Diversity Of Heavy-metal Resistant Microorganisms In The Hydrothermal Sulfide Of The Eastern Pacific Ocean And The Microbial Community Structures Of The Surface Seawater Across The South Mid-Atlantic Ridge

This study mainly focused on the biodiversity of the heavy-metal resistant microorganisms in the hydrothermal sulfide of the Eastern Pacific Ocean, and the microbial community structures of the surface seawater across the South Mid-Atlantic Ridge.Part ⅠHeavy metal pollution is thought to be the huge threat on human health and ecosystem. While, previous reports indicated microorganisms are able to absorb and eliminate heavy-metals from environments using various mechanisms. To study and analysis the biodiversity and resistance mechanism of the heavy-metal resistant bacteria, will benefit to bioremediation of the heavy-metal pollution marine environments.In this study, two hydrothermal vent sulfide samples (TC and VS) from the Eastern Pacific Ocean were separately enriched with seven kinds of heavy-metal (Zn2+、Ni2+、Mn2+、Cu2+、Hg2+、Co2+and Cr7+), and fourteen enriched consortia were obtained in total. PCR-DGGE results indicated that the dominant bacteria in Zn-, Ni-, Mn-, Cu-, Co-and Cr-enriched consortia were:Halomonas and Paracoccus; Microbacterium and Alcanivorax; Halomonas, Marinobacter and Georgenia; Achromobacter and Serratia; Halomonas and Idiomarina; Idiomarina and Bacillus, respectively.With agar plates of MA medium, a total of16bacteria and8fungi were isolated and identified from the fourteen consortia mentioned above. Phylogenetic analysis showed that these bacteria belong to13species within10genera, including Salegentibacter, Dietzia, Paracoccus, Alcanivorax, Georgenia, Halomonas, Microbacterium, Salinicola, and Thalassospira. Among them, Halomonas; Microbacterium and Alcanivorax; Georgenia; Halomonas were the dominant bacteria identified by PCR-DGGE from the Zn-, Ni-, Mn-and Co-enriched consortia, respectively. In addition, eight fungi isolated from the Cu-, Co-, Zn-and Ni-enriched consortia were all identified as Rhodotorua minuta using the internal transcribed spacer (ITS) sequences.Moreover, a Zn-resistant bacterium named VCZn-1was isolated from the Zn-enriched consortium from the VC sample. Phylogenetic analyses revealed that strain VCZn-1belongs to the genus Halomonas, and its16S rRNA gene sequence shared100%similarity with the type strain Halomonas zincidurans B6T. Strain VCZn-1can grow in5-25%of NaCl (optimum13%) at4-37℃(optimum28℃); and fit at pH5-10(optimum8-9). It even can grow at27mmol/L Zn2+in the MA medium. Atomic absorption spectrometric results showed that strain VCZn-1can removal49±3.42%Zn2+from the medium within3days. The possible Zn-resistant mechanism is that strain VCZn-1secreted the alkaline secondary metabolites in cell stable phase, as a result, Zn2+was precipitated with the increase of the pH in the medium. In addition to Zn2+, strain VCZn-1also was found to be able to resist other kinds of heavy-metal, such as Mn(9mmol/L), Cu(1mmol/L) and Ni(1mmol/L). The genome sequencing results indicated that its genomic size is370,983,8bp with a GC content of64.04%. In the genome, many heavy-metal resistant related genes were found, such as ATPase transporters and RND family transporter genes, which may be the molecular bases for the heavy-metal resistance mechanism of the strain VCZn-1.Part ⅡTo investigate the community structures of the surface seawater across the South Mid-Atlantic Ridge, fourteen surface seawater samples were subjected to454pyrosequencing. A total of177,364raw reads of V5-V8regions of the bacterial and archaeal16S rRNA genes were obtained. After filtering the low-quality reads and chimeras, the remained164,784clean reads with the average length of286bp were analyzed by using the QIIME (v1.7.0) pipeline. Shannon and Chaol indexes showed that the biodiversity of these samples is quite rich and no obvious differences among them. Unifrac and UPGMA analyses showed that all the samples clustered according to their sampling geographical locations, which meant these microorganisms have the biogeographic characteristics. Among these communities, the microorganism composition showed quite similar. In detail, the dominant archaea in all samples belonged to the Marine group Ⅱ and Marine Group Ⅲ within Euryarchaeota. Correspondingly, the most dominant bacteria were affiliated to the SAR86clade (11.93%～18.24%of total reads of one sample) of the order Oceanospirillales within the class Gammaproteobacteria. The second dominant bacterial group was SAR11surface1clade (9.48%～16.19%) of the order Rickettsiales within the class Alphaproteobacteria. At genus level, only20.9%～29.1%percentage of total reads among the samples were assigned into the known genera. The top ten genera were Candidatus Portiera, Alteromonas, Pseudoalteromonas, Fluviicola, Thalassomonas, Oleibacter, Vibrio, Hyphomonas, Alcanivorax and Pelagibaca.Meanwhile, in total of116cultivable bacteria were isolated by using eutrophic2216E and oligotrophic R2A media from these fourteen surface seawater samples. Phylogenetic analysis indicated that they belong to24genera within Gamma-, Alpha-, Epsilonproteobacteria, Bacteroidetes, Bacilli and Actinobacteria. Among them, most of isolates (63.79%of total cultivable bacteria) belonged to Gammaproteobacteria and were affiliated to the following genera:Vibrio, Pseudoalteromonas, Alteromonas, Marinobacter and Halomonas. The other isolates mainly belonged to genera of Erythrobacter, Pseudoruegeria and Sulfitobacter within the class Alphaproteobacteria. Noteworthily, some genera identified by pyrosequencing, such as Alteromonas, Alcanivorax, Marinobacter, Pelagibaca, Photobacterium, Pseudoalteromonas, Tenacibaculum and Vibrio, were isolated by using the two media mentioned above.To sum up, this thesis analysis the biodiversity of heavy-metal resistant bacteria in the hydrothermal sulfide of the Eastern Pacific Ocean and the microorganism community structures of the surface seawater from the South Mid-Atlantic Ridge. Moreover, the Zn-resistant mechanism of a strain isolated from the Zn-enriched consortia was explored. These results will benefit to development and utilization of the heavy-metal resistant bacteria, and help us better understanding the biodiversity of the surface seawaters across the South Mid-Atlantic Ridge.