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SATISH PRADHAN DNYANASADHANA COLLEGE
ARTS, COMMERCE AND SCIENCE, THANE
2017-18
A Project Report
on
“ISOLATION AND SCREENING OF CELLULOLYTIC
FUNGI FROM THANE CREEK ”
Submitted by
DR. MANDAKINI INGLE
Department of Botany
Satish Pradhan Dnyanasadhana College of Arts Commerce, Thane
INDEX
R. NO CONTENT PAGE NO.
01 Introduction 01-02
02 Review of Literature 03-04
03 Material and Method 05
04 Results 06-11
05 Discussion 12
06 Bibligraphy 13-14
CHAPTER 1
INTRODUCTION
Cellulolytic enzymes are synthesized by a number of micro-organisms. Fungi and bacteria are
the main natural agents of cellulose degradation (Lederberg, 1992). Fungi are well-known
agents of decomposition of organic matter, in general, and of cellulosic substrate in particular.
The initial step in cellulose degradation is enzymatic hydrolysis of polymers.
Cellulases are the third most significant commercial enzyme in the world market. Cellulases,
solely or in a mixture with other enzymes, involved in several industries including
biofuel,food, feed, beverages, agriculture etc (Kuhad et al.2011; Siti Lusi Sari et al. 2017).
Isolation and characterisationn of cellulolytic fungi provide a good starting point for the
discovery of beneficial enzymes (Rathnan et al.2012; Siti Lusi Sari et al. 2017). Most of the
cellulolytic fungi belong to Ascomycota and Basidiomycota phylums.
Mangroves are the coastal wetland forests generally found near the intertidal regions of
estuaries between creeks, lagoons, marshes etc. Mangroves provide a unique ecological site to
different microbes. Because of richness in carbon and other nutrients mangrove ecosystem
harbors diverse microbial communities which can adapt themselves in the extreme conditions
there. Microorganisms forms integral part of the mangrove ecosystem. Mangrove ecosystem
shows diversity of microbes such as bacteria, fungi, actinomycetes etc. The microorganisms of
mangrove are essential in the productivity, conservation, and rehabilitation of mangrove
ecosystems (Holguin et al., 2001).
The creek, known as “Thane Creek” separates the Island City of Mumbai in the west from the
mainland in the east and houses industrial areas at a distance of about 25 Km north-east of
Mumbai city. Thane Creek lies in the southern part of the Deccan belt of India between
latitude 18º 53’ to 19º 04’ N longitude 72º 48’ to 72º 53’E. It is a triangular mass of brackish
water which widens out and opens to the Arabian Sea in the South. The creek is narrow at the
Northern end, were it is fed partially by river Ulhas. Soil from thane creek is very rich in
micro-organisms like bacteria and fungi. Fungi can produce both intra-cellular as well as
extra-cellular enzymes. All fungi are hetrotropic, and rely on carbon compounds synthesized
by other living organisms.
Fungi as decomposers, play a most important role in our economy because fertility of soil
greatly depends on microbial activity.A wide variety of microbes including bacteria, fungi,
actinomycetes and protozoa are involved in the decomposition of cellulose, fungi have been
generally considered as main organisms’ responsible (Cowling, 1958). So the rate of
utilization of plant materials and its transformation into humus is largely dependent on the
active soil mycoflora. This is the reason why scientists are studying the fungi at molecular
level, trying to discover more cellulolytic fungi.
The aims and objectives of the present study include:
I) Isolation and identification of cellulose degrading fungi from soils of
Thane creek.
i) Preparation of large inoculum of each form to study cellulose
degradation capacity.
ii) Frequency distribution of cellulose decomposing fungi.
CHAPTER 2
REVIEW OF LITERATURE
Efficient hydrolysis of ligno-cellulose to simple sugars that can then be adequately
used as raw materials for production of ethanol and various industrial products by use
of chemicals is not cost effective (Sharada et al., 2013). Biological pretreatment of
lignocellulose via microorganisms is safe, environment friendly alternative (Okano et
al., 2005; Ravichandra et al., 2013). Many microorganisms are capable of degrading
and utilizing celluloses, hemicelluloses and other polysaccharides as carbon and
energy sources naturally in ecosystem. These organisms can produce a coordinate
battery of lingo-cellulolytic enzymes that are capable of degrading cellulose as well as
other plant cell-wall polymers. The lingo-cellulolytic enzymes comprises of a large
group of extracellular proteins including hydrolytic enzymes (cellulases.
hemicellulases, pectinases, amylases, mannases etc.) and lignolytic enzymes (Mtui,
2012).
