ISSN: 2320 – 0774

IJBAF, January, 2014, 2(1): 1-7
ISSN: 2320 – 0774
STUDIES ON LIGNIN MODIFING MICROORGANISMS ISOLATED FROM THE
COASTAL AREAS OF SOUTH INDIA
PRASAD MP1*, SETHI R2, PADMAVATHI T2 AND JAIN AK1
1: Sangenomics Research Lab, Domlur Layout, Bangalore 560071, India
2: Department of Microbiology, Jain University, Bangalore, India
* Corresponding Author: E Mail- [email protected]; Ph.: 9844357929
ABSTRACT
In nature, lignocellulose accounts for the major part of biomass and consequently, its
degradation is essential for the operation of the global carbon cycle. Lignin is a major cell wall
component which provides structural support to the plant and makes the polymer resistant to
enzymatic degradation by microorganisms. In the present investigation, marine samples like
sediments, water and floating plant material were collected for isolation of the microorganisms
capable of oxidizing lignin. On plating the samples on marine agar and modified Potato dextrose
agar over ninety bacterial species and around forty fungal species were isolated. These organisms
were subjected to lignin modifying assay to screen for the production of ligninases. The result
showed more bacterial species capable of oxidizing compared to fungi. This indicates the
abundant presence of organic matter in the marine environment which have created the micro
environment to thrive breaking down lignin and utilizing as a sole source of carbon. Such
isolates allow the utilization of bound cellulose and hemicellulose which have applications in
industries related to paper, textile, feed and also in the conversion of cellulose to biofuel.
Keywords: Lignocellulose, Ligninases, Marine sediment, Agro wastes, LBM Medium
INTRODUCTION
In recent years marine microorganisms have
Among the three major habitats of the
become important in the study of novel
biosphere, the marine realm which covers
microbial products exhibiting antimicrobial,
70% of the earth’s surface provides the largest
antiviral, antitumor as well as anticoagulant
inhabitable
and cardio active properties [1-3].
particularly microbes. Marine microbes thrive
space
for
living
organisms,
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IJBAF, January, 2014, 2(1)
Prassad MP et al
Research Article
not only in the surface waters of the sea, but
bacteria are manganese peroxidase, most
also in the lower and abyssal depths from
marine environments contain only dilute
coastal to the offshore regions, and from the
substances that can be used for metabolism
general oceanic to the specialized niches like
and growth. In contrast, natural surfaces tend
blue waters of coral reefs to black smokers of
to collect and concentrate nutrients by
hot thermal vents at the sea floor [4].
hydrophobic interactions [6].
Microorganisms have a great role in the
Lignin is a complex chemical compound,
mineral cycle in the marine environment. The
most commonly derived from wood and
element carbon which forms the basis of all
cross-linked racemic macromolecule with
organic matter undergoes a constant cycle in
molecular masses in excess of 10,000 units. It
nature by various heterotrophic bacteria.
is relatively hydrophobic and aromatic in
A dramatic increase in ethanol production
nature and present in plants and some algae,
from cornstarch-based technology may not be
and is one of the most abundant organic
practical because corn is used as food and
polymers on Earth,
feed. The other source for low-cost ethanol
cellulose lignin peroxidase and laccase [7]
production
crop
whereas the cellulose degrading enzymes are
residues, grasses, sawdust, wood chips, and
endo-1,4-beta-D-glucanase (endoglucanase),
solid animal waste. Cellulosic resources such
exo-1,4-beta-D-glucanase (exoglucanase) and
as paper,
beta-glucosidase.
is
lignocellulose
from
cardboard, wood, agricultural
exceeded only
by
residues, waste papers and fibrous plant
Lignin is closely associated with cellulose and
materials from forests which comprise about
hemicellulose in hardening and strengthening
80% of the world’s biomass [5].
of plant cell wall. Lignin provides protection
Lignin fills the spaces in the cell wall between
to cellulose from degradation, as lignin itself
cellulose,
is extremely resistant to chemical and
components
hemicellulose,
for
covalently
and
pectin
to
biological degradation and only a few
hemicellulose and polysaccharides, forming
microorganisms are able to mineralize [8] and
the
oxidise it. The biodegradation of lignin is
lignocellulosic
linking
substrates.
These
substrates are degraded by lignolytic and
unusual.
cellulolytic enzymes which are secreted by
biopolymers
certain
lignin
dimensional structure, not having respective
degrading enzymes present in these fungi and
linkage between the monomeric building
fungi and
bacteria.
