ARTICLE

Biohydrogen Production From Wheat Straw

Hydrolysate by Dark Fermentation Using Extreme
Thermophilic Mixed Culture
Prawit Kongjan,1 Sompong O-Thong,1,2 Meher Kotay,1 Booki Min,1,3 Irini Angelidaki1
1

Department of Environmental Engineering, Technical University of Denmark, DK-2800

Lyngby, Denmark; telephone: 45-4525-1429; fax: 45-4593-2850; e-mail: ria@env.dtu.dk
2
Faculty of Science, Department of Biology, Thaksin University, Phathalung, Thailand
3
Department of Environmental Science and Engineering, Kyung Hee University,
Yongin-si, Korea
Received 3 July 2009; revision received 13 September 2009; accepted 9 November 2009
Published online 7 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22616

ABSTRACT: Hydrolysate was tested as substrate for hydrogen production by extreme thermophilic mixed culture
(708C) in both batch and continuously fed reactors. Hydrogen was produced at hydrolysate concentrations up to
25% (v/v), while no hydrogen was produced at hydrolysate
concentration of 30% (v/v), indicating that hydrolysate at
high concentrations was inhibiting the hydrogen fermentation process. In addition, the lag phase for hydrogen
production was strongly influenced by the hydrolysate
concentration, and was prolonged from approximately
11 h at the hydrolysate concentrations below 20% (v/v) to
38 h at the hydrolysate concentration of 25% (v/v). The
maximum hydrogen yield as determined in batch assays was
318.4
5.2 mL-H2/g-sugars (14.2
0.2 mmol-H2/g-sugars)
at the hydrolysate concentration of 5% (v/v). Continuously
fed, and the continuously stirred tank reactor (CSTR),
operating at 3 day hydraulic retention time (HRT) and
fed with 20% (v/v) hydrolysate could successfully produce
hydrogen. The hydrogen yield and production rate were
178.0
10.1 mL-H2/g-sugars (7.9
0.4 mmol H2/g-sugars)
and 184.0
10.7 mL-H2/day Lreactor (8.2
0.5 mmol-H2/
day Lreactor), respectively, corresponding to 12% of the
chemical oxygen demand (COD) from sugars. Additionally,
it was found that toxic compounds, furfural and hydroxymethylfurfural (HMF), contained in the hydrolysate were
effectively degraded in the CSTR, and their concentrations
were reduced from 50 and 28 mg/L, respectively, to undetectable concentrations in the effluent. Phylogenetic analysis
of the mixed culture revealed that members involved hydrogen producers in both batch and CSTR reactors were
phylogenetically related to the Caldanaerobacter subteraneus,
Thermoanaerobacter subteraneus, and Thermoanaerobacterium thermosaccharolyticum.
Correspondence to: I. Angelidaki
Contract grant sponsor: Ministry of Science and Technology of the Royal Thai
Government
Contract grant sponsor: Danish Innovation and Research Council
Contract grant sponsor: The Strategic Research Program for Energy and Environment
(DSF)
Contract grant number: 2104-06-0004

2009 Wiley Periodicals, Inc.

Biotechnol. Bioeng. 2010;105: 899908.

2009 Wiley Periodicals, Inc.
KEYWORDS: hemicelluloses; biohydrogen; mixed culture
fermentation; microbial community dynamics; extreme
thermophilic conditions

Introduction
Hydrogen is a promising energy carrier because it has high
energy yield of 122 kJ/g, which is 2.75 times higher than that
of hydrocarbon-based fuels and is a clean energy, generating
only water in either the internal combustion engine or
the fuel cell system (van Groenestijn et al., 2002).
Biohydrogen production of organic wastes through anaerobic dark fermentation is recognized as an environmental
friendly, cost effective, and sustainable process for energy
production along with treatment of organic wastes and/or
residues (Hawkes et al., 2007; Li and Fang, 2007). During the
dark fermentation process, a diverse group of bacteria
having multienzyme systems involved in two steps of
hydrolysis and acidogenesis can produce hydrogen along
with CO2 and organic acids. Chemical oxygen demand
(COD) removal efficiencies of 3055% have been reported
by the dark fermentation with different wastewaters (Guo
et al., 2008; O-Thong et al., 2008). As reviewed previously by
Hawkes et al. (2007), the residual organic acids after the
fermentation could be further utilized to either methane by
traditional anaerobic digestion or to hydrogen by photofermentation or to electricity by microbial fuel cells system.
Fermentative biohydrogen production has recently been
focusing on using mixed culture at extreme thermophilic
conditions (Kongjan et al., 2009; Kotsopoulos et al., 2006;

