Bioresource Technology 113 (2012) 30–36

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Fermentative biohydrogen production from lactate and acetate

Chao-Wei Wu a, Liang-Ming Whang a,b,c,⇑, Hai-Hsuan Cheng a, Kan-Chi Chan a
a
Department of Environmental Engineering, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan
b
Sustainable Environment Research Center (SERC), National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan
c
Research Center for Energy Technology and Strategy (RCETS), National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: In this study, a continuous-flow stirred tank reactor (CSTR) fed with lactate and acetate was operated to
Received 15 October 2011 enrich hydrogen-producing bacteria. By varying the influent substrate concentrations and hydraulic
Received in revised form 25 December 2011 retention times (HRT), the volumetric loading rate (VLR) of 55.64 kg-COD/m3/day seemed to be optimum
Accepted 26 December 2011
for this enriched culture for fermentative hydrogen production from lactate and acetate. The results of
Available online 5 January 2012
batch experiments confirmed that the enriched culture tended to fulfill the e equiv requirement for cell
growth at a lower VLR condition (21.77 kg-COD/m3/day), while it could largely distribute the e equiv for
Keywords:
hydrogen production at a higher VLR condition. However, a maximum lactate/acetate concentration
Bioenergy
Hydrogen production
allowed for enriching this culture existed, especially at a lower HRT condition in which wash-out can
Lactate be an issue for this enriched culture. Finally, the results of cloning and sequencing indicated that Clostrid-
Acetate ium tyrobutyricum was considered the major hydrogen-producing bacteria in the CSTR fed with lactate
Clostridium tyrobutyricum and acetate.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
The importance of renewable energy sources increases as the is-
sues of fossil fuel exhaustion and global warming become serious.
Considering our dependence on fossil fuel energy and the global
environment issues, there is an urgent need in developing a clean
and renewable energy such as hydrogen. Hydrogen can be pro-
duced biologically from potential renewable materials such as car-
bohydrate-containing biomass and organic wastes (Das and
Veziroglu, 2001; Lee et al., 2010). Among these H2-generating pro-
cesses, the dark fermentation process is considered more favorable
because it offers a potential means to produce H2 without requiring
a significant energy input while partially stabilizing a variety of
complex organic wastes (Davila-Vazquez et al., 2008; Lee et al.,
2010).
Hydrogen production from anaerobic waste treatment benefits
both organic wastes reduction and renewable energy production
at the same time, but it also creates challenges because the waste
materials usually are composed of a variety of substrates that can
be used by different species of microorganisms (Whang et al.,
2006; Li and Fang, 2007; Li et al., 2010). Fermentative biohydrogen
has been studied for the organic fraction of municipal solid wastes
⇑ Corresponding author at: Research Center for Energy Technology and Strategy
(RCETS), National Cheng Kung University, No. 1, University Road, Tainan 701,
Taiwan. Tel.: +886 6 2757575x65837; fax: +886 6 2752790.
E-mail address: whang@mail.ncku.edu.tw (L.-M. Whang).
(Lay et al., 1999), spoiled wheat grains (Kalia et al., 1993), municipal
wastewater and sludge (Kim et al., 2004; Van Ginkel et al., 2005),
potato wastes (Zhu et al., 2008), and food wastes (Han and Shin,
2004; Kim et al., 2004; Chu et al., 2008; Lee et al., 2008b), but results
suggest that hydrogen production is more efficient from carbohy-
drates than other materials (Li and Fang, 2007). In fact, fermentative
hydrogen production from simple sugars, such as sucrose and glu-
cose, can be easily achieved at high conversion efficiencies and its
metabolism has been widely studied (Li and Fang, 2007; Lin et al.,
2007). In addition to carbohydrates, fermentative biohydrogen pro-
duction from lactate/acetate-containing wastes has been discussed
recently (Jo et al., 2008a,b; Lee et al., 2008b; Juang et al., 2011), but
detailed information is rather limited. Although utilization of lac-
tate and acetate for production of butyrate and hydrogen by a num-
ber of microorganisms including Clostridium acetobutylicum,
Clostridium tyrobutyricum, Clostridium beijerinckii, and Butyribacte-
rium methylotrophicum has been previously reported (Balows
et al., 1992; Diez-gonzalez et al., 1995; Hashsham et al., 2000; Jo
et al., 2008a,b), this occurrence, however, has been reported only
for batch experiments.
In this study, we operated a continuous-flow stirred tank reac-
tor (CSTR) fed with lactate and acetate in order to enrich lactate/
acetate-utilizing and hydrogen-producing consortia and evaluate
their hydrogen production capacity under different volumetric
loading rate (VLR) conditions. In addition to CSTR operation, batch
experiments were conducted to evaluate effects of pH and initial
substrate-to-biomass (S0/X0) ratio on fermentative biohydrogen
production of the enriched culture. Finally, molecular methods

