Application of infrared spectroscopy to study carbon-deuterium kinetics and isotopic spectral shifts at the single-cell level

Microbial communities play crucial roles in shaping natural ecosystems, impacting human well-being, and driving advancements in industrial biotechnology. However, associating specific metabolic functions with bacteria proves challenging due to the vast diversity of microorganisms within these communities. In the past decades stable isotope probing (SIP) approaches, coupled with vibrational spectroscopy, have emerged as a novel method for revealing microbial metabolic roles and interactions in complex communities. In this study, we employed various combinations of heavy stable isotopes (D, ¹³C, ¹⁵N, and ¹⁸O), to evaluate all possible isotopic spectral shifts in the mid-IR region using Fourier-Transform Infrared (FT-IR) and Optical Photothermal Infrared (O-PTIR) spectroscopy, at both community and single-cell levels. Additionally, we conducted a time-course study to explore the kinetics of CD vibration in Escherichia coli bacteria, allowing time-based sampling and assessment of isotopic labeling kinetics. The FT-IR and O-PTIR, along with the second derivative spectra of E. coli cells cultured in minimal medium supplemented with various combinations of heavy isotopes exhibited notable similarities. Several spectral shifts in primary vibrational peaks were observed due to the incorporation of heavy isotopes into various biomolecules. Remarkably, the incorporation of deuterium into amide groups, resulting in the formation of nitrogen-deuterium bonds, caused a shift in amide A and B into the silent region, overlapping with CD signature peaks. The incorporation of ¹⁸O into the ester group of lipids and the carbonyl group of proteins resulted in a notable shift to the lower wavenumber region. Additionally, the second derivative of FT-IR spectral data highlighted the integration of ¹⁸O into α-helix and β-sheet structures. Furthermore, the spectra, second derivative, and PC-DFA scores and loadings plot of FT-IR data collectively illustrated the practicality of monitoring ¹³C and D incorporation into E. coli bacterial cells within the first 30-min incubation period. The findings of this study suggest that FT-IR and O-PTIR can serve as efficient tools for monitoring the incorporation of heavy isotopes into bacteria at both the population and single-cell levels. Additionally, the SIP approach allowed us to assign two new deuterium-associated vibrational peaks to their corresponding functional groups, which to the best of our knowledge have not been reported previously.