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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6

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Batch fermentative hydrogen production utilising

sago (Metroxylon sp.) starch processing effluent by
enriched sago sludge consortia

Nurleyna Yunus a, Jamaliah Md Jahim b,c,*, Nurina Anuar b,

Siti Rozaimah S. Abdullah b, Norhisham T. Kofli b
a
Downstream Technology Division, CRAUN Research Sdn Bhd, 93055 Kuching, Sarawak, Malaysia
b
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment,
Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia
c
Fuel Cell Institute, Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia

article info abstract

Article history: Sago starch processing effluent (SSPE) is an ideal bio-resource that can be utilised as a
Received 11 August 2014 substrate for fermentative reactions due to its relatively high organic content. Annually in
Received in revised form Malaysia, about 2.5 million tonnes of effluent are generated from the processing of sago
1 October 2014 starch. In this study, the potential use of SSPE as a substrate for fermentative hydrogen
Accepted 3 October 2014 production was confirmed under all the experimental conditions studied. The maximum
Available online 29 October 2014 hydrogen production and volumetric hydrogen production rate were 575 mL H2/L SSPE and
57.54 mL H2/hr.L SSPE, respectively, from cultures with an initial pH of 7 and substrate
Keywords: concentration of 11 g soluble carbohydrate/L SSPE. The final soluble metabolites were
Anaerobic fermentation comprised mainly of acetate (24e43%), butyrate (4e20%), propionate (1e7%) and ethanol
Biohydrogen production (44e66%), suggesting an acetic acid-ethanol type fermentation pathway.
Sago starch processing effluent Copyright 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Initial pH reserved.
Initial substrate concentration

industry, is a carbohydrate-rich liquid waste comprised

Introduction
The sago palm (Metroxylon spp.) is an important food security
crop in tropical Asia. Sago starch, which accumulates in the
stem of the palm, is an important food resource and industrial
raw material used throughout the world. Malaysia, is the
largest exporter of sago starch to the world with an annual
production of approximately 51,000 tonnes of dry starch. The
sago starch processing effluent (SSPE), generated by this
mainly of macromolecules in the form of polysaccharides
(starch and hemicelluloses). However, improper management
of these wastes have resulted in vigorous research to solve the
current predicament facing Malaysian sago industry. Past
findings have shown the potential of SSPE as a substrate for
the development of value-added products such as algae
cultivation [1] and biomethane generation via anaerobic
fermentation [2].

