一株聚羟基脂肪酸酯合成菌的筛选及鉴定

Screening and identification of a polyhydroxyalkanoate- synthesizing strain

摘要:

【背景】传统石油基塑料产品给人类和环境带来的危害日益严重，聚羟基脂肪酸酯(polyhydroxyalknoates, PHA)作为新型可降解塑料原料越来越受到青睐。但PHA生产成本过高，使其推广应用严重受限。筛选适合大规模生产PHA的高产菌株是解决这一问题的重要途径。【目的】以挖掘合成PHA的菌种资源为目标，从极端环境筛选和鉴定新的高产PHA合成菌。【方法】通过尼罗蓝平板分离法和PCR法分离纯化菌株，采用16S rRNA基因鉴定并通过MEGA 6.0软件构建系统发育树，分析菌株的进化关系，最后通过尼罗红染色定性分析和气相色谱法定量测定该菌株在不同时期的PHA积累量。【结果】从盐碱地垃圾沉积物中分离得到了一株高产PHA的菌株，PhaC的PCR扩增结果证实了该菌株是PHA合成菌，经16S rRNA基因鉴定为Pseudomonas brassicacearum，将其命名为NP-2，进一步优化了菌株NP-2的培养条件，在培养48 h时PHA积累量最大，达到3.78 mg/mL。【结论】NP-2属于Pseudomonas brassicacearum，能高产PHA。本研究为生产PHA提供了极端环境的高产PHA微生物菌种资源，为进一步挖掘高产PHA菌株积累了数据。

Abstract:

[Background] Due to the aggravating harm of petroleum-based plastic products to humans and the environment, polyhydroxyalkanoates (PHA) are becoming increasingly popular as new biodegradable plastic materials. However, the high production cost restricts the popularization and application of PHA. Screening the strains with high PHA yield for mass production is a key solution to this problem. [Objective] To explore the strain resources capable of synthesizing PHA and screen out and identify the high PHA-yielding bacteria from extreme environments. [Methods] The strain was isolated and purified by nile blue A plate culture and PCR methods. The 16S rRNA gene was sequenced for the identification of the strain and the construction of the phylogenetic tree in MEGA 6.0. Finally, the accumulation of PHA in the strain under different conditions was quantified by Nile red staining and gas chromatography. [Results] A strain with high yield of PHA was isolated from the garbage sediment of a saline-alkali land. The amplification of PhaC confirmed that the strain had the ability to synthesize PHA. Based on the 16S rRNA gene, the strain was identified as Pseudomonas brassicacearum and named NP-2. Furthermore, the culture conditions of strain NP-2 were optimized and the maximum accumulation of PHA reached 3.78 mg/mL at the time point of 48 h. [Conclusion] NP-2 belongs to P. brassicacearum and has high production of PHA. This study provides resources of high PHA-yielding strains in extreme environment for the production of PHA and accumulates data for further mining high PHA-yielding strains.