Volume 1 Number 1 (2017) 1 – 9

Sustinere
Journal of Environment and Sustainability
Article history: Print ISSN 2549-1245
Received 01 December 2016 Online ISSN 2549-1253
Received in revised form 01 January 2017 http://sustinerejes.com
Accepted 01 February 2017 E-mail: sustinere.jes@iain-surakarta.ac.id
Published 01 June 2017

Isolation and characterization of lignocellulolytic

microbes from oil palm empty fruit bunches (EFB)
Ariana1,*, Krishna P. Candra2
1 Institut Agama Islam Negeri (IAIN) Surakarta, Sukoharjo, Indonesia
2 Universitas Mulawarman, Samarinda, Indonesia

Abstract. Oil palm empty fruit bunches (EFB) is one of the major by-products of palm oil
production. This lignocellulosic biomass is commonly used as a fertilizer at oil palm
plantations. Unfortunately, the composting process of EFB is very slow. This study aimed to
identify potential lignocellulosic microbes isolated from EFB. This information is essential
for improving EFB biodegradation process by reducing the decomposition time. Samples of
approximately 6, 12, and 24-month-old EFB were obtained from two palm oil mills in East
Kalimantan, Indonesia. The isolation of lignocellulytic microbes utilized selective medium
cellulose congo red agar (CCRA) while its characterization used lignin agar (LgA) and oil
palm empty fruit bunches agar (EFBCRA). As much as 430 isolates were successfully
collected and 12 of them exhibited promising capability to synthesize lignocellulolytic
enzyme, the key for FEB degradation.

Keywords: Lignocellulose, lignocellulases, microbe characterization, oil palm empty fruit

bunches (EFB)

1. Introduction
Palm oil production process generates approximately 45 % of solid waste consist of
manure, dust, fiber, fruit shell, and oil palm empty fruit bunches (EFB). EFB constitutes nearly
50 % of those solid wastes. This lignocellulosic biomass, composed of cellulose, hemicellulose,
lignin and other extractive materials, has great potential to be processed as bioenergy, fertilizer
and other products with higher economic value. Because of the high content of lignin of up to 21
%, EFB carries similar hardness as wood (Kumar et al., 2009). EFB also contains energy of
18795 kJ kg-1, makes it one of the choices for a source of renewable energy (Prihandana &
Hendroko, 2008). At the palm oil plantation itself, EFB is commonly applied as fertilizer; it
biodegrades and becomes compost that helps to enrich the soil.

Degradation of lignocellulosic biomass, with high lignin concentration, requires a

pretreatment process that can be performed through a physical, chemical, mechanical, as well as
biological method using microbes or enzymes (Atlas & Bartha, 1986). The purpose of
pretreatment is to remove or to break down lignin structure to increase the digestibility of
cellulose fraction so that the decomposition process is more efficient. Natural decomposition
process of lignocellulosic biomass, instead of by a certain microorganism, is performed by a
consortium of many types of microorganism.

*Correspondingauthor. E-mail: ariana@iain-surakarta.ac.id

DOI 10.22515/sustinere.jes.v10i23.2 x

Sustinere | Volume 1 Number 1 (2017) 1

 White rot fungi, for example, is the fungi that capable of degrading lignocellulosic biomass
by producing extracellular enzymes that destroy cellulose and lignin. Cellulose and
hemicellulose are utilized by white rot fungi for growth substrate, while lignin is degraded
during nutrient (such as nitrogen, carbon, and sulphur) deficiency conditions (Hatakka, 2005).
Other examples are Phanerochaeta porigens and Phanerochaeta chrysosporium (Goenadi &
Away, 1993; Hatakka, 2005). Additionally, Trichoderma spp, Fusarium oxysporum, Aspergillus
niger, Penicillium citrinum, Rhizopus oryzae, Aspergillus nidulans are known as types of fungi that
mostly play role in the decomposition of lignocellulosic biomass (Gunam et al., 2010; Mukhlis et
al., 2013; Taherzadeh & Karimi, 2007; Tristianti et al., 2013). From bacterial microorganism,
Bacillus circulans and Sphingomonas paucimobilis are known to be able to produce
lignocellulolytic degrading enzymes (Kurakake et al., 2007) such as cellulases, hemicellulases,
and lignin-degrading enzymes.

