Research Article

Received: 28 May 2013

Revised: 31 July 2013

Accepted article published: 21 August 2013

Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.4197

Influence of temperature on the bioconversion

of palm oil mill effluent into volatile fatty acids
as precursor to the production of
polyhydroxyalkanoates
Wee Shen Lee, Adeline Seak May Chua, Hak Koon Yeoh
and Gek Cheng Ngoh
Abstract
BACKGROUND: The focus of wastewater management has evolved from treatment technology into resource recovery, which
enables simultaneous waste minimization and value-added product generation. This study utilized palm oil mill effluent (POME)
for volatile fatty acids (VFA) production by acidogenic fermentation at mesophilic (30 C and 40 C) and thermophilic (55 C)
temperatures. The viability of using the VFA produced to generate biodegradable plastics polyhydroxyalkanoates (PHA) was
also determined.
RESULTS: The VFA production at mesophilic temperature outperformed thermophilic temperature considerably, with a degree
of acidification of 48% at both 30 C and 40 C but only 7% at 55 C. These results were in agreement with the substrate
consumption profiles whereby the percentage of substrate consumption at 55 C was six times lower than those at 30 C and
40 C. The VFA produced from POME could be used for PHA production, achieving 17 wt% PHA of sludge dry weight.
CONCLUSION: Mesophilic acidogenic fermentation of POME is preferable due to its better VFA production. Since palm oil
mills are located in tropical countries, the fermentation can be conducted under ambient conditions (2532 C) and without
temperature control. The potential of VFA-rich fermented POME for PHA production is recognized, but optimization of the PHA
production conditions is required for higher PHA content.
c 2013 Society of Chemical Industry


Keywords: volatile fatty acids; palm oil mill effluent; acidogenic fermentation; polyhydroxyalkanoates; mesophilic; thermophilic

NOTATION
sCODinitial
sCODfinal
VFAinitial
VFAfinal
vvm

Concentration of sCOD measured at the beginning

of fed-batch operation
Concentration of sCOD measured at the end of
fed-batch operation
Concentration of VFA measured at the beginning
of fed-batch operation
Concentration of VFA measured at the end of
fed-batch operation
Gas volume flow per reactor working volume per
minute

INTRODUCTION
Palm oil is one of the main vegetable oils traded in the global market
due to its versatile applications in food, oleochemicals and energy
industries. Although the palm oil industry is of great economic
importance, it has been recognized to be highly polluting because
of the massive generation of palm oil mill effluent (POME) in the
milling process. It is estimated that more than 2.5 tonnes of POME
could be generated from one tonne of palm oil production.1
J Chem Technol Biotechnol (2013)

POME is an acidic brownish colloidal suspension containing

large amounts of organic substances with chemical oxygen
demand (COD) in a range of 35 00057 000 mg L-1 . Such
high COD implies the need for proper POME management to
avoid severe environmental pollution. In general, most palm
oil mills have employed ponding systems for treating POME.2
A ponding system, as depicted in Fig. 1, consists of a series
of open ponds whereby POME generated from the mill is first
gathered in the collection pond for waste palm oil recovery.
After that, POME undergoes primary treatment in the anaerobic
pond. It is further treated in either the facultative or aerobic
pond (with surface aerators) before being discharged into the
environment. However, this treatment-oriented management
approach neglects the potential of POME as a feedstock for
the production of various chemicals such as antibiotics, solvents

Correspondence to: Adeline Seak May Chua, Department of Chemical

Engineering, Faculty of Engineering, University of Malaya, Lembah Pantai,
50603 Kuala Lumpur, Malaysia. Email: adeline@um.edu.my
Department of Chemical Engineering, Faculty of Engineering, University of
Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia

www.soci.org

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www.soci.org

WS Lee et al.

