Abstract: Marine microorganisms in the tidal flat sediment from Zhenhai were isolated with pure culture method and bacterial community and diversity were analyzed. The tidal flat sediment sample was collected from Zhenhai, Zhejiang Province of China in June, 2008. The diluted sediment was plated on modified ZoBell medium agar plates. After incubation aerobically at 25 °C and 37 °C, strains were selected and purified. The 16S rRNA genes were amplified with primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5’-GGTTACCTTGTTACGACTT-3') and sequenced. The sequences obtained were compared with closely related sequences of described bacteria from the EzTaxon-e online service. Sequence data were aligned with Clustal W and phylogenetic tree were constructed by the neighbor-joining method with the MEGA 5 program package.
Totally, 39 bacterial strains were isolated. Analysis of the 16S rRNA gene sequences show that the isolates were closely related to described bacteria and fell into four lineages: Firmicutes (20 strains, 51. 3%), Proteobacteria (12 strains, 30. 8%),Bacteroidetes (6 strains, 15. 4%) and Actinobacteria (1 strain, 2. 6%). Firmicutes strains belong to genera Bacillus, Ornithinibacillus and Thalassobacillus; Proteobacteria strains belong to genera Roseicyclus, Rhodobacter, Erythrobacter, J annaschia and Ruegeria of Alphaproteobacteria and genera Acinetobacter and Pseudomonas of Gammaproteobacteria; Bacteroidetes strains belong to genus Algori phagus and family Flavobacteriaceae; and Actinobacteria strains belong to genus Gordonia.
Bacilli and Alphaproteobacteria were the largest group in Firmicutes and Proteobacteria, respectively. They might play an important role in biogeochemical cycles in the coastal sediment ecosystem. Bacilli strains are heterotrophic bacteria, and their occurrence indicates that the tidal flat environment is rich in organic nutrition. 15 out of 20 (75%) of the isolated Bacilli strains showed closer phylogenetic relationship with the bacteria of land origin rather than bacteria of marine origin. In addition, strain ZH04 as well as ZH55 shared the highest 16S rRNA gene sequence similarities to the arsenic-resistant bacterium Bacillus indicus Sd/3T (99.9%); strain ZH41 as well as ZH43 shared 99.7% sequence similarity to the iron-and manganese reducing bacterium Bacillus subterraneus DSM 13966T; strain ZH56 were most related to Bacillus thioparans U3, which can oxidize manganese and remove cadmium, zinc, copper and lead. The discovery of strains related to absorption and oxo-reduction of heavy metals indicates heavy metal pollution of the offshore environment resulted from large amount of heavy metal sewage discharge in recent years and the impact of human activities on land on the coastal ecological environment. Of the 10 strains assigned to Alphaproteobacteria, up to 8 strains were assigned to Roseobacter lineage, including ZH40,ZH15, ZH32,ZH18, ZH36, ZH39,ZH50 and ZH17. The Roseobacter lineage is one of the richest marine microorganisms, and the bacteria of this group have multiple pathways for energy and carbon acquisition, including carbon monoxide and hydrogen sulfide oxidation, CO2 fixation, aerobic anoxygenic phototrophy and aromatic compound degradation, etc. Thus, the bacteria of Rhoseobacter lineage is called as‘ecological generalist’, which play important roles in the carbon and sulfur biogeochemical cycles of the offshore ecosystem.
In addition, strains ZH09,ZH15, ZH17 and ZH38 shared low 16S rRNA gene sequence similarities with Robiginitalea biformata HTCC2501T (93.6%),Rhodobacter aestuarii JA29T (97.1%),Ruegeria pomeroyi DSS-3T(97.2%) and Acinetobacter indicus A648T (96. 8%),respectively. The relatively low sequence similarities with described bacteria indicate that these strains may represent novel phylogenetic lineages in the offshore environment.
The research deepens our understanding of the diversity of culturable bacteria in the coastal sediment of Zhejiang Province and provides material for future exploitation and utilization of marine microorganism resources.

Key words: Zhenhai, tidal flat sediment, bacteria, culturable, phylogenetic analysis