基于分子水平的降解型生物膜形成机理与生物膜强化处理机制

Biofilm is the most frequent mode for the growth of bacteria. The highly structured, matrix-enclosed, and functionally differentiated community provides the inner bacterial cells with wholesome protection in an inhospitable environment, and biofilm-mediated pollution remediation has been attracting worldwide attentions. However, there are still multiple critical knowledge gaps in understanding biofilm with degrading functions, such as the behavioral patterns of degrading bacteria on surface, formation and degradation mechanisms of such biofilm. In this proposed project, using a model bacterium for biofilm study and several degrading bacteria isolated and conserved in my laboratory, we will distinguish the key impact factors for the adhesion of degrading bacteria on the surface of a submerged material and the formation of degrading biofilm under different cultural conditions; we will analyze the intercellular signal molecules for quorum sensing and the metabolic products during the degradation of nitrogen heterocyclic compounds (NHCs); we will detect the function genes that regulate and control those biochemical reactions; and consequently, we will establish a new theory for the formation of degrading biofilm. Taking an actual industrial wastewater containing NHCs and a municipal sewage receiving such industrial wastewater as two scenarios, we will cultivate NHCs-degrading biofilms and enhance their degradation capacity in a lab-scale experiment of wastewater treatment; we will observe the pollutants' removal efficiency of the biofilm technology, and on the other hand, track the temporal and spatial variation of bacterial community structures of the biofilms; we will then discuss the relationship between the variation of bacterial community structures and the removal of organic and inorganic pollutants, and finally from the molecular level, propose a novel method to enhance the degradation performance of biofilm-augmented treatment. The outputs of the project will establish the theoretical framework for a new approach to manipulate degrading biofilm formation and develop the empirical guidelines of biofilm-augmentation for the purpose of pollution remediation.

生物膜是细菌最常见的生长方式。结构有序、功能分化的生物膜群落为内部细菌提供了在污染环境中生存的庇护，其治理污染功效也日益受到关注。然而，降解菌在材料表面的行为、生物膜形成机理与降解机制尚有较多理论空白。本项目选择生物膜模式细菌和实验室分离保藏的降解菌，在不同培养条件下，识别细菌在浸水材料表面粘附和形成降解型生物膜的关键因子，分析细胞合成的群体感应信号物质和降解氮杂环芳烃化合物（NHCs）的产物，检测调控这些生化反应的功能基因，建立降解型生物膜形成的理论；针对实际含NHCs的工业废水及收纳该工业废水的城市污水两种情景，培养和强化具有NHCs降解功能的生物膜，开展废水处理模拟试验研究，考察生物膜法处理效率，解析生物膜菌群结构的时空变化，探讨菌群结构演替与污染物去除的关系，提出从分子水平强化生物膜整体降解能力的方法。研究成果将创新和完善具有降解功能的生物膜形成机理和生物膜强化处理技术的理论体系。

本项目基于生物膜发生装置和检测技术，评价了降解菌的生物膜形成能力，识别了影响其生物膜形成的关键环境因子，解析了动态培养系统中降解菌生物膜的特征和微观结构，最后在掌握降解菌成膜机制的基础上，探索发展了人工调控成膜过程的方法。主要研究结论有：.1）采用改良微孔板法对吡啶和喹啉高效降解菌的生物膜形成能力进行评价。结果显示，不同降解菌株的成膜能力有显著差异，Pseudomonas sp. ZX08等假单胞菌属降解菌相比于Arthrobacter sp. ZX07等节杆菌属降解菌更容易成膜。胞外蛋白分泌量、胞外多糖分泌量和由鞭毛参与的游动能力与细菌成膜能力之间存在显著的正相关关系，这些特征可能在成膜过程中发挥了重要作用。.2）考察环境因素对降解菌生物膜形成的影响。结果显示，降解菌生物膜的形成受到pH、温度、转速、培养时间的影响，存在显著差异（P<0.05）；BC026对pH的变化较为敏感，随着pH升高，生物膜量减少速度远远高于浮游态； BC026和BW004对温度的变化较为敏感，在较高温度（30-40oC）条件下，生物膜态都有逐渐占据优势的趋势；而BW001形成的生物膜对pH和温度的响应都不敏感，而对转速最为敏感。.3）考察目标污染物浓度对降解菌生物膜形态和结构的影响。结果表明，高浓度的污染物会影响降解菌生物膜的形态、分布特征，并抑制生物膜的发生发展以及生物膜内的细胞活性：200 mg/L浓度下，降解菌团聚结构分布较为零散，但具有一定厚度；800 mg/L浓度下，降解菌团聚结构明显变小，在表面的分布更为均匀；200 mg/L浓度下的膜内活菌百分比是800 mg/L浓度下的2.1倍左右。.4）利用特殊化学物质调控降解菌成膜的结果显示：一定浓度的Ca2+可以显著促进降解菌ZX01和ZX07形成厚实致密的生物膜。Ca2+发挥促进作用的阶段不在初始粘附阶段，而是促进了后续过程中细菌细胞之间的粘附。外加微量的AHLs也可以促进降解菌ZX01的成膜；另外，利用群体感应淬灭菌QX14可以降解AHLs的特性，发现体系中存在QX14时可以显著降低降解菌ZX08 的成膜量，这也反证了AHLs对于降解菌成膜的重要作用。

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