goutam dissertation

S.no Contents Page no 1 Introduction

1.1 Coprophilous Fungi 1-2

1.2 Bio Diversity of Fungi 2-4

1.3 Cellulose Bio Conversion 4-5

1.3.a Cellulolysis 6-7

2 Review of Literature

2.1 Coprophilous Fungi 9-11

2.1 Dung Physiology 11-14

2.3 Coprophilous Fungal Diversity 14-18

2.4 Secondary Metabolites From Coprophilous Fungi 18-22

2.5 Cellulose Bio Conversion 22-26

3 Material and Methods

3.1 Requirements 27-30 3.2 Collection of Sample 31 3.3 Isolation of Coprophilous Fungi 31 3.3.a Moist Chamber Method 31 3.3.b Dilution Plating 32-33 3.4 Sub Culturing Technique 33-34 3.5 Lactophenol Mounting of Fungi 34 3.6 Shake Flask Culturing of Fungus 35 3.7 Test for Cellulase Activity 35 3.7.a Benedict’s Test 36-37

3.7.b DNS Reagent Test 37-39

3.8 Culture Media 40

3.8.a Potato Dextrose Agar Medium 40

3.8.b Czapeck’s Modified Broth Medium 40-41

4 Results

4.1 Part-1 Bio Diversity of Coprophilous Fungi 42-46 4.2 Part-2 Shake Flask Culturing of Fungus 46-47 4.3 Part-3 Cellulose Bio Conversion 47 4.3.a Benedicts Test 47-48 4.3.b DNS Reagent test 49-50

5 Discussion 51

6 Summary 52-53

7 Bibliography 54-56

DEPARTMENT OF BioSciences

SRI SATHYA SAI UNIVERSITY

(Established under Section 3 of the UGC Act, 1956)

Accredited by NAAC at A++ level

Vidyagiri, Prasanthi Nilayam – 515 134, Anantapur District, Andhra Pradesh, India

CERTIFICATE

This is to certify that the Dissertation entitled “Studies On

Coprophilous Fungi From The Dung Temple Elephant(Elephas

Maximus.L) Of Vidyagir Complex , Prashanthi Nilaym And Its

Environments. ” submitted by Sri Chintala Goutam for the award of

the degree of Master of Sciences (Biosciences) is a bonafide record of

Research work carried out by him in the Department of Biosciences, Sri

Sathya Sai University, Prasanthi Nilayam, under my guidance. The work

is original and has not formed the basis for the award of any degree,

diploma or any other such title by this or any other university.

Prasanthi Nilayam, Anantapur Dt. (A.P), India. – 515 134.

Prof.S.Krupanidhi

(Head of the Department)

Dr. B.S.Vijaya Kumar

(Dissertation Guide)

Place: Prashanthi Nilayam

Date:

Sri Sathya Sai University, Prasanthi Nilayam – 515134, Anantapur Dt., A.P., India.

DEPARTMENT OF BIOSCIENCES

SRI SATHYA SAI UNIVERSITY (Established under Section 3 of the UGC Act, 1956)

PRASANTHI NILAYAM CAMPUS PRASANTHI NILAYAM - 515134

DECLARATION

This dissertation entitled “Studies On Coprophilous Fungi From The

Dung Of Temple Elephant (Elephas maximus) Of Vidyagiri Complex,

Prashanthi Nilayam And Its Environments” is an original work done by

me under the supervision of Dr B. S Vijaya Kumar, Department of

Biosciences, Sri Sathya Sai University, Prasanthi Nilayam, in partial

fulfillment of the requirements for the award of the degree of Master of

Science in Biosciences of this University, and has not formed the basis for

the award of the degree, diploma or any other such title of this University or

any other University.

Chintala Goutam

(Regd No. 08151)

Place: Prasanthi Nilayam

Date:

Acknowledgement

Acknowledgement

I place my heartfelt gratitude at the Divine Lotus Feet of my Beloved Bhagawan

Sri Sathya Sai Baba, without his Grace this dissertation would not have been

accomplished. His invisible hand was a guiding force that was felt at every stage of

this dissertation.

I take this opportunity to acknowledge with thanks, the Guidance, Encouragement

and Support received from my respected guide, Dr.B.S.Vijaya Kumar, Department

of Biosciences, throughout the course of the dissertation. His dedication and

commitment to the subject has inspired me and has left an indelible mark in my

memory.

I am gratefully indebted to the head of the department

Prof. S.Krupanidhi and to all my teachers in the department, for their unstinted

support and guidance.

I am indebted to DR.H.V.Batra sir for allowing me to do summer project under

him and I take this opportunity express my sincere thanks to him for teaching me

various microbial techniques.

My thanks to Sri Renju Raghuveeran and sri Prakash chittaranjan for providing

the computer and internet facilities which helped me a lot in collection of relevant

literature.

Acknowledgement

I wish to specially thank Sri Robin sharma, Sri Sai malliswar, Sri K.N.Narsh , Sri

Sujit Kumar, Sri Anand and Sri Sai Krishna for their selfless help extended to me

in spite of their pressing academic and non-academic commitments.

My heartfelt thanks are also due to my project mate Rajesh and my classmates

Vijay sai, Goutham nag, Somanth and Raja and my roommates for their

encouragement and help during the study.

I also wish to thank M. Sai Ram for his support rendered in using MS Office and

A.Sunil Kumar helping me in taking print out of this dissertation.

I thank all those who helped me directly or indirectly in the completion of this

work, especially our beloved temple Elephant “Krishna Geeta”.

Last, but not the least, I express my gratitude to my mother who constantly

supported and helped me in this endeavor to finish this project.

CHINTALA GOUTAM

Introduction

Studies On Coprophilous Fungi From Dung Of Elephant Page 1

1. INTRODUCTION

1.1 COPROPHILOUS FUNGI

Fungi are ubiquitous organisms which grow on variety of substrates. Some

depend on the dead and decaying mater for source of carbon others survive as

pathogens on plants and animals. Coprophilous fungi are associated with

herbivore dung which means dung loving fungi; they are type of saprobic fungi

that grow on animal dung. They play an important role in the ecosystem,

responsible for recycling the nutrients in animal faeces. Herbivorous animals

grazing on vegetation ingest many fungal spores along with their food. Some of

the fungi will be coprophilous, and these usually have thick-walled, pigmented

spores that require passage through the gut of an animal to germinate. The high

temperature and enzymes in the digestive tract of the animal will kill most of

the other fungal spores they ingest. Once the dung is voided, the viable fungal

spores will germinate, grow and fruit on the dung. The spores are usually

forcibly discharged onto the vegetation surrounding the dung; another grazing

animal comes along, eats the vegetation and the cycle is repeated.

Dung is a great substrate for isolating a wide range of fungi. It consists of

remains of plant material plus the micro biota associated with its digestion.

Much of the material consists of readily available carbohydrate in addition to

cellulose and lignin. The material is complex and includes fatty acids, vitamins

and amino acids. Since the coprophilous fungi have got complex materials such

as cellulose and lignin as source of food, it contains strong hydrolytic enzymes

which hydrolyze them. This group is an important source of antibiotics,

enzymes, and biological control agents (Sayanh). Coprophilous fungi may be

useful indicators of habitat diversity (Richardson, 2001). Coprophilous fungi

have already been shown to produce interesting secondary metabolites (Gloer,

1995).These rarely-studied fungi are ecologically, morphologically and

Introduction


taxonomically distinctive, and they commonly display antagonistic effects

against ohter fungi. Our search for antifungal metabolites from these species is

based on a systematic, ecology-based approach to microorganism selection that

represents a departure from traditional random microbial screening programs.(

James B Gloer). Most previous ecological studies of coprophilous Ascomycetes

have been done in tropical and warm arid regions (Elshafie, 2005; Masunga et

al. 2006; Jeamjitt et al. 2007), or on domesticated animals and rabbits

ingrasslands (Wicklow et al., 1980; Angel and Wicklow, 1983). Dung from

wild borealanimals, and especially forest-living species, have been much less

studied. The aim of this work is to study the diversity of coprophilous fungi

from a tamed, temple, Asian elephant and identify few for cellulase enzyme

production. Cellulase production is the most important step in the

economicalproduction of ethanol and other chemicals from renewable cellulosic

materials. (Md. Munir H. Khan and etal.)

1.2 BIO DIVERSITY OF COPROPHILOUS FUNGI

The distribution of coprophilous fungi is closely linked to the distribution of the

herbivores on which they rely, such as rabbits, deer, cattle, horses and sheep.

Some species rely on a specific species for dung; for instance, Coprinus

radiatus and Panaeolus campanulatus grow almost exclusively on horse feces,

while others, such as Panaeolus sphinctrinus, can grow on any feces or even just

particularly fertile soil. Further, some species (such as Conocybe rickenii) can

be found in large numbers in areas where manure has been used as a soil

fertilizer, such as in gardens. Some coprophilous fungi are also known to grow

from the dung of omnivores (such as Chaetomium globisporum from rat

droppings) or even carnivores (such as Chaetomium rajasthanense, from tiger

feces).

Introduction


Food choice is another important factor influencing the species richness of

Coprophilous Ascomycetes, and that some species are more associated with

habitat and food choice of the herbivore, rather than a specific dung type/animal

species. The composition of species on the different dung types is also

discussed. Results suggest that the coprophilous mycota in the boreal forest is

poorly known. So many coprophilous species are ubiquitous, while others have

high preferences for a particular dung type (Lundqvist, 1972), and dung from

closely related herbivores generally have similar species composition

(Richardson, 2001). This suggests that the digestive system of the herbivore

may influence species composition and richness, as differences in digestion

could affect both the passage of the spores through the gut, dung moisture and

nutrient content.