Biotechnology and Bioengineering, Vol. 105, No. 5, April 1, 2010

899

Liu et al., 2008a,b; Yokoyama et al., 2007, 2009; Zheng et al.,

2008). Recently, Yokoyama et al. (2009) have reported a
hydrogen yield of 413.4 mL-H2/g-glucose (83% of theoretical yield of 498 mL-H2/g-sugar), which is the highest value
reported so far by mixed culture fermentation. The
fermentation by mixed culture has no requirements for
sterilization of media, offers better adaptation capacity due
to its high microbial diversity, offers the possibility of mixed
substrates co-fermentation, and allows continuous fermentation processes (Kleerebezem and van Loosdrecht, 2007).
Although the robustness of the process is facilitated when
mixed cultures are used as inoculum, one drawback is that
reproducibility of experiments in other laboratories, with
mixed cultures from different sources, could be difficult
(Kongjan et al., 2009; Liu et al., 2008b; Zhao et al., 2009). In
the mean time, fermentation by extreme thermophiles at
temperatures around 70808C could possibly result in
higher hydrogen yields compared to fermentation at lower
temperatures, due to favorable thermodynamics and lower
variety in soluble by-products (van Groenestijin et al., 2002).
However, by comparing the hydrogen yields obtained at
mesophilic or thermophilic temperatures from various
studies (reviewed in Li and Fang, 2007), it becomes obvious
that besides temperature, other important factors such as
reactor configurations, substrate, inoculum, and tolerance
to toxicity can significantly influence the hydrogen yields (Li
and Fang, 2007). Due to higher hydrolysis activity, better
pathogenic destruction, and less risk contamination from
methanogens over mesophilic or thermophilic fermentation
(Lu et al., 2008; van Groenestijn et al., 2002), extreme
thermophilic fermentation seems more attractive for
lignocellulosic substrates containing mainly in organic
wastes or agricultural residues.
One drawback of operating at extreme thermophilic
temperatures is the low cell densities, requiring technologies
where cells are retained in the reactors, such by cell
immobilization (Kotsopoulos et al., 2006; Zheng et al.,
2008). Extra energy costs for maintaining the high
temperature should also be considered. In some cases, such
as for hydrolysate, extreme thermophilic fermentation
would not add any energy cost as the hydrolysate from
the hydrothermal pretreatment of straw is already very hot.
In other situations, extreme thermophilic fermentation can
be combined with sanitation of wastes, which according to
EU regulation requires treatment at 708C for 1 h (Angelidaki
et al., 2003).
During hydrothermal pretreatment, a process applied for
releasing of sugars from lignocellulosic material for
bioethanol production, two fractions are produced; a solid
fraction, mainly containing cellulose and a liquid fraction,
containing pentoses (xylose and arabinose) and small
amount of hexose (glucose), called hydrolysate. Wheat
straw hydrolysate is today a wastewater process and cannot
be efficiently used for ethanol production (Thomsen et al.,
2008). Therefore, utilization of the hydrolysate as a substrate
for biohydrogen production is attractive and alternative that
would increase the overall economy of the process.

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Biotechnology and Bioengineering, Vol. 105, No. 5, April 1, 2010

Furthermore, the effluents from the fermentation steps

(ethanol, hydrogen) can be used for methane production.
Such a biorefining process would result in multibiofuels
(ethanol, hydrogen, and methane) production and give
possibilities for exploitation of different market demands
(Kaparaju et al., 2009). It has been previously shown that
xylose, the main sugar in hemicelluloses, could be
successfully converted to hydrogen by either the pure
extreme thermophile Caldicellulosiruptor saccharolyticus
achieving a relatively high yield of 333.4 mL-H2/g-xylose
(Kadar et al., 2004) or mixed culture of extreme
thermophiles achieving a yield of 218.2 mL-H2/g-xylose
(Kongjan et al., 2009). In addition, Kongjan et al. (2009)
reported that continuous hydrogen production can be
established successfully in continuously stirred tank
reactor (CSTR) by using mixed culture at extreme
thermophilic temperature (708C) with a yield of 203 mLH2/g-xylose.
Although many investigations have dealt with utilization
of lignocellulosic substrates for biological hydrogen production (Bagi et al., 2007; Datar et al., 2007; de Vrije et al., 2009;
Fan et al., 2006; Ivanova et al., 2008, 2009; Kadar et al., 2004;
Li and Chen, 2007; Pattra et al., 2008), no investigation
for hydrogen production from hemicellulose hydrolysate
by using mixed extreme thermophilic fermentation has
been reported. In particular, conditions for successful
dark fermentation of hydrolysate are not elucidated.
Additionally, the effect of hydrolysate, which often
contains toxic compounds on the microbial composition of mixed hydrogenogenic culture has not been
investigated. This can give important information about
the robustness of specific microbial strains that can be
adapted to efficiently ferment sugars from harsh substrates,
such as hydrolysate.
In this study, hydrogen production from hydrolysate
obtained from hydrothermal pretreatment of wheat straw
was investigated in both batch and CSTR reactors using
mixed culture at extreme thermophilic temperature (708C).
The bacterial diversity was also identified both for batch
cultivation and in continuously fed reactor operation for
identifying the main hydrogenogens in the reactors.

Materials and Methods

Substrate and Basic Anaerobic (BA) Medium
A hemicellulose-rich hydrolysate was kindly supplied by
Dong Energy (Kalundborg, Denmark) and stored at 208C
until further use. Its chemical composition is presented in
Table I. The hydrolysate was generated from pilot scale
hydrothermal pretreatment facility treating wheat straw in
two steps process with 100 kg/h capacity (Thomsen et al.,
2008). Basic anaerobic (BA) medium was prepared as
described by Angelidaki and Sanders (2004) and was
amended with 1 g/L of yeast extract.

Table I.

Chemical composition of the hydrolysate.

