Accepted Manuscript

Co-composting of palm empty fruit bunch and palm oil mill effluent: Microbial diversity
and potential mitigation of greenhouse gas emission

Yuvaneswaran Krishnan, Cassendra Phun Chien Bong, Nadia Farhana Azman,

Zuriati Zakaria, Nor’Azizi Othman, Norhayati Abdullah, Chin Siong Ho, Chew Tin Lee,
Sune Balle Hansen, Hirofumi Hara
PII: S0959-6526(16)31273-2
DOI: 10.1016/j.jclepro.2016.08.118
Reference: JCLP 7910

To appear in: Journal of Cleaner Production

Received Date: 10 January 2016

Revised Date: 11 August 2016
Accepted Date: 23 August 2016

Please cite this article as: Krishnan Y, Bong CPC, Azman NF, Zakaria Z, Othman N, Abdullah N, Ho
CS, Lee CT, Hansen SB, Hara H, Co-composting of palm empty fruit bunch and palm oil mill effluent:
Microbial diversity and potential mitigation of greenhouse gas emission, Journal of Cleaner Production
(2016), doi: 10.1016/j.jclepro.2016.08.118.

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 ACCEPTED MANUSCRIPT

Co-composting of palm empty fruit bunch and palm oil mill effluent: Microbial diversity

and potential mitigation of greenhouse gas emission

Yuvaneswaran Krishnana,b§, Cassendra Phun Chien Bongc§, Nadia Farhana Azmana, Zuriati

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Zakariaa, Nor’Azizi Othmand, Norhayati Abdullaha, b, Chin Siong Hoe, Chew Tin Leec, Sune

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Balle Hansenb, and Hirofumi Haraa,*

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a
Department of Environmental Engineering and Green Technology, Malaysia-Japan

International Institute of Technology (MJIIT), Universiti Teknologi Malaysia (UTM), Kuala

Lumpur, Malaysia
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b
UTM Palm Oil Research Center, UTM, Kuala Lumpur, Malaysia
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Faculty of Chemical & Energy Engineering, UTM, Johor Bahru, Malaysia
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d
Department of Mechanical Precision Engineering, MJIIT, UTM, Kuala Lumpur, Malaysia
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e
UTM Low Carbon Asia Research Centre, Faculty of Built Environment, UTM, Johor Bahru,
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Malaysia.

§: Both authors contributed equally to this work.

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Corresponding author: Hirofumi Hara

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E-mail: hhara@utm.my
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Tel: (+60) 3-2203-1264; Fax: (+60) 3-2203-1266

Department of Environmental Engineering and Green Technology, Malaysia-Japan International
Institute of Technology, University Teknologi Malaysia, Jalan Sultan Yahya, 54100, Kuala
Lumpur, Malaysia

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ABSTRACT

The identification of microbial diversity is important to speed up the co-composting process of

EFB and POME from the palm oil mills, as an effective and cost-efficient way, to treat the

residues and reducing the greenhouse gas emission. This study was conducted to identify the

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microbial diversity from EFB-POME co-compost and to evaluate its potential mitigation of

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greenhouse gas emission. The physiochemical properties such as temperature (°C), pH and

moisture content of the EFB-POME co-compost were measured. The microbial diversity was

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identified by 16S rRNA and 18S rRNA gene metagenomic sequencing analysis. The

temperature, pH and moisture content recorded for the surface compost and inside compost were

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30 °C, 7.43 and 58.76 %, and 45 °C, 7.94 and 60.56 %, respectively. Based on the 16S rRNA
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gene sequencing for the identification of bacteria, the dominant genera in the surface compost

were Nitriliruptor, Delftia, Filomicrobium, Steroidobacter, and Ohtaekwangia; the dominant

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genera in the inside compost were Steroidobacter, Nitriliruptor, Anaeromyxobacter,

