Enhanced Biogas Production from Palm Oil Mill

Effluent Supplemented with Untreated Oil Palm Empty

Fruit Bunch Biomass with a Change in the Microbial
Community

= Japan journal of food engineering

ISSN 13457942

Ali, A.A.Md.
Zainudin, Md.H.Md.
Idris, A.
Baharuddin, A.S.
Sulaiman, A.

Matsui, T.
Osaka, N.
Oshibe, H.
Hassan, Md.A.
Shirai, Y.
/ 133
p. 37-41

20129

Tsukuba Office, Agriculture, Forestry and Fisheries Research Council Secretariat
 Japan Journal of Food Engineering, Vol. 13, No.3, pp. 37 - 41, Jun. 2012

000 Note 000

Enhanced Biogas Production from Palm Oil Mill Effluent Supplemented with
Untreated Oil Palm Empty Fruit Bunch Biomass with a Change
in the Microbial Community

Ahmad Amiruddin Mohd ALI\ Mohd Huzairi Mohd ZAINUDIN2 , Azni IDRIS3,
Azhari Samsu BAHARUDDIN3, Alawi SULAIMAN4, Toru MATSUI5, Noriko OSAKA5,
Hiroshi OSHIBE5, Mohd Ali HASSAN2, and Yoshihito SHIRAIl, t

1 Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology,
2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196,Japan
2 Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
3 Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
4 Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA,
40450 Shah Alam, Selangor, Malaysia
5 Fundamental Technology Department, Technical Research Institute, Tokyo Gas Co., Ltd.
1-7-7 Suehiro-cho Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

The biogas and biomethane production in a 50 litre closed stirred tank anaerobic bioreactor
treating palm oil mill effluent (PO ME) supplemented with oil palm biomass in the form of oil
palm empty fruit bunch (OPEFB) under mesophilic condition was evaluated. With OPEFB
supplementation, the biogas and biomethane generation increased by 63% and 52%, respectively.
During this process, we found changes in the OPEFB morphology and microbial community through
microbiota analysis using 16S rRNA gene clone library method, after OPEFB was added, suggesting
that the increased biogas and biomethane production would be due to enhanced lignocellulosic
biomass degradation.
Key words: Biogas, biomethane, microbial community, oil palm empty fruit bunch (OPEFB), palm
oil mill effluent (POME)

1. Introduction anaerobic fermentation of POME has been extensively

Biogas and biomethane fermentation of lignocellulosic
biomass have previously been attempted but with varied
success due to the rate limiting hydrolysis of the materi-
als which affects both fermentation rate and extent [1].
Without any recourse to pretreatments, the presence of
lignin is one of the major drawbacks in lignocellulosic
fermentation, as it renders the lignocellulose resistant to
biological and chemical degradation, thus hampering bio-
gas and biomethane productivity [2, 3]. The palm oil
industry represents the largest agro-economic sector in
Malaysia. The iu.dustry produces abundant biomass
wastes with 19.8 million tonnes of oil palm empty fruit
bunch (OPEFB) and more than 50 million tonnes of palm
oil mill effluent (POME) being generated from over 400
mills in Malaysia annually [4,5]. Biogas production from
(Received 17 May. 2012: accepted ll]uly. 2012)
t Fax: +81-93-695-6060. Email: shirai@life.kyutech.ac.jp.
studied [6-10]. Recently, co-digestion of POME with
OPEFB pretreated with alkali, acid and steam were
reported to increase biogas productivity in thermophilic
PO ME fermentation [11, 12]. However, presently no
study has been carried out to identify the changes in
additional OPEFB and microbial consortia related to
increased biogas and biomethane productivity in such
systems. This study deals with the biogas and biometh-
ane enhancement by OPEFB supplementation as well as
the changes in the OPEFB morphology and microbial
community during the fermentation.
2. Materials and methods
2.1 POME
Raw PO ME was collected from Seri Ulu Langat Palm
Oil Mill, Dengkil, Selangor, Malaysia, and preserved in <
4C prior to use.
38 Ahmad AmifUddin Mohd AU, Mohd Huz~ri Mohd ZAlNUDIN, Ami !DRlS, Azhari Samsu BAHARUDDlN, A1awi SUIAIMAN, TofU MATSUI, Noriko OSAKA, Hiroshi OSHIBE, MohdAli HASSAN, and Yoshihito SHIRAI

