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Industrial Crops and Products xxx (2016) xxxxxx

Contents lists available at ScienceDirect

Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Thermophilic solid-state anaerobic digestion of solid waste residues

from palm oil mill industry for biogas production
Wantanasak Suksong a , Aminee Jehlee a , Apinya Singkhala a , Prawit Kongjan b ,
Poonsuk Prasertsan c , Tsuyoshi Imai d , Sompong O-Thong a,e,
a
Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung 93210, Thailand
b
Chemistry Division, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand
c
Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkhla 90112, Thailand
d
Division of Environmental Science and Engineering, Graduated school of Science and Engineering, Yamaguchi University, Yamaguchi 755-8611, Japan
e
Research Center in Energy and Environment, Faculty of Science, Thaksin University, Phatthalung 93210, Thailand

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

Article history: Thermophilic solid-state anaerobic digestion (SS-AD) of biomass from palm oil mill industry, including
Received 20 July 2016 empty fruit bunches (EFB), oil palm fronds (OPF) and oil palm trunks (OPT) were investigated under
Received in revised form 25 October 2016 different feedstock-to-inoculum ratios (F:I), carbon-to-nitrogen ratios (C:N) and total solids contents
Accepted 2 November 2016
(TS). The optimal condition for biogas production was achieved at F:I ratio of 2:1, C:N ratio of 40:1
Available online xxx
and TS content of 16%. The highest methane yield from EFB, OPF and OPT were 223, 207 and 161 m3
CH4 tonne1 VS, respectively. Bacterial community structure in thermophilic SS-AD of oil palm biomass
Keywords:
was dominated by Thermoanaerobacterium sp. and Clostridium sp. Archaeal community structure was
Solid-state anaerobic digestion
Thermophilic condition
dominated by Methanosaeta sp. and Methanoculleus sp. The highest methane production of 45 m3 CH4
Methane production tonne1 fresh biomass was achieved from EFB. Solid waste residues from palm oil mill industry have
Microbial community great potential for methane production via thermophilic SS-AD.
Oil palm biomass 2016 Elsevier B.V. All rights reserved.

1. Introduction
Oil palm is one of the most important commercial crops for edi-
ble oil production. The process for extracting oil from palm fruit
generates large amounts of solid wastes such as 0.32 t of empty
fruit bunches (EFB), 0.12 t of mesocarp ber, 0.03 t of decanter cake
and 0.05 t of shell for every 1 t of fresh fruit bunches (Najafpour
et al., 2005). Plantation area also generates large amounts of solid
waste such as oil palm trunks (OPT) and oil palm fronds (OPF). In
2013, the palm oil industry in Thailand produced approximately
26.3 106 t per year of solid wastes from the milling process and
11.3 106 t per year from plantation area (Mukherjee and Sovacool,
2014). Empty fruit bunches were used for compost production
with aerobic digestion process, while ber and shells were directly
burned to produce steam for sterilizing fresh fruit bunches. Solid
waste residues from the palm oil industry are composed of cel-
lulose (3040%), hemicelluloses (2535%), and lignin (1525%)
(Mohammed et al., 2011; Kong et al., 2014). Solid waste from the
Corresponding author at: Biotechnology Program, Department of Biology, Fac-
ulty of Science, Thaksin University, Phatthalung 93210, Thailand.
E-mail addresses: sompong@tsu.ac.th, sompong.o@gmail.com (S. O-Thong).
palm oil industry such as EFB, OPF and OPT have high moisture
content (6575%). It is not technically economical to use as fuel for
steam production (Foo and Hameed, 2009). Empty fruit bunches, oil
palm fronds and oil palm trunks could be utilized as a substrate for
biogas production (Suksong et al., 2016; Chaikitkaew et al., 2015).
Empty fruit bunches, oil palm fronds and oil palm trunks have a
high content of organic matter with the high potential to be used
for biogas production (O-Thong et al., 2012). Solid-state anaerobic
digestion (SS-AD) of solid wastes from the palm oil industry in a
single treatment step would simplify the technical and economic
requirements for the transformation of solid wastes into biogas and
compost (Yang et al., 2015).
Solid-state anaerobic digestion is usually operated with high
solid contents (more than 15% total solids) feedstock (Motte et al.,
2014). Solid-state anaerobic digestion can operate at a higher
organic loading rate, smaller reactor volume, lower energy demand
for heating with higher volumetric methane productivity, and less
wastewater generation (Yang et al., 2015). Brown et al. (2012)
reported that the methane productivity of SS-AD was 27 times
greater in liquid anaerobic digestion (L-AD). Liew et al. (2012)
reported that the economic methane yield of SS-AD was achieved
at the F:I ratio of 2:1 from corn stover (81.2 L kg1 VS), wheat
straw (66.9 L kg1 VS), yard waste (40.8 L kg1 VS) and leaves

