i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 1 7 3 3 e1 1 7 3 7

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Technical Communication

Anaerobic treatment of cassava stillage for hydrogen

and methane production in continuously stirred tank reactor
(CSTR) under high organic loading rate (OLR)

Gang Luo a, Li Xie a,*, Zhonghai Zou a, Wen Wang a, Qi Zhou a, Hojae Shim 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
Department of Civil and Environmental Engineering, Faculty of Science and Technology, University of Macau, Macau SAR 999078, PR China

article info abstract

Article history: Anaerobic hydrogen and methane production from cassava stillage in continuously stirred
Received 24 April 2010 tank reactor (CSTR) were investigated in this study. Results showed that the heat-
Available online 9 September 2010 pretreatment of inoculum did not enhance hydrogen yield compared to raw inoculum
under mesophilic condition after continuous operation. However, the hydrogen yield
Keywords: increased from about 14 ml H2/gVS under mesophilic condition to 69.6 ml H2/gVS under
Anaerobic thermophilic condition due to the decrease of propionate concentration and inhibition of
Hydrogen homoacetogens. Therefore, temperature was demonstrated to be more important than
Methane pretreatment of inoculum to enhance the hydrogen production. Under high organic
Cassava stillage loading rate (OLR) (>10 gVS/(L$d)), the two-phase thermophilic CSTR for hydrogen and
Organic loading rate (OLR) methane production was stable with hydrogen and methane yields of 56.6 mlH2/gVS and
249 mlCH4/gVS. The one-phase thermophilic CSTR for methane production failed due to
the accumulation of both acetate and propionate, leading to the pH lower than 6. Instead of
propionate alone, the accumulations of both acetate and propionate were found to be
related to the breakdown of methane reactor.
ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Anaerobic hydrogen production from organic wastes is
reported to be an efficient process that has potential to be an
economical and sustainable way for renewable hydrogen
production [1e3]. It has been found that pretreatment of the
inoculum is very important to inhibit methanogens and
enrich hydrogen producers for higher hydrogen production,
and most studies have evaluated inocula with different
pretreatments on hydrogen production under batch mode [4].
However, our previous study found that inoculum with and
without pretreatments did not show any differences in
thermophilic continuous hydrogen production from cassava
stillage [5]. Whether the above result is applicable to hydrogen
production from cassava stillage at mesophilic temperature
still needs further investigation. Contrary results on hydrogen
production under different temperatures have been reported
recently. Lee et al. [6] noticed that mesophilic condition (37 ! C)
was preferable for hydrogen production from cassava starch
compared to thermophilic condition (55 ! C) with hydrogen

* Corresponding author. Tel.: þ86 21 65982692; fax: þ86 21 65986313.

