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-c 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.rnTotally, 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, Jannaschia and Ruegeria of Alphaproteobacteria and genera Acinetobacter and Pseudomonas of Gammaproteobacteria; Bacteroidetes strains belong to genus Algoriphagus and family Flavobacteriaceae; and Actinobacteria strains belong to genus Gordonia.rnBacilli 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.rnIn addition, strains ZH09, ZH15, ZH17 and ZH38 shared low 16S rRNA gene sequence similarities with Robiginitalea biformata HTCC2501T (93. 6% ) , Rhodohacter 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.rnThe 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.%针对浙江镇海潮间带沉积物样品,采用纯培养法分离培养海洋微生物,并基于16S rRNA基因序列,开展系统发育学研究,分析沉积物细菌群落结构及多样性.分离获得细菌39株,16S rRNA基因序列分析表明,这些菌株分别属于厚壁菌门Firmicutes (51.3％)、变形杆菌门Proteobacteria (30.8％)、拟杆菌门Bacteroidetes(15.4％)和放线菌门Actinobacteria(2.6％)4个类群.厚壁菌门和变形杆菌门的菌株主要归属于芽孢杆菌纲Bacilli和α-变形杆菌纲Alphaproteobacteria,它们可能在近海海洋生态系统的元素地球化学循环中发挥了重要作用.此外,部分菌株16S rRNA基因序列与已报道物种的相似性较低,可能代表了新的分类单元.研究丰富了浙江近海微生物种质资源,并拓展了对浙江近海可培养细菌多样性的认识,为今后开发利用海洋微生物资源积累了资料.
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