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.

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.

3.1. 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.

Figure 1: Microbial diversity on skimmed milk agar plate. [Click here to view]

Figure 2: Zone of clearance around the bacterial colonies after 48 h of incubation. [Click here to view]

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. [Click here to view]

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

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.

Table 5: Biochemical characterization of keratinolytic bacteria.

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. [Click here to view]

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. [Click here to view]