Assessment of feather degrading activity of thermophilic bacilli isolated from Armenian geothermal springs

Assessment of feather degrading activity of thermophilic bacilli isolated from Armenian geothermal springs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of feather degrading activity of thermophilic bacilli isolated from Armenian geothermal springs Mane Tadevosyan, Armine Margaryan, Hovik Panosyan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5090232/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jan, 2025 Read the published version in Biologia → Version 1 posted 5 You are reading this latest preprint version Abstract The aim of this study was to isolate and characterize keratinolytic thermophilic aerobic bacilli from Armenian geothermal springs. In total 20 thermophilic aerobic bacilli strains have been isolated using chicken feather enrichment cultures. Among these, four strains affiliated as Bacillus licheniformis (95–97% similarity) and Bacillus borbori (> 99% similarity) demonstrated the capability to completely degrade chicken feathers at 55°C. The highest rate of feather hydrolyses in mono-species cultures was observed with 40 g L − 1 substrate. Notably, enhanced keratin weight loss (≥ 80%) was observed in dual co-cultures involving B. borbori M14, highlighting superior degradative potential of this strain. Keratinolytic enzyme production was dedected during the late exponential growth phase, reached its maximum activity (0.013 U mL − 1 ) during the stationary phase, suggesting growth-associated enzyme synthesis. High-performance liquid chromatography (HPLC) of the hydrolysis end products revealed that aspartic acid and isoleucine were the predominant amino acids, followed by leucine, phenylalanine, alanine, tyrosine and glutamic acid. These findings confirm that the newly isolated strains are promising sources of keratinolytic proteases, with potential applications in circular bioeconomy based processes. Feather keratin keratinolytic thermophilic bacilli Bacillus borbori Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Keratin is a recalcitrant structural protein and the third most abundant polymer globally, following cellulose and chitin (Lange et al. 2016). It forms the structural basis of various biological materials, including skin, feathers, wool, hair, hooves, and horns. Keratin is classified into two types based on its secondary structure: α-keratin and β-keratin. α-Keratin is found in mammalian epidermal materials such as hair, skin, and wool, whereas β-keratin, which contains more cysteine residues and forms stronger disulfide bonds, is found in bird feathers and reptile scales. This structural stability makes feather keratin particularly resistant to protease degradation (Wang et al. 2016, Tesfaye et al. 2017, Qiu et al. 2020). Despite its abundance, keratin’s potential as a source of amino acids and oligopeptides remains underutilized (Gupta et al. 2013; Wang et al. 2017; Qiu et al. 2020; Zhou et al. 2023). Feather waste, a major by-product of the poultry industry, accounts for 5–7% of chicken body weight (Li 2019). With millions of tons produced annually feathers offer an ideal source of keratin due to their high protein content 90% (Verma et al. 2017; da Silva 2018). However, untreated feather waste poses environmental and health risks, serving as a habitat for pathogenic microbes and releasing pollutants such as nitrous oxide, ammonia, and hydrogen sulfide (Tamreihao et al. 2019). Processed feathers have diverse applications including decorative materials, medical devices, bedding materials, and feedstock (Li 2019). Traditional methods, such as chemical treatment or stem pressure cooking, are economically unviable and often degrade essential amino acids (Papadopoulos 1989; Latshaw et al. 1994; Wang and Parsons 1997; Kumar et al. 2021). Consequently, biotechnological approaches such as the use of keratinolytic enzymes offer a promising alternative․ The biological degradation of keratin by keratinolytic proteases represents cost-effective, and environmentally friendly method for its decomposition. Keratinases (EC 3.4.-.- peptide hydrolases) are hydrolytic enzymes that break down the hard-to-degrade keratin efficiently (Verma et al. 2017). Various bacteria bacteria ( Bacillus, Streptomyces, Nocardiopsis ) and fungi (non-pathogenic and dermatophytic pathogenic) secrete keratinolytic enzymes, enabling the conversion of feather waste into value-added products (Li 2019; Hassan et al. 2020; Tadevosyan et al. 2022). Thermophilic microbes, with their unique genomic adaptations, represent an untapped resource for thermostable keratinases with broad industrial applications (Cavello et al. 2018; Qiu et al. 2020; Javier-Lopez et al. 2023). Keratinase, from thermophilic microbes, due to their thermostability and wide substrate specificity, are considered as a better catalyst in feed, biofertilizer, detergent, leather, and pharmaceutical/biomedical industries (Gupta et al. 2013; Li 2019; Zhou et al. 2023). Thermophilic keratinolytic protease producing microbes are phylogenetically very diverse. The majority of studied keratinolytic microbes such as Fervidobacterium pennivorans (Friedrich and Antranikian 1996), Fervidobacterium islandicum AW-1 (Nam et al. 2002), Thermoanaerobacter keratinophilus 2KXI (Riessen and Antranikian 2001), Caldanaerobacter sp. strain 1523-1 (Kublanov et al. 2009), Clostridium sporogenes bv. pennavorans (Ionata et al. 2008), Fervidobacterium pennivorans subsp. keratinolyticus strain T (Javier-Lopez et al. 2023) are anaerobic microorganisms. Reports on thermophilic aerobic keratinase producers are scare. Only few reports are available about thermophilic aerobic keratinase producing bacteria isolated from geothermal areas or hot springs (Yamaoka et al. 2014; Cavello et al. 2018). Numerous high-altitude geothermal springs of different geotectonic origins, and with different physicochemical properties, are found within the territory of Armenia and Nagorno-Karabakh (Henneberger et al. 2000; Panosyan et al. 2018). Initial studies based on both metagenomics and culture-dependent analyses of the microbiota from certain Armenian geothermal springs have revealed diverse thermophilic bacteria and archaea with hydrolytic potential (Panosyan et al. 2020; Saghatelyan et al. 2021; Burkhardt et al. 2024). However, the diversity and biotechnological potential of keratinase producing thermophilic bacilli from these springs remain largely unexplored This study focuses on isolating and characterizing keratinolytic thermophilic aerobic bacilli from high-elevated Armenian geothermal springs. It demonstrates the synergistic keratin-degrading capabilities of bacterial consortia and highlights their potential for sustainable applications in the bio-based circular economy. Materials and methods Sample collection Sludge samples or sediments were aseptically collected from the outlet of geothermal springs located in Karvachar (40°17′41.00" N; 46°27′50.00" E), Zuar (40°02′47.60′’ N; 46°14′09.30′’ E) and Arzakan (40°27′36.10″ N, 44°36′17.76″ E) in 2020. The samples were placed in sterilized thermostatic bottles to maintain habitat temperature until further processing for incubation and isolation. Temperature, pH and conductivity of the spring water were measured in situ with a HANNA HI98129/HI98130 portable instrument. The geographical locations and elevations of the springs were determined using a portable global positioning system (GPS) device (GARMIN 64 s). The map and images of the sampling locations are shown on Fig. 1 S, and the main characteristics of the collected samples are listed in Table S1 , Enrichment and isolation The watery sludge or sediment samples (1 g) were suspended in 10 mL sterile water and mixed for 1 min to obtain the enrichment cultures. The supernatant was pasteurized at 80°C for 10 min to isolate only endospore-forming bacilli. Aliquots (1.0 mL) were inoculated in 100 mL Erlenmeyer flask containing 5.0 g of chicken feathers as the keratin source, along with 100 mL media with following composition (g L − 1 ): yeast extract, 0.5; peptone, 0.5; NaNO 3 , 5.0; K 2 HPO 4 , 5.0; NaCl, 10.0; MgSO 4 •7H 2 O, 0.4; CaCl 2 •H 2 O, 0.2, at pH 7.2. Chicken feathers (white, brown, and black colored) from Yerevan broiler chickens were collected from local slaughterhouses and private hen hatcheries. Feathers were first washed with tap water and cut into 5mm 2 sections and treated by shaking in 70% ethanol for 5 min at room temperature. They were then soaked in 0.9% bleach for 30 min, rinsed with distilled water, washed with a 10% sodium bicarbonate solution, rinsed again with distilled water and air-dried. Alternatively, feathers were sterilized by autoclaving at 121°C for 15 min to preserve the native structure of the feather. To assess the impact of acidic and alkaline conditions on feather degradation 1 g of native feathers were treated with 20 mL of 12% HCl solution (pH 2) or 1 M NaOH solution (pH 9) and stored in a closed container for 30 min (Istrate et al. 2013). Untreated feathers sereved as controls. The treated feathers were then washed with sterile distillated water to neutralize the acid/base. Both treated and untreated feathers were dried in an oven. Enrichments were incubated in an orbital shaker at 180 rpm at 55°C until feather degradation was visually confirmed (1–5 days). Turbid cultures (0.1 mL, and the standard serial dilution 1/10, 1/100) were streaked on nutrient nutrient broth (NB, Liofilchem, Italy) agar (1.5%) plates and incubated at 50, 55, 60 and 65°C for 24 h. Single colonies exhibiting different morphologies were isolated and subcultured by streaking onto the same medium at least three times to obtain pure monoculture. Subcultures were considered pure after microscopic examination revealed a single morphological type per culture. The purity and cell morphology were determined by phase-contrast microscopy (OMAX, M8311 USA) of freshly prepared wet mounts. All isolates were stored at 4°C, continuously subcultured, and preserved at − 80°C in nutrient broth containing 20% glycerol. All isolates are maintained in the extremophilic microbial culture collection at the Department of Biochemistry, Microbiology and Biotechnology of Yerevan State University, Armenia. DNA extraction, 16S rDNA amplification and phylogenetic characterization Total bacterial DNA was extracted using the GenEluteTM Bacterial Genomic DNA Kit (Sigma, Germany) following the manufacturer’s protocol. The DNA was eluted with 50 µL of Tris–EDTA buffer (TE) and used as a template for amplifying the 27-1492 region of the 16S rRNA gene via polymerase chain reaction (PCR). The universal primers 16SF (5'- GAGTTTGATCCTGGCTCAG-3') and 16SR (5'-GAAAGGAGGTGATCCAGCC-3') ( Escherichia coli numbering) were used for amplification. PCR mixtures used for amplification of sequences contained 10 ng of DNA, 5 µL 10 × PCR buffer, 5 µL 10 mM dNTP (dATP, dGTP, dCTP and dTTP), 1 µL of each primer (25 pmol µL − 1 ), 0.2 µL Taq DNA polymerase and sterile water to make up a final volume of 50 µl. PCR amplification was conducted using a DNA Engine thermocycler (BIO-RAD Thermo Scientific Arktik Thermal Cycler). First, the templates were denaturized for 3 min at 96°C, then 30 cycles of the following steps were completed: denaturation for 30 s at 96°C, annealing for 30 s at 55°C and extension for 2.5 min at 72°C. The 30 cycles were followed by a final 10 min extension at 72°C. PCR products were viewed under UV light after standard ethidium bromide gel electrophoresis. PCR products were purified using the GenElute™ PCR Cleanup Kit (Sigma, Germany). 16S rDNA sequencing of bacterial amplicons was performed using the ABI PRISM capillary sequencer, according to the protocol of the ABI Prism Big-Dye Terminator kit (Perkin Elmer), using above mentioned primers. Raw data of the DNA sequences were analyzed using the BioEdit software. Chimeric sequences were identified using the DECIPHER web tool ( https://decipher.cee.wisc.edu/FindChimeras.html ). A nucleotide sequences were subjected to a BLAST search to find phylogenetically closest relatives (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/Blast ) (Altschul et al. 1997). The assembled 16S rRNA gene sequences were aligned with a representative set of 16S rRNA gene sequences from GenBank database using ClustalW (Thompson et al. 1994). A phylogenetic tree was constructed using the neighbor-joining method with the MEGA X software ((Saitou and Nei, 1987; Kumar et al. 2018). Confidence in branching points was determined by bootstrap analysis (1000 replicates). Phenotypic characterization The morphological, physiological and biochemical (tests for oxidase, anaerobic growth, Voges–Proskauer reaction, hydrolysis of starch and gelatin, citrate utilization, nitrate reduction, H 2 S and indole production) characteristics of the isolate were investigated as described by Smibert and Krieg (1981). Each experiment was conducted in triplicates. Cell morphology, sizes, endospore formation and its location, and motility were determined by phase-contrast microscopy (Nikon; Eclipse E400 microscope). The temperature range for growth was determined by incubating the strains in NB medium from 30 to 80°C at 5°C temperature intervals. The effect of pH on growth was examined at optimal growth temperature in the range 5.0–12.0, with increments of 0.5 pH units adjusted with 1 M NaOH or 1 M HCl. The range of NaCl concentrations for growth was tested by supplementing 0.5–5% (w v − 1 ) NaCl at intervals of 0.5% w v − 1 ). Growth was assessed by measuring optical density (OD) at λ 540 nm. Catalase activity was evaluated by bubble formation in 3% hydrogen peroxide solution (Tindall et al. 2007). Anaerobic growth was checked by inoculation of microbes in tubes containing NB medium supplemented with agar (2%, w v − 1 ). The ability to utilize various carbon sourses (D-glucose, L-arabinose, D-xylose, D-sucrose, D-fructose, inositol, mannitol) and produce acids was assessed in the medium containing (g L − 1 ) carbon source, 10; (NH 4 ) 2 HPO 4 , 1; KCl, 0.2; MgSO 4 •7H 2 O, 0.2; yeast extract, 0.2 by the addition of 15 mL 0.04% w v − 1 solution of bromocresol purple. Proteolytic (caseolytic) activity was detected by the formation of clear zones on 0.5% skim milk agar (pH 7.0) (Kim et al. 2001). Feather degradation in mono-species and co-cultures The ability to the strains to degrade keratin was assessed by inoculating 5% of 16 h old cultures into 100 mL Erlenmeyer flask containing 5.0 g of chicken feathers as the sole carbon and nitrogen sources, along with 100 mL of mineral media with following composition (g L − 1 ): NaNO 3 , 5.0; K 2 HPO 4 , 5.0; NaCl, 10.0; MgSO 4 •7H 2 O, 0.4; CaCl 2 •H 2 O, 0.2, pH 7.2. Incubaction was performed at 50°C or 65°C for 96 h and checked visually for feather degradation. Different substrate amounts (5, 10, 20, 30, 40, 50 and 60 g L − 1 ) were tested to investigate feather-degrading activity. Keratin mass loss was determined by filtering the bacterial cultures through pre-weighed filter paper (12–15 µm particle retention) (Frisenette APS, Denmark). The feather was washed to remove attached bacteria and the filter paper was dried at 50˚C for 48 h and re-weighed. Media without bacteria served as controls (Nasipuri et al. 2020). Keratin degradation in co-cultures of most active four isolates was performed using equal volumes (1 mL)of turbid mono-cultures in 250 mL shake flasks containing feather and mineral medium for 3 days at 55˚C, with shaking at180 rpm. Degradation from co-cultivation was evaluated for all combinations of dual-species cultures. Microbial growth and keratinolytic protease production kinetics were monitored in batch culture at 55°C, pH 7.0, with shaking at 240 rpm, by sampling 10 mL of culture broth every 1 h during 12 h. Microbial growth was estimated by recording optical density (OD) at 560 nm. Keratinolytic protease enzyme assay Keratinolytic protease activity was studied using an overnight culture in a minimal nutrient medium where feather served as the sole source of carbon and nitrogen. Three samples were taken every hour for 12 h. The first samples was used to measure OD of microbial cells at 560 nm. In the second sample, the protein concentration was determined using the Bradford method (Bradford 1976). Standard curve for albumin (using 0.1, 0.5, 1, 2.5, and 5 mg mL − 1 solutions of albumin) was prepared. 