ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING Asia-Pac. J. Chem. Eng. (2011) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI:10.1002/apj.

550

Research Article

Optimization of biomethane production by anaerobic digestion of palm oil mill efuent using response surface methodology
A. F. Saleh,1 E. Kamarudin,1 A. B. Yaacob,1 A. W. Yussof2 and M.A. Abdullah1 *
1 2

Department of Chemical Engineering, Universiti Teknologi Petronas, Bandar Seri Iskandar, 31750 Tronoh, Perak D. R., Malaysia Palm Oil Mill, FELCRA Nasaruddin, KM37, Jalan Tronoh, 32600, Bota, Perak, Malaysia

Received 25 August 2010; Revised 25 November 2010; Accepted 14 December 2010

ABSTRACT: This study investigated the effects of factors namely temperature, palm oil mill efuent (POME) volume, inoculum volume, and co-substrate addition such as oil palm empty fruit bunch (EFB) and palm kernel on the anaerobic digestion process for biogas and methane production. Response surface methodology by the Box-Behnken design veried that the specic biogas production rate and methane yield were mainly affected by operating temperature and co-substrate addition. The optimal conditions for the maximum specic biogas production rate (0.0574 m3 / kg chemical oxygen demand per day) and methane yield (25.6%) have been predicted by multiple response optimization and veried experimentally at 47.8 C operating temperature, 50.4 mL POME volume, and 5.7 g EFB addition. The error percentage between experimental and predicted values which were around 5% for methane composition and 12% for specic biogas production rate suggests the good predictability of the model. 2011 Curtin University of Technology and John Wiley & Sons, Ltd. KEYWORDS: palm oil mill efuent; anaerobic digestion; biomethane; carbon dioxide; response surface methodology

INTRODUCTION
Bioenergy is an important form of renewable energy. Stored in a biological material such as wood, manure, straw, and other agricultural products, bioenergy is one of the key options on shorter and medium term to mitigate greenhouse gas (GHG) emissions and substitute fossil fuels.[1] It can be used to generate heat or electricity or to produce transport fuel.[2 4] Annually, some 590880 million tons of methane are released worldwide into the atmosphere through microbial activity and about 90% come from biogenic sources. Methane is over 20 times more effective in trapping heat in the atmosphere than carbon dioxide (CO2 ) over a 100-year period.[5] Hence, capturing and utilizing methane as a renewable energy can prevent its release to the atmosphere and can be used to obtain Certied Emission Reduction (CER) credit by Clean Development Mechanism (CDM) under the Kyoto protocol.[6] Conversion of biomass into biogas can be achieved when the microorganisms digest the organic materials in the absence of oxygen (anaerobic digestion). Biogas can
*Correspondence to: M.A. Abdullah, Department of Chemical Engineering, Universiti Teknologi Petronas, Bandar Seri Iskandar, 31750 Tronoh, Perak D. R., Malaysia. E-mail: azmuddin@petronas.com.my
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be produced in landlls, or from dairy efuent on farms, or at sewage plants. Anaerobic digestion has successfully been demonstrated for its ability to recycle biological wastes and produce biogas.[7,8] Agricultural waste and sewage especially contain many nutrients for the anaerobes. Typical composition of biogas from anaerobic digestion is methane (CH4 ) 60%, carbon dioxide (CO2 ) 35%, hydrogen sulde (H2 S) 3%, hydrogen (H2 ) 1%, and ammonia (NH3 ) or other gases 5%.[9 11] The left over is called digestate, rich in nutrients and can be a good source of soil amendments or liquid fertilizers. There are several digester types used conventionally such as Covered Lagoon, Complete Mix, Plug Flow, and Fix Film.[3] Provided the system is operated at optimum operating conditions, the anaerobic digestion process is highly stable, economical, and requires relatively small space. It has low and stabilized sludge with high dewaterability and high tolerance toward xenobiotics, and 1020% of chemical oxygen demand (COD) is removed.[12 14] In biogas production, the level of putrescibility is the key factor for successful application.[15] Substrate composition determines methane yield and production rates. Other important parameters include temperature, pH, organic loading rate (OLR), mixing, solid retention time (SRT), and hydraulic retention time (HRT).

