Functional Analysis Of Bacillus Cabrialesii BCH For Deodorization And Application In Pig Manure Composting

With the improvement of people’s living standard,livestock and poultry breeding industry develops rapidly,but at the same time,it also produces a large number of livestock and poultry feces,which brings environmental pollution problems.Compost fermentation is an important disposal method of livestock and poultry manure.The fermented and decomposed products can be used as organic fertilizers in farmland soil to improve the level of soil organic matter and the quality and safety of agricultural products.However,in the traditional composting process,there are some problems such as long cycle,incomplete decomposition and release of odorous substances,which affect the speed and quality of the harmless treatment of livestock and poultry manure.Studies on compost fermentation of livestock and poultry manure showed that adding highly active deodorizing strains isolated from livestock and poultry manure into livestock and poultry manure can promote the fermentation process of compost and reduce the emission of odors.In the early stage of this research group,a Bacillus strain BCH with deodorization effect was isolated and purified from pig manure compost.It was further identified as Bacillus cabrialesii,and its growth temperature range,growth p H range,enzyme production ability,ammonia nitrogen utilization rate,and removal ability of NH₃and H₂S in pig manure were determined.On this basis,the whole genome of strain BCH was sequenced for three generations.KEGG and CAZyme databases were used for sequence annotation and analysis,and genes related to nitrogen and sulfur metabolism were mined.Pig manure was used as composting raw material,and two treatments were set up:the experimental group inoculated with strains and the control group CK without inoculation.The effects of strains on livestock and poultry manure composting were determined,including temperature,p H,seed germination rate,total phosphorus,total potassium,total nitrogen,ammonia nitrogen content and other major nutrients.The main findings are as follows:（1）Identification,deodorization ability and enzyme production characteristics of deodorizing strainsA strain of deodorizing Bacillus was obtained from fermented pig manure and identified as B.caboratesii by morphology and molecular biology.The deodorizing ability and enzyme production characteristics were studied.In terms of growth characteristics,the strain can grow normally in the range of 30°C～60°C and p H4.0～9.0;the strain produced acid in the medium with glucose,fructose and sucrose as the sole carbon source,and the p H of the corresponding medium decreased by 0.90,0.72 and 1.20,respectively.In terms of deodorization ability,the ammonia nitrogen utilization rate of the strain reached 45%.In the indoor pig manure fermentation test,the removal rate of ammonia nitrogen reached27%and the removal rate of hydrogen sulfide reached 26%.The determination of enzyme activity showed that the strain showed a variety of degrading enzyme activities.The highest lipase activity was 23.99 U/m L,and the highest amylase activity was 2.52 U/m L.The highest protease activity was 36.44 U/m L,and the highest carboxymethyl cellulase activity was 36.52 U/m L.The highest urease activity was 18.02 U/m L,and the highest catalase activity was 39.42 U/m L.（2）Whole genome sequencing of B.cabrialesii BCHThe strain BCH was subjected to three generations of whole genome sequencing analysis,and 2822,876,20 and 21 genes were annotated from the KEGG,CAZyme,CARD and VFDB databases,respectively.The KEGG database showed that carbohydrate metabolism contained 172 genes in the pathways related to metabolism.A total of 18 genes were annotated to the nitrogen metabolism pathway,and 20 genes were matched to the sulfur metabolism pathway.A total of 876 genes were annotated in the carbohydrate active enzyme database,including 536 glycoside hydrolase genes（GH）,27 carbohydrate binding modules（CBM）,169 glycosyltransferases（GT）,62 carbohydrate esterases（CE）,53polysaccharide lyases（PL）,and 26 auxiliary enzymes（AA）.In the nitrogen metabolism and sulfur metabolism pathways,the protein structure of the two genes hao and sox B was predicted.The HAO amino acid residues were 1032,and the protein contained a molecular weight of 83.87 k Da and a theoretical isoelectric point of 5.11.SOXB has 681 amino acids with a molecular weight of 54.68 k Da and a theoretical isoelectric point of 5.15.（3）Application of B.cabrialesii BCH strain in pig manure compostingThrough the analysis of the composting indexes and flora of the experimental group with the addition of exogenous strain BCH and the CK blank group,the organic matter mass fraction（dry basis）of the experimental group was 64.35%,the total nutrient（N+P₂O₅+K₂O）mass fraction（dry basis）was 4.66%,and the seed germination index（GI）was 151.64%,which were better than the corresponding physical and chemical indexes of the CK blank group.After the temperature of the two groups tended to be consistent,the odor release of the bacteria group was lower than that of the CK group,and the nitrogen loss of the experimental group was less than that of the control group.After analyzing the changes of microbial flora,it was found that Firmicutes was the dominant flora during the whole composting period at the phylum level.At the genus level,Sphingobium was the dominant flora in the two groups of composts at the initial stage of composting fermentation.Only the abundance of Bacillus in the experimental group was the top ten,and the abundance of bacteria with nitrification ability such as Ureibacillus and sulfur oxidation ability such as Pseudoxanthomonas increased in the experimental group.The abundance of nitrate reduction in the experimental group was higher than that in the CK group,and the abundance of fermentation was higher than that in the CK group.In this study,B.cabrialesii BCH has deodorization and a variety of enzyme activities.It can grow normally in the range of 30℃～60℃and p H4.0～9.0.Its genome has a relatively complete metabolic pathway of ammonia nitrogen and hydrogen sulfide.Adding deodorant strains to pig manure compost can promote the maturation of compost fermentation,reduce the discharge of ammonia and hydrogen sulfide,and reduce the relative abundance of potential pathogens in the pile,so that compost products can reach the maturation standard faster.The research provides basis and resources for livestock manure fermentation and deodorant bacterials.