Treatment-oriented approach Ponding system

Anaerobic
pond(s)

POME collection
pond(s)

Facultative or
aerobic pond(s)

Treated
effluent

POME
Palm oil mill
Resource recovery approach
Bioplastics - polyhydroxyalkanoates
Acidogenic
fermentor

VFA

Bioenergy - hydrogen, electricity, methane

Biological nutrient removal

Figure 1. Treatment-oriented versus resource recovery management approaches to handling POME.

and organic acids.3 It is apparent that the transformation

of POME into value-added products is a better management
approach. In this study, we are interested in converting POME
into volatile fatty acids (VFA) because VFA have a broad range
of applications such as in the production of biodegradable
plastics polyhydroxyalkanoates4 (PHA) and bioenergy,5 and in
the biological removal of nutrient from wastewater,6 as shown in
Fig. 1.
In the literature, VFA has predominantly been produced via the
acidogenic fermentation process from a variety of wastes such as
paper mill wastewater7 and sewage sludge.8 In this bioprocess,
complex organic substrates in the wastes are hydrolyzed and
fermented into VFA under anaerobic conditions. The production
of VFA by acidogenic fermentation can be affected by a number
of key factors such as pH, temperature, hydraulic retention time,
sludge retention time and organic loading rate.9 11 While each
of these has been studied extensively, the effect of temperature
is of particular interest due to the nature of POME, which is
typically discharged at rather high temperatures of 6070 C.
As such, it could potentially be used for VFA production at
both mesophilic and thermophilic temperatures without requiring
high heating energy to maintain the fermentation temperature.
Since studies related to the production of VFA from POME12,13
were often conducted at 2830 C and there is little reported
information about the influence of higher temperature, this
study aims to compare the performance of VFA production
from POME at mesophilic (30 C and 40 C) and thermophilic
(55 C) temperatures. In addition, this study also tests the
applicability of VFA produced from POME to microbial production
of PHA.

MATERIALS AND METHODS

Characterization of POME
Raw POME was collected every 23 weeks from the outlet of a
POME collection pond in a local palm oil mill. The POME was
allowed to settle at 4 C for 24 h to reduce its solid content.
After settling, the supernatant was separated from the settled
solid and stored at 4 C for preservation before it was used in
acidogenic fermentation. Both the raw POME and the supernatant
of the settled POME were characterized for pH, total chemical
oxygen demand (TCOD), soluble chemical oxygen demand (sCOD),
total suspended solids (TSS), volatile suspended solids (VSS) and
VFA.

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Operation of the anaerobic reactors

Three anaerobic reactors (each with a working volume of 1.5 L)
were used for the production of VFA from POME at room temperature (30 1 C), 40 1 C and 55 1 C. These reactors were seeded
with sludge collected from different POME treatment ponds with
temperatures closest to the operating temperatures of the reactors. The seed sludge for reactor operating at 55 C was sampled
from a POME collection pond with a temperature of 5363 C. On
the other hand, the reactor operating at 40 C was inoculated with
a mixture of sludge taken from the first and the second anaerobic
ponds with temperatures of 45 C and 33 C, respectively.
Meanwhile, the sludge seeded into the reactor operating at 30 C
was collected from the aforementioned second anaerobic pond.
Except operating temperature, all three reactors were operated
under similar fed-batch mode with a feeding interval of 5 days.
At the end of each fed-batch operation, 1.2 L of mixed liquor
was withdrawn from each reactor to maintain the hydraulic and
sludge retention times at approximately 6 days. The pH in all
reactors was not controlled. To create and maintain the anaerobic
conditions, nitrogen gas was introduced into the reactor at the
beginning of each fed-batch operation, and during sampling and
mixed liquor withdrawal. All three reactors were operated for 60
days, corresponding to a total of 12 fed-batch operations.