Enzymatic hydrolysis of cellulose and other related oligo-saccharides is catalyzed by
cellulase. The cellulase enzyme system is a mixture of hydrolytic enzymes including
exoglucanases, endoglucanases and β-glucosidases acting in a synergistic manner
(Enari, 1983; Acharya et al., 2008).
Shobana and Maheshwari (2013) stated that cellulose producing fungi cultures using
agricultural wastes soil as substrate by culturing them on media plate and isolating
cellulolytic fungi for the study of maximum cellulase enzyme production activity by
the fungi.Fusarium oxysporum isolated from tomato plant parts produces maximum
cellulose at optimum parameters. (Ramananthan, etal., 2010).
Soma Mrudula and Rangasamy Murugammal ,have reported that the physical and
nutritional parameters of fermentation like pH, substrate, temperature, carbon and
nitrogen sources for cellulase production byAspergillus nigerduring submerged
fermentation and solid state fermentation play an important role. Kale and Zanwar
(2016) isolated Trichoderma and Aspergilluswere from different sources and screened
for their ability to degrade cellulose.Sumathi.et al.,(2016), investigated the efficacy of
fungal cultres isolated from plastic dumped soils as a substrate by using six different
culture mediums by serial dilution method. Cellulase producing fungi were isolated on
other specific culture media. Thorn et al ., (1996) screened 67 basidiomycetes from
40 of the 64 soil samples and 8 of 11 habitats and 17 of the 35 plots sampled.
Bhat and Shewade(2013), stated in their study by collecting mangrove soil samples
from five different stations of CBD Belapur, Navi Mumbai. Among the soil samples
screened, all gram positive isolates were obtained. 14 distinct isolates were
characterized. Isolates demonstrated L-asparaginase activity.
Noor Ashiqin Jamroo et al. (2015) stated that the thermostable cellulases from fungi
has high potential for industrial applications. The fungal species such as Trichoderma
sps. and Aspergillus sps., have most widely been used for production of cellulolytic
enzymes. Out of 26 fungal isolates, only eight isolates were selected for screening and
showed the abilities to secrete cellulases by forming distinct halo zones on selective
agar plates under various temperature. Study was conducted to isolate and screen of
cellulolytic fungi from Salacca leaf litter. There were about 12 fungal isolates which
were obtained in this research and 8 isolates showed cellulolytic activity. Based on
morphological characters, these cellulolytic fungi were identified as belonging to 4
genera, i.e. Penicillium, Aspergillus, Paecilomyces, and Thielaviopsis. Based on clear
zone formation, some isolates showed highest cellulolytic activity (Siti Lusi Sari et
al., 2017).
CHAPTER 3
MATERIALS AND METHODS
Isolation of fungi: Soil samples were collected from two different locations from
Thane creeks and stored in sterile containers. The fungi were isolated from the soil
samples using serial dilution technique. Various fungal strains were obtained on each
of the culture media .
Identification of fungi:
The macroscopic characters like the morphological characteristics of fungal strains
including color, texture of mycelia and growth pattern were observed. Microscopic
characters like septate or aseptate mycelium, sporulation were observed under light
microscope using lactophenol cotton blue staining.
Qualitative Screening of Cellulase Producing Alkalophilic Fungi:
Cellulose degrading fungi were screened on Carboxymethyl cellulose(CMC) agar
based on modified composition of Mendel basal medium(1975). The screening of
cellulolytic fungi was carried out by Congo red dye degradation of carboxymethyl
cellulose (Pointing, 1991).
CHAPTER 4
RESULTS
Isolation and Identification of fungi: The fungi were isolated from soil samples
collected from two different locations of Thane creek . The isolated strains were grown
on Potato dextrose agar plates and identified by macroscopic and microscopic characters
using Handbook of Soil Fungi. Four different strains of Aspergillus and Rhizopus were
identified (Table 4.1, Figures 4.1).
Screening of Cellulase Producing Alkalophilic Fungi:
The alkalophilic fungi were screenedby growing the fungal isolates on PDA plates with
varying pH 6-9. The fungi those gre at pH8 were screened for celluase production by
Congo red dye degradation method. The plates were incubated with mycelia plugs
obtained from one week old pure culture. These plates were incubated at ±26ºC for 5-6
days. Diameter of each plates were measured in terms of millimeter after 6 days to study
the cellulase production.(Table 4.2, Figures 1-9).
