Collection of samples
The soil samples were collected from waste disposal sites of Hazaribagh tannery (23.7361 N, 90.3631 E), Dhaka, in the month of September, 2018. A total of six samples were collected from two different waste disposal sites, three from each site. Collected samples were taken in a sterile ziplock bag and transported directly to the laboratory.
Isolation of bacteria
Isolation of soil bacteria was performed by spread plate method. One gram of soil from each of the six samples was poured into 9 ml of sterile distilled water and homogenized by vortexing. Serial dilutions (10−1–10−5) was performed using deionized distil water, and 100 μl of diluted samples was plated in nutrient agar using an L-shaped glass rod. The plates were incubated overnight at 37 °C followed by picking of single colony and sub-culturing on nutrient agar.
Screening for the keratinase-producing bacteria
Screening on skim milk agar
Skim milk agar (SMA) (Himedia, India) medium was used for the screening of keratinolytic bacteria . A total of 50 single colonies were cultured in SMA and incubated at 37 °C for 48 h. Trichloroacetic acid (10%) was poured in the media to visualize the zone of clearance. The bacteria that showed a clear zone in the plate were primarily considered as positive for keratinolytic activity.
Screening on casein agar
Casein agar medium (Himedia, India) was used for screening keratinolytic bacteria according to the method described earlier . A total of 50 single colonies were streaked on the casein agar plate and incubated at 37 °C for 48 h. Proteolytic activity was confirmed by a zone of clearance in the agar plate.
Screening on feather meal agar
The feather meal was prepared from native chicken feathers as described earlier . Briefly, finely pieced feather was defatted in chloroform to methanol (1:1 v/v) for 48 h, followed by chloroform to acetone to methanol (4:1:3 v/v/v) for another 48 h, and then rinsed in sterile water, dried for 24 h at 37 °C, and ground in a mortar-pestle to obtain a powdered feather meal. This agar medium was used for the confirmation of keratinase-producing bacteria. The composition and concentration of feather agar plate was maintained according to the method described earlier (1.0% feather meal, 0.05% of NH4Cl and NaCl, 0.04% K2HPO4, 0.03% KH2PO4, 0.01% of MgCl2, 0.01% yeast extract, pH 7.5) . A total of 16 single bacterial colonies were transferred to a feather meal agar after checking the activity on skim milk agar medium and casein agar. The culture plates were was incubated at 37 °C for 48 h.
Identification of potent keratinolytic bacterial isolates
The identification of prospective keratinolytic bacteria was carried out by morphological and biochemical tests according to Bergey’s Manual Systematic Bacteriology  as well as 16S rRNA amplification using universal primers.
The primary identification of 50 bacterial colonies was performed based on Gram’s test, Gram staining, catalase, oxidase, indole production, nitrate reduction, gelatin and starch hydrolysis, salt tolerance, citrate utilization, and growth on MacConkey agar.
DNA extraction and PCR amplification
Bacterial DNA from overnight culture was extracted using phenol-chloroform isoamyl alcohol (25:24:1) . Extracted DNA was stored at – 20 °C until further use. PCR amplification of bacterial 16S rRNA was carried out with 27F (5′-AGA GTT TGA TCC TGG CTG AG-3′) and 1492R (5′-GGC TAC CTT GTT ACG ACT T-3′) universal primers. PCR master mix was prepared as 25 μl final volume with 12.5 μl 2X master mix (Thermo Fisher Scientific, USA), 1 μl of each forward and reverse primers, 2 μl of template DNA, and 8.5 μl of nuclease-free water. A total of 40 cycles of amplification reactions was carried out in an ASTEC thermal cycler (Gene Atlas, Japan). The amplification conditions was maintained as follows: initial denaturation at 95 °C for 30 s, denaturation 95 °C for 1 min, annealing at 56 °C for 30 s, extension at 72 °C for 1.5 min, and a final extension at 72 °C for 10 min. PCR products were separated in 1.5% agarose gel with 1-kb DNA ladder (Thermo Fisher Scientific, USA) and visualized under gel documentation system.
The initial quality of Sanger sequence was checked using FastQC pipeline. Editing and removing of low-quality bases and de-novo assembly was performed using Geneious (vR11.1) bioinformatics software. ClustalW Multiple sequence alignment (MSA) and neighbor-joining phylogenetic tree contraction were performed in MEGA 7.0 with 1000 bootstrap replications .