PHA production by using fermented POME

PHA production was carried out in an aerobic reactor (with a
working volume of 0.5 L) at room temperature (30 1 C) for 24 h.
The aerobic conditions were achieved by supplying air at around 1
vvm to the reactor. Activated sludge taken from a lab-scale reactor
operating on the enhanced biological phosphorus removal
process was employed as the inoculum. Since the experiment
was preliminary, the inoculum was not specially cultivated and
was whatever available during the time of the experiment. In
this study, fermented POME obtained from the anaerobic reactor
operating at 30 C was utilized as the sole carbon substrate. Prior
to the experiment, the ammonium and phosphate concentrations
in fermented POME were measured. After the measurement,
the fermented POME was filtered through 1.2 m filter paper to
remove coarse solid particles and its pH was adjusted to 7 to avoid
the detrimental effect of acidic pH on PHA production.14 Then it
was added into the reactor at hours 0 and 8 to a concentration of
750 mg VFA-C L-1 and 300 mg VFA-C L-1 , respectively, to ensure
sufficient VFA was available for PHA production.

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Analytical methods
TSS and VSS were determined according to the standard
methods.15 The COD was measured in accordance with HACH
method 8000 using a colorimeter (DR/890, HACH, USA). With
ion chromatography (861 Advanced Compact IC, Metrohm,
Switzerland), the concentrations of VFA (formate, acetate,
propionate, butyrate and valerate) were measured using the
Metrosep Organic Acids 250/7.8 column, the ammonium
concentration with the Metrosep C 4 150/4 column, and the
phosphate concentration with the Metrosep A Supp 5 150/4
column. Standard solutions used in VFA analysis were prepared
from concentrated formic (98%, Merck), acetic (99.8%, Riedelde-Haen), propionic (>99%, Merck), butyric (99%, Merck) and
valeric acids (98%, Merck). Ammonium and phosphate standard
solutions were prepared by dissolving ammonium chloride
(99.8%, Merck) and potassium dihydrogen phosphate (99.5%,
Merck) in ultrapure water (Arium 611UF, Sartorius), respectively.
The PHA was extracted from the sludge according to the
method described by Satoh et al.16 The concentration of the
extracted PHA was determined using gas chromatography
(GC2010, Shimadzu, Japan) equipped with a capillary column
(J&W, DB-Wax) and flame ionization detector. Two types of PHA
were quantified in this study namely 3-hydroxybutyrate (3HB)
and 3-hydroxyvalerate (3HV) by employing poly(3-hydroxybutyric
acid-co-3-hydroxyvaleric acid) (Natural origin, PHV content 12
wt%, Sigma-Aldrich) as the standard.
Performance evaluation
Degree of acidification (DA), as originally defined by Equation (1),
is a parameter commonly used to assess the performance of
VFA production from wastewater.10 DA signifies the percentage
conversion of soluble organic substrates available in wastewater
into VFA.
DA =

VFAfinal
VFA produced
100% =
100%
Initial substrate
sCODinitial

(1)

In this study, to account for the existence of VFA in POME prior

to acidogenic fermentation, DA was computed differently as in
Equation (2).
DA =

VFAfinal VFAinitial
VFA produced
100% =
100%
Initial substrate
(sCOD VFA)initial
(2)

Here it was recognized that VFA would contribute to the sCOD

measured, so its concentration was deducted from sCODinitial
to avoid overestimating the availability of fermentable soluble
organic substrates.
For the same reason, the percentage of substrate consumption
in acidogenic fermentation of POME was calculated according to
Equation (3).
Substrate consumed
100%
Initial substrate
(sCOD VFA)initial (sCOD VFA)final
=
100%
(sCOD VFA)initial

%substrate consumption =

(3)

The PHA production performance was evaluated by using PHA content which is the weight percentage of PHA of sludge dry weight.
The sludge dry weight is represented by the mixed liquor VSS.
J Chem Technol Biotechnol (2013)

Table 1. Characteristics of raw POME and supernatant of settled

POME recovered after 24 h gravitational settling (standard deviation
due to different batches of POME collected from the mill)
Parameters

Raw POME

pH
TSS (mg L-1 )
VSS (mg L-1 )
TCOD (mg L-1 )
sCOD (mg L-1 )
VFA (mg L-1 )
sCOD/TCOD
VFA/sCOD

