Producción de Metano e Hidrógeno A Partir de Biomasa

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Production of Methane and Hydrogen

from Biomass through Conventional and
High-Rate Anaerobic Digestion Processes
a b a b
Burak Demirel , Paul Scherer , Orhan Yenigun & Turgut T. Onay
b
a
Hamburg University of Applied Sciences, Lifetec Process
Engineering , Lohbrgger Kirchstrasse 65, 21033, Hamburg, Germany
b
Bogazici University, Institute of Environmental Sciences , Bebek,
34342, Istanbul, Turkey
Published online: 28 Jan 2010.
To cite this article: Burak Demirel , Paul Scherer , Orhan Yenigun & Turgut T. Onay (2010)
Production of Methane and Hydrogen from Biomass through Conventional and High-Rate Anaerobic
Digestion Processes, Critical Reviews in Environmental Science and Technology, 40:2, 116-146, DOI:
10.1080/10643380802013415
To link to this article: http://dx.doi.org/10.1080/10643380802013415
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Critical Reviews in Environmental Science and Technology, 40:116146, 2010
Copyright Taylor & Francis Group, LLC
ISSN: 1064-3389 print / 1547-6537 online
DOI: 10.1080/10643380802013415
Production of Methane and Hydrogen from

Biomass through Conventional and High-Rate
Anaerobic Digestion Processes
BURAK DEMI REL,1,2 PAUL SCHERER,1 ORHAN YENI GUN,2

AND TURGUT T. ONAY2
1
Hamburg University of Applied Sciences, Lifetec Process Engineering, Lohbrugger

Kirchstrasse 65, 21033, Hamburg, Germany

2
Bogazici University, Institute of Environmental Sciences, Bebek, 34342, Istanbul, Turkey
Anaerobic digestion processes have often been applied for biologi-

cal stabilization of solid and liquid wastes. These processes generate
energy in the form of biogas. Recently, high-rate methane and hy-
drogen fermentation from renewable biomass has drawn much
attention due to current environmental problems, particularly re-
lated to global warming. Therefore, laboratory-scale research on
this topic has significantly accelerated. The primary aim of this
review paper is to summarize the most recent research activities
covering production of methane and hydrogen via both conven-
tional single and high-rate two-phase anaerobic digestion processes
of natural sources of biomass.
KEY WORDS: anaerobic digestion, biogas, biomass, hydrogen,

methane, renewable energy, two-phase anaerobic digestion
INTRODUCTION
Conventional single-phase and high-rate two-phase anaerobic digestion pro-

cesses have frequently been employed in order to treat both soluble and
solid types of domestic and industrial wastes. The most significant outcome
of anaerobic digestion processes is that they generate energy in the form of
This study was supported by the Boazici University Research Fund, project number
02S103.
Address correspondence to Orhan Yenigun, Bogazici University, Institute of Environ-
mental Sciences, Bebek, 34342, Istanbul, Turkey; E-mail: yeniguno@boun.edu.tr
116
Production of CH4 and H2 through Anaerobic Digestion 117
biogasnamely, methane and hydrogen. Therefore, due to current imper-

ative environmental issues such as global warming, ozone depletion, and
formation of acid rain, substitution of renewable energy sources produced
from biomass, such as methane and hydrogen, produced through anaerobic
digestion processes will definitely affect the demand and consumption of
fossil-fuel derived energy soon.
Biomass is a flexible feedstock that can be converted to solid, liquid, and
gaseous fuels by chemical and biological processes.1 Furthermore, according
to the European Union, biomass will contribute 83% to the increased use of
renewable sources by the year 2010, and will have a major role in substitution
of fossil fuels with renewable resources.2 In particular, the production of
methane through anaerobic digestion of biomassnamely, energy crops
and organic wasteswould benefit society by providing a clean fuel from
renewable feedstocks.3 Presently, there exist 20 centralized and more than 35

farmscale biogas plants in Denmark, where digestion of manure and organic
wastes have been carried out to produce renewable energy.4
Special emphasis was initially focused on anaerobic digestion of munici-
pal solid wastes for bioenergy production almost a decade ago.57 Biological
conversion of biomass to methane (CH4 ) by anaerobic digestion processes
including hand- and mechanically sorted municipal solid waste, various types
of fruit and vegetable solid wastes, leaves, grasses, woods, weeds, and ma-
rine and freshwater biomasshas previously been discussed.8 Essentially, in
order to reduce carbon dioxide (CO2 ) emissions according to the Kyoto pro-
tocol, the applications of anaerobic digestion processes have recently been
evaluated more significantly in detail.912
In addition to methane, hydrogen (H2 ) can also be produced biologically
from renewable sources, such as biomass and/or industrial wastewater efflu-
ents. Hydrogen is not chemically bound to carbon; therefore, using hydrogen
produced from renewable sources will not contribute to CO2 emissions, acid
rain, or ozone depletion.13,14
A vast amount of literature already exists about the applications and
benefits of the anaerobic digestion processes for waste treatment, particularly
focusing on upgrading process efficiency and performance. Consequently,
the effects of both operational and environmental parameters on process
performance were often explored in order to obtain high treatment effi-
ciency. As the world seeks clean energy source alternatives nowadays, more
attention is currently being directed toward biological production of methane
and hydrogen from biomass using anaerobic digestion processes, as biomass
seems a feasible source for renewable energy production at the moment. For
example, it was recently reported that Canada generated approximately 1.45
108 t of residual biomass annually, which was estimated to contain an
approximate energy value of 2.28 109 GJ. This value accounted for about
22% of Canadas current annual energy use.15
118 B. Demirel et al.
The primary objective of this review paper is to summarize the most

recent research activities covering biological production of methane and hy-
drogen via conventional single- and high-rate two-phase anaerobic digestion
processes from various feedstocks/biomass and wastewater types. In the pa-
per, the biological production of methane through single- and two-phase
anaerobic digestion processes will be summarized. Then, biological produc-
tion of hydrogen via anaerobic fermentation will be discussed. Finally, areas
where further attention required will be presented.
PRODUCTION OF METHANE BY ANAEROBIC DIGESTION

PROCESSES
Single-Phase Anaerobic Digestion Process

Conventional single-phase anaerobic digestion process is often employed to
recover bioenergy (methane) from biomass (energy crops), various types of
solid wastes, and industrial wastewaters. A summary of these recent research
activities will now be summarized in this section of the paper. An overview
of these studies is also presented chronogically in Table 1.
In a former laboratory-scale study, in which dairy wastewater was used
as the substrate, the incorporation of a biofilm support in continuous stirred
tank reactors (CSTRs) was evaluated.16 CSTRs with biofilm support systems
provided 20% better improvement in methane yield, in comparison to the
digesters operated without biofilm support systems. According to the authors,
the biofilm support system improved the efficiency of the digesters without
any provision of biomass recycling.
Manure is often used during co-digestion with other organic wastes
or energy crops. Methane production characteristics of a low-concentration
liquid swine waste was investigated, using a conventional dispersed growth
anaerobic fermenter operated at 35 C, and in a hydraulic retention time
(HRT) range between five and two days.17 Methane productivity ranged
from 0.36 to 0.22 L CH4 /g VS added, for the five and two days of HRT,
respectively. Mean percentage of the methane in digester biogas ranged
between 63.4 and 65.2%, at five and three days HRT, respectively. However,
the digester indicated stress conditions during the operation at a HRT of two
days.
In a similar work, anaerobic conversion of a mixture of pig manure,
fish oil waste, and waste from bentonite of edible oil filtration process for
biogas production was investigated using a continuously stirred laboratory-
scale anaerobic digester operating at 30 C and a HRT of 15 days.18 An
average methane content of 65% was obtained during the experiments, with
a maximum methane production of around 74% in digester biogas.
TABLE 1. Applications of conventional single-phase anaerobic digestion processes for bioenergy recovery
Temperature HRT Methane Methane

