international journal of hydrogen energy 34 (2009) 245–254

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/he

A bench scale study of fermentative hydrogen and methane

production from food waste in integrated two-stage process

Xing Wang*, You-cai Zhao

The State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Siping road 1239, Shanghai 200092, China

article info abstract

Article history:
Received 11 August 2008
Received in revised form
27 September 2008
Accepted 28 September 2008
Available online 28 November 2008
Keywords:
Integrated two-stage process
Fermentative hydrogen production
None-heat treatment of inoculum
Indigenous microbial cultures
A two-stage fermentation process combining hydrogen and methane production for the
treatment of food waste was investigated in this paper. In hydrogen fermentation reactor,
the indigenous mixed microbial cultures contained in food waste were used for hydrogen
production. No foreign inoculum was used in the hydrogen fermentation stage, the
traditional heat treatment of inoculum was not applied either in this bench scale experi-
ment. The effects of the stepwise increased organic loading rate (OLR) and solid retention
time (SRT) on integrated two-stage process were investigated. At steady state, the optimal
OLR and SRT for the integrated two-stage process were found to be 22.65 kg VS/m3 d (160 h)
for hydrogen fermentation reactor and 4.61 (26.67 d) for methane fermentation reactor,
respectively. Under the optimum conditions, the maximum yields of hydrogen
(0.065 m3 H2/kg VS) and methane (0.546 m3 CH4/kg VS) were achieved with the hydrogen
and methane contents ranging from 29.42 to 30.86%, 64.33 to 71.48%, respectively. Biode-
gradability analysis showed that 5.78% of the influent COD was converted to the hydrogen
in H2-SCRD and 82.18% of the influent COD was converted to the methane in CH4-SCSTR
under the optimum conditions.
ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.

1. Introduction economic feasibility of waste treatment. Moreover, biological

Hydrogen has been widely recognized as a clean energy
carrier of the future. It can be used as an important indus-
trial raw material in hydrogenation processes [1]. Among
various hydrogen-producing methods, anaerobic dark
fermentation might be the most promising one since the
rate of hydrogen is higher than that of photo fermentation
and bio-photolysis [2].
Dark fermentation is a preferred method for resource
recovery and energy conversion from food waste. It has
several advantages, including volume reduction, waste
stabilization, and biogas recovery. In particular, the two-stage
process including bio-hydrogen and methane production with
phase separation has considerable potential to enhance the
methods produce hydrogen from various renewable resources
and are less energy-intensive than chemical or electro-
chemical ones since they are carried out at ambient temper-
ature and pressure.
Two-phase anaerobic digestion system is often used for
fermentative hydrogen and methane fermentation. The
system refers to the development of unique acid-formers and
methane formers in two separate reactors [3–5]. In such
a system, only slow-growing acidogens and hydrogen
production microbes are found in the first phase and involve
the production of volatile fatty acids (VFAs) and hydrogen,
while slow-growing acetogens and methanogens are found in
the second phase, in which VFAs are converted to methane
and carbon dioxide.

* Corresponding author. Tel.: þ86 021 65980609.

