Applied Energy 87 (2010) 3710–3717

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Fermentative hydrogen production from cassava stillage by mixed anaerobic

microflora: Effects of temperature and pH
Gang Luo a, Li Xie a,*, Zhonghai Zou a, Qi Zhou a, Jing-Yuan Wang b
a
Key Laboratory of Yangtze River Water Environment, Ministry of Education (Tongji University), UNEP-Tongji, Tongji University, Siping Road No. 1239, Shanghai 200092, PR China
b
School of Civil and Environmental Engineering, Nanyang Technological University, N1-01b-45, 50 Nanyang Avenue, 639798 Singapore

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

Article history: Fermentative hydrogen production from cassava stillage was conducted to investigate the influences of
Received 13 February 2010 temperature (37 °C, 60 °C, 70 °C) and initial pH (4–10) in batch experiments. Although the seed sludge
Received in revised form 30 June 2010 was mesophilic anaerobic sludge, maximum hydrogen yield (53.8 ml H2/gVS) was obtained under ther-
Accepted 2 July 2010
mophilic condition (60 °C), 53.5% and 198% higher than the values under mesophilic (37 °C) and extreme-
Available online 24 July 2010
thermophilic (70 °C) conditions respectively. The difference was mainly due to the different VFA and eth-
anol distributions. Higher hydrogen production corresponded with higher ratios of butyrate/acetate and
Keywords:
butyrate/propionate. Similar hydrogen yields of 66.3 and 67.8 ml H2/gVS were obtained at initial pH 5
Fermentative hydrogen production
Temperature
and 6 respectively under thermophilic condition. The total amount of VFA and ethanol increased from
pH 3536 to 7899 mg/l with the increase of initial pH from 4 to 10. Initial pH 6 was considered as the optimal
Cassava stillage pH due to its 19% higher total VFA and ethanol concentration than that of pH 5. Homoacetogenesis and
Mesophilic anaerobic sludge methonogenesis were very dependent on the initial pH and temperature even when the inoculum was
Homoacetogenesis and methanogenesis heat-pretreated. Moreover, a difference between measured and theoretical hydrogen was observed in
this study, which could be attributed to homoacetogenesis, methanogenesis and the degradation of
protein.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction [9,10]. Thus cassava stillage should be of interest as a potential

Ethanol is an important renewable fuel that can contribute to
the reduction of negative environmental impacts by fossil fuels
[1]. China is the third largest ethanol producer in the world [2],
and cassava-derived ethanol production has been increasing due
to its economic benefits compared to other ethanol-producing
crops in the country [3]. However, large amounts of wastewater
(cassava stillage), containing high concentrations of organics and
suspended solids are produced during the ethanol production pro-
cess. In addition, the waste temperature could be as high as 90 °C
after distillation separation. Our previous study showed that cas-
sava stillage is suitable for thermophilic anaerobic treatment and
that the contained organics could be converted to fatty acids,
hydrogen, carbon dioxide and consequently to methane [4]. Com-
pared with methane, the intermediate product, hydrogen is a more
important industrial commodity which is widely used for synthe-
ses of ammonia, alcohols and aldehydes [5]. Hydrogen could be
produced by fermentation from organic wastes such as household
solid waste [6], sewage sludge [7] and winery wastewater [8]. It
was reported that the carbohydrate contained in the wastes was
effective and reliable for fermentative hydrogen production
* Corresponding author. Tel.: +86 21 65982692; fax: +86 21 65986313.
E-mail address: sally.xieli@tongji.edu.cn (L. Xie).
substrate for hydrogen production.
Considering the high temperature of cassava stillage, thermo-
philic and extreme-thermophilic conditions may be more suitable
without additional costly pre-cooling facilities. Fermentative
temperature can affect hydrogen production by influencing the
microorganism composition and activity of enzymes such as
hydrogenases [11]. It was reported that the hydrogen yield ob-
tained under thermophilic condition could be as high as 2.4 mol
H2/mol hexose, much higher than the value of less than 2.0 mol
H2/mol hexose from mesophilic condition [12–15]. The maximum
hydrogen yields of 2.68 and 2.47 mol H2/mol hexose were obtained
at 75 °C [16] and 70 °C [17], respectively. However, when using cas-
sava starch as substrate, Lee et al. [18] found that the hydrogen pro-
duction obtained at 37 °C was six times higher than that of 55 °C.
The suitable temperature range for hydrogen production from cas-
sava stillage is still unknown. During the fermentative hydrogen
production, high concentrations of volatile fatty acids such as
butyric and acetic acids can be accumulated in the system and
result in the drop of pH if not properly controlled. pH variation
may alter the quantity of hydrogen production, aqueous product,
substrate utilization efficiency and microbial community. Many
studies were conducted on the effect of initial pH under mesophilic
condition [19–21], but the obtained results were inconsistent and
the optimal pH differed from one study to another. For example,

