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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)
9
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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[2] White, T., Burns T., Lee, S. and Taylor, J. (1990). Amplification and Direct Sequencing of
RNA genes for phylogenetics- In PCR Protocols, pp 315-322, Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, San Diego Academic Press.
[3] TAPPI (Technical Association of paper and pulp industry). (1992).
Technical Association of Pulp and Paper Industry, Atlanta, Georgia, USA.
[4] Teramoto, Y., Tanaka, N., Lee, S. H., and Endo, T., (2008). Pretreatment
of eucalyptus woodchips for enzymatic saccharification using combined sulfuric acid-free ethanol cooking and ball milling. Biotechnology and
bioengineering, 99: 75-85.
[5] Rowell, M. R. (1992). Opportunities for lignocellulosic materials and
composites. In: Emerging technologies for material and chemicals from
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Chemical Society. pp 26-31. [6] Fan, L. T., Gharpuray, M. M., and Lee, Y. H. (1987). Cellulose
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[7] Sun, Y., and Cheng, J., (2002). Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource technology. 83: 1-11.
[8] Camarero, S., Galletti, G. C., and Martinez, and A. T. (1994).
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