Research Article | Volume 11, Issue 3, May, 2023

Isolation and characterization of keratinolytic bacteria from poultry waste soils of Himachal Pradesh

Richa Vema Vijay Kumar   

Open Access   

Published:  Apr 04, 2023

DOI: 10.7324/JABB.2023.112620
Abstract

The aim of the present study was to isolate and screen keratinase producing bacteria from poultry waste dumping sites of Himachal Pradesh that can be used to degrade chicken feathers. The poultry farming industry creates millions of tons of chicken feathers each year, which are the primary source of protein waste and environmental pollution. Thirty poultry waste soil samples were collected from the different areas of Himachal Pradesh. A total of 64 bacterial isolates were obtained from different poultry waste soil samples. These isolates were primarily screened for proteases activities on skimmed milk agar plates. Out of 64, 17 isolates showed proteolytic activity. All 17 isolates were, further, screened and confirmed to possess the keratinolytic activity on feather basal medium. The test showed almost complete degradation of feathers within 15 days. Four bacterial isolates which have potential of keratinase activity were characterized and identified using 16S rRNA gene sequencing. Results of molecular characterization revel that all four isolates belong to Bacillus genera. Isolates TP1, JP1, and PP1 were identified as Bacillus cereus, whereas isolate VP4 was confirmed as Bacillus thuringiensis.


Keyword:     16S rRNA sequencing Feather degradation Keratinase Keratinolytic bacteria Poultry waste


Citation:

Vema R, Kumar V Isolation and characterization of keratinolytic bacteria from poultry waste soils of Himachal Pradesh. J App Biol Biotech. 2023;11(3):172-179. https://doi.org/10.7324/JABB.2023.112620

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. INTRODUCTION

Poultry industries are increasing rapidly day by day. The growth of this sector is good as it provides career opportunities to the people, but problems regarding waste management also come with it. Poultry waste includes chicken feathers, chicken poop, bedding material, split feed, and hatchery material. Keratin comes primarily from the poultry industry. In actuality, the global poultry business processes about 400 million chickens/week, resulting in approximately 5 million tons of feathers [1]. These cause the problem regarding waste disposal. As a result of their wide range of uses, keratinase are earning a lot of interest. They are used in waste treatment, agricultural, pharmaceutical, and biomedical areas right now and in the future. Nanobiotechnology, leather, cosmetics, and detergent manufacturing industries are some of the other sectors. Several microbial strains with exceptional feather-degrading abilities could be valuable in the management of feather waste. There is also environmental pollution due to the recalcitrant character of keratinaceous substances [2].

Keratin, an insoluble protein macromolecule with exceptionally high stability and a low breakdown rate, is the main component of chicken feathers. Hair, feathers, nails, wool, and horns are the primary components that contain keratin [3]. Keratin waste contains a high amount of protein content, which can be utilized as a source of amino acids and proteins by recycling it. The feathers can be recapitulated, which may provide an alternative and inexpensive protein meal. Furthermore, this protein meal stuff can be used for animals’ fodder. Yet, keratin is not digested easily as it is difficult to recycle [4]. An extracellular enzyme, keratinase, is used for the biodegradation of keratin. Normally, microbial keratinase is generated in the presence of a keratin substrate. To degrade keratin substrate, this enzyme primarily destroys its disulfide bond. Microorganisms that produce keratinase are capable of destroying chicken feathers, hair, nails, wool, and other materials [5].

In soil collected from sites where poultry waste is dumped, keratinolytic bacteria were isolated and identified. Selvam et al. (2013) show that keratin is resistant to peptidases such as pepsin, papain, and trypsin [6]. The protein chains in keratin are tightly packed either into ?-helix or into β-sheet structures that fold into three-dimensional form [7]. One of the important characteristics of keratin is that it has high mechanical stability due to the presence of hydrogen and disulfide bonds [8]. Keratin is also a prime component of the epidermis and its appendages, such as nails, horns, hoofs, hair, feathers, scales, and wool. According to their sulfur content, keratins are classified into two types; one is hard keratins and the other is soft keratins. The hard keratins are found in appendages, while the soft keratins are found in skin and callus. In soft keratin, there is a low content of disulfide bonds [6]. Keratinolytic enzymes are generated by bacteria, fungi, and actinobacteria that have been commonly isolated from soils, where keratinous substances are accrued. Normally, thermophilic microorganisms are related with increased keratinolytic activity, microorganisms that decompose keratin wastes [9].

