International Journal of Hydrogen Energy 32 (2007) 3274 – 3283

www.elsevier.com/locate/ijhydene

Hydrogen production from diluted molasses by anaerobic hydrogen

producing bacteria in an anaerobic baffled reactor (ABR)
Jianzheng Li a,∗ , Baikun Li b , Gefu Zhu a , Nanqi Ren a , Lixin Bo a , Junguo He a
a School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China
b Department of Civil and Environmental Engineering, University of Connecticut, Storrs, CT 06269, USA

Received 19 December 2006; received in revised form 10 April 2007; accepted 10 April 2007
Available online 8 June 2007

Abstract
Hydrogen production from diluted molasses by anaerobic fermentation bacteria was investigated in a three-compartment anaerobic baffled
reactor (ABR) with an effective volume of 27.48 L. After being inoculated with aerobic activated sludge and operated at chemical oxygen
demand (COD) of 5000 mg/L and temperature of 35 ◦ C for 26 days, the ABR achieved stable ethanol-type fermentation. The liquid fermentation
products, including volatile fatty acids (VFAs) and ethanol, stabilized at 1254, 2053, and 2761 mg/L in the three compartments, respectively.
Effluent pH, oxidation–reduction potential (ORP), and alkalinity ranged at 4.3–4.4, −241 to −249 mV, and 306.334 mg CaCO3 /L, respectively.
The hydrogen yield of the ABR was 32.51 L/d at the stable operation status, specific hydrogen production rate of anaerobic activated sludge
was 0.13 L/g MLVSS d, and the substrate conversion rate was 0.13 L/g COD. Hydrogen yields, fermentation types, and acclimatization durations
varied in each compartment, with the 1st compartment having lowest hydrogen yield but longest acclimatization duration and the 2nd and
3rd compartments having higher hydrogen yields but shorter acclimatization durations. The study found that the individual compartment
configuration in the ABR system provided a favorable environment for different types of anaerobic bacteria. Compared with complete stirring
tank reactor (CSTR), the ABR system had a better operation stability and microbial activity, which led to higher substrate conversion rate and
hydrogen production ability.
䉷 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Keywords: Hydrogen production; Anaerobic baffled reactor (ABR); pH; Alkalinity; Oxidation–reduction potential (ORP); Ethanol-type fermentation;
Acidogentic bacteria; Acetogenic bacteria

1. Introduction
Energy is vital to global prosperity, yet the reserves of the
fossil fuel are limited. The excessive use of fossil fuels is
one of the primary causes for global warming and acid rain,
which have affected the earth’s climate, weather, vegetation,
and aquatic ecosystems [1]. In order to reduce global pollution
and save the existing energy reserves, non-polluting and renew-
able energy need to be developed. Hydrogen is expected to be
a substitute for fossil fuel as clean energy in the 21st century,
since it only generates water when burning [2]. Hydrogen can
be produced in anaerobic treatment of organic wastes, which
employs renewable biomass and high concentrated wastewater.
∗ Corresponding author. Tel.: +86 451 86283761.
E-mail address: ljz6677@163.com (J. Li).
0360-3199/$ - see front matter 䉷 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2007.04.023
The production of clean energy from waste treatment meets the
requirement of sustainable strategy and has become worldwide
competitive new technology [3–7].
Anaerobic hydrogen production is achieved in the acidogenic
phase in fermentation processes. Extensive studies have been
carried out to enhance hydrogen production by pure bacteria
and mixed cultures with various types of reactors [5,8–17].
Most of anaerobic hydrogen bioreactors tested are traditional
complete stirring tank reactors (CSTR) [8,10–12,14,15,18].
Although stirring/mixing could improve the efficiency of mass
transfer and increase hydrogen productivity in CSTRs, it con-
sumed electric energy and had potential problem of biogas leak-
ing. A high volumetric loading rate (VLR) has been found as a
critical factor to improve hydrogen yield, but it caused the accu-
mulation of liquid fermentation products (e.g. acetic acid, pro-
pionic acid, and butyric acid) [18,19], which led to a significant
 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283 3275