100 µL of the second sample was mixed with 1000 µL of Bradford solution. After incubation for 5–10 min, OD was measured at 595 nm. To create a standard curve for tyrosine, solutions of tyrosine (0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg mL − 1 ) were used. A100 µL aliquot from the third sample was mixed with 500 µL of β-casein solution (65 mg mL − 1 in phosphate buffer pH-7․38). After 20 min of incubation at 55°C, 400 µL of Na 2 CO 3 solution and 130 µL of Folin and Ciocalteu's reagent (Sigma, Germany) were added, followed by a further 30 min incubation at 37°C. OD was measured at 660 nm, and standard curves were constructed using Excel. The protease activity was calculated using the formula: $$\:\frac{\text{U}\text{n}\text{i}\text{t}\text{s}}{\text{m}\text{L}\:\text{e}\text{n}\text{z}\text{y}\text{m}\text{e}}=\frac{\left({\mu\:}\text{m}\text{o}\text{l}\:\text{t}\text{y}\text{r}\text{o}\text{s}\text{i}\text{n}\text{e}\:\text{e}\text{q}\text{u}\text{i}\text{v}\text{a}\text{l}\text{e}\text{n}\text{t}\text{s}\:\text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e}\text{d}\right)\cdot\:\left(\text{a}\right)}{\left(\text{b}\right)\cdot\:\left(\text{c}\right)\cdot\:\left(\text{d}\right)}$$ Where: a, total assay volume (mL); b, volume of enzyme used (mL); c, assay time (min); d, volume used in colorimetric determination (in mL). The enzyme activity is expressed as units (U) , where one unit is defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per min under the assay conditions. High-performance liquid chromatography (HPLC) analyses of hydrolysis products A 500 µL aliquot was taken from the decomposed sample, where the feather was the sole source of carbon and nitrogen. This sample was then centrifuged to remove the feather particles. Amino acids determination was carried out following the method previously described by Aghajanyan et al. (2024). The amino acid analysis was conducted using a Shimadzu Nexera X2 amino acid analyzer (Shimadzu Corporation, Kyoto, Japan), equipped with an RF-20A fluorescence detector. Separation of the amino acids was achieved on a SUPELCOSIL LC-DABS chromatographic column (3 µm, 15 cm × 4.6 mm). The mobile phase consisted of two components: (A) acetonitrile:methanol:water (45:40:15, v/v/v) and (B) a phosphate buffer at pH 6.8. Analysis was performed at a flow rate of 0.3 mL/min, with the column maintained at 30°C. Fluorescence detection was carried out with excitation at 350 nm and emission at 450 nm. Nucleotide sequence accession numbers . The 16S rRNA gene sequences were deposited in GenBank and the assigned accession numbers are as follows: PQ097580 - PQ097583. The B. botbori M14 strain was deposited in the Microbial Depository Center of Armenia under accession numbers MDC 11863. Results Enrichment Three watery sludge and sediment samples were analyzed to evaluate the abundance of thermophilic aerobic keratinolytic bacilli. For the primary enrichment of keratin degrading bacteria, the media mentioned above was supplemented with pretreated white, brown, and black colored chicken feathers. After incubation at 55°C in aerobic conditions, almost complete degradation of the feathers was observed within 4 days in the enrichment culture (Fig. 1 ). The feather color had no significant effect on degradation. Feathers collected from both local slaughterhouses and private hen hatcheries were hydrolyzed similarly. To enhance the degradation efficiency of the enrichment culture, feathers were treated with alkaline (NaOH, pH 9), and acidic (HCl, pH 2) solutions for primary hydrolysis. After alkaline solution treatment, the feathers were completely hydrolyzed within 96 h in an aerobic enrichment culture, whereas acid-treated feather required 120 h for complete degradation. Untreated feather similar to those treated with alkaline solutions, also required 96 h for complete degradation (Fig. S2). Identification of isolated strains based on polyphasic approach A primary culture of aerobic thermophilic bacteria capable of degrading feathers was successfully developed from watery sludge and sediment samples collected from the geothermal springs in Karvachar, Zuar, and Arzakan. A total of twenty isolates with different colony morphologies were obtained on NB agar plates and were characterized. The isolates were designated as M1-M20. A large number of isolates with varied colony morphologies and cell shapes were obtained from Karvachar (70°C) and Arzakan (44°C) geothermal springs, while only one or two morphotypes were observed from the Zuar (42°C) hot spring (Table 1 ). Table 1 Keratin degrading thermophilic isolates originating from geothermal spring samples. Sample no. Location of hot spring Sample origin Hot spring water temperature, °C Hot spring water pH Number of isolates obtained Strain name NB agar Feather enrichment 1 Karvachar Water with fine clay 70 7.3 10 2 M3, M2, M10, M11, M13, M14, M17-M20 3 Arzakan Silicate sand 44 7.2 7 1 M1, M5, M6-M8, M12, M15 2 Zuar Watery Sand/clay 42 7.0 2 1 M4, M16, M9 Colonies of the isolates were transparent, creamy, white, milky in color, opaque or translucent, with rough, smooth or glossy surfaces and regular or irregular edges (Fig. S3). All isolates were rod-shaped, Gram-positive, endospore-forming, and catalase and oxidase-positive bacteria. Four isolates designated as M4 (Zuar), M12 (Arzakan), M3 and M14 (Karvachar) were selected prioritizing their ability to grow in enrichment media with feathers as a sole carbon and nitrogen source and to degrade feathers completely. All studied strains were motile Gram positive, endospore forming, and tested positive for both oxidase and catalase. The Voges–Proskauer test was positive for the isolate. Isolates M3 and M4 were shown growth under anaerobic conditions and can be described as facultative aerobes, while the growth of M12 and M14 strains in anaerobic conditions was weak. At optimal growth conditions, all isolates were capable of reducing nitrate. The specific phenotypic properties that distinguish these strains from related reference type strains include differences in growth conditions (such as temperature and pH ranges), enzyme production and ability to utilize various carbon sources. None of the isolates were able to grow below 30°C, or above 65–70°C. Optimal growth temperature for M3 and M4 was observed at 50°C, while M12 and M14 grew best at 65°C, thus categorizing them as moderate thermophiles. No growth was observed when the NaCl concentration exceeded 5% (w v − 1 ). In comparison to closely related strains, all isolates displayed a broad pH growth range. None of the studied strains could utilize the D-xylose and citrate, produce indole or H 2 , or synthesize urease. The strain M14 could not assimilate sucrose, while strain M12 could not utilize arabinose. Unlike the type strain of Bacillus borbori type strain, strains M12 and 14 showed the ability to hydrolase casein. The nitrate reduction to nitrite, Voges–Proskauer (VP) reaction, production of H 2 S and indole, citrate utilization, and enzymatic activities such as β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease and tryptophan deaminase were tested for strain M14 strain using an API 20 E strip (bioMérieux)., according to the manufacturer’s instructions The strain was capable to reduce nitrate to nitrite and was positive for the VP reaction. However, it was negative for urease, H 2 S,and indole production, and lacked β-galactosidase, and ornithine-decarboxylase activities. It exhibited arginine dihydrolase, lysine-decarboxylase, gelatinase activity, and assimilated tryptophan. The identification of the four isolates was confirmed by sequencing the almost complete 16S rRNA genes. The sequences revealed that all isolates were closely related to the genus Bacillus (Table 2 ). Isolates M3 and M4 shared 97.02% and 95.85% similarity, respectively, to Bacillus licheniformis , suggesting that they may represent a potential new species. Table 2 BLAST results of 16S rRNA gene sequences of feather degrading thermophilic bacilli and accession numbers. Isolate Sequence length (bp) Closest match taxonomic affiliation, phylotype accession no % Similarity to closest match Accession no M3 1475 Bacillus sp. MK418572.1 Bacillus licheniformis LT669756.1 97.22 97.02 PQ097581 M4 1496 Bacillus sp. MK418572.1 Bacillus licheniformis LT669758.1 95.85 PQ097583 M12 1456 Bacillus borbori NR132725.1 99.52 PQ097580 M14 1462 Bacillus borbori NR132725.1 99.72 PQ097582 The other two isolates were related to Bacillus borbori with 99.52% and 99.72% similarity, respectively. The isolates associated with Bacillus borbori were obtained from Karvachar and Arzakan geothermal areas, while those associated with the B. licheniformis were isolated from samples Karvachar and Zuar geothermal areas. The phylogenetic tree based on the 16S rRNA genes of isolated strains and selected reference sequences from GenBank was constructed using the MEGA-X program (Fig. 2 ). High bootstrap values (above 90%) for the clades with strains M3, M4 and B. licheniformis and M12, M14 with B. borbori , supports the current clade structure. Further studies are necessary to confirm the taxonomic affiliation of these isolates. Phenotypic characteristics of keratin degrading isolates were compared with those of reference strains: Bacillus subtilis ATCC6051 T (Schleifer, 2009), Bacillus borbori DX-4 T (Wang et al. 2013) and Bacillus licheniformis ATCC 14580 T (Schleifer 2009). Results are reported in Table 3 . Table 3 Comparison of phenotypic properties of thermophilic feather degrading bacilli isolated from Arzakan geothermal spring with its nearest phylogenetic neighbor Bacillus species type strains and type species of genus Bacillus (1, B. subtilis ATCC6051 T ; B. borbori DX-4 T ; 3, B. licheniformis ATCC 14580 T ). Characteristics Strains M3 M4 M12 M14 1 a 2 a 3 b Cell size (µm) 0.6–0.8 × 1.6–2.8 0.4–0.9 × 1.3-3.0 0.3–0.5 × 1.0-2.1 0.2–0.6 × 1.5–2.1 0.7–0.8 × 2.0–3.0 0.2–0.5 × 1.2-2.0 0.6-08 × 1.5-3.0 Motility + + + + + + + Spore form/location E/C E/C E/S or T E/S or T E/C E/S E/C Swell sporangia - - + + - + - Temperature range (T opt ) (°C) 30–65 (50–55) 30–65 (50–55) 30–70 (55) 30–70 (55) 20 − 5 (28–30) 30–65 (55) 20–55 (Nd) NaCl range (optimum) (%) 0–5.0 (> 1.0) 0–5.0 (> 1.0) 0–5.0 (1.0) 0–4.0 (1.0) 0–7.0 (> 7) 0–6.0 (Nd) 2.0–7.0 (Nd) pH range (optimum) 5.0-10.5 (7.5-8.0) 5.0-9.5 (7.5-8.0) 6.0–9.0 (7.5) 5.0–9.0 (7.5) 5.5–8.5 (7.0) 6.0-8.5 (7.0-7.5) ND (5.7–6.8) Anaerobic growth + + ± ± - + + Voges-Proskauer + + ± + + + + Catalase + + + + + + + Oxidase + + + + v + v Acid from D-glucose + + + + + + + D-Fructose- + + + + + + Nd L-Arabinose + + - + + + + D-Xylose - - - - + - + D-Sucrose + + + - Nd + ± Inositol Nd Nd - - Nd - + Mannitol Nd Nd - - ± - + Hydrolysis of Casein + + + + + - + Starch + + + + + + + Gelatin + + + + + + + Utilization of Citrate - - Nd - + - + Nitrate reduction + + Nd + + + + Production of Indole - - Nd - Nd - Nd Urease - - Nd - Nd - ± H 2 S - - Nd - Nd - Nd a Data from Schleifer (2009); b Data from Wang et al, (2013); +, Positive; –, negative; ±, weakly positive result; v, variable; Nd, not determined; E, ellipsoidal; C, central; T, terminal; S, subterminal. Feather degradation in mono-species and co-cultures The pure cultures of M12 and M14 isolates completely degraded the feather after 72 h. The M14 strain proving to be the most effective in breaking down feathers (Fig. 3 ). The hydrolytic activity of the most potent feather-degrading bacilli strains was investigated in case of different amount of the substrate. Results are shown in Fig. 4 . The highest hydrolytic activity was observed when the feather amount in the enrichment culture was 40 g L − 1 . In community-based degradation experiments, feather degradation was compared to that of single species cultures to explore whether the community-intrinsic properties could enhance the process. Feather degradation was evaluated in dual-species co-cultures (Fig. S4)․ Co-cultures containing B. borbori M12 or B. borbori M14 exhibited enhanced keratin degradation compared to the individual strains. Feather degradated reached 86% in the co-culture of M12 and M14 after 48 h incubation at optimal growth conditions. Co-cultures of M4 with M12 or M14 also displayed significantly improved decomposition of the feather with rates of 82–85%. The lowest degradation rate (30%) was observed in co-cultures of M3 and M4 strains (Fig. S5). Among all strains tested B. borbori M14 was the best keratin degrade showing high culture density and keratin degradation potential. Based on these findings subsequent experiments on keratinolytic enzymatic activities were conducted using only on B. borbori M14. The growth and production of keratinolytic proteases in B. borbori M14 were monitored over the 12 h period at the optimum growth temperature of 55°C under aerobic conditions (240 rpm). Growth was measured by optical density (OD) at 560 nm and maximum growth was observed at 9 h (Fig. 5 ). Enzymatic activity increased significantly from 8 h onwards (late exponential phase) reaching its peak at 0. 013 U mL − 1 during the stationary phase indicating a growth-associated production of enzymes. HPLC analysis feather hydrolysis products indicated that aspartic acid and isoleucine were the main end products, followed by leucine, phenylalanine, alanine, tyrosine and glutamic acid (Fig. S6, Table S2). Discussion Bioconversion of poultry feathers into value-added products such as biofertilizers and high nutrient animal feeds is an attractive strategy, particularly through the use of thermostable enzymes. Thermophilic bacteria that produce keratinolytic enzymes, such as keratinases, are especially important for the bioconversion of keratin, which is highly resistant to degradation. The demand for thermostable keratinolytic enzymes from industries like detergent, leather, textile, food, and pharmaceuticals continues to rise, highlighting the ongoing need for robust and specific enzymes (Gupta et al. 2013; Qiu et al. 2020; Zhou et al. 2023). Thermostable keratinases found in high temperature environments like geothermal springs have emerged as key sources of these enzymes. Recent studies have shown that high altitude hot springs distributed on the territory of Armenia and Nagorno Karabakh harbor thermophilic bacilli with promising hydrolytic activity indicating their potential for biotechnological applications (Panosyan et al. 2020; Saghatelyan et al. 2021). Inspired from this evidence, the keratinase producing bacterial diversity and the abundance of resident bacteria in Armenian geothermal springs were studied. Despite the growing body of research on thermophiles, few reports focus on microbes from hot springs capable of degrading native feathers. Most feather degrading obligate thermophilic microbes isolated from hot springs, geothermal vents and volcanic areas were strict anaerobes, which hinder their practical utilization (Friedrich and Antranikian 1996; Riessen and Antranikian 2001; Nam et al. 2002; Ionata et al. 2008; Kublanov et al. 2009; Cavello et al. 2018; Javier-Lopez et al. 2023). Aerobic feather degrading bacteria such as thermoalkalophilic Bacillus halodurans AH-101 (Takami et al. 1999), Bacillus pseudofirmus FA 30 − 01 (Kojima et al. 2006) Bacillus sp. JB 99 (Johnvesly et al. 2002; Shrinivas and Naik, 2011) have been isolated from environmental samples (soil, waste streams, sugarcane molasses and poultry wastes) but few come from hot springs. One exception is Meiothermus ruber H328 isolated from a hot spring (Yamaoka et al. 2014). More recently studies in Patagonia, Argentina have explored the diversity of thermophilic aerobic bacilli capable of the few published efforts to examine the keratinase-producing diversity of thermophilic aerobic bacilli in geothermal springs (Cavello et al. 2018). This study represents one of the few published efforts to examine keratinase-producing diversity of thermophilic aerobic bacilli in geothermal springs In this study, samples from three Armenian geothermal springs were analyzed for their ability to degrade chicken feathers. Keratin is not soluble protein and its bioconversion is time-consuming process. To address this the effects of acidic (HCl) and alkaline (NaOH) treatments on feathers were evaluated, with alkaline treatment proving more affective (Fitriyanto et al. 2022). However untreated feathers required 96 h for complete degradation, which was similar to the alkaline-treated samples, prompting the use of untreated feathers for further experiments. A total 20 aerobic thermophilic bacilli strains have been isolated from chicken feather enrichment cultures. Four of these strains were capable of completely degrading feathers at 55°C, confirming their moderate