Bio-utilization of keratinous waste biomass for the production of keratinolytic proteases by Chryseobactreium aquifrigidense isolated from poultry waste dumpsite

Keratin is an insoluble and a fibrous protein that is mostly found in feathers, animal wool, and hair, making them mechanically stable. Avian feathers are the most ubiquitously generated keratinous waste biomass from the poultry processing plants. Keratinous waste biomass valorization could produce amino acids and bioactive peptides. Hence, the bio-recycling of keratin-rich wastes bears an advantage over the chemical and thermal means. In recent times, the microbial keratinases continues to gain traction due to the litany of their potential significance in green technology. Consequently, this study assessed chicken feather degrading and keratinase production potentials of bacteria isolated from a local poultry dumpsite. Soil samples were collected from the poultry dumpsite. Bacteria were isolated using basal salt medium and screened for keratinolytic activity. The identification of potent chicken feather degrading bacterial isolates was through 16S rRNA gene sequence analysis. Keratinase production efficiency of isolates on chicken feather constituted medium was optimized. Hydrolysate's amino acid compositions were quantified, and the keratinases produced was characterized. Out of 22 bacteria isolated from the soil samples, 12 showed a varying degree of proteolytic activity on skimmed milk agar. Four (4) isolates, coded as FPS-01, FPS-07, FPS-09, WDS-06, out of the 12 proteolytic strains further displayed remarkable degradation of the intact chicken feather; percentage degradation ranged from 69 percent for FPS-01 to 88 percent for FPS-09. The extracellular keratinase activity ranged from 610.91 ± 56.57 U/mL for WDS-06 and 834.55 ± 66.86 U/mL for FPS-07. Additionally, the sulfhydryl group concentration quantified from the fermentation broth was 2.22 ± 0.37 (mM), 0.22 ± 0.08 (mM), 2.27 ± 0.09 (mM), and 2.76 ± 0.79 (mM) for FPS-01, FPS-07, FPS-09 and WDS-06, respectively. Based on 16S rRNA gene sequencing and phylogenetic analyses, the isolates FPS-07 and FPS-09 were identified as Chryseobacterium aquifrigidense FANN1 and Chryseobacterium aquifrigidense FANN2. Following the optimization process, the improved fermentation conditions were pH 6, inoculum side (4 percent, v/v), temperature (30 oC), and chicken feather (0.5-1.5 percent, w/v) for FANN1. FANN2 optimal fermentation conditions were pH 6, inoculum (5 percent, v/v), temperature (30 oC), and chicken feather (1.5 percent, w/v). Both bacterial isolates showed the highest extracellular keratinase production after 72 h of the fermentation time. Analysis of the hydrolysates generated from the bacteria fermentation showed a high concentration of arginine, serine, glutamic acid, glycine, proline, valine, and leucine at a respective concentration of 1.13, 1.02, 0.83, 0.94, 0.85, 0.84, and 0.86 (g/100g sample) against FANN1. Similarly, FANN2 generated hydrolysate showed high concentrations of glutamic acid, arginine, serine, aspartic acid, and glycine at this respective abundance 2.52, 1.92, 2.12, 2.25 and 1.9 (g/100g sample). Keratinases from FANN1 and FANN2 showed optimal catalytic efficiency at pH 8 and temperature between 40-50 oC. The enzyme was considerably thermostable at 40 oC and 50 oC after 120 min of preheating. Both FANN1 and FANN2 showed variable residual activity in the presence of the different metal ions. Keratinase from FANN1 recorded the following residual activity of Fe3+ (120 ± 5.06 percent), Ca2+ (100 ± 10.33 percent), Na+ (122 ± 2.95 percent), Al3+ (106 ± 10.33 percent). Likewise, FANN2 keratinase showed remarkable stability against Na+ (108 ± 13.71 percent), Ba2+ (102 ± 0.86 percent), Al3+ (105 ± 2.57 percent), and Ca2+ (96 ± 2.99 percent). Keratinase from FANN1 was catalytically activated after 60 min of pre-treatment with the following detergents, Sunlight (129 percent), Ariel (116 percent), MAQ (151 percent), and Surf (143 percent) compared to the control. FANN2 keratinase showed less stability with laundry detergents after 60 min of preincubation. FANN1 keratinase showed remarkable stability in the presence of chemical agents tested, with residual activity of 90 ± 0.18 percent, 105 ± 7.55 percent, 108 ± 4.31 percent, 123 ± 1.44 percent, 132 ± 1.26 percent, 96 ± 7.19 percent, and 101 ± 3.06 percent for DTT, hydrogen peroxide, DMSO, acetonitrile, triton X-100, tween-80, and SDS, respectively. The enzyme activity was also considerably inhibited by PMSF and EDTA, which suggested a mixed type of protease. Furthermore, keratinase from FANN2 was inhibited by EDTA, and such inhibition pattern grouped it as a metallo-type of protease. The enzyme was also stable in the presence of other chemical agents tested. Therefore, the findings suggest the isolates and their enzymes' relevance to sustainable recycling of recalcitrant keratinous wastes into high-value products with immense application potentials. The remarkable stability shown by keratinases from FANN1 and FANN2, post detergent and chemical agents pre-treatment, indicates promise for the biotechnology and industrial sector.Thesis(MSc) (Microbiology) -- University of Fort Hare, 202