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Filomicrobium, and Truepera; and the dominant genera in the POME were Parabacteroides,
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Bellilinea, Levilinea, Smithella, and Prolixibacter. Based on the 18S rRNA gene sequencing for

the identification of fungal, the dominant genera in the surface compost were Remersonia,
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Inonotus, Kluyveromyces, Chaetomium, Thermomyces, and Candida; the dominant genera in the

inside compost were Remersonia, Inonotus, Saccharomycopsis, Chaetomium, and Saccobolus;

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and the dominant genera in the POME were Kluyveromyces, Inonotus, Kazachstania, Candida,
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andCystofilobasidium. The co-composting of EFB-POME is estimated to reduce up to 76 % of

greenhouse gas emission by avoiding the methane gas emission from the EFB open dumping and

POME treatment ponds, as well as from the replacement of chemical fertilizers. The microbial

diversity identified from EFB-POME compost and POME may enhance the effectiveness of co-

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composting due to the ability to synthesize ligninolytic and cellulolytic enzymes and reduce the

emission of greenhouse gases from the oil palm plantations.

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1. INTRODUCTION

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The palm oil industry is the fourth largest revenue-generating sector in Malaysia (Chua, 2010)

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with oil palm plantations covering 15 % of Malaysia’s land area in 2013 (MPOB, 2014). The

production of 19 Mt of crude palm oil (CPO) in 2013 (MPOB, 2014) resulted in the generation

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of over 83 Mt of solid biomass by the Malaysian palm oil industry in 2013. This volume is
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projected to increase to 85–110 Mt by 2020. Similarly, palm oil mill effluent (POME) volumes

are expected to increase from 60 Mt in 2012 to 70–110 Mt by 2020 (Melssen, 2013).

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Sustainability of the palm oil sector is becoming a key area for the economic development of
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Malaysia and South East Asia (Hansen et al., 2015).

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Empty fruit bunches (EFB) and POME compose the largest residual fractions from palm

oil mills. EFB is generated after extracting palm oil off fresh fruit bunches, while POME is
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wastewater generated mainly from sterilization and pressing of the fruits. Untreated EFB is

commonly used as mulch in the surrounding oil palm plantations and provides nutrients and
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carbon to the soil as it decomposes. In rare cases, EFB is left to decompose in dump sides,
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potentially resulting in anaerobic conditions with large emissions of the strong greenhouse gas

(GHG) methane; or it may be burned without energy recovery to minimize the bulk before

landfilling. EFB from some mills is used for energy recovery, often following palletization.

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POME has high content of suspended and dissolved organic matter and has a chemical oxygen

demand (COD) of about 15,000-100,000 mg /L, averaged at 51,000 mg/L (Madaki & Seng,

2013). It is mostly treated using open lagoon technology, which generates large quantities of

methane. Approximately half of the total GHG emission from conventional palm oil production

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is from methane emission. While a number of palm oil mills are implementing methane capture

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plans, many are hesitating due to the capital and operational costs involved. Only a few of the

mills that lack a methane capture facility, take advantage of the energy content of the gas to

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generate heat or electricity. Most often methane is flared without energy recovery to convert it

into the less harmful form–carbon dioxide.

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Composting can be defined as controlled biological decomposition of organic substrates
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carried out by successive microbial populations combining both mesophilic and thermophilic

activities. Co-composting is controlled degradation of organic substrates using more than one
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material (Rosnani et al., 2014). Co-composting of POME and EFB is a cost-efficient way to treat
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the palm mill residues while recovering valuable nutrients and generating a value added product
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for fertilizer application in the plantations or as a commercial product. It was observed that the

addition of urea as a source of nitrogen to composting mixtures rich in lignocellulosic residues

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favored the degradation of cellulose and hemicellulose, mainly by reducing the composting time

compared to mixtures of lignocellulosic materials alone (Monica et al., 2014). EFBs composted
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alone may have disadvantages related to increased time of composting and poor fertilizer value
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of the compost produced, because of its high lignocellulosic content. The addition of POME

provides nitrogen that favors the degradation of cellulose and hemicellulose in EFB and reduces

the composting time. Singh et al. (2010) have suggested that co-composting of EFB and POME

is a viable waste management option in palm oil mills. Hayawin et al. (2012) reported that

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vermicomposting of EFB and POME can accelerate the mineralization of nutrients and suggested

that the palm oil sludge can be utilized as an efficient soil conditioner. Mohammad et al. (2013)

studied an effective co-composting process of EFB with POME identifying optimized process

parameters and a compatible multi-enzymatic fungal system.