0306-2619/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2010.07.004
 G. Luo et al. / Applied Energy 87 (2010) 3710–3717
Khanal et al. [19] reported that acidic initial pH was favorable for
hydrogen production, while Lee et al. [20] found that alkaline initial
pH was suitable for hydrogen production through batch tests. The
disagreements in the optimal temperature and pH in different stud-
ies may be resulted from the differences in substrates, seed sludge
and operation conditions. In addition, there are few studies focus-
ing on the effect of pH on hydrogen production under thermophilic
condition. It is necessary to investigate the effects of temperature
and pH on hydrogen production from the same substrate by certain
seed sludge.
Currently, few reports are available on the systematical investi-
gation of hydrogen producing characteristics of mesophilic anaer-
obic sludge from real wastes under different temperature and pH
ranges. Mesophilic anaerobic sludge as seed sludge for hydrogen
production can easily be obtained since most of the anaerobic reac-
tors were operated under mesophilic conditions [22–24]. By pre-
treatments (e.g. heat, acid and base), the methanogens in the
mesophilic anaerobic sludge can be inhibited while hydrogen pro-
ducing bacteria can be enriched [25]. Homoacetogens and metha-
nogens are two hydrogen consuming microorganisms and will lead
to the lower hydrogen yields if they are not inhibited [7]. However,
the inhibition effectiveness of pretreatment on their activities is
still questionable; as hydrogen consumption was still observed
even when the inoculum was pretreated [26]. Further study is nec-
essary to elucidate the activities of homoacetogens and methano-
gens under different fermentation conditions.
This study was conducted to explore the feasibility of hydrogen
production from cassava stillage by mesophilic anaerobic sludge.
The effects of temperature (mesophilic, thermophilic and ex-
treme-thermophilic) and initial pH (4–10) on the hydrogen pro-
duction were investigated and the involved mechanisms were
discussed. In addition, the activities of homoacetogens and metha-
nogens in the hydrogen production process were also discussed.
2. Materials and methods
2.1. Cassava stillage composition and seed sludge
Cassava stillage used in this study was obtained directly from
Taicang cassava ethanol plant (Jiangsu, China). The characteristics
of cassava stillage are as follows: total solids (TS) 47.9 g/l, volatile
solids (VS) 40.5 g/l, total carbohydrate 28.2 g/l, soluble carbohy-
drate 4.3 g/l and total protein 5.7 g/l. After collection, cassava stil-
lage was stored at 4 °C before usage.
The seed sludge was taken from a mesophilic upflow anaerobic
sludge bed (UASB) from the same ethanol plant. Concentrations of
volatile suspended solids and total suspended solids, as well as the
pH value of the inoculum were 42 g/l, 70 g/l, and 7.5, respectively.
Before use, the anaerobic sludge was heated at 90 °C for 1 h to en-
rich hydrogen-producing anaerobes.
2.2. Experimental design and procedure
Batch hydrogen production tests were performed in 250 ml
vials. The vials were similar to those described in [27]. Seed sludge
of 40 ml and raw cassava stillage of 140 ml were added to each
bottle and the working volume was adjusted to 200 ml with dis-
tilled water. The initial pH value of the mixed solution in each bot-
tle was adjusted by 2 N HCl or 2 N NaOH. Nitrogen gas was purged
into bottles for 5 min to provide anaerobic conditions. The capped
bottles with rubber stoppers were placed in a water bath shaker
(Kexing, China) preset at 150 rpm. First, the temperature was pre-
set at 37 °C (mesophilic), 60 °C (thermophilic) and 70 °C (extreme-
thermophilic) with initial pH 7 to find the optimal fermentation
temperature by anaerobic sludge. Then, experiments on the effects
3711
of initial pH were conducted in the range of 4–10 at an interval of 1
at the optimal temperature. Each test was carried out in triplicates
and analysis of variance (ANOVA) at 0.05 level was used to analyze
the data.
The evolved biogas was collected through a vapor trap in an alu-
minized polyethylene bag (GL Sciences, Japan). The amount of bio-
gas in the gas bags was determined every 5–10 h using a syringe
(50 ml). Volume determinations were made by allowing the syr-
inge plunger to move and equilibrate between the gasbag and
atmospheric pressures [28]. Then, the syringe was held horizon-
tally for measurement. The readings for the biogas volume were ta-
ken at room temperature. At the same time the compositions of the
biogas in the gas bag and headspace of the bottles were measured.
The measured hydrogen volume was calibrated to the volume at
STP (standard temperature 0 °C and pressure 1 atm). The cumula-
tive hydrogen production was calculated by the following
equation:
V h;i ¼ V h;i1 þ C h;i V hg;i þ ðC hh;i  C hh;i1 ÞV he ð1Þ
where Vh,i and Vh,i1 are cumulative hydrogen gas volumes at the
current (i) and previous (i  1) time intervals, Ch,i and Vhg,i are the
fractions of hydrogen gas and biogas volumes in the gas bag at
the current (i) time interval, Chh,i and Chh,i1 are the fractions of
hydrogen gas in the headspace of the bottle at the current (i) and
previous (i  1) time intervals, Vhe is the total volume of headspace
in the reactor.
The cumulative hydrogen production process in the batch tests
were simulated by the modified Gompertz equation as follows:
  