4.7 0.3
20000 3000
18000 2000
47000 7000
20000 3500
2600 900
0.42 0.03
0.18 0.12

Supernatant of settled POME

4.6 0.3
2400 900
2100 800
22000 4000
20000 3500
2700 1100
0.89 0.05
0.19 0.14

RESULTS AND DISCUSSION

Characteristics of POME
The characteristics of raw POME and the supernatant of settled
POME are compared in Table 1. It can be seen that raw POME is
an acidic, organic-rich wastewater containing a lot of suspended
solids. To reduce its solid content, raw POME was allowed to settle
for 24 h. It was found that about 88% of the TSS in raw POME could
be removed by this simple solid separation technique, resulting in
a final TSS concentration of 2400 mg L-1 .
The removal of such a great amount of TSS resulted in a
lower TCOD concentration. However, the supernatant of settled
POME was still considered a suitable feedstock for VFA production
because it remained very rich in organic matter with TCOD at
22 000 mg L-1 . More importantly, most of the organic matter is
soluble as indicated by the high ratio of sCOD/TCOD at 0.89. This
ratio was double that of the raw POME at 0.42. The higher fraction
of soluble organic matter in the supernatant of settled POME is
advantageous to microorganisms as they could more easily utilize
the organic matter for VFA production.
According to Table 1, VFAs were detected in the supernatant
of settled POME with an average concentration of 2700 mg L-1 .
Nonetheless, they were not the main fraction of the soluble organic
matter in POME, as indicated by the low ratio of VFA to sCOD of
0.19. Consequently, there is room to maximize the utilization of
POME by further converting the remaining organic matter into
VFA via acidogenic fermentation.
Another piece of useful information derived from the
characterization study was that pH control might not be required
during the acidogenic fermentation of POME. This is because
the acidic nature of POME (pH 4.6) could suppress the activity
of methanogens which are active within a narrow pH range of
6.47.8.17 Methanogens, if present in the reactor, could consume
the VFA for methane formation. Without pH control, VFA can
be produced at lower cost in line with the economic advantage
of using waste for the production of a value-added product.
Collectively, the results obtained from the characterization study
suggest that POME is suitable for VFA production.
Influence of temperature on degree of acidification
A total of 12 fed-batches of acidogenic fermentation of POME
were executed at 30 C, 40 C and 55 C and their respective DA
profiles are depicted in Fig. 2(a). There was not much difference
in the performance of VFA production conducted at 30 C and
40 C, as indicated by the fairly similar DA obtained at these
two temperatures. On average, the DA attained at 30 C and
40 C was 48 4% and 48 5%, respectively. However, these

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(a)

WS Lee et al.

Table 2. Average concentrations of VFA obtained at the end of

fed-batch acidogenic fermentation of POME at 30 C, 40 C and 55 C
Temperature ( C)
30
40
55

Final VFA concentration (mg L-1 )

8300 1000
8500 1000
3700 1300

(b)

Figure 2. (a) Degree of acidification and (b) percentages of substrate

consumption in the acidogenic fermentation of POME at 30 C, 40 C and
55 C.

