Identification of Keratinophilic Fungi in Urban Waste and Cattle Field Soil of Kanpur, India for Environmental Pollution Management

Kanpur is a city which has huge number of leather product units and leather processing plants. These units are one of major contributors of keratinous waste and produces keratinous material as waste in the form of hairs, hides, dermis. During the present study 83 keratinophilic fungi were isolated from 40 soil samples of urban waste and cattle field habitat of various localities. From 20 samples of urban waste, 44 keratinophilic fungi were isolated, 39 fungi recorded from Cattle field. The frequency of genera Chrysosporium was recorded in urban waste (29.54%) and cattle field soil (20.51%). Maximum (13.83%) frequency was recorded in the case of Chrysosporium indicum in urban waste.


INTRODUCTION
Soils are natural reservoir of keratinophilic fungi due to presence of keratinous waste materials which is most suitable for the growth [1]. Keratinophilic fungi are present in the environment with in consistent al location which depends on as human and animal presence [2].

Original Research Article
The existence of keratinophiles in the soil is also influenced by the presence of other microbes namely the bacteria and actinomycetes and fungal components which exert an antagonistic effect on keratinophilic fungi [3][4][5].
Soil of various habitats of Kanpur city were studied for occurrence of keratinophilic fungi; indoor environment [6][7], house dust [8][9], birds [10], house sweeping dust, sand [11][12][13], potted indoor plant [14], parks [15] aquatic habitats [16] and sewage slug [17] Few other recent reports of occurrence of keratinophiles from India and other parts of Worlds are Hilly soil [2], Ladak [18], Sewage sludge of Vishakhapatnam [19], Vidarbha region of Maharashtra [20], poultry site of Shivamogga Karnataka [21], predatory birds [22], Parks of Jaipur [23]. Climate and keratinous waste in environments make Kanpur city appropriate study area. Kanpur is city situated at the bank of river Ganga and famous for their leather-based industry. Leather product units and leather processing plants are one of major contributors of keratinous waste and produces keratinous material as waste in the form of hairs, hides, dermis. Soil of Kanpur is thoroughly screened for these fungi in other habitats but particular emphasis was not given to the urban waste and cattle fields. The objective of this study to isolate new strains of these fungi.

Collection of Soil Samples and Keratinous Substrate
A total 40 soil samples were collected from 20 each from cattle fields and urban waste soil were taken from superficial soil layer depth not exceeding 2 to 3 inches with a sterilized plastic spoon in sterilized polythene bags. Human hair, Chicken feather, Human nail, Horn and cattle hair were washed and cut into pieces of 1-2 cm autoclaved for 15 min at 15 lbs pressure and used as keratinous substrates.

Isolation Methods
Ten to twenty gm of soil from each collected sample was collected in pre sterilized poly bags. Petri dishes and moistened with 10 ml of sterilized distilled water by hair baiting method [24] (Fig 1A). These samples were baited by keratinous substrates. Human, horse, and buffalo hair, human nails, chicken feathers and cow horns were used as keratinous substrate. When fungal colony observed on bait it is isolated on Potato dextrose agar and Sabouraud's dextrose agar (Fig. 1B) and maintained as culture in tube, water cultures and dry herbarium are also maintained.

Purification and Identification of Fungi
In the Petri dishes, when a fungal colony was seen for the first time, it is transferred to other dishes for purification. To ensure the purity of cultures, all the isolated cultures studied are derived from a single spore raised through the dilution method. After ensuring the complete purity of cultures, the descriptions are made. Measurement for each fungus is taken by culturing it on a suitable medium. Identification of the isolated fungi will be confirmed with the help of literature available in this department and secured through the courtesy of various mycologists from India and abroad. Living cultures were deposited in DST sponsored Germplasm Centre for Keratinophilic Fungi (GPCK), Department of Botany, Christ Church College, Kanpur.