Rm  e
HðtÞ ¼ P  exp  exp ðk  tÞ þ 1 ð2Þ
P
where H (ml) is the cumulative H2 production, P (ml) is the H2 pro-
duction potential, Rm (ml/h) is the maximum H2 production rate, k
(h) is the lag-phase time. The meaning of each parameter was de-
scribed in [29]. The values of P, Rm and k for each batch can be deter-
mined by best fitting the H2 production data for Eq. (2) using the
origin 7.5.
2.3. Analytical methods
Samples were centrifuged at 3500 rpm for 10 min and filtrated
through 0.45 lm filters to determine soluble components. TS, VS
and ammonia were analyzed in duplicates in accordance with
Standard APHA Methods [30]. The determinations of carbohy-
drates and proteins were described in the study of [31].VFA (C2-
C5) and ethanol were determined by a gas chromatograph
(HP6890II) equipped with a flame ionization detector and a
CPWAX52CB column (30 m  0.25 mm  0.25 lm). The tempera-
ture of the injector and FID were 200 °C and 220 °C, respectively.
Nitrogen was the carrier gas with a flow rate of 50 ml/min. The
GC oven was programmed to begin at 70 °C and remain there for
2 min, then increase at a rate of 10 °C/min to 220 °C, and remain
at 220 °C for an additional 2 min. The sample injection volume
was 1.0 ll. The biogas composition (H2, CH4 and CO2) was deter-
mined using a gas chromatograph (Shimadzu GC-14B) equipped
with a thermal conductivity detector and a stainless steel column
packed with Carbosive S II (diameter of 3.2 mm and 2.0 m length).
Injector, detector and column temperatures were kept at 100 °C,
105 °C and 60 °C, respectively. The carrier was nitrogen and the
flow rate used was 30 ml/min. The sample injection volume was
0.2 ml.
3712 G. Luo et al. / Applied Energy 87 (2010) 3710–3717

3. Results and discussion hydrogen production from cassava stillage, indicating that the
mixed inoculum contained various microbial communities which
3.1. Effects of temperature on hydrogen production could function at different temperatures. Previous studies showed
that thermotolerant species could be detected in PCR-DGGE bands
Fermentative hydrogen production at three typical tempera- of mesophilic sludge [32,33].
tures 37 °C, 60 °C and 70 °C, representing mesophilic, thermophilic Fig. 1b describes the variations of hydrogen compositions in the
and extreme-thermophilic fermentation respectively, was con- gas phase of the reactors with fermentation time. The produced
ducted at initial pH 7 with mesophilic anaerobic sludge as inocu- biogas only contained hydrogen and carbon dioxide. Initially, the
lum. Fig. 1a illustrates the time courses of hydrogen production hydrogen composition increased with fermentation time. After
during 85 h fermentation at different temperatures. The cumula- 20 h fermentation, the hydrogen composition in the gas phase be-
tive hydrogen production data were well fitted to the modified gan to decrease at the temperature of 37 °C and 60 °C, while it sta-
Gompertz equation with R2 > 0.99 (Table 1) and the obtained bilized at 70 °C. No biogas was collected after the highest hydrogen
parameters were strongly temperature dependent. The highest P production was achieved for all conditions. Hydrogen consumption
value of 307 ml was observed at 60 °C, which was 53.5% and was also observed at fermentation temperatures of 60 °C or 25 °C
198% higher than the values at 37 °C and 70 °C, respectively. Corre- [34] and homoacetogenesis played an important role [26]. It is
spondingly, 60 °C gave the highest hydrogen yield and Rm. The va- obvious that heat-pretreatment could not inhibit homoacetogene-
lue of k at 37 °C and 60 °C were only 8.4 h and 9.7 h while at 70 °C, sis and the fermentation conditions (37 °C or 60 °C with initial pH
it was extended to 27.6 h, suggesting that microorganisms need 7) are favorable for homoacetogenesis. The hydrogen consumption
more time to adapt to the extreme-temperature condition. is further studied by changing pH in subsequent sections.
Although mesophilic anaerobic sludge was used as seed sludge, The amount and component of VFA/ethanol reflect the metabo-
thermophilic fermentation was demonstrated to be favorable for lism of hydrogen-producing anaerobes. The different hydrogen
Cumulative hydrogen production (ml)

400
o a
37 C
o
60 C
o
300 70 C

200

100

0
Hydrogen content in the gas phase (%)

60
b
50

40

30

20

10

0
0 20 40 60 80 100
Time (h)
Fig. 1. Hydrogen production at different temperature with initial pH 7: (a) Variation of cumulative hydrogen production with fermentation time. (b) Variation of hydrogen
content in the gas phase of the bottle with fermentation time.