E-mail address: wangxing24@126.com (X. Wang).
0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2008.09.100
246 international journal of hydrogen energy 34 (2009) 245–254
Nomenclature
TS total solid
VS volatile solid
HRT hydraulic retention time
VFA volatile fatty acid
NH4-N ammonia-nitrogen
C0 VS concentration of the influent (g/kg)
r(c) substrate removal rate (kg VS/m3 d)
q organic loading rate (kg VS/m3 d)
m0 feeding rate (kg/d)
mH theoretic maximum H2 yield (m3/kg VS)
mM theoretic maximum CH4 yield (m3/kg VS)
kH first order constant for hydrogen
fermentation stage (d1)
kM first order constant for methane
fermentation stage (d1)
H2-SCRD semi-continuous rotating drum
CH4-SCSTR semi-continuous stirred tank type
reactor
SRT solid retention time
COD chemical oxygen demand
t time (d).
Ct VS concentration in the reactor at time t (g/kg)
Vm volume of the reactor (m3)
k first order constant (d1)
m theoretic maximum biogas yield (m3/kg VS)
y biogas yield (m3/kg VS)
With regard to two-stage fermentation process, the first stage
(fermentative hydrogen production process) does not signifi-
cantly reduce the organic content of the feed. Usually, chemical
oxygen demand (COD) removal is below 20% during hydrogen
production process [6], corresponding to a mean hydrogen
production of 2.5 mol/mol glucose. The undegraded COD can be
removed in a subsequent methane fermentation stage with the
conversion of organic content to CH4. Moreover, the first stage
(hydrogen fermentation stage) can also be used as an indepen-
dent hydrogen production unit but not as a precursor/pretreat-
ment for the methanogenic reactor. The present study focuses
on the exploitation of unsterilized food waste as a source for
hydrogen and subsequent methane production.
It has been reported that in two-phase anaerobic digestion,
H2 and CO2 were produced in the first stage, the effluent of first
stage was transferred to the second stage to be converted to
CH4, few measurements on hydrogen production were
reported in the literature [7]. Previous study found that only
small amount of hydrogen was produced since the hydrogen
was utilized by methanogens [8].
To produce hydrogen from a dark fermentation metabo-
lism, the blocking of the methanogenesis in the anaerobic
pathway is one of the key considerations, due to the conver-
sion of hydrogen to methane in this step. The inhibition of the
methanogenic activity can be achieved by controlling pH,
temperature (heat treatment) or using additives. Of the
various parameters, heat treatment of the inoculum (seed
sludge) was usually applied because high temperature can
inactivate hydrogentrophic methanogens, enrich hydrogen-
producing bacteria and select spore-forming anaerobes [9,10].
Although thermal treatment of the inoculum can, to some
extent, improve the fermentative hydrogen production,
however, it consumes energy, increases the operation cost
and complicates the operation procedure since the inoculum
should be first heated at 100  C for more than 15 min before
fermentation. Except for the heat treatment of inoculum,
hydrogen production by fermentative bacteria is also highly
dependent on the conditions of the process, such as solid
retention time (SRT), organic loading rate (OLR) and gas partial
pressure, which affect the microbial metabolic balance and
subsequently the fermentation end products [6]. The SRT
determines the substrate uptake efficiency, microbial pop-
ulation, and metabolic pathway. In H2 fermentation, it is
generally assumed that a high SRT causes the growth of H2
consumers, including methanogens, and competitors for
substrates, such as non-H2-producing acidogens [11].
However, a low SRT may reduce substrate uptake efficiency,
active biomass retention, and therefore, the overall process
efficiency [12]. Changing the OLR can increase the H2 yield.
However, there is disagreement in the literature as to whether
higher H2 yields are achieved with lower or higher OLRs. In
some cases higher OLRs decreased the H2 yield whereas in
others higher OLRs increased the H2 yield. In the latter case, as
OLRs increased the H2 yield usually became constant or
eventually began to decrease thereby providing an optimal
OLR (maximum H2 yield) [13,14].
Based on the above information, we developed a two-stage
process combined by a mesophilic hydrogen production
reactor and a methane production reactor. In hydrogen
fermentation reactor, the heat treatment was not applied in
hydrogen fermentation. The indigenous mixed microbial
culture contained in food wastes was used for hydrogen
production. No other foreign seed organisms, such as anaer-
obic sludge, waste activated sludge was added into the
hydrogen fermentation reactor.
The objectives of this study were to investigate the (a)
fermentative production of hydrogen from food waste in
semi-continuous rotating drum (H2-SCRD) at various OLR and
SRT using an indigenous mixed microbial culture contained in
food waste as the hydrogen producer and (b) subsequent
anaerobic treatment of the effluent of the H2-SCRD with the
simultaneous production of methane in a semi-continuous
stirred tank type reactor (CH4-SCSTR). The two-stage process
was operated at semi-continuous and integrated mode. The
effects of the stepwise increased organic loading rate (OLR)
and solid retention time (SRT) on integrated two-stage process
were also investigated in this bench scale study. A simple
kinetic model for anaerobic digestion based on mass balance
equation was used to evaluate the performance of the
hydrogen and methane fermentation phase.
2. Materials and methods
2.1. Feedstock and inoculum for methane fermentation
The indigenous microbial cultures contained in food waste
were used for fermentative hydrogen production. Food waste
was collected from the restaurants near the experiment
site, and crushed into small particles (2–4 mm). The chemical
 international journal of hydrogen energy 34 (2009) 245–254
and physical characteristics of the food waste and inoculum
are presented in Table 1.
An anaerobic granular sludge obtained from a UASB tank
treating cassava waste was used as inoculum for methano-
genic reactor. The inoculum has been sufficiently adapted to
the food waste for over 1 year in another experiment.
2.2. Integrated two-stage fermentation system
An integrated two-stage fermentation system was designed
for bench scale study (Fig. 1). The system consisted of
a hydrolysis/acetogenesis rotating drum for hydrogen
production (total volume and working volume was 0.45 and
0.2 m3, respectively, stainless steel), a methane fermentation
tank (total volume and working volume was 1.5 and 0.8 m3,
respectively, stainless steel). The system was operated at the
integrated mode. Two buffer tanks were installed in this
system, which were used to temporarily hold the food waste
and effluent of hydrogen fermentation reactor before they are
pumped into H2-SCRD and CH4-SCSTR. Food waste was first
added into a buffer tank and then feed into H2-SCRD with
a peristaltic pump for fermentative hydrogen production,
subsequently the effluent of the H2-SCRD was pumped into
the other buffer tank and immediately drained into
CH4-SCSTR to be converted to methane. In order to save the
energy consumption and better reflect the large-scale
processes, the heat treatment was not applied in the first stage
of hydrogen fermentation.
The rotating speed of H2-SCRD was set at 5 rpm and pro-
grammed to rotate periodically for 3 min, 5 times per hour by
Siemens Micromaster Vector 420. CH4-SCSTR was equipped
with an impeller fixed in the center of the tank at the speed of
120 rpm. Due to safety consideration, the biogas was not
combusted to generate heat for the bioreactors. The H2-SCRD
and CH4-SCSTR were constructed with water jacket (8 cm
thick) to maintain stable temperature for the fermentation
process, in which the water was heated by super-heated
steam supplied from a nearby steam factory. The temperature
in water-jackets was set at 40  2  C and controlled by
temperature sensors. In addition, the whole system was
equipped with other facilities such as food waste grinder,
peristaltic pump, biogas bag, and gas meter.
2.3. Semi-continuous experiments for fermentative
hydrogen and methane production
The semi-continuous experiments were carried out with
four OLRs (feeding rate). For start-up, the H2-SCRD (working
volume 0.2 m3) was filled with food waste, operated
Table 1 – Characteristics of food waste and inoculum
Parameter Food waste Inoculum
TS (%) 17.60  0.35 5.78  0.42
VS/TS (%) 85.91  1.27 63.66  1.68
pH 4.68  0.14 7.04  0.27
Total-N (%) 2.34  0.08 0.42  0.15
TCOD (g O2/kg)a 211.79  11.13 93.114  4.36
a In wet weight.
247
anaerobically at batch mode for about 72 h to activate the
hydrogen producer and subsequently switched to the semi-
continuous mode at designated OLR. The whole process
was conducted with OLR increased in steps and maintained
for each OLR level of about 30 days. The four levels of OLR
for H2-SCRD were designed to be 15.10, 22.65, 30.20,
37.75 kg VS/m3 d, respectively. The corresponding solid
retention time (SRT) for H2-SCRD was 240, 160, 120, 96 h,
respectively. VS content of effluent from H2-SCRD was
determined periodically, and the OLR for CH4-SCSTR was
calculated to be 2.94, 4.61, 6.28, 8.15 kg VS/m3 d. The corre-
sponding SRT for methane fermentation step was 40, 26.67,
20, 16 d, respectively. In order to better evaluate the
performance of the two-stage anaerobic digestion system,
the pH in both fermentation stages was not adjusted in this
study.
2.4. Analytic method
The VFAs were determined with gas chromatography (GC).
The samples were centrifuged at 10000 rpm for 10 min and
filtered through a 0.45-mm membrane before the VFA deter-
mination. The filtrate was collected in a 1.5 ml GC vial, and
acidified with 3% H3PO4 to pH 4.0 before assayed on an Agilent
6890N GC with flame ionization detector and DB-WAXETR
column (30 m  1.0 mm  0.53 mm). Nitrogen was the carrier
gas and the flux was 25 ml/min. The injection port and the
detector were maintained at 220 and 250  C, respectively. The
sample injection volume was 1.0 ml. The oven of GC was pro-
grammed to begin at 110  C and to remain there for 2 min,
then to increase at a rate of 10  C/min to 220  C, and to hold at
220  C for an additional 2 min.
The concentration of lactate was determined using
a liquid chromatograph (Agilent, 1200) equipped with ultra-
violet detector. ODS Hypersil (4.6 mm  250 mm  5 mm) was
used for the column. Chromatography was performed using
binary mobile phase composed of phosphoric acid (0.01 mol/
l NaH2P04) and methanol, 98:2 (v/v), pH 3.0. UV detection
was accomplished at 214 nm. The analysis was carried out
at a detector temperature of 25  C, flow velocity of 1.0 ml/
min, and UV 210 nm. The retention time for lactate was
4.8 min.
TS and VS were analyzed according to the standard
methods [15]. The biogas produced from H2-SCRD and
CH4-SCSTR was measured using a wet gas meter collected in
gas bags. CH4 and CO2 were determined in a 200 ml sample by
gas chromatography (SHIMADZU GC-14B) with a stainless
steel column packed with Carbosive SII (diameter of 3.2 mm
and 2.0 m length) and thermal conductivity detector (TCD).
The temperature of the injection, column and detector was set
at 40, 80 and 90  C, respectively. The carrier was nitrogen and
the flow rate used was 30 ml/min. A gas standard consisting of
60% (v/v) CH4 and 40% of CO2 was used for gas chromatog-
raphy calibration.
2.5. Kinetic models for two-step anaerobic digestion
The mass balance equation with equal mass flow of input/
output and the derivation methodology for the first order
kinetic constant described by Linke [16] was used in this study.
248 international journal of hydrogen energy 34 (2009) 245–254