Twenty-one dung samples were collected, from Puerto Rico, US Virgin Islands

(St John), Guadeloupe, Dominica, and St Lucia (Table 1). On incubation they

yielded a total of 199 records of 54 species. The composition of the mycota was

very similar to those found elsewhere. The average number of characteristically

Coprophilous species recorded from a sample was nine, with a range from 3-15.

The average is slightly lower than the 10-12 for analogous dung types (e.g.

excluding samples from lagomorphs and herbivorous birds) reported from much

larger collections worldwide (Richardson 2001a)

Fifty-seven species of coprophilous fungi are recorded from 14 dung samples

collected from the Souss Valley area of southern Morocco that were incubated

in moist chambers. Several new records for Morocco are reported. Evidence for

reduced diversity due to the severely degraded nature of the habitats in which

the samples were collected is discussed. (Richardson)

A preliminary investigation was made of coprophilous fungi from Khao Yai

National Park. Dung of sambar deer (Cervus unicolor), common barking deer

Introduction


(Muntiacus muntjak) and Asian elephant (Elephas maximus) were collected and

incubated in moist chambers. These yielded over 90 fungi, 68 belong to

Coprinus sp., Cunninghamella echinata, Delitschia pachylospora, Idriella

lunata, Penicillium claviforme, Pilobolus sp., Podospora communis, Podospora

sp., Poronia gigantea, Saccobolus citrinus, S. thaxteri, Scopulariopsis brumptii,

Stilbella sp., Syncephalastrum racemosum, Volutella cilliata, Wiesneriomyces

laurinus, and Zygospermella sp. The remaining species are undergoing

characterization and identification.

1.3 CELLLOSE BIO CONVERSION

Cellulose is an organic compound with the formula (C6H10O5)n, a

polysaccharide consisting of a linear chain of several hundred to over ten

thousand β(1→4) linked D-glucose units. Cellulose is the structural component

of the primary cell wall of green plants, many forms of algae and the

oomycetes. Cellulose is the most common organic compound on Earth. About

33 percent of all plant matter is cellulose (the cellulose content of cotton is 90

percent and that of wood is 50 percent).

For industrial use, cellulose is mainly obtained from wood pulp and cotton. It is

mainly used to produce paperboard and paper; to a smaller extent it is converted

into a wide variety of derivative products such as cellophane and rayon.

Converting cellulose from energy crops into biofuels such as cellulosic ethanol

is an alternative fuel source.

Cellulosic ethanol is a biofuel produced from wood, grasses, or the non-edible

parts of plants. It is a type of biofuel produced from lignocellulose, a structural

material that comprises much of the mass of plants. Lignocellulose is composed

mainly of cellulose, hemicellulose and lignin. Corn stover, switch grass,

miscanthus, woodchips and the byproducts of lawn and tree maintenance are

some of the more popular cellulosic materials for ethanol production.

http://en.wikipedia.org/wiki/Organic_compound

http://en.wikipedia.org/wiki/Chemical_formula

http://en.wikipedia.org/wiki/Carbon

http://en.wikipedia.org/wiki/Hydrogen

http://en.wikipedia.org/wiki/Oxygen

http://en.wikipedia.org/wiki/Polysaccharide

http://en.wikipedia.org/wiki/Glucose

http://en.wikipedia.org/wiki/Cell_wall

http://en.wikipedia.org/wiki/Green_plants

http://en.wikipedia.org/wiki/Algae

http://en.wikipedia.org/wiki/Oomycetes

http://en.wikipedia.org/wiki/Cotton

http://en.wikipedia.org/wiki/Wood

http://en.wikipedia.org/wiki/Wood_pulp

http://en.wikipedia.org/wiki/Cotton

http://en.wikipedia.org/wiki/Paperboard

http://en.wikipedia.org/wiki/Paper

http://en.wikipedia.org/wiki/Cellophane

http://en.wikipedia.org/wiki/Rayon

http://en.wikipedia.org/wiki/Energy_crop

http://en.wikipedia.org/wiki/Biofuel

http://en.wikipedia.org/wiki/Cellulosic_ethanol



http://en.wikipedia.org/wiki/Lignocellulose

http://en.wikipedia.org/wiki/Cellulose

http://en.wikipedia.org/wiki/Hemicellulose

http://en.wikipedia.org/wiki/Lignin

http://en.wikipedia.org/wiki/Corn_stover

http://en.wikipedia.org/wiki/Switchgrass

http://en.wikipedia.org/wiki/Miscanthus

http://en.wikipedia.org/wiki/Wood_pulp

Introduction


Production of ethanol from lignocellulose includes a process called as

cellullolysis. It involves hydrolysis on pretreated lignocellulosic materials, using

enzymes to break complex cellulose into simple sugars such as glucose and

followed by fermentation and distillation.

The cellulases needed for breaking down cellulose so far have come from fungi,

in particular from Trichoderma reesei . NREL scientists have investigated other

sources, such as the bacterium Acdiothermus cellulolyticus , which they found

in the hot springs of Yellowstone National Park. But bacterial exoglucanases are

not usually as good as the fungal ones, though they tolerate high temperatures.

A next step is to combine high temperature tolerance with the efficiency of the

fungal enzyme. NREL and DOE have contracted the world's largest enzyme

companies, Genecor International and Novozymes to reduce the cost of

producing cellulases down to a range of $.10-$.20 per gallon of ethanol, and

they have succeeded .

A further improvement involves the simultaneous action of enzyme and

fermenting microbes, so that as the sugars are produced by the cellulases, the

microbes ferment the glucose to ethanol .

Iogen Corporation based in Ottawa, Canada was the first to develop the enzyme

process for getting ethanol from cellulose.(ISIS Report 15/03/06 Ethanol from

Cellulose Biomass Not Sustainable nor Environmentally Benign Major

technical and economic hurdles remain in getting ethanol from plant wastes,

while burning ethanol produces carcinogens and increases ozone levels in the

atmosphere.( Dr. Mae-Wan Ho )

http://en.wikipedia.org/wiki/Hydrolysis

http://en.wikipedia.org/wiki/Enzyme

http://en.wikipedia.org/wiki/Sugars

http://en.wikipedia.org/wiki/Glucose

http://en.wikipedia.org/wiki/Ethanol_fermentation

http://en.wikipedia.org/wiki/Distillation

Introduction


1.3. a CELLULOLYSIS

Cellulolysis is the process of breaking down cellulose into smaller

polysaccharides called cellodextrins or completely into glucose units; this is a

hydrolysis reaction. Because cellulose molecules bind strongly to each other,

cellulolysis is relatively difficult compared to the breakdown of other

polysaccharides.

Mammals do not have the ability to break down cellulose directly. Some

ruminants like cows and sheep contain certain symbiotic anaerobic bacteria

(like Cellulomonas) in the flora of the gut wall, and these bacteria produce

enzymes to break down cellulose; the breakdown products are then used by the

mammal. Similarly, lower termites contain in their hindguts certain flagellate

protozoa which produce such enzymes; higher termites contain bacteria for the

job. Fungi, which in nature are responsible for recycling of nutrients, are also

able to break down cellulose.

Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and

protozoans that catalyze the cellulolysis (or hydrolysis) of cellulose. Several

different kinds of cellulases are known, which differ structurally and

mechanistically. Other names include 'endoglucanases' are: endo-1,4-beta-

glucanase, carboxymethyl cellulase (CMCase), endo-1,4-beta-D-glucanase,

Beta-1,4-glucanase, Beta-1,4-endoglucan hydrolase, Celludextrinase. The other

types of cellulase belong to Excocellulases. The reaction involves Hydrolysis of

1,4-beta-D-glycosidic linkages in cellulose, lichenin and cereal beta-D-glucans.

The cellulolytic activtiy of some soil fungi isolated from soil and decomposing

pieces plant material was observed to depend upon the genera of fungi , the

nature of substrate and the temperature, the cellulase produced in the presence

of cellulose powder was active against CMC this observation was given by

R.Lal and M.M.Mishra. Dept of microbiology, Haryana Agricultural University,

http://en.wikipedia.org/wiki/Cellodextrin


http://en.wikipedia.org/wiki/Ruminant

http://en.wikipedia.org/wiki/Symbiosis

http://en.wikipedia.org/wiki/Anaerobic

http://en.wikipedia.org/w/index.php?title=Cellulomonas&action=edit&redlink=1

http://en.wikipedia.org/wiki/Enzyme

http://en.wikipedia.org/wiki/Termite

http://en.wikipedia.org/wiki/Hindgut

http://en.wikipedia.org/wiki/Flagellate

http://en.wikipedia.org/wiki/Protozoa

http://en.wikipedia.org/wiki/Fungus

http://en.wikipedia.org/wiki/Enzymes

http://en.wikipedia.org/wiki/Fungi

http://en.wikipedia.org/wiki/Bacteria

http://en.wikipedia.org/wiki/Protozoan

http://en.wikipedia.org/wiki/Catalyze


http://en.wikipedia.org/wiki/Glycosidic_linkage

Introduction


Hissar, India. (cellulolytis activity of some solil fungi, folia microbiol 23,68-

71(11978).

Most fungal cellulases have a two-domain structure with one catalytic domain,

and one cellulose binding domain, that are connected by a flexible linker. This

structure is adaption for working on an insoluble substrate and it allows the

enzyme to diffuse two-dimensionally on a surface in a caterpillar way.

