利用含油培养基富集、分离和评估海洋产聚羟基脂肪酸酯细菌

Enrichment, isolation, and evaluation of polyhdroxyalkanoate-producing marine bacteria from oil-containing culture medium

摘要:

[背景] 细菌可通过脂肪酸代谢途径耦合聚羟基脂肪酸酯（Polyhydroxyalkanoate，PHA）合成途径实现合成中长链PHA；其拉伸强度、玻璃化温度等加工特性均优于短链PHA，是未来工业化PHA材料的发展方向。[目标] 为了获得新的可代谢油类的产PHA细菌，使用3种分离策略从海洋沉积物中分离和鉴定细菌，并评估菌株产PHA的能力。[方法] 采集深圳大鹏湾近海沉积物样品；样品通过直接分离，5周连续培养分离，或10周连续提高盐度（3.5%-12.5%）和含油量（1%-10%）富集培养分离，纯化得到菌株；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聚合酶（PhaC）基因鉴定细菌合成PHA的能力；通过测定基因组框架图，分析细菌的PhaC类型、代谢通路及系统进化关系；通过气相色谱分析细菌产PHA的含量及组成。[结果] 从深圳大鹏湾近海底泥样品中分离得到96株细菌，phaC基因阳性率达38%，其中包含9个此前未通过产物证实可产PHA的属，包括尖球菌属（Acuticoccus）、海洋源菌属（Idiomarina）、盐芽孢杆菌属（Halobacillus）、微泡菌属（Microbulbifer）、海棍状菌属（Maritimibacter）、硝酸盐还原菌属（Nitratireductor）、公海橄榄菌属（Pelagibaca）、假海栖菌属（Pseudooceanicola）和海旋菌属（Thalassospira）。除了微泡菌属，其他8个属的细菌通过含油富集法分离获得，说明含油富集方法有利于发现新的产PHA细菌资源。此外，获得2株胞内PHA含量高的菌株。Nitratireductor aquimarinus SY-2-4在营养肉汤中培养可累积细胞干重35.0%的PHA；Roseibium aggregate SN13-21以丙酮酸盐为碳源，可累积细胞干重19.9%的PHA。[结论] 大鹏海域蕴藏着丰富的产PHA细菌资源，值得进一步挖掘研究。梯度增加含油量和盐度的富集方法有利于分离区系不同的产PHA细菌，适用于分离PHA资源。获得2株潜在PHA高产菌，未来将对菌株进行发酵优化研究。

Abstract:

[Background] Bacteria can synthesize medium-chain-length polyhydroxyalkanoates (MCL-PHA) through fatty acid metabolic pathway coupled with PHA synthetic pathway. MCL-PHA’s tensile strength, glass transition temperature, and other processing properties better than the short-chain-length PHA, are the promising candidate for PHA industrialization. [Objective] In order to obtain new PHA-producing bacteria that can metabolize oils, we used three methods to isolate bacteria from marine sediments, identified them, and then evaluated the PHA-producing ability. [Methods] We collected the offshore sediment sample of Dapeng Bay in Shenzhen, and isolated bacteria directly from the sample, each week during the 5-week incubation, or during the 10-week enrichment with stepwise increase of oil content (1%–10%) and salt content (3.5%–12.5%). Then, we identified the strains based on 16S rRNA gene similarity and phylogenetic analysis, tested the PHA-producing ability by sequencing the gene of PHA synthase (PhaC), and determined the types, metabolic pathways, and phylogenetic relationship of PhaC according to the draft genome. The content and composition of PHA were detected by gas chromatography. [Results] A total of 96 strains were isolated and 38% had the phaC gene. Among them, 9 genera had not been reported to produce PHA: Acuticoccus, Idiomarina, Halobacillus, Microbulbifer, Maritimibacter, Nitratireductor, Pelagibaca, Pseudooceanicola and Thalassospira, and 8 of the 9 genera except Microbulbifer were enriched with oil. Thus, the enrichment with oil helped to discover new PHA-producing bacterial resources. In addition, we obtained 2 strains with high intracellular PHA content: N. aquimarinus SY-2-4 could accumulate PHA of 35.0% dry cell weight when cultured in nutrient broth, while Roseibium aggregate SN13-21 could use pyruvate as sole carbon source to accumulate PHA of 19.9% dry cell weight. [Conclusion] The offshore environment of Dapeng Bay harbors rich PHA-producing bacterial resources that are worthy of further investigation. The enrichment method of stepwise increase of oil content and salt content is beneficial to isolating different PHA-producing bacteria. Next, fermentation optimization of the 2 high-PHA-content strains will be further investigated.