Characteristics
pH
TS (%)
VS (%)
VFA (g/L)
Furfural (mg/L)
Phenols (mg/L)
HMF (mg/L)
Sugar
Glucose (g/L)
Xylose (g/L)
Arabinose (g/L)

monitored. Liquid samples were taken at the 1st week

(day 3), 2nd week (day 10), 3rd week (day 17), 4th week (day
26), and 5th week (day 35) for microbial community
analysis.
Liquid samples were also taken at the end of the stationary
phase from batch reactor and daily from CSTR for further
analysis of volatile fatty acids (VFAs), alcohols, lactate,
formate, and total sugars.

Value
4.9
4.4
3.3
0.7
250
140
140
Free sugar
1.15
1.1
0.5

Total sugar
2.9
11.3
1.3

Extracted from Kaparaju et al. (2009).

Reactors Operation and Monitoring

The batch experiments were carried out in 250 mL serum
vials with a working volume of 100 mL and at temperature of
708C. The initial inoculum was an enriched hydrogenogenic
culture from a lab scale CSTR fed with xylose concentration
of 1 g/L operated at 708C and 3 days hydraulic retention
time (HRT) that had been operated for at least 1 year
(Kongjan et al., 2009). In order to adapt this culture to the
hydrolysate, batch cultivations were carried out with
addition of hydrolysate at different concentrations: 5%,
10%, 20%, 25%, and 30% (v/v). Preparation of the batch
cultivations was as follows: the serum vials were first filled
with 80 mL of hydrolysate diluted at different concentrations with BA medium, and the vials were thereafter sealed
with butyl stoppers and aluminium crimps. Afterwards,
the sealed vials were purged with a gas mixture (20/80)
of CO2/N2 with needles inserted through the butyl stoppers
for 10 min, and were preheated in an incubator for 15 min
before transferring 20 mL of inoculum by a syringe into the
sealed vials. Control vials containing only water, yeast
extract, vitamins, and inoculum were included, in order to
account for possible background hydrogen production from
these additions. The background hydrogen was subtracted
from hydrogen produced in vials with hydrolysate. All
experiments were done in triplicate. The hydrogen
concentration in the gas phase was monitored periodically.
A 1 L CSTR reactor with 700 mL working volume was first
started up as batch reactor filled with 140 mL of inoculum
and 560 mL of 20% (v/v) hydrolysate in BA medium. The
inoculum used for starting-up the reactor was a mixed
culture cultivated as batch with 20% (v/v) hydrolysate. The
operation of the CSTR reactor was shifted to continuous
feeding mode at a HRT of 3 days, 5 days later, when the
hydrogen production ceased. The hydrolysate of 20% (v/v)
diluted in BA medium was fed into the CSTR, corresponding to an influent sugars, furfural, and hydroxymethylfurfural (HMF) concentrations of 3.1 g/L, 50 mg/L, and 28 mg/
L, respectively. The temperature was controlled at 708C by
hot water circulating in a water jacket. All gas components
(H2, CH4, and CO2) from CSTR reactor were routinely

Analyses
All biogas components (H2, CH4, and CO2) were measured
by gas chromatography (GC; MicroLab, Arhus, Denmark)
equipped with a thermal conductivity detector (TCD). The
VFAs and alcohols were determined using a GC (GC-2010
Shimadzu, Kyoto, Japan) equipped with a flame ionization
detector (FID). Lactate and formate were analyzed by
suppressed ion exclusion chromatography equipped with a
(high-performance liquid chromatography (HPLC) pump
L2100 HITATHI (Kongjan et al., 2009). Sugars were
determined by HPLC equipped with refractive index (RI)
detector, and both furfural and HMF were analyzed by
HPLC fitted with ultraviolet (UV) detector (Kaparaju et al.,
2009).
The modified Gompertz equation (Eq. 1) was used to fit
cumulative hydrogen production in the batch experiments
(Datar et al., 2007)




Rm e
Ht P exp exp
l  t 1
P

(1)

where H(t) is the cumulative hydrogen volume at time t (h)

and was obtained by measuring accumulated hydrogen
content from the fixed headspace volume at time t (h), P
(mL-H2) is the hydrogen potential and obtained during
stationary phase, Rm (mL-H2/h) is the maximum hydrogen
production rate and is obtained during exponential phase,
and l (h) is lag time. Those parameters were estimated by
function solver in Microsoft excel 2003. The specific
hydrogen potential, Ps (mL-H2/g-sugarsadded) and the
specific maximum hydrogen production rate, Rs (mL-H2/
g-sugarsadded h) were obtained by dividing P and Rm by
sugar added. Batch fermentation performance for hydrogen
conversion from different initial hydrolysate concentrations
was compared based on lag time, Ps, and Rs.
COD balance of xylose degradation products was made
based on the measured concentrations. The cell mass
concentration of the extreme thermophiles (assumed
formula C5H7O2N) used in COD balance was assumed to
be 15% of the sugars degraded (Kotsopoulos et al., 2006).
The hydrogen yield (mL-H2/g-sugarsadded) was calculated as
the total volume of hydrogen produced divided by g-sugars
added (pentoses and hexose). The yields that we report from
the batch experiments are average of triplicates.