thermophilic nature. Two strains (M3 and M4) were closely related to B. licheniformis (95–97% similarity), while other two strains (M12 and M14) shared over 99% similarity to B. borbori . The low (< 97%) similarity, of the M3 and M4 strains to B. licheniformis suggests that they may represent a new species. Starins of B. licheniformis are known for their hydrolytic activity (Manczinger et al. 2003; Muras et al. 2021) and previous reports have documented keratinase production by B. licheniformis PWD-1 (Cheng et al. 1995). However, to our knowledge, this is the first report of keratin degradation by B. borbori strains. Substrate concentration is an important factor in bioconversion processes, as it can influence enzyme activity. Evaluation of influence of the feather amount revealed that 40 g L − 1 of feather in enrichment was optimal for feather degradation. Under the tested conditions, the strains synthesized keratinolytic proteases during the late growth stationary phase. As a thermophilic process feather degradation occurred effectively at 55°C within a relatively short period compared to mesophilic processes. When compared to other bacteria such as B. licheniformis PWD-1 (Lin et al. 1997), B. licheniformis K-508 (Manczinger et al. 2003), B. licheniformis FK14 (Suntornsuk et al. 2005), B. licheniformis ER-15 (Tiwary and Gupta, 2010), Bacillus amyloliquefaciens S13 (Hamiche et al. 2019), Actinomadura viridilutea DZ50 (Ben Elhoul et al. 2016), Caldicoprobacter algeriensis Bouacem et al. 2016) and Actinomadura keratinilytica strain Cpt29 (Habbeche et al. 2014) the keratin degradation rate of B. borbori M14 was comparable, with no significant difference in degradation time. Studies comparing optimal growth temperatures and degradation times for various bacteria are summarized in Fig. 6 . While most studies have focused on single-strain systems for feather degradation, using microbial consortia may offer a more effective approach. The synergy between different strains can accelerate degradation and improve efficiency. In this study, co-cultures of B. borbori strains demonstrated up to 1.5 times greater degradation of feathers compared to mono-cultures. This enhanced degradation in mixed cultures suggests that community intrinsic properties, such as a switch from sulfitolytic to proteolytic activity, may play a role in more efficient keratin breakdown (Nasipuri et al. 2020). Feathers are rich in essential amino acids such as cysteine, glutamine, proline, and serine (Tesfaye et al. 2017) making them a valuable resource for microbial cultures. The feather hydrolysis products can serve not only protein or amino feeds for animals but also as important carbon and nitrogen sources for microbial culture (Williams et al. 1991). HPLC analysis of feather hydrolysis products by B. borbori M14 revealed significant amount of essential amino acids, including isoleucine, leucine, phenylalanine, and nonessential amino acids such as aspartic acid, glutamic acid, glutamic acid, alanine and tyrosine. This highlights the potential for bioconversion of feathers into high quality animal feed or amino acids. In contrast, physical and chemical treatments of feathers often result in the loss of important amino acids like methionine, lysine and tryptophan (Kumar 2021). Therefore, microbial keratinases present a more sustainable and nutritionally beneficial method for feather conversion. The results of this study confirm that the newly isolated strains from Armenian geothermal springs have significant potential for use in biotechnological applications, including the production of keratinolytic proteases for feather meal or amino acid recovery. This work contributes valuable insights into the microbial diversity of geothermal springs and their potential for circular bioeconomy processes. Abbreviations HPLC high-performance liquid chromatography GPS global positioning system TE Tris-EDTA buffer PCR polymerase chain reaction VP Voges-Proskauer NB nutrient broth OD optical density Declarations Ethical approval This article does not contain any studies involving human participants or animals. Consent for publication All the authors consented to the publication of this work. Conflict of interest The authors declare no competing interests. Funding The research was conducted with support from The Higher Education and Science Committee of RA within the scope of the research projects No. 21T-1F191. Author Contributions: Conceptualization, HP; Field sampling, HP and AM; Experimental design and data collection, HP and MT; Data acquisition and interpretation, MT and AM, Manuscript writing—original draft preparation, TM, HP and AM; Writing—review and editing, HP; Visualization, MT and AM, Supervision, HP; Funding acquisition, HP All authors have read and agreed to the published version of the manuscript. Acknowledgments We acknowledge Ella Minasyan for HPLC analyses. Data availability All data generated or analyzed during this study are included in this published article. References Aghajanyan AE, Hambardzumyan AA, Minasyan EV, Hovhannisyan GJ, Yeghiyan KI, Soghomonyan TM, Avetisyan SV, Sakanyan VA, Tsaturyan AH (2024) Efficient isolation and characterization of functional melanin from various plant sources. Int J Food Sci Technol 59(6):3545–3555. https://doi.org/10.1111/ijfs.17016 Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402. https://doi.org/10.1093/nar/25.17.33894o Ben Elhoul M, Zaraî Jaouadi N, Rekik H, Omrane Benmrad M, Mechri S, Moujehed E, Kourdali S, El Hattab M, Badis A, Bejar S, Jaouadi B (2016) Biochemical and molecular characterization of new keratinolytic protease from Actinomadura viridilutea DZ50. Int J Biol Macromol 92:299–315. https://doi.org/10.1016/j.ijbiomac.2016.07.009 Bouacem K, Bouanane-Darenfed A, Zaraî Jaouadi N, Joseph M, Hacene H, Ollivier B, Fardeau ML, Bejar S, Jaouadi B (2016) Novel serine keratinase from Caldicoprobacter algeriensis exhibiting outstanding hide dehairing abilities. Int J Biol Macromol 86:321–328. https://doi.org/10.1016/j.ijbiomac.2016.01.074 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1006/abio.1976.9999 Burkhardt C, Baruth L, Meyer-Heydecke N, Klippel B, Margaryan A, Paloyan A, Panosyan H, Antranikian G (2024) Mining thermophiles for biotechnologically relevant enzymes - evaluating the potential of European and Caucasian hot springs. Extremophiles 28:5. https://doi.org/10.1007/s00792-023-01321-3 Cavello I, Urbieta MS, Segretin AB, Giaveno A, Cavalitto S, Donati ER (2018) Assessment of keratinase and other hydrolytic enzymes in thermophilic bacteria isolated from geothermal areas in Patagonia, Argentina. Geomicrobiol J 35(2):156–165. https://doi.org/10.1080/01490451.2017.1339144 Cheng SW, Hu HM, Shen SW, Takagi H, Asano M, Tsai YC (1995) Production and characterization of keratinase of a feather-degrading Bacillus licheniformis PWD-1. Biosci Biotechnol Biochem 59(12):2239–2243. https://doi.org/10.1271/bbb.59.2239 da Silva RR (2018) Keratinases as an alternative method designed to solve keratin disposal on the environment: its relevance on agricultural and environmental chemistry. J Agric Food Chem 66:7219–7221. https://doi.org/10.1021/acs.jafc.8b03152 Fitriyanto NA, Ramadhanti Y, Rismiyati, Rusyadi I, Pertiwiningrum, Prasetyo RA, Erwanto Y (2022) Production of poultry feather hydrolysate using HCl and NaOH as a growth medium substrate for indigenous strains. IOP Conf Ser Earth Environ Sci 951:012064. https://doi.org/10.1088/1755-1315/951/1/01206 Friedrich AB, Antranikian G (1996) Keratin degradation by Fervidobacterium pennavorans , a novel thermophilic anaerobic species of the order Thermotogales . Appl Environ Microbiol 62:2875–2882. https://doi.org/10.1128/aem.62.8.2875-2882.1996 Gupta R, Tiwary E, Sharma R, Rajput R, Nair N (2013) Microbial keratinases: diversity and applications. In: Satyanarayana T, (eds) Thermophilic microbes in environmental and industrial biotechnology: biotechnology of thermophiles, pp 881–904. https://doi.org/10.1007/978-94-007-5899-5_33 Habbeche A, Saoudi B, Jaouadi B, Haberra S, Kerouaz B, Boudelaa M, Badis A, Ladjama A (2014) Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 isolated from poultry compost. J Biosci Bioeng 117(4):413–421. https://doi.org/10.1016/j.jbiosc.2013.09.006 Hamiche S, Mechri S, Khelouia L, Annane R, El Hattab M, Badis A, Jaouadi B (2018) Purification and biochemical characterization of two keratinases from Bacillus amyloliquefaciens S13 isolated from marine brown alga Zonaria tournefortii with potential keratin-biodegradation and hide-unhairing activities. Int J Biol Macromol 122:758–769. https://doi.org/10.1016/j.ijbiomac.2018.10.174 Hassan MA, Abol-Fotouh D, Omer AM, Tamer TM, Abbas E (2020) Comprehensive insights into microbial keratinases and their implication in various biotechnological and industrial sectors: A review. Int J Biol Macromol 154:567–583. https://doi.org/10.1016/j.ijbiomac.2020.03.116 Henneberger R, Cooksley D, Hallberg J (2000) Geothermal resources of Armenia. In: Proceedings World Geothermal Congress. Kyushu-Tohoku, 1217–1222 Ionata E, Canganella F, Bianconi G, Benno Y, Sakamoto M, Capasso A, Rossi M, La Cara F (2008) A novel keratinase from Clostridium sporogenes bv. pennavorans bv. nov., a thermotolerant organism isolated from solfataric muds. Microbiol Res 163(1):105–112. https://doi.org/10.1016/j.micres.2006.08.001 Istrate D, Popescu C, Er Rafik M, Möller M (2013) The effect of pH on the thermal stability of fibrous hard alpha-keratins. Polym Degrad Stab 98(2):542–549. https://doi.org/10.1016/j.polymdegradstab.2012.12.001 Javier-Lopez R, Mandolini E, Dzhuraeva M, Bobodzhanova K, Birkeland N-K (2023) Fervidobacterium pennivorans subsp. keratinolyticus subsp. nov., a novel feather-degrading anaerobic thermophile. Microorganisms 11:22. https://doi.org/10.3390/microorganisms11010022 Johnvesly B, Manjunath BR, Naik GR (2002) Pigeon pea waste as a novel, inexpensive substrate for production of a thermostable alkaline protease from thermoalkalophilic Bacillus sp. JB-99. Bioresour Technol 82(1):61–64. https://doi.org/10.1016/s0960-8524(01)00147-x Kim SS, Kim YJ, Rhee IK (2001) Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674. Arch Microbiol 175(6):458–461. https://doi.org/10.1007/s0020301002824 Kojima M, Kanai M, Tominaga M et al (2006) Isolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA30-01. Extremophiles 10:229–235. https://doi.org/10.1007/s00792-005-0491-y Kublanov IV, Perevalova AA, Slobodkina GB et al (2009) Biodiversity of thermophilic prokaryotes with hydrolytic activities in hot springs of Uzon Caldera, Kamchatka (Russia). Appl Environ Microbiol 75(1):286–291. https://doi.org/10.1128/AEM.00607-08 Kumar J (2021) Microbial hydrolyzed feather protein as a source of amino acids and protein in the diets of animals including poultry. In: Advances in poultry nutrition research. Intech Open. https://doi.org/10.5772/intechopen.96925 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096 Lange L, Huang Y, Busk PK (2016) Microbial decomposition of keratin in nature—a new hypothesis of industrial relevance. Appl Microbiol Biotechnol 100:2083–2096. https://doi.org/10.1007/s00253-015-7262-1 Latshaw JD, Musharaf N, Retrum R (1994) Processing of feather meal to maximize its nutritional value for poultry. Anim Feed Sci Technol 47:179–188. https://doi.org/10.1016/0377-8401(94)90122-8 Li Q (2019) Progress in microbial degradation of feather waste. Front Microbiol 10:2717. https://doi.org/10.3389/fmicb.2019.02717 Lin X, Wong S-L, Miller ES, Shih JCH (1997) Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis . J Ind Microbiol Biotechnol 19(2):134–138. https://doi.org/10.1038/sj.jim.2900440 Manczinger L, Rozs M, Vágvölgyi C, Kevei F (2003) Isolation and characterization of a new keratinolytic Bacillus licheniformis strain. World J Microbiol Biotechnol 19:35–39. https://doi.org/10.1023/A:1022576826372 Muras A, Romero M, Mayer C, Otero A (2021) Biotechnological applications of Bacillus licheniformis . Crit Rev Biotechnol 41(4):609–627. https://doi.org/10.1080/07388551.2021.1873239 Nam G-W, Lee D-W, Lee H-S, Lee N-J, Kim B-C, Choe E-A et al (2002) Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch Microbiol 178:538–547. https://doi.org/10.1007/s00203-002-0489-0 Nasipuri P, Herschend J, Brejnrod AD, Madsen JS, Espersen R, Svensson B, Burmølle M, Jacquiod S, Sørensen SJ (2020) Community-intrinsic properties enhance keratin degradation from bacterial consortia. PLoS ONE 15(1):e0228108. https://doi.org/10.1371/journal.pone.0228108 Panosyan H, Margaryan A, Birkeland N (2020) Geothermal springs in Armenia and Nagorno-Karabakh: potential sources of hydrolase-producing thermophilic bacilli. Extremophiles 24:519–536. https://doi.org/10.1007/s00792-020-01173-1 Panosyan H, Margaryan A, Poghosyan LA, Saghatelyan A, Gabashvili E, Jaiani E, Birkeland NK (2018) Microbial diversity of terrestrial geothermal springs in Lesser Caucasus. In: Egamberdieva D, Birkeland NK, Panosyan H, Li WJ (eds) Extremophiles in Eurasian ecosystems: ecology, diversity, and applications. Springer, Singapore, pp 81–117 Papadopoulos MC (1989) Effect of processing on high-protein feedstuffs: a review. Biol Wastes 29(2):123–138. https://doi.org/10.1016/0269-7483(89)90092-X Qiu J, Wilkens C, Barrett K, Meyer AS (2020) Microbial enzymes catalyzing keratin degradation: classification, structure, function. Biotechnol Adv 44:107607. https://doi.org/10.1016/j.biotechadv.2020.107607 Riessen S, Antranikian G (2001) Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity. Extremophiles 5:399–408. https://doi.org/10.1007/s00792010020 Saghatelyan A, Margaryan A, Panosyan H, Birkeland N-K (2021) Microbial diversity of terrestrial geothermal springs in Armenia and Nagorno-Karabakh: a review. Microorganisms 9:1473. https://doi.org/10.3390/microorganisms9071473 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 Schleifer K-H (2009) Phylum XIII. Firmicutes Gibbons and Murray. In: De Vos P et al (eds) Bergey’s Manual® of Systematic Bacteriology: Volume Three: The Firmicutes. Springer, New York, pp 19–1317. https://doi.org/10.1007/978-0-387-68489-5 Shrinivas D, Naik GR (2011) Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity. Int Biodeterior Biodegrad 65(1):29–35. https://doi.org/10.1016/j.ibiod.2010.04.013 Smibert R, Krieg N (1981) General characteristics. In: Gerhardt P, Murray R, Costilow R, Nester E, Wood W, Phillips G (eds) Manual of Methods for General Bacteriology, 3rd edn. American Society for Microbiology, Washington, pp 7–243 Suntornsuk W, Tongjun J, Onnim P, Oyama H, Ratanakanokchai K, Kusamran T, Oda K (2005) Purification and characterization of keratinase from a thermotolerant feather-degrading bacterium. World J Microbiol Biotechnol 21:1111–1117. https://doi.org/10.1007/s11274-005-0078-x Tadevosyan M, Yeghiazaryan S, Ghevondyan D, Saghatelyan A, Margaryan A, Panosyan H (2022) Microbial thermostable hydrolases (amylases, lipases, and keratinases) and polymerases: biology and applications. In: Arora NK, Agnihotri S, Mishra J (eds) Extremozymes and Their Industrial Applications. Elsevier Academic, pp 177–204. https://doi.org/10.1016/B978-0-323-90274-8.00007-1 Takami H, Nogi Y, Horikoshi K (1999) Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans . Extremophiles 3:293–296. https://doi.org/10.1007/s007920050130 Tamreihao K, Mukherjee S, Khunjamayum R, Devi LJ, Asem RS, Ningthoujam DS (2019) Feather degradation by keratinolytic bacteria and biofertilizing potential for sustainable agricultural production. J Basic Microbiol 59:4–13. https://doi.org/10.1002/jobm.201800434 Tesfaye T, Sithole B, Ramjugernath D (2017) Valorisation of chicken feathers: a review on recycling and recovery route—current status and future prospects. Clean Technol Environ Policy 19:2363–2378. https://doi.org/10.1007/s10098-017-1443-9 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice. Nucleic Acids Res 22(22):4673–4680. https://doi.org/10.1093/nar/22.22.4673 Tindall BJ, Sikorski J, Smibert RA, Krieg NR (2007) Phenotypic characterization and the principles of comparative systematics. In: Reddy CA et al (eds) Methods for General and Molecular Microbiology. American Society for Microbiology, Washington, DC, pp 330–393. https://doi.org/10.1128/9781555817497.ch15 Tiwary E, Gupta R (2010) Extracellular expression of keratinase from Bacillus licheniformis ER-15 in Escherichia coli . J Agric Food Chem 58:8380–8385. https://doi.org/10.1021/jf100803g Verma A, Singh H, Anwar S, Chattopadhyay A, Tiwari KK, Kaur S et al (2017) Microbial keratinases: industrial enzymes with waste management