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Understanding the involvement of various microorganisms is essential to determine the

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effectiveness of composting and to produce high quality compost. As a result of the humid,

tropical climate with high temperatures, abundant sunshine and rainfall all year round, Malaysia

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has a large microbial diversity including both fungi and bacteria.

This study aimed to identify the aerobic and anaerobic microorganisms from the EFB-

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POME co-compost that are capable of degrading complex organic compounds. This detailed
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knowledge on the active microorganisms will enable further research to improve the speed of the

composting process and the quality of the final compost through the addition of specialized
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microbial species. The accelerated composting time and improved compost quality can make
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composting a more attractive option for palm oil mills, with economic and environmental
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benefits. The study also aimed to determine the importance of composting to reduce the emission

GHG, which is generated abundantly from open EFB dumps and POME treatment ponds.
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2. MATERIALS AND METHODS

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2.1 Collection of sample

The compost samples of EFB and POME were taken at week 6 of the composting process from

Felda Maokil, Labis, Johor. The compost windrow was 1 m in height and made up of with the

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ration of 40 t EFB: 120 t POME. Turnover of the compost pile was done once in every 3 days

with special vehicle to provide aeration. A total of 5 kg compost material was collected and

sampled at its surface (surface compost) and 0.5 m inside (inside compost). These samples were

used for microbiological studies and physicochemical analysis and stored at -80°C for any

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further analysis. Microbiological studies were also performed on POME sample.

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2.2 Moisture content

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10 g of compost samples was dried in an incubator at 105 °C. Dry weights of the samples were

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taken until each sample reached a constant weight. The percent moisture was expressed as shown
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in Eq. (1):
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Moisture % = (W1 W2)/ W1 × 100 (1),

where, W1 = Weight of soil before oven drying; and W2 = Weight of soil after oven drying.
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2.3 pH of soil sample

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The compost samples were dried at 105 °C. 2.5 g of compost was dissolved in 25 mL of distilled

water and vortexed for 5 min at 120 rpm. pH was measured using a pH meter.
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2.4 16S rRNA and 18S rRNA Sequencing

The 16S rRNA and 18S rRNA gene sequences from the genomic DNA isolated from the

compost were used to identify microbial strains in the samples. DNA was extracted using Power

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Soil DNA Isolation Kit (MO BIO Laboratories, California, USA). The extracted genomic DNA

was observed by agarose gel to check its integrity and concentration. Qubit 2.0 DNA Kit (Life

Technologies, Carlsbad, CA, USA) was used to precisely quantify the genomic DNA for PCR

reaction. The universal primers of MiSeq sequencing platform, V3-16S primer, 341F (5′

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CCTACGGGNGGCWGCAG 3′), and 805R (5′ GACTACHVGGGTATCTAATCC 3′), were

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used to identify the bacterial strains. Fungal diversity was identified using the universal 18S

rRNA fungal gene primers NS1 (5′ GTAGTCATATGCTTGTCTC 3′) and Fung (5′

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ATTCCCCGTTACCCGTTG 3′). The PCR products were tested by agarose gel electrophoresis

and recovered using Sangon agarose recovery kit (Sangon Biotech, Shanghai, China).

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Subsequently, the recovered PCR products were quantified and mixed in 1:1 ratio based on DNA
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concentration using Qubit 2.0 Fluorometer (Life Science). Metagenomic sequencing analysis

was conducted using MiSeq (Illumina) at Sangon Biotech Co., Ltd. (Shanghai, China).
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The sequence data from this research have been deposited in the NCBI’s Sequence Read Archive
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database (http:// www.ncbi.nlm.nih.gov/sra) with the temporary submission ID of

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(SUB1750450).
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3. RESULTS AND DISCUSSIONS

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3.1 Physicochemical Analysis

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The results of the physicochemical analysis are tabulated in Table 1. The magnitude of all the

parameters tested was different for the samples from the surface compost compared to inside

compost. The moisture content of the surface compost and inside compost was 58.76% and

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60.56% respectively. The addition of POME caused the high moisture content of the compost.