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.12.130
 C.-W. Wu et al. / Bioresource Technology 113 (2012) 30–36
were applied to investigate microbial ecology of lactate/acetate-
utilizing and hydrogen-producing bacteria enriched in the CSTR.
2. Methods
2.1. Operation of the hydrogen fermentation bioreactor
A continuous stirred tank reactor-type (CSTR) bioreactor was
operated in this study in order to enrich lactate/acetate-utilizing
and hydrogen-producing consortia. The total volume of the biore-
actor was 4 L with a working volume of 2 L and bioreactor was
equipped with a mechanic propeller for completely mixing at an
agitating speed of 160 rpm. The influent medium solution, contain-
ing predetermined concentrations of lactate and acetate, was
stored at 4 °C in a refrigerator and continuously fed into the biore-
actors using a peristaltic pump. Each liter of the influent feed was
supplemented with the following chemicals (in mg): resazurin,
0.175; CaCl26H2O, 32.32; MgCl26H2O, 232.26; KCl, 167.81;
MnCl24H2O, 63.87; CoCl26H2O, 3.87; H3BO3, 0.74; CuCl2H2O,
0.35; Na2MoO42H2O, 0.33; ZnCl2, 0.27; FeCl24H2O, 10.62; sodium
thioglycolate, 217.35; KH2PO4, 119 (Liu et al., 2011). The pH in both
bioreactors was controlled at 6 ± 0.1 using a pH controller by the
addition of 5% H3PO4and 10% NaOH throughout the experiments.
The seeding microorganism was obtained from a lab-scale contin-
uous flow bioreactor fed with wasted residues from a bioethanol
fermentation process (Juang et al., 2011). In each run, the data
were collected from the bioreactors after operation of a period
passing through more than 10 reactor volumes. The amount of
gases produced in the bioreactor was measured with a wet-gas
flow meter (Shinagawa W-NK-0.5B, Tokyo, Japan). The bioreactors
and the off-gas flow meter were kept in an air-bath incubator and
the temperature was maintained at 35 °C.
2.2. Fermentative biohydrogen tests
Fermentative biohydrogen batch tests conducted in this study
were a modified version of biochemical methane potential test
originally developed by Owen et al. (1979). The test was carried
out in a series of 1 L glass bottles equipped with pH/ORP monitor-
ing and gas collection systems. To each bottle with a liquid work-
ing volume of 800 mL, predetermined concentrations of lactate/
acetate and biomass taken from the CSTR were added as fermenta-
tion substrate and seeding sludge, respectively. In addition, each
bottle contained many chemicals as growth nutrients (Liu et al.,
2011). The bottles were incubated at 35 °C in a water bath tank
equipped with magnetic stirring at a rotational rate of 120 rpm.
The pH of the mixed liquor was controlled by feeding with either
NaOH (10%) or H3PO4 (5%). The total volume of gases produced
during fermentation was determined using a gas collection system
with water displacement, and the gas composition was analyzed
using gas chromatography (GC). Samples were frequently taken
throughout the batch experiments for the determination of organic
acids using high-performance liquid chromatography (HPLC).
2.3. Analytical methods
The composition of biogas collected from bioreactors and
batches was analyzed using gas chromatograph (China GC 8900,
Taipei, Taiwan) equipped with a thermal conductivity detector
(TCD). A 2 m stainless column was packed with Hayesep Q (60/
80 mesh) and installed in a 60 °C oven. The operational tempera-
tures of the injection port, the oven, and the detector were all set
at 60 °C. Nitrogen was used as the carrier gas at a flow rate of
15 mL/min. Volatile fatty acids (VFAs), alcohols, and sugars were
determined using HPLC (Hitachi D2000 system, Tokyo, Japan)
31
equipped with an ICSep COREGEL-87H3 column (7.8  300 mm)
in a 42 °C oven and a RI Range 16 detector. The eluent usedwas
0.008 N sulfuric acid at a flow rate of 0.6 mL/min. The carbohydrate
was analyzed using the phenol–sulfuric acid method (Herbert
et al., 1971). The pH, ORP, NH4+–N and volatile suspended solids
(VSS) were measured according to standard methods (APHA,
1998).
2.4. Calculations of electron-equivalent balance
We used Eq. (1) proposed in reference (Lee and Rittmann, 2009;
Lee et al., 2008a) for establishing the electron-equivalent (e equiv)
balance in all experiments.
eHLa;HAc ¼ eSEP þ eH2 þ ebiomass þ eundet ð1Þ
 
