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

1. Ali, M. I. A., Neveen, M., Khalil and Abd El-Ghany (2012 ). Biodegradation of

some polycyclic aromatic hydrocarbons by Aspergillus terreus. African Journal of

Microbiology Research, 6(16): 3783-3790.

2. Alexander, A. Kamnev and Danie¨l van der Lelie, (2000). Chemical and

Biological Parameters as Tools to Evaluateand Improve Heavy Metal

Phytoremediation. Bioscience Reports, 20 : 239-258.

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

Partial Purification of cellulase by Aspergillus niger fermented with paper and timber

sawmill industrial wastes, Journal of Microbiology and Biotechnology

Research,2(1):120-128.6. Gupta, N., Das, S. and Basak, U.C., (2007). Useful extracellular activity of

bacteria isolated from Bhitarkanika mangrove ecosystem of Orissa coast, Malaysian

Journal of Microbiology, 3(2):15-18.7. Kuhad, R.C., Singh, A. and Eriksson, K. E. (1997). Microorganisms enzymes

involved in the degradation of plant fiber cell walls. Adv. Biochem. Eng. Biotechnol.

57: 45-125.

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

optimization of Cellulase from Fusarium oxysporum by Submerged

Fermentation.Journal of Scientific and Industrial Research. 69:454-459.10. Reese, E.T. and Mandels, M. (1984) Rolling with the time: production and

applications of Trichoderma reesei cellulase. Annual Report of Fermentation

Processes. 7:1–20.

11. Sahoo K. and Dhal, N.K. (2009), Potential microbial diversity in mangrove

ecosystem, A review Indian journal of Marine Sciences, 38(2): 249-256.

12. Sharada, R., Venkateswarlu, G., Venkateshwar, S. and Anand Rao, M. (2013).

Production of cellulase - A review. International Journal of Pharmaceutical, Chemical

and Biological Sciences, 3(4): 1070-1090.13. Singh, A., Singh, N. and Bishnoi, N.R. (2009). Production of Cellulases by

Aspergillus Heteromorphus from Wheat Straw under Submerged Fermentation.

International Journal of Civil and Environmental Engineering, 1:23-26.

Web bibliography

http://www.indianexpress.com/news/elevated-churchgatevirar-corridor-still-

stuck/1017250/0

http://scholarsresearchlibrary.com/archive.html