INTRODUCTION:

Feather is generated in bulk quantities as a by-product in the poultry industry globally. It is a very rich source of protein with β-keratin constituting 91% of feather protein. The presence of keratin makes feather recalcitrant to most common proteases like trypsin, pepsin, papain, and so forth, thus slowing down its degradation process in nature [Mabrouk, M.E.M. 2008]. Typically, each bird has up to 125 gm of feather and with more than 400 million chickens being processed every week worldwide, the daily accumulation of feather waste reaches five million tons [Han, M., Luo, W., Gu, Q. and Yu, X. 2012]. The bulk of feather waste is poorly recycled in nature and has limited utility due to the chemically unreactive nature of keratin.

Conventionally, this waste has been converted into feed supplement, resulting in feed of poor quality which is nonviable economically [Acda, M.N. 2010]. Thus, recycling of this by-product is neither profitable nor environmentally friendly. The disposal of this waste is a global environmental issue leading to pollution of both air and underground water resources [Lin, X., Lee, C.G., Casale, E.S. and Shih J.C.H. 1992]. In recent years, feather treated with microbial keratinase is attracting wide attention with several applications. Keratinase-treated feather is increasingly considered as a viable source of dietary protein in food and feed supplements, as the enzyme-treated end product retained high nutritive value. Keratinases are projected to generate a potential worldwide market similar to other proteases. Diverse groups of microorganisms are reported to produce keratinase like fungi (Doratomyces microsporus, Alternaria radicina, Trichurus spiralis, Aspergillus sp., Rhizomucor sp., Absidia sp., Stachybotrys alba, etc.), actinomycetes (Streptomyces pactum, S. alvs, S. thermoviolaceus, S. fradiae, Thermoactinomyces candidus etc.), and several bacterial species (Fervidobacterium islandicum, Pseudomonas aeruginosa, Microbacterium sp., and many species of Bacillus including Bacillus licheniformis and B. pumilus) earlier [Han, M., Luo, W., Gu, Q. and Yu, X. 2012; Deivasigamani, B. and Alagappan, K.M. 2008; Mukhopadhyay, R.P. and Chandra, A.L. 1993; Nam, G.W., Lee, D.W., Lee H.S. 2002; Suneetha, V. and Lakshmi, V.V. 2005]. However, the full commercial potential of keratinases is yet to be realized. Major component of feather is keratin which is insoluble fibrous protein. Keratin is highly resistance to hydrolysis by week acids, alkalies, ethanol or salt solution (Page, 1950) and also to enzymatic digestion (Weary et al., 1965). The durability of Keratin is due to cross binding of closely packed polypeptide chain in which cystine molecules are held together by disulphide bonds(S-S). However, the keratinophilic fungi have been frequently isolated from soil, where they colonize various keratinous substrates, degrade them and add the mineral content to the soil (Kunert, 1989).

Feathers of birds are most suitable substrate for the survival of much fungus in nature (Sarangi, S.M. and Ghosh, G.R.1990). Ramesh (1996) isolated a number of pathogenic and non pathogenic fungi from keratin substrates and studied their intensity and the type of hair degradation. However, there is no detailed investigation on the degradation of feathers. Therefore, the present investigation was envisaged to study the biodegradation of feathers. Currently, almost all the habitats of the world have been surveyed for the presence of keratinophilic fungi (Kushwaha, R. K. S., 2000). Most of these fungi belong to families Arthrodermataceae and Onygenaceae, order Onygenales in Ascomycetes (Currah, R. S., Mycotaxon, 1985).

METHODOLOGY:

Method of sample collection and isolation of potential fungal colonies

Feathers of broiler chicken were collected from Jaggi poultry farm, Mandir Hasaud, Raipur. Fungi were isolated by feather baiting technique. Feathers of hens were cut and finely powdered and sterilised by using 70 % sodium hypochlorite solution for 5 minutes. These sterilised feathers were then inoculated on Sabouraud Dextrose Agar (SDA) media to obtain culture of fungal colonies. Six different colonies obtained were inoculated in 50 ml mineral media (Sodium Nitrate, 3g; Dipotassium Hydrogen phosphate, 1g; Potassium Chloride, 0.5g; Magnesium Sulphate 0.5g, Ferrous Sulphate 0.01g) along with 500mg of feathers as a sole source of Nitrogen and Carbon (Day et al 1968) in 250 ml flasks and incubated for observation.

Identification of isolates

Fungus was identified by Lacto phenol cotton blue staining method. A drop of Lacto phenol cotton blue was taken on a slide. A fungal hyphae was picked from plate with the help of a needle and placed on the slide containing Lacto phenol. Then the Slide was covered with a cover slip and observed under the microscope.

