Research ArticleReceived: 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 bioconversionof palm oil mill effluent into volatile fatty acidsas precursor to the production ofpolyhydroxyalkanoatesWee Shen Lee, Adeline Seak May Chua,∗ Hak Koon Yeohand Gek Cheng Ngoh

Abstract

BACKGROUND: The focus of wastewater management has evolved from treatment technology into resource recovery, whichenables 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) wasalso determined.

RESULTS: The VFA production at mesophilic temperature outperformed thermophilic temperature considerably, with a degreeof acidification of 48% at both 30◦C and 40◦C but only 7% at 55◦C. These results were in agreement with the substrateconsumption profiles whereby the percentage of substrate consumption at 55◦C was six times lower than those at 30◦C and40◦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 oilmills are located in tropical countries, the fermentation can be conducted under ambient conditions (25–32◦C) and withouttemperature control. The potential of VFA-rich fermented POME for PHA production is recognized, but optimization of the PHAproduction 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

NOTATIONsCODinitial Concentration of sCOD measured at the beginning

of fed-batch operationsCODfinal Concentration of sCOD measured at the end of

fed-batch operationVFAinitial Concentration of VFA measured at the beginning

of fed-batch operationVFAfinal Concentration of VFA measured at the end of

fed-batch operationvvm Gas volume flow per reactor working volume per

minute

INTRODUCTIONPalm oil is one of the main vegetable oils traded in the global marketdue to its versatile applications in food, oleochemicals and energyindustries. Although the palm oil industry is of great economicimportance, it has been recognized to be highly polluting becauseof the massive generation of palm oil mill effluent (POME) in themilling process. It is estimated that more than 2.5 tonnes of POMEcould be generated from one tonne of palm oil production.1

POME is an acidic brownish colloidal suspension containinglarge amounts of organic substances with chemical oxygendemand (COD) in a range of 35 000–57 000 mg L-1. Suchhigh COD implies the need for proper POME management toavoid severe environmental pollution. In general, most palmoil mills have employed ponding systems for treating POME.2

A ponding system, as depicted in Fig. 1, consists of a seriesof open ponds whereby POME generated from the mill is firstgathered in the collection pond for waste palm oil recovery.After that, POME undergoes primary treatment in the anaerobicpond. It is further treated in either the facultative or aerobicpond (with surface aerators) before being discharged into theenvironment. However, this treatment-oriented managementapproach neglects the potential of POME as a feedstock forthe production of various chemicals such as antibiotics, solvents

∗ Correspondence to: Adeline Seak May Chua, Department of ChemicalEngineering, 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 ofMalaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia

J Chem Technol Biotechnol (2013) www.soci.org c© 2013 Society of Chemical Industry

www.soci.org WS Lee et al.

Treatment-oriented approach – Ponding system

POME collection pond(s)

Anaerobic pond(s)

Facultative or aerobic pond(s)

Treated effluent

Palm oil mill

POME

Resource recovery approach

Acidogenic fermentor

VFA Bioenergy - hydrogen, electricity, methane

Bioplastics - polyhydroxyalkanoates

Biological nutrient removal

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

and organic acids.3 It is apparent that the transformationof POME into value-added products is a better managementapproach. In this study, we are interested in converting POMEinto volatile fatty acids (VFA) because VFA have a broad rangeof applications such as in the production of biodegradableplastics polyhydroxyalkanoates4 (PHA) and bioenergy,5 and inthe biological removal of nutrient from wastewater,6 as shown inFig. 1.

In the literature, VFA has predominantly been produced via theacidogenic fermentation process from a variety of wastes such aspaper mill wastewater7 and sewage sludge.8 In this bioprocess,complex organic substrates in the wastes are hydrolyzed andfermented into VFA under anaerobic conditions. The productionof VFA by acidogenic fermentation can be affected by a numberof key factors such as pH, temperature, hydraulic retention time,

sludge retention time and organic loading rate.9–11 While eachof these has been studied extensively, the effect of temperatureis of particular interest due to the nature of POME, which istypically discharged at rather high temperatures of 60–70◦C.As such, it could potentially be used for VFA production atboth mesophilic and thermophilic temperatures without requiringhigh heating energy to maintain the fermentation temperature.Since studies related to the production of VFA from POME12,13

were often conducted at 28–30◦C and there is little reportedinformation about the influence of higher temperature, thisstudy aims to compare the performance of VFA productionfrom POME at mesophilic (30◦C and 40◦C) and thermophilic(55◦C) temperatures. In addition, this study also tests theapplicability of VFA produced from POME to microbial productionof PHA.