Substrate type Application status Digester type ( C) (day) productivity content (%) Reference
Liquid swine waste Dispersed growth 35 532 0.36 L CH4 /g VS 63.4 (5 day HRT) 17
anaerobic added (5 day 65.2 (3 day HRT)
fermenter HRT)
Pig manure + fish oil Laboratory-scale Continuously stirred 30 15 73.6 18
waste + waste from anaerobic
bentonite of edible digester
oil filtration process
Brewery wastewater Laboratory-scale Anaerobic contact 0.28.035 m3 6779 21
with ultra CH4 /kg CODrem.
filtration
Cattle dung + digested Laboratory-scale Batch fermenter 2023 5560 24
slurry
Hog + poultry waste Laboratory-scale Batch fermenter 35 130 20 ml 25
CH4 /g VS
destroyed
Pharmaceutical Pilot-scale UASB 3036 4.5 67 cm3 /CH4 g 26
VSS/day
Fodder beet silage Laboratory-scale Continuous 5.515 40.1 2 m3 5862 28
anaerobic CH4 /ton fodder
digester beet silage
Fruit and vegetable Laboratory-scale Semi-continuous 35 20 64 29
waste tubular digester
Olive mill solid waste Laboratory-scale CSTR 35 408.3 1.243.79 L CH4 / 30
(d L)
Pineapple peel Laboratory-scale 30 0.67 m3 /kg VS 65 33
added
Pineapple peel Pilot-plant 2533 25 4365 33
Municipal garbage Laboratory-scale 2529 6272 34
Fruit and vegetable Laboratory-scale Tubular anaerobic 55 5862 35
waste digester
Fodder beet silage Laboratory-scale Continuous 42 8.3 L/(d L) (total 37
gas production)
119
(Continued on next page)
TABLE 1. Applications of conventional single-phase anaerobic digestion processes for bioenergy recovery (Continued)
120
Temperature HRT Methane Methane
Substrate type Application status Digester type ( C) (day) productivity content (%) Reference
Potato tuber and Laboratory-scale CSTR 35 0.13-0.15 m3 /kg 42

potato byproducts VSadded
Waste activated sludge Full-scale 2040 0.50.6 m3 /kg 44
VSdestroyed
(specific gas
production)
Full-scale biogas plant 35 20 3101 m3 /d (total 46
gas prod.)
Cow manure Laboratory-scale 11 and 35 6.433.61 CH4 /kg 50
VS (11 C)
49.6131.31
CH4 /kg VS
(35 C)
Llama manure Laboratory-scale 11 and 35 3.319.31 CH4 /kg 50
VS (11 C)
35.684.11
CH4 /kg VS
(35 C)
Barley waste 363 L CH4 /kg VS 51
Dairy manure Pilot-scale 55 13 56 52
Dairy manure 125166 L CH4 /kg 53
VS
Potato waste CSTR Thermo 0.650.85 L/g 5850 57
(biogas yield)
Food waste 1028 348435 ml/g VS 73 (ave.) 58
Sugar beet top Lab/pilot Single-stratified bed 0.310.36 m3 59
CH4 /kg VSadded
Sugar beet silage Laboratory-scale Continuous flow 42 25 0.72 L/gVS/d (as 63 (ave.) 60
spec. GPR)
Whey solution was used as the substrate for methane production, which
was actually a simple type of substrate for anaerobic digestion.19 An anaero-
bic fixed-bed reactor was operated in this study, at HRTs of 15 and 10 days,
at 37 C. During continuous fermentation at 10 and 15 days of HRT, about
90% of chemical oxygen demand (COD) was converted to biogas.
Laboratory-scale anaerobic digesters were operated for the anaerobic
conversion of municipal grey waste to biogas.20 The methane content in
digester biogas varied between 60 and 65% during the entire experimental
work.
In addition to dairy wastewater, energy recovery from anaerobic treat-
ment of brewery wastewater was also investigated in a pilot-scale anaerobic
contact digester coupled with an UF (ultrafiltration) membrane unit.21 The
anaerobic contact reactor was operated in a pH range between 6.9 and 7.2,
and at 36 1 C, up to an organic loading rate (OLR) of 28.5 kg COD/m3 /day.

The percentage of methane in digester biogas ranged between 67 and 79%,
with a corresponding methane yield of 0.280.35 m3 CH4 /kg CODremoved .
Activities of various microorganisms and hydrolytic enzymes were eval-
uated in a laboratory-scale work in order to investigate anaerobic digestion
of damaged wheat grains.22 Utilization of Aspergillus and Bacillus and hy-
drolytic enzymes resulted in a methane production of 155 to 220 L/kg TS
(total solids).
Applications of response surface methods (RSM) in anaerobic co-
digestion of multi-compenent agro-wastes was evaluated in laboratory-scale
digesters operated at 35 C.23 The authors reported that RSM could success-
fully be used to predict the optimum mixing ratios during anaerobic co-
digestion of multi-waste components.
Anaerobic conversion of pure cattle dung and cattle dung mixed with
10% digested slurry was investigated in batch fermenters, at an ambient
temperature of 2023 C, for the production of biogas.24 According to the
experimental findings of this study, the addition of digested slurry to cattle
dung resulted in a higher rate of biogas production and shorter digestion
periods. The authors recommended mixing digested slurry for increasing
biogas production from cattle dung. Because anaerobic digestion is com-
monly used to treat animal wastes, this recommendation could be useful to
increase biogas production from various animal wastes.
In addition to continuous systems, anaerobic batch experiments were
also carried out to co-digest hog and poultry wastes at 35 C.25 Biogas yield
and methane production were determined as 200 30 ml/g VSdestroyed and
130 20 ml/g VSdestroyed , respectively. According to the authors, superior
biogas and methane yields indicated that co-digestion of hog and poultry
wastes seemed a feasible option for waste disposal and bioenergy recovery
from these wastes.
High-rate anaerobic treatment of industrial wastewaters is another fea-
sible option to produce biogas. An upflow anaerobic sludge blanket (UASB)
reactor was used to treat fermentation-based pharmaceutical wastewater.26

The reactor was operated up to an OLR of 10.7 kg COD/m3 /day, reaching
about 94% COD removal. With an OLR of 6.1 kg COD/m3 /day, a yield of
67 cm3 CH4 /g VSS/day could be obtained. Through specific methanogenic
activity test, it was found that the yield corresponded to 94% of the potential
acetoclastic methane production rate.
The effects of certain antibiotics, which are commonly used in the treat-
ment of pigs, were studied on the anaerobic digestion of pig waste slurry.27
As a result of batch experiments conducted at 37 C, the authors concluded
that the presence of antibiotics in pig waste slurry could cause problems, par-
ticularly from a biogas production point of view, during anaerobic treatment
of this wastewater type. However, information regarding both the degree
of inhibition of methane production and the part of the anaerobic micro-
bial community adversely affected by antibiotics were not presented by the

researchers.
Continuous laboratory-scale anaerobic digesters were run in order to
investigate continuous biogas production from fodder beet silage as a mono-
substrate without the addition of manure.28 The methane content in digester
biogas ranged between 62 and 64%, resulting in a methane yield of 40.1
2 m3 CH4 /ton fodder beet silage.
Experiments were carried out to study the anaerobic conversion of fruit
and vegetable waste into biogas, using a semi-continuous mixed mesophilic
tubular digester, operated at a HRT range between 12 and 20 days.29 At a
HRT of 20 days and a feed concentration of 6% total solids (TS), the methane
content of the digester biogas was about 64%.
Anaerobic digestion of a two-phase olive mill solid waste was investi-
gated in a laboratory-scale completely stirred tank reactor at 35 C, operated
at HRTs of 40 and 8.3 days, with influent substrate concentrations of 34.5,
81.1 and 113.1 g COD/L.30 For substrate concentrations of 34.5, 81.1, and
113.1 g COD/L, the maximum volumetric methane production rates were
determined to be 1.24, 3.30, and 3.79 L CH4 STP/(L d), respectively.
Anaerobic digestion of cattle manure and a mixture of cattle manure
with glycerol trioleate was evaluated in laboratory-scale CSTRs, operated
at 37 C and a HRT of 15 days.31 The authors observed that the anaerobic
digester treating manure and lipids exhibited a better performance than that
of the digester treating only cattle manure, in terms of both specific methane
yield and volatile solid (VS) removal.
The effect of temperature variations on anaerobic digestion of biomass
is another topic that has received much attention lately. Laboratory-scale ex-
periments were conducted in order to compare thermophilic and mesophilic
anaerobic digestion of sludge.32 A cylindrically shaped well-mixed anaer-
obic reactor with a working volume of 20 litres was operated at a HRT
range between 1 and 10 days. According to the experimental results ob-
tained, the authors concluded that thermophilic digestion was much faster
than mesophilic digestion, producing more biogas in a shorter HRT or with