Kongjan et al.: H2 Production From Wheat Straw Hydrolysate

Biotechnology and Bioengineering

901

Microbial Community Analysis

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to study microbial
community structure in the mixed culture. Two millilitres
of the mixed culture was taken from batch and continuous
operation, then, centrifuged at 5,000g for 5 min. The
pelletized cells were subsequently re-suspended in 50 mL
sterile MilliQ water. Genomic DNA was extracted and
purified using QIAamp DNA Stool Mini Kit (QIAgen,
Hilden, Germany). Although this Kit cannot extract DNA
from spores directly, it can extract DNA from vegetative cells
which are produced by both spore-forming and non-sporeforming bacteria during the active cultivation. For
eubacteria, universal primer 1492r and bacteria-specific
primer 27f were used. In the mean time, Arch21f and
Arch958r were also applied to identify archaea (Lane, 1991).
For the first PCR, no PCR product was obtained with
archaea primers, indicating very low detected archaea
population in the mixed culture. Only PCR of eubacteria
was then performed in a 50 mL (total volume) reaction
mixture containing 50 mM KCl, 20 mM TrisHCl (pH 8.4),
5 mM MgCl2, each deoxynucleotide triphosphate at a concentration of 200 mM, 1 mL of Taq polymerase (2 U/mL;
Sigma-Aldrich, St. Louis, MO), 10 pmol of each primer, and
1 mL of DNA extract. The thermal cycling program used for
first amplification was as follows: predenaturation at 958C
for 5 min; 25 cycles of denaturation at 958C for 30 s,
annealing at 528C for 40 s, elongation at 728C for 90 s, and
postelongation at 728C for 5 min. The reactions were
subsequently cooled to 48C. The size of the amplicon was
estimated on 1.5% agarose gel. Primer 518r and 357f (with
40 bp GC clamp at the 50 end; Muyzer et al., 1993) were used
to amplify the 200 bp fragment of the V3 region in second
PCR. The amplicon was used as DNA template to
incorporate a GC clamp in the DNA fragment prior to
DGGE (Muyzer and Smalla, 1998). The second PCR
program corresponded to 20 cycles of three steps: 948C
for 1 min, 658C for 0.75 min, and 728C for 1 min, 10 cycles of
three steps: 948C for 1 min, 558C for 0.75 min, and 728C for
1 min followed by a final step at 728C for 10 min. PCR
products were stored at 48C and analyzed on 1.5% agarose
before DGGE.
DGGE analysis of the amplicons obtained from second
PCR was performed as previously described by Zoetendal
et al. (2001) using the Dcode Universal Mutation Detection
system (Bio-Rad, Hercules, CA) with 8% (v/v) polyacrylamide gels and a denaturant gradient of 3060%. A 100%
denaturing solution was defined as 7 M urea and 40%
formamide. Electrophoresis was performed for 16 h at 70 V
in a 0.5 TAE buffer at 608C. The DGGE Marker II set
(Nippon Gene, Tokyo, Japan) was co-electrophoresed with
the samples. DGGE gels were stained with SYBR Green for
15 min and analyzed on GelDoc XR 1708170 system (BioRad Laboratories, Hertfordshire, UK). DGGE profiles were
compared using the Quantity One software package (version
4.6.0; Bio-Rad Laboratories). Most of the bands were excised

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Biotechnology and Bioengineering, Vol. 105, No. 5, April 1, 2010

from the gel and re-amplified with primer 357f without a GC

clamp and the reverse primer 518r. After re-amplification,
PCR products were purified using E.Z.N.A Cycle Pure Kit
(Omega Bio-tek, Doraville, GA) and sequenced using
primer 518r and an ABI PRISM Big Terminator Cycle
Sequencing Kit Version 3.1 (Applied Biosystems, Foster
City, CA) in accordance with the manufacturers instructions. Closest matches for partial 16S rRNA gene sequences
were identified by database searches in Gene Bank using
BLAST (Altschul et al., 1997). Quantitative PCR was not
performed, however, as the same loading quantity of PCR
products for each lane was applied, different intensities of
each band at different lanes revealed by DGGE should
therefore indicate the relative difference of dominances of
microbial community.

Results
Batch Hydrogen Production at Different Hydrolysate
Concentrations
Hydrogen production from the batch cultivations at
different hydrolysate concentrations of 5%, 10%, 20%,
and 25% (v/v) as well as the theoretical calculated hydrogen
production according to Gompertz equation is shown in
Figure 1. Hydrogen was produced successfully with
hydrolysate, although increasing hydrolysate concentrations
were inhibiting the hydrogen fermentation process, as seen
by decreasing hydrogen yields, increasing lag phase, lower
final pH, and lower growth rates (Fig. 1 and Table II). The
hydrogen yields experimentally determined from the batch
assays were 318.4
5.2, 179.4
5.8, 178.4
9.42, and
141.8
4.2 mL-H2/g-sugars (corresponding to 14.2
0.2,

Figure 1. Hydrogen production profiles during batch fermentation at the different hydrolysate concentrations.

Table II. Gompertz equation kinetic parameters, final pH, sugar consumption, and remaining furan during batch experiments at different hydrolysate
concentrations.