MATERIALS AND METHODSCharacterization of POMERaw POME was collected every 2–3 weeks from the outlet of aPOME collection pond in a local palm oil mill. The POME wasallowed to settle at 4◦C for 24 h to reduce its solid content.After settling, the supernatant was separated from the settledsolid and stored at 4◦C for preservation before it was used inacidogenic fermentation. Both the raw POME and the supernatantof the settled POME were characterized for pH, total chemicaloxygen demand (TCOD), soluble chemical oxygen demand (sCOD),total suspended solids (TSS), volatile suspended solids (VSS) andVFA.

Operation of the anaerobic reactorsThree anaerobic reactors (each with a working volume of 1.5 L)were used for the production of VFA from POME at room tempera-ture (30 ± 1◦C), 40 ± 1◦C and 55 ± 1◦C. These reactors were seededwith sludge collected from different POME treatment ponds withtemperatures closest to the operating temperatures of the reac-tors. The seed sludge for reactor operating at 55◦C was sampledfrom a POME collection pond with a temperature of 53–63◦C. Onthe other hand, the reactor operating at 40◦C was inoculated witha mixture of sludge taken from the first and the second anaerobicponds with temperatures of 45◦C and 33◦C, respectively.Meanwhile, the sludge seeded into the reactor operating at 30◦Cwas collected from the aforementioned second anaerobic pond.

Except operating temperature, all three reactors were operatedunder similar fed-batch mode with a feeding interval of 5 days.At the end of each fed-batch operation, 1.2 L of mixed liquorwas withdrawn from each reactor to maintain the hydraulic andsludge retention times at approximately 6 days. The pH in allreactors was not controlled. To create and maintain the anaerobicconditions, nitrogen gas was introduced into the reactor at thebeginning of each fed-batch operation, and during sampling andmixed liquor withdrawal. All three reactors were operated for 60days, corresponding to a total of 12 fed-batch operations.

PHA production by using fermented POMEPHA production was carried out in an aerobic reactor (with aworking volume of 0.5 L) at room temperature (30 ± 1◦C) for 24 h.The aerobic conditions were achieved by supplying air at around 1vvm to the reactor. Activated sludge taken from a lab-scale reactoroperating on the enhanced biological phosphorus removalprocess was employed as the inoculum. Since the experimentwas preliminary, the inoculum was not specially cultivated andwas whatever available during the time of the experiment. Inthis study, fermented POME obtained from the anaerobic reactoroperating at 30◦C was utilized as the sole carbon substrate. Priorto the experiment, the ammonium and phosphate concentrationsin fermented POME were measured. After the measurement,the fermented POME was filtered through 1.2 µm filter paper toremove coarse solid particles and its pH was adjusted to 7 to avoidthe detrimental effect of acidic pH on PHA production.14 Then itwas added into the reactor at hours 0 and 8 to a concentration of750 mg VFA-C L-1 and 300 mg VFA-C L-1, respectively, to ensuresufficient VFA was available for PHA production.

wileyonlinelibrary.com/jctb c© 2013 Society of Chemical Industry J Chem Technol Biotechnol (2013)

Temperature effects on bioconversion of palm oil mill effluent www.soci.org

Analytical methodsTSS and VSS were determined according to the standardmethods.15 The COD was measured in accordance with HACHmethod 8000 using a colorimeter (DR/890, HACH, USA). Withion chromatography (861 Advanced Compact IC, Metrohm,Switzerland), the concentrations of VFA (formate, acetate,propionate, butyrate and valerate) were measured using theMetrosep Organic Acids – 250/7.8 column, the ammoniumconcentration with the Metrosep C 4 – 150/4 column, and thephosphate concentration with the Metrosep A Supp 5 – 150/4column. Standard solutions used in VFA analysis were preparedfrom concentrated formic (≥98%, Merck), acetic (99.8%, Riedel-de-Haen), propionic (>99%, Merck), butyric (≥99%, Merck) andvaleric acids (≥98%, Merck). Ammonium and phosphate standardsolutions 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 themethod described by Satoh et al.16 The concentration of theextracted 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 PHAwere quantified in this study namely 3-hydroxybutyrate (3HB)and 3-hydroxyvalerate (3HV) by employing poly(3-hydroxybutyricacid-co-3-hydroxyvaleric acid) (Natural origin, PHV content 12wt%, Sigma-Aldrich) as the standard.