smaller digester volumes.
Anaerobic digestion of pineapple processing waste for methane gener-
ation was evaluated in both laboratory- and pilot-scale studies.33 During the
laboratory-scale work, digester biogas contained 65% methane, with a biogas
yield of 0.67 m3 /kg VS added, using ensilaged pineapple peel as substrate
(at 30 C). During anaerobic conversion of fresh and dried pineapple peels
as substrates, the methane content in digester biogas varied between 41 and
51%. In pilot-plant studies, at a loading rate of 60 kg TS/m3 /day and a HRT of
25 days, methane content in biogas ranged from 43 to 65% for a temperature
range between 25 and 33 C.
The organic fraction of municipal solid waste (OFMSW) has always
been an attactive substrate for production of methane by anaerobic digestion
process. However, pre-treatment of MSW prior to digestion is the initial step.

A lab-scale batch digestion of municipal garbage was investigated at average
temperatures of 25 and 29 C, with a substrate concentration range between
45 and 135 g TS/L.34 During the study, bioprocess conversion efficiency was
determined to be around 85%. The methane content of the biogas produced
from the reactors varied between 62 and 72%.
In a similar study, the performance of a lab-scale anaerobic tubular di-
gester treating fruit and vegetable waste was compared, under psychrophilic,
mesophilic, and thermophilic temperature ranges.35 The digester was oper-
ated semi-continuously, at a HRT range between 10 and 20 days, with sub-
strate concentrations of 4, 6, 8 and 10% TS. The authors reported that the
performance of thermophilic digestion was higher than that of psychrophilic
or mesophilic digestion, by 144 and 41%, respectively, in terms of average
biogas production. The best biogas production was obtained at a HRT of 10
days and 55 C, with a feed concentration of 4% TS.
In addition to investigating the effect of environmental and operational
parameters on the performance of anaerobic biogas digesters, process mon-
itoring and control have also drawn much attention recently. A new control
strategy was proposed to operate anaerobic digesters efficiently at high load-
ing rates.36 This strategy consisted of measuring pH and biogas production
rate and changing the organic loading rate (OLR) by manipulating the influ-
ent flow rate. Furthermore, a Fuzzy logic control system was also developed
for continuous biomethanization of renewable sources and other organic
substrates.37 A stable and reliable fermentation of fodder beet silage as the
sole substrate could be carried out at a HRT of 6.5 days and an OLR of 14.3
kg VS/m3 /d, with a volumetric gas production of 8.3 L/(d L).
Anaerobic batch digestion of potato waste and co-digestion of potato
waste with sugar beet was investigated in a lab-scale work.38 The authors
reported that the co-digestion of potato waste with sugar beet leaves resulted
in a higher methane yield between 31 and 62%, as compared with digestion
of potato waste alone. For the potato waste, the highest methane yield was
determined to be 0.32 litres CH4 /g VSadded , while for co-digestion of potato

waste and sugar beet leaves, the highest methane production was 1.63 litres
(for 24% potato waste + 16% beet + 60% TS inoculum). In addition to oper-
ational and environmental factors, it also seems clear that the characteristics
of a substrate are another significant parameter that affects the performance
of a biogas digester.
Laboratory-scale anaerobic batch digestion of waste activated sludge
was investigated, after recovery of phosphorus (P) from sludge.39 The exper-
imental results obtained indicated that the sludge processing for P recovery
improved digestion efficiency and methane productivity, both at mesophilic
(37 C) and thermophilic (55 C) temperatures.
Methane productivity of manure, straw, and solid fractions of manure
was studied in laboratory-scale anaerobic batch experiments at 35 C.40 The
volumetric methane yield of straw was found to be higher than that of

the total manure and the solid fraction of manure, as it contained a higher
percentage of VS.
The influence of temperature (50 and 60 C) on the performance of
CSTRs digesting cow manure was investigated, at HRT levels of 10 and 20
days.41 The results demonstrated that the methane production rate at 60 C is
lower than that of at 50 C, at all HRT levels applied. Furthermore, the authors
also reported that the concentration of free ammonia affects the performance
of the acetate-utilizing bacteria, and the hydrolysis and acidification stages
of anaerobic digestion process as well.
Anaerobic semi-continuous co-digestion of potato tuber and its indus-
trial byproducts (potato stillage and potato peels) with pig manure was
investigated in a laboratory-scale study, using CSTRs operated at 35 1 C.42
At a loading rate of 2 kg VS/m3 /d, the methane yields were determined to
vary between 0.13 to 0.15 m3 /kg VSadded .
The effect of various kinds of inorganic adsorbent zeolites (morden-
ite, clinoptilolite, zeolite 3A, zeolite 4A), clay mineral (vermiculite), and
manganese oxides (hollandite, birnessite) on methane production from
ammonium-rich organic sludge during anaerobic digestion was investigated
in batch tests at 35 C.43 The natural mordenite was found to enhance
methane production during anaerobic digestion of ammonium rich sludge.
In a full-scale work, the performance of mesophilic anaerobic digesters
treating waste activated sludge was evaluated.44 The anaerobic digesters
were operated at a HRT range between 20 and 40 days, and at an organic
loading rate (OLR) of about 1 kg VS/m3reactor /day. For a feed substrate con-
centration between 2.6 and 3.9% TS, the specific gas production was found
to vary between 0.5 and 0.6 m3 /kg VSdestroyed .
Batch, continuous single-phase, and continuous two-phase anaerobic
digestion of fruit and vegetable wastes were discussed in detail in a more
recent review paper, particularly in terms of energy production.45 The au-
thors concluded that among those processes, continuous two-phase systems
seemed a promising method for treating these wastes with high efficiency,
in terms of degradation yield and biogas productivity.
A comprehensive analysis, in terms of technical-economical aspects, of
a full-scale biogas power plant was recently reported.46 For a feed substrate
concentration of 6.794% TS and an input of 6202 kg TS/d, the biogas plant
could produce a total gas volume of 3101 m3 /d, at a residence time of 20
days and 35 C.
Anaerobic codigestion of two different wastes (fresh vegetable waste
and precooked food waste) with agro-industrial wastewater treatment sludge
was investigated in a laboratory-scale work.47 Co-digestion of fresh vegetable
waste and sludge mixture provided higher methane yields after start-up (37%
at high organic load and 57% at low organic load).
An anaerobic CSTR and an anaerobic filter were used for the produc-
tion of biogas from steam-treated municipal solid waste wastewater.48 In

