【作者】 张静；

【导师】 晏鹏； 许小伟；

【作者基本信息】 重庆大学 ， 工程（环境工程）（专业学位）， 2022， 硕士

【摘要】 纳米银材料具有良好的导电性能和抗菌性能,在各个领域展现出极为广阔的应用前景。纳米银颗粒的广泛使用,使大量纳米银颗粒随着污水进入到城市污水处理厂,对污水处理系统中的功能微生物产生生理毒性,进而影响生物除磷脱氮效率。好氧反硝化菌在好氧条件下进行反硝化作用,使得硝化反应和反硝化反应可以在同一反应器中进行,从而可以降低设备投入和操作管理成本。好氧反硝化已成为近年来生物脱氮领域的研究热点。研究好氧反硝化菌对纳米银颗粒毒性作用的适应,能够为从工艺层面维持高效生物脱氮能力提供理论支撑。目前,尽管已有研究关注纳米银颗粒对好氧反硝化菌的抑制及其对生物脱氮性能的影响,但研究结论并不一致,纳米银颗粒对好氧反硝化菌的抑制机制仍有待进一步揭示。此外,Ag NPs在实际使用过程中存在不同的形态,目前少有研究关注Ag NPs的形态效应对微生物毒性的影响,因此,开展不同形态Ag NPs对好氧反硝化菌的胁迫机制研究,既符合实际工程情况,也能丰富好氧反硝化菌对Ag NPs胁迫的适应机制。本研究基于Scene模型,结合分形维数对Ag NPs的形态特征进行了评估,系统研究了Ag NPs形态效应对典型好氧反硝化菌-施氏假单胞菌生理代谢特征和生物膜反应器性能的影响机制,采用分子生物学的手段,深入解析了施氏假单胞菌适应不同形态纳米银颗粒的微观机制,为Ag NPs胁迫好氧反硝化菌自适应机制提供新的视角。本文的研究工作和成果可以归纳为以下5个方面:(1)通过对球状和片状纳米银颗粒的表面形貌、表面电荷、吸附性和分形维数等进行表征发现:球状Ag NPs呈规则光滑的球形,粗糙度小;片状Ag NPs呈现不规则的扁平片状,粗糙度高于球状颗粒。片状Ag NPs的表面Zeta电位绝对值较大,性质更加稳定,疏水性更高。从二维和三维两个方式计算两种颗粒的分形维数,得到一致的结论:片状Ag NPs的分形维数要高于球状Ag NPs,这表明片状颗粒的表面不规则程度更高。(2)施氏假单胞菌在不同形状Ag NPs胁迫下的生理响应主要体现在:抑制细菌的生长和脱氮能力,细菌的表面会产生凹陷及膜破裂的现象,同时也会产生褶皱现象,且片状组相比球状组会造成更多的机械损伤,对细菌具有更严重的损害作用。使用表面等离子体成像系统观察了细菌在Ag~+作用下的运动能力,发现Ag~+对细菌运动活性的影响呈现明显的剂量效应,浓度越高对细菌垂直方向运动的幅度抑制越强,活性也越来越低,会直接影响到菌株的生理功能及避害性。(3)施氏假单胞菌在不同形状Ag NPs胁迫下的生化响应主要是:两种形状颗粒胁迫均会导致胞内ROS增加,但片状组细菌胞内ROS增幅更高,片状纳米银不利于细菌抵抗Ag NPs导致的ROS压力。产生EPS是细菌对抗Ag NPs胁迫的响应机制之一,片状颗粒具有棱角直接造成细胞膜损伤,诱发细菌EPS分泌量增多。同时,NR和Ni R酶活有下降的趋势,与细胞代谢相关的电子传递链活性受到抑制,细菌的脱氮功能与生理代谢活性降低,片状颗粒由于表面形貌的不规则会对细菌造成更大的损伤,这也是Ag NPs对细菌毒性的形态效应表现。(4)通过探究Ag NPs对施氏假单胞菌生物膜反应器的脱氮性能、碳源利用率、生物膜生物量等指标进行测定发现,采用海绵填料能实现快速挂膜;在Ag NPs胁迫下,脱氮效果、生物膜生物量、碳源利用率均受到严重影响,片状组受的影响更为严重。这是由于片状颗粒表面粗糙和形状不规则,易吸附在生物膜上阻碍了微生物的传质及利用,同时降低胞外EPS的保护作用。(5)基于原核转录组学发现:在Ag NPs的胁迫下,细菌会上调嘧啶核碱基分解代谢和尿嘧啶分解代谢过程,同时下调亚硝酸还原酶活性、氧化还原酶活性、以及硝酸盐代谢过程,纳米银导致细菌的氮物质代谢和能量代谢过程受到抑制。此外,细菌在两种形状Ag NPs胁迫下的基因代谢特性存在差异性表达,这是由颗粒的形状效应所导致;相比于球状颗粒,片状颗粒的形状不规则性更高,其对细菌会产生更多关键代谢通路的下调,尤其在脱氮能力、代谢活性、运动响应方面。更多还原