pH drop in the reactor. The pH could drop to 3.8 and inhibit the Biogas meter
activity of hydrogenic anaerobic bacteria, which was referred Pump Waterlock
to as “over-acidification” [20]. Previous study [11] found that
the pH in an anaerobic hydrogen-producing CSTR system was Biogas outlet
4.3 at a VLR of 6 kg COD/m3 d without pH adjustment in in-
fluent. pH in the CSTR was still as low as 4.3 at a VLR of Baffle Effluent
20.48 kg COD/m3 d, even the influent pH was increased to 9
by adding lime [21]. When the system was over acidified, the
activity of anaerobic hydrogen-producing sludge was severely
Sampling port
impacted. The hydrogen productivity could not recover even Feed tank
though pH was later adjusted to above 4.0 by lowering influ-
ent loading or adding lime, since the microbial community had
changed dramatically during over-acidification period [5]. Solid outlet
On the other hand, anaerobic baffled reactor (ABR), which
has been developed over 20 years for wastewater treatment, Fig. 1. Schematic diagram of the anaerobic baffled reactor system.
possesses several advantages over CSTR [22]. The ABR com-
prises a series of vertical baffles to force wastewater to flow Table 1
under and over them, and wastewater can flow through ac- Composition of the normal molasses (w/w)
tivated sludge beds in each compartments [23,24]. Although Component Percentage (%)
the mass transfer efficiency may be limited without stirring, it
can be compensated by a sufficient contact between activated Dried materials 78–85
sludge and wastewater. The compartmentalized ABR consists Total sugar 48–58
TOC 28–34
of three zones (acidification, methanation, and buffer zones). TKN 0.2–2.8
High activated sludge concentrations (> 20 g biomass/L) in the P2 O 5 0.02–0.07
reactor could enhance the system’s stability [22,25]. It was CaO 0.15–0.8
found [26] that a steady increase in the feed loading from MgO 0.01–0.1
4.8 to 18 kg COD/m3 d did not significantly reduce the sub- K2 O 2.2–4.5
SiO2 0.1–0.5
strate removal efficiency in a three-compartment ABR system. Al2 O3 0.05–0.06
However, no study has been conducted on hydrogen anaerobic Fe2 O3 0.001–0.02
production in an ABR system. Ash content 4–8
There are several parameters affecting the stability and ef-
ficiency of anaerobic hydrogen production systems, such as
VLR, hydraulic retention time (HRT), pH, oxidation–reduction The trapped solids were discharged periodically from the reac-
potential (ORP), and alkalinity [13–15,18,20,27,28]. This tor. Each compartment was equipped with sampling ports that
study investigated the changes of operational parameters in a allowed biological solids, gas, and liquid samples to be with-
three-compartment ABR system with diluted molasses as the drawn. The reactor was wrapped by electrothermal wire and
substrate. The hydrogen production yield (LH2 /d), specific the temperature was maintained at 35 ± 1◦ C. The evolved bio-
hydrogen production rate per biomass (LH2 /g-biomass d), gas was collected separately from the upper part of each com-
substrate conversation efficiency (LH2 /g substrate), hydrogen partment. Biogas volumes for each compartment were daily
percentage in biogas (%), and liquid fermentation products measured using waterlocks and wet gas meters (Model LML-
were compared in each compartment. The hydrogen produc- 1, Changchun Filter Co., Ltd.). Waterlocks and wet gas meters
tion and operation stability of the ABR system was compared had been filled with water of pH 3 in order to prevent the bio-
with CSTR systems and the difference was analyzed at both gas from dissolution.
biochemical and ecological levels.
2.2. Feeding solution and seed sludge
2. Materials and methods