the anaerobic CSTR, biogas production was observed to be between 0.02
and 0.29 kg CH4 /m3 /day, while for the anaerobic filter, biogas production
ranged between 0.04 and 0.47 kg CH4 /m3 /day. However, as the CSTR re-
ceived a wastewater containing suspended solids, the anaerobic filter re-
ceived a wastewater relatively free from suspended solids, which probably
affected the performance of both reactors in terms of biogas production and
composition.
The presence of fats and lipids has often caused several problems dur-
ing anaerobic digestion processes. Anaerobic co-digestion of a simulated
fraction of MSW and fats of animal and vegetable origin was conducted in
a semi-continuous pilot-scale mesophilic plant at a HRT of 17 days.49 After
a short period of adaptation, total fat removal was found to be over 88%.
Co-digestion of OFMSW and fat-containing wastes appear to be a promising
method to eliminate such wastes and obtain biogas as a renewable energy.
Biogas production from llama and cow manure was studied at high al-
titude using semi-continous lab-scale bioreactors.50 The effects of pressure
(495 and 760 mmHg), temperature (11 and 35 C), and HRT (20 and 50 days)
and the content of manure in slurry (10, 20, and 50%) on biogas production
were investigated. Temperature was found to be the most significant param-
eter, while HRT and the manure content seemed to have fewer effects on
the process performance. In addition, the pressure for the range investigated
also seemed to have no significant effect on the process performance.
Attempts were carried out in order to enhance production of methane
from barley waste, which resulted from production of instant coffee
substitutes.51 Anaerobic co-digestion of barley waste (40%) with kitchen
waste (60%) resulted in a methane production of 363 L CH4(STP) /kg VS,
along with a TS and TVS reductions of 61 and 67%, respectively.
A pilot-scale thermophilic anaerobic digester was operated in a cold re-
gion to produce biogas from dairy manure.52 At an average OLR of 6.75
kg/m3 /day and a HRT of 13 days, the average biogas production was
150 m3 /day, with 56% methane in digester biogas, at an average ambient

temperature of 23 C.
In a similiar work, manures received from dairy systems were anaer-
obically digested to produce biogas as a renewable energy source.53 The
methane yield obtained varied from 125 to 166 L CH4 /kg VS, which de-
pended on the milk yield and diet of the dairy cow.
Anaerobic co-digestion of dairy manure with sugar beets was also stud-
ied using continous-flow lab-scale digesters operated under thermophilic
conditions.54 At a HRT of 20 days, the average methane yield for dairy ma-
nure and 40% beet top mixture was 1.49 times more than that of 100% dairy
manure.
The effect of oleate on the performance of biogas reactors treating
mixtures of cattle and pig manure was studied using thermophilic CSTRs.55
The addition of 2 g/L oleate severely inhibited the process, as indicated by

a sudden increase in VFA production and an immediate drop in methane
production. However, after 20 days of acclimation, the reactors exhibited a
lower VFA production and a higher methane production.
The degradation efficiency of biogas plants in Denmark was investi-
gated, which processed manure and food wastes to generate biogas.56 The
findings of this study indicated that the residual biogas potential in the main
digestion effluent is originating mainly from undegraded particulate matter in
the biomass, which probably resulted due to insufficient HRT for hydroliza-
tion to take place.
Potato processing wastes were anaerobically digested to produce bio-
gas, using thermophilic CSTRs.57 In an OLR range from 0.8 to 3.4 g/L/d,
biogas yields and methane composition were determined to be 0.850.65
L/g, and 5850%, respectively. Both biogas yield and methane percentages
decreased with an increase in OLR.
Food waste was characterized for its potential use as a feedstock for
anaerobic digestion.58 After digestion at retention times of 10 and 28 days,
the methane yield was determined to be 348 and 435 ml/g VS, respectively.
The average methane content was 73%, with an average VS destruction of
81% at 28 days of digestion. The findings of this study indicated that the
food waste was a highly desirable substrate for anaerobic digestion, due to
its high biodegradability, nutrient content, and methane yield.
Anaerobic high-solids single-stage stratified bed digesters have been
found to be simple and flexible designs for small-scale reactors, which are
located in medium- to low-technology environments.59 Fed-batch experi-
ments using sugar beet tops in both pilot and lab-scale studies at an average
loadings of 2 kg VS/m3 /d resulted in average biogas production rates of 1.2
to 1.4 m3 /d and methane yields of 0.31 to 0.36 m3 /kg VSadded , respectively.
Single-phase mesophilic continuous anaerobic digestion of sugar beet
silage (without addition of manure) was investigated in a lab-scale work,
with a HRT range of between 95 and 15 days and an OLR range between
0.937 and 6.33 g VS/L/d.60 The highest specific gas production rate of 0.72
L/gVS/d was obtained at a HRT of 25 days and a pH of 6.80. The methane
content of biogas was around 63%.
Two-Phase Anaerobic Digestion Process

A two-phase anaerobic digestion system includes an acidogenic reactor as
the first phase, which is followed by a methanogenic reactor as the second
phase in series. The most important advantage of a two-phase anaerobic
digestion system is that it is possible to produce hydrogen during the first
acidogenic phase, and subsequently to produce methane during the second
methanogenic phase.61 Therefore, two-phase anaerobic digestion processes
can easily be employed for production of methane from various sources of
biomass. In this section, recent research activities on two-phase anaerobic

digestion process are exclusively discussed. Additionally, a summary of the
findings of these studies is also given in Table 2.
Two-phase anaerobic treatment of cheese whey was investigated in a
laboratory-scale system, including a CSTR as the acid phase reactor and an
upflow anaerobic filter reactor as the methane phase reactor.62 At a HRT of
4 days (for the methane phase reactor) and 35 1 C, the system provided
a biogas yield of 0.55 m3 /kg CODremoved .
A pilot-scale two-phase anaerobic digestion system was tested to treat
food wastes for methane production.63 The acidification reactor was operated
with a retention time of 5 days and at a pH of 6.5, while the methane phase
reacor was operated with a retention time of 15 days and in a pH range of
between 7.4 and 7.8. Maximum organic loading rate was determined to be
7.9 kg VS/m3 /day, and the methane content in biogas was around 70%.
Conventional high-rate and two-phase anaerobic digestion of municipal
solid waste-sludge blend was investigated in another earlier research.64 The
authors reported that a pilot-scale two-phase anaerobic digestion process
provided a higher methane yield and a higher methane-containing digester
biogas than those obtained by the single-phase high-rate process.
Anaerobic batch digestion of banana stem waste with a TS concentration
of between 2 and 16% was studied under both mesophilic (3740 C) and
thermophilic (5055 C) conditions.65 Under mesophilic conditions, for a feed
TS concentration of 24%, the final biogas yield ranged between 267 and 271
L/kg TS fed, while under thermophilic conditions, final biogas yield varied
between 212 and 229 L/kg TS fed, for a feed TS concentration of 28%.
Methane content in total biogas ranged between 59 and 79%.
The performance of a laboratory-scale mesophilic (35 C) two-phase
anaerobic digestion system was evaluated using sugar beet pulp as the
substrate.66 The acidification reactor was operated in a pH range of be-
tween 4.0 and 4.5 and a HRT of 4 days, while the methanogenic reactor was
operated in a pH range of 6.7 to 7.2 and a HRT of 8.9 to 13.3 days. Methane
TABLE 2. Applications of two-phase anaerobic digestion processes for bioenergy recovery
128
Application Temperature Methane Methane
Substrate type status Digester types ( C) HRT (day) productivity content (%) Reference
Cheese whey Laboratory-scale CSTR (AR) 35 1 (AR) 4 (MR) 0.55 m3 62

Anaerobic filter biogas/kg
(MR) CODremoved
Food waste Pilot-scale 5 (AR) 15 (MR) 70 63
Banana stem Laboratory-scale Batch digestion 3740 5055 5979 65
waste
Sugar beet pulp Laboratory-scale 35 4 (AR) 8.913.3 363 ml/g VS 280 71.9 66
(MR) ml/g COD
Spent tea leaves Laboratory-scale 10 (AR) 73 68
Food waste UASB (MR) 2.31 m3 /m3 /day 69
Grass Pilot-scale Solid bed (AR) 0.15 m3 CH4 /kg 71 70
Anaerobic filter substrate
(MR)
Food waste Laboratory-scale UASB (MR) 35 1 0.25 L CH4 /g VS 6870 71
Coffee wastes Laboratory-scale Completely mixed 80 72
Distillery waste Laboratory-scale Anaerobic filter 36 1.5 1019 (AR) 5575 73
(AR) UASB 2039 (MR)
(MR)
Sewage sludge Laboratory-scale CSTR (AR) UASB 56 (for CSTR) 36 0.024 dm3 /g VSS >60 74
+ OFMSW (MR) (for UASB) added
Fruit and Laboratory-scale ASBR 35 3 (AR) 10 (MR) 320 L/kg COD 6971 75
vegetable input
waste
Food waste Laboratory-scale Leaching-bed 37 6 (AR as SRT) 0.21 m3 /kg 76.4 76
(AR) UASB 0.6 (MR) VSadded
(MR)
Willow sugar Pilot-scale 0.16, 0.38, 0.39 77
beet grass (m3 /kg
silage VSadded )
Cheese whey Stirred 1 (AR) 4 (MR) >70 78
AR = acidification reactor, MR = methane reactor, OFMSW = organic fraction of municipal solid waste, SRT = sludge retention time.
content in biogas was determined to be around 72% for the two-phase diges-
tion system. Specific biogas and methane production levels were observed
to be around 504 ml/g VS and 363 ml/g VS, respectively.
Two-phase anaerobic digestion of spent tea leaves was investigated
for biogas and manure generation.67 The system provided an average
biogas yield of 0.48 m3 /kg CODremoved and 73% of methane content in
biogas.
Two-phase semi-continuous methane production from mud sediment
was studied in laboratory-scale research using UASB reactor systems as aci-
dogenic and methanogenic reactors, both operated at 37 C.68 The system
resulted in a methane production of 110 mmol from the methanogenic reac-
tor.
A novel anaerobic processnamely, multi-step sequential batch two-
phase anaerobic compostingwas used to recover methane from food

