentially grow
microorganisms that are similar to Thiobacillus denitrificans. Other
investigations in Table 1 used sulfide as the electron donor with filter-sterilized
produced water from the oil field that was being studied [51, 71]. The filtered
produced water undoubtedly contained some dissolved organic compounds, so it
would support the growth of heterotrophic NRB and autotrophic NRB. The
medium used by Telang et al. [72] in Table 1, contained only inorganic
compounds except for acetate, with sulfide serving as the electron donor.
Telang et al. [72] in Table 1 described the isolation and characterization
of two autotrophic NR-SOB from an oil field in Saskatchewan, Canada. One
was designated Thiomicrospira strain CVO (formerly Campylobacter strain
CVO, [51 ]) and the other was designated Arcobacter strain FWKO B. The DNA
from these two isolates has been used extensively with a method known as
reverse sample genome probing (RSGP), first described by Voordouw et al.
[73]. Using RSGP, Telang et al. [51] (Table 1), demonstrated that the abundance
of strain CVO increased after the waterflooded oil field was treated with nitrate.
This molecular technique corroborated the increase in NR-SOB numbers
determined by the MPN method. The high specificity of the RSGP for NR-SOB
precluded the detection of other NRB in samples from four additional oil fields
321
from western Canada and west Texas [72], although culture methods detected
NRB (Table 1).
Eckford et al. [74], in Table 1, surveyed five oil fields in western Canada
for various types of NRB. Different media formulations were used to selectively
enumerate thiosulfate-oxidizing NRB, heterotrophic NRB, or NR-SOB. None of
the 18 water samples contained detectable numbers of thiosulfate-oxidizing
NRB. As was observed by Adkins et al. [70], the numbers of NRB were very
low or non-detectable near the wellheads [74]. However, NRB were detected in
source and preinjection waters, and in samples from water storage tanks and free
water knock out units. Although much of the work on NRB in oil field waters
has neglected the heterotrophic NRB, the numbers of heterotrophic NRB were
greater than the numbers of autotrophic NRB in 12 of the 15 samples compared.
In one oil field, heterotrophic NRB were found, but no autotrophic NRB were
detected (Ref. 74, Table 1).
NRB were detected in biofilms on coupons in the anaerobic part of the
water injection system of the Veslefrikk field in the North Sea [75], (Table 1).
The medium used to enumerate these attached bacteria contained organic acids
as carbon sources, providing counts of heterotrophic NRB. These numbers
increased dramatically after nitrate injection (Table 1).
The literature surveyed in Table 1 represents 15 different oil fields that
have been examined for NRB. Each of the oil fields contained detectable
numbers of NRB at one or more sampling locations. Thus, each field had a
microbial community containing NRB with the potential to be stimulated by
nitrate amendment.
6. CONTROLLING MICROBIAL PRODUCTION OF SULFIDE WITH
NITRATE ADDITION
6.1. Microbial mechanisms leading to the control of sulfide concentrations
after nitrate addition
There appear to be five mechanisms by which sulfide concentrations can
be controlled in the presence of nitrate and sulfate. The first involves the
competition between heterotrophic NRB and SRB for a common electron donor.
For example, acetate serves as an electron donor for NRB [76] and for several
genera of SRB [34]. Equations (5) and (6) illustrate that if acetate is available,
nitrate reduction yields more energy per mol of electron donor or acceptor than
does sulfate reduction [77].
322
Table 1
Detection and enumeration of NRB in oil field waters.
Refs.
Oil
70
fields
Methods
Comments
Oklahoma,
USA
71
Saskatchewan,
Canada
Single-bottle MPN
using filter-sterilized
oil field water
supplemented with
nitrate
51
Saskatchewan,
Canada
Single-bottle MPN
using filter-sterilized
oil field water
supplemented with
nitrate
51
Saskatchewan,
Canada
RSGP
NR-SOB
strain
CVO became
dominant community member after
nitrate injection into reservoir.
72
72
14
Oklahoma,
USA and
Alberta, Canada
Method likely selected for thiosulfateoxidizing NRB, but may have grown
heterotrophic NRB. Counts were
typically <500 mL"1.
74
Alberta and
Saskatchewan,
Canada
No
thiosulfate-oxidizing
NRB
detected in any of 18 samples. Other
NRB detected in 16 samples. Number
of heterotrophic NRB greater than
number of NR-SOB in 12 of 15
samples.
75
Veslefrikk in
the North Sea
323
324
325
Table 2
Laboratory and field studies using nitrate to control sulfide production in wastewaters
Ref.
Summary
83
Three pulp mills discharged sulfite wastes into the Androscoggin River in Maine
U.S.A. This resulted in H2S production in the river and odor problems in nearby
towns. In 1949, a total 641 tons (582 Mg) of NaNC>3 were added to the river. This
controlled H2S production and odors. Most of the nitrate was reduced to ammonium.
84
To control odor, waste sodium nitrate liquor (containing both nitrate and nitrite) was
added to a storage lagoon that held aerobically digested waste activated sludge.
Initially, the redox potential of the water was near -lOOmv, but after several months of
nitrate addition, it rose to near +300 mV. There was low odor potential when the
redox was above +100. Acetate concentrations decrease in the lagoon, and N2
production from denitrification provided mixing within the sludge.
78
Laboratory studies were done with a 10-fold dilution of sewage sludge amended with
20 mM sulfate and one of three electron donors: glucose, acetate, or H2. The addition
of 59 mM nitrate completely inhibited sulfide production. Nitrate, nitrite and N2O
were detected in the inhibited samples, and the oxidation of the redox indicator,
resazurin, was attributed to the presence of N2O. The numbers of SRB decreased with
prolonged incubation of the oxidized medium.
85
Oily sludge from a settling tank at the U.S. Navy Craney Island Fuel Depot in
Virginia was placed in serum bottles and amended with nitrate, stimulating indigenous
NRB. Sulfate reduction was diminished with 50 mM nitrate, and sulfide accumulation
was prevented with as little as 16 mM nitrate. Nitrite and nitrous oxide were products
of nitrate reduction. Sulfide was oxidized to sulfur or sulfate. The results indicated
that nitrate would be useful for preventing sulfide formation in oily wastes produced
onboard marine vessels.
58
This paper reviewed bench-scale processes developed for the sulfide removal from
gases and aqueous solutions by Thiobacillus denitrificans. When H2S was introduced
to batch anoxic or aerobic cultures of T. denitrificans, the H2S was immediately
metabolized. Oxidation of H2S to sulfate was accompanied by growth. T. denitrificans
was immobilized by co-culture with floc-forming heterotrophs and this mixture was
used to treat water that was contaminated with sulfide. The sulfide-active floe was
stable for 5 months of operation with no external organic carbon required to support
the growth of the heterotrophs. T. denitrificans strain F, which tolerates higher sulfide
concentrations, was also used in some studies.
326
Table 3
Laboratory studies using nitrate to control sulfide production columns or cores
Ref.
Summary
59
This study investigated the efficacy of nitrate and the sulfide-tolerant Thiobacillus
denitrificans strain F in controlling H2S concentrations in cores of sandstone.
Formation water from a gas storage facility in Redfield, Iowa, U.S.A. was injected
into two core systems, with hydraulic retention times (HRTs) of 3.2 h and 16.7 h.
With the addition of nitrate alone, no thiobacilli were cultured from the core system,
but nitrate was consumed and the concentrations of sulfide in effluent decreased by
about 40% in the core with the shorter HRT, and 98% with the longer HRT. Thus, an
indigenous microbial community capable of oxidizing sulfide while using nitrate as
the electron acceptor was present. Inoculation with strain F reduced the effluent
sulfide by about 80% in the core with the shorter HRT.
60
The test materials for this study included core material from the St. Peter formation at
Redfield, Iowa, U.S.A. and water from the same formation, supplemented with
acetate and enriched with SRB to 107 cells ml/ 1 . The core material did not contain
large numbers of organisms capable of using nitrate, and no strain F-like organisms
were detected. When nitrate and strain F were injected into the core, sulfide
concentrations decreased, demonstrating the ability of strain F to control sulfide in
the core.
86
Brine from an oil field near Coleville, Saskatchewan, Canada was filtered,
supplemented with phosphate and nitrate and pumped into a porous (1288 mD)
ceramic core 19.1 cm long. When 5 mM nitrate was shut in the column, all of the
sulfide was removed in 3 d and the numbers of NRB increased. Under various flow
regimes, with sulfide-containing brine, sulfide removal was between 87 and 100%.
Elemental sulfur, bacteria and CaCC>3 were produced, but there was no significant
permeability changes across the core following all treatments.
49
327
Four of the five studies in Table 3 detected NRB in the cores or produced
waters used in the experimental systems. In the fifth study, [49] the investigators
inoculated the column with a mixture of enrichment cultures, including NRB.
Two of the studies, Refs. 59 and 60, focused on the activities of thiobacilli.
None were detected in the cores or waters, similar to the findings of Eckford and
Fedorak [74]. Inoculating these two cores with Thiobacillus denitrificans strain
F stimulated sulfide reduction when nitrate was injected into the cores (Refs. 5960, Table 3).
Two of the studies [2, 86], (Table 3) relied solely on the formation water
as the source of NRB. One study supplemented the medium with short-chain
organic acids [86], whereas the other study did not supplement with organic
compounds [2]. Thus, these studies likely enriched for different nutritional types
of NRB. Nonetheless, souring was inhibited in both studies. Indeed, sulfide
production was controlled in each of the five studies summarized in Table 3.
6.4. Laboratory studies using natural microbial communities in produced
waters
Produced waters from various oil fields have been used as sources of
planktonic microorganisms in studies of the ability of nitrate to control sulfide
formation in these waters. Table 4 summarizes four of these investigations in
chronological order. In each study, sulfide removal was stimulated by nitrate
addition. In three of the reports, no organic supplementation was required to
stimulate sulfide removal. However in one case [87], two of the four oil field
waters did not respond to amendments with inorganic nutrients (nitrate and
phosphate). Sulfide removal was only stimulated after the addition of acetate or
formate plus vitamins or yeast extract, indicating that in some cases
heterotrophic NRB play an important role in the process of sulfide removal.
Eckford and Fedorak [15] demonstrated that heterotrophic NRB can be
stimulated by simply adding nitrate. This is illustrated in Figs. 5 and 6. A
produced water sample was collected from the free water knock out at the
Coleville field that has a severe souring problem. This water was used for a
serum-bottle microcosm study. Initially, the microcosm contained 2.7 mM
sulfide which increased to 3.1 mM by day 1 and then dropped below detection
by day 3 in the nitrate-amended microcosm (Fig. 5a). The sulfate concentration
increased noticeably over the first 14 d of incubation, with a total increase of 3.5
mM by day 38, closely matching the 3.1 mM decrease in sulfide. The nitrite
concentration was at a maximum of 1.8 mM on day 3 and then gradually
decreased to 0.2 mM by day 38. Figure 5b shows the results of chemical
analyses of a microcosm that was not supplemented with nitrate. The sulfide
increased to 4 mM by day 5, and the sulfate remained fairly steady at from 0.68
mM to 0.54 mM throughout the testing period. Neither nitrate nor nitrite was
detected in the microcosms. The huge increase in numbers of heterotrophic NRB
328
(Fig. 6a) during the time that sulfide was removed (Fig. 5a) suggests that these
bacteria play a role in this process. However, their role has not be elucidated.
Table 4
Laboratory studies on controlling sulfide production in produced waters by adding nitrate to
stimulate natural microbial communities.
Ref.
Summary
71
&
2
87
Waters from four west Texas oil fields were used to determine which amendments
were required to stimulate sulfide removal. In two of the samples, addition of 40
mM nitrate and phosphate was not sufficient to promote microbial removal of
sulfide over a 28-d incubation. However, sulfide removal was observed when
acetate or formate plus vitamins or yeast extract were added to these two waters that
had been supplemented with nitrate and phosphate. These results illustrate the
importance of heterotrophic activity in sulfide removal.
14
Two waterflooded, souring oil fields in Oklahoma, U.S.A. and Alberta, Canada were
studied. SRB and NRB were found in produced waters from both oil fields. The
majority of the sulfide production appeared to occur after the oil was pumped
aboveground, rather than in the reservoir. Sulfide production was greatest in the
water storage tanks in the Alberta field. Laboratory experiments showed that adding
5 and 10 mM nitrate to produced waters from the Oklahoma and Alberta oil fields,
respectively, decreased the sulfide content to negligible levels and increased the
numbers of NRB.
15
Produced waters from three sulfide-containing western Canadian oil fields were
amended with nitrate only. In less than 4 d, the sulfide was removed from the waters
from two of the oil fields (designated P and C), whereas nearly 27 d were required
for sulfide removal from the water from the third oil field (designated N). Nitrate
stimulated large increases in the numbers of the heterotrophic NRB and NR-SOB in
the waters from oil fields P and C, but only the NR-SOB were stimulated in the
water from oil field N. These data suggest that the stimulation of the heterotrophic
NRB is required for rapid removal of sulfide from some oil field produced waters.
329
Fig. 5. Chemical analyses of microcosms that contained produced water from the Coleville oil
field in Canada. Nitrate amended (a), unamended (b). From Ref. 15.
330
incubation, the heterotrophic NRB numbers remained high, whereas the NRSOB numbers dropped to near their original count (Figs. 6a and 6b). The SRB
numbers did not change in the nitrate-amended microcosm and showed a slight
increase in the unamended microcosm with a maximum at day 7 (Fig. 6c).
Fig. 6. Heterotrophic NRB (a), NR-SOB (b) and SRB (c) counts is samples from microcosms
that contained produced water from the Coleville oil field in Canada (Fig. 5). Error bars show
95% confidence intervals. From Ref. [15].
331
332
Table 5
Laboratory studies using co-cultures and nitrate to control sulfide production.
Ref.
Summary
72
Mixtures of strains CVO and FWKO B were incubated in medium with different
concentration of sulfide. Using RSGP, it was demonstrated that CVO dominated in
co-cultures with low (1 mM) sulfide, but FWKO B dominated with high (15 mM)
sulfide. CVO or FWKO B were co-cultured with Desulfovibrio strain Lac6. Sulfide
drop from 1 mM to 0 mM in 24 h in the presence of CVO. Over a 277-h incubation,
sulfide remained between 1 and 2 mM in the presence of FWKO B.
91
Strain CVO was added to cultures of Desulfovibrio strain Lac6 that were growing in
various concentrations of nitrate or lactate. In pure culture, sulfate reduction by the
Desulfovibrio sp. was unaffected by the nitrate concentrations up to 10 mM. Sulfide
concentrations decreased rapidly after the addition of CVO. This effect was due to the
increase in the redox potential of the medium, as indicated by the oxidation of
resazurin.
89
68
Strain CVO was grown in co-cultures with four different Desulfovibrio strains. Two
of these did not have nitrite reductase, and their growth was stopped in the presence of
CVO as it produced nitrite and elevated the redox potential of the medium. However,
two of the strains had nitrite reductase, and they reduced the nitrite formed by strain
CVO. The SRB decreased the redox potential and continued to produce sulfide. This
illustrated that the action of strain CVO cannot inhibit SRB that possess nitrite
reductase.
333
334
Table 6
Field studies and operations using nitrate to control sulfide production.
Ref.
Summary
12
Ammonium nitrate (45 T) was injected into a souring oil field at the Southeast Vassar Verta
Sand Unit in Oklahoma, U.S.A. At the time of injection, no nitrate was detected in three
adjacent production wells. Forty-five days after injection, nitrate was detected at these wells,
and the sulfide concentrations were reduced by 40 to 60%.
71
In 1994, a solution of NH4NO3 and NaH2PO4 was injected into three wells in the Coleville field
in Saskatchewan, Canada. Prior to treatment, the produced waters from these wells contained
between 52 and 160 mg sulfide L"1. After injection, there were shut-in periods of between 24
and 70 h before pumping resumed. The sulfide concentrations dropped by as much as 98% of
the initial concentrations, with ranges between 40% and 60% being sustained for several hours.
The numbers of NRB increased by 100- to 10,000-fold.
88
&
13
In 1996, a solution of NH4NO3 and NaH2PO4 was injected into two injection wells in the
Coleville field for 50 d. Two producer wells were monitored for 90 d after the injection began.
After 10 d, the sulfide in the producers decreased by as much as 50 to 60% of the initial
concentrations of 60 and 40 mg L"1. The cumulative sulfide removal from the two producers
were estimated to be 50 and 70 kg over the 90-d test period. The numbers of NRB increased at
least 1,000-fold during the time of nitrate injection.
51
Samples were taken from the Coleville field in 1996. These were taken 8 d before and 20, 55,
and 82 d after the injection of a solution of NH4NO3 and NaH2PO4 began. RSGP analyses,
using 47 DNA standards, showed that strain CVO became the dominant community member
immediately after injection. The abundance of CVO decreased within 30 d after completion of
nitrate injection.
11
Studies were done in the Skjold oil field in the North Sea in 2000. Three injection strategies
were used. In each case, the highest nitrate concentrations were used at the beginning of the
treatment, then the concentration was decreased. First, nitrate (4.5 to 1.7 mM) was injected into
one well for 1 month; second, nitrate (3.8 to 1.8 mM) was injected into this well plus another
well for 2 months; third, nitrate (4.4 mM to a mean of 2.8 mM) was injected into all of the
other wells for 3 months. Only one of the monitored production wells showed marked
reduction in H2S. This well was in the highly fractured zone of the reservoir, and nitrate
reached it within 24 h of the start of injection. The amount of H2S in the produced gas dropped
from 240 ppm to between 30 to 60 ppm. After nitrate addition, the numbers of mesophilic NRB
and NR-SOB increased about 10,000- and 1,000-fold, respectively.