However, the slightly higher moisture content of inside compost was due to the less exposure to

sunlight and external environment when compared to the surface compost.

The increase in temperature of the compost was due to the microbial activities, which

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generates heat. The inside compost temperature was 45 °C, probably due to the activity of

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thermophilic bacteria, while the surface compost temperature was 30 °C, with the main activity

being from mesophilic bacteria. The slightly alkaline pH averaging at 7.7 might be due to the

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production of ammonia by the biochemical reaction of nitrogen-containing materials during

composting (Baharuddin et al., 2010).

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3.2 16S rRNA and 18S rRNA Sequencing
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Table 2 and Table 3 show the number of reads obtained for the three samples (surface compost,
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inside compost, and POME) for 16S rRNA and 18S rRNA metagenomic sequencing analysis.
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The filtered number of reads shows the final number of reads obtained. The results of 16S rRNA

and 18S rRNA sequencing were valid as the total number of filtered reads exceeded 10,000,
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which is sufficient to identify the microbial diversity. Based on the α-diversity index report in

Table 3 for bacterial diversity and Table 4 for fungal diversity, OTUs (Operational Taxonomic
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Unit), where one OTU denotes a sequence with an identity value equal to or higher than 97%,
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showed higher number of fungal diversity in surface but opposite in inside of EFB-POME

compost.

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3.3 Bacterial Diversity

Fig. 1 shows the results of 16S rRNA sequencing identifying the distribution of bacterial genera

in the surface compost, inside compost and POME. The dominant bacterial genera in the surface

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compost were identified as Nitriliruptor (7.9 %), Delftia (4.6 %), Filomicrobium (3.2 %),

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Steroidobacter (2.9 %), and Ohtaekwangia (2.1 %). The dominant bacterial genera in the inside

compost were identified as Steroidobacter (5.0 %), Nitriliruptor (3.9 %), Anaeromyxobacter (2.9

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%), Filomicrobium (2.5 %), and Truepera (1.4 %). The dominant bacterial genera in POME

were Parabacteroides (5.0 %), Bellilinea (4.7 %), Levilinea (3.5 %), Smithella (3.5 %), and

Prolixibacter (2.5 %).

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There were two thermophilic isolates belonging to a new species of a novel genus

Truepera from the phylum Deinococcus, which were identified from hot spring runoffs on the
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Island of São Miguel in the Azores (Albuquerque et al., 2005). The study showed that the
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bacteria from Truepera genus are thermophilic in nature. These bacteria are predicted to be one
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of the candidates contributing to the higher temperature of the inside compost (45°C). The

bacteria from the genera Delftia and Filomicrobium, found mainly in the surface compost of
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EFB-POME, are gram negative and aerobic in nature. Bacteria from the genus

Anaeromyxobacter identified in the inside compost sample, are gram negative and facultative
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anaerobic in nature. This showed that there are more anaerobic bacteria in the inside compost
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than the surface compost, as the inside compost lacks oxygen for aerobic respiration.

One of the bacterial genus identified from the inside compost is Bacillus, a facultative

anaerobe. In a study done by Acharya et al. (2012), Bacillus subtilis, a potent thermophilic

cellulolytic bacterium, was isolated from compost piles in Nepal. The cellulase enzyme activity

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of B. subtilis was optimum at pH 7.2 at 50 °C. This is consistent with the current study where

Bacillus was identified in the inside compost sample, with almost similar pH and temperature

conditions of 7.94 and 45 °C, respectively; these conditions being optimal for the breakdown of

cellulose, which constitutes 45.8 % of EFB-POME compost. Besides this, in a study by Sabiha et