* Corresponding author. Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti
Kebangsaan Malaysia, 43600 Bangi, Malaysia. Tel.: 60 3 8921 6427; fax: 60 3 8921 6148.
E-mail address: jamal@eng.ukm.my (J.M. Jahim).
http://dx.doi.org/10.1016/j.ijhydene.2014.10.015
0360-3199/Copyright 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
19938 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6
An alternative prospect is to utilize the effluent as a sub-
strate for fermentative hydrogen production; this was the
purpose of this study. However, to effectively make use of this
waste as a substrate for fermentation reactions, the effluent
needs to be pre-treated by either acid or enzymatic hydrolysis
to cleave the polysaccharides into simple sugar form. Ac-
cording to a review by Kapdan and Kargi [3], simple sugars
such as glucose, sucrose and xylose are readily digestible and
are the preferred substrates for hydrogen production. This is
because bacterial metabolism of macromolecules (such as
polysaccharides) differs from those of smaller molecular
weight compounds (e.g. monosaccharides) as a consequence
of physical and biological factors. Typically, compounds with
molecular weights in excess of 1000 atomic mass units need to
be hydrolysed into smaller polymers before they can be
transported across the bacterial cell wall and used for energy
production [4].
It has been claimed that Clostridia sp. has the ability to
directly digest polysaccharides (starch particles) for bio-
hydrogen production [5]. However, the process of digestion
and rate of biohydrogen production is impeded due to the
slow starch hydrolysis process [6]. To eliminate the rate-
limiting starch hydrolysis step, Chen et al. [7] attempted to
hydrolyse starch with amylase prior to utilising it as an energy
source for biohydrogen production via dark fermentation.
Meanwhile, Lean ~ o and Babel [8] were able to improve bio-
hydrogen production from cassava wastewater by pre-
treating the wastewater with commercial enzymes to in-
crease the simple sugar concentration in the substrate.
The production of biohydrogen from starch-containing
synthetic wastewater has been reported extensively in the
past [8e10]. However, studies on biohydrogen production
from real starch processing wastewater using mixed cultures
are limited, and most of these studies used cassava/tapioca
wastewater [11e15]. No studies have documented the use of
real sago starch processing wastewater/effluent for this
purpose.
The purpose of this study was to ascertain the feasibility of
utilising SSPE as a substrate for fermentative hydrogen pro-
duction through a series of batch assays. This was achieved by
an initial attempt to increase the soluble carbohydrate con-
centration in the SSPE through in situ enzymatic treatment
utilising a-amylase and g-amylase. Following to this, an
investigation was conducted to study the effects of the initial
substrate concentration and cultivation pH (over a pertinent
range) on the hydrogen production potential seeded with
heat-treated sago sludge inocula. The kinetics of hydrogen
production were analysed using the modified Gompertz
equation.
Materials and methods
SSPE preparation and pre-treatment
SSPE is easily fermented during storage as a result of its car-
bohydrate content. For this reason, in addition to stand-
ardising the soluble carbohydrate concentration, all SSPE used
throughout this study were processed under laboratory
conditions in accordance with the traditional extraction
method normally carried out by sago farmers in Sarawak.
Sago logs, obtained from the Paya Paloh Sago Research
Station in Kota Samarahan, Sarawak, Malaysia, were manu-
ally debarked and the pith was chopped into smaller pieces,
bagged and stored frozen. When required, the chopped pith
was thawed to room temperature and liquefied in a heavy
duty blender with tap water, then filtered through muslin
cloth followed by settling of the starch particles. The super-
natant was then drawn off for another round of filtration and
settling. This process was repeated three times. The final su-
pernatant was then collected, kept in capped bottles and
stored at 4  C to be used within 3 days. In order to obtain
similar COD/soluble carbohydrate concentrations as effluents
from a sago mill, a standard 1:4 ratio of pith to water was
applied.
Characterisation on the basic properties of SSPE was per-
formed for SSPE sampled from a sago mill and from those
prepared under laboratory conditions to compare the
variability.
SSPE pre-treatment with a-amylase and g-amylase
In order to naturally increase the soluble carbohydrate
composition in SSPE, comparisons were made by pre-treating
with a-amylase (Termamyl 120 L, Novo Nordisk, Denmark,
with declared enzyme activity of 120 KNU/g) alone, and with a
cocktail of a-amylase and g-amylase (AMG 300 L, Novo Nor-
disk, Denmark, with declared enzyme activity of 300 AGU/mL)
at equal concentrations ranging from 0.2% to 1% (equivalent to
26.4e144 KNU/g and 60e300 AGU/mL for a-amylase and g-
amylase, respectively). Upon addition of the enzyme, the SSPE
solution was stirred until it was homogeneous and its pH was
adjusted to 6.0 with 1 M NaOH or 1 M HCl. The solution was
then left to incubate at room temperature for 10 min.
Additionally, further trials were also conducted to study
the effect of increasing, in equal treatment concentrations,
the enzyme cocktail of a-amylase and g-amylase ranging from
0.2% to 5% (equivalent to 26.4e739 KNU/g and 60e1500 AGU/
mL for a-amylase and g-amylase, respectively). The tested
range of enzyme treatment concentration were aimed to
sufficiently increase the soluble carbohydrate concentrations
in the SSPE for a subsequent anaerobic fermentative reaction.
Sampling was performed from each treatment for soluble
carbohydrate analysis. To ascertain the storage stability of the
SSPE after enzyme treatment, the mixtures were refrigerated
at 4  C for 48 h and sampled every 24 h to determine the sol-
uble carbohydrate concentration.
Hydrogen producing seed
Sago sludge consortia (sampled from the waste drain of a sago
mill) that have been previously heat-treated in oven at 100  C
for 1 h were used as inoculum source. The consortia were first
adapted to an SSPE-based substrate. Preliminary analyses
suggested that SSPE lacks the necessary nutrients for the
growth of anaerobic microorganisms, specifically in terms of
N, P and micronutrients (Table 1). Therefore, the Endo nutrient
solution (adapted from the formulation by Endo et al. [16] as
described by Lin and Lay [17]) was employed in this study to
 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6 19939