Where e 
HLa;HAc = e equiv of initial substrate, eSEP = e equiv of all

soluble end products (SEP), e H2 = e equiv of cumulative hydrogen

gas during the incubation period, e biomass = e equiv of growth bio-

mass during batch experiment, and e undet = e equiv of not detected
in the measured sinks. All values were experimentally measured
except eundet . The chemical formula for biomass was assumed to
be C5H7O2N (Rittmann and McCarty, 2001). SEP includes formate,
acetate, lactate, butyrate. The conversion of e equiv values fol-
lows: 1 mol acetate 8 e equiv, 1 mol butyrate 20 e equiv, 1 mol
lactate 12 e equiv, 1 mol H2 2 e equiv, and 1 mol biomass
(C5H7O2N) 20 e equiv(Lee et al., 2008a; Rittmann and McCarty,
2001).
2.5. DNA extraction, polymerase chain reaction (PCR),cloning,
sequencing and phylogenetic analysis of [Fe–Fe] hydrogenase gene
The genomic DNA samples in biomass taken from the CSTR were
extracted using the UltracleanTM Soil DNA Isolation Kit (MoBio Lab-
oratories, Inc., Solana Beach, CA). After DNA extraction, a forward
primer HydH1f (50 -TTIACITSITGYWSYCCIGSHTGG-30 ) and a reverse
primer HydH3r (50 -CAICCIYMIGGRCAISNCAT-30 ) (Schmidt et al.,
2010) was used for amplifying an approximate 500-bp fragment
of [Fe–Fe]-hydrogenase functional gene for the clone library analy-
sis. The thermal profile used for the amplification was as follows: a
hotstart at 95 °C for 10 min, 30 cycles of denaturation (45 s at
94 °C), annealing (45 s at 55 °C) and extension (90 s at 72 °C), and
a final extension at 72 °C for 3 min. The PCR products of HydH1f-
HydH3r were purified by electrophoresis in a 1.5% (wt/vol) agarose
gel, and used in the construction of the clone library. The purified
PCR products were ligated into the pGEM-T Easy vector (Promega,
Madison, WI) and the ligation products were used to transform
Escherichia coli DH5a competent cells following the manufacture’s
protocol (Invitrogen, Groningen, Netherlands). Plasmids of clones
were extracted by Mini-M plasmid DNA extraction system (Vio-
gene, Sunnyvale, CA, USA). DNA sequencing reactions were per-
formed using ABI 3100 and 3730 capillary sequencers (Applied
Biosystems, Foster City, CA, USA). BioEdit was used to align cloned
and published sequences in GenBank using the Basic Local Align-
ment Search Tool program developed by US National Center for Bio-
technology Information (Altschul et al., 1990).
3. Results and discussion
3.1. Performance of the hydrogen fermentation bioreactor
The experimental results of the hydrogen fermentation bioreac-
tor obtained under five different operational conditions by varying
the influent substrate concentrations and HRTs are summarized in
Table 1. It was evident that the microorganisms enriched in the
32 C.-W. Wu et al. / Bioresource Technology 113 (2012) 30–36

Table 1
Summary of operation and performance of the fermentative biohydrogen bioreactor.