Methane
hot water tank

CH4-SCSTR
food waste H2-SCRD:
Semi-continuous
rotating drum

CH4-SCSTR:
Semi-continuous
H2-SCRD stirred tank type
buffer tank Residue
reactor

Fig. 1 – Schematic diagram of integrated two-stage fermentation process.

The mass balance equation for each fermentation stage can be (d1); m0 the feeding rate (kg/d); m the theoretic maximum
presented as biogas yield (m3/kg VS); y the biogas yield (m3/kg VS); t the
dc time (d).
V ¼ m0 $c0  m0 $c þ V$rðcÞ (1) Results from bench-scale experiment were used to apply
dt
the model. Parameter values of mH, mM, kH and kM can be esti-
The rate of degradation of the substrates was assumed to
mated using non-linear regression fit to the hydrogen and
follow first-order kinetics, thus the following equations were
methane production data with Eq. (8).
used to describe the H2/CH4 production and substrate degra-
dation in semi-continuous operation
dc 2.6. Determination of biodegradability in H2/CH4 step
 ¼ rðcÞ ¼ k$c (2-1)
dt
Biodegradability can better reflect the conversion rate of the
y ¼ uð1  eÞkt (3) substrate into biogas. In present study, the anaerobic biode-
By integrating Eq. (2-1), we obtained gradabilities of the substrate in hydrogen and methane
fermentation stages were calculated from the maximum
cðtÞ ¼ c0 $ekt (2-2) H2/CH4 yield and the COD of the substrate [17], according to
According to the following combination of equations, we
Hy $0:714
obtained Eqs. (4)–(8) BDH ¼ 100 (a)
WH $CODH
9
dc >
>
¼ 0ðsteady stateÞ >
>
dt >
>
>
>
dc >
> My $2:85
Vm ¼ m0 $c0  m0 $c þ Vm $rðcÞ ð1Þ >
= c0 $k$ct
BDM ¼
WM $CODM
100 (b)
dt q¼ ð5Þ;
dc >
> c0  ct
 ¼ rðcÞ ¼ kc ð2-1Þ >
> where BDH and BDM represent the biodegradability in
dt >
>
>
> hydrogen and methane fermentation reactor at a specific OLR,
c0 >
>
Vm ¼ m0 $ >
;
q respectively. Hy and My represent the hydrogen and methane
q$c0 production at a specific OLR(m3), respectively. CODH repre-
cðtÞ ¼ (6)
q þ c0 $k sents the COD of the food waste (in kg O2/kg VS) and CODM
represents the COD of the effluent of H2-SCRD (in kg O2/kg VS)