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al. (2015) naturally ligninolytic bacterial flora from the Dhamdha agro-fields of Bhilai-Durg

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using an industrial lignin residue, Bacillus sp. was isolated and showed positive ligninolytic

enzyme activity ( laccase, manganese peroxidase and lignin peroxidase). This result concluded

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that Bacillus sp. is able to degrade lignin substrate, the second most abundant waste material in

the world. EFB-POME compost consists of 28.3 % lignin. Therefore, based on the two previous

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studies, it is strongly predicted that Bacillus species contributes highly to the breakdown of
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lignin and cellulose in the EFB-POME compost.

All the genera identified in POME are anaerobic in nature as POME is an oily liquid that
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is treated in anaerobic open pond system that favors the growth of anaerobic bacteria. Bellilinea
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is one of the dominant genera identified in POME. Yamada et al. (2007) identified Bellilinea
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caldifistulae gen. nov., sp. nov., an obligate anaerobe, in the thermophilic sludge of sewage and

domestic garbage. POME showed the highest number of filtered reads of 16S rRNA sequencing,
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suggesting that it has the highest bacterial diversity amongst the three samples. Thus, the

addition of POME to EFB contributes to effective co-composting due to the increase in bacteria
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that can degrade cellulose, hemicellulose, and lignin.

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3.4. Fungal Diversity

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Fig. 2 shows the result of 18S rRNA sequencing to identify the fungal diversity in the surface

compost, inside compost and POME. The dominant fungal genera in the surface compost were

identified as Remersonia (33.9 %), Inonotus (19.4 %), Kluyveromyces (9.1 %), Chaetomium (5.0

%), Thermomyces (4.1 %), and Candida (4.1 %). The dominant fungal genera in the inside

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compost were identified as Remersonia (52.5 %), Inonotus (13.6 %), Saccharomycopsis (3.3 %),

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Chaetomium (3.3 %) and Saccobolus (3.3 %). The dominant fungal genera in POME were

Kluyveromyces (57 %), Inonotus (21.1 %), Kazachstania (15.7 %), Candida (2.1 %), and

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Cystofilobasidium (2.1 %).

The genus Remersonia was identified from both surface and inside of the EFB-POME

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compost. Keith et al. (1997) isolated a thermophilic mold from compost that belongs to the genus
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Remersonia. The study revealed that the mold showed thermotolerant growth. This could also

explain the greater abundance of Remersonia in the inside compost (52.5 %) compared to surface
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compost (33.9 %) in our experiments, as the temperature inside compost was higher compared to
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surface temperature. Another dominant fungal genus identified from the surface compost, inside
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compost and POME was Inonotus. Inonotus sp. SP2, a new species of white rot fungi was

identified in southern Chile and was characterized as producing extracellular enzymes with
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ligninolytic and cellulolytic activity, including manganese peroxidase, lignin peroxidase, and

laccase (Garees et al., 2012). This supports our observation also as we found Inonotus in
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abundance in EFB-POME composts and POME, where it may assist to break the complex lignin
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and cellulose, which act as a source of carbon for energy production.

Kluyveromyces is the major fungus found in POME and one of the dominant fungus in

the surface compost of EFB-POME. A recent study analyzed the complete genome of the yeast

Kluyveromyces marxianus proving that it is a thermotolerant fungus (Noppon et al., 2015).

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POME is released at high temperature from the palm oil mills before entering and cooling down

in the open ponds, possibly explaining why this thermotolerant fungus is abundant in POME

(57%). Another fungal genus with interesting characteristics that has been identified in this study

is Thermomyces. Singh et al. (2003) reported that the fungal species Thermomyces lanuginosus is

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capable of producing high levels of cellulose free L-xylanase and low extracellular levels of other

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hemicellulases involved in hemicellulose hydrolysis. Therefore, the fungus from this genus is

believed to play an important role for the degradation of hemicellulose in EFB-POME compost.

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EFB comprises of 17.1 % of hemicellulose (Baharuddin et al., 2010).