Feathers are generally composed of over 90% protein and are produced in large quantities as a waste product by poultry processing industries worldwide. Feathers accumulating in the environment may lead to wastage of feather protein [10]. Various keratinous substances such as feathers, wool, and hair can be used as substrates for the production of keratinase [5]. Bacterial keratinase has potential for the degradation of exclusive insoluble keratin substrates and on wide kinds of protein materials [11]. This enzymes function as pioneer biocatalysts which have various avail in different sectors such as in cosmetics industries, detergent industries, leather industries, and textile industries. This enzyme also works on drug delivery systems, production of biodegradable integument, or bio-processing of used X-ray integument and is also used in agroindustrial waste degradation [12,13].

In many ecological environments, a cluster of microbes that can produce keratinase enzymes and break down keratinous waste includes bacteria, saprophytic fungus, actinomycetes, and dermatophytes [14]. Therefore, the present research work was an attempt to isolate keratinase-producing microorganisms from the selected 30 poultry waste dumping soils, screen, and characterize keratinase-producing microbes from the poultry dumping sites of Himachal Pradesh.


2. MATERIALS AND METHODS

2.1. Collection of poultry waste soil samples

A total of 30 poultry waste soil samples were collected from local poultry processing industries in Himachal Pradesh, India. The rotted feathers and poultry waste soil samples were collected from regular poultry waste dumping sites. From a depth of 3–4 cm, samples of poultry soil with deteriorated feathers were taken and put into sterile plastic bags [15]. The samples of poultry waste soil were brought to a laboratory for further processing. The details of sample collection are shown in Table 1.

Table 1: List of poultry waste soil samples collected from different locations in Himachal Pradesh.

S. No.Sampling siteSoil Sample Name
1.Hills Poultry Farm, Dharwad Pater Talwara Chowk, Kuthera Distt.-Bilaspur H.P. 174026.KP
2.Hemraj Poultry Farm, VPO- Hartlyalagar, Distt.- Bilaspur.HT
3.Him Chicken Corner, Bus Stand, Ghumarwin, Bilaspur H.P. 174021HC
4.Jai Maa Kali, Kadaknath Poultry Farm Jahu, Mandi (H.P.) 177118JP
5.Chauhan Poultry and Hatchery, Vill- Lunapani, P.O. Bhangrotu, MandiCP
6.Him Hatchery Govt. Hatchery, Sundernagar, Mandi, (H.P.)HH
7.The Happy Village Hens Farm and Hatchery, Khungan, P.O. – Kaphara, Bilaspur, (H.P.)HV
8.Vishal’s Farm Near, Dakri Chowk, Ghumarwin Distt.- Bilaspur (H.P)VP
9.Chauhan Poultry Farm, Township Dhanotu Chowk, Mandi (H.P.) 174402DP
10.Sandha Desi Murgi Poultry Farm, Hamirpur, (H.P.)SD
11.Poultry Farm (Haroli) Chicken Hatchery, Palakwah (H.P.)HR
12.Akib Poultry Farm Mahadev,(H.P.) 175019AP
13.Birbal Poultry Farm, Gagal, Mahadev Nagar, Mandi 175121BP
14.Naitre Singh Poultry Farm, Dugrain Teh. & P.O.- Chachiot 175020NP
15.Mahinder Singh Poultry Farm, Naugraon, Near Abhilashi University P.O.- Chailchowk, Mandi, (H.P.)MP
16.Thakur Poultry Farm, Hamirpur H.P. 177301TP
17.VK Poultry Farm Vill.- Loharli P.O.- Jahallan Nadaun Hamirpur 177301VK
18.Pride dairy &Poultry Farm Galore, Hamirpur 177001PDP
19.Kaku Poultry Farm & Poultry House PalampurKPF
20.Shiv G Poultry Farm Sundernagar (H.P.)SP
21.Poultry Farm Haroli, Una Palakwah. 177220PH
22.Dr. Kuldeep’s desi Poultry and Kadaknath PoultryDKP
23.Kadaknath Poultry Farm, Man Simble, Uparla, H.P. 176083MS
24.Chicken Neck Poultry Farm, Raakh, Baijnath, Kangra (H.P.) 176071CNP
25.Namay Desi Poultry Farm, Ladraur, Bari Chowk (H.P.) 174028LD
26.Rajput Abhishek Jamwal Poultry Farm, Vill. Samlohal Teh.- Ghumarwin, Bilaspur (H.P.) 174029SG
27.Patyal Poultry Farm, Vill. Bhallu P.O. Berthin Teh.- Jhandutta, Bilaspur (H.P.) 174029PP
28.Anurag Poultry and Fish Farm, Soyad Khad near Sandyar Androli, Chhat, H.P. 174029SA
29.AL Nadeem Kadknath Farm Danoh, Bilaspur H.P. 174001AB
30.AQSA Chicken Shop Ghumarwin, Bilaspur, (H.P.) 174021AQG

