JOURNAL OF THE CHINESE

Article CHEMICAL SOCIETY

Biohydrogen Production from Textile Wastewater by Mixed Microflora in an

Intermittent-flow, Stirred Tank Reactor: Effect of Feeding Frequency

Chyi-How Lay,a,b,†* Biswarup Sen,a,b,c,† Szu-Yu Kuo,a Chin-Chao Chend and Chiu-Yue Lina,b,c
a
Master Program of Green Energy Science and Technology, Feng Chia University, Taichung 407, Taiwan
b
Green Energy Development Center, Feng Chia University, Taichung 407, Taiwan
c
Department of Environmental Engineering and Science, Feng Chia University, Taichung 407, Taiwan
d
Department of Landscape Architecture, Chung Chou University of Science and Technology, Changhua 510, Taiwan
(Received: Oct. 17, 2013; Accepted: Apr. 7, 2014; Published Online: ??; DOI: 10.1002/jccs.201300524)

An intermittent-flow, stirred tank reactor (IFSTR) was used to produce biohydrogen from textile waste-
water (TW) by activated sludge. The IFSTR was operated at pH 5.5 under various feeding frequencies and
TW concentrations. The first intermittent peak biogas production rate of 17.5 L biogas/L-d and HPR of 10
L H2/L-d were obtained at feeding frequency of 12 times/d with TW concentration of 33.1 g hexoses/L.
Butyrate and acetate were the major soluble metabolites at peak HPR, whereas lactate and ethanol accu-
mulation decreased the HPR. Close resemblance of bacterial communities in periods of peak HPR with
hydrogen producer Clostridium butyricum was observed.

Keywords: Activated sludge; Hydrogen production rate; Biogas; Fermentation; Microbial commu-
nity.

INTRODUCTION wastewater generated from textile processing contain high

Sustainable energy resources have become crucial
nowadays because of the concerns and issues of energy se-
curity caused by the growing energy demands worldwide.1
Advanced biofuel for e.g., biohydrogen, is an important
sustainable fuel source due to its renewable, pollution free,
and zero emission characteristics.2 However, biohydrogen
produced from food and lignocellulosic based feedstocks
could lead to the food vs. fuel competition and indirect
land-use change. In addition, the process of biohydrogen
production from these feedstocks must involve a pretreat-
ment step, which makes the overall process cumbersome
and complex at commercial scale. Therefore, the explora-
tion of producing hydrogen from wastewaters with concur-
rent wastewater treatment3,4 is an attractive and effective
way of tapping clean energy from renewable resources in a
sustainable manner. This provides dual environmental ben-
efits in the direction of wastewater treatment along with
sustainable bioenergy generation.5,6
Wastewaters from industries are very complex and
some are known to contain several toxic compounds in ad-
dition to high chemical oxygen demand (COD). Among the
various industrial wastewaters, textile wastewater (TW) is
highly toxic and difficult to degrade. The high amounts of
COD, dyes, sizing agents, salts, and dyeing auxiliaries; and
contributes significantly to the pollution of water bodies
and environment.7-9 The starch-rich textile wastewater is a
good feedstock for biohydrogen production by dark fer-
mentation.10 Previous studies have demonstrated the feasi-
bility of hydrogen production from textile wastewater by
using sludge as the inoculum in batch operation.10,11 Up to
now, there are no studies which report the hydrogen pro-
duction from textile wastewater in a continuous operation
mode. Owing to the toxic nature of the textile wastewater, a
continuous operation might not be effective due to accumu-
lation of slow biodegradable dyes leading to microbial
growth inhibition.
To overcome the microbial growth inhibition and re-
duce the toxicity levels in the fermentor; an intermittent-
flow, stirred-tank reactor (IFSTR) was conceived and oper-
ated with a fill-and-draw strategy. This kind of strategy is
less energy consuming as compared with the continuous
feeding using a peristaltic pump. Such strategy has been
used to ferment kitchen waste to produce biohydrogen effi-
ciently at mesophilic and thermophlic conditions.12,13 This
study investigated the hydrogen production performance
of IFSTR by varying the frequency of feeding at constant

Special Issue for Green & Sustainable Chemistry

* Corresponding author. E-mail: kfc0612@gmail.com
†
Both authors have equal contribution