http://dx.doi.org/10.1016/j.indcrop.2016.11.002
0926-6690/ 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: Suksong, W., et al., Thermophilic solid-state anaerobic digestion of solid waste residues from palm
oil mill industry for biogas production. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.002
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(55.4 L kg1 VS). The methane yield from solid wastes via the SS-AD
process depended on the solid wastes composition. High cellulose
containing in feedstocks were related to high methane production
by SS-AD (Brown et al., 2012). Brown et al. (2012) also showed
that methane yields from corn stover, wheat straw, and switch-
grass were 25 times higher than those from yard waste, maple,
and pine biomass.
Several process parameters can be optimized to operate ther-
mophilic SS-AD such as feedstock-to-inoculum ratio (F:I), initial
total solids concentration (TS), carbon-to-nitrogen ratio (C:N) and
type of feedstock (Yan et al., 2015). The F:I ratio and C:N ratio are
two of the most important factors for the onset of a balanced micro-
bial population. Li et al. (2011) found that when operated at high
F:I ratio (4.58 and 7.41) signicant decreasing amounts of biogas
were observed and stop on the rst few days. High F:I ratio was
resulted in high acidity in the reactor probably due to the overpro-
duction of volatile fatty acids (VFAs) and methanogenic archaea
were subsequently inhibited. Furthermore, excessive TS loading
could result in rapid hydrolysis as well as overproduction of VFAs
and thus reduce the process stability of SS-AD reactors. Neverthe-
less, proper TS loading was also necessary for the SS-AD process, as
TS overloading could delay the start-up of the digestion and thus
affect the performance of the SS-AD process (Yan et al., 2015). The
effect of F:I ratio, C:N ratio and TS content on SS-AD performance
for biogas production from oil palm biomass under thermophilic
conditions has not been investigated.
The objective of the present study was to investigate the
methane production from solid waste residues from palm oil mill
industry using thermophilic SS-AD under different F:I ratios, C:N
ratios and TS content. Microbial community during thermophilic
SS-AD was investigated to better understand the thermophilic SS-
AD.
2. Materials and methods
2.1. Feedstock and inoculum
Solid waste residues of palm oil mill (EFB, OPF, OPT) were col-
lected from United Palm Oil Industry Public Company Limited,
Krabi province. Oil palm biomass was oven dried at 95 C in a con-
vection oven to less than 10% moisture, ground with a hammer
mill to pass through a 5 mm screen, and stored in airtight con-
tainers prior to use. The characteristics of the solid waste residues
were analyzed for total solids, volatile solids, total Kjeldahl nitrogen
(TKN), C:N ratio, cellulose, hemicellulose, lignin and lipid content.
The thermophilic methane production sludge was obtained from
Microbial Resource Management Research Unit, Faculty of Science,
Thaksin University. The thermophilic methane production sludge
was cultivated with palm oil mill efuent (POME) supplemented
with 0.01% (w/v) oil palm ash. Prior to using the sludge inoculum
was starved for 1 week to reduce the effect of organic materi-
als contained in microbial sludge. The sludge having the volatile
solids (VS) concentration of higher than 8.0% (w/v) was used in the
thermophilic SS-AD experiments.
2.2. Thermophilic SS-AD for methane production
Solid-state anaerobic digestion batch reactors were set up by
mixing feedstock with the inoculums to obtain F:I ratios of 2:1, 3:1,
4:1 and 5:1 (based on VS), a xed initial total solid content of 16%
(w/v) in batch assays under thermophilic conditions (55 C). Bio-
gas production was determined in batch assays under thermophilic
conditions as described previously by Angelidaki et al. (2011). The
effect of TS and C:N ratio on methane production via thermophilic
SS-AD was investigated with optimum F:I ratio. Total solids
Please cite this article in press as: Suksong, W., et al., Thermophilic solid-state anaerobic digestion of solid waste residues from palm
oil mill industry for biogas production. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.002
concentrations of 16%, 25% and 35% with C:N ratios of 20:1, 30:1 and
40:1 were chosen to evaluate their effect on the biogas production
via thermophilic SS-AD. Urea (CH4 N2 O) was added to adjust the C:N
