Study On The Prokaryotic Diversity And PAH-degrading Bacteria In Deep-Sea Sediments Of Lau Basin Hydrothermal Vent Field Of Southwest Pacific

Deep-sea hydrothermal systems are frequently found in mid-ocean ridges and back-arc basins,and characterized by marvelous physical and chemical gradients.The hydrothermal vent activity provides unique niches for vent lives,with sharp changes of temperature,dissolved oxygen levels,heavy metal content and reduced substrates.Such environments have shaped special microbial communities of high diversity in taxonomy, physiology and metabolism.By far,the well-studied hydrothermal microbial communities were usually located at the Mid-Pacific Ridge and Mid-Atlantic Ridge.However,there were few reports about the microbial communities retrieved for a submarine volcano back-arc basin.Here,three hydrothermal sediments(TVG9,TVG2 and TVMC2) were sampled from the southern and northern hydrothermal regions of Lau basin,during the cruise of DY 115-19 of R/V Da-Yang Yi-Hao.Through constructing the 16S rRNA gene and amoA gene libraries,the in situ bacteria and archaea communities were investigated in these sites,which possibly involved in the biogeochemical cycle.In addition,the diverisity of PAH-degrading bacteria in these sediments was also anlyzed after enrichment using PAHs as the only carbon source.Meanwhile,a selected PAH-degrading bacterium named strain Novosphingobium sp.TVG9-Ⅶwas studied in more details at molecular level.The main results were as follows:The profile of microbial community structures in southern and northern hydrothermal sediments of Lau baisn were investigated by culture-independent molecular phylogenetic methods.The investigation data indicated that:1) the bacteria strain resource was novel in Lau basin.About 95%OTUs in three bacterial 16S rRNA gene libraries shared less 97%similarity with the known microorganism.Moreover,the low coverage of three bacteria libraries(＜70%) indicated that these libraries didn't reach saturation.So,all these data showed that there were many kinds of new and unique microbiology inhabited in the hydrothermal sediments of Lau basin.2) ammonia-oxidizing archaea dominated in all three archaea community.The ratios of potential ammonia-oxidizing archaea clones in all three archaea 16S rRNA libraries were more than 55%.They had Nitrosopumilus maritimus as the closest cultured neighbor with 92%16S rDNA sequence similarity.Specially,the OTU YI-2D shared 97.92%similarity with N.maritimus and occupied 58.59%clones in TVG9 library.3) The structures of microbial community had a clear characteristic with sampling sites.Theε-,ζ-proteobacteria and Euryarchaeota were merely detected in TVG9 site,although the Proteobacteria and marine Crenarchaeota were the dominate member in all three site.4) The hydrothermal activities had a significant influence on the microbial community structure.According to the statistical results of 16S rRNA gene libraries,not only in bacteria but in archea libraries,there was a clear trend in microbial diversity aspect TVMC2＞TVG2＞TVG9.In particular,the Libshuff results of three archaea 16S rRNA gene libraries indicated that TVG9 archaeal library was significantly different with TVG2 (P=0.001) or TVMC2-1(P=0.017) archaeal libraries.The soxhlet extraction and GC-MS quantitative analysis of PAHs in three sediments showed that there were different kinds of PAHs in three sediments.The concentration of total PAHs were 114.52 ng/g,409.65 ng/g,277.67 ng/g dry weight for TVG9,TVG2 and TVMC2,respectively.After enrichment with a PAH mixture(naphthalene,phenanthrene and pyrene),three degrading consortia were obtained from these three sediments.In these consortia,the cultivable degrading bacteria belonged to genus Novosphingobium ofα-proteobacteria and genus Pseudomonas ofγ-proteobacteria;the cultivable non-degrading bacteria belonged to genus Halomonas and genus Alcanivorax ofα-proteobacteria,and genus Thalassospira ofγ-proteobacteria.According to DGGE and 16S rRNA gene libraries results,the bacteria belonging to genera Novosphingobium, Halomonas,Thalassospira and Alcanivorax were confirmed as the dominate member both in TVG9 and TVG2 consortia.In contrast,the major member in TVMC2 consortium belonged to genera Pseudomonas,Alcanivorax,Halomonas,Psychroflexus and Alteromonas.Although community structures dynamically changed spatially and temporally,bacteria closely affiliated to Novosphingobium,Halomonas and Thalassospira most frequently occurred.The genus Novosphingobium bacteria usually became the dominate member in middle-stage and then held this to the end during the consortium growth course.However,the non-PAHs degrading bacteria,such as Halomonas and Thalassospira,only dominated in initial stages and were substituted by PAHs degraders.In addition,the degrading characteristics for various PAHs of the strain TVG9-Ⅶ, the dominate member of TVG9 consortium,were confirmed.It could completely degrade the naphthalene(200 mg/L) and phenanthrene(100 mg/L),and more than 60% fluoranthene(20 mg/L) and pyrene(20 mg/L) in three weeks.The 16S rDNA sequencing result indicated strain TVG9-Ⅶbelonged to genus Novosphingobium.According to the cloned PAHs initial dioxygenase gene sequences in genus Novosphingobium,we designed a degenerate prime and cloned four different initial dioxygenase gene fragments.Then,a fosmid library of strain TVG9-Ⅶgenomic DNA was constructed,and a completive degrading gene cluster was obtained from this library.The sequencing result indicated that the arrangement of degrading genes was the same as the one found in other strains in this genus.The gene expression analysis results indicated that the expression of these four initial dioxygenase genes induced by phenanthrene was increased significantly and there was no expression of them induced by high molecular weight fluoranthene or pyrene. These data indicated a multi-copy strategy of degrading genes was applied in strain TVG9-Ⅶto degrade phenanthrene,and also implied there were some other new potential genes in TVG9-Ⅶgenome responding to the degradation of high molecular weight PAHs.In this report,the microbial community structures were investigated for the first time in Lau basin hydrothermal region,and the PAHs-degrading bacteria in this ecosystem were also detected.These results will help to study and utilize the gene resource retrieved from Lau basin hydrothermal vent field,and to reveal the biogeochemical distribution of PAH-degrading bacteria inhabited in hydrothermal ecosystem.