| Biogas digestion technology has been widely used to solve the problems of environment, agriculture and energy all over the world, and brought huge benefits in ecology, society and economy. Generally, biogas digestion experienced four biochemical processes which are hydrolysis, acidogenesis, acetogenesis and methanogenesis; a series of prokaryotes(bacteria and archaea) played key roles in these biochemical processes. Most of these prokaryotes above have not yet been cultured, so, culture-dependent method was not feasible to study these prokaryotes roles in biogas digestion; however, the culture-independent methods, especially a series of methods based on 16 S rRNA gene has been widely used to research the structure and dynamic of anaerobic digestion microbial community. At present,research concerning the structure and dynamic of microbial community in household biogas digester are rarely reported. Therefore, the systemic research about the ecological problems in household biogas digester had important theoretical and applicated value.Based the analysis above, we studied the prokaryotic community dynamics in household biogas degester from Yunnan subtropical region with lab-scale batch biogas reactors. In this study, anaerobic sludge from household digester and livestock dung from the farmers at Yunnan subtropical region are used as the inoculas and substrate repectively. PCR-DGGE and Illumina high-throughput sequencing based on16 S r RNA gene amplicons are performed to study microbial community dynamic,SPSS software are used to analyze the correlation between microbial community and environmental factors. The results were shown as following.At 15 ℃ conditions, the initial structure of prokaryotic community was different significantly from fermentation process. In fermentation process, the community structures of bacteria and archaea changed constantly, the microbial communities changed largely from 0 to 24 days, and were stable relatively after 32days; most dominant species always had been maintained in whole process, and the abundance of these species increased further at biogas production peak periods. At the phylum level, the main group of bacteria was Firmicutes, followed by Bacteroides,Proteobacteria; the main group of archaea was Euryarchaeota, followed by Crenarchaeota. At the species level, the top 10 groups of bacteria were OTU1(97 %similarity to Clostridium sartagoforme), OTU209(98 % similarity to Clostridium sartagoforme), OTU2(99 % similarity to Terrisporobacter glycolicus), OTU4(100 %similarity to Clostridium butyricum), OTU3(93 % similarity to Alkaliflexus imshenetskii), OTU5(97 % similarity to Advenella mimigardefordensis), OTU200(100 % similarity with Sphingomonas sp.), OTU366(98 % similarity to Clostridium beijerinckii), OTU6(99 % similarity with Turicibacter sanguinis), and OTU8(99 %similarity with Clostridium sp. Ade.TY) repectively. The top 5 sepecies of archaea were OTU1(82 % similarity to Staphylothermus hellenicus), OTU7(96 % similarity to Methanobacterium formicicum), OTU3(96 % similarity to Methanobacterium formicicum), and OTU5(82 % similarity to Staphylothermus hellenicus), and OTU2(100 % similarity to Methanosaeta concilii) repectively.The results of different temperatures(9 to 55 ℃) batch fermentation showed that,with temperature increased, microbial communities has undergone an obvious change,the abundance of most of dominant species reduced and a few increased. During the 9to 45 ℃, the dominance of most of dominant species changed slightly; during the45–55 ℃, the dominant species has undergone a fundamental change. At phylum level, the structures of bacterial and archaeal community were similar with that of15 ℃ batch fermentation process. At species level, the top 10 species of bacteria were OTU1(97 % similarity to Clostridium sartagoforme), OTU2(99 % similarity to Terrisporobacter glycolicus), OTU5(90 % similarity to Candidatus Cloacamonas acidaminovorans), OTU620(98 % similarity to Bifermentans), OTU3(86 %similarity to Zhouia amylolytica), OTU9(90 % similarity to Clostridium sticklandii),OTU6(100 % similarity to Clostridium butyricum), and OTU450(86 % similarity to Galbibacter marinus) repectively. The top 5 groups of archaea were OTU1(99 %similarity to Methanocorpusculum labreanum), OTU6(100 % similarity to Methanosarcina thermophila), OTU2(82 % similarity to Staphylothermus hellenicus),and OTU3(100 % similarity to Methanosaeta concilii)(84 % similarity to Methanobrevibacter sp.) repectively.At the biogas production peak period of different temperature batch reactors, the microbial community structures and metabolic pathways were very similar during15–35 ℃(the major reason was that bacterial(archaeal) species of hydrolysis,acidogenesis, acetogenesis and methanogenesis were the same). When the temperature dropped from 15 to 9 ℃, there were some differences in metabolic pathways(the major reason was that all of the methane almost came fromhydrogentrophic pathway at 9 ℃ while the methane came from both hydrogentrophic and acetotrophic pathways at 15–35 ℃). When the temperature rised from 35 to 45 ℃,metabolic pathway changed in some extent(the major reason was that, compared with35 ℃, the species of hydrolyzed, acetogenic bacteria and methanogens increased in some extent). When the temperature rised from 45 to 55 ℃, metabolic pathways were changed significantly(the main reason was that, compared with 15–45 ℃, the species of hydrolic bacteria increased, the species of acidogenic bacteria decreased, the species of acetogenic bacteria were changed, the acetotrophic matebolism became the main pathway for methane production). Even the microbial communities were the same, the fermentation efficiencies were largely different during 15–35 ℃, mainly due to temperature difference. Based on the analysis above, we believed that the microbial communities and metabolic pathways were the same during 15–35 ℃;45 ℃ was the turning point of the metabolic pathways and microbial commnities from mesophilic to thermophilic; the microbial community and metabolic pathway of9 ℃ were different with that of 15– 35 ℃.Overall, in batch-type biogas fermentation process,(1) between repetitions, the shift of microbial communities and the change of abiotic factors were substantially simultaneous; prokaryotic communities at start-up time were significantly different from that of fermentation process; the microbial community always changed in fermentation process; the key species of bacteria and archaea at biogas production peak at different temperatures were significantly different.(2) The combination of metabolic pathway analysis based OTU(species level) and high-throughput sequencing method of 16 S rRNA gene amplification can reveal complete metabolic pathways in biogas fermentation process. Based the method above, we believed that,for the biogas fermentation used lignocellulosic feedstock, although microbial diversity was very rich(more than 400 kinds of prokaryote), the key species(the bacteria of hydrolysis, acidogenesis, acetogenesis and methanogen) were very small(not more than 20 kinds), and most of these key species were very closed to culturable strains.(3) Generally, bacteria which can hydrolyze cellulose, hemicellulose accupied the most dominant aboundance; and the acetogenic bacteria had lower species and abundance. A few dominant species of bacteria and archaea which played leading roles in the fermentation have significantly positive correlation each other, and formd stable combination; the greater the temperature difference, the greater the difference between the combinations above. Very few dominant groups of bacteria and archaeahad very significant positive correlation with biogas production rate, and these species had potential as the marker which indicated the performance of biogas reactor. |
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