Studies on the Physiology of some wood
rotting fungi for Bioremediation of Dyes
and Effluents
Sushma Negi
Research scholar
Dept of Environment Studies
Panjab university
INTRODUCTION
India’s environment is becoming fragile and environmental pollution is
one of the undesirable side effects of industrialization, urbanization,
population growth and unconscious attitude towards the environment.
v Industrialization and development in agriculture are necessary to
meet the basic requirement of people, at the same time it is necessary
to preserve the environment.
v Textile industry in India is a fast growing industry and its wastewater
is rated as the most polluting among all industrial sectors considering
both volume and composition of effluent.
v Wastewater released from textile and dye industry can cause serious
environmental effects due to the presence of toxic dyes and dark
coloration
Contd……..
v It technologies for decolorization of textile dyeing effluents like
adsorption, precipitation, membrane filtration, chemical degradation
and photochemical degradation are relatively expensive and
commercially unattractive.
v Microbial decolorization is a potential and an effective alternative
for the decolorization of wastewater.
v White-rot fungi such as Phanerochaete chrysosporium, Trametes
versicolor, Bjerkandera adusta, Pycnoporus cinnabarinus and
Phanerochaete sordida have been shown to decolorize textile dyes or
colour effluents
REVIEW OF LITERATURE
Ø Nasreen et al., (2007) tested the ability of four different species of white rot fungi
[Link] versicolor, Termetomyces sp, Pleurotus ostreatus and Schizophyllum
commune to remove azo dyes from aqueous solutions. Studies indicate that C.
versicolor has the potential to remove colour from aqueous solutions and may be
used as an efficient biological agent for the decolouration of dyes in industrial
effluents.
Ø Mario et al., (2008) studied the ligninolytic system of Grammothele subargentea,
extracellular ligninolytic enzyme activities and ability to degrade synthetic dyes as
well as Eucalyptus globus wood. It showed resistance to the inhibitory action of
synthetic dyes. It also has ability to decolorize different chromophore types. The
recycled high-kappa-number pulp strengthens when treated with laccase extract of
Grammothele subargentea which suggest that this fungus might be a useful tool in
the biotechnology field such as the pulp industry and bioremediation.
Ø Selvam and Priya (2012) found removal of azo dyes from aqueous solution by
Schizophyllum commune (50 µm) concentration showed 96.86% of colour removal
in congored, 97.57% in methylorange and 97.40% in erichrome black-T . Lenzites
eximia decolourised congo red by 95.50%, methylorange by 94.79% and erichrome
black-T by 95.36% at (50 µm) concentration respectively.
Contd………………….
Ø Poojary et al., (2012) reported the production of ligninolytic enzymes by indigenous
basidiomycetes. Two fungal strains such as Peniophora sp and Phellinus sp are reported for the
first time as the efficient producers of laccase. Strain Peniophora sp. showed more than 80%
of decolorization of six out of 14 textile dyes, whereas Phellinus sp showed nine out of 14
dyes. The isolates showed good performance in the decolorization of textile dye which
reinforces the potential of these fungi for environmental decontamination.
Ø Celebi et al., (2013) observed that, decolorization of cationic dye effiency was high (90 %)
with Trametes versicolor for 7 days, whereas little decolorization was observed with
Horseradish peroxidase enzyme.
OBJECTIVES
v Study of optimum physical factors for growth, lingo-cellulolytic
enzyme production.
v Study of optimum nutritional requirements for growth, enzyme
production. These will include effect of :
(i) Carbon source
(ii) Nitrogen source
(iii) Micro-nutrients/trace elements.
v Studies on the decolorization of textile dyes and bioremediation of
industrial effluents
Material and Methods
AREA: Himachal Pradesh.
SUBSTRATUM: Dead twigs,
bark, decaying wood.
INFORMATION: Place of
collection, date and other
relevant information was
immediately recorded and
maintained if possible the
name of the host was also
recorded.
Method of Taxonomic study-The specimen has
been taxonomically described using the following
mountants/stains(Krick et al.,2008)
ü Amann’s Lactophenol: (used for mounting of microscopic structures).
50%.
ü Glycerine: (used for mounting of microscopic structures).
ü 4% Potassium hydroxide: (used for microchemical tests and softening
of the study materials).
ü Melzer’s Iodine: (used to check amyloidity of the sporulating
structures).
ü Distilled water: (for observing the natural color of the microscopic
structures).
ü 1 or 2%Congo-red in 50% ammonia: (used for observing septation in
spores and mycelium
ü Cotton blue: (used to stain the cytoplasm of the fungal cells and also
to observe cyanophilly of the microscopic structures.
In vitro conservation and
preservation
v The fungal isolations have been done on
PDA (Potatoes 200g, Dextrose 20 g,
Agar 20 g, distilled water to make 1000 ml)
and MEA (Malt Extract- 20 g, Agar- 18/20 g,
distilled water- up to 1000 ml) media
by single spore isolation or single.
hyphal tip technique at 24OC in an incubator.
v The isolated fungi have been maintained
at ± 4OC for preservation.
Lignin modifying enzyme(LME)assay
Azure-B agar clearance method is used to detect the activity of
lignin modifying enzyme. For this LME Basal medium
consisting of: KH2PO4 -1g, C4H12N2O6 -0.5g, MgSO4.7H2O -
0.5g, CaCl2.2H2O -0.01g, Yeast extract -0.01g, CuSO4.5H2O -
0.001g, Fe2(So4)3 -0.001g, MnSO4.H2O -0.001g and distilled
water to make 1000ml was supplemented with 0.01% w/v
Azure-B and 1.6% w/v agar and autoclaved at 15 lbs psi for 15-
20 minutes. One ml of separately sterilized 20% glucose
solution was aseptically added to each 100 ml of the medium.
Twenty five ml of the medium was poured into petri plates.
Plates were then inoculated with the test fungi and incubated
for 10 days at 24°C. After 10 days, discoloration of blue
colored medium was recorded which indicate the activity of
lignin modifying enzymes.
Cellulolytic enzyme assay
For this dye staining of CMC (Carboxy Methyl Cellulose) agar
method is used. Cellulolysis basal medium (CBM) consisting of:
C4H12N2O6 -5g, KH2PO4 -1g, MgSO4.7H2O -0.5g, Yeast extract -
0.1g,CaCl2.2H2O -0.001g and distilled water to make 1000ml has
been used. Prepared CBM medium was supplemented with 2% w/v
CMC and 1.6% w/v agar and autoclaved at 15 lbs psi for 15-20
minutes and aseptically poured into petri plates. These plates were
then inoculated with the test fungi and incubated for 3-4 days at
24°C. These plates were then stained with 2% w/v aqueous solution
of congo-red when the colony diameter was about 30 mm and left
for 15 minutes and washed with distilled water. After that plates
were flooded with 1M NaCl to destain for 15 minutes. Appearance
of yellow-opaque color around the colony indicated the positive
results for degradation of cellulose.
SCREENING OF LIGNIN AND CELLULOSE
DEGRADING ENZYME
These species have been screened for lignocellulolytic enzyme
using Azure B method (after Archibald, 1992) and CMC
method (after Pointing, 1999).
Lignin Modifying Enzyme Activity Cellulose Degrading Enzyme Activity
Fig-(a) Fig-(b)