2.1. Experiment apparatus and processes
The continuous fermentative bio-producing hydrogen reac-
tor was constructed from 10 mm thick transparent perspex,
with internal dimensions of 100 cm × 10 cm × 110 cm. The
reactor contained three compartments of same size with the
total effective volume of 27.48 L (Fig. 1). Each compartment
was constituted with an up-flow zone and a down-flow zone. A
sedimentation tank with a volume of 1.5 L was attached to the
last compartment for water level control and trapping solids.
The influent was pumped to the reactor by a peristaltic pump.
Molasses were collected from a local beet sugar refinery and
its characteristics are given in Table 1. Molasses were diluted
by water to a chemical oxygen demand (COD) of 5000 mg/L,
and the COD:N:P was maintained at 300–500:5:1 by adding
synthetic fertilizer to supply microorganisms with adequate ni-
trogen and phosphorus. The pH and alkalinity of the feeding so-
lution were adjusted to 6.6 and 270 mg CaCO3 /L by NaHCO3
powder. The ABR system was operated at a HRT of 13.5 h and
a VLR of 8.89 kg COD/m3 d.
The reactor was inoculated with excess sludge taken from
a secondary settling tank in a local brewing wastewater
3276 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283
treatment plant. There were three reasons that the sludge
was not inoculated from anaerobic digesters. First, anaerobic
digested sludge contains more methanogens than hydrogen-
producing bacteria and most of them are embedded in granules.
Since the pH in the ABR system was maintained below 5.0 for
hydrogen production, these methanogens would not survive
at this low pH condition. Second, even if some methanogens
could survive at the ABR system, they would consume hydro-
gen produced by acidogens and resulted in a lower hydrogen
production. Third, most of microbes in anaerobic digested
sludge are obligate anaerobic with low reproduction rates,
which would prolong the inoculation period for hydrogen
production.
The ratio of mixed liquor volatile suspend solid (MLVSS)
to mixed liquor suspend solid (MLSS) was 0.71 in the in-
oculated sludge. After being diluted to a concentration about
6.00 g MLVSS/L, the activated sludge was pumped into the
three compartments one by one. After the sludge inoculation,
the ABR system was started up at a HRT of 13.5 h with a VLR
of 8.89 kg COD/m3 d, and the temperature was raised from 18
to 35◦ C within 24 h. The sludge concentrations of each com-
partment on the first day were 6.07, 6.32, and 6.27 g MLVSS/L,
respectively.
2.3. Analytical methods
COD, MLSS, MLVSS, pH, alkalinity, and ORP were mea-
sured according to standard methods [29]. The biogas con-
stituents (H2 and CO2 ) were analyzed by a gas chromatogram
(Model SC-7, Shandong Lunan Instrument Factory) equipped
with a thermal conductivity detector and a 2 m stainless
column packed with Porapak TDS201 (60/80 mesh). The con-
centrations of the volatile fatty acids (VFAs) were measured
using another gas chromatograph (Model GC-122, Shanghai
Analytical Apparatus Corporation) with a flame ionization de-
tector and a 2 m stainless column packed with Porapak GDX103
(60/80 mesh). COD, pH, alkalinity, ORP, VFAs, biogas yield,
and its constituents were measured or monitored daily. MLSS
and MLVSS were measured once every 3 days in the first 33
days and once a week in the later experimental period.
3. Results
3.1. Biogas and hydrogen production
The three compartments showed variations of biogas and
hydrogen yields during the acclimatization period (Fig. 2). For
the 1st compartment, the yields of biogas and hydrogen fluctu-
ated greatly in the first 7 days (Fig. 2a). Biogas yield reached
the first peak of 4.56 L/d on the 3rd day, but gradually dropped
to 1.00 L/d on the 13th day. After 14 days, biogas yield rapidly
increased and reached 14.01 L/d on the 25th day. The 1st com-
partment stabilized afterwards with an average biogas yield
of 14.06 L/d. Based on the biogas yield of the 1st compart-
ment, there were three stages for acclimatization, with 1–13
days as lag stage, 14–25 days as acceleration stage, and 26–55
days as stabilized stage. The percentage of hydrogen in biogas
kept increasing in the 1st compartment at the lag stage (1–13
days) (Fig. 2a), indicating that anaerobic hydrogen produc-
ing bacteria started adapting to the ABR system. Both biogas
yield and hydrogen percentage began to increase significantly
from the 14th day and stabilized on the 25th day. During the
stabilized stage (25–55 days), hydrogen average yield was
7.17 L/d and hydrogen percentage in biogas was 51% in the 1st
compartment.
The 2nd and 3rd compartments showed the similar trend
as the 1st compartment, but had shorter lag stages and higher
hydrogen yields (Fig. 2b and c). Biogas and hydrogen yields
gradually increased on the 6th day in these two compartments,
while there was no obvious increase of biogas and hydrogen
yields in the 1st compartment till the 13th day. The possible
reason for the shorter lag stage in 2nd and 3rd compartments
was the more stable environment achieved by the buffering ef-
fect of the 1st compartment. During the stabilized stage (26–55
days), the hydrogen yield in the 2nd and 3rd compartments
were 15.11 and 10.23 L/d, with the percentage of hydrogen in
biogas of 60% and 51%, respectively.
3.2. Liquid fermentation products
Along with the biogas and hydrogen production, liquid
fermentation products also underwent variations during the
acclimatization period. In the 1st compartment, the to-
tal concentration of liquid fermentation products ranged at
725–930 mg/L before the 20th day, then rapidly increased, and
stabilized at 1220–1296 mg/L after the 26th day (Fig. 3a). The
constituents of liquid fermentation products also significantly
changed before the 20th day. On the 6th day, the concen-
trations of ethanol, acetic acid, propionic acid, and butyric
acid were 191, 137, 145, and 215 mg/L, which indicated a
mixed-acid fermentation. On the 10th day, ethanol, acetic acid,
propionic acid, and butyric acid changed to 159, 311, 177, and
263 mg/L. The total amount of acetic acid and butyric acid was
574 mg/L, counting for 63% of total liquid products, which
was a typical butyric acid fermentation [30]. From the 11th
day, the production of butyric acid gradually decreased while
ethanol and propionic acid increased. On the 26th day, the 1st
compartment reached a stabile stage, with the concentrations
of ethanol, acetic acid, propionic acid, butyric acid, and valeric
acid of 443, 432, 245, 107, and 27 mg/L. The total amount
of ethanol and acetic acid was 876 mg/L, counting for 70%
of the total liquid products, which indicated an ethanol-type
fermentation [20]. According to the study of Li and Ren [20],
an ethanol-type fermentation was obtained when the mass per-
centage of ethanol and acetic acid reached above 60% in the
total fermentation end-product. Ethanol-type fermentation had
a higher hydrogen production ability than mixed acid-, butyric
acid-, and propionic acid-type fermentations [14,15]. These
shifts of fermentation types corresponded with hydrogen pro-
duction (Fig. 2), which showed hydrogen yields significantly
increased after 26 days in the 1st compartment. In addition,
the variation of liquid fermentation products during the ac-
climatization and stabilized stage was found to be related with
the shifts of microbial community [11,12,18].
 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283 3277