Another important genus identified in all the three samples is Candida. Jantaporn et al.

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(2014) isolated a thermotolerant yeast in Thailand known as Candida easanensis JK-8. This
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yeast can tolerate up to 40 °C and has the potential to synthesize cellulase and xylanase enzymes.

Candida species is capable of degrading cellulose and hemicellulose, including xylan, a

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hemicellulose abundant in plant cell walls, in the EFB-POME co-compost.

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3.5 Benefits of the co-composting of EFB-POME

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Co-composting of EFB and POME can offer economic and environmental benefits to

palm oil mills. In terms of economic feasibility, composting seems to offer greater advantages
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than the installation of gas capturing and power generation units from the anaerobic digestion
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(AD) of POME. Nowadays, in the rapid developing countries like Malaysia and Thailand,

utilization of POME for biogas to generate electricity is getting popular. The advantages of self-

generating electricity and sale of excess electricity to the national grid are promising and can

over-shadowed the use of POME to be co-composted with EFB. However, the capital cost for

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biogas utilization is relatively high, especially for small and medium scale plantation. For

example, based on the CDM case study in the Felda Maokil plantation in Malaysia (2009), the

crude palm oil factory has a capacity of 40 t FFB /hr and generation of up to 0.62 m3 POME per t

FFB. The plant has two anaerobic ponds with a capacity of 46,500m3 per pond and the retention

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time of 133 d. The heat and electricity generation system consists of three units of boiler, two

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units of turbine and two units of diesel generator sets. Their report showed an overall cost of

MYR 2,969, 250 for the total investment on construction and equipment for the biogas capturing

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system, and another MYR 1,681,000 the for biogas to electricity generation set. In total, such a

medium scale biogas project at Felda Maokil gives a capital cost of close to MYR 5M.

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In addition to the high investment cost, the utilization of biogas to renewable energy is
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also dependent on the connection to grid. Burning of CH4 into CO2 is a highly damaging issue as

it releases additional CO2 and wasting energy source. Joint venture between the local plant
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operators with private or foreign investment could alleviate the financial burden. The biogas
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project in Felda Maokil has involved the cooperation with counterparts from the United
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Kingdom of Great Britain and Northern Ireland. In addition, the local government could

implement more financial schemes such as the feed-in-tariff (FiT), soft loan, low interest rate to
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attract more investors. Government could provide adequate infrastructure for the efficient grid

connection to ensure the plant owner is eligible to claim FiT by supplying energy to the grid.
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In terms of environmental benefits, the co-composting of EFB and POME can lead to the
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reduction in GHG emission due to the avoided emission from the open-dumping of EFB,

uncaptured CH4 from the POME digestion and production of chemical fertilizer. It also offers

additional savings in terms of C sequestration through the use of compost to land and crops (Saer

et al., 2013).

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The GHG emission and carbon savings from the conventional practices and the co-

composting of EFB and POME were calculated based on the data from the literature. In the oil

extraction process, about 1 t of crude palm oil (CPO) is extracted from 5 t of fresh fruit bunches

(FFB), leaving 0.23 t of EFB and 0.65 t of POME as residues per t of FFB processed (Yusoff,

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2006). From the work by Stichnothe and Schuchardt (2011), IPCC model (2006) was used to

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calculate the emission result for the LCA of oil palm system. Their work used the

recommendation value by IPCC (2006) for garden and park waste disposal as there is no

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emission data currently available for the EFB dumping site. The model was based on the GaBi

and Ecoinvent database, where 80% of anaerobic condition is assumed as the worst case scenario

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as suggested by the IPCC model (2006) for garden and park waste. It is estimated that if EFB is
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open dumped, it would generate about 230 kg CO2-eq/t FFB in the worst-case scenario

