Pilot Studies Of Microbiome Methodology Based On Synchrotron FTIR Microspectroscopy

The microbiome refers to the total microbial communities occupy a certain habitat boundaries,which determines the overall functions of microbiota.Since in-deep analysis of its amount and distribution would be the key to solve the problems in energy,agricultural and medical fields,the microbiome is becoming a hot topic.Due to the fact that the microbiome research not only requires to distinguish the species composition but also needs to explore the interactions among them as well as with their hosts and/or environment,there remains higher requirements and big challenges for the research techniques.Infrared spectroscopy is able to detect total chemical molecules in microorganisms and provide a specific fingerprint spectrum for microbial identification.The motivation of this thesis is to develop the methodology for microbiome by using synchrotron radiation based Fourier transform infrared(SR-FTIR)microspectroscopy.SR-FTIR microspectroscopy,combined with statistics methods,such as principle component analysis(PCA)and hierarchical cluster analysis(HCA),was applied to analyze the individual microbial system composed by 16 kinds of pathogens and a simple microbiome system containing 3 mixed pathogens,respectively.The methodology for discriminating and classifying microbiome was preliminarily established.The main results are as follows:First,SR-FTIR microspectroscopy-based method was developed for differentiating microbiome composed individual pathogens at the genus,species and subspecies level.We found that the whole spectral region(3000-2800,1800-900cm-1)as well as four subdivided regions: lipid region(3000-2800cm-1),protein region(1800-1500cm-1),mixed region(1500-1200cm-1)and polysaccharide region(1200-900cm-1)can be used to discriminate bacteria at species and subspecies level,but the whole spectra should be given preference at the genus level.Second,a SR-FTIR microspectroscopy based technique for classifying single microbiome was established.We found that both the most specific information and the best cluster results could be achieved from the spectral region 1300-1000cm-1,mainly representing polysaccharides in the cell wall and nucleic acids.Therefore,we refer to this region as the fingerprint region.Third,SR-FTIR microspectroscopy was used to discriminate a simple microbiome model constructed by several bacteria.After detailed analyzing the positions of the absorption peaks,the methods to discriminate the microbiome with 2 or 3 mixed pathogens were preliminarily established.In conclusion,taking the advantages in high spatial resolution,fast detection speed and good reproducibility,SR-FTIR microspectroscopy combined with statistical methods such as PCA and HCA,can be used not only to classify microbes at taxa level,but also to differentiate mixture systems,thus providing great promise as an important tool for microbiome.