wastes.69 A UASB reactor was utilized as the methanogenic reactor. The
system could be operated at a loading of 10.9 kg VS/m3 /day, yielding a
methane gas production rate of about 2.31 m3 /m3 /day.
A two-phase pilot-scale anaerobic digestion system was operated for
energy recovery from grass.70 The system produced an average of 0.15 m3
CH4 per kg of grass. The average methane content in biogas was 71%.
A hybrid anaerobic solid-liquid (HASL) bioreactor, operated as a two-
phase anaerobic digestion system, was employed for the digestion of food
waste in a laboratory-scale study.71 In this system, a UASB reactor was used
as a methanogenic reactor, and both acidogenic and methanogenic reactors
were operated at 35 1 C. During the operation, the digestion system
removed 5960% of VS added, providing a methane yield and a methane
content of 0.25 L/g VS and 6870%, respectively.
Laboratory-scale completely mixed anaerobic reactors were employed in
a two-phase anaerobic digestion system for methanization of coffee wastes.72
The acidogenic and methanogenic reactors were operated at OLRs of 5
and 0.5 g COD/L/d, respectively. Overall, the two-phase anaerobic system
produced biogas with a methane content of 80%.
Anaerobic digestion of distillery waste was studied in a two-stage anaer-
obic laboratory-scale treatment system, consisting of an anaerobic filter as
the acidogenic reactor and a UASB reactor as the methanogenic reactor, both
operated at 36 1.5 C.73 The system yielded biogas with a methane content
of between 55 and 75%.
Laboratory-scale anaerobic co-digestion of sewage sludge and organic
fraction of municipal solid waste (OFMSW) using a two-phase anaerobic
digestion system operated in quasi-continuous mode resulted in a methane
production over 60%.74 A CSTR was employed as the acidogenic reactor
operated at 56 C, and a UASB reactor was employed as the methanogenic
reactor operated at 36 C. The system provided the highest specific methane
yield as 0.024 dm3 /g VSSadded .
Two-phase anaerobic digestion of fruit and vegetable wastes for bio-

gas production was studied using two coupled laboratory-scale anaerobic
sequencing batch reactors (ASBR) operated at 35 C.75 The acidogenic ASBR
was operated at a HRT of 3 days and in an OLR range between 3.7 and
10.1 g COD/L/d, while the methanogenic ASBR was operated at a HRT of 10
days and in an OLR range between 0.72 and 1.65 g COD/L/d. Biogas pro-
ductivity [L/(d L)], biogas yield (L/kg COD input), and methane content in
the methanogenic reactor biogas varied between 0.26 0.01 to 0.74 0.02,
363.1 16.5 448.5 19, and 69 2-71 2, respectively. The authors
reported a high methane productivity of 320 L CH4 /kg COD input.
Laboratory-scale two-phase anaerobic conversion of food waste to
methane was investigated using leaching-bed reactors for acidification and
a UASB for methanization, both operating at 37 1 C.76 The acidogenic
phase had an OLR and a sludge retention time (SRT) of 11.9 kg VS/m3 /d
and 6 days, respectively, while the methanogenic phase was operated at an
OLR and a HRT of 5.4 kg COD/m3 /d and 0.6 days, respectively. The sys-
tem resulted in VS reduction, CH4 recovery (from VSremoved ), CH4 production
rate, and CH4 yield values of 73%, 70% COD, 1.75 m3 /m3 /d, and 0.21 m3 /kg
VSadded , respectively. The percentage of methane in the UASB reactor biogas
was slightly over 76%.
In a pilot-scale application, two-stage anaerobic digestion of energy
crops (i.e., willow, sugar beet, and grass silage) were investigated.77 The
specific methane yields observed were 0.16, 0.38, and 0.39 m3 /kg VSadded
for willow, sugar beet, and grass silage, respectively, which corresponded to
annual gross energy yields of 15, 53, and 26 MWh per hectare, respectively.
Recently, two-phase anaerobic digestion of cheese whey was studied,
using a stirred acidogenic reactor, followed by a stirred methanogenic reactor
coupled with a membrane filtration system.78 The acidogenic reactor was
operated at a HRT of 1 day, while the methanogenic reactor was operated at
a HRT of 4 days and up to an organic load of 19.78 g COD/L/d. The methane
content in biogas was greater than 70%.
Two-phase systems have the advantage to produce hydrogen and
methane, respectively; however, strict process control must be carried out.
In addition, the construction and operation of two separate reactor con-
figurations should also be considered beforehand. On the other hand, the
adjustment of pH and buffering capacity for the methane reactor is relatively
easier than that for a conventional single-phase reactor system.
PRODUCTION OF HYDROGEN BY ANAEROBIC DIGESTION
During anaerobic treatment of organic wastes, acidogenesis is the sec-

ond phase of the process, after initial hydrolysis, when volatile fatty acids
(VFAs), alcohols, and hydrogen (H2 ) are produced. Recently, the biological
production of hydrogen from various organic wastes through anaerobic aci-

dogenesis has drawn significant attention. Thus, these research activities are
discussed in this section of this paper, with an extensive summary displayed
in Table 3 as well.
Two anaerobic chemostat-type digesters were operated at 37 1 C in
order to investigate production of hydrogen from glucose.79 The first digester
was operated at a pH of 5.7, and in a SRT range of between 0.25 and 2 days,
while the second digester was operated in the same SRT range, but at a pH
of 6.4. For an OLR range of between 52 and 416 mmol glucose/dm3 /d, the
first and the second digesters provided hydrogen production rates varying
between 33.3 and 711, and 46.8 and 574 mmol H2 /dm3 /d, respectively. The
hydrogen content in biogas resulting from the first and the second digesters
ranged from 43.1 to 48.8%, and 43.8 to 53.3%, respectively. The first digester
provided the highest hydrogen productivity as 1.76 mol H2 /mol glucose, and
a specific hydrogen production rate of 456 mmol H2 /g VSS/d.
Batch experiments were carried out in order to determine the biological
hydrogen production potential of individual organic fraction of municipal
solid wastes, including rice, cabbage, carrot, egg, lean meat, fat, and chicken
skin.80 Biological hydrogen potential of some individual carbohydrates
namely, cabbage, carrot and ricewere determined to vary between 26.3
and 61.7 ml/g VS, 44.9 and 70.7 ml/g VS, and 19.3 and 96.0 ml/g VS, respec-
tively. The percentages of hydrogen in the total gas amount produced from
cabbage, carrot, and rice were found to be between 33.9 and 55.1%, 27.7
and 46.8%, and 44.0 and 45.6%, respectively.
An anaerobic chemostat reactor was operated to produce hydrogen
from starch.81 A maximum hydrogen production rate of 1600 L/m3 /d could
be achieved, under an OLR of 6 kg starch m3 /d, at a pH of 5.2 and a HRT
of 17 hours. During the experimental study, the percentage of hydrogen in
digester biogas was detected to be around 60%. Hydrogen could be produced
within a pH range of between 4.7 and 5.7 at a HRT of 17 hours.
Laboratory-scale continuous anaerobic fermenters were operated in a
HRT range between 13.3 and 6 hours (corresponding to a dilution rate
of 0.075 to 0.167/h), and at a pH of 6.7 and a temperature of 35 C, for
production of hydrogen from sucrose.82 Operation at dilution rates of 0.075 to
0.167/h seemed favorable for H2 production, resulting in a H2 concentration
of about 0.02 mol/L, with an optimum hydrogen production rate of 0.105
mol/h at a dilution rate of 0.125/h. The authors also reported that the product
formation in continuous hydrogen-producing cultures was essentially a linear
function of biomass concentration.
Mesophilic batch experiments were performed using a sucrose-rich syn-
thetic wastewater in order to investigate the effects of varying pH (4.57.5)
and substrate concentration (1.544.8 g COD/L) on hydrogen gas produc-
tion from wastewaters.83 The highest hydrogen production rate of 74.7 ml
H2 /L/h occured at a pH of 5.5 and a substrate concentration of 7.5 g COD/L,
132
TABLE 3. Production of hydrogen by anaerobic acidogenesis
Application Digester Temperature Hydrogen Hydrogen

