Succession And Interactions Of Microbial Communities In Cow Manure And Corn Stalk Composting

Composting is the mainstream technology of the comprehensive utilization of agricultural solid waste.Microorganisms play an important role in the composting process.In the previous researches,the study of microbial community in compost mainly focused on the internal structure of the community,ignoring the study of the ecological function and the interaction between communities,resulting in the limitations in understanding the microecological law of composting process.Therefore,in this study,cattle manure and corn straw were used as raw materials for composting,and the community structure of bacteria and fungi at the DNA level were detected during composting by high-throughput sequencing technology,and the community structure of denitrifying and nitrifying bacteria were detected at the DNA and c DNA levels respectively.The changes in the abundance of the target genes were measured by fluorescence quantitative technique.The microbial community succession,ecological function succession,key bacterial groups in the community and interaction relationships between communities were analyzed through software such as R,database such as PICRUSt.The correlations between microbial community structure and gene abundance with physicochemical factors were analyzed.This study can provide a theoretical basis for further understanding of the mechanism of microecology in composting process.The main findings are as follows:?1?The composting process lasted 59 days and the piles entered thermophilic phase on the 6th day?>55??and lasted 32 days.During composting process,the C/N value gradually decreased,and the electrical conductivity?EC?and seed germination index?GI?gradually increased.At the end of composting,these three indexes were 17.02,2.16 ms/cm and 89.0%,indicating that the compost had completely matured.?2?Permutation multivariate analysis of variance and non-metric multidimensional analysis showed that there are significant differences in bacterial community composition at different stages of composting?p=0.001?.The PICRUSt function prediction analysis of the bacterial community found 23 secondary function types and 217 tertiary function types.The peak value of carbohydrate metabolism,energy metabolism and lipid metabolism was observed at cooling stage,the peak value of amino acid metabolism was observed at maturation stage.KEGG predicts that the dehydrogenase content is highest during composting,followed by protease,urease and?-glucoside hydrolase.Biomarkers of thermophilic phase were belonged to Chloroflexi and Bacillales,biomarkers of maturation phase were belonged to Actinomycetales.Mental test showed temperature,water content,NH₄⁺-N,NO₃^--N,TN and C/N all significantly affected the succession of bacterial communities.?3?Non-metric multi-dimensional and Permutation multivariate analyses showed that the composition of the fungal community was significantly different between the initial,mesophilic and thermophilic phases and the cooling and maturation satges?p=0.025?.unclassified?Microascaceae and Anthostomella were biomarkers of cooling and maturation stages and no biomarker was found at thermophilic phase.The fungal community FUNGuild nutritional type predictions found nutritional types in saprotroph,pathotroph and symbiotroph,each trophic fungal community had significant succession during composting,and the relative abundance of plant pathogens was less than 1%at the end of composting.Mental test showed that only TN,Org N and water content had a significant effect on the succession of fungi community.?4?Co-occurrence network analysis showed that the construction of bacteria,fungi and bacterial-fungal co-occurrence networks were non-random,has small-world,scale-free,and modular characteristics.In the bacterial co-occurrence network,63.14%of the connections were positively correlated.There were 14 connectors and 8 module hubs.The dominant bacteria in the modules belong to Proteobacteria and Firmicutes.In the fungal co-occurrence network,90.65%of the connections were positively correlated,4 connectors and 2 module hubs.The dominant fungi in the modules belong to Ascomycota and Basidiomycota.In the bacterial-fungal co-occurrence network,69.64%of the connections were positively correlated.Intra-bacterial connections were72.25%,and intra-fungal connections were 25.36%.However,there were only 33 bacterial-fungal connections,which accounted for 2.39%of the total connections,and were mainly negatively related.Ascomycetes were found in the main modules.?5?Non-metric multi-dimensional analysis showed that the distances between the sample points of the nir S-type denitrifying bacteria community at different stages were different at the two levels of DNA and c DNA.The analysis of permutation multivariate showed that the composition of the community at each stage of composting was significantly different only at the DNA level?p=0.028?.Seven of the 51 nir S gene OTUs were unique at the DNA level.At the DNA level Pseudomonas was indicator of initial and mesophilic stages,Azoarcus was indicator of maturation stage at the c DNA level.Mental test showed that TN,C/N,and water content had significant effects on the nir S-type denitrifying bacterial community at the DNA level.The influence of physicochemical factors on nir S-type denitrifying bacterial communities at c DNA level was not detected.?6?Non-metric multi-dimensional analysis showed that the nos Z-type denitrifying bacterial community formed different clusters of sample points at each stage at the two levels of DNA and c DNA,and the permutation multivariate analysis showed that the difference of community composition at each stage of composting was significant at the c DNA level?p=0.001?.Two of the49 nos Z gene OTUs were unique at the DNA level.Thauera,Hyphomicrobium and Pseudomonas were indicators of mesophilic stage at DNA and c DNA levels,Ochrobactrum was indicator of maturation stage at the c DNA level.Mental test showed that the succession of the nos Z-type denitrifying bacterial community was significantly affected by TN and water content at the DNA level,and it is significantly affected by NO₃^--N,TN,C/N,temperature and moisture content at the c DNA level.?7?Non-metric multi-dimensional analysis showed that the composition of the ammonia-oxidizing bacteria community at each stage of compost formation at the two levels of DNA and c DNA is different.Permutation multivariate analysis showed the difference in community composition of ammonia-oxidizing bacteria at each stage of composting was significant at DNA and c DNA levels?p=0.003,p=0.001?.No specific OTUs at the DNA level were found in the 21 amo A gene OTUs.Nitrosomonas and Nitrosospira were indicators of mesophilic stage at the c DNA level.Mental test showed that the succession of ammonia-oxidizing bacterial communities was affected by TN,C/N and water content at the DNA level and were significantly affected by NO₃^--N,NH₄⁺-N,TN,C/N,and water content at the c DNA level.?8?Analysis of the co-occurrence network of functional group revealed that there in the interaction relationship between the functional groups and the keystone bacteria in communities at DNA and c DNA levels were different.According to the intermediate centrality index,Pseudomonas,Azoarcus and Rubrivivax;Azohydromonas,Thiohalomonas and Paracoccus were keystone species of nir S-type denitrifying bacteria communities at DNA and c DNA levels reapectively.Thauera,Paracoccus,and Paramesorhizobium;Taoer,Pseudomonas,and Agrobacterium were nos Z-type denitrifying bacteria communities at DNA and c DNA levels.Nitrospira and Nitrosomonas were the keystone species in the network of ammonia-oxidizing bacteria at the DNA level,and Nitrosspira was the keystone species at the c DNA level.The network of functional group of nitrogen transformation was dominated by positive connection,Leptothrix,Thauera and Nitrosospira were keystone species at the c DNA level.?9?Non-metric multi-dimensional and Permutation multivariate analyses showed that the difference of composition of anaerobic ammonia-oxidizing bacterial communities was not significant at each stage of composting.The 20 anaerobic 16S r RNA gene OTUs belonged to Candidatus Scalindua,and the Candidatus Scalindua brodae cluster was the dominant cluster of the Candidatus Scalindua genus.Candidatus Scalindua brodae was indicator of mesophilic and cooling phase,and Candidatus Scalindua marina was indicator of thermophilic,cooling and maturation stages.Mental test showed that NH₄⁺-N,TN,C/N and water content had significant effects on the composition of anaerobic ammonia-oxidizing bacteria,and C/N could significant affect the anaerobic ammonia-oxidizing bacteria community succession without interacting with other environmental variables.