30 70
Biogas yield Hydrogen yield
Hydrogen concentration 60

Hydrogen concentration (%)

25

Biogas yields (L/d)

50
20
40
15
30
10
20
5 10

0 0
30 70

60

Hydrogen concentration (%)

25
Biogas yields (L/d)

50
20
40
15
30
10
20
5 10

0 0
30 70

60

Hydrogen concentration (%)

25
Biogas yields (L/d)

50
20
40
15
30
10
20
5 10

0 0
0 5 10 15 20 25 30 35 40 45 50 55
Time (d)

Fig. 2. Biogas and hydrogen yields of each compartment in the ABR system (a, 1st compartment; b, 2nd compartment; c, 3rd compartment).

Like the 1st compartment, the 2nd and 3rd compartments
also experienced the transition of fermentation types and finally
stabilized at ethanol-type fermentation (Fig. 3b and c). The
2nd compartment showed a mixed-acid-type fermentation in
the first 5 days and butyric-acid-type fermentation during 6–16
days, and stabilized at ethanol-type fermentation at 26–55 days
(Fig. 3b). The 3rd compartment had the same transition as the
2nd compartment and stabilized at ethanol-type fermentation
(Fig. 3c).
3.3. The variations of pH, alkalinity, and ORP in the ABR
system
Previous studies [12,20,31,32] have showed that pH, alka-
linity, and ORP are important factors for anaerobic hydrogen
production. The changes of these three factors affected not only
the anaerobic hydrogen production ability, but also the micro-
bial community and fermentation types. It was found that all
three compartments underwent significant variations of pH, al-
kalinity and ORP in the first 25 days (Table 2), which was
concurrent with the fluctuation of hydrogen and liquid fermen-
tation productions (Figs. 2 and 3). The pH and alkalinity of the
1st compartment dropped from 4.8 and 310 mg/L on the 1st
day to 3.5 and 0 mg/L (the lowest values) on the 15th day. The
pH and alkalinity of 2nd and 3rd compartments dropped to the
lowest values on the 10th day, with pH of 3.7 and alkalinity
of 0 mg/L for the 2nd compartment, and pH of 3.9 and alka-
linity of 0 mg/L for the 3rd compartment. As for ORP values,
it rose from −510 mV on the 1st day to −190 mV on the 20th
day in the 1st compartment, and then dropped to −230 mV af-
ter the 25th day. In the mean time, ORP of the 2nd and 3rd
3278 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283

1500

1200

VFAs (mg/L) 900 ethanol acetate

butyrate propionate
valerate total
600

300

0
2500

2000
VFAs (mg/L)

1500

1000

500

0
3500

3000

2500
VFAs (mg/L)

2000

1500

1000

500

0
0 5 10 15 20 25 30 35 40 45 50 55
Time (d)

Fig. 3. The variation of VFAs and ethanol concentrations in each compartment of the ABR system (a, 1st compartment; b, 2nd compartment; c, 3rd compartment).

compartments ranged from −490 to −210 and from −440 to
−250, respectively. After 25 days, the pH, alkalinity and ORP of
the three compartments stabilized with pH of 4.3–4.4, alkalin-
ity of 306.334 mg CaCO3 /L, and ORP of −240 to −250 mV,
which had been demonstrated as the optimal condition for
ethanol-type fermentation [12,20].
Ethanol-type fermentation has been found to occur at pH of
4.0–4.5 and ORP of −200 to −400 mV [30–32]. ORP values
were mainly affected by pH in an anaerobic system. Because the
ABR system was operated in a continuous mode and pH under-
went consistent variations in each compartment, it was difficult
to derive a clear correlation between ORP and pH. However, it
could still be seen from Table 2 that ORP was inversely related
with pH in most cases, with low ORP corresponding to high pH.
Kang and Yin [33] proposed a kinetic model of ORP values in
acidogenic fermentation systems: Eh = −0.06pH.0.031 gP H2
(Eh is the oxidation–reduction potential, and P H2 is the par-
tial pressure of hydrogen in the acidogenic reactor). This model
 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283 3279

Table 2
The variation of pH, alkalinity, and ORP in the ABR system during 55 days operation

1st day 5th day 10th day 15th day 20th day 25th day 35th day 45th day

Influent pH ALK (mg CaCO3 /L) ORP (mV) 6.8 6.6 6.7 6.7 6.7 6.5 6.7 6.8
270 280 290 270 260 270 270 270
— — — — — — — —

1st compartment pH ALK (mg CaCO3 /L) ORP (mV) 4.8 4 3.8 3.5 3.9 4.4 4.4 4.4
310 120 70 0 60 270 330 320
−510 −230 −220 −260 −190 −230 −240 −240

2nd compartment pH ALK (mg CaCO3 /L) ORP (mV) 4.4 4.1 3.7 3.9 3.9 4.3 4.4 4.4
1400 190 0 120 40 260 330 320
−490 −320 −310 −400 −210 −240 −250 −250