Substrate type status types ( C) HRT productivity content (%) Reference
Glucose Laboratory-scale Chemostat 35 0.5 (as SRT) 1.76 mol H2 /mol 45.3 79
glucose
Starch Laboratory-scale Chemostat 17 (hour) 1.29 L H2 /g 60 81
starch-COD
Sucrose Laboratory-scale Continuous 35 13.36 (hour) 0.105 mol 82
fermenter H2 /hour
Sucrose rich Laboratory-scale Batch 37 74.7 ml H2 /L/hour 83
synthetic
wastewater
Glucose Laboratory-scale Completely mixed 37 39.1 (hour) 4548 91
continuous
fermenter
Sucrose Laboratory-scale Fixed-bed 35 15 (hour) 0.4151.32 L 2535 92
H2 /h/L
Rice winery Laboratory-scale Upflow 55 2 (hour) 9.33 L H2 /g VSS/d 5361 93
wastewater
Wheat starch Laboratory-scale CSTR 3035 1812 (hour) 1.3 mole H2 /mole 31.354.8 94
co-product hexose
Glucose Membrane 5760 96
bioreactor
Sucrose Laboratory-scale Anaerobic 35 412 (hour) 1535 97
sequencing
batch reactor
Food processing Laboratory-scale Batch system 0.12.8 l H2 /L 60 105
wastewaters wastewater
OFMSW Laboratory-scale Semi-continuous Meso-Thermo 360 Nml H2 /g 58 (Thermo) 106

fermenter VSrem. (Thermo) 42 (Meso)
- 165 Nml H2 /g
VSrem. (Meso)
Food waste CSTR Thermo 5 days 1.0 L H2 /(L d) 107
MSW + 34 35 days 52.5-71.3 N L/kg 109
slaughterhouse VSrem.
waste
Palm oil mill 357 days 0.42 L/g CODdest. 57 (at 7 days 110
effluent (ave.) HRT)
Glucose, sucrose, Laboratory-scale Membrane CSTR 1 hour 1.48, 2.07, 116
fructose 2.75 L/(h L)
Laboratory-scale Continuous flow 15 L/d 117
At a pH of 5.7. OFMSW = Organic fraction of municipal solid waste.
133
with a conversion efficiency of 38.9 ml H2 /g COD/l. The highest conver-

sion efficiency was found to be 46.6 ml H2 /g COD/L. Furthermore, the
authors also concluded that the seed sludge used for inoculation and sub-
strate concentration were two important factors to be considered to start up
a hydrogen-producing anaerobic reactor.
Batch experiments were conducted to investigate biohydrogen produc-
tion from cellulose by anaerobic fermentation.84 During the experiments, the
hydrogen content in headspace was determined to be greater than 50%, and
no methanogenesis was observed.
The effect of iron concentration on hydrogen production was investi-
gated using a mixed culture and a sucrose solution at 37 C.85 The concentra-
tion of iron (Fe) ranged between 0 and 4000 mg FeCl2 /L. At 4000 mg FeCl2 /L,
the maximum specific hydrogen production rate was determined to be 24
ml/g VSS/h, while at 800 mg FeCl2 /L, the maximum hydrogen production
yield of 132 ml/g sucrose was obtained.
The effect of pH on microbial hydrogen fermentation was investigated
in laboratory-scale batch experiments performed at 37 C.86 At pH values
of 3, 11, and 12, no hydrogen production could be observed, while some
hydrogen production did occur at pH values of 5 and 5.5. The maximum
specific production yield of hydrogen and the maximum specific hydrogen
production rate were determined to be 126.9 cm3 /g sucrose (at a pH of 9)
and 37 cm3 /g VSS/h, respectively.
The influence of acid-base enrichment (by sludge pH adjustment) on
the anaerobic hydrogen-producing microorganisms were investigated, carry-
ing out batch experiments at 35 C.87 The hydrogen-production potential of
the sludge with acid or base enrichment was 200 and 333 times higher than
that of sludge not enriched, when the enrichment pH was 10 and 3, respec-
tively. According to the authors, the enhancement was due to a shortening
of the microorganisms lag-time, which occured at a proper cultivation-pH
level.
In an earlier review paper, information from continuous laboratory-
scale works on fermentative hydrogen production was given.88 The authors
suggested that for laboratory-scale work on continuous processes, operating
temperature, pH, and HRT should be 30 C, 5.5, and between 8 and 12 hours,
respectively, for simple type of substrates.
A hydrogen-producing anaerobic sludge degraded 99% of glucose sub-
strate at 36 C and a pH of 5.5, producing a methane-free biogas with a
hydrogen content of 64%.89 The yield and production rate were determined
to be 0.26 L H2 /g glucose and 4.6 L H2 /g VSS/d, respectively.
The effect of pH (4.07.0) on conversion of glucose to hydrogen by a
mixed culture of bacteria was evaluated at 36 C.90 At a pH of 5.5 and a HRT
of 6 hours, the biogas comprised 64 2% hydrogen. The yield and specific
production rate were computed to be 2.1 mol H2 /mol glucose and 4.6 L H2 /g
VSS/d, respectively.
A complete mixing anaerobic acidogenic fermentor was operated at

37 C and in a pH range of from 5 to 8, using glucose as substrate.91 The
highest production of hydrogen gas (4548%) was attained at a pH of 7 and
within a retention time range of between 3.0 and 9.1 hours.
Anaerobic production of hydrogen was studied using fixed-bed reactors
operated at 35 C and an initial pH of 6.7 with sucrose as the substrate.92
At an influent sucrose concentration of 20 g COD/L and 2 hours of HRT,
the expanded-clay reactor produced H2 at a rate of 0.415 L/(h L), while the
activated carbon reactor exhibited a H2 production rate of 1.32 L/(h L), at a
HRT of 1 hour. The biogas produced from both reactors contained 2535%
H2 .
Continuous production of hydrogen from anaerobic acidogenesis of a
high-strength rice winery wastewater using a mixed anaerobic culture was
investigated in a laboratory-scale upflow reactor.93 The effects of HRT (2