J. Chin. Chem. Soc. 2014, 61, 000-000 © 2014 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1
Article
optimum pH and hydraulic retention time (HRT). In addi-
tion, the microbial community shifts due to the effect of dif-
ferent feeding frequencies was also monitored to rational-
ize the IFSTR performance.
EXPERIMENTAL
Seed inoculum and substrate: The activated sludge and
TW were collected from the wastewater treatment plant in a tex-
tile factory located in central Taiwan, and were used as the seed
inoculum and feedstock, respectively. A heat pretreatment (95 °C
for 40 min) was used to inhibit hydrogen consuming bacteria in
the activated sludge. The wastewater was stored at 4 °C before
use. The characteristics of the textile wastewater were: total
COD, 10-50 g/L; total carbohydrate, 10-40 g/L; volatile sus-
pended solid (VSS), 1-2 g/L; alkalinity, below 4 g/L as CaCO3;
and pH 6-8. The nutrient formulation contained sufficient inor-
ganic nutrients necessary for microorganisms (mg/L): NH4HCO3,
5240; K2HPO4, 125; MgCl2×6H2O, 15; FeSO4×7H2O, 25; CuSO4×
5H2O, 5; CoCl2×5H2O, 0.125; NaHCO3, 6720. 14
Experimental procedures: The experiments were carried
out in a 2.5 L glass reactor vessel keeping a working volume of 2.0
L including 1.0 L of nutrient solution and 1.0 L of pretreated
sludge for start-up. The temperature of the reactor vessel was con-
trolled at 35 °C by a water jacket around the vessel, and was agi-
tated at a rate of 150 rpm by a magnetic stirrer. The reactor was
initially gassed with argon to remove oxygen from the headspace
and maintain an anaerobic environment, prior to inoculation. The
volume and composition of biogas and the concentrations of vola-
tile fatty acid (VFA) were determined daily. The experimental de-
signs to investigate the effect of cultivation pH and feeding fre-
quency are described below.
Effect of cultivation pH: Batch fermentation was carried
out at pH range of 5.5 to 7.0 with a 0.5 interval, temperature 35
°C, and TW concentration of 15 g hexoses/L.
Effect of feeding frequency: Table 1 shows the description
of the feeding frequency for operating the IFSTR. The various
feeding frequencies (6 to 12 times/d) and substrate concentrations
(17.8 and 33.1 g hexoses/L), represented the organic loading rate
range (OLR) of 1.32 to 1.65 g hexoses/L-h in the five different
runs. For example, in Run 1, the bioreactor was fed 12 times per
day and every feeding solution contained TW (33.1 g hexose/L)
and nutrient formulation of 0.166 L. The IFSTR pH was kept con-
stant at 5.5 using 0.1 M NaOH or HCl solution with an automated
peristaltic pump. The HRT and reactor vessel temperature were
set at 24 h and 35 °C, respectively.
Monitoring and microbial analysis: During the hydrogen
fermentation, the monitoring parameters were pH, ORP (oxida-
2 www.jccs.wiley-vch.de © 2014 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Lay et al.
Table 1. Feeding strategy in intermittent-flow stirred-tank reactor
(IFSTR)
Parameters Run 1 Run 2 Run 3 Run 4 Run 5
Feeding frequency 12 8 6 6 6
(times/d)
Feeding volume (L/feed) 0.166 0.25 0.33 0.4/0.32 0.33
Substrate conc. 33.1 33.1 33.1 33.1 17.8
(g hexoses/L)
Organic loading rate 1.38 1.38 1.38 1.65/1.32 1.46
(g hexoses/L-d)
tion-reduction potential), alcohols, VFA, and biogas. The biogas
volume in the gas bag was determined by a gas-tight syringe at
room temperature and pressure (760 mm Hg). The analytical pro-
cedures of Standard Methods15 were used to determine pH, ORP,
and VSS. Alcohols and VFA were analyzed with a gas chromato-
graph which is described in our previous study. 16 Anthrone-
sulphuric-acid method was used to measure the concentration of
total carbohydrates (g hexoses/L).17 The microbial analysis was
done as per method described in our previous study.18
RESULTS AND DISCUSSION
Effect of fermentation pH on hydrogen production
from textile wastewater
Fermentation pH plays a crucial role in the generation
of biohydrogen; therefore, finding the optimum pH is of
prime importance for producing biohydrogen at the maxi-
mum rate. 19 The optimum pH varies with the type of
feedstock and the source of microorganisms used. In the
present study, batch experiments were conducted to find
the optimum pH for TW fermentation by activated sludge.
As shown in Table 2 there was a drop in the hydrogen pro-
duction with the increase in pH levels. The total biogas and
hydrogen productions increased from 0.95 to 3.65 L
biogas/L and 0.46 L to 1.89 L H2/L, respectively, with the
decrease in the pH level from 7.0 to 5.5. Besides, the degra-
dation of carbohydrates in TW also increased from 83.3%
to 96% with the decrease in pH levels. However, the pH
levels tested in this study did not influence the hydrogen
content in the biogas significantly; the H2 content ranged
from 48.2 to 55.6%. Hydrogen is a product of carbohydrate
metabolism by bacteria present in the activated sludge and
constitutes the major sink of electrons and protons gener-
ated during the catabolism of hexoses. The hexoses in TW
originated from starch which is used while processing of
textiles. The hydrogen content in biogas varies in the range
of 10-60% depending on the bioreactor conditions. Our ex-
J. Chin. Chem. Soc. 2014, 61, 000-000
 JOURNAL OF THE CHINESE
Biohydrogen Production from Textile Wastewater CHEMICAL SOCIETY

