Enrichment And Reconstruction Of Tetrahydrofuran-degrading Microbial Culture And Interactions Between Degrading Bacterium ZM07 And Non-degrading Bacteria

Tetrahydrofuran（THF）is a widely used organic solvent,causing persistent pollution and global environmental problems.Microbial degradation processes are universally considered to be one of the best ways to reduce THF pollution.In this research,an enrichment culture,H-1,with a stable THF-degrading ability and microbial community structure was enriched from activated sludge.And a thiamine auxotrophic THF-degrading bacterium,Rhodococcus ruber ZM07,was isolated from it.Then,we used composite strains of H-1 to constructed THF-degrading microbial communities and tested their degradation efficiencies and microbial structures.These results provide available resources for THF biodegradation in practical applications.Furthermore,based on the“Black Queen Hypothesis”,we used ZM07 and its cooperators to explore the interaction mechanisms between THF-degrading and non-THF-degrading bacteria in the cocultured systems.Main achievements of this study are shown below:（1）An efficient THF-degrading bacterial culture,H-1,was enriched from activated sludge.The THF degradation performance,stress resistance ability,biodegradation substrate spectrum,and microbial community structure of H-1 were investigated.The results showed that the maximum growth rate and maximum THF degradation rate were0.143 h^-1 and 208.522 mg THF h^-1 g^-1 dry weight for H-1,respectively,which were higher than that of the reported pure strains;H-1 could maintain efficient THF degradation ability under harsh environmental conditions,e.g.,high concentrations of substrate,contamination of heavy metal ions,high temperature,etc.;additionally,the THF degradation could be further improved by replenishment of appropriate carbon sources;H-1 could degrade a variety of organic pollutants,including THF,methylbenzene,ethyl acetate,methanol,1,4-butylene glycol andγ-butyrolactone;and the 16S rRNA sequencing results indicated that Rhodococcus and Hydrogenophaga were the dominant species in H-1.Those two genera were negatively correlated with each other according to Spearman’s correlation analysis.Notably,these species may cooperate with each other to degrade THF in a sustainable and effective manner.（2）Thiamine functions as a cofactor for key enzymes in cellular carbohydrate metabolism,and plays an important role in regulating and controlling the life activities of organisms.The THF-degrading bacterium ZM07 isolated from H-1 was proved to be a thiamine auxotrophic strain according to KEGG metabolic pathway analysis and experiments.It lacks the genes thi F,thi H and thi I,which are required for 4-methyl-5-（beta-hydroxyethyl）-thiazole synthesis,and cannot synthesize thiamine autonomously.Furthermore,we found that ZM07 could cooperate with Escherichia coli K12 through syntrophism without physical contact using two-phase reactors.Strain K12 provides thiamine as a public good to ZM07 in exchange for available metabolites of THF（likely substances that are easily used,such as succinate）.The results showed that:strain ZM07could form a stable symbiotic system with trace amounts of K12 through interspecies cooperation,with THF as the sole carbon source;and it cannot be invaded by the artificially constructed strain Escherichia coli K12（35）thiE（which can neither degrade THF nor produce thiamine）.We also found that in thiamine-rich systems,the growth of K12 was significantly inhibited compared with that in systems without thiamine,indicating that ZM07 would compete niches when it does not need cooperators providing thiamine.（3）H-1 were passaged with or without exogenous thiamine for 40 transfers,and the several response mechanisms of THF-degrading and non-THF-degrading bacteria in H-1 were revealed through 16S rRNA gene sequencing and metatranscriptomic sequencing.First,exogenous thiamine could instantaneously improve the THF degradation performance,increase the total biomass,and affect a large number of genes’expression（the significant difference genes（DEGs）are mainly distributed on metabolism）of H-1.Second,the community structure and transcriptional level of H-1were significantly changed after 40 transfers with exogenous thiamine,it helped strain ZM07,but at the same time it inhibited other microbial metabolism of the community,and the responsiveness abilities of H-1 to environment changes were also reduced.Last,network-directed efficiently isolated ZM07’s cooperator Hydrogenophaga intermedia ZM11,which was significantly negatively correlated with ZM07;these two strains are the core members of H-1,and exogenous thiamine provoked ZM07 and suppressed ZM11 in microbial abundance and even the transcription of the whole microbial community.The results showed that the DEGs were mostly up-regulated in ZM07 and down-regulated in ZM11,indicating that the ecological niches of ZM11 was deprived after losing its value.（4）THF-degrading bacteria ZM07 and 5 non-THF-degrading bacteria,isolated from H-1,were used to construct synthetic microbial consortia.The results showed that Rhizobium daejeonense ZM10 and Hydrogenophaga intermedia ZM11 were the best two cooperators of ZM07 during THF degradation.And the THF degradation performance of the two-strain system of ZM07 and ZM10 was even better than six-strain system.In addition,simultaneous absence of strains ZM10 and ZM11 would significantly reduce the synthetic microbial communities’THF degradation ability.Otherwise,we found that there are some other metabolic exchanges between THF-degrading bacterium ZM07 and non-THF-degrading bacteria ZM10 and ZM11 except thiamine and THF intermediate metabolites through KEGG metabolic pathway analysis.These results provided reference to further exploration of the interaction mechanism between THF-degrading and non-THF-degrading microorganisms.On one hand,this research expands the repertories of functional microorganisms and provides references for THF bioremediation by enriching and constructing bacterial cultures.On the other hand,we focused on the theoretical research of this topic.By exploring the mechanisms of microbial communities in response to exogenous growth factors,this study may shed light on the potential mechanisms of the functional microbial communities’succession.