Performance evaluationDegree of acidification (DA), as originally defined by Equation (1),is a parameter commonly used to assess the performance ofVFA production from wastewater.10 DA signifies the percentageconversion of soluble organic substrates available in wastewaterinto VFA.

DA = VFA produced

Initial substrate× 100% = VFAfinal

sCODinitial× 100% (1)

In this study, to account for the existence of VFA in POME priorto acidogenic fermentation, DA was computed differently as inEquation (2).

DA = VFA produced

Initial substrate× 100% = VFAfinal − VFAinitial

(sCOD − VFA)initial× 100%

(2)

Here it was recognized that VFA would contribute to the sCODmeasured, so its concentration was deducted from sCODinitial

to avoid overestimating the availability of fermentable solubleorganic substrates.

For the same reason, the percentage of substrate consumptionin acidogenic fermentation of POME was calculated according toEquation (3).

%substrate consumption = Substrate consumed

Initial substrate× 100%

= (sCOD − VFA)initial − (sCOD − VFA)final

(sCOD − VFA)initial× 100% (3)

The PHA production performance was evaluated by using PHA con-tent which is the weight percentage of PHA of sludge dry weight.The sludge dry weight is represented by the mixed liquor VSS.

Table 1. Characteristics of raw POME and supernatant of settledPOME recovered after 24 h gravitational settling (standard deviationdue to different batches of POME collected from the mill)

Parameters Raw POME Supernatant of settled POME

pH 4.7 ± 0.3 4.6 ± 0.3

TSS (mg L-1) 20000 ± 3000 2400 ± 900

VSS (mg L-1) 18000 ± 2000 2100 ± 800

TCOD (mg L-1) 47000 ± 7000 22000 ± 4000

sCOD (mg L-1) 20000 ± 3500 20000 ± 3500

VFA (mg L-1) 2600 ± 900 2700 ± 1100

sCOD/TCOD 0.42 ± 0.03 0.89 ± 0.05

VFA/sCOD 0.18 ± 0.12 0.19 ± 0.14

RESULTS AND DISCUSSIONCharacteristics of POMEThe characteristics of raw POME and the supernatant of settledPOME are compared in Table 1. It can be seen that raw POME isan acidic, organic-rich wastewater containing a lot of suspendedsolids. To reduce its solid content, raw POME was allowed to settlefor 24 h. It was found that about 88% of the TSS in raw POME couldbe removed by this simple solid separation technique, resulting ina final TSS concentration of 2400 mg L-1.

The removal of such a great amount of TSS resulted in alower TCOD concentration. However, the supernatant of settledPOME was still considered a suitable feedstock for VFA productionbecause it remained very rich in organic matter with TCOD at22 000 mg L-1. More importantly, most of the organic matter issoluble as indicated by the high ratio of sCOD/TCOD at 0.89. Thisratio was double that of the raw POME at 0.42. The higher fractionof soluble organic matter in the supernatant of settled POME isadvantageous to microorganisms as they could more easily utilizethe organic matter for VFA production.

According to Table 1, VFAs were detected in the supernatantof settled POME with an average concentration of 2700 mg L-1.Nonetheless, they were not the main fraction of the soluble organicmatter in POME, as indicated by the low ratio of VFA to sCOD of0.19. Consequently, there is room to maximize the utilization ofPOME by further converting the remaining organic matter intoVFA via acidogenic fermentation.

Another piece of useful information derived from thecharacterization study was that pH control might not be requiredduring the acidogenic fermentation of POME. This is becausethe acidic nature of POME (pH 4.6) could suppress the activityof methanogens which are active within a narrow pH range of6.4–7.8.17 Methanogens, if present in the reactor, could consumethe VFA for methane formation. Without pH control, VFA canbe produced at lower cost in line with the economic advantageof using waste for the production of a value-added product.Collectively, the results obtained from the characterization studysuggest that POME is suitable for VFA production.