2.2. Isolation of Microbes from Poultry Soils

Microorganisms were isolated from poultry waste soil samples by the serial dilution and plating method on skimmed milk agar. Ten grams of poultry waste soil samples were mixed thoroughly in 90 mL of sterilized feather basal medium. For 24 h, the flasks were placed in a rotator shaker incubator at 120 rpm. The sample was serially diluted up to a 10-7 dilution. The diluted sample in a 0.1 mL volume was inoculated in skimmed milk medium and spread evenly on petri plates using a spreader. Plates had a 24- to 48-h incubation period. At 37°C [16], the colonies were purified using the streak plate method. A pure culture was obtained by further subculturing the isolated colonies on nutrient agar [17].

2.3. Primary Screening of Proteolytic Bacteria

The skimmed milk agar medium was then inoculated with the microbial isolates. The inoculated plates were incubated in the incubator at 37°C for 24 h, and the observed positive appearance of a clear zone around the colony suggested the proteolytic activity [18]. The diameter of each zone was recorded. The microbial colonies that have produced large zones of hydrolysis were, further, selected for secondary screening. Pure bacterial isolates were kept for further research on nutrient agar medium slants at 4°C.

2.4. Secondary Screening of Keratinolytic Bacteria

The bacterial isolates with a clear zone around the inoculum were further used for secondary screening. The isolates were put in a base medium that also contained chicken feathers. The cultures were inoculated, the pH was adjusted to 8.0, and they were incubated at 37°C on a rotary shaker at a speed of 150 rpm [19].

2.5. Characterization of Isolates

The isolated bacteria were identified based on their molecular makeup as determined by 16S rRNA sequencing, Gram staining, biochemical assays, and culture features. The color, shape, size, kind of colony, and pigmentation of the colony were all noted [20]. The bacterial isolates were gram stained and examined in a light microscope with a high power magnifying lens [21]. The bacterial isolates were characterized biochemically by the Indole test, methyl red test, Voges–proskauer’s test, Citrate utilization test, motility test, catalase test, Oxidase test, Nitrate Reductase, Urease, H2S production, and Carbohydrate test [22]. The selective isolates with maximum keratinase producing activity were further characterized for 16S rRNA gene sequencing and phylogenetic analysis.

2.6. Isolation of Bacterial Genomic DNA

In 5 ml of sterile feather basal media, purified bacterial strains were inoculated and allowed to grow overnight. The next day, the cells were collected by centrifugation for 5 min at 10,000 rpm. In 1 mL of extraction buffer (100 mM Tris HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl, 0.07% -mercaptoethanol, and 20 mg/ml lysozyme), the bacterial cell pellets were once again re-dissolved. Following this, 50 μl of 10% SDS was added, carefully mixed, and then incubated for 30 min at 65°C. The samples were spun at 10,000 rpm for 20 min, and the supernatant was then poured into a fresh tube. Each sample was treated with RNase (1–2 μL of 1 mg/mL stock) and, then, incubated at 37°C for an hour. The samples were extracted 3 times using an equal volume of chloroform/tris-saturated phenol (1:1). DNA was precipitated by mixing 2.5 volumes of pure ethanol and 1/10 sample volume of 5 M NaCl. The samples were thoroughly mixed before being centrifuged for 20 min at 12,000 rpm after being incubated at −20°C for 2 h. The DNA pellet was cleaned with 1 mL of 70% ethanol, allowed to air dry, and then dissolved in TE (10 mM Tris HCl, 1mM EDTA) buffer. It was, then, stored at −20°C until it was needed again [23].