Inoculum preparation and fermentation
Pure cultures of eight identified potent keratinolytic bacteria were used as inoculant for shake flask fermentation of keratinase where each sample was run in triplicate. First, bacterial inoculum was prepared in nutrient broth culture by incubating overnight at 37 °C in a rotary shaking incubator. Submerged fermentation was carried out in an Erlenmeyer shake flask fermenter. Basal modified medium was used for keratinase production containing feather meal powder (10 g/l), NH4Cl (1.0 g/l), NaCl (1.0 g/l), KH2PO4 (0.8 g/l), K2HPO4 (0.6 g/l), MgCl2.6H2O (0.5 g/l), yeast extract (0.2 g/l), and pH (7.5) with 5% bacterial inoculum . The fermented organisms were cultivated in 250-ml cotton-plugged Erlenmeyer flasks after 72 h shaking at 150 rpm. After incubation, fermented broth was centrifuged at 8000 rpm for 15 min at 4 °C. Cell-free supernatant was collected and preserved for the estimation of keratinase activity as described previously .
Assay for keratinase activity
Keratinase activity was assayed with keratin azure (Sigma Aldrich, Germany) as a substrate . Briefly, the reaction mixture containing 1.0 ml enzyme preparation was transferred into 1.0 ml 50 mM Tris-HCl buffer suspended into 5 g keratin azure powder (pH 8.0). The mixture was incubated for 30 min at 50 °C, and the reaction was then stopped by the addition of 2 ml of 10% (w/v) trichloroacetic acid (TCA). After centrifugation at 1500 rpm for 30 min, the supernatant was evaluated for the release of azo dye at 595 nm using spectrophotometer (Thermo Fisher Scientific, USA). Enzyme control (1.0 ml buffer + keratin azure suspension) was also prepared along with enzyme sample and incubated for 30 min at 50 °C, followed by addition of 2.0 ml TCA + 1.0 ml enzyme solution. One unit (U/ml) of keratinolytic activity was defined as an increase of corrected absorbance (0.01) at 595 nm, in relation to control using the formula:
$$ U=4\times n\times A595/\left(0.01\times 10\right) $$
where n is the dilution rate, 4 is the final reaction volume (ml), and 10 is the incubation time (min). Observations were obtained in triplicate for each sample.
Selection of best conditions for keratinase activity
The selection screening was done using the basal modified medium and following previously described methods . Fermentation was carried out at 37 °C for 72 h with 150 rpm. The pH and volume of the medium were maintained at 7.5 and 50 ml, respectively. Three replicates were used for each experiment.
Effect of chicken feather concentrations
Various concentrations of feather meal were used in production media to identify the best concentration for maximum production of enzyme. The concentrations of feather meal were set to 0.25%, 0.5%, 1%, 1.5%, and 2% (w/v).
Effect of organic nitrogen sources
In the screening of various organic nitrogen sources, yeast extract (0.02%) in the basal media was replaced with tryptone, peptone, beef extract, and gelatin at the same concentration, individually.
Effect of inorganic nitrogen sources
In the screening of various inorganic nitrogen sources, NH4Cl (0.1%) in the basal media was replaced with NH4NO3, KNO3, NH4H2PO4, NH4SO4, and NaNO3 at the same concentration, individually.
Effect of carbon supplements
Additional 1% (w/v) carbon supplements were used in the fermentation process to find out the favorable carbon supplement. The supplements were starch, glucose, fructose, carboxymethyl cellulose (CMC), lactose, and sucrose.
The culture conditions and replicates were maintained as described earlier in optimization for production medium . Three replicates were used for each experiment.
Effect of incubation temperature and period
The effect of temperature on enzyme production was carried out by fermentation at different incubation temperatures from 25 to 45 °C (25, 30, 35, 40, and 45 °C). The effect of incubation period on the production of keratinase was investigated by fermenting the medium from 24 to 96 h at 37 °C (24, 48, 72, and 96 h). The sample was collected in every 24 h to observe the changes in keratinase production. The culture conditions were maintained as described earlier in optimization for production medium.
Effect of culture volume
The effects of different volumes of inoculum was investigated using 2%, 3%, 4%, 5%, 6%, and 7% inoculum in fermentation for the production of keratinase. The culture conditions were maintained as described earlier in optimization for production medium.
Dehairing of goat skin
Goat skin was collected from a local slaughter house followed by washing with deionized distill water. The skin was treated with salt to protect the skin from rotting and dried in the presence of sunlight. Skin pieces (1 in. × 1 in.) were incubated in 20 ml crude keratinase at 37 °C for 72 h. Besides, a control dehairing was performed with physiological phosphate buffered saline (PBS) at the same conditions.
One-way ANOVA with Tukey’s post hoc test was used to analyze all numerical data for this study. R studio was used for data analysis and plotting. In all cases, a P value of less than 0.05 was considered statistically significant.