However, there are also cellulases (mostly endoglucanases) that lacks cellulose

binding domain. These enzymes might have a swelling function. Many reports

are there regarding production of cellulase enzymes by fungi like T.L. Highley

and Barbara L. Illman

U.S. Department of Agriculture, Forest Service, and Forest Products Laboratory

had reported that brown rot fungi deteriorate wood, by primarily utilizing the

cellulose and hemicellulose components of wood by production of cellulasese at

the contact point. The effect of brown-rot fungi on wood strength properties

reflects cellulose depolymerisation. Shortly after colonizing wood, brown-rot

fungi cause a sharp reduction in the degree of polymerization (DP) of cellulose

(1800-2000 glucosyl units to 150-200 units) at low weight loss without

removing the lignin (Cowling, 1961). E. Yague and M. P. Estevez reported

that The epiphytic lichen Evernia prunastri (L.) Ach. synthesizes both a

constitutive and a carboxymethycellulose-inducible P-l,4-glucanase (cellulase).

The production of endoglucanase(EC 3.2.1.4) and exoglucanase (EC 3.2.1.91)

enzyme was studied during penetration of the host and development of the

VAM fungus Glomus mosseae in the roots of lettuce(Lactuca sativa) and

onion(Allium cepa) was rported by J.M Garcia and etal.

Introduction


OBJECTIVES

1. To isolate coprophilous fungi from temple elephant (Elephas maximus.L)

dung.

2. To culture and identify different species of coprophilous fungi from

Elephant dung in laboratory.

3. To Study cellulolytic activity of crude enzyme extracted from Coprophilous

fungi from Elephant (Bio prospecting).

PLAN OF WORK

1. Collection of Elephant dung form the Vidyagiri complex and its

environments of Puttaparthi.

2. Incubation of the collected dung.

3. Observation for 4 to 5 weeks for fungal fruiting bodies and spores.

4. Medium preparation.

5. Plating and slant preparation.

6. Isolation and identification of Coprophilous fungi.

7. Preparation of pure culture.

8. Growing in shake flask culture to obtain crude metabolite.

9. Bio prospecting.

Lot of work has been done on Coprophilous fungi around the world but no work

has been done in the Coprophilous Fungi of temple elephant of Prasanthi

Nilayam , hence this preliminary investigation forms a original contribution in

the field of Mycology and fungal biotechnology.

Review of Literature


2. REVIEW OF LITERATURE

2.1 COPROPHILOUS FUNGI

Coprophilous fungi (literally dung-loving fungi are a type of saprobic fungi that

grow on animal dung. The hardy spores of coprophilous species are unwittingly

consumed by herbivores from vegetation, and are excreted along with the plant

matter. The fungi then flourish in the dung, before releasing their spores to the

surrounding area .Coprophilous fungi release their spores to the surrounding

vegetation, which is then eaten by herbivores. The spores then remain in the

animal as the plants are digested, pass through the animal's intestines and are

finally defecated. The fruiting bodies of the fungi then grow from the animal

dung. It is essential that the spores of the species then reach new plant material;

spores remaining in the feces will produce nothing. As such, some species have

developed means of discharging spores a large distance. An example of this is

the genus Pilobolus. Fruiting bodies of Pilobolus will suddenly rupture, sending

the contents over 2 metres away.

Coprophilous Ascomycetes are a diverse group of saprobes including taxa from

most major taxonomical groups. Some species are strictly coprophilous while

others may germinate on several substrates. The Coprophilous ascospores are

spread by various dispersal mechanisms from the dung pile to the surrounding

vegetation. The spores are often surrounded by mucilage or have gelatinous

appendages, and attach easily to the plant parts on which they land (Wicklow,

1981). Feeding herbivores ingest the spores that often are darkly pigmented and

well protected against both gastric juices and the harmful UV-light of the sun.

With the digestive system of herbivores, spore germination can even be

triggered by gastric juices (Webster, 1970). Some species are ubiquitous, while

others have high preferences for a particular dung type (Lundqvist, 1972), and



dung from closely related herbivores generally have similar species composition

(Richardson, 2001). This suggests that the digestive system of the herbivore

may influence species composition and richness, as differences in digestion

could affect both the passage of the spores through the gut, dung moisture and

nutrient content. Richardson (2005) compared dung of sheep and hare that were

feeding the same vegetation, and found marked differences in species

compositions; differences that he attributed to differences in digestion.

Differences in ascomycete species richness and composition may also reflect

differences in feeding habits and/or food choice between herbivores. Ebersohn

and Eicker 74 Most previous ecological studies of coprophilous ascomycetes

have been done in tropical and warm arid regions (Elshafie, 2005; Masunga et

al. 2006; Jeamjitt et al. 2007), or on domesticated animals and rabbits in

grasslands (Wicklow et al., 1980; Angel and Wicklow, 1983). Dung from wild

boreal animals, and especially forest-living species, have been much less

studied.

Coprophilous fungi are usually associated with herbivore dung. This group has

been a source of biological control agents, enzymes, antibiotics, etc. A

preliminary investigation was made of coprophilous fungi from Khao Yai

National Park. Dung of sambar deer (Cervus unicolor), common barking deer

(Muntiacus muntjak) and Asian elephant (Elephas maximus) were collected and

incubated in moist chambers. These yielded over 90 fungi, 68 belong to

Coprinus sp., Cunninghamella echinata, Delitschia pachylospora, Idriella

lunata, Penicillium claviforme, Pilobolus sp., Podospora communis, Podospora

sp., Poronia gigantea, Saccobolus citrinus, S. thaxteri, Scopulariopsis brumptii,

Stilbella sp., Syncephalastrum racemosum, Volutella cilliata, Wiesneriomyces

laurinus, and Zygospermella sp (Sayanh Somrithipol) .



Pilobolus species found on herbivore dung from the São Paulo Zoological Park,

Brazil). A study of Pilobolus species from 168 dung samples of various

herbivoresous animals, collected in the São Paulo Zoological Park, was carried

out. Ten species were found, illustrated, described, and a key for their

identification is provided Acta bot. bras. 22(3): 614-620. 2008 Pilobolus

species found on herbivore dung from the São Paulo Zoological Park, Brazil

Aírton Viriato.

2.2 DUNG PHYSIOLOGY

The animal dung provides an environment rich in nitrogenous material, which

has been largely sterilised by the high temperature, as well as the enzymes in

the animal's digestive system. The spores themselves survive digestion by being

particularly thick-walled, allowing them to germinate in the dung with

minimum competition from other organisms. This thick wall is often broken

down during digestion, readying the spore for germination. The spores are so

hardy that samples of dried dung can later be rehydrated, allowing the fungus to

fruit weeks later.

Dung is source of organic matter and a potential home for saprotrophs. From a

fungal point of view, herbivore dung is the more interesting, since bacteria are

largely responsible for the breakdown of carnivore and omnivore dung.

Herbivore dung supports a wide variety of coprophilous fungi. Herbivore dung

typically contains plant material digested to varying extents

Dung is a great substrate for isolating a wide range of fungi. There are even

several books available just for the identification of dung fungi. The collection

and incubation of dung is quite easy. The best dung to work with is herbivore

dung; any type of dung will work, but the dung of omnivores and carnivores

gets quite disgusting after a week or so of incubation, and this type of dung do



not yields that many different types of fungi. The dung of omnivore and

carnivores also is more likely to broken down by bacteria than fungi (Bell,

1983). There are good results with dung from elk, deer, cows, horses, llamas,

sheep and rabbits. In general the dung from pets which are fed pelleted food is

disappointing (Bell, 1983.The dung should be as fresh as possible, and once

collected, brought immediately into the laboratory and placed on moist filter

paper in a Petri dish.

The dung is incubated at room temperature. Natural light is often beneficial in

inducing sporulation of coprophilous fungi. Start examining the dung with a

dissecting microscope after several days. It is important to examine the dung on

a regular basis because a succession of fungi will sporulate, and many of the

dung fungi produce ephemeral fruiting bodies. The best way for isolating the

dung fungi is to pick off individual fruiting bodies with a sterile minuten pin,

and transferring the fruiting body directly to antiobiotic agar. Alternatively,

perithecia can be placed in a drop of sterile water on a clean microscope slide,

crushed and the spores streaked out on agar. Some of the more interesting and

beautiful cup fungi require several weeks‟ incubation to develop. These cup

fungi can be isolated by picking one of the fruiting bodies (apothecia) off the

dung with a minuten pin, and suspending the apothecium on the inner lid of a

Petri dish with a small amount of petroleum jelly. The spores will be forcible

discharged down onto the agar, where they can be picked off and transferred to

a clean dish of agar. Many of the coprophilous fungi forcibly discharge their

spores, and a good place to look for the spores is on the inside of the Petri dish

lid. Also look for colonies of nematodes on the lid; some nematodes are known

to “hitch a ride” on the forcibly discharged propagules of Pilobolus and these

nematodes, as previously noted, may also have an unusual group of fungi

parasitizing them. Among the succession of fungi developing on dung are the



tiny basidiocarps of Coprinus species. These mushrooms are often less than 1

cm in height and collapse shortly after maturity. Coprophilous fungi producing

perithecia and apothecia are also very common, but may not be very

conspicuous until you really start looking. I find that it is easy to spend hours

scanning my „dung scape‟ for different fungi. Dung cultures can be maintained

for several weeks as long as they are not allowed to dry out. Herbivore really

doesn‟t smell that bad, and maintaining cultures for long periods of time is not a

problem.The most useful are those with keys and descriptions for identification

by Bell (1983), Ellis and Ellis (1988) and Seifert et al (1983).

