1 Introduction

Keratin biomass is one of the most complicated biological materials. It is responsible for the majority of skin appendages such as hair, nails, tortoiseshells, horns, beaks, claws, and feathers [1]. For instance, millions of tonnes of keratinous materials are produced each year as waste or byproducts of animal meat manufacturing [2, 3]. According to a survey by the US Department of Agriculture, the output of meat produced in 2020 was about 100.5 million tonnes which had the parallel result of producing more than 4.7 million tonnes of poultry feathers around the world [4]. According to the United Nations of Food and Agriculture Organization (FAO), more than 24.8 billion chickens will be produced annually in 2030 over the world, and 37.0 billion in 2050 [5]. Keratin is introduced as the third most abundant polymer in nature, following cellulose and chitin [6].

Disulfide bridges produced between cysteine residues in keratin polypeptides are primarily responsible for the great stability and tight conformation of keratin [7]. Improper disposal and management of this “difficult to decompose” keratinous waste is one of the most pressing problems facing industries [8]. Because the excessive dumping rate of these wastes leads to nitrate intrusion into groundwater and phosphorus runoff into adjacent water bodies, it also emits different pollutants including hydrogen sulfide, nitrous oxide, and ammonia, which have negative health and environmental consequences. Furthermore, feather waste can be a habitat for pathogenic organisms such as Vibrio and Salmonella, as well as promote the growth of bacterial or viral pathogens in lakes and streams [9,10,11].

Chicken feathers are the most significant abundant byproduct, which consists of keratin (~ 91%), moisture (~ 8%), and lipid (~ 1%), in addition to representing 5–7% of the chicken body weight [12, 13]. Keratinous compound materials are unique materials because they are very abundant in certain amino acids, especially the sulfur-containing amino acid, cysteine, and other amino acids like proline, arginine, glycine, and the essential amino acids threonine, valine, and leucine [14, 15]. In addition, they have high mineral contents like N, P, K, Fe, Mg, Ca, Mn, Zn, and Cu [14].

The process of dissolving and extracting keratin is complicated in comparison with extracting other natural polymers such as collagen, chitosan, and starch. Therefore, the widespread use of keratin relies on cost-effective and fast extraction hydrolysis methods. The current extraction strategies include physical, chemical, and biological methods [16]. The generation of hydrolyzed keratin by physical or chemical treatment requires a high temperature. In this way, amino acids (e.g., tryptophan, lysine, and methionine) that are sensitive to heat could be destroyed. After that, the dietary value of hydrolysates would be reduced, resulting in low solubility and value [17].

Furthermore, these high-temperatures, high-pressure treatments produce a significant amount of sulfur and ammonia waste gasses, making physical or chemical treatments inefficient and polluting [18]. Therefore, biological degradation with the keratinase represents a promising alternative technique and is eco-friendly for recycling keratinous wastes, especially poultry feathers, leading to the formation of highly nutritious value products [19]. Keratinase enzyme is naturally produced by a variety of microorganisms including bacteria (e.g., Bacillus licheniformis, B. subtilis, B. paralicheniformis, Microbacterium sp., and Pseudomonas sp.), fungi (e.g., Fusarium, Aspergillus, Trichoderma, Penicillium, and Doratomyces), yeasts (e.g., Trichosporon and Candida sp.), and actinomycetes (e.g., Thermoactinomyces candidus and Streptomyces sp.) [20, 21].

Microbial keratinases have been accepted for numerous potential applications in the medical, biotechnological, and industrial fields. It has been found that using keratinase to degrade feathers produces beneficial feather hydrolysates (which contain peptides and free amino acids) that can be used as a highly protein-rich meal for animals and as a nitrogenous fertilizer for plants [9]. Keratinase has been widely employed in the sector of detergents, after their introduction as a detergent ingredient in 1914; their use in detergent composition accounts for a large part of their total sales (89%) [22]. Tanneries and leather manufacturing have accepted the use of keratinase because it is an active enzyme for dehairing techniques and can be an effective replacement for harmful chemicals [13, 23, 24]. Furthermore, advanced biodegradation technologies for chicken feathers may provide a cost-effective, more efficient, and environmentally friendly method for processing feathers. Microbial digestion of keratinous biomass has become one of the preferable approaches for the sustainable conversion of cheap and readily available keratin-rich agricultural wastes into valuable products [25, 26].

Therefore, the present study aimed to isolate effective keratinolytic bacteria showing rapid feather degradation from keratin waste dump sites. It also aims to optimize keratinase activity using Plackett–Burman methodology and investigate its potential applications in various fields as an effective thermostable enzyme. The current research provides significant implications for the enzyme in biofertilizers in agriculture, in addition to the detergent and leather industries.

