Stable Isotope Raman Microspectroscopy (SIRM) for Quantitative and Nondestructive 2D & 3D Analysis of Biofilms: Microbial Degradation of Microplastic

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Stable isotope-based analytical methods gain increasing relevance in different scientific fields. Although mass spectrometry-based (MS) methods enable sensitive analysis of bulk samples (e.g., isotope ratio mass spectrometry, IRMS) or provide a spatial resolution down to 50 nm (e.g., nanoscale secondary ion mass spectrometry, NanoSIMS), these methods are destructive and require time-consuming sample preparation. Here, a combination of Raman microspectroscopy (RM) with the stable isotope approach – stable isotope Raman microspectroscopy (SIRM) – can extend the capabilities of the well-established techniques with a nondestructive, quantitative and spatially-resolved analysis. SIRM provides characteristic fingerprint spectra of samples with the spatial resolution of a confocal optical microscope, containing information on stable isotope-labeled substances and the amount of a label (based on red shift of bands of the labeled substances). Simultaneously, these spectra deliver information on the chemical composition and structure of samples. Furthermore, this method requires no or limited sample preparation, and can be performed in situ and in vivo without spectral interference of water.The feasibility of SIRM for the quantitative 2D & 3D studies of different samples, incl. humic substances, microorganisms and biofilms, has been demonstrated during the first phase of the applicant project. SIRM has been also successfully combined with resonance and surface-enhanced Raman scattering (SERS) effects, to improve its sensitivity. The achieved results as well as current studies of other researchers indicate a high potential of SIRM for the characterization of microbial communities at the single cell level. This method provides information on the carbon metabolism / flow and the cell activity, and has already been tested for the analysis of the degradation of environmental pollutants. However, no research has been performed so far on the microbial degradation of a most prominent emerging pollutant in the aquatic environment – microplastic (MP).The goal of this follow-up project is to develop and evaluate a SIRM-based method for quantitative and nondestructive 2D & 3D analysis of biofilms involved in the biodegradation of MP. SIRM in combination with SERS has a potential to provide novel information on the carbon assimilation and general metabolic activity of microorganisms from plastic-degrading biofilms. Besides the use of stable isotope-labeled polymers which are expensive or even unavailable, alternative approaches – D2O- and reverse-labeling SIRM – will be applied. The obtained SIRM results will be validated with IRMS and NanoSIMS data. Altogether, this should help us to establish reliable method for the analysis of biodegradation of (micro)plastic in the aquatic environment, thus providing us with (direct) information at the single cell level.