Operational parameter Unit Run 1 Run 2 Run 3 Run 4 Run 5

HRT h 23 18 18 12 12
Lactate/acetate conc. mg/L 15000/4500 15000/4500 30000/9000 30000/9000 15000/4500
VLR kg-COD/m3/day 21.77 27.82 55.64 83.46 41.73
Biogas production L-biogas/day 3.25 ± 0.60 5.94 ± 1.92 19.40 ± 0.50 27.90 ± 2.20 11.61 ± 1.17
H2 content % 20 ± 3 25 ± 10 40±1 40 ± 2 29 ± 2
Specific H2 production rate mmol/g-VSS/h 0.48 ± 0.11 1.59 ± 1.05 4.27 ± 0.20 4.72 ± 0.73 3.08 ± 0.74
Volumetric H2 production rate mmol/L/h 0.56 ± 0.56 1.40 ± 0.90 6.60 ± 0.36 9.48 ± 0.93 2.86 ± 0.47
H2 yield mmol/mmol-HLa 0.09 ± 0.02 0.16 ± 0.02 0.42 ± 0.02 0.39 ± 0.04 0.21 ± 0.03

bioreactor were capable of producing hydrogen by utilizing lactate
and acetate. In addition, the hydrogen production performance of
the bioreactor seemed to be a function of the calculated volumetric
loading rate (VLR). As the VLR increased from 21.77 to 83.46 kg-
COD/m3/day, the specific hydrogen production rate increased from
0.48 to 4.72 mmol/g-VSS/hr. The trend was similar for the hydro-
gen content and the hydrogen yield, but there was no increase of
these two parameters as the VLR increased from 55.64 to
83.46 kg-COD/m3/day. It is likely that the maximum VLR for the
microorganisms enriched in the bioreactor has been reached, since
an incomplete utilization of lactate was occurred at the VLR of
55.64 kg-COD/m3/day.
Table 2 summarizes the yields (based on per mol of lactate) of
lactate/acetate fermentation end products in the bioreactor under
five different VLR conditions. In general, the enriched microorgan-
isms utilized lactate and acetate for biomass production, with
butyrate as the major SEP. Production of hydrogen and carbon
dioxide that accompanies the formation of butyrate from lactate/
acetate fermentation has been reported previously (Diez-gonzalez
et al., 1995; Matsumoto and Nishimura, 2007). Our results, with a
satisfactory recovery in carbon and electron balance, confirmed the
proposed metabolism of lactate and acetate for hydrogen produc-
tion that was previously discussed in several hydrogen fermenta-
tion bioreactors (Jo et al., 2008a; Lee et al., 2008b; Juang et al.,
2011). Furthermore, the stoichiometric equations obtained at dif-
ferent VLR conditions were summarized as follows:
Run 1 ðVLR ¼ 21:77 kg  COD=m3 =dayÞ
HLa þ 0:27 HAc þ 0:03 NH3 ! 0:07 C5 H7 O2 N þ 0:09 H2
þ 0:36 CO2 þ 0:65 HBu
Run 2 ðVLR ¼ 27:82 kg  COD=m3 =dayÞ
HLa þ 0:28 HAc þ 0:02 NH3 ! 0:06 C5 H7 O2 N þ 0:16 H2
þ 0:43 CO2 þ 0:69 HBu
Run 3 ðVLR ¼ 55:64 kg  COD=m3 =dayÞ
HLa þ 0:42 HAc þ 0:02 NH3 ! 0:056 C5 H7 O2 N þ 0:47 H2
þ 0:7 CO2 þ 0:71 HBu
Run 4 ðVLR ¼ 83:46 kg  COD=m3 =dayÞ
HLa þ 0:38 HAc þ 0:02 NH3 ! 0:08 C5 H7 O2 N0:45 H2
þ 0:67 CO2 þ 0:68 HBu ðEq: 4Þ
Run 5 ðVLR ¼ 41:73 kg  COD=m3 =dayÞ
HLa þ 0:16 HAc þ 0:02 NH3 ! 0:05 C5 H7 O2 N þ 0:21 H2
þ 0:51 CO2 þ 0:66 HBu ðEq: 5Þ
where HLa is lactate, HAc is acetate, and HBu is butyrate. In
view of these stoichiometric equations, the enriched culture fer-
mented lactate and acetate to produce biomass, butyrate, isobuty-
rate, CO2 and H2, although the yield for hydrogen production
varied with the VLR condition.
The calculated fractions of e equiv distributions (in percent-
age) for biomass and hydrogen at different VLR and food-to-bio-
mass (F/M) conditions are presented in Fig. 1. As the VLR
increased from 21.77 to 55.64 kg-COD/m3/day, the e equiv distri-
bution toward to hydrogen increased linearly, while the e equiv to
biomass decreased correspondingly. Similar trends were also
found for biomass and hydrogen when the F/M ratio increased
from 17.1 to 32.4 kg-COD/kg-VSS/day. It is hypothesized that at a
lower VLR or F/M condition with limited e equiv available the en-
riched culture distributes a higher percentage of e equiv for cell
synthesis, while more excess e equiv is available for hydrogen
production at a higher VLR or F/M condition in which the e equiv
requirement for cell synthesis can be easily satisfied.
Indeed, at the VLR of 55.64 kg-COD/m3/day or the F/M ratio of
ðEq: 1Þ 32.4 kg-COD/kg-VSS/day in the Run 3, there was about 2500 mg/L
of residual lactate remained in the effluent, due presumably to that
the maximum VLR or the maximum F/M ratio for the enriched cul-
ture has been reached. Compared to the operational condition of
the Run 3, similar influent concentrations of lactate (30000 mg/L)
and acetate (9000 mg/L) but a lower HRT (12 h) were applied in
ðEq: 2Þ
the Run 4 with the VLR of 83.46 kg-COD/m3/day or the F/M ratio
of 36.2 kg-COD/kg-VSS/day, resulting in a higher e equiv demand
for cell synthesis. Although in the Run 4 about 3500 mg/L of resid-
ual lactate still remained available for hydrogen production, no fur-
ðEq: 3Þ ther increase of e equiv toward to hydrogen production was
observed as expected. This is likely that either the maximum VLR