75
Data were presented after 32 months of adding nitrate to water injected from the Veslefrikk
platform in the North Sea. Glutaraldehyde injection was stopped in January 1999, and replaced
by continuous 0.25 mM nitrate injection. Microbial counts in biofilms were monitored and
corrosion was measured by weight loss from C-steel biocoupons. After 32 months, the
numbers of SRB decreased 20,000-fold and after 18 months, the number of NRB increased
60,000-fold. Most of the NRB were heterotrophic facultative anaerobes. Sulfate-reducing
activity (measured using 35S-sulfate) decrease 50-fold. Prior to nitrate treatment, the corrosion
rate was 0.7 mm y"1. This fell to 0.02 mm y"1 after 4 months of nitrate injection.
335
Table 7
Examples of United States patents for the control of sulfide through the application of NRB.
Patent no.
Inventors and
year
Title
4,879,240
Sublette et al.
1989
4,880,542
Sublette
1989
5,405,531
Hitzman et al.
1995
5,686,293
Jenneman et al.
1997
Sulfide-oxidizing bacteria
5,750,392
Hitzman et al.
1998
5,789,236
Jenneman
1998
336
for offshore oil fields. The costs did not include the cost of transporting the
chemicals. The estimated prices per litre of the chemicals were: US$0.25 for
nitrate (as a 40% solution of CaNO3), $2.50 for glutaraldehyde (as a 50%
solution), and $4.00 for THPS (as a 50% solution). Although the cost of nitrate
was lower, the solution was continuously injected at a dose of 60 mg L"1. In
contrast, the two biocides were injected for 1 h, twice per week at a dose of
500 mg L"1. Based on treating 200,000 barrels of produced water per d, the
yearly costs for chemicals were US$575,000 for nitrate, $345,00 for
glutaraldehyde, and $500,000 for THPS. Per 100 barrel of water treated, these
costs become US$0.79, and $0.47, and $0.68, respectively.
From these two cost analyses, the use of nitrate for sulfide control is
competitive with other chemicals. The cost of treating 100 barrels of water
calculated from the data given by Jenneman et al. [88] is higher than that
reported by Herbert [92], because Jenneman et al. [88] also injected
monosodium phosphate, which is 8 times as expensive as the ammonium nitrate.
Herbert [92] used only calcium nitrate. The need to add a phosphate source to
stimulate NRB would have to be evaluated for each oil field.
Besides the cost, other factors must be considered when choosing
chemicals for controlling sulfide in produced waters. Most notably, workers
safety and potential environmental impact of spilled chemical must be
considered. Nitrate salts are far less toxic than the biocides commonly used in
oil fields, and therefore its use presents few safety issues for oil field workers.
Spilled biocides have negative affects on the environment. In contrast, nitrate is
widely used as an agricultural fertilizer, so spills on land present no major
problem. Nitrate is listed as a substance that poses little or no risk to the marine
environment [75]. However, caution must be used to avoid contamination of
fresh surface waters or potable ground waters with nitrate (or any biocide).
7. CONCLUDING REMARKS
The use of nitrate to control microbially-produced sulfide in oil fields is a
proven biotechnology that is grossly under-used by the petroleum industry. Its
effectiveness has been demonstrated in many laboratory investigations and in
some field studies. The microbiology is adequately well-understood, although it
is not clear whether heterotrophic or autotrophic NRB play the more important
role. This may vary from oil field to oil field. Nonetheless, from the results in
the literature, nitrate amendment (and in some cases phosphate or organic acid
amendment) stimulates NRB in the oil field waters, and there appears to be little
need to add an inoculum of NRB.
Nitrate has replaced biocides in some of the oil fields in the North Sea,
and the results have been very positive. Besides controlling sulfide levels, there
is also preliminary evidence that corrosion rates are reduced [75]. In addition,
337
there are plans to use nitrate in the Gulf of Mexico when sea water injection
begins in the near future (Stephen Maxwell, Commercial Microbiology Inc.,
personal communication). In contrast, there is little or no use of nitrate in landbased souring oil fields in North America. It is now very clear that land-based
oil field operators should seriously consider using this proven biotechnology to
control, and possibly eliminate, microbially-induced souring and the problems
associated with H2S formation.
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Chapter 12
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any aromatic substrate, the xylS gene is expressed at low levels from the a70dependent promoter Ps2, ensuring the presence of basal levels of XylS protein.
This protein as such is not able to activate transcription. When a substrate of the
meta pathway, e.g. 3-methylbenzoate, is present in the growth medium, the XylS
protein interacts with it and becomes active to promote transcription from the
Pm promoter, which controls expression of the meta pathway. Expression from
Pm requires RNA polymerase with either a32 in the early exponential phase or
a38 thereafter. The XylR protein, which regulates its own transcription from two
o70-dependent promoters, is synthesized in sufficient amounts under all growth
conditions. When a substrate of the upper pathway, e.g. toluene, is present in the
culture medium, the binding of this effector to the protein triggers a series of
molecular events that result in the activation of transcription from two o5 dependent promoters: Psl for the xylS gene, and Pu, which drives expression of
the upper pathway. This latter activation requires the integration host factor
(IHF). As a consequence of Psl activation, the XylS protein is overproduced,
and even in the absence of a meta pathway effector, transcription from Pm
occurs. The current knowledge of the molecular biology of each step on the
regulatory pathway is reviewed in detail below.
Fig. 2. The TOL pathway regulatory network. Elliptical boxes indicate the inactive form of
the regulatory proteins. Shaded square boxes indicate the active form of the regulatory
proteins. Lines represent the connections between regulatory proteins and promoters, where
(+) is activation of transcription and (-) is inhibition of transcription; GR, global regulation.
The dotted line indicates transcription activation of overproduced XylS in the absence of
effector. The sigma factor(s) involved in transcription initiation are indicated above each
promoter. Aromatic substrates of the pathways that act as effectors of the regulatory proteins
are indicated. The regulatory circuits are explained in the text.
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348
transition from the inactive to the active form may be mediated by effector
binding. Recent studies further pinpointed residues Asp 137 and His 153 as
crucial for interactions with the effector molecule [50]. In addition to
influencing effector specificity, these two residues were shown to contact
specific residues in the RNA polymerase a subunit carboxy terminal domain (aCTD) [50] .
XylS mutants such as XylSArg41Cys, XylSPro37Gly XylSGly44Ser,
XylSSer229Ile, XylSAsp274Val, and XylSAsp274Glu mediated transcription
from Pm in the absence of effectors [46, 47]. These results support the
hypothesis that XylS exists in vivo in a dynamic equilibrium between an inactive
and an active form, with respect to transcriptional stimulation. Within the
family, some regulators such as MarA are present in solution as monomers,
whereas most of the members of the family are found as dimers [51-54]. XylS is
likely active as a dimer and in vivo and in vitro assays have shown that Leu 193
and Leul94 in XylS play a crucial role in dimerization [55].
It is predicted that the DNA binding domain of XylS consists of seven ahelix units which fold to assemble two helix-turn-helix (HTH) motifs that
interact with two neighboring major grooves on one face of the target DNA.
Involvement of the XylS C-terminal domain in DNA binding was first predicted
after the finding of mutations in this domain that rendered mutant regulators able
to promote high transcription levels in the absence of effectors [47, 48].
Mutation analysis of the predicted conserved positions of the HTH motifs of
XylS showed that the most conserved positions in the family seem to be
essential to preserve the structure of this domain [56]. Deletion of the 209 Nterminal residues of XylS rendered a C-terminal domain-protein able to bind Pm
promoter and, when overproduced, able to activate transcription in vivo to levels
similar to those in the wild type protein. However, activity was clearly reduced
when the C-terminal fragment was synthesized at physiological levels. As
expected, the truncated protein was not responsive to effector-mediated control
[57].
2.2.2. The Pm promoter
XylS-mediated transcription activation from Pm requires a DNA fragment
extending to position -70 upstream from the transcription start site. The DNA in
this region exhibits a 40 bend centered between positions -41 and -46 [58]. The
XylS binding site in the Pm promoter was first defined through site-directed
mutagenesis [59-63] and further confirmed by in vitro and in vivo footprint
assays [60, 64, 65]. The XylS binding site in Pm consists of two directed repeats
(5'-TGCAN6GGNTA-3') spanning positions -34 to -68, and overlapping the
RNA polymerase biding site by 1 bp [58, 60]. This overlap with the RNA
polymerase binding site is also observed in several other members of the family
[66-68].
349
350
receptor from the environment and the direct sensor of the aromatic molecule.
This was surmised from the ability of the protein to activate transcription from
the Pu promoter in the presence of a wide range of toluene derivatives, and by
experiments with XylR mutants with altered effector specificity [75, 76, 77, 78].
These data, obtained in the heterologous host E. coli, led to the conclusion that
XylR was directly activated via interaction with the effector. XylR is closely
related to the DmpR regulator for phenol degradation in Pseudomonas sp.
CF600, which recognizes phenol and derivatives, but not toluene, as an effector
[79]. Further evidence for the direct interaction of the A-domain of these
proteins with the effector molecule came from the construction of a chimeric
protein in which the receptor domain of DmpR was replaced by the
corresponding domain of XylR, resulting in a hybrid regulator that responded to
toluene for activation of the Vo promoter of the phenol degradation pathway
[80]. DNA shuffling assays to create hybrid A-domains between DmpR and
XylR confirmed that the residues 110 to 186 of both proteins were responsible
for the effector profile of these regulators [80].
The A-domain operates as an intramolecular repressor of the central
activating domain of the protein [81, 82]. In fact, a XylR derivative in which the
A domain has been deleted is able to activate Pu in the absence of an aromatic
effector. The truncated derivative of XylR depleted of the A domain and
therefore unable to respond to effector-dependent modulation showed intrinsic
ATP binding and hydrolysis activity, located in the central activation domain
(C-domain). This activity was stimulated by the presence of a DNA fragment
containing the native XylR binding site in Pu (UAS) [83]. Furthermore, binding
of ATP to this truncated protein alone was able to induce conformational
changes in the protein. Initially, a cyclic model to explain XylR activation of Pu
was proposed by Perez-Martin and de Lorenzo [83], according to which ATP
binding to the XylR central domain led to multimerization of the regulator
bound to its UAS in Pu, followed by ATP hydrolysis. This in turn triggered a54dependent transcription initiation in Pu, allowing the system to return to its
initial disassembled state [83]. Recently, Shingler and co-workers studied the
analogous regulator DmpR, and suggested an alternative mechanism to explain
effector-dependent activation of a54-dependent promoters. According to their
model, DmpR dimers are activated after binding of the effector molecule to the
A domain, followed by a conformational change that allows ATP binding to the
central domain and oligomerization to a hexameric conformation, probably
required to promote transcription initiation. Finally, ATP hydrolysis leads to
dissociation of the hexameric structure and dissociation of the effector [83].
2.3.2. The Pu promoter
Pu promoter belongs to the class of promoters dependent on the
alternative sigma factor o54 (Fig. 2), and shows the typical architectural
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352
nonactivated XylR with the Psl UAS [94], and an ATP-dependent repression
level resulting from the cooperative oligomerization of activated XylR at the
UAS in Psl [89]. As soon as the protein is activated and the UAS is strongly
bound by the regulator, XylR expression is minimized, thus limiting the period
of time during which the Ps 1 and Pu promoters of the TOL plasmid are in an
activated state [89].
The role of IHF in Psl expression deserves special attention. Analysis of
Psl activity in isogenic IHF-plus and minus backgrounds showed that in the
presence of toluene, the highest levels of expression were achieved in the
absence of IHF [97]. This may reflect a better access of either XylR to its
binding site or of o54-RNA polymerase to the -12/-24 region of Psl, or both. On
the other hand, it may be the consequence of structural hindrance, as the DNA
bending induced by IHF bound to two sites may give rise to a highly ordered
structure that restricts the access of regulatory proteins to the corresponding
promoters. The high level of expression from Psl in the IHF-minus background
in the presence of effectors contrasts with the diminished expression from the
TOL plasmid Pu promoter for the upper pathway in an IHF-deficient
background. The most noticeable difference between the two promoters is the
position of the IHF binding site, which in Pu lies between the UASs and the 12/-24 box. In addition to affecting Psl expression, the close proximity of the
regulatory sequences in the intergenic region results is a high expression level
from Ps2 in the absence of a54, i.e., when RNA polymerase is unable to bind to
the Psl promoter [97].
In general, the physiological consequence of this organization is that in
the absence of any effector in the culture medium, Ps2, Prl and Pr2 promoters
are slightly repressed. In the presence of toluene, activation of Psl causes a
stronger repression of both xylR promoters. As a result, the level of XylR
decreases at approximately 30 monomers per cell [98], which are apparently
sufficient to promote high expression of both xylS and the upper pathway. Under
these conditions the XylS protein is overproduced, which allows induction of
expression from the Pm promoter even in the absence of meta pathway
substrates. Therefore in the presence of toluene or a substituted derivative, both
the upper and the meta pathways are coordinately expressed to optimize total
degradation of the aromatic (Fig. 2).
2.5. Integration in the bacterial metabolism
The expression of the TOL pathways is tightly regulated according to the
carbon sources available for growth [99-104]. The regulation is exerted mainly
at the level of the two o54-dependent promoters Pu and Psl, and was first
observed as a delay in the induction of expression from these promoters when
cells were induced in a rich complex medium [102, 103]. Because both
promoters were silent in this medium during rapid exponential growth, and
353
expression appeared only at the end of the exponential phase, this behavior was
named exponential silencing [105]. However, it is worth noting that exponential
silencing is only observed in rich medium; in defined minimal media with
succinate (for example) as a carbon source, expression of both Psl and Pu is
observed immediately after induction [102, 103, 106]. Growth rate as a
determinant of Pu and Psl expression was ruled out through a series of
continuous culture experiments that compared different growth rates controlled
by different limiting substrates. The results led to the conclusion that repressive
conditions correlated with a high energy status of the cells [99, 100]. In other
words, in all conditions tested where excess carbon was available, the system
was repressed. However, when oxygen was the growth-limiting substrate, a
situation where carbon was also present in excess, a certain degree of
derepression was observed although activity never reached maximum values
(Fig. 3).
Fig. 3. Integration of cell signals that lead to modulation of the expression of the Pm and Pu
promoters.
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356
Fig. 4. Organization of the tod genes and its regulatory circuit. Top. The tod genes are
organized in two operons expressed from sigma-70 dependent promoters upstream from todX
and todS. The TodS protein (O) is synthesized in an active form that in the presence of
toluene phosphorylates TodT ( ) , which functions as the activator of the todX...
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358
Fig. 5. Regulation of toluene degradation in P. mendocina KR1. The upper part of the figure
summarizes the oxidation of toluene to protocatechuate and the genes needed for each
reaction. The lower part shows a scheme of the cluster of tmolpculpob genes in this strain.
tmoABCDEF, toluene-4-monooxygenase genes; c, putative cytochrome c gene; pcuRCAXB,
/?-cresol utilization genes; pobRl and pobAl, regulator and p-hydroxybenzoate hydroxylase,
respectively; tmoST, two-component signal transduction system; p-HBOH, />-hydroxybenzyl
alcohol; />-HBHO, p-hydroxybenzyl aldehyde; p-HBA, p-hydroxybenzoate; PCA,
protocatechuate. Reproduced with permission from [132].
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363
Fig. 6. Organization of the ttgABC (A), ttgDEF (B) and ttgGHI (C) operons and their
respective regulatory genes. The regulatory regions of each gene cluster are zoomed. TtgR
(A), TtgT (B) and TtgV (C) DNA binding regions, deduced from DNAsel footprinting, are
shadowed. Putative palindromic (arrows) or symetric (bold and underlined) recognition sites
for each repressor are indicated. The +1 and the direction of transcription are marked with
small triangles for each promoter (except for ttgT one, which distance from
indicated). The -10 and the -35 positions of each promoter are also shown.
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365
mutant was 100 times lower than in the wild type. Expression studies of the
ttgDEF operon at the transcriptional level revealed that this pump is not
expressed during growth under normal laboratory conditions, and demonstrated
its inducible character in the presence of organic solvents (toluene or styrene)
(Table 1) [147].
The wild type multiple antibiotic resistance was not affected in a TtgDEFdeficient strain; moreover, no increase in antibiotic resistance was obtained by
pre-inducing the culture with toluene [147], suggesting that the substrate
specificity of this pump is limited to organic solvents. There was also no
induction of the ttgDEF operon in the presence of several antibiotics in the
culture media (Teran et al., unpublished).
Upstream from the ttgDEF operon and divergently transcribed, there is an
open reading frame whose product shares homology with several members of
the IclR family of transcriptional regulators. This gene, called ttgT, encodes for
a protein 70% identical to the SrpS-negative regulator of SrpABC solvent efflux
pump of P. putida S12 (see below).
A ttgT knockout mutant showed a small increase in ttgDEF expression
under non-inducing conditions, suggesting its involvement in the negative
regulation of this operon. The fact that in this mutant strain there was still a
strong induction of the ttgDEF expression in the presence of organic solvents
suggested that TtgT is not the only protein involved in the induction of this
operon by organic solvents (Teran et al., unpublished).
Differently from ttgR, ttgT gene expression remained unaltered regardless
of the organic solvent present in the growth medium, which suggested that
expression from ttgDEF and ttgT promoters was not coordinated. Moreover, in
the TtgT-deficient mutant, the activity of the ttgT promoter was similar to that of
the wild-type, indicating that TtgT does not regulate its own transcription (Teran
et al., unpublished).
Gel shift experiments showed that TtgT was able to specifically bind a
DNA fragment containing the ttgT-ttgDEF intergenic region (Teran et al.,
unpublished). DNAse I footprint assays revealed a single binding site along the
ttgT-ttgDEF intergenic region which covers only the ttgDEF promoter region (37 to +5 from the transcription start point) and not the ttgT one, consistent with
the in vivo expression studies described above. Therefore TtgT is directly
involved in ttgDEF operon repression, probably by competing with the RNA
polymerase for access to the efflux pump promoter. Analysis of the operator
sequence does not reveal the presence of a clear single inverted repeat. Recent
results of our laboratory suggests the induction of this efflux pump in a ttgTdeficient background by organic solvents is mediated regulator by the TtgV
regulator.