cðtÞ ¼ c0 $ekt ð2-2Þ
 c0 m at a specific OLR. WH represents the feed rate of H2-SCRD at
kt ¼ (4)
y ¼ u$ 1  e ð3Þ cðtÞ m  y a specific OLR (in kg VS). Wm represents the feed rate of CH4-
SCSTR at a specific OLR (in kg VS). The constants 0.714 and 2.85
9
c0 m correspond to the COD (kg O2) of 1 m3 H2 and CH4, respectively.
¼ ð4Þ > >
>
>
cðtÞ m  y >
>
>
=
c0 $k$ct m$k$cðtÞ
q¼ ð5Þ y¼ ð7Þ;
c0  ct >
> q
>
>
q$c0 >
> 3. Results and discussion
cðtÞ ¼ ð6Þ >
;
q þ c0 $k
m$k$c0 3.1. Biogas production
y¼ ð8Þ
k$c0 þ q
where c0 is the VS concentration of the influent (g/kg); ct the VS The integrated two-stage process for fermentative hydrogen
concentration in the reactor at time t (g/kg); r(c) the substrate and methane production was operated for about 4 months.
removal rate (kg VS/m3 d); Vm the volume of the reactor (m3); q Using indigenous microbial cultures contained in the food
the organic loading rate (kg VS/m3 d); k the first order constant waste as the hydrogen producer, fermentative hydrogen was
 international journal of hydrogen energy 34 (2009) 245–254
successfully produced in the first stage without foreign inoc-
ulation and heat treatment.
After the operation of 34 days, steady state was achieved
for the integrated two-stage process. Biogas yield ranged from
0.167 to 0.236 m3/kg VS in H2-SCRD, the content of H2 was
28.03–33.02%, CO2 46.68–51.47%, and CH4 was not detected. In
CH4-SCSTR, biogas yield reached 0.760–0.874 m3/kg VS at
steady state, the content of CH4 lied between 58.32% and
71.48%, CO2 between 26.34% and 40.73%. In CH4-SCSTR, the
hydrogen content was determined to be less than 0.2%
through the entire experiment even when operated at the
maximal OLR (8.15 kg VS/m3 d) where large amount of
hydrogen producer entered the CH4-SCSTR with the effluent
of hydrogen fermentation reactor. This result suggested that
methanogens can quickly dominate the culture although the
H2 producers were present at high OLR [18].
The stepwise increased OLR caused the fluctuation of
biogas yields in hydrogen and methane fermentation reactor
as shown in Fig. 2, the contents of hydrogen and methane in
different reactors were also observed to decrease with the
increase of OLR. An increase of lactic acid concentration was
observed (from 2345.62 to 4425.56 mg/l) as OLR increased from
15.10 to 37.75 kg VS/m3 d. It has been reported that lactic acid
has inhibition effect on hydrogen production [19]. In the
present study, the pattern of changes in lactic acid concen-
tration seemed to be related with hydrogen production. As the
concentration of lactic acid increased from 2345.62 to
4425.56 mg/l, the hydrogen yield dropped from 0.071 to
Fig. 2 – Biogas production in (a) H2-SCRD and
(b) CH4-SCSTR.
249
0.049 m3/kg VS, which was confirmed to be statistically
significant (ANOVA, P < 0.05).
In this bench scale test, the pH was between 5.2 and 5.8 in
H2-SCRD (Fig. 3(a)) without manual interference, the higher
hydrogen yield (0.071 m3/kg VS) was obtained as OLR was kept
at 15.10 kg VS/m3 d with the pH varying from 5.62 to 5.74, in
comparison with the hydrogen yield of 0.065, 0.054, 0.049 m3/
kg VS at the OLR of 22.65, 30.20, 37.75 kg VS/m3 d, respectively.
The pH variation in the present study agrees with much of the
work published regarding fermentative hydrogen production
in which a pH value between 5.0 and 6.0 is considered favor-
able [21]. It has been reported that at pH 5.5 a peak of hydrogen
production rate was observed. In addition, VS removal and
total VFAs production were higher inside the pH range of
5.0–6.0 [22].
It is obvious that the mixed acid fermentation occurred in
hydrogen fermentation phase in this study. It is also observed
that the formation of hydrogen was accompanied with that of
the VFAs. According to the compositions of the determined
VFAs, it can be concluded that butyrate-producing pathway
was the dominant hydrogen fermentation pathway at low
OLR, which subsequently shifted to lactic acid-producing
pathway when OLR increased to high level. In this bench scale
test, neither heat treatment of inoculum nor foreign inocula-
tion was applied in the start-up of H2-SCRD. From a practical
point of view, it is notable that the indigenous food waste
microbial cultures can be used as the mixed microflora inoc-
ulum in hydrogen fermentation stage. This can be beneficial
to the operation of full-scale plant since no extra energy would
be consumed for the heat treatment, moreover the operation
of the whole process can be facilitated.
3.2. Effects of OLR and SRT on biogas production
The OLRs for hydrogen fermentation stage and methane
fermentation were both kept at four levels, as shown in
Table 2. Each OLR lasted for 30 day. Negative effects of higher
OLR were exhibited as it stepwise increased, resulting in the
decrease in both hydrogen and the subsequent methane
yields. An increase of OLR from 15.10 to 22.65 kg VS/m3 d
caused an insignificant decrease of hydrogen yield from 0.071
to 0.065 m3/kg VS d (ANOVA, P > 0.05), however, the further
increase of OLR from 22.65 to 30.20 kg VS/m3 caused consid-
erable decline of hydrogen yield from 0.065 to 0.054 kg VS/m3,
which was found to be statistically significant (ANOVA,
P < 0.05).
The methane fermentation, using the effluent from H2-
SCRD as substrate, was performed in CH4-SCSTR at OLR
varying from 2.94 to 8.15 kg VS/m3 d. The methane yields of
0.551  0.001, 0.546  0.014, and 0.525  0.003 m3/kg VS were
found at the lower OLR of 2.94, 4.61, 6.28 kg VS/m3 d, respec-
tively. The statistical difference was found to be insignificant
(ANOVA, P > 0.05). This result indicated that the OLR ranging
from 2.94 to 6.28 kg VS/m3 d did not exhibited significant
inhibition on methanogens in CH4-SCSTR. However, with an
increase of OLR from 6.28 to 8.15 kg VS/m3 d, methane yield
rapidly decreased by 8.57% from 0.525  0.003 to
0.480  0.021 m3/kg VS, the statistical difference was found to
be significant (ANOVA, P < 0.05).
250 international journal of hydrogen energy 34 (2009) 245–254

4 -N (mg/l)

881.65  40.81
1417.82  65.63
2134.68  43.54
2625.45  96.14
4 -N(mg/l)
258.14  24.62
350.87  29.51
422.41  31.95
427.52  30.25

NHþ
NHþ

Lactic acid (mg/l)

Lactic acid (mg/l)