2.2 POME sludge clone library method. Firstly, DNA was extracted using
Matured and substrate-acclimatized POME sludge DNA extraction kit (UltraClean Soil DNA Isolation Kit,
inoculum was obtained from the settling tank of a 500 m3 MO-BIO). The DNA was extracted based on the manu-
closed anaerobic digester located at FELDA Serting Hilir facturer's instruction except for modification as
Mill Biogas Plant, Negeri Sembilan, Malaysia, where sev- described below. Firstly, 0.3 g of sample was added into 2
eral studies on POME treatment were previously done ml bead solution tube. After that, 500 ,LII of lysozyme
[8, 10]. (l00 mg/ml) was added into the same tube. The bead
solution tube containing sample and el1jYmes was incu-
2.30PEFB bated at 37C for 30 min. The subsequent steps were con-
Press-shredded OPEFB was obtained from Seri Ulu tinued according to the manufacturer's instruction.
Langat Palm Oil Mill, Dengkil, Selangor, Malaysia. The Polymerase Chain Reaction (PCR) analysis was then con-
OPEFB was then dried and ground using a hammer mill ducted whereby the 16S rRNA fragments was amplified
(Hsiangtai CW-1) and passed through a 2.0 mm sieve with primer 27F and 1492R. The PCR amplification and
prior to use. construction of 16S rRNA gene clone library were car-
ried out based on methodology by Zhu et al. [14]. The
2.4 Methane fermentation set up partial 16S rRNA sequence were sent to First Base
The system comprised of two 50 litre closed stirred Laboratories Sdn. Bhd., a sequencing company for
bioreactors. One was used as a control experiment while sequence determination. The 750R sequences primer
the other was used for the test experiment. A 40 litre was used for sequence analysis. Sequence similarity to
working volume was used with mesophilic condition (37 closest relative searches were conducted by matching
2C) maintained throughout the experimental period. the sequence database at National Center for
Wet gas meters (Shinagawa Seiki Co., OSK 14608) were Biotechnology Information (NCBI) using nucleotide-
used to record biogas flowrate. Fourty litres of PO ME nucleotide Basic Local Alignment Search (BLASTn) and
sludge was fed into the bioreactors for the start up stage Ribosomal Database Project II (RDPII) using sequence
to stabilise the fermentation system and deplete all bio- match program.
gas that may be contributed from residual organic matter
in the sludge inoculum. Four litres of raw PO ME was 3. Results and discussion
then fed into the control bioreactor. Simultaneously, 4
litres of raw POME and OPEFB was fed into the test bio- 3.1 Biogas and biomethane productivity
reactor. TS level within the control bioreactor after Figure 1 shows that with only PO ME fed into the sys-
POME feeding was 39 gil, i.e. 3.9%. The TS level was tem, 152litres of biogas was produced. After POME with
increased to 5.4% by adding OPEFB into the test bioreac- OPEFB was fed, the biogas generated was approximately
tor. The fermentation was continued until the biogas pro- 248 litres within the same duration, giving an increase in
duction from the control bioreactor had ceased. biogas generation by 63%. With OPEFB addition, 134
litres of biomethane was produced compared to 88 litres
2.5 Analytical methods
Composition of CH 4 and CO 2 gases were analysed
using a gas chromatograph (GC) (TCD Shimadzu 300
GC2014). Chemical oxygen demand (COD) analyses """ 250
=
'0
were conducted in accordance to the APHA Standard > 200
VI
ro
Methods [13]. All biogas and biomethane values are pre- Cl 150
0
sented as values at Standard Temperature and Pressure :.c 100
<Il
(STP, i.e., OC, 760 mmHg). OPEFB morphology was ana- .,f; 50
..!!!
::; 0
lysed using Scanning Electron Microscope E o 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
(SEM) (Hitachi S-3400N). (5
Time (d)

