Abstract—The potentials of two new native fungi Trichoderma

aureoviride UPM 09 JN811061and Fusarium equiseti UPM 09

JN811063 isolated from Asian elephant dung for their ability to

digest lignin and hemicellulose was exploited using two pretreatment

methods, submerged cultivation (SMC) and solid state cultivation

(SSC). The pretreatment effect (% loss on lignin and hemicellulose

determined after treatment) on rice husk (RH), rubber wood saw dust

(RW) and oil palm empty fruit bunch (EFB) using SMC and SSC by

T. aureoviride UPM 09 JN811061 was statistically significantly

(P<0.05) higher than by F. equiseti UPM 09 JN811063. However, the

result of this study, therefore, showed that the fungi T. aureoviride

UPM 09 JN811061 and F. equiseti UPM 09 JN811063 both have

great selectivity for lignin with T. aureoviride UPM 09 JN811061

having greater selectivity.

Keywords—Pretreatment, Fungal, Biomass, Lignocellulose

I. INTRODUCTION

OTENTIALLY valuable lignocellulosic biomass

materials were treated as waste in many countries in the

past, and are still today treated same in most developing

countries, which raise many environmental concerns. Sanchez

(2009) [1] reported that lignocellulosic residues from wood,

grass, agricultural, forestry wastes and municipal solid wastes

are particularly abundant in nature and have a potential for

bioconversion. Lignocellulosic feedstock require aggressive

pretreatment to yield a substrate easily hydrolyzed by

commercial cellulolytic enzymes, or by enzyme producing

microorganisms, to release sugars for fermentation (Agbor et

al., 2011).

The purpose of this study is to explore the ability of native

fungi to digest lignin and hemicellulose in the biomass

materials in order to expose cellulose which can be converted

into glucose for subsequent fermentation into Biofuels. This

will save our environment from air pollution resulting from

burning the biomass wastes, on one hand, and also help reduce

reliance on fossil fuels that also pollute the environment, on

the other.

Ahmadu Ali1. Farouq was with the Laboratory of Industrial Biotechnoogy, Institute of Bioscience, Universiti Putra Malaysia, Serdang,

Darul Ehsan, 43400, Malaysia phone. He is now with the Department of

Microbiology, Faculty of Science, Usmanu Danfodiyo University, Sokoto.Nigeria. (+2348161653400; fax: 23605159; e-mail:

ahmedalifaq@yahoo.com)

Dzulkefly Kuang2. Abdullah2 is with the Laboratory of Industrial

Biotechnoogy, Institute of Bioscience, Universiti Putra Malaysia, Serdang,

Darul Ehsan, 43400, Malaysia; e-mail: dzul2240@gmail.com).

II. MATERIALS AND METHODS

Lignocellulose biomass materials sourced locally were

ground and sieved into 1mm size before treating with two

novel strains of fungi isolated in our laboratory from Asian

elephant dung using molecular assay [2], namely, T.

aureoviride UPM 09 (JN811061) and Fusarium. equiseti

UPM 09 (JN811063) (accession numbers in parenthesis were

assigned by Gen Bank Database USA). The fungi were further

screened for cellulolytic activities and used individually and in

consortium for the pretreatment of lignocellulose biomass

materials, namely, rice husk (RH), rubber wood saw dust

(RW) and oil palm empty fruit bunch (EFB) using submerged

(SMC) and solid state cultivation (SSC) with the untreated

biomass used as control. Both the composition of the treated

and untreated lignocellulose biomass and lignin,

hemicellulose, and cellulose reduction were determined [3, 4,

5] and analyzed by two-way ANOVA using JMP, Version

9.0.3 SAS Institute Inc. Cary, NC, 1989-2010. When ANOVA

was found to be significant, Tukey’s test was used to

determine the difference between the groups

III. RESULTS

Fig.1: Mean cellulose loss (%) of treated RH = Rice Husk, RW =

Rubber Wood and EFB = Empty Fruit Bunch determined after

pretreatment using submerged cultivation SMC and solid state

cultivation (SSC).

The Potentials of Novel Native Fungi in

Delignification of Lignocellulose

Biomass Wastes

Ahmadu Ali Farouq, and Dzulkefly Kuang Abdullah

P

3rd International Conference on Biotechnology, Nanotechnology and its applications (ICBNA'2014) March 19-20, 2014 Abu Dhabi (UAE)

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Fig. 2: Mean hemicellulose loss (%) composition of fungal treated

and untreated (U-control) RH = Rice Husk, RW = Rubber Wood and

EFB = Empty Fruit Bunch determined after pretreatment using

submerged cultivation (agitated) SMC and solid state cultivation

(SSC).