Table 1
Hydrogen production performances at different temperatures with initial pH 7.

Temperature (°C) P (ml) Rm (ml/h) k (h) R2 Hydrogen yield Final pH Ammonia Calculated hydrogen Percentage of measured
(ml H2/gVS) (mg/l) production (ml) hydrogen/calculated hydrogen (%)
37 200 10.3 8.4 0.995 35.3 ± 2.6 5.3 ± 0.1 256 ± 30 521 38
60 307 14.1 9.7 0.997 53.3 ± 1.8 5.5 ± 0.1 333 ± 53 632 49
70 103 4.6 27.6 0.998 17.8 ± 1.4 5.4 ± 0.1 455 ± 61 245 42
 G. Luo et al. / Applied Energy 87 (2010) 3710–3717
Individual VFA/Ethanol Concentration (mg/L)
5000
Ethanol
Total VFA/Ethanol
4000
3000
2000
1000
0 0
o o
37 C
Fig. 2. Total and individual VFA and ethanol concentration at different temperatures with initial pH 7.
production under different temperatures could be explained from
the aspect of VFA and ethanol distribution. At 37 °C, acetate, propi-
onate and butyrate were predominant metabolites, while at 60 °C
butyrate and ethanol concentrations were significantly enhanced
(ethanol: p = 0.0003 < 0.05; butyrate: p = 0.017 < 0.05) and acetate
and propionate production were inhibited. Although the total
amounts of VFA and ethanol were similar at 37 °C and 60 °C, the ra-
tios of butyrate/acetate (B/A) and butyrate/propionate (B/P) were
2.58 and 8.6 respectively at 60 °C, much higher than the values
of 1.67 and 2.2 at 37 °C. Higher B/A and B/P ratios were proven
to be correlated with higher hydrogen production in previous stud-
ies [35,36]. At 70 °C, propionate and butyrate concentrations de-
creased sharply while acetate and ethanol concentrations were
similar with those at 60 °C. The total amount of VFA and ethanol
(Fig. 2) at 70 °C was much lower than that at 37 °C and 60 °C, which
was well in accord with its lower hydrogen yield. The hydrogen
production at 70 °C was not as efficient as that at 60 °C or 37 °C
though previous studies have demonstrated that extreme-thermo-
philic hydrogen production from the wastes is superior compared
with mesophilic or thermophilic hydrogen production [37,17,34].
The above inconsistence might be attributed to the difference in
seed sludge because only cow manure or digested cow manure
was used as inocula for efficient extreme-thermophilic hydrogen
production. Nevertheless, the present study focused on the effects
of temperature on hydrogen production from widely existed mes-
ophilic anaerobic sludge and thermophilic hydrogen fermentation
was demonstrated to be more efficient than mesophilic or ex-
treme-thermophilic fermentation.
3.2. Effects of initial pH on hydrogen production
The variations of thermophilic hydrogen production with fer-
mentation time at different initial pH are shown in Fig. 3a. The data
were also fitted to the Gompertz model (Table 2). The P values fluc-
tuated with the variations of initial pH and peaked at pH 6 with the
value of 383 ml. The maximum Rm obtained at pH 6 coincided with
maximum cumulative hydrogen production. The cumulative
hydrogen production at initial pH 4 and 10 were 253 ml and
195 ml respectively, much lower than the value at initial pH 6.
However, no significant difference (p = 0.43 > 0.05) in cumulative
hydrogen production was found between pH 5 (376 ml) and pH
6 (383 ml). The result indicated that hydrogen could be produced
efficiently in the initial pH range of 5–6. In this study, the k was
also greatly affected by the pH and shorter k was obtained at the
initial pH 7–10. The k was as long as 27.1 h at initial pH 4 while
the value was only 6.8 h at initial pH 9.
3713
Total VFA/Ethaonl concentration (mg/L)
10000
Acetate Propionate Butyrate
8000
6000
4000
2000
o
60 C 70 C
Fig. 3b shows the variations of hydrogen compositions in the
gas phase of the reactors with fermentation time. Initially, the
hydrogen composition increased with fermentation time over
the initial pH range investigated. When no biogas was collected
in the gas bag, the hydrogen composition in the gas phase of the
reactor began to decrease at the initial pH 6–10 due to the
hydrogen consumption. Moreover, methane was detected at the
initial pH 9 and 10 at the end of the fermentation (data not shown).
The obtained result indicated that only heat-pretreatment could
not inhibit methanogenesis efficiently, and the cultivation pH
might be a crucial factor to inhibit methanogenesis. The inhibition
of homoacetogenesis was observed with initial pH range 4–5 since
a decrease in hydrogen composition was not detected, which has
not been reported before. It is obvious that both homoacetogenesis
and methanogenesis could be completely inhibited at 60 °C with
initial pH 4 and 5.
Fig. 4 shows the variation of the total amount of VFA and etha-
nol with pH under thermophilic condition. The trend was different
with that of hydrogen production. The VFA and ethanol production
increased with the increase of initial pH while hydrogen produc-
tion peaked at initial pH 5 and 6. The phenomenon was different
from reports on fermentative hydrogen production from pure sub-
strates. When starch was used as the substrate for hydrogen pro-
duction, the total amount of VFA and ethanol peaked at initial
pH 6 corresponding with the highest cumulative hydrogen produc-
tion [5]. However, in a UASB reactor treating synthetic sucrose
wastewater for hydrogen production, Zhao and Yu [38] found that
the total amount of VFA and ethanol did not change significantly
with pH variation. The different results from the above studies
may be due to the different substrates. Compared with starch
and sucrose, cassava stillage contains high SS. Organics from SS
could be solubilized at higher initial pH condition and the newly
dissolved organics could induce higher production of VFA and eth-
anol [39]. As shown in Fig. 4, the variations of individual VFA com-
ponents during fermentation did not follow the same trend as the
amount of total VFA. The increase of initial pH stimulated the
production of acetate and propionate which could consume hydro-
gen, while inhibiting the production of butyrate. Therefore, the
hydrogen production decreased with further increase of initial
pH above 6.
3.3. Theoretical hydrogen production under different temperature and
pH
The theoretical hydrogen production can be calculated from the
metabolic products since carbohydrate was the main composition
3714 G. Luo et al. / Applied Energy 87 (2010) 3710–3717

