Journal of Sustainability Science and Management

Volume 9 Number 2, December 2014: 114-118

ISSN: 1823-8556
Penerbit UMT

ISOLATION AND IDENTIFICATION OF LOCALLY ISOLATED LIGNIN

DEGRADING BACTERIA
ZUHARLIDA TUAN HARITH*, NOOR AZLINA IBRAHIM AND NORAZILA YUSOFF
Faculty of Agro Based Industry, Universiti Malaysia Kelantan (Jeli Campus), Locked Bag No. 100, 17600 Jeli, Kelantan.
*Corresponding author: [email protected]

Abstract: Lignocellulosic biomass is a renewable and abundant resource with great potential for
bioconversion to value added bioproducts. In Malaysia, this biomass become a major problem to
the environment since most of these materials come from the waste of palm oil industry. However,
the conversion of the lignocellulosic material becomes a hurdle due to lacking of biocatalysts that
can overcome the biorefining process. Therefore, this study is aimed to isolate and identify locally
isolated bacteria that are able to degrade lignin. Twenty two bacterial strains were isolated from
decayed plants in Agro Park, Universiti Malaysia Kelantan Jeli Campus, Kelantan. Out of twenty
two isolates, eight potential strains designated as ZA1, ZA2, ZA32, ZA42, ZA5, ZA71, ZA72 and
ZA9 were found capable to effectively degrade the lignin through screening on selective agar plate
contaning alkaline lignin as sole carbon source. Subsequently, partial sequence of 16S rRNA gene
identified strains ZA1, ZA32 and ZA72 as Klebsiella sp., isolates ZA2, ZA42, ZA5 and ZA9 as
Enterobacter sp. and isolate ZA71 as Bacillus cereus. From phylogenetic tree analysis, isolate ZA1
was found to be closely related to isolate ZA72 and isolate ZA2 was found to be closely related to
isolate ZA42.
KEYWORDS: Lignin-degrading bacteria, identification.
Introduction
The expansion of agro-industry activity in
Malaysia has led to an accumulation of agro
industrial wastes. About 4.49 million ha of land is
planted with palm oil trees throughout Malaysia.
In the process of extracting the palm oil from the
fruit, various solid wastes are produced which
include empty fruit bunch (OPEFB), seed shells
and fibre from mesocarp. Approximately 15
million tonnes of OPEFB are generated annually
throughout Malaysia (Rahman et al., 2006). In
practice, the waste is burned which subsequently
will cause environmental pollution. In Malaysia,
this biomass become a major problem to the
environment since most of these materials come
from the waste of palm oil industry. Hence,
there is an urgent need for a sustainable waste
management system to tackle these wastes.
Lignocellulosic materials are composed of
cellulose, hemicelluloses and lignin. Cellulose
and hemicelluloses are polysaccharide that can be
hydrolyzed to produce simple sugar. Many factors

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such as lignin, cellulase crystallanity, degree

of polymerization limit the digestability of the
hemicelluloses and cellulose (Zhao et al., 2011).
It is a major carbon source and has significant
concentration of simple carbohydrates. A good
understanding of these chemical constituents of
the cell wall components is important to develop
a good mechanism for the conversion of these
constituents into more value added products
such as low price chemicals (eg; xylitol, xylose),
biofibres, biopulps and ruminant feed. The
conversion of these lignocellulosic materials
into value added end products becomes a hurdle
due to lacking of biocatalysts that can overcome
the biorefining process (Yang et al., 2011).
There are high potential of lignin-degrading
bacteria and the chacterization of lignin degrading
enzymes may give potential benefits for biofuel
production from lignocellulosic material.
Previously, white rot fungi was considered to
be the primary lignin degrader. However, the
ability to degrade lignin by bacteria may provide

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ISOLATION AND IDENTIFICATION OF LOCALLY ISOLATED LIGNIN DEGRADING BACTERIA 115

a lot of advantages compared to fungi such as

the ability adapted better in anaerobic conditions
(Huang et al., 2013), higher potential due to its
environmental adaptibility and biochemical
versatility (Abd-Elsalam, 2009).

plates were incubated at 32 oC for 24 hours. The

plates were monitored for the growth and the
development of decolorization zones.

The conversion of lignocellulosic materials

to its derivatives using whole microbial cells or
enzymes has been suggested as an economically
feasible process and potential to reduce the use
of fossil fuels and reduce economic pollution.
The advantages of this process over chemical
pretreatment includes low energy requirement,
mild reaction, high substrate specificity, high
yield of products and high hydrolysis efficiency.
However, the pretreatment process conditions
must be tailored to the specific chemical and
structural composition of the various and
variable sources of lignocellulosic materials
(Mosier et al., 2005).