Estimation of keratin degradation

Filtrates were collecetd in an interval of 5 days starting from the 10^th day of incubation. Filtrate was collected by filtering the incubated media by arranging 2-3 Wattman filter paper on one above another. This filtrate does not contain any feathers, fungal culture and mineral media. Change in pH was measured by using digital pH meter with a glass electrode. Determination of nitrate release (NO₃) was done by the method of Goldsmith (Goldsmith et. al, 1973). Determination of cysteine and cystine was done by the method of Ramakrishna (Ramakrishna et. al, 1979). Determination of methionine was done by the method of Timothy (Timothy, E. McCarthy and Sullivan, M.X. 1940).

RESULT AND DISCUSSION:

Fig: 1- Trichoderma

Fig: 2- Gliocladium

Fig: 3- Fusarium

Fig: 4- Syncephalastrum

Fig: 5- Aspergilus

Fig: 6- Mucor

Table 1: No. of filtrates

Filtrate	Microorganism
Filtrate 1	Trichoderma
Filtrate 2	Gliocladium
Filtrate 3	Fusarium
Filtrate 4	Syncephalastrum
Filtrate 5	Aspergillus
Filtrate 6	Mucor

Increase in pH

During the process of bio degradation there was gradual increases of pH in to the alkaline phase for feathers till 25 day incubation. However, the pH increases from 10^th day , 20^th day for 1^st to 4^th culture and increases continuously from 10^th to 25^th day in case of 5^th and 6^th culture. Such an alkalinisation of the medium may be due to excretion of excess nitrogen via deamination and ammonium excretion. Keratin degradation involves rupturing the disulphide linkage between the peptide chain of keratin molecules by some extra and intra cellular enzymes collectively called keratinise.

Fig 7: Increase in pH

Increase in Nitrate

The liberation of nitrogenous compound gradually increases from 10^th day to 20^th day for 1^st to 4^th culture and increases continuously from 10^th day to 25^th day in case of 5^th and 6^th culture.

Fig: 8 Increase in Nitrate

Increase in Methionine

Methionine release in the filtrate increases from 10^th to 20^th day in 1^st to 5^th culture and increases continuously from 10^th to 25^th day way in case of 6^th culture.

Fig: 9 Increase in Methionine

Increase in Cystine:

Accumulation of cystine may be direct reduction of disulphide bridges of keratin (Noval and Nickerson, 1959). Chicken feathers, a type of eukeratin which connected histidine, lysine, and arginine in a definite proportion of 1:4:12 and 3-5% of sulphur nearly all of which is in the form of cystine (Singh et al., 1995). In filtrate 1, 2, 3 and 6 cystine release increases from 10^th day to 25^th day where as increases continuously in 4^th and 5^th culture.

Fig 10: Increase in cystine

Increase in Cysteine

The release of sulphydryl compounds namely, cysteine release increases from 10^th day to 20^th day and decreases in 25^th day in all the culture except in 5^th culture in which cysteine release increases continuously. During the process of degradation the -S- sulfo groups (-S.SO₃H) and Sulfhydryl groups were formed by Sulfitolysis (Kunert, 1976).

The fungus degrades this highly resistant Keratin of feather. With an increasing world-wide concern for the environment it is possible to use these 6 fungus for the degradation of enormous quantity of waste feathers. Biodegradation leads to recycle the wastes and thus maintaining the environmental quantity of the biosphere.

Fig 11: Increase in cysteine

Current and future Use

Wastage of protein-rich reserve is ultimately converted into feather meal using keratinolytic fungi (Shih, J. C. H., 1993, and Bertsch, A. and Coello, N., 2005). The addition of microbial digested feather meal to the animal feed improves digestibility and bolstered growth of poultry. Nutritional enhancement can also be achieved by hydrolysis of raw feathers using these keratinolytic fungi. Microbial-digested feather meal is also used as slow nitrogenreleasing fertilizer. Keratinophilic fungi are used for the production of biodegradable films, coatings and glue from keratinous waste. Keratinases of these fungi are utilized in enzyme-based detergents which are used in the removal of keratinous soils, common in the laundry, on collars of shirts, etc. These enzymes are also used for cleaning up of drains clogged with keratin waste. These keratinases are also employed in the leather industry in hairsaving dehairing in place of chemical based dehairing. Recently, these keratinases have been found to degrade prion protein leading to the prevention/cure of mad cow disease (Wang, J. J., Borwornpinyo, R., Odetallah, N. and Shih, J. C. H., 2005). Further, keratinases are applied in the modification of silk and wool fibers, for acne or psoriasis, for making vaccines of dermatophytosis and has addictives in skin-lightening agents. In addition to the keratinases, these fungi have the potential to generate natural gas for fuel from poultry-waste degradation.

Waste material, hen feather, a bio sorbent, was successfully utilized in removing a water-soluble hazardous triphenylmethane dye, Brilliant Blue FCF from wastewater. Chicken feathers could help save trees by taking the place of wood pulp in air filters, paper products, and other uses, according to chemist Walter Schmidt of the U.S. Agricultural Research Service. Replacing half the wood-pulp content of composite paper with chicken feathers means only half as many trees. Chicken feathers can also to be use for the production of fuel.

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