Bacterial Diversity Of Microbial Fermentation Bed Based On Metagenomic Analysis

A large number of animal feces produced in the large-scale livestock and poultry farming industry have caused a significant problem in environment.The pig feces are mixed with the litters and decomposed by microbes simultaneously in microbial fermentation bed(MFB).Importantly,the manure mixture decomposting can occur within the MFB without waste emission and odor spreading,achieving zero discharge of pig farming.Moreover,the completely fermented pig feces and litter mixture can be further transformed into the qualified bio-fertilizers.During the decomposting process,it is believed that bacteria play pivotal roles.Therefore,the main objective of this study is to better understand the dynamics of bacterial communities in MFB,and to provide appropriate information for biodegradation of pig manures.In the light of the above considerations,diversity and dynamics of bacterial community in the MFB system were evaluated by Illumina sequencing of 16S rRNA gene V3-V4 hypervariable region.Our research provides theoretical guidance for the maintenance and replacement of mattress in MFB.1 Bacterial community diversity and dynamics of the litters in summer and winter762,923 sequences were obtained from samples of two seasons,containing 34 phyla,70 classes,260 famlies and 1843 OTUs with above 90%shared OTUs.The main phyla in the MFB of those two seasons were Bacteroidetes,Firmicutes,Proteobacteria and Actinobacteria.Litters had different bacterial community structure.First of all,more valid sequencing reads and operational taxonomic units(OTUs)and richer diversity of bacterial taxa could be found in the litters in summer than in winter.Contents of the Phyla Actinobacteria(19.8%)and Deinococcus-Thermus(7.4%)in the summer samples were much higher than those in the winter litters.At the genus level,the contents of the genera Truepera and Moheibacter in the summer samples were higher than those in the winter samples.More importantly,the high contents of those bacteria would be associated to the biodegradation of pig manure in high temperature environment in summer.Oppositely,the contents of the Phyla Bacteroidetes(31.6%)and Proteobacteria(22.3%)in the winter samples were higher than those in the summer samples.At the genus level,the contents of the genera Proteiniphilum,Brumimicrobium and Flavobacterium of Bacteroidetes in the winter litters were 1.8,2.1 and 5.2 times of those in the summer samples.the contents of the genera Lactobacillus,Pseudomonas and Thiopseudomonas(Proteobacteria)in the winter litters were higher than those in the summer samples.The high contents of those bacteria would be associated to the biodegradation of pig manure in low temperature environment in winter.2 Bacterial community diversity and dynamics of the litters with different fermentation levels1,198,467 sequences were obtained,containing 33 phyla,272 famlies,600 genera and 1839 OTUs(1348 shared OTUs).Bacterial community diversity of the litters with different fermentation levels showed that Bacteroidetes,Proteobacteria,Actinobacteria and Firmicutes were the main phyla.The bacterial community structures were related with fermentation levels.The contents of the Families Dermabacteraceae,Microbacteriaceae,Brevibacteriaceae and Staphylococcaceae decreased with increase of the fermentation level,suggesting that the MFB could bring biocontrol effect on pathogens to protect the health of pigs.Organic degradation bacteria were different in the three stages of fermentation.In the mildly fermented samples,Xanthomonadaceae and Intrasporangiaceae were the main degradation bacteria,while Saprospiraceae(Proteobacteria)Flavobacteriaceae(Bacteroidetes),Corynebacteriaceae(Actinobacteria)and Trueperaceae(Deinococcus-Thermus)were the decomposers in the mildly fermented litters.In the deeply fermented litters,however,Ruminococcaceae(Firmicutes),Porphyromonadaceae(Bacteroidetes),Pseudomonadaceae and Alcaligenaceae(Proteobacteria)were the main decomposers.The contents of the Ruminococcaceae from manure increased with fermentation,which can be a singal to replace the litters in MFB.Another important finding was that an unclassified bacterium was found on phylum level,suggesting new microorganism resource in MFB.3 Bacterial community diversity and dynamics of the litters from different depths1,045,225 sequences were obtained,containing 32 phyla,203 famlies,609 genera and 1834 OTUs(1482 shared OTUs).The shallow litters had most abundant bacteria,while middle litters had greatest diversity.The microbial community was obviously related to the depth of the litters.In the shallow litters,contents of the Phyla Proteobacteria(25.9%)and Actinobacteria(10.2%)were higher than other litters.The shallow litters had high content of Bacteroidetes(27.8%),Proteobacteria(25.1%)and Firmicutes(17.0%).However,the contents of the Phyla Bacteroidetes(33.3%)and Spirochaetae(33.3%)in the deep litters were higer than those in the shallow litters.Importantly,our reults showed that contents of the organic matter-decomposing bacteria participating possibly in the degradation of pig manures displayed a feature with numerous species and high abundance in the shallow litters,and the contents of the anaerobic bacteria increased with the increase of the litter depth.It is worthing mentioning that Cryomorphaceae from ocean was firstly found in terrestrial habitats,and further research is neeed to reveal the function of those bacteria in MFB.4 Diversity and dynamics of the bacterial community participating possibly in the degradation of pig manures in the MFB315,761 sequences were obtained.Diversity and dynamics of the bacterial community participating possibly in the degradation of pig manures in the in situ MFB were further investigated.It was found that the Phyla Firmicutes,Actinobacteria,Bacteroidetes and Proteobacteria were the most dominant groups among all the fermented pig manure samples.According to the heatmap which depicted the relative percentage of each bacterial family within each sample,the pig manure samples could be definitely clustered into three basic categories:fresh,short-term fermented,and long-term fermented pig manures.The most abundant OTUs at the phylum level were Firmicutes,Actinobacteria and Proteobacteria in the long-term fermented pig manure samples,whereas Firmicutes,Bacteroidetes,and Proteobacteria were found to predominate in the short-term fermented ones.At the genus level,the organic matter-decomposing bacteria such as Corynebacterium,Bacillus,Virgibacillus,Pseudomonas,Actinobacteria,Lactobacillus,and Pediococcus were were the dominate genera in the long-term fermented samples,but they were rare in the short-term fermented samples.Furthermore,the association analysis indicated that Firmicutes,Actinobacteria,Acidobacteria and Proteobacteria might be closely relevant to increase of the pig manure C/N ratios and decrease of unpleasant odor substance 3-MI concentration in the litters,suggesting their roles in the degradation of pig manures.5 Development of Fermentation Bacillus sp.for MFBTo develop Bacillus that can be used in MFB,biological characteristics and antifungal capacity of the biosurfactant-producing Bacillus sp.FJAT-14262 and FJAT-28592 were analyzed.The crude extracts of the strain FJAT-14262 was analyzed by Fourier Transforms Infrared Spectroscopy,Reverse-Phase High-Performance Liquid Chromatography(RP-HPLC)and Liquid Chromatography-Mass Spectrometry(LC-MS),respectively.Moreover,the genome of the strain FJAT-14262 was sequenced,and bioinformatic analysis revealed it contained the complete surfactin peptide biosynthesis genes.All these results demonstrated that the lipopeptide from FJAT-14262 was surfactin with polar amino acids and hydrophobic fatty acid chains.The crude extracts of the strain FJAT-28592 was compared with the reference standards of Iturin A by HPLC.Moreover,the Iturin A synthesis related genes could be cloned by PCR.The chemical and molecular genetic evidence thus indicated that the biosurfactant from FJAT-28592 was a kind of iturin A.Finally,the antimicrobial experiments confirmed that the strains Bacillus sp.FJAT-14262 and FJAT-28592 exhibited strong inhibitory activities against Fusarium oxysporum,inferring their good biocontrol prospects in MFB.