Influence of temperature on degree of acidificationA total of 12 fed-batches of acidogenic fermentation of POMEwere executed at 30◦C, 40◦C and 55◦C and their respective DAprofiles are depicted in Fig. 2(a). There was not much differencein the performance of VFA production conducted at 30◦C and40◦C, as indicated by the fairly similar DA obtained at thesetwo temperatures. On average, the DA attained at 30◦C and40◦C was 48 ± 4% and 48 ± 5%, respectively. However, these

J Chem Technol Biotechnol (2013) c© 2013 Society of Chemical Industry wileyonlinelibrary.com/jctb

www.soci.org WS Lee et al.

(a)

(b)

Figure 2. (a) Degree of acidification and (b) percentages of substrateconsumption in the acidogenic fermentation of POME at 30◦C, 40◦C and55◦C.

values were about six times higher than that achieved at 55◦Cof 7 ± 6%. The poorer VFA production at 55◦C was caused bythe lower microbial activity in the reactor, as the percentagesubstrate consumption at 55◦C was also approximately six timeslower than those at 30◦C and 40◦C (Fig. 2(b)). This reductionin microbial activity might result from either poorer substrateuptake ability of the thermophilic VFA-forming microorganismsor relatively fewer such microbes in the reactor. A similar findinghad been reported by Zhuo et al.11 whereby the consumption ofsubstrate decreased with increase in temperature from 37◦C to55◦C during the acidogenic fermentation of ultrasonic-pretreatedwaste activated sludge, leading to lower VFA production at 55◦C.

Based on this study, it is obvious that mesophilic temperaturesof 30◦C and 40◦C favor the production of VFA from POMEcompared with thermophilic temperature of 55◦C. Furthermore,the comparable VFA production at 30◦C and 40◦C suggests thattemperature control will not be required in actual industrialapplication because most of the palm oil producers such asIndonesia, Malaysia and Thailand are tropical countries withambient temperatures at around 25–32◦C. Nevertheless, POME,which is commonly discharged at 60–70◦C, has to be cooled to40◦C before use in acidogenic fermentation. Such cooling of POMEis presently accomplished in palm oil mills by using one or twoopen ponds in series, hence no additional cooling facilities arenecessary to retrofit the mills for VFA production.

It is notable that the DA achieved in this study (48%) wascomparable with that attained in the acidogenic fermentationof wood mill effluent,18 pharmaceutical wastewater,10 and dairywastewater19 at 42%, 44% and 56%, respectively. This implies thatPOME is another promising wastewater for VFA production withsimilar performance. In addition, this is further supported by thehigh VFA concentration obtained at the end of the acidogenicfermentation of POME, as summarized in Table 2.

In this study, pH inside the anaerobic reactor was in the range4.2–5.3. In the literature, there were studies20,21 reporting thathigher DA could be attained by increasing the pH to 6–7. However,operating the reactor at pH 6–7 requires implementation of a pHcontrol system, which would make the acidogenic fermentation of

Table 2. Average concentrations of VFA obtained at the end offed-batch acidogenic fermentation of POME at 30◦C, 40◦C and 55◦C

Temperature (◦C) Final VFA concentration (mg L-1)

30 8300 ± 1000

40 8500 ± 1000

55 3700 ± 1300

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

the inherently acidic POME less cost effective due to the additionalequipment and chemical costs.

Influence of temperature on VFA compositionThe influence of temperature on the composition of VFA obtainedat the end of acidogenic fermentation of POME is illustrated inFig. 3. Regardless of the operating temperature, acetic acid was theprimary VFA in the fermented POME. Furthermore, it was observedthat the relative abundance of propionic, butyric and valeric acidsobtained at 30◦C and 40◦C did not differ much. However, at 55◦C,the percentage of propionic and valeric acids in the fermentedPOME was comparatively lower. Formic acid was detected at 55◦Cbut its percentage was insignificant compared with the otherVFA.