potential. Crit Rev Biotechnol 37:476–491. https://doi.org/10.1080/07388551.2016.1185388 Wang B, Yang W, McKittrick J, Meyers MA (2016) Keratin: structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration. Prog Mater Sci 76:229–318. https://doi.org/10.1016/j.pmatsci.2015.06.001 Wang J, Hao S, Luo T, Cheng Z, Li W, Gao F et al (2017) Feather keratin hydrogel for wound repair: preparation, healing effect and biocompatibility evaluation. Colloids Surf B Biointerfaces 149:341–350. https://doi.org/10.1016/j.colsurfb.2016.10.038 Wang X, Parsons C (1997) Effect of processing systems on protein quality of feather meals and hog hair meals. Poult Sci 76:491–496. https://doi.org/10.1093/ps/76.3.491 Wang YQ, Yuan Y, Yu Z, Yang GQ, Zhou SG (2013) Bacillus borbori sp. nov., isolated from an electrochemically active biofilm. Syst Appl Microbiol 67(6):718–724. https://doi.org/10.1007/s00284-013-0426-2 Williams CM, Lee CG, Garlich JD, Shih JC (1991) Evaluation of a bacterial feather fermentation product, feather-lysate, as a feed protein. Poult Sci 70:85–94. https://doi.org/10.3382/ps.0700085 Yamaoka A, Kataoka M, Kawasaki K, Kobayashi E, Shigeri Y, Watanabe K (2014) Meiothermus ruber H328 enhances the production of membrane vesicles for feather degradation. Biosci Biotechnol Biochem 78(9):1623–1625. https://doi.org/10.1080/09168451.2014.918488 Zhou B, Guo Y, Xue Y et al (2023) Comprehensive insights into the mechanism of keratin degradation and exploitation of keratinase to enhance the bioaccessibility of soybean protein. Biotechnol Biofuels 16:177. https://doi.org/10.1186/s13068-023-02426-9 Supplementary Material Supplementary Materials are not available with this version. Supplementary Files RevisedversionofSupplimentarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 28 Jan, 2025 Read the published version in Biologia → Version 1 posted Reviewers agreed at journal 10 Dec, 2024 Reviewers invited by journal 10 Dec, 2024 Editor assigned by journal 09 Dec, 2024 First submitted to journal 06 Dec, 2024 Editorial decision: Minor revisions 23 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5090232","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":388374099,"identity":"8dce3fee-dcf2-4b87-8624-104211bce15e","order_by":0,"name":"Mane Tadevosyan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Mane","middleName":"","lastName":"Tadevosyan","suffix":""},{"id":388374100,"identity":"4ca77fa7-aba0-4aa7-86cf-009f0ed6896c","order_by":1,"name":"Armine Margaryan","email":"","orcid":"","institution":"Yerevan State University","correspondingAuthor":false,"prefix":"","firstName":"Armine","middleName":"","lastName":"Margaryan","suffix":""},{"id":388374101,"identity":"2f0e9a21-30c3-4519-8735-16b088887d21","order_by":2,"name":"Hovik Panosyan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACAziLvYFkLTwHSNYikUCkFnPp5mcPPvw5LG8u+cb4Mw+DXTRBLZZzjpkbzmw7bLhzdo6ZNA9Dcm4DQYfdSDCT5m04zLjhdo4ZMw8DMzFa0r9J8/w5bL/h5hmQw+qJ0QJyD9vhxA03eAyADjtMhJY7Z8okZ7alJ284k1YmOcfgOBFabrdvk/jwx9p2w/HDmz+8qagmrIVBAkw2AzGHAXI0EdRSB8TsD4hRPwpGwSgYBSMQAADYID/GdlY5xQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4891-0922","institution":"Yerevan State University","correspondingAuthor":true,"prefix":"","firstName":"Hovik","middleName":"","lastName":"Panosyan","suffix":""}],"badges":[],"createdAt":"2024-09-14 17:51:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5090232/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5090232/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11756-025-01877-9","type":"published","date":"2025-01-28T15:58:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71165238,"identity":"87134145-44f5-4a80-beab-6d27ad6f8297","added_by":"auto","created_at":"2024-12-11 17:21:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":330852,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent pattern of feather decay in enrichment culture. A. 24th h of incubation B. 72nd h of incubation C. 96th h of incubation.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5090232/v1/aae72cad615b91acaf228639.png"},{"id":71165241,"identity":"f4e8fd47-06c8-483b-b4ca-864a9b1ac50a","added_by":"auto","created_at":"2024-12-11 17:21:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93947,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on nearly complete 16S rRNA gene sequences, showing the relationships between isolated of bacilli strains (indicated by circle) obtained from geothermal springs of Armenia and Nagorno-Karabakh and closely related type strains. Evolutionary analyses were conducted in MEGAX using the neighbor-joining method. The percentage of rep licate trees (\u0026gt; 55%) in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. \u003cem\u003eThermobacilus composti\u003c/em\u003e and \u003cem\u003eThermobacillus xylanilyticus\u003c/em\u003e were used as out-groups to root the tree. Scale bar represents 0.02 substitutions per site.\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5090232/v1/4f91d948b845232be00981ea.png"},{"id":71165239,"identity":"2008b92c-3852-4edf-b2c7-79012228a04f","added_by":"auto","created_at":"2024-12-11 17:21:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":828030,"visible":true,"origin":"","legend":"\u003cp\u003eFeather-degrading potential of pure isolates. Cultivation was performed at optimal growth temperature for 72 h and visually checked for feather degradation. A. Non-inoculated control was incubated at the same conditions; B. M3; C. M4; D. M12; E. M14.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5090232/v1/62c776498cecc92340ebc4ff.png"},{"id":71165865,"identity":"cb72548d-9e6c-476b-9d9e-adc790ec234e","added_by":"auto","created_at":"2024-12-11 17:29:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22127,"visible":true,"origin":"","legend":"\u003cp\u003eThe feather-degrading activity of the strains depending on the substrate concentration.\u003c/p\u003e\n\u003cp\u003eThis figure illustrates the relationship between substrate concentration and the percentage of feather decay after enzymatic degradation. The percentage of substrate hydrolysis was calculated by measuring the dry mass of feathers before and after degradation. The initial dry mass of the feathers was recorded, followed by the dry mass post-degradation.\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5090232/v1/acfe8bf68fb221c225360cad.png"},{"id":71165866,"identity":"b8c57356-9cb9-4f5f-8f66-caa7ceb3309d","added_by":"auto","created_at":"2024-12-11 17:29:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":220081,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of growth and keratinolytic proteases activity by strain \u003cem\u003eB. borbori\u003c/em\u003e M14. at 55 °C, pH 7, 240 rpm in the medium A. Samples were taken in 1 \u0026nbsp;h interval and assayed for growth (blue line) and enzyme activity (red line)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5090232/v1/d31aac9570f8e7b7418567d1.png"},{"id":71166037,"identity":"ea32cd88-4a71-4850-9792-f6ab3e6edf58","added_by":"auto","created_at":"2024-12-11 17:37:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":230823,"visible":true,"origin":"","legend":"\u003cp\u003eA comparative overview of optimal growth temperature and the duration needed for feather degradation by different bacteria.\u003c/p\u003e","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-5090232/v1/5e427126081aa45ef99d6a4c.png"},{"id":75351347,"identity":"660d29d9-ba7d-4a84-a627-2da52dd5a42c","added_by":"auto","created_at":"2025-02-03 16:09:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3653316,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5090232/v1/515a6ab0-31cb-4b03-8e99-4f72837911b8.pdf"},{"id":71165259,"identity":"4180c846-afa0-4a08-826f-7573e64c2a61","added_by":"auto","created_at":"2024-12-11 17:21:22","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3606708,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedversionofSupplimentarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5090232/v1/6885eb3a7e6bd503d6d2ee8d.docx"}],"financialInterests":"","formattedTitle":"Assessment of feather degrading activity of thermophilic bacilli isolated from Armenian geothermal springs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKeratin is a recalcitrant structural protein and the third most abundant polymer globally, following cellulose and chitin (Lange et al. 2016). It forms the structural basis of various biological materials, including skin, feathers, wool, hair, hooves, and horns. Keratin is classified into two types based on its secondary structure: α-keratin and β-keratin. α-Keratin is found in mammalian epidermal materials such as hair, skin, and wool, whereas β-keratin, which contains more cysteine residues and forms stronger disulfide bonds, is found in bird feathers and reptile scales. This structural stability makes feather keratin particularly resistant to protease degradation (Wang et al. 2016, Tesfaye et al. 2017, Qiu et al. 2020).\u003c/p\u003e \u003cp\u003eDespite its abundance, keratin\u0026rsquo;s potential as a source of amino acids and oligopeptides remains underutilized (Gupta et al. 2013; Wang et al. 2017; Qiu et al. 2020; Zhou et al. 2023). Feather waste, a major by-product of the poultry industry, accounts for 5\u0026ndash;7% of chicken body weight (Li 2019). With millions of tons produced annually feathers offer an ideal source of keratin due to their high protein content 90% (Verma et al. 2017; da Silva 2018). However, untreated feather waste poses environmental and health risks, serving as a habitat for pathogenic microbes and releasing pollutants such as nitrous oxide, ammonia, and hydrogen sulfide (Tamreihao et al. 2019). Processed feathers have diverse applications including decorative materials, medical devices, bedding materials, and feedstock (Li 2019).\u003c/p\u003e \u003cp\u003eTraditional methods, such as chemical treatment or stem pressure cooking, are economically unviable and often degrade essential amino acids (Papadopoulos 1989; Latshaw et al. 1994; Wang and Parsons 1997; Kumar et al. 2021). Consequently, biotechnological approaches such as the use of keratinolytic enzymes offer a promising alternative․ The biological degradation of keratin by keratinolytic proteases represents cost-effective, and environmentally friendly method for its decomposition.\u003c/p\u003e \u003cp\u003eKeratinases (EC 3.4.-.- peptide hydrolases) are hydrolytic enzymes that break down the hard-to-degrade keratin efficiently (Verma et al. 2017).\u003c/p\u003e \u003cp\u003eVarious bacteria bacteria (\u003cem\u003eBacillus, Streptomyces, Nocardiopsis\u003c/em\u003e) and fungi (non-pathogenic and dermatophytic pathogenic) secrete keratinolytic enzymes, enabling the conversion of feather waste into value-added products (Li 2019; Hassan et al. 2020; Tadevosyan et al. 2022). Thermophilic microbes, with their unique genomic adaptations, represent an untapped resource for thermostable keratinases with broad industrial applications (Cavello et al. 2018; Qiu et al. 2020; Javier-Lopez et al. 2023). Keratinase, from thermophilic microbes, due to their thermostability and wide substrate specificity, are considered as a better catalyst in feed, biofertilizer, detergent, leather, and pharmaceutical/biomedical industries (Gupta et al. 2013; Li 2019; Zhou et al. 2023).\u003c/p\u003e \u003cp\u003eThermophilic keratinolytic protease producing microbes are phylogenetically very diverse. The majority of studied keratinolytic microbes such as \u003cem\u003eFervidobacterium pennivorans\u003c/em\u003e (Friedrich and Antranikian 1996), \u003cem\u003eFervidobacterium islandicum\u003c/em\u003e AW-1 (Nam et al. 2002), \u003cem\u003eThermoanaerobacter keratinophilus\u003c/em\u003e 2KXI (Riessen and Antranikian 2001), \u003cem\u003eCaldanaerobacter\u003c/em\u003e sp. strain 1523-1 (Kublanov et al. 2009), \u003cem\u003eClostridium sporogenes\u003c/em\u003e bv. \u003cem\u003epennavorans\u003c/em\u003e (Ionata et al. 2008), \u003cem\u003eFervidobacterium pennivorans\u003c/em\u003e subsp. \u003cem\u003ekeratinolyticus\u003c/em\u003e strain T (Javier-Lopez et al. 2023) are anaerobic microorganisms. Reports on thermophilic aerobic keratinase producers are scare. Only few reports are available about thermophilic aerobic keratinase producing bacteria isolated from geothermal areas or hot springs (Yamaoka et al. 2014; Cavello et al. 2018).\u003c/p\u003e \u003cp\u003eNumerous high-altitude geothermal springs of different geotectonic origins, and with different physicochemical properties, are found within the territory of Armenia and Nagorno-Karabakh (Henneberger et al. 2000; Panosyan et al. 2018). Initial studies based on both metagenomics and culture-dependent analyses of the microbiota from certain Armenian geothermal springs have revealed diverse thermophilic bacteria and archaea with hydrolytic potential (Panosyan et al. 2020; Saghatelyan et al. 2021; Burkhardt et al. 2024). However, the diversity and biotechnological potential of keratinase producing thermophilic bacilli from these springs remain largely unexplored\u003c/p\u003e \u003cp\u003eThis study focuses on isolating and characterizing keratinolytic thermophilic aerobic bacilli from high-elevated Armenian geothermal springs. It demonstrates the synergistic keratin-degrading capabilities of bacterial consortia and highlights their potential for sustainable applications in the bio-based circular economy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eSludge samples or sediments were aseptically collected from the outlet of geothermal springs located in Karvachar (40\u0026deg;17\u0026prime;41.00\" N; 46\u0026deg;27\u0026prime;50.00\" E), Zuar (40\u0026deg;02\u0026prime;47.60\u0026prime;\u0026rsquo; N; 46\u0026deg;14\u0026prime;09.30\u0026prime;\u0026rsquo; E) and Arzakan (40\u0026deg;27\u0026prime;36.10\u0026Prime; N, 44\u0026deg;36\u0026prime;17.76\u0026Prime; E) in 2020. The samples were placed in sterilized thermostatic bottles to maintain habitat temperature until further processing for incubation and isolation. Temperature, pH and conductivity of the spring water were measured \u003cem\u003ein situ\u003c/em\u003e with a HANNA HI98129/HI98130 portable instrument. The geographical locations and elevations of the springs were determined using a portable global positioning system (GPS) device (GARMIN 64 s). The map and images of the sampling locations are shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS, and the main characteristics of the collected samples are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e,\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnrichment and isolation\u003c/h3\u003e\n\u003cp\u003eThe watery sludge or sediment samples (1 g) were suspended in 10 mL sterile water and mixed for 1 min to obtain the enrichment cultures. The supernatant was pasteurized at 80\u0026deg;C for 10 min to isolate only endospore-forming bacilli. Aliquots (1.0 mL) were inoculated in 100 mL Erlenmeyer flask containing 5.0 g of chicken feathers as the keratin source, along with 100 mL media with following composition (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): yeast extract, 0.5; peptone, 0.5; NaNO\u003csub\u003e3\u003c/sub\u003e, 5.0; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 5.0; NaCl, 10.0; MgSO\u003csub\u003e4\u003c/sub\u003e\u0026bull;7H\u003csub\u003e2\u003c/sub\u003eO, 0.4; CaCl\u003csub\u003e2\u003c/sub\u003e\u0026bull;H\u003csub\u003e2\u003c/sub\u003eO, 0.2, at pH 7.2. Chicken feathers (white, brown, and black colored) from Yerevan broiler chickens were collected from local slaughterhouses and private hen hatcheries. Feathers were first washed with tap water and cut into 5mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e sections and treated by shaking in 70% ethanol for 5 min at room temperature. They were then soaked in 0.9% bleach for 30 min, rinsed with distilled water, washed with a 10% sodium bicarbonate solution, rinsed again with distilled water and air-dried. Alternatively, feathers were sterilized by autoclaving at 121\u0026deg;C for 15 min to preserve the native structure of the feather.\u003c/p\u003e \u003cp\u003eTo assess the impact of acidic and alkaline conditions on feather degradation 1 g of native feathers were treated with 20 mL of 12% HCl solution (pH 2) or 1 M NaOH solution (pH 9) and stored in a closed container for 30 min (Istrate et al. 2013). Untreated feathers sereved as controls. The treated feathers were then washed with sterile distillated water to neutralize the acid/base. Both treated and untreated feathers were dried in an oven.