2 Materials and methods

2.1 Keratin-substrate

Chicken feathers collected from a local poultry manufacturer were used as a keratin substrate [27]. The feathers were washed properly with tap water to remove impurities and blood residue, and then washed with a detergent; after that, it was washed with distilled water and dried at 60 °C for 24 h. The dried feathers were crushed into small fragments using a blender; fine feather powder was obtained by sieving with a pore size of 160 µm and then stored in the refrigerator in an airtight container for use as a substrate for enzyme assay. The grinded feathers were used for fermented media (Figure S1).

2.2 Samples collection and bacterial isolation

Samples of soil were collected from a chicken farm’s waste and a leather storehouse in Hosh Essa, EL Beheira, Egypt. 0.1 g of each sample was vortexed in 10 ml sterile distilled water; 1 ml of soil suspension was cultivated in 25-ml Erlenmeyer flask containing 50-ml chicken feather medium (CFM) [28], composed of (g/L): K2HPO4, 3.0; KH2PO4, 1.0; MgSO4, 0.2; pH 7; supplemented with the feather, 10 (chicken feather cut into small pieces) as sole carbon and nitrogen source and incubated in shaking incubator at 200 rpm for a week at 30 °C for enrichment. From the flask that showing turbidity and feather disintegration, 1 ml was withdrawn and mixed with 9 ml of sterilized saline solution. Then, 1 ml of the supernatant was spread on the CFM agar plates and incubated at 30 °C for 4 days. Colonies that showed different growth and morphology were selected and purified by repeated screening on Luria–Bertani medium (LB) [29] and preserved on slants of the same medium at 4 °C for further study.

2.3 Screening for proteolytic activity by bacterial isolates

The capacity of selected isolates to hydrolyze casein in skimmed milk (SM) was performed according to the method of Riffel and Brandelli [30] with some modifications. Proteolytic capabilities were investigated on CFM agar using 50 ml pasteurized skimmed milk (containing solid protein 0.03 g/ml) instead of feather. The plates were cultivated with bacterial isolates using toothpicks and then incubated at 37 °C for 48 h, measuring the halo zone formed around the colony every 6 h, then determining the clear zone index by dividing the clear zone diameter by the colony diameter.

2.4 Keratin degradation by bacterial isolates

Bacterial isolates were grown in a submerged fermentation performed in 100-ml Erlenmeyer flasks with 20-ml chicken feather medium (CFM) pH 7.0; each flask inoculated with 2% (v/v) of bacterial suspension (OD600 ≈ 1.6) was used as a fresh inoculum from 24 h slant [27]. Cultivated flasks were incubated in an orbital shaker (200 rpm) for 48 h at 37 °C. pH was measured immediately after the completion of the fermentation using a pH meter (MARTINI Mi 150 pH). The fermentation broth was sampled for analysis of protein concentration and percentage of feather degradation by the standard assay method.

The cultures were filtered via pre-weighed filter paper Whatman no. 1 to recover unutilized feathers. The residues were then cleaned using distilled water to eliminate bacterial cells, and the remaining chicken feathers were oven dried at 70 °C for 24 h to reach a constant weight, which was utilized to calculate the degradation percentage [31]. The following equation was used to estimate the degradation percentage:

$$\mathrm{Feather}\;\mathrm{degradation}\;\left(\%\right)=\left(m_i-m_{\mathrm f}\right)/m_i\times100$$

where mi is the dry feather’s initial weight before fermentation, and mf is the feather’s weight after fermentation. While the filtrates were centrifuged for 10 min at 4000 rpm, and the clear supernatant was used for determining the protein concentration [32]. The isolate L10 exhibiting the highest feather degradation and extacellular protein content was selected as a promising isolate in the present study.

2.5 Molecular identification of keratin-degrading bacteria

The isolate L10 was molecularly identified by sequencing the partially amplified 16S rRNA gene, using polymerase chain reaction (PCR). The 16S rRNA gene was amplified using the forward primer 5′-AGAGTTTGATCCTGGCTCAG-3′ and reverse primer 5′-GGTTACCTTGTTACGACTT-3′. The PCR mixture contained 8 μL DNA templates, 1 µl forward primer (20 Pico mol), 1 µl reverse primer (20 Pico mol), 25 µl My Taq Red Mix, and 15 μl nuclease-free water. Steps of the thermal cycler condition were carried out with 94 °C initial denaturation for 6 min, followed by 35 cycles of 94 °C denaturation for 45 s, 56 °C annealing for 45 s, 72 °C extension for 1 min, and 72 °C final extension for 5 min. The sequencing of the 16S rRNA gene was then carried out by Sigma Company (St. Nazeeh Khalifa, Heliopolis, Cairo, Egypt). The nucleotide sequences were compared to reference sequences in the database using the basic local alignment search tool (BLASTn) software system from the National Center for Biotechnology Information (NCBI) [33].