3rd compartment pH ALK (mg CaCO3 /L) ORP (mV) 4.5 4.7 3.9 4.2 4.2 4.3 4.4 4.4
1240 290 0 360 260 380 370 360
−350 −440 −330 −380 −430 −250 −250 −250

could explain the inverse relation between ORP and pH in the
ABR system.
The mixed liquor pH in an anaerobic system was determined
by VFAs concentration and alkalinity. Because alkalinity was
affected by the balance between [CO2 ] and [HCO− 3 ], and the
majority of alkalinity was [HCO− 3 ] at pH lower than 5, low pH
and alkalinity were expected at high VFA concentration due to
the consumption of HCO− 3 . In the first 15 days of ABR oper-
ation, the average total amounts of VFAs were 832, 1521, and
1688 mg/L in each compartment, respectively, and did not have
distinct changes (Fig. 3, Table 3). During this period, mixed
liquor pH was below 4.7 (Table 3) and determined by alka-
linity, which mainly consisted HCO− −
3 . [HCO3 ] was positively
correlated with CO2 produced by anaerobic fermentation bac-
teria. Biogas production was low in the first 15 days (Table 3),
which meant that [HCO− 3 ] in mixed liquor was low. There-
fore, both alkalinity and pH underwent steady decrease in the
first 15 days (Table 3). After 15 days, both biogas production
(Fig. 2, Table 3) and VFAs (Fig. 3, Table 3) increased, indicat-
ing anaerobic bacteria had been adapted to the ABR system.
With more CO2 being produced, [HCO− 3 ] became higher and
alkalinity increased correspondingly. A higher alkalinity en-
hanced the system neutralization capability for VFAs and led
to a stable pH value. Thereby, even though more VFAs were
produced after the 15th day, pH stabilized at 4.4 in all three
compartments.
3.4. COD removal
In a traditional anaerobic wastewater treatment process, COD
was mainly removed through the conversion of intermediate
products (e.g. acetic acid) to methane by the methanogenic
microbes [34]. In an ABR system where acidogenic bacteria
were dominant, COD was removed through the cytogenesis and
gas releases (mainly CO2 and H2 ), while a significant amount
of COD was converted to liquid intermediate products (e.g.
ethanol, butyrate, and propionate) and stayed in the system.
Therefore, COD removal efficiency in acidogenic phase was
lower than methanogenic phase. The study showed that COD
in the ABR system gradually decreased along each compart-
ment (Table 4). In the initial days of the start-up period, the
COD removal efficiency was higher due to the activity of in-
oculated aerobic activated sludge and the absorption of sludge
floc. Influent COD was 5389 mg/L on the 2nd day of inoc-
ulation, the COD of each compartment was 4308, 3807, and
3353 mg/L. The average COD removal efficiency was 24.3% in
the first 5 days and then gradually decreased to 11.8% in 6–25
days. It stabilized at 9.4% in 26–55 days, during which influ-
ent COD was 5270 mg/L, and the COD concentrations of each
compartment were 5097, 4946, and 4776 mg/L. After combin-
ing with hydrogen production yield of 32.51 L/d at 26–55 days
(Fig. 2), it was obtained that substrate conversation efficiency
(LH2 /g substrate) was 0.03, 0.06, and 0.04 L/g COD for each
compartment.
3.5. Biomass
Based on the measurement of hydrogen yield, liquid fer-
mentation products, pH, alkalinity, and ORP values (Figs. 2, 3
and Table 2), the ABR system reached a stable status on the
26th day. However, biomass concentration (MLVSS) exhibited
a different trend (Fig. 4). The biomass concentration dropped
during the starting-up period and became the lowest on the 6th
day with 5.40, 5.47, and 5.36 g MLVSS/L in each compart-
ment. Afterwards, it gradually increased to 7.31 g MLVSS/L
on the 18th day in the 1st compartment and stabilized in the
later period. However, the concentrations of liquid fermenta-
tion products, which indirectly reflected the shift of microbial
community structure, did not reach a stable status until the 26th
day (Fig. 3a). In contrast, biomass concentration of the 2nd
and 3rd compartments stabilized around 10.00 g MLVSS/L
on the 33rd day (Fig. 4), while the microbial community
structure had already reached the stable status on the 26th
day according to the liquid fermentation products (Fig. 3b
and c). These results indicated that microbial growth (indicated
by MLVSS) and the succession of microbial community
(indicated by liquid fermentation products) occurred at differ-
ent rates.
3280 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283

Table 3
The variation of pH, alkalinity, ORP, VFAs, and CO2 in the ABR system during 55 days operationa

