Research Of Syntrophic Propionate-degrading Consortia From Anaerobic Digestion Of Vinasse

Volatile fatty acids(VFAs)such as propionate and acetate are important intermediate product in the process of anaerobic digestion.Factors such as complex materials and organic overload will lead to accumulation of VFAs,which in turn will cause acidification of the system and inhibit the growth of microorganisms,thereby affecting the stable operation of the anaerobic digestion system.Reactor acidification has become an important factor restricting the popularization and application of organic solid waste anaerobic digestion technology.Thermodynamically,anaerobic digestion of propionate is a highly energy-absorbing biochemical reaction,which usually accumulates in the anaerobic digestion system and may even cause the anaerobic digestion process failure.Since the degradation of propionate depends on the symbiosis of propionate-oxidizing bacteria and methanogens under anaerobic conditions,the separation and cultivation of anaerobic propionate-oxidizing bacteria is very difficult.At present,there are few studies on the propionate-degrading community and there is insufficient information about the microorganisms of the propionate oxidizing bacteria in the biogas project.Therefore,the study of the propionate-degrading community in the anaerobic digestion system is of great significance for controlling the accumulation of propionate and ensuring the stable operation of the biogas plant.This study combines traditional culture technology with high-throughput sequencing technology to study the propionate-degrading community,and uses vinasse biogas slurry as the research object to provide theoretical and scientific basis for the stable operation of of vinasse biogas plant.The main contents of the thesis specifically included the following aspects:(1)Analysis of microbial community of vinasse biogas plant based on 16 S rRNA gene-based high-throughput sequencing technology.To investigate microbial community of vinasse biogas plant,primary enrichments were made with vinasse as sole carbon source with two stages of the vinasse biogas slurry as inoculum.Two stable mesophilic methanogenic vinasse degrading enrichments JO and JN were obtained after half year of successive transfers.Batch experiments were conducted with vinasse with different substrate concentrations(TS,4.3% or 7.8%)as carbon source.During anaerobic degradation of vinasse,volatile acids and aromatic compounds(p-cresol,phenylpropionate,phenol and benzoate)were detected as transient intermediates.The bacterial and archaeal community structures were investigated with a 16 S rRNA gene-based high-throughput sequencing technology.The results showed that phylum Bacteroidetes,Firmicutes,Candidatus Cloacimonetes,and Synergistetes were the most frequently.Candidatus Cloacimonetes members W5 and W27 genera account for 13.8% ? 30.67%,indicating that they play an important role in the anaerobic degradation of vinasse.(2)Enrichment and phylogenetic analysis of syntrophic propionate-degrading consortia from anaerobic digestion of vinasse.To investigate the propionate-degrading community of vinasse biogas plant,primary enrichments were made with propionate as sole carbon source with vinasse biogas slurry JO and JN as inoculum.Two stable mesophilic methanogenic propionate-degrading enrichments JO-P and JN-P were obtained after one year of successive transfers.Propionate was converted to methane and acetate within 1.5?2 months.Acetate accumulation occurred in culture JO-P,and the concentration remained around 10.5 mmol/L at the end of the experiment.Acetate accumulation is an important factor that affects the degradation rate and conversion rate of propionate in culture JO-P.The results showed that the dominant propionate-degrading bacteria in the two enrichments were different.In culture JO-P,three types of cultured propionate-degrading species Smithella propionica,Syntrophobacter wolinii and Desulfobulbus propionicus,which accounted for 37.61%,10.40% and 1.34% of the total number of bacterial community,The archaeal community in two cultures was also different.In JO-P culture,hydrogenotrophic methanogen Methanoculleus receptaculi and acetotrophic methanogen Methanosaeta concilii accounted for 95.09% and 4.85% of the total archaeal community,respectively.Hydrogenotrophic methanogens Methanoculleus receptaculi was dominant in JN-P culture,which accounted for 99.98%.The cause of the accumulation of acetate in the culture JO-P is the lack of acetotrophic methanogens.(3)To investigate the propionate-degrading community of vinasse biogas plant,primary enrichments were made with propionate and mixed amino acid as composite carbon source with vinasse biogas slurry as inoculum.A mesophilic methanogenic propionate-degrading enrichments J-AP were obtained after one year of successive transfers.Propionate was converted to methane and acetate within 20 days and propionate removal rate is 100%.The analysis results of bacterial community analysis showed that there was a propionate-degrading species Pelotomaculum schinkii in J-AP and JT-AP,accounting for 16.7% and 17.4% of the cultured bacteria,respectively.The archaeal community in two cultures was also different.In JO-P culture,hydrogenotrophic methanogen Methanoculleus receptaculi and acetotrophic methanogen Methanosaeta concilii accounted for 37.2% and 58.6% of the total archaeal community,respectively.Hydrogenotrophic methanogens Methanoculleus receptaculi was dominant in JN-P culture,which accounted for 83.8%.The degradation of propionate in the vinasse biogas plant is coordinated by a variety of microbial communities.The use of different isolation and culture strategies can help to analyze the community structure and function of different propionate-degrading bacterial communities.