The VFA composition is important to PHA production becauseit can affect the types of PHA produced and hence theirmechanical properties. It had been reported that acetic andbutyric acids favored the formation of 3HB, meanwhile propionicand valeric acids encouraged the generation of 3HV.22,23 Sincecopolymer of P(3HB-co-3HV) is more flexible and tougherthan homopolymer P(3HB),24 it is thus preferred to use thefermented POME obtained at 30◦C or 40◦C for PHA productionbecause of the higher percentage of propionic and valericacids.

Specific characteristics of fermented POME for PHAproductionIn PHA production, instead of DA which relates to the performanceof the upstream acidogenic fermentation, here one is concernedwith the fraction of sCOD that exists as VFA. The fermented POMEhad high VFA content in which 69% of the sCOD was VFA. Thisresult was comparable with that reported by Bengtsson et al.7

whereby VFA accounted for 74% of the sCOD in the fermentedpaper mill wastewater used for PHA production. The high VFAcontent obtained in this study is advantageous because VFA havebeen identified as the main carbon substrates leading to theformation of PHA. Furthermore, the fermented POME had a highmolar ratio of VFA-C:N:P at 147:2:1, indicating that it was rich in

wileyonlinelibrary.com/jctb c© 2013 Society of Chemical Industry J Chem Technol Biotechnol (2013)

Temperature effects on bioconversion of palm oil mill effluent www.soci.org

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

Table 3. Net amount of VFA consumed and net amount of PHAgenerated in the 24 h PHA production experiment

VFA

Net amount

of VFA

consumed

(mgC L-1) PHA

Net amount

of PHA

produced

(mgC L-1)

Acetic acid 362 3HB 86

Butyric acid 272

Propionic acid 264 3HV 115

Valeric acid 121

Total 1019 Total 201

carbon but deficient in nutrients. This characteristic is consideredbeneficial to PHA production as it would favor the utilization ofcarbon for PHA production over microbial growth.7 These twopositive characteristics imply that fermented POME is a potentialfeedstock for PHA production.

PHA production using fermented POMETo test the abovementioned deduction, a PHA productionexperiment was carried out using fermented POME as the solesubstrate. Figure 4 shows the concentration profiles of PHA andVFA obtained in this experiment. In the first 8 h, it was observedthat the PHA concentration increased along with the consumptionof VFA. This affirms the viability of using fermented POME forPHA production. After that, despite the replenishment of VFA,the PHA concentration reached a plateau even though there wascontinuous uptake of VFA. The maximum PHA production capacityby the activated sludge seemed to have been reached.

Since VFA – well-known as favourable substrates for PHAproduction7,23 – are the dominant organic compounds in thefermented POME, it is expected that VFA are the main substratesleading to the formation of PHA in this study. The remainingorganic compounds in the fermented POME are most likelythe organic leftover that did not undergo acidification, whichare larger molecules that could not be taken up directly bymicroorganisms for PHA production. In this experiment, about20% of the consumed VFA were converted to PHA, as detailed inTable 3.

In terms of composition, PHA consisting of 45 mol% 3HBand 55 mol% 3HV was produced. This suggests that the PHA-accumulating organisms in the sludge preferred to utilize the VFAfor the production of 3HV. Following a report by Lemos et al.,22

the 3HV produced in this study was attributed to propionic andvaleric acids mainly, as butyrate resulted in 3HB only while acetate

resulted in PHA with a minor fraction of 3HV. Such correspondencesurvives scrutiny via carbon balance in Table 3.

The PHA content achieved at the end of the experiment was 17wt% of sludge dry weight. By using activated sludge enriched withthe PHA-accumulating organisms as the inoculum and optimizingthe PHA production conditions, PHA contents of 40–77wt% have

been reported.12,25–27 Through these strategies, it is believed thatthe production of PHA using fermented POME could be maximized.Seen as a whole, these results strongly suggest that fermentedPOME is a promising carbon substrate for PHA production.