Exploration of microbial diversity can be done in-situ at the waste location and ex-situ in the
laboratory. The information obtained from such exploration is essential to achieve an effective
EFB biodegradation (Baharuddin et al., 2009). However, little research has been done to explore
the diversity of lignocellulolytic-microbial inhabiting EFB. Studies on the decomposition of EFB,
especially in Indonesia, predominantly used non-indigenous microbes instead of isolates from
EFB itself (Andayani, 2011). Bio-activator is commonly used in the decomposition of cellulose
waste to accelerate the process (Suriadikarta et al., 2006) even though indigenous
microorganism (IMO) has a higher tolerance to the environmental conditions of its original
habitats (Oktavia, 2010). It has been proven that the use of IMO speeds up EFB
vermicomposting process (Azizah et al., 2014). The purpose of this study was to identify
potential lignocellulolytic microbes in EFB. This research will be used as basic information in
developing a better EFB biodegradation process by diminishing decomposition time.

2. Method
2.1. Screening of cellulolytic microbial
EFB was derived from Longkali palm oil mills (POM) and Samuntai POM PTPN XVI Tanah
Grogot, East Kalimantan, Indonesia. Samples from each location were divided into three
categories based on the age of EFB, i.e. 6, 12, and 24 months old. One gram of the sample was
added to 9 mL NaCl 0.85 % sterile and diluted up to 1010 time. The microbial screening used
pour plate method. As much as 0.5 mL sample from each dilution were poured onto the medium
of cellulose congo red agar (CCRA), modified from Kausar et al. (2012), that contained 0.5 g L-1
KH2PO4, 0.25 g L-1 MgSO4, 2.0 g L-1 Carboxy Methyl Cellulose (CMC), 15 g L-1 agar, 0.2 g L-1 congo
red, and 2.0 g L-1 gelatin. Samples were incubated at room temperature for 48 hours. Cellulolytic
microbial colonies were indicated by a clear zone around the colony.

2.2. Isolation and characterization of lignocellulolytic microbes

Colonies that have the highest cellulolytic index were subsequently regrown using CCRA.
The lignocellulolytic activity of selected isolates was then characterized using lignin agar (LgA)
and empty fruit bunch cellulose congo red agar (EFBCRA) in order to observe their capability to
degrade lignin and natural substrate (EFB powder).

The LgA consisted of 0.5 g L-1 KH2PO4, 0.25 g L-1 MgSO4, 2.0 g L-1 lignin, 15 g L-1 agar, and 2.0
g L-1 gelatin; while EFBCRA contained 0.5 g L-1 KH2PO4, 0.25 g L-1 MgSO4, 2.0 g L-1 EFB, 15 g L-1
agar, 0.2 g congo red, and 2.0 g L-1 gelatin. Cellulolytic index, lignin index, and lignocellulolytic
index are the ratio between diameters of the clear zone surrounding colonies with diameter of
the colonies. It illustrates the capability of microbes to degrade lignocellulosic materials.

2 Sustinere | Volume 1 Number 1 (2017)

 Microscopic morphology of bacteria and fungi was observed using electron microscope
with 100x and 400x magnification. For bacteria, the colonies were subjected to gram-negative
staining procedure prior to microscope observation.

3. Result and Discussion

3.1. Screening of cellulolytic microbial
This study successfully isolated 430 microbes from Longkali POM and Samuntai POM. The
isolation to obtain cellulolytic microbes used selective medium CCRA. The microbes grow on
this medium are only they that can use CMC, an amorphous type of cellulose substrate, as the
carbon source. CMC is hydrolyzed into oligosaccharide due to the activity of endo 1.4-β-
gluconate which breaks the bond of the β-1,4-glycosidic polymer of glucose (Meryandini et al.,
2009). In this experiment, all isolates were capable of cellulose degradation; indicated by the
presence of clear zone around the colonies. This zone evidence the capability of the respective
microbe to produce cellulose enzyme (Figure 1).