Elephant dung is a very complex system, with so many possible chemical

agents which might act as stimulators or inhibitors of germination and/or seed

growth. These may be the metabolic products of the elephant‟s own

physiological system and chemicals from the very large amount of

undigested/partially digested vegetal remains, a characteristic feature of

elephant and rhino dung. Such dung is expected to contain phenolics. Since a

large quantity and variety of phenolic substances occur in the plant world, we

have investigated the possibility of phenolics in elephant dung exerting an

influence on seedling growth. This would be apart from any nutritive effect that

the dung as a source of manure might exert. (Mandal, August 2002)

As carbon losses occur in the form of CO, mineral elements which do not have

a gaseous phase or which are efficiently conserved by the microflora, such as

nitrogen, should increase in their percentage composition. The decreases in

nitrogen, phosphorus and possibly magnesium content of the exposed dung by

day 14 therefore reflect real reductions in the amounts of these elements. The

increase in percent potassium content suggests that potassium was not lost

during decomposition and may have shown a real increase due to the



contamination of the dung by dust and soil. The slight increase in carbon

content may not be significant but is also indicative of the differential removal

of other elements. The decreases of nitrogen, phosphorus and magnesium could

be caused by the activities of the dung fauna or by leaching.

Nitrogen could also have been lost by denitrification and/or ammonification but

it is more likely that it was conserved by incorporation into microbial tissues.

2.3 COPROPHILOUS FUNGAL DIVERSITY

The distribution of coprophilous fungi is closely linked to the distribution of the

herbivores on which they rely, such as rabbits, deer, cattle, horses and sheep.

Some species rely on a specific species for dung; for instance, Coprinus

radiatus and Panaeolus campanulatus grow almost exclusively on horse feces,

while others, such as Panaeolus sphinctrinus, can grow on any feces or even

just particularly fertile soil. Further, some species (such as Conocybe rickenii)

can be found in large numbers in areas where manure has been used as a soil

fertilizer, such as in gardens. Some coprophilous fungi are also known to grow

from the dung of omnivores (such as Chaetomium globisporum from rat

droppings) or even carnivores (such as Chaetomium rajasthanense, from tiger

feces).

If you incubate a dung sample and observe it for many weeks you will see a

sequence of fruiting bodies appearing on it. Some people may refer to this as a

succession of fungi appearing on the dung and make analogies with a plant

succession in a disturbed area. First come the pioneering plants and these are

later displaced by other species as fresh seeds arrive or as buried seeds

germinate.

../../wiki/Rabbit

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../../wiki/Cattle

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../../wiki/Sheep

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../../wiki/Panaeolus_campanulatus

../../wiki/Panaeolus_sphinctrinus

../../wiki/Fertility_(soil)

../../wiki/Conocybe_rickenii

../../wiki/Manure

../../wiki/Fertilizer

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../../wiki/Carnivore

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One thing to be very careful of when talking about fungal succession is that the

visible fruiting bodies are not the whole story. There are the mycelia, out of

sight inside the dung, and the visible sequence of fruiting bodies need not reflect

a similar sequence of fungal colonization of the dung. If the fruiting bodies of a

particular fungus appear a couple of months after the dung was dropped, is it

because the spores of that particular fungus only arrived later? Or was the

mycelium of that fungus present early on, but needing a long time to build up

the mass necessary for the production of fruiting bodies? If the mycelium was

present fairly early, along with the mycelia of other species, you can certainly

talk about fruiting body succession, but there has been no fungal succession.

The spores of many dung fungi are on the dung at the time it is dropped by an

animal, for the animal will have swallowed many fungal spores in the course of

feeding. Once released from their dung-inhabiting fruiting bodies, the spores of

many dung fungi end up falling onto grass and leaves. Many species of dung

fungi have spores with thick walls, which weaken during passage through an

animal‟s gut and so ready the spores for germination, once they have been

deposited with the animal‟s droppings.

At the time the dung drops to the ground there are likely to be a number of

fungal species with spores ready to germinate. Many of these germinate at much

the same time but the mycelia then grow at varying speeds. Thus, in some cases

the sequence of fruiting body appearances reflects the speed of mycelial growth,

and how quickly a mycelium can accumulate enough resources to allow the

production of fruiting bodies. Some dung fungi, though slow growing, are very

antagonistic to other species and able to destroy or severely inhibit other

mycelia.



One promising approach in the search for fungi producing biologically active

secondary metabolites is to focus on species capable of colonizing rich and

highly contested substrates. One of the richest natural substrates is the dung of

herbivores which is in habited by a wide range of specialized coprophilous

fungi, in addition to opportunistic colonizers from the surrounding soil (Dix and

Webster, 1995; Richardson, 2001). A succession of different groups of fungi on

fresh dung has been characterized by the order of appearance of their fruit-

bodies (Webster, 1970). Early colonizers (mostly zygomycetes) grow and fruit

rapidly but soon give way to the more slowly-growing species of asco- and

basidiomycetes. We are interested especially in these late colonizers because

they must displace the mycota already present on the dung and are therefore

more likely producers of bioactive secondary metabolites. Some Coprophilous

fungi have already been shown to produce interesting secondary metabolites

(Gloer, 1995), but many remain to be examined. Although data on the

production of antibiotic substances in situ are still sparse, antibiosis is thought

to be an important determinant of the succession on dung. A small number of

species of Xylariaceae (Ascomycota) have adopted a coprophilous lifestyle, and

among them Poronia punctata has been shown to produce a range of

biologically active sesquiterpenes . Podosordaria tulasnei, which is associated

with rabbit dung in dry habitats such as sand dunes, spreading into the soil and

colonizing new pellets by means of rhizomorphs (Webster and Weber, 2000).

When we incubate a dung sample and observe it for many weeks we will see a

sequence of fruiting bodies appearing on it. Some people may refer to this as a

succession of fungi appearing on the dung and make analogies with a plant

succession in a disturbed area. First come the pioneering plants and these are

later displaced by other species as fresh seeds arrive or as buried seeds

germinate. One thing to be very careful of when talking about fungal succession



is that the visible fruiting bodies are not the whole story. There are the mycelia,

out of sight inside the dung, and the visible sequence of fruiting bodies need not

reflect a similar sequence of fungal colonization of the dung. If the fruiting

bodies of a particular fungus appear a couple of months after the dung was

dropped, is it because the spores of that particular fungus only arrived later? Or

was the mycelium of that fungus present early on, but needing a long time to

build up the mass necessary for the production of fruiting bodies? If the

mycelium was present fairly early, along with the mycelia of other species, you

can certainly talk about fruiting body succession, but there has been no fungal

succession. The spores of many dung fungi are on the dung at the time it is

dropped by an animal, for the animal will have swallowed many fungal spores

in the course of feeding. Once released from their dung-inhabiting fruiting

bodies, the spores of many dung fungi end up falling onto grass and leaves.

Many species of dung fungi have spores with thick walls, which weaken during

passage through an animal‟s gut and so ready the spores for germination, once

they have been deposited with the animal‟s droppings. At the time the dung

drops to the ground there are likely to be a number of fungal species with spores

ready to germinate. Many of these germinate at much the same time but the

mycelia then grow at varying speeds. Thus, in some cases the sequence of

fruiting body appearances reflects the speed of mycelial growth, and how

quickly a mycelium can accumulate enough resources to allow the production

of fruiting bodies. Some dung fungi, though slow growing, are very antagonistic

to other species and able to destroy or severely inhibit other mycelia. Of course

there can be a fungal succession - some spores may germinate later, spores from

elsewhere may land on the dung and then germinate and grow there or mycelia

from elsewhere may move into the dung. As well as fungi various bacteria,

nematodes, mites and flies also make use of dung and in the wild the moisture



content of the dung may fluctuate. All in all, dung is a complex environment

and the interactions between all these organisms (and the weather) will also

influence the appearance of fruiting bodies. (Bell, 1983)

Harper & Webster (1964) attempted to explain the consistency in the onset of

sporulation within species of coprophilous fungi by suggesting that each species

required a minimum amount of time to produce fruit-bodies. Thus according to

Rayner & Todd (1979) citing work done by Harper & Webster, „the observed

succession [of fruit-bodies] could at least partly be explained simply by the

increasing amounts of time required by succeeding fungi to fruit‟. on equine

dung

The variability in conidiogenesis of the coprophilous Basifimbria aurea,

typespecies of the genus, is redescribed and illustrated, and is similar to that of

B. spinosa. The distinction of the species from Stenocephalopsis subalutacea

(syn. Rhinotrichum subalutaceum) is emphasized. (GL, may 2005)

2.4 SECONDARY METABOLITES FROM COPROPHILOUS FUNGI

Coprophilous fungi. These rarely-studied fungi are ecologically,

morphologically and taxonomically distinctive, and they commonly display

antagonistic effects against other fungi. The search for antifungal metabolites

from these species is based on a systematic, ecology-based approach to

microorganism selection that represents a departure from traditional random

microbial screening programs. A variety of new antifungal agents, including

many with unusual chemical structures, have been isolated from coprophilous

fungi. The results provide compelling evidence that these fungi show

considerable promise as sources of novel antifungal natural products. Because

of the increasingly urgent need for the new treatment effective against

opportunistic fungal infection in humans, screening effort will focus exclusively



on searches for new metabolites with activity against medically relevant fungi.

Pure compounds will ultimately be tested for other activities, with high priority

assigned to cancer-relevant assays. (James B Gloer)

Tulasnein , a new metabolite with strong antimicrobial and weaker cytotoxic

and phytotoxic activity, was isolated from culture filtrates of three strains of the

xylariaceous coprophilousfungus Podosordaria tulasnei. The producing strains

were identified by their rhizomorphsand by ITS rDNA sequence analysis. A

second new metabolite, podospirone , was alsoproduced by all three strains

whereas the weakly cytotoxic (+)-3,4-anhydroshikimic acidmethyl ester was

detected in only one strain.Tulasnein and Podospirone from the Coprophilous

Xylariaceous FungusPodosordaria tulasnei (Daniela C. Ridderbuscha)

The N-methylated antifungal cyclic tridecadepsipeptide Petriellin A was

isolated from the coprophilous fungus Petriella sordid (UAMH 7493) by Gloer

et al.,1 who used 1D and 2D NMR experiments to identify this compound.