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370
371
372
373
Chapter 13
1. INTRODUCTION
One of the greatest challenges faced by the modern world is the dissociation
from the heavy dependency of the energy technologies upon the chemical bonds
of hydrocarbons. The imminent exhaustion of conventional oil sources, ranging
from a pessimistic ultimate recovery volume of 0.6 trillions of barrels to a highly
optimistic volume of 3.9 trillions of barrels [4], results in a stringent requirement
for the development of alternative technologies. It is important to note that the
world is not to run out of hydrocarbons, given the substantial amount of lowgrade, hard-to-extract supplies such as the Canadian tar sands or the abundant
heavy oil reservoirs in Venezuela and Mexico. Nevertheless, exploiting these
reservoirs is likely to be much more expensive financially, energetically,
politically and especially environmentally.
Biotechnology has greatly impacted modern industry, from the now
conventional production of goods by the use of fermentations to the novel
synthesis of valuable fine chemicals using enzymes [5, 6, 7, 8]. Notwithstanding
its enormous potential, the incorporation of biotechnological tools into the oil
industry has faltered [9]. In particular, the search of alternative hydrocarbon
sources through biotechnological media has not been assessed. Here, I compile
information regarding the biological production of hydrocarbons by bacteria and
explore its potential, not only as an environmentally-friendly fuel supply, but
also as a renewable source for basic petrochemicals.
374
375
Table 1
Taxonomical distribution of eubacterial and archaeal species able to
synthesize and accumulate hydrocarbons.
Phyllum
Class
Genus
Reference
Proteobacteria
a Proteobacteria
Rhodopseudomonas sphaeroides
Rhodospirillum rubrum
Chromatium
Escherichia coli
Rhodimicrobium vannielii
Vibrio furn issii
Serratia marinorubrum
Vibrio marinus
Vibrio ponticus
Vibrio furnissii
Desulfovibrio desulfuricans
Desulfovibrio Essex
Desulfovibrio Hildenborough
[33,30]
[33,30]
[29]
[33,30]
[30]
[17]
[23]
[34]
[23]
[17]
[35]
[30]
[30]
Clostridium acidiurici
Clostridium tetanomorphum
Sarcinaflava
Sarcina lutea
Sarcina subflava
Staphylococcus sp.
Bacillus sp.
[30]
[30]
[18]
[18,28]
[18]
[18]
[36]
y Proteobacteria
8/E Proteobacteria
Firmicute
Clostridia
Bacilli
Actinobacteria
Actinobacteria
Micrococcus lysodeikticus
Micrococcus sp.
Mycobacterium sp.
Corynebacterium sp.
Arthrobacter sp.
[18,33,30]
[36]
[36]
[36]
[36]
Cyanobacteria
Nostococales
Oscillatoriales
Nostoc muscorum
Nostoc sp.
Phormidium luridum
[30]
[36]
[30]
Chlorobi
Chlorobia
Chlorobium
[30]
Euryarchaeota
Thermoplasmata
Methanomicrobia
Halobacteria
Thermoplasma sp.
Methanosarcina barkeri
Halobacterium cutirubrum
[32]
[32]
[32]
Crenarchaeota
Thermoprotei
Sulfolobus sp.
[31]
This ability is not restricted to eubacteria. Some archeal species from the
genus Sulfolobus, Thermoplasma, Methanosarcina and Halobacterium, have
been demonstrated able to synthesize and accumulate hydrocarbons such as
squalene and other acyclic isoprenoids (C20-C25). [31, 32]. Furthermore,
individual species that produce hydrocarbons as major components have been
isolated from mesophilic, thermophilic, psycrophilic, acidophilic, alkalinophilic,
376
Fig. 1 Metabolic pathways for aliphatic hydrocarbon biosynthesis (Modified from [3] and
[1]). LCFA - Long Chain Fatty Acids
377
378
Fig. 2. Multiple alignment of fatty acyl CoA reductases from the proteobacteria Acinetobacter
calcoaceticus [43] and Photobacterium leiognathi [44] and from the plant Simmondsia
chinensis (jojoba) [42]. Residues conforming the predicted catalytic triad are highlighted.
379
Fig. 3. Deduced Fatty Acyl CoA reductases are organized in three different groups on
the basis of sequence similarity. Sequence identification numbers in parenthesis.
380
Fig. 5 Relationship between isoleucine and acetate, the anteiso C15 fatty acid and the
C29 hydrocarbon with anteiso branch methyls in both ends. Modified from [1].
381
All this information leads to the idea that ability of bacteria to synthesize
hydrocarbons is widespread and that it probably occurs by more than one
mechanism which are significantly different compared to those described in
plants.
4. DOWNSTREAM PROCESSING
The isolation and refinement of bacterial hydrocarbons has not been approached
at production scale. Nevertheless, there is abundant information regarding the
operations developed for a similar procedure with B. braunii. The process of
extracting hydrocarbons from these cells can be thought of as consisting of three
major operations. The first is that of harvesting the cells from the growth
medium. This involves the concentration or flocculation of cells from the liquid
where it is grown. This operation can be achieved through a variety of means
that include filtration, mechanical centrifugation or concentration, gravitational
concentration or chemical flocculation. The most efficient method for largescale hydrocarbon recovery is chemical flocculation. For efficient extraction, the
cells must be concentrated to a semi-dry paste.
The second step is that of the actual physical extraction of the
hydrocarbon fuel from the cells. Under suitable conditions, up to 70% of the
total hydrocarbon content can be released by 30 min of contact with solvents.
The selected solvent should be immiscible with water, with a density
significantly different than water, should be non-toxic and reusable. In view of
these considerations, hexane appears to be the solvent of choice [59]. Growth
and hydrocarbon production are not affected by repeated extraction with hexane.
In fact, a higher content of hydrocarbons has been observed in hexane-treated
biomass relative to controls. Nevertheless, recovery yields are influenced by the
physiological status of the culture. Scale up of the extraction can be difficult,
given the algae propensity to aggregate. Extensive clumping shields a large
fraction of the biomass from exposure to solvent. Alternative methods aimed to
increase the oil extraction yield have been explored. Recovery yields are
markedly increased relative to freely suspended controls when cells immobilized
by adsorption in polyurethane foam were continuously extracted with hexane
[60]. Supercritical fluid extraction is another technology that has been applied
[61].
The third operation would be the collection and concentration of the
hydrocarbon product. Although biosynthetic hydrocarbons can be directly used
in internal combustion engines after extraction with hexane, its performance is
improved by further modification. Cellular hydrocarbons can be converted to
gasoline (60 to 70%), light cycle oil (10 to 15%), heavy cycle oil (2 to 8%) and
coke (5 to 10%) after catalytic cracking [62]. The yield of gasoline obtained by
382
383
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3 85
Chapter 14
386
wells that have never been water-flooded [6, 7], strongly indicating an
indigenous origin. The presence of an indigenous microbial community is
further supported by the isolation of novel species that never has been recovered
from any other sources, and by the physiological characteristics of some isolates
indicating a close adaptation to the respective in situ reservoir conditions.
Recovery of closely related strains from remote oil fields [7-9] also supports the
existence of a widespread microbial biosphere in oil-bearing strata. However,
the problems associated with recovery of biological samples from oil wells are
extensive. Sampling from wellheads is the only way of collecting samples from
petroleum reservoirs, and the possible sources of contamination are numerous. It
is furthermore possible that exogenous mesophilic bacteria can propagate in top
facilities of the oil field installations. Occasionally, aerobic and microaerophilic
bacteria are recovered from produced oil-well water, but available chemical data
suggest that oxygen is absent in oil reservoirs, and these isolates should thus not
be considered as being truly indigenous to deep oil wells. In addition to SRB
and fermentative bacteria, Voordouw et al. [10] detected several aero- and
microaerophilic bacteria in a 600 m deep water flooded oil reservoir in Canada.
Nitrate-reducing bacteria were recovered from a similar shallow oil field [11]. It
is postulated that oxygen and nitrate is able to reach these shallow oil-bearing
formations through diffusion or convection from surface layers, giving support
to a limited community of bacteria respiring with nitrate or oxygen [10, 11].
The number of bacterial cells in water produced from oil reservoirs is
highly variable. Total bacterial counts demonstrated the presence of more than
106 cells per ml in water from a non-water flooded reservoir in California [6],
and from sulfide-rich production water from a German water-flooded petroleum
reservoir up to 6.3 x 106 colony-forming units of sulfate-reducing bacteria per
ml has been obtained [12]. Although only a few bacteria per ml have been
detected in water produced from some oil wells, these results show that the
bacterial density can be significant. In the present chapter our current knowledge
of these microorganisms is reviewed.
2. SULFATE-REDUCING BACTERIA AND ARCHAEA (SRB)
Sulfate-reducing prokaryotes constitute a diverse physiological group of sulfideproducing microorganisms able to carry out anaerobic respiration with sulfate as
a terminal electron acceptor. They are widespread in anaerobic environments
were sulfate is available. Typically, they oxidize organic acids either to acetate,
or by complete oxidation of acetate or other acids to CO2, but autotrophic
species using H2 and CO2 as energy and carbon-sources are also common. A
wide range of organic acids, e.g. acetate, propionate, butyrate, pentanoate and
hexanoate, at concentrations up to 20mM has been found in oil reservoirs [13,
14]. On the other hand, the concentration of sulfate seems to be rather low in
387
388
389
validated [24]. The genus Thermodesulfobacterium is the third deepestbranching phylogenetic lineage in the bacterial domain. Thermodesulfobacterium commune was originally isolated from a geothermal area in
Yellowstone National Park [25], but later it has been recovered from a
continental non-water flooded reservoir in the Paris Basin [6]. T. thermophilum
has been recovered also from the Paris Basin and the North Sea [26]. This
widespread occurrence indicates a cosmopolitan subterranean distribution of
these species which obviously must be indigenous to oil reservoirs. Both species
can utilize H2, formate, lactate and pyruvate as energy sources. The organic
acids are oxidized incompletely to CO2.
Two thermophilic SRB belonging to novel genera of the delta subdivision
of proteobacteria, Desulfacium infernum and Thermodesulforhabdus norvegicus
have been isolated from North Sea oil wells [27, 28]. Both species can utilize a
range of organic acids, including acetate, which they oxidize completely to CO2.
Table 1
Sulfate-reducing prokaryotes recovered from oil field production waters
Species
Archaeoglobus fulgidus strain 7324
'Archaeoglobus lithotrophicusM
Archaeoglobus profundus
Desulfacinum infernum
Desulfobacter vibrioformis
Desulfobacterium cetonicum
Desulfobulbus rhabdoformis
Desulfomicrobium sp.
Desulfomicrobium apsheronum
Desulfotomaculum spp.
Desulfotomaculum halophilum
Desulfotomaculum kuznetsovii
Desulfotomaculum nigrificans
Desulfotomaculum thermocisternum
Desulfovibrio gabonensis
Desulfovibrio longus
Desulfovibrio vietnamensis
Thermodesulfobacterium
thermophilum
Thermodesulfobacterium commune
Thermodesulforhabdus norvegicus
a
Temperature
optimum [C]
76
nd
nd
60
33
30-35
31
25-35
25-30
65
35
60-65
60
62
30
35
37
65
70
60
Location of
oil
field
North Sea
North Sea
North Sea
North Sea
North Sea
Russia
North Sea
North Sea
Apsheron
North Sea
Paris Basin, France
Russia
Russia
North Sea
Gabon, WestAfrica
offshore
Paris Basin, France
Vietnam, offshore
Caspian Sea
North Sea
Paris Basin, France
North Sea
Complete
oxidizer
+
nd
+
+
+
+
+
-
Ref.
80
19
26,
81
6
28
5
4
4
27
73
74
75
76
77
2
20
22
78
79
15
390
391
at 65C and has an upper growth-limit at 80C, has been recovered from oilfields in Tataria and Western Siberia [38]. Two other thermophilic methanogens
have been recovered from oil wells, which both share with M.
thermoalcaliphilum, the ability to use hydrogen as energy source and CO2 as
carbon source (Table 3). Although positive enrichments of acetate-utilizing
methanogens at 60C have been obtained, it has not yet been possible to obtain
pure cultures of acetoclastic thermophilic methanogens from oil wells [37, 38,
39]. A plausible reason is that thermophilic methane production from acetate in
these environments might be a result of interspecies hydrogen transfer [37].
Several
mesophilic
methanogens
have
been
isolated,
including
hydrogenotrophic types as well as strains utilizing methylamines and acetate
(Table 3).
4. FERMENTATIVE BACTERIA AND ARCHAEA
Fermentative organisms are able to utilize organic substances such as
carbohydrates and peptides for growth producing organic acids, ammonium and
hydrogen as fermentation products. In contrast to SRB and methanogens, these
organisms do not use any external electron acceptor in their energy-yielding
reactions, thus maintaining an internal red-ox balance. Occasionally,
fermentative microbes can transfer excess reduction power to sulfur compounds
like thiosulfate or elemental sulfur, which both are reduced to sulfide, thereby
improving growth rate and substrate utilization. However, the sulfur compounds
only serve as electron "sinks", and the sulfur-reducing reactions do not appear to
be linked to any energy-conserving mechanism. This sulfur-reducing ability can
be an important step in the geochemical cycling of sulfur in these anaerobic
thermal environments. Fermentative bacteria from a great variety of
phylogenetic lineages have been isolated from oil reservoirs, especially during
the recent years (Table 4).
Table 2
Examples of methanogenic reaction and their standard free energy changes.
Reaction
4 H 2 + CO 2 -> CH 4 + 2H 2 O
4 Methanol - 3CH 4 + CO 2 + 2H 2 O
4 Methylamine + 2H 2 O -> 3CH 4 + CO 2 + 4NH 4 +
Acetate -> CH 4 + CO 2
AG 0 ' [KJ/mol CH 4 ]
-135.6
-104.9
-75.0
-31.0
Table 3
Methanogens recovered from oil reservoirs
Species
Methanobacterium bryantii
Methanobacterium ivanovii
Methanobacterium
thermoaggregans
Methanobacterium
thermoalcaliphilum
Methanobacterium
thermoautrophicum
'Methanocalculus
halotolerans'
Methanococcus
thermolithotrophicus
Methanohalophilus euhalobius
''Methanoplanus petrolearius'
Methanosarcina mazei
Methanosarcina siciliae
Temperature
optimum [C]
37
45
60
65
Location
Tatarstan and Western
Siberia
Tatarstan
California
60
Tatarstan
Siberia
Tatarstan
38
France
60
North Sea
and Western
28-37
37
Western Siberia
Gulf of Guinea
37
40
Tatarstan
Gulf of Mexico
Substrates used
[methane produced from]
H2
References
H2
H2
34,82
H2
38
H2
36
H2, formate
84
H2
39
methylamines
H2 + CO2, formate, 2propanol + CO2
Acetate, methylamines
methylamines
85
86
38
83
87
88
393
394
Table 4
Fermentative bacteria and Archaea recovered from oil reservoirs
Species
Acetoanaerobium romashkovii
Anaerobaculum thermoterrenum
Dethiosulfovibrio peptidovorans
Fusibacter paucivorans
Geotoga petraea
Geotoga subterranea
Haloanaerobium acetoethylicum
Haloanaerobium congolense
Haloanaerobium kushneri
Haloanaerobium salsuginis
Petrotoga mexicana
Petrotoga miotherma
Petrotoga mobilis
Petrotoga olearia
Petrotoga sibirica
Spirochaeta smaragdinae
Thermoanaerobacter
acetoethylicus
Thermoanaerobacter subterraneus
Thermoanaerobacter brockii
Thermococcus sp.
Thermococcus sibericus
Thermosipho geolei
Thermotoga elfii
Thermotoga hypogea
Thermotoga maritima Ml2597
Thermotoga naphthophila
Thermotoga petrophila
Thermotoga subterranea
Optimal
Temp.
37
55
42
37
50
45
34
42
35-40
40
55
55
58-60
55
55
37
Nd
65
55-60
85
78
70
66
70
nd
80
80
70
Location
reservoir
of
oil
Reduction of sulfur
compounds
S2O3"
S
Western Siberia
Utah
Congo, offshore
Congo, offshore
Oklahoma/Texas
Oklahoma/Texas
Gulf of Mexico
Congo, offshore
Oklahoma
Oklahoma
Gulf of Mexico
Oklahoma/Texas
North Sea
Western Siberia
Western Siberia
Congo, offshore
Western Siberia
Nd
+
+
+
+
+
Nd
+
Nd
+
+
+
+
+
+
Nd
France
France
Niigata, Japan
Western Siberia
Western Siberia
Africa
Cameroon
Western Siberia
Niigata, Japan
Niigata, Japan
Paris Basin,
France
+
Nd
+
+
Nd
+
+
-
nd
+
+
+
nd
nd
nd
+
-?
nd
+
nd
+
+
Nd
+
Nd
nd
+
+
Nd
[weak +]
+
+
Ref.
59
56
58
89
44
44
48
49
51
50
40
44
90
41
41
53
68
55
54
46
47
42
95
97
68
43
43
98
4.2. Archaea
Hyperthermophilic fermentative Archaea belonging to the genus
Thermococcus were first isolated from Japanese oil reservoirs in 2000 [46].
Although the isolates were not described at the species level, they were
nutritionally very similar to other thermococci, growing on proteinaceous
substrates, yeast extract and amino acids. Although the in situ reservoir
temperature ranged from 50 to 58C, the optimal temperature of the isolates was
above 80C [43, 46]. The number of hyperthermophilic cocci that were present
395
in produced water from the oil wells was estimated to be up to 4.6 x 104
cells/ml. The organisms were not able to grow in produced water due to lack of
required nutrients, but under starved conditions at 50C the viable cell count was
stable for 200 days, indicating that they have developed an amazing ability to
survive prolonged periods under starved conditions. This feature is probably
important for the continued existence in a hot subterranean oil reservoir where
the supply of nutrients is limited [43]. A novel thermococcal species,
Thermococcus sibiricus, has been isolated from an oil reservoir in Western
Siberia [47], and a novel species has also recently been recovered from a North
Sea oil well (Birkeland, unpublished). This indicates that the high-temperature
oil reservoir biosphere is also inhabited by indigenous hyperthermophilic
Archaea. There is also evidence for their presence in other oil wells [4, 7, 8].