Hydrolysate
(v/v)
5%
10%
20%
25%

Sugars
conc.
(g/L)

Ps
(mL-H2/g-sugarsadded)

Rs
(mL-H2/g-sugarsadded h)

l (h)

H2 conc.
in the
headspace
(%)

0.8
1.5
3.1
3.9

317.6
183.5
186.5
147.7

9.8
4.6
2.9
2.1

11.9
11.0
10.4
38.7

17
19
37
37

8.0
0.3, 7.9
0.06, and 6.3
0.2 mmol-H2/g-sugars)

at the hydrolysate concentrations of 5%, 10%, 20%, and
25% (v/v), respectively. Those experimental yields are
slightly different from the specific hydrogen potential ( Ps) in
Table II, where the yields are derived from the curve fitting
of the modified Gompertz equation. No hydrogen production was observed when 30% (v/v) hydrolysate was used
during 6-day period (data not shown), indicating initial
inoculum was presumably unable to adapt to the toxicants
and sugars containing in the hydrolysate concentration of
30% (v/v). The lag phase was prolonged significantly from
approximately 11.6
0.9 h for the inoculation with 520%
(v/v) hydrolysate to 38 h with 25% (v/v) hydrolysate. The
maximum specific hydrogen production rate (Rs) was also
affected and decreased with increasing initial hydrolysate
concentrations. The highest specific hydrogen potential ( Ps)
of 317.6 mL-H2/g-sugars was obtained from the fermentation with 5% (v/v) hydrolysate.
Metabolic products concentrations and COD balance
during batch experiments at different initial hydrolysate
concentrations are presented in Table III. Accumulation of
soluble products was higher at fermentation with higher
hydrolysate concentrations. Metabolic products were
mainly acetate, followed by ethanol, lactate, and formate.
The COD balance for all batch fermentations was below
8% error, indicating that the measurements of metabolic
products were quite accurate. The ethanol to lactate COD
ratio was significantly increased with hydrolysate concentration, leading in a decrease of the hydrogen production.
Table III.

Remaining
toxic compounds
(mg/L)

Final
pH

Sugar
consumption
(%)

HMF

Furfural

6.9
6.6
5.3
5.2

97.1
96.6
93.6
89.7

0.3
0.5
0.6
0.8

0.0
0.6
2.0
7.0

Bacterial Community Composition in Batch Cultivation

DGGE was performed to unveil the effect of hydrolysate
on the bacterial community composition. The DGGE profile
of initial inoculum and samples of different hydrolysate concentrations showed three strongly stained distinctive
bands that phylogenetically related to Caldanaerobacter
subteraneus, Thermoanaerobacter subteraneus, and Thermoanaerobacterium thermosaccharolyticum (Fig. 2). Thermoanaerobacterium species and Thermoanaerobacter species
were detected in our experiment are spore-forming
clostridia, confirming that QIAamp DNA Stool Mini Kit
(Qiagen, 51504) can extract DNA from active spore-forming
bacteria. Thermoanaerobacter subteraneus was present in all
batch cultivations independent the hydrolysate concentration. Caldanaerobacter subteraneus was dominant in samples
where the initial hydrolysate concentration was 510% (v/
v), while T. thermosaccharolyticum was dominant in samples
with 2025% (v/v) initial hydrolysate concentration.

Hydrogen Production in CSTR

The variation of volumetric hydrogen production rate,
reactor pH, and concentration of soluble fermentation
products over time are shown in Figure 3. The hydrogen
decreased slightly during the first 6 days to a minimum
hydrogen production rate of 130 mL-H2/day Lreactor. The
reactor pH was approximately 5.5 during the 1st week of the
operation and decreased to approximately 5.2 in the last

Metabolic product concentrations and COD balance during batch experiments at different hydrolysate concentrations.
Concentration (mM)

Consuming sugar
Acetate
Butyrate
Propionate
Ethanol
Lactate
Formate
Hydrogen
cell mass (C5H7O2N)a
Balance

COD (mg-COD/L)

COD distribution (%)

10

20

25

10

20

25

10

20

25

4.9
4.6
0.4
0.1
0.5
0.5
0.34
11.2
0.8

9.7
6.3
0.4
0.2
3.0
2.6
0.7
12.4
1.5

18.7
12.9
0.7
0.5
5.7
4.9
1.4
25.3
2.6

22.5
13.3
0.6
0.2
7.3
7.5
2.1
24.5
3.1

803.3
292.7
62.4
15.6
51.9
46.8
5.4
184.4
120.5
23.6

1597.0
401.4
63.5
26.1
287.7
246.1
11.3
198.5
239.6
123.1

3098.7
825.0
118.4
59.7
545.8
472.6
21.6
404.2
464.8
186.5

3718.4
916.8
89.1
23.5
696.6
718.1
33.1
392.3
557.8
291.1

100
36.4
7.8
1.9
6.5
5.8
0.7
23.0
15.0
2.9

100
25.1
4.0
1.6
18.0
15.4
0.7
12.4
15.0
7.7

100
26.6
3.8
1.9
17.6
15.3
0.7
13.1
15.0
6.0

100
24.7
2.4
0.6
18.7
19.3
0.9
10.6
15.0
7.8

5, 10, 15, 20, and 25 represent hydrolysates concentrations (%).

a
Assumed value: according to the previous study (Kotsopoulos et al., 2006).

Kongjan et al.: H2 Production From Wheat Straw Hydrolysate

Biotechnology and Bioengineering

903

Figure 2.