E-mail address: sally.xieli@gmail.com (L. Xie).
0360-3199/$ e see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2010.08.033
11734 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 1 7 3 3 e1 1 7 3 7
production potentials of 1447 ml and 241 ml, respectively,
while other studies reported that thermophilic condition was
more favorable [7,8]. The comparison of continuous hydrogen
production under both mesophilic and thermophilic condi-
tions is important to clearly understand the different mech-
anisms involved.
After hydrogen production, the effluent contains high
content of organic acids. Anaerobic digestion for methane
production is an ideal way to utilize metabolites (volatile fatty
acids (VFA), and alcohols) from hydrogen production process for
additional energy production. Currently, cogeneration of
hydrogen and methane in two phase anaerobic process have
been reported using food waste or pure substrate like glucose as
substrate [2,9,10]. The two-phase process separates and
enriches acidogens and methanogens in different reactors that
may improve the process stability and efficiency compared to
traditional one-phase methane production process. Although
hydrogen and methane production from waste under lower OLR
(between 2 and 5 gCOD/(L$d)) has been reported [2,11], perfor-
mance of the two-phase process under higher OLR was seldom
investigated. Moreover, there are few reports on the comparison
of performances of two-phase (hydrogen and methane) and
one-phase (methane) CSTR under higher OLR (>10 gVS/(L$d)).
Investigation on the process performance of two-phase CSTR
under higher OLR may accelerate its application.
The objective of the present study was therefore to inves-
tigate the impacts of pretreatment and temperature on
continuous hydrogen production firstly. And then the
performances of two-phase and one-phase CSTR under higher
OLR (>10 gVS/(L$d)) were compared and discussed. Cassava
stillage, a kind of high concentration organic wastewater from
an ethanol plant, was used as the substrate.
2. Materials and methods
2.1. Feed stock and inoculum
Cassava stillage used in this study was collected from cassava
ethanol plant,in Jiangsu province. Two batches of cassava
stillage taken at different sampling time were used as feed-
stocks. The first batch (pH 4.1, TS 47.4 g/L, VS 40 g/L, TCOD
48 g/L, SCOD 22 g/L, soluble carbohydrate 7.5 g/L, total carbo-
hydrate 26.9 g/L, acetate 200 mg/L) was used for the temper-
ature and pretreatment experiments and the second batch
(pH 4.0, TS 62.2 g/L, VS 53 g/L, TCOD 65 g/L, SCOD 25.6 g/L,
soluble carbohydrate 8.3 g/L, total carbohydrate 29.5 g/L,
acetate 169 mg/L) was used for the two-phase (hydrogen and
methane) and one-phase (methane) fermentation. After
sampled, the cassava stillage was stored at 4 ! C before usage.
The seed sludge for hydrogen reactor was anaerobic sludge
(pH 7.5, TS 70 g/L, VS 42 g/L) from UASB reactor. To accelerate
the start-up of methane reactors, thermophilic digested cas-
sava stillage (pH 7.8, TS 25.3 g/L, VS 14.9 g/L) from CSTR in the
same plant was used as seed sludge.
2.2. Experimental set-up
Three 250 ml identical bottles (R1, R2, R3) were used for
hydrogen production with working volume 200 ml. R1 and R2
were operated under mesophilic condition (37 ! C) while R3
was operated under thermophilic condition (60 ! C). Heat
pretreatment is the most widely used method for hydrogen
producing bacteria enrichment [4]. Therefore, R1 was seeded
with raw sludge while the seed sludge for R2 was heat-pre-
treated at 90 ! C for 1h. R3 was also seeded with raw sludge
according to the results of previous study [12]. Before contin-
uous experiment, a batch operation was carried out for each
reactor. The seed sludge of 40 ml and raw cassava stillage
140 ml were added to each reactor. The working volume was
adjusted to 200 ml using distilled water. The reactors were
then put in water bath shakers (37 ! C and 60 ! C, respectively)
and rotated at 150 rpm. After the hydrogen production ceased,
the reactors were converted to semi-continuous mode and
operated as continuously stirred tank reactor (CSTR) with HRT
1 d. Half of the mixtures in the bottles were removed and fresh
cassava stillage were added at every 12 h. Sodium bicarbonate
(5 g/L) was added to raw cassava stillage to maintain the
suitable pH for hydrogen production.
According to the results obtained from the above exper-
iments, the two-phase and one-phase CSTR were operated
under thermophilic condition. R3 was still used for hydrogen
production and the effluent was fed to the subsequent
methane reactor (R4, total volume 1.5 L with working
volume 800 ml, 55 ! C, HRT 4 d). One-phase fermentation was
carried out in reactor R5 with raw cassava stillage as
substrate (total volume 1.5 L with working volume 1000 ml,
55 ! C, HRT 5 d). Thus the two-phase and one-phase CSTR
had the same HRT. The alkalinity of cassava stillage for R5
was also the same as that for two-phase process. Initially,
the two methane reactors were filled up with inoculum.
After two days’ acclimation, R4 and R5 were fed at every 12 h
with corresponding substrates. In this experiment, the
second batch cassava stillage was used and the OLR was
about 10.6 gVS/(L$d).
The biogas produced from all the reactors was collected by
gas bags. The amount of biogas was determined by 100 ml
glass syringe and the composition of the biogas was also
measured. The effluent pH of each reactor was measured
daily. The steady-state in this study was defined as a sus-
tained biogas production within #10% deviation, and in this
period the average values of parameters (pH, VFA, hydrogen
production, methane production et al.) for six consecutive
days were reported. Analysis of variance (ANOVA) at 0.05 level
was used to analyze the data.
2.3. Analysis
The collected samples from each reactor were centrifuged at
3500 rpm for 10 min, and filtrated through 0.45 mm filters to
determine soluble components. Total solid (TS), volatile solid
(VS), Total and soluble COD (TCOD and SCOD), were analyzed
in duplicate in accordance with Standard APHA Methods [13].
Total and soluble carbohydrates were determined using phe-
nolesulfuric acid method [14]. The concentrations of ethanol
and VFA (C2eC5), and biogas composition were determined by
gas chromatograph and detailed information could be found
in Ref. [15]. The measured biogas volume was adjusted to
volume at STP (standard temperature 0 ! C and pressure
1 atm).
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 1 7 3 3 e1 1 7 3 7 11735