24 hour), COD concentration of influent wastewater (1436 g COD/L), pH
(4.56.0), and temperature (2055 C) on the performance of the anaerobic
reactor was studied. Under all the conditions investigated, the reactor biogas
contained 53 to 61% of H2 . An optimum H2 production rate of 9.33 L H2 /g
VSS/d was attained at a HRT of 2 hours, with an influent COD concentration
of 34 g/L, pH of 5.5, temperature of 55 C, and a hydrogen yield that ranged
between 1.37 and 2.14 mol/mol-hexose. The authors also stated that the spe-
cific hydrogen production rate increased with the wastewater concentration
and temperature, but with a decrease in HRT.
Laboratory-scale anaerobic CSTRs were used to produce hydrogen from
a wheat starch coproduct using a mixed microflora in HRT, pH, and temper-
ature ranges of 1812 hours, 4.55.2, and 3035 C, respectively.94 In contin-
uous operations, hydrogen yields of around 1.3 mol H2 /mole hexose could
be obtained, and H2 content in digester biogas varied between 31.3 and
54.8%.
Biological production of hydrogen from sucrose was studied using
anaerobic sequencing batch reactors operated at a pH of 6.7 and at 35 C.95
In a HRT range from 4 to 12 hours and an OLR range between 40 and 120
kg COD/m3 /d, the H2 content of digester biogas varied between 15 and
35%. At a HRT of 8 hours and an OLR of 0.23 mol sucrose/dm3 /d, each
mole of sucrose produced 2.6 mole of hydrogen, and each gram of biomass
produced 0.069 mole of hydrogen per day.
A cross-flow membrane was coupled to a chemostat anaerobic mem-
brane bioreactor for biological hydrogen production, using glucose as the
substrate.96 Under all the conditions tested, the system produced biogas with
a H2 content of 5760%.
Batch experiments were carried out to analyze the influence of alkaline
pretreatment and initial pH value on hydrogen production from sewage
sludge.97 At an intial pH of 11, the maximum hydrogen yield could be
observed. In addition, the hydrogen yield from the alkaline pretreated sludge
was determined to be 16.6 ml H2 /g dried solids, higher than of 9.1 ml H2 /g

dried solids value obtained for the raw sludge. The authors concluded that
combination of the high initial pH and alkaline pretreatment would lead to
an enhanced biohydrogen production by maintaining a suitable pH range
for the growth of dominant H2 -producing anaerobes and also inhibiting the
growth of H2 -consuming anaerobes.
The effects of carbonate and phosphate concentrations on biological
hydrogen production was investigated by batch experiments, using CSTRs
operated at 35 C fed with sucrose.98 The authors reported that by using a
proper carbonate and phosphate concentration formulation, the hydrogen
production rate can be enhanced almost two-fold, as compared with an
acidogenic nutrient formulation.
Hydrogen production from food waste by mesophilic and thermophilic
acidogenic culture was studied by batch tests performed semi-continuously,

at a HRT of 5 days and pH of 5.6.99 The maximum hydrogen content was
around 69%, and the hydrogen yield ranged between 0.9 and 1.8 mol H2 /mol
hexose.
The influence of pH and intermediate products on biological production
of hydrogen was investigated by batch tests, using sucrose and starch as
substrates.100 The lowest pH level of about 4.5 provided the highest specific
hydrogen production potential of 214 ml H2 /g COD.
Batch experiments were conducted to examine production of H2 and
VFAs from glucose by an enriched anaerobic culture, in the presence of
copper (Cu) and zinc (Zn).101 At a dosage of from 50 to 100 mg Cu/dm3 or
10 to 250 mg Zn/dm3 , the specific hydrogen production rate was enhanced.
However, over a dosage of 200 mg Cu/dm3 or 500 mg Zn/dm3 , the specific
hydrogen production rate was inhibited.
The effect of carbon/nitrogen (C:N) ratio on biological hydrogen pro-
duction was studied in batch experiments, using sucrose as the substrate.102
At a C:N ratio of 47, the hydrogen productivity and hydrogen produc-
tion rate were 4.8 mol H2 /mol sucrose and 270 mmol H2 /L/d, respec-
tively. The hydrogen production ability of the seed sludge was found to
depend on the influent C:N ratio, and the proper C:N ratio on hydro-
gen production enhancement was accomplished by shifting the metabolic
pathway.
Inhibitory effects of butyrate addition on hydrogen production from glu-
cose was investigated, performing batch experiments with anaerobic mixed
cultures.103 Butyrate concentrations of 4.18 and 6.27 g/L only slightly af-
fected hydrogen production, while the addition of between 8.36 and 12.54
g/L of butyrate imposed a moderate inhibitory effect. Strong inhibitory ef-
fects of butyrate could be pronounced at a concentration of 25.08 g/L, with
a maximum hydrogen production rate of 59.3 ml/g SS/h.
Anaerobic sewage sludge acclimated with sucrose in a CSTR operated
at 35 C was employed as the seed in batch experiments in order to exploit
nutrient formulation for biological production of hydrogen by anaerobic

culture.104 The seed sludge enriched with the proposed nutrient formulation
provided a hydrogen productivity of 3.43 mol H2 /mol-sucrose, about 30%
higher than those of control and an acidogenic nutrient formulation.
Wastewaters with COD concentrations of 9 g/L (apple processing),
21 g/L (potato processing), and 0.6 and 20 g/L (confectioners A and B)
were used in batch tests to investigate biohydrogen production from these
substrates.105 Biogas produced from all of these wastewaters consistently
contained 60% H2 , and the overall H2 gas conversions were determined to
be between 0.7 and 0.9 l H2 /L wastewater for the apple wastewater, 0.1 L/L
for confectioner A, 0.42.0 L/L for confectioner B, and 2.12.8 L/L for potato
wastewater.
Laboratory-scale experiments were performed to analyze the influence
of temperature (mesophilic versus thermophilic) on semi-continuous acido-

genic solid substrate anaerobic digestion of the organic fraction of municipal
solid waste (OFMSW).106 The thermophilic mode of operation produced a
higher percentage of H2 (58%) than that of the mesophilic mode (42%).
Furthermore, the thermophilic operation provided a significantly higher H2
yield than that of mesophilic mode (360 versus 165 Nml H2 /g VSremoved ).
The effects of HRT, OLR, and pH on conversion of food waste to hydro-
gen was investigated using a thermophilic CSTR.107 The optimum operational
conditions for continuous hydrogen production could be attained at a load-
ing of 8 g VS/L/d, five days of HRT, and a pH of 5.5.
In order to enhance production of hydrogen, nitrate was introduced in
an anaerobic reactor.108 At a KNO3 concentration of 1000 mg/L and more
in the digester, hydrogen yield was almost 1 H2 -mol/glucose-mol, and there
was no production of methane.
Hydrogen production from MSW and slaughterhouse waste was investi-
gated using a mesophilic two-phase fermentation process.109 In a HRT range
between three and five days, the amount of H2 generated varied from 52.5
to 71.3 N L/kg VSremoved , with no methane production during the first phase
of the digestion.
Palm oil mill effluent was treated anaerobically to produce hydrogen,
at a pH value of 5.0 and with an influent COD concentration from 5000
to 59300 mg/L.110 At HRT values of 3, 5, and 7 days, the average biogas
generation was determined to be 0.42 L/g CODdestroyed , with a H2 content of
57% at 7 days of HRT. The biogas contained no methane.
The growth kinetics of hydrogen producing bacteria using three different
substratesnamely, sucrose, non-fat dry milk, and food wastewere inves-
tigated in dark fermentation through a series of batch experiments.111 The
hydrogen production rate seemed to increase with an increasing substrate
concentration. In addition, pH values lower than 4.0 inhibited production
of hydrogen and resulted in a lower fermentation of carbohydrate at higher
substrate concentrations.
The preparation of inoculation for biological production of hydrogen or