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Oil palm wastes are extensively studied as the source of biomass for anaerobic digestion technology. In oil palm mills, the treatment of the wastes not only reduces pollution, but also produces clean-renewable energy and the electricity needs of the workers, and can be sold to energy companies.[16] Malaysia is among the worlds largest producer and exporter of palm oil and its products with yearly production of more than 13 million tons of crude palm oil (CPO). In 2003, more than 3.79 million hectares of land are under oil palm cultivation, occupying more than one-third of the total cultivated area in Malaysia and 11% of the total land area.[17,18] The main product is the CPO and various forms of solid and liquid wastes such as empty fruit bunches (EFB), palm press ber (PPF), palm kernel cake (PKC), palm kernel shell (PKS), sludge cake (SC), and palm oil mill efuent (POME).[19] POME, a thick brownish colloidal suspension with pH 45, is not toxic but has unpleasant odor.[6] It also consists of 9596% water, 0.60.7% oil, 4.45% total solids, and 24% suspended solids.[20] In 2002, an estimated 35.7 million m3 of POME, nearly three times the quantity of CPO, have been reportedly generated.[18,21] Typically, 1 t of CPO production requires 57.5 t of water, over 50% of which ends up as POME, mainly from clarication (60%), sterilization (36%), and hydrocyclone (4%) units.[16,22] POME wastewater has biological oxygen demand (BOD) level at 25 000 mg/L and COD at 50 000 mg/L.[23] The Malaysian Department of Environment (DOE) BOD discharge standard is 100 mg/L. Response surface methodology (RSM) is a collection of mathematical and statistical techniques for modeling analysis in which a response of interest is inuenced by several variables and the objective is to optimize this response.[24] In order to optimize the variables, the main effects and two-factor interactions from low to high level has to be evaluated. For this purpose, response surface design includes center points located in the middle of tested level to test the curvature. Usually center points are more than one because additional center points run improve estimates of quadratic effects and also provide additional degree of freedom for error.[25] There are a number of surface response designs available but only the central composite, facecentered cube (derived from central-composite), and BoxBehnken design are mostly used. The aim of this study was to identify and optimize factors such as temperature, volume of sludge as inoculum, POME volume, and co-substrate addition, for an anaerobic digestion process to produce biomethane. The co-substrate combination tested includes the oil palm EFB, kernel, and shell. Waste characterization such as BOD, COD, total suspended solids (TSSs), and elemental analyses (carbon, nitrogen, phosphorus, and sulfur) was also carried out.
2011 Curtin University of Technology and John Wiley & Sons, Ltd.