Table 2. Effect of different pH levels on batch fermentation of Table 3. Distribution of soluble metabolic products (SMPs) at
textile wastewater different fermentation-pH levels
Total biogas H2 Carbohydrate HY EtOH[a] BuOH[b] HAc[c] HPr[d] HBu[e] EtOH/HAc HAc/HBu
H2 pH
pH production production degradation (mol H2/mol
(%) (mg COD/L)/(% of SMP[f]) (mol/mol)
(L) (L/L) (%) hexoses)
7.0 1107 6 1362 N.D. 1767 0.81 0.77
7.0 0.95 48.3 0.46 83.3 0.27 (0.1) (32.1) (0) (41.7)
(26.1)
6.5 2.20 55.6 1.22 83.6 0.72
6.5 1265 13 1419 N.D. 1934 0.89 0.73
6.0 2.60 48.2 1.25 95.5 0.64
(27.3) (0.3) (30.6) (0) (41.8)
5.5 3.65 52.0 1.89 96.0 0.97
6.0 840 7 946 35 3169 0.88 0.29
(16.8) (0.1) (18.9) (0.7) (63.4)
5.5 759 3 1120 187 3180 0.67 0.35
perimental evidence suggested pH 5.5 as the optimum pH (14.5) (0.1) (21.3) (3.6) (60.6)
for high volumetric biohydrogen production from TW by [a] EtOH: ethanol; [b] BuOH: butanol; [c] HAc: acetate; [d] HPr:
activated sludge. propionate; [e] HBu: bytyrate; [f] SMP: soluble metabolic
The main soluble metabolic products (SMP) in TW products.
batch fermentation by activated sludge were acetate and
butyrate, at all the pH levels; and represented a maximum ever, the HPR gradually declined over prolonged operation
of 82.3% of SMP at pH 6 (Table 2). Hawkes et al.20 reported in Run 1 conditions. A second peak of HPR (6.18 L H2/L-d)
that hydrogen would be generated when acetate and butyr- was observed on 2nd day which later declined gradually to a
ate are the end-products of fermentation; however, the hy- negligible level on 8th day. Several reasons could be attrib-
drogen production could be inhibited when ethanol is pro- uted to the cessation of biogas production in Run 1. Accu-
duced as the end-product. Therefore, the cultivation pH 5.5
was used in the further studies with IFSTR. The Etha-
nol/Acetate (EtOH/HAc) ratio and Acetate/Butyrate (HAc/
HBu) ratio are the important indices in monitoring the met-
abolic behavior of microorganisms in hydrogen production
system.21 The ratios of EtOH/HAc and HAc/HBu close to
one are a good indication of efficient hydrogen produc-
tion.22 At pH 5.5, the EtOH/HAc ratio and HAc/HBu ratio
were 0.67 and 0.35, respectively (Table 2) and were the
threshold values for maximum hydrogen production from
TW in batch operation.
Effect of IFSTR’s feeding frequency on TW fermen-
tation for hydrogen production
Li et al. 12 developed an intermittent-continuously
stirred tank reactor (I-CSTR) which was operated with a
fill-and-draw process to avoid slurry substrates clog in the
tube. I-CSTR has been successfully used to produce bio-
hydrogen from biowaste.12,13,23,24 In the present study, the
working principle of the IFSTR was similar to the I-CSTR.
Various feeding frequencies (12 times/d, 8 times/d, and 6
times/d) with substrate concentrations of 33.1 and 17.8 g
hexoses/L at HRT 24 h and pH 5.5 were used as the opera-
tion conditions for producing hydrogen from TW in the
IFSTR. As shown in Fig. 1, the first peak BPR of 18.2 L
Fig. 1. Variations of ORP (-mV), pH, H2 (%), HPR (L
biogas/L-d and HPR of 9.95 L H2/L-d were obtained in Run H 2 /L-d), and BPR (L biogas/L-d) in intermit-
1 by applying the feeding frequency of 12 times/d with sub- tent-flow, stirred-tank reactor (IFSTR) fed with
strate concentration of 33.1 g hexoses/L at 21.5 h. How- textile wastewater.