2.6 Significant factors that affected the keratinase production by Plackett–Burman design

Plackett–Burman design (PBD) was used to screen key components in terms of their main effects and significance [34]. B. cereus L10 keratinase production was optimized by screening ten factors with two levels, namely, feather (X1), K2HPO4 (X2), KH2PO4 (X3), MgSO4 7H2O (X4), NaCl (X5), yeast extract (X6), incubation time (X7), inoculum size (X8), pH (X9), and agitation (X10). The levels of each factor are listed in Table 1 with the coded and uncoded form of high (+ 1) and low (− 1) values by using the statistical software Minitab 16 (Stat-Ease, Minneapolis, MN, USA). Keratinase activity (U/ml) was evaluated as a response.

Table 1 Actual and coded values of factors for screening at two levels with Plackett–Burman design for keratinase production
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2.7 Keratinase assay

The keratinase activity was measured by using tiny chicken feather powder as a substrate. The reaction mixtures containing feather powder (4 mg) suspended in 750 µl of tris–HCl buffer 0.05 M (pH 8), and 750 µl of culture supernatant was incubated for 30 min in a water bath at 50 °C. The enzyme reaction was stopped by placing the reaction mixture in ice for 15 min, followed by cooling centrifugation at 5000 rpm for 15 min. Afterward, the resulting supernatant was used to measure the amount of amino acids released by using Lowry et al. (1951) with tyrosine as a standard against a control having only the substrate and inactived enzyme. The amount of enzyme required to release 1 µg of tyrosine per minute per ml of the enzyme was defined as a unit of keratinase activity (U) [35]. All assays were performed in triplicate. The following equation was used to calculate keratinase activity:

$$\mathrm{Keratinase}\;\mathrm{activity}\;(\mathrm U/\mathrm{ml})=\frac{\lbrack\mathrm{Tyrosine}\;\mathrm{concentration}\;(\mathrm\mu\mathrm g/\mathrm{ml})\times\mathrm{Reaction}\;\mathrm{volume}\;(\mathrm{ml})\rbrack}{\lbrack\mathrm{Incubation}\;\mathrm{time}\;(\min)\times\mathrm{Enzyme}\;\mathrm{volume}\;(\mathrm{ml})\rbrack}$$

2.8 Validation of the predicted optimal conditions

The optimized feather medium (OFM) and culture conditions obtained from Plackett–Burman design was inoculated with 4% (v/v, O.D600 ≈ 1.7) B. cereus L10 of 24 h seed culture grown in nutrient broth and incubated with shaking (200 rpm) at 37 °C for 168 h. Aliquots were withdrawn from the fermentation medium periodically at 12 h intervals for estimation keratinase activity, protein production, and residual feathers remaining to determine the optimum time for maximum enzyme production.

2.9 Degradation of different keratinous sources by B. cereus L10

The ability of B. cereus L10 to degrade different keratinous substrates (duck feather, turkey feather, horn and hoof of cows, sheep wool, nails, and human hairs) was investigated. All of the substrates were cleaned as described previously and cut into small pieces ≈ 1 cm. An optimized medium of 20 ml pH 7.0 in a 100-ml Erlenmeyer flask supplemented separately with 1% of each keratinous substrate autoclaved and inoculated with 4% (v/v, O.D600 ≈ 1.6) of bacterial inoculum. Fermentation was carried out at 37 °C, 200 rpm for 120 h. The culture was inspected for visible degradation of keratinous substrates against control a sterile optimized medium containing keratinous substrates. The samples were collected, centrifuged, and subjected to extracellular keratinase activity and extracellular protein content.

2.10 Thermal stability of keratinase of B.cereus strain L10

The influence of temperature on enzyme stability was performed by incubating the enzyme solution over a range of temperatures (10 to 100 °C) for 1 h; it was possible to determine how temperature affected the stability of the enzyme [36]. A standard enzyme assay was used to determine the residual enzyme activity. The enzyme activity at room temperature (20 °C) was considered 100%.