Fig. 3: Mean lignin loss (%) composition of fungal treated RH =

Rice Husk, RW = Rubber Wood, EFB = Empty Fruit Bunch

determined after pretreatment using submerged cultivation SMC and

solid state cultivation (SSC).

Fig.4: Individual and consortium of T. aureoviride UPM 09

(JN811063) and F. equiseti UPM 09 (JN811O61) interwoven on

rubber wood saw dust. (A.) Rubber wood treated with T. aureoviride

UPM 09 (JN811063, (B.) Rubber wood treated with F.equiseti UPM

09 (JN811O61) (C.) Rubber wood treated with consortium of T.

aureoviride UPM 09 (JN811063 and F. equiseti UP M 09

(JN811O61) (D.) Untreated RW.

Fig.5: Consortium of T. aureoviride UPM 09 (JN811063) and F.

equiseti UPM 09 (JN811O61) interwoven on EFB. (A.) EFB treated

with T. aureoviride UPM 09 (JN811063, (B.) EFB treated with F.

equiseti UPM 09 (JN811O61) (C.) EFB treated with Consortium of

T. aureoviride UPM 09 (JN811063 and F. equiseti UPM 09

(JN811O61) (D.) Untreated EFB.

Fig. 6: Consortium of T. aureoviride UPM 09 (JN811063) and F.

equiseti UPM 09 (JN811O61) interwoven on rice husk (RH). (A.)

Untreated RH. (B.) RH treated with Consortium of T. aureoviride

UPM 09 (JN811063 and F. equiseti UPM 09 (JN811O61) (C.) RH

treated with T. aureoviride UPM 09 (JN811063 (D.) RH treated with

F. equiseti UPM 09 (JN811O61).

.

3rd International Conference on Biotechnology, Nanotechnology and its applications (ICBNA'2014) March 19-20, 2014 Abu Dhabi (UAE)

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TABLE I COMPOSITION OF UNTREATED BIOMASS MATERIALS

Composition

Percentage (%)

RH RW EFB

α – Cellulose 34.93 ±0.0067 42.97±0.0033 50.73

±0.2136

Hemicellulose

24.97±0.0033 31.93± 0.0667 21.77 ± 0.1856

Lignin

19.93± 0.066 21.17±0.166 11.17 ± 0.441

Extractives

3.00±0.033 2.70± 0.0033 3.47 ± 0.0033

Ash

16.9± 0.0033

0.79± 0.0033

11.0 ±

0.0287

IV. DISCUSSION

The general ideas in various pretreatment technologies are

to alter or remove lignin and hemicelluloses. Lignin and

hemicelluloses are degraded, altered or displaced as goals of

pretreatment (Figs. 2.4, 2.5) in order to expose and expand the

cellulose thereby increasing the surface area and decreasing

the crystallinity of cellulose (Galbe and Zacchi, 2002,

Jorgensen et al., 2007; Wyman et al., 2005) for enzymatic

hydrolysis (Mosier et al., 2005).

Lignin is one of the main components in plant cell wall that

limits enzymatic hydrolysis of lignocellulosic biomass by

cross linking with cellulose and hemicelluloses fibers [6].

Reducing the lignin content of the biomass helps to expose the

highly ordered crystalline structure of cellulose and facilitates

substrate access by hydrolytic enzymes [7]. The ability of

fungi to produce cellulase enzymes is responsible for their

capability to digest the lignocellulose in biomass materials [1].

As indicated in this study as shown in Table 1, rice husk (RH),

rubber wood saw dust (RW) and empty fruit bunch (EFB)

contain a considerable amount of lignocellulose fibers that

may likely be preferred by the fungi.

The pretreatment effect (% loss) on lignin and

hemicellulose determined after treatment) on RH, RW and

EFB using SMC and SSC by T. aureoviride UPM 09

JN811061 was statistically significantly (P<0.05) higher than

by F. equiseti UPM 09 JN811063. Similarly, the effect of

pretreatment on RH, RW and EFB using SMC and SSC by the

consortium of the two fungi was also correspondingly

statistically significantly (P<0.05) higher than either of the two

fungi individually based on the % mean lignin and

hemicellulose determined after pretreatment (Figs. 1, 2 and 3)

with cellulose relatively conserved. However, on the average,

the % lignin and hemicellulose determined after the

pretreatment in RH (35-37 %, 41-44 %) and RW (26-35 %,

31-36 %) using SSC by the individual and consortium of the

two fungi was higher than pretreated RH (29-37%, 35-42% )