J. Chin. Chem. Soc. 2014, 61, 000-000 © 2014 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.jccs.wiley-vch.de 3
Article
mulation of toxic dyes in TW inside the reactor might have
taken place, leading to growth inhibition of hydrogen pro-
ducers; or high feeding frequency (12 times/d) might have
caused accumulation of starch (constituent of TW) which
is a slow biodegradable substrate. Therefore, the feeding
frequency was lowered to 8 times/d in Run 2 with a feeding
volume of 0.25 L/feed to increase the time for biodegrada-
tion of TW and eliminate the inhibitions. Interestingly,
again two peaks of BPR, HPR and hydrogen content were
noted in Run 2. The first peak in BPR (6.4 L biogas/L-d),
HPR (2.6 L H2/L-d) and hydrogen content (40.7%) were
observed on about 10th day; whereas, the second peak in
BPR (4.9 L biogas/L-d), HPR (2.0 L H2/L-d) and hydrogen
content (40.6%) were noted on about 14th day.
The feeding frequency and feeding volume were in-
creased to 6 times/d and 0.33 L/d, respectively, in Run 3.
The peak BPR of 6.7 L biogas/L-d, HPR of 3.4 L H2/L-d,
and hydrogen content of 51% in Run 3 were higher than in
Run 2. But the biogas production decreased gradually
along with carbohydrate degradation (about 39%) after
prolonged fermentation. The feeding volume in Run 4 was
then changed to 0.4 L at first feeding and 0.32 L at others
with the feeding frequency of 6 times/d to support enough
substrate for hydrogen producers. As a result, the OLR
changed to 1.65 and 1.32 g hexoses/L-h. This strategy,
however, did not improve the IFSTR performance, and the
hydrogen production was still low and unstable in Run 4. In
Run 5, the OLR was decreased to 1.46 g hexoses/L-h with
substrate concentration of 17.8 g hexoses/L. The Run 5
also did not show any improvement in the hydrogen pro-
duction efficiency of the IFSTR.
The results of Run 1 to Run 3 showed an intermittent
production of hydrogen from TW, and this pattern of
IFSTR performance was further supported by the results
Fig. 2. Variations of HPR, VSS and carbohydrate deg-
radation in intermittent-flow, stirred-tank reac-
tor (IFSTR) fed with textile wastewater.
4 www.jccs.wiley-vch.de © 2014 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Lay et al.
shown in Fig. 2. As evident from Fig. 2, the carbohydrate
degradation and VSS showed good correlation with the
HPR. Since carbohydrate degradation and VSS represents
the starch breakdown and microbial growth; it could be
clearly stated that the hydrogen production performance in
the IFSTR was intermittent and non-steady owing to the
complex and heterogeneous nature of the TW. The in-
creased OLR in Run 3 caused an overloading of the sub-
strate along with the toxic compounds leading to low car-
bohydrate degradation and VSS (Fig. 2). Increased OLR
led to drastic inhibition in activity or death of the hydrogen
producers or shift in metabolic pathways which were prob-
ably irreversible. Therefore the performance of the IFSTR
could not be recovered even when the OLR was reduced in
Run 5.
In the initial phases (Run 1) of fermentation, butyrate
and acetate were major soluble metabolites as seen in the
batch experiments. The concentration of butyrate was 5305
mg COD/L (69.2% of SMP) and acetate was 1418 mg
COD/L (18.5% of SMP) at the second peak HPR in Run 1.
In Runs 2 and 3, the butyrate concentration was in the range
of 2779-3333 mg COD/L representing 21.9-32.3% of SMP.
However, during Run 2 to Run 5, lactate started to accumu-
late and became the main soluble metabolite with concen-
tration ranging from 3713 to 9724 mg COD/L, representing
32.3 to 69% of SMP. In addition to lactate, ethanol was also
noted in Run 4 and Run 5 with a concentration of 2149 mg
COD/L and 1011 mg COD/L. The accumulation of lactate
only occurs when the glycolytic flux is high or there is a
growth of lactic acid producing bacteria in the fermentor.
Consequently, the NADH flux which is consumed in lac-
tate and ethanol production is not available for hydro-
genase complex to produce hydrogen. In fact, Gottschalk25
has shown that the hydrogen production will be inhibited
when the metabolic pathway shifts to produce ethanol and
lactate.
Microbial community shifts
In order to verify the shifts in the microbial commu-
nity resulting from the different feeding frequencies; mo-
lecular analysis of the metagenomic DNA isolated from the
samples collected at various intervals of IFSTR operation
was performed. PCR-DGGE analysis of the metagenomic
DNA of day 0, 4, 8, 10, 12, 14, 17, 20, and 23 revealed
prominent shifts in the microbial community in the IFSTR
(Fig. 3a). Further cluster analysis of the DGGE patterns
showed linkages of communities with >70% resemblance
in their patterns (Fig. 3b). Cluster analysis showed that the
J. Chin. Chem. Soc. 2014, 61, 000-000