MATERIALS AND METHODS

Waste characterization Sample preparation Wastes were collected from Palm Oil Mill Felcra Nasaruddin in Bota, Perak, Malaysia. The samples used as feedstocks were from oil palm solid and liquid wastes. The sludge for seeding the anaerobic digester was obtained from the sludge pit where all the efuent accumulated before being sent to the conventional anaerobic pond. The solid wastes were EFB, ber, shell, and kernel, while POME was from the conventional anaerobic pond. They were sealed in a container and stored in a cold room at temperature 4 C, until used. Before the experiment, the solid samples were dehydrated in the oven at 110 C for about 3 h. The samples were then pounded using mortar and pestle and grinded into small pieces using an electrical grinder. Chemical analysis pH of POME was measured using a Mettler Toledo320 pH probe. BOD measurement was carried out using BODTrak (Hach) according to the standard method provided by Hach. One milliliter of POME was diluted with distilled water at 1 : 100 ratio. Sample of 95 mL was heated to 20 C and then poured into the BODTrak sample bottle. Four samples were prepared and 3.8 cm magnetic stirring bar was placed in each sample bottle. One BOD Nutrient Buffer Pillow was added for optimum bacterial growth. Lithium hydroxide powder was then added to the seal cup. Stopcock Grease was applied to the seal lip of the bottle and to the top of the seal cup to prevent vaporization. The setup was placed in an incubator at 20 C. The test duration of 5.25 days and the range of 0700 mg/L were selected from the Hach program installed. After 5 days, the BOD reading was taken. COD measurement was carried out using Spectrophotometer DR2800, according to 8000-Reactor Digestion Method provided by Hach. The DRB200 reactor was preheated to 150 C. One milliliter of sample was diluted for POME-to-deionized water ratios of 1 : 50, 1 : 100, and 1 : 250. Diluted POME of 2 mL was added to the high range COD digestion reagent vial and the mixture inverted several times to ensure well mixing. Deionized water of 2 mL was added to another high range COD digestion reagent vial to serve as blank. The samples were placed in the reactor for 2 h. Both vials were cooled to room temperature before reading. The Hach program 435 COD HR was used. The TSSs were analyzed based on the standard method provided by APHA.[26] A lter paper (Whatmann No. 4) was weighed (B mg). Ten milliliter of the POME was ltered under vacuum through the preweighed lter paper and the lter paper was dried in an
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oven at 105 C, cooled to room temperature in a desiccator, and weighed until constant weight (A mg). The TTS was calculated as: TTS (mg/L) = [(B A)/Sample volume] 1000 (1) The elemental analysis was performed on the EFB and the palm kernel samples using CHNS-932 analyzer. Analyses of carbon, hydrogen, nitrogen, and sulfur contents were carried out according to the standard operating procedure for CHNS-932. Prior to the analyses, the samples were dehydrated in an oven at 105 C and crushed into powder form. Both samples and sulfamethazine (as a standard) were weighed (x mg) to 1.5 > x > 2 and placed in a tin capsule. Sulfamethazine was tested to ensure that the percentage of CHNS reading met the requirement of the standard, or otherwise the equipment would be recalibrated. The results were shown as mean values of triplicates with standard errors.

bath was stable and no bubble detection in the owmeasuring cell, the challenge environmental system (CES) program was started. The cell counters and timers from the control screen of the computer program were reset and data acquisition was initiated. The experiment was left for 3 days. Gases produced by the anaerobic digestion ow through each cell under the inuence of a slight pressure buildup caused by gas production in the reaction vessel and bubbles of a xed volume are formed in the lower section of the cell. These bubbles in turn passed through a detection section and were detected by the photocell and the sensor in the cell base. The signals were processed by the interface module and the computer.

Anaerobic digestion experiments Anaerobic test setup The CHALLENGE AER-200 Respirometer System for anaerobic digestion experiments consists of eight 250 mL reaction vessels, a stirring base for mixing the samples, a water bath to control the temperature of reaction vessel, a cell base containing eight ow-measuring cells, an interface module, and a computer program. The system can be operated aerobically or anaerobically. The test bottles and related parts were cleaned using deionized water and rinsed thoroughly before autoclaving at 121 C, 20 min, to ensure no contamination from previous experiments. The subsequent procedures were carried out under non-sterile environment to establish the results as it would be applied in the eld. The Teon -coated magnetic stirring bar was added and measured samples at designated volumes (the POME and co-substrate and inoculum) were transferred into the bottles. pH was adjusted to 6.27.2 by adding 2 mg sodium bicarbonate. Each of the bottles was purged with nitrogen gas for adequate duration before the screw cap with inserted butyl rubber septum was quickly tightened to ensure anaerobic environment. The test bottles were then placed into a MS8-300 magnetic stirring base water bath and the temperature was set to 35 C. Stirring rate was adjusted to 100 rpm to provide adequate mixing. The test bottles were vented by inserting a clean 20gage needle through the septum. This venting prevented over-pressure due to a gas buildup in the bottle during set up. Four test bottles were attached to the tubing connected to a ow-measuring cell to analyze the total gas production and its production rate and another four test bottles were connected to the gas bags for biogas composition analyses. After the temperature of water
2011 Curtin University of Technology and John Wiley & Sons, Ltd.

