biomass and bioenergy 34 (2010) 42–47

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Thermophilic biohydrogen production from palm oil mill

effluent (POME) using suspended mixed culture

Isnazunita Ismail a,b,*, Mohd. Ali Hassan b, Nor Aini Abdul Rahman b, Chen Sau Soon a
a
Environment & Bioprocess Technology Centre, SIRIM Berhad, 40911 Shah Alam, Selangor D.E., Malaysia
b
Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor D.E., Malaysia

article info abstract

Article history: A batch study was conducted to determine the fate of carbohydrate and oil that are present
Received 7 January 2009 in palm oil mill effluent (POME) during the biohydrogen fermentation process. Sucrose and
Received in revised form crude palm oil (CPO) were chosen as substrates and the kinetic profile indicated that
24 September 2009 mainly sucrose was metabolised by the mixed sludge. The hydrogen yield based on the
Accepted 25 September 2009 COD of sucrose added was 146 cm3 g1 which is equivalent to a hydrogen to hexose mole
Available online 28 October 2009 ratio of 2.5. The free fatty acids from hydrolysed CPO were not metabolised further which
render insignificant generation of hydrogen and volatile fatty acids from oil-based
Keywords: substrate. The average continuous biohydrogen production rate (HPR) from a unit volume
Biohydrogen of POME under thermophilic condition at 55  C was 2.64 m3 m3 d1 at a hydraulic retention
Anaerobic fermentation time (HRT) of 4 days. Hydrogen constitutes up to 52% of the total biogas and methane was
Palm oil mill effluent not detected over the 60 day continuous operation. The hydrogen yield (i.e. based on mole
Thermophilic ratio of hydrogen to hexose) was 1.72 with an average carbohydrate conversion efficiency
of 58%. These limit the potential of recovering more hydrogen energy from POME under
current operating conditions.
ª 2009 Elsevier Ltd. All rights reserved.

1. Introduction still unknown on the influences of the chemical properties of

Research in dark fermentation for hydrogen (H2) production is
on the increase in recent years but many utilise typical simple
sugars or starch [1,2] which are not economically feasible due
to their high cost. The new strategy of market-driven research
is to focus on using cheap, organic waste-based feedstocks,
employing indigenous mixed cultures and improving the
hydrogen production yield. Palm oil mill effluent (POME) is
constantly associated with environmental burden due to the
voluminous discharge of the wastewater during milling
process. POME is also considered as high strength complex
wastewater with total chemical oxygen demand that can reach
up to 94 kg m3 [3]. Many researchers have recognised the
potential of harnessing hydrogen from POME [4,5] but much is
POME in converting to hydrogen using mixed microflora.
Many studies have focused on manipulating the operating
conditions by pre-treating either the mixed microflora or the
non-sterile substrate with chemicals [6,7], sonication [8] or
working at low temperatures [9] to prevent the growth of
hydrogen-consuming methanogens. At the same time, treat-
ing the wastewater at high temperatures will enhance
hydrogen evolution rate due to suppression of propionate
formation [10] and low hydrogen partial pressure in the liquid
phase. The solubility of hydrogen in water decreases with
increasing temperature with minimal solubility at tempera-
tures from 50 to 60  C [11]. Propionate fermentation is known to
produce liquid by-products without significant gas production
and hydrogen is not involved in the fermentative pathway

* Corresponding author at: Environmental & Bioprocess Technology Centre, SIRIM Berhad, 40911 Shah Alam, Selangor Darul Ehsan,
Malaysia. Tel.: þ60 3 5544 6559; fax: þ60 3 5544 6590.
E-mail address: isnazunita_ismail@sirim.my (I. Ismail).
0961-9534/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2009.09.009
 biomass and bioenergy 34 (2010) 42–47 43

