Isolation,Identification,Fermentation Of Keratin-degrading Strains

Feather is an important protein source which composed mainly of keratin protein(over 80%). However, keratin is poorly biodegradable and hard to be used directly because of its strong resistance. In China, millions of tons of feather waste are produced every year, which will continue to increase in the future. It is very significant to achieve the conversion and recycling of keratin waste, for the scarcity of protein resources and China’s economic development. Traditional ways to degrade feathers using physical and chemical methods not only consume a lot of energy but also cause serious secondary pollution. Biodegradation of feathers by keratinase from microorganisms may provide a viable alternative.The aim of this study was to identify some newly isolated feather-degrading bacterium strains and to optimize the conditions for keratinase production in feather substrates. The basic medium used for isolation and fermentation of the feather-degrading microorganisms contained casein or feathers as the sole carbon source and nitrogen source. Eight efficient strains, from poultry carcasses buried soil, flower beds and ponds, were isolated on the basic of casein degrading-circles and feather degradation effect, and named F-T1-2, X1, D1, T1, T3, Y2, T2 and B1-2, respectively. Identification of strains was conducted through morphological observation, biochemical identification tests, 16 S r DNA sequencing and phylogenetic analysis. Strain F-T1-2, T1 and T3 were identified as Bacillus cereus. Strain X1 and D1 were identified as Pseudomonas beteli. Strain Y2 and T2 were identified as Stenotrophomonas maltophilia. Strain B1-2 was identified as Pseudomonas aeruginosa. We measured the keratinase enzyme activity of fermentation broth after 24 hours’ degradation, and got results as T1: 49.9 U/m L, T3: 34.3 U/m L, F-T1-2: 32.05 U/m L, D1: 23.1 U/m L, X1: 35.6 U/m L, Y2: 35.45 U/m L, T2: 28.65 U/m L. To our knowledge, it is the first report to isolate feather-degrading Pseudomonas beteli in the world, and the first report to isolate feather-degrading Pseudomonas aeruginosa in China.We used 1% feather as substrate to optimize fermentation conditions and degradation efficiency of isolated strains. Results showed that optimum initial p H values of the strains are: D1 and T2’s were p H=10, T3 and B1-2’s were p H=11, Y2’s was p H=9, F-T1-2’s was p H=7. And optimum temperatures during fermentation were: D1 and T2’s were 27℃, F-T1-2, B1-2 and Y2’s were 37℃, T3’s was 32℃.To characterize the degradation of keratin-containing substrates, the culture broths were observed by naked eyes, and feather samples were scanned by electron microscopy before and after the treatment. Visual observation and SEM showed that part barbules and barbs dropped, and surface structure of feather stems began to flake off in 24h; most barbules and barbs broke off, and fibrous structure under feathers stem scale exposed in 48h; barbs fractured severely, barbules almost disappeared, lots of cracks occurred on feather stems and internal honeycomb structure exposed in 72h;barbs and barbules has completely disappeared,internal honeycomb structure of feather stems was fully exposed and began to dissolve; fermentation broth became cloudy and brown during the feather degradation. We concluded that the degradation of feathers begins from barbules, followed by barbs, the last is feather stem. In summary, feather-degrading bacterium adhered on feathers at first, and then barbules completely degraded, barbs completely degraded later, feather stems completely degraded in the end.This study enriches the contents of keratin-degrading bacterium. The newly isolated strains have great potential for degradation and utilization of feather keratin.