Analytical method The rate of biogas production and total volume of biogas were recorded automatically by the CES program. The number of bubbles was measured as cumulative volume and ow rate.[27] The lowest volume of measurement using the standard anaerobic cell was one bubble or about 0.15 mL; the upper range was 23 bubbles/s or about 2025 mL/min. High-sensitivity cells having about 0.05 mL per bubble or 810 mL/min were also available. The composition of biogas was determined by using gas chromatography (Shimadzu, GC-2010) under the following conditions: GS-Q column (J&W Scientic), helium as a carrier gas at a ow rate of 54 mL/min, the column temperature of 250 C, the injector temperature of 150 C, the detector temperature of 200 C, using thermal conductivity detector (TCD). Two milliliter of the gas sample was injected to analyze the main composition of biogas; namely CH4 and CO2 . Statistical design and analysis Screening experiments were divided into two parts. In the rst part, 2k factorial design of eight experimental runs was carried out, with all possible combination of values for each experimental factor (x ), namely POME volume (x1 ), palm kernel substrate (x2 ), and inoculum volume (x3 ). The levels tested were as follows: x1 = 50, 100 mL; x2 = 4, 8 g; and x3 = 40, 90 mL. In the second part, the factors were POME volume (x1 ), EFB (x2 ), and inoculum volume (x3 ) and the levels were x1 = 100, 400 mL; x2 = 0, 30 g; and x3 = 25, 100 mL. For RSM, the BoxBehnken design was used. All experiments were carried out in a randomized order to minimize the error in the response due to miscellaneous factors. The experiment consists of 15 experimental runs, with all possible combination of values for each experimental factor (x ), namely temperature (x1 ), POME volume (x2 ), and EFB addition (x3 ). The independent variables at low, medium, and high levels are shown in Table 1. In both screening and RSM experiments, the responses studied were the specic biogas production
Asia-Pac. J. Chem. Eng. (2011) DOI: 10.1002/apj

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Table 1. BoxBehnken experimental design and responses.

Responses Independent variables Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 T ( C) x1 20 20 35 20 50 35 50 35 50 35 20 35 50 35 35 POME (mL) x2 3100 350 350 375 375 375 350 3100 375 375 375 375 3100 350 3100 EFB (g) x3 3 3 6 0 0 3 3 6 6 3 6 3 3 0 0 Specic biogas production rate, y1 Experimental 0.0145 0.0159 0.0569 0.0111 0.0366 0.0419 0.0527 0.0501 0.0438 0.0434 0.0136 0.0499 0.0395 0.0468 0.0360 Predicted 0.0148 0.0170 0.0547 0.0085 0.0356 0.0451 0.0523 0.0487 0.0464 0.0451 0.0147 0.0451 0.0384 0.0482 0.0382 Methane (%), y2 Experimental 2.44 7.55 25.6 5.66 14.4 15.1 21.2 13.9 18.6 16.0 5.88 18.8 12.2 20.0 11.6 Predicted 1.96 8.52 24.2 5.03 14.2 16.6 21.6 14.2 19.2 16.6 6.02 16.6 11.3 19.7 12.7

rate (y1 ) and percentage of methane (y2 ). The specic biogas production rate was calculated as follows: Specic biogas production rate (m3 /kg COD per day) = Total volume of biogas produced (m3 ) COD load Time (kg COD per day) (2)

The statistical analysis was performed by using Statgraphic Version 5, Rockville, USA. A quadratic polynomial regression model was used to predict both responses as follows:
3 3 2 3

y = o +
i =1

i xi +
i =1

ii xi2 +
i =1 j =i +1

ij xi xj (3)

where o , i , ii , and ij are intercept, linear, quadratic, and interaction regression coefcient terms, respectively, and xi and xj are independent variables. The goodness of t of the model was evaluated by the analysis of variance (ANOVA). Pareto charts and contour surface response were constructed to evaluate the interaction that has signicant effects.