2.6 Microbiota analysis ---- POME Only ~POME+OPEFB

Microbiota profiles of day 14 samples from both con- Fig. 1 Cumulative biogas generation of PO ME only and PO ME
trol and test bioreactors were analysed using 16S rRNA with OPEFE.
 Enhanced biogas production
of biomethane generated with PO ME only, which gave a
52% increase in biomethane volume. In terms of biometh-
ane yield from soluble COD, 0.48g CH 4/g SCOD
removed was obtained after OPEFB addition. Judging
from stoichiometric analysis of methane fermentation,
obtained yield (0.48) should be too high, indicating that
during hydrolysis stage of the fermentation, more solu-
ble COD became available due to OPEFB degradation,
which were subsequently converted into biogas and bio-
methane by the microbes. In the first three days, biogas
production was almost the same for both experiments.
However, from the fourth day onwards, this trend began
to change whereby the biogas continued to increase in
the test experiment, while gradually decreasing in the
control experiment. This biogas profile change sug-
gested the effect of additional lignocellulosic biomass
degradation due to the activity of potential cellulolytic
microbes.
3.2 Substrate Degradation
SEM micrographs in Fig. 2 show the structural mor-
phology of the OPEFB before and after fermentation. It
can be observed that raw OPEFB had a solid and intact
structure. However, after fermentation, the OPEFB had
visibly decayed and exhibited a brittle, flaky and porous
appearance. This suggested that some parts of the
OPEFB had been degraded which might contribute
towards the enhanced biogas and biomethane productiv-
ity.
3.3 Microbiota analysis
The microbial population in both the control and test
samples are shown in Table 1. In the Table, [No. of
picked up clones/plates] means positive clones/plates,
Fig. 2 SEM analysis: (a) OPEFB before fermentation; (b) OPEFB after fermentation
(all images under 500x magnification).
39
and also means the target clones identified by the micro-
biota method. In the control sample of PO ME only,
Clostridium group was identified as a potential bacterium
responsible for lignocellulosic biomass degradation with
2 clones. In the test sample of POME and OPEFB, the
same group of potential cellulolytic bacteria was also
found with 5 clones identified. Clostridium species was
previously identified in anaerobic treatment of dairy-pro-
cessing wastewater and are known for carbohydrate, lac-
tate or ethanol fermentation as well as homoacetogenesis
functions [15]. However, in contrast with the control
sample, another type of bacteria potentially capable of lig-
nocellulose degradation was identified in the test sample
of POME and OPEFB. This bacterium was related to
Ruminofilibacter xylanolyticum with 4 clones identified.
This microbe is a rumen bacterium involved in the xylan
degradation previously detected in a biogas plant fed
with maize silage, green rye and liquid manure [16]. A
colonization study of activated zeolite by microorganism
during biogas production from grass silage has indicated
that this bacterium showed hydrolytic activity of xyla-
nase shortly after the reincubation in sterilised sludge on
model substrate [17]. Among other bacteria found in
both control and test experiments were Bacillus and
Pseudomonas groups that have been known to produce
lipase which could contribute to the biogas productivity
due to the degradation of residual oil content in the
OPEFB [18]. The biogas production increased by 63%
with OPEFB addition due to the change in the microbial
community particularly the potential cellulolytic and
hemicellulolytic bacteria responsible for lignocellulosic
biomass degradation. OPEFB addition could also have
triggered the release of organic matters such as carbohy-
drate and organic acids via degradation of lignocellulosic
40 Ahmad Amiruddin Mohd ALI, Mohd Huzairi Mohd ZAlNUDlN, Azni IDRlS, Azhan Samsu BAHARUDDlN, Alaw SUWMAN, Tom MATSUI, Noriko OSAKA, Hiroshi OSHIBE, Mohd Ali HASSfu'l, and Yoshihito SHIRAI

material for utilization by dominant anaerobic microbes Acknowledgements

detected such as Trichococcus sp., Sedimentibacter sp.,
and Acidovorax sp. which indirectly support the increase The authors would like to acknowledge Kyushu
in biogas generation. In the PO ME samples however, Institute of Technology, Japan, Universiti Putra Malaysia,
biogas generation was comparatively low due to unavail- Tokyo Gas Co., Ltd., Federal Land Development
ability of significant amounts of lignocellulosic matter, Authority (FELDA) Palm Industries Sdn. Bhd., FELDA
which limited the activity of these bacteria. Serting Hilir Palm Oil Mill and Seri Ulu Langat Palm Oil
Mill for their financial and technical support towards this
4. Conclusion research. Special thanks also to Prof. Dr. Kenji Sakai of
Kyushu University, Japan, as well as to Mr. Mohd
1. We confirmed the increase in biogas and biomethane Ridzuan Othman and Ms. Halimatun Saadiah Hafid for
production during PO ME anaerobic fermentation by sup- their invaluable input during this study.
plementation of untreated OPEFE.
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Table 1 Microbiota before and after GPEFB supplementation.

Clone count
[No. of picked up clones/plate]*
No. Species
POMEwith
POME only
OPEFB

Trichococcus sp. 23 12
2 Uncultured bacteria 16 54
3 Sedimentibacter sp. 3 9
4 Alcaligenes sp. 2 8
5 Acidovorax sp. 0 8
6 Parasporobacterium paucivorans 11
7 Carnobacteriaceae bacterium 7 3
8 Clostridium group 2 5
9 Bacillus group 1 4
10 Pseudomonas group 5 3
11 Ruminofilibacter xylanolyticum 0 4
12 Others 8 13

* Total number of positive clones for each microbe that were randomly selected between 80-120 clones
from 3 plates for control and test samples, each. Then from that amount of clones selected, we
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