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Transcript of 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
Studies On Coprophilous Fungi From Dung Of Elephant Page 2
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 3
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 4
(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.
Introduction
Studies On Coprophilous Fungi From Dung Of Elephant Page 5
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 )
Introduction
Studies On Coprophilous Fungi From Dung Of Elephant Page 6
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,
Introduction
Studies On Coprophilous Fungi From Dung Of Elephant Page 7
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 8
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 9
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
Review of Literature
Studies On Coprophilous Fungi From Dung Of Elephant Page 10
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) .
Review of Literature
Studies On Coprophilous Fungi From Dung Of Elephant Page 11
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
Review of Literature
Studies On Coprophilous Fungi From Dung Of Elephant Page 12
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
Review of Literature
Studies On Coprophilous Fungi From Dung Of Elephant Page 13
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
Review of Literature
Studies On Coprophilous Fungi From Dung Of Elephant Page 14
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.
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Studies On Coprophilous Fungi From Dung Of Elephant Page 15
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.
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Studies On Coprophilous Fungi From Dung Of Elephant Page 16
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
Review of Literature
Studies On Coprophilous Fungi From Dung Of Elephant Page 17
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
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Studies On Coprophilous Fungi From Dung Of Elephant Page 18
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
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Studies On Coprophilous Fungi From Dung Of Elephant Page 19
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-
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Studies On Coprophilous Fungi From Dung Of Elephant Page 20
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
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Studies On Coprophilous Fungi From Dung Of Elephant Page 21
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)
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Studies On Coprophilous Fungi From Dung Of Elephant Page 22
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
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Studies On Coprophilous Fungi From Dung Of Elephant Page 23
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
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Studies On Coprophilous Fungi From Dung Of Elephant Page 24
(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
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Studies On Coprophilous Fungi From Dung Of Elephant Page 25
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
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Studies On Coprophilous Fungi From Dung Of Elephant Page 26
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.
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Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 27
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
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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
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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
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 30
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.
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Studies On Coprophilous Fungi From Dung Of Elephant Page 31
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.
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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.
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 33
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 .
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 34
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.
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 35
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
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 36
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.
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 37
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 :
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 38
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.
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 39
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.
Materials and Methods
Studies On Coprophilous Fungi From Dung Of Elephant Page 40
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
Materials and Methods
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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
Studies On Coprophilous Fungi From Dung Of Elephant Page 42
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 43
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 44
Rhizopus nigricans (100X)
The dilution plating results of the fungal cultures are as follows:
1/1000 dilution plate 1/10 dilution plate
Results
Studies On Coprophilous Fungi From Dung Of Elephant Page 45
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 46
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 47
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 48
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 49
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 50
The graph was
Discussions
Studies On Coprophilous Fungi From Dung Of Elephant Page 51
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 52
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 52
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
Studies On Coprophilous Fungi From Dung Of Elephant Page 53
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.
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Studies On Coprophilous Fungi From Dung Of Elephant Page 54
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