Research On Microbial Community Structure And Their Function Of Rural Household Biogas Digesters In Qinghai

Biogas is a promising clean energy source,and the extended application of biogas provides an effective approach to solve rural energy supply problems and environmental protection issues,to establish a circular agricultural system,to achieve sustainable agricultural development,and to promote the construction of a new socialist countryside.An efficient and stable microbial ecosystem is the key to the stable operation of biogas digesters.Due to the cold winters in northern China,most of the biogas digesters undergo low production of gas and low utilization efficiency.In this study,the rural household biogas digesters in Qinghai Province were taken as samples to study the characteristics of microbial community under different temperature using Illumina MiSeq high-throughput sequencing and PCR-DGGE.The effect of temperature on the microbial community structure in the biogas fermentation system has been studied.Metagenome sequencing technology is used to analyze the gene function and metabolic pathways of microbial communities under different temperature,and to explore the key enzymes and functions of the biogas microorganisms in these metabolic processes.The present study lays the foundation for the identification of microbial groups with specific functions,and provides theoretical basis and technical support for improving the efficiency of biogas digesters in Qinghai.1.We exploited PCR-DGGE technique to study the microbial community structures of biogas digesters in different regions(Datong,Huangyuan and Ledu counties)of Qinghai Province.The results showed that the bacteria and archaea collected from all the 15 biogas digesters can be classified into taxon-rich groups.The bacteria belonged to 10 phyla and 38 genera with three dominant phylum(Bacteroidetes,Firmicutes and Proteobacteria)and the most dominant genus Mangroviflexus.The archaea belonged to 6 orders and 10 genera with the most dominant group Methanogenium(Methanomicrobiales order).Significant differences in bacteria and archaea community structures were observed in the samples collected from different regions.The microbial community structures in the samples collected from Datong and Huangyuan areas were relatively similar.I lowever,the microbial community structures in Ledu samples were significantly different from Datong and Huangyuan samples.RDA analysis showed that fermentation temperature and altitude were the major factors affecting the composition and diversity of microbial communities,and the formcr was the most important to influence the Unclassified Marinilabiliaceae,Proteiniclasticum,Unclassified Clostridiales,Methanogeniuwn and Thermomyrmomas.2.Statistical analysis of the correlation between the inside and outside whole-year temperature of biogas digesters and biogas production found that fermentation temperature was affected by ambient temperature.The occurrence of the special "extreme delay" phenomenon revealed that the ambient temperature and biogas production were positively correlated.The microbial community structures obtained by high-throughput sequencing were generally consistent with those obtained by PCR-DGGE.However,the Shannon-Wiener index(H)' obtained by high-throughput sequencing was significantly higher than that of DGGE,demonstrating the higher sensitivity of high-throughput sequencing than the latter method.3.Illumina Miseq high-throughput sequencing generated 651,037 bacterial and 29,248 archaeal sequences in total,comprising 91,286 bacterial taxa OTUs and 5,207 archaeal OTUs from all biogas-digester samples annully collected at Ledu region.The bacteria belonged to 21 phyla and 345 genera,and the archaea belonged to 6 orders and 17 genera.Finnicutes,Bacteroidetes,and Proteobacteria were classified as the most important functional bacterial communities in the present biogas fermentation system.The abundance of Synergistetes was significantly affected by the change of temperatures and positively associated with biogas production,suggesting the functional importance of Synergistetes in our biogas fermentation system.At genus level,Proteiniphilum,Clostridium sensu stricto 1,Petrimonas,Pseudomonas and Fastidiosipila have been identified as the top five dominant groups in all samples,simultaneously playing important roles in the biogas fermentation.As for archaea,Methanomirobiales and Methanobacteriales constituted the major groups with Methanomirobiales the most dominant.At genus level,the aboundance of Methanogeniwn(the most dominant archaea group in all the samples)regularly changed with the fluctuation of fermentation temperature,suggesting the first functional contribution of Methanogenium to the biogas production.The abundance of Thermogymnomoncas also to certain extent reflected the production efficiency of the biogas system.4.Metagenome sequencing of LD-G2,LD-B2,LD-G5,and LD-B5 samples produced 6.38 Gb,5.80 Gb,4.68 Gb and 5.36 Gb clean data,respectively.A total number of 199467,184181,145803 and 142638 ORFs were acquired for LD-G2,LD-B2,LD-G5 and LD-B5 samples,respectively.The relationship between temperature change and carbon metabolism of microbial communities was further explored using KEGG analysis.The results revealed that the functional genes to support primary nutrient metabolisms including glycolysis,pentose phosphate pathway and fatty acid metabolism remained intact in the four samples.Interestingly,their contributions to specific key enzme genes were different as LD-G2>LD-B2>LD-G5>LD-B5,indicating the promotion of higher temperature on the ability of microorganisms to degrade carbon compounds.Meanwhile,the abundance of UDP-glucose 6-dehydrogenase(the key enzyme for bacterial synthesis of extracellular polymers)were increased at higher temperatures in the 4 samples,demonstrating the stimulating effect of higher temperature on the bacterial ability to produce extracellular polymers that facilitated degradation of various carbon sources.5.The relationship between temperature change and microbial community nitrogen and sulfur metabolism was investigated using KEGG analysis.The results showed that all the enzyme genes required for amino acid metabolisms and sulfate reduction pathway were essentially complete in the investigated samples.Among these 4 samples,the genetic abundances of amino acid decarboxylases(such as glutamate decarboxylase and aspartate 4-decarboxylase)and enzymes of sulfur metabolism(sulfate adenylyltransferase,adenylyl-sulfate kinase,phosphoadenylyl-sulfate reductase,and assimilatory sulfite reductase)were exclusively ordered as LD-G2>LD-B2>LD-G5>LD-B5,indicating the accelerating effect of higher temperature on amino acid catabolism and sulfur metabolism.In addition,our results also suggested the crucial role of temperature in regulating urease(an important enzyme in urea metabolism)transcript aboundace and the related denitrification(a major inorganic nitrogen metabolism process)efficiency.KEGG analysis further revealed the positive correlation of Mcr gene(encoding Methyl-CoM reductase,a key enzyme in methane metabolism)aboundance from methanogens with temperature increasing,the intact metabolic pathways to reduce CO2 by hydrogenotrophic methanogens in all the 4 samples,and the equal contribution of several important enzymes(i.e.formylmethanofuran-tetrahydromethanopterin N-fonnyltransferase,methenyltetrahydromethanopterin cyclohydrolase,methylenetetrahydromethanopterin dehydrogenase,5,10-methylenetetrahydromethanopterin reductase and Mcr)to the temperature-controlled biogas production,indicating the positive correlation between temperature and the ability of methanogens to reduce CO2 to CH4.No information on carbon monoxide dehydrogenase and acetyl-CoA synthetase complex(Codh/Acs)genes was found in any of the four samples,suggesting the lacking of acetate-utilizing methanogens in the present methane fermentation.Thus our study demonstrated the predominant role of CO2-reducing methanogenesis in CH4 production for Qinghai biogas digesters.Up to date,it has been rarely reported on microbial community structures and functions of rural biogas digesters at Qinghai plateau and more wide northern cold area of China.In summary,the present study reported a special "extreme delay" phenomenon,indicating the positive contribution of fermentation temperature to Qinghai plateau biogas production;and further molecularly identified microbial community structure characteristics of rural biogas digesters under Qinghai alpine cold conditions and to the end elucidated specific functional gene activities and related metabolic processes to support the identified microbial communities at different working temperatures,providing theoretical foundation to develop effective microbial agents to optimize biogas fermentation at alpine cold regions.