\u003c/p\u003e \u003cp\u003eEnrichments were incubated in an orbital shaker at 180 rpm at 55\u0026deg;C until feather degradation was visually confirmed (1\u0026ndash;5 days). Turbid cultures (0.1 mL, and the standard serial dilution 1/10, 1/100) were streaked on nutrient nutrient broth (NB, Liofilchem, Italy) agar (1.5%) plates and incubated at 50, 55, 60 and 65\u0026deg;C for 24 h. Single colonies exhibiting different morphologies were isolated and subcultured by streaking onto the same medium at least three times to obtain pure monoculture. Subcultures were considered pure after microscopic examination revealed a single morphological type per culture. The purity and cell morphology were determined by phase-contrast microscopy (OMAX, M8311 USA) of freshly prepared wet mounts.\u003c/p\u003e \u003cp\u003eAll isolates were stored at 4\u0026deg;C, continuously subcultured, and preserved at \u0026minus;\u0026thinsp;80\u0026deg;C in nutrient broth containing 20% glycerol. All isolates are maintained in the extremophilic microbial culture collection at the Department of Biochemistry, Microbiology and Biotechnology of Yerevan State University, Armenia.\u003c/p\u003e\n\u003ch3\u003eDNA extraction, 16S rDNA amplification and phylogenetic characterization\u003c/h3\u003e\n\u003cp\u003eTotal bacterial DNA was extracted using the GenEluteTM Bacterial Genomic DNA Kit (Sigma, Germany) following the manufacturer\u0026rsquo;s protocol. The DNA was eluted with 50 \u0026micro;L of Tris\u0026ndash;EDTA buffer (TE) and used as a template for amplifying the 27-1492 region of the 16S rRNA gene via polymerase chain reaction (PCR). The universal primers 16SF (5'- GAGTTTGATCCTGGCTCAG-3') and 16SR (5'-GAAAGGAGGTGATCCAGCC-3') (\u003cem\u003eEscherichia coli\u003c/em\u003e numbering) were used for amplification. PCR mixtures used for amplification of sequences contained 10 ng of DNA, 5 \u0026micro;L 10 \u0026times; PCR buffer, 5 \u0026micro;L 10 mM dNTP (dATP, dGTP, dCTP and dTTP), 1 \u0026micro;L of each primer (25 pmol \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), 0.2 \u0026micro;L Taq DNA polymerase and sterile water to make up a final volume of 50 \u0026micro;l. PCR amplification was conducted using a DNA Engine thermocycler (BIO-RAD Thermo Scientific Arktik Thermal Cycler). First, the templates were denaturized for 3 min at 96\u0026deg;C, then 30 cycles of the following steps were completed: denaturation for 30 s at 96\u0026deg;C, annealing for 30 s at 55\u0026deg;C and extension for 2.5 min at 72\u0026deg;C. The 30 cycles were followed by a final 10 min extension at 72\u0026deg;C. PCR products were viewed under UV light after standard ethidium bromide gel electrophoresis. PCR products were purified using the GenElute\u0026trade; PCR Cleanup Kit (Sigma, Germany).\u003c/p\u003e \u003cp\u003e16S rDNA sequencing of bacterial amplicons was performed using the ABI PRISM capillary sequencer, according to the protocol of the ABI Prism Big-Dye Terminator kit (Perkin Elmer), using above mentioned primers. Raw data of the DNA sequences were analyzed using the BioEdit software. Chimeric sequences were identified using the DECIPHER web tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://decipher.cee.wisc.edu/FindChimeras.html\u003c/span\u003e\u003cspan address=\"https://decipher.cee.wisc.edu/FindChimeras.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A nucleotide sequences were subjected to a BLAST search to find phylogenetically closest relatives (National Center for Biotechnology Information, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Blast\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Altschul et al. 1997). The assembled 16S rRNA gene sequences were aligned with a representative set of 16S rRNA gene sequences from GenBank database using ClustalW (Thompson et al. 1994). A phylogenetic tree was constructed using the neighbor-joining method with the MEGA X software ((Saitou and Nei, 1987; Kumar et al. 2018). Confidence in branching points was determined by bootstrap analysis (1000 replicates).\u003c/p\u003e\n\u003ch3\u003ePhenotypic characterization\u003c/h3\u003e\n\u003cp\u003eThe morphological, physiological and biochemical (tests for oxidase, anaerobic growth, Voges\u0026ndash;Proskauer reaction, hydrolysis of starch and gelatin, citrate utilization, nitrate reduction, H\u003csub\u003e2\u003c/sub\u003eS and indole production) characteristics of the isolate were investigated as described by Smibert and Krieg (1981). Each experiment was conducted in triplicates.\u003c/p\u003e \u003cp\u003eCell morphology, sizes, endospore formation and its location, and motility were determined by phase-contrast microscopy (Nikon; Eclipse E400 microscope). The temperature range for growth was determined by incubating the strains in NB medium from 30 to 80\u0026deg;C at 5\u0026deg;C temperature intervals. The effect of pH on growth was examined at optimal growth temperature in the range 5.0\u0026ndash;12.0, with increments of 0.5 pH units adjusted with 1 M NaOH or 1 M HCl. The range of NaCl concentrations for growth was tested by supplementing 0.5\u0026ndash;5% (w v\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) NaCl at intervals of 0.5% w v\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Growth was assessed by measuring optical density (OD) at λ 540 nm. Catalase activity was evaluated by bubble formation in 3% hydrogen peroxide solution (Tindall et al. 2007).\u003c/p\u003e \u003cp\u003eAnaerobic growth was checked by inoculation of microbes in tubes containing NB medium supplemented with agar (2%, w v\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The ability to utilize various carbon sourses (D-glucose, L-arabinose, D-xylose, D-sucrose, D-fructose, inositol, mannitol) and produce acids was assessed in the medium containing (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) carbon source, 10; (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1; KCl, 0.2; MgSO\u003csub\u003e4\u003c/sub\u003e\u0026bull;7H\u003csub\u003e2\u003c/sub\u003eO, 0.2; yeast extract, 0.2 by the addition of 15 mL 0.04% w v\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e solution of bromocresol purple.\u003c/p\u003e \u003cp\u003eProteolytic (caseolytic) activity was detected by the formation of clear zones on 0.5% skim milk agar (pH 7.0) (Kim et al. 2001).\u003c/p\u003e\n\u003ch3\u003eFeather degradation in mono-species and co-cultures\u003c/h3\u003e\n\u003cp\u003eThe ability to the strains to degrade keratin was assessed by inoculating 5% of 16 h old cultures into 100 mL Erlenmeyer flask containing 5.0 g of chicken feathers as the sole carbon and nitrogen sources, along with 100 mL of mineral media with following composition (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): NaNO\u003csub\u003e3\u003c/sub\u003e, 5.0; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 5.0; NaCl, 10.0; MgSO\u003csub\u003e4\u003c/sub\u003e\u0026bull;7H\u003csub\u003e2\u003c/sub\u003eO, 0.4; CaCl\u003csub\u003e2\u003c/sub\u003e\u0026bull;H\u003csub\u003e2\u003c/sub\u003eO, 0.2, pH 7.2. Incubaction was performed at 50\u0026deg;C or 65\u0026deg;C for 96 h and checked visually for feather degradation.\u003c/p\u003e \u003cp\u003eDifferent substrate amounts (5, 10, 20, 30, 40, 50 and 60 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were tested to investigate feather-degrading activity.\u003c/p\u003e \u003cp\u003eKeratin mass loss was determined by filtering the bacterial cultures through pre-weighed filter paper (12\u0026ndash;15 \u0026micro;m particle retention) (Frisenette APS, Denmark). The feather was washed to remove attached bacteria and the filter paper was dried at 50˚C for 48 h and re-weighed. Media without bacteria served as controls (Nasipuri et al. 2020).\u003c/p\u003e \u003cp\u003eKeratin degradation in co-cultures of most active four isolates was performed using equal volumes (1 mL)of turbid mono-cultures in 250 mL shake flasks containing feather and mineral medium for 3 days at 55˚C, with shaking at180 rpm. Degradation from co-cultivation was evaluated for all combinations of dual-species cultures.\u003c/p\u003e \u003cp\u003eMicrobial growth and keratinolytic protease production kinetics were monitored in batch culture at 55\u0026deg;C, pH 7.0, with shaking at 240 rpm, by sampling 10 mL of culture broth every 1 h during 12 h. Microbial growth was estimated by recording optical density (OD) at 560 nm.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKeratinolytic protease enzyme assay\u003c/h2\u003e \u003cp\u003eKeratinolytic protease activity was studied using an overnight culture in a minimal nutrient medium where feather served as the sole source of carbon and nitrogen. Three samples were taken every hour for 12 h. The first samples was used to measure OD of microbial cells at 560 nm. In the second sample, the protein concentration was determined using the Bradford method (Bradford 1976). Standard curve for albumin (using 0.1, 0.5, 1, 2.5, and 5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e solutions of albumin) was prepared. 100 \u0026micro;L of the second sample was mixed with 1000 \u0026micro;L of Bradford solution. After incubation for 5\u0026ndash;10 min, OD was measured at 595 nm.\u003c/p\u003e \u003cp\u003eTo create a standard curve for tyrosine, solutions of tyrosine (0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were used. A100 \u0026micro;L aliquot from the third sample was mixed with 500 \u0026micro;L of β-casein solution (65 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in phosphate buffer pH-7․38). After 20 min of incubation at 55\u0026deg;C, 400 \u0026micro;L of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution and 130 \u0026micro;L of Folin and Ciocalteu's reagent (Sigma, Germany) were added, followed by a further 30 min incubation at 37\u0026deg;C. OD was measured at 660 nm, and standard curves were constructed using Excel.\u003c/p\u003e \u003cp\u003eThe protease activity was calculated using the formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\frac{\\text{U}\\text{n}\\text{i}\\text{t}\\text{s}}{\\text{m}\\text{L}\\:\\text{e}\\text{n}\\text{z}\\text{y}\\text{m}\\text{e}}=\\frac{\\left({\\mu\\:}\\text{m}\\text{o}\\text{l}\\:\\text{t}\\text{y}\\text{r}\\text{o}\\text{s}\\text{i}\\text{n}\\text{e}\\:\\text{e}\\text{q}\\text{u}\\text{i}\\text{v}\\text{a}\\text{l}\\text{e}\\text{n}\\text{t}\\text{s}\\:\\text{r}\\text{e}\\text{l}\\text{e}\\text{a}\\text{s}\\text{e}\\text{d}\\right)\\cdot\\:\\left(\\text{a}\\right)}{\\left(\\text{b}\\right)\\cdot\\:\\left(\\text{c}\\right)\\cdot\\:\\left(\\text{d}\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere: a, total assay volume (mL); b, volume of enzyme used (mL); c, assay time (min); d, volume used in colorimetric determination (in mL).\u003c/p\u003e \u003cp\u003eThe enzyme activity is expressed as \u003cb\u003eunits (U)\u003c/b\u003e, where one unit is defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per min under the assay conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHigh-performance liquid chromatography (HPLC) analyses of hydrolysis products\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA 500 \u0026micro;L aliquot was taken from the decomposed sample, where the feather was the sole source of carbon and nitrogen. This sample was then centrifuged to remove the feather particles. Amino acids determination was carried out following the method previously described by Aghajanyan et al. (2024). The amino acid analysis was conducted using a Shimadzu Nexera X2 amino acid analyzer (Shimadzu Corporation, Kyoto, Japan), equipped with an RF-20A fluorescence detector. Separation of the amino acids was achieved on a SUPELCOSIL LC-DABS chromatographic column (3 \u0026micro;m, 15 cm \u0026times; 4.6 mm). The mobile phase consisted of two components: (A) acetonitrile:methanol:water (45:40:15, v/v/v) and (B) a phosphate buffer at pH 6.8. Analysis was performed at a flow rate of 0.3 mL/min, with the column maintained at 30\u0026deg;C. Fluorescence detection was carried out with excitation at 350 nm and emission at 450 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNucleotide sequence accession numbers\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe 16S rRNA gene sequences were deposited in GenBank and the assigned accession numbers are as follows: PQ097580 - PQ097583.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eB. botbori\u003c/em\u003e M14 strain was deposited in the Microbial Depository Center of Armenia under accession numbers MDC 11863.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEnrichment\u003c/h2\u003e \u003cp\u003eThree watery sludge and sediment samples were analyzed to evaluate the abundance of thermophilic aerobic keratinolytic bacilli. For the primary enrichment of keratin degrading bacteria, the media mentioned above was supplemented with pretreated white, brown, and black colored chicken feathers. After incubation at 55\u0026deg;C in aerobic conditions, almost complete degradation of the feathers was observed within 4 days in the enrichment culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The feather color had no significant effect on degradation. Feathers collected from both local slaughterhouses and private hen hatcheries were hydrolyzed similarly.\u003c/p\u003e \u003cp\u003eTo enhance the degradation efficiency of the enrichment culture, feathers were treated with alkaline (NaOH, pH 9), and acidic (HCl, pH 2) solutions for primary hydrolysis. After alkaline solution treatment, the feathers were completely hydrolyzed within 96 h in an aerobic enrichment culture, whereas acid-treated feather required 120 h for complete degradation. Untreated feather similar to those treated with alkaline solutions, also required 96 h for complete degradation (Fig. S2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of isolated strains based on polyphasic approach\u003c/h2\u003e \u003cp\u003eA primary culture of aerobic thermophilic bacteria capable of degrading feathers was successfully developed from watery sludge and sediment samples collected from the geothermal springs in Karvachar, Zuar, and Arzakan. A total of twenty isolates with different colony morphologies were obtained on NB agar plates and were characterized. The isolates were designated as M1-M20. A large number of isolates with varied colony morphologies and cell shapes were obtained from Karvachar (70\u0026deg;C) and Arzakan (44\u0026deg;C) geothermal springs, while only one or two morphotypes were observed from the Zuar (42\u0026deg;C) hot spring (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKeratin degrading thermophilic isolates originating from geothermal spring samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLocation of hot spring\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample origin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHot spring water temperature, \u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHot spring water pH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eNumber of isolates obtained\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrain name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eNB agar\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eFeather enrichment\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKarvachar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWater with fine clay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eM3, M2, M10, M11, M13, M14, M17-M20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArzakan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSilicate sand\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eM1, M5, M6-M8, M12, M15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZuar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWatery Sand/clay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eM4, M16, M9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eColonies of the isolates were transparent, creamy, white, milky in color, opaque or translucent, with rough, smooth or glossy surfaces and regular or irregular edges (Fig. S3). All isolates were rod-shaped, Gram-positive, endospore-forming, and catalase and oxidase-positive bacteria.\u003c/p\u003e \u003cp\u003eFour isolates designated as M4 (Zuar), M12 (Arzakan), M3 and M14 (Karvachar) were selected prioritizing their ability to grow in enrichment media with feathers as a sole carbon and nitrogen source and to degrade feathers completely.