2.7. Sequence Analysis of the 16S rRNA Gene

Strain analysis was done through a comparative genomic method using 16S rRNA gene sequencing. Gene sequencing was performed by Shankarnarayan Life Sciences, Ltd. Bengaluru. The primers used for 16S rDNA amplification were fP1 (5’-GGGGGATCCAGAGTTTGATCCTGGCTCA-3’) and rP2 (3’- GGGGGATCCAGAGTTTGATCCTGGCTCA-5’) (Weisburg et al. 1991). The thermocycling settings included a preliminary denaturation stage at 94 °C for 5 min, 33 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min, and a concluding extension step at 72°C for 10 min. MEGA 11 used the Neighbor-Joining (N-J) method to build the phylogenetic tree [24,15].

2.8. Submission of gene sequences

The 16S rDNA sequence of potential keratinase producing bacterial isolates TP1, JP1, VP4, and PP1 were deposited in the GenBank database under the accession number OP748783, OP748782, OP420549, and OP748781 respectively.


3. RESULTS

3.1. Isolation of keratinase producing bacteria from poultry waste soil samples

The poultry waste soil samples were collected from 30 different local poultry farming sites in Himachal Pradesh [Table 2]. For isolation of microbes 10 g of poultry, waste soil sample was transferred to 90 mL of sterilized feather basal medium. 1 mL of soil suspension serially diluted up to 7 times. The diluted sample in 0.1 mL volume from each dilution was transferred to skimmed milk agar plates. After 24 h of incubation, the microbial diversity appears on the skimmed milk agar plates, as shown in Figure 1. A total of 64 well-formed single colonies were selected on the basis of their morphological differences. All the 64 isolates were subjected to primary screening on skimmed milk agar plates. Out of 64 isolates, 17 isolates named KP1, HT2, HT3, JP1, HH1, VP3, VP4, AP2, BP1, NP2, MP1, TP1, SP3, DKP1, LD1, and PP1 demonstrated a clear zone on skimmed milk agar plates after 24 h of incubation at 37°C. The zone size was recorded in mm after 48 h of incubation [Figure 2 and Table 3].

Table 2: Details of bacterial isolates isolated from different soil samples.

S. No.Soil SampleIsolated bacteria on nutrient agarIsolates showing keratinolytic activity on skimmed milk agar
1.KPKP1, KP2, KP3KP1
2.HTHT1, HT2, HT3HT2, HT3
3.HCHC1Not Detected
4.JPJP1, JP2JP1
5.CPCP1Not Detected
6.HHHH1, HH2, HH3HH1
7.HVHV1Not Detected
8.VPVP1, VP2, VP3, VP4VP3, VP4
9.DPDP1, DP2Not Detected
10.SDSD1, SD2Not Detected
11.HRHR1Not Detected
12.APAP1, AP2AP2
13.BPBP1, BP2, BP3BP1, BP3
14.NPNP1, NP2, NP3NP2
15.MPMP1, MP2, MP3MP1
16.TPTP1, TP2, TP3TP1
17.VKVK1, VK2Not Detected
18.PDPPDP1, PDP2Not Detected
19.KPFKPF1, KPF2Not Detected
20.SPSP1, SP2, SP3SP3
21.PHPH 1, PH 2Not Detected
22.DKPDKP1, DKP2, DK3DKP1
23.MSMS1, MS2Not Detected
24.CNPCNP1Not Detected
25.LDLD1, LD2LD1
26.SGSG1, SG2Not Detected
27.PPPP1, PP2PP1
28.SASA1Not Detected
29.ABAB1Not Detected
30.AQGAQG1, AQG2Not Detected
Total isolates6417
Figure 1: Microbial diversity on skimmed milk agar plate.



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Figure 2: Zone of clearance around the bacterial colonies after 48 h of incubation.



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3.2. Secondary Screening

Secondary screening was done to find out the feather degradation capability of selected seventeen isolates. For secondary screening, basal media enriched with feathers were inoculated with KP1, HT2, HT3, JP1, HH1, VP3, VP4, AP2, BP1, BP3, NP2, MP1, TP1, SP3, DKP1, LD1, and PP1. The isolates use feathers as a single carbon and nitrogen source. After 15 days of incubation periods at 37°C, it was found that there was complete degradation of feathers by the isolates [Figure 3 and Table 3].

Figure 3: Feather degradation test for selected keratinolytic bacterial isolates (a) initial stage (b) after 15 days of incubation.



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Table 3: Bacterial isolates showing keratinolytic activities on skimmed milk agar and feather basal media.