and RW (27-34, 32-38 %) by SMC. The reason may be as a

result of the spreading and penetration of the mycelial mat of

the fungi on and deep into the softer fibers of RH and RW as

opposed to EFB due to structural and compositional

differences in the feedstocks. Reverse was, however, the case

for EFB where the % mean lignin and hemicellulose reduction

after pretreatment was significantly (P<0.05) higher in SMC

for T. aureoviride UPM 09 JN811061 and in consortium with

F. equiseti UPM 09 JN811063 than in SSC as shown in Figs.

4.12 and 4.13 where the liquid medium may reduce such

trespassing by the two fungi.

As can be seen in Figs. 2 and 3, the % mean loss or

reduction of lignin and hemicellulose determined after

pretreatment by the consortium of the two fungi pretreatment

using SSC for RH, RW and EFB was significantly (P<0.05)

less than in the corresponding pretreatment by SMC, this is

because the mycelium of T. aureoviride UPM 09 JN811061

quickly spread over the surface of the biomass substrates,

especially on RH and RW thereby entangling the mycelium of

F. equiseti UPM 09 JN811063 and hence arresting the total

growth and spread of F. equiseti UPM 09 JN811063.

In both the methods of pretreatment employed in this study

of the three biomass substrates [ rice husk (RH), rubber wood

saw dust (RW) and empty fruit bunch (EFB)] separately using

the individual fungus and their consortium (2) (T. aureoviride

UPM 09 JN811061 and F. equiseti UPM 09 (JN811063), it

was found that the difference in hemicelllulose and lignin

percentage reduction was statistically significant (P<0.05)

between the individual fungus and the consortium of the two

fungi in both SMC and SSC pretreatments as shown in Figs. 2

to 3.

The percentage cellulose loss determined in EFB after

pretreatment was significant (P< 0.05) and was higher than in

RH and RW (Fig.1, Table 1) between the individual and

consortium of the two fungi in both SMC and SSC, more

probably because it had higher cellulose content. The

percentage hemicellulose and lignin reduction as shown in

Figs. 2 and 3 were between 27-38 % and 32-40 % for SMC

and SSC, respectively.

Higher selectivity means better prospects for preferential

delignification of the three substrates pretreated by the fungi in

this study (Fig.3) as reported by [8] and low selectivity value

meant relatively high cellulose loss during the biological pre-

treatment. The result of this study, therefore, showed that the

fungi T. aureoviride UPM 09 JN811061 and F. equiseti UPM

09 JN811063 had great selectivity for lignin with T.

aureoviride UPM 09 JN811061 having greater selectivity.

The effect of fungal pretreatment on the morphology of RH,

RW and EFB was examined using scanning electron

microscopy. Figures (4, 5 and 6) show the SEM micrograph of

untreated and pretreated using individual and consortium of

the fungi. SEM analysis revealed that the pretreated biomass

have a rugged and partially broken face, while the untreated

have a continuous even and smooth flat surface area, which

resulted from the removal of lignin and breaking of

lignocelluloses networks during the pre-treatment. SEM

analysis also showed that in all the treated samples, branched

hyphae covered the surface of the wood chips. Mycelia mass

increased during degradation. The hyphae penetrated the chips

through the lumen and pit fiber cells of the biomass. In

addition, during the pretreatment by the consortium of the

3rd International Conference on Biotechnology, Nanotechnology and its applications (ICBNA'2014) March 19-20, 2014 Abu Dhabi (UAE)

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fungi T. aureoviride UPM 09 (JN811063) hindered and

stemmed the growth spread of F. equiseti UPM09 (JN811061)

by its rapid growing mycelia by penetrating into its cells.

Therefore, the results of this research show that native fungi

have the potential to digest lignocellulose biomass waste that

is being generated in tonnes annually from our countries and if

bioprocessed can be harnessed and used as feedstock for

Biofuel production.

ACKNOWLEDGMENT

The authors are indebted to Institute of Bioscience,

Universiti Putra Malaysia (UPM), for the research facilities,

Ministry of Higher Education, Malaysia, and ERGS (project

number:1/11/STG/UPM/01/27) for the research funding. The

authors would also like to thank Dr. I. A. Anka for guiding us

on statistical analysis he authors are grateful to Institute of

Bioscience, Universiti Putra Malaysia for providing research

facilities for this research.

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[5] Rowell, M. R. (1992). Opportunities for lignocellulosic materials and

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