Kara Müller successfully defended her PhD-Thesis – Congratulations!

Raman Microspectroscopy Combined with Deuterium-Labelling to Study Microbial Assimilation of (Micro)Plastics at the Single-Cell Level and Complementary Bulk Fatty Acid Analysis

Abstract:

Plastics are ubiquitous in soils, particularly in agricultural systems where full recovery of polymer products is often impossible. This creates a pressing need for biodegradable alternatives. Assessing biodegradability requires different analytical methods to capture mineralization and assimilation into biomass. Current methods for studying incorporation of polymer-derived elements into microbial biomass focus on bulk stable isotope measurements, which mask cell-tocell differences, or on NanoSIMS single-cell approaches, which are laborious and unsuitable for high throughput applications.
This thesis establishes a multi-scale, stable isotope–based framework with the aim of investigating microbial plastic assimilation into microbial biomass. Deuterium-labeled polylactic
acid (dPLA) served as a model polymer for non-destructive single-cell analysis using stable isotope Raman microspectroscopy (SIRM). The method exploits C–D vibrations in the Ramansilent spectral region. SIRM enabled quantification of deuterium incorporation into individual cells of the bacterium Sphingomonas koreensis. Pronounced heterogeneity was observed: some cells incorporated substantial polymer-derived deuterium, while others remained unlabeled. Characteristic labeling patterns, including preferential lipid over protein deuteration, were detected. Deuteration also resulted in pronounced shifts in carotenoid resonance Raman spectra, illustrating potential for high-throughput imaging and cell sorting. Transfer of the developed approach to soil bacterial isolates confirmed that environmental communities assimilate polymer-derived deuterium within two weeks. Complementary lipid stable isotope probing (FAME-GC-MS) was applied to microbial cultures grown on different carbon sources (glucose, glucose-d12, or lactate) in the presence of deuterium (D2O or glucose-d12) to benchmark and validate the interpretation of single-cell SIRM data. Systematic comparison of SIRM and lipid-SIP showed strong agreement and revealed complementary insights into cell heterogeneity, substrate utilization, and isotopologuespecific labeling. While FAME-GC-MS has not yet been applied to dPLA, the framework, where it is combined with SIRM, is recommended for cross-validated plastic biodegradability
assessments.
Together, these findings demonstrate that single-cell SIRM directly quantifies incorporation of polymer-derived deuterium into individual microbial cells, revealing cellular heterogeneity and metabolic preferences, while lipid-SIP can provide complementary information at thebulk level. The integration of both approaches establishes a coherent analytical framework
that captures the fraction of active degraders as well as their heterogeneous metabolic responses. The framework is well positioned for future application in plastic biodegradability studies, where it could enable robust, high-throughput and environmentally relevant assessment, with potential applications in environmental monitoring, sustainable polymer design, and plastic certification.