Cultivation Of Acidogenic Microbiota From Pit Mud And Characterization Of A Novel Caproate-producing Bacterium In Chinese Strong-aroma Liquor Fermentation

Pit mud quality determines the quality of Chinese strong-aroma liquor.Acidogenic microbiota in pit mud have an important effect on pit mud quality as well as quality of the liquor.Nowadays,many researches related to pit mud are mainly focused on microbial community structure.However,little is known about the carbon source cycle in pit mud,as well as the dynamic variation rule of microbiota and its relation to pit mud quality.Microbiota in pit mud belongs to anaerobic system.The difficulty is that a large number of microorganisms which have important metabolic function are hard to be cultivated in laboratories.Therefore,it is of vital importance to build up suitable culture technology,based on which metabolic functions of pit mud microbiota can be further studied.Metabolic profiles of pit mud microbiota have a reflection on their practical function.This study built up a cultivation model system concerning caproate production.We established a quick method to enrich uncultured caproate-producing microbiota,and also studied the metabolic features of caproate-producing microbiota in pit mud through this model system.Meanwhile,High-throughput sequencing technique was applied to analyzing microbial community composition in the cultivation system.We isolated the microbiota and obtained a novel caproate-producing strain which could utilize glucose to produce caproate.Furthermore,we studied the strain’s metabolic features through fermentation.It is expected to be beneficial to other researches in pit mud caproate-producing microbiota.The main results of this study are as follows:（1）Through pit mud microbiota fermentation,we explored and studied the selection of different carbon sources and microbiota treatment measures.The results showed that pit mud microbiota could directly use glucose to produce considerable caproate,and it was a common phenomenon in different pit muds from different areas.The highest caproate production could reach up to 6.68 g?L^-1 in fed batch fermentation of pit mud.Compared to ethanol,choosing glucose as the carbon source could dramatically improve substrate utilization,biomass growth as well as caproate production.Spore-forming microbiota during later period of fermentation showed more stable caproate-producing ability than early period microbiota.Clostridium confirmation in pit mud microbiota by PCR showed that genomic DNA of Clostridium kluyeri was not found in pit mud fermentation microbiota.It indicated that there were other caproate-producing microorganisms apart from Clostridium kluyeri in the liquor fermentation system.（2）During the fermentation process of pit mud acidogenic microbiota,metabolites such as butyrate and caproate had an obvious changing rule.During early period（3 d）of pit mud fermentation,butyrate concentration was generally high in all samples.At 7 d of fermentation or later,caproate was apparently increased while butyrate concentration was declined.It indicated that caproate-producing microbiota could use butyrate to produce caproate.16S rRNA gene sequencing showed that unclassified Clostridium 1,uncultured Caproiciproducens,Clostridium butyricum,unclassified Clostridium 12 were effectively enriched in the cultivation system,whose relative abundance reached up to 81.75%,96.06%in AH1,SC1 microbitoa,repectively.Especially,uncultured Caproiciproducens could be effectively enriched.（3）We obtained a novel caproate-producing strain named strain H2 which could utilize glucose to produce caproate.The strain was short,spindle-shaped,spore-forming bacteria of1.5-2.5μm length and 0.5μm width.Transmission electron micrographs also showed that strain H2 was a peritrichous bacterium with capsule outside.Electron acceptors such as acetate and butyrate could promote biomass growth.Butyrate was a more effective electron acceptor than acetate involving in caproate formation.（4）Whole genome sequencing of strain H2 was carried out.The results showed that its genome size was 2996201 bp,48.48%GC content.COG function classification showed that carbohydrate transport and metabolism had the most matched genes.KEGG pathway annotation showed that carbohydrate metabolism and amino acid metabolism had 117 and 100gene numbers.