(assuming 80 % anaerobic conditions), and 140 kg CO2-eq/t FFB will be released from the
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POME pond treatment system. However, during the co-composting of EFB and POME, a
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significant reduction of 76 % (460 reduced to 110 kg CO2-eq/t FFB) of the total GWP was
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achieved due to the avoidance of methane emission from the EFB dumping sites and POME

ponds.
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In addition to the potential for mitigating GHG emission, co-composting technology also

offers potential for GHG savings by avoiding fossil fuel consumption needed to produce
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chemical fertilizers. The final product from the co-composting of EFB and POME is reported to
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have a nutrient value of 2.2 % N, 1.28 % P, and 2.79 % K (Baharuddin et al., 2009). Hence, 1 t

of EFB compost is reported to be equivalent to the chemical fertilizer, i.e., 47.8 kg of N fertilizer,

25.6 kg P fertilizer, and 46.5 kg K fertilizer (Chiew and Shimada, 2013).

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As seen in Table 6, substantial GHG emission can be mitigated through the utilization of

EFB-POME compost, especially in terms of N fertilizer, the most important player in the

fertilizer market. The application of compost can help to build up soil organic matter, which

contributes to the improved soil structure and porosity, increases the availability of nutrients, and

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improves the water holding capacity of the soil for better plant and soil health (Bot and Benites,

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2005).

There is a general concern on the health issue of EFB-POME compost due to the

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presence of heavy metals in POME. The POME has a high COD of 15,000 – 100,000 mg /L and

metals concentration (e.g. B 7.6 mg /L, Fe 46.5 mg /L, Mn 2.0 mg /L, Cu 0.89 mg /L and Zn 2.3

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mg /L) (Madaki and Seng, 2013). Co-composting of POME and EFB can serve as a clean
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technology by reducing the bioavailability of the heavy metals due to the mechanisms such as

metal binding, microbial immobilization and oxidation (Greenway and Song, 2002), and OM
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humification (Wong and Selvam, 2006) . The heavy metals can be concentrated along the
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composting process due to the loss of OM and water (Farrell and Jones, 2009a). Farrell and
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Jones (2009b) reported that the heavy metals concentrated along the composting process of

MSW is not available for plants and thus poses limited risk. By profiling the microbial
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community in the POME and EFB-POME compost, future research such as the use of specific

microbial species to increase the effectiveness of composting, e.g. identification of species with
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higher oxidation and immobilization of toxic heavy metals, can be incorporated to improve the
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cleanliness of EFB and POME co-composting.

Prolonged composting period seems to be a major hurdle to encourage composting

practices among oil palm plantation. The composting period of EFB and POME is reported to be

around 60 d (Baharuddin et al., 2009), which is economically not attractive. Only 30 % of the

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palm oil mills in Malaysia engage in some form of reuse or recycling of the palm oil mill

residues (Roslan et al., 2011). The identification and isolation of the microbes involved in the co-

composting of EFB and POME could provide novel and valuable insights into the mechanism

and help to improve the efficiency and efficacy of the composting process, such as shorter

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maturation time, higher process stability, and improved compost quality in terms of nutrient and

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carbon sequestration ability.

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4. CONCLUSION

In conclusion, the physicochemical properties such as temperature and pH influence the

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distribution of microorganisms in the surface compost, inside compost, and POME, as there are
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thermophilic, mesophilic, acidic, and alkaline bacteria in the EFB-POME co-compost. The

microbial diversity of EFB-POME co-compost and POME are promising, and may enhance the
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effectiveness of composting and its future applications. Many of the bacteria and fungus
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identified in the three samples synthesize enzymes that degrade lignin, hemicellulose, and
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cellulose, all of which are abundant in EFB and POME. To our knowledge, this is the first report

working on the profiling of the prokaryotic and eukaryotic microbial diversity in the co-compost
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of EFB and POME using pyrosequencing analysis.

The addition of specialized microbes to EFB-POME co-compost may help to reduce the
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magnitude of waste biomass generated from palm oil mills and provide crop fertilizers. Several
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bacteria and fungi have been isolated for further analysis to enhance the speed and quality of

composting to suit agricultural purposes. Furthermore, the co-composting of EFB-POME is

considered as a cleaner process as it is estimated to reduce up to 76 % of the GHG emission by

avoiding the emissions generated from the open dumps of EFB and POME treatment ponds. This

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method offers both the economic and environmental benefits, further research would help in its

effective implementation.

