Isolation and Application of keratinophilic Fungi collected from
Agricultural Lands of Kota, Rajasthan
Under the Supervison of
Dr. Pallavi Sharma
Dr. Shweta Gupta

Introduction
• Keratin is a natural fibrous protein forming the outermost keratinized layer of human and animals . Various keratinous
substrates occur in nature in various forms such as hair, wool, features, nails, claws,quills, scales, horns, hooves, and tortoise
shell and in the outer layer of skin.
• Keratinophilic fungi colonize different keratinous substrates and degrade them to components of low molecular weight. These
fungi can be divided into
(1) keratinophilic fungi (grow on keratinic materials)
(2) keratinolytic fungi ( capable of decomposing keratin completely )
( 3 ) Dermatophytes ( belong to the genera Trichophyton, Microsporum, and Epidermophyton ) .
• Keratinophilic fungi include a variety of filamentous fungi mainly belonging to the order Onygenales and other saprophytic
fungi of hyphomycetes, and several other taxonomic groups. All the dermatophytes are keratinolytic in nature.
• Based on infection these can be anthropophilic (infecting human ) , zoophilic ( infecting animals ) , and geophilic (reported
from soil) .

The soils represents the main reservoir of fungi. Some soil fungi are potential pathogen to both human and
animal.The potentially pathogenic keratinophilic fungi and allied geophilic dermetophytic species are widespread
worldwide.
The forest farmyard, park soils, as well as sediments of the rivers and oceans contained humus and organic
material are the best candidate for growth of keratinolytic and saprophytic fungi (Mohamed et al ., 2000).
Therefore hygienic and ecological interest have led us to study the keratinophilic mycoflora of farm yards and
forests, where farmers, tourists, and animals spend a large proportion of their time and may be exposed to
pathogenic fungi ( Mahadevi Omram , 1991) .
This would help us to know the distribution and occurrence of dermetophytes and other keratinophilic fungi and
risk of human dermatophytosis in those region , which could have a role in degradation of keratinous material as
a industrial point of view ( Nasery Bande Gharey , 1992) .

Materials&Methods
Collected of soil samples
A total of 20 soil samples were collected from different area of agricultural land in Kota (
Rajasthan) during the period June- July 2022 . Different soil samples in sterile polythene
bags and brought to the laboratory for further microbiological analysis.
Physical properties of soil samples
The collected soil samples for the isolation were analyzed for the following
physiochemical parameter like pH, electronic conductivity, total dissolve solids (TDS) ,
Salinity, by using soil analysis kit and other like dissolved oxygen (DO) by adopting
Standard procedure.

Vanbreuseghem’s Hair Bait Technique
The keratinolytic nature of these fungi makes it possible to isolate them from soil by implanting hair, the‘hair baiting’ technique initially
developed by R Vanbreuseghem, a Belgian mycologist in 1952. Since then, a number of modifications have been developed, but the basic
principle remains the same i.e. use of natural keratin substrate as baits to recover these fungi from soil.
Collection of Soil: Soil can be collected in sealed polythene bags using sterile spatula/spoon from habitats where keratin and hence
keratinolytic fungi are present
eg. barber’s dump, animal burrow, cattle or poultry sheds, sewage and garbage
Hair Baiting:
I Half fill sterile Petri dishes with the soil samples.
II Spread short (2-3 cm) strands of sterilized defatted* human hair or horsehair over the surface of the soil
III. Add 10-15 ml of sterile water to the soil to facilitate germination of fungal spores.
IV Some antibiotic to prevent bacterial growth may also be added.
V. Incubate the preparations at room temperature (20-25 C) in the dark, for 4-6 weeks. Examine the plates periodically for
the development of mycelium using a Stereo binocular microscope.
Note: while opening the plates make sure that there is no free moving air, because the spores are dispersed by air and many
keratinolytic fungi are pathogenic
VI. Remove hairs with fungus growth or take inoculum and place it on plate of Sabouraud’s dextrose agar.
VII After one or more week, check the colonies and identify the fungus. Pure cultures can now be prepared. *Defatting can
be done by soaking the hair for 24 hrs in either diethyl ether or in a chloroform/methanol(1:1) mixture. Later rinse 4-5 times
with distilled water and air dry

Hair baiting technique

Fungal isolates
Chrysosporium spp
Cultural and morphological features
Aspergillus flavus Colonies are granular, flat often with radial grooves yellow at first but quickly become bright to dark yellow
green with age. Conidiophores stripes are hyaline and coarsely roughened, often more noticeable near the
vesicle. Conidia are globosetosubglobose) pale green and conspicuous Colonies are granular, flat, often with
radial grooves, yellow at first but quickly becoming bright to dark yellow green.
Trichophyton rubrum Colonies are flat to slightly raised, white to cream, suede like to downy, with a yellow brown to wine red
reverse. Most cultures show scanty to moderate numbers of slender clavate to pyri form microconidia.
Rhizopus stolonifer Colony whitish becoming greyish brown due to brownish sporangiophores, sporangia globose to subglobose .
Penicillium species Blue green fluffy growth on plate. Blue-green conidiospore borne in multilink chains .
Microsporum canis Rapid growth rate texture, silky to coarsely fluffy. Thallus color are white, reverse, deep yellow, slow growing
surface no reverse pigment, macroconidia absent. Long rough, thick-walled macroconidia with asymmetrical
knob on end.
Fusarium species Flat to slightly raised colonies on SDA, white to cream, suede like to downy, with a yellow-brown to wine red
reverse. Pyriformmicronidia along unbranched hypae .
Aspergillus niger Colonies were circular, opaque, smooth and flat with small black spots on short white hyphae. Septate
hyphae, branched condiophore with secondary branches. The condiophore is enlarged at the tip forming
rounding vesicle like chains.

