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

基于稳定同位素的分析方法在不同的科学领域获得越来越多的相关性。虽然基于质谱（MS）的方法能够对大量样品进行敏感分析（例如，同位素比质谱法，IRMS）或提供低至50纳米的空间分辨率（例如，纳米级二次离子质谱法，NanoSIMS），但这些方法具有破坏性并且需要耗时的样品制备。在这里，拉曼微光谱（RM）与稳定同位素方法的结合-稳定同位素拉曼微光谱(SIRM) -可以通过非破坏性，定量和空间分辨分析扩展成熟技术的能力。SIRM提供具有共聚焦光学显微镜空间分辨率的样品特征指纹光谱，包含稳定同位素标记物质的信息和标记量（基于标记物质波段的红移）。同时，这些光谱提供了样品的化学成分和结构信息。此外，该方法不需要或有限的样品制备，并且可以在原位和体内进行，没有水的光谱干扰。在申请项目的第一阶段，SIRM对不同样品（包括腐殖质物质、微生物和生物膜）进行定量二维和三维研究的可行性已经得到证明。SIRM还成功地与共振和表面增强拉曼散射（SERS）效应相结合，以提高其灵敏度。已取得的结果以及其他研究人员目前的研究表明，SIRM在单细胞水平上表征微生物群落方面具有很高的潜力。该方法提供了碳代谢/流动和细胞活性的信息，并已用于分析环境污染物的降解。然而，迄今为止，尚未对水生环境中最突出的新兴污染物-微塑料（MP）的微生物降解进行研究。这个后续项目的目标是开发和评估一种基于sirm的方法，用于定量和无损的二维和三维分析参与MP生物降解的生物膜。SIRM结合SERS有可能为塑料降解生物膜中微生物的碳同化和一般代谢活性提供新的信息。除了使用昂贵甚至无法获得的稳定同位素标记聚合物外，还将采用其他方法- D2O和反向标记SIRM。得到的SIRM结果将用IRMS和NanoSIMS数据进行验证。总之，这将有助于我们建立可靠的方法来分析（微）塑料在水生环境中的生物降解，从而为我们提供单细胞水平的（直接）信息。

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.