不同寡营养培养条件下南海水体细菌群落结构及其对碳源的利用特征

Bacterial community structure and its utilization characteristics of carbon sources in water of South China Sea under different low-nutrient culture conditions

【背景】绝大多数海洋微生物不可培养,为挖掘海洋生态系统中可培养的微生物资源,研究者尝试寡营养培养等方法。【目的】比较不同寡营养培养条件下南海水体细菌数量、群落结构及其对碳源的利用特征差异。【方法】采用原2216E培养液(Y)、稀释10倍(Y-10)和稀释50倍(Y-50)的2216E培养液培养南海海水样品,用荧光定量PCR法和16S rRNA基因检测细菌数量和菌群结构;利用平板计数法计数异养细菌的数量,纯化鉴定可培养细菌;采用Biolog EcoPlateTM微板法分析不同培养基中细菌群落对碳源的利用特征。【结果】Y组细菌总数高于Y-10组和Y-50组,差异不显著(P>0.05),但异养细菌数量显著高于Y-10组和Y-50组(P<0.05)。16S rRNA基因测序结果表明,不同稀释倍数下的细菌群落结构差异明显,Y组检测出10门193属,优势类群为Proteobacteria(56.44%)和Bacteroides(37.27%);Y-10组检测出15门220属,优势类群为Proteobacteria(40.30%)、Bacteroides(36.91%)和Firmicutes(17.30%);Y-50组检测出14门226属,优势类群为Proteobacteria(45.19%)、Bacteroides(25.29%)、Planctomycetes(13.58%)和Firmicutes(11.21%)。通过平板培养,Y组和Y-10组均分离到6属14株优势菌,Y-50组分离到7属13株优势菌,其中,Bacillus为其共有的优势菌属,稀释10倍培养液筛得的Microbacterium(1株)、Vibro(1株)、Idiomarina(1株)、Halobacillus(1株)共4株优势菌和稀释50倍培养液筛得的Alcanivorax(1株)、Sulfitobacter(1株)、Alteromonas(1株)、Pseudomonas(1株)、Exiguobacterium(2株)、Vibro(3株)共9株优势菌不同于原培养液。通过寡营养培养,可培养细菌群落的代谢活性和McIntosh指数显著增加(P<0.05),其对聚合物、羧酸、氨基酸、糖类的利用率也显著提高(P<0.05)。【结论】通过寡营养培养能增加细菌群落的丰富度和多样性,提高可培养细菌的代谢活性和对碳源尤其是聚合物、羧酸、氨基酸和糖类的利用率,分离纯化可获得原培养基未筛选得到的细菌。因此,在南海远洋海域可培养细菌样品的采集及复苏时,可通过寡营养培养法获得更丰富的南海可培养微生物资源。

[Background] Most marine microorganisms are uncultivated. In order to excavate the culturable microbial resources in the marine ecosystem, we tried some methods, such as oligotrophic culture. [Objective] To compare the differences in bacterial population, community structure and utilization characteristics of carbon sources of South China Sea under different low-nutrient culture conditions. [Methods] The seawater samples from South China Sea were cultured in the original 2216 E medium(group Y), diluted 10 times(group Y-10) and 50 times(group Y-50) in 2216 E culture medium. The number of bacteria and the structure of bacterial communities were detected by real-time PCR and 16 S rRNA gene. The number of heterotrophic bacteria was also counted by plate counting method, and the culturable bacteria were separated and identified. The utilization characteristics of carbon sources of bacterial communities in different media were analyzed by Biolog EcoPlateTM method. [Results] The total number of bacteria in Y group was higher than that in Y-10 group and Y-50 group(P>0.05), but the number of heterotrophic bacteria was significantly higher than that in Y-10 group and Y-50 group(P<0.05). The sequencing of 16 S rRNA gene shows that the bacterial community structure was significantly different under different dilution multiples, in the Y group, 10 phyla and 193 genera were detected, and the dominant groups were Proteobacteria(56.44%) and Bacteroides(37.27%);in the Y-10 group, 15 phyla and 220 genera were detected, and the dominant groups were Proteobacteria(40.30%), Bacteroides(36.91%) and Firmicutes(17.30%);in the Y-50 group, 14 phyla and 226 genera were detected, and the dominant groups were Proteobacteria(45.19%), Bacteroides(25.29%), Planctomycetes(13.58%) and Firmicutes(11.21%). Through plate culture, 14 dominant bacteria of 6 genera were isolated from Y group and Y-10 group, 13 dominant bacteria of 7 genera were isolated from Y-50 group. Among them, Bacillus is the common dominant bacteria. Four dominant bacteria(1 strain of Microbacterium, 1 strain of Vibro, 1 strain of Idiomarina, 1 strain of Halobacillus) screened by diluting 10 times of culture medium and nine dominant bacteria(1 strain of Alcanivorax, 1 strain of Sulfitobacter, 1 strain of Alteromonas, 1 strain of Pseudomonas, 2 strains of Exiguobacterium, 3 strains of Vibro) screened by diluting 50 times of culture medium were different from the original culture medium. Through low-nutrient culture, the metabolic activity and McIntosh index of culturable bacterial communities increased significantly(P<0.05), and its utilization rate of polymers, carboxylic acids, amino acids and carbohydrates also increased significantly(P<0.05). [Conclusion] Low-nutrient culture can increase the richness and diversity of bacterial community, improve the metabolic activity of culturable bacteria and the utilization of carbon sources, especially polymers, carboxylic acids, amino acids and carbohydrates. Therefore, in the collection and recovery of culturable bacteria samples in the pelagic area of South China Sea, more abundant culturable microbial resources can be obtained through low-nutrient culture method.