Bio-degradation of Feathers Waste by the Staphylococcus epidermidis P3A

Development of industrialization is directly proportional to the development of nations. Rising industrializations also increase the pollutions. One of them is poultry industry which discharges a huge amount of keratinous waste. The present study focused on the selection of feather degrading bacteria from Barshi region of Maharashtra. Bacterial strains were grown in whole feather broth medium. Out of 16 proteolytic bacterial isolates, 9 feather degrading isolates were selected from the Parande road side feather waste dumping area. Among these isolates, P3A was selected and classified on the basis of morphological and biochemical analysis. 16S rRNA studies confirmed that the isolated strain was Staphylococcus epidermidis. Staphylococcus epidermidis P3A shows 84% feather degradation and 72 U/ ml maximum keratinase activity after 96 hrs. The isolate shows potential use in feather waste disposal methods which are safe and environment friendly.


INTRODUCTION
Disposal of environmental waste is becoming a significant issue across the world and it majorly polluted water sources and land [1][2][3][4] . The waste byproducts resulting from food processing are keratinous substances including animal hairs, chicken feathers, wool and horns, and vast amounts of certain byproducts are generated yearly. For example, during the year 2019 it has been approximated that over 4.7 million tons of waste feathers were generated by poultry industries alone 5 .
Waste material from the poultry sector which always includes excreta, wasted feed, cracked eggs, dead birds, feathers and offal may appear as significant environmental pollutants. Feathers are excellent sources of essential amino acids and could be processed into necessary products such as bio-fuel, recycled plastics, feather food and organic fertilizers 6 . Keratinous components are special since they are composed of essential amino acids including glycine, arginine proline, leucine, valine and threonine. Nevertheless, the robust structure, elevated keratin cohesion are especially due to disulphide bonds formed between cysteine chains within as well as among keratin polypeptide chains 7 but existing feather waste management strategies such as incineration, soil filling or processing to animal food by high pressure steam cooking often need a lot of energy inputs also generates handling, storage, pollution control problems 8 . Development of keratin hydrolysates through physical or chemical methods usually requires a high temperature and therefore effects in deterioration of heat susceptible amino acids, such as lysine, tryptophan and methionine which reduce in the nutritional value of the hydrolysates 9 . Feather contains more than 90% of protein, the primary element of which is beta-keratin, insoluble and fibrous structural protein that bridges widely interconnected by disulphide bonds, this makes them very rigid to digestion by livestock, foremost to severe problem for discarding 10 . Keratin belongs to the class of scleroprotein is highly stable and have low rate of degradation 11,12 .
The Bacteria secretes enzymes which specifically breakdown the beta-keratin part of feathers 13 . Keratinase is an extra cellular enzyme only secreted when keratinous materials such as hairs, wool, feather, horn nails etc are present and act on the disulphide bond of keratin 14,15 .
In spite of their foremost ability of withstand, keratins do not mount up in environment because of the numerous microorganisms has ability to hydrolyze them. Keratinolytic activity was widely recognized for strains of the genera Bacillus as well as Streptomyces among bacteria [16][17][18] . Bacterial keratinase enzyme may have significant uses during the expansion of non-polluting processes in biotechnological processes relating to keratinous waste from the poultry and leather productions. Biodegradation of feathers using keratinase producing microorganisms attracts several researchers as it can offer an effective technique to dispose feather waste that can be combined with useful product formation 19 . The purpose of this study was to select novel bacteria for degradation of chicken feather from dumping sites of feather waste in Barshi region of Maharashtra.

Collection of soil samples
For the purpose of selection of feather bio-degradable micro-organisms soil samples from the depths of approximately 3.0 to 4.0 cm were collected from various chicken feather waste dumping sites.

Processing of Feathers
Waste feathers were thoroughly cleaned in potable water to eliminate the blood particles, dust and eventually washed with distilled water. Feathers were dehydrated in sunlight and after that at 60°C for 48 hrs and stored at 5°C for further study 20 .

Isolation of bacteria
Five-fold series dilution was prepared from soil samples, enriched in whole feather broth medium and incubated for 7 days at 37°C with 120 rpm.