12.46  2.54
8.24  1.27
11.38  3.12
9.78  2.68
2345.62  54.32
3216.78  34.53
3645.38  48.17
4425.56  51.68

Metabolites in the effluent from CH4-SCSTR

Butyric acid (mg/l)

Butyric acid (mg/l)

45.32  12.63
48.16  16.44
67.98  15.98
76.25  17.43
Metabolites in the effluent from H2-SCRD

2641.08  54.65
2307.43  74.01
2411.23  63.22
2235.54  74.83

Propionic acid (mg/l)

Propionic acid (mg/l)

76.73  21.16
87.14  16.76
105.21  19.32
112.80  17.71
1241.13  39.67
1861.35  47.49
1743.84  56.34
1758.65  48.65

Acetic acid (mg/l)

322.66  38.87
338.28  65.43
398.41  54.32
432.09  65.76
Fig. 3 – Profiles of pH, VS concentration and OLR in H2 (a)/
Acetic acid (mg/l)

CH4 and (b) fermentation step of two-stage process,

Table 2 – Operation parameters and profiles of metabolites in H2-SCRD/CH4-SCSTR

4355.41  89.74
4153.46  93.45
3623.16  75.29
3426.17  64.76

respectively.

Different letters indicate a significant difference at 5% level Duncan’s multiple range test.
Ethanol (mg/l)

A decrease of VS removal efficiency was observed as the

OLR was gradually increased to higher level, indicating the
reduction of hydrolysis efficiency in hydrogen fermentation
Ethanol (mg/l)

2320.55  57.43
2102.68  64.39
1748.62  48.27
1412.80  72.63

stage. As shown in Fig. 3(a), VS was destructed from 15.81%

N/A
N/A
N/A
N/A

(influent) to 11.77% at OLR of 15.10 kg VS/m3 d with a VS

removal efficiency of 25.55%. The VS content was further
CH4 yield (m3/kg VS)

increased from 11.77% (OLR 15.10 kg VS/m3 d) to 13.14% at the

OLR of 37.75 kg VS/m3 d with a VS removal efficiency of
b
a
a
a
0.551  0.001
0.546  0.014
0.525  0.003
0.480  0.021

16.89%. The decrease of VS removal efficiency was probably

0.054  0.002 b
0.049  0.001 b
0.071  0.001 a
0.065  0.002 a
(m3/kg VS)

attributed to the increasingly greater OLR, since higher OLR

H2 yield

can lead to shorter solid retention time (SRT) thus cause

insufficient time for hydrolysis of insoluble substrates. Bio-
hydrogen is known to be produced as a by-product in the
hydrolysis of various substrates, e.g., carbohydrate. It should
Feed rate

be noticed that the hydrolysis of substrate is commonly

Feed rate

(kg/d)
(kg/d)

20
30
40
50

known as the rate-limited step, therefore any parameter that

20
30
40
50

can negatively affect the hydrolysis process would decrease

the hydrogen yield, such as the parameter of SRT in the
OLR in CH4-SCSTR

present study. From the perspective of hydrolysis efficiency, it

OLR in H2-SCRD

can be emphasized that the lower OLR (from 15.10 to

22.65 kg VS/m3 d) is more suitable for hydrogen fermentation
(kg VS/m3 d)

(kg VS/m3 d)