2.11 Application

2.11.1 Plant growth promoting activities B. cereus L10

Production of plant growth promoting hormone indole acetic acid (IAA) by B. cereus L10 grown on optimal feather medium was investigated. IAA in the supernatants was estimated by mixing 2 ml of Salkowski reagent (0.5 M FeCl3, 35% H2SO4; (1:49)) with 1 ml of the culture supernatant; development of a pink color was indicative of IAA production [37].

2.11.2 Feather hydrolysate as biofertilizer

Feather hydrolysate has been applied for its effect on the germination of wheat seeds and the promotion of growth [38]. The experiment was carried out in plastic cups with 300 g of sandy soil washed with HCl and dried, then planted with 50 seeds of wheat (Triticum aestivum). The addition of 60 ml diluted feather hydrolysate in the ratio of 1:4 along with water for seed germination and frequently irrigating with 30 ml diluted hydrolysate requires for plant growth in the test, and the control group received the same amount of pure water sprayed on the plants instead of feather hydrolysate. All seeded cups were incubated in laboratory under ambient conditions for 10 days. Thereafter, germinated seeds were counted to calculate the germination percentage. Lengths of seedlings root and shoot and fresh weight and the number of lateral roots were also recorded. The dry weight was recorded after drying at 60 °C to a constant weight.

2.11.3 The cleaning properties of B. cereus L10 keratinase

Evaluation of the feasibility and compatibility of B. cereus L10 keratinase as a laundry detergent additive elucidated according to the procedure of Paul and Emran [22, 39]. Cotton fabrics and medical aprons (surgical cloths made from polyester fabric) pieces (5 cm2) were stained with 0.1 ml of fresh blood and a milk product with chocolate flavor (Nesquik, Nestle, Borg Al Arab, Alexandria, Egypt) and dried for 1 h at 70 °C. Separate flasks were used to hold the stained cloth pieces separately. One flask containing only 20 ml of tap water as a negative control; the second flask contained tap water (20 ml) with detergent as a positive control, while the third contained tap water (20 ml) with (7 mg/ml) Persil inactivated detergent (endogenous enzymes free, was conducted by boiling the detergents for 30 min), whereas the fourth contained only (20 ml ≈ 9.50 U/ml) crude enzyme solution. Finally, the flask contained tap water (10 ml) with (7 mg/ml) detergent enzymes free with 10 ml crude enzyme solution (≈ 9.50 U/ml). All flasks were maintained for 30 min at 36 °C in a shaking water bath, then washed with tap water and dried for 1 h in the oven at 70 °C. The dried cloth pieces were used to visually test the crude enzyme’s ability to remove stains. Water-treated cloth stained pieces were taken as control.

2.11.4 Dehairing performance of B. cereus L10 keratinase

Fresh raw cowhides were obtained from a local slaughterhouse, cleaned with concentrated sodium chloride and washed several times with water to remove blood, mud, and other impurities before being dried in the air. The obtained hides were cut into square-shape pieces of approximately 5 cm2 by a sharp scissor and then drained. The cowhide pieces were separately placed into a petri dish containing 40 ml of enzyme solution (9.50 U/ml). While the rest was soaked in 40 ml of distilled water as a negative control. In addition, pieces treated separately with 40 ml chemical agents such as sodium sulfide (Na2S) (2%) and calcium oxide (CaO) (2%) were used as a positive control to compare dehairing efficiency [40]. After incubating for 40 h at room temperature (30 °C). The cowhide pieces were washed with tap water and observed for dehairing by a blunt scalpel to check the depilation potential of crude enzyme. The general appearance of skin surface and the quality of the dehairing were observed visually.

3 Results

3.1 Isolations

The soil samples used to isolate bacteria on chicken feather medium (CFM) were investigated. The inoculated flasks that showed turbidity and feather disintegration were observed (Figure S2); after purification, a total of 10 bacterial isolates were obtained; five isolates were isolated from feather waste soil designated as F1–F5, and the other five were designated as L6–L10 were isolated from the leather storehouse. The ability of bacterial colonies to grow in a keratin-selective media proved that they are keratinase-producing bacteria.

3.2 Screening of proteolytic activity

All selected isolates displayed significant protease (caseinase) activity on skimmed milk agar plates by forming a zone of clearance with varying halo zone generation. The proteolytic activity has been produced in the first 6 h of incubations. The isolates F1, F5, and L10 showed strong proteolytic activity after 48 h (Fig. 1). The proteolytic activity of these 10 isolates was used as a primary indicator in subsequent experiments such as various biotechnological applications.