Table 2
Summary of fermentation end product yields observed under different operational HRT conditions.

HRT Unit Run 1 Run 2 Run 3 Run 4 Run 5

h 23 18 18 12 12
Operation VLR kg-COD/m3/day 21.77 27.82 55.64 83.46 41.73
Substrate Lactate mol/mol 1.00 1.00 0.90 0.87 0.99
Consumption Acetate mol/mol 0.27 0.28 0.38 0.33 0.16
Gas Hydrogen mol/mol 0.09 ± 0.02 0.16 ± 0.10 0.42 ± 0.02 0.39 ± 0.04 0.21 ± 0.03
Carbon dioxide mol/mol 0.36 ± 0.07 0.43 ± 0.10 0.63 ± 0.01 0.58 ± 0.05 0.51 ± 0.04
Biomass Biomass mol/mol 0.07 ± 0.02 0.06 ± 0.01 0.05 ± 0.00 0.07 ± 0.01 0.05 ± 0.01
Organic acids Isobutyrate mol/mol 0.04 ± 0.00 0.03 ± 0.01 0.01 ± 0.01 0.02 ± 0.00 0.03±
Butyrate mol/mol 0.61 ± 0.02 0.66 ± 0.05 0.63 ± 0.02 0.57 ± 0.03 0.63±0.03
De equiv % –3.5 –6.9 –5.0 –6.1 –10.3
C recovery % 93.2% 96.0% 99.9% 99.1% 96.8%
 C.-W. Wu et al. / Bioresource Technology 113 (2012) 30–36 33

60

Accumulated gas (mmol/L)

50
H2
40

30

20 CO2
10

0
0 5 10 15
Time (h)

120

Concentration (mmol/L)
100
Lactate
80
Butyrate
60

40
Acetate
20

0
0 5 10 15
Time (h)