evaluate the effects of the independent variables (i.e. initial pH

Table 1 e Comparison between sago starch processing
and substrate concentration) as well as any interaction be-
effluents sampled from a sago mill and processed under
laboratory conditions. tween these two variables on the response variables. The
hydrogen production rate (HPR) and hydrogen yield (HY) were
Parameter mg/L
defined as the response variables. The use of this experi-
Mill Lab mental design had been similarly employed in the past
pH 4.7 0.3 4.2 0.1 [20e22] to study the effects of independent variables such as
Total COD 12,409 262 10,642 157 the initial pH and substrate concentration on biohydrogen
Soluble COD 9530 198 8013 112 production utilising substrates from various waste and
TS 6542 157 6047 109
wastewater sources.
TSS 1516 221 1339 197
VSS 2340 257 1998 91
A thirteen trial design matrix was constructed to cover the
TKN 124 6 114 8 two independent variables, each at five levels. All trials were
Soluble carbohydrate 683 32 557 13 done in triplicate to ensure the reproducibility of the results.
Trace elements P ND ND The substrate concentrations, expressed in terms of soluble
K 331 7.9 207 3.8 carbohydrate (SC), were varied from 3 to 11 g SC/L, with a
Cu 0.46 0.01 0.37 0.06
central value of 7 g SC/L. The initial cultivation pH varied from
Zn 0.83 0.07 1 0.01
6.0 to 8.0 with a central value of 7.0.
Ca 143 6.2 119 3.4
Mg 58 4.1 31 2.2
Mn 4 0.12 5.4 1.5 Experimental apparatus and procedure
Fe 1 0.08 3 0.57 The experiments were conducted in a series of 60 mL serum
Na 48 0.89 55 2.3 bottles with a 50 mL working volume. The substrate was made
Ni 0.09 0.01 ND up of enzyme-treated SSPE and the Endo nutrient solution (as
Cr ND ND
described in Section Hydrogen producing seed). A 10% inoc-
Al 1 0.2 ND
ulum concentration was employed throughout this study. Upon
B ND ND
inoculation, the pH in each serum bottle was adjusted accord-
ingly with 1 M NaOH or HCl. The bottles were then purged with
provide sufficient nutrients to the SSPE for anaerobic bacterial
nitrogen gas for 3 min (to create anaerobic conditions) and
growth. The Endo nutrient formulation has been widely used
capped with a rubber septum. A hypodermic syringe (Terumo,
by researchers for fermentative biohydrogen production
Japan) was fitted to equilibrate the pressure inside each bottle to
seeded with mixed microflora utilising synthetic and real
ambient pressure during the gas production phase. The cul-
waste materials/wastewater as the substrate [11,17,18].
tures were then placed in a dark incubator shaker at 37  C and
The substrate was made up of enzyme-treated SSPE with
100 RPM. During the course of the experiment, biogas samples
an approximate concentration of 5 g/L soluble carbohydrate
were collected routinely for biogas composition analysis. Liquid
and supplemented with the Endo nutrient solution containing
samples from each serum bottle were drawn at the end of the
5240 mg/L NH4HCO3, 125 mg/L K2HPO4, 15 mg/L MgCl2$6H2O,
trial and analysed for primary volatile fatty acids and related
25 mg/L FeSO4$7H2O, 5 mg/L CuSO4$5H2O, 0.125 mg/L
solvents, soluble carbohydrate and pH.
CoCl2$5H2O and 6720 mg/L NaHCO3. Cultivation was con-
ducted in 60 mL serum bottles with a 50 mL working volume at
Analytical methods
a 10% inoculant concentration. The incubation temperature
was set at 37  C with a stirring rate of 100 RPM. Two successive
Biogas production was measured periodically throughout the
transfers of 48 h cultures as the inoculum (10%) to a fresh
duration of the experiments by logging the volume indicated on
substrate were made to obtain a stable hydrogen producing
the fitted syringes. The biogas was also sampled from the
microbial consortium. The developed cultures were then used
headspace with a 500 mL gas-tight syringe (Hamilton, USA) and
as hydrogen-producing seed stock in the following batch
analysed for the gas composition using a gas chromatograph
experiments.
(GC; Hewlett Packard 5890) equipped with a thermal conduc-
tivity detector (TCD). The GC was fitted in series with a stainless
Batch fermentative hydrogen production using SSPE as the steel molecular sieve column (10 ft 45/60) and Porapak Q packed
substrate column (9 ft 80/100). The injector, oven and detector tempera-
tures were set at 90  C, 50  C and 200  C, respectively. Helium
Experimental design was used as the carrier gas with a flow rate of 18.7 mL/min.
Batch experiments were carried out to investigate the Primary volatile fatty acids (VFA) and related solvents were
hydrogen production potential of SSPE as the main substrate analysed using a GC (Hewlett Packard 5890) equipped with a
component. However, due to the diverse organic components flame ionisation detector (FID). An HP-FFAP column was used
present in SSPE, it would be difficult to standardise the type of (50 m L  0.2 mm ID  0.33 mm film) with helium as the carrier
substrate used as the energy source for fermentation re- gas. The injector and detector temperature were maintained
actions. Therefore, throughout this study, the concentration at 270  C and 200  C, respectively.
of substrate for fermentation is generally identified by its The total chemical oxygen demand (COD), total solids (TS),
soluble carbohydrate concentration. total suspended solids (TSS) and volatile suspended solids
A fractional factorial central composite design and (VSS), total Kjeldahl nitrogen (TKN) and trace elements con-
response surface methodology (RSM) [19] were employed to centrations were analysed in accordance with standard
19940 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6
methods [23]. Meanwhile, the determination of total carbo-
hydrate was performed according to the method developed by
Dubois et al. [24], while soluble carbohydrate analysis was
carried out using the Nelson-Somogyi method as described by
Refs. [25], with D-glucose (Sigma Aldrich, USA) as the standard.
Data analysis
Cumulative hydrogen production curves were constructed
according to the method described by Van Ginkel [26]. The gas
composition in the headspace of the serum bottle and the
total volume of the biogas produced at each time interval were
measured and applied to the mass balance equation (Eq. (1)):



VH;i VH;i1 CH;i VG;i  VG;i1 VH CH;i  CH;i1
where VH,i and VH,i1 are cumulative hydrogen gas volumes at
the current (i) and previous (i1) time interval; VG,i and VG,i1
are the total biogas volumes at the current (i) and previous
(i1) time interval; CH,i and CH,i1 are the fraction of hydrogen
gas in the headspace of the bottle as determined by gas
chromatography in the current and previous interval; and VH
is the total volume of headspace in the bottle.
Each of the cumulative hydrogen production curves were
fitted with the modified Gompertz equation (Eq. (2)) using
Sigmaplot Software (Version 11, Systat Software Inc., USA).
   