Coprophilous fungi are uniquely adapted to herbivore dung, where they play an

important role in recycling the nutrients in animal faeces. They are also a good

source of antibiotics, enzymes and biological control agents. Petriellin A,

produced by antagonistic fungi, is heavily N-methylated, containing two N-

methyl-threonines, two N-methyl-valines and one N-methyl-isoleucine. Similar

to Cyclosporin A, it has a lactone backbone linkage but contains the relatively

rare structures: N-methyl-threonine, N-methylisoleucine and R configured

phenyllactic acid. In the original report the chirality of three of the amino acid

residues were not determined. Petriellin A is a novel cyclic depsipeptide

antifungal compound consisting of nine L-configured residues, one D-

phenyllactic acid (PhLac) and three unknown chiral centres: two N-methyl-



threonines (MeThr1 & MeThr2) and one N-methyl-isoleucine (MeIle). Solution

structures by NMR of a novel antifungal drug: Petriellin A† (Jason Dang L. A.)

(+)-Isoepoxydon has been established as the major causative agent of

interference competition between Poronia punctata (NRRL 6457), a late fungal

colonist of cattle dung, and two early-occurring dung colonists, Ascobolus

furfuraceus (NRRL 6460) and Sordaria fimicola (NRRL 6459). This compound

was isolated from ethyl acetate extracts of liquid cultures of P. punctata by

silica gel chromatography and identified by mass spectrometry and proton and

carbon nuclear magnetic resonance spectroscopy. (Jason Dang L. A.)

The enantioselective synthesis of the originally proposed structure of

communiol C, an antibacterial 2,4-disubstituted tetrahydrofuran natural product

from the coprophilous fungus Podospora communis, (Enomoto M)

Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus

were screened for peroxidase activities by using selective agar plate tests and

complex media based on soybean meal. Two species, Coprinus radians and

C.verticillatus, were found to produce peroxidases, which oxidized aryl alcohols

to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-

thiolatehaloperoxidases) (Anh DH)

Antiamoebins I, III and XVI as well as several others in minor amounts were

produced by four strains of the coprophilous fungus Stilbella

erythrocephala(syn. S. fimetaria) in its natural substrate and in liquid culture.

The total antiamoebin concentration in dung was 126-624 microg g(-1) fresh

weight, with minimum inhibitory concentrations against most other

coprophilous fungi being at or below 100 microg mL(-1). Myrocin B, not

previously described from S. erythrocephala, was also produced, but only at

low, nonfungicidal levels (< 5.3 microg g(-1)). No other antifungal substances



were detected. It is concluded that antiamoebins are responsible for antibiosis in

dung colonized by S.erythrocephala. (Lehr NA)

Rabbit pellets collected from the field were colonized by Podospora pleiospora

atthe exclusion of other coprophilous fungi, suggesting antibiosis. In liquid

culture, P. pleiospora produced sordarin (1); sordarin B (2), a new compound in

which sordarose is replaced by rhamnose; hydroxysordarin (3); and sordaricin

(4). The major compounds 1 and 2 exhibited minimum inhibitory

concentrations of 0.5-2.5 microg ml(-1) against the yeasts Nematospora coryli

and Sporobolomyces roseus, but showed little or no activity against bacteria or

Coprophilous filamentous fungi. In liquid culture, the production of 1 and 2

together amounted to 2.7 microg ml(-1), whereas in rabbit dung only 1 was

produced at a similar concentration (2.3 microg g(-1) fresh weight) (Weber

RW)

Communiols E-H (1-4), four new polyketide-derived natural products

containing furanocyclopentane, furanocyclopentene, cyclopentene, or gamma-

lactone moieties, have been isolated from two geographically distinct isolates

of the coprophilous fungus Podospora communis. The structures of these

compounds were determined by analysis of NMR and MS data. (Che Y)

Decipinin A (1), a new compound with antifungal and antibacterial activity,

hasbeen isolated from liquid cultures of the coprophilous fungus Podospora

decipiens(JS 270). Two new tetracyclic sesquiterpene lactones, decipienolides

A (2) and B (3), were also obtained from this isolate as an inseparable mixture

of epimersthat showed antibacterial activity. The structures of 1-3 were

elucidated by analysis of 1D and 2D NMR data, aided by chemical shift

comparisons to related compounds. (Che Y G. J., 2002 jun)



Reinvestigation of the fermentation broth and mycelium of the coprophilous

fungus Guanomyces polythrix, grown in static conditions, led to the isolation of

severalphytotoxic compounds, including two new naphthopyranone derivatives,

namely (2S, 3R)-5-hydroxy-6,8-dimethoxy-2,3-dimethyl-2,3-dihydro-4H-

naphtho[2,3-b]-pyran-4-one and (2S,3R)-5-hydroxy-6,8,10-trimethoxy-2,3-

dimethyl-2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one. The structures of the

new compounds were established by spectral andchiroptical methods. In

addition, the structure of 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylic

acid methyl ester wasunambiguously determined by X-ray analysis. The isolates

caused significantinhibition of radicle growth of two weed seedlings

(Amaranthus hypochondriacusand Echinochloa crusgalli) and interacted with

both spinach and bovine braincalmodulins. (Macías M, 2001 nov)

2.5 CELLULOSE BIO CONVERSION

Cellulose is the most abundant polymer in the biosphere with its

estimated synthesis rate of 1010 tonnes per year (Schlesinger, 1991;

Singh and Hayashi, 1995; Lynd et al., 2002). Cellulose-rich plant biomass

is one of the foreseeable and sustainable source of fuel, animal feed

and feed stock for chemical synthesis (Bhat, 2000). The utilization of

cellulosic biomass continues to be a subject of worldwide interest in

view of fast depletion of our oil reserves and food shortages (Kuhad et al.,

1997; Gong et al., 1999).

The conversion of cellulosic mass to fermentable sugars through

biocatalyst cellulase derived from cellulolytic organisms has been

suggested as a feasible process and offers potential to reduce use of

fossil fuels and reduce environmental pollution (Dale, 1999; Lynd et al.,

1999). Complete enzymatic hydrolysis of enzymes requires synergistic



action of 3 types of enzymes,namely cellobiohydrolase,

endoglucanase or carboxymethylcellulase (CMCase) and β-glucosidases

(Bhat, 2000).

However, the high cost of production of these enzymes has hindered the

industrial application of cellulose bioconversion. One of the different

approaches to overcome this hindrance is to make continuous search for

organisms with secretion of cellulase enzymes in copious amounts and to

optimize enzyme production with them. In this paper, effects of nutrient

on cellulase production by Aspergillus niger, a local isolate, in submerged

fermentation in a laboratory study are presented. (Narasimha et al).

The control and the improvement of edible fungus cultures have provoked

considerable interest in the past few years because mushroom production is

economically important. Pleurotus spp.is third place in worldwide

production of edible mushrooms, after Agaricus bisporus and Lentinula

edodes (Chang 1999). Mycelial growth of Pleurotus spp. is fast, and various

lignocellulosic waste products can be used as culture substrate (Yildiz et al

2002).

The aim of commercial mushroom substrate preparation is to produce a

substrate that is optimal and selective for vegetative mycelial growth. In the

case of A. bisporus, the white button mushroom, this is accomplished

largely by microbial activities during composting. In the case of Pleurotus

spp., a wood-rot fungus, this is achieved by the application of various heat

treatments to eliminate competitive fungi. Trichoderma spp., soil filamentous

fungi, are antagonists that can cause extensive losses in mushroom production



(Badham 1991, Jandaik and Guleriai 1999). These fungi produce several

enzymes involved in degradation of the fungal cell walls that may contain

chitinases and glucanases (Sivan and Chet 1989, Geremia et al 1993, Ait-

Lahsseni et al 2001).

There has been considerable research into the biodiversity of tropical micro

fungi, and the most frequently studied are those inhabiting lignocellulose

substrates (Hyde 1997). Recent research has included detailed investigations

of saprobic fungal biodiversity on various non-wood lignocellulose substrates

such as bananas (Photita et al., 2001), bamboo (Hyde et al., 2001), grasses

(Wong and Hyde, 2001) and palms (Yanna et al., 2001).

Relatively little, however, is known about the physiology of substrate

utilization by such fungi. All lignocellulosic materials are formed

predominantly of three components: cellulose, a structural carbohydrate

responsible for strength and flexibility; lignin, a polyaromatic heteropolymer

conferring decay resistance and hardness; and hemicellulose, a structural

carbohydrate intimately associated with lignin (Eaton and Hale, 1993). The

composition of bananas, bamboo, grasses and palms, differs only slightly from

wood in both morphology and association of these components (Fengel and

Wegener, 1989).

The only major chemical difference lies in the incorporation of coumaryl,

sinapyli and vanilyl monomers in the lignin of grasses and

gymnosperm/angiosperm wood. Most of our knowledge on lignocellulose

substrate utilization is from studies of those species involved in the decay of

commercially important timber in temperate egions (Eaton and Hale, 1993).

The degradation of lignocellulose by such fungi is well understood. Cellulose

is attacked predominantly by hydrolytic cellulases although oxidative



enzymes may also be involved in cellobiose and glucose utilization.

Hemicellulose breakdown is less well understood but is thought to involve

hydrolytic endo-type hemicellulases. The mineralization of lignin is achieved

by a group of peroxidase and phenoloxidase enzymes known as lignin-

modifying enzymes (LME's). They produce highly reactive radicals that

oxidize phenolic and non-phenolic lignin components (Pointing, 2001).