Fig. 1. The calculated fractions of e equiv distributions (in percentage) for biomass 1400
and hydrogen at different VLR and food-to-biomass (F/M) conditions. h: Biomass,
j: hydrogen. 1200
Biomass (mg-VSS/L)

or the maximum F/M ratio for the enriched culture has been
reached, especially at such a lower HRT in which wash-out can be
an issue for this enriched culture, forcing this culture to distribute
more e equiv for growth. In comparison with the operational con-
dition of the Run 4, the same HRT but a lower influent concentra-
tions of lactate (15000 mg/L) and acetate (4500 mg/L) were
applied in the Run 5, resulting in a high F/M ratio of 42.9 kg-COD/
kg-VSS/day and a drop in e equiv for hydrogen production. Under
such a low HRT and insufficient substrate (lactate) condition, wash-
out is a serious issue for this enriched culture, forcing this culture to
distribute more e equiv for growth although the substrate (lactate)
was obviously insufficient. In summary, the operational condition
with a VLR of 55.64 kg-COD/m3/day or the F/M ratio of 32.4 kg-
COD/kg-VSS/day seemed to be optimum for this enriched culture
for fermentative hydrogen production from lactate and acetate.
3.2. Effect of pH on fermentative biohydrogen
Batch experiments were conducted to evaluate fermentative
biohydrogen production potential at different pH conditions.
Fig. 2 shows the results of fermentative biohydrogen test for the en-
riched culture at pH 6.5, including the concentrations of lactate,
acetate, biomass, SEPs, and the cumulative gas production. The re-
sults confirmed the utilization of lactate and acetate by the en-
riched culture for biomass growth, formation of butyrate, and
production of hydrogen. Once the lactate was depleted, biomass
growth, butyrate formation, and gas production also ceased, despite
that acetate remained available. Production of hydrogen that
accompanies the formation of butyrate from glucose fermentation
is a typical metabolic pathway by many clostridial species (Ljung-
dahl et al., 1989). Only a few clostridial species such as C. acetobu-
tylicum and C. tyrobutyricum as well as other species like
Butyribacterium methylotrophicum have been related to the meta-
1000 Biomass
800
600
400
200
0
0 5 10 15
Time (h)
Fig. 2. Fermentative biohydrogen performance of the enriched culture at pH 6.5.
bolic reaction described in this study (Hashsham et al., 2000; Jo
et al., 2008a).
Table 3 summarizes the H2 yield and specific H2 production rate
(SHPR) in fermentative biohydrogen tests conducted at different
pH conditions. In the batch1 experiment, the results indicated that
among examined pH conditions (pH 5–6.5) pH 5.5 was favored for
achieving anoptimum hydrogen production potential with the
highest H2 yieldof 0.54 mol-H2/mol-HLa and a second highest
SHPR of 215 mL-H2/g-VSS/hr. In batches 2–4, the results showed
that pH 6 was favored over pH 7 for attaining higher H2 yield
and SHPR in the three initial substrate-to-biomass ratios examined
(S0/X0 = 5, 15, 50), with a higher initial biomass concentration the
more significant in the difference. The calculated e equiv distribu-
tions (in percentage) for hydrogen, biomass and butyrate obtained
in the batch 1 experiment are summarized in Fig. 3. As the pH in-
creased from pH 5 to pH 6.5, the e equiv toward to biomass in-
creased from 2.4% to 7.2%, due presumably to a preferred growth
condition at a neutral pH. As a result, a lower e equiv was distrib-
uted toward to hydrogen production (Liu et al., 2011). These find-
ings agreed with previous study that higher hydrogen production
and SHPR were attained at pH 5.5 and 6 when using ethanol fer-
mentation residues as the substrate in which utilization of lactate
34 C.-W. Wu et al. / Bioresource Technology 113 (2012) 30–36