Rm  e
H t Hmax  exp  exp: l  t 1
Hmax
where H(t) is the cumulative hydrogen production (mL); Hmax is
the hydrogen production potential (mL); Rm is the maximum
hydrogen production rate (mL H2/hr); l is the duration of the lag
phase (h); e is 2.71828 and t is the incubation time (h). Each curve
was best fitted using the MarquardteLevenberg algorithm by
minimising the sum of square differences between the observed
and predicted values of the dependent variable.The response
variables (HPR and HY) were calculated from the data, followed
by analysis using RSM to enhance the visual understanding of
the type of trends that exist for the variables as a function of the
experimental treatments. The response variables (HPR and HY)
were fitted using a quadratic model to correlate each of the
response variables to the independent variables (initial cultiva-
tion pH and substrate concentration). The mathematical form of
each quadratic equation is as described in Eq. (3):
Y A0 A1 X1 A2 X2 A1;2 X1 X2 A1;1 A21 A2;2 X22
where X1, X2 are the actual values of the independent vari-
ables, Y is the corresponding response variable, A0 is the
constant of the model, A1 and A2 are the linear coefficient, A1,2
is the interactive coefficient and A1,1 and A1,2 are the quadratic
coefficients. RSM analyses were performed using Design-
Expert software (Version 6, Stat-Ease Inc. USA).
Results and discussion
Characterisation of SSPE
Comparisons on the characteristics of SSPE were made be-
tween those collected from a sago mill and those processed
under laboratory conditions (Table 1). The average percentage
of variability obtained between the two sources of SSPE was
25% (for detectable data only). The highest percentage of
variability was mainly attributed to trace element concen-
trations, particularly K, Mg, Mn and Fe, which respectively
were well above 30%. Nevertheless, this finding revealed that
the quality of the SSPE processed under laboratory conditions
is sufficiently comparable with those processed in the mill.
The basic properties of SSPE can be generally categorised
as acidic with a pH range of 4.2e4.7. The total COD had an
approximate range of 10,000 to 12,000 mg/L while the soluble
carbohydrate concentration was relatively low at 500e700 mg/
L. This was attributed to the fact that most of the carbohydrate
content in the effluent is in the form of starch particles and
(1) thin-walled parenchyma fibres/tissues which had penetrated
the filter during the filtration process. These are macromole-
cules which need to be hydrolysed before they can be trans-
ported across the anaerobic bacteria cell wall. This hydrolysis
step is rate-limiting and will impede the rate of biohydrogen
production unless pre-treatment was conducted to facilitate
in the breakdown of the carbohydrate components into sim-
ple sugar form prior to initiating the fermentation reaction.
Effect of in situ enzymatic treatment of SSPE on its soluble
carbohydrate concentration
Effect of a-amylase with and without g-amylase combination
(2)
Comparisons were made in treating the SSPE with a-amylase
alone and with a combination of a-amylase with g-amylase at
different treatment concentrations (Fig. 1). From the results,
the enzyme cocktail was found to be more effective in
increasing the soluble carbohydrate concentration by
approximately two-fold or higher in comparison to a-amylase
alone. This increase could be attributed to the added advan-
tage of g-amylase as it is able to act on available poly-
saccharides, dextrins and sugars by cleaving a-1, 4- and a-1, 6-
glycosidic linkages, thereby releasing stepwise from the end of
the compound chain. Treatment with a-amylase alone will
only hydrolyse the a-1,4 glycosidic linkages in polysaccharides
to yield dextrins, oligosaccharides and monosaccharides [8].
Additionally, as the treatment concentration of the enzymes
was increased, significant improvements (at a 0.05) in the
soluble carbohydrate concentrations were observed, specif-
ically with the cocktail of a-amylase and g-amylase at a 1%
(3) concentration, which achieved about 6000 mg/L soluble car-
bohydrate compared to about 3000 mg/L achieved by treat-
ment with a-amylase alone.
Effect of the enzyme cocktail (a-amylase and g-amylase) at
different treatment concentrations
Additional comparisons were made to identify the effect of
treating SSPE with a combination of a-amylase and g-amylase
at various treatment concentrations from a low of 0.2% to a
high of 5% (v/v). The results, as shown in Fig. 2, show that the
soluble carbohydrate concentration in SSPE improved signifi-
cantly (at a 0.05) with increasing enzyme treatment con-
centration. An increase from approximately 800 mg/L to
11,000 mg/L soluble carbohydrate was observed when SSPE
was treated at the 0.2% and 2% treatment concentrations,
respectively. Meanwhile, treatment at 2.5% achieved a
 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6 19941

Fig. 1 e Effect of treatment with and without g-amylase on the soluble carbohydrate concentration in SSPE.

marginal increase to approximately 12,000 mg/L as compared
to the results obtained at 2%. A further trial at a high enzyme
treatment concentration of 5% showed approximately a two-
fold increase to 24,000 mg/L in the soluble carbohydrate
concentration.
Kinetic analysis of hydrogen production
Examples of the cumulative hydrogen production curves ob-
tained from each trial are presented in Fig. 3. Methane gas was
not detected in the biogas produced in any of the experiments.
Following a lag period that ranged from approximately 5 to
7 h, depending on the initial cultivation pH and substrate
concentration, biohydrogen production started at different
rates and to different extent. Cultures with a lower initial
cultivation pH (i.e. pH 6.0 and 6.5) were observed to have
longer lag phase before gas production began as compared to
those with a higher initial cultivation pH (i.e. pH 7.0e8.0).
Similar observations have been reported in the past in studies
utilising starch as the substrate in dark fermentation [27].
To further analyse the experimental data, the modified
Gompertz model was used to fit the cumulative hydrogen
production data from each experiment to obtain the param-
eters of interest, namely Hmax, Rm and l. The average results
for all measured and calculated parameters are presented in
Table 2. The coefficients of determination (R2) achieved for all

Fig. 2 e Effect of enzymatic (cocktail of amylase and g-amylase) treatment concentrations on the soluble carbohydrate
concentration in SSPE.
19942 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6