The
Lignin differs
by
its
from all other
seemingly
three-
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IJBAF, January, 2014, 2(1)
Prassad MP et al
Research Article
blocks. In spite of its potentially high energy
improve the digestibility of highly ligniﬁed
content, it is not used as a sole source of
plant tissues [14].
energy by any known living organism. Thus,
MATERIALS AND METHODOLOGY
lignin forms a barrier against microbial
Sampling and Isolation
destruction
readily
Marine back water sediment samples were
assimilable polysaccharides.In the course of
collected from coastal areas of Tamil Nadu
evolution, only one gr up of organisms – the
and Karnataka. The samples were collected in
basidiomycetes – has developed the ability to
sterile bottles which were preserved in
degrade lignin (>20% lignin) substantially,
refrigerator
i.e., to mineralize it into CO2 and water [9,
Standard
10]. Wood colonizing fungi causing white rot
followed for the purpose of isolation [15].
and certain litter decomposing fungi degrade
One ml of the desired dilution was transferred
lignin in the wood and in the soil, respectively
aseptically into Potato Dextrose Agar (PDA)
[11, 12].
and Marine agar plates for fungi and bacteria
Lignin degradation is mainly occurring due to
respectively. Plates were incubated for 24-48
certain
non-speciﬁc
hrs for bacterial and 5-7 days for fungal
oxidoreductase enzymes like MnP, LiP and
growth. The isolates thus obtained were sub
laccase secreted by fungi. The production of
cultured and the pure cultures were subjected
MnP
for screening.
by
protecting
extracellular
is
apparently
limited
the
to
certain
until
further
microbiological
investigation.
methods
were
basidiomycetes fungi, which secrete MnP
Lignin Modifying Enzyme Assays
mostly
their
Actively growing culture was used to
environment. However, lignin cannot be
inoculate each assay medium, for bacteria 24-
degraded as asole source of carbon and
48 h culture was used and for fungi 5-7 days
energy. Degradation of lignin by white rot
old culture was used to inoculate each assay
fungi enables themto gain accesst o the
medium individually.
holocellulose,which is their actual carbon and
Sterile LBM medium was prepared and 1 ml
energy source [13]. Fungi that selectively
of separately sterilized 20% w/v aqueous
remove lignin without loss of appreciable
glucose solution is added to each 100ml of
amounts of cellulose are extremely attractive
LBM growth medium and was supplemented
for use in biological pulping processes and to
with 0.25% w/v lignin and 1.6% w/v agar.
in
multiple
forms
into
The media was then aseptically transferred to
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IJBAF, January, 2014, 2(1)
Prassad MP et al
Research Article
sterile petri dishes. The media was then
Gram positive bacillus followed by Gram
inoculated with the test organism. The
positive cocci and Gram negative rods.
inoculated media was incubated at room
The fungal isolates were found in the
temperature for 48 h for bacteria and 5-7 days
sediment samples from the backwaters from
for fungi. After the incubation period, the agar
all the places of sampling. This indicates the
plates were stained with 1% ferric chloride
availability of organic substances for the
and potassium ferri cyanide prepared freshly
growth of these fungi which could have
before use. Clear zones around colonies
originated from the vegetation of terrestrial
indicated oxidation of phenolic compounds
origin.
against blue green undegraded lignin. Phenols
The screening activity was assessed based on
in undegraded lignin will stain blue-green
dye staining and zone of hydrolysis similar to
with clear zone around colonies indicating
the findings of [17] and [18]. The lignin
oxidation of phenolic compounds [16].
degradation capacity was limited to very few
RESULTS AND DISCUSSION
isolates. A total of 15 bacterial, 11 fungal
The bacterial and fungal species were isolated
isolates, 2 actinomycetes showed degradation
which were numbered based on the place and
in cuddalore sediment samples and 14
type of sample. A total number of 137 isolates
bacterial,
were obtained from Mangalore sediment
actinomycetes in mangalore sediment samples
samples followed by 128 isolates from
showed lignin oxidation (Figure 3).
Cuddalore sediment samples (Figure 1, 2).
Lignin forms an irregular non-crystalline
This clearly indicates that the presence of a
network in plant cell wall to protect cellulose
large number of organisms occurred at the
and hemicelluloses which is highly resistant
bottom of the sea or the backwaters and not in
to biodegradation. Organisms cleave lignin
sea water itself. Mangalore samples showed
barrier
results similar to that of samples from
enzymes,
Cuddalore with the sediment samples. The
oxidases [19, 20, 21].
8
fungal
through
isolates
diffusible
ligninperoxidases
and
1
ligninolytic
and
phenol
maximum number of isolates was found to be
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IJBAF, January, 2014, 2(1)
Prassad MP et al
Research Article
Figure 1: Fungal Isolates on PDA
Figure 2: Bacterial Cultures on Marine Isolation Agar
Figure 3: Lignin Hydrolysis on LBM Medium
[4] Qasim SZ, The Indian Ocean: Images
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