Table 3 Table 4
Hydrogen performancein fermentative biohydrogen tests conducted with enriched Fermentative biohydrogen performance under different X0 and S0/X0.
culture under different pH conditions.
X0 (mg- S0 (HLa/HAc) S0/ Yield (mol-H2/ SHPR(mL-H2/ Lag T
pH S0 (HLa/ X0 Yield SHPR* VSS/L) (mg-HLa/L) X0 mol-HLa) g-VSS/h) (h)
HAc)
300 7500/2250 25 0.51 153 3.3
mg/L mg-VSS/ mol-H2/mol- mL-H2/g-VSS/
300 15000/4500 50 0.53 188 4.89
L HLa h
300 30000/9000 100 0.49 128 9.7
Batch 1 5 10000/3000 600 0.48 142 1000 7500/2250 7.5 0.57 119 1.53
5.5 10000/3000 600 0.54 215 1000 15000/4500 15 0.59 151 2.91
6 10000/3000 600 0.42 207 1000 30000/9000 30 0.45 148 8.27
6.5 10000/3000 600 0.47 227 1700 7500/2250 4.5 0.76 181 0.3
Batch 2 6 15000/4500 300 0.53 188 1700 15000/4500 9 0.79 265 0.77
7 15000/4500 300 0.51 153 1700 30000/9000 18 0.58 235 1.96
Batch 3 6 15000/4500 1000 0.59 151
7 15000/4500 1000 0.45 129
Batch 4 6 15000/4500 1700 0.79 265
7 15000/4500 1700 0.61 169
tion at a higher VLR condition (55.64 kg-COD/m3/day). S0, on the
S0: initial substrate, X0: initial biomass. other hand, slightly affect the e equiv distributions of biomass
*
SHPR: Specific hydrogen production rate.
and hydrogen, but the S0 (HLa/HAc) at 30000/9000 mg/L did show
inhibition on biological activities in growth and hydrogen produc-
tion. This observation suggests that a maximum lactate/acetate
and acetate was found to be the mechanism for hydrogen produc-
concentration allowed for enriching this culture in a CSTR is ex-
tion (Juang et al., 2011).
isted, especially at a lower HRT condition in which wash-out can
be an issue for this enriched culture.
3.3. Fermentative biohydrogen tests at different S0/X0 ratio and X0

In Table 3, in addition to pH, the S0/X0 ratio seemed to affect the
hydrogen production potential as well. Therefore, fermentative
biohydrogen tests were conducted in order to evaluate the effect
of initial biomass (X0) and substrate (S0) on hydrogen production
and the results are presented in Table 4. In general, increase in
H2 yield and SHPR but decrease in lag time were associated with
an increase in X0. However, increase of S0 (HLa/HAc) from 7500/
2250 to 15000/4500 slightly increased H2 yield and SHPR, but a
noticeable decrease in H2 yield occurred at the S0 (HLa/HAc) of
30000/9000. In association with the S0/X0 ratio, the trend was less
clear but the hydrogen production performance generally de-
creased with an increased S0/X0 ratio. These observations agreed
with previous findings that the increase of the S0/X0 ratio in batch
experiments led to a decrease in the hydrogen production (Juang
et al., 2011; Van Ginkel et al., 2001).
Fig. 4 presents the dependences of the calculated e equiv dis-
tributions (in percentage) for hydrogen and biomass on S0 and
X0, and the corresponding fitted surfaces by regression. The en-
riched culture, at a higher X0 value, tended to distribute more e
equiv toward to hydrogen than biomass, while more e equiv
was distributed toward to biomass at a lower X0 value. These re-
sults supported the findings observed in the bioreactor that the en-
riched culture tended to fulfill the e equiv requirement for cell
growth at a lower VLR condition (21.77 kg-COD/m3/day), while it
could start to largely distribute the e equiv for hydrogen produc-
Fig. 3. The calculated e equiv distributions (in percentage) for hydrogen, biomass
and butyrate obtained in the batch 1 experiment.
3.4. Microbial community in continuous cultures fed with lactate and
acetate medium
Microbial community of the enriched culture fed with lactate
and acetate was evaluated based on the results of cloning and
sequencing for the [Fe–Fe] hydrogenase functional gene. The re-
sults of cloning and sequencing indicated that one dominant oper-
ational taxonomic unit (OTU), representing 93% of the 130 clones
retrieved, was identified to be closely related to C. tyrobutyricum
JM1 (Jo et al., 2008b), with a similarity of 99%. Therefore, C. tyrobu-
tyricum was considered the major hydrogen-producing bacteria in
the bioreactor fed with lactate and acetate.
C. tyrobutyricum has been widely reported for its capability of
producing hydrogen from utilizing different sugars such as glucose
and xylose (Ljungdahl et al., 1989; Balows et al., 1992; Jo et al.,
2008ab; Zhu and Yang, 2004; Lin et al., 2007; Liu et al., 2011;
Whang et al., 2011).Only a few reports have mentioned that C. tyro-
butyricum is also capable of utilizing lactate as the major substrate
for growth but acetate is required for hydrogen production (Balows
et al., 1992; Diez-Gonzalez et al., 1995). In fact, it hasbeen shown
that the conversion of lactate and acetate to butyrate and hydrogen
is thermodynamically favorable (Hashsham et al., 2000; Matsum-
oto and Nishimura, 2007; Jo et al., 2008a). Indeed, by examining
several isolated clostrdial species including C. acetobutylicum, C.
diolis, C. butyricum, and C. beijerinckii, previous studies (Diez-Gonz-
alez et al., 1995; Matsumoto and Nishimura, 2007) have confirmed
fermentative biohydrogen ability of these species from utilizing
lactate and acetate, although the growth rate of C. acetobutylicum
on lactate and acetate (0.05 h1) is much lower than that on glu-
cose (0.67 h1) (Diez-Gonzalez et al., 1995). In addition, Diez-
Gonzalez et al. (1995) has proposed the metabolic pathway of C.
acetobutylicum strain P262 for the utilization of lactate and con-
firmed that acetate was required as an electron acceptor.
Table 5 summarizes stoichiometric equations for hydrogen and
butyrate production from lactate and acetate utilization proposed
in previous studies. These equations suggest that approximately
1 mol of lactate and 0.43–0.5 mol of acetate utilization formed
0.5–0.6 mol of hydrogen and 0.7–0.83 mol of butyrate. In compar-
ison with stoichiometric equations obtained in this study
(Eqs (1)–5), relatively less acetate was consumed (0.16–0.42 mol)
and less hydrogen was produced (0.09–0.47 mol), especially at
lower VLR conditions (VLR < 41.73 kg-COD/m3/day). As discussed
 C.-W. Wu et al. / Bioresource Technology 113 (2012) 30–36 35