Table 2, the pH, BOD, and COD values were slightly higher but comparable to typical values as reported for the raw POME feeding to the anaerobic digestion and aerobic/faculative treatment system which were 4.7, 20 00025 000 mg/L, and 45 00050 000 mg/L, respectively.[16] The COD/BOD ratio can be used to assess the biodegradability of the organic matter. The COD/BOD ratio around 1.52.0 suggests that the organic matter is biodegradable.[29] As the COD/BOD ratio of this sample was about 2.2, it may suggest that the organic matter was biodegradable and had high potential for biogas production anaerobically. However, the TSS was much lower than the values reported in the range of 18 00040 500 mg/L. This may suggest the need for substrate addition as high suspended solids in POME also indicate high potential for gas production.[6] From Table 2, the C : N ratio of 27 : 1 for EFB and 34 : 1 for palm kernel from CHNS analysis suggested that the nutrients and minerals required for bacterial growth were present. The optimum C : N ratio for the microbial food requirement in the anaerobic digestion is in the range of 2030 : 1.[30] Therefore, both EFB and palm kernel were potential co-substrate addition to the anaerobic digestion of POME to enhance the biogas production.

RESULTS AND DISCUSSION

Waste characterization
Waste characterization enables the prediction of the biogas production performance because different palm oil mills have different characteristics of efuent, depending on the performance of the processes in producing CPO and also the mill operation.[28] As shown in
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Screening experiments
In the rst part of screening experiments with palm kernel (Fig. 1), both POME volume and palm kernel addition had signicant effects on total biogas production, but none had signicant effect on methane. The
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Table 2. Characteristics of POME, EFB, and palm kernel.

Analysis pH BOD COD TSS Elemental composition

mg/L 4.55.2 27 766 61 994 7 860 (%) EFB Palm kernel Carbon, C 45.5 56.3 Hydrogen, H 6.1 9.0 Nitrogen, N 1.7 1.7 Sulfur, S 0.14 0.18

had resulted in not only zero methane, but also only around 25100 mL of biogas production.

Optimization experiment
On the basis of the fresh fruit bunch composition, EFB actually constitutes 59% while only 1% of it is the palm kernel.[19] Out of this, 49% could end up as dried EFB. Hence, EFB contributes more as a solid waste and is the best candidate to study its potential as a substrate in anaerobic digestion. The design of optimization experiment and the responses are as shown in Table 1. The volume of inoculum (POME sludge) as a source of bacteria was set at a constant volume of 150 mL. The biogas production was initiated after 3 h at 35 and 50 C operating temperatures, while at 20 C, it took 10 h. When comparing according to respective operating temperature (results not shown), the working volume with high EFB addition resulted in, on average, 16.9% increase in the gas production rate. The highest production rate of 56.5 mL/h or 529 mL total volume of biogas and the highest methane percentage of 25.6% were achieved at 35 C with 6 g EFB. The Pareto charts as shown in Fig. 2 describe the relative importance of the factor and also the effect of

lowest methane production was 5.6% with total biogas production of 826 mL while the highest methane yield was 20% with total biogas of 1175 mL (results not shown). In general, the higher the POME volume and the kernel, the higher the biogas and methane produced, but higher inoculum could enhance methane but not biogas production. In the second part with EFB (results not shown), both POME volume and EFB addition had signicant effects on total biogas production at 417 mL and methane at 11.4%. At 400 mL POME, 50 mL inoculum and without co-substrate addition, a total biogas production of 1813 mL was attained, but methane remained at 4.2%. There was an optimum co-substrate level (around 510 g) as the higher level did not necessarily translate into higher methane or biogas. It also conrmed earlier nding that within the range of 40100 mL inoculum addition, no signicant effects on biogas or methane production were observed. In addition, pH 6.6 was conducive for anaerobic digestion as pH at 4.6 and 7.8

Figure 1.