Fig. 1
figure 1

Clear zone index on skimmed milk agar medium after hydrolysis by proteolytic bacteria at different times: A 6 h, B 12 h, C 24 h, and D 48 h. Values are presented as mean and standard deviation. The values without the same superscript letters above the column are significantly different (p < 0.05)

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3.3 Determination of keratinolytic activity of bacterial isolates

After 48 h of fermentation on basal chicken feather medium (CFM), the degradation potential of feathers varied depending on the tested isolate, ranging from 6.2% for isolate F4 to 72.7% for isolate L10; also, total protein concentrations were estimated to be 146.0 µg/ml and 406.83 µg/ml for isolate L6 and L10, respectively (Fig. 2). The fermentation broth of L10 turned yellow with powder-like consistency, and feather residue had unrecognizable structures remaining. The pH of the fermentation media of all tested strains was in the neutral range except for strain L10 shifted to alkaline (pH 7.8) (Table S1). So strain L10 was shown to be the most promising isolate with high feather degradation and total protein yield.

Fig. 2
figure 2

Feather degradation and protein concentration of bacterial isolates on a basal chicken feather media after 48 h of incubation period. Values are presented as mean and standard deviation. Different letters in the same test indicate significant differences according to one way ANOVA test (p ≤ 0.05)

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3.4 Molecular identification of keratin-degrading bacteria

According to the results of PCR amplification and subsequent partial 16S rRNA gene sequencing, the determined 16S rRNA nucleotide sequence of isolate L10 is about 420 base pairs long. The analysis of the sequence using basic local alignment search tool (BLASTn) results revealed that isolate L10 belonged to the genus Bacillus with a high similarity percentage (> 99.5%) to the B. cereus group. As a result, the L10 isolate was identified as B. cereus L10 and was submitted to the GenBank under accession no. OK483474. A phylogenetic tree was designed using a neighbor-joining approach with related gene sequences from the GenBank (NCBI) database employing the closest homologous sequences in a phylogenetic analysis with the MEGA 11 software (Figure S3).

3.5 Plackett–Burman design

The design pattern for selecting significant parameters for the production of keratinases and the relevant responses (keratinase activity (U/ml) has been shown in Table 2; numerous variables with high and low degrees of impact in various combinations cause response variance (0.38–7.32 U/ml), with the 11th run showing the highest keratinase production. p-values of the test ≤ 0.05 were used to determine which factors had a significant impact on the response. The F value of the model (27.04) implies that it was significant. The accuracy of determination coefficient R2 (99.63%,) and adjusted R2 (95.95%) reveals the adequacy and fitness of the experimental model. The analysis of variance (ANOVA) was performed to check the statistical significance of the regression model analysis at the significance level (p ≤ 0.05) (Table S2). Among all of examined variables, only fermentation time displayed the significance effect (p = 0.044) on the yield of keratinase. The Pareto chart was identifed as a useful tool for determining the most signifcant effects in the order of significance (Fig. 3).

Table 2 PBD matrix for screening of independent process variables with actual and coded values influencing on keratinase production
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Fig. 3
figure 3

Pareto chart illustrating the order and significance of variables affecting keratinase activity produced by Bacillus cereus L10 using Minitab 16 software and Plackett–Burman design

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3.6 Verification experiment of the predicted optimal conditions

A verification experiment was used to compare the predicted optimal levels of independent factors with anti-optimized and basal condition settings to determine the efficiency of the applied Plackett–Burman statistical design (Table 3). Figure 4 shows the growth of B. cereus L10 on the optimized feather medium (OFM) with the following composition: chicken feather, 1% (w/v); K2HPO4, 0.3% (w/v); KH2PO4, 0.05% (w/v); MgSO4 7H2O, 0.01% (w/v); yeast extract, 0.05% (w/v); inoculum size 4% (v/v) with pH 7 and incubation period 2 days at 35 °C. The high level of keratinolytic activity reached 7.317 U/ml. The effect of incubation time on keratinase production under previously optimized conditions was investigated, and the results revealed that the keratinolytic activity reached a maximum titer (9.602 U/ml) after 120 h, with increased by 4.56-fold ≈ 456.15% when compared with that obtained under the original medium fermentation conditions (2.105U/ ml). Feather degradation reached 94% and protein content is 521.17 µg/ml (Fig. 5).