S. No.IsolatesDiameter of zone of clearance around the colonies after 48 h on skimmed milk agar (mm)Rate of feather degradation on feather basal broth media (after 15 days)*
1.KP124+++++
2.HT224+++++
3.HT328+++++
4.JP128+++++
5.HH126++++
6.VP332++++
7.VP428+++++
8.AP230++++
9.BP132++++
10.BP330++++
11.NP224++++
12.MP128++++
13.TP120+++++
14.SP314+
15.DKP132++++
16.LD128+++++
17.PP132+++++

* (+) low degradation; (++++) moderate degradation; (+++++) complete degradation

3.3. Biochemical and Morphological Identification of Keratinolytic Bacteria

The characterization of all seventeen keratinase producer microbes was done on the basis of morphological and biochemical tests. Seven isolates belong to Gram-positive, rod-shaped bacteria and ten isolates were identified as Gram-negative, rod-shaped bacteria Table 4. The morphological and biochemical characterization were compared with Bergey’s manual of determinative bacteriology [Table 5].

Table 4: Colony morphology and microscopic characterization of keratinolytic bacterial isolates.

Colony morphology and microscopic characterization

IsolatesColony CharacteristicsGram staining

SizeShapeMarginTextureElevationOpacityColor
KP1LargeIrregularUnevenSmoothRaisedOpaqueWhite+Rod Shaped
HT2LargeIrregularUnevenRoughRaisedOpaqueWhite+Rod Shaped
HT3SmallIrregularEvenSmoothRaisedOpaqueWhite+Rod Shaped
JP1MediumRoundEvenSmoothRaisedOpaqueCream+Rod Shaped
HH1MediumRoundUnevenRoughRaisedOpaqueWhite-Rod Shaped
VP3SmallIrregularEvenSmoothRaisedOpaqueCream-Rod Shaped
VP4MediumIrregularEntireSmoothRaisedOpaqueWhite+Rod Shaped
AP2MediumIrregularUnevenSmoothRaisedOpaqueCream-Rod Shaped
BP1MediumRoundEvenRoughFlatOpaqueWhite-Rod Shaped
BP3LargeRoundUnevenSmoothRaisedOpaqueWhite-Rod Shaped
NP2MediumRoundUnevenSmoothUmbonateOpaqueWhite-Rod Shaped
MP1SmallIrregularUnevenRoughUmbonateOpaqueWhite-Rod Shaped
TP1LargeIrregularUnevenSmoothUmbonateOpaqueWhite+Rod Shaped
SP3MediumRoundUnevenSmoothRaisedOpaqueWhite-Rod Shaped
DKP1LargeRoundUnevenSmoothRaisedOpaqueWhite-Rod Shaped
LD1MediumRoundUnevenSmoothFlatOpaqueWhite-Rod Shaped
PP1LargeRoundUnevenSmoothFlatOpaqueWhite+Rod Shaped

+: Gram-positive; -: Gram-negative

Table 5: Biochemical characterization of keratinolytic bacteria.

IsolatesIndole testMethyl Red testVogas Proskaur testCitrate TestMotility TestCatalase TestOxidase TestNitrate ReductaseUreaseH2S ProductionDextrose fermentationLactose fermentationSucrose fermentation
KP1-+--+--+--+--
HT2++--+--+--+--
HT3++--+-+++-+--
JP1--++++-+--+--
HH1++--+--+--+--
VP3-+--+--+--+--
VP4++-++-++-----
AP2-+--+--+-----
BP1++--+--+--+--
BP3++--+--+--+--
NP2-+--+-+++-+--
MP1-+-+++++-----
TP1--++++-++----
SP3+---++++-----
DKP1+++-+--+-----
LD1-+--+--+-----
PP1--++++-+--+--

+: Positive, -: Negative

3.4. Molecular Identification of Keratinase Producing Bacteria

The bacterial isolates, namely, JP1, TP1, VP4, and PP1 exhibited maximum keratinase producing activity and were selected for molecular characterization through 16S rRNA gene sequencing and phylogenetic analysis [Figures 4 and 5]. Using the Bioedit tool, the forward and reverse primer results were combined to extract the entire aligned sequences. The NCBI BLAST was performed to confirm the identity of specimens [25]. Utilizing BLAST, the 16S rRNA gene sequence was compared to the Gen-bank database. The isolates TP1, JP1, VP4, and PP1 displayed high levels of sequence similarity with members of the genus Bacillus in their 16S rRNA gene sequences. The NCBI database was queried using the “BLASTn” tool to assign sequences, and the highest-scoring hit from each query was used to identify the bacterial species. A phylogenetic tree was constructed using MEGA 11 [Figure 5]. The bacterial strains JP1, TP1, and PP1 were identified as Bacillus cereus, while strain VP4 was identified as Bacillus thuringiensis.