\u003c/p\u003e \u003cp\u003eAll studied strains were motile Gram positive, endospore forming, and tested positive for both oxidase and catalase. The Voges\u0026ndash;Proskauer test was positive for the isolate. Isolates M3 and M4 were shown growth under anaerobic conditions and can be described as facultative aerobes, while the growth of M12 and M14 strains in anaerobic conditions was weak. At optimal growth conditions, all isolates were capable of reducing nitrate. The specific phenotypic properties that distinguish these strains from related reference type strains include differences in growth conditions (such as temperature and pH ranges), enzyme production and ability to utilize various carbon sources. None of the isolates were able to grow below 30\u0026deg;C, or above 65\u0026ndash;70\u0026deg;C. Optimal growth temperature for M3 and M4 was observed at 50\u0026deg;C, while M12 and M14 grew best at 65\u0026deg;C, thus categorizing them as moderate thermophiles. No growth was observed when the NaCl concentration exceeded 5% (w v\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). In comparison to closely related strains, all isolates displayed a broad pH growth range. None of the studied strains could utilize the D-xylose and citrate, produce indole or H\u003csub\u003e2\u003c/sub\u003e, or synthesize urease. The strain M14 could not assimilate sucrose, while strain M12 could not utilize arabinose. Unlike the type strain of \u003cem\u003eBacillus borbori\u003c/em\u003e type strain, strains M12 and 14 showed the ability to hydrolase casein. The nitrate reduction to nitrite, Voges\u0026ndash;Proskauer (VP) reaction, production of H\u003csub\u003e2\u003c/sub\u003eS and indole, citrate utilization, and enzymatic activities such as β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease and tryptophan deaminase were tested for strain M14 strain using an API 20 E strip (bioM\u0026eacute;rieux)., according to the manufacturer\u0026rsquo;s instructions The strain was capable to reduce nitrate to nitrite and was positive for the VP reaction. However, it was negative for urease, H\u003csub\u003e2\u003c/sub\u003eS,and indole production, and lacked β-galactosidase, and ornithine-decarboxylase activities. It exhibited arginine dihydrolase, lysine-decarboxylase, gelatinase activity, and assimilated tryptophan.\u003c/p\u003e \u003cp\u003eThe identification of the four isolates was confirmed by sequencing the almost complete 16S rRNA genes. The sequences revealed that all isolates were closely related to the genus \u003cem\u003eBacillus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Isolates M3 and M4 shared 97.02% and 95.85% similarity, respectively, to \u003cem\u003eBacillus licheniformis\u003c/em\u003e, suggesting that they may represent a potential new species.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBLAST results of 16S rRNA gene sequences of feather degrading thermophilic bacilli and accession numbers.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClosest match taxonomic affiliation, phylotype accession no\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Similarity to closest match\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAccession no\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus sp.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eMK418572.1\u003c/p\u003e \u003cp\u003e\u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/p\u003e \u003cp\u003eLT669756.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.22\u003c/p\u003e \u003cp\u003e97.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePQ097581\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus sp.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eMK418572.1\u003c/p\u003e \u003cp\u003e\u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/p\u003e \u003cp\u003eLT669758.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePQ097583\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1456\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus borbori\u003c/em\u003e\u003c/p\u003e \u003cp\u003eNR132725.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePQ097580\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBacillus borbori\u003c/em\u003e\u003c/p\u003e \u003cp\u003eNR132725.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePQ097582\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe other two isolates were related to \u003cem\u003eBacillus borbori\u003c/em\u003e with 99.52% and 99.72% similarity, respectively. The isolates associated with \u003cem\u003eBacillus borbori\u003c/em\u003e were obtained from Karvachar and Arzakan geothermal areas, while those associated with the \u003cem\u003eB. licheniformis\u003c/em\u003e were isolated from samples Karvachar and Zuar geothermal areas.\u003c/p\u003e \u003cp\u003eThe phylogenetic tree based on the 16S rRNA genes of isolated strains and selected reference sequences from GenBank was constructed using the MEGA-X program (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). High bootstrap values (above 90%) for the clades with strains M3, M4 and \u003cem\u003eB. licheniformis\u003c/em\u003e and M12, M14 with \u003cem\u003eB. borbori\u003c/em\u003e, supports the current clade structure. Further studies are necessary to confirm the taxonomic affiliation of these isolates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhenotypic characteristics of keratin degrading isolates were compared with those of reference strains: \u003cem\u003eBacillus subtilis\u003c/em\u003e ATCC6051\u003csup\u003eT\u003c/sup\u003e (Schleifer, 2009), \u003cem\u003eBacillus borbori\u003c/em\u003e DX-4\u003csup\u003eT\u003c/sup\u003e (Wang et al. 2013) and \u003cem\u003eBacillus licheniformis\u003c/em\u003e ATCC 14580\u003csup\u003eT\u003c/sup\u003e (Schleifer 2009). Results are reported in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of phenotypic properties of thermophilic feather degrading bacilli isolated from Arzakan geothermal spring with its nearest phylogenetic neighbor \u003cem\u003eBacillus\u003c/em\u003e species type strains and type species of genus \u003cem\u003eBacillus\u003c/em\u003e (1, \u003cem\u003eB. subtilis\u003c/em\u003e ATCC6051\u003csup\u003eT\u003c/sup\u003e; \u003cem\u003eB. borbori\u003c/em\u003e DX-4\u003csup\u003eT\u003c/sup\u003e; 3, \u003cem\u003eB. licheniformis\u003c/em\u003e ATCC 14580\u003csup\u003eT\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c3\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eM12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eM14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell size (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.6\u0026ndash;0.8 \u0026times;\u003c/p\u003e \u003cp\u003e1.6\u0026ndash;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4\u0026ndash;0.9 \u0026times;\u003c/p\u003e \u003cp\u003e1.3-3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3\u0026ndash;0.5 \u0026times;\u003c/p\u003e \u003cp\u003e1.0-2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2\u0026ndash;0.6 \u0026times;\u003c/p\u003e \u003cp\u003e1.5\u0026ndash;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.7\u0026ndash;0.8 \u0026times; 2.0\u0026ndash;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.2\u0026ndash;0.5 \u0026times;\u003c/p\u003e \u003cp\u003e1.2-2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.6-08 \u0026times;\u003c/p\u003e \u003cp\u003e1.5-3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpore form/location\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eE/C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eE/C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eE/S or T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE/S or T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eE/C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eE/S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eE/C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSwell sporangia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature range (T\u003csub\u003eopt\u003c/sub\u003e) (\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e30\u0026ndash;65\u003c/p\u003e \u003cp\u003e(50\u0026ndash;55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u0026ndash;65\u003c/p\u003e \u003cp\u003e(50\u0026ndash;55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30\u0026ndash;70 (55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30\u0026ndash;70 (55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u0026thinsp;\u0026minus;\u0026thinsp;5\u003c/p\u003e \u003cp\u003e(28\u0026ndash;30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e30\u0026ndash;65\u003c/p\u003e \u003cp\u003e(55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20\u0026ndash;55\u003c/p\u003e \u003cp\u003e(Nd)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl range (optimum) (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0\u0026ndash;5.0 (\u0026gt;\u0026thinsp;1.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026ndash;5.0 (\u0026gt;\u0026thinsp;1.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026ndash;5.0 (1.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026ndash;4.0 (1.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u0026ndash;7.0\u003c/p\u003e \u003cp\u003e(\u0026gt;\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u0026ndash;6.0\u003c/p\u003e \u003cp\u003e(Nd)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.0\u0026ndash;7.0\u003c/p\u003e \u003cp\u003e(Nd)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH range (optimum)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e5.0-10.5 (7.5-8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0-9.5 (7.5-8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.0\u0026ndash;9.0 (7.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.0\u0026ndash;9.0 (7.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.5\u0026ndash;8.5\u003c/p\u003e \u003cp\u003e(7.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.0-8.5\u003c/p\u003e \u003cp\u003e(7.0-7.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eND\u003c/p\u003e \u003cp\u003e(5.7\u0026ndash;6.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnaerobic growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVoges-Proskauer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ev\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ev\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcid from\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Fructose-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Arabinose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Xylose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Sucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInositol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMannitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydrolysis of\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCasein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGelatin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUtilization of\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrate reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProduction of\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ea\u003c/sup\u003eData from Schleifer (2009);\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003eb\u003c/sup\u003eData from Wang et al, (2013);\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e+, Positive; \u0026ndash;, negative; \u0026plusmn;, weakly positive result; v, variable; Nd, not determined; E, ellipsoidal; C, central; T, terminal; S, subterminal.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFeather degradation in mono-species and co-cultures\u003c/h2\u003e \u003cp\u003eThe pure cultures of M12 and M14 isolates completely degraded the feather after 72 h. The M14 strain proving to be the most effective in breaking down feathers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe hydrolytic activity of the most potent feather-degrading bacilli strains was investigated in case of different amount of the substrate. Results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The highest hydrolytic activity was observed when the feather amount in the enrichment culture was 40 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn community-based degradation experiments, feather degradation was compared to that of single species cultures to explore whether the community-intrinsic properties could enhance the process. Feather degradation was evaluated in dual-species co-cultures (Fig. S4)․ Co-cultures containing \u003cem\u003eB. borbori\u003c/em\u003e M12 or \u003cem\u003eB. borbori\u003c/em\u003e M14 exhibited enhanced keratin degradation compared to the individual strains. Feather degradated reached 86% in the co-culture of M12 and M14 after 48 h incubation at optimal growth conditions. Co-cultures of M4 with M12 or M14 also displayed significantly improved decomposition of the feather with rates of 82\u0026ndash;85%. The lowest degradation rate (30%) was observed in co-cultures of M3 and M4 strains (Fig. S5).\u003c/p\u003e \u003cp\u003eAmong all strains tested \u003cem\u003eB. borbori\u003c/em\u003e M14 was the best keratin degrade showing high culture density and keratin degradation potential. Based on these findings subsequent experiments on keratinolytic enzymatic activities were conducted using only on \u003cem\u003eB. borbori\u003c/em\u003e M14.\u003c/p\u003e \u003cp\u003eThe growth and production of keratinolytic proteases in \u003cem\u003eB. borbori\u003c/em\u003e M14 were monitored over the 12 h period at the optimum growth temperature of 55\u0026deg;C under aerobic conditions (240 rpm). Growth was measured by optical density (OD) at 560 nm and maximum growth was observed at 9 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Enzymatic activity increased significantly from 8 h onwards (late exponential phase) reaching its peak at 0. 013 U mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during the stationary phase indicating a growth-associated production of enzymes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHPLC analysis feather hydrolysis products indicated that aspartic acid and isoleucine were the main end products, followed by leucine, phenylalanine, alanine, tyrosine and glutamic acid (Fig. S6, Table S2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBioconversion of poultry feathers into value-added products such as biofertilizers and high nutrient animal feeds is an attractive strategy, particularly through the use of thermostable enzymes. Thermophilic bacteria that produce keratinolytic enzymes, such as keratinases, are especially important for the bioconversion of keratin, which is highly resistant to degradation. The demand for thermostable keratinolytic enzymes from industries like detergent, leather, textile, food, and pharmaceuticals continues to rise, highlighting the ongoing need for robust and specific enzymes (Gupta et al. 2013; Qiu et al. 2020; Zhou et al. 2023).\u003c/p\u003e \u003cp\u003eThermostable keratinases found in high temperature environments like geothermal springs have emerged as key sources of these enzymes. Recent studies have shown that high altitude hot springs distributed on the territory of Armenia and Nagorno Karabakh harbor thermophilic bacilli with promising hydrolytic activity indicating their potential for biotechnological applications (Panosyan et al. 2020; Saghatelyan et al. 2021). Inspired from this evidence, the keratinase producing bacterial diversity and the abundance of resident bacteria in Armenian geothermal springs were studied. Despite the growing body of research on thermophiles, few reports focus on microbes from hot springs capable of degrading native feathers. Most feather degrading obligate thermophilic microbes isolated from hot springs, geothermal vents and volcanic areas were strict anaerobes, which hinder their practical utilization (Friedrich and Antranikian 1996; Riessen and Antranikian 2001; Nam et al. 2002; Ionata et al. 2008; Kublanov et al. 2009; Cavello et al. 2018; Javier-Lopez et al. 2023). Aerobic feather degrading bacteria such as thermoalkalophilic \u003cem\u003eBacillus halodurans\u003c/em\u003e AH-101 (Takami et al. 1999), \u003cem\u003eBacillus pseudofirmus\u003c/em\u003e FA 30\u0026thinsp;\u0026minus;\u0026thinsp;01 (Kojima et al. 2006) \u003cem\u003eBacillus\u003c/em\u003e sp. JB 99 (Johnvesly et al. 2002; Shrinivas and Naik, 2011) have been isolated from environmental samples (soil, waste streams, sugarcane molasses and poultry wastes) but few come from hot springs. One exception is \u003cem\u003eMeiothermus ruber\u003c/em\u003e H328 isolated from a hot spring (Yamaoka et al. 2014). More recently studies in Patagonia, Argentina have explored the diversity of thermophilic aerobic bacilli capable of the few published efforts to examine the keratinase-producing diversity of thermophilic aerobic bacilli in geothermal springs (Cavello et al. 2018). This study represents one of the few published efforts to examine keratinase-producing diversity of thermophilic aerobic bacilli in geothermal springs\u003c/p\u003e \u003cp\u003eIn this study, samples from three Armenian geothermal springs were analyzed for their ability to degrade chicken feathers. Keratin is not soluble protein and its bioconversion is time-consuming process. To address this the effects of acidic (HCl) and alkaline (NaOH) treatments on feathers were evaluated, with alkaline treatment proving more affective (Fitriyanto et al. 2022). However untreated feathers required 96 h for complete degradation, which was similar to the alkaline-treated samples, prompting the use of untreated feathers for further experiments.