DGGE profile of 16S rRNA gene fragments. The fragments were PCRamplified from total DNA extracted of batch cultivation used for hydrogen production
with hydrolysate. Lanes: 1, inoculum; 2, sample taken from 5% hydrolysate cultivation;
3, sample taken from 10% hydrolysate cultivation; 4, sample taken from 20% hydrolysate cultivation; 5, sample taken from 25% hydrolysate cultivation; 6, DGGE marker.

week of the operation. Acetate and propionate were

increased and stabilized after the 3rd week of the CSTR
operation, while lactate and formate became less
dominant. Butyrate and ethanol were rather stable
during the whole operation period. During days 3239
(steady state conditions), hydrogen content in the produced
biogas was 36.5
0.7%, the rest being carbon dioxide.
Hydrogen yield and hydrogen production rate were
178.0
10.1 mL-H2/g-sugars (7.9
0.4 mmol-H2/g-sugars)
and 184.0
10.7 mL-H2/day Lreactor (8.2
0.5 mmol-H2/
day Lreactor), respectively. The soluble end-products consisted mainly of acetate (14.7
0.5 mM), while the
concentration of butyrate, ethanol, and propionate was
almost three times lower (<4.8
0.04 mM). Lactate and
formate were found at even lower concentrations (0.6 mM).
Additionally, the toxic compounds, furfural and HMF were
undetectable. Removal of the influent sugars (days 3239)
was nearly complete (98%), and, therefore, influent sugar
concentration could be assumed to be the growth limiting
factor. COD balance during the stable period between
days 32 and 39 revealed that sugars were converted to 30%
acetate followed by 24% butyrate, and to 12% hydrogen
(Table IV).

Bacterial Community Composition Dynamics in CSTR

Microbial community composition dynamics was studied
during hydrolysate fermentation in CSTR aiming in
obtaining insight into the hydrogen fermentation microbiology. The DGGE analysis (Fig. 4) showed two distinct
banding pattern viz. progressive bands and regressive bands
corresponding to the bacterial species becoming more
enriched and disappearing, respectively. The mixed culture
in the CSTR reactor underwent both of a progressive growth
of C. subteraneus, T. subteraneus, T. thermosaccharolyticum,
Dysgonomonas wimpennyi, and Dysgonomonas mossii, and a
regressive growth of Lactobacillus lactis, Lactobacillus sp.,
Pseudomonas sp., Bacteriodes sp., and gamma proteobacteria. Among the species enriched, T. subteraneus was the
dominant taxon which is assumed to be the responsible
microorganism for hydrogen production in the CSTR.

Discussion

Figure 3.

Profiles of CSTR performance operated at 708C, 3 days HRT and

influent sugars concentration of 3.1 g/L. AA, acetate; BA, n- and iso-butyrate; ETOH,
ethanol; LA, lactate; FA, formate; PA, propionate. Arrows indicate when liquid samples
were taken for microbial community analysis.

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Almost all sugars in hydrolysate were oligomeric and mainly

dominated by pentoses (xylose and arabinose), accounting
for 81% of total sugars in hydrolysate (Table I). Hydrolysis
was therefore needed for further utilization of the oligomeric
sugars contained in hydrolysate as fermentation substrate
for hydrogen production. Extreme thermophilic temperature (708C) applied in this study would be advantageous to
accelerate the rate of oligosaccharides hydrolysis by the
extreme thermophiles (Lu et al., 2008). Besides sugars,
hydrolysate also contained compounds formed during
hydrothermal pretreatment of the straw components.

Table IV.

Product concentrations and COD balance of CSTR at steady state conditions (days 3239).
Concentration (mM)

Consumed sugar
Acetate
Butyrate
Propionate
Ethanol
Butanol
Lactate
Formate
Hydrogen
Cell mass (C5H7O2N)a
Balance

COD (mg-COD/L)

20.0
1.6
14.7
0.5
4.8
0.04
2.2
0.09
3.2
0.3
0.3
0.01
0.4
0.02
0.4
0.04
23.8
1.7
2.9
0.24

% COD distribution

3136.0
250.9
940.0
34.9
760.2
53.0
251.2
9.9
302.8
28.5
64.44
1.0
42.2
1.4
7.5
0.6
381.5
26.6
470.4
37.6
84.3

100
30.0
24.2
8.0
9.7
2.1
1.3
0.2
12.2
15a
2.7

Assumed value: according to the previous study (Kotsopoulos et al., 2006).

These compounds including acetic acid from the hydrolysis

of acetyl groups contained in hemicelluloses, phenolics,
dominated by vanillin, 2-furoic, coumaric acid, and ferulic
acid, from lignin decomposition, and HMF and furfural
from sugar conversion are considered as microbial toxicants
inhibiting the fermentation process (Datar et al., 2007; de
Vrije et al., 2009; Kaparaju et al., 2009).
Hydrolysate Concentration Influencing Batch
Hydrogen Production
The modified Gompertz equation could describe
hydrogen formation from batch hydrolysate cultivation at
525% (v/v) well. This equation has been previously used
successfully to predict hydrogen production from corn

Figure 4. DGGE analyzes of the samples representing progressive and regressive bands during CSTR operation. Lanes: 1, sample taken from the 1st week (day 3); 2,
sample taken from 2nd week (day 10); 3, sample taken from 3rd week (day 17); 4,
sample taken from 4th week (day 26); 5, sample taken from 5th week (day 35).
[Color figure can be seen in the online version of this article, available at www.
interscience.wiley.com.]