Table 3 Verification test used to compare the optimal levels of independent factors with anti-optimized medium and basal medium
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Fig. 4
figure 4

Verification experiments of the applied Plackett–Burman statistical design for comparing keratinase production by B. cereus L10 grown on basal, optimized, and anti-optimized media. Values are presented as mean and standard deviation; different letters indicate significant differences according to one way ANOVA test (p ≤ 0.05)

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Fig. 5
figure 5

Keratinase activity, feather degradation, and protein concentration of Bacillus cereus L10 during the course of fermentation

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3.7 Effect of different substrates on keratinase production

B. cereus L10 can degrade different types of keratinous substrats (Figure S4). High keratinase activity (10.553 U/ml) was produced in the case of sheep wool and protein content (561.83 µg/ml). Human nails showed the lowest keratinase activity (6.239 U/ml), with protein content (300.00 µg/ml). Other types of keratin ranged between these two values (Fig. 6).

Fig. 6
figure 6

Effect of different substrates on keratinase production and protein concentrations using optimum feather medium after 120 h of incubation period. Values are presented as mean and standard deviation; different letters in the same test indicate significant differences according to one way ANOVA test (p ≤ 0.05)

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3.8 Thermal stability of keratinase

The thermal stability profile of B. cereus L10 keratinase (Fig. 7) manifestly showed that the enzyme was highly stable at various temperature ranges up to 80 °C, maintaining greater than 97.66% of original activity after incubation for 1 h at 100 °C.

Fig. 7
figure 7

The effect of temperature on keratinase activity, keratinase achieved thermal stability at 80°C for 1 h and retained 97.66% of its residual activity at 100 °C

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3.9 Application

3.9.1 Plant growth promoting activities B. cereus L10

One of the interesting results was the production of indole acetic acid (IAA) (54.6 ± 3.8 µg/ml), observed after 5 days of incubation in the improved feather medium without supplementation of L-tryptophan (Figure S5). The capability of B. cereus L10 to form feather hydrolysate and synthesize IAA is an important feature to be considered a plant growth-promoting bacteria (PGPB).

3.9.2 Feather hydrolysate as a biofertilizer

Feather hydrolysate was used in the soil to assess seed germination, growth, and development of Triticum aestivum (wheat). The seed germination was faster in soil treated with feather hydrolysate (98%) in comparison to the control (23%). Germination was early in treated soil and started in the third day compared to the control without feather hydrolysate. This control seemed weak and started germination in the fourth day. The germination rate continued to increase over the 10 days. Furthermore, the application of feather hydrolysate (leads to a signifcant increase in wheat seedling length, fresh and dry weights, and the number of laterall roots weight) significantly increased shoot and root lengths, plant fresh and dry weights, and the number of laterall roots compared to the control without treatment (Fig. 8; Table 4).

Fig. 8
figure 8

Utilization of the feather hydrolysate as a biofertilizer and study and the germination of wheat seeds after 10 days for wheat seedlings: A, C control seedlings and B, D seedlings treated with feather hydrolysate

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Table 4 Effect of feather hydrolysate as a biofertilizers on Triticum aestivum
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3.9.3 The Cleaning properties of B. cereus L10 keratinase

The crude enzyme was used to remove blood and a chocolate-flavored milk product stains. The results demonstrated the enzyme’s effectiveness in eliminating stains with or without the use of inactivated detergent, as shown in Fig. 9. The addition of prepared enzyme (9.50 U/ml) with detergent (Persil) greatly increases the cleaning quality of the chocolate-flavored milk stains from cotton fabrics and resulted in the chocolate stain remaining slightly in the cotton fabrics; however, it was seen that using detergent only failed in removing chocolate stain and it still fully retained in cotton fabrics. Also, keratinolytic enzyme is very effective in removing blood stains on cotton fabrics and medicinal aprons which were washed with detergent combined with keratinase enzyme. The results revealed that B. cereus L10 keratinase displayed stability and compatibility with the detergent. Furthermore, keratinase independently could remove the blood stains with excellent results in copmparison to that obtained with detergent only.

Fig. 9
figure 9

Washing performance of keratinase enzyme

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3.9.4 Evaluation of dehairing efficiency

The dehairing efficacy of the B.cereus L10 keratinase on cowhides was assessed. After 40 h of incubation with 40 ml of enzyme solution (9.50 U/ml), keratinase could effectively remove hair completely from cowhides, and hair could be manually scraped away without harming the skin’s quality compared to the positive and negative control (Fig. 10). In addition, the dehaired pelt exhibited a smooth, velvety, and white color surface, good flexibility, without observable damage, and no scars. On the other hand, chemical dehairing (2% Na2S) was unable to completely remove hair from cowhides after 40 h, and portions of hair could still be seen on the skin with a harsh, thickened, hard in touch, whereas chemical treatment hides treated with 2% CaO, showed that the dehaired pelts were not only yellow, but also have hard, cracked, and wrinkled appearance. The control cowhide, incubated in water under the same conditions, showed no sign of hair removal (Fig. 10). The results revealed that keratinase from B. cereus L10 could efficiently accomplish the dehairing process and with satisfactory properties. The amount of soluble protein resulting from the dehairing solution was measured for improving sustainable benefits (Table S3).

