Microscopic Analyses On Interactions Of Bacterial Interface

The bacteria-pollutant and bacteria-surface interfacial interactions are the foundation of wastewater biotreatment processes and affect the treatment efficiency substantially.Hence,thorough investigations into these bacterial interfacial interactions are conducive to the understanding of the mechanisms of wastewater biotreatment,and also facilitate the development of novel biotreatment processes.In this dissertation,we investigated the bacterial interfacial interactions with multidiscipline analytical tools such as microscopy,microbiology,and electrochemistry.First,we explored the biodegradation and biosorption of pollutants on bacterial interface.Second,we probed the bacterial adhesion and electron transfer to surfaces at the single-cell level.The main results of this dissertation are as follows:1.Extracellular biodegradation of pollutants on the bacterial interface of electrochemically active bacteria(EAB).Because of its electrochemical activity,the bacterial interface of EAB makes a tremendous contribution to the extracellular degradation of pollutants.To verify such a role of the bacterial interface in the bioreduction of azo dyes,Geobacter sulfurreducens PCA,a model EAB,was used for the bioreduction of methyl orange(MO),a typical azo dye.G.sulfurreducens PCA efficiently reduced MO into amines.Kinetic results show that G.sulfurreducens PCA has the highest decolorization efficiency among the currently known MO reducing strains.The results of mass and electron balances,fluorescent probing,and proteinase K treatment indicate that the biodecolorization of MO by G.sulfurreducens PCA is a process on the bacterial interface.The outer-membrane cytochrome Cs(OMC)were identified as the key outer-membrane proteins for the extracellular MO reduction.This work deepens our understanding of EAB physiology and is useful for the decontamination of environments polluted with azo dyes.2.Pollutants biosoiption on the bacterial interface of EAB.The redox-related charge on the bacterial interface of EAB can enhance the electrostatic force between EAB and pollutants,thus improves the biosorption processes of pollutants.Here,the biosorption of Cd(?)by Shewanella oneidensis MR-1 is explored under different redox conditions.The results of batch experiments and in-situ surface plasmon resonance microscopic(SPRM)analysis show that the S.oneidensis MR-1 incubated in the reducing environment is substantially superior over the strain incubated in the oxidizing environment in both Cd(II)biosorption capacity(22.48 mg/gdw,2.2 times)and biosorption rate(2.13 times).The results of spectral and mutant strain experiments reveal the important role of the OMC in the biosorption enhancement.The reduced OMC endows S.oneidensis MR-1 with a more negative zeta potential on the bacterial interface and improves its complexation and binding with Cd(II).These results deepen our understanding of the biosorption of heavy metals by microbes and broaden the environmental applications of EAB for the bioremediation of metal-contaminated environments.3.Plasmonic probing of the adhesion strength of single microbial cells.Probing the binding between a microbe and surface is critical for understanding biofilm formation processes,developing biosensors,and designing biomaterials,but it remains a challenge due to the complexity of the bacterial interface.Here,we demonstrate a method to measure the interfacial forces of bacteria attached to the surface.We tracked the intrinsic fluctuations of individual bacterial cells using an interferometric plasmonic imaging technique.Unlike the existing methods,this approach determined the potential energy profile and quantified the adhesion strength of single cells by analyzing the intrinsic fluctuations.The calculated binding constant can be used to quantitatively determine the single-cell binding strength and is positively related to the bacterial adhesion rates.This method provides new insights into biofilm formation and can also serve as a promising platform for investigating biological entity/surface interactions.4.Spatiotemporal imaging of the transition from reversible to irreversible adhesion reveals the nanoscaled step of microbial adhesion.As the prerequisite of biofilm formation,bacterial adhesion has a profound influence in multiple fields such as wastewater biotreatment and infection control.Among the stages of bacterial adhesion,the initial adhesion is the most decisive step,during which the bacteria undergo the transition from the reversible to irreversible adhesion.In spite of the fundamental importance,limited by analytical methods,the microscopic mechanism of adhesion transition remains elusive.Here,using SPRM,we imaged the transition process from the reversible to irreversible bacterial adhesion with high spatiotemporal resolution.By analyzing the SPRM images,we reported the first observation of the bacterial nanoscaled step-like approach towards the substrate,indicating the discontinuous nature of the transition from the reversible to irreversible adhesion.Furthermore,we explored the mechanism of the step-like transition and attributed it to the discontinuous interactions between extracellular polymeric substances patches and the substrate.Our work thus provides a revised picture of bacterial adhesion transition,and has an eminent value for both the theoretical explanation and practical regulation of biofilm formation.5.Rapid assessment of water toxicity by plasmonic nanomechanical sensing.The ability to rapidly and accurately detect water toxicity is crucial for monitoring water quality and assessing toxic risk,but such detection remains a great challenge.Here,we present a plasmonic nanomechanical sensing(PNMS)system for the rapid assessment of water toxicity.This technique is based on the plasmonic sensing of the nanomechanical movement of single bacterial cells that could be inhibited upon exposure to potential toxicants.By correlating the amplitude of nanomechanical movement with bacterial activity,we detected a variety of toxicants in water.The direct readout of bacterial activity via PNMS allowed for a high sensitivity to toxicants in water,thereby enabling us to evaluate the acute toxicological effect of chemical compounds rapidly.The PNMS method has a stable response to different toxicants(both organic and inorganic)and is robust to real wastewater samples.The PNMS method is promising for online alerts of water quality safety and for assessing chemical hazards.6.Probing electrochemical activity of single microbial cells by plasmonic imaging.The electrochemical activity of microbial cells is the indispensable foundation of microbial extracellular electron transfer(EET)that has a wide application in energy recovery and environmental remediation.However,the detailed mechanism of EET remains unclear.The high-throughput analytical tool for the single-cell electrochemical activity is still lacking.To address these challenges,here,we develop a plasmonic electrochemical imaging system(PEIS)to characterize the electrochemical activities of single microbial cells.Taking advantage of the high sensitivity of PEIS to refractive index change,we can image the electrochemistry-related refractive index change of individual cells and then obtain the single-cell electrochemical parameters(apparent formal potential and number of electrons transferred)with high-throughput.Considering its ease of handling and scalability,we anticipate that the present approach would be applied in the rapid screen of EAB strains and functional identification of key proteins in EET,thus paving ways for the research and optimization of bioelectrochemical systems.