Cumulative hydrogen production (ml)

400 pH 4
pH 5 a
pH 6
300 pH 7
pH 8
pH 9
pH 10
200

100

0
Hydrogen concentration in gas phase (%)

60
b
50

40

30

20

10

0
0 20 40 60 80 100
Time (h)
Fig. 3. Hydrogen production at different initial pH with fermentation temperature 60 °C pH 7: (a) Variation of cumulative hydrogen production with fermentation time. (b)
Variation of hydrogen content in the gas phase of the bottle with fermentation time.

Table 2
Hydrogen production performances at different initial pH with fermentation temperature 60 °C.

Initial pH P (ml) Rm (ml/h) k (h) R2 Hydrogen yield Final pH Ammonia Theoretical hydrogen Percentage of measured
(ml H2/gVS) (mg/l) production (ml) hydrogen/theoretical hydrogen (%)
4 253 15.8 27.1 1 44.4 ± 2.1 4.4 ± 0.1 240 ± 24 348 73
5 376 12.7 13.7 0.982 66.3 ± 1.9 4.9 ± 0.1 235 ± 31 479 78
6 383 24 13 0.999 67.8 ± 1.8 5.4 ± 0.1 277 ± 41 550 70
7 307 14 9.7 0.997 53.3 ± 1.8 5.5 ± 0.1 333 ± 53 632 49
8 251 12.8 7 0.997 44.1 ± 2.6 5.7 ± 0.1 350 ± 49 652 38
9 222 10.1 6.8 0.995 39.0 ± 1.9 5.7 ± 0.1 390 ± 62 700 32
10 195 11.9 9.5 0.993 34.5 ± 2.3 5.9 ± 0.1 420 ± 58 748 26

of cassava stillage. As shown in equations 1–3, different amounts of A possible explanation for such a difference was that part of the
hydrogen could be obtained when glucose was biodegraded to dif- acetate was produced by homoacetogenesis accompanying with
ferent end products. Eq. (5) could be used to estimate the theoret- hydrogen consumption, but not from the degradation of glucose.
ical hydrogen production. As shown in Tables 1 and 2, the Another reason was that the produced hydrogen was consumed
measured hydrogen production was lower than theoretical hydro- by methanogenesis which led to the lower measured hydrogen
gen production under different temperatures and initial pH. since methane was detected at initial pH 9 and 10 at 60 °C. How-
ever, it is difficult to explain the difference between theoretical
C6 H12 O6 þ 2H2 O ! 4H2 þ 2CH3 COOH þ 2CO2 ð3Þ
and measured hydrogen production at 70 °C with initial pH 7
C6 H12 O6 þ 2H2 ! 2CH3 CH2 COOH þ 2H2 O ð4Þ and at 60 °C with initial pH 4–5, because decrease of hydrogen
composition and/or production of methane were not observed in
C6 H12 O6 ! 2H2 þ CH3 CH2 CH2 COOH þ 2CO2 ð5Þ the whole process. It was speculated that the acetate obtained at
70 °C might be not only be from the degradation of glucose but also
C6 H2 O6 ! 2CH3 CH2 OH þ 2CO2 ð6Þ from the degradation of protein contained in cassava stillage. By
stickland reaction, the degradation of protein can lead to the pro-
HydrogenðmolÞ ! 2  ðbutyrateÞ þ 2  ðacetateÞ duction of acetate without hydrogen production [40]. The higher
ammonia concentration (455 mg/l) at 70 °C with initial pH 7
 ðpropionateÞ ð7Þ compared with initial value (242 mg/l) in the fermented liquid
 G. Luo et al. / Applied Energy 87 (2010) 3710–3717 3715