Fig. 3 e Cumulative hydrogen production curves reported at 25  C and 1 atm. Each curve represents one of the replicates
from each trial run.

the fittings were more than 0.98, indicating that the modified
Gompertz model could successfully describe the progress of
cumulative hydrogen production in the batch trials in this
study. Hydrogen yield (HY) was calculated by normalising
Hmax with respect to mL H2 per litre SSPE, converted to H2
molar concentration and further fractionated with the molar
concentration of substrate consumed (with the assumption
that glucose was the sole carbohydrate present in the SSPE).
Meanwhile the parameter Rm was normalised with respect to
mL H2 per litre SSPE to attain the calculated values to
hydrogen production rate (HPR).
The highest hydrogen production (normalised per litre
SSPE) recorded was at 575 mL H2/L SSPE with a corresponding
volumetric hydrogen production rate of 57 mL H2/hr.L SSPE
attained from the cultures with an initial cultivation pH of 7.0
and a substrate concentration of 11 g SC/L. Meanwhile, cul-
tures with an initial cultivation pH of 6.0 at 3 g SC/L substrate
had the lowest hydrogen production at 201 mL H2/L SSPE with
a corresponding volumetric hydrogen production rate of
23.6 mL H2/hr L SSSPE. Therefore, this study was able to
demonstrate the ability of SSPE to be utilised as a substrate for
fermentative biohydrogen production after successful bio-
hydrogen production was achieved within the range of con-
ditions studied.
A comparison of biohydrogen production yield and rate
achieved in the present study with literature on real starch-

Table 2 e Average values for measured and calculated kinetic parameters.

Run Initial Substrate Modified Gompertz equation parameter values HPR HY
pH concentration
Hmax Rmax l R2
g SC/L SSPE mL H2 mL H2/h h mL H2/h.L mol H2/mol
SSPE glucoseconsumed
1 8 3 18.8 0.3 1.8 0.03 5.2 0.046 0.99 0.002 35.3 0.6 1.09 0.03
2 6 7 15.8 0.5 1.7 0.07 7.2 0.112 0.99 0.001 33.7 1.4 0.39 0.01
3 6.5 5 15.6 0.8 1.8 0.15 7.3 0.049 0.99 0.0004 36.7 2.9 0.53 0.02
4 6.5 9 19.3 3.2 2.1 0.18 7.3 0.137 0.99 0.003 41.8 3.6 0.34 0.07
5 7 3 15.5 2.3 1.3 0.01 5.3 0.173 0.99 0.005 27.0 0.3 0.83 0.13
6 8 7 22.7 2.8 2.1 0.27 5.7 0.365 0.99 0.002 42.5 5.4 0.62 0.12
7 7 7 17.7 0.5 2.7 0.27 6.7 0.175 0.99 0.002 53.9 5.5 0.47 0.01
8 8 11 23.9 0.5 2.5 0.07 5.8 0.242 0.99 0.002 49.2 1.5 0.39 0.02
9 7 11 27.9 1.2 2.8 0.04 6.5 0.069 0.99 0.001 57.0 0.8 0.40 0.02
10 7.5 5 18.0 0.7 2.4 0.21 5.8 0.181 0.98 0.0004 48.9 4.2 0.61 0.04
11 6 11 12.4 0.5 1.9 0.24 7.1 0.055 0.99 0.002 37.2 4.9 0.53 0.16
12 7.5 9 25.1 0.004 3.3 0.28 6.2 0.153 0.99 0.0004 66.1 5.5 0.44 0.02
13 6 3 10.4 0.5 1.2 0.03 6.7 0.129 0.99 0.007 23.6 0.6 0.64 0.01

SC, soluble carbohydrate.

 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6 19943

based wastewater as substrate is summarized in Table 3. The
highest HY achieved in this study is found comparable with
the results obtain for cassava starch processing wastewater
[11], starch containing textile wastewater [20] and potato
processing wastewater [26]. All of which were within the
range of 0.94e1.09 mol H2/mol glucose. Meanwhile, data for
highest HPR for SSPE at 502.5 mL H2/L SSPE was much lower
compared to cassava starch processing wastewater at 1.25 L
H2/L [12], starch-containing textile wastewater at 1.14 L H2/L
[20], and potato processing wastewater at 2.8 L H2/L [26]. This
was mainly attributed by the different loading rate applied in
the respective studies.
Response surface analysis
Effect of initial cultivation pH and substrate concentration on
hydrogen yield (HY)
In order to examine the relationship between the initial
cultivation pH and substrate concentration on HY, the design
matrix with the corresponding calculated results in Table 2
was subjected to stepwise regression and generated the
following equation:
HY 1:750:46X1 0:045X2 0:056X21 0:01X22 0:034X1 X2
where the response HY is in its original units and the X1, X2 are
the actual values of the initial cultivation pH and substrate
concentration, respectively.
The analysis of variance (ANOVA) on the fitting model was
considered to be a good representation of the HY data with a p-
value of the F test less than 0.0001, while the regression co-
efficient (R2) was 0.9780, indicating that the model was able to
explain 97.8% of the variability in the response variable. These
findings show that Eq. (4) could successfully describe the ef-
fect of the initial cultivation pH and substrate concentration
on HY in this study.
The ANOVA results also indicated that most of the terms
(i.e. the linear effect of the initial cultivation pH and substrate
concentration, the quadratic effect of substrate concentration,
and the interactive effect between the initial cultivation pH
and substrate concentration) were highly significant,
achieving p-values less than 0.05, thus suggesting that these
terms have a great impact on the HY performance. Further-
more, a significant interaction between the two independent
variables suggested that the heat-treated sago consortia fav-
oured different initial cultivation pH levels when they were
allowed to grow at different substrate concentrations during
fermentative hydrogen production.
Following to this, a two-dimensional contour plot was
constructed using Eq. (4), as presented in Fig. 4. This plot clearly
displays the tendency for HY to increase with increasing initial
cultivation pH and decreasing substrate concentration. The
highest HY obtained for this study was 1.09 mol H2/mol glu-
coseconsumed at an initial cultivation pH and substrate con-
centration of pH 8 and 3 g SC/L, respectively. The hydrogen
yield was found to be lower at higher substrate concentrations
(between 7 and 11 g SC/L) at all the pH levels tested. This trend is
in agreement to a certain extent with those reported by Lay
et al. [20] and Ginkel et al. [22]. The reason for this could be
(4) attributed to the fact that a higher substrate concentration
resulted in a sharp decrease in the fermentation broth pH due
to the higher accumulation of volatile fatty acids (VFA), which
consequently led to the inhibition of the microorganisms
involved in hydrogen production [22].
Effect of initial cultivation pH and substrate concentration on
the hydrogen production rate (HPR)
Stepwise regression analysis was also conducted for the data
on HPR to evaluate its relationship with the initial cultivation
pH and substrate concentration. The following quadratic
equation was generated:

Table 3 e A comparison on biohydrogen yield and production rate from various starch-based wastewater via dark
fermentation.
Wastewater (WW) Organism Substrate Hydrogen yield (HY)a Hydrogen production Reference
concentration rate (HPR)
Sago (Cassava) starch MC 10.6 g COD/L 0.94 mol H2/mol glucose e Sen and Suttar (2012)
processing WW
Cassava WW MC 32 g COD/L 847.71 mol H2/mol glucose e ~ o and Babel (2012)
Lean
Cassava processing WW PC 5 g COD/L 2.41 mol H2/mol glucose e Cappelletti et al. (2011)
Cassava starch processing WW MC 5 g starch/L 0.18 mol H2/mol glucose 1246 mL/L O-Thong et al. (2011)
Cassava Stillage MC 60.1 g VS/L 65.3 mL H2/g VS e Luo et al. (2010)
Cassava WW MC 30 g COD/L 3.29 mol H2/mol glucose 524 mL H2/g VSS.d Sreethawong et al. (2010)
Cassava starch manufacturing MC 22.6 g COD/L 0.06 mol H2/mol glucose 4.42 mL H2/g VSS. hr Sangyoka et al. (2007)
WW
Cassava WW MC 20 g COD/L 1.20 mol H2/mol glucose 940.90 mL H2/g VSS Reungsang et al. (2007)
Starch containing textile WW MC 20 g COD/L 0.97 mol H2/mol hexose 1.14 L H2/L WW/d Lay et al. (2012)
Potato processing WW MC 21 g COD/L 1.06 mol H2/mol glucose 2.8 L H2/L Van Ginkel et al. (2005)
Sago (Metroxylon sp.) starch MC 3 g SC/L 1.09 mol H2/mol glucose 375 mL H2/L SSPE This study
processing effluent
Sago (Metroxylon sp.) starch MC 9 g SC/L 0.44 mol H2/mol glucose 502.5 mL H2/L SSPE This study
processing effluent

MC, mixed culture; PC, pure culture.

a
The published HY were standardised to mol H2/mol glucose with the assumption that 1 g/L COD 1.067 g/L glucose and 1 g starch 10.3 g/L
COD [11].
19944 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6
Fig. 4 e Response surface contour plot for hydrogen yield (HY).
HPR 569:1 164:3X1 2:45X2  11:2X21
where the response HPR is in its original units, while X1 and X2
represent the actual values of the initial pH and substrate
concentration, respectively.
The significance of Eq. (5) was estimated using the same
statistical approach that was used for Eq. (4), achieving a p-
value of 0.008 and R2 of 0.7644. These findings indicate that the
regression model was suitable to adequately represent the
experimental data. Through analysis of variance, it was found
that the linear and quadratic effect of the initial cultivation pH
and the linear effect of the initial substrate concentration had
a significant impact (p < 0.05) on HPR performance. Mean-
while, the quadratic term for the initial substrate concentra-
tion and the interaction between the two independent
variables were found to be statistically insignificant (p > 0.05),
indicating that these terms had little impact on HPR.
In order to have a graphical understanding of the distri-
bution of HPR based on the independent variables, a two-
dimensional contour plot was constructed using Eq. (5), as
shown in Fig. 5. Generally, it can be observed that HPR was
found to increase with an increasing substrate concentration.
An increase in the initial cultivation pH also led to higher HPR
with peak values obtained within the pH range of 7.0e7.5. At
higher substrate concentrations, an improved HPR can be
observed on the higher side of the pH range (i.e. pH 7.0e7.5).
The optimal HPR predicted by Eq. (5) was 58.4 mL H2/hr.L SSPE
obtained from the cultures with an initial cultivation pH of 7.0
and a substrate concentration of 11 g RS/L. Similar observa-
tions were also documented [20] where the optimal HPR was
observed within the initial cultivation pH range of 6.5e7.5 and
initial substrate concentration between 9 and 17 g COD/L,
utilising starch-containing textile wastewater as the substrate
(5)
seeded with heat-treated cow dung.
Soluble metabolites distribution
The results regarding the volatile fatty acids (VFA), alcohol
concentrations, pH and percentage of substrate consumed
after 48 h at various initial cultivation pH levels and substrate
concentrations are presented in Table 4.
Based on these results, acetic acid and ethanol were the
major metabolites produced in all the trials, while butyric acid
and propionic acid were also present but in smaller quantities.
The concentrations of these soluble metabolites (acetic acid,
butyric acid and ethanol) were also found to generally in-
crease with an increase in the initial substrate concentration
for all the initial cultivation pH levels trialled. Numerous
studies have reported that the fermentation pathway of
hydrogen production differs with the type of substrate used in
addition to bacterial metabolism. A study conducted by O-
Thong et al. [12] utilising cassava starch processing waste-
water obtained a similar dominance of acetic acid and ethanol
out of the total metabolites produced. In another study, Sen
et al. [11] reported acetate and butyrate production fermen-
tation pathways utilising cassava wastewater as the substrate
seeded with heat-treated cow dung. Additionally, Luo et al.
[28] documented butyrate/acetate and butyrate/propionate
pathways for the fermentation of cassava stillage seeded with
anaerobic sludge obtained from an ethanol-producing reactor.
The findings obtained through this study suggest that the
glycolysis of soluble carbohydrates present in SSPE are prone
to the acetic acid/ethanol production fermentation pathway.
According to studies by Li et al. [29] and Ren et al. [30], acetic
acid/ethanol type fermentation occurs when the mass
 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6 19945