[12,13]. Continuous production of biohydrogen from POME
requires understanding on the effective conversion of multiple
components in the wastewater. Substrates studied for
fermentative hydrogen production have mostly been rich in
carbohydrates and the impact on the conversion due to the
presence of oil is still unclear. Lipid hydrolysis hardly occurs in
the absence of methanogens due to the syntrophic association
of long chain fatty acid oxidisers with hydrogenotrophic
methanogens to maintain low level of hydrogen concentration.
In the present work, the effect of the chemical properties of
POME is investigated to determine the viability of using POME
as substrate for the continuous generation of biohydrogen by
anaerobic digested sludge under thermophilic condition. Batch
study using chemically defined substrates namely sucrose and
CPO to model the carbohydrate and fat in POME, provided some
preliminary results that supported the experimental set up for
the continuous study.
2. Materials and methods
2.1. POME substrate
Fresh POME was collected from the receiving tank of an oil
palm mill (2 540 20.2700 N, 101 20 35.3500 E) in Selangor Darul
Ehsan, Malaysia. The sampling took place in 6 batches over
a year from August 2007 to July 2008. The temperature of the
discharged POME range from 70 to 75  C and raw POME was in
the form of acidic colloidal mixture with pH 4.52  0.21. The
POME was fully characterised, as presented in Table 1, and
kept in cold room at 4  C prior to use.
2.2. Seed sludge
A digested sludge originating from a mesophilic digester-
treating POME was used as inoculum. The initial pH, volatile
suspended solid (VSS) and total suspended solid (TSS)
concentration of the sludge was 6.80, 8.0 and 11.4 kg m3,
respectively. The sludge was sieved through mesh size of
600 mm to remove sand, fibres and coarse particulates and
stored in phosphate buffer pH 5.5 at 4  C. The specific
hydrogen production rate per unit VSS of the sludge was pre-
determined at 20 cm3 g1 h1 in 5 g dm3 glucose under batch
study condition of pH 5.5 and temperature 55  C.
2.3. Batch experiment
2.3.1. Biohydrogen fermentation from model or synthetic
substrates
The hydrogen production experiments were conducted in
a series of 122 cm3 serum vials with final seed concentration of
4.4–5.0 g dm3. A medium containing (g dm3); 1.78 NaH-
PO4.2H2O, 0.024 Na2HPO4.2H2O, 1.25 NH4Cl, 0.375 CaCl2.2H2O,
0.556 MgCl2.H2O, and trace elements of (in mg dm3);
5 FeCl2.4H2O, 0.425 CoCl2.6H2O, 0.42 ZnSO4.7H2O, 0.15 H3BO3,
1.25 MnCl2.4H2O, 0.1 NiCl2.6H2O, 0.1 CuSO4.5H2O, 1.25
(NH4)6Mo7O24.4H2O, 12.5 EDTA disodium salt and 0.63 resa-
zurin was prepared. POME is a complex wastewater; rich in
carbohydrate and fats. Therefore, in order to model the
generation of hydrogen from POME, sucrose (20 g dm3) and
premium grade Crude Palm Oil (CPO) (4 g dm3) were used as
sources of carbohydrate and fats, respectively. 1 g dm3
NaHCO3 was added to buffer the reaction. The serum vials
were purged with nitrogen gas, sealed with butyl rubber
stoppers and the pH in each serum vials was adjusted to
5.5  0.1 using 1 N NaOH or 1 N HCl. The vials were incubated
in reciprocating shaker water bath at 150 rpm and 55  1  C.
During the test, biogas samples were collected routinely and
analysed for hydrogen, carbon dioxide and methane contents.
Before each sampling event, the pressure inside the vials was
equilibrated to the ambient pressure and the volume recorded
was added to the headspace volume. Mixed liquor samples
were analysed for chemical oxygen demand (COD), VSS,
volatile fatty acids (VFAs) and long chain fatty acids (LCFAs).
2.4. Continuous experiment
2.4.1. Reactor set up and operation
The sludge with VSS and TSS of 0.6 and 0.8 g dm3, respec-
tively, was seeded into a completely stirred tank reactor

Table 1 – Composition of raw POME.

Parameter Unit [3] This studya

pH 4 (0.10) 4.50 (0.21)

COD kg m3 total 93.6 (3.6) 94.4 (2.2)
sol 40.9 (2.2) 39.6
Carbohydrate kg m3 (COD basis) total 22.5 (3.4) 21.2 (4.7)
sol 13.7 (0.4) 15.5 (0.4)
Protein kg m3 (COD basis) total 26.9 (2.0) 24.2 (4.8)
sol 10.9 (1.1) 11.6 (1.0)
O&G kg m3 (COD basis) total 23.4 (0.9) 10.1 (1.3)
TKN kg m3 total 2.1 (0.1) 0.8 (0.3)
VFAs kg m3 (COD basis) C2 0.64C3 0.04; C4 0.01 C2 0.71; C3 0.08; C4 0.06
Free LCFAs kg m3 (COD basis) N.A C16 0.37; C18 0.04; C18:1 0.19; C18:2 0.06
SS kg m3 36.4 (8.0) 27.8 (10.0)
VSS kg m3 31.4 (6.4) 23.5 (6.3)
C/N ratio 18.5 (1.3) 24.8 (4.1)
Mineral content kg m3 sol N.A Al:0.16; Ca:0.17; Fe:0.05; K:2.26; Mg:0.49; Mn:0.005; P:0.25

a Figure in parenthesis means standard deviation, n ¼ 6.