(a) (b)
Figure 1. (a) Microbe isolation: 1 is the colony, 2 is the clear zone around microbe colony; (b)
fungi isolation: 1 is the colony and the white spore, 2 is the clear zone around fungi
colony (isolation condition: CCRA medium, 25oC, 48 hours)

The screening result showed that colonies were dominated by fungi with distinct
morphological features of having (powder-like) spore that gradually amassed on the surface of
the medium (Figure 1b). The observation of fungi colonies under a microscope revealed the slim
and branched mycelium (Figure 2a). Previous studies reported that fungi can decompose
lignocellulosic materials more quickly and intensively than bacteria. The species of fungi (white
rot fungi) such as Phanerochaeta chrysosprorium, Phlebiaradiata, Tremetes versicolor,
Ceriporiopsis subvermispora have the ability to degrade lignocellulosic materials (Suriadikarta et
al., 2006).

Other colonies appeared to be irregularly shaped, velvety yellowish mucus (Figure 3a).
After gram-staining and microscope observation, isolates were revealed to be gram-negative
cocci bacteria (Figure 3b). Suriadikarta et al. (2006) reported that some types of bacteria such
as Pseudomonas spp, Bacillus spp, Streptomyces spp, Flavobacterium spp, Clostridium spp, spp
Thermonospora capable of lignocellulosic materials decomposition, even though not as fast as
fungi.

Sustinere | Volume 1 Number 1 (2017) 3

 (a) (b)
Figure 2. Microscopic morphology of fungi: (a) 100x magnification shows branched mycelium;
(b) 400x magnification shows reproductive organs, 1 is sporangium and 2 is
sporangiophore

(a) (b)
Figure 3. (a) Irregularly-shaped bacteria colony: 1 is the colony, 2 is the clear zone around the
colony; (b) the morphology of the bacteria colony from 400x magnification
microscope observation (isolation condition: CCRA, 25oC, 48 hours)

3.2. Isolation and characterization of lignocellulolytic microbes

The capability of microbes to facilitate the decomposition of lignocellulosic biomass is
measured with cellulolytic, lignolytic, and lignocellulolytic indices. These indices are essentially
diameter ratio of the microbes’ colony and the clear zone around the respective colony. Thus,
the clear zone test is only considered as a preliminary test. This test is particularly difficult due
to the often irregularly shaped colony and clear zone around it.

The clear zone indicates the level of solubility of the enzyme. The higher the level of
solubility of the enzyme, the greater the clear zone formed. The diameter of clear zone is
generally larger than the diameter of the colonies because the enzyme is secreted into the
surrounding environment (Zverlov et al., 2003).

3.2.1. Cellulolytic activity

Cellulolytic microbial selection using CCRA media defined 12 isolates, consist two
bacterium and 10 fungi, to be investigated further for identification of their potential of
cellulolytic activity. The selection was based on the morphology and cellulolytic index that have
been characterized. The higher the cellulolytic index, the higher the capability to degrade the

4 Sustinere | Volume 1 Number 1 (2017)

cellulose material (higher cellulolytic activity). The cellulolytic activities from the lowest to the
highest are presented in Table 1.

Table 1. Cellulolytic index of microbes derived from EFB wastes

Types of
Location Isolate code Colonies morphology Cellulolytic index
microbes
Samuntai POM B1-1 yellow, mucus 0.5 Bacteria
Longkali POM A3-1 yellow, mucus 0.25 Bacteria
Longkali POM A1-2 white spore 1.5 Fungi
Longkali POM A1-1 white spore 2 Fungi
Longkali POM A3-2 white spore 3 Fungi
Longkali POM A2-1 white spore 3.25 Fungi
Samuntai POM B3-2 white spore 4.7 Fungi
Samuntai POM B1-2* Red 8.5 Fungi
Samuntai POM B2-1* Red 10 Fungi
Samuntai POM B3-1* white spore 10.7 Fungi
Longkali POM A2-2* white spore 14 Fungi
Samuntai POM B2-2* white spore 14 Fungi
* Five isolates with the best cellulolytic activity