Individual VFA&Ethanol Concentration (mg/L)

Total VFA&Ethaonl concentration (mg/L)

6000 10000
Ethanol Acetate Propionate Butyrate
5000 Total VFA/Ethanol
8000

4000
6000
3000
4000
2000

2000
1000

0 0
4 5 6 7 8 9 10

Fig. 4. Total and individual VFA and ethanol concentration at different initial pH with fermentation temperature 60 °C.

supported this speculation (Table 1). The increase of ammonia con- observed. Meanwhile ammonia concentration increased from
centration was not observed at pH 4–5 at 60 °C. However, the deg- 242 mg/l to 306 mg/l, indicating that the acetate was produced
radation of protein might really occur considering the produced from the degradation of protein without hydrogen production.
ammonia nitrogen is an essential nutrient for bacterial growth After 12 h of fermentation, ammonia concentration remained rel-
which could be simultaneously consumed [41]. atively stable, while the acetate concentration decreased gradu-
ally with increase of ethanol concentration. The result was
3.4. Hydrogen production characteristics under optimal fermentation consistent with the finding that the acetate could be converted
condition to ethanol at higher hydrogen partial pressure [26]. After fermen-
tation for about 58 h, the acetate concentration increased again,
Initial pH 6 was thought to be the optimal condition consider- and the trend correlated with the variations of hydrogen compo-
ing more VFA and ethanol production (18% higher than pH 5), sition in the gas phase as described in Fig. 3b, further indicating
although hydrogen production at initial pH 5 was nearly equal that the decrease of hydrogen content in gas phase resulted from
to that at pH 6 and homoacetogenesis was inhibited. To better the consumption of hydrogen to acetate by homoacetogenesis.
understand the hydrogen production process under optimal con- The fermentation pH was in the range 5.2–6.0, which was also
ditions, the variations of VFA and ethanol production, mixture suitable for hydrogen production.
pH and cumulative hydrogen production with fermentation time Under optimal condition, the hydrogen yield from cassava stil-
were monitored. The result is shown in Fig. 5. It is obvious that lage by anaerobic granular sludge was 67.8 ml H2/gVS added or
butyrate was the main metabolic byproduct and the hydrogen 0.78 mol H2/mol hexose. The yield was relatively lower (<2 mol
production increased significantly with the increase of butyrate H2/mol hexose) since most carbohydrate was in the suspended sol-
concentration. Although acetate increased from 0 to 668 mg/l at ids and difficult to be utilized (the soluble carbohydrate was only
fermentation time 0–12 h, no obvious hydrogen production was 4.3 g/l while total carbohydrate was 28.2 g/l). However, the yield
Individual VFA&Ethanol Concentration (mg/L)

5000 10
Cumulative hydrogen production (ml)

Ethanol
Acetate 400
4000 Propionate
Butyrate
H2 300 8
3000
pH
pH

200
2000
6
1000 100

0 0 4
0 20 40 60 80 100
Time (h)