Fig. 5 e Response surface contour plot for the hydrogen production rate (HPR).

percentage of acetic acid and ethanol are above 60% in the
total fermentation end products.
Meanwhile, from the data in Table 4, it was also observed
that a higher initial cultivation pH level (between pH 6.5e8.0)
allowed the fermentation reaction to continue thoroughly,
thus improving on the percentage of substrate consumed.
Additionally, the percentage of substrate consumed and the
final pH were found to decrease with an increase in the initial
substrate concentration at a lower initial pH. This could be
attributed to the greater accumulation of volatile fatty acids
which depleted the buffering capacity of the medium, leading
to bacterial growth inhibition due to an acidic environment.
According to the findings of Dabrock et al. [31], a gradual
decrease in pH inhibits hydrogen production due to the acidic
environment which affects the activity of the iron-containing
hydrogenase enzyme. Meanwhile, Roychowdhury [32] docu-
mented that hydrogen production by Clostridium sp. is
inhibited in the pH range of 4.0e5.0.
Conclusion
Preliminary trials have demonstrated the possibility of
increasing the soluble carbohydrate concentration in SSPE via in

Table 4 e Soluble metabolites at the end of the batch experiments.

Run Initial pH Substrate concentration Volatile fatty acids Alcohols Substrate consumed Final pH
AA PA BA EtOH
g SC/L SSPE mg/L mg/L %
1 8 3 66925 301 39711 90039 925.4 6.430.03
2 6 7 1740117 376 82069 134533 990.7 5.080.15
3a 6.5 5 1315284 19030 3718 1437108 990.7 5.930.03
4a 6.5 9 9816 18631 551106 200791 980.1 4.910.31
5 7 3 111695 15313 34321 1130136 970.3 5.200.27
6 8 7 82733 301 28714 193266 961.6 6.140.06
7 7 7 953235 17363 40735 1427125 970.2 6.130.01
8 8 11 103440 330 33116 236680 862.4 4.990.04
9a 7 11 106485 14534 24151 2946270 980.9 5.070.33
10 7.5 5 107856 5517 31711 144573 970.5 6.450.01
11 6 11 1099127 313 30016 1184109 347.8 4.880.01
12a 7.5 9 1449160 6444 40328 2116185 990.3 6.280.03
13 6 3 7058 270 25312 108918 850.5 5.500.01
a
Propanol present at concentration 36 mg/L.
19946 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 9 ( 2 0 1 4 ) 1 9 9 3 7 e1 9 9 4 6