4.3. Halophilic bacteria
Some oil reservoirs contain highly saline brines with salinity above 20%.
Anaerobic fermentative bacteria that can grow at this high salinity have been
isolated from such oil wells in Africa, the Gulf of Mexico and USA [48, 49, 50,
51]. These bacteria belong to the genus Haloanaerobium, one of a few genera of
anaerobic fermentative bacteria that are adapted to high-salt conditions. These
organisms have frequently been isolated from bottom sediments of hypersaline
lakes and lagoons. They are typically saccharolytic bacteria, fermenting a range
of carbohydrates. Only mesophilic members, with growth optima between 34
and 42C, have so far been recovered from oil reservoirs, but related
thermophilic bacteria have been isolated from other sources. In contrast to other
halophilic bacteria, which accumulate organic osmotic solutes in order to
maintain an osmotic balance between the surrounding medium and the
cytoplasm, the members of the Haloanaerobium genus accumulate Na+, K+ and
Cl as compatible solutes [48, 52]. Accumulation of inorganic ions is a property
they share with extremely halophilic Archaea, which accumulate up to 3M KC1.
Another unusual feature of the Haloanaerobium genus is the nature of their cellwall structure as compared to their phylogenetic position. They stain Gramnegative when subjected to the Gram-staining procedure, but electron
micrographs show the presence of a typical Gram-negative cell wall. However,
based on the sequence of the 16S rRNA they form a deep-branching cluster
within the phylum of Gram-positive bacteria. This is taken as evidence that
certain descendants of the ancestors of the Gram-positive bacteria maintained
their Gram-negative cell wall structure, which is also the case with certain other
strict anaerobic relatives.
A moderately halophilic spirochete, Spirochaeta smaragdinae, growing
optimally at a salinity of 5% has been isolated from an offshore oil well in
Congo [53]. This bacterium is nutritionally very versatile, fermenting
396
carbohydrates, glycerol, fumarate, peptides and yeast extract, and is the only
spirochete so far isolated from the deep subsurface. Evidence for the presence of
a closely related spirochete in North Sea oil wells has, however, been obtained
using direct molecular techniques (Birkeland, unpublished).
4.4. Other mesophilic and thermophilic fermentative bacteria
Thermoanaerobacter subterraneus and Thermoanaerobacter brockii
subsp. lactiethylicus are Gram-positive thermophilic carbohydrate-fermenting
bacteria isolated from French oil wells [54, 55]. They grow optimally at 65 and
55-60C, respectively. T. brockii forms endospores. Spores have not been
observed in cultures of T. subterraneus, but because this organism can survive
autoclaving for 45 minutes, the presence of heat-resistant forms has been
suggested [55]. Anaerobaculum thermoterrenum is a Gram-positive bacterium
isolated from an oil well in Utah [56]. It defines a novel moderately
thermophilic genus, Anaerobaculum, phylogenetically related to Thermoanaerobacter. It is nutritionally versatile, growing on a wide range of
carbohydrates including cellulose, as well as peptone and organic acids like
citrate. It is able to utilize crotonate as electron acceptor, reducing it to butyrate
[57]. Although it groups within the phylum of Gram-positive bacteria, A.
thermoterrenum has a Gram-negative cell wall, a feature it shares with the
halophilic genus Haloanaerobium. A mesophilic thiosulfate-reducing bacterium
termed Dethiosulfovibrio peptidovorans, which can only utilize peptides and
amino acids for growth, has been isolated from a corroding offshore oil well in
Congo [58]. The isolate was shown to cause a strongly enhanced corroding
activity of steel in the presence of thiosulfate, indicating that apart from SRB,
thiosulfate-reducing bacteria can contribute to this process. Phylogenetically,
this organism groups within Gram-positive bacteria of the clostridial group, but
shares with Anaerobaculum a multilayered cell wall ultrastructure typical of
Gram-negative bacteria. Together with Anaerobaculum and a few other small
genera, Dethiosulfovibrio spp. form a separate phylogenetic cluster in the
Clostridium group of Gram-positive bacteria. Another Gram-positive bacterium,
Fusibacter paucivorans, isolated from an African oil well, defines a novel genus
within the Clostridium phylum. F. paucivorans is a mesophilic and halotolerant
Gram-positive bacterium fermenting a limited number of carbohydrates. Spores
have never been observed.
Acetoanaerobium romashkovii is a homoacetogenic mesophilic bacterium
isolated from a Siberian oil field in 1992 [59]. Homoacetogenic bacteria is a
group of anaerobes that can use CO2 as an electron sink and reduce it to acetate
as a fermentation product via the carbon monoxide dehydrogenase pathway.
Hydrogen can be used as energy source, but as is the case for A. romashkovii,
various one-carbon compounds, amino acids and sugars can also be utilized.
397
398
6. CULTURE-INDEPENDENT APPROACHES
Direct analyses of uncultured natural microbial communities based on
fluorescence microscopy using fluorescent antibodies (FA) or oligonucleotide
probes directed against specific bacterial groups, and amplification and analyses
of genes from DNA extracted from environmental samples have contributed
significantly to an improved understanding of the structural complexity of
natural microbial communities during the last decade. The development of the
polymerase chain reaction (PCR), automated DNA sequencing and DNA
microchip technologies has provided efficient tools for culture-independent
analyses of microbial diversity.
Genus specific antibodies directed against the hyperthermophilic
Archaeoglobus, and the thermophilic genera Desulfotomaculum and
Thermodesulforhabdus have been used for analyzing the distribution of SRB in
produced oil reservoir waters sampled at different dates and from different wells
in the Gullfaks field in the North Sea [18]. Archaeoglobus and
Thermodesulforhabdus strains were detected in 4 of 16 samples, but in most
samples the numbers were below the detection limit. The number of cells varied
from 400 to 2 x 104 per ml. Desulfotomaculum strains were only detected in one
of the wells. In 3 wells only one of the three types could be detected. This
investigation demonstrated that the distribution of these SRB in the Gullfaks
field is subject to strong spatial and temporal variations.
Oligonucleotide microchips containing specific 16S rRNA probes
targeting selected microbial groups encompassing key genera of thermophilic
bacteria and Archaea were used for probing the diversity in water samples from
the Samotlor high-temperature oil reservoir in Western Siberia [37]. The results
confirmed the presence of organisms identified by culture-based methods, but
organisms that had not been identified by culture-dependent methods were also
detected. These organisms included representatives of the aerobic genus
Thermus and the microaerophilic Aquifwales group, as well as anaerobes
belonging to the genera Desulfurobacterium and Thermovibrio. None of these
groups have previously been detected in oil reservoirs. Orphan et al. [7] made
16S rDNA libraries from total DNA from water produced from hightemperature petroleum reservoirs in California, using either universal or archaeal
primer sets, 83 unique clones were identified from the universal library, and
sequence analysis revealed that the majority of the clones grouped within the
bacterial domain, with only 8.8% of the library affiliated with the domain
Archaea. The dominating bacterial phylotypes were close relatives of Grampositive fermentative genera Acidaminococcus and Thermoanaerobacter, and to
the halophilic proteobacterium Halomonas. The genus Acidaminococcus has
never been isolated from petroleum reservoirs. Clones related to aerobic bacteria
were also identified. The archaeal library was dominated by methanogen-like
399
400
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405
Chapter 15
406
407
408
409
2. TEST FIELD
The test field, Fuyu oilfield, is located in the northeastern area of China (Fig. 2
A and B). Oil production in this area began in 1973, and waterflooding was
instituted in 1983. Current production is conducted using sucker rod pumping.
410
Table 1
Reservoir data for test area
Block
Reservoir Areas
East 24-23
[km2]
East 24-26
0.562
0.219
Res. Depth
[m]
300-450
320-450
Res. Temperature
[C]
28.0
28.0
Res. Thickness
[m]
15.2 (net)
14.8 (net)
[md]
240
240
[%]
27
27
[kg cm"2]
28.8
18.6
[%]
73.8
69.7
Permeability
Porosity
Current Pressure
Water Cut
(1995end)
The reservoir data for test area is shown in Table 1. The target reservoir is
sandstone and its depth is from 300 to 450 m. The temperature of the reservoir is
approximately 30 C. The average permeability is approximately 240 md and
porosity is 27%. The present water cut of most wells averages more than 90%
and there are heterogeneous fracture zones between each injection well and
production well.
3. THE COLLECTION OF SCIENTIFIC KNOWLEDGE OF THE
FUNDAMENTAL MICROBIOLOGY RELATED TO MEOR
3.1. Development of investigation technique of microbes related to MEOR.
Fig. 3 shows the analytical protocol of microbes related to MEOR. The
authors developed a biotechnological tool, a combination of plating and
PCR-RFLP analysis [11] to estimate the behavior of microbes which are able to
propagate in the reservoir using molasses. Those microbes unable to propagate
using molasses were not relevant to our study and therefore did not need to be
analyzed. The RFLP profile described in the present study refers to the profile of
411
412
In the comparison of RFLP profile of microbes from #J15 with those from
#J16, 15 to 20% of RFLP profiles from #J16 matched those of #J15, and 20 to
25% of RFLP profiles from #J16 are closely related species (that is, two RFLP
profiles were exact matches) to microbes from #J15. Almost all microbes of
these microbes were detected at 102to 105 cfu g"1, and some grew to more than
108 cfu ml"1 using molasses.
These results indicate that aerobes and facultative anaerobes isolated from
the reservoir rock are trapped in high permeability zones and highly saturated
water zones, such as fractures created by hydraulic fracturing operations. The
aerobes and facultative anaerobes have also been carried into the reservoir from
the surface by water flooding operations, and have accumulated over a long
period of time. During these operations, the injection water, including these
microbes, is apt to enter into the high permeability zones. Therefore, in the
development of MEOR techniques, we must consider that microbes injected into
the reservoir will need to co-exist with these indigenous microbes. It is
necessary to monitor the indigenous microbes that make use of molasses,
particularly those microbes' potential to suppress the growth of in situ microbes
injected into the reservoir.
413
414
Fig. 5. Injection process of molasses solution by Huff & Puff injection method
415
Table 2
The number of species distinguished by RFLP profile
(in ground water, molasses, reservoir brine)
The number of
species detected
in the samples
Ground water
Molasses
Reservoir brine from
#26-231
#24-24,
#20-28
#T-59
7 (10-103)
5 (102-103)
3
1
(10-104)
(102-103)
(104-105)
(102-103)
0
1
0
0
2
8
4
6
Table 3 shows the microbes detected in the injected fluid before injection.
These injected fluids were collected from tank trucks at each well site. Within
the four injection fluid samples, the number of species distinguished by their
RFLP profile was three to five, and the concentration of viable cells of each
species was 104 to 107 cfu ml"'. Of these, almost all species grew to more than
107 cfu ml"1 using molasses. Moreover, based on their RFLP profiles, almost all
microbes detected in the injected fluid matched microbes which were isolated
from the ground water.
Table 4 shows microbes detected in the production water after the
injection test. In samples obtained from the four wells, three predominant
species are distinguished by their RFLP profiles. These species, in viable cell
concentrations of 103 to 107 cfu ml"1 were also detected in the 5 to 13 samples of
production water collected daily throughout the 20-day test period. Moreover,
based on their RFLP profiles, these microbes matched microbes which were
isolated from the ground water.
Fig. 6 (A, B, C and D) shows the production history of the producing
wells in which the molasses solution was injected. It is apparent from these data
that molasses injection into the target reservoir did not result in increased oil
recovery.
416
The number of
species detected
in the samples
Injected fluid for
#26-23,
#24-24,
#20-28
#T-59
4 (104-107)
4 (104-106)
3 (106-107)
5 (106-107)
4
4
3
5
RFLP profile
A
B
C
Frequency of
/* . , . .
detection (times)
5 (104-106)
8 (103-105)
13 (103-107)
417
: Total liquid
o : Water cut
418
419
420
Fig. 9. Growth and production of insoluble polymer of strain CJF-002 (After 1 day
incubation).
Fig. 9 shows the growth and production of insoluble polymer from the
strain CJF-002. It is apparent from these data that one of the notable features of
CJF-002 is its ability to grow and produce insoluble polymer when fed molasses.
Moreover, the cells of the strain CJF-002 are small enough to pass through the
pore throat of average sandstone.
Fig. 10 shows the visual image (A) and SEM images (B and C) of
insoluble polymer. Cellulase can degrade this insoluble polymer.
Results of sugar composition (Table 5) and methylation analysis (Table 6)
also indicated that the insoluble polymer produced by strain CJF-002 is a
cellulose derivative.
5.1.2. Development of the monitoring technique of viable strain CJF-002
propagating in the reservoir
Conventional culture-based bacteriological methods for detecting
microbes in environment samples depend on their recovery from each sample
and therefore on their culture conditions. However, these methods are not
suitable for MEOR because their lower selectivity can not distinguish target
microbes from the various microbes inhabiting the reservoir fluid. These
methods also require several days to produce results. The process of MEOR
421
Fig. 10. Visual image and SEM images of insoluble polymer by strain CJF-002
(A);Visual image, (B and C);SEM images (After 1 day incubation)
Table 5.
Sugar composition of insoluble polymer
Sugar
Detected substance
composition
Glucose
97.31
Mannose
1.32
Arabinose
0.42
Galactose
0.95
Uronic acid
0.0
422
Table 6.
Result of methylation analysis of insoluble polymer
Peak
No.
Retention
time (MS)
partially methylated
sugar alcohol
Binding
location
Peak
area
ratio
19'09"
1,5diO-acethyl-2,3,4,6-tetra-Omethylglucitol
nonreduced
end Glc
2840
1.00
20' 50"
1,4,5-tri-O-acethyl-2,3,6-tri-Omethylglucitol
^4Glc
88917
29.22
20' 57"
l,5,6-tri-O-acethyl-2,3,4-tri-Omethylglucitol
-+6Glc
310
0.10
21' 27"
l,3,4,5-tetra-O-acethyl-2,6-diO-methylglucitol
^3,4Glc
706
0.22
21'38"
l,2,4,5-tetra-O-acethyl-3,6-diO-methylglucitol
2,4Glc
1572
0.48
22' 01"
1,4,5,6-tetra-O-acethyl-2,3-diO-methylglucitol
^4,6Glc
1549
0.48
423
distilled water (SDW) and Chelex 100 resin, as per the manufacturer's
instructions. PCR amplification was performed under the following conditions.
PCR mixtures contained 2 ul of 10x PCR buffer (500 mM KC1, 100 mM
Tris-HCl [pH 8.3], 15 mM MgCl2), 2 ul of 25 mM MgCl2, 2 ul of a
deoxynucleotide triphosphate (dNTP) mixture (concentration of each dNTP, 2.5
mM), 10 pM of each primer, 5 ul of the extracted DNA sample and 0.4 U of Taq
DNA Polymerase (Takara Shuzo Co., Ltd., Kyoto, Japan), in a total volume of
20 ul.
After the solution was overlaid with 30 ul of mineral oil (Chill-out 14
Liquid Wax, MJ Research Inc., Watertown, MA), the PCR program was initiated
with a preincubation at 94C for 30 s. The amplification profile is 94C for 45s,
58C for 50 s, and 72C for 60 s. PCR products were electrophoresed in a 1.5 %
agarose gel and visualized by UV transillumination after being stained in
ethidium bromide solution (5 ug ml"1).
A primer annealing temperature close to the theoretical primer melting
point, that is, 58C, allowed amplification of a single 280 bp product only in
strain CJF-002 (see Table 7), according to the direct PCR protocol. The band
size of the amplificates matched the expected size. These results demonstrated
that the 16S-23S spacer sequence of strain CJF-002 is sufficiently
species-specific for the derivation of PCR primers used to identify strain
CJF-002.
5.1.3. Biofilm formation test
A biofilm formation test was performed as follows. Sliced reservoir rock
was set vertically inside a bottle filled with the 4% molasses solution,
synthetic brine, and strain CJF-002 (see Fig. 11). The components of the
synthetic brine were established previously based on the components of
reservoir brine obtained from the test field (see Table 8). The molasses medium
with strain CJF-002 was then stirred 1.7cm per second and incubated at 30 C.
The medium predominantly affected one face of the rock during the test period.
After one day, the surface of the sliced reservoir rock was covered with
biofilm consisting of the insoluble polymer produced by strain CJF-002 (see Fig.
12). These results suggest that the insoluble polymer produced by strain
CJF-002 will adhere to the surface of reservoir rock in the target reservoir.
424
Table 7.
Summary of PCR amplification for various species by spacer primers
Species
CJF-002
Acinetobacter calcoacelis
Aeromonas hydrophila
Alcaligenes faecalis
Azotobacter vinelandii
Bacillus subtilis
Rhodococcus erythropolis
Staphylococcus aureus
Pseudomonas fluorescens
Enterobacter cloacae
Citrobacter freundii
Escherichia coli
Erwinia carotovora
Klebsiella pneumoniae
Clostridium acetobutylicum
Clostridium butyricum
Strain No.
IFO 12552
IFO 13286
IFO 14479
IFO 12018
IFO 3134
IFO 12320
IFO 12732
IFO 14160
IFO 13535
IFO 13546
IFO 13898
IFO 14082
IFO 13541
IFO 13948
IFO 13949
425
Teble 8.
The components of reservoir brine
Synthetic brine
NaCl :
KC13
NaHCO
CaCl2
MgCl2
FeCl3
KH2PO4
NaHSO4
(1000ml)
1210 (mg)
23
2820
140
53
2
10
3
426
Table 9.