Figure 4: Polymerase chain reaction amplification of 16S rDNA fragment from bacterial samples.



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Figure 5: Phylogenic tree of strain VP4, JP1, TP1, and PP1 constructed by Neighbor-Joining method derived from analysis of the 16S rRNA gene sequences and related sequences obtained from NCBI. Scale bar, 0.050 substitutions per nucleotide.



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4. DISCUSSION

Feathers have more than 90% protein. Due to insoluble nature of keratinous materials such as feather, hair, and wool, they are resistant to degradation by common microbial proteases. Thus, the annual production of several million tons of feathers by the chicken sector causes troublesome environmental contamination and waste of protein-rich reserve [10,26].

It has been observed that keratinase enzymes are a powerful enzyme for breaking down feathers, hair, and poultry waste. In this current research work, a total of 64 bacterial isolates were isolated from poultry waste soil samples from 30 different poultry farming industries in Himachal Pradesh, India. Another author covered the isolation of the Bacillus spp. from the wastewater of a poultry processing plant, as well as its growing conditions and keratinolytic activity [8].

Seventeen isolates from poultry waste has been shown to degrade feather keratin. Seventeen isolates exhibited the zone of clearance around the colonies on skimmed milk agar plates. On the basis of the zone of clearance, the isolates were grown in basal media, in which chicken feathers were used as a source of carbon and nitrogen. Although these 17 isolated strains have shown the significant property to degrade the feathers which are sophisticated to achieve. Isolates KP1, HT2, HT3, JP1, HH1, VP3, VP4, AP2, BP1, NP2, MP1, TP1, SP3, DKP1, LD1, and PP1 completely degraded the chicken feathers. However, in comparison to other isolates, strain SP3 has demonstrated dissimilar characteristics in the same feather basal medium as it has taken longer time for feather degradation. The degradation rate has been introduced by many authors as an indicator evidently correlated to the keratinase activity of a microbial strain [27].

After biochemical tests, these microorganisms were confirmed to belong to the Bacillus and Pseudomonas species. The previous studies have documented the isolation of keratinase-producing strains of Bacillus licheniformis, B. subtilis, B. pumilis, B. cereus, B. halodurans, B. pseudoformis [28], and Pseudomonas sp. [29]. In another study, Arthrobacter sp. was also reported to produce keratinase [30]. Arthrobacter creatinolyticus KP015744 was observed to be a better keratinase producer microorganism [31]. All biochemical tests for results are equivalent to those reported by various authors who proved that Gram-positive bacteria, inclusive of Bacillus, can hydrolyze keratin [5]. Likewise, species of Bacillus are considered the best producers of keratinase. Bacillus sp. has been identified as a potential keratinolytic organism in the previous research for the isolation of keratinolytic organisms from soil and other natural sources [32]. These studies suggest that this organism may be used in field studies for the biodegradation of feathers in feather processing units. Keratinases also have applications in personal care products to form skin whitening and bleaching creams [33].

The 16S rRNA sequencing results of these isolates demonstrate high levels of sequence similarity with genus Bacillus. For phylogenetic analysis, small subunit of rRNA is used that is extremely faithful method to determinate the taxonomic relationships between microorganisms [34]. According to both biochemical characterization and 16S rRNA gene sequence analysis, the JP1, TP1, PP1, and VP4 strains were identified as B. cereus and B. thuringenesis, respectively.


5. CONCLUSION

These keratinolytic isolates have the potential to degrade the feather keratin, which can be used to make animal feed proteins, can be used to make slow nitrogen-releasing fertilizers, and can also overcome the problems regarding waste management, because poultry waste causes environmental pollution. Therefore, the microorganisms isolated in this study exhibited potential for biotechnological uses.


6. ACKNOWLEDGMENT

I am grateful to Dr. Vijay Kumar, Assistant Professor, Career Point University, Hamirpur, for giving the possibility to complete this research work. I would like to give special thanks to Kehloor Biosciences and Research Center, Ghumarwin, India for their assistance in the practical work.


