Studies On Microorganisms And Hydrogenase Diversity From Marine Hot Springs

Thermotolerant hydrogenases are usually found in deep-sea hydrothermaland shallow water hot springs. Microbial fermentation is an important technology forhydrogen production. The hydrogen-producing microorganisms in the environment ofhydrothermal vents are thermophilic and salt-tolerant. Recent findings of H₂redox-active thermophilic bacteria in the thermophilic environment indicates thatthere is some unknown and may be cultured hydrogen-producing microorganism.In this thesis, microbial communities in costal hot springs of Kalianda,Indonesia were investigated using culture-independent16S rRNA gene clone analysis.Community analysis of the vent fluid and mat revealed high diversity of bacterialcommunities. Most sequences have high similarity with the16S rDNA sequence ofknown bacterial （88%¹00%）. Attributable to the Alphaproteobacteria,Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria of the Proteobacteria,Clostridia, Bacilli, Deferribacteres_c, Verrucomicrobiae and other systems taxon. Inthe KWB, the γ-proteobacteria Proteobacteria （total clone41.88%）, β-Proteobacteria（total clone19.23%）, α-Proteobacteria （8.97%of total clones） are the dominantbacterial populations. Cyanobacteria Chroobacteria （clone55.29%of total）,Bacteroides （total clone13.53%）, α-Proteobacteria （10%of the total clones） is thedominant bacterial populations in the KMB. Deferribacteres_c （28.21%of totalclones）, the α-Proteobacteria （total clone12.85%）, γ-Proteobacteria （total clone10.03%）, β-Proteobacteria （total clones8.78%）, δ-Proteobacteria （8.78%of totalclones） is a marine sediment advantage hot Springs bacterial groups.Using different catalase primers, environmental DNA as template toamplify，to construct catalase gene library, to analyze the catalase diversity of hotspring microbial. Three pairs of primers were used for amplification and marinesediment DNA or mat DNA were used as a template. Target fragments were achievedby both of them. By using primer of HydA-F/HydA-R, six different genotypeswere obtained in HKM samples, Four different genotypes were obtained in HKSsamples. Gene coverage of HKM samples and HKS samples were98.04%and97.62%, respectively. The similarity of amino acid sequence to Ignavibacteriumalbum JCM16511FeFe acid bacteria group hydrogen enzymes were about75%-96%. Ignavibacterium album and is a kind of chemical mesophilic heterotrophic bacteria isolated from algae bed of land springs. These Fe-Fe hydrogen bacteriaenzymes group evolutionary relationship of amino acid sequences was in the range of72%-99%. By using FeFe-272F/FeFe-427R as primers, there were14kinds of FeSgenotypes. Its coverage is62.3%. there were24genotypes abtaioned in bacterial mat（FeM） samples and the coverage was65.2%. With HoxH-f/HoxH-r primer, therewere seven genotypes in bacterial mat, which reached65%coverage. As for soilsamples, there was only1genotype, and its coverage was95%.However, up to date, there was no report on the environment specifically forthermophilic bacteria which produce H₂through Fe–Fe hydrogenases （H₂ase）. Usingheterotrophic medium, several microflora from a seaweed bed associated with marinehot springs were enriched and analyzed for H₂production. A hydrogen-producingmicroflora was obtained from Sargassum sp.,16S rRNA genes and Fe-Fehydrogenase （H₂ase） diversities of this enrichment were also analyzed. Based on16S rRNA genes analysis,10phylotypes were found in the microflora showing90.0%to99.5%similarities with known species, and belonged to Clostridia,Gammaproteobacteria and Bacillales. Clostridia were the most abundant group.Three phylotypes were closest to known hydrogen producers such as Anaerovoraxodorimutans （94.0%identity）, Clostridium papyrosolven （98.4%identity） andClostridium tepidiprofundi （93.1%identity）. For Fe-Fe H₂ases, seven phylotypes wereobtained showing63%to97%similarities with known Fe-Fe H₂ases, and fell intofour distinct clusters. Phylotypes HW55-3and HM55-1belonged to thermophilic andsalt-tolerant hydrogen-producing Clostridia, Halothermothrix orenii-like Fe-Fe H₂ases（80%identity）, and cellulolytic hydrogen-producing Clostridia, Clostridiumpapyrosolvens-like Fe-Fe H₂ases （97%identity）, respectively. Both16S rRNA genesand Fe-Fe H₂ases suggested that the thermophilic and halotolerant hydrogenproducing microflora represented previously unknown hydrogen producers, and havepotential application for hydrogen production.