enrichment of mixed cultures to maximize hydrogen production has recently
been an attractive field of activity for researchers. Production of hydrogen by
an immobilized culture grown on granular activated carbon in an anaerobic
fluidized bed reactor was investigated, at a pH of 4.0 and 37 C, using glu-
cose as substrate.112 The system was operated at a HRT range from 4 to 0.5 h
and at 10 g/L influent strength, or by increasing the influent concentration
of glucose from 10 to 30 g/L at 1 h HRT. The biogas produced was com-
posed of H2 and CO2 and free of CH4 . The hydrogen production rate and
the specific hydrogen production rate were determined to be 2.36 L/(h L)
and 4.34 mmol H2 /g VSS/h, respectively. The authors concluded that a sub-
stantial quantity of retained biomass would enable the reactor to run at the
high organic loading rates, thereby enabling higher hydrogen gas production
rates.
In a recent work, heat, acid, and alkaline pre-treatment methods were
used to suppress methanogenic mixed cultures to enrich H2 -producing
bacteria.113 The highest H2 yield of 2.00 mol-H2 /mol-glucose was achieved
with the heat-treated sludge, while lowest yield of 0.48 mol-H2 /mol-glucose
was obtained with the alkaline-treated sludge. A butyrate-type fermentation
was found out for both heat- and alkaline-treated sludge, while a mixed-type
fermentation occurred for the acid-treated sludge.
The biological sludge from an animal wastewater treatment plant was
also treated to enrich H2 -producing bacteria, and the effects on hydrogen
yield were further investigated in another work.114 Enrichment was carried
out on the inoculum withing a pH range of 3 to 5, and with and without addi-
tional heat treatment. The main effects of heat treatment and pH enrichment
were significantly observed on thermophilic hydrogen production.
Bacillus coagulans strain IIT-BT S1 isolated from anaerobically digested
activated sewage sludge was investigated for its capability to produce H2
from glucose-based medium using different environmental parameters.115
The highest H2 yield (2.28 mol H2 /mol glucose) was achieved at an initial
glucose concentration of 2% (w/v), pH 6.5, temperature 37 C, inoculum
volume of 10% (v/v), and inoculum age of 14 h. Cell growth rate and rate of
hydrogen production decreased when glucose concentration was increased
above 2% w/v, indicating substrate inhibition.
A membrane bioreactor was operated to produce H2 at low HRTs, using
glucose, sucrose, and fructose as substrates.116 The system exhibited hydro-
gen production rates of 1.48, 2.07, and 2.75 L/(h L), respectively, for using
glucose, sucrose, and fructose as the sole carbon source, at a HRT of 1 hour.
The optimum operating conditions in continuous flow anaerobic acido-
genic reactors was evaluated, in order to maximize the biological production
of hydrogen, using mixed cultures.117 A stable reactor operation could be
attained up to an OLR of 86.1 kg COD/m3 /d. The maximum hydrogen pro-
duction reached up to around 15 L/d.
CONCLUSIONS
Conventional single-phase and high-rate two-phase anaerobic digestion pro-

cesses have been recently employed to produce renewable biogas from solid
types of substrates, such as various food wastes and the organic fraction of
municipal solid wastes, and from high-strength organic wastewaters, such
as agro-industry wastewaters, in bench, pilot, and full-scale applications.
Two-phase anaerobic digestion processes can be useful for special cases,
especially for substrates with a very low pH and buffering capacity (sugar
beet) and with high concentrations of ammonia (NH3 ). Furthermore, in a
two-phase system, hydrogen can also be produced in the acid-phase reactor,
while methane can be generated in the subsequent methane phase reactor,
from the same substrate. Therefore, more attention should be directed to-
ward ultimate bioenergy recovery using two-phase anaerobic digestion pro-

cesses from various types of substrates. Particularly, microbiology of both
acid and methane phase reactors should clearly be understood to improve
degradation efficiencies and biogas yields. The effects of operational and en-
vironmental parameters on the performances of both single- and two-phase
anaerobic processes have often been investigated in detail until now, and
in the last decade, more attention has been directed toward the behavior of
the microbial ecology in anaerobic digesters. These efforts should be aimed
to develop digester performances, in terms of obtaining a higher digestion
efficiency and higher biogas yields from solid and liquid wastes. It is clear
that understanding and predicting the activity and behaviour of bacteria are
the key issues.
Mostly complex types of substrates have been employed for production
of methane in both single- and two-phase anaerobic digestion processes.
However, relatively simple-soluble types of substrates were mostly utilized
for biohydrogen production until now. Economic ways of hydrogen pro-
duction from complex types of industrial wastes by anaerobic acidogenesis
should particularly be investigated in more detail. The operation of simple
single-phase digesters for the conversion of various agricultural wastes to
methane in rural areas seems another promising alternative for production
of renewable and clean energy, especially for developing countries but also
for developed countries as well. Economic aspects of constructing, operating,
and maintaining anaerobic digesters treating agro-industry wastes should be
evaluated more comprehensively.
A lack of organic waste to be digested in biogas plants is a parameter
that affects the performances of continuous processes. Co-digestion can be
an alternative method to solve this problem. Some problematic wastes, such
as high fat-containing industrial wastes, can be digested together with other
types of organic wastes in biogas plants. Therefore, more research should
be conducted to investigate these opportunities.
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On: 25 May 2014, At: 09:32

Critical Reviews in Environmental

Production of Methane and Hydrogen

To link to this article: http://dx.doi.org/10.1080/10643380802013415

PLEASE SCROLL DOWN FOR ARTICLE

Production of Methane and Hydrogen from

BURAK DEMI REL,1,2 PAUL SCHERER,1 ORHAN YENI GUN,2

Kirchstrasse 65, 21033, Hamburg, Germany

Anaerobic digestion processes have often been applied for biologi-

KEY WORDS: anaerobic digestion, biogas, biomass, hydrogen,

Conventional single-phase and high-rate two-phase anaerobic digestion pro-

biogasnamely, methane and hydrogen. Therefore, due to current imper-

renewable feedstocks.3 Presently, there exist 20 centralized and more than 35

The primary objective of this review paper is to summarize the most

PRODUCTION OF METHANE BY ANAEROBIC DIGESTION

Single-Phase Anaerobic Digestion Process

Temperature HRT Methane Methane

Potato tuber and Laboratory-scale CSTR 35 0.13-0.15 m3 /kg 42

and at 36 1 C, up to an organic loading rate (OLR) of 28.5 kg COD/m3 /day.

reactor was used to treat fermentation-based pharmaceutical wastewater.26

bial community adversely affected by antibiotics were not presented by the

than mesophilic digestion, producing more biogas in a shorter HRT or with

process. However, pre-treatment of MSW prior to digestion is the initial step.

determined to be 0.32 litres CH4 /g VSadded , while for co-digestion of potato

volumetric methane yield of straw was found to be higher than that of

tion of biogas from steam-treated municipal solid waste wastewater.48 In

150 m3 /day, with 56% methane in digester biogas, at an average ambient

The addition of 2 g/L oleate severely inhibited the process, as indicated by

Two-Phase Anaerobic Digestion Process

biomass. In this section, recent research activities on two-phase anaerobic

TABLE 2. Applications of two-phase anaerobic digestion processes for bioenergy recovery

Cheese whey Laboratory-scale CSTR (AR) 35 1 (AR) 4 (MR) 0.55 m3 62

phase anaerobic compostingwas used to recover methane from food

Two-phase anaerobic digestion of fruit and vegetable wastes for bio-

PRODUCTION OF HYDROGEN BY ANAEROBIC DIGESTION

During anaerobic treatment of organic wastes, acidogenesis is the sec-

production of hydrogen from various organic wastes through anaerobic aci-

Application Digester Temperature Hydrogen Hydrogen

OFMSW Laboratory-scale Semi-continuous Meso-Thermo 360 Nml H2 /g 58 (Thermo) 106

with a conversion efficiency of 38.9 ml H2 /g COD/l. The highest conver-

A complete mixing anaerobic acidogenic fermentor was operated at

investigated in a laboratory-scale upflow reactor.93 The effects of HRT (2

was determined to be 16.6 ml H2 /g dried solids, higher than of 9.1 ml H2 /g

acidogenic culture was studied by batch tests performed semi-continuously,

nutrient formulation for biological production of hydrogen by anaerobic

of temperature (mesophilic versus thermophilic) on semi-continuous acido-

The preparation of inoculation for biological production of hydrogen or

Conventional single-phase and high-rate two-phase anaerobic digestion pro-

ward ultimate bioenergy recovery using two-phase anaerobic digestion pro-

improvement of anaerobic digestion of waste biomass. Biotechnology Letters,

leaves. Renewable Energy, 29, 1811.

co-digestion of dairy manure and sugar beets. International Congress Series,

lective production of organic acids in anaerobic acid reactor by pH control.

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