Study On The Soil Microbial Community In Feather Degradation And The Isolation,Indentification And Degradation Mechanism Of Bacillus Sp.JY-23

As the main component of feathers,keratin has a large number of cross-linked chemical bonds such as disulfide,hydrogen and salt bonds,which are difficult to be effectively degraded and place great pressure on the environmental governance and resource utilization.Keratinase is a multifunctional enzyme capable of degrading keratin,it is not only green and environmental protection,but also can not destroy the natural structure of amino acids in feathers,and can achieve recycling of resources.In this study,a soil sample with obvious degradation characteristics for feathers was selected.The microbial diversity of the soil was dynamically analyzed at the same time a Bacillus sp.JY-23 with high-efficiency degradation effect on feathers was isolated and screened,and it is enzymatic properties of keratinase,feather biodegradation dynamics,and feather degradation mechanism were studied.In order to provide theoretical basis for the practical application of the strain in the degradation of waste feathers.The main results are as follows:1.From the soil samples collected from Jiujiang City,Shangrao City,Nanchang City of Jiangxi Province and Sanming City of Fujian Province,a soil sample with significant degradation effect on feathers was selected.The microbial diversity during soil degradation of feathers was studied by using Illumina miseq high-throughput sequencing technology,it was found that a total of 194 bacterial OTUs were obtained from 32 samples and divided into 11 phylums and 83 genus.In the 120h degradation solution,Proteobacteria,Bacteroides,and Cyanobacteria were the dominant phylums during the degradation of the feathers,while Acidovorax,Stenotrophomonas,Chitinophaga and Comamonas were the dominant genus at the genus level.2.Using the coating casein solid medium and feather as the sole carbon and nitrogen source liquid culture,five strains with obvious hydrolysis circle in casein solid medium and obvious degradation of feather were obtained by preliminary screening.Then three strains were identified by polyphasic taxonomy as Bacillus acidiceler,one strain was Ochrobactrum intermedium,and the other strain may be a new species of Bacillus sp.（according to the number named Bacillus JY-23）.Further re-screening found that Bacillus sp.JY-23 had the best feather degradation effect among all the initial screening strains,and it is feather degradation rate reached more than 90%at 72 h.3.Preliminary studies on the enzymatic properties of keratinase produced by Bacillus sp.JY-23 showed that the optimal temperature and pH of keratinase by keratinase produced by strain were 50℃ and pH 8.0,the enzyme is relatively stable under the condition of pH 6.0-10.0 and belongs to moderate temperature alkaline protease.What’s more,metal ions Na⁺,Co²⁺,Ca²⁺,Fe²⁺ and Fe³⁺ can promote the protease,while Ni²⁺,Zn²⁺,Sn²⁺,Ba²⁺,Ag+,Cu²⁺and Mn²⁺will inhibit the activity of the keratinase.And Reagents EDTA and NaClO can reduce enzyme activity,indicating that the active center of the enzyme may have metal ions.In addition,the michaelis constant Km value in the kinetics of the reaction obtained using feather meal and the maximum reaction rate Vmax were 11.87 mg/mL and 26.67 U/（mL·min）,respectively.4.The kinetics of batch fermentation of Bacillus sp.JY-23 feathers showed that the growth,soluble protein production,and feather degradation of Bacillus sp.JY-23 can be fitted using Logistic,Luedeking-Piret,and Luedeking-Piret-Like models respectively.The correlation coefficients of the models are 0.9875,0.9713,and 0.9541,respectively.Furthermore,these models have outstanding fitting effect.The model explained the relationship among the growth of bacteria,the induced secretion of keratinase and the bioavailability of degradation products during the biodegradation of feathers by Bacillus sp.JY-23.That is to say,Bacillus sp.JY-23 can make feathers as the only carbon and nitrogen source to realize the growth of bacteria and induce the secretion of keratinase,thus promoting the degradation of feathers,and the degradation solution correspondingly shows the accumulation of soluble protein.5.By studying the physicochemical and biochemical mechanism of degradation of feather by Bacillus sp.JY-23,it is found that the keratinase had weak amide bond in the feather,which can degrade the barb rapidly and gradually transfer to the denser barb axis,thus producing significant feather degradation effect;The keratin in feather was degraded into soluble protein,amino acid and amino nitrogen under the action of bacteria;Under the action of keratinase produced by the bacteria,a large number of sulfur elements on the cross-linked disulfide bond in keratin are degraded and synthesized in sulfite and peptide containing sulfhydryl group,and the main free amino acids in the degradation solution are proline,tyrosine,lysine and glycine.