Fig. 1. A. Growth of Chrysosporium indicum B. Growth of C. indicum on Potato dextrose agar
For quantitative analysis, following parameters were considered to estimate fungal population.
Total number of samples examined Frequency of isolation (%) = Number of strains of a given species Total number of fungal strains

RESULTS AND DISCUSSION
During the present study, 40 soil samples collected from the different habitat of various localities yielded 83 keratinophilic fungi. Isolated fungi were morphological identified (Fig. 2).
Isolated strains belong to 21 genera and 45 species. Results of the incidence of keratinophilic fungi are given in Table 1.  Auxarthron conjugatum (anamorph of Malbranchea sp.) showed its highest percentage of distribution in urban waste. Arthroderma simii (anamorph of Trichophyton simii) showed its maximum distribution in urban waste. Among all perfect forms, the maximum percent frequency was recorded in urban waste. The fast-growing nondermatophytic keratinophilic fungi isolated from keratinous substrates and found in various habitats. Alternaria alternata was recorded in urban waste. Five species of Aspergillus was isolated during the present study Aspergillus sparus was isolated from street sweeping soil with 10.52% distribution. Aspergillus sydowii revealed the 5.56%% distribution in urban waste. Penicillium was next dominant genus among fast growing keratinophilic fungi. Penicillium griseofulvum was isolated from three habitats. Two species of Fusarium belonging to 8 isolates were isolated from various habitats. Fusarium oxysporum and Fusarium proliferatum exhibited same pattern of distribution and isolated from both habitats.
Urban waste represents polluted field soil while sand represents water habitats. Urban waste and sand allow the growth of several dermatophytes and non dermatophytic keratinophilic fungi. Urban waste and street sweepings are polluted habitats as comparison to normal habitats. Street sweepings are a component of municipal solid waste. The urban waste was found to be rich in keratinolytic fungi and the genera Chrysosporium predominated among the isolates. In sweepings, Epidermophyton and Microsporum were predominated. The quantitative and qualitative composition in the sweepings was associated with pH, the content of heavy metals and particle size [25]. The diversity of keratinophilic fungal communities in field soils and waste water habitats was studied b [26]. Cattle field samples were rich with a high content of keratin in the form of cattle hairs, horns. [27] Isolated Aphanoascus terreus, Apinisia queenslandica, Chrysosporium indicum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium queenslandicum from cattle soil. All the habitats discussed above are hygienic and epidemiological importance. However, in reports many fungi are used for feather waste utilization for biofertilizers [28][29][30][31][32][33][34].
Out of forty-three keratinophilic fungi, twenty-one isolates of Chrysosporium were observed as dominating fungi. Chrysosporium queenslandicum was isolated from two habitats. Chrysosporium pannicola was found in the soil of urban waste. Chrysosporium sulphureum was restricted in its distribution and isolated from cattle field soil. The frequency of Chrysosporium recorded in various habitats was as follows: urban waste (29.53%) and cattle field soil (17.94 %). A Maximum (15.90%) frequency was recorded in the case of Chrysosporium indicum in urban waste ( Table 2). The frequency of Microsporum recorded in urban waste (10.00 %), cattle field (5.00%). Trichophyton ajelloi was isolated from soils collected from urban waste.
Trichophyton rubrum was isolated from cattle field was 10.00 % in its distribution. In some soil samples keratinophilic fungi also developed ascostoma of their corresponding anamorph. Aphanoascus terreus (anamorph of Chrysosporium indicum) was isolated from urban waste where it was 5.00 % in its distribution. Aphanoascus fulvescens (anamorph of Chrysosporium sp.) showed 5.00 % distribution in urban waste while Aphanoascus keratinophilus (anamorph of Chrysosporium keratinophilum) showed the 10.00 %, distribution in cattle field soil.

CONCLUSION
The keratinolytic fungi can be the bioindicators of environmental pollution with waste. Fungal indices also show the contamination hazard related with pollution of the environment with potential fungal pathogens. Given these findings, it can be concluded that urban waste and cattle fields are rich in keratinophilic fungi as well as dermatophytes. Therefore, cleanliness actions should be taken to control the spread of these fungi in the environment and check fungal infections.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.