Study On The Diversity Of Denitrifying Bacteria Of Changjiang Estuary Sediments And Extracellular Enzyme Producing Bacteria Isolated From Southern Okinawa Trough Deep-sea Sediments

As the third biggest estuary in the world, Changjiang Estuary plays an important role in marine nitrogen cycle of the globe. The diversity of denitrifying bacterial nirS gene from the sediments of Changjiang Estuary was investigated by using a PCR-based cloning approach. A total of 633 clones were obtained from 7 stations. Restriction enzyme digestion analysis of cloned nirS showed that these clones fall into 364 OTUs (Operational Taxonomic Unit,OTU). The number of OTUs each station was ranged from 59 to 75 and the percentages of OTUs shared by any station with the others varied between 21.7 and 57.4. One representative from each OTU was sequenced. The similarity of the nirS sequences obtained with those deposited in NCBI database varied from 61 to 100. Molecular phylogenetic analysis of nirS sequences retrieved from the sediments or column of 12 sea areas including Baltic Sea and Gulf of Mexico assigned the nirS sequences into 7 major clusters (Marine ClusterⅠ-Ⅶ). With 50% similarity, Marine ClusterⅦwas divided into 8 sub-clusters, of which Marine ClusterⅦg andⅦh were specific for Changjiang Estuary reflecting their unique characteristics. Cluster Environment, Jackknife Cluster and Principal Coordinates Analysis (PCA) based on the percentage of OTUs and nirS sequences indicated that the denitrifying community was more similar between geographically closer stations. The geographic location appears to be a key factor controlling the community structure of denitrifying bacteria.The Southern Okinawa Trough is the major sediment area of particulate organic matter (POM) exported from the East China Sea shelf and the island of Taiwan. Cultivation-depending method was used to investigate the diversity of extracellular hydrolytic enzymes producing bacteria from the deep-sea sediment of station MD05-2907 of southern Okinawa Trough. A total of 98 bacterial isolates were characterized based on 16S rRNA gene phylogenetic analysis. These isolates were grouped into 4 bacterial divisions or phyla, including Proteobacteria, Firmicutes or low G+C gram positive group, Actinobacteria or high G+C gram negative group and Bacteroidetes. The isolates are quite diverse at the genus level, a total of 14 genera can be identified, including Alcanivorax, Bacillus, Cobetia, Halomonas, Methylarcula, Micrococcus, Myroides, Paracoccus, Planococcus, Pseudomonas, Psychrobacter, Sporosarcina, Sufflavibacter and Wangia. Bacteria of theγ-Proteobacteria lineage, especially those from Halomonas and Psychrobacter dominate the area. The screening of bacteria with diverse extracellular hydrolytic enzyme activities showed that most of the isolates produced at least one kind of the hydrolase, such as amylases, protease, lipase and deoxyribonuclease. Extracellular chitinase producing bacteria were not found. A large portion of isolates produced extracellular hydrolytic enzyme at a low temperature. Most of the strains showing extracellular hydrolytic enzymatic activity belonged toγ-Proteobacteria, Firmicutes, (low G+C gram positive group) and Bacteroidetes. Some isolates of Halomonas could produce all kinds of the tested extracellular enzymes at both 28℃and 4℃. Some cultures of Psychrobacter could excreted all kinds of extracellular tested at 28℃and/or 4℃. In this study, a new species named Wangia profunda gen. nov., sp. nov., aslo a new genus, was found. The study of diverse extracellular hydrolytic enzymes producing bacteria from the deep-sea sediment of southern Okinawa Trough provided basis for understanding the envirnmental characteristics of sediments and utilizing microbial resource.