| The interactions between different species could affect the structure,assembly,succession,and ecological function of the microbial community.The mud cellar is the fermentation container of Chinese strong-flavor baijiu,and the microbial community in pit mud plays an important role in the formation of the flavor compound precursors(butyric and caproic acid).Syntrophic bacteria(such as Syntrophococcus and Syntrophomonas)and Clostridium are the dominant functional genuses in pit mud.Their metabolic activities are closely related to the production of short-/medium-chain fatty acids(S/MCFAs)such as butyric and caproic acid.At present,based on the analysis of microbial community structure in pit mud by amplicon sequencing,metagenome and multivariate statistical analysis,researchers have predicted the interaction between microorganisms,including syntrophic bacteria and Clostridium,but there was no direct evidence to explain the complex interaction between species,and the interaction between syntrophic bacteria and Clostridium and its impact on the production of S/MCFAs is not clear.In this paper,Novisyntrophococcus fermenticellae JN500902(N.902)and different Clostridium strains isolated from pit mud were studied.Firstly,comparative genome analysis was used to research the functional characteristics of the pan genome and core genome and compare their S/MCFAs metabolism pathways.Then,N.902 was co-cultured with various Clostridium strains,and the differences in growth and S/MCFAs accumulation in mono-and co-cultures were evaluated to determine the interaction types of different combinations.The combination that promoted growth and acid production,N.902 and C.fermenticellae JN500901(C.901),was further selected to study the interaction mechanism between them by transcriptomics.This study is conducive to deepening our understanding of the mechanism of pit mud microorganisms’ collaborative fermentation to promote aroma production.The main results are as follows:(1)Comparative genomic analysis was carried out on N.902 and different Clostridium species(C.fermenticellae,C.tyrobutyricum,C.scatologenes,C.luticellii,C.aciditolerans,C.guangxiense,and C.beijerinckii).The results showed that Clostridium’s core genome was annotated for metabolism function the most,while specific genes were mainly annotated for carbohydrate metabolism,energy metabolism,and amino acid metabolism.We further analyzed the annotation results of the metabolic pathways of KEGG debates and constructed the S/MCFAs metabolism pathway of the above microorganisms.N.902 was annotated to complete glycolysis,pyruvate metabolism,and the Wood-Ljungdahl pathway to potentially produce acetic acid.Clostridium species were found to be capable of producing acetic acid,butyric acid,valeric acid,and caproic acid by glycolysis,pyruvate metabolism,and reverse β-oxidation.Different Clostridium species have different metabolic pathways for S/MCFAs.Among them,C.luticellii and C.tyrobutyricum mainly produce butyric acid through the but(butyryl-Co A:acetate-Co A transferase)pathway,while other Clostridium species have both but and buk(butyrate kinas)pathways.Only the genomes of C.aciditolerans,C.luticellarii,and C.scatologenes have the complete pathway of converting glycerone-phosphate to propionyl-Co A,the precursor of odd carbon fatty acids,while other species have mainly annotated the genes related to the production of propionyl-Co A from acetyl Co A.(2)The types of interactions between N.902 and various pit mud Clostridium strains(C.901,C.tyrobutyricum Clb01,C.scatologenes Cls01,C.luticellarii Clu07,C.aciditolerans Claci01,C.guangxiense Clgx01 and C.beijerinckii Clb01)were investigated.N.902 cocultured with C.901 or Clu07 significantly promoted growth and metabolism.Glucose consumption and biomass increased in co-culture.In addition,the accumulation of butyric acid and caproic acid increased by about 0.44 and 0.63 times in the N.902-C.901 co-culture,and the yield of butyric acid and valeric acid increased by 0.77 and 2.63 times in the N.902-Clu07 coculture,respectively.The co-culture of N.902 with two valeric acid producing strains(Cls01and Claci01)had no significant effect on the growth,but the N.902-Cls01 combination promoted the production of valeric acid,while the N.902-Claci01 combination inhibited the accumulation of valeric acid.Coculture of N.902 with three butyric acid producing-strains,C.beijerinckii Clb01,C.guangxiense Clgx01,and C.tyrobutyricum Clt01,showed a higher growth rate and biomass than that of mono-culture in the early stage(before 24 h),but the accumulation of butyric acid decreased to varying degrees at the end of coculture fermentation.In conclusion,the interaction between N.902 and different Clostridium strains showed specieslevel diversity.(3)The interaction mechanism of co-culture of N.902 and C.901 to promote the production of S/MCFAs was analyzed.q PCR analysis showed that the co-culture of N.902 and C.901 could promote the growth of both sides,and the type of interaction was protocooperation.The growth and S/MCFAs metabolism of the combination showed stability and robustness in co-culture experiments with different inoculation ratios and continuous passage.A transwell system was used to co-culture N.902 and C.901 separately.The results showed that the interaction between them did not depend on physical contact but mainly on the exchange of metabolites.The metabolic exchange mechanism of N.902 and C.901 was predicted by transcriptome analysis.The results of the differential expression genes revealed that C.901 genes involved in pyruvate metabolism and formic acid production were up-regulated,as were genes involved in the reverse-oxidation pathway,which could convert acetic acid to butyric acid and caproic acid.N.902 was capable of transferring formic acid to acetic acid via the Wood Ljungdahl pathway,and almost all genes in this pathway were up-regulated.The interation mechanism was verified by RT-q PCR and by experiments involving the deletion or addition of intermediate metabolites.The results showed that the formic acid metabolized by C.901 in co-culture could be used by N.902 via the Wood-Ljungdahl pathway to promote the production of acetic acid,and that the acetic acid produced by N.902 could be used by C.901 via the reverse β-oxidation pathway to promote the accumulation of butyric acid and caproic acid.In addition,considering the environmental characteristics of pit mud in situ,the effects of lactic acid and hydrogen on N.902-C.901 co-culture were studied.We found that high concentrations of lactic acid obviously inhibited the growth of N.902 and the accumulation of acetic acid,butyric acid,and caproic acid in co-culture.The reduction of hydrogen partial pressure did not affect the growth of the co-culture but significantly inhibited the production of butyric acid and caproic acid. |
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