\u003c/p\u003e \u003cp\u003eA total 20 aerobic thermophilic bacilli strains have been isolated from chicken feather enrichment cultures. Four of these strains were capable of completely degrading feathers at 55\u0026deg;C, confirming their moderate thermophilic nature.\u003c/p\u003e \u003cp\u003eTwo strains (M3 and M4) were closely related to \u003cem\u003eB. licheniformis\u003c/em\u003e (95\u0026ndash;97% similarity), while other two strains (M12 and M14) shared over 99% similarity to \u003cem\u003eB. borbori\u003c/em\u003e. The low (\u0026lt;\u0026thinsp;97%) similarity, of the M3 and M4 strains to \u003cem\u003eB. licheniformis\u003c/em\u003e suggests that they may represent a new species. Starins of \u003cem\u003eB. licheniformis\u003c/em\u003e are known for their hydrolytic activity (Manczinger et al. 2003; Muras et al. 2021) and previous reports have documented keratinase production by \u003cem\u003eB. licheniformis\u003c/em\u003e PWD-1 (Cheng et al. 1995). However, to our knowledge, this is the first report of keratin degradation by \u003cem\u003eB. borbori\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003eSubstrate concentration is an important factor in bioconversion processes, as it can influence enzyme activity. Evaluation of influence of the feather amount revealed that 40 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of feather in enrichment was optimal for feather degradation. Under the tested conditions, the strains synthesized keratinolytic proteases during the late growth stationary phase. As a thermophilic process feather degradation occurred effectively at 55\u0026deg;C within a relatively short period compared to mesophilic processes.\u003c/p\u003e \u003cp\u003eWhen compared to other bacteria such as \u003cem\u003eB. licheniformis\u003c/em\u003e PWD-1 (Lin et al. 1997), \u003cem\u003eB. licheniformis\u003c/em\u003e K-508 (Manczinger et al. 2003), \u003cem\u003eB. licheniformis\u003c/em\u003e FK14 (Suntornsuk et al. 2005), \u003cem\u003eB. licheniformis\u003c/em\u003e ER-15 (Tiwary and Gupta, 2010), \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e S13 (Hamiche et al. 2019), \u003cem\u003eActinomadura viridilutea\u003c/em\u003e DZ50 (Ben Elhoul et al. 2016), \u003cem\u003eCaldicoprobacter algeriensis\u003c/em\u003e Bouacem et al. 2016) and \u003cem\u003eActinomadura keratinilytica\u003c/em\u003e strain Cpt29 (Habbeche et al. 2014) the keratin degradation rate of \u003cem\u003eB. borbori\u003c/em\u003e M14 was comparable, with no significant difference in degradation time. Studies comparing optimal growth temperatures and degradation times for various bacteria are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile most studies have focused on single-strain systems for feather degradation, using microbial consortia may offer a more effective approach. The synergy between different strains can accelerate degradation and improve efficiency. In this study, co-cultures of \u003cem\u003eB. borbori\u003c/em\u003e strains demonstrated up to 1.5 times greater degradation of feathers compared to mono-cultures. This enhanced degradation in mixed cultures suggests that community intrinsic properties, such as a switch from sulfitolytic to proteolytic activity, may play a role in more efficient keratin breakdown (Nasipuri et al. 2020).\u003c/p\u003e \u003cp\u003eFeathers are rich in essential amino acids such as cysteine, glutamine, proline, and serine (Tesfaye et al. 2017) making them a valuable resource for microbial cultures. The feather hydrolysis products can serve not only protein or amino feeds for animals but also as important carbon and nitrogen sources for microbial culture (Williams et al. 1991). HPLC analysis of feather hydrolysis products by \u003cem\u003eB. borbori\u003c/em\u003e M14 revealed significant amount of essential amino acids, including isoleucine, leucine, phenylalanine, and nonessential amino acids such as aspartic acid, glutamic acid, glutamic acid, alanine and tyrosine. This highlights the potential for bioconversion of feathers into high quality animal feed or amino acids. In contrast, physical and chemical treatments of feathers often result in the loss of important amino acids like methionine, lysine and tryptophan (Kumar 2021). Therefore, microbial keratinases present a more sustainable and nutritionally beneficial method for feather conversion.\u003c/p\u003e \u003cp\u003eThe results of this study confirm that the newly isolated strains from Armenian geothermal springs have significant potential for use in biotechnological applications, including the production of keratinolytic proteases for feather meal or amino acid recovery. This work contributes valuable insights into the microbial diversity of geothermal springs and their potential for circular bioeconomy processes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglobal positioning system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTris-EDTA buffer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVoges-Proskauer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enutrient broth\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies involving human participants or animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors consented to the publication of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe research was conducted with support from The Higher Education and Science Committee of RA within the scope of the research projects No. 21T-1F191.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e\n\u003cp\u003eConceptualization, HP; Field sampling, HP and AM; Experimental design and data collection, HP and MT; Data acquisition and interpretation, MT and AM, Manuscript writing\u0026mdash;original draft preparation, TM, HP and AM; Writing\u0026mdash;review and editing, HP; Visualization, MT and AM, Supervision, HP; Funding acquisition, HP All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe acknowledge Ella Minasyan for HPLC analyses.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAghajanyan AE, Hambardzumyan AA, Minasyan EV, Hovhannisyan GJ, Yeghiyan KI, Soghomonyan TM, Avetisyan SV, Sakanyan VA, Tsaturyan AH (2024) Efficient isolation and characterization of functional melanin from various plant sources. Int J Food Sci Technol 59(6):3545\u0026ndash;3555. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ijfs.17016\u003c/span\u003e\u003cspan address=\"10.1111/ijfs.17016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltschul SF, Madden TL, Sch\u0026auml;ffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389\u0026ndash;3402. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/25.17.33894o\u003c/span\u003e\u003cspan address=\"10.1093/nar/25.17.33894o\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBen Elhoul M, Zara\u0026icirc; Jaouadi N, Rekik H, Omrane Benmrad M, Mechri S, Moujehed E, Kourdali S, El Hattab M, Badis A, Bejar S, Jaouadi B (2016) Biochemical and molecular characterization of new keratinolytic protease from \u003cem\u003eActinomadura viridilutea\u003c/em\u003e DZ50. Int J Biol Macromol 92:299\u0026ndash;315. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2016.07.009\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2016.07.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouacem K, Bouanane-Darenfed A, Zara\u0026icirc; Jaouadi N, Joseph M, Hacene H, Ollivier B, Fardeau ML, Bejar S, Jaouadi B (2016) Novel serine keratinase from \u003cem\u003eCaldicoprobacter algeriensis\u003c/em\u003e exhibiting outstanding hide dehairing abilities. Int J Biol Macromol 86:321\u0026ndash;328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2016.01.074\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2016.01.074\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248\u0026ndash;254. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/abio.1976.9999\u003c/span\u003e\u003cspan address=\"10.1006/abio.1976.9999\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurkhardt C, Baruth L, Meyer-Heydecke N, Klippel B, Margaryan A, Paloyan A, Panosyan H, Antranikian G (2024) Mining thermophiles for biotechnologically relevant enzymes - evaluating the potential of European and Caucasian hot springs. Extremophiles 28:5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00792-023-01321-3\u003c/span\u003e\u003cspan address=\"10.1007/s00792-023-01321-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCavello I, Urbieta MS, Segretin AB, Giaveno A, Cavalitto S, Donati ER (2018) Assessment of keratinase and other hydrolytic enzymes in thermophilic bacteria isolated from geothermal areas in Patagonia, Argentina. Geomicrobiol J 35(2):156\u0026ndash;165. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/01490451.2017.1339144\u003c/span\u003e\u003cspan address=\"10.1080/01490451.2017.1339144\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng SW, Hu HM, Shen SW, Takagi H, Asano M, Tsai YC (1995) Production and characterization of keratinase of a feather-degrading \u003cem\u003eBacillus licheniformis\u003c/em\u003e PWD-1. Biosci Biotechnol Biochem 59(12):2239\u0026ndash;2243. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1271/bbb.59.2239\u003c/span\u003e\u003cspan address=\"10.1271/bbb.59.2239\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva RR (2018) Keratinases as an alternative method designed to solve keratin disposal on the environment: its relevance on agricultural and environmental chemistry. J Agric Food Chem 66:7219\u0026ndash;7221. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.8b03152\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.8b03152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitriyanto NA, Ramadhanti Y, Rismiyati, Rusyadi I, Pertiwiningrum, Prasetyo RA, Erwanto Y (2022) Production of poultry feather hydrolysate using HCl and NaOH as a growth medium substrate for indigenous strains. IOP Conf Ser Earth Environ Sci 951:012064. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/1755-1315/951/1/01206\u003c/span\u003e\u003cspan address=\"10.1088/1755-1315/951/1/01206\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedrich AB, Antranikian G (1996) Keratin degradation by \u003cem\u003eFervidobacterium pennavorans\u003c/em\u003e, a novel thermophilic anaerobic species of the order \u003cem\u003eThermotogales\u003c/em\u003e. Appl Environ Microbiol 62:2875\u0026ndash;2882. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aem.62.8.2875-2882.1996\u003c/span\u003e\u003cspan address=\"10.1128/aem.62.8.2875-2882.1996\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta R, Tiwary E, Sharma R, Rajput R, Nair N (2013) Microbial keratinases: diversity and applications. In: Satyanarayana T, (eds) Thermophilic microbes in environmental and industrial biotechnology: biotechnology of thermophiles, pp 881\u0026ndash;904. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-94-007-5899-5_33\u003c/span\u003e\u003cspan address=\"10.1007/978-94-007-5899-5_33\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabbeche A, Saoudi B, Jaouadi B, Haberra S, Kerouaz B, Boudelaa M, Badis A, Ladjama A (2014) Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete \u003cem\u003eActinomadura keratinilytica\u003c/em\u003e strain Cpt29 isolated from poultry compost. J Biosci Bioeng 117(4):413\u0026ndash;421. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jbiosc.2013.09.006\u003c/span\u003e\u003cspan address=\"10.1016/j.jbiosc.2013.09.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamiche S, Mechri S, Khelouia L, Annane R, El Hattab M, Badis A, Jaouadi B (2018) Purification and biochemical characterization of two keratinases from \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e S13 isolated from marine brown alga \u003cem\u003eZonaria tournefortii\u003c/em\u003e with potential keratin-biodegradation and hide-unhairing activities. Int J Biol Macromol 122:758\u0026ndash;769. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2018.10.174\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2018.10.174\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassan MA, Abol-Fotouh D, Omer AM, Tamer TM, Abbas E (2020) Comprehensive insights into microbial keratinases and their implication in various biotechnological and industrial sectors: A review. Int J Biol Macromol 154:567\u0026ndash;583. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2020.03.116\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2020.03.116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenneberger R, Cooksley D, Hallberg J (2000) Geothermal resources of Armenia. In: Proceedings World Geothermal Congress. Kyushu-Tohoku, 1217\u0026ndash;1222\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIonata E, Canganella F, Bianconi G, Benno Y, Sakamoto M, Capasso A, Rossi M, La Cara F (2008) A novel keratinase from \u003cem\u003eClostridium sporogenes\u003c/em\u003e bv. pennavorans bv. nov., a thermotolerant organism isolated from solfataric muds. Microbiol Res 163(1):105\u0026ndash;112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micres.2006.08.001\u003c/span\u003e\u003cspan address=\"10.1016/j.micres.2006.08.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIstrate D, Popescu C, Er Rafik M, M\u0026ouml;ller M (2013) The effect of pH on the thermal stability of fibrous hard alpha-keratins. Polym Degrad Stab 98(2):542\u0026ndash;549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.polymdegradstab.2012.12.001\u003c/span\u003e\u003cspan address=\"10.1016/j.polymdegradstab.2012.12.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJavier-Lopez R, Mandolini E, Dzhuraeva M, Bobodzhanova K, Birkeland N-K (2023) \u003cem\u003eFervidobacterium pennivorans\u003c/em\u003e subsp. keratinolyticus subsp. nov., a novel feather-degrading anaerobic thermophile. Microorganisms 11:22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms11010022\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms11010022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnvesly B, Manjunath BR, Naik GR (2002) Pigeon pea waste as a novel, inexpensive substrate for production of a thermostable alkaline protease from thermoalkalophilic \u003cem\u003eBacillus\u003c/em\u003e sp. JB-99. Bioresour Technol 82(1):61\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0960-8524(01)00147-x\u003c/span\u003e\u003cspan address=\"10.1016/s0960-8524(01)00147-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SS, Kim YJ, Rhee IK (2001) Purification and characterization of a novel extracellular protease from \u003cem\u003eBacillus cereus\u003c/em\u003e KCTC 3674. Arch Microbiol 175(6):458\u0026ndash;461. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s0020301002824\u003c/span\u003e\u003cspan address=\"10.1007/s0020301002824\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKojima M, Kanai M, Tominaga M et al (2006) Isolation and characterization of a feather-degrading enzyme from \u003cem\u003eBacillus pseudofirmus\u003c/em\u003e FA30-01. Extremophiles 10:229\u0026ndash;235. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00792-005-0491-y\u003c/span\u003e\u003cspan address=\"10.1007/s00792-005-0491-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKublanov IV, Perevalova AA, Slobodkina GB et al (2009) Biodiversity of thermophilic prokaryotes with hydrolytic activities in hot springs of Uzon Caldera, Kamchatka (Russia). Appl Environ Microbiol 75(1):286\u0026ndash;291. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00607-08\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00607-08\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar J (2021) Microbial hydrolyzed feather protein as a source of amino acids and protein in the diets of animals including poultry. In: Advances in poultry nutrition research. Intech Open. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5772/intechopen.96925\u003c/span\u003e\u003cspan address=\"10.5772/intechopen.96925\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547\u0026ndash;1549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msy096\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msy096\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLange L, Huang Y, Busk PK (2016) Microbial decomposition of keratin in nature\u0026mdash;a new hypothesis of industrial relevance. Appl Microbiol Biotechnol 100:2083\u0026ndash;2096. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-015-7262-1\u003c/span\u003e\u003cspan address=\"10.1007/s00253-015-7262-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLatshaw JD, Musharaf N, Retrum R (1994) Processing of feather meal to maximize its nutritional value for poultry. Anim Feed Sci Technol 47:179\u0026ndash;188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0377-8401(94)90122-8\u003c/span\u003e\u003cspan address=\"10.1016/0377-8401(94)90122-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q (2019) Progress in microbial degradation of feather waste. Front Microbiol 10:2717. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2019.02717\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2019.02717\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin X, Wong S-L, Miller ES, Shih JCH (1997) Expression of the \u003cem\u003eBacillus licheniformis\u003c/em\u003e PWD-1 keratinase gene in \u003cem\u003eB. subtilis\u003c/em\u003e. J Ind Microbiol Biotechnol 19(2):134\u0026ndash;138. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sj.jim.2900440\u003c/span\u003e\u003cspan address=\"10.1038/sj.jim.2900440\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManczinger L, Rozs M, V\u0026aacute;gv\u0026ouml;lgyi C, Kevei F (2003) Isolation and characterization of a new keratinolytic \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain. World J Microbiol Biotechnol 19:35\u0026ndash;39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1022576826372\u003c/span\u003e\u003cspan address=\"10.1023/A:1022576826372\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuras A, Romero M, Mayer C, Otero A (2021) Biotechnological applications of \u003cem\u003eBacillus licheniformis\u003c/em\u003e. Crit Rev Biotechnol 41(4):609\u0026ndash;627. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/07388551.2021.1873239\u003c/span\u003e\u003cspan address=\"10.1080/07388551.2021.1873239\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNam G-W, Lee D-W, Lee H-S, Lee N-J, Kim B-C, Choe E-A et al (2002) Native-feather degradation by \u003cem\u003eFervidobacterium islandicum\u003c/em\u003e AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch Microbiol 178:538\u0026ndash;547. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00203-002-0489-0\u003c/span\u003e\u003cspan address=\"10.1007/s00203-002-0489-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNasipuri P, Herschend J, Brejnrod AD, Madsen JS, Espersen R, Svensson B, Burm\u0026oslash;lle M, Jacquiod S, S\u0026oslash;rensen SJ (2020) Community-intrinsic properties enhance keratin degradation from bacterial consortia. PLoS ONE 15(1):e0228108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0228108\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0228108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanosyan H, Margaryan A, Birkeland N (2020) Geothermal springs in Armenia and Nagorno-Karabakh: potential sources of hydrolase-producing thermophilic bacilli. Extremophiles 24:519\u0026ndash;536. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00792-020-01173-1\u003c/span\u003e\u003cspan address=\"10.1007/s00792-020-01173-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanosyan H, Margaryan A, Poghosyan LA, Saghatelyan A, Gabashvili E, Jaiani E, Birkeland NK (2018) Microbial diversity of terrestrial geothermal springs in Lesser Caucasus. In: Egamberdieva D, Birkeland NK, Panosyan H, Li WJ (eds) Extremophiles in Eurasian ecosystems: ecology, diversity, and applications. Springer, Singapore, pp 81\u0026ndash;117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapadopoulos MC (1989) Effect of processing on high-protein feedstuffs: a review. Biol Wastes 29(2):123\u0026ndash;138. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0269-7483(89)90092-X\u003c/span\u003e\u003cspan address=\"10.1016/0269-7483(89)90092-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu J, Wilkens C, Barrett K, Meyer AS (2020) Microbial enzymes catalyzing keratin degradation: classification, structure, function. Biotechnol Adv 44:107607. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2020.107607\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2020.107607\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiessen S, Antranikian G (2001) Isolation of \u003cem\u003eThermoanaerobacter keratinophilus\u003c/em\u003e sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity. Extremophiles 5:399\u0026ndash;408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00792010020\u003c/span\u003e\u003cspan address=\"10.1007/s00792010020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaghatelyan A, Margaryan A, Panosyan H, Birkeland N-K (2021) Microbial diversity of terrestrial geothermal springs in Armenia and Nagorno-Karabakh: a review. Microorganisms 9:1473. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms9071473\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms9071473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406\u0026ndash;425. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/oxfordjournals.molbev.a040454\u003c/span\u003e\u003cspan address=\"10.1093/oxfordjournals.molbev.a040454\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchleifer K-H (2009) Phylum XIII. \u003cem\u003eFirmicutes\u003c/em\u003e Gibbons and Murray. In: De Vos P et al (eds) Bergey\u0026rsquo;s Manual\u0026reg; of Systematic Bacteriology: Volume Three: The Firmicutes. Springer, New York, pp 19\u0026ndash;1317. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-0-387-68489-5\u003c/span\u003e\u003cspan address=\"10.1007/978-0-387-68489-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShrinivas D, Naik GR (2011) Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic \u003cem\u003eBacillus halodurans\u003c/em\u003e JB 99 exhibiting dehairing activity. Int Biodeterior Biodegrad 65(1):29\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ibiod.2010.04.013\u003c/span\u003e\u003cspan address=\"10.1016/j.ibiod.2010.04.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmibert R, Krieg N (1981) General characteristics. In: Gerhardt P, Murray R, Costilow R, Nester E, Wood W, Phillips G (eds) Manual of Methods for General Bacteriology, 3rd edn. American Society for Microbiology, Washington, pp 7\u0026ndash;243\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuntornsuk W, Tongjun J, Onnim P, Oyama H, Ratanakanokchai K, Kusamran T, Oda K (2005) Purification and characterization of keratinase from a thermotolerant feather-degrading bacterium. World J Microbiol Biotechnol 21:1111\u0026ndash;1117. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11274-005-0078-x\u003c/span\u003e\u003cspan address=\"10.1007/s11274-005-0078-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTadevosyan M, Yeghiazaryan S, Ghevondyan D, Saghatelyan A, Margaryan A, Panosyan H (2022) Microbial thermostable hydrolases (amylases, lipases, and keratinases) and polymerases: biology and applications. In: Arora NK, Agnihotri S, Mishra J (eds) Extremozymes and Their Industrial Applications. Elsevier Academic, pp 177\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B978-0-323-90274-8.00007-1\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-323-90274-8.00007-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakami H, Nogi Y, Horikoshi K (1999) Reidentification of the keratinase-producing facultatively alkaliphilic \u003cem\u003eBacillus\u003c/em\u003e sp. AH-101 as \u003cem\u003eBacillus halodurans\u003c/em\u003e. Extremophiles 3:293\u0026ndash;296. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s007920050130\u003c/span\u003e\u003cspan address=\"10.1007/s007920050130\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamreihao K, Mukherjee S, Khunjamayum R, Devi LJ, Asem RS, Ningthoujam DS (2019) Feather degradation by keratinolytic bacteria and biofertilizing potential for sustainable agricultural production. J Basic Microbiol 59:4\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jobm.201800434\u003c/span\u003e\u003cspan address=\"10.1002/jobm.201800434\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTesfaye T, Sithole B, Ramjugernath D (2017) Valorisation of chicken feathers: a review on recycling and recovery route\u0026mdash;current status and future prospects. Clean Technol Environ Policy 19:2363\u0026ndash;2378. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10098-017-1443-9\u003c/span\u003e\u003cspan address=\"10.1007/s10098-017-1443-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice. Nucleic Acids Res 22(22):4673\u0026ndash;4680. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/22.22.4673\u003c/span\u003e\u003cspan address=\"10.1093/nar/22.22.4673\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTindall BJ, Sikorski J, Smibert RA, Krieg NR (2007) Phenotypic characterization and the principles of comparative systematics. In: Reddy CA et al (eds) Methods for General and Molecular Microbiology. American Society for Microbiology, Washington, DC, pp 330\u0026ndash;393. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/9781555817497.ch15\u003c/span\u003e\u003cspan address=\"10.1128/9781555817497.ch15\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTiwary E, Gupta R (2010) Extracellular expression of keratinase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e ER-15 in \u003cem\u003eEscherichia coli\u003c/em\u003e. J Agric Food Chem 58:8380\u0026ndash;8385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf100803g\u003c/span\u003e\u003cspan address=\"10.1021/jf100803g\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma A, Singh H, Anwar S, Chattopadhyay A, Tiwari KK, Kaur S et al (2017) Microbial keratinases: industrial enzymes with waste management potential. Crit Rev Biotechnol 37:476\u0026ndash;491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/07388551.2016.1185388\u003c/span\u003e\u003cspan address=\"10.1080/07388551.2016.1185388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B, Yang W, McKittrick J, Meyers MA (2016) Keratin: structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration. Prog Mater Sci 76:229\u0026ndash;318. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pmatsci.2015.06.001\u003c/span\u003e\u003cspan address=\"10.1016/j.pmatsci.2015.06.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Hao S, Luo T, Cheng Z, Li W, Gao F et al (2017) Feather keratin hydrogel for wound repair: preparation, healing effect and biocompatibility evaluation. Colloids Surf B Biointerfaces 149:341\u0026ndash;350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.colsurfb.2016.10.038\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfb.2016.10.038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Parsons C (1997) Effect of processing systems on protein quality of feather meals and hog hair meals. Poult Sci 76:491\u0026ndash;496. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ps/76.3.491\u003c/span\u003e\u003cspan address=\"10.1093/ps/76.3.491\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang YQ, Yuan Y, Yu Z, Yang GQ, Zhou SG (2013) \u003cem\u003eBacillus borbori\u003c/em\u003e sp. nov., isolated from an electrochemically active biofilm. Syst Appl Microbiol 67(6):718\u0026ndash;724. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00284-013-0426-2\u003c/span\u003e\u003cspan address=\"10.1007/s00284-013-0426-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams CM, Lee CG, Garlich JD, Shih JC (1991) Evaluation of a bacterial feather fermentation product, feather-lysate, as a feed protein. Poult Sci 70:85\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3382/ps.0700085\u003c/span\u003e\u003cspan address=\"10.3382/ps.0700085\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamaoka A, Kataoka M, Kawasaki K, Kobayashi E, Shigeri Y, Watanabe K (2014) \u003cem\u003eMeiothermus ruber\u003c/em\u003e H328 enhances the production of membrane vesicles for feather degradation. Biosci Biotechnol Biochem 78(9):1623\u0026ndash;1625. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/09168451.2014.918488\u003c/span\u003e\u003cspan address=\"10.1080/09168451.2014.918488\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou B, Guo Y, Xue Y et al (2023) Comprehensive insights into the mechanism of keratin degradation and exploitation of keratinase to enhance the bioaccessibility of soybean protein. Biotechnol Biofuels 16:177. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13068-023-02426-9\u003c/span\u003e\u003cspan address=\"10.1186/s13068-023-02426-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Material","content":"\u003cp\u003eSupplementary Materials are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Feather, keratin, keratinolytic thermophilic bacilli, Bacillus borbori","lastPublishedDoi":"10.21203/rs.3.rs-5090232/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5090232/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study was to isolate and characterize keratinolytic thermophilic aerobic bacilli from Armenian geothermal springs. In total 20 thermophilic aerobic bacilli strains have been isolated using chicken feather enrichment cultures. Among these, four strains affiliated as \u003cem\u003eBacillus licheniformis\u003c/em\u003e (95\u0026ndash;97% similarity) and \u003cem\u003eBacillus borbori\u003c/em\u003e (\u0026gt;\u0026thinsp;99% similarity) demonstrated the capability to completely degrade chicken feathers at 55\u0026deg;C. The highest rate of feather hydrolyses in mono-species cultures was observed with 40 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e substrate. Notably, enhanced keratin weight loss (\u0026ge;\u0026thinsp;80%) was observed in dual co-cultures involving \u003cem\u003eB. borbori\u003c/em\u003e M14, highlighting superior degradative potential of this strain. Keratinolytic enzyme production was dedected during the late exponential growth phase, reached its maximum activity (0.013 U mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) during the stationary phase, suggesting growth-associated enzyme synthesis. High-performance liquid chromatography (HPLC) of the hydrolysis end products revealed that aspartic acid and isoleucine were the predominant amino acids, followed by leucine, phenylalanine, alanine, tyrosine and glutamic acid. These findings confirm that the newly isolated strains are promising sources of keratinolytic proteases, with potential applications in circular bioeconomy based processes.\u003c/p\u003e","manuscriptTitle":"Assessment of feather degrading activity of thermophilic bacilli isolated from Armenian geothermal springs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-11 17:21:17","doi":"10.21203/rs.3.rs-5090232/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-12-10T08:11:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-10T06:19:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-09T09:51:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2024-12-06T19:12:16+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revisions","date":"2024-10-23T05:34:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"015adfa6-9c65-4cc4-a90b-fa989493beac","owner":[],"postedDate":"December 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T16:03:53+00:00","versionOfRecord":{"articleIdentity":"rs-5090232","link":"https://doi.org/10.1007/s11756-025-01877-9","journal":{"identity":"biologia","isVorOnly":false,"title":"Biologia"},"publishedOn":"2025-01-28 15:58:04","publishedOnDateReadable":"January 28th, 2025"},"versionCreatedAt":"2024-12-11 17:21:17","video":"","vorDoi":"10.1007/s11756-025-01877-9","vorDoiUrl":"https://doi.org/10.1007/s11756-025-01877-9","workflowStages":[]},"version":"v1","identity":"rs-5090232","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5090232","identity":"rs-5090232","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}