44 biomass and bioenergy 34 (2010) 42–47
Table 2 – Kinetic parameters for hydrogen production from different substrates.
Substrate H R
(cm3) (cm3 h1)
Sucrose 129.6 2.3  0.3
Sucrose and CPO 146.2 2.2
(CSTR) of 4 dm3 working volume with the following operating
conditions; pH, temperature and agitation were controlled at
5.5, 55  C  2 and 200 rpm, respectively. The substrate was
refrigerated at the controlled temperature of 4  C and the
POME continuously homogenised with an agitator. At start up,
2.5 dm3 of seed sludge was fed into the reactor, sparged with
nitrogen gas and acclimatised to the operating conditions.
Substrate of 0.5 dm3 volume was fed everyday until the
experimental volume reached 4 dm3. Subsequently, draw and
fill method was employed over a week to ensure the active
biomass is retained sufficiently prior to continuous feeding.
Under the continuous mode, POME was fed and mixed liquor
withdrawn intermittently by time-controlled peristaltic
pumps. The reactor start up began with HRT of 8 day and
decreased stepwise to 2 day upon reaching steady state at
each run. The gas volumes were corrected to standard
temperature and pressure (STP)(273.15 K and 101.325 kPa).
2.4.2. Monitoring
The daily monitoring parameters during the continuous
hydrogen production experiments were pH, biogas production
and hydrogen content. Sampling for influent and effluent
were conducted three times per week and the samples were
kept at 4  C prior to analysis.
2.5. Analyses
Every batch of the collected POME was analysed for pH, TSS,
VSS, COD, Kjedhal nitrogen (TKN), oil and grease (O&G) and
minerals according to the APHA Standard methods [14].
Carbohydrate was measured according to the phenol-sul-
phuric method with glucose as standard for calibration.
Protein was analysed according to the Folin Lowry method
using albumin as standard. For the quantification of soluble
carbohydrate, protein and other soluble metabolites, samples
Fig. 1 – Kinetic profiles of hydrogen production from
different substrates. ( ) CPO; ( ) mixture of sucrose
and CPO; and ( ) sucrose.
l Hydrogen yield Specific hydrogen
(h) (cm3 g COD1) production rate
(cm3 g VSS1 d1)
12.3  1.9 145.9 240.5  58.3
10.1 110.4 193.4
were centrifuged at 10,000 rpm for 10 min and filtered with
0.45 mm cellulose-acetate membrane filter prior to analyses.
Organic acids were quantified using HPLC (Shimadzu, LC10AS)
by post labelled method. The sample in the mobile phase;
3 mM perchloric acid was eluted in the column (Shim-
pack,SCR-1021) and post labelled with mixed solution of
bromo thymol blue prior to detection by UV–vis at 439 nm
wavelength. The free LCFAs were determined by re-dissolving
the weighed O&G residue in 99.9% GC grade n-hexane. The
analysis was carried out using gas chromatograph (Hewlett
Packard 5890 series II) with a flame ionization detector and
capillary column (HP-FFAP 30 m, inner diameter 0.53 mm, film
1 mm). The oven was programmed from 160  C (hold for 2 min)
and to 230  C (hold for 11 min) at a rate of 4  C min1. Helium
was used as carrier gas at constant flow and both the injector
and detector temperature was set at 260  C. For alcohol
analysis, 1 cm3 of sample acidified with 0.03 cm3 20% H2SO4,
was analysed using GC-FID and capillary column of the same
model and type, respectively. Minerals were quantified by
Inductively Coupled Plasma Mass Optical Emission Spectro-
photometer (ICP-OES, Perkin Elmer Optima, 2000DV) after
digestion with conc. HNO3. The C and N were determined
using CHNSO Elemental Analyser (Fison 1108). The biogas
composition was quantified with a gas chromatograph (Shi-
madzu, GC-2014) equipped with a thermal conductivity
detector and a stainless steel packed column (Unibeads C,
2 m  ø3 mm, 60/80- mesh) with argon as carrier gas.
2.6. Data analyses of batch experiment
In this study, cumulative hydrogen production curves with
respect to time were obtained firstly from the hydrogen
production experiments; then the modified Gompertz equa-
tion was applied to determine the hydrogen production
potential (H ), hydrogen production rate (R) and lag phase (l).
  
