Design And Preparation Of Hydrophobic Adsorption Fluorescent Probes For Labelling Microthrix Parvicella And Quantitative Evaluation

Microthrix parvicella is the most important filamentous bacterium that causes sludge bulking,but its physiological characteristics and life rule are still not very clear because it is difficult to be cultured successfully.Therefore,it is of great significance to study M.parvicella’s growth rule and the corresponding change behavior of other bacterial community in the process of excessive reproduction for the fundamental understanding of filamentous sludge bulking.At present,the recognized accurate quantitative methods of M.parvicella are FISH and quantitative PCR,but both of these methods have the characteristics of complex experimental process,time-consuming and expensive.Therefore,the establishment of a new method for rapid identification and quantification of M.parvicella is great significance for studying the characteristics of M.parvicella and the early warning of sludge bulking.In this study,two novel fluorescent probes were designed,synthesized and characterized,based on M.parvicella being hydrophobic and preferring oleic acid（18 carbonic acid）,for the labeling of M.parvicella in activated sludge.The two kinds of probe’s molecular structure as the fluorescent molecular matrix of carbazole derivatives,modified by 18 carbon alkyl and 18 carbonic acid,respectively.M.parvicella was identified in situ,and a quantization method has been preliminarily established.The enrichment culture of M.parvicella in SBR reactor was used to analyze the influence of temperature on the growth of M.parvicella and the relationship between the growth of M.parvicella and seasonal alternation.Changes of bacterial community during sludge bulking caused by overgrowth of M.parvicella were analyzed.The main research results are as follows:（1）Both probes A and B have excellent spectral characteristics and optical stability.When the probe concentration was less than or equal to 0.01 mmol/L,the mean fluorescence density of highlighted M.parvicella was positively correlated with the probe concentration（R²=0.99）,and 0.01 mmol/L was determined as the optimal concentration for the probe to identify M.parvicella.Probe A has stronger biological stability and better"tolerance"than probe B.In addition,the quantitative relationship between sludge volume index（SVI）and M.parvicella’s integrated fluorescence density（IOD）was preliminarily established by using Image Pro Plus6.0 analysis.When SVI was about 123 m L/g,M.parvicella grew the fastest in activated sludge floc.（2）Study found that M.parvicella grows mainly in winter,the summer even if under the condition of low temperature（14±1℃）,M.parvicella cannot grow,but in winter,low temperature condition is（14±1℃）was more advantageous for M.parvicella growing.（3）In the process of sludge bulking caused by excessive reproduction of M.parvicella,the abundance of Proteobacteria in the dominant population decreased significantly while that of Actinomycetes increased.The decrease of the abundance of functional bacteria was the main reason for the accumulation of NH₄⁺-N and NO₂^--N during suldge bulking period.Neisser staining results showed that M.parvicella contained a large number of polyphosphate granules,which is the main reason for no significant change in PO₄^3--P concentration in the effluent water.