New identified Bacillus licheniformis isolates ensure enzymatic biodegradation of feathers into a feed additive

Feather waste biodegradation by keratinolytic microorganisms is an alternative to convert feather into nutritionally valuable feed additives and an attractive way to prevent environmental pollution. Cytolytic proteases are used to produce rare amino acids (proline, serine and cysteine). Bacterial hydrolysate of feathers can serve as an ingredient in animal feed or as an organic fertilizer, thereby reducing the impact of poultry waste on the environment. In this work, for the first time, it was found that with this method of pen processing by the Bacillus licheniformis BAL-2 strain, which uses the feather as the only carbon source, free keratin is completely converted nto digestible peptides and essential amino acids. The aim of the work is to study the morphological, physiological-biochemical and molecular-genetic properties of Bacillus licheniformis strains BAL-1 and BAL-2, their identification and use for fermentation of broiler chicken feathers. Bacillus licheniformis strains BAL-1 (VKPM B-14243) and BAL-2 (VKPM B-14244) are natural isolates from sediments of the Gulf of Finland of the Baltic Sea, Kandikul village. Ross-308 chicken feather (PJSC Cherkizovo Group Poultry Processing Complex Konstantinovo, Moscow Province, Ramenskoye District, Konstantinovo village) obtained from an industrial bird plucking line, had blood inclusions, humidity 75 % and was used for fermentation 24 hours after delivery. The morphotypes of B. licheniformis colonies were determined on Trypton soybean agar. The genomic DNA was isolated using a GeneJET Genomic DNAPurification Kit («Thermo Scientific», Lithuania). The 16S rDNA fragment was amolified by PCR with primers specific to the 16S rRNA genes of eubacteria: 8f 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492r 5'-TACGGHTACCTTGTTACGACTT-3'. The analysis of the identity of nucleotide sequences was performed using the BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST). Feather (5 g) was added to a flask with 100 cm3 of mineral-nutrient medium. B. licheniformis BAL-1 or BAL-2 was introduced into the mineral medium to a final concentration of 1×106 CFU/cm3. Fermentation was carried out for 72 hours at 28 °C and constant stirring. Keratinase activity was determined in the culture fluid using a keratin suspension. The enzyme solution (1 cm3) was added to the keratin suspension (1 cm3) in the reaction buffer. After 1 hour of incubation at 37 °C, the reaction was stopped by adding 2 cm3 of 5 % TCA and the solution was filtered. The fermented feather was examined by scanning electron microscopy. Two-dimensional gel electrophoresis was performed according to O'Farrell's procedure with isoelectrofocusing in the ampholine pH gradient (IEF-PAGE); proteins were detected by staining with Coomassie Brilliant Blue R-250 and silver nitrate. For 2D electrophoresis, 100 mg portion of the crushed feather was homogenized in 2 cm3 of the lysing solution. The homogenate was clarified by centrifugation, the supernatant fraction containing protein extract was used for fractioning in equal applications of 50-75 µl. After trypsinolysis, protein fractions were identified by MALDI-TOF and MS/MS mass spectrometry (Ultraflex mass spectrometer, Bruker Daltonics GmbH & Co. KG, Germany) with a UV laser (λ = 336 nm) in the detection mode of positive ions in the mass range 500-8000 Da. Thin-layer paper chromatography was performed according to GOST 28366-89. The total nitrogen content in the dried feather after fermentation was evaluated by the Kjeldahl method. Genomic fingerprint was carried out by rep-PCR with primers Rep1R-I and Rep2-I ERIC-PCR (a set of primers ERIC1R and ERIC2), ERIC2-PCR, the primer ERIC2, BOX-PCR, the primer BOX A1R, and (GTG)5-PCR, the primer (GTG)5. To prepare DNA matrices, individual colonies were selected, suspended in 20 µl of deionized water, and processed for 2 minutes in a microwave oven at maximum power. The resulting preparations werestored at 20 °C, then thawed and used in PCR. According to the results of the analysis of the nucleotide sequence of the 16S rRNA gene, it was found that the strains BAL-1 and BAL-2 belong to the species Bacillus licheniformis . The strains are able to grow up to 1½108 CFU/cm3 on a mineral medium with chicken feather as a single carbon source. By serial dilutions, 16 morphotypes of colonies were identified, 10 for B. licheniformis BAL-1, and 6 for B. licheniformis BAL-2. The preservation of the identified morphotypes in culture fluid was confirmed by PCR-based genomic fingerprint. The keratinase activity of B. licheniformis BAL-1 was 16.2±0.3 KA/cm3, of BAL-2, 18.3±0.2 KA/cm3. The ability of B. licheniformis keratinases to convert indigestible chicken feather keratin into digestible peptides occurred due to a complete decomposition of free keratin by BAL-2 strain and partial decomposition by BAL-1 strain. It was shown by 2D electrophoresis and mass spectrometric identification.