ratio (Yan et al., 2015). The inoculums and oil palm biomass feed-
stocks were mixed with a hand-mixer and ushed with nitrogen
gas to generate anaerobic conditions. All tests were conducted in
0.5 L serum bottles with a working volume of 0.3 L. Duplicate serum
bottles were run at each condition. The serum bottles were closed
with butyl stoppers and placed in a 55 C incubator for 45 days.
Biogas volume and composition were daily monitored by displace-
ment method and gas chromatography. The weight of digestated
in each serum bottle was determined and digestated samples were
taken for composition analysis at the end of the experiments.
2.3. Analytical methods
Chemical and physical composition of EFB, OPF, OPT, inoculums,
and digestated were analyzed for pH, total Kjeldahl nitrogen (TKN)
and alkalinity according to APHA (1998). The TS content of each
sample was measured after drying 20 g of fresh sample at 95 C for
48 h, followed by 3 h of calcinations (550 C) for VS content determi-
nation (Motte et al., 2014). The lignin, cellulose, and hemicellulose
contents were determined according to the procedures proposed
by Van Soest et al. (1991). Volatile fatty acids (VFAs) were mea-
sured at the start and at the end of each experiment. At the end of
the experiment, 0.5 g of sludge samples were diluted in 9.5 mL of
deionized water and mixed for 10 min. Solutions were centrifuged
at 12,000 rpm for 10 min. The supernatant was collected and l-
tered through 0.2 m nylon membrane. Filtrated samples were
used to measure the VFAs. Volatile fatty acids concentrations were
determined with a gas chromatograph Shimadzu GC-17A with a
stabilwax -DA fused silica 30 m column connected to a ame ion-
ization detector at 240 C and helium at 30 mL min1 as the carrier
gas. The daily biogas production for each experiment was recorded
using the water displacement method (Yan et al., 2015). The bio-
gas composition was measured by gas chromatography equipped
with thermal conductivity detectors (TCD). Methane, carbon diox-
ide, hydrogen, and nitrogen were analyzed by GC-TCD tted with
1 m stainless steel column packed with Shin Carbon (60/80 mesh).
Argon was used as the carrier gas at a ow rate of 14 mL min1 .
The temperatures of the injection port, oven and detector were
at 120, 40 and 100 C, respectively. The gas sample of 0.5 mL was
injected in duplicate. The theoretical methane potential was cal-
culated according to Bushwells formula that is derived from the
stoichiometric conversion of the organic compound to CH4 , CO2 ,
and NH3 . It was assumed that the averaged chemical compositions
of VS in EFB, OPF and OPT were VScarbohydrate (C6 H10 O5 ). The energy
yield from biogas was calculated by using an energy factor of 36 MJ
per m3 CH4 described by O-Thong et al. (2012).
2.4. Microbial community analysis
Polymerase chain reaction-denaturing gradient gel elec-
trophoresis (PCR-DGGE) was used to study microbial community
structure in the thermophilic SS-AD as previously described by
Prasertsan et al. (2009). Most of the bands were excised from the gel
and re-amplied. After re-amplication, PCR products were puri-
ed and sequenced by Macrogen Inc. (Seoul, Korea). The closest
matches for partial 16S rRNA gene sequences were identied by
database searches in Gene Bank using BLAST (Maidak et al., 1996).
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Table 1 Table 3
Characteristics of oil palm biomass and inoculum. Comparison of methane yield from thermophilic and mesophilic SS-AD of oil palm
biomass.
Characteristics EFBa OPTa OPFa Inoculum
Oil palm F:I ratio Thermophilic methane Mesophilica methane yield
Total solid (%) 99.7 0.31 100.0 0.48 100.0 0.45 12.4 0.29
biomass yield (m3 tonne1 VS) (m3 tonne1 VS)
Volatile solid (%) 77.3 1.75 78.3 1.58 83.1 2.01 8.5 1.8
Nitrogen content (%) 1.1 0.02 0.5 0.00 0.3 0.21 3.3 0.11 2:1 166.6 68.0
Lipids (%) 4.7 1.64 1.3 0.98 1.2 0.28 0.1 0.03 3:1 147.7 62.5
EFB
Cellulose (%) 40.3 1.44 41.2 2.08 46.0 1.82 N.D. 4:1 141.5 67.5
Hemicelluloses (%) 24.2 1.07 29.8 1.77 19.1 1.49 N.D. 5:1 147.3 57.5
Lignin (%) 20.5 1.29 16.0 1.26 19.9 0.44 N.D. 2:1 89.8 46.7
C:N ratio 72:1 170:1 282:1 2.6:1 3:1 102.2 44.5
OPF
a
4:1 83.6 43.0
Base on dry weight.
5:1 91.4 12.7
2:1 63.5 57.8
3:1 63.8 57.1
3. Results and discussion OPT
4:1 60.7 51.4
5:1 59.1 44.6
3.1. Composition of oil palm biomass and thermophilic inoculums a
Suksong et al. (2016).