Fig. 5. Variation of VFA and ethanol, pH and cumulative hydrogen production with fermentation time under optimal fermentation condition (initial pH 6, temperature 60 °C).
3716 G. Luo et al. / Applied Energy 87 (2010) 3710–3717
was still comparable or even higher than some actual wastes. The
hydrogen yield was 68.6 ml H2/gVS from acid-pretreated beer lees
at 36 °C [42], while the maximum hydrogen yield from alkaline-
pretreated waste sludge was only 14.4 ml/g VS at 37 °C [7]. Higher
hydrogen yield could be obtained if the cassava stillage is properly
pretreated. On the other hand, the hydrogen production process
could be coupled with methane production as the second step to
further utilize the VFA in the effluent. It has been demonstrated
that the methane production in a two-stage (hydrogen + methane)
process was higher than traditional single methane production
from household solid waste [6]. It is suggested to further study
and optimize the two-stage process for hydrogen and methane
production from cassava stillage to obtain more energy.
4. Conclusions
Thermophilic hydrogen production from cassava stillage by
mesophilic anaerobic sludge was proven to be more efficient than
that of mesophilic and extreme-thermophilic fermentation condi-
tions in this study. The higher hydrogen production under thermo-
philic condition corresponded with the enhanced butyrate
production and lower propionate production. Moreover, the full
inhibition of butyrate under extreme-thermophilic condition led
to much lower hydrogen production. Experimental results of pH
influence showed that initial pH 6 was optimal for hydrogen pro-
duction at 60 °C while lower or higher initial pH resulted in lower
hydrogen production. The higher pH resulted in higher total VFA,
ethanol and acetate concentration. The increase of acetate concen-
tration at higher initial pH could be attributed to the higher activ-
ity of homoacetogens and degradation efficiency of protein.
Homoacetogenesis was inhibited at lower initial pH 4–5. In addi-
tion, heat-pretreatment of anaerobic sludge was not sufficient to
fully inhibit the activity of methanogens, and a combination of
heat-pretreatment and low cultivation pH was required to prevent
methanogenesis.
Acknowledgements
This research was financially supported by the Foundation of
Key Laboratory of Yangtze River Water Environment, Ministry of
Education (Tongji University), China (No. YRWEY1003), the Bayer
Sustainable Development Foundation, and National Water Pollu-
tion Control and Management of China (2008ZX07316-002). The
authors also wish to express their appreciation to Nanyang Tech-
nological University (NTU) in Singapore for its support in this joint
collaboration between NTU and Tongji University.
References
[1] Balat M, Balat H. Recent trends in global production and utilization of bio-
ethanol fuel. Appl Energy 2009;86:2273–82.
[2] Yan J, Lin T. Biofuels in Asia. Appl Energy 2009;86:S1–S10.
[3] Jansson C, Westerbergh A, Zhang JM, Hu XW, Sun CX. Cassava, a potential
biofuel crop in (the) People’s Republic of China. Appl Energy 2009;86:S95–9.
[4] Luo G, Xie L, Zhou Q. Enhanced treatment efficiency of an anaerobic
sequencing batch reactor (ASBR) for cassava stillage with high solid
contents. J Biosci Bioeng 2009;107:641–5.
[5] Zhang T, Liu H, Fang HHP. Biohydrogen production from starch in wastewater
under thermophilic condition. J Environ Manage 2003;69:149–56.
[6] 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.
[7] Cai ML, Liu JX, Wei YS. Enhanced biohydrogen production from sewage sludge
with alkaline pretreatment. Environ Sci Technol 2004;38:3195–202.
[8] Yu HQ, Zhu ZH, Hu WR, Zhang HS. Hydrogen production from rice winery
wastewater in an upflow anaerobic reactor by using mixed anaerobic cultures.
Int J Hydrogen Energy 2002;27:1359–65.
[9] Lay JJ, Fan KS, Chang JI, Ku CH. Influence of chemical nature of organic wastes
on their conversion to hydrogen by heat-shock digested sludge. Int J Hydrogen
Energy 2003;28:1361–7.
[10] Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T. Enhancement of
hydrogen production from glucose by nitrogen gas sparging. Bioresour
Technol 2002;73:59–65.
[11] Valdez-Vazquez I, Poggo-Varaldo HM. Hydrogen production by fermentative
consortia. Renew Sustain Energy Rev 2009;13:1000–13.
[12] Ueno Y, Kawai T, Sato S, Otsuka S, Morimoto M. Biological production of
hydrogen from cellulose by natural anaerobic microflora. J Ferment Bioeng
1995;79:395–7.
[13] 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.
[14] Fang HHP, Liu H. Effect of pH on hydrogen production from glucose by a mixed
culture. Bioresour Technol 2002;82:87–93.
[15] Hwang MH, Jang NJ, Hyun SH, Kim IS. Anaerobic bio-hydrogen production
from ethanol fermentation: the role of Ph. J Biotechnol 2004;111:297–309.
[16] Yokoyama H, Moriya N, Ohmori H, Waki M, Ogino A, Tanaka Y. Community
analysis of hydrogen-producing extreme thermophilic anaerobic microflora