7. AUTHORS’ CONTRIBUTIONS

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Richa and Vijay Kumar. The first draft of the manuscript was written by Richa and second author commented on previous versions of the manuscript. All authors read and approved the final manuscript.


8. FUNDING

The authors received no financial support for this research work.


9. CONFLICTS OF INTEREST

The authors report no financial or any other conflicts of interest in this work.


10. ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.


11. DATA AVAILABILITY

Most of the data supporting our findings included in the manuscript.


12. PUBLISHER’S NOTE

This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

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21.  Sen SK, Raut S, Satpathy S, Rout PR, Bandyopadhyay B, Mohapatra PK. Characterizing novel thermophilic amylase producing bacteria from Taptapani hot spring, Odisha, India. Jundishapur J Microbiol 2014;7:e11800. [CrossRef]

22.  Uddin ME, Maitra P, Faruquee HM, Alam MF. Isolation and characterization of proteases enzyme from locally isolated Bacillus sp. Am J Life Sci 2014;2:338-44. [CrossRef]

23.  Allpress JD, Mountain G, Gowland PC. Production, purification and characterization of an extracellular keratinase from Lysobacter NCIMB 9497. Lett Appl Microbiol 2002;34:337-42. [CrossRef]

24.  Saitou N, Nei M. The neighbor-joining method:A new method for reconstructing Phylogenetic trees. Mol Biol Evol 1987;4:406-25.

25.  Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-10. [CrossRef]

26.  Onifade AA, Al-Sane NA, Al-Musallam AA, Al-Zarban S. A review:Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresource Technol 1998;66:1-11. [CrossRef]

27.  Veenayohini K, Sangeetha D. Isolation and identification of keratinolytic bacteria from poultry waste and assessment of its keratinase activity on chicken feathers. Int J Appl Res 2016;2:396-402.

28.  Gessesse A, Hatti-Kaul R, Gashe BA, Mattiasson BO. Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb Technol 2003;32:519-24. [CrossRef]

29.  Riffel A, Brandelli A. Keratinolytic bacteria isolated from feather waste. Braz J Microbiol 2006;37:395-9. [CrossRef]

30.  Pereira JQ, Lopes FC, Petry MV, Medina LF, Brandelli A. Isolation of three novel Antarctic psychrotolerant feather-degrading bacteria and partial purifcation of keratinolytic enzyme from Lysobacter sp. A03. Int Biodeterior Biodegrad 2014;88:1-7. [CrossRef]

31.  Kate S, Pethe A. Study of efficiency of keratinase production by Arthrobacter creatinolyticus KP015744 isolated from leather sample. Int J Adv Res 2014;11:992-9.

32.  Mazotto AM, de Melo AC, Macrae A, Rosado AS, Peixoto R, Cedrola SM, et al. Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp. World J Microbiol Biotechnol 2011;27:1355-65. [CrossRef]

33.  Gupta R, Rajput R, Sharma R, Gupta N. Biotechnological applications and prospective market of microbial keratinases. Appl Microbiol Biotechnol 2013;97:9931-40. [CrossRef]

34.  Iwamoto K, Yoshikawa T, Sakata T, Maeda H. Phylogenetic analysis of marine algicidal filamentous bacteria inferred from SSU rDNA and intergenic spacer regions. Microbes Environ 2001;16:213-21. https://doi.org/10.1264/jsme2.2001.213

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2. Lateef A, Oloke JK, Kana EG, Sobowale BO, Ajao SO, Bello BY. Keratinolytic activities of a new feather-degrading isolate of Bacillus cereus LAU 08 isolated from Nigerian soil. Int Biodeterior Biodegradation 2010;64:162-5. https://doi.org/10.1016/j.ibiod.2009.12.007

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10. Gousterova A, Braikova D, Goshev I, Christov P, Tishinov K, Tonkova VE, et al. Degradation of keratin and collagen containing wastes by newly isolated thermo actinomycetes or by alkaline hydrolysis. Lett Appl Microbiol 2005;40:335-40. https://doi.org/10.1111/j.1472-765X.2005.01692.x

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15. Prasad VH, Kumar G, Kartik L, Rao KV. Screening of extracellular keratinase producing bacteria from feather processing areas in Vellore, Tamil Nadu, India. J Sci Res 2010;2:559. https://doi.org/10.3329/jsr.v2i3.4567

16. Jayasudha J, Kumar G, Karthik L, Rao KV. Biological control of vibriosis by antagonistic actinobacteria-an in vitro study. J Agric Technol 2011;7:271-80.