stover biomass pretreated with a steam explosion process

(Datar et al., 2007) and sugarcane bagasse hydrolysate
(Pattra et al., 2008). Higher initial hydrolysate concentrations lowered batch fermentation performance as indicated
by increasing lag time, decreasing specific hydrogen
potential ( Ps), and the specific maximum hydrogen
production rate (Rs), suggesting that the inhibition of
hydrogenogenic activity. Additionally, no hydrogen was
produced from the batch fermentation with 30% (v/v)
hydrolysate, corresponding to initial sugars concentration
of 4.65 g/L. This initial sugars concentration was below
inhibition level for hydrogen dark fermentation. Yokoyama
et al. (2007) had previously found successful hydrogen
production from 5.8 g/L initial xylose concentration by
mixed culture fermentation at extreme thermophilic
temperature (758C). This could confirm that the inhibition
observed at high hydrolysate concentrations was not due to
sugars overloading but rather due to the toxic compounds in
hydrolysate, such as phenolics, furfurals, and HMF.
Based on stoichiometric conversions of xylose to
hydrogen, only acetate and butyrate pathways could result
in hydrogen production with 498.0 and 249.0 mL-H2/gxylose, respectively (Kongjan et al., 2009). Other metabolic
pathways such xylose conversion to ethanol, lactate,
propionate, and formate do not result directly in hydrogen
production (Kadar et al., 2004; Kongjan et al., 2009). Lower
hydrogen yields were coincident with higher hydrogen
partial pressure and accumulation of lactate and ethanol
than hydrogen (Tables II and III). Kongjan et al. (2009) and
van Niel et al. (2003) have previously reported that higher
hydrogen partial pressure could shift the metabolic pathway
of sugars to more reduced metabolic products than acetate,
along with lower hydrogen production.
The hydrogen yield obtained by fermentation of
25% (v/v) hydrolysate with mixed cultures was
141.8
4.2 mL-H2/g-sugars. Levels between 84.0 and
330.0 mL-H2/g-sugars obtained by utilizing mixed culture
fermentation at extreme thermophilic conditions have been
previously reported and are in good correspondence with
our results (Kongjan et al., 2009; Liu et al., 2008b; Yokoyama
et al., 2007; Zheng et al., 2008). Dark fermentation using

Kongjan et al.: H2 Production From Wheat Straw Hydrolysate

Biotechnology and Bioengineering

905

pure extreme thermophiles obviously results in higher

hydrogen yields (334.0473.0 mL-H2/g-sugars; de Vrije
et al., 2009; Ivanova et al., 2008, 2009; Kadar et al., 2004;
van Niel et al., 2002) than that using mixed cultures. The
higher yields are however, counteracted by the much higher
operational costs combined with sterile substrate for
fermentations (Kleerebezem and van Loosdrecht, 2007).

Continuous Process for Hydrogen Production

From Hydrolysate
A continuous process is more economical over batch or
semi-batch processes (van Groenestijn et al., 2002), and can
achieve stable hydrogen production rate which is essential
for commercial applications (Hawkes et al., 2007).
Moreover, continuous fermentation, rather than batch
can be applied to avoid accumulation of hydrogen in the
headspace of the reactors, which could inhibit hydrogenogenic activity (van Niel et al., 2003). In this study, we
demonstrated that stable and relatively high hydrogen-yield
fermentation from hydrolysate at extreme thermophilic
temperatures was possible. We observed that low and
unstable hydrogen production was associated with high
lactate concentration during initial stage of our CSTR
operation. During steady state period (days 3239), acetate
concentration was increased, while lactate concentration
was significantly decreased, which resulted in higher
hydrogen production with a yield of 178.0
10.1 mL-H2/
g-sugars. Similar tendencies reported in the literature,
moderate- to high hydrogen yields (78.0414.0 mL-H2/gsugars) were achieved when acetate was the dominant
metabolic pathway (Kongjan et al., 2009; Kotsopoulos et al.,
2006; Yokoyama et al., 2009; Zheng et al., 2008), while lower
hydrogen yields of 21.2 and 52.3 mL-H2/g-sugars, respectively, were associated with higher lactate concentration and
coincident with unstable or overloading conditions
(Koskinen et al., 2008; Liu et al., 2008a).
Lignocellulosic biofuel production is not yet economically
competitive with fossil fuels, therefore, a successful
utilization of all sugars is important for improving the
overall economy (Hallenbeck et al., 2009). Utilization of the
hemicellulosic hydrolysate fraction for fermentative hydrogen production was successfully demonstrated in our
investigation. Extreme thermophilic fermentation is advantageous in this process, as the hydrolysate is hot after the
hydrothermal pretreatment of straw, and cooling of the
hydrolysate before fermentation could be reduced
(Thomsen et al., 2008). The dark fermentation process
was shown to utilize only part of the energy content of
hydrolysate (approx. 20%). A subsequent step such as
biomethanation or photofermentation or microbial fuel cell
should be coupled to the process for utilizing the full energy
potential.
In the present study, we supplemented media with yeast
extract and vitamins for ensuring optimal conditions for
biohydrogen production at lab scale. For industrial

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Biotechnology and Bioengineering, Vol. 105, No. 5, April 1, 2010

applications, however, the need for these supplements

should be further investigated, in order to reduce the
operational cost and increase hydrogen production.