Result of competitive vulture test
(A) with indigenous microbes in the reservoir brine
Incubation period
Screened
place
initial
lday
3 days
5 days
lOdays
20days
Strain A
Japan
x 1O s
xlO6
xlO6
xlO 5
OlO3
OlO3
Strain B
Japan
xlO6
xlO7
xlO4
DOlO 3
OlO 3
NT
CJF-002
China
xio 6
xlO8
xlO 8
xlO8
xlO 8
xlO4
Screened
place
initial
lday
3 days
5 days
lOdays
20days
Strain A
Japan
xlO6
xlO8
xlO 8
xlO8
xlO8
OlO3
Strain B
Japan
xlO5
xlO7
OlO3
OlO 3
OlO 3
OlO3
CJF-002
China
xlO6
xlO9
xlO8
xlO8
xlO8
OlO3
427
428
429
430
Teble 10.
Result of Huff & Puff Test by strain CJF-002
Before injection
Well*1)'2)
1. #22-27
2. #24-23 4
3.#22-26 4
4. #26-231
5. #26-254
6. #24-24 j
Water R t
cut
[%]
[t day"1]
99
97-98
98
98
97
99
0.2
0.1-0.2
0.2-0.3
0.2
0.2-0.3
o.l
Condition of injection
Molasses
cone.
[%]
Injection system
(CJF-002and
molasses)
1.9-3.0
0.6-1.5
3.3-5.7
4.2
4.6-7.4
4.4-8.0
Separately
Separately
Separately
Separately
Simultaneously
Simultaneously
After injection
Microbe
Oil
WC*5)
3)
4)
in Prod.* Prod.*
r 0/1
L J
[%]
83.3
85.7
88.9
57.1
62.5
85.7
?9
?9
*1) 1.-4.
10m3 of CJF-002 culture broth & 80m3 of molasses were injected separately.
*2) 5. and 6. 10m3 of CJF-002 culture broth & 80m3 of molasses were injected
simultaneously.
*3) The ratio of samples with CJF-002 to samples of produced water analyzed.
*4) Oil production; Increase , Same level
*5) Water cut; Decrease
, Same level
431
432
433
434
435
o Other microbes
436
o other microbes
437
A 10% molasses solution was then injected along with strain CJF-002.
After two months of CJF-002 and molasses injections, water injection was
continued as usual. The strain CJF-002 and other microbes were measured as in
the first trial, described above.
The microbial concentrations in injection water taken from the manifold
are shown in Fig. 25. The strain CJF-002 and non-CJF-002 microbial
concentrations were at almost same levels during injection of the strain CJF-002
and molasses. Theoretical concentration, that is, approximately 105 cfu ml" of
strain CJF-002 was routinely detected in the injection water. In other words,
results of this microbial monitoring indicate that the strain CJF-002 can increase
in cell number and produce the insoluble polymer in the reservoir.
Figure 26 shows the results of the microbial monitoring of production
water from well #J18. Notably, the concentration of strain CJF-002 detected in
the production water was relatively high, approximately 103 to 106cfu ml"1, for
20 days following the initial injection. Consequently, the strain CJF-002 was
detected in all producing wells in this test area. Parts of insoluble polymer which
may have come off the reservoir rock were also detected in all producing wells
by HPLC analysis combined with cellulase degradation. This result proves that
the strain CJF-002 has the ability to produce insoluble polymer in porous media
in the reservoir. Results also show that the concentrations of the injected
CJF-002 and molasses during the second trial are enough to sustain the strain
CJF-002's competitiveness and survivability.
The oil production of all wells in the test area is shown in Fig. 27.
Eventually, the oil production increased by more than two times for at least one
year, and incremental oil production reached 3,392 tons [approximately 24,521
bbls] after microbial injection. Total water cut also fell from 88% to 65%.
Notably, a dramatic increase in oil production was observed approximately 20
days after beginning injections. Even after the final CJF-002 and molasses
injection, the great improvements in oil production and water cut continued, and
the oil recovery rate after one year was still doubled. These results indicate that
438
the increased oil production is related to the high concentration of the strain
CJF-002 which was detected at the manifold in the 20 days following the initial
injection.
The authors believe that the principal cause of the sustained increase in oil
recovery over the following year is the stability of the insoluble polymer in the
reservoir. Degradation by strain CJF-002 and the other microbes inhabiting the
reservoir is one factor that may affect the stability of the insoluble polymer.
Some microbes producing cellulase, such as Clostridium sp., are generally
well-known, and some researchers have reported that the microbes belonging to
these species inhabit reservoirs. In our preliminary experiment, however, we
confirmed that the insoluble polymer is not degradable by any microbes
inhabiting the target reservoir or by the strain CJF-002 (data not shown).
Another factor influencing insoluble polymer stability is the possible
absorption of insoluble polymer into reservoir rocks. Considering the results of
the biofilm formation test described above, it is presumed that the insoluble
polymer produced by strain CJF-002 will adhere to the surface of reservoir rock
in the target reservoir.
439
440
Figure 28 shows the carbon number of production oil from well #24-254'
after microbial treatment. The carbon number of production oil shifted to a low
molecular weight which can be recovered easily. This result indicates that the
production oil after microbial treatment was produced from previously unswept
zones.
In addition, the effectiveness of the microbial treatment was evaluated
through a comparison of tracer tests using NH4SCN solution. After the first and
second microbial treatment, 30,000 ppm of NH4SCN solution was injected into
the reservoir through two injecting wells. The cumulative quantity of that tracer
was approximately 500 kg. In well #22-27, tracer could not be detected before
the treatment, but could be detected after the treatment (see Fig. 29 (A)). In
contrast, in well #26-25, tracer could be detected before, but not after the
treatment (see Fig. 29 (B)). Furthermore, in the other wells, changes in tracer
peak or time of detection were observed. These results show that the microbial
treatment modified the sweep pattern of injection water.
The results described above prove that the insoluble polymer increases the
volumetric sweep efficiency by diverting injection water from the most highly
permeable zones to previously unswept oil-bearing zones. Moreover,
considering the results of the second trial, injection of strain CJF-002 and
molasses for 20 days when the oil production rate decreases may be effective in
continuing to increase oil recovery.
441
Fig. 29. Result of tracer test at well 22-27 and well 26-25 (2nd trial)
O Before microbial treatment A After microbial treatment
442
443
5) It is clear that the following points are very important for development of the
MEOR technique:
a) Understanding of microbes related to MEOR (including microbes
inhabiting the ground water, molasses and reservoir environments, in addition to
the injecting microbe).
b) Monitoring of behavior of these microbes at field trials.
c) Designing of field operations which reflect those monitoring data.
d) Establishing techniques for transplanting microbes and demonstrating
microbial metabolic function in the reservoir.
In the present study, valuable data to demonstrate the MEOR effect were
successfully collected and the results obtained from this research support the
theory that MEOR can effectively increase oil recovery. Though the results are
seen in only a few cases yet at most, the authors believe that this is an example
of a successful application of MEOR to a broad range of reservoirs.
6.2. Contribution of biotechnology to the development of the MEOR
technique.
In the examination of microbes related to MEOR processes, PCR-RFLP
analysis of the 16S rDNA of microbes was useful for investigating microbes
inhabiting the ground water, molasses, reservoir brine and reservoir rock. This
method is also useful for confirming a state of sterilization in equipment, such as
incubation tanks, tank trucks, and fluid lines, during field operations.
As demonstrated in this study, the Direct-PCR method, with primers
including sequences in the ribosomal spacer region between the 16S and 23S
rDNA, is a powerful tool for screening the injection microbe. The screening
involves several tests, such as competitive culture tests, on multiple diverse
microbes. The Direct-PCR method is also useful for monitoring injection
microbes in the incubation tanks, tank trucks, manifolds and well sites (injection
wells and production wells) during field operations.
Therefore, biotechnologies contribute to promoting MEOR because the
MEOR process must be designed based on the features of microbes and
geological characteristics in each oil field or reservoir.
Acknowledgements
We would like to thank Japan National Oil Corporation, PetroChina
Company Limited Jilin Oilfield Company and Chugai Technos. Company
Limited for the permission to present this paper. We also thank Tohoku
University and KRI Inc. for their constant support in microbial analysis at Fuyu
oilfield. We are also very grateful to fellows for their continuous assistance
during this collaborative research project.
444
REFERENCES
[I] J. W. Beckman, Ind. Eng. Chem., November, 10 (1926) 3.
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Advances, MEOR Field trials carried out over the world during the last 35 years,
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[3] J. B. Clark, D. M. Munnecke, G. EJanneman, Dev. Ind. Microbiol., 22 (1981) 695.
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[6] H. Yonebayasi, H. Enomoto, T. Chida and K. Fujiwara, Proceeding of 17th Workshop of
the International Energy Agency, Sydney Australia, September, 1996
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Petroleum Engineers 38070 (1997).
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leads to MEOR Field Pilot in Fuyu-oilfield, Chaina, Proceeding of 9th European
Symposium on Improved Oil Recovery, The Hague, October 20-22, 1997
[9] K. Ono, S. Maezumi, H. K. Sarma, H. Enomoto, C-X. Hong, S-C. Zhou and K. Fujiwara,
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Hong, Proceeding of 20th Workshop of the International Energy Agency,
Enghien-les-Bains (Paris), France, September 22-24, 1999
[II] K. Fujiwara, S. Tanaka, M. Ohtsuka, N. Ichimura, H. Yonebayashi, C. X. Hong and H.
Enomoto, Sekiyu Gakkaishi (J. Jpn. Petrol. Inst.), 42 (1999) 342.
[12] K. Fujiwara, S. Tanaka, M. Ohtsuka, K. Nakaya, S. Maezumi, N. Yazawa, C. X. Hong, T.
Chida and H. Enomoto, Sekiyu Gakkaishi (J. Jpn. Petrol. Inst.), 43 (2000) 274.
[13] K. Fujiwara, S. Tanaka, M. Ohtsuka, H. Yonebayashi and H. Enomoto, Sekiyu
Gakkaishi (J. Jpn. Petrol. Inst.), 43 (2000) 43.
[14] K. Fujiwara, Proceeding of International Symposium on Research and Education in the
21st Century, Sendai, Japan, August 18-25, 2000
[15] H. Enomoto, K. Fujiwara, H. Yonebayashi Sekiyu Gakkaishi (J. Jpn. Petrol. Inst.), 43
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[16] K. Nagase, S. T. Zhang, H. Asami, N. Yazawa, K. Fujiwara, H. Enomoto, C. X. Hong
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Hong, J. Jap. Associ. Petrol. Technol., 68 (2003) 271.
445
447
Chapter 16
1. INTRODUCTION
1.1. Common Target Contaminants
Total petroleum hydrocarbons (TPH) comprise a diverse mixture of
hydrocarbons that occur at petrochemical sites and storage areas, waste disposal
pits, refineries and oil spill sites. TPHs are considered persistent hazardous
pollutants, and include compounds that can bioconcentrate and bioaccumulate in
food chains [1], are acutely toxic [2], and some such as benzene [3] and
benzo[a]pyrene are recognized mutagens and carcinogens [4]. Since this group
includes chemicals that have physical and chemical characteristics that vary over
orders of magnitude, TPHs are divided into two categories (Fig. 1). Gasoline
range organics (GRO) corresponds to small chain alkanes (C6-Ci0) with low
boiling point (60-170 C) such as isopentane, 2,3-dimethyl butane, rc-butane and
fl-pentane, and volatile aromatic compounds such as the monoaromatic
hydrocarbons benzene, toluene, ethylbenzene, and xylenes (BTEX). Diesel
range organics (DRO) includes longer chain alkanes (Cio-C4O) and hydrophobic
chemicals such as polycyclic aromatic hydrocarbons (PAH).
Whereas most of these contaminants do have natural sources,
concentration and release of contaminants through anthropogenic activities has
led to significant contamination of soil and groundwater. The extent of
petroleum hydrocarbon contamination throughout the United States is reflected
by the large number of Superfund sites and Leaking Underground Storage Tanks
(LUST) sites that contain these contaminants (Fig. 2 and 3). These sites often
contain high concentrations of contamination. However, individual
contaminants behave differently. Some contaminants such as BTEX compounds
are highly mobile in the environment, while others such as PAHs tend to bind
448
strongly to soil particles near the source or remain entrapped within an organic
phase.
449
Fig. 2. United States Superfund sites containing petroleum hydrocarbon contamination for
FY1982 to FY1999 (834 total projects, [6]).
Fig. 3. Total United States underground storage tank corrective actions (FY 1992 to FY 2003,
[7]).
450
451
Table 1
Advantages and disadvantages of phytoremediation over traditional technologies such as
pump and treat of contaminated groundwater and soil excavation and above-ground treatment.
Advantages
Relatively low cost
Easily implemented and maintained
Several mechanisms for removal
Environmentally friendly
Aesthetically pleasing
Reduces landfilled wastes
Harvestable plant material
Disadvantages
Longer remediation times
Climate dependent
Effects to food web might be unknown
Ultimate contaminant fates might be unknown
Results are variable
452
Fig. 5. Plan view of trees planted on a line (similar to an interdiction well field) to capture a
shallow groundwater plume (Modified after [9]).
453
(1)
(2)
Phase I - Conversion
Phase II - Conjugation
Phase III - Compartmentation
454
Fig. 6. Estimated transpiration stream concentration factors (TSCF) for BTEX using Eq. 2.
455
456
lipophilicity of a pesticide determines its fate in a barley plant [11]. High Kow
values (an indicator of hydrophobicity) corresponded to a greater possibility that
the compound would be retained in the roots (Eq. 3). Burken and Schnoor
(1998) published similar results for the sorption of a wide range of organic
contaminants to roots of hybrid poplar plants grown hydroponically (Eq. 4) [12].
log (RCF - 0.82) = 0.77 log Kow -1.52
(3)
(4)
Where the Root Concentration Factor (RCF) (L/kg dry roots) is the ratio
of organic chemical sorbed on the root (mg/kg of fresh root tissue) to that in
hydroponic solution (mg/L). This equilibrium partitioning coefficient has
generally proved to be a good indicator of whether a plant retains a contaminant
in the root, which increases the probability of microbial degradation (not
withstanding significant bioavailability limitations). However, a few exceptions
exist such as phenol and aniline, which bind irreversibly to the root (especially
aniline) and are chemically transformed. They are not appreciably desorbed
because they are covalently bound as metabolic products in plant tissue [35].
Fig. 7. Estimated Root concentration factors (RCF) for PAHs using Eq. 4.
457
Figure 7 uses Eq. 4 to estimate RCF values for a few common PAHs. The
hydrophobic (high sorption) characteristics of PAHs and other DRO compounds
result in high retention in the root zone. Fortunately, the rhizosphere of most
plants promotes a wealth of microorganisms that can contribute significantly to
the degradation of petroleum hydrocarbons during phytoremediation. Thus,
though a plant may not directly act upon these contaminants, a plant can
influence the microbial community within its root zone to a great extent.
Potential rhizosphere interactions that may be important for
phytoremediation of petroleum hydrocarbons include:
1.
2.
3.
4.
5.
458
459
460
Table 2
Potential clean-up mechanisms during phytoremediation of hydrocarboncontaminated sites based on physical properties of the target pollutants such as
octanol-water partitioning coefficient (KoW) and Henry's dimensionless constant (KH).
Contaminants
Sources
KoW*
. ,
{ K H
TITCT
KH*
^1A4
>10
n m
ln-4
-s
< 2 x l 1A
O
Potential Removal
Mechanisms
Hydraulic Control
Pnytovolatihzation
Hydraulic Control
Phytovolatilization
, .
,. ..
Rhizoremediation
Site Treatability
Plant selection and planting density
Irrigation, agronomic inputs and maintenance
Cost Estimation
Mathematical Modeling
Clean-up time required
Analysis of failure modes
461
462
may pose problems for phytoremediation when deciduous vegetation loses its
leaves, transformation and uptake cease, and soil water is no longer transpired.
However, a combination of grasses can be used to prolong the growing period.
4.2. Plant Selection Criteria
Plants should be selected according to the needs of the application, the
contaminants of concern and their potential to thrive on contaminated soil.
Design requirements should include the use of native plants to avoid
introduction of invasive species. Apart from this, vegetation should be fast
growing, hardy, easy to plant and maintain. The main aim is to ensure that roots
expand throughout the entire contaminated zone. In temperate climates with
shallow contaminated aquifers, phreatophytes, such as Populus sp. (hybrid
poplar, cottonwood, aspen) and Salix sp. (willow) are often selected because of
fast growth, deep rooting ability down to the surface of groundwater, large
transpiration rates, and the fact that they are native throughout most of the
country. Among tropical plants tested for use in Pacific Islands, three coastal
trees, kou {Cordia subcordata), milo (Thespesia populnea), and kiawe (Prosopis
pallida) and the native shrub beach naupaka (Scaevola sericd) tolerated field
conditions and facilitated clean-up of soils contaminated with diesel fuel [59].
Grasses are often planted in tandem with trees at sites with organic
contaminants as the primary remediation method. They provide a tremendous
amount of fine roots in the surface soil, which is effective at binding and
transforming hydrophobic contaminants such as TPH, BTEX, and PAHs.
Grasses are often planted between rows of trees to provide for soil stabilization
and protection against wind-blown dust that can move contaminants off-site.
Legumes such as alfalfa (Medicago sativa), alsike clover (Trifolium hybridum ),
and peas (can be used to restore nitrogen to poor soils. Fescue (Vulpia myuros),
rye (Elymus sp.), clover {Trifolium sp.) and reed canary grass (Phalaris
arundinacea) have been used successfully at several sites, especially
petrochemical wastes. Once harvested, the grasses can be disposed off as
compost or burned.