Empty fruit bunches (EFB), oil palm fronds (OPF) and oil palm
trunks (OPT) had high TS content of 99.7-100% and VS content of
77.383.1%. EFB, OPF, and OPT had C:N ratios of 72:1, 170:1 and
282:1, respectively (Table 1). Oil palm biomass has a considerably
high C:N ratio and unsuitable C:N ratio for anaerobic digestion. Lig-
nocellulosic biomass generally had a C:N ratio higher than 50:1
(Yang et al., 2015). Suksong et al. (2016) recommended that optimal
C:N ratio in anaerobic digestion systems should be 30:1. Optimum
C:N ratio could enhance the growth of bacterial, hydrolysis activity
and methane production from oil palm biomass. High TS content
and high C:N ratio may cause hydrolytic inhibition, rapid accumu-
lation of VFAs and nitrogen limitation during SS-AD (Sheets et al.,
2015). In this study, oil palm biomass also had substantial lignin
content (16.5-20%), which has been shown to be recalcitrant to
cellulolytic degradation (Mohammed et al., 2011). The cellulose
and hemicelluloses content of oil palm biomass were 40.3-46.0%
and 19.1-29.8%, respectively. Cellulose, hemicellulose and lignin
content in oil palm biomass in Thailand was similar to Malaysia
with the range of 38.349.8, 31.835.3 and 22.125.7, respectively
(Mohammed et al., 2011). C:N ratio, TS, VS and lipid content in ther-
mophilic inoculum was 2.6:1, 12.4%, 8.5% and 0.1%, respectively.
Sludge inoculum considers as low C:N ratio, when oil palm biomass
was mixed with sludge inoculum resulting in decreased C:N ratio
in thermophilic SS-AD systems.
3.2. Effect of F:I ratio on methane production
The daily methane production rates of oil palm biomass at the
different F:I ratios (2:1, 3:1, 4:1, 5:1) were shown in Fig. 1. Highest
methane production rates were observed from EFB at the F:I ratio
2:1 on the rst week and trend to decreased during continuous
incubation. The methane production rate began to increase dra-
matically and reached maximum rates of 20.6 m3 t1 VS d1 for EFB,
18.1 m3 t1 VS d1 for OPF and 11.4 m3 t1 VS d1 for OPT (Fig. 1).
Shi et al. (2013) reported that thermophilic SS-AD shown faster
and greater reductions of cellulose and hemicellulose during the
rst 12 days compared to mesophilic SS-AD. The daily methane
production rate of oil palm biomass has patterns similar to corn
stover that reported by Li et al. (2011). The daily methane pro-
duction rate of corn stover increase dramatically during the rst
9 and reached a maximum rate of 27.9 m3 t1 VS d1 at F:I ratios
of 2.43. The nal pH and alkalinity from thermophilic SS-AD of
OPF and OPT at F:I ratios 4:15:1 were ranged of 6.36.4 and
5.26.4 g CaCO3 kg1 , respectively, while nal alkalinity from ther-
mophilic SS-AD of EFB was ranged of 8.49.0 g CaCO3 kg1 at F:I
ratios of 4:15:1 (Table 2). The rapid hydrolysis at high F:I ratio
resulted in an accumulation of VFAs (21212459 mg L1 ), indicat-
ing a relatively high acidity in the digesters, probably due to the
overproduction of VFAs (Yang et al., 2015). The methane yields of
EFB were 38.3% higher than of OPF and 50.1% higher than OPT,
respectively. Daily methane contents in the biogas produced by
thermophilic SS-AD from EFB, OPF and OPT were ranged 5560%
during the steady state. The highest methane yields achieved from
EFB, OPF and OPT were 229.8, 138.3 and 110.2 m3 t1 VS, respec-
tively, corresponding to 53.3, 32.6 and 27.4 m3 t1 fresh biomass,
respectively at F:I ratios of 2:1. These results indicate that ther-
mophilic SS-AD could operate at F:I ratio between 2:1 to 4:1. The
F:I ratio of 3:1-4:1 is preferable since reducing the amount of inocu-
lums for large scale application (Cui et al., 2011). The methane yield
for the 25-day incubation from thermophilic SS-AD of oil palm
biomass (63166 m3 t1 VS) was higher than the methane yield
from mesophilic SS-AD (5768 m3 t1 VS) (Table 3). Thermophilic
solid-state anaerobic digestion usually shows a better startup per-
formance than mesophilic SS-AD due to an accelerated hydrolysis
process, the major rate-limiting step during AD (Yang et al., 2015). It
was found that feedstock degradation was faster in a thermophilic
digester than in a mesophilic digester, and 95% of the methane

