Construction Of Microbial Sensing Interface And Its Application In Water Toxicity Assessment

With the rapid development of modern industry and agriculture,it is urgent to develop rapid,simple,sensitive water toxicity detection methods and equipments owing to more and more severe water pollution.The ubiquitous and diverse microorganisms have become the focus of attention in the field of water toxicity detection.Because of the advantages of abundant bacteria,not affected by chromaticity and turbidity,the electrochemical method for water toxicity detection has become a research hotspot in this field.However,there are many problems to be solved,such as complicated procedures and low sensitivity,compared with luminescent bacteria method and other technologies.In this paper,several new principles and new methods of water toxicity detection were developed by building microbial sensor interface,which provided new ideas for the development of simple,rapid and sensitive devices of water toxicity detection.The research works can be summarized into three parts:（1）Preparation of micro-nano electrode and its application in water toxicity detection.Micro-nano electrodes play an important role in the development of rapid toxicity detection methods in water.Firstly,a novel nanoribbon electrode（NRE）has been prepared by electrodeposition of a polyallylphenol（2-allylphenol）insulating layer on indium tin oxide（ITO）glass.NRE has the advantages of fast mass transfer rate of microelectrode and high current of millimeter electrode.Potassium ferricyanide（K₃[Fe（CN）₆]）was used as the mediator and E.coli was used as the biological subject.The half inhibitory concentration(IC₅₀)of the standard toxic substance,3,5-dichlorophenol（DCP）,was 3.01 mg/L at a constant potential of 450 m V.Secondly,according to the similar linear thermal expansion coefficient between glass and metal Pt,and the maximum size of the microelectrode（50μm in diameter）,a Pt ultramicroelectrode（UME）with high detection signal and good stability was prepared.At the same time,the water toxicity detection procedure was also simplified using mixed microorganisms cultured with LB medium preparaed with tap water itself through chemical sterilization.The IC₅₀ values of wastewater containing heavy metal ions of Cd²⁺,Cu²⁺and Ni²⁺were 3.99 mg/L,1.16 mg/L and2.37 mg/L,respectively.The order of toxicity was Cu²⁺>Ni²⁺>Cd²⁺.Thirdly,a biosensor of p-benzoquinone（BQ）-K₃[Fe（CN）₆]was constructed by using microelectrode array（MEA）and mixed microorganism cultured in situ.BQ was accidentally found to be highly toxic with an IC₅₀ value of 0.89 mg/L,which explained that the reason that the introduction of lipophilic mediator BQ could neither improve the detection current nor the detection sensitivity.It has important guiding significance for the mediator scientific screening in the field of water toxicity detection.（2）Development of new methods for colorimetric detection of toxicity in water.First,a new double-signal toxicity detection method based on the rapid colorreaction between E.coli and BQ was developed.The toxicity of wastewater containing Cu²⁺,IC₅₀=0.75 mg/L,was obtained by two-step method,which was more sensitive than that of Cu²⁺,IC₅₀=0.95 mg/L by one-step method in water toxicity detection.At the same time,the method has the function of colorimetric detection,and the lowest concentration（LC）of 3.2 mg/L could be visually estimated by naked eye.This biosensor expanded the application range of BQ as a mediator,and it provided a fast and sensitive double-signal detection method for toxicity in water.The detection results were more reliable,which had potential application value.Secondly,a recoverable,visualized and self-powered biosensor of water toxicity detection was developed based on microbial fuel cells（MFC）combining the biological anode and prussian blue（PB）cathode.The PB cathode had excellent stability and reversibility.According to the electrochromic display of PB cathode,the toxicity of wastewater containing 1 mg/L heavy metal ions of Cd²⁺,Co²⁺,Pb²⁺and Cu²⁺was detected by colorimetric method.The absorbance inhibition ratios（IA）obtained by the change of absorbance were 28.4%,11.0%,33.8%and 66.6%,respectively.The order of toxicity from small to large was Co²⁺<Cd ²⁺<Pb²⁺<Cu²⁺.The biosensor has certain advantages and application prospects in field emergency water toxicity detection.（3）Application of microbial electrolytic cell（MEC）-biosensor in online monitoring of water toxicity.Using cheap 3D graphite felt as anode and cathode（loaded with Pt/C）electrodesubstrate,an online toxicity monitoring device based on a single chamber MEC-biosensor was successfully constructed.The effects of rinsing time,concentration of phosphate buffer saline（PBS）and glucose concentration on the current signal stability of the MEC-biosensor were investigated in detail.The optimized results showed that the rinsing time of PBS was 30 min,the concentration of PBS was 20 m M,and the concentration of glucose was 300 mg/L.The reactor had a high degradation efficiency for organic matter without adding filler.The online toxicity monitoring experiment using Cu²⁺as toxic substance simulated water sample polluted by Cu²⁺,which showed that the reactor had a sensitive stress response for 1 mg/L Cu²⁺,and the inhibition ratio of 2 mg/L Cu²⁺was 19.45%.