Primary screening
Suspension from enrichment culture was evaluated for proteolytic activity on skim milk agar. Well-grown Clear zone forming colonies were selected for the study of keratinolytic potential. Secondary screening 24 hrs old cultures were prepared from the promising isolates selected in primary screening and inoculated in sterile whole feather broth Medium composed as (gm/l): Feathers-5.0, K 2 HPO 4 -0.3, KH 2 PO 4 -0.4, NaCl-0.5 and pH-7.5. The flasks were incubated on shaker for 7days at 37°C.

Identification of isolates
The promising strains were identified in accordance with the taxonomic scheme of Bergey's manual based on their morphological, cultural and physiological characters. The tests carried out include gram-nature, motility, catalase, coagulase, oxidase, urease, indole production, production of hydrogen sulfide, methyl red, Voges-Proskauer, nitrate reduction, oxidative fermentative test 21 .
Further identification was confirmed by 16S rRNA sequencing. Bacterial DNA was isolated using DNA isolation kit. Gene fragment was amplified by the PCR. A single specific PCR amplicon band was selected and unincorporated PCR primer and dNTPs from PCR products were removed by purification of the column. The PCR amplicon DNA sequencing reaction was performed with 27F/1492R primers using the BDT v3.1 Cycle sequencing kit on the ABI 3730xl Genetic Analyzer. The gene sequence was used to perform BLAST with the database of NCBI Genbank database. On the basis maximum similarity score, initial 10 sequences were selected and matched using multiple alignment software programs and the phylogenetic tree was constructed using the Neighbor-Joining method.

Determination of keratinase activity
24 hrs of old culture of isolated strain was inoculated in whole feather broth medium incubated at 37°C on shaker with 120 rpm for production of keratinase enzyme. Aliquot of the broth culture was withdrawn after 24 hrs of interval; crude enzyme was extracted by filtration and centrifugation. Keratinase activity was measured with Keratin Azure (K8500 Sigma Aldrich) as substrate with slight changes of methods described by (22, 23, 15 and 24). In brief, the reaction mixture prepared as 0.8 ml 50mM potassium phosphate buffer of pH 7.5 contain keratin azure (0.4% w/v) added with the 0.2 ml crude form of enzyme and incubated at 50°C for 1 hr with 200 rpm shaking condition. The enzyme substrate reaction was stopped by addition of 0.2 ml 10% trichloroacetic acid. Fallowed by 15 minute's centrifugation at 15000 rpm and supernatant was utilized to measure the absorbance at 595 nm as compare with control sample. The control sample was prepared by similar procedure used for preparation of test sample except 10% trichloroacetic acid was added before crude enzyme in reaction mixture. 1 unit of enzyme activity equal to the quantity of enzyme that responsible for 0.01 absorbance's changes at 595 nm under the defined reaction conditions.

Determination Feather degradation
Degradation of feather was determined by visual observation further percentage of feather degradation was calculated, after biodegradation process, the remaining feathers particles were accumulated from whole feather broth medium by filtering through the whatman filter paper No 3. The collected feathers particles were dehydrated at 50°C up to its weight stabilized to constant value and compared to initial weight of feather taken in preparation of whole feather broth medium. The Isolates which effectively degraded feather were selected for further analysis. The percentage of feather degradation was determined using the formula: Feather degradation (%) = (Initial weight of feathers -Final weight of feathers)/Initial weight of feathers×100 25,26 .