Table 2
Changes of pH and total alkalinity from thermophilic SS-AD of oil palm biomass.

Oil palm biomass F:I ratio Initial pH Final pH Initial alkalinity (g CaCO3 kg1 ) Final alkalinity (g CaCO3 kg1 )

2:1 7.57 7.89 13.22 8.42

3:1 7.51 7.35 14.86 8.81
EFB
4:1 7.30 7.21 14.44 9.00
5:1 7.36 7.28 13.24 8.45
2:1 7.45 7.01 16.46 6.22
3:1 7.37 7.02 12.83 5.66
OPF
4:1 7.26 6.42 14.72 5.31
5:1 7.75 6.47 14.82 5.23
2:1 7.73 7.03 16.61 7.63
3:1 7.65 7.05 16.48 7.21
OPT
4:1 7.57 6.45 12.85 5.41
5:1 7.57 6.38 14.73 5.31

Please cite this article in press as: Suksong, W., et al., Thermophilic solid-state anaerobic digestion of solid waste residues from palm
oil mill industry for biogas production. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.002
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25
F:I=2:1
EFB
F:I=3:1

Methane production rate

20

(m3 tonne-1 VS d-1)

F:I=4:1
15 F:I=5:1

10

25
F:I=2:1
OPF
Methane production rate

20 F:I=3:1
(m3 tonne-1 VS d-1 )

F:I=4:1
15 F:I=5:1

10

25
F:I=2:1
OPT
Methane production rate

20 F:I=3:1
(m3 tonne-1 VS d-1)

F:I=4:1
15 F:I=5:1

10

0
0 5 10 15 20 25 30 35 40 45
Days

Fig. 1. Methane production rate from thermophilic SS-AD of oil palm biomass at different F:I ratio.

yield was achieved in 20 days under thermophilic while 45 days
for mesophilic conditions (Ge et al., 2016; Yang et al., 2015)
3.3. Effect of TS and C:N ratio on methane production
Organic loading in terms of TS and C:N ratio was investigated
under optimal F:I ratio (2:1) for each biomass. Both TS and C:N
ratio affected on methane yields of thermophilic SS-AD from oil
palm biomass was investigated. The optimal TS and C:N ratio for
biogas production on thermophilic SS-AD from EFB, OPF, and OPT
were 16% and 40:1, respectively. Under optimal conditions, EFB,
OPF, and OPT had methane yields of 223.3, 207.0 and 160.5 m3 t1
VS, respectively (Fig. 2). The high TS (35%) and low C:N ratio (20:1)
of EFB, OPF and OPT have 6996% methane yields lower than the
optimal condition. The TS content of 35% and 20:1 of C:N ratio of
EFB, OPF and OPT had cumulative methane yields of 31.5, 62.4 and
5.5 m3 t1 VS, respectively. Cumulative methane yield tended to
decrease when TS content was increased. The maximum methane
yield of 223 m3 t1 VS was achieved from EFB at 16% of TS and
40:1 of C:N ratio. The highest methane yield from OPF and OPT was
207 and 161 m3 t1 VS, respectively (Fig. 3). In this study, a strong
inverse linear relationship was observed between the methane
yield and the TS content of feedstocks with an r2 of 0.996 for EFB,
r2 of 0.974 for OPF and r2 of 0.975 for OPT (Fig. 4a). Mass trans-
fer or diffusion coefcients have been found to decrease drastically
with increases of TS content (Abbassi-Guendouz et al., 2012). The
low water content at a higher TS content might have inhibited
microbial activity. The total methane yield decreased signicantly
by 2538% when the TS content increased from 22% to 30% in SS-
AD of yard trimmings and corn stover feedstock (Lin et al., 2014;
Yang et al., 2015). The high solids content (>16%) in SS-AD could
also produce dead zones in the reactor leading to reductions of
methane yield and causing reactor overloading (Sawatdeenarunat
et al., 2015). The methane production rate of EFB, OPF and OPT

Please cite this article in press as: Suksong, W., et al., Thermophilic solid-state anaerobic digestion of solid waste residues from palm
oil mill industry for biogas production. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.002
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250

EFB
200

Cumulative methane yield

(m 3 tonne -1 VS)
150

100

50

250

OPF
200
Cumulative methane yield
(m3 tonne-1 VS)

150

100

50

250

OPT
200
Cumulative methane yield
(m3 tonne-1 VS)

150

100

50

0
0 10 20 30 40 50
days

(16%TS,40:1C:N) (16%TS,30:1C:N) (16%TS,20:1C:N)

(25%TS,40:1C:N) (25%TS,30:1C:N) (25%TS,20:1C:N)
(35%TS,40:1C:N) (35%TS,30:1C:N) (35%TS,20:1C:N)

Fig. 2. Cumulative methane yield from thermophilic SS-AD of oil palm biomass at different TS contents and C:N ratios.

Please cite this article in press as: Suksong, W., et al., Thermophilic solid-state anaerobic digestion of solid waste residues from palm
oil mill industry for biogas production. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.002
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300

250

(m3 CH4 tonne-1 VS)

Methane yield
200

150

100

50

0 (16%TS,40:1C:N)
(16%TS,30:1C:N)
(16%TS,20:1C:N)

(25%TS,30:1C:N)
(25%TS,20:1C:N)
(35%TS,40:1C:N)

(16%TS,30:1C:N)

(25%TS,40:1C:N)

(16%TS,30:1C:N)

(25%TS,40:1C:N)

(25%TS,20:1C:N)
(35%TS,40:1C:N)
(25%TS,40:1C:N)

(16%TS,40:1C:N)

(16%TS,20:1C:N)

(25%TS,30:1C:N)
(25%TS,20:1C:N)
(35%TS,40:1C:N)

(16%TS,40:1C:N)

(16%TS,20:1C:N)

(25%TS,30:1C:N)
(35%TS,30:1C:N)
(35%TS,20:1C:N)

(35%TS,30:1C:N)
(35%TS,20:1C:N)

