Hydrolysis of Chicken Feathers by Microbial Keratinase for Production of Value-added Compounds

Abstract

This study was aimed to isolate and screen efficient keratinolytic bacterial strains for bioconversion of chicken feathers waste into value-added products. Based on screening results C1M isolate was the best to hydrolyze feathers keratin and was identified as Pseudomonas aeruginosa.-C1M. By conventional optimization, chicken feathers (carbon source), urea (nitrogen source), sodium sulfite (sulfur source), and zinc chloride (salts) were significant media components, while 37°C temperature and pH 8 were significant physiological factors for the enhanced keratinase production. Further optimization was carried out through Plackett-Burman Design, chicken feathers were found to be the most substantial nutritional factor and specific activity of keratinase was found as 14.64 U/mg. Feathers keratin was converted into hydrolysate through keratinase of Pseudomonas aeruginosa C1M, and bioactive peptides having antioxidant properties were obtained. Keratinase was purified from the fermentation media through ammonium sulfate precipitation followed by column chromatography. After partial purification of keratinase from Pseudomonas aeruginosa C1M, specific activity was found 25.06 U/mg on 60% ammonium sulfate saturation. However, after purification from gel filtration chromatography 74U/mg specific activity was obtained. Keratinase characterization revealed its stability at broad range of temperature (30-50°C) and pH (6-9). Besides feather keratin, the enzyme was active upon the soluble proteins keratin azure, casein, and bovine serum albumin (BSA); however, no conspicuous activity was observed in the presence of insoluble hair. Keratinase displayed high compatibility and stability with detergents by efficiently removing the proteinaceous stains from fabric pieces and exhibited activity improvements 40-73% compared with commercial detergents. Crude keratinase from Pseudomonas aeruginosa-C1M was used for the biogenic synthesis of zero-valent silver nanoparticles (AgNPs). These particles appeared transparent to dark brown with single, distinctive peak of UV-Visible spectroscopy was observed at ˷450nm. FTIR inference proved that silver nanoparticles were capped with proteins (crude keratinase). X-ray diffraction indicated that biogenic synthesized AgNPs were crystalline in nature and lattice planes were face-centered and cubic. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that DRSML QAU silver nanoparticles (AgNPs) were found as mono-dispersed spheres of variable sizes. Likewise, Dynamic light scattering (DLS) analysis showed that the diameter of AgNPs was ~ 119 nm. A high negative zeta potential value supports good colloidal properties, long-term stability, and high dispersity of the particles. Green synthesized silver nanoparticles exhibited antibacterial activity against pathogenic strains of Staphylococcus aureus and Escherichia coli with zone of inhibition of 22 and 25mm, respectively. The synthesized zero-valent silver nanoparticles (AgNPs) aided in the decontamination of azo-dyes in the presence of the reductant sodium borohydride (NaBH4) and reduced azo dyes (methyl orange, methyl red) to a less hazardous form. Similarly, bioremediation of other dyes (Safranin O, and methyl violet) by silver nanoparticles was carried out through light catalyzed processes. Keratin hydrolysate produced as a result of keratinase from Pseudomonas aeruginosa C1M was used in different biotechnological applications such as biogas and biofertilizers. Hydrolysate consists of polypeptides and amino acids and therefore has the ability to be used as precursor for biogas and biofertilizers production. For the biogas production, chicken feathers, rice husk and green grocery waste served as substrates in batch and continuous reactors. Pretreatment of chicken feathers through microbial keratinolytic Pseudomonas aeruginosa-C1M increased biogas yield. The pretreatment of rice husk RH significantly increased the biogas yield by 23.8%. The co-digestion process also addressed the enhancement and stability of anaerobic digestion as biogas production. The highest biogas yield was obtained in a batch reactor with co-digestion of pretreated rice husk and microbial pretreated chicken feathers (333.6 N ml/g VS) and it significantly increased 34% from calculated value (249Nml/g VS). Co-digestion of chicken feathers hydrolysate with green-grocery waste in continuous fermentation mode has also enhanced the biogas yield compared to the mono-digestion (Chicken feather hydrolysate and green-grocery waste) and therefore improves the efficiency of the overall process. Similarly, keratin hydrolysate potential was also evaluated on the growth improvement of spinach plant as a bio-fertilizer. Beside hydrolysate reactor digestate, a byproduct obtained after biogas production from hydrolysate and grocery waste, was also used as biofertilizers. A greenhouse study was carried out to examine the comparative efficacy of DRSML QAU reactor digestate and feather hydrolysate on the growth of spinach plants. The application of keratin hydrolysate-based fertilizers observed a remarkable increase in the agronomic parameters. Plants treated with dry hydrolysate indicated a 50% increase in leaf breadth as compared to control plant provided with normal fertilizer. Plant height, number of leaves per plant, root length, chlorophyll content, protein content, biomass, and nutrient content in the spinach plant were also considerably enhanced with keratin hydrolysate based fertilizers. However, results were more promising with applying Pseudomonas aeruginosa-C1M derived hydrolysate, indicating its application as a potential bio stimulant in the agroindustry. This study concluded that chicken feathers waste was efficiently converted into keratin hydrolysate with the help of the keratinase of Pseudomonas aeruginosa-C1M. The potential of keratinase was exploited for bioactive peptide extraction and as a bio-additive in detergent formulations. Moreover, keratinase was used for the biogenic synthesis of silver nanoparticles which served as antibacterial agents and in dye degradation studies. The prospect of keratin hydrolysate was used for biogas generation and bio-stimulant for plant growth.