ReSUlTS AND DISCUSSION Isolation and identification
Sixteen isolates exhibited protease activity were confirmed by skim milk agar plates, which were selected from soil sample collected from the feather waste dumping yard on the side of Parande road, out of them nine isolates showed potential for degrading feathers and therefore they were used for further studies. The isolate P3A showed significant feather degradation (Fig  1) was identified by various morphological and biochemical characterizations ( Table 1). The strain was Circular, Creamy white, Raised, Translucent, viscid, Gram positive, non motile. According to 16S rRNA analysis the selected bacteria was identified as Staphylococcus epidermidis and designated as Staphylococcus epidermidis P3A. DDBJ had received the isolates under the accession number LC530604. Phylogenic tree showed the detailed relationship between the isolated strain and other homologous species of the genus Staphylococcus epidermidis (Fig 3). Feather degradation process mostly carried out by the gram-positive bacteria 27 also the feather-degradation by gram-negative Journal of Pure and Applied Microbiology bacteria is noted in few reports [28][29][30] . Our results of isolation of feather degrading bacteria support the results of Lin et al. 31 they isolated seven proteolytic bacteria among them five bacterial strain had potential of feather degradation identified as Bacillus licheniformis. Several Bacillus sp. including Bacillus subtilis, Bacillus coagulans, Bacillus cereus and Bacillus pumilus have been recognized to be prospective sources of keratinases 32 . In addition to these, vast variety of keratinase producing microorganism also exists such as Nocardiopsis dassonvillei NRC2aza 33 , Stenotrophomonas 34 , Chryseobacterium sp. 35 , Vibrio sp. kr2 36 and Arthrobactercreatinolyticus KP015744 37 .
There are very few reports available on feather degradation by the staphylococcus sp. Venkata and Divakar 38 isolated the Staphylococcus aureus, Bacillus cereus and Bacillus licheniformis from poultry waste dumping yard. Feather waste dumping sites are good habitat and best resource for isolation of feather degrading bacteria.

Determination of keratinase activity
Promising isolate P3A effectively degraded the feather by secreting extracellular keratinase enzyme. The maximum keratinase production recoded as 72 U/ml after 96 hrs in whole feather broth medium. The enzyme activity was decreases as per increases the incubation time after 96 hrs ( Table 2). Mursheda akhter et al. 39 recorded highest keratinase activity 60 U/ml after 96 hrs from the Bacillus cereus and 44 U/ml after 72 hrs from Pseudomonas sp using chicken feather. Similar work of Jahan et al. 40 revealed that isolate Z4 showed highest keratinase activity 22.3 U/ml after 72 hrs and complete degradation of feather after 7 days

Percentage of feather degradation
Isolate P3A exhibited the 84% feather degradation after 96 hrs in whole feather broth medium (Fig 2). Nonso et al. 43 39 revealed that complete degradation of feather was obtained within 5 days. Jendri Mamangkey et al. 45 reported newly isolated strain Azotobacter chroococcum B4 capable of degrading whole feathers after 5 days. Similar work done by Mehta et al. 46 they reported that entire degradation of feather waste within 7 days by the Bacillus sonorensis strain NRRLB-23154 isolated from poultry farm. While Shah and Vaidya 8 recorded 70% degradation after 6 days by Acinetobacter sp. PD12 isolated from feather waste dumping area. In the current reports we have attempted to isolate and classify a novel bacterial strain capable for degradation of keratinous wastes into soluble and useful products. Many bacterial members from the genus Bacillus 47 and Streptomyces sp. 48,18,49 were selected from poultry chicken feathers waste, these bacteria degrade feather keratin for utilization carbon, nitrogen and sulfur as primary source for their growth. Similar work done by Suntornsuk et al. 50 they observed that the isolate Bacillus licheniformis FK14 degrade the feather within 5 days at 50°C. Giongo et al. 51 reported that keratinolytic Bacillus sp. selected from Brazilian Amazon basin shows significant degradation after 72 hrs. We conclude from the results of this study that chicken feather waste dumping sites are the rich source of feather degrading bacteria. Feather degrading bacteria utilize the feather keratin protein for their growth.

CONClUSION
From the discussion it might be concluded that a novel gram positive Staphylococcus epidermidis P3A was isolated from feather waste discarding area from Barshi town. The bacteria effectively utilize feather keratin substrate as the predominant resource of energy, nitrogen, carbon, and sulphur. The isolated strain can be applicable for reduction of environmental pollution arises due to poultry chicken feather waste.

ACKNOWleDGMeNTS
Authors sincerely thanks to Triyat Genomics Laboratory Nagpur India for 16S rRNA sequencing analysis. ARJ acknowledges to BARTI Pune for PhD fellowship for the year 2015-2017.

CONFlICT OF INTeReST
The authors declare that there is no conflict of interest.

AUTHORS' CONTRIBUTION
ARJ: concept of the main idea, complied information from the literature, performing