(35%TS,30:1C:N)
(35%TS,20:1C:N)
EFB OPF OPT

Fig. 3. Methane yield from oil palm biomass at different TS contents and C:N ratios by thermophilic SS-AD.

was low at start period. The methane production rate of EFB, OPF Meanwhile, the cumulative VFAs at TS content of 35% and 20:1
and OPT was 3.03, 4.94, 3.71 m3 t1 VS d1 , respectively. The low of C:N ratio of EFB, OPF, and OPT were 20.6-45.1% higher than at
methane production rate could be caused by slow adaptation of optimal condition. The highest total ammonia nitrogen (TAN) con-
inoculum to the substrate type and the operating conditions (Yan centration of 2300, 2630 and 2480 mg L1 was obtained from EFB,
et al., 2015). Microorganisms were gradually adapting to the SS-AD OPF, and OPT, respectively at C:N ratio of 20:1. A strong linear rela-
environment and methane production rate of EFB, OPF and OPT was tionship was observed between the methane yield and the C:N ratio
greatly increased linearly to18.09, 21.09 and 13.33 m3 tonnee1 VS of feedstock with an r2 -0.999 of EFB, r2 -0.982 of OPF and r2 -0.983
d1 , respectively at day 58. of OPT (Fig. 4b). At lower C:N ratio causes ammonia accumula-
The highest total solid removal of 49.6%, 45.9%, and 35.6% was tion,which is toxic to methanogenic archaea. In contrast, a high
obtained from EFB, OPF, and OPT, respectively at 16% TS content C:N ratio is an indication of rapid consumption of nitrogen by bac-
condition. This study shows that total solid removal in the ther- teria and methanogens with results in lower gas production (Yan
mophilic SS-AD process was low when increasing TS content from et al., 2015). Excessive VFAs and TAN were inhibitors that impair
16% to 35%. The TS removal of EFB, OPF, and OPT at TS content digester stability and methane yield. Volatile fatty acids and TAN
of 35% were 715%, 1115%, and 312%, respectively (Table 4). can be produced from carbohydrate-rich and protein-rich waste
feedstock (Yang et al., 2015). The previous report shows that a C:N
ratios of 40:1-30:1 being the most common in SS-AD (Yen and
250 Brune., 2007; Yan et al., 2015). Degradation of cellulose in ther-
(A) EFB (R = 0.9963) mophilic SS-AD at the F:I ratio of 2:1 was determined at day 45
Methane yield (m3 tonne-1VS)

200 OPF (R = 0.9738)

OPT (R = 0.9748) Table 4
150 Total solid removal from thermophilic SS-AD of oil palm biomass.

Oil palm biomass TS content (%) C:N ratio TS removal (%)

100
EFB 16 40:1 49.63
OPF 16 30:1 47.84
50 16 20:1 19.61
OPT
25 40:1 31.51
0 EFB 25 30:1 20.94
0 5 10 15 20 25 30 35 40 25 20:1 3.46
35 40:1 15.18
Initial total solid (%)
35 30:1 8.83
35 20:1 7.01
250 16 40:1 45.99
(B)
Methane yield (m3 tonne-1 VS)

16 30:1 24.61
200 16 20:1 33.87
25 40:1 27.19
OPF 25 30:1 16.91
150 25 20:1 16.83
35 40:1 15.43
100 35 30:1 11.90
EFB (R = 0.9991)
EFB 35 20:1 13.86
OPF (R = 0.9826) 16 40:1 35.67
50 OPF
16 30:1 31.15
OPT OPT (R = 0.9834)
16 20:1 4.71
0 25 40:1 21.73
0 10 20 30 40 50 OPT 25 30:1 11.87
25 20:1 5.08
C:N ratio
35 40:1 12.90
35 30:1 3.33
Fig. 4. Relations between methane yield and initial total solid (A), methane yield
35 20:1 1.22
and C:N ratio (B).

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oil mill industry for biogas production. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.002
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16 methane production rate using the PCR-DGGE. Bacterial communi-

Initial cellulose (%) Final cellulose (%) EFB ties in the thermophilic SS-AD were clearly dominated by members
14 of the genus Thermoanaerobacterium sp., Ruminococcus sp. and
12 Clostridium sp. (Fig. 6a). Moreover, the minor bacterial members
in thermophilic SS-AD of EFB were comprised of Desulfovibrio sp.,
Cellulose (%)

10 Sporobacterium sp. and Acidithiobacillus sp. Bacterial community in

thermophilic SS-AD of OPF and OPT were comprised of Desulfobater
8
sp. and Desulfonatronovibrio sp. Thermophilic bacteria in original
6 thermophilic inoculum were dominated by Clostridium sp. and
Thermoanaerobacterium sp., which is usually involved in hydrolysis
4 and acidogenesis process. Clostridium sp. is able to ferment various
carbohydrates to acetate, butyrate and hydrogen (Lee et al., 2010).
2
Clostridium is an important cellulolytic bacterial group in degra-
0 dation of lignocellulosic biomass under anaerobic conditions with
versatile fermentation capability. Thermoanaerobacterium sp. is an
16
anaerobic hemicellulolytic bacterium, in class Clostridia, capable of
Initial cellulose (%) Final cellulose (%) OPF pentose fermentation to ethanol and hydrogen at high tempera-
14
ture (Wongwilaiwalin et al., 2010). Cellulolytic bacteria consisted
12 of Clostridium sp. and Ruminococcus sp. were found predominantly
Cellulose (%)

