Research On Microbial Community With High Throughput Sequencing Method And Disease Resistance In Livestock Poultry Waste Aerobic Composting

The essence of static aerobic composting is material transformation under the action of microorganisms.The microbial community variation has been a research hotspot.The materials of static aerobic compoting have spatio-temporal difference,so it is unreasonable to group samples only in accordance with time,temperature or space.At present,the high-throughput sequencing data are analyzed for revealing the differences of microbial communities from samples according to dimension reduction methods or Euclidean distance methods.However,these methods are not accurate enough to analyze the samples with discreteness or different sequencing size.In this study,samples are collected from different time and depth in static aerobic samples.Illumina high-throughput sequencing method is used.The Dirichlet Multinomial Mixtures（DMM）is applied for analyzing microbial community data in order to more fully and accurately revealing the temporal and spatial variation of microbial communities.Under the premise of fully understanding the dominant microbial communities during the static aerobic composting,the effective heat resistance strain is isolated and screened and we also have optimized the fermentation conditions.The effective heat resistance strain is inoculated into compost for enhancing the colonization ability and improving the stability of biological organic fertilizer products.The results are as follows:（1）The study results showed that the physical and chemical indexes of the composting process are different with time and space variation.The depth of compost affects temperature,pH and GI.（2）The high-throughput sequencing results showed that there were 5372 bacterial Operational Taxonomic Units（OTUs）including 42 archaea OTUs and 670 fungi OTUs detected during the static aerobic composting process.The top four OTU number of bacterial phylum level were Bacteria unclassified phylum（1553）,Proteobacteria phylum（1232）,Firmicutes phylum（915）and Actinobacteria phylum（456）.The top four OTU relative abundance were Bacteria unclassified phylum（23.56%）,Firmicutes phylum（19.91%）,Chloroflexi phylum（13.14%）and Proteobacteria phylum（12.74%）;Ascomycota phylum was dominant in fungi community and the OTU number and relative abundance were 317 OTUs and 94.17%,respectively.（3）Using the DMM-based approach,compost samples fell into three community types（type I,II,III）.Most of the samples from compost initial stage were grouped together in in type I.The top three relative abundance OTUs were Thermus sp.（13.37%）,Thermaerobacter sp.（12.81%）and Planifilum sp.（9.07%）;Composting medium stage samples were in type II.Chloroflexi unclassified sp.（8.04%）,Bacillus sp.（7.62%）and Thermopolyspora sp.（5.95%）had high relative abundance in type II.Type III was mainly composed of compost samples from late stage of composting and Chloroflexi unclassified sp.was dominant OTU（6.18%）.（4）Accroding to the fungi community,compost samples fell into four community types（type FI,FII,FIII and FIV）.Most of the samples from compost initial stage were grouped together in type FI,and Saccharomycetales sp.was dominant OTU;Type FIV was mainly composed of compost samples from late stage of composting.Scedosporium minutisporum,Aspergillus cibarius and Thermomyces lanuginosus were top three OTUs relative abundance;The samples from medium stage of composting distributed in community type FII and FIII.The dominant OTUs in type FII and FIII were Sordariales sp.and Acremonium alcalophilum,Saccharomycetales sp.and Scedosporium minutisporum,respectively.（5）Depth had remarkable effects on the dominant microbial OTUs in static aerobic composting.These OTUs were Planifilum sp.,Thermaerobacter sp.,Thermus sp.,Acidobacteria Gp6 sp.,Bacillus sp.,Chloroflexi unclassified sp.and three Bacteria unclassified sp.The relative abundance of Scedosporium minutisporuma also was affected by depth.Therefore,the microbial community variation was affected not only by time,but also by depth.（6）Bacillus sp.was the only OTU which could have over 1.0%during the whole static aerobic composting process.The dominant Actinobacteria OTUs mainly existed in bacterial type II.Methanosarcina sp.was the only one Archaea OTU which had over 1%relative abundance in type I.（7）In this study,we isolated and screen one strain（G68）with good genetic stability,wide antibacterial spectrum,high temperature resistant ability from dominant Bacillus genus in static aerobic composting.It was identified as bacillus subtilis.Single factor method and response surface analysis were combined applied to optimize the fermentation medium and culture conditions.The optimum fermentation culture medium（1L）was as follows:Maltose 2.18%,Soybean powder 1.45%,MnSO₄ 0.03%,CaCl₂ 0.02%,MgSO₄0.03%,FeSO4 0.02%.The optimum fermentation conditions were as follows:initial pH 7.5,inoculation amount 5.0%,revolution 180 rpm/min,temperature 40°C and fermentation time 36 hours.We produced the organic fertilizer with G68 addition.The spores were not decreased significantly after the high temperature fermentation which indicated that the G68 has good heat resistance and can be used for high temperature aerobic composting.The protection effect of organic fertilizer with G68 addtion was significantly improved.