5th day 15th day 25th day 35th day 45th day 55th day

pH 4–4.7 3.6–4.3 4.2–4.4 4.4 4.4 4.3–4.4

Alkalinity (mg CaCO3 /L) 120–290 0–360 360–380 320–360 300–330 240–250
ORP (mV) −230 to −440 −260 to −400 −230 to −250 −240 to −250 −240 to −250 −240 to −250
Total VFAs (mg/L) 731–1650 815–1647 1231–2837 1294–2736 1213–2851 1260–2769
Biogas yield (L/d) 0.81–2.00 3.21–10.01 14.01–24.66 14.11–25.49 13.85–24.87 14.37–25.13
CO2 yield (L/d)b 1.09–2.02 2.44–7.61 6.86–10.09 6.91–10.45 6.51–9.70 6.75–10.05
a The
data ranged from the lowest and highest values in three compartments in the ABR system.
b Biogas
yields were highest in the 1st compartment and lowest in the 3rd compartment during the first 5 days operation period. Afterwards, biogas yields
became highest in the 2nd compartment and lowest in the 1st compartment.

Table 4
The variation of COD in each compartment of the ABR system during 55 days operation

Time (d) Influent (mg/L) 1st compartment (mg/L) 2nd compartment (mg/L) 3rd compartment (mg/L) Total COD removal (%)

2–5 5150 ± 166 4600 ± 298 4305 ± 370 3890 ± 365 24.3 ± 9.1
6–25 5249 ± 275 4991 ± 225 4834 ± 245 4625 ± 291 11.8 ± 3.9
26–55 5270 ± 214 5097 ± 242 4946 ± 277 4776 ± 313 9.4 ± 3.7

12 products (e.g. ethanol, propionate, and butyrate) increased due

to the degradation of molasses in the 1st compartment (Fig. 1),
10 which provided a better condition for the growth of acetogenic
biomass (gMLVSS/L)