Comparison of POME management by acidogenicfermentation and by a conventional ponding systemCompared with a conventional ponding system, the bioconversionof POME into VFA via acidogenic fermentation is a more sustainablealternative for managing POME. One key reason is that the use ofanaerobic open ponds in POME treatment causes the release ofmethane, which is generated from VFA via methanogenesis, intothe atmosphere. Since methane is a greenhouse gas, long-termoperation of the ponding system can contribute substantiallyto global warming. In contrast, there is no obvious sign of theoccurrence of active methanogenesis in this study, as suggestedby the fairly low percentage of COD reduction of 7% on average.Therefore, acidogenic fermentation of POME can possibly helpto reduce the emission of methane, thus reducing the carbonfootprint of the palm oil industry.

Another advantage of managing the POME via acidogenicfermentation rather than a ponding system is space savings.In practice, a large area of land is needed to implement aponding system because of the long retention time (20–200days).2 Conversely, the production of VFA from POME can beaccomplished at a relatively shorter retention time, i.e. 6 days asin this study, thus reducing the reactor volume and the land arearequired.

More importantly, for acceptance by commercial players, theacidogenic fermentation of POME produces VFA which arevaluable carbon substrates for PHA production, as demonstrated inthe present study. When the PHA production process is optimized,conceivably the existing land for the ponding system couldbecome a revenue centre for PHA production.

CONCLUSIONSThis study demonstrated that conducting acidogenic fermentationof POME at mesophilic temperatures of 30◦C and 40◦C led tobetter VFA production than at a thermophilic temperature of55◦C. The VFA production performance at both 30◦C and 40◦C wascomparable, suggesting the possibility to conduct fermentationat ambient temperatures of 25–32◦C and without temperaturecontrol. This study also showed that fermented POME, whichhad high VFA content and high molar ratio of VFA-C:N:P, was asuitable feedstock for PHA production. Although the PHA contentachieved at this time was 17 wt% of sludge dry weight, this canbe improved by fine-tuning and optimizing the PHA productionconditions.

ACKNOWLEDGEMENTSThe authors would like to acknowledge the University of MalayaPostgraduate Research Grant (PV028-2012A) and the University ofMalaya Research Grant (RP002C-13AET) for funding this study.

J Chem Technol Biotechnol (2013) c© 2013 Society of Chemical Industry wileyonlinelibrary.com/jctb

www.soci.org WS Lee et al.

REFERENCES1 Ahmad AL, Ismail S and Bhatia S, Water recycling from palm oil

mill effluent (POME) using membrane technology. Desalination157:87–95 (2003).

2 Poh PE and Chong MF, Development of anaerobic digestion methodsfor palm oil mill effluent (POME) treatment. Bioresource Technol100:1–9 (2009).

3 Wu TY, Mohammad AW, Jahim JM and Anuar N, A holistic approach tomanaging palm oil mill effluent (POME): biotechnological advancesin the sustainable reuse of POME. Biotechnol Adv 27:40–52 (2009).

4 Valentino F, Brusca AA, Beccari M, Nuzzo A, Zanaroli G and Majone M,Start up of biological sequencing batch reactor (SBR) and short-termbiomass acclimation for polyhydroxyalkanoates production. J ChemTechnol Biotechnol 88:261–270 (2013).

5 Zhao J, Li X, Ren Y, Wang X and Jian C, Electricity generation from TaihuLake cyanobacteria by sediment microbial fuel cells. J Chem TechnolBiotechnol 87:1567–1573 (2012).

6 Tong J and Chen Y, Enhanced biological phosphorus removal drivenby short-chain fatty acids produced from waste activated sludgealkaline fermentation. Environ Sci Technol 41:7126–7130 (2007).

7 Bengtsson S, Werker A, Christensson M and Welander T, Productionof polyhydroxyalkanoates by activated sludge treating a paper millwastewater. Bioresource Technol 99:509–516 (2008).

8 Liu X, Liu H, Chen Y, Du G and Chen J, Effects of organic matter andinitial carbon–nitrogen ratio on the bioconversion of volatile fattyacids from sewage sludge. J Chem Technol Biotechnol 83:1049–1055(2008).

9 Miron Y, Zeeman G, Lier JBV and Lettinga G, The role of sludge retentiontime in the hydrolysis and acidification of lipids, carbohydrates andproteins during digestion of primary sludge in CSTR systems. WaterRes 34:1705–1713 (2000).