values were about six times higher than that achieved at 55 C

of 7 6%. The poorer VFA production at 55 C was caused by
the lower microbial activity in the reactor, as the percentage
substrate consumption at 55 C was also approximately six times
lower than those at 30 C and 40 C (Fig. 2(b)). This reduction
in microbial activity might result from either poorer substrate
uptake ability of the thermophilic VFA-forming microorganisms
or relatively fewer such microbes in the reactor. A similar finding
had been reported by Zhuo et al.11 whereby the consumption of
substrate decreased with increase in temperature from 37 C to
55 C during the acidogenic fermentation of ultrasonic-pretreated
waste activated sludge, leading to lower VFA production at 55 C.
Based on this study, it is obvious that mesophilic temperatures
of 30 C and 40 C favor the production of VFA from POME
compared with thermophilic temperature of 55 C. Furthermore,
the comparable VFA production at 30 C and 40 C suggests that
temperature control will not be required in actual industrial
application because most of the palm oil producers such as
Indonesia, Malaysia and Thailand are tropical countries with
ambient temperatures at around 2532 C. Nevertheless, POME,
which is commonly discharged at 6070 C, has to be cooled to
40 C before use in acidogenic fermentation. Such cooling of POME
is presently accomplished in palm oil mills by using one or two
open ponds in series, hence no additional cooling facilities are
necessary to retrofit the mills for VFA production.
It is notable that the DA achieved in this study (48%) was
comparable with that attained in the acidogenic fermentation
of wood mill effluent,18 pharmaceutical wastewater,10 and dairy
wastewater19 at 42%, 44% and 56%, respectively. This implies that
POME is another promising wastewater for VFA production with
similar performance. In addition, this is further supported by the
high VFA concentration obtained at the end of the acidogenic
fermentation of POME, as summarized in Table 2.
In this study, pH inside the anaerobic reactor was in the range
4.25.3. In the literature, there were studies20,21 reporting that
higher DA could be attained by increasing the pH to 67. However,
operating the reactor at pH 67 requires implementation of a pH
control system, which would make the acidogenic fermentation of

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Figure 3. Average composition of VFA obtained at the end of 12 fedbatches of acidogenic fermentation of POME at 30 C, 40 C and 55 C.

the inherently acidic POME less cost effective due to the additional
equipment and chemical costs.
Influence of temperature on VFA composition
The influence of temperature on the composition of VFA obtained
at the end of acidogenic fermentation of POME is illustrated in
Fig. 3. Regardless of the operating temperature, acetic acid was the
primary VFA in the fermented POME. Furthermore, it was observed
that the relative abundance of propionic, butyric and valeric acids
obtained at 30 C and 40 C did not differ much. However, at 55 C,
the percentage of propionic and valeric acids in the fermented
POME was comparatively lower. Formic acid was detected at 55 C
but its percentage was insignificant compared with the other
VFA.
The VFA composition is important to PHA production because
it can affect the types of PHA produced and hence their
mechanical properties. It had been reported that acetic and
butyric acids favored the formation of 3HB, meanwhile propionic
and valeric acids encouraged the generation of 3HV.22,23 Since
copolymer of P(3HB-co-3HV) is more flexible and tougher
than homopolymer P(3HB),24 it is thus preferred to use the
fermented POME obtained at 30 C or 40 C for PHA production
because of the higher percentage of propionic and valeric
acids.
Specific characteristics of fermented POME for PHA
production
In PHA production, instead of DA which relates to the performance
of the upstream acidogenic fermentation, here one is concerned
with the fraction of sCOD that exists as VFA. The fermented POME
had high VFA content in which 69% of the sCOD was VFA. This
result was comparable with that reported by Bengtsson et al.7
whereby VFA accounted for 74% of the sCOD in the fermented
paper mill wastewater used for PHA production. The high VFA
content obtained in this study is advantageous because VFA have
been identified as the main carbon substrates leading to the
formation of PHA. Furthermore, the fermented POME had a high
molar ratio of VFA-C:N:P at 147:2:1, indicating that it was rich in

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resulted in PHA with a minor fraction of 3HV. Such correspondence
survives scrutiny via carbon balance in Table 3.
The PHA content achieved at the end of the experiment was 17
wt% of sludge dry weight. By using activated sludge enriched with
the PHA-accumulating organisms as the inoculum and optimizing
the PHA production conditions, PHA contents of 4077wt% have
been reported.12,25 27 Through these strategies, it is believed that
the production of PHA using fermented POME could be maximized.
Seen as a whole, these results strongly suggest that fermented
POME is a promising carbon substrate for PHA production.

Figure 4. Concentration profiles of PHA and VFA in PHA production.

