Effects Of Ceftazidime On Activated Sludge Bacteria By Raman Spectroscopy

Antibiotic is one of the most widely used drugs in the world.Because of its bacteriostatic efficacy,antibiotics are widely used in bacterial infection control,livestock and aquaculture.But most antibiotics are difficult to be degraded by human and other organisms,and wastewater containing antibiotics has also become a kind of wastewater hard to be degraded with biological toxicity.Antibiotics are heavily emitted into the environment,causing great pollution to the environment and threatening the diversity of microorganisms.For a long time,the problem of antibiotic wastewater treatment has not been well solved.Many researchers have discussed the problem from the technological level,and put forward some physicochemical methods to deal with antibiotic wastewater,but the effect is not ideal.Biological treatment is still an important way of antibiotic wastewater treatment,but the related mechanism Is relatively scarce,especially the microscopic mechanism of the interaction between antibiotics and microorganisms.As a fingerprint spectrum,Raman spectroscopy is of great importance in medical science and biology because of its advantages of non-destructive,non-invasive and available in water environment.Therefore,Raman spectroscopy is a powerful tool for studying the microbiological process of microorganisms,and can be applied to the microscopic mechanism research of biological processes in antibiotic wastewater treatment.In order to explore the mechanism of the interaction between antibiotics and microorganisms,this thesis uses Raman spectroscopy to monitor the changes of substances in bacteria under antibiotic stress,and further explores the way of antibiotics influencing on bacteria.The following work has been completed:(1)Screening target bacteria with resistance.Ceftazidime has been selected as the antibiotic used in the experiment.Screening ceftazidime resistant strains from the ordinary activated sludge,and then these strains were cultured in ceftazidime solution.Liquid chromatography was used to analyze the residual ceftazidime in solution,which is to determine ceftazidime degradation ability of these bacteria.Finally,four bacteria strains with a certain ability to degrade ceftazidime were obtained,to be used as the target strain of the follow-up experiment.The four strains were identified by 16SrRNA gene sequencing,and the results show that they belong to Aeromonas,bacillus and rhodotococcus,respectively.(2)Using Raman spectroscopy to test the characteristic peaks of the screened four strains.First,the optimum conditions for the Raman spectra of bacteria were investigated.Ordinary glass,quartz and CaF2 were selected to be the substrate to test the Raman spectra of the bacteria.The result shows that the fluorescence background of ordinary glass substrate is the strongest,not suitable as substrate for bacterial Raman test.And the fluorescence background of CaF2 is the weakest,is the ideal substrate for Raman test.At the same time,the Raman spectra of single bacterium and multi-bacteria of the same strain were compared.Although the same strain,the Raman spectra of the two samples were not exactly the same.The Raman spectra signal of single bacterium is weaker,and with a low signal-to-noise ratio,and the Raman spectra are not the same at different sampling points.On one hand,it shows the heterogeneity of individual bacteria,on the other hand it also shows Raman spectra can distinguish information of different parts of individual bacterium.The Raman spectra of multi-bacteria is relatively stronger,with low noise.However,signals of different sampling point are approximately the same,indicating that the Raman spectra of multi-bacteria reflects average signal of bacteria.Using quartz chip as the substrate and multi-bacteria of each strain as sample,the Raman spectra of four strains were obtained.The Raman signals of protein and nucleic acid were strong,accounting for most of the information,while the signals of lipids and saccharides were fewer.The principle component analysis(PCA)was used to distinguish four strains.The results were consistent with that of gene sequencing,indicating that Raman spectroscopy has the ability to identify bacteria.(3)Exploring the biochemical processes in the bacteria under ceftazidime stress by Raman spectroscopy and Raman imaging.Firstly,the Raman spectral difference between bacteria under and without antibiotic stress was analyzed by PCA analysis,and the characteristic peaks were extracted.The result shows that amino acids first change and the lipid reduce at the same time when bacteria suffer ceftazidime stress.Meanwhile,in Raman imaging,when bacteria were subjected to antibiotic stress,the peak 1002cm-1 which belong to phenylalanine increased,while the peak of 1172cm-1,which represented lipid weakened.It is concluded that when the bacteria are stimulated by antibiotics,on one hand,the intracellular lipid reduced,which may suggest the change of the membrane permeability,and on the other hand,bacteria will resist the external stimuli of antibiotic by regulating the activity of related enzymes,so the content of certain amino acids will increase.This thesis studied the interaction between antibiotics and bacteria by Raman spectroscopy,and preliminarily discussed the feasibility of applying Raman spectroscopy to research on microbial mechanism of antibiotics and microorganisms in wastewater treatment.Also,foundation is laid for the dynamic study of microbial biochemical processes on greater depth.