Cellulase production was carried out by solid state bioconversion (SSB) method

using rice straw, a lignocellulosic material and agricultural waste, as the

substrate of three Trichoderma spp. and Phanerochaete chrysosporium in lab-

scale experiments. The results were compared to select the best fungi among

them for the production of cellulase. Phanerochaete chrysosporium was found

to be the best among these species of fungi, which produced the highest

cellulase enzyme of 1.43 IU/mL of filter paper activity (FPase) and 2.40 IU/mL

of carboxymethylcellulose activity (CMCase). The “glucosamine” and

“reducing sugar” parameters were observed to evaluate the growth and substrate

utilization in the experiments. In the case of Phanerochaete Chrysosporium, the

highest glucosamine concentration was 1.60 g/L and a high concentration of the

release of reducing sugar was measured as 2.58 g/L obtained on the 4th day of

fermentation. (Md. Munir H. Khan1, 2007)

The cellulolytic activtiy of some soil fungi isolated from soil and decomposing

pieces plant material was observed to depend upon the genera of fungi , the

nature of substrate and the temperature, the cellulase produced in the

presesnce of cellulose poweder was was active against CMC. (M.M.Mishra,

1978)

The epiphytic lichen Evernia prunastri (L.) Ach. synthesizes both a constitutive

and a carboxymethycellulose-inducible P-l,4-glucanase (cellulase). While

the inducible enzyme is readily released into the incubation medium, some



activity is always found in the intact thallus (Yagiie et al. 1984). Since cellulase

might be located in either or both symbionts, we have assayed separated algal

and fungal cells from recently collected E. prunastri, in order to determine

cellulase location under natural, uninduced conditions. (Estevez*, 1989)

The production of endoglucanase(EC 3.2.1.4) and exoglucanase (EC 3.2.1.91)

enzyme was studied during penetration of the host and development of the

VAM fungus Glomus mosseae in the roots of lettuce(Lactuca sativa) and

onion(Allium cepa) was rported by J.M Garcia and etal.

The fungus Penicillium brasilianum IBT 20888 was cultivated on three

different carbon sources to investigate the effect of the carbon source on the

enzyme production. The carbon sources used were Sigmacell cellulose

(SC),steam pretreated spruce (SPS) and a mixture of SC, oat spelts xylan and

birchwood xylan (SCXX). Enzymatic assays and capillary electrophoresis

revealed clear differences among three enzyme preparations produced-both in

activity levels and in the distribution between enzymes within the same class.

The hydrolysis efficiency of the resulting enzyme preparations was studied on

SC and SPS. The three enzyme preparations performed equally well on SPS

using an enzyme loading of 25 FPU (g cellulose)-1

. ((1) & Lisbeth, 2006)

A new celllulase producing sps of Penicillium named Penicillium ireiense has

bemnn isolated cultres of thois fungus is liquid media continning cellulose as

carbon source, excrete in to medium as complex medium ale to degrade both

soluble an dinsoluble forms of cellulose. This comlex has bemnn seperated

into five protein fungctions . 3 of then are endddowed with cmc cellulase

activity one containd sa cellulobiose aand one contains a c1 like factoer .thease

fractions show moderate synergism in the atack of cotton fibre.

Materials and Methods


3. MATERIALS AND METHODS

3.1 REQUIREMENTS

Following is a list of basic requirements which is required in a laboratory for

microscopic examination, isolation or culturing and identification of a

microorganism as well as to study its structure, function and application.

A. Instruments and Appliances

1. Bunsen burner or spirit lamp

2. Laminar flow safety hood

3. Microscope and immersion oil

4. Oven

5. Incubators

6. Refrigerator

7. Autoclave or pressure cooker

8. Hotplate/Heater

9. Centrifuge

10. pH meter

11. Spectrophotometer

12. Camera lucida

13. Balances

B. Tools

1. Transfer needle

2. Inoculating loop

3. Dissecting needles



4. Forceps

5. Scissors

6. Hemostat

7. Ocular micrometer

8. Thermometers

C. Glassware

1. Petri dishes

2. Conical flasks

3. Culture tubes without screw caps

4. Screw-capped tubes for media

5. Durham fermentation tubes

6. Beakers

7. Funnels

8. Graduated cylinders

9. Graduated pipettes

10. Glass cover slips

D. Miscellaneous

1. Culture media

2. Test tube rack

3. Cotton plugs

4. Stains

5. Disinfectant

6. Distilled water



7. Blotting paper

8. Rubber bands

9. Soap solution

LAMINAR AIR FLOW (LAF)

The laminar flow hood is used for reducing the danger of infection while

working with infectious microorganisms and for preventing contamination of

sterile materials. It works on the principle of application of high-efficiency

particulate air (HEPA) filters (fibreglass filters) instead of membrane filters

(membrane filters are thin pieces of synthetic material, usually cellulose

acetate or polycarbonate, that contain very small openings or pores, so small

that microbial cells cannot pass through them in air filtration. Room air is

filtered before entering the working chamber and moves in a single direction.

AUTOCLAVE

The autoclave is an apparatus in which saturated steam under pressure effects

sterilization (autoclaving). The pressure increases the boiling point of water,

thereby increasing the temperature to which water can be heated. Cells are

destroyed by the higher temperature and not by the pressure. Sterilization in

an autoclave is done with saturated steam under pressure.

HOT-AIR OVEN

An oven is based on the principle where sterilization is accomplished by dry

heat or hot air. Dry heat of a given temperature is not nearly as effective a



sterilizing agent as moist heat of the same temperature, in other words, the

sterilization process in an oven is longer than autoclaving.

Hot-air ovens are most commonly used for sterilizing glassware like Petri

dishes, test tubes, pipettes, metal instruments that can tolerate prolonged

heat exposure.

An oven consists of an insulated cabinet which is held at a constant

temperature by means of an electric heating mechanism and thermostat. It is

fitted with a fan to keep the hot air circulating at a constant temperature and

thermometer for recording the temperature of the oven. For proper circulation

of the hot air the shelves are perforated. For normal sterilization work, the

oven should be operated at 160oC and most glassware will require a period of

two hours for total sterilization.

SPECTROPHOTOMETER

A spectrophotometer instrument is used for counting population of bacteria,

based on the principle of turbidity determination. Turbidity or optical density is

the cloudiness of the suspension. The more turbid a suspension, the less light

will be transmitted through it. In other words, the amount of light absorbed

and scattered is proportional to the mass of calls in the light path. As bacteria

grow in a broth, the clear broth becomes turbid. Since the turbidity increases

as number of cells increase, this indicator is used as an indicator of bacterial

density in the broth. Turbidity is also useful for standardizing the population

densities of bacterial cultures of clinical significance.



3.2 COLLECTION OF SAMPLE

Biological material is dung from temple Elephant (Elephas maximus. The dung

sample was collected from the Asian Elephant (Elephas maximus) residing at

the Vidyagiri complex. Samples of dung that appear to be relatively fresh are

collected and incubated within a day of collection, if samples could not be

incubated shortly after collection, they were air-dried until Incubation.

Incubation is done under sterile conditions and under ambient light at room

temp of 20-24°C .The sample was normally kept for 4-12 weeks for continuous

examination and recording. Microscopic observation and photography was

carried out using phase contrast microscope with digital camera.

3.3 ISOLATION OF COPROPHILOUS FUNGI

3.3. a MOIST CHAMBER METHOD

Incubation of the dung samples were carried out as suggested by G.S Masunga

etal . 20 cm diameter glass Petri-plates are used as moist chambers. The Petri-

plates were first sterilized and then prepared by placing within sterilized filter

paper. High moisture content was maintained by moisturizing the filter paper

with sterile water periodically. Precautions were taken to prevent the filter

papers being flooded. 50gm of dung sample was spread evenly on the sterile

paper towel and sterile water was sprinkled to create moist environment

conducive for fungal growth. Container were closed and kept in ambient light.

A stereo microscope was used to scan the surface of the dung for fungal fruiting

bodies.



Elephant dung in sterile Petri plates.

3.3. b SERIAL DILUTION-AGAR PLATING METHOD

The serial dilution-agar plating method is one of the commonly used

procedures for the isolation and enumeration of fungi, bacteria and

actinomycetes. This method is based upon the principle that when material

containing microorganisms is cultured each viable microorganism will

develop into a colony; hence the number of colonies appearing on the plates

represents the number of living organisms present in the sample.

Procedure

1. Collect fresh dung samples from a field.

2. Air dry them for one week.

3. Label 90ml sterile water blanks as 1,2,3,4,5,6 and 7.

4. Add 10g sample of finely pulverized, air dried dung into no.1 test tube to

make 1:10 dilution.

5. After shaking vigorously transfer 10ml of the suspension from t.t. no.1 to

t.t.no. 2 with a pipette under aseptic condition to make 1:100 dilution.



6. Prepare another dilution 1:1000 by pipetting 10ml of the suspension

into t.t.no.3, by afresh sterile pipette and shake it.

7. Make further dilutions by pipetting 10ml suspension into additional

water blanks (4,5,6 etc.) as prepared above.

These dilutions are used as a seed for growing microorganism. For fungi PDA

medium supplemented with amphicillin.

3.4 SUB CULTURING (OR PICKING OFF) TECHNIQUE

After incubation has been completed in streak-plate, pour-plate, or spread-

plate, techniques ad appearance of the discrete, well separated colonies has

been examined, the next step is to subculture some of the cells from one of

the colonies to separate agar plates or nutrient agar slants with a sterilized

needle or loop for further examination and use.