10 Oktem YA, Ince O, Donnelly T, Sallis P and Ince BK, Determination ofoptimum operating conditions of an acidification reactor treatinga chemical synthesis-based pharmaceutical wastewater. ProcessBiochem 41:2258–2263 (2006).

11 Zhuo G, Yan Y, Tan X, Dai X and Zhou Q, Ultrasonic-pretreated wasteactivated sludge hydrolysis and volatile fatty acid accumulationunder alkaline conditions: effect of temperature. J Biotechnol159:27–31 (2012).

12 Salmiati, Ujang Z, Salim MR, Din MFM and Ahmad MA, Intracellularbiopolymer productions using mixed microbial cultures fromfermented POME. Water Sci Technol 56:179–185 (2007).

13 Hassan MA, Shirai Y, Kusubayashi N, Karim MIA, Nakanishi K andHashimoto K, Effect of organic acid profiles during anaerobictreatment of palm oil mill effluent on the production ofpolyhydroxyalkanoates by Rhodobacter spaeroides. J Ferment Bioeng82:151–156 (1996).

14 Chua ASM, Takabatake H, Satoh H and Mino T, Production ofpolyhydroxyalkanoates (PHA) by activated sludge treating municipalwastewater: effect of pH, sludge retention time (SRT), and acetateconcentration in influent. Water Res 37:3602–3611 (2003).

15 APHA, Standard Methods for the Examination of Water and Wastewater,18th edn. APHA, USA (1992).

16 Satoh H, Ramey WD, Koch FA, Oldha WK, Mino T and Matsuo T,Anaerobic substrate uptake by the enhanced biological phosphorusremoval activated sludge treating real sewage. Water Sci Technol34:9–16 (1996).

17 Grady CPL, Daigger GT, Love NG and Filipe CDM, Biological WastewaterTreatment, 3rd edn. CRC Press, Boca Raton (2011).

18 Ben M, Mato T, Lopez A, Vila M, Kennes C and Veiga MC, Bioplasticproduction using wood mill effluents as feedstock. Water Sci Technol63:1196–1202 (2011).

19 Demirel B and Yenigun O, Anaerobic acidogenesis of dairy wastewater:the effects of variations in hydraulic retention time with no pHcontrol. J Chem Technol Biotechnol 79:755–760 (2004).

20 Yu HQ and Fang HHP, Acidogenesis of gelatin-rich wastewater in anupflow anaerobic reactor: influence of pH and temperature. WaterRes 37:55–66 (2003).

21 Dinopoulou G, Rudd T and Lester JN, Anaerobic acidogenesis of acomplex wastewater. I. The influence of operational parameters onreactor performance. Biotechnol Bioeng 31:958–968 (1988).

22 Lemos PC, Serafim LS and Reis MAM, Synthesis of polyhydroxyalka-noates from different short-chain fatty acids by mixed culturesubmitted to aerobic dynamic feeding. J Biotechnol 122:226–238(2006).

23 Albuquerque MGE, Eiroa M, Torres C, Nunes BR and Reis MAM,Strategies for the development of a side stream process forpolyhydroxyalkanoate (PHA) production from sugar cane molasses.J Biotechnol 130:411–421 (2007).

24 Holmes PA, Applications of PHB – a microbially produced biodegrad-able thermoplastic. Phys Technol 16:32–36 (1985).

25 Din MFM, Mohanadoss P, Ujang Z, van Loosdrecht MCM, Yunus SM,Chelliapan S, Zambare V and Olsson G, Development of Bio-PORecsystem for polyhydroxyalkanoates (PHA) production and its storagein mixed cultures of palm oil mill effluent (POME). Bioresource Technol124:208–216 (2012).

26 Jiang Y, Marang L, Tamis J, van Loosdrecht MCM, Dijkman H andKleerebezem R, Waste to resource: converting paper mill wastewaterto bioplastic. Water Res 46:5517–5530 (2012).

27 Albuquerque MGE, Torres C and Reis MAM, Polyhydroxyalkanoate(PHA) production by a mixed microbial culture using sugar molasses:effect of the influent substrate concentration on culture selection.Water Res 44:3419–3433 (2010).

wileyonlinelibrary.com/jctb c© 2013 Society of Chemical Industry J Chem Technol Biotechnol (2013)