SUMMARY
Keratinophilic fungi are natural colonizers of keratinic substrates. Some are kera-tinolytic and play an important
ecological role in decomposing α-keratins, the insoluble fibrous proteins. Because of the tight packing of their
polypeptide chains in α-helix structures and their linkage by disulphide bridges, they are poorly biodegradable.
Two main forms of attack have been identified: surface erosion and radial penetration. In surface erosion, the
sequence of degradation proceeds as the level of keratinisation (the cystine crosslinks) of the components of the
keratinic matrix increases. In radial penetration, on the other hand, specia- lized hyphae can penetrate like a drill
throught the matrix, irrespective of the degree of keratinisation. This may illustrate how the growth can change
direction and how secretory activity may concentrate at the tips of the penetrating hyphae.
KEYWORDS
Keratinophilic fungi, Role in nature, Keratin decomposition.

Figure : 2 Showing isolted fungul culture of
Aspergillus niger and Trichophyton spp

CONCLUSION AND FUTURE SCOPE OF THE WORK
Keratinophilic fungi are reported in various part of India, but the numbers of samples collected were limited in
comparison to the area of country; hence there is a need for systemic survey for prevalence of these fungi.
From India only 28 new fungi were recorded so far but the systematic survey will definitely add significant
numbers to the list of new fungi.
The molecular approach for identification will also help to obtain novel fungi. The whole- genome sequencing
and transcriptome analysis will aid in identifying genes that encode keratinases. Recombinant strains containing
potent keratinase genes will decompose feathers or other keratinous waste in soil and increase nutritional
profile.
Keratinophilic fungal provisions improve the degradation of feather waste by keratinase activities and increase
nutrient availability in soil. Crop- specific research is necessary on these aspects. Keratinases from these fungi
could be used as additives in detergent application for cleaning of clogged drains. Fungal enzymes can be useful in
the dehairing process of leather. These fungi could be major pollution controllers for the leather industry because
all waste of leather treatment contains keratinous material. Partially hydrolyzed keratin can be used in making
glue, biodegradable films, and coating from keratinous waste. Partially hydrolyzed feathers are good adsorbents of
heavy metals.

This could aid in pollution abatement. Keratinophilic fungi survive in
various extreme environmental conditions and are known to produce a
number of bioactive metabolites including enzymes, indole compounds,
and antimicrobial, antiviral, cytotoxic, cytoprotective, and other
substances.
The overall metabolic potential of keratinophilic fungi is immense
because of their specific nature. Keratinophilic fungi, if explored
extensively for metabolite production, can be novel sources of many
promising molecules of medical, biotechnological, and agrochemical
utility. There is a need for systematic sampling of large areas to recover
forms that have very restricted distribution. Selection of keratin-rich
habitats is also a key in obtaining rare onygenalean forms, many of which
are still to be describe
It will be beneficial to decompose feather waste in agricultural
fields in a short duration. There are a few reports on bacterial
strain development carrying multiple keratinase gene copies in
the chromosome of Bacil-lus licheniformis for increasing the
production of keratinase (Wang et al. 2004).

References
Anbu, P., Hilda, A., Gopinath, S.C. 2004. Keratinophilic fungi of poultry farm and feather dumping soil in Tami Nadu, India.
Mycopathologia, 158(3): 303 9.
Deshmukh, S.K., Mandeel, Q.A., Verekar, S.A. 2008. Keratinophilic fungi from selected soils of Bahrain. Mycopathol., 165(3):
143 147. Jain, N., Sharma, M. 2011.
Mycosis, 54: 52 8. Khanam, S.J.P., Jain, P.C. 2002. Isolation of keratin degrading fungi from soil of Damoh, India. Asian J.
Microbiol. Biotechnol. Environ. Sci., 4: 251254. Kunert, J.R.K.S., Kushwaha, Guarro, J. 2000. Revista Iberoamericana de
Micologia, 9: 77 85. Mahdavi Omran, S. 1991.
In: Kushwaha, R.K.S., Guarro, J. (Eds.) Biology of dermatophytes and other keratinophilic fungi. Revista Iberoamericana de
Micologia, Spain, Bilbao. Pp. 86 92. Mohamed, S., Ali-Shtayeh., Rana, M.F., Jamous. 2000.
Keratinophilic fungi and related dermatophytes in Int.J.Curr.Microbiol.App.Sci (2015) 4(7): 229-237polluted soil and
water habitats. In: Kushawaha, R.K.S., Guarro. J. (Eds). Biology of dermatophytes and other keratinophilic fungi.
Revista Iberoamericana de Micologia, Spain. Pp. 51 59. Nasery Bande Gharaey, A. 1992.