Fig. 10
figure 10

Comparative study of dehairing assay after 40 h of treatment period: A control with water, B dehaired with keratinase enzyme, C conventional method dehaired with 2% Na2S, and D conventional method dehaired with 2% CaO

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4 Discussion

Green technology is concerned with sustainability for a clean environment and has accelerated the quest for microbes with efficient biotechnological and industrial potential [41,42,43]. The bioconversion of feather waste into nutritional valuable products with a microbial enzyme keratinase is an excellent strategy for dealing with such obstinate environmental wastes [43]. Based on feather keratin growth and distinct zone hydrolysis on casein agar plates, 10 different colonies were selected for chicken feather degradation. Screening on skimmed milk was an important step in identifying isolates with proteolytic potential [30, 44]. The isolate L10 was found to degrade chicken feathers with the highest efficiency (73%) within 48 h of fermentation accompanied by the highest level of protein content 407 µg/ml and identified as B. cereus L10 by sequencing the 16S rRNA. Similarly, different strains of Bacillus sp. are employed for the degradation of keratinous wastes [31, 45,46,47,48]. The continued exploration of new keratinolytic strains of the B. cereus group often leads to the potentiality for discovering new keratinases with varied properties for novel vistas of keratinous material exploitation [49]

There is no definite medium for maximizing the induction of enzymes because different microbial strains require different culture conditions required for maximum productivity of enzymes. The Plackett–Burman design)PBD (was a major indicator for choosing the process variables that had a significant influence on keratinase production. The incubation time was considered the most effective factor influencing the yield of B.cereus L10 keratinase. The optimal conditions were validated resulting in the increment of keratinase yield by 9.602 U/ml ≈ 4.56-fold. The results in this study are in agreement with those recorded by several researchers after statistical optimization of process parameters, e.g., Sharma et al. [46] achieved about 4.92-folds of keratinase production by B. velezensis NCIM 5802; Jana et al. [45] showed 5 fold enhanced titers of keratinase. However, Ramnani and Gupta [50] obtained a 3.5-fold increase in keratinase output by Bacillus licheniformis RG1.

Besides, the percentage of feather degradation would be optimized and reached 94% after 5 days; this result was significantly faster than the other keratinolytic strains previously reported by Park and Son [51]; reported complete degradation of chicken feathers by Bacillus megaterium was achieved upon 7 days of incubation. According to Williams et al. [52], the ability of Bacillus licheniformis PWD-1 to hydrolyzed chicken feathers completely required 10 days. Hong et al. [53] detected 65% of feather weight was lost within 2 days by Chryseobacterium sp. P1-3, while Kshetri et al. [28] observed that Chryseobacterium sediminis RCM-SSR-7 recorded degraded 84% feather degradation in 84 h.

B.cereus L10 keratinase exhibited extremely thermal stability in the temperature range of 20–80 °C and retained 97.66% of the activity at 100 °C for 60 min. As a result, this protein is categorized as a thermo-stable keratinase and could be widely used in various biotechnological applications. The keratinase activity of B. cereus L10 exceeds Bacillus aerius NSMk2 whose activity was only stabilized at 50 °C and retained 50% activity at 90 °C after 30 min [36].

B. cereus L10 could utilize different keratinous substrates such as chicken feather, duck feather, turkey feather, horn, hoof, sheep wool, nails, and human hairs with no need for another carbon or nitrogen source, and the results revealed that sheep wool was the promising substrate for keratinase induction, followed by the other keratin substrates. B.cearus L10 showed different levels of keratinolytic activities when cultured on different keratinous substrate; these may be due to the difference in the percentage of protein content in each substrate, for example, feather dry matter contains ~ 90% protein [54, 55], while wool contains up to 95% by weight of pure keratin [56].

An important implication of our study is that B. cereus L10 was investigated for its ability to produce indole-3-acetic acid (IAA). IAA is a significant plant hormone that has a significant impact on plant growth and development, by enhancing cell division, cell elongation, cell differentiation, embryogenesis, apical dominance, and root growth and stimulating water and nutrient uptake [48, 57,58,59]. Several microorganisms have been found to produce IAA utilizing various precursors, such as tryptophan-mediated IAA synthesized by Pseudomonas fluorescens [60], and Fusarium species can produce the plant hormone IAA from tryptophan via the intermediary of indole-3-acetamide (IAM) [61].