stage when the indigenous food waste microbial cultures were

used as hydrogen producer.
15.10
22.65
30.20
37.75

2.94
4.61
6.28
8.15

In methane fermentation stage, the suitable OLR ranged

from 2.94 to 4.61 kg VS/m3 d since higher OLR caused the
 international journal of hydrogen energy 34 (2009) 245–254
Table 3 – Comparable H2 yields from different substrates
Substrate SRT (h) Reactor pH
Kitchen waste 96–240 Rotating drum 5.2–5.8
Food waste 126 ASBR 5.3
Food waste 120 CSTR 5.5
Household solid waste 48 CSTR 4.8–5.2
Household solid waste 28.8 CSTR 5.8–6.0
a m3/kg, VS.
b Theoretical maximum yield.
accumulation of NHþ 4 -N in CH4-SCSTR. It is known that the
methane production with good performance is strictly regu-
lated by environmental factors, such as pH, temperature, VFA
concentration, and NH4þ-N concentration. High concentra-
tion of VFA and NHþ 4 -N can inhibit the methane production. As
shown in Table 3, the total concentration of VFAs in CH4-
SCSTR slightly fluctuated from 457.17 to 630.92 mg/l, the pH
varied in the range of 6.7–7.3. With the low concentration of
VFAs and the stable fluctuation in pH, their effects on
methane fermentation can be ruled out from the possibilities
that may result in the decrease of methane yield. At this point,
the decrease of methane yield was most likely to be caused by
the accumulation of NHþ 4 -N. As OLR increased in steps, the
concentration of NHþ 4 -N considerably increased from 0.551 to
2.625 g/l in CH4-SCSTR. High NHþ 4 -N concentration is toxic for
anaerobes [23,24]. Methanogens are the least tolerant to
ammonia and most likely to cease growth due to ammonia
inhibition [25]. McCarty reported that NHþ 4 -N concentrations
in excess of 3000 mg/l were expected to be toxic at any pH
value [26]. Hobson and Shaw reported that 235 mmol
(3290 mg/l) of NHþ 4 -N completely prevented the growth of
Methanobacterium formicicum in the pure culture [27].
In H2 fermentation, it is generally assumed that a high SRT
causes the growth of H2 consumers, including methanogens
and non-methanogenic H2-consuming or non-H2-producing
acidogens [11]. However, in the present study, higher SRT
seemed to be beneficial to H2 fermentation since high SRT can
provide sufficient time for the hydrolysis of substrate and
reduce the influx of lactic acid-producing microorganisms.
Besides, using indigenous food waste microbial cultures as the
hydrogen producer avoided the existence and prevalence of
methanogens, which was commonly thought to be the main
obstacle in fermentative hydrogen. It was found in this study
that SRT ranging from 160 to 240 h yielded higher hydrogen
production, which was statistically greater than that at lower
SRT. Previous studies reported the similar observations that
a higher SRT was required in bio-hydrogen fermentation due
to the slowly degradable organic compounds [20,28]. The H2
yield in this test was comparable to other reported values in
H2 production using organic solid waste, as shown in Table 3.
It should be noticed that under the conditions where none-
heat treatment was applied (as in the present study) in
hydrogen fermentation, the SRT was relatively longer than in
conventional studies which used heat treatment to select
spore-forming bacteria and inactivate the hydrogen-
consumer.
SRT is an important parameter in determining the
fermentation efficiency and the size of fermenter. Present
251
Temperature ( ) H2 yield (ml/g VS) References
a b
40 0.049–0.071 (0.118 ) Present study
35 80.9 [12]
55 125.0 [28]
37 43.0 [9]
60 46.3 [35]
study showed a significant increase ( p < 0.05) in hydrogen
yield (as shown in Table 2) when SRT increased from 120 to
160 h (corresponding to OLR decreasing from 30.20 to
22.65 kg VS/m3 d). The further increase of SRT from 160 h (OLR
22.65 kg VS/m3 d) to 240 h (OLR 15.10 kg VS/m3 d) caused an
insignificant ( p > 0.05) increase in hydrogen yield. With regard
to the methane production performance, the statistical
difference was also confirmed to be insignificant ( p > 0.05)
among the methane yields when SRT was kept at 40 d
(2.94 kg VS/m3 d), 26.67 d (4.61 kg VS/m3 d) and (6.28 kg VS/
m3 d), respectively. Therefore, based on the above observa-
tions on the hydrogen and methane yields and the consider-
ation of fermenter size, the optimal SRT and OLR for the
integrated two-stage process were considered to be 160 h
(22.65 kg VS/m3 d) and 26.67 d (4.61 kg VS/m3 d), respectively.
3.3. Metabolic products
The distribution of metabolites is a crucial signal in the
assessment of the efficiency of hydrogen-production course.
The concentrations of the main metabolic products measured
during bench scale tests are presented in Table 3. In the
effluent of H2-SCRD, the dominant metabolic products were
ethanol, acetic acid and butyric acid. The sum of which
accounted for 72.20% (9317.04 mg/l) of the determined
metabolites (12903.79 mg/l, NH4-N not included) as OLR was
kept at 15.10 kg VS/m3 d, revealing the dominance of ethanol,
acetic acid and butyric acid producing bacteria in hydrogen
fermentation reactor. As the OLR further increased to
37.75 kg VS/m3 d, the sum percentage of ethanol, acetic acid
and butyric acid considerably declined to 7074.51 mg/l,
amounting up to 53.36% of the total determined metabolites
(13258.72 mg/l). It was found that as the concentrations of
acetic acid, butyric acid and ethanol were reduced, the pro-
pionic acid and lactic acid concentrations increased. There-
fore, it can be inferred that branch reactions for the
production of propionic and lactic acids occurred.
At the OLR of 37.75 kg VS/m3 d, the lactic acid concentra-
tion reached the maximum of 4425.56 mg/l, accounting for
33.37% of the determined VFAs, which was close to the sum
percentage of ethanol and acetate (36.49%). This result
demonstrated the bloom of lactic acid-producing microor-
ganism at the highest OLR of 37.75 kg VS/m3 d since at higher
OLR more lactic acid producer may enter the hydrogen reactor
with the influx of food waste.
As mentioned above, the stepwise increased OLR led to the
declining rate in hydrolysis of substrate, which was probably
the main cause for the considerable drop of hydrogen yield.
252 international journal of hydrogen energy 34 (2009) 245–254

Besides that, the increase of lactic acid concentration might be

another cause for the decrease of hydrogen yield. The initial
concentration of lactic acid in food waste was 1435.56 mg/l,
revealing the presence of lactic acid-producer before its
entering into H2-SCRD. In other words, large unexpected
influx of lactic acid producer may enter the H2-SCRD at high
OLR, which converted glucose contained in the food waste
into lactic acid, therefore led to the increase of lactic acid
concentration and the reduction in hydrogen yield. According
to the metabolic pathway of glucose, hydrogen producer, i.e.
Clostridium can hardly produce hydrogen from lactic acid as
a sole carbon source [29]. In the present study, an increase of
lactic acid concentration (from 2345.62 to 4425.56 mg/l) was
observed with the OLR from 15.10 to 37.75 kg VS/m3 d. It was
reported that the conversion efficiency of lactic acid to
hydrogen (0.5%) was much lower than that of glucose or
sucrose [30]. Accordingly, the observed increase of lactic acid
concentration was probably another cause for the decrease of
hydrogen production. In addition, the decrease of hydrogen
yield was also likely to be affected by the increase in propio-
nate concentration, since hydrogen cannot be produced in the
propionate production pathway. As shown in Table 2, the
concentration of propionate was 1241.13 mg/l at the lowest
OLR of 15.10 kg VS/m3 d, which was gradually increased to
1758.65 mg/l with OLR increasing up to 37.75 kg VS/m3 d.
Hence, it was inferred that the propionic acid was an impor-
tant indicator of the variation of bio-hydrogen process. It has
been reported that at pH 6.0  0.1 the propionate producers
were active and competed with the ethanol and butyrate
producers for the substrate in hydrogen fermentation [31].