bacteria. Although acidogenic bacteria could still grow in these

8
6 teria. However, since the growth rates of acetogenic bacteria
4 1st compartment 2nd compartment
2 3rd compartment
0
0 5 10 15 20 25 30 35 40 45 50
time (d)
Fig. 4. The variation of biomass concentration (MLVSS) in each compartment
of the ABR system during 55 days operation.
This disparity between microbial growth and the microbial
community succession could be explained by the different reac-
tion rates and growth rates of anaerobic bacteria. There are four
major groups of anaerobic bacteria: acidogenic, acetogenic, ho-
moacetogenic bacteria, and methanogenic bacteria. Acidogenic
bacteria grow fast and degrade complex organic compounds
(e.g. hydrocarbonate, fatty acid, and protein), while acetogenic
and homoacetogenic bacteria grow slow and can only degrade
simple compounds (e.g. ethanol, propionate, butyrate, CO2 , and
H2 ) [30,35–37]. In the 1st compartment of the ABR system,
sufficient substrates (mainly molasses) in influent provided a
favorable condition for the fast growth of acidogenic bacteria.
They reached a stable growth on the 18th day. Although pH,
alkalinity, and ORP kept changing until the 26th day, the mi-
crobial community in the 1st compartment had been dominated
by acidogenic bacteria since the 18th day. In the 2nd and 3rd
compartments, the concentrations of intermediate fermentation
two compartments, they were outnumbered by acetogenic bac-
were lower than acidogenic bacteria, it took longer time for
them to achieve a stable status. This was the reason that the
biomass kept increasing until the 33rd day in the 2nd and 3rd
compartments, while fermentation types (Fig. 2) and other oper-
ational parameters (Table 2) had become stable on the 26th day.
55 Based on the results at the stable period (33–55 days), the
biomass concentrations of each compartment were 7.29, 10.42,
and 10.00 g MLVSS/L, and the hydrogen productions were
7.18, 15.10, and 10.21 L/d, respectively. Therefore, the specific
hydrogen production rates for each compartment were 0.11,
0.16, and 0.11 L/g MLVSS d, with the average specific rate of
0.13 L/g MLVSS d in the ABR system. This was higher than a
previous study by Ren et al. [15], in which the maximal spe-
cific hydrogen production rate of a CSTR system was only
0.08 L/g MLVSS d.
4. Discussion
4.1. Comparison of ABR and CSTR in terms of hydrogen
production
Previous study [15] showed that a CSTR system reached
the maximal specific hydrogen production rate of 0.08 L/g
MLVSS d and the maximal substrate conversion efficiency of
0.10 L/g COD when operated at influent COD of 5000 mg/L,
HRT of 8 h, and temperature of 356 ◦ C. In this study, the ABR
system tested under a longer HRT of 13.5 h. Longer HRT was
supposed to lead to a lower specific hydrogen production rate
due to less substrate flowing into the system per hour. However,
 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283
the ABR system achieved a higher hydrogen production with
specific hydrogen production rate of 0.13 L/g MLVSS d and
substrate conversion efficiency of 0.13 L/g COD. There were
two major reasons for the higher hydrogen production in the
ABR system.
(1) The configuration of the reactor: The variations of influ-
ent quality and quantity affected hydrogen production in anaer-
obic systems [16,18,25,26]. Although the stirring in CSTRs
diluted the influent and reduced shocks, this dilution could
not eliminate shocks. The pH, alkalinity, and ORP values of
CSTRs substantially fluctuated and inhibited hydrogen produc-
tion [18,31,32]. It was found that hydrogen yield of a CSTR
system varied from 9.75 to 14.99 L/d even at stable ethanol-
type fermentation [15].
In the ABR system, the 1st compartment handled influent
variations, which was the main reason for the good stability
of the system. Its hydrogen production ranged at 6.65–7.61 L/d
during the stable operation period (26–55 days) (Table 5). Be-
sides the dilution of influent, high concentration of floated
sludge floc in the sludge bed also played an important role in
the 1st compartment. High content of microorganisms in these
sludge flocs interacted with wastewater and formed a stable
micro-ecological system. When there was a shock in influent
quality and quantity, the system could still maintain the sta-
bility through internal adjustment [23,25,26]. This buffering
function in the 1st compartment protected the 2nd and 3rd com-
partments from shocks. Therefore, these two compartments ex-
hibited much stable hydrogen production with 15.72 ∼ 16.00
and 9.60 ∼ 10.86 L/d, respectively (Table 5).
(2) The fermentation microbial community: Complex organic
substances (e.g. starch) in wastewater were first hydrolyzed to
single organic (e.g. glucose). Glucose is further broken down in
two-step reactions sequentially carried out by acidogenic fer-
mentation bacteria (Reaction (1)) and hydrogen-producing ace-
togenic bacteria (Reactions (2)–(4)) under anaerobic condition
[34–37]. Ren et al. [30,38] have found that 1 mol glucose gen-
erated 1 mol acetic acid, 1 mol ethanol as well as 2 mol H2 and
2 mol CO2 at ethanol-type fermentation (Reaction (1)).
C6 H12 O6 + H2 O → CH3 COOH + CH3 CH2 OH
+ 2CO2 + 2H2 ,
CH3 CH2 COOH + 2H2 O → CH3 COOH + CO2 + 3H2 ,
CH3 CH2 CH2 COOH + 2H2 O → 2CH3 COOH + 2H2 ,
CH3 CH2 OH + H2 O → CH3 COOH + 2H2 .
The stirring in CSTRs provided a favorable condition for the
fast growing bacteria (acidogenic fermentation bacteria), while
the slow growing bacteria (hydrogen-producing acetogenic bac-
teria) were at disadvantageous status. Therefore, the hydrogen
production in CSTRs was mainly achieved through Reaction
(1) by acidogenic fermentation bacteria, and the intermediate
liquid products could hardly produce hydrogen through Reac-
tions (2)–(4) due to the low population of hydrogen-producing
acetogenic bacteria.
The growth of different types of bacteria in individual com-
partments separated microorganisms with different growth rates
3281
in an ABR system [39]. A previous study used single-strand
conformation polymorphism (SSCP) technique targeting the
V1.V3 region of 16S rDNA genes to characterize the micro-
bial community of different compartments in the ABR system
[40]. The results showed that the structure of bacterial commu-
nity did not vary with time during the stable period, but signif-
icantly varied along the three compartments. According to the
retrieved results of the operational taxonomic units (OTUs) by
BLAST (the National Center for Biotechnology Information,
NCBI), acidogenic fermentation bacteria were dominant in the
1st compartment while the population of hydrogen-producing
acetogenic bacteria increased in the 2nd and 3rd compartments.
We thought the substrate selectivity might be the main rea-
son for this shift of microbial community. In the ABR sys-
tem, hydrogen was not only produced from glucose through
Reaction (1) in the 1st compartment, but also produced from
liquid intermediate products through Reactions (2)–(4) in the
2nd and 3rd compartments. This was the reason that ABR had
a higher substrate conversation rate (0.13 LH2 /g COD) than
CSTR (0.10 L/g COD) (Table 5).
4.2. The variation of liquid fermentation products in the
ABR system
The concentrations of liquid fermentation products varied in
each compartment (Table 6). The total concentration of VFAs
increased along the three compartments with 1255, 2053, and
2761 mg/L, respectively. This might be caused by the accumu-
lation effect, which means that VFAs was carried over from the
1st compartment to the 2nd compartment and then to the 3rd
compartment. The net increase of VFAs was highest in the 1st
compartment (1254 mg/L), followed by the 2nd compartment
(798 mg/L) and 3rd compartment (707 mg/L). Most of the liq-
uid fermentation products increased along compartments, ex-
cept propionic acid, which was 245, 388, and 324 mg/L in each
compartment. The reason for the high level of propionic acid
in the 2nd compartment might be that propionic acid was car-
ried over from the 1st compartment. But this accumulation ef-
fect did not apply to the 3rd compartment where propionic acid
concentration dropped. This indicated that the conversion rate
(1) from propionic acid might be greater than the generation rate of
propionic acid in the 3rd compartment. Propionic acid could be
(2)
converted to acetic acid and hydrogen by hydrogen-producing
(3) acetogenic bacteria (Reactions (2)–(4)). Although the concen-
trations of ethanol and butyric acid gradually increased from 1st
(4)
to 3rd compartments, their net yields became lower in 2nd and
3rd compartments, indicating that a portion of ethanol and bu-
tyric acid might be converted to acetic acid and hydrogen. The
conversion of intermediate products (ethanol, propionic acid,
and butyric acid) to hydrogen by hydrogen-producing aceto-
genic bacteria led to higher hydrogen productions and lower
net VFSs concentrations in the 2nd and 3rd compartments than
that in the 1st compartment (Tables 5 and 6).
Both operational parameters and liquid fermentation prod-
uct results showed that ABR system provided a favorable
condition for both acidogenic fermentation bacteria and
hydrogen-producing acetogenic bacteria. Organic substances in
3282 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283