Plant tolerance to high contaminant concentrations is also a very
important factor to keep in mind. The phytotoxicity of petroleum hydrocarbons
is a function of the specific contaminant composition, its concentration, and the
plant species used. Major adverse effects typically include reduced germination
and growth if contaminant concentrations are sufficiently high. In general, TPH
values of 15 percent or greater can result in significant reductions in plant
growth and in some cases mortality. Compared with uncontaminated soil, soils
with 2% TPH reduced alfalfa yields by 32 percent [61]. Production of biomass
by ryegrass was reduced 46 percent at a soil concentration of 0.5 percent (5000
mg/kg) hydrocarbons [47]. It was found that plants pre-grown in clean soil and
subsequently transplanted to the contaminated soil grew nearly as well as the
463
control, showing that toxicity was associated with germination and/or early plant
growth. Similarly, poor rooting of ryegrass compared to legumes appeared to
adversely affect the removal of TPH from Gulf War-contaminated soils [62].
Also, although the germination of sunflower seeds and beans was greater than
that of maize, vegetative growth was greater for maize than beans,
demonstrating that germination and later plant growth may be affected
differently [63].
Aged spills tend to be much less phytotoxic than fresh ones, possibly
because of the lower bioavailability of toxic compounds in the aged spills.
However, the speciation of petroleum hydrocarbons is also very important in
determining phytotoxicity. A fuel oil with 30 percent aromatics resulted in LC50
germination (oil concentration lethal to 50 percent of test plants) values of
7 percent (70,000 mg/kg) for sunflower seeds. The volatile fraction can prove
most toxic to plants. Aromatic volatile petroleum hydrocarbons such as benzene
have been used as herbicides in the past years, illustrating their phytotoxicity
when applied to plant leaves [64]. In contrast, no phytotoxic effects were
observed in hybrid poplar trees exposed to a simulated groundwater containing a
mixture of VOCs including BTEX, chlorinated aliphatics, and alcohols at a total
concentration of 169mg/L [65]. Reduction of the volatile fraction may be
accomplished through management, such as by tillage of the soil. If initial
efforts at plant establishment at a site fail, replanting the area may ultimately
lead to success as concentrations or bioavailability of the more phytotoxic
components decline.
Solution-phase concentrations of hydrocarbons are also important,
particularly for aquifer remediation applications of phytoremediation. Additional
components with phytotoxic effects include various unsaturated hydrocarbons
and acidic hydrocarbons such as alicyclics with carboxylic acid groups
(naphthenic acids) [64].
A screening test and knowledge from the literature of plant attributes is
essential for selection of plants. Most experts recommend a mixture of grasses
or legumes to address surface soils contaminated with petroleum hydrocarbons.
However, design engineers should work in interdisciplinary teams that include a
botanist and/or agricultural specialist to identify and select plants that will grow
well at the site. Preliminary greenhouse studies should also be used to identify
plants that can thrive and enhance transformation of contaminants of concern to
non-toxic or less toxic products.
The U.S. Department of Agriculture also provides two databases on plants
(http://Plant-Materials.nrcs.usda.gov/
and
http://plants.usda.gov/).
For
information specifically pertaining to plants used for phytoremediation of
petroleum hydrocarbons, the Phytopet database compiled by the Department of
Soil Science, University of Saskatchewan in co-operation with Environment
Canada is available at http://www.phytopet.usask.ca.
464
465
466
Table 3
Macro- and Micro-nutrients required for healthy plant growth.
Macronutrientsa (-100 ppm)
Micronutrientsb (~1 ppm)
Nitrogen (N)
Iron (Fe)
Phosphorus (P)
Boron (B)
Potassium (K)
Zinc (Zn)
Magnesium (Mg)
Copper (Cu)
Calcium (Ca)
Manganese (Mn)
Sulfur (S)
Molybdenum (Mo)
http://extension.oregonstate.edu/mg/botany/table3.html
b
http://extension.oregonstate.edu/mg/botany/table4.html
467
468
Including all these costs, the start-up cost for phytoremediation at $10,000
- 25,000/ acre is still considerably less expensive than other competing
technologies (Table 4). However, since phytoremediation usually requires five
or more years, it is very important to make sure that funding for operation and
maintenance is available during the life of the project.
4.5. Operation and Maintenance Issues
Operation and maintenance (O & M) is vital to ensure vigorous growth of
plants. Some of the major problems in the field have been weeds, killing frosts
or drought, insect or disease infestation, beaver or deer browse, and damage by
voles. It has been estimated that at least 30 percent of the plants may need to be
replanted in the second or third year. Phreatophytic trees are also a source of
concern since there is a potential for the expanding roots to enter and restrict
flow of subsurface drains and sewers and break power and communication
cables and small pipelines. Further, mowing, pruning, harvesting, monitoring
vegetation for contaminants, irrigation and fertilizer costs should be included in
the initial estimated costs. Jordahl, et al. (2002) provides a good summary of key
siting and O&M issues that occur during the life of a field-scale project [85].
Table 4.
Five-Year Cost Comparison of Phytoremediation by Hybrid Poplar Trees versus
Conventional Pump and Treat [84]
1. Phytotransformation
Design and Implementation
$ 50,000
Monitoring Equipment
Capital
10,000
Installation
10,000
Replacement
5,000
5-Year Monitoring
Travel and administration
50,000
Data collection
50,000
Reports (annual)
25,000
Sample analysis 50,000
TOTAL
$ 250,000
2. Pump and Treat (3 wells and Reverse Osmosis System)
Equipment
$ 100,000
Consulting
25,000
Installation/Construction
100,000
5-Year Costs
Maintenance
105,000
Operation (electricity)
50,000
Waste disposal
180,000
Waste disposal liability
100,000
TOTAL
$ 660,000
469
5. Mathematical Modeling
5.1. Groundwater Capture and Transpiration
One must understand where the water is moving at a site in order to
estimate contaminant fate and transport. For applications involving groundwater
remediation, a simple capture-zone calculation [86] can be used to estimate
whether the phytoremediation "pump" can be effective at intercepting and
extracting the plume of contaminants. Trees can be grouped for consideration as
average withdrawal points. The goal of such a phytoremediation effort is to
create a water table depression where contaminants will flow to the vegetation
for uptake and treatment or volatilization. It is important to realize that organic
contaminants are not taken-up at the same concentrations that are present in the
soil or groundwater. Rather, there is a transpiration stream concentration factor
(a fractional efficiency of uptake) that accounts for the partial uptake of
contaminant (due to membrane barriers at the root surface).
U = (TSCF) (T) (C)
(5)
where:
U = uptake rate of contaminant, mg/day
TSCF = transpiration stream concentration factor, dimensionless
T = transpiration rate of vegetation, L/day
C = aqueous phase concentration in soil- or ground-water, mg/L
A method for estimating the Transpiration Stream Concentration Factor
(TSCF) for eq. (5) was given by eq. (1) and (2).
If the contaminant plume is not completely taken-up by the vegetation,
the plume that remains could be evapoconcentrated; i.e., the mass of
contaminant in the plume will be less due to uptake by vegetation, but the
concentration remaining will actually be greater due to preferential uptake of
water over the contaminants. This is a potential concern for phytoremediation of
groundwater plumes or created wetlands, where a relatively hydrophilic
contaminant can be concentrated on the downstream side of the phytosystem.
Mature phreatophyte trees (poplar, willow, cottonwood, aspen, ash, alder,
eucalyptus, mesquite, bald cypress, birch and river cedar) typically can transpire
3-5 acre-ft of water per year (36-60 inches of water per year). This is equivalent
to about 600-1000 gallons of water per tree per year for a mature species planted
at 1500 trees per acre. Transpiration rates in the first two years would be
somewhat less, about 200 gallons per tree per year, and hardwood trees would
transpire about half the water of a phreatophyte. Two meters of water per year is
a practical maximum for transpiration in a system with complete canopy
coverage (a theoretical maximum would be 4 m/yr based on the solar energy
470
(6)
where:
k = first order rate constant for uptake, yr"1
U = contaminant uptake rate, kg/yr
Mo = mass of contaminant initially, kg
Then, an estimate for mass remaining at any time is expressed by equation (7)
below.
M = Moe"kt
where:
M = mass remaining, kg
t = time, yr
Solving for the time required to achieve clean up of a known action level:
t = -(lnM/M 0 )/k(8)
where:
t = time required for clean-up to action level, yr
M = mass allowed at action level, kg
Mo = initial mass of contaminant, kg
(7)
471
472
degradation rates. Also, it cannot simulate growth of multiple plant species that
might be used in field-scale applications.
6. REGULATORY ISSUES
Compliance with regulatory concerns is a critical factor when considering
remediation of a site. State and federal acceptance of the technology has been
slow but is the product of input by the Interstate Technology and Regulatory
Cooperation Work Group (ITRC), the Superfund Innovative Technology
Evaluation (SITE) program and the Research Technologies Demonstration
Forum (RTDF) program of EPA. The Phytotechnologies Work Team, a part of
the ITRC (www.itrcweb.org), published a Decision Tree (1999) and a Guidance
Document (2001) as a first approximation for whether phytoremediation is
suitable for a particular site. The latter guidance document in combination with
the USEPA document titled "Introduction to Phytoremediation " (EPA 600-R99-107) should be useful in guiding industrial site managers.
Apart from the ITRC, the SITE program and RTDF were also designed to
evaluate the potential of phytoremediation for field-scale purposes.
Phytoremediation has been the subject of six SITE investigations and over 25
field trials by RTDF (http://www.rtdf.org). SITE is a formal program established
by EPA's Office of Solid Waste and Emergency Response (OSWER) and the
Office of Research and Development (ORD) in response to the Superfund
Amendments and Reauthorization Act of 1986 (SARA). Consultants are
responsible for operating the innovative system on site and are expected to pay
the costs of the demonstration, together with site owners. EPA is responsible for
project planning, sampling and analysis, quality assurance and quality control,
preparing reports, disseminating information, and transporting and disposing of
treated waste materials.
Under Superfund laws, EPA (1998) [88] lists nine criteria for
consideration:
1. Overall protection of human health and the environment
2. Compliance with Applicable and Relevant and Appropriate Requirements
3. Long-term effectiveness and permanence
4. Reduction of contaminant toxicity, mobility, or volume
5. Short-term effectiveness (including the length of time needed to
implement the technology and associated risks to workers, residents, and the
environment during that time)
6. Implementability (including availability of goods and services)
7. Cost including capital, operation and maintenance, and monitoring
8. State (and federal) acceptance of the technology and its evaluation of its
performance
473
474
475
476
REFERENCES
[I]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[II]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
477
478
[61] C.C. Wiltse, W. L. Rooney, Z. Chen, A. P. Schwab, and M. K. Banks, J. Environ. Qual.,
27 (1998) 169.
[62] Yateem, A., A.S. El-Nawawy, and N. Al-Awadhi,. Soil and Groundwater Cleanup,
1999, pp. 31-33.
[63] C. H. Chaineau, J. L. Morel, and J. Oudot, J. Environ. Qual., 26 (1997) 1478.
[64] Baker, J.M. 1970. Environ. Pollut., 1 (1970) 27.
[65] Ferro, J. Kennedy, R. Kjelgren, J. Rieder, and S. Perrin, Int. J. Phytoremediation, 1
(1999) 9.
[66] J. Giddens, J. Environ. Qual., 5 (1970) 179.
[67] Amadi, A. A. Dickson, and G.O. Maate, Wat. Air Soil Pollut., 66 (1993) 59.
[68] S. L. Hutchinson, N. K. Banks, and A. P. Schwab, J. Environ. Qual., 30 (2001) 395.
[69] E. J. Joner, S. C. Corgie, N. Amellal, and C. Leyval, Soil Biol. Biochem., 34 (2002)
859.
[70] M. R. T. Palmroth, J. Pichtel, and J. A. Puhakka, Bioresource Technol, 84 (2002) 221.
[71] J. L. Walworth, C. R. Woolard, and K. C. Harris, Cold Regions Sci. Technol., 37 (2003)
81.
[72] T. M. Phillips, A. G. Seech, D. Liu, H. Lee, and J. T. Trevors, Environ. Toxicology, 15
(2000) 99.
[73] D. Breeveld, and M. Sparrevik, Biodegradation, 11 (2000) 391.
[74] D. W. Graham, V. H. Smith, D. L. Cleland, and K. P. Law, Wat., Air, and Soil Poll.,
I l l (1999)1.
[75] L. M. Carmichael, and F. K. Pfaender, Biodegradation, 8 (1997) 1.
[76] C.R. Johnson and K.M. Scow, Biodegradation, 10 (1999) 43.
[77] R.W. Lee, S.A. Jones, E.L. Kuniansky, G. Harvey, B.S. Lollar, and G.F. Slater, Int. J.
Phytoremed., No. 2 (2000) 193.
[78] L. E. Erickson, M. K. Banks, L. C. Davis, A. P. Schwab, N. Muralidharan, K. Reilley,
and J.C. Tracy, Environ. Progress, No. 13 (1993) 226.
[79] J. A. Rentz, B. Chapman, P. J. J. Alvarez, and J. L. Schnoor, Int. J. Phytoremed., No. 5
(2003) 57.
[80] J. M. Lynch, The Rhizosphere, New York, Wiley, 1990.
[81] D. S. Neuman, M. Wagner, J. H. Braatne, and J. Howe, Stress Physiology - abiotic, In:
Biology of Populus and its implications for management and conservation, (eds. R. F.
Stettler, H. D. Bradshaw, Jr., P. E. Heilman, and T. M. Hinckley), NRC Research Press,
National Research Council of Canada, Ottawa, ON, 1996, pp. 423-458.
[82] Ferro, J. Chard, R. Kjelgren, B. Chard, D. Turner, and T. Montague, Int. J. Phytoremed.,
No. 3 (2001) 105.
[83] S. S. Koenigsberg and R. D. Norris, Accelerated Bioremediation Using Slow Release
Compounds, Regenesis Bioremediation Products, San Clemente, CA, 1999.
[84] E.G. Gatliff, Phytoremediation. Ground Water Monitoring Review, Winter/1996.
[85] J. L. Jordahl, M. F. Madison, H. M. E. Smesrud, and M. Q. Motte, (Eds. S. C.
McCutcheon and J. L. Schnoor), In: Phytoremediation- Degradation and Control of
Contaminants, Wiley fnterscience, New York, NY, 2002.
[86] P. A. Domenico and F. W. Schwartz, Physical and Chemical Hydrogeology, NY, John
Wiley & Sons, Inc., 1998.
[87] G. J. Thoma, T. B. Lam, and D. C. Wolf, Int. J. Phytoremed., 5 (2003) 47.
[88] Environmental Protection Agency (EPA), Phytoremediation Handbook for Site
Manager, 1998 Draft.
479
Chapter 17
Department of Process Engineering, Universidad Autonoma MetropolitanaIztapalapa (UAM-I). Apdo. Postal 55-534, 09340 Mexico D.F., Mexico
b
480
481
Although the basic-transfer and reaction mechanisms are the same for all
the BATS, there are different equipment configurations. These can be grouped,
as shown in Table 1, depending on the state of biomass and liquid phase in the
reactor.
2.2. Types of reactors
The principal reactors described in Table 1 are:
2.2.1. Biofllter (BF).
In biofilters (Fig. 3), the polluted air percolates through a moist packed
bed, which supports the microorganisms, that grow on its surface and crevices,
forming a biofilm. Pollutant transformation rates depends on the microbial
density and activity, its bioavailability and the environmental conditions, such as
temperature, nutrients, pH and humidity. The biofilm humidity is one of the
most critical condition to maintain a proper performance, since biological
activity is highly dependent on the water activity (aw). Drying occurs by
incoming air with low humidity and by the heat generated by the biological
reaction [5]. Increased drying rates are obtained with dry air and high
elimination capacity, therefore the air is generally pre-humidified and the
support periodically water sprayed. Biofilters have generally a high void fraction
to limit pressure drop and to reduce ventilation costs.
The supports can be natural and bioactive, inert or a mixture. The natural
bioactive supports are soil, peat, compost, bark etc. They are relatively
482
inexpensive and abundant, and have been used for many applications. They can
retain water and generally contain an initial microbial population with enough
mineral nutrients [6]. Suitable structural characteristics are obtained by mixing
with a coarser fraction, including plastics or ceramics, to prevent high pressure
drop and to limit bed compaction. The natural supports may degrade with time
and lose their structure and water-retaining capacity, inducing channeling and
performance loss [7]. In some cases, re-mixing the support with some fresh
material and nutrients allows to recover the activity [8], but eventually, it will
need to be replaced. With proper maintenance, the support can be used for
several years. As mentioned above, biofilters can use inert, natural or synthetic
supports such as activated carbon, ceramics, lava rock, polyurethane foam,
vermiculite and perlite. On one hand, these supports lack the nutrients required
to sustain the microbial activity, therefore it is necessary to intermittently add
them. On the other hand, they are not degraded and, in theory, could be
engineered to have optimal properties such as controlled head loss, porosity,
adsorptive capacity, etc. This remains an area of active research [2, 3, 4].
The high surface and low water content favor degradation of the lesshydrophilic pollutants (Henry's constant, H >10). Empty bed retention time
(EBRT) is generally between 30 seconds and 2 minutes. Due to the type of
supports used, the height of the packed bed is generally about 0.8 to 1.2 m,
making thus necessary to have a large footprint, which may be a disadvantage
for situations where space is limited.
2.2.2. Biotricklingfilters (BT).
In BT, the polluted air (Fig. 4) flows upflow or downflow through a
packed column where liquid is continuously recirculated. The pollutant is first
solubilized in the falling liquid film and then transferred to the biofilm
developed on the support. The liquid provides moisture, nutrients, pH control to
the biofilm and allows the removal of inhibiting products and excess biomass.
Table 1.
Classification of biological reactors
Biomass
Fixed on a support
Fixed on a support
Liquid phase
Stationary
Flowing
Suspended
Suspended or fixed
Fixed on a membrane
Flowing
Stationary
Flowing
Reactor
Biofilter, BF
Biotrickling filter, BT
Rotating contactors, RC
Bioscrubber, BS
Suspended growth, SR
Membrane, MR
483
484
485
(1)
Where
H: dimensionless Henry's constant
Cg: gas phase concentration (g L"1)
Q: liquid phase concentration (g L"1)
In Table 2, some Henry's coefficients are reported for different pollutants.
