Parametrically optimized feather degradation by Bacillus velezensis NCIM 5802 and delineation of keratin hydrolysis by multi-scale analysis for poultry waste management

Abstract

Enormous amounts of keratinaceous waste make a significant and unexploited protein reserve that can be utilized through bioconversion into high-value products using microbial keratinases. This study was intended to assess the keratinase production from a newly isolated B. velezensis NCIM 5802 that can proficiently hydrolyze chicken feathers. Incubation parameters used to produce keratinase enzyme were optimized through the Response Surface Methodology (RSM) with chicken feathers as substrate. Optimization elevated the keratinase production and feather degradation by 4.92-folds (109.7 U/mL) and 2.5 folds (95.8%), respectively. Time-course profile revealed a direct correlation among bacterial growth, feather degradation, keratinase production and amino acid generation. Biochemical properties of the keratinase were evaluated, where it showed optimal activity at 60 °C and pH 10.0. The keratinase was inhibited by EDTA and PMSF, indicating it to be a serine-metalloprotease. Zymography revealed the presence of four distinct keratinases (Mr ~ 100, 62.5, 36.5 and 25 kDa) indicating its multiple forms. NMR and mass spectroscopic studies confirmed the presence of 18 free amino acids in the feather hydrolysates. Changes in feather keratin brought about by the keratinase action were studied by X-ray diffraction (XRD) and spectroscopic (FTIR, Raman) analyses, which showed a decrease in the total crystallinity index (TCI) (1.00-0.63) and confirmed the degradation of its crystalline domain. Scanning electron microscopy (SEM) revealed the sequential structural changes occurring in the feather keratin during degradation. Present study explored the use of keratinolytic potential of the newly isolated B. velezensis NCIM 5802 in chicken feather degradation and also, unraveled the underlying keratin hydrolysis mechanism through various analyses.

Figure 1

Response surface 3D plots showing the interaction of factors affecting keratinase production where, (A) pH and temperature (°C), (B) inoculum size (% v/v) and substrate concentration (%) and feather degradation where, (C) pH and temperature (°C), (D) inoculum size (%) and substrate concentration (%).

Figure 2

Time course profile of keratinase production (A) time course of decomposition of chicken feathers by B. velezensis NCIM 5802 (%), keratinase activity (U/mL), bacterial growth (OD₆₀₀), soluble protein content (μg/mL), amino acid content (μg/mL) and pH (optimized culture conditions: initial pH 7.0, time 96 h, 3.5% v/v inoculum, 0.625% w/v substrate concentration at 40 °C and 200 rpm). Temperature and pH profile of keratinase produced by B. velezensis NCIM 5802 (B) pH optima and stability (C) temperature optima and stability. Values represent average of three independent replicates ± standard deviation.

Figure 3

Feather degradation by B. velezensis NCIM 5802. Control (0 h); colonization of bacterial cells on feather surface (12–24 h); initialization of vane degradation (36 h); curling and exposure of secondary fibers (48–60 h); degradation of feather barbs, barbules, and invasion of bacterial cells in the shaft (72–84 h); complete disruption of feather rachis and shaft (96 h) by the colonized bacteria. Feather samples were harvested from the submerged cultures, gold coated and viewed at 5000 X in FEI Scanning electron microscope.

Figure 4

(A) Functional group characterization and structural changes in intact (blue) and hydrolyzed feather samples at 48 h (black) and 96 h (red) using ATR-FTIR spectra. (B) Chemical fingerprinting of hydrolyzed and unhydrolyzed chicken feathers using Raman spectra of intact feather (blue) and hydrolyzed feathers at 48 h (black) and 96 h (red) incubation.

Figure 5

X-ray diffraction patterns of the intact (blue) and hydrolyzed feather samples at different time intervals 48 h (black) and 96 h (red) of incubation.

Figure 6

¹H NMR spectra showing decomposition of native chicken feathers during the cultivation time of 96 h by B. velezensis NCIM 5802 and the release of amino acids.

Figure 7

Keratinase zymogram showing NCIM 5802 keratinase during the course of fermentation at different time intervals (0 h-48 h), M: standard protein marker. The semi-native PAGE contained 1% feather keratin. The gel was stained with Coomassie Blue after 24 h of incubation at 40 °C.