10 in thermophilic SS-AD of wheat straw (Heeg et al., 2014) and corn

stover (Shi et al., 2013; Li et al., 2015). These species can pro-
8 duce thermoactive hydrolytic extracellular enzymes (e.g., protease,
lipase and cellulase). A recent study on SS-AD of lignocellulosic
6
biomass showed that thermophilic SS-AD led to greater reductions
4 of cellulose (614%) during the rst 1220 days when compared to
mesophilic SS-AD, due to the 1050 times larger populations of cel-
2 lulolytic microbes at thermophilic conditions (Heeg et al., 2014; Li
et al., 2015; Yang et al., 2015). Lomans et al. (2001) reported that the
0
genus Sporobacterium sp. able to ferment various hydroxylated aro-
16 matic residue (gallate, oil) were converted to acetate and butyrate.
14
Initial cellulose (%) Final cellulose (%) OPT Sporobacterium sp. was dominant in the thermophilic SS-AD of EFB
due to the lipid content (4.7 g L1 ) in EFB was higher than the OPF
12 and OPT (1.21.3 g L1 ).
Cellulose (%)

The archaeal community in thermophilic SS-AD of oil palm

10
8 of 40:1 and 16% TS) were comprised of Methanospirillum sp.,
6 sp. (Fig. 6b). The archaeal community in thermophilic SS-AD of
4
2 are mainly responsible for methane production in thermophilic SS-
0
Fig. 5. Cellulose degradation of oil palm biomass at different TS contents and C:N
ratios in thermophilic SS-AD.
of incubation. This study shows that cellulose removal in the ther-
mophilic SS-AD process was low, when increased TS content from
16% to 35% and decreased C:N ratio from 40:1 to 20:1. The highest
cellulose degradation of 50% was observed in thermophilic SS-AD
of EFB followed by OPF (46%) and OPT (36%) at TS content of 16%
and 40:1 of C:N ratio (Fig. 5). The degradation of cellulose was neg-
atively related to the lignin content of the lignocellulosic biomass
(Liew et al., 2012).
3.4. Microbial community in thermophilic SS-AD
The bacterial and archaeal community structure at genus level
in thermophilic SS-AD of oil palm biomass (EFB, OPF, and OPT)
under optimum condition (F:I ratio of 2:1, C:N ratio of 40:1
and TS of 16%) were investigated at the exponential phase of
biomass under optimum conditions (F:I ratio of 2:1, C:N ratio
Methanosaeta sp., Methanoculleus sp. and Methanomassiliicoccus
EFB, OPF and OPT were not differences during 20 days incuba-
tion. This indicates that Methanosaeta sp. and Methanoculleus sp.
AD. Methanosaeta sp. was distinguished from members of the other
genera by their exclusive use of acetate as a substrate for producing
methane. Methanosaeta sp. always prefers a low acetate concen-
tration for its growth (Narayanan et al., 2009). In the thermophilic
SS-AD reactors, the other detected non-acetoclastic methanogens
included the genera Methanospirillum sp. and Methanoculleus sp.,
which all belong to the class Methanomicrobia. Methanospiril-
lum sp. is a genus of strictly hydrogenotrophic methanogens. Li
et al. (2015) reported that the genus Methanothermobacter sp.
Methanoculleus sp. was dominated in the thermophilic SS-AD reac-
tors for biogas production from corn stover.
3.5. Feasibility of using oil palm biomass to produce methane by
thermophilic SS-AD
A thermophilic SS-AD process for methane production from
oil palm biomass was demonstrated that technically feasible. The
optimal condition for methane production from oil palm biomass
(EFB, OPF, and OPT) by thermophilic SS-AD was F:I ratio of 2:1,
C:N ratio of 40:1 and TS content of 16%. The methane content
in the biogas was 5060%. Oil palm biomass in plantation area
or palm oil mill factory have a moisture content of 7075%.
Technical methane yield for EFB, OPF, and OPT was 223, 207,
and 161 m3 t1 VS, respectively. The calculated methane produc-
tion from fresh EFB, OPF and OPT were 45, 42 and 35 m3 CH4

Please cite this article in press as: Suksong, W., et al., Thermophilic solid-state anaerobic digestion of solid waste residues from palm
oil mill industry for biogas production. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.002
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Fig. 6. DGGE prole of bacterial community (A) and archaea community (B) in thermophilic SS-AD of oil palm biomass.