The positive isolates were then identified by 16S

rRNA gene identification. The genomic DNA
was extracted using DNeasy Tissue Handbook
(Qiagen, Germany) according to manufacturers
instructions and the 16S rRNA gene was amplified
by PCR using the universal primers (Rahman et
al., 2007); forward primer: 5-GAG TTT GAT
CCT GGC TCA-3 and reverse primer: 5-CGG
CTA CCT TGT TAC GAC TT- 3. Amplification
reactions were performed in a total volume of 50
l. The reaction mixtures contained 5 l DNA,
6 l of each forward and reverse primer, 25 l
2X Taq Master Mix and 8 l sterile distilled
water. After 4 min pre-denaturation at 94 oC,
30 cycles of PCR including 1 min at 94 oC
(denaturation), 1 min at 58 oC (annealing) and
1 min at 72 oC (extension) were performed.
This was followed by one cycle of 7 min at 72
o
C and held at 4 oC. The PCR products were
examined by electrophoresis and detected using
ethidium bromide fluorescence. QIAquick PCR
Purification Kit (Qiagen, Germany) methods
were used for the purification of PCR product
and the purified PCR product was sent for
sequencing. The sequence for DNA homology
was matched with the Genebank database that
was available at http://www.ncbi.nlm.nih.gov/
BLAST/).

Therefore, this study is aimed to screen,

isolate and identify novel bacteria producing
lignin degrading enzymes that have the potential
to degrade lignocellulosic biomass from decayed
plants.
Methodology
Bacterial Sources
Decayed plants from UMK Agro Park area were
collected as microbial sources. The samples
were inoculated in 10 ml Nutrient Broth (NB)
medium and incubated overnight at 32 oC.
The cultures were serially diluted and plated
on Nutrient Agar (NA) for isolating bacterial
strains. The pure bacterial strains were isolated
by repeated sub cultures and the pure cultures
were maintained on nutrient agar plates and
stored at 4 oC, while the stock was maintained in
15% glycerol at -80 oC.
Screening of Lignin Degrading Bacteria
Each isolates was further screened on selective
agar containing malt extract agar (48 g/L),
alkaline lignin (4 g/L), calcium carbonate (2
g/L) and toluidene blue (0.025 g/L). The agar

Bacterial Identification

Phylogenetic Tree Analysis

The multiple sequence alignments analysis
between the isolates and other bacteria strains
were conducted using Clustal Omega (http://
www.ebi.ac.uk). Phylogenetic tree was
constructed based on the multiple sequence
alignment analysis and displayed using
TreeView program.

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Zuharlida Tuan Harith et al.

Results and Discussion

Screening of Lignin Degrading Bacteria
Based on qualitative screening on the selective
agar, 8 out of 24 isolates showed positive results
(Figure 1). The decolourization of toluidine
blue dye indicates the positive strain for
lignin degradation. The positive isolates were
designated as ZA1, ZA2, ZA32, ZA42, ZA5,
ZA71, ZA72 and ZA9.
Bacterial Identification
The rRNA sequence comparisons help to
construct a universal tree of life, dividing all life
on earth into three equidistant domains namely
Eukarya, Bacteria and Archea (Woese, 1998).
From the analysis, isolates ZA1, ZA32 and
ZA72 were identified as Klebsiella sp., isolates
ZA2, ZA42, ZA5 and ZA9 were identified as
Enterobacter sp. and isolate ZA71 was identified
as Bacillus cereus.
Phylogenetic Tree Analysis
The phylogenetic tree was constructed based
on comparison of 16S rRNA sequences of the
positive strains with other bacterial strains that
were extracted from GeneBank database (http://
www.ncbi.nlm.nih.gov). Ten of 16S rRNA
sequences from different strains were compared.
All sequences were aligned with Clustal Omega
and phylogenetic tree was constructed.