3.2.2. Lignolytic activity

Using LgA media, we were able to obtain 12 isolates that have lignin degradation capability,
evidenced by the presence of clear zone around the colonies (Figure 4). The lignolytic activities,
from the lowest to the highest, are presented in Table 2. Lignin is a noncarbohydrate fraction
and the most difficult to degrade lignocellulosic component (Kuhad et al., 2007). Lignin is an
aromatic phenolic polymer of coniferyl, sinapyl and p-coumaryl alcohol (Urairuj et al., 2003). It is
three-dimensionally structured, branched, complex, and has a high molecular weight. Although
the mechanism of decomposition of lignin has not been clearly known, many studies reported
that fungi can degrade lignin to CO2 and H2O (Risna & Suhirman, 2002). Isolates that degrade
lignin can be used for lignocellulosic pretreatment process, to reduce lignin matter and change
its chemical and physical structure so decomposition process occurs more efficient (Isroi et al.,
2011).

(a) (b)
Figure 4. (a) The colonies of fungi: 1 is the colony, 2 is the clear zone around the fungi colony;
(b) the colonies of bacteria: 1 is the colony, 2 is the clear zone around the bacteria
colony (isolation condition: LgA media, 25oC, 48 hours)

Sustinere | Volume 1 Number 1 (2017) 5

 Table 2. The lignolytic activity of microbes derived from EFB wastes
Location Isolate code Lignolytic Index Types of microbes
Samuntai POM B3-2 1 Fungi
Longkali POM A3-1 1 Bacteria
Samuntai POM B1-1 1.1 Bacteria
Longkali POM A1-2 1.7 Fungi
Longkali POM A1-1 2.4 Fungi
Samuntai POM B3-1 3 Fungi
Longkali POM A3-2 3 Fungi
Samuntai POM B2-2* 4 Fungi
Longkali POM A2-2* 9 Fungi
Samuntai POM B2-1* 21 Fungi
Longkali POM A2-1* 16.5 Fungi
Samuntai POM B1-2* 26.5 Fungi
* Five isolates with the best lignolytic activity

3.2.3. Lignocellulolytic activity

The 12 isolates with highest lignocellulolytic activity are presented in Table 3. These
isolates were obtained using EFBCRA media, and the clear zones around the colonies were also
distinctly observable as depicted in Figure 5. The size of the clear zone will be affected by the
lignocellulolytic activity.

Table 3. Lignocellulolytic activities of 12 isolate microbes

Location Isolate code Lignocellulolytic Index Types of microbe
Samuntai POM B1-1 0.2 Bacteria
Longkali POM A1-2 0.3 Fungi
Longkali POM A3-1 0.5 Bacteria
Longkali POM A1-1 0.6 Fungi
Longkali POM A3-2 1.5 Fungi
Samuntai POM B3-1 1.5 Fungi
Samuntai POM B3-2 2.7 Fungi
Longkali POM A2-1* 4 Fungi
Longkali POM A2-2* 4 Fungi
Samuntai POM B2-2* 4 Fungi
Samuntai POM B2-1* 7 Fungi
Samuntai POM B1-2* 7 Fungi
* Five isolates with the best lignocellulolytic activity

(a) (b)

Figure 5. (a) The colonies of fungi: 1 is the colony, 2 is the clear zone around the fungi colony;
(b) the colonies of bacteria: 1 is the colony, 2 is the clear zone around the bacteria
colony (isolation condition: EFBCRA media, 25oC, 48 hours)

6 Sustinere | Volume 1 Number 1 (2017)

 The presence of clear zone is an early suggestion that the isolates have capability as
lignocellulosic biomass degrading microbes. These microbes are able to break down the
complex of lignocellulosic biomass into monomers which can be used as the main carbon source
for their metabolism.

