Swine Manure Compost Technic And Microorganism Screening, Purification And Fermentation

Compost is a primary treatment for the waste of livestock. This method is efficiency and resourceful to deal with organic solid wste, but it also have some disadvatages, for exampl, long time for fermentation and low qulity. In order to improve the quality of compost products, shorten the composting time and improve the composting efficiency, this study reserch in the choice of compost conditioner, screening for compost microbial strains and the impact of the strains for microbial community in compost mainly.The results are summarized as follows:1. Different Conditions on aerobic composting of pig manureRespectively, sawdust, rice bran and wood chipper were used as the conditioner for swin composting. The initial carbon and nitrogen ratio was 25:1. the moisture content of the mixture was 60% and the ventilation method was turning pig manure composting. We compared the difference in temperature, moisture, pH, organic carbon, germination.index, total nitrogen and nitrogen ratio. Three piles experienced a rapid warming stage, high temperature stage and slow cooling stage. All cmpost piles meet the national standard for fecal harmless. Different Conditions influenced the temperature of composting pile. With sawdust as bulking agents in the pile heat up the fastest, followed by rice bran. Wood chips was the slowest. With rice bran as the conditioning agent experenced the longest high-temperature stage, followed by sawdust, wood chips is the shortest one. The trend of moisture content of three piles reduced with the composting process. All piles reduced to about 30% from 60%. Use different conditioning agents in the pig manure composting lead to a significantly different (P<0.05) content of nitrogen. Total nitrogen content of compost with sawdust, rice bran and wood chipers were 2.12%,2.10% and 1.90% respectely.2. Screening, isolation and purification for endo-glucanase enzymes produced strains in compostAfter growing in screening medium in which CMC-Na was the only carbon source we get 17 different microorganisms. All this microorganisms were screened again by filter paper medium we finally obtained an CMCase produced strain. After observed by the culture, Gram staining, spores staining and compareing 16S rRNA with NCBI database the screening strain was identified as Bacillus subtilis and named Subtilis bacillus dcy-1, and preserved in the China Center for Type CultureCollection (CCTCC), preservation code:CCTCC M 208122. The best liquid medium for enzyme production was:wheat bran 1.5%, soybean meal 1.5%, NaCl 0.5%, CaCl2 0.05%, MgSO4 0.05%, yeast extract 0.1%. The best fermentation conditions were:fermentation temperature is 30℃, initial pH 7.0 and the fermentation time were 40h. The best reaction temperature for enzym activity was 55℃, the best pH value was 7.5, Na-, Ca2+, Ni2+ would improve enzyme activity and Cu2+ and Mn2+ would inhibit enzyme activity. Endo-glucanse was good heat stability in less than 60℃and pH 5-8.3. Isolated and identified Egl gene from subtilis bacillus which separated from pig manure composting, complete CDS sequence obtained, GenBank database accession number is, predicted structure and properties of the gene by bioinformatics analysis software were.Constructed recombinant prokaryotic expression vector pGEX-KG of the Egl gene of Bacillus subtilis dcy-1 was constucted. The optimum conditions for expression were:the concentration of IPTG was 0.6mmol/L and the induction time was 3.5h. The activitys of the expressed products were 121.23U/ml.4. Improvement for DNA Extraction method of microorganism in compostBased on the strong resistance of microorganism in compost, designed that samples of compost were digested by protease and helicase. An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical-chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA was amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme Hhal.5. Adding exogenous microbial communities of bacteria in compostThe temperature of-compost can significantly increase when inoculated the microorganisms. Reserched the diversity of bacterial communities in compost by RFLP technique. Compared the difference on bacerial species and qualtity between inoculated and without inoculated composte piles. Results showed that the microorganisms communities can be changed when inoculated exogenous bacterial to cmopost. The exogenous bacterial can grow rapidly in compost and improve to be predominated communities.35 sequences were found, have no similarity with synxenic in th genbank data, this new sequeces may be repreasentation of new spices and the role they played in compst would be worth for further research.