For high values of Henry's coefficient (H), the pollutant is slightly soluble in
water. For example, with hexane concentration in air of 1 g m"3, corresponding
486
Henry's coefficient
(non-dimensional)
Methane
41.3 U)
Hexane
30.9
Oxygen
29.1
Hydrogen sulfide
0.92
Toluene
0.25
0.22
Benzene
MTBE (methyl t- butyl ether) [19]
0.026
Ethanol
0.0012
Ammonia
0.0005
(1) http://www.epa.gov/regionl/measure/Natatten.pdf
487
J=-D^l
(2)
ax
where:
J: mass flux (g m"2 s"1)
D :diffusion coefficient (m2 s"1)
Cl: liquid concentration (g m"3)
x: distance in the biolayer (m)
In biofilms, diffusion coefficient may be strongly reduced by the presence
of biomass and EPS. Most of the effective diffusion coefficients in biofilms are
evaluated by either empirical or semi-empirical correlations. In terms of
concentration gradient, two typical cases of biofiltration are found:
1) No diffusion limitation exists and the biofilm is fully active. In this
case, the biological reaction is the limiting step.
2) Diffusion limitation occurs and the biofilm is not fully active, this
condition is referred as diffusion limited. In this situation, a reaction-free zone is
predicted.
2.3.3. Biodegradation of the pollutant in the biofilm
In most of BATS, pollutants are degraded by microorganisms structured
in biofilms mainly composed of microbial cells and EPS, which promote
adhesion. EPS may account up to 90 % of the total. While the biofilm is often
represented as an homogeneous mass with constant thickness, it has, in reality, a
complex structure comprising diverse biological (presence of bacteria, yeasts
and fungi, EPS,...) and physical (cavities, detachment of the biofilm, microbesupport interactions, less-dense zones, etc.) components. Biofilm structure and
ecology are poorly known.
In BATS, organic pollutants serve as carbon and energy sources, and the
dissolved oxygen as electron acceptor. Degradation occurs not only during
growth phase, but also when the net growth rate is zero. The energy released
during degradation is used for growth and maintenance metabolism. The
pollutant uptake rate can be defined as:
(3)
where:
(a.: net cell growth (h"1)
X: biomass concentration (gxL"')
Yxs: biomass yield from the pollutant (gx g poiiutant"1)
m: maintenance coefficient (g poiiutant gx' h"1)
488
Specific growth rate (a) depends on the concentration of the limiting substrate:
(4)
where:
|imax: maximum cell growth (h"1)
Ks: half saturation constant (g L"1)
In BATS, mixed populations are generally found and their behavior can
be described by a net growth rate as:
*$=Mx
(5)
BATS are open biological systems as the air is never filtered. The large
variety of microorganisms (fungi, yeast, and bacteria) come from the initial
inoculum, from the biofilter packing material (compost, peat, etc..) and from the
incoming air [2, 3, 4, 12]. The microorganisms present in the biofilters are
similar to those found in the natural ecosystems or other biological processes
such as compost or water treatment plants. Although bacteria are dominant in
biofilters, fungi are frequently observed. Recent studies showed that some fungi
[20, 21, 22, 23] can degrade toluene and hexane vapors at higher rates than
bacteria.
For a successful biological air treatment, the pollutants have to be
biodegradable. In general, organic compounds with low molecular weight,
highly soluble in water and simple-bond structures are the best candidates.
Alcohols, aldehydes, ketones, and some simple aromatics have very good
biodegradability, while phenols, aliphatic and chlorinated hydrocarbons show
moderate to slow degradation (Table 4). Ethers, like diethyl and dimethyl ethers
are generally easily degraded while MTBE (Methyl tert butyl ether) is reported
to be very recalcitrant.
Table 4
Biodegradability of pollutants [Ref. 24].
Rapidly
489
490
alkanes. Recent studies showed that biofilter performance was improved using
filamentous fungi [22]. An elimination capacity of toluene of 258 g m"3 h"1 (RE =
98 %) was attained. This value was higher than those obtained for bacterial
biofilters [27-28]. It is hypothesized that the aerial fungal mycelia, which are in
direct contact with the gas, give a larger superficial area and allow a direct mass
transfer of the volatile compound. Poor degradation of hydrophobic n-alkanes in
biofilter could strongly be increased using filamentous fungi as shown in Ref.
[23].
Very few reports have been published addressing MTBE biofiltration.
Except for the results obtained by Fortin et al, 1999 [29], elimination capacities
of MTBE are generally lower than 10 g m"3 h"1. MTBE is a highly soluble
compound in water but the ether bond and the ter/-butyl moiety have been
shown to limit MTBE biodegradability. Cometabolism has proved to be a good
way to increase biomass production and MTBE degradation. Microbial consortia
[30], can degrade MTBE by cometabolism with n-alkanes (hexane, pentane and
heptane) present in gasoline. Cometabolism with pentane using a single bacteria
(Pseudomonas aeruginosa) can be used to degrade MTBE in a biofilter packed
with vermiculite [31].
Biofilters used to treat gasoline from a soil vapor extraction operation
showed that higher molecular weight compounds were almost completely
removed while lower molecular weight were less degraded [32]. The
predominant compounds remaining in the outlet of the biofilter were tentatively
identified as methyl-substituted alkanes and cycloalkanes in the C6 to C9 range.
A pilot-scale biofilter system treating gasoline vapors presented total
hydrocarbon-elimination capacities of 16 g m~3 h"1 [33]. Linear alkanes and
aromatics were rapidly degraded, while branched alkanes had lower removal
efficiencies. Pilot-scale biofilter elimination capacities for hexane, isooctane and
toluene were 3.2 ghexane m"3 h"1, 3.1 g,so-octanc m"3 h"1, and 1.5 gtoiucne m"3 h"1,
respectively. Removal efficiencies for toluene were the highest and the most
stable.
4. CONCLUSIONS
BATS are among the established technologies that can be applied to control
VOC and odor emissions. For their choice, the characteristics of the stream
(flow, temperature, presence of particles, humidity, etc.), the pollutant
(composition, concentration, reactivity, solubility and biodegradability) and the
required performance have to be considered. BATS are applicable for a wide
range of volatile pollutants found in the petroleum industry and their
applications are growing continually based on scientific and technological
developments.
491
Table 5
Examples of treatment of volatile petroleum hydrocarbons by BATS
Compounds
BATS
Packing
Cgi,,(gm-3)
EBRT (min)
EC
Ref.
(grnV)
Efficiency
Gasoline
BT
Gasoline
BT
Ethers
MTBE
BF
Pall rings
MTBE a
BT
Vermiculite
MTBE
BT
Celite R-635 b
MTBEC
BT
Aliphatics
Isopentane
BT
Peat moss
Pentane
BT
Vermiculite
Hexane
BT
Hexane
BT
Compost/perlite
50:50 (v/v)
Granular expanded clay
Hexane
BF
Cyclohexane
BT
BF
Aromatics
Benzene
Xylenes
BT
Peat
Toluene
BT
Toluene
BT
Vermicul ite/Granular
activated carbon 85:15 (v/v)
Peat
Toluene
BT
Peat
1000 ppmv
1
140-490 ppmv
2.3
16d
45
0.8
0.9
5
66
0.13 (35ppmv)
1
0.21
1
50
90
0.8
18
7.8
99
3.8
30
5 (1700 ppmv)
3
17.4
35
0.7 (200ppmv)
3
12
26
10
6.3
0.004 (1.2 ppmv)
2
50
40
12
40
21
99
150
>80
80
89
[33]
[32]
89
0.5
Q
O
5U
8.2
1.7
5
1.2
1.5
1
6.2
1.3
66
23
258
98
25
31
165
70
[29]
[31]
[38]
[33]
[34]
[31]
[35]
[23]
[39]
[36]
[37]
[28]
[22]
[27]
[28]
492
REFERENCES
[I]
H. Rafson (ed.), Odor and VOC Control Handbook. McGraw Hill Professional. USA
1998.
[2]
J.S. Devinny, M. Deshusses and T. S. Webster, Biofiltration for air pollution control.
CRC Press, Boca Raton, Fl. USA, 1999.
[3]
C. Kennes and M. C. Veiga MC (eds.), Bioreactors for waste gas treatment. Kluwer
Academic Publishers, The Netherlands, 2001.
[4]
Z. Shareefdeen and A. Singh (eds.) Biotechnology for Odour and Air Pollution,
Springer-Verlag, Germany (in press).
[5]
[6]
[7]
[8]
[9]
[10] J. W. Van Groenestijn, In C. Kennes and M. C. Veiga MC (eds.), Bioreactors for waste
gas treatment. Kluwer Academic Publishers, The Netherlands, (2001) 133.
[II] S. P. P. Ottengraf In: Biotechnology 8, Rehm HJ and Reed G (eds), VCH
Verlagsgesellschaft Weinheim, Germany, (1986) 426.
[12] J. W. Van Groenestijn and P. G. Hesselink, Biodegradation 4 (1993) 283.
[13] S. Ergas, In C. Kennes and M. C. Veiga MC (eds.), Bioreactors for waste gas treatment.
Kluwer Academic Publishers, The Netherlands, (2001) 163.
[14] A. Bielefeldt, In C. Kennes and M. C. Veiga MC (eds.), Bioreactors for waste gas
treatment. Kluwer Academic Publishers, The Netherlands, (2001) 215.
[15] R. P. Bowker, In H. Rafson (ed.), Odor and VOC Control Handbook. McGraw Hill
Professional. USA (1998)8.192.
[16] R. von Rohr and P. Ruediger In C. Kennes and M. C. Veiga MC (eds.), Bioreactors for
waste gas treatment. Kluwer Academic Publishers, The Netherlands, (2001) 201.
[17] B. Davison, J. Barton, T Klasson and, A. Francisco Biotechnol. Bioeng. 68, (2000) 279.
[18] T. Card, In H. Rafson (ed.), Odor and VOC Control Handbook. McGraw Hill
Professional. USA (1998)2.1.
[19] A. Fischer, M. Muller and J. Klasmeier, Chemosphere 54 (2004) 689.
[20] H. H. J. Cox, R. E. Moerman, S. Vanbaalen, W. N. Vanheiningen, H. J. Doddema and
W. Harder, Biotechnol. Bioeng. 53 (1997) 259.
493
495
Chapter 18
1. INTRODUCTION
Crude oils are the liquid fossil residues of aquatic algae, sometimes with minor
contributions from terrestrial plants, that grew in the distant past. Then as now,
we can imagine that most of this material was biodegraded and recycled on an
essentially annual timescale, but a small fraction became buried and underwent
diagenesis and catagenesis to become oil [1]. This process usually took millions
of years, and was dependent on the depth of burial and the temperature. Some
oil dates from the Precambrian (>570 million years ago), but most is rather
younger; the average age of commercially important oil is about 100 million
years, with the majority being from the Jurassic and Cretaceous (180 to 85
million years ago) [2].
Commercially important oil has migrated from its source rock to a
reservoir, and it is not unusual for these reservoirs to leak. If the leak reaches the
surface, it is known as an oil seep. Humans have used material from such seeps
for thousands of years. Early uses include hafting stone axes to their handles, as
an embalming agent, and as a medical nostrum. Genesis (11,3) says that bitumen
was used as the mortar for the Tower of Babel, and Exodus (2,3) that Moses'
basket was made waterproof with a bitumen daub. It seems likely that several
religions started around natural gas seeps, either as eternal flames or as sources
of hallucinogenic vapors [3]. But these were only very minor uses, and it is only
in the last century and a half that oil has come to play a truly central role in
modern society [4]. Terrestrial seeps were the first locations to be drilled when
oil production began in earnest in the nineteenth century, such as the 1860 Drake
well in Pennsylvania, but marine seeps were drilled by the end of the century.
496
497
Fig. 1. Map of the major oil seeps into the World's oceans. Data taken from reference [5].
498
Fig. 2. Oil input into the World's oceans. Data taken from reference [5].
499
500
molecules are either degraded photochemically [23] or are washed from the
atmosphere in rain and then biodegraded [24], likely far from the spill site.
Under particularly aggressive aeration in water, such as in surf, evaporation may
extend to molecules with >30 carbon atoms [25], but evaporation is more
usually limited to molecules with less than about 15 carbon atoms [21]. Thus
evaporation is the likely fate of most of a gasoline spill, three-quarters or more
of a diesel spill, and perhaps 20-40% of a typical crude oil. Heavy fuels, such as
the Bunker fuels used in ships, and bitumen emulsions (Orimulsion, [26]) do
not contain a significant volatile fraction.
Two competing emulsification processes occur as water and oil mix;
water can become entrained in the oil to form an emulsion known as mousse
[27], or oil can disperse into the water column as a suspension of small droplets,
as happened during the 1993 Braer spill off the Shetland Islands [28]. Mousses
are remarkably persistent, and are thought to be the precursors of tarballs that
can last for decades [29]. As we shall discuss below, chemical dispersants that
break emulsions and stimulate the natural dispersion process are effective tools
in the oil spill response "toolkit".
Oil also interacts with small mineral particles in a process originally
termed "Clay-oil flocculation" [30], and now termed "Oil-Mineral Fines
Interactions" [31]. Like dispersion, this dramatically increases the surface area
of the oil.
Aliphatic hydrocarbons are remarkably insoluble, but small aromatics,
particularly the notorious BTEX (benzene, toluene, ethylbenzene and the
xylenes) and small polar molecules such as naphthenic acids dissolve from
floating slicks or dispersed oil, and even from oils immobilized on shoreline
sediments and particles [32]. Again, their eventual fate is biodegradation.
Aromatic hydrocarbons can be photochemically oxidized [33], converting
them to polar products that are probably polymerized species. The process is
most effective on the larger and more alkylated forms, and although such
hydrocarbons are only a minor component of crude oils [2], they have important
toxicological properties [34], and are on the USEPA list of priority pollutants
[35] and the EU list of priority substances in the field of water policy [36]. Since
light cannot penetrate very far into a dark oil slick, photooxidation has little
effect on the bulk properties of spilled oil. Nevertheless it may be important in
generating a polymerized "skin" that enhances the stability of tarballs and
"pavements" on beaches. Layers of immobile, hardened oil and sediment,
termed pavements, form when oil reaches a shoreline as a heavy, thick slick. Oil
becomes trapped in the sediment, and the oil and the sediment become saturated
with each other [37]. Oil incorporated into such pavements is effectively
preserved from weathering processes until this heavy, solidified material is
physically disrupted, so a major goal of spill clean-up operations is to prevent
the formation of pavements.
501
502
503
6. SPILL RESPONSE
6.1. At sea:
When oil is spilled at sea, deployment of mechanical equipment designed
for containment and recovery is often a slow and inefficient, if not ineffective,
response. The rapid spreading of the oil, the slow rate at which mechanical
equipment (once deployed) can encounter spreading oil, and interference from
waves and currents often limits recovery effectiveness to less than 20% of the
oil spilled, significantly less under conditions of severe wind and weather [55,
56]. Unrecovered oil remains in the environment, and undergoes the weathering
processes described above, with the most severe environmental consequences
resulting when oil strands on shorelines [57, 55]. Beached oil increases the
likelihood of contamination for habitats and animals found in subtidal, intertidal
and supratidal areas, which include some of the most productive and diverse
portions of the marine environment.
Burning spilled oil in a contained and controlled manor, so as not to
jeopardize the bulk of remaining cargo or other response assets, can rapidly
remove bulk oil from the water surface. However, it is a logistical and
operational challenge to contain the oil, arrange and control its placement out of
the immediate area of spill response activity, and ensure sufficient oil thickness
to sustain an efficient burn [40]. Many of the logistical and physical constraints
working against efficient mechanical containment and recovery also confound
attempts to collect and burn oil on water. When the oil does burn, the unburned
residue is comprised mostly of the heavy, longer chain hydrocarbons, which are
relatively resistant to ready microbial degradation [58].
Dispersants are widely recognized by many regulatory agencies as an
effective at-sea response that provides a net environmental benefit when
compared to reliance on mechanical recovery alone (see chapter 9). Application
of chemical dispersants facilitates the breakup of the oil slick, moving oil from
the water surface into the water column as neutrally buoyant oil droplets ranging
from 1 to 100 micrometers in diameter, due to the mixing action of waves and
currents. Subsequently, this plume of oil droplets rapidly distributes throughout
the water column, mixing into lateral and deeper water masses and reducing oil
concentrations below levels of concern for marine life. The rate and
effectiveness of this process depends on the nature of the spilled oil (its API
gravity and viscosity, degree of weathering, extent of emulsification, and pour
point) and the ability of the dispersant formulation to mix with the oil.
Dispersants have been an effective aspect of oil spill response over the
past 30 years, with applications to major and smaller oil spill incidents in many
of the world's oceans (Fig. 3). From 1970 through 1998, dispersants have been
used on approximately 37% of oil spills covered in a worldwide database by the
Oil Spill Intelligence Reporter [59]. In addition to countless small-scale tests
504
that have been conducted in laboratories around the world, critical assessments
of dispersant performance have been organized by private and government
research organizations, often cooperatively, using controlled releases of large
volumes of oil and dispersant applications under real world conditions (Fig. 3).
These studies have led to modern dispersant formulations with improved
effectiveness and greater environmental safety. A range of dispersant products
are stockpiled around the world for spill response, and a few have been shown to
be effective over a broad range of oil types and environmental conditions [60].
An important environmental consideration associated with dispersant use
is assessing the environmental tradeoff between intentionally exposing water
column plants and animals to dispersed oil and the often significant effects of
unrecovered oil left to drift at sea to potentially strand on a shoreline. In most
cases, these considerations demonstrate a net environmental benefit to the use of
dispersants because the short-term, transient exposure of water column
communities has much less ecological effect than the prolonged, wide-spread
contamination of oil reaching shorelines [57, 55, 61].