The acetate produced by homoacetogens was estimated
based on the study of Arooj et al. [16]. The VFA and ethanol
concentrations in the reactors reported here have subtracted
the initial concentrations in raw cassava stillage.
3. Results and discussion
3.1. Effects of pretreatment and temperature on
continuous hydrogen production
As presented in Table 1, similar hydrogen yields were
obtained in R1 and R2 under mesophilic condition ( p > 0.05)
and methane was not detected in both hydrogen reactors.
Moreover, the distributions of VFA/ethanol in R1 and R2 were
similar with butyrate, acetate and propionate as the dominant
metabolic species, which was corresponded with the similar
hydrogen yields. Previous study contributed the importance of
heat-pretreatment of inoculum to the inhibition of homo-
acetogens under mesophilic condition [17]. However, in this
study the calculated acetate produced from homoacetogens
did not present any significant difference ( p > 0.05) in R1 and
R2 (as shown in Table 1), indicating that heat-pretreatment
had no effect on the homoacetogens in the continuous
experiments. Oh and his co-workers [18] also noticed that
heat-pretreatment could not inhibit homoacetogens but was
essential to eliminate methanogens to enhance the hydrogen
production in batch study. The absence of methane in R1
inoculated with raw sludge might be due to the lower HRT
(higher OLR) and lower pH, which were enough to inhibit the
methanogens [2,19]. Thus, mesophilic hydrogen production
from cassava stillage was not affected by heat-pretreatment
of inoculum in continuous experiment.
Fermentation temperature played an important role in
hydrogen production process and hydrogen yield at meso-
philic and thermophilic temperature was compared in this
study. The hydrogen yield (69.6 ml H2/gVS) in R3 was signifi-
However, the distribution of VFA was quite different. Butyrate
was the main metabolite under thermophilic condition which
was significantly higher than that under mesophilic condition
( p < 0.05). The propionate concentration at mesophilic
temperature was about 5 times higher than that at thermo-
philic temperature. Propionate is not suitable for hydrogen
production because it could consume hydrogen (as presented
in equation (3)). Moreover, the acetate concentration under
mesophilic condition was also significantly higher ( p < 0.05).
Such higher acetate concentration might be generated by
homoacetogens through hydrogen consumption [18]. As
shown in Table 1, the acetate produced by homoacetogens
under mesophilic condition was significantly higher than that
under thermophilic condition ( p < 0.05). The present study
demonstrated that continuous hydrogen production from
cassava stillage depended on the cultivation condition instead
of inoculum pretreatment.
C6H12O6 þ 2H2 ¼ 2CH3CH2COOH þ 2H2O (3)
3.2. Hydrogen and methane production and comparison
with one-phase fermentation system
To further utilize the residual organic acids, the effluent of R3
was fed into R4 for continuous methane production. Fig. 1
describes profiles of methane production, pH and VFA/
ethanol content with operation time. pH could be maintained
above 7.0 in the methane reactor (R4). After one day accli-
mation, methane production increased and reached steady-
state fast. The methane yield was 249 mlCH4/g VS with VS
removal efficiency of 44%. Most organics was degraded and
removed in the methane reactor compared to the VS removal
efficiency of 17% in hydrogen reactor. The methane yield was
comparable with the methane yield 192 mlCH4/gVS from
Methane production rate (L/(L.d)