116

The phylogenetic tree in Figure 2

demonstrated the linkage of the eight positive
strains with other ten bacteria strains. From
the phylogenetic tree of 16S rRNA sequences,
Klebsiella sp. ZA1 was found to be closely
related to Klebsiella sp. ZA72 and Enterobacter
sp. ZA2 was found to be closely related to
Enterobacter sp. ZA42. Obtained result also
revealed that the sequence of Klebsiella sp. ZA32
showed the highest similarity with Klebsiella
sp. SZH11(GU384262), and the sequence of
B. cereus ZA71 showed the highest similarity
with B. cereus cr-50 (JF895490). Meanwhile,
Enterobacter sp. ZA5 was found to be
closely related to Enterobacter sp. B25(2012)
(JX941520) and Enterobacter sp. ZA9 was
found to be closely related to Enterobacter
sp. DC6 (HM625774). The phylogenetic
tree analysis also indicated that all of these
positive strains except B. cereus ZA71 were
phylogenetically distant from other bacterial
species such as Aeromonas sp. H1 (AM179893),
Pseudomonas
stutzeri
SP1402(U26418),
Xanthomonas sp. EB5(HF566369) and
Agrobacterium tumefaciens RV3(AJ389903).
Gene identification using 16S rRNA has
been done for more than a decade to analyse
evolutionary relationships between organisms.
Ribosomal RNAs are ancient molecules,
functionally constant, universally distributed
and moderately well conserved across broad
phylogenetic distance.

Figure 1: Decolourization Zones in Toluidine Blue-containing Plates After 24 h of Incubation. ZA1, ZA2,
ZA32, ZA42, ZA5, ZA71, ZA72 and ZA9: bacterial isolates; control: no bacteria
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ISOLATION AND IDENTIFICATION OF LOCALLY ISOLATED LIGNIN DEGRADING BACTERIA 117

Figure 2: Phylogenetic Tree Showing the Relationship between Bacterial Strains ZA1, ZA2, ZA32, ZA42,
ZA5, ZA71, ZA72 and ZA9 to Other Bacteria Strains
*AM179893 (Aeromonas sp. H1), U26418 (Pseudomonas stutzeri SP1402), HF566369 (Xanthomonas sp. EB5),
AJ389903 (Agrobacterium tumefaciens RV3), ZA71 (Bacillus cereus ZA71), JF895490 (Bacillus cereus cr-50), JN987863
(Enterobacter sp. FF3), ZA9 (Enterobacter sp. ZA9), HM625774 (Enterobacter sp. DC6), ZA5 (Enterobacter sp. ZA5),
JX941520 (Enterobacter sp. B25(2012)), GU272393 (Enterobacter sp. WP2ME), ZA42 (Enterobacter sp. ZA42), ZA2
(Enterobacter sp. ZA2), ZA32 (Enterobacter sp. ZA32), GU384262 (Klebsiella sp.SZH11), ZA1 (Klebsiella sp. ZA1), ZA72
(Klebsiella sp. ZA72), JX005889 (Klebsiella sp. SO22-4051), U31075 (Klebsiella sp. ZMMO).

In this study, the isolated strains have been

identified as Klebsiella sp, Enterobacter sp and B.
cereus according 16S rRNA gene identification.
Lignin degradation by the bacteria was observed
to have the ability to breakdown the lignin in the
media containing lignin as sole carbon source.
These isolates formed clearing zones on the
media containing dye in this case, toluedine
blue as colour indicator. Bacillus sp. had been
reported to be a potential species to degrade
lignin by Abd-Elsalam (2009) and Bandounas
et al., (2011). Besides that, Achromobacter sp.,
Flavobacterium sp., Pseudomonas sp. as well
as nitrifying bacteria such as Nitrobacter and
Nitrosomonas has been reported to have the
ability to degrade lignin (Sharafi-Yazdi, 2001).

Previously, the UMK Agro Park was a

tropical forest, then it was used for rubber
plantation for a period of 20 years after which
it is now under Agro Park. Still, it carries a lot
of biodiversity in microorganisms living in
the soils. According to Parton et al., (2007),
microorganisms isolated from rainforest soils
have been identified as the fastest decomposers
of the plant biomass compared to other.
There are clear evidances that four enzymes
are actively involved in lignin degradation
namely lignin peroxidase (LiP) [E.C.1.11.1.14],
Mn-peroxidase (MnP) [E.C.1.11.1.13], versatile
peroxidase (VP) [EC1.11.1.16] and laccase (Lac)
[E.C.1.10.3.2]. In addition, other enzymes such
as H2O2 producing enzymes, glyoxaloxidase

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Zuharlida Tuan Harith et al.

(GLox) and aryl alcohol oxidase (AAO) [EC

1.1.3.7] have their roles to degrade lignin.
Furthermore, these enzymes can be used in wider
field such as in pulp and paper industry, textile
industry, bioconversion, feed, food processing,
chemical industry and biosensors.
Conclusion
This study identified 3 bacterial isolates which
can be used to degrade lignin from decayed
plants more effectively which in bioconversion
of lignocellulosic biomass into other value
added end products such as as biofuel, sugar
productions and management of waste to wealth.
Acknowledgement
The authors would like to thank the Ministry
of Education Malaysia for funding this project
under Research Acculturation Grant Scheme
(R/RAGS/A07.00/00669A/001/2012/00095).
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