Table 5
The average values of hydrogen production parameters in the ABR system at stable status (26–55 days)

Influent 1st compartment 2nd compartment 3rd compartment Average Total

Biogas yield (L/d) — 14 25 20 20 59

H2 percentage (%) — 51 60 51 54 —
H2 yield (L/d) — 7.17 15.11 10.23 10.84 32.51
Biomass (g) — 67 95 92 85 254
Special H2 production rate (L/g MLVSS d) — 0.11 0.16 0.11 0.13 —
COD removal (%) — 3.9 3.6 0.9 2.8 8.5
H2 yield from COD (L/g COD) — 0.03 0.06 0.04 0.04 0.13

Table 6
The average concentrations and net yields of VFAs and ethanol in the ABR at stable status (26–55 days)

Ethanol Acetic acid Propionic acid Butyric acid Valeric acid Total

1st compartment Concentration (mg/L) 443 432 245 108 26 1255

Percentage (%) 35 34 20 9 2 —
Net increase (mg/L) 443 432 24 107 26 1255

2nd compartment Concentration (mg/L) 634 767 388 181 84 2053

Percentage (%) 31 37 19 9 4 —
Net increase (mg/L) 190 335 143 73 57 799

3rd compartment Concentration (mg/L) 908 1094 324 269 166 2761
Percentage (%) 33 40 12 10 6 —
Net increase (mg/L) 274 326 −64 88 83 708

wastewater were converted to hydrogen through reactions
carried out by different types of bacteria in each compart-
ment, thus achieved a higher substrate conversion rate of
0.13 LH2 /g COD (Table 5).
It should be noted that although ABR systems had a
higher hydrogen production rate than CSTRs, it was only
0.13 LH2 /g COD. In this study, hydrogen anaerobic produc-
tion was achieved by the acidogenesis of reductant sugar by
acidogens. The concentration of reductant sugar in the mo-
lasses was only 28%, and there are many other components
(e.g. cellulose, xylogen, and pectin) in molasses, which could
not be effectively degraded by acidogens within a HRT of
13.5 h. Therefore, even though the influent COD was as high
as 5000 mg/L, the specific hydrogen conversation rate by COD
was still low due to the presence of other non-easily-degraded
components. There might be two approaches to increase the
hydrogen production rate: (1) to find out a process to biode-
grade cellulose and xylogen effectively to monosaccharide;
(2) to enrich hydrogen-producing acetogens in fermentation
systems to enhance the conversation rate of volatile fatty acids
(except acetic acid) produced by acidogens to acetic acid and
hydrogen.
5. Conclusion
Based on the study of hydrogen production in an ABR system
with three compartments, several conclusions were drawn:
(1) With diluted molasses as the substrate, the ABR system
reached stable ethanol-type fermentation after 26 days of
acclimatization. The total liquid products content stabi-
lized at 1200, 2000, and 2800 mg/L in the three com-
partments, respectively. Effluent pH, ORP, and alkalinity
ranged from 4.2 to 4.4, −230 to −250 mV, and 240 to
350 mg CaCO3 /L.
(2) Compared with CSTR systems, the individual com-
partment configuration in the ABR achieved a separa-
tion of different anaerobic bacteria. Organic substrates
in influent were efficiently converted to hydrogen in
each compartment. ABR system achieved a hydrogen
yield of 32.51 L/d and a substrate conversion rate of
0.13 L/g COD.
(3) Acidogenic fermentation bacteria dominated in the 1st
compartment, due to the presence of sufficient organic
compounds, while hydrogen-producing acetogenic bacte-
ria became dominant in the 2nd and 3rd compartments
where simple organic compounds had been produced
from the degradation of molasses. The ABR system ex-
hibited a better stability and higher microbial activity than
CSTRs.
Acknowledgements
The authors would like to thank the National Natural Sci-
ence Foundation of China (Contract No. 50378025), the Na-
tional High Technology Research and Development Program of
China (863 Program, Grant No. 2006AA05Z109), and Provin-
cial Science Foundation of Heilongjiang (Grant No. E0305) for
their supports for this study.
 J. Li et al. / International Journal of Hydrogen Energy 32 (2007) 3274 – 3283
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