Table 3. Net amount of VFA consumed and net amount of PHA

generated in the 24 h PHA production experiment

VFA
Acetic acid
Butyric acid
Propionic acid
Valeric acid
Total

Net amount
of VFA
consumed
(mgC L-1 )
362
272
264
121
1019

PHA

Net amount
of PHA
produced
(mgC L-1 )

3HB

86

3HV

115

Total

201

carbon but deficient in nutrients. This characteristic is considered

beneficial to PHA production as it would favor the utilization of
carbon for PHA production over microbial growth.7 These two
positive characteristics imply that fermented POME is a potential
feedstock for PHA production.
PHA production using fermented POME
To test the abovementioned deduction, a PHA production
experiment was carried out using fermented POME as the sole
substrate. Figure 4 shows the concentration profiles of PHA and
VFA obtained in this experiment. In the first 8 h, it was observed
that the PHA concentration increased along with the consumption
of VFA. This affirms the viability of using fermented POME for
PHA production. After that, despite the replenishment of VFA,
the PHA concentration reached a plateau even though there was
continuous uptake of VFA. The maximum PHA production capacity
by the activated sludge seemed to have been reached.
Since VFA well-known as favourable substrates for PHA
production7,23 are the dominant organic compounds in the
fermented POME, it is expected that VFA are the main substrates
leading to the formation of PHA in this study. The remaining
organic compounds in the fermented POME are most likely
the organic leftover that did not undergo acidification, which
are larger molecules that could not be taken up directly by
microorganisms for PHA production. In this experiment, about
20% of the consumed VFA were converted to PHA, as detailed in
Table 3.
In terms of composition, PHA consisting of 45 mol% 3HB
and 55 mol% 3HV was produced. This suggests that the PHAaccumulating organisms in the sludge preferred to utilize the VFA
for the production of 3HV. Following a report by Lemos et al.,22
the 3HV produced in this study was attributed to propionic and
valeric acids mainly, as butyrate resulted in 3HB only while acetate
J Chem Technol Biotechnol (2013)

Comparison of POME management by acidogenic

fermentation and by a conventional ponding system
Compared with a conventional ponding system, the bioconversion
of POME into VFA via acidogenic fermentation is a more sustainable
alternative for managing POME. One key reason is that the use of
anaerobic open ponds in POME treatment causes the release of
methane, which is generated from VFA via methanogenesis, into
the atmosphere. Since methane is a greenhouse gas, long-term
operation of the ponding system can contribute substantially
to global warming. In contrast, there is no obvious sign of the
occurrence of active methanogenesis in this study, as suggested
by the fairly low percentage of COD reduction of 7% on average.
Therefore, acidogenic fermentation of POME can possibly help
to reduce the emission of methane, thus reducing the carbon
footprint of the palm oil industry.
Another advantage of managing the POME via acidogenic
fermentation rather than a ponding system is space savings.
In practice, a large area of land is needed to implement a
ponding system because of the long retention time (20200
days).2 Conversely, the production of VFA from POME can be
accomplished at a relatively shorter retention time, i.e. 6 days as
in this study, thus reducing the reactor volume and the land area
required.
More importantly, for acceptance by commercial players, the
acidogenic fermentation of POME produces VFA which are
valuable carbon substrates for PHA production, as demonstrated in
the present study. When the PHA production process is optimized,
conceivably the existing land for the ponding system could
become a revenue centre for PHA production.

CONCLUSIONS
This study demonstrated that conducting acidogenic fermentation
of POME at mesophilic temperatures of 30 C and 40 C led to
better VFA production than at a thermophilic temperature of
55 C. The VFA production performance at both 30 C and 40 C was
comparable, suggesting the possibility to conduct fermentation
at ambient temperatures of 2532 C and without temperature
control. This study also showed that fermented POME, which
had high VFA content and high molar ratio of VFA-C:N:P, was a
suitable feedstock for PHA production. Although the PHA content
achieved at this time was 17 wt% of sludge dry weight, this can
be improved by fine-tuning and optimizing the PHA production
conditions.

ACKNOWLEDGEMENTS
The authors would like to acknowledge the University of Malaya
Postgraduate Research Grant (PV028-2012A) and the University of
Malaya Research Grant (RP002C-13AET) for funding this study.

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