5. ACKNOWLEDGEMENT

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The authors gratefully acknowledge the funding support for this work provided by

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Ministry of Education, Malaysia and Universiti Teknologi Malaysia (UTM) under the Grant Vot

number of R.J1300000.7301.4B145 and Japan International Cooperation Agency (JICA) under

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the scheme of SATREPS Program (Science and Technology Research Partnership for

Sustainable Development) for the project entitled “Development of Low Carbon Scenario for

Asian Region”.
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Table 1. Physico-chemical Parameters of EFB-POME Compost from FELDA Maokil, Johor.

Sample Temperature (°C) pH Moisture Content (%)

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Surface Compost 30 7.43 58.76

Inside Compost 45 7.94 60.56

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POME 28 7.96

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Table 2. Number of Reads for 16S rRNA Sequencing.

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Sequence Out target Chimeras Filtered

Sample
Number Number Number Number
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POME 40,794 38 9,053 31,703

Surface Compost 31,712 1 1,535 30,176

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Inside Compost 32,551 4 2,898 29,649

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Table 3. Number of Reads for 18S rRNA Sequencing.

Out Target
Sample Sequence Number Chimeras Number Filtered Number
Number

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POME 99,902 6 6,835 93,061

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Surface Compost 110,948 441 8,495 102,012

Inside Compost 85,945 294 4,615 81,036

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Table 4.The List of Alpha Diversity Index Cumulative Results for 16s rRNA Sequencing

Sample Sequence OUT Shannon ACE Index Chao Index Coverage

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Number Number Index

Surface 30176 2507 5.677 8018.545 5321.240 0.955

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Compost
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Inside 29649 4227 6.318 17129.480 10739.980 0.911

compost

POME 31703 6024 6.483 35967.740 18713.330 0.868

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Table 5.The List of Alpha Diversity Index Cumulative Results for 18s rRNA Sequencing

Sample Sequence OUT Shannon ACE Index Chao Index Coverage

Number Number Index

Surface 102012 3395 4.224 7520.280 6026.976 0.985

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Compost

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Inside 81036 2524 3.700 6315.630 4805.988 0.985

Compost

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POME 93061 2434 3.082 6711.396 4788.397 0.987

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Table 6. Savings of kg CO2 eq through the application of 1t of EFB compost.

Chemical Composition of GHG emission from production Savings from replacement

fertilizer element (kg) of 1kg chemical fertilizer (kg through EFB compost (kg

type (Chiew & CO2 eq/ kg element) CO2 eq/kg element)(taking

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Shimada, 2013) (Boldrin et al., 2009) the minimum value)

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N
47.8kg N 4.7-13.0 224.66 (47.8 * 4.7)
fertilizer

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P
25.6kg P 0.5-3.1 12.8 (25.6 * 0.5)
fertilizer

K
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46.5kg K 0.4-1.5 18.6 (46.5 * 0.4)
fertilizer
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Nitriliruptor
Delfia

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Filomicrobium
Steroidobacter

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Ontaekwangia
Others

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A
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Steroidobacter
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Nitriliruptor
Anaeromyxobacter
Filomicrobium
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Truepera
Others
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Parabacteroides
Bellilinea

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Levilinea
Smithella
Prolixibacter

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Others

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Fig. 1. a)of bacteria in genus level for surface compost; b) distribution of bacteria in
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genus level for inside compost c) distribution of bacteria in genus level for POME.
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Remersonia
Inonotus
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Kluyveromyces
Chaetomium
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Thermomyces
Candida
Others

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Remersonia
Inonotus
Saccharomycopsis

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Chaetomium
Others

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B
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Kluyveromyces
Inonotus
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Kazachstania
Candida
Others
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Fig.2. a) distribution of fungus in genus level for surface compost; b) distribution of

fungus in genus level for inside compost; c) distribution of fungus in genus.

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