Fig. 4. The dependences of the calculated e equiv distributions (in percentage) for hydrogen and biomass on S0 and X0 (upper), and the corresponding fitted surfaces by
regression (bottom).

Table 5
improvement, this study was the first one, using a continuous flow
Stoichiometric equations for hydrogen and butyrate production from lactate and bioreactor, producing hydrogen from lactate and acetate
acetate utilization proposed in previous studies. utilization.
Equation Reference
1 Lactate + 0.5 acetate ? 0.75 Matsumoto and
butyrate + CO2 + 0.5 H2 + 0.5 H2O Nishimura (2007) 4. Conclusions
2 Lactate + 0.43 acetate ? 0.7 Diez-Gonzalez et al.
butyrate + CO2 + 0.57 H2 + 0.7 H2O (1995)
This study demonstrates fermentative hydrogen production
3 Lactate + 0.4 acetate + 0.7 H+ ? 0.7 Hashsham et al. (2000)
butyrate + CO2 + 0.6 H2 + 0.4 H2O from lactate and acetate utilization in a continuous flow bioreactor.
4 Lactate + 0.5 acetate ? 0.83 butyrate + 0.75 Juang et al. (2011) The VLR of 55.64 kg-COD/m3/day or the F/M ratio of 32.4 kg-COD/
CO2 + 0.5 H2 + 0.83 H2O kg-VSS/day seems to be optimum for the enriched culture for fer-
mentative hydrogen production from lactate and acetate. However,
a maximum lactate/acetate concentration allowed for enriching
this culture in a CSTR exists at a lower HRT condition, in which
previously, it is likely that the VLR condition or the S0/X0 ratio has wash-out can be an issue for this enriched culture. Finally, the re-
an effect on the e equiv distribution into biomass and hydrogen, sults of cloning and sequencing indicate that C. tyrobutyricum is
resulting in the difference in stoichiometry. Despite the fact that considered the major hydrogen-producing bacteria in the bioreac-
the hydrogen yield was slightly lower and required further tor fed with lactate and acetate.
36 C.-W. Wu et al. / Bioresource Technology 113 (2012) 30–36
Acknowledgements
The authors would like to acknowledge the financial support
from the National Science Council of Taiwan under Grant NSC
98-3114-E-006-013 and NSC100-3113-E-006-017, and NSC 101-
3113-E-006-016.
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