cantly higher than that in R1 and R2 ( p < 0.05). The total 4.0 8.0
concentration of VFA/ethanol was about 6900 mg/L in R3, 3.5 7.5
similar with the value of 7400 mg/L in both R1 and R2 ( p > 0.05). 3.0
7.0
2.5
pH

2.0 6.5
Table 1 e Performances of hydrogen production in CH4
1.5 6.0
pH
different reactors under steady-state. 1.0
5.5
Parameter R1 R2 R3 0.5
0.0 5.0
pH 5.43 # 0.03 5.41 # 0.03 5.78 # 0.02
Hydrogen yield 14.1 # 1.2 13.8 # 0.9 69.6 # 1.3 6000
(mlH2/gVS) Ethanol
Concentration (mg/L)

VFA/ethanol (mg/L) 7316 # 887 7400 # 835 6909 # 711 5000 Acetate
Ethanol (mg/L) 236 # 52 250 # 41 446 # 98 Propionate
4000 Butyrate
Acetate (mg/L) 1600 # 250 1490 # 310 689 # 143
Propionate (mg/L) 1760 # 285 1980 # 220 367 # 150 3000 Valerate
Butyrate (mg/L) 3450 # 285 3390 # 250 5407 # 320
2000
Valerate (mg/L) 270 # 15 290 # 14 /
Acetate production by 816 # 121 752 # 140 165 # 58 (24%) 1000
homoacetogens (51%)a (50.5%)
0
(mg/L) 0 2 4 6 8 10 12 14 16 18
Time (d)
“/” not detectable.
a The value in the bracket represents the ratio of acetate produced
Fig. 1 e Profiles of methane, pH and VFA/ethanol
by homoacetogens/total acetate.
distributions of R4 in the two-phase process.
11736 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 1 7 3 3 e1 1 7 3 7