Each of these new culture represents the growth of a single species and is

called a pure or stock culture. Sub-culturing is the term used to describe the

procedure of transferring of microorganisms from their parent growth source

to a fresh one or from one medium to another.

Procedure

1. Sterilize the inoculating loop by holding it in the hottest portion of the

Bunsen burner flame.

2. Flame until the entire wire becomes red hot.

3. allow the loop to cool for a few minutes or cool it by dipping in a fresh

agar medium.

4. Touch the tip of the loop to the surface of a selected colony .



5. Remove the plug of the agar slants, grasp the plug with the little finger

of the left hand and pass the neck of the tube rapidly over the flame.

Insert the loop into the subculture tube and inoculate it by drawing it

lightly over the hardened surface in a straight or zig-zag line and recap

the tube.

6. Reflame the inoculating loop to destroy existing organism.

7. Incubate the cultures at 25c for 48 to 72 hours.

3.5 LACTOPHENOL COTTON BLUE MOUNTING OF FUNGI

Lactophenol cotton blue is a stain commonly used for making semi permanent

microscopic preparation of fungi. It stains the fungal cytoplasm and provides a

light blue background. It contains four constituents: phenol, which serves as a

fungicide; lactic acid, as a clearing agent; cotton blue, as a stain for cytoplasm;

glycerine, which gives a semipermanent preparation.

Procedure

1. Place a drop of lactophenol cotton blue on a clean slide.

2. Transfer a small tuft of the fungus, using a flamed, cooled needle.

3. Gently tease the material using the two mounted needles.

4. Mix gently the stain with the mold structures

5. Place a cover glass over the preparation taking care to avoid trapping air

bubbles in the stain.

6. Seal lactophenol mounts: to keep the slides for many years cover slip is

sealed with nail-polish or DPX mountant.



Slides of the fungal fruiting bodies which were made with lacto phenol blue

(Ritchie 2002) and were viewed under light micro scope and following are the

observations.

3.6 SHAKE FLASK CULTURING OF FUNGUS

Inoculam Preparation:

The isolated cultures of Penicillium sps were maintained as stock cultures in

PDA. The pure cultures were grown on PDA slants. They were grown at 30°C

for 5 days and stored at 4°C. Conidial suspensions were prepared from slants by

flooding the surface of the cultures with sterile water and gently rubbing with a

inoculation needle. The innoculum was kept in shaker for(150rpm) before it was

used for fermentation process.

Fermentation Process Using Shake Flask Culture:

To the sterilized Czapek’s broth medium 1ml of the dense spore and mycelia

mat suspension Penicillium sps was added as innoculum for each 500ml

Erlenmeyer flask. The inoculated cultures were incubated at 25°C on rotary

shaker at 140rpm. Flasks were withdrawn after 7-day incubation period and

fungal culture was filtered through Whatman no. 1 filter paper to separate

mycelia mat and culture filtrate.

3.7 TEST FOR CELLULAS ACTIVITY

The amount of enzyme secreted by the pathogen to degrade the cellulose in the

medium was measured. The enzymatic activity of the cellulases produced by the

fungus was measured

1.Qualitatively by using Benedict’s test for reducing sugars (Sadasivam S)

2.Quantitatively by estimation of reducing sugars due to cellulolytic activity



using Dinitrosalicylic (DNS reagent) acid method by preparation of standard

glucose graph (Denison, D A and Koehn, R D)

3.7. a BENEDICT’S TEST

The Benedict's test allows us to detect the presence of reducing sugars (sugars

with a free aldehyde or ketone group). All monosaccharides are reducing

sugars; they all have a free reactive carbonyl group. Some disaccharides have

exposed carbonyl groups and are also reducing sugars. Other disaccharides

such as sucrose are non-reducing sugars and will not react with Benedict's

solution. Starches are also non-reducing sugars. The copper sulfate (CuSO4)

present in Benedict's solution reacts with electrons from the aldehyde or ketone

group of the reducing sugar to form cuprous oxide (Cu2O), a red-brown

precipitate.

CuSO4 Cu++

+ SO4-

2 Cu++

+ Reducing Sugar Cu+

(electron donor)

Cu+

Cu2O (precipitate)

The final colour of the solution depends on how much of this precipitate was

formed, and therefore the colour gives an indication of how much reducing

sugar was present. Increasing amounts of reducing sugar are green yellow

orange brown.



Procedure:

4ml of culture filtrate containing the enzyme is added to 5ml each of 1% and

5% starch and cellulose solutions in four different test tubes and incubated for

5min, for the enzyme to act on substrates (1% and 5% starch and cellulose).Two

test tubes were taken as control i.e. Tube containing culture filtrate without

starch and cellulose. Equal volume of Benedict’s reagent was added to all test

tubes and this mixture was then incubated in boiling water bath for 25 min.

Entire experiment was repeated with three replicas.

3.7. b DINITROSALICYLIC ACID REAGENT TEST FOR ESTIMATION OF REDUCING

SUGARS

Hydrolysis of cellulose is a complex process. A minimum of three different type

of enzymes are believed to be involved.

1. Endo-β1, 4 glucanase (cellulase)

2. Endo-β1, 4 glucanase (cellulase)

3. β-glucosidase (cellobiase)

Initiation of hydrolysis of native cellulose is effected by C1 enzyme. This

enzyme is exo-β1, 4 glucanase. Exo-glucanase splits alternate bonds from the

non reducing end of cellulose chain yielding cellobiose. The endo glucanse is

distinguished by by mechanism of their attack on carboxy methyl cellulose. It

does not act on the native cellulose. . β-glucosidase play an important function

in th degradation of cellulose by hydrolysing cellobiose by which is an inhibitor

of exo-glucanase. Only organisms producing C1-celllose are capable of

hydrolysing native cellulose (filter paper, cotton etc.)

Principle :



The production of reducing sugars (glucose) due to celluloytic activity is

measured by Dinitrosalicylic acid method.

Requirements:

Sodium citrate buffer 0.1M(ph5.0)

Filter paper disc

Dinitrosalicylic acid (DNS) reagent

40% Rochelle salt solution (Potasium sodium tartarte)

Test tubes

Preparation:

DNS reagent:

Dissolve by stirring 1gm of Dinitrosalicylic acid,200mg of crystalline phenoland

50mg sodium sulphite in 100ml of 1%NaOH. Store at 4°C. Since the reagent

deteriorates due to sodium sulphate. If long storage is required, sodium

sulphite may be added at the time of use.

40%Rochelle salt:

Dissolve 40gm of potassium sodium tartarte in100ml of distilled water.

Whatman filter paper no.1:

Cut the filter paper with a paper punch to ensure the same surface area of

substance in a reaction tube.



Procedure:

Sample:

1. 1. Add 2ml of enzyme extract to 32mg of dry whatman no.1 filter paper.

2. Incubate the mixture for 1hr at 50°C.

3. Add 3ml of DNS reagent.

4. Heat the mixture in boiling water bath for 5min.

5. While the contents of the sample are still hot add 1ml of 40% Rochelle

salt solution.

6. Add water to make volume to 5ml.

7. Measure the absorbance at 540nm.

Glucose standard curve:

Standard glucose: stock-100mg in 100mlof water. So 1ml consists of 1gm or

1000µgm.

1. Standards are prepared taking 0.1, 0.2, 0.5, 0.8 and1.0ml in four test

tubes labelled tube1, 2, 3and 4 respectively. A blank was maintained by

taking 2ml of distilled water into a tube labelled `B’.

2. Make up the volume to 2ml in all tubes including the sample tube by

adding distilled water.

3. Add 2ml of DNS reagent

4. Heat the contents in boiling water bath for 5min.

5. When the contents of the tube are still warm,add1ml of 40%Rochelle

salt solution.

6. Cool and read the absorbance at 540nm.



3.8 CULTURE MEDIA

3.8.a CZAPEK’S MODIFIED BROTH MEDIUM

(pH 6.5) for fungi

Dipotassium hydrogen phosphate 1.0g

Sodium nitrate 2.0g

Magnassium sulphate 0.5g

Potassium chloride 0.5g

Ferrous sulphate 0.01g

Cellulose powder 2.5g

Dist. Water 500.0ml.

Dissolve all the ingredients except phosphate in half of the water and add

sucrose. Dissolve phosphate separately and add to the rest. Make volume

to 1 litre. Sterilize by autoclaving at 1210C for 15 minutes.

3.8.b POTATO DEXTROSE AGAR(PDA)

pH 5.6

Potato (peeled) 200.0g

Dextrose 20.0g

Agar 15.0g



Peel off the skin of potatoes, cut into small pieces and boil in 500ml of water,

till they are easily penetrated by a glass rod. Filter through cheesecloth. Add

dextrose to the filtrate. Dissolve agar in water and bring up to the required

volume by adding of water. Autoclave at 15lb pressure for 15 minutes.

Results


4. RESULTS

4.1 BIO DIVERSITY OF COPROPHILOUS FUNGI ISOLATED FROM

ELEPHANT DUNG FUNGI FROM PUTTAPARTI MANDAL

Biological diversity or biodiversity means the variability among living

organisms from all sources including, terrestrial, marine and other aquatic

ecosystems and the ecological complexes of which they are part; this includes

diversity within species and between species. Fungi are known to colonize,

multiply and survive in diversified habitats, viz. water, soil, air, litter, dung,

foam, etc. Fungi are ubiquitous and cosmopolitan in distribution covering

tropics to poles and mountain tops to the deep oceans. Coprophilous fungi are

known to colonize in different types of dung based on association with the

herbivores on which they rely, such as rabbits, deer, cattle, horses, sheep and

Elephant, another important factor is food choice which influences the species

richness of Coprophilous Ascomycetes, and that some species are more

associated with habitat and food choice of the herbivore, rather than a specific

dung type/animal species.