R:e
HðtÞ ¼ Hexp  exp ðl  1Þ þ 1 (1)
H
where H(t) is cumulative hydrogen production (cm3) at time t;
l is time of lag phase (h); H is hydrogen production potential
(cm3); R is hydrogen production rate (cm3 h1); and e is exp (1)
i.e. 2.71828.
3. Results and discussion
3.1. Kinetic profile of model substrates
The kinetic parameters estimated based on Eq. (1) are listed in
Table 2. Hydrogen yield and specific hydrogen production rate
(SHPR) were calculated from H and added substrate and from
R and biomass concentration, respectively. The hydrogen
 biomass and bioenergy 34 (2010) 42–47
Fig. 2 – Profiles of (a) LCFAs, ( ) capric; ( ) lauric; ( )
palmitic; ( ) stearic and ( ) oleic acids and (b) VFAs, ( )
acetic and ( ) butyric acids; production in the fermentation
broth.
yield based on of the COD of sucrose added was 146 cm3 g1
which is equivalent to a hydrogen to hexose mole ratio of 2.5
when fed with sucrose of 20 g dm3. The Clostridium pasteur-
ianum was known to generate high hydrogen yield at
a hydrogen to hexose mole ratio of 2.07 from sucrose with
COD of 40 g dm3 [15]. The results also indicated that i) CPO
results in low generation of hydrogen as shown in Fig. 1 and is
therefore not a significant substrate for hydrogen production
even when digester sludge for treating POME was used as
inoculum, ii) the substrates containing a mixture of sucrose
and CPO and sucrose only, showed the same kinetic profile
with the hydrogen production rate at 2.3 cm3 h1. The result
infers that the hydrogen generated during the first 100 h was
from the fermentation of sucrose.
The free LCFAs in the fermentation broth (Fig. 2a) that was
analysed towards the end of the experimental study showed
a significant reduction of saturated fatty acids, namely pal-
mitic acid and concomitant increase in concentration of
shorter chain fatty acids such as lauric and capric acids. This
was also supported by the increase in butyric acid (0.5 kg m3)
and acetic acid (0.2 kg m3) in vials fed with sucrose and CPO
(Fig. 2b). No significant increase of VFAs was detected in the
fermentation broth containing CPO even after 300 h incuba-
tion. In the thermophilic fermentation; major fraction of oil
was hydrolysed to release free fatty acids (an average of
3.1  0.1 kg m3) and the glycerol was further converted to
hydrogen and VFAs. Obligate hydrogen producing aceto-
genesis is an acetate producing reaction that can only oxidise
45
free fatty acids while reducing hydrogen ions to hydrogen.
Fatty acid is oxidised sequentially via b-oxidation to produce
fatty acids with one pair less of carbon atoms for each cycle.
As the reaction was controlled at pH lower than the hydro-
genotrophic methanogens growth range of pH 6.5–7.0 [16],
further metabolism of fatty acids was not able to proceed as
illustrated in the kinetic profile. Therefore in the subsequent
POME fermentation studies, the hydrogen yield was moni-
tored solely on unit volume of hydrogen produced per gram of
carbohydrate consumed.
3.2. Continuous hydrogen production in stirred tank
reactor
The raw POME used in this study contained high concentra-
tions of organic matters with carbohydrate and protein
contributing to 23% and 26% of the total COD, respectively.
The values are comparable to those reported by other
researchers [3]. The effluent also contained essential nutri-
ents, fatty acids and minerals. Soluble minerals such as K, Mg,
P, Ca, Al and Fe were present above trace quantities and in
agreement with other reported findings [17]. These minerals
are readily available to microorganisms and Mg was deter-
mined as the most important element that affect hydrogen
production [18]. Mg, Na, Zn and Fe are trace elements that
relate to the bacterial enzyme co-factor, transport processes
and dehydrogenases.
Fig. 3 describes the performance of the CSTR in generating
hydrogen using untreated sludge obtained from a methane
digester. The average HPR of the CSTR as per unit volume of
POME was determined at 2.64 m3 m3 d1 with hydrogen
composition ranging from 48% to 51%. The POME used in this
study had a C/N ratio of 25 with soluble P and Fe at 0.25 and
0.05 kg m3, respectively. The low ratio of C/N/P in POME has
been considered as one of the factors attributed to the low
hydrogen yield. A medium optimisation study of C/N and C/P
at the ratios of 74 and 599, respectively from a nutrient-sup-