Standardized Pareto chart for (a) total biogas production and (b) methane production using palm kernel as a co-substrate. This gure is available in colour online at www.apjChemEng.com.

Figure 2. Standardized Pareto charts for (a) specic biogas production rate and (b) methane percentage using EFB as a co-substrate. This gure is available in colour online at www.apjChemEng.com.
Asia-Pac. J. Chem. Eng. (2011) DOI: 10.1002/apj

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factor setting adjustment, by displaying the most inuencing factor followed by the least one. Temperature has the most signicant positive effect, followed by EFB addition, on the specic biogas production rate and methane production. However, at xed inoculum level of 150 mL, the increasing POME volume within the range of 50100 mL had negative effects on both. Too much increment in temperatures also could have deleterious effects. From the response surface plots (Fig. 3), the responses increased upon increasing the temperature and EFB addition, but decreasing with POME volume. At considerably high inoculum, moderate levels of temperature and POME with high level of EFB may be favorable for biogas and methane production. By applying multiple regression analysis on the experimental data, the second-order polynomial equation can be developed to represent the specic biogas production rate (Eqn 4) and methane production

(Eqn 5): y1 = 0.06375 + 0.00703x1 0.00074x2 + 0.00016x3 0.000008x1 x2 + 0.000026x1 x3 + 0.000013x2 x3 0.000079x 1 2 + 0.000005x 2 2 0.000111x 3 2 y2 = 21.3 + 2.42x1 0.145x2 + 0.026x3 0.00254x1 x2 + 0.0221x1 x3 + 0.01x2 x3 0.0275x 1 2 + 0.0006x 2 2 0.075x 3 2 (5) (4)

Figure 3. Response contour and surface plots for (a) specic

biogas production rate and (b) methane percentage using EFB as a co-substrate. This gure is available in colour online at www.apjChemEng.com.

The predicted optimum levels of temperature, POME volume, and EFB addition as shown in Table 1 were obtained by applying regression analysis to Eqns 4 and 5. The optimum values were predicted at 43 C, 50 mL POME volume, and 6 g of EFB addition to obtain about 0.0597 m3 / kg COD per day of specic biogas production rate. For methane, the optimum values were predicted at 44 C, 50 mL POME volume, and 6 g of EFB addition to obtain about 26.5% of methane. The values of the adjusted coefcient of determination, R 2 , at 94.3 and 93.5%, respectively, indicate good agreement between experimental and predicted values. By performing multiple response optimization, in which both responses, specic biogas production rate and percentage of methane, were taken into consideration, the optimum conditions were predicted at operating temperature 47.8 C, 50.4 mL POME volume, and 5.7 g EFB addition to obtain about 0.0574 m3 / kg COD per day of specic biogas production rate and 25.6% methane. Experiments were carried out to verify this as shown in Table 3. The error percentage between experimental and predicted values which are around 5% for methane composition and 12% for specic biogas production rate suggests that the model could predict well the responses at optimum conditions. In the process of degrading POME into methane, carbon dioxide, and water, there is a sequence of reactions involved: hydrolysis, acidogenesis (including acetogenesis), and methanogenesis. Hydrolysis is where complex molecules (i.e. carbohydrates, lipids, proteins) are converted into sugar, amino acid, etc. In the acidogenesis step, acidogenic bacteria will break down these sugar, fatty acids, and amino acids into organic acids

Table 3. Summary for total biogas production and CH4 composition using optimum condition.

