Rapid Quantification Of Pseudomonas Sp. And Analysis Of Its Interaction With Surfactants For Whole Cell Applications

Pseudomonas is an important genus both for environmental biotechnology and industrial biotechnology. In order to control the bacteria of this genus and intensify the metabolism of the whole cells, rapid and accurate monitoring of cell number and cellular characteristics is indispensable.This thesis aimed at establishing rapid and simple tools for quantification and monitoring of the whole cells and cellular activities of Pseudomonas sp., by means of fluorescence and bioluminescence techniques.To study the rapid quantification of Pseudomonas sp., the system of drinking water with its purifier was chosen, for its bacterialogical quality is closely related to the human health. The water purifier was found to be capable of simplifying the structure of bacterial community in drinking water by API kits identification. While, the biofilm formed inside the purifier would significantly increase the total number of bacteria (HPC) and the formation was closely related to the configuration and temperature of the purifier. P. putida was detected in the drinking water, but became non-detectable either in the filtered water or the biofilm formed inside the purifier.A novel immunoassay-based bacterial quantification method was developed by using the KinExA instrument, showing the superior performances with high sensitivity, high accuracy, high reproducibility, simple manipulation and short detection time, etc. By this method, the sensitivity and dynamice range for P. aeruginosa detection were close to or even better than those of the best immunological detection method reported so far, and the sensitivity was 100 times higher than that of ELISA.To analyze the whole cell characteristics and intracellular metabolic activities under the presence of cationic surfactants, a genetically engineered strain, P. putida BLU-CFP, was constructed by employing the LUX-CFP dual bioremarkers. Results showed that LUX reflected the metabolic activity of the cells, while CFP could estimate the membrane permeability, the shape integrity of the cells and also the activities of most intracellular proteins. By using this dual bioremarkers of LUX-CFP, the monitoring of whole cell catalysis would be much simpler.The action process of DTAB (one of the cationic surfactants) on the whole cell of P. putida could be divided into five stages according to the changes of the cells by the treatment of DTAB from low to high concentrations: normalcells resting metabolic activity inhibited cell viability lost lysed. And according to the changes of intracellular enzymes, the DTAB effect was described as normal partly inhibited, while mass transfer speed enhanced leaked outside the cell completely denatured. In addition, the hydrophobic group of quarternary ammonium compounds was found playing a key role in the action of cationic surfactant on the whole cells and its intracellular enzymes.