Biohydrogen Production from Textile Wastewater
Fig. 3. (A) DGGE patterns of the PCR amplified 16S
rRNA genes from metagenomic DNA of reac-
tor samples at different time-points, Lanes
marked D0 to D23 represents samples collected
on day 0, 4, 8, 10, 12, 13, 17, 20, and 23 of
IFSTR operation with textile wastewater; (B)
Cluster analysis of the DGGE patterns using
Euclidean distance and UPGMA linkage algo-
rithm with cut off value of 0.7 for grouping in a
single cluster. Lane 1-8 represents D0-D23
samples in similar order.
communities (day 2, 6, and 8) at peak HPRs were closely
related. Sequence analysis of the prominent bands revealed
the presence of Clostridium butyricum, Clostridium sp.,
and Megasphaera sp. in the IFSTR fed with TW. Earlier
studies have demonstrated the dominance of Clostridium
spp. in heat-treated sludges, and are the key hydrogen pro-
ducers.26,27 C. butyricum is a well-known hydrogen pro-
ducing bacteria.28-30 In fact, our study also found the pres-
ence of C. butyricum at the periods of peak HPRs (Fig. 3a).
On the other hand, Megasphaera sp. has been detected in
I-CSTR, and it could convert saccharides and lactate to
produce hydrogen.31,32 However, we did not observe effi-
cient lactate conversion to hydrogen in this study; probable
reason could be presence of non-hydrogen producing
Megasphaera sp. or too high lactate accumulation leading
to substrate inhibition. Further studies on the growth condi-
tions of Megasphaera sp. might give important clue to
cultivate this species in the reactor for hydrogen production
from lactate.
CONCLUSIONS
1. pH 5.5 was the optimum pH for high volumetric
biohydrogen production from TW by activated sludge.
2. The hydrogen production performance in the
IFSTR was intermittent and non-steady owing to the com-
plex and heterogeneous nature of the TW.
3. Butyrate and acetate were the major soluble metab-
olites along with higher H2 production performance. On the
J. Chin. Chem. Soc. 2014, 61, 000-000 © 2014 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
contrary, lower H2 production performance was associated
with lactate and ethanol accumulation.
4. The key microorganisms were Clostridium butyr-
icum, Clostridium sp., and Megasphaera sp. in the IFSTR
during TW fermentation.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial
support by Taiwan’s Bureau of Energy (102-D0616), Tai-
wan’s National Science Council (NSC-102-2221-E-035-
002-MY3, 102-2622-E-035-007) and Feng Chia Univer-
sity (FCU-10G27101).
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