plemented POME reported a maximum yield of H2 per unit
volume of POME of 6.33 m3 m3 [19]. However, in that partic-
ular study it was also noted that by supplementing Fe2þ at
0.26 kg m3 to the raw POME resulted in an increase of
corrosive hydrogen sulphide (H2S) gas from 60 to 80 ppmv. H2S
content in the biogas is detrimental as this will decrease the
lifespan of hydrogen fuel cell. Substantial amount of peptone
Fig. 3 – Hydrogen evolution rate and biogas composition
from the fermentation of POME in CSTR under thermophilic
condition. ( ) CH4; ( ) H2; ( ) CO2 and ( ) hydrogen
production rate.
46 biomass and bioenergy 34 (2010) 42–47
Table 3 – Soluble and gaseous metabolites obtained at steady state condition.
HRT (d) Soluble metabolites (kg m3)
VFAs Alcohols
8 7.5  1.6 0.7  0.3
4 8.6  1.1 0.8  0.3
2 9.0 N.A
is also required to maintain the C/N ratio to the optimum
value. It is worthwhile to mention that protein fermentation
was not considered in this study as the net hydrogen
production is zero due to the combination of the generated
hydrogen with nitrogen to directly produce ammonia.
Methane was not detected throughout the 60 days opera-
tion indicating repression of methanogenic activities under
controlled pH of 5.5. However, low HPR was also observed due
to inefficiency in total carbohydrate consumption averaging
58  18%. POME contains very high particulates or VSS
(23 kg m3) which require long hydrolysis time. Hydrolysis is
known to be the rate-limiting step for carbohydrate conver-
sion [20]. Only 12 kg m3 of the total COD (primarily from
carbohydrate fraction) of the POME was converted to the
gaseous metabolites and other liquid by-products. Similar
experimental data analysis from other researchers [19]
showed 13.5 kg m3 of carbohydrates (i.e 14.4 kg m3 COD
equivalent) were consumed by the thermophilic mixed
cultures to generate biohydrogen with total COD degradation
of 36%. In another study, a carbohydrate-rich waste namely,
food waste with carbohydrate COD of 85 kg m3 reported
higher hydrogen production potential with 58% COD removal
and over 0.1 m3 of cumulative hydrogen obtained at HRT of
5.3 h [21].
The main soluble metabolites of the fermentation were
acetate and butyrate (3.7 kg m3 and 7.6 kg m3on COD basis,
respectively) with low amount of formic and propionic acid.
Ethanol and lactic acid were detected but both products were
less than 10% of the total soluble fermentation products
(Table 3). From the HBu/HAc molar ratio of 0.8, the stoichi-
ometry of hydrogen production from POME in this study is
proposed as Eq. (2):
6C6H12O6 þ 2H2O / 5C2H4O2 þ 4C4H8O2 þ 12H2 þ 10CO2 (2)
Equation (2) estimates a theoretical molar ratio of hydrogen
to hexose of 2.0 and the average experimental hydrogen yield
of the current work is 1.72 mol H2 mol1 hexose. It is proposed
that part of the carbohydrate was converted to biomass as
hydrogen was produced during the exponential growth phase.
4. Conclusion
The potential of generating biohydrogen from POME is investi-
gated in this study and the variation was analysed based on the
chemical composition of the wastewater. The kinetic profile
derived from using synthetic substrates and mixed microflora
under thermophilic condition confirms the soluble carbohy-
drate assimilation process and the insignificant production of
hydrogen from fats. Irrespective of the carbon source used
Gaseous metabolites (%)
Lactic H2 CO2
0.008 37.3  8.0 55.5  5.4
0.003 47.9  3.2 51.5  3.4
N.A 51.4  2.0 48.3  1.5
(sucrose or POME); butyrate and acetate were the predominant
soluble metabolites. Continuous fermentation of POME by
a mixed microflora generated hydrogen mainly from the
conversion of soluble carbohydrate with hydrogen to hexose
mole ratio of 1.72 at HRT 4d. Only 13% of the total organic
contents of POME was utilised in the fermentation which
renders this substrate unfavourable when compared to other
carbohydrate-rich waste.
Acknowledgements
The CPO and POME samples supplied by West Oil Mill, Sime
Darby Plantation are greatly appreciated. The authors gratefully
acknowledge the financial support from Ministry of Science,
Technology and Innovation of Malaysia through grant No:
07-01-04-EB009. Gratitude is also extended to Mr. Zairulnaim
Yeop and Mr.Firdaus Abdul Rashid of SIRIM Berhad for the
technical support in ICP-OES and CHNSO elemental analysis,
respectively.
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