3.4. H2/CH4 theoretical maximum yield and first order

kinetic constant determination

Fig. 4 – Linear correlation between OLR and H2 (a)/CH4 (b)

The estimated curves with theoretical maximum yield of H2/
CH4 (mH/mM) plotted against OLR show that the first-order
kinetic model described well the degradation of food waste in
H2/CH4 fermentation, as shown in Fig 4. mH and mM were
obtained by curve fitting from Eq. (8), which resulted in
0.118  0.001 m3 H2/kg VS and 0.611  0.015 m3 CH4/kg VS. The
theoretical maximum yield of H2/CH4 (mH/mM) was equivalent
to the ultimate anaerobic biodegradability and results when
OLR was near zero.
The range of the first-order constant for putrescible
components in methane fermentation has been reported to be
between 0.10 and 0.35 d1. The first-order constant kM for
methane fermentation was determined to be
0.264  0.046 d1in the present study. Compared with
a previous study, a similar constant for methane fermentation
was obtained for the swine wastes anaerobic digestion
(k ¼ 0.259 d1). k-value was also found to be 0.211–0.331 d1
during the anaerobic digestion of swine wastes with zeolite
added in the range of 0–0.5 g zeolite/g VS [32]. Jokela et al.
reported much lower constant for the grey wastes
(0.021–0.058 d1). It shall be noticed that the grey wastes are
rich in cellulose, which are relatively difficult to be hydrolyzed
compared with putrescibles [33]. In this case, the high value of
first-order constant in this study was attributed to the fact
that the hydrogen fermentation stage pre-hydrolyzed the food
waste, breaking the molecular structure of organic particles,
yield.
thus enhanced the efficiency of methane fermentation stage
and increased the hydrolysis rate since hydrolysis is recog-
nized as a rate-limited step where the substrate consists of
particles [34]. With regard to the first-order constant in
hydrogen fermentation, mH (0.173  0.007 d1) was much lower
than that of methane fermentation, indicating slower hydro-
lysis rate in hydrogen fermentation step under the circum-
stance of no foreign inoculation.
3.5. Biodegradability in H2-SCRD and CH4-SCSTR
The biodegradability of the substrate in H2-SCRD and
CH4-SCSTR indicated the ratio of substrate decomposed in
each step of two-stage fermentation process. As shown in
Fig. 5, the biodegradability in both H2-SCRD and CH4-SCSTR
decreased linearly with OLR up to 37.75 and 8.15 kg VS/m3 d,
respectively. Linear fit of biodegradability calculated for
different SRT in each fermentation stage allowed to obtain
regression coefficient R2 of 0.986 and 0.997, respectively,
which gave a good description of linear correlation between
OLR and biodegradability. The obtained results, calculated
from Eqs. (a) and (b), demonstrated that at optimum
 international journal of hydrogen energy 34 (2009) 245–254
Fig. 5 – Biodegradabilities at different OLR in hydrogen and
methane fermentation stages.
conditions (OLRH2: 22.65 kg VS/m3 d, OLRCH4: 4.61 kg VS/m3 d)
5.78% of the influent COD was converted to the hydrogen in
H2-SCRD and 82.18% of the influent COD was converted to the
methane in CH4-SCSTR, therefore a total of 87.96% of
substrate was converted to biogas in the integrated two-stage
process.
Based on the experimental results, the mass balance in the
process is summarized as below. Under the optimum condi-
tions (OLRH2: 22.65 kg VS/m3 d, OLRCH4: 4.61 kg VS/m3 d), 50 kg
of wet food waste (VS 15.12%) could produce hydrogen-rich
biogas of 1.64 m3 with hydrogen content of around 29.95%
from hydrogen production reactor, and methane-rich biogas
of 4.98 m3 with methane content of around 67.50% from
methane production reactor by using the two-stage fermen-
tation process.
4. Conclusion
In this bench scale test, the fermentative H2 and CH4 were
successfully produced from food waste in integrated two-
253
stage process with none-heat treatment of inoculum. The
conventional heat treatment was replaced by using indige-
nous food waste microflora as hydrogen producer. The bench
scale test demonstrated that the application of indigenous
food waste microflora was applicable for the H2 and CH4
production in the integrated two-stage fermentation process.
From an industrial and commercial perspective, this can
facilitate the hydrogen fermentation process and save energy
consumption.
The bench scale study focused on the exploitation of
unsterilized food waste as a source for hydrogen and subse-
quent methane production, the indigenous food waste
microflora was used as inoculum. At lower OLR (<22.65 kg VS/
m3 d), acetic acid and butyric acid producing pathway were
the dominant hydrogen fermentation pathway, the hydrogen
yield was not significantly fluctuated. At higher OLR
(>22.65 kg VS/m3 d), a decrease in hydrolysis rate of substrate
and an increase of propionic and lactic acids were observed,
which were considered as the main causes for the decrease in
hydrogen yield when the system was operated at high OLR.
The optimal OLR and SRT for the integrated two-stage
process were considered to be 22.65 kg VS/m3 d (160 h) for
H2-SCRD and 4.61 (26.67 d) for CH4-SCSTR, respectively. Under
the optimum conditions, the maximum yields of H2 and CH4
were 0.065 and 0.546 m3/kg VS d with its composition in biogas
of 29.95% and 67.50%, respectively.
With the application of simple mass balance equation, the
theoretic maximum yield for H2 and CH4 were determined to
be 0.118  0.001 m3 H2/kg VS and 0.611  0.015 m3 CH4/kg VS,
respectively. Under the optimum conditions, biodegradability
analysis showed that 5.78% of the influent COD was converted
to the hydrogen in H2-SCRD and 82.18% of the influent COD
was converted to the methane in CH4-SCSTR, thus a total of
