| Drinking water safety is an outstanding environmental issue in our country and directly affects the life and health of the people.Bacterial microbes' contamination in water is much higher than that of chemical pollution.The traditional biochemical analysis methods are intermittent and random sample collection and complex and time-consuming laboratory analysis,it can not achieve rapid,real-time online detection of pathogenic bacteria in water,can also not achieve comprehensive and continuous water quality monitoring and early warning,and provides the best opportunity for timely response processing.Multi-wavelength transmission spectroscopy technology has the advantages of comprehensive measurement information,fast measuring speed,no reagent assistance,and the multi wavelength transmission spectra of bacteria contain information of bacterial concentration,size,shape,composition and structure,which provides potential for rapid identification and detection of bacteria in water.Therefore,multi-wavelength transmission spectra analysis methods of bacterial microbes in water are studied.Which can not only provides a means for scientific research of structural composition,physiology and mechanism of bacterial microorganisms,but also provide a method for rapid identification and early warning of typical pathogenic bacteria in drinking water sources.This thesis selected the common bacteria(Escherichia coli,Klebsiella pneumoniae,Staphylococcus aureus and Salonella typhimurium)in water as research object,started with multi wavelength transmission spectra characteristics analysis of bacteria,built the multi wavelength transmission spectra interpretation model of bacteria microbes in water,stressed on the obtained method of characteristic parameters of bacteria microbes in water,and finally studied the changing rule of bacterial characteristic parameters in different growth stages.The main research results are as follows:(1)Different bacteria microbes spectral characteristics analysis and spectra pretreatment:the transmission spectra of four bacteria microbes of Escherichia coli,Klebsiella pneumoniae,Staphylococcus aureus and Salmonella typhimurium at different concentrations were measured,the peak position,the correlation between the concentration and the optical density of the transmission spectrum,and the similarity between transmission spectra at different concentrations were analyzed.The results showed that the peak position of the transmission spectra of bacteria with different concentrations was basically the same;the linearity of bacteria concentration and their corresponding transmission spectra was better,and their correlation coefficients were all greater than 0.99;the same bacteria,spectra similar degrees of different concentrations are all above 0.99.The dretreatment of transmission spectrum eliminates spectral changes caused by bacterial concentrations,resulting in differences between different bacterial microbes.The pretreatment method of multi wavelength transmission spectra of E.coli suspension at different concentrations was studied based on particles concentration normalization,maximum normalization,integral normalization,sum normalization and average normalization.The results showed that in the region from 200 to 900 nm the relative standard deviations of different methods can be expressed as particle concentration normalization>maximum normalization>integral normalization>sum normalization= average normalization,that the relative standard deviations of the sum normalization and average normalization is the minimum,its average value is 0.041%,which provides a basis for multi wavelength transmission spectra interpretation of bacteria microbes in water.(2)The analytical model of multi wavelength transmission spectrum of bacteria microbes in water was constructed.Based on Mie scattering theory,combining the physiological structure of bacteria and using the nonlinear least square algorithm,the refractive index of the external structure and internal structure of bacteria is calculated.Based on the UV absorption characteristics of the main chemical components of microorganism(nucleic acid,protein,etc.),the complex refractive index of the main chemical components of the bacteria was calculated through the Kramers-Kronig(K-K)relationship.On this basis,the multi wavelength transmission spectra analysis model of bacteria microbes in water was constructed.The multiwavelength transmission spectra of escherichia coli,klebsiella pneumoniae and staphylococcus aureus were predicted.The results show that for Escherichia coli,Klebsiella pneumonia and Staphylococcus,the maximum relative errors between the calculated spectrum and the measured spectrum are 4.05%,7.72%and 3.22%,The mean relative errors are respectively 0.06%?0.22%and 0.27%.It is shown that there was high consistency between the calculated spectra and the measured spectra,the proposed model can reproduce the features of experimentally measured spectra.(3)The obtained method of characteristic parameters of bacteria microbes in water was studied.The multi wavelength transmission spectrum of Escherichia coli samples was collected.In order to eliminate the influence of the bacterial concentration,the total normalization is used to preprocess the spectral data.Then,based on the Mie scattering model,the transmission spectrum in the visible band was analyzed by using the nonlinear least square optimization algorithm,to obtain bacteria structure parameters;Then,the multi-wavelength transmission reference spectra of single bacterium was calculated by using the bacterial structure parameters,and the bacteria concentration was inferred from the correlation between the measurement spectrum and the reference spectrum of single bacterium;Finally,based on Beer-Lambert law,the absorption part of the transmission spectrum in the best sensitive band(250-300nm)was analyzed to obtain the average nucleic acid content and protein concent of single bacteria.The calculated bacteria volume was 1.6899 and the relative error of bacterial volume measured by the microscope was only 7.90%.The bacterial concentration was 44649000 cells/ml,compared with the plate counting method,the relative error was 1.02%.The average nucleic acid content of the single bacteria is 7.1335 x 10"14g/cell and the protein content is 1.1764*10-13 g/cell.The results are consistent with the literature data.(4)The change of the characteristic parameters of water bacteria in different growth stages were studied.According to the above characteristic parameters obtained method,the transmission spectrum of Escherichia coli with different culture time was analyzed,and the change characteristics of in the structure,chemical composition,and concentration of E.coli cells at different growth stages were studied.The results showed that the spectra of the bacteria evolved as function of time;the bacterial cell size in E.coli suspension first increased with the extension of culture time,and then tended to fluctuate,The result was consistent with the observations by the microscope;the change of bacteria concentration is basically consistent with the result of the plate count method;the nucleic acid content of single bacteria is 1.1120 x 10-3-3.0920 x 10-13g/cell,Consistent with data obtained from confocal microscopy by Valkenburg et al.Multi-wavelength transmission spectroscopy can use bacteria moving-tracking detection during growth,and obtain morphological characteristics and chemical composition of bacteriaThe results showed that the multi wavelength transmission spectroscopy can not only provide a new measure for bacterial microbial scientific research,but also provide method for rapid identification,detection and earin warning of bacteria microbes in water. |
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