Diversity Of Clostridia Communities And Fermentation Characteristics Of Clostridia With High Yield Of Butyric Acid In Chinese Strong-flavor Liquor Pit Muds

As one of the four basic flavor liquor in China,strong-flavor liquor shares more than70% of the total liquor market,and is deeply appreciated.Pit mud is the necessary material of strong-flavor liquor production,and the species and quantity of microbes in the pit mud greatly affect the flavor and taste of liquor.The clostridia community is one of the dominators in pit mud,which provides the direct source of important flavor substances(such as ethyl hexanoate)or their precursors(such as butyric acid and hexanic acid)in liquor.In addition,butyric acid is a very important raw material for the synthesis of aroma substances and other fine chemical products,which has been applied to various industries such as food,chemical,feed and pharmaceuticals with huge market demands.At present,the effects of clostridia community on formation of the flavor substances of liquor and the clostridia with high yield of butyric acid in strong-flavor Liquor cellar mud are seldom studied.In this paper,the 50years' pit muds in a strong-flavor liquor distillery in Henan was taken as materials,the diversity of clostridia community in pit muds was analyzed by both high-throughput sequencing and traditional culture technology.The volatile metabolites in the fermentation broth of culturable clostridia were extracted and detected by headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry(HS-SPME-GC-MS).The volatile metabolite maps of clostridia bacteria that can be cultivated in pit mud were clearly clarified.The clostridia with high yield of butyric acid were screened and identified and the fermentation conditions were optimized.The study provides a theoretical and technical support for strengthening the formation of liquor flavor substances by bioaugmentation to improve the quality of pit mud and expanding the production of butyric acid by fermentation.The main results are listed as follows:(1)The high throughput sequencing technology and traditional separation and purification technology were used to analyze the community diversity of the uncultivable and cultivable clostridia in the 50 years' pit muds of a famous strong-flavor liquor distillery in Henan province.The study revealed 2 dominant order: Clostridiales,Natranaerobiales;6dominant families of cultivable clostridia: Clostridiaceae,Tissierellaceae,Anaerobrancaceae,Eubacteriaceae,Mogibacteriaceae and Lachnospiraceae;7 dominant genera:Caldicoprobacter,Sedimentibacter,Clostridium,Pseudoramibacter_Eubacterium,Garciella,Caloramator and Oxobacter.The 14 sequences of relative content greater than 1% are selected as the main OTU,and they have the highest similarity to the Caloramator quimbayensis,Christensenella timonensis,C.tagluense,Dethiobacter alkaliphilus,C.cellobioparum,Ihubacter massiliensis,Tissierella creatinini,Tissierella praeacuta,Alkalibaculum bacchi,Sedimentibacter acidaminivorans.88 anaerobic or facultative anaerobic microbes were isolated from the pit of strong-flavor liquor liquor.By sequencing and phylogenetic analysis of the 16 S rRNA gene sequence,it was found that 75 of the pure cultures belonged to 8 phylogenetic clusters(cluster I-VIII),and the highest clostridia resemble of different developmental clusters were C.tyrobutyricum,C.butyricum,C.indolis,C.acetobutylicum,C.bifermentans,C.sporogenes,C.cadaveris and C.kluyveri,in which C.bifermentans and C.cadaveris were isolated in the pit mud for the first time,to a certain extent,enriched the species diversity of clostridia in pit mud.(2)The 14 sequences with relative content greater than 1% were selected as main OTU,which had the highest similarity to Caloramator quimbayensis,Christensenella timonensis,C.tagluense,Dethiobacter alkaliphilus,C.cellobioparum,Ihubacter massiliensis,Tissierella creatinini,Tissierella praeacuta,Alkalibaculum bacchi and Sedimentibacter acidaminivorans.Total of 88 anaerobic or facultative anaerobic microbes were isolated from the pit muds of strong-flavor liquor.Sequencing and phylogenetic analysis of the 16 S rRNA gene sequence found that the 75 isolates belonged to 8 phylogenetic clusters(cluster I-VIII),and the highest resembles in different clusters were C.tyrobutyricum,C.butyricum,C.indolis,C.acetobutylicum,C.bifermentans,C.sporogenes,C.cadaveris and C.kluyveri,among which C.bifermentans and C.cadaveris were isolated in the pit mud for the first time.The study enriched the species diversity of clostridia in pit muds.(3)The capacity of butyric acid production of the representative strains from each phylogenetic cluster were assayed by gas chromatography.The results found that RL1(C.tyrobutyricum)was the best strain producing butyric acid.The culture conditions and medium composition of butyric acid production for the strain were optimized by single factor coupled with orthogonal test and response surface methodology,respectively.The optimum culture conditions were as follows: the initial pH value was 6.8,the liquid amount was 100%,the culture temperature was 34?,the inoculation amount was 3%(v/v),the best deoxidizer wassodium thioglycolate and its dosage was 0.5 g/L.After optimization,the concentration of butyric acid in fermentation broth reached 10.66 g/l,which increased by 41.76% compared with 7.52 g/l before optimization.The best medium composition was as follows(g/l): glucose85.29,yeast extract 7.5,peptone 9.37,sodium chloride 7.5,sodium acetate 4.5,hydrogen phosphate two potassium 2.5,seven hydrated Magnesium Sulfate 0.3,ammonium sulfate 1.5,seven hydrate ferrous sulfate 0.015,sodium thioglycolate 0.5,and calcium carbonate 11.6.The concentration of butyric acid in the optimized medium was 22.96 g/l,which increased by61.35% compared with the 14.23 g/l of the basal medium.In conclusion,by optimizing the culture conditions and medium composition of RL1 strain,the yield of butyric acid increased from 7.52 g/l to 22.96 g/l,which showed ideal effect of optimization.