situ enzymatic treatment without requiring a long incubation
period. The experimental results utilising treated SSPE showed
that biohydrogen production was achieved under all the con-
ditions studied, thus confirming the potential use of SSPE for
fermentative hydrogen production. Improved HY can be ach-
ieved by setting higher initial cultivation pH with low substrate
concentration. On the contrary, to achieve higher HPR requires
higher substrate concentration within initial cultivation pH of
7.0e7.5. The overall final soluble metabolite concentrations
indicated that the digestion of SSPE by the mixed consortia lead
to an acetic acid/ethanol type fermentation pathway.
Acknowledgements
The authors wish to thank the Sarawak State Government for
financial support of this work through the grant provided
under Sago Biomass Utilisation.
references
[1] Phang SM, et al. Spirulina cultivation in digested sago starch
factory wastewater. J Appl Phycol 2000;12:395e400.
[2] Nurleyna Y, Azhar AR. Biogas generation potential via
anaerobic treatment of sago mill effluent. In: 2nd ASEAN
Sago Symposium. Kuching, Sarawak: Universiti Malaysia
Sarawak; 2012.
[3] Kapdan IK, Kargi F. Bio-hydrogen production from waste
materials. Enzyme Microb Technol 2006;38(5):569e82.
[4] Confer DR, Logan BE. Molecular weight distribution of
hydrolysis products during the biodegradation of model
macromolecules in suspended and biofilm cultures. II.
Dextran and dextrin. Water Res 1997;31(9):2137e45.
[5] Yokoi H, et al. H2 production from starch by a mixed culture
of Clostridium butyricum and Enterobacter aerogenes.
Biotechnol Lett 1998;20(2):143e7.
[6] Lo Y-C, et al. Dark H2 fermentation from sucrose and xylose
using H2-producing indigenous bacteria: feasibility and
kinetic studies. Water Res 2008;42(4e5):827e42.
[7] Chen S-D, et al. Batch and continuous biohydrogen
production from starch hydrolysate by Clostridium species.
Int J Hydrogen Energy 2008;33(7):1803e12.
[8] Lean~ o EP, Babel S. Effects of pretreatment methods on
cassava wastewater for biohydrogen production
optimization. Renew Energy 2012;39(1):339e46.
[9] Cappelletti BM, et al. Fermentative production of hydrogen
from cassava processing wastewater by Clostridium
acetobutylicum. Renew Energy 2011;36(12):3367e72.
[10] Su H, et al. Improving hydrogen production from cassava
starch by combination of dark and photo fermentation. Int J
Hydrogen Energy 2009;34(4):1780e6.
[11] Sen B, Suttar RR. Mesophilic fermentative hydrogen
production from sago starch-processing wastewater using
enriched mixed cultures. Int J Hydrogen Energy
2012;37(20):15588e97.
[12] O-Thong S, et al. Biohydrogen production from cassava
starch processing wastewater by thermophilic mixed
cultures. Int J Hydrogen Energy 2011;36(5):3409e16.
[13] Sreethawong T, et al. Hydrogen production from cassava
wastewater using an anaerobic sequencing batch reactor:
effects of operational parameters, COD: N ratio, and organic
acid composition. Int J Hydrogen Energy 2010;35:4092e102.
[14] Sangyoka S, Reungsang A, Moonamart S. Repeated-batch
fermentative for bio-hydrogen production from cassava
starch manufacturing wastewater. Pak J Biol Sci
2007;10(11):1782e9.
[15] Reungsang A, et al. Factors affecting hydrogen production
from cassava wastewater by a co-culture of anaerobic sludge
and Rhodospirillum rubrum. Pak J Biol Sci
2007;10(20):3571e7.
[16] Endo G, Noike T, Matsumoto J. Characteristics of cellulose
and glucose decomposition in acidogenic phase of anaerobic
digestion (in Japanese) Proc Jpn Soc Civ Eng 1982;325:61e8.
[17] Lin CY, Lay CH. A nutrient formulation for fermentative
hydrogen production using anaerobic sewage sludge
microflora. Int J Hydrogen Energy 2005;30:285e92.
[18] O-Thong S, Prasertsan P, Birkeland N-K. Evaluation of
methods for preparing hydrogen-producing seed inocula
under thermophilic condition by process performance and
microbial community analysis. Bioresour Technol
2009;100:909e18.
[19] Box GEP, Hunter WG, Hunter JS. Statistics for experimenters:
an introduction to design, data analysis and model building.
New York: Wiley; 1978.
[20] Lay C-H, et al. Fermentative biohydrogen production from
starch-containing textile wastewater. Int J Hydrogen Energy
2012;37(2):2050e7.
[21] Skonieczny MT, Yargeau V. Biohydrogen production from
wastewater by Clostridium Beijerinckii: effect of pH and
substrate concentration. Int J Hydrogen Energy
2009;34:3288e94.
[22] Van Ginkel S, Sung S, Lay JJ. Biohydrogen production as a
function of pH and substrate concentration. Environ Sci
Technol 2001;35(24):4726e30.
[23] APHA. Standard methods for the examination of water and
wastewater. 19th ed. Washington D. C.: American Public
Health Association; 1995.
[24] DuBois M, et al. Colorimetric method for determination of
sugars and related substances. Anal Chem 1956;28(3):350e6.
[25] Nelson N. A photometric adaptation of the Somogyi method
for the determination of glucose. J Biol Chem
1944;153:373e80.
[26] Van Ginkel S. Optimization of biohydrogen production from
food processing wastewater, in Department of Civil and
Environmental Engineering. Pennsylvania State University;
2005.
[27] Khanal SK, et al. Biological hydrogen production: effects of
pH and intermediate products. Int J Hydrogen Energy
2004;29(11):1123e31.
[28] Luo G, et al. Fermentative hydrogen production from cassava
stillage by mixed anaerobic microflora: effects of
temperature and pH. Appl Energy 2010;87(12):3710e7.
[29] Li J, et al. Anaerobic biohydrogen production from
monosaccharides by a mixed microbial community culture.
Bioresour Technol 2008;99(14):6528e37.
[30] Ren N, Wang B, Huang J-C. Ethanol-type fermentation from
carbohydrate in high rate acidogenic reactor. Biotechnol
Bioeng 1997;54(5):428e33.
[31] Dabrock B, Bahl H, Gottschalk G. Parameters affecting
solvent production by clostridium pasteurianum. Appl
Environ Microbiol 1992;58(4):1233e9.
[32] Roychowdhury S, Cox D, Levandowsky M. Production of
hydrogen by microbial fermentation. Int J Hydrogen Energy
1988;13:407e10.