The current study presented an innovative ability of strain L10 to synthesize IAA in feather medium without the need to supplement tryptophan, and an increased IAA synthesized was obtained with increasing the concentration of chicken feathers, which is since the hydrolysis of feather resulted in tryptophan biosynthesis from keratin which serves as an inducer for IAA synthesis [62]. According to these results, the culture broth of B. cereus L10 could be useful as a biofertilizer. Many studies show that feather hydrolysate can promote plant development, suggesting that it could be used in agriculture to enhance agricultural output and quality [63].

The finding was quite surprising using the feather hydrolysate of B.cereus L10 as a soil biofertilizer in comparison to the control trial. Through a study of the germination of Triticum aestivum (wheat), the seeds were selected owing to its importance in crops in all worlds; soil supplemented with feather hydrolysate showed earlier seed germination with a very high germination percentage of 98% versus 23% control. Our finding was in agreement with others stated that feather hydrolysate achieved through microbial degradation has been reported as promising fertilizer for various plants [14, 64,65,66,67].

The feather hydrolysate greatly enhanced the growth of plants over control plants grown without feather hydrolysate supplements. This might be due to the presence of many types of amino acids, nutrients, and plant hormones in feather hydrolysate which has a great effect on plants. The supernatant of Bacillus cereus-treated feathers has been shown to contain around 17 amino acids eight of which were essential amino acids, namely, lysine, leucine, methionine, isoleucine, valine, phenylalanine, threonine, and tryptophan [68, 69]. In addition, Mabrouk [70] recorded the total amount of free amino acids in Streptomyces sp. MS-2 feather hydrolysate was nearly 51 times greater than the control. Moreover, Sobucki et al. [71] recorded that feather hydrolysate contained a high amounts of nitrogen, calcium, potassium, iron, magnesium, copper, and zinc produced by the activity of Bacillus sp.. Furthermore, the presence of amino acids and proteins is essential in the genetic and physiological processes of plants [72, 73].

Another important implication of these findings is that the use of detergent supplemented with crude keratinase on stained cloth resulted in a better stain removal than the use of detergent alone. Commercial detergent contains several additives as bleaching agents (formic acid, sodium chlorite, sodium nitrate, hypochlorite, and peroxides), softening builders and surfactants. These additives have several drawbacks, containing toxic fume products, high energy, time consumption, fabric strength reduction, and the potential for significant fiber damage. So enzymatic washed of fabrics was a common procedure in the textile sector to achieve special finishing effects [22]. It was worth to mention that B.cereus L10 keratinase can be use as alternative to detergent for removal of blood stains. As a reason, hemoglobin in the blood (containing globin protein) was broken down into peptides as a result of proteolytic by the keratinase [22]. Our study concluded that the keratinase enzyme produced by B. cereus L10 has a great capacity for eliminating protein stains from cloth; it might be employed as a keratinolytic protease additive in detergent powders or solutions. Generally, it is speculated that the high stability of enzymes with detergents indicated its suitability for use as a detergent additive. There have also been several reports on the compatibility of microbial keratinases with commercial laundry detergents which validated its suitability for potential use as a detergent additive [74].

In the current study, it was discovered that B. cereus L10 keratinase can be a useful leather dehairing agent that has no negative effects on the leather (does not cause skin collagen damage) and could completely dehair leather after 40 h without the use of any chemical. Whereas chemicals have noxious consequences such as cause damage, harsh texture, and lower quality of leather. As a result, the proposed poultry waste–degrading keratinase outperforms chemicals in terms of not only dehairing but also leather quality. Furthermore, enzymatic dehairing reduces reliance on toxic chemicals (sulfide, lime, and amines) commonly employed in tanneries, hence protecting human health and wildlife by reducing pollution in soil, water, and the overall environment [13].

Enzymatic dehairing has a greater benefit over typical chemical techniques in that it removes hair from the basis without hurting the collagen of the skin [36, 75, 76]. The use of biocatalysts to replace inorganic sulfide demonstrated significant benefits in terms of environmental protection and unhairing efficiency. Keratinolytic protease is a good biocatalyst for hydrolyzing disulfide bond-rich proteins in hair, and it does not harm leather [77, 78], compared to the chemicals that had bad effects on the quality of skin making severely cut without removing the hair completely, making keratinase a good alternative to the chemical dehairing techniques now utilized in industry. Because of its great properties, this enzyme is a great option for the development of eco-friendly cleanliness and smart leather processing techniques that produce high-quality leather in a short time.