Based on enzyme biosynthesis ability, isolates A2-1, A2-2, B2-2, B2-1, and B1-2 were
selected as the best isolates as it can synthesize cellulase and ligninase enzymes. These enzymes
will accelerate the degradation of EFB substrate. Some studies demonstrated that adding
cellulolytic microorganisms can accelerate the decomposition of organic matter. Therefore,
employing indigenous lignocellulolytic microbes will accelerate the EFB decomposition process.
Gusmawartati (1999) and Wahyuni (2008) reported that using one of cellulolytic
microorganism or consortium of bacteria, fungi, or actinomycetes combined with the addition of
chicken manure can rapidly diminish C/N ratio during the decomposition of EFB.

4. Conclusion
This study has isolated 430 microbes from EFB wastes sampled from Longkali POM and
samuntai POM, in East Kalimantan, Indonesia. After selected the best 12 isolates having the
highest indices, five isolates of fungi (A2-1, A2-2, B2-2, B2-1, and B1-2) found to have the best
lignocellulolytic activity. This result will be followed up with a more comprehensive study to
produce data and information that can be used for an industrial base lignocellulosic wastes
processing.

Reference
Andayani, R. (2011). Pembuatan Bioetanol dari Tandon Kosong Kelapa Sawit Melalui Proses
Fungal Treatment oleh Aspergillus niger dan Fermentasi oleh Zygomonas mobilis. Institut
Teknologi Sepuluh Nopember.

Atlas, R. M., & Bartha, R. (1986). Microbial ecology: fundamentals and applications. Menlo Park:
Benjamin-Cummings Pub. Co.

Azizah, S. N., Muzakhar, K., & Arimurti, S. (2014). Screening of Cellulolytic bacteria from
Vermicomposting Empty Fruit Bunch of Palm Oil. Berkala Sainstek, 2(1), 26–30.

Baharuddin, A. S., Kazunori, N., Abd-Aziz, S., Tabatabaei, M., Abdul Rahman, N. A., Hassan, M. A.,
… Shirai, Y. (2009). Characteristics and Microbial Succession in Co-Composting of Oil Palm
Empty Fruit Bunch and Partially Treated Palm Oil Mill Effluent. The Open Biotechnology
Journal, 3(1), 87–95. http://doi.org/10.2174/1874070700903010087

Goenadi, H. D., & Away, Y. (1993). Seleksi dan Isolasi Fungi Pelapuk Putih dari TKKS. Menara
Perkebunan, 63, 88–101.

Gunam, I. bagus W., Buda, K., & Guna, I. M. Y. S. (2010). Pengaruh perlakuan delignifikasi dengan
NaOH dan konsentrasi substrat jerami padi terhadap produksi enzim selulase dari
aspergillus niger NRRL-AII, 264. Jurnal Biologi, 14(2), 55–61.

Gusmawartati. (1999). Pengaruh Pemberian Mikroorganisme Selulolitik dan Kotoran Ayam

Terhadap Dekomposisi Tandan Kosong Kelapa Sawit. Universitas Sumatera Utara.

Hatakka, A. (2005). Biodegradation of lignin. In Biopolimers Online.

Isroi, Millati, R., Syamsiah, S., Niklasson, C., Cahyanto, M. N., Lundquist, K., & Taherzadeh, M. J.
(2011). Biological pretreatment of lignocelluloses with white-rot fungi and its
applications: A review. BioResources, 6(4), 5224–5259.
http://doi.org/10.15376/biores.6.4.5224-5259

Sustinere | Volume 1 Number 1 (2017) 7

Kausar, H., Sariah, M., Ismail, M. R., Saud, H. M., & Habib, S. H. (2012). Development of a potential
lignocellulolytic resource for rapid bioconversion of rice straw. African Journal of
Biotechnology, 11(38), 9235–9242. http://doi.org/10.5897/AJB10.212

Kuhad, R. C., Kuhar, S., Kapoor, M., Sharma, K. K., & Singh, A. (2007). Lignocellulolytic
microorganisms, their enzymes and possible biotechnologies based on lignocellulolytic
microorganisms and their enzymes. In R. C. Kuhad & A. Singh (Eds.), Lignocellulose
Biotechnology: Future Prospects. New Delhi: IK International Pvt Ltd, 3-22.

