Isolation And Characterization Of Anaerobic Bacteria From Syntrophic Alkane-Degrading Communities

Crude oil is the main energy source and chemical raw material of China.However,more than two thirds of crude oil can't be recovered efficiently.Research on biodegradation of residual crude oil into biogas by methanogenic microbial communities is the frontier in energy fields.Petroleum hydrocarbon is the main component of crude oil,and the process of methanogenic degradation of petroleum hydrocarbon is a multiplex syntrophic metabolism,such as the initial degradation of hydrocarbon and oxidation of intermediate fatty acids,which needs cooperation among different types of syntrophic bacteria and methanogens.Syntrophic metabolism is an limited step during methanogenic degradation of petroleum hydrocarbons.Giving the difficulties on isolation of syntrophic bacteria through traditional culture-dependent methods,Culture-independent techniques have been applied to study the functional microorganisms and methanogenic pathway in the degradation of petroleum hydrocarbon.However,it has become a stumbling-block to unravel the molecular mechanism of syntrophic degradation of hydrocarbon for the lacking of pure cultures of synrophic bacteria.It's a low efficiency to isolate syntrophic bacteria through traditional Hungate anaerobic technique,it is also difficult to isolate targeted bacteria for the strategy is selective enrichment first,which would make the targeted microorganism disappeared before isolation.In this study,a high-throughput isolation technique has been developed,which was applied to isolate anaerobic microorganisms involved in methanogenic hydrocarbon degradation.Furthermore,we characterized and proposed two novel families.(1)Syntrophic alkane-degrading communities incubated under different temperature conditions(25-55?)have the ability to utilize VFAs(include formate,acetate,propionate and butyrate)and generate methane.:The predominant syntrophic bacteria are different under different incubated temperature and VFA addition conditions.The members related to Tepidanaerobacter(formate as a sole carbon source),Thermosyntropha(acetate/propionate/butyrate as sole carbon sources),Thermotoga and Pelotomaculum(formate/acetate/propionate/butyrate as a sole carbon source)became dominant in relative abundance under high temperature conditions;Tepidanaerobacter(acetate/propionate as sole carbon source)was the dominant known syntrophic bacteria under moderate temperature conditions;Syntrophomonas(butyrate as a sole carbon source)was the predominant syntrophic bacteria under low temperature conditions;a relative high abundance of unclassified phylotypes was also enriched in the cultures with VFAs addition.(2)A total of 265 anaerobic strains was isolated through the high throughput isolation technique.Among of them,142 strains could be classified into 12 families and 13 genera,11 strains belong to Thermodesulfovibrio yellowstonii capable of syntrophic lactate oxidation;while the residual 123 strains probably represent 18 novel taxa for their 16S rRNA gene sequence similarity is less than 97%to known species.(3)A rod-shaped,Gram-stain-positive,obligately anaerobic,xylan-degrading bacterium SK-Y3~T was isolated from thermophilic,methanogenic alkane-degrading culture SK.Optimum growth occurred at 50? under neutral pH conditions without NaCl addition.The predominant cellular fatty acids of SK-Y3~T were C15:0 iso,C15:0 anteiso and C17:0 iso.The genomic DNA G+C content is 37.2%.Based on distinct physiological and phylogenetic differences from the aforementioned described taxa,strain SK-Y3~T is proposed as the type strain of a novel species of a new genus Petroclostridium xylanilyticum gen.nov.,sp.nov.Further analysis through 16S rRNA gene-,ribosomal protein-and whole genome-sequences indicated that clostridial cluster III members could be reclassified into four novel genera:Hungateiclostridium gen.nov.,Thermoclostridium gen.nov.,Ruminiclostridium gen.nov.,and Psedodoclostridium gen.nov.,clostridial cluster III members formed a monophyletic clade within the order Clostridiales but was clearly distinguished from other Ruminococcaceae member,which is proposed as a novel family Hungateiclostridiaceae fam.nov.(4)A rod-shaped,Gram-stain-negative,obligately anaerobic,acetate-producing bacterium SK-G1~T was isolated from thermophilic,methanogenic alkane-degrading culture SK.The strain was motile by means of one lateral flagellum,optimum growth occurred at 55? under pH 7.0–7.5 conditions.The genomic DNA G+C content is 43.9%.Genomic analysis revealed the syntrophic butyrate oxidation pathway.Based on distinct physiological and phylogenetic differences from the aforementioned described taxa,strain SK-G1~T is proposed as the type strain of a novel species of a new genus Biomaibacter acetigenes gen.nov.,sp.nov.Further analysis through 16S rRNA gene-,ribosomal protein-and whole genome-sequences indicated that family III Incertae sedis of order Thermoanaerobales and Biomaibacter gen.nov formed a monophyletic clade,which is proposed as a novel family Biomabacteraceae fam.nov..