The environmental risks of dispersed oil are further decreased by the
rapid degradation of the small, dispersed oil droplets moving through the water
column, compared to the persistence observed for bulk oil stranded on
shorelines and incorporated into sediments. The large surface to volume ratio
characteristic of micron-sized dispersed oil droplets provides a colonizing
substrate for oil degrading bacteria and a source of degradable hydrocarbon to
support their growth. And, because the small oil droplets are widely dispersed in
the water column, the supply of nitrogen and phosphorus nutrients needed to
support bacterial degradation of the diluted oil is sufficient to maintain a viable
degrader community in association with the oil droplet. Furthermore, laboratory
studies have shown that some dispersants can enhance the initial rate of oil
degradation due to the presence of constituents that serve as initial substrates for
nascent bacterial growth [62, 63].
Laboratory studies of the fate of dispersed oil droplets have characterized
the process by which it becomes a physical substrate for supporting a microbial
community as well as a chemical substrate to support their growth. Within 2 to
4 days, the dispersed oil droplet becomes colonized by oil degrading microbes
[63-65]. Subsequently, this can become a full floating heterotrophic community
consisting of oil, bacteria, protozoa and even nematodes. Macnaughton et al.
[65] reported that by day 16, the size of the clusters increased and sank in test
microcosms, most likely the result of reduced buoyancy due to oil
biodegradation and increased biomass associated with the droplets.
505
Fig. 3. Dispersant response to oil spills. Data taken from reference 59.
6.2. On shore:
If oil reaches shore then the first response is to collect it [66]. Oil typically
lands on only the upper portion of the intertidal zone, and on sandy beaches it
may be possible to collect the oiled sand with mechanical equipment. This was
done, for example, with the spill from the Sea Empress [67]. Particularly heavy
oils may be best picked up by hand, as in the case of the spill from the Prestige
[68]. On rocky beaches it may be possible to wash oil back into the sea where it
can be collected with skimmers, as was done following the spill from the Exxon
Valdez [69].
Once the bulk oil has been removed by physical techniques, residual oil is
eventually naturally biodegraded. Bioremediation aims to stimulate the rate of
natural biodegradation, without causing any additional adverse impact, by at
least partially alleviating whatever is limiting microbial growth. In most porous,
and therefore aerobic shorelines, the most likely limitation is biologically
available nitrogen and phosphorus, and effective bioremediation protocols have
applied various forms of fertilizers to deliver these nutrients.
Research on this topic has been going on for decades in many parts of the
world (Fig. 4; reviewed in 42, 44, 70-77). The simplest approach is to alleviate
the nutrient limitation of oil biodegradation by adding fertilizers. This was the
basis for the successful bioremediation of the spill from the Exxon Valdez [78-
506
80]. Two different fertilizers were used, an oleophilic fertilizer, Inipol EAP22,
designed to adhere to oil and deliver nutrients at the oil-water interface [81] and
a slow-release granular agricultural product (Customblen) that would release
nutrients over many weeks through the beach gravel. Inipol EAP22 is a
microemulsion with an external oil phase of oleic acid and trilaureth-4phosphate, containing an internal phase of urea in aqueous solution, cosolubilized with butoxy-ethanol to adjust the viscosity. It contains 7.4% nitrogen
and 0.7% phosphorus by weight, and was applied with airless sprayers
transported on small shallow-draft catamarans. Customblen is a high quality
agricultural fertilizer designed to release its nutrients over several weeks. It
consists primarily of ammonium nitrate, calcium phosphate and ammonium
phosphate, encapsulated in polymerized linseed oil. Customblen contains 28%
nitrogen and 3.5% phosphorus by weight, and was applied by workers walking
the beaches with broadcast spreaders. An extensive monitoring program
demonstrated that the fertilizer applications were successful at stimulating the
rate of biodegradation some two- to five-fold [78-80]
A quite similar approach was used on a limited scale following the spill
from the Sea Empress [82]. Much of this spill was treated with dispersants while
at sea, and most of the residue that landed on shore was collected by work
crews, but some oil landed on a relatively steep (gradient 10-12.5%) shingle and
pebble beach at Bulwell Bay. Because the beach was so steep, slow-release
fertilizer was placed in 1 m mesh bags, and secured to the beach with steel pegs.
Again, the rate of biodegradation was stimulated more than two-fold on the
fertilized part of the beach.
To our knowledge, these are the only two occasions when bioremediation
by the addition of relatively simple fertilizers was used following a spill, but
there have been field and laboratory tests all over the world that have found
similar results (see Figure 4). All sorts of fertilizers have been used, usually with
success, including soluble and slow release forms of inorganic and organic
nitrogen. Our most recent experiments were on Spitsbergen, the largest island of
Svalbard, Norway, (approximately 78 N, 17' E.) [83, 84]. Slow release and
soluble fertilizers were applied in much the same way they were in Alaska, and
they led to an approximate doubling of the rate of biodegradation, even in this
cold, Arctic environment.
A slightly more complex approach has been championed by Rosenberg
and colleagues [71, 85, 86]. In this case the fertilizer is an insoluble polymer of
urea and formaldehyde, and it is applied together with an oil-degrading bacterial
inoculum that can use this nitrogen source. The approach was apparently able to
stimulate the biodegradation of a small spill (100 tons) of a heavy crude oil on a
sandy beach between Haifa and Acre in Israel in the early 1990's [71, 86].
507
Fig. 4. Bioremediation response to oil spills. Data taken from references 70 - 80.
Others have suggested that what really limits oil biodegradation in the
environment is the absence of effective oil degrading microorganisms, and they
therefore seek to add such organisms. Most recently this has been attempted on
heavy oil spilled by the Nakhodka in the Sea of Japan [87, 88]. Assessing this
work is problematic. The published analyses of the field work rely on digital
photography of representative oiled rocks, and no detailed chemical analyses
have been presented that can be compared with what has been found in other
spills. Earlier microbial inocula did not perform well in standardized tests [89].
An important corollary to any oil spill remediation is that it should have a
net environmental benefit [90]. By aiming to stimulate natural processes,
bioremediation is likely to have minimal adverse effects if carried out carefully
and conscientiously, but there are obvious potential risks that must be evaluated.
Potential adverse impacts include the possibility that the fertilizer applications
might be acutely toxic to marine biota, might stimulate nearshore algal blooms,
might cause the production of biosurfactants that could result in increased
removal of oil from the shorelines by tidal flushing and lead to broader shoreline
impacts, or might generate toxic by-products. Careful monitoring following the
spill from the Exxon Valdez [91] and a field trial in the Arctic [92] failed to
detect any adverse environmental impact from the careful application of
fertilizers, while the rate of hydrocarbon biodegradation was stimulated two- to
five-fold.
508
7. CONCLUSIONS
Oil spill bioremediation technologies epitomize modern environmental
techniques: working with natural processes to remove spilled oil from the
environment while minimizing undesirable environmental impacts. If a floating
oil slick cannot be collected or burnt, chemical dispersants will cause the oil to
move into the water column as tiny droplets with a dramatically increased
surface area that allows rapid biodegradation. If oil reaches a shoreline and
cannot be removed physically, the careful addition of fertilizers will stimulate
oil biodegradation without adverse environmental impact. These two tools are
thus an important part of the toolkit for dealing with accidental and deliberate
releases of oil into the marine environment.
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513
Chapter 19
514
515
Table 1
Main water-soluble contaminants generated by the petroleum
industry.
Family
Aromatic hydrocarbons
Oxygenated compounds
Sulfur compounds
Nitrogen compounds
Compounds
Benzene
Toluene
Ethylbenzene
Xylene
Phenols
Organic acids
Aldehydes
Metyl tert-butyl ether
Hydrogen sulfide
Mercaptans
Ammonium
Amines
Urea
Products
>
N2 + CO2 + Biomass
N 2 + SO 4 (S) + Biomass
->
NO3" + CO 2 + Biomass
->
-
SO 4 (S) + Biomass
H2O + CO2 + Biomass
516
517
The average phenol and alkyl phenols concentrations are 30.5 g 1" and 28.2 g 1' ,
respectively.
There are several reports about the toxic effect produced by phenolic
compounds on the acetoclastic methanogenic activity (AMA) of the granular
sludge. Table 3 shows the inhibitory concentrations that reduce in 50% (IC50)
the AMA. In general, the susceptibility of a granular sludge to the inhibitory
effect of phenolic compounds is affected by the impact of its "acclimation
history". The phenol-degrading acid-forming bacteria are more susceptible to
phenol inhibition than the methanogens [5]. Most granules have a layered
structure that protects bacteria, particularly methanogens. In the case of a
phenol-acclimated granular sludge, it is possible that a phenol-degraders layer
develops in the external zone of the granules, preventing the inward diffusion of
the toxic compounds. This outer layer can prevent the methanogens deactivation
either by reducing the exposure level or by a partial or complete
biotransformation into nontoxic intermediates such as volatile fatty acids [5].
The selection and multiplication of an acetoclastic flora more resistant to those
toxic compounds might be another protection mechanism.
The inhibitory mechanism of the phenolic compounds is governed by their
hydrophobicity that increases the ability of these compounds to solubilize into
the lipid bacterial membranes, altering the membrane functions, such as ion
transports causing cellular lysis. High linear correlations of methanogenic
toxicity data to the logarithm of octanol-water partition coefficients of phenolic
compounds (log P) have been proposed as shown in Fig. 1. This simple model
adequately estimates the IC50 values for anaerobic granular sludge in the
presence of phenolic compounds.
Table 3
Inhibitory concentrations that reduce in 50%
(IC50) the acetoclastic methanogenic activity
of granular sludge (phenol-acclimated and
non-acclimated) in batch assays [6-8].
Compound
Phenol
o-cresol
m-cresol
p-cresol
3,4-dimethylphenol
2-ethylphenol
4-methylphenol
4-ethylphenol
IC50
(mg I"1)
470 - 7802
433 - 844
443 - 919
389-1525
329-378
195-207
657
289
518
Fig. 1. Relationship between IC50 of phenolic compounds and the octanol/water partition
coefficient (Log P). Synthetic "spent-caustic phenols mixture" (X), data from reference [6]
(), data from reference [7] () and data from reference [8]. (A). (1), phenol; (2), 4-methylphenol; (3), 4-ethylphenol; (4), o-cresol; (5), m-cresol; (6), /)-cresol; (7), 3,4-dimethylphenol;
(8), 2-ethylphenol; (9), "synthetic phenols mixture".
Log (l/ICso) = 0.77 Log P - 2.28, r2 = 0.90
519
under methanogenic conditions. A reversible reaction from 2-ethylphenol to 3hydroxy-4-ethylphenol seems to take place, but no further degradation has been
described.
2.1.2. Anaerobic treatment systems for the biodegradation of phenol
Anaerobic treatment of phenolic-bearing wastewaters produced from the
petroleum industry is a viable option. The bioreactor system most commonly
used for the anaerobic treatment of phenolics is the UASB, operating to a certain
organic loading rate (OLR), usually referred to chemical oxygen demand
(COD).
Lab scale UASB reactors have been applied to treat single phenolic
compounds at OLR as high as 6 and 7.2 g COD I"1 d"1 for phenol and />-cresol,
respectively, showing high compound removal efficiencies [11, 12]. However,
effluents from the petroleum industry are expected to contain mixtures of phenol
and cresols as the main COD bearing fractions. Thus, a successful treatment of
these effluents would require a simultaneous degradation of the major phenolic
substrates. Table 4 shows some results of anaerobic treatment of phenolic
compounds mixtures.
Table 4.
Continuous anaerobic treatment results of mixtures of phenolic
compounds treated in upflow anaerobic sludge bed reactors.
Mixture
OLR
(g COD I ' d 1 )
Reference
Phenol
p-Cresol
94
[9]
7.1
91
[9]
2.95
81.8
[9]
8.12
85
[10]
0.66
85
[13]
4.3
[15]
Phenol
/?-Cresol
Phenol
jt?-Cresol
o-Cresol
Phenol
p-Cresol
Phenol
/>-Cresol
o-Cresol
Phenol
m-Cresol
520
521
522
523
(1)
(2)
The reactor configuration, to promote both sulfur formation and
accumulation, was evaluated and reported by Janssen et al. [27] and Alcantara et
al. [28]. The configuration consisted mainly in the separation of aeration process
from the bioreactor. Thus the liquid saturated with oxygen from the aerator
vessel is sent to the reactor (reaction vessel) at a specific rate, which allows the
control of stoichiometric molar ratio between oxygen and sulfide (theoretical
molar ratio, Rmt, O2/S2~). When Rmt is close to 0.5, the sulfide oxidation is driven
to elemental sulfur formation, while a Rmt close to 2 promotes sulfate as the
main product. The performance of the system reported by Alcantara et al. [28]
was inoculated with a sulfoxidizing consortium and it is shown in Fig. 2.
Sulfide oxidation was studied under different dilution rates at steady state
conditions of 0.5, 1, 1.5, 2 and 3 d"1 (zones A, B, C and D, respectively),
maintaining a constant sulfide concentration in the feed solution at 4.0 g I"1.
Elemental sulfur was produced at dilution rates of 0.5, 1, 1.5 and 2. The
maximum sulfur formation occurred at Rmt of 0.5, where 85% of the total sulfur
added to the reactor as sulfide was transformed to elemental sulfur and 92% of it
was recovered from the bottom of the reactor.
Fig. 2. Performance of the recirculation reactor system under different culture conditions.
Capital letters corresponds to the following dilution rates (d"1): A, 0.5; B, 1; C, 2 and D, 3.
Subtitle letters show the Rmt evaluated: 2: b, c; 1.5: d; 1, e, k; 0.75: f, 1: m; 0.5: a, g; 0.35: h;
0.25: i; 0.15: j . Sulfide influent (), sulfate (), elemental sulfur (A), thiosulfate (o) and
sulfide effluent (A).
524
(3)
(4)
Sour waste streams, including sour water, sour gases and refinery spentsulfidic caustics, have been successfully treated using Thiobacillus denitrificans.
For instance, the organic compounds such as benzene, toluene and phenol are
525
526
527
528
Fig. 3. Proposed metabolic pathway for aerobic MTBE biodegradation Adapted from Fayolle
et al. [49] and Steffan et al. [57].
529
Table 5
Technology Performance for MTBE biological removal
Treatment
Scale
Microorganism
MTBE initial
concentration
Treatment period or
removal rate
Reference
Field
Field
MC-100
Native microorganisms
PM-1
ENV425
Native microorganisms
7 mg I"1
1.5 mgT 1
150-200 days
4 days
[62]
[63]
320 mg r 1
19.6 mgl- 1
90 days
60 days
[65]
[66]
Hydrogenophaga flava
Mixed culture
Cytophaga-Flexibacter-Bacteroides
1000 mgl"1
5 mg T1
42 mg I"1 h"1
2.5 mg I"1 rf1
[67]
[68]
ENV735
10 mg T1
10 mg 1"' in 15 min
[65]
10-50 mgT 1
29 mg r 1 h '
[69]
9 mg r 1 If1
In situ treatment
Field
Field
Ex situ treatment
Laboratory
Membrane
Laboratory
Fluidized
bioreactor
Laboratory
Biotrickling filter
Biofilter
Biofilter
N.S. not specified
Field
Laboratory
Field
Mixed culture
Mixed bacterial culture
N. S.
9.6 mg r 1
8.25 mg I"1
10 mgl"' and
15mgr'TBA
Laboratory
Laboratory
Laboratory
F-consortium
PM-1
P. aeruginosa
0.8 mg I"1
100 mg 1"'
1.1-12.3 mgl"1
50 mg r1 h"1
58 mg r1 h"1
l.Smgr'h' 1
ls.smgr'h"
[69]
[70]
[71]
[72]
[73]
[74]
531
532
similar to biofilters, but they have an aqueous phase trickling over the
packed bed. The liquid contains essential nutrients and it is usually
recycled. Biotrickling filters are more complex than biofilters but are
usually more effective, especially for the treatment of compounds that
generate acidic by-products (see chapter 17).
5. PERSPECTIVES
It is expected that more stringent environmental regulatory actions will be
taken by governments, worldwide. As water is the most important resource
for human, animal and plant life, holistic environmental wastewater
management will continue to gain in importance with time [75].
During the last decade significant efforts were devoted to the
development of technologies for process integration targeting energy
conservation and waste reduction. Great efforts have been done in industries
in order to increase the water conservation and reduce wastewater [76].
However, these integrated technologies will produce less and more
concentrated wastewater whose characteristic would lead to a complete
redesign of the biological wastewater treatment processes that are currently
applied on the process industry. Consequently, facility upgrading,
innovative and sustainable treatment technologies would reshape the
petroleum industry.
The anaerobic processes for the treatment of organic compounds in
industrial wastewater offer important advantages over conventional aerobic
processes. To date, less than 15% of the nearly 1600 full-scale anaerobic
wastewater treatment systems are used by the chemical and petrochemical
industry. However, as the range of compounds that are found to be
biodegraded under anaerobic conditions has increased enormously lately, a
large potential expansion seems possible in the future [22]. Thanks to a
combination of a simple construction and a high volumetric treatment
capacity, the UASB reactor is the dominant concept in the industrial
anaerobic wastewater treatment and it probably will keep reigning in the
future. Nonetheless, higher loaded expanded granular sludge bed reactors
will gradually replace at least part of the UASB applications.
In the case of wastewater streams rich in reduced sulfur compounds,
the new sulfur biotechnology has allowed the development of reactor
systems to remove sulfide producing elemental sulfur. This technology has
been adapted for the sweetening of natural gas [30] and more recently for
liquefied petroleum gas (LPG), which contains predominantly sulfide and
lower alkylthiols [77]. The latter process involves three steps: 1) extraction
of the sulfur compounds from the liquefied hydrocarbon phase to a mild
533
Fig. 4. Schematic representation of the close water cycle in the petroleum industry.
534
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537
STUDIES IN SURFACE SCIENCE AND CATALYSIS
Advisory Editors:
B. Delmon, Universite Catholique de Louvain, Louvain-la-Neuve, Belgium
J.T. Yates, University of Pittsburgh, Pittsburgh, PA, U.S.A.
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