potato waste [3], but lower than 500 mlCH4/gVS from house- be one reason for the different hydrogen yields. It should be
hold solid waste [2]. The VFA concentration also decreased noticed that the addition of NaHCO3 to the influent is neces-
from 6744 mg/L in hydrogen reactor to 2171 mg/L in the sary which could maintain the suitable pH (around 5.5) in the
subsequent methane reactor. Obviously, VFA was not hydrogen reactor. From economical view point, cheaper
completely utilized and the residual dominant composition chemicals like CaCO3 should be used and also the effluent
was propinate (about 2000 mg/L). After day 17, the two-phase (with neutral pH) of methane reactor could be recycled to
CSTR was operated for another one month and similar hydrogen reactor to reduce the addition of such chemicals.
performance was observed with hydrogen and methane yield For two-phase process, the effluent contained high content
of 56.6 mlH2/g VS and 249 mlCH4/g VS. However, the perfor- of propionate while the other VFA were very low. The propi-
mance of one-phase CSTR (R5) was unstable. As shown in onate concentration in this study was about 2000 mg/L and it
Fig. 2, methane production fluctuated with operation time and did not affect the process performance as reflected by the
the production rate was only about half of that in R4. higher methane yield and stable pH even after long term
Accompanied with the decrease of pH from 6.5 to 5.5, methane operation. The results were contrary to the study of Barredo
production continuously decreased and ceased on day 14. and Evison [20], who predicted that the propionate concen-
Hydrogen production was observed thereafter. The distribu- tration between 1500 and 2220 mg/L would result in the
tion of VFA in R5 was found to be different with that in R4. The inhibition of methanogens and further lead to the failure of
total concentration of dominant species of acetate and the reactor. Our results suggested that the failure of anaerobic
propionate increased gradually to nearly 8000 mg/L on day 12. process was not so dependent only on higher propionate
Accompanied with the decrease of pH to 5.5 and hydrogen concentration. In the study of Wiegant et al. [21], higher
production, butyrate became the main metabolite. propionate concentration (2000e6000 mg/L) under thermo-
The organics in hydrogen reactor could be effectively philic condition was also found, but the anaerobic process was
converted to methane in the subsequent methane reactor. still stable and the effluent quality did not deteriorate with
Biogas production in the two-phase CSTR process was rela- increasing loading rates from 17 to 98 kgCOD/m3.d in UASB.
tively more stable than that in the one-phase CSTR at the OLR Our further study noticed that propionate decreased to below
of 10.6 gVS/(L$d) (or 13 gCOD/(L$d)). Moreover, TCOD and VS 200 mg/L if the HRT of R5 increased from 4 d to 10 d (data not
removal efficiency in the two-phase CSTR process could be shown), indicating that propionate could be controlled if the
58% and 61%, respectively. The values were comparable with OLR is lowered. In one-phase process, when acetate was also
the study of Zhu et al. [3], who found that 64% of TCOD and accumulated, the breakdown of system occurred. The higher
70% of VS removal from potato wastes was obtained in the concentration of acetate probably indicated the imbalance of
two-phase process, while relatively low OLR (2.57 gVS/(L$d)) methanogens and acidogens. The accumulation of both
was applied in their study. The hydrogen yield in the two- acetate and propionate might be an important parameter for
phase process was 56.6 ml H2/gVS. It was higher than 43 ml indication of the process imbalance.
H2/gVS from household solid waste in the study of Liu et al. [2],
but lower than 80.9 ml H2/gVS from food wastes in the study of
Kim et al. [1]. The different characteristics of the wastes might 4. Conclusion

Heat-pretreatment of inoculum was not necessary and could

Methane/Hydrogen production rate

4.0 8.0
not improve the hydrogen production from cassava stillage
3.5 7.5 under mesophilic condition in continuous experiment.
3.0 CH4
7.0 Moreover, inhibition of homoacetogens by the heat-pretreat-
2.5
(L/(L.d))

H2
pH ment of inoculum was not observed. Fermentation under
pH

2.0 6.5
thermophilic condition enhanced hydrogen yield from about
1.5 6.0 14 ml H2/gVS to 69.6 ml H2/gVS due to the lower propionate
1.0
5.5 production and lower activity of homoacetogens. Under
0.5
OLR > 10 gVS/(L$d), two-phase thermophilic CSTR for
0.0 5.0
hydrogen and methane production was stable with hydrogen
and methane yield of 56.6 mlH2/g VS and 249 mlCH4/g VS
6000
Ethanol respectively. The one-phase thermophilic CSTR for methane
Concentration (mg/L)

5000 Acetate production failed due to the accumulation of high concen-

Propionate tration of acetate and propionate. The accumulation of both
4000 Butyrate
Valerate acetate and propionate instead of propionate alone was
3000
probably related to the breakdown of anaerobic methane
2000 process.
1000
0
0 2 4 6 8 10 12 14 16 18
Acknowledgements
Time (d)

Fig. 2 e Profiles of methane, hydrogen, pH and VFA/ethanol This research was financially supported by the Foundation of
distributions of R5 in the one-phase process. Key Laboratory of Yangtze River Water Environment, Ministry
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 1 7 3 3 e1 1 7 3 7 11737

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