Identification:

Identification of the coprophilous fungal sps was done using a manual known as

“Identification of Fungi” by Nagamani and Manoharachary et.al, 2004. A total

of 4 fungal species were identified as Coprophilous fungal species of the

samples analyzed.

List of copophilous fungal species identified are as follows:

1. Penicillium sps

2. Fusarium oxysporum

3. Dsechelera hawiiensis

Results


4. Rizopus nigricans

Predominantly penicillium sps has been growing is all the culture plates.

Penicillium sps(100X) Penicillium sps (100X)

Conidial head(100X) Ascospores(100X)

Dsechelera hawiiensis (100X) Fusarium oxysporum (100X)

Results


Rhizopus nigricans (100X)

The dilution plating results of the fungal cultures are as follows:

1/1000 dilution plate 1/10 dilution plate

Results


Pure culture of Penicllium sps

green mycelia.

Penicillium sps:

Penicillium (from Latin penicillus: paintbrush) is a genus of Ascomycetous

fungi of major importance in the natural environment as well as food and drug

production. It produces penicillin, a molecule that is used as an antibiotic, which

kills or stops the growth of certain kinds of bacteria inside the body. The thallus

, (mycelium) typically consists of a highly branched network of multinucleate,

septate, usually colorless hyphae. Many-branched conidiophores sprout on the

mycelia, bearing individually constricted conidiospores. The conidiospores, are

the main dispersal route of the fungi, and often green. These are clearly evident

from the slides above.

Reproduction:

Sexual reproduction involves the production of ascospores, commencing with

the fusion of an archegonium and an antheridium, with sharing of nuclei. The

irrregularly distributed asci contain eight unicellular ascospores each. The

Results


ascospores are brown in colour which can be sen in the slide fig 1.2. it is the

mode of sexual reproduction in these fungi.

Uses:

Several species of Penicillium play a central role in the production of cheese

and of various meat products. Penicillium camemberti and Penicillium

roqueforti are the molds on Camembert, Brie, Roquefort and many other

cheeses. Penicillium nalgiovense is used to improve the taste of sausages and

hams and to prevent colonization by other moulds and bacteria. In addition to

their importance in the food industry, species of Penicillium and Aspergillus

serve in the production of a number of biotechnologally produced enzymes and

other macromolecules, such as gluconic, citric and tartaric acids, as well as

several pectinases, lipase, amylases, cellulases and proteases. Most importantly,

they are the source of major antibiotics, particularly penicillin and griseofulvin.

4.2 SHAKE FLASK CULTURING OF FUNGUS

Culturing Of Pencillium Sps In In Rotary Shaker With

Czapeck’s Broth Medium:

In shake culture, penicillium sps was aseptically inoculated in a conical flask

which contained Czapeck’s broth medium. Instead of CMC, cellulose powder

was used. It was grown for one week. pH 6.5 was maintained.

After 6 days visible change in the colour of the medium and also the growth of

the mycelium was observed. The rotary shaker showed a green mycelial

mat with conidial suspension. The crude metabolite was obtained by filtration

using Whatman filter paper.

Results


Czapeck’s modified medium with cellulose powder as substrate for fungus

4.3 CELLULOSE BIO CONVERSION

The crude enzyme extract obtained from the culture filtrate convert the starch

and cellulose of the substrate to more simple sugars like glucose (reducing

sugars). Formation of the reducing sugars is visualized by treating with

Benedict’s reagent and estimated using DNS Reagent test by preparation of

standard graph of glucose.

4.3. a BENEDICTS TEST

The Benedict's test allows us to detect the presence of reducing sugars (sugars

with a free aldehyde or ketone group). All monosaccharides are reducing

sugars; they all have a free reactive carbonyl group. The activity of the

cellulolytic enzymes present in the filtrate was measured indirectly by

Results


measuring the formation of reducing sugars using Benedict’s reagent. The

change of colour from blue to dark green and then to yellow is the proof of

presence of reducing sugars. The enzymes from the filtrate convert the starch

and cellulose of the substrate to more simple sugars like glucose (reducing

sugars). .. It was observed that by visual colour change of the Benedict’s

reagent from blue to dark green colour took 25 min and from dark green to

yellow 3hrs after the addition of Benedict’s reagent to the mixture. The

presence of maximum activity was observed in cellulose followed by starch. It

was thought that enzymes were equally active on both 5% starch and cellulose

and 1% starch and cellulose, only in later being more active can be made out by

deep colour change.

Yellow colour of the precipitate indicates the presence of reducing sugars

which is seen in both 1% and 5% starch and cellulose respectively. Blue

coloured solution indicates control.

Results


4.3. b DNS REAGENT TEST

The presence and estimation of the reducing sugars were confirmed by the

estimation of reducing sugars by Dinitrosalysilic acid (DNS) Reagent and by

preparation of standard graph of glucose. Absorbance of the following samples

was measured using spectrophotometer.

The following table shows the conc. of glucose µgm/ml and their respective

absorbance along with sample,

Sl.No Test tube ml. Conc. In µgm Absorbance

1 blank 1 0 0

2 tube1 0.1 100 0.07

3 tube 2 0.2 200 0.145

4 tube3 0.5 500 0.304

5 tube 4 0.8 800 0.596

6 tube5 1.0 1000 0.786

7 sample 1.0 X 0.243

The sample’s Absorbance was intercepted on the graph to deduce its conc.

Which was 325µgm/ml. since before measuring it was diluted 10 times the

original conc. is 325x10=3250µgm/ml, which is 3.25 mg/l

Calculation of reducing sugars:

For 1 ml its 3.25 mg/ml

For 6 ml of the sample volume it is =19.5 mg.

Results


The graph was

Discussions


5. DISCUSSION

The elephant dung supports wide variety of fungi known as coprophilous fungi,

majority of them are belonging to Ascomycetes like Sordaria sps, Sporormiella

australis ,Podospora sps, Ascobolus sps. Few of the Basidiomycota are

Coprinus sps, Coemansia sps and some of Zygomycota include Pilobolus sps

Dung is very rich medium for fungal growth. It consists of remains of plant

material plus the micro biota associated with its digestion. Much of the material

consists of readily available carbohydrate in addition to cellulose and lignin.

Coprophilous fungi nutrition is saprophytic in nature thus they produce enzymes

extracellular to digest the food material. In the present study Penicillium species

was dominant coprophilous fungi isolated from dung of temple elephant of

Prasanthi Nilayam and it is an ascomycete because it is producing ascospores

hence our studies goes hand in hand with the reports of earlier workers in the

field of coprophilous fungi. In the present investigation it was revealed That

Ppenicillium sps under our study responded similarly by producing cellulase

enzymes with respect to the cellulosic filter paper in the czapeck’s broth

medium. Thus the crude enzyme extract was collected and Benedict’s as well as

DNS Reagent test was done to confirm the presence of cellulases in the extract,

positive results were obtained in both the cases. Hence it was very clear that our

present investigation reports the production of cellulase enzyme by the

Penicillium sps isolated from dung of temple Elephant.

Only very few organisms belonging to the genera Aspegillus and Trichoderma

etc., were used for commercial production of starch and cellulose degrading

enzymes. The production of enzymes by penicillium sps has to be compared

with already exploited species like Aspergillus niger etc., and only then the use

of penicillium sps for commercial enzyme production can be validated.

Discussions


The production of cellulase enzyme by Pencillium sps such as iriense and

brasilianum was reported by Giulia Borelti and etal and Jørgensen Henning, etal

respectively. The present study using penicillium sps for the production of cell

wall degrading enzymes with czapeck’s broth is in accordance with the ongoing

search for better and efficient way of enzyme production.

Summary


6. SUMMARY

Coprophilous fungi are the dung loving fungi having great bio diversity. Many

important secondary metabolites namely bio active compounds are reported

from this group.

The study of Coprophilous fungi from dung of elephant from Puttaparthi

mandal is an endeavour to study their habitat diversity and obtain few species

Which may be able to produce novel bio active compounds.

The biological sample for our study was dung of Temple Elephant residing in

the Vidyagiri complex of Puttaparthi mandal. Isolation of the Coprophilous

fungi was carried out using moist chamber method and dilution plating method

and later cultured on potato dextrose medium.

Four Coprophilous species were identified namely Penicillium sps, Fusarium

oxysporum, Dsechelera hawiiensis, Rhizopus nigrecans. Pure culture of

penicillium sps was used as the inoculum and grown in shake flask culture

containing modified Czapeck’s broth media and cellulose powder as substrate.

The crude extract obtained from the filtrate was filtered using whatman no.1

filter paper and it was tested for cellulose degrading activity. Benedict’s test for

reducing sugars was used to confirm the qualitative testing of cellulases on 1%

and 5% starch and cellulose respectively. Positive results were obtained by

visible colour change form blue to dark green and then to yellow. In the present

investigation it was very clearly found out that the cellulase activity of the

enzyme was confirmed quantitatively by using DNS reagent for estimation of

reducing sugars and by preparation of glucose standard graph. The present

investigation reveals that the production of cellulase enzyme by Penicillium sps

of coprophilous fungi isolated from dung of the elephant.

Summary


This present preliminary investigation gives the significance of Coprophilous

fungi and its importance in biodegradation and biogeochemical cycling .This

present study is novel and original because it is explored the dung fungi from

the temple Elephant of Prasanthi Nilayam and also reported the Cellulase

activity of Penicillium sps isolated from Dung of temple elephant of Prasanthi

Nilayam. Hence this present preliminary investigation forms original and

significant contribution in the field of fungal biotechnology.

Bibliography


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