Microorganisms Enriched in Batch and Continuous

Operation Mode for Hydrogen Production
Both inoculum and batch cultivation at different hydrolysate concentrations (Fig. 2) was dominated with the same
species, which was as expected, as not significant change of
the initial species can take place during batch cultivation.
The initial inoculum dominated by Thermoanaerobacter
subterraneus,
Caldanaerobacter
subterraneus,
and
T. thermosaccharolyticum was adapted to ferment xylose
to hydrogen and was able to successfully ferment sugars in
hydrolysate. Although microbes are not washed out during
batch cultivation, some changes in the relative growth rates
of the different microorganisms cultivated with the
hydrolysate might be detected in the strength of the
DGGE bands. In that way, the different banding pattern
shown in DGGE, suggesting the different dominance of the
microbial community composition achieved from both
initial inoculum and batch cultivation at different hydrolysate concentrations was attributed to the different
substrates and their concentrations used, and, thereby
caused different generation of hydrogen and soluble end
products. The separation of PCR-amplified DNA fragments
by DGGE (PCR-DGGE) is an extensively used screening
method for fast assessment of microbial community
diversity and dynamics, coupling high sample throughput
with DNA-based phylogenetic resolution for an entire target
community (Fischer and Lerman, 1979; Muyzer et al., 1993).
However, limitations arise when PCR-DGGE is applied to
study microbial community. Mainly, due to the 16S rRNAbased PCR-DGGE approaches used, single set of conserved
primers do not cover all recognized bacteria (Joulian et al.,
2001). PCR assays involving GC-clamped primers, as
required for DGGE analyzes, also suffer from sensitivity
limitations (Vanbroekhoven et al., 2004) that might
represent a bottleneck when dealing with low target
abundance samples from natural environments.
Thermoanaerobacter subterraneus and C. subterraneus
are extreme thermophiles which can produce hydrogen
along with acetate as the major soluble product
during carbohydrate degradation (Yokoyama et al., 2007).
Thermoanaerobacterium thermosaccharolyticum saccharolyticum is a thermophile with optimal growth temperature at
608C and is also able to convert carbohydrate to hydrogen
with butyrate as the end soluble product (O-Thong et al.,
2008). Its presence in both batch and continuous cultivations suggests that this microorganism could tolerate
extreme thermophilic temperature and was able to compete
with other extreme thermophiles.
Gradual enrichment of the mixed culture in CSTR
resulted in decrease in the diversity of bacterial communities. Microbial community changed to progressive growth

of high hydrogen-yielding species, and was consistent

with higher hydrogen yields, and acetate. In the same way,
the culture showed significant regressive growth of nohydrogen producing acidogenic species of Lactobacillus sp.,
which probably grow in the feeding pipe at room
temperature, and, consequently, appeared into the reactor
during the 1st 3 weeks of operation. Their disappearance
after the 3rd week of operation confirmed that Lactobacillus
species could not tolerate the extreme thermophilic
environment. This was also consistent with the significantly
reduced lactate concentration after the 3rd week of the
CSTR experiments. The increase of the hydrogen production along with the continuous operation of the CSTR
reactor could be attributed to the shift of the microbial
composition with higher growth of high hydrogenyielding producers (T. subteraneus, C. subterraneus, and
T. thermosaccharolyticum) and decrease of the growth of
lactate producers, Lactobacillus sp. (Fig. 4). The second
dominant organisms in the enriched population were
Dysgonomonas species. Dysgonomonas mosii found in the
mammalian gut can produce only acids from sugars and its
growth was obtained at 258C but not 428C (Lawson et al.,
2002), while D. mosii was detected from microbial fuel cell
operated at 258C (Zhang et al., 2009). Presumably, its
growth at extreme thermophilic conditions is not possible.
Therefore, its presence in the samples could possibly be
explained as contamination.
The microbial studies along with the reactor performance
data underline the importance of the microbial composition
in mixed culture fermentation processes. Establishment of
the right microbial composition containing high hydrogenproducing species is therefore essential for the efficiency of
the hydrogen fermentation process. Furthermore, operational conditions (pH, HRT, temperature) should be
adjusted to optimize proliferation of hydrogen producing
microorganisms such as T. subteraneus, while exclude
growth of lactate-producing species of Lactobacillus.
As aforementioned, these results suggest that the right
inoculum and right operational conditions should be
carefully monitored and maintained in order to favor the
preferred metabolic pathway for fermentative hydrogen
production.

Conclusions
Hydrogen containing biogas, free of methane, could be
successfully produced by extreme thermophilic fermentation of hemicelluloses rich hydrolysate in both batch and
continuous mode operations by using mixed culture.
Different hydrolysate concentrations can lead to different
microbial community composition, subsequently leading to
generation of different products. Stable hydrogen production rate of 184.0
10.7 mL-H2/day Lreactor was achieved in
the CSTR reactor operated at a 3 days HRT and 20% (v/v)
hydrolysate fed. Thermoanaerobacter subteraneus, an excellent hydrogen producer well adapted to xylose as carbon and

energy sources, was enriched dominantly in CSTR during

steady state conditions in the last week of operation. The
results from the present study show that there is a great
potential to combine fermentative hydrogen production
with second-generation ethanol production from lignocellulosic material.
This research was financially supported by a PhD grant from the
Ministry of Science and Technology of the Royal Thai Government
and by the Danish Innovation and Research Council, The Strategic
Research Program for Energy and Environment (DSF) Project No.
2104-06-0004. The authors would like to thank Hector Garcia and
Jens S. Srensen for technical assistance.

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