87.96% of substrate was converted to biogas in the integrated
two-stage process.
references
[1] Das D, Veziroglu TN. Hydrogen production by biological
processes: a survey of literature. Int J Hydrogen Energy 2001;
26:13–28.
[2] Benemann J. Hydrogen biotechnology: progress and
prospects. Nat Biotechnol 1996;14:1101–3.
[3] Azbar BN, Speece RE. Two-phase, two-stage, and single-stage
anaerobic process comparison. J Environ Eng 2001;127:240–7.
[4] Demirel B, Yenigun O. Two-phase anaerobic digestion
processes: a review. J Chem Technol Biotechnol 2002;77:
743–55.
[5] Fox P, Pohland FG. Anaerobic treatment applications and
fundamentals: substrate specificity during phase separation.
Water Environ Res 1994;66:716–24.
[6] Antonopoulou G, Gavala NH, Skiadas IV, Angelopoulos K,
Lyberatos G. Biofuels generation from sweet sorghum:
Fermentative hydrogen production and anaerobic digestion
of the remaining biomass. Bioresour Technol 2008;99:110–9.
[7] Liu DW, Liu DP, Zeng RJ, Angelidaki I. Hydrogen and methane
production from household solid waste in the two-stage
fermentation process. Water Res 2006;40:2230–6.
[8] Van GS, Sung SW, Lay JJ. Biohydrogen production as
a function of pH and substrate concentration. Environ Sci
Technol 2001;35:4726–30.
254 international journal of hydrogen energy 34 (2009) 245–254
[9] Mu Y, Wang G, Yu HQ. Response surface methodological
analysis on biohydrogen production by enriched anaerobic
cultures. Enzyme Microb Technol 2006;38:905–13.
[10] Chang JJ, Wu JH, Wen FS, Hung KY, Chen YT, Hsiao CL, et al.
Molecular monitoring of microbes in a continuous hydrogen-
producing system with different hydraulic retention time.
Int J Hydrogen Energy 2008;33:1579–85.
[11] Hawkes FR, Dinsdale R, Hawkes DL, Hussy I. Sustainable
fermentative hydrogen production: challenges for process
optimisation. Int J Hydrogen Energy 2002;27:1339–47.
[12] Kim SH, Han SK, Shin HS. Optimization of continuous
hydrogen fermentation of food waste as a function of solids
retention time independent of hydraulic retention time.
Process Biochem 2008;43:213–8.
[13] Wu SY, Hung CH, Lin CN, Chen HW, Lee AS, Chang JS.
Fermentative hydrogen production and bacterial community
structure in high-rate anaerobic bioreactors containing
silicone-immobilized and self-flocculated sludge. Biotechnol
Bioeng 2006;93:934–46.
[14] Yang HJ, Shao P, Lu TM, Shen JQ, Wang DF, Xu ZN, Yuan X.
Continuous bio-hydrogen production from citric acid
wastewater via facultative anaerobic bacteria. Int J Hydrogen
Energy 2006;31:1306–13.
[15] APHA. AWWA, WEF: standard methods for the examination
of water and wastewater. Washington, USA:: American
Public Health Association; 1998.
[16] Linke B. Kinetic study of thermophilic anaerobic digestion of
solid wastes from potato processing. Biomass Bioenerg 2006;
30:892–6.
[17] Veeken AHM, Hamelers BVM. Effect of temperature on
hydrolysis rates of selected biowaste components. Bioresour
Technol 1999;69:249–54.
[18] Cooney M, Maynard N, Cannizzaro C, Benemann J. Two-
phase anaerobic digestion for production of hydrogen-
methane mixtures. Bioresour Technol 2007;98:2641–51.
[19] Noike T, Takabatake H, Mizuno O, Ohba M. Inhibition of
hydrogen fermentation of organic wastes by lactic acid
bacteria. Int J Hydrogen Energy 2002;27:1367–71.
[20] Lay JJ. Modelling and optimization of anaerobic digested
sludge converting starch to hydrogen. Biotechnol Bioeng
2000;68:269–78.
[21] Fang HHP, Liu H. Effect of pH on hydrogen production from
glucose by a mixed culture. Bioresour Technol 2002;82:87–93.
[22] Jaime MN, Richard D, Alan G. Hydrogen production from
sewage sludge using mixed microflora inoculum: effect of pH
and enzymatic pretreatment. Bioresour Technol 2008;99:
6325–31.
[23] Sprott GD, Patel GB. Ammonia toxicity in pure cultures of
methanogenic bacteria. Syst Appl Microbiol 1986;7:358–63.
[24] Hendrinksen HV, Ahring BK. Effects of ammonia on
growth and morphology of thermophilic hydrogen-
oxidizing methanogenic bacteria. FEMS Microbiol Lett
1991;85:241–6.
[25] Kayhanian M. Performance of a high-solids anaerobic
digestion process under various ammonia concentrations.
J Chem Technol Biotechnol 1994;59:349–52.
[26] McCarty PL, McKinney RE. Salt toxicity in anaerobic
digestion. J. Water Pollut Control Fed 1961;33:399–415.
[27] Hobson PN, Shaw BG. Inhibition of methane production by
Methanobacterium formicicum. Water Res 1976;10:849–52.
[28] Shin HS, Youn JH. Conversion of food waste into hydrogen by
thermophilic acidogenesis. Biodegradation 2005;16:33–44.
[29] Jo JH, Jeon CO, Lee DS, Park JM. Process stability and
microbial community structure in anaerobic hydrogen-
producing microflora from food waste containing kimchi. J
Biotechnol 2007;131:300–8.
[30] Logan BE, Oh SE, Van Ginkel SW. Biological hydrogen
production measured in batch anaerobic respirometers.
Environ Sci Technol 2002;36:2530–5.
[31] Hwang MH, Jang NJ, Hyun SY, Kim IS. Anaerobic bio-
hydrogen production from ethanol fermentation: the role of
pH. J Biotechnol 2004;111:297–309.
[32] Montalvo S, Guerrero L, Borja R, Travieso L, Sánchez E,
Dı́az F. Use of natural zeolite at different doses and dosage
procedures in batch and continuous anaerobic digestion of
synthetic and swine wastes. Resour Conserv Recy 2006;47:
26–41.
[33] Jokela JPY, Vavilin VA, Rintala JA. Hydrolysis rates, methane
production and nitrogen solubilisation of grey waste
components during anaerobic degradation. Bioresour
Technol 2005;96:501–8.
[34] Eastman PA, Rico JL, Polanco FF. Anaerobic treatment of
cheese whey in a two-phase reactor. Environ Technol 1981;
12:355–62.
[35] Ueno Y, Fukui H, Goto M. Operation of a two-stage
fermentation process producing hydrogen and methane
from organic waste. Environ Sci Technol 2007;41:1413–9.