红树林土壤中产聚羟基脂肪酸酯细菌的分离及其评估

Isolation and evaluation of polyhdroxyalkanoate producing bacteria from mangrove soil

摘要:

[背景] 细菌能通过合成聚羟基脂肪酸酯（Polyhydroxyalknoates，PHA）在细胞内储存物质和能量，提高对环境的适应能力。在红树林中，由于土壤周期性受海水浸没，形成营养物质种类丰富和含量波动大的特殊生境，为细菌进化出特殊的PHA合成途径提供了条件。[目标] 为了增加对红树林产PHA细菌资源的了解，获得产PHA细菌，使用纯培养方法分离和鉴定细菌，并评估菌株的产PHA能力。[方法] 采集红树植物海桑根系和红树滩涂土壤样品，连续5周培养、分离纯化获得细菌菌株；通过16S rRNA基因相似性及系统进化分析鉴定细菌分类地位，利用PHA合成酶基因（phaC）鉴定细菌合成PHA的能力；通过基因组草图测序，分析细菌的phaC基因种类、代谢通路及系统进化关系；通过气相色谱分析细菌产PHA的累积量及组成。[结果] 从红树林土壤样品中分离得到97株细菌，其中13株带有phaC基因，包括坚强芽孢杆菌（Cytobacillus firmus）、弯曲芽孢杆菌（Bacillus flexus）、除烃海杆菌（Marinobacter hydrocarbonoclasticus）和酯香微杆菌（Microbacterium esteraromaticum）。B. flexus MN15-19以丙酮酸盐为碳源，可累积细胞干重11%的PHA，同时具有固碳功能的还原性三羧酸循环通路，有开发成为固碳产PHA工程菌株的潜力。酯香微杆菌可产PHA，但是其phaC基因结构特殊，基因组注释未能识别出任何已知phaC基因。[结论] 研究发现红树林土壤可培养细菌中存在未知的PHA合成途径，说明红树林生态系统中的细菌具有资源挖掘的重要价值。

关键词:红树林生态系统;细菌;聚羟基脂肪酸酯;代谢通路;phaC基因

Abstract:

[Background] Bacteria can synthesize polyhydroxyalkanoates (PHA) as intracellular carbon and energy storage compounds in order to better adapt to the environment. In mangroves, the soil is periodically submerged by seawater, which forms a special habitat with abundant nutrients and fluctuating contents, providing conditions for bacteria to evolve a special PHA synthesis pathway. [Objective] In order to improve the understanding of PHA-producing bacterial resources, we isolated and identified the PHA-producing bacteria, and evaluated their ability to produce PHA. [Methods] We collected soil samples from shoal and Sonneratia caseolaris rhizosphere, isolated bacteria during the 5 weeks of incubation, identified them by 16S rRNA gene sequence similarity and phylogenetic tree, and determined the PHA producing ability by testing the PHA synthase gene (phaC). Based on the draft genome sequence data, we determined the class of phaC gene, the metabolic pathways and the phylogenetic relationship among different strains. We analyzed the PHA content and composition using gas chromatography. [Results] A total of 97 strains of bacteria were isolated, among which 13 strains were PHA-producing bacteria, including Cytobacillus firmus, Bacillus flexus, Marinobacter hydrocarbonoclasticus and Microbacterium esteraromaticum. B. flexus MN15-19 using pyruvate as the sole carbon source could accumulate 11% PHA per dry cell weight. This strain had reverse tricarboxylic acid cycle, which is considered as carbon fixation pathway. Therefore, based on B. flexus MN15-19, PHA production technique directly using CO₂ could be developed. Mi. esteraromaticum was a PHA-producing bacterium, whose phaC gene was too unique to be identified as any known phaC genes. [Conclusion] Bacteria isolated from mangrove ecosystem have unknown PHA synthase pathway, which indicates that mangrove bacterial biological resources deserve further study.

Key words:mangrove ecosystem;bacteria;polyhydroxyalkanoates;metabolism pathways;phaC