The Change Of Microbial Community Diversity In Deep-Litter System And The Isolation And Identification Of Bacillus

With the rapid increase in the proportion of large-scale pig, the new problems that bring environmental pollution have become an urgent need to solve. By in situ fermentation of pig excrement in the litter, deep-litter system can effectively solve the problem of environmental pollution that caused by pig production.The key technology of deep-litter system is activity of microbial communities in the litter, so dominant bacterium of deep-litter system in the litter and the microbial community diversity become research hotspots in recent years.In this study, efficient bacillus strains were isolated by using pure culturing methods and the microbial community diversity were studied by denaturing gradient gel electrophoresis (DGGE). The results are listed as follows:1The isolation and identification of efficient bacillus strains(1)15bacillus strains, which have unique morphological features, were isolated from litter of deep-litter system.(2)5efficient bacillus strains, which have the higher productivity of amylase and protease, were screened from15bacillus strains.(3)Though the observation of colonial morphology, Gram staining, spore staining, physiological-biochemical characteristic experiments and16S rDNA sequencing analysis,5efficient bacillus strains were identified as Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus stearothermophilus.2molecular biology analysis with DGGE(1)Extraction of total microbial genomic DNA,16S rRNA V6-V8genes were amplified by PCR. Through the perpendicular gradient gel fingerprint analysis and parallel DGGE tests, to determine the denaturant concentration range45%to65%for parallel DGGE. Then, we obtain the DGGE fingerprint of bacteria on deep-litter system sample in the nursery stage and rearing stage.(2)Through the analysis of DGGE fingerprint by Quantity One software, we found that a) The DGGE bands which from each year, thickness and layer of the rearing stage samples were significantly more than the nursery stage samples. b)2years samples of the rearing stage and the nursery stage have highest microbial community diversity in each thickness. c) Small differences of microbial community diversity between each layer of the same pigs feeding stage and service life of padding. d) Small differences of microbial community diversity between shallow layer and deep layer of deep-litter system sample in the rearing stage.(3) The specific bands were cloned and sequenced, we found that a) There are a large number of microorganisms which are not or difficult to be isolated and cultured in deep-litter system. These microorganisms also play a certain role that should to be cognitive in the further. b) Although many of the opportunistic pathogens were detected, swine reared in deep-litter system have low incidence, this may be due to the antagonism of other beneficial microorganisms and pigs’ higher immunity. c) Bacteria and actinomycetes may play a key role in degradation of swine manure, such as Bacillus subtilis. d) Salinicola salarius strain can survive in the high-salt environment. e) Copper ions in deep-litter system were adsorbed and accumulated by dyella terrae strain, thus, the heavy metal pollution was removed.