enriched from cow manure with five substrates. Appl Microbiol Biotechnol
2007;77:213–22.
[17] Kotsopoulos TA, Zeng RJ, Angelidaki I. Biohydrogen production in granular
up-flow anaerobic sludge blanket (UASB) reactors with mixed cultures
under hyper-thermophilic temperature (70 °C). Biotechnol Bioeng 2007;94:
296–302.
[18] Lee KS, Hsu YF, Lo YC, Lin PJ, Lin CY, Chang JS. Exploring optimal environmental
factors for fermentative hydrogen production from starch using mixed
anaerobic microflora. Int J Hydrogen Energy 2008;33:1565–72.
[19] Khanal SK, Chen WH, Li L, Sung SW. Biological hydrogen production: effect of
pH and intermediate products. Int J Hydrogen Energy 2004;29:1123–31.
[20] Lee YJ, Miyahara T, Noike T. Effect of pH on microbial hydrogen fermentation. J
Chem Technol Biotechnol 2002;77:694–8.
[21] Lin CY, Hung CH, Chen CH, Chung WT, Cheng LH. Effects of initial cultivation
pH on fermentation hydrogen production from xylose using natural mixed
cultures. Process Biochem 2006;41:1383–90.
[22] Valdez-Vazquez I, Ríos-Leal E, Muñoz-Páez KM, Carmona-Martínez A, Poggi-
Varaldo HM. Effect of inhibition treatment, type of inocula, and incubation
temperature on batch H2 production from organic solid waste. Biotechnol
Bioeng 2006;95:342–9.
[23] Valdez-Vazquez I, Ríos-Leal E, Esparza-García F, Cecchi F, Poggi-Varaldo HM.
Semi-continuous solid substrate anaerobic reactors for H2 production from
organic waste: mesophilic versus thermophilic regime. Int J Hydrogen Energy
2005;30:1383–91.
[24] Kotsopoulos TA, Fotidis IA, Tsolakis N, Martzopoulos GG. Biohydrogen
production from pig slurry in a CSTR reactor system with mixed cultures
under hyper-thermophilic temperature (70 °C). Biomass Bioenergy
2009;33:1168–74.
[25] Wang JL, Wan W. Experimental design methods for fermentative hydrogen
production: a review. Int J Hydrogen Energy 2009;34:235–44.
[26] Oh SE, Ginkel SV, Logan BE. The relative effectiveness of pH control and heat
treatment for enhancing biohydrogen gas production. Environ Sci Technol
2003;37:5186–90.
[27] Angelidaki I, Alves M, Bolzonella D, Borzacconi L, Campos JL, Guwy AJ, et al.
Defining the biomethane potential (BMP) of solid organic wastes and energy
crops: a proposed protocol for batch assays. Water Sci Technol
2009;59:927–34.
[28] Owen WF, Stuckey DC, Healy HB, Young LY, Mccarty PL. Bioassay for
monitoring biochemical methane potential and anaerobic toxicity. Water
Res 1979;13:485–92.
[29] Ginkel SV, Sung S. Biohydrogen production as a function of pH and substrate
concentration. Environ Sci Technol 2001;35:4726–30.
[30] APHA. Standard methods for the examination of water and wastewater. 19th
ed. New York, USA: American Public Health Association; 1995.
[31] Yuan HY, Chen YG, Zhang HX, Jiang S, Zhou Q, Gu GW. Improved bioproduction
of short-chain fatty acids (SCFAs) from excess sludge under alkaline
conditions. Environ Sci Technol 2006;40:2025–9.
[32] Kim W, Hwang K, Shin SG, Lee S, Hwang S. Effect of high temperature on
bacterial community dynamics in anaerobic acidogenesis using mesophilic
sludge inoculum. Bioresour Technol 2009;101:S17–22.
[33] Luo G, Xie L, Zou ZH, Wang W, Zhou Q. Exploring optimal conditions for
thermophilic fermentative hydrogen production from cassava stillage. Int J
Hydrogen Energy 2010;35:6161–9.
[34] Yokoyama H, Waki M, Moriya N, Yasuda T, Tanaka Y, Haga K. Effect of
fermentation temperature on hydrogen production from cow waste slurry by
using anaerobic microflora within the slurry. Appl Microbiol Biotechnol
2007;74:474–83.
[35] Arooj MF, Han SK, Kim SH, Kim DH, Shin HS. Effect of HRT on ASBR converting
starch into biological hydrogen. Int J Hydrogen Energy 2008;33:6509–14.
[36] Arooj MF, Han SK, Kim SH, Kim DH, Shin HS. Continuous biohydrogen
production in a CSTR using starch as a substrate. Int J Hydrogen Energy
2008;33:3289–94.
[37] Pakarinen O, Lehtomäki A, Rintala J. Batch dark fermentation hydrogen
production from grass silage: the effect of inoculum, pH, temperature and VS
ratio. Int J Hydrogen Energy 2008;33:594–601.
[38] Zhao QB, Yu HQ. Fermentative H2 production in an upflow anaerobic sludge
blanked reactor at various pH values. Bioresour Technol 2008;99:1353–8.
[39] Torres ML, Llorens MDE. Effect of alkaline pretreatment on anaerobic digestion
of solid wastes. Waste Manage 2008;28:2229–34.
 G. Luo et al. / Applied Energy 87 (2010) 3710–3717 3717

[40] Karlsson A, Vallin L, Ejlertsson J. Effects of temperature, hydraulic retention [41] Li CL, Fang HHP. Fermentative hydrogen production from wastewater and
time and hydrogen extraction rate on hydrogen production from the solid wastes by mixed cultures. Crit Rev Env Sci Technol 2007;37:1–39.
fermentation of food industry residues and manure. Int J Hydrogen Energy [42] Fan YT, Zhang GS, Guo XY, Xing Y, Fan MH. Biohydrogen-production from beer
2008;33:953–62. lees biomass by cow dung compost. Biomass Bioenergy 2006;30:493–6.