【Abstract】 Nano-silver materials have good electrical conductivity and antibacterial properties,showing extremely broad application prospects in various fields.The widespread use of nano-silver particles makes a large number of nano-silver particles enter the urban sewage treatment plant with sewage,which produces physiological toxicity to the functional microorganisms in the sewage treatment system,thereby affecting the biological phosphorus and nitrogen removal efficiency.Aerobic denitrifying bacteria perform denitrification under aerobic conditions,so that nitrification and denitrification can be carried out in the same reactor,thereby reducing equipment investment and operation and management costs.Aerobic denitrification has become a research hotspot in the field of biological denitrification in recent years.Studying the adaptation of aerobic denitrifying bacteria to the toxicity of silver nanoparticles can provide theoretical support for maintaining high-efficiency biological denitrification from the process level.At present,although there have been studies focusing on the inhibition of aerobic denitrifying bacteria by nano-silver particles and its effect on biological denitrification performance,the research conclusions are inconsistent,and the inhibitory mechanism of nano-silver particles on aerobic denitrifying bacteria still needs to be further revealed.In addition,there are different forms of Ag NPs in the actual use process.At present,few studies have paid attention to the effect of the morphological effect of Ag NPs on microbial toxicity.Therefore,the study of the stress mechanism of different forms of Ag NPs on aerobic denitrifying bacteria is consistent with the actual engineering situation and can also enrich the adaptive mechanism of aerobic denitrifying bacteria to Ag NPs stress.In this study,the morphological characteristics of Ag NPs were evaluated based on the Scene model combined with fractal dimension,and the morphological effects of Ag NPs on the physiological and metabolic characteristics and biofilm reactor performance of the typical aerobic denitrifying bacteria-Pseudomonas stutzeri were systematically studied.Using the means of molecular biology,we deeply analyzed the microscopic mechanism of Pseudomonas stutzeri adapting to different forms of silver nanoparticles,providing a new perspective for the adaptive mechanism of Ag NPs stressing aerobic denitrifying bacteria.The research work and results of this paper can be summarized in the following five aspects:(1)By characterizing the surface morphology,surface charge,adsorption,and fractal dimension of spherical and flaky silver nanoparticles,it is found that spherical Ag NPs are regular and smooth spherical with small roughness;flaky Ag NPs are irregular.Flat flakes with higher roughness than spherical particles.The absolute value of surface Zeta potential of sheet Ag NPs is larger,the properties are more stable,and the hydrophobicity is higher.The fractal dimensions of the two kinds of particles were calculated from two-dimensional and three-dimensional methods,and a consistent conclusion was obtained: the fractal dimension of the flake Ag NPs was higher than that of the spherical Ag NPs,which indicated that the surface irregularity of the flake particles was higher.(2)The physiological response of Pseudomonas stutzeri under the stress of Ag NPs of different shapes is mainly reflected in: inhibiting the growth and denitrification ability of the bacteria,the surface of the bacteria will produce depressions and membrane ruptures,and wrinkles will also occur and the flake group caused more mechanical damage than the spherical group,and had more serious damage to bacteria.The motility of bacteria under the action of Ag+ was observed using a surface plasmon imaging system,and it was found that the effect of Ag+ on bacterial motility showed a significant dose effect.The higher the concentration,the stronger the inhibition of the vertical movement of bacteria,and the more active low,it will directly affect the physiological function of the strain and its ability to avoid harm.(3)The biochemical responses of Pseudomonas stutzeri under the stress of Ag NPs of different shapes are mainly: the two shapes of particles both lead to the increase of intracellular ROS,but the increase of intracellular ROS in the flaky group is higher,and the flaky nanoparticle has a higher increase in intracellular ROS.Silver is not conducive to bacterial resistance to ROS stress caused by Ag NPs.The production of EPS is one of the response mechanisms of bacteria against Ag NPs stress.The edges and corners of the flaky particles directly cause cell membrane damage and induce the increase in bacterial EPS secretion.At the same time,the enzymatic activities of NR and Ni R tended to decrease,the activity of the electron transport chain related to cell metabolism was inhibited,the denitrification function and physiological metabolic activity of bacteria were reduced,and the irregular surface morphology of flaky particles would cause more damage to bacteria.Large damage,which is also the morphological effect of Ag NPs on bacterial toxicity.(4)By exploring Ag NPs to determine the denitrification performance,carbon source utilization,biofilm biomass,and other indicators of Pseudomonas stutzeri biofilm reactor,it is found that the use of sponge filler can achieve rapid film hanging;under Ag NPs stress,the denitrification effect,biofilm biomass,carbon source utilization rate were all seriously affected,and the flake group was more seriously affected.This is due to the rough surface and irregular shape of the flaky particles,which are easily adsorbed on the biofilm,hindering the mass transfer and utilization of microorganisms,and at the same time reducing the protective effect of extracellular EPS.(5)Based on prokaryotic transcriptomic findings: Under the stress of Ag NPs,bacteria up-regulate pyrimidine nucleobase catabolism and uracil catabolism,while down-regulating nitrite reductase activity,oxidoreductase activity,and nitrate metabolism During the process,nano-silver leads to the inhibition of nitrogen metabolism and energy metabolism of bacteria.In addition,the gene metabolism characteristics of bacteria under the stress of two shapes of Ag NPs were differentially expressed,which was caused by the shape effect of particles;There will be more down-regulation of key metabolic pathways,especially in denitrification capacity,metabolic activity,and exercise response.更多还原