Study On Construction,Application And Enzymatic Properties Of Compound Bacteria For Degradation Of Dead Swine Carcass

The harmless composting treatment of dead swine carcasses has the advantages of simple operation and low cost.However,due to the lack of microorganisms,traditional composting has some defects such as low dead swine degradation rate,slow fermentation and poor composting quality.In order to improve the harmless treatment efficiency of dead swine carcass compost,protease-producing bacteria and lipase-producing bacteria with strong pork degradation ability and no mutual antagonism were screened from dead pig carcass compost samples,and constructed as a compound bacterial agent.After optimizing the enzyme production conditions and adjusting the technical parameters,the compound bacterial agent was inoculated into the dead swine carcass composting to evaluate the application effect,and the regulation effect of the compound bacterial agent on the bacterial community structure in composting was analyzed by high-throughput sequencing technology.In order to study the way of the compound bacterial agent decomposes the dead swine carcasses,the protease produced by strain DB1 was purified and identified,and the binding mode of protease and actin was analyzed by molecular docking.The main research contents are as follows:（1）Serratia marcescens DB1 with high protease production ability and Bacillus velezensis ZF2 with high lipase production ability were screened by identification medium screening,enzyme activity test and pork degradation test.Oxford cup antagonism test indicated that there was no antagonismin the two strains,and they could be used to construct compound bacterial agents.（2）The enzyme production conditions of the screened strains were optimized by single factor experiment and response surface experiment.The results showed that the highest protease activity of strain DB1 could reach 54.32 U/m L while culture conditions were 5 g/L glucose,7g/L urea,1.46 mmol/L Mg²⁺,31.59℃,p H 7.86,inoculation amount 5%,60 h.The highest lipase activity of Strain ZF2 could reach 33.09 U/m L while cultured conditions were 3 g/L sucrose,7 g/L ammonium sulfate,0.86 mmol/L Mn²⁺,26.33℃,p H 6.19,inoculation amount5%,48 h.（3）After optimizing the technical parameters of the compound bacterial agent,the compound bacterial agents was inoculated into compost to evaluate the application effect.According to the optimization results,the compound bacterial agent was inoculated with 0%,1%,3%,and 5%of the material wet weight as the control group,low dose group,medium dose group,and the high dose group.The results showed that the high temperature period of the control group and the low,medium and high dose groups was maintained for 7,14,20 and 15days respectively,and the maximum temperature reached 60,64,69 and 62℃respectively.At the end of composting,the degradation rates of dead swine carcasses in the control group,low,medium and high dose groups were 78.31%,93.15%,98.03%,94.96%respectively,the water contents were 38.22%,33.64%,28.73%,31.46%respectively,the p H values were 8.17,8.47,8.62,8.53 respectively,the E₄/E₆ values were 2.82,2.37,2.09,2.52 respectively,and the TOC were 40.51%,36.26%,31.86%,35.37%respectively,the TN were 1.28%,1.34%,1.45%,1.32%respectively,the NH₄-N were 1.30,1.32,1.43,1.44 mg/g respectively,the NO₃-N were0.65,0.60,0.71,0.59 mg/g respectively,and the GI was 75.43%,86.55%,96.78%,92.40%.In conclusion,inoculating compound bacteria in compost could increase the temperature,accelerate the decomposition of corpses,promote the maturity and improve the product quality.Among them,the treatment effect of the medium inoculum was the best.After composting,the pathogens in each group samples were completely sterilized,and the hygienic indicators of the samples in each group met the standard of harmless treatment.（4）Through high-throughput sequencing technology,it was revealed that the bacterial community structure of the composting,and the correlation between bacterial community structure and environmental factors was research through RDA analysis.Strain DB1 was the key to the function of compound bacterial agent.Compound bacterial agent mainly accelerates the decomposition of dead swine carcass by improving the relative abundance of strain DB1.The compound agent accelerated the compost fermentation and decomposition of dead swine carcasses by improving the abundance of Serratia.Cerasibacillus and Myroides dominated fermentation and warming in the early stage of composting,Firmicutes＿Unclassified,pseudogracilibacillus and Bacillus dominated the decomposition of organic matter at thermophilic stage and maturity stage.The treatment of the medium dose group increased the abundance of Gammaproteobacteria＿unclassified,Ochrobactrum,Cerasibacillus,and Pusillimonas,thereby accelerating the heating of compost,promoting the decomposition of dead swine carcasses and increasing NO₃-N content.（5）The compound bacteria agent mainly decompose dead swine carcass through strain DB1 in compost.In order to explore the way of decomposing dead swine carcass by strain DB1,the protease produced by strain DB1 was purified and the binding mode between the protease and animal protein was analyzed.After purification,the specific activity of the protease reached1982.77 U/mg,the purification multiple was 46.37 times,and the molecular weight was about112 Kda.This protease is a hydrophilic thiol-dependent thermostable alkaline metalloserine protease with resistance to surfactants and organic solvents.And this protease combines with the protein in the dead swine carcass through the active residues of GLU,ARG,SER,ASP and TYR,thereby realizing the decomposition of the dead swine carcass.