tonne1 biomass, accounting for the energy yield of 1620, 1513
and 1260 kJ t1 biomass, respectively. Overall, methane produc-
tion in the oil palm industry achieved from EFB, OPF and OPT were
207, 25 and 84 m3 CH4 ha1 y1 , respectively (Fig. 7). The elec-
tricity generation from biogas of EFB, OPF and OPT would be 828,
100 and 336 KW h ha1 y1 , respectively. It was calculated from the
conversion efciency of approximately 40% in a gas motor. The
study shows that maximum methane and energy recovery achieved
from EFB via thermophilic SS-AD. In addition, the economic feasi-
bility of thermophilic SS-AD of oil palm biomass could be improved
by upgrading digestated from thermophilic SS-AD to fertilizers
(Chaikitkaew et al., 2015). At the end of thermophilic SS-AD, the

Please cite this article in press as: Suksong, W., et al., Thermophilic solid-state anaerobic digestion of solid waste residues from palm
oil mill industry for biogas production. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.002
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Electricity production
EFB ( 828 kwh ha-1 y-1)

Provincial Electricity OPF (100 kwh ha -1 y-1)

Authority OPT (336 kwh ha -1 y-1)

Oil palm plantationa,b
FFB (16.4 tonne ha -1 y-1)
OPF (0.6 tonne ha-1 y-1)
OPT (2.4 tonne ha -1 y-1)
Palm oil mill industrial a,b Methane production
EFB (207 m3 ha-1 y -1)
EFB (4.6 tonne ha-1 y-1)
OPF (25 m3 ha-1 y-1)
Crude oil (3.2 tonne ha-1 y-1)
OPT (84 m3 ha-1 y-1)
Compost production
from digested
EFB (2.3 tonne ha-1 y-1)
OPF (0.3 tonne ha-1 y-1)
OPT (1.5 tonne ha-1 y-1)

Shell (1.3 tonne ha-1 y-1)

Thermophilic SS-AD
(F:I ratio of 2:1, C:N ratio of 40:1, 16% of TS)
Fiber (1.9 tonne ha-1 y -1)

Heat generation with an incineration

Nitrogen 0.05 %(w/w)
Phosphorus 0.02 %(w/w)
Potassium 0.04%(w/w)

Fig. 7. Feasibility diagram of the mass and energy balance for methane production from oil palm biomass by the thermophilic SS-AD process.
source: a Mukherjee and Sovacool (2014) and b Kong et al. (2014).

overall organic removal (VS) efciency of EFB, OPF, and OPT were
50, 46 and 36%, respectively. The remaining organic matters (VS) of
EFB, OPF, and OPT were an approximately 2.3, 0.3 and 1.5 t ha1 y1 ,
respectively. The thermophilic SS-AD digestated has rich in nutri-
ent comprising of nitrogen 0.0150.115 t ha1 y1 , phosphorus
0.0060.046 t ha1 y1 and potassium 0.0120.092 t ha1 y1 . The
study shows that oil palm biomass has a high potential to be con-
verted into renewable energy and fertilizers. The EFB, OPF, and OPT
could be efciently transformed into biogas, then the energy short-
age in rural areas of Thailand, as well as CO2 emission problems in
the oil palm mill plant will be alleviated to a certain extent.
Acknowledgements
This work was supported by the Core-to-Core Program, which
was nancially supported by Japan Society for the Promotion of Sci-
ence (JSPS), National Research Council of Thailand (NRCT), Vietnam
Ministry of Science and Technology (MOST), the National Univer-
sity of Laos, Beuth University of Applied Sciences and Brawijaya
University, Research and Development Institute Thaksin Univer-
sity (RDITSU), Agricultural Research Development Agency (ARDA)
through grant number POP5705020660, Research Group for Devel-
opment of Microbial Hydrogen Production Process from Biomass,
Khon Kaen University and Thailand Research Fund through grant
number RRI MSD57I0103, PHD57K0042 and RTA5780002.

4. Conclusion
Thermophilic solid-state anaerobic digestion of oil palm
biomass was investigated. The optimal condition for biogas pro-
duction was achieved at F:I ratio of 2:1, C:N ratio of 40:1 and
TS of 16%. Optimal condition was effectively improved methane
production from oil palm biomass by thermophilic SS-AD. The
maximum methane yield of 223 m3 t1 VS was achieved from EFB
under optimal condition. The highest methane yield from OPF and
OPT was 207 and 161 m3 t1 VS. The highest methane production
from EFB, OPF and OPT were 45.0, 42.3 and 34.7 m3 CH4 tonne1
fresh biomass, respectively. Empty fruit bunches were easily bio-
logically converted to methane. Solid waste residues from palm
oil mill industry have great potential for methane production via
thermophilic SS-AD. Thermoanaerobacterium sp. and Clostridium sp.
were responsible for a hydrolytic process in thermophilic SS-AD,
while Methanoculleus sp. and Methanosaeta sp. were responsible
for the methanogenic process.
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