Specic biogas production rate (m3 / kg COD per day) Sample 1 2 Total biogas produced (mL) 475.88 533.35 Experimental value 0.0512 0.0573 % Error 12.1 0.17

Methane composition (%) Experimental value 26.8 27.4 % Error 4.48 6.57

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which mainly consist of acetic acid (from acetogenesis) together with hydrogen and carbon dioxide. Hydrogen and carbon dioxide will be utilized by hydrogenotropic methanogens while acetic acid and carbon dioxide will be utilized by acetoclastic methanogens to give methane as the nal product.[6] The low percentage of methane in this study as compared to theoretical value of biogas (60% CH4 , 40% CO2 ) is possibly due to the process being conducted in a batch mode. As there is no continuous inow and outow of the organic material, the accumulation of high volatile fatty acid concentration will cause a drop in pH inside the digester which inhibited methanogenesis.[31] In fact, methanogenic bacteria grow much slower than acidogenic bacteria and are extremely pH sensitive (pH 6.87.4 optimum).[32] If an excess of organic material is fed to a digester, the acidogens will grow rapidly and produce an excess of volatile acids. This can lead to increased acid concentrations. As the methanogens cannot keep up with this change and degrade acids as fast as they are generated, the methane produced will be lower. If methanogenic activity is under steady state, the CO2 gas produced will also be converted into methane. Optimal operation occurs when the methanogens use all the acids at approximately the same rate as the acidogens produce them. The need to keep the right temperature is critical as the effect of organic substrates on methanogenic activity is very much dependant on maintaining the right operating temperature.[33,34] Generally, there are three distinct operating temperature ranges of anaerobic digestion process to occur for biogas production and each range of temperature will have different type of bacteria that can work for digestion process.[3] Mesophilic bacteria are optimally active at the temperature range of 3243 C, thermophilic bacteria at the range of 4960 C, and psychrophilic bacteria at a room temperature of about 32 C and below. The temperature in the anaerobic digestion process must not be lower than 1720 C, or otherwise the digestion process will slow down or completely stop. In the palm oil mill processing system, the efuent is discharged at relatively high temperatures of around 8090 C,[35] making it feasible to perform the anaerobic process either in mesophilic or thermophilic temperatures. Methanogenesis is the rate limiting step in anaerobic digestion of POME. In a batch process with an organic removal efciency of 35% associated with a 91% decrease in the bacterial 16S rRNA gene concentration, several bacterial species such as Fusibacter-related, Clostridium-like, and Syntrophuslike organisms, appear to be responsible for acidogenesis or syntrophic acid degradation, while both hydrogenotrophic and aceticlastic methanogens appear to have been involved in the methanogenesis with the acidogenic products.[36] As such, conventional anaerobic digesters require large reactors and long retention
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time to ensure complete digestion of treated inuent. High-rate anaerobic bioreactors have been proposed to reduce reactor volume, shorten retention time as well as capture methane gas for utilization.[6] Another strategy is in the application of adsorbent such as natural zeolite, which has reportedly promoted the organic matter removal by 1720% more than control without zeolite and with an increase in daily methane production by 11.130.8%. It is demonstrated that when the adsorbent is added, the digester does not require frequent cleaning.[37] In our study, even though high CO2 content have been produced, further optimization need to be carried out with regards to operating temperature and co-substrate addition such that methanogenic activity can be increased to convert the CO2 gas into methane. Thermal treatments and adsorbent application together with semicontinuous or continuous mode can be used to enhance the efciency of anaerobic digestion.[37,38] Anaerobic digestion method would denitely give benets of recovery of methane gas for renewable energy as well as reducing the GHG emission and better management of POME.

CONCLUSION
The study veried the effects of temperature, inoculum, POME, and co-substrate addition such as EFB and palm kernel in an anaerobic digestion for the production of biogas and methane. Using the BoxBehnken statistical experimental design, the optimal conditions to obtain 25.6% methane has been predicted and veried at a temperature of 47.8 C, 50.4 mL POME volume, and 5.7 g EFB addition. Further optimization work is required to enhance methanogenic activity to improve methane yield.

Acknowledgement
The authors would like to thank Universiti Teknologi Petronas for the research facilities to carry out the study.

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