Kumar, P., Barret, D. M., Delwiche, M. J., & Stroeve, P. (2009). Methods for pretreatment of
lignocellulosic biomass for efficient hydrolysis and biofuel production. Industrial and
Engineering Chemistry Research, 48(8), 3713–3729.

Kurakake, M., Ide, N., & Komaki, T. (2007). Biological pretreatment with two bacterial strains for
enzymatic hydrolysis of office paper. Current Microbiology, 54(6), 424–428.
http://doi.org/10.1007/s00284-006-0568-6

Meryandini, A., Widosari, W., Maranatha, B., Sunarti, T. C., Rachmania, N., & Satria, H. (2009).
Isolasi bakteri selulolitik dan karakterisasi enzimnya. Makara Sains, 13(1), 33–38.
http://doi.org/10.7454/mss.v13i1.369

Mukhlis, Mohd Saud, H., Sariah, M., Razi Ismail, M., Habib, S. H., & Kausar, H. (2013). Potential
lignocellulolytic Trichoderma for bioconversion of oil palm empty fruit bunches.
Australian Journal of Crop Science, 7(3), 425–431.

Oktavia, B. (2010). Kajian Kekayaan Bakteri Indigenous Indonesia untuk Boremidiasi Limbah.
Yogyakarta: Jurusan Pendidikan Biologi, Universitas Negeri Yokyakarta.

Pérez, J., Muñoz-Dorado, J., De La Rubia, T., & Martínez, J. (2002). Biodegradation and biological
treatments of cellulose, hemicellulose and lignin: An overview. International Microbiology,
5(2), 53–63. http://doi.org/10.1007/s10123-002-0062-3

Prihandana, R., & Hendroko, R. (2008). Energi Hijau: Pilihan Bijak Menuju Negeri Mandiri Energi.
Jakarta: Penebar Swadaya.

Risna, R. A., & Suhirman. (2002). Ligninolytic enzyme production by Polyporaceae from Lombok,
Indonesia. Fungal Diversity, 9, 123–134.

Suriadikarta, D. A., Simanungkalit, R. D. M., Saraswati, R., Setyorini, D., & Hartatik, W. (2006).
Pupuk Organik dan Pupuk Hayati. Bogor: Balai Besar Litbang Sumber Daya Lahan
Pertanian.

Taherzadeh, M. J., & Karimi, K. (2007). Enzyme-based hydrolysis processes for ethanol from
lignocellulosic materials: A review. BioResources, 2(4), 707–738.
http://doi.org/10.15376/BIORES.2.3.472-499

Tristianti, S. Y., Sarjono, P. R., & Mulyani, N. S. (2013). Aktivitas Fusarium oxyporum dalam
menghidrolisis enceng gondok (eichhornia crassipes) dengan variasi waktu fermentasi.
Chem Info, 1(1), 265–273.

Urairuj, C., Khanongnuch, C., & Lumyong, S. (2003). Ligninolytic enzymes from tropical
endophytic Xylariaceae. Fungal Diversity, 13(June 2015), 209–219. Retrieved from
http://www.scopus.com/scopus/inward/record.url?eid=2-s2.0-
2342445666&partnerID=40&rel=R7.0.0

Wahyuni, M. (2008). Laju Dekomposisi Aerob dan Mutu Kompos Tandan Kosong Kelapa Sawit
dengan Penambahan Mikroorganisme Selulolitik, Amandemen dan Limbah Cair Pabrik
Kelapa Sawit. Universitas Sumatera Utara.

8 Sustinere | Volume 1 Number 1 (2017)

Zverlov, V. V., Höll, W., & Schwarz, W. H. (2003). Enzymes for digestion of cellulose and other
polysaccharides in the gut of longhorn beetle larvae, Rhagium inquisitor L. (Col.,
Cerambycidae). International Biodeterioration and Biodegradation, 51(3), 175–179.
http://doi.org/10.1016/S0964-8305(02)00139-7

Sustinere | Volume 1 Number 1 (2017) 9