17. Sunita JV, Dolly PR, Bateja S, Vivek NU. Isolation and screening for hydrocarbon utilizing bacteria (HUB) from petroleum samples. In J Curr Microbiol Appl Sci 2013;2:48-60.

18. Jeong JH, Park KH, Oh DJ, Hwang DY, Kim HS, Lee CY, et al. Keratinolytic enzyme-mediated biodegradation of recalcitrant feather by a newly isolated Xanthomonas sp. P5. Polym Degrad Stab 2010;95:1969-77. https://doi.org/10.1016/j.polymdegradstab.2010.07.020

19. Al Hasan MM. Isolation and Identification of Extracellular Keratinase Producing Bacteria from Soil. Bangladesh: BRAC University; 2018.

20. Ashwini K, Gaurav K, Karthik L, Rao KV. Optimization, production and partial purification of extracellular α-amylase from Bacillus sp. Marini Arch Appl Sci Res 2011;3:33-42.

21. Sen SK, Raut S, Satpathy S, Rout PR, Bandyopadhyay B, Mohapatra PK. Characterizing novel thermophilic amylase producing bacteria from Taptapani hot spring, Odisha, India. Jundishapur J Microbiol 2014;7:e11800. https://doi.org/10.5812/jjm.11800

22. Uddin ME, Maitra P, Faruquee HM, Alam MF. Isolation and characterization of proteases enzyme from locally isolated Bacillus sp. Am J Life Sci 2014;2:338-44. https://doi.org/10.11648/j.ajls.20140206.12

23. Allpress JD, Mountain G, Gowland PC. Production, purification and characterization of an extracellular keratinase from Lysobacter NCIMB 9497. Lett Appl Microbiol 2002;34:337-42. https://doi.org/10.1046/j.1472-765X.2002.01093.x

24. Saitou N, Nei M. The neighbor-joining method: A new method forreconstructing Phylogenetic trees. Mol Biol Evol 1987;4:406-25.

25. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-10. https://doi.org/10.1016/S0022-2836(05)80360-2

26. Onifade AA, Al-Sane NA, Al-Musallam AA, Al-Zarban S. A review: Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresource Technol 1998;66:1-11. https://doi.org/10.1016/S0960-8524(98)00033-9

27. Veenayohini K, Sangeetha D. Isolation and identification of keratinolytic bacteria from poultry waste and assessment of its keratinase activity on chicken feathers. Int J Appl Res 2016;2:396-402.

28. Gessesse A, Hatti-Kaul R, Gashe BA, Mattiasson BO. Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb Technol 2003;32:519-24. https://doi.org/10.1016/S0141-0229(02)00324-1

29. Riffel A, Brandelli A. Keratinolytic bacteria isolated from feather waste. Braz J Microbiol 2006;37:395-9. https://doi.org/10.1590/S1517-83822006000300036

30. Pereira JQ, Lopes FC, Petry MV, Medina LF, Brandelli A. Isolation of three novel Antarctic psychrotolerant feather-degrading bacteria and partial purifcation of keratinolytic enzyme from Lysobacter sp. A03. Int Biodeterior Biodegrad 2014;88:1-7. https://doi.org/10.1016/j.ibiod.2013.11.012

31. Kate S, Pethe A. Study of efficiency of keratinase production by Arthrobacter creatinolyticus KP015744 isolated from leather sample. Int J Adv Res 2014;11:992-9.

32. Mazotto AM, de Melo AC, Macrae A, Rosado AS, Peixoto R, Cedrola SM, et al. Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp. World J Microbiol Biotechnol 2011;27:1355-65. https://doi.org/10.1007/s11274-010-0586-1

33. Gupta R, Rajput R, Sharma R, Gupta N. Biotechnological applications and prospective market of microbial keratinases. Appl Microbiol Biotechnol 2013;97:9931-40. https://doi.org/10.1007/s00253-013-5292-0

34. Iwamoto K, Yoshikawa T, Sakata T, Maeda H. Phylogenetic analysis of marine algicidal filamentous bacteria inferred from SSU rDNA and intergenic spacer regions. Microbes Environ 2001;16:213-21. https://doi.org/10.1264/jsme2.2001.213

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