活性污泥中群体感应淬灭菌的分离纯化及功能验证

Isolation and characterization of indigenous quorum quenching bacteria from activated sludge

摘要:

[背景] 近年来，群体感应淬灭（Quorum Quenching，QQ）技术在膜生物污堵防控中的应用研究受到了广泛关注。然而，目前已成功分离纯化的高效QQ菌有限，更多高效QQ菌资源亟待挖掘。[目的] 从实际运行的膜生物反应器（Membrane Bioreactor，MBR）活性污泥中采样，分离并富集高效QQ菌。[方法] 以根瘤农杆菌（Agrobacterium tumefaciens） A136为报告菌株，使用指示琼脂平板法测定各菌株的N-辛酰基高丝氨酸内酯（N-Octanoyl-DL-Homoserine Lactone，C8-HSL）降解能力。以紫色色杆菌（Chromobacterium violaceum） VIR24为报告菌株，定量测定所得QQ菌降解N-己酰高丝氨酸内酯（N-Hexanoyl-DL-Homoserine Lactone，C6-HSL）信号分子的能力。通过微生物形态、生理生化及16S rRNA基因序列测定、构建系统发育树、扫描电子显微镜形态观测等方法对菌株进行分类学鉴定。用共培养法分析QQ菌对生物膜形成的抑制能力，通过聚乙烯醇和海藻酸钠包埋固定化QQ菌。[结果] 筛选出了6株高效QQ菌，其中对C8-HSL分解能力最强的为杆状、革兰氏阴性戴尔福特菌属（Delftia sp.） JL5。定量分析结果表明菌株JL5能在10 h内完全降解C6-HSL。菌株JL5显著抑制铜绿假单胞菌（Pseudomonas aeruginosa） PAO1和菠萝泛菌（Pantoea ananatis） SK-1生物膜的形成。固定化后的JL5微球仍具有高效的C6-HSL和C8-HSL信号分子分解能力，而且分解速度较被广泛报道的红球菌（Rhodococcus sp.） BH4更快。[结论] 研究分离得到了高效的QQ菌，能够有效抑制N-酰基高丝氨酸内酯（N-Acyl-Homoserine Lactones，AHL）型群体感应菌生物膜的形成，固定化后仍然具有强QQ活性，具备广泛的应用前景，为后续QQ膜生物污堵防控技术的实践应用奠定了基础。

关键词:群体感应;群体感应淬灭;细菌分离;膜防污

Abstract:

[Background] In recent years, the application of quorum quenching (QQ) technology in the prevention and control of membrane biofouling has received extensive attention. However, limited QQ bacteria have been successfully isolated and identified. More efficient signal molecule degrading bacteria awaits identification and further investigation. [Objective] Isolate more efficient QQ bacteria from native real membrane bioreactor (MBR) activated sludge and extending QQ bacteria resources. [Methods] Agrobacterium tumefaciens A136 was used as the reporter strain, to test isolated strain's C8-HSL (N-octanoyl-DL-homoserine lactone) degradation. Reporter Chromobacterium violaceum VIR24 was used to quantify signal molecules degradation of QQ bacteria. 16S rRNA gene sequencing was used to identify isolated bacteria, phylogenetic trees taxonomic of the isolates were then constructed. Scanning electron microscopy (SEM) was used to determine the morphology of bacteria. Coculture of QQ strain and typical biofilm forming bacteria was conducted to analyze biofilm inhibition ability of isolates. QQ beads was prepared using polyvinyl alcohol and sodium alginate. [Results] Six QQ strains were successfully isolated and identified, among which a Gram negative rod strain Delftia sp. JL5 showed the highest efficiency in C8-HSL degradation. Besides, our results showed that JL5 significantly inhibited biofilm formation of both Pseudomonas aeruginosa PAO1 and Pantoea ananatis SK-1, which are two typical N-acyl-homoserine lactones (AHL) dependent biofilm forming bacteria. Furthermore, JL5 remained high AHL degrading activity after being entrapped in QQ beads. Its AHL degrading efficiency was higher than the widely reported Rhodococcus sp. BH4. [Conclusion] We successfully isolated QQ bacteria. The isolates showed high C6/C8-HSL degrading ability. The bacteria inhibited biofilm formation efficiently. This research set solid foundation for further application of QQ bacteria in biofouling control.

Key words:quorum sensing;quorum quenching;bacterial isolation;anti-biofouling