Table 4.1:Morphological features of cellulolytic alkalophilic fungal isolates :
Fungal
isolates
Growth
pattern
Configurations Margins Color Of
Mycelium
Aspergillus terreus Median Irregular and
spreading
Smooth Aerial-Light brown
Reverse-Brownish Yellow
Aspergillus flavus Median Irregular and
spreading
Smooth Aerial-Olive green
Reverse-Greyish green
Aspergillus sp. Median Irregular and
spreading
Smooth Aerial-Olive brown
Reverse-Yellowish brown
Aspergillus
fumigatus
Fast Irregular and
spreading
Smooth Aerial-Grey
Reverse-Greyish yellow
Rhizopus Fast Irregular and
radiating
margin
Ciliate Aerial-White
Reverse-Yellowish white
Figure 4. 1. A-D: Aspergillus fumigates. A: Culture plate front side, B:
Reverse side of plate, C, D: Microscopic feature showing Conidiophore,
Conidia and Spores.
H
A
G
A
FE
A
Figure 4.1. E-H: Rhizopus. E: Culture plate, F: Reverse Side of plate,G: Microscopic
feature showing Conidiophore, Conidia and Spores
Figure 4.1. I-K: Aspergillus. I: Culture plate, J: Reverse Side of plate, K:
Microscopic feature showing Conidiophore, Conidia and Spores
JI
K
N
ML
Figure 4.1. L-N: Aspergillus terreus L: Culture plate, M: Reverse Side of
plate, N: Microscopic feature showing Conidiophore, Conidia
Table 4.2: Congo red dye degradation by Cellulase (mm)
Fungal
isolates
Plate1 Plate 2 Plate3
Aspergillus
terreus
42 74 52
Aspergillus
flavus
- 60 50
Aspergillus 68 50 44Aspergillus
fumigatus
- - 40
Rhizopus - - -
PO
Figure 4.1. O & P: Aspergillus flavus P: Culture plate, O: Microscopic
feature showing Conidiophore, Conidia.
CHAPTER 5
DISCUSSION
21
54
3
Figures 1-9: Plates showing cellulase degradation by Congo red assay
Figure1-3:Aspergillus terreus; Figure4-5:Aspergillus flavus; Figure6-8:
Aspergillus; Figure9: Aspergillus fumigates.
9
876
The soil samples obtained from Thane creek were found to be alkaline. From the soil
samples, Aspergillus species and Rhizopus species were isolated. This indicated that these
species were dominant and alkaliphilic.
Screening for cellulase producing fungal strains was carried out and it was found that
among the various species of Aspergillus isolated, Aspergillus terreus and A. flavus
were highly potent for cellulase production under alkaline conditions.
The aquatic systems containing tremendous organic matter becomes polluted if the
organic matter is not degraded. Also, the enzymatic hydrolysis for breakdown requires
acidic conditions. Thus the enzymes that function under alkaline condition would be a
boon for sewage treatment.
CHAPTER 6
BIBLIOGRAPHY
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some polycyclic aromatic hydrocarbons by Aspergillus terreus. African Journal of
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3. Bhat M. R. and Shewade, L. (2013). Isolation and Characteristics of micro-
organisms from mangrove soil of CBD Belapur creek, Navi Mumbai, MS India.4. C.J Alexopoulos et al., (1979). Book for Introductory mycology.5. Charitha Devi, M., and M.Sunil Kumar (2012). Production, Optimization and
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sawmill industrial wastes, Journal of Microbiology and Biotechnology
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bacteria isolated from Bhitarkanika mangrove ecosystem of Orissa coast, Malaysian
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involved in the degradation of plant fiber cell walls. Adv. Biochem. Eng. Biotechnol.
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8. Lowry, O.M., Rosebrough, N. J., Farr, A.L. and Randall, R.J. (1951). Protein
measurement with Folin-phenol reagent. Journal of Biological Chemistry. 193:265-
275.9. Ramanathan, G., Banupriya, S., and Abrirami, D. (2010). Production and
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Fermentation.Journal of Scientific and Industrial Research. 69:454-459.10. Reese, E.T. and Mandels, M. (1984) Rolling with the time: production and
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11. Sahoo K. and Dhal, N.K. (2009), Potential microbial diversity in mangrove
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12. Sharada, R., Venkateswarlu, G., Venkateshwar, S. and Anand Rao, M. (2013).
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Web bibliography
http://www.indianexpress.com/news/elevated-churchgatevirar-corridor-still-
stuck/1017250/0
http://scholarsresearchlibrary.com/archive.html