Influence of Carbon Sources on Quantification of Deuterium Incorporation in Heterotrophic Bacteria: A Raman-Stable Isotope Labeling Approach

Abstract

A rapid and reliable method for the differentiation between active and inactive bacteria at single cell level is urgently needed in many fields including clinical diagnosis and environmental microbiology, to understand the contribution of metabolically active bacteria in fundamental processes triggering environmental and public health risks. Here, using heavy water (D 2 O) with Raman-stable isotope labeling (Raman-D 2 O), we evaluated the reliability of the quantification of deuterium uptake, a well-known indicator for the general metabolic activity of bacteria. For this purpose, we based our study on the quantification of deuterium assimilation from heavy water into single bacterial cells to check the influence of carbon source and bacterial identity on the deuterium uptake. We show that compared to complex carbon substrates, the deuterium assimilation is higher in the presence of simpler substrates such as sugars but differs significantly among bacterial isolates. Despite this variability, the developed classification models could differentiate deuterium labeled and nonlabeled single cells with high sensitivity and specificity. Highlighting the variability between single bacterial cells, the study emphasizes the challenges in establishing a threshold in terms of deuterium uptake to distinguish deuterium labeled and nonlabeled cells. Overall, we show that the Raman-D 2 O approach, when coupled with chemometrics, constitutes a powerful approach for monitoring single bacterial cells.

Raman 18O‐labeling of bacteria in visible and deep UV‐ranges

“…isotope labeling is based on the replacement of single atoms (eg, 12 C, 14 N, 1 H and 16 O) by their stable heavier isotopes ( 13 C, 15 N, 2 H and 18 O) in biomolecules, such as proteins, lipids and nucleic acids [7,21]. This isotopic substitution leads to a red-shift of the vibrational frequency of the functional groups involved, that is, a shift toward lower wavenumbers, due to increased atomic mass [7,21].…”

SRS-FISH: High-Throughput Platform Linking Microbiome Function to Identity at the Single Cell Level

“…2). The higher levels of D incorporation observed for growth on M9 (where only a defined carbon source is present) compared to a complex medium has been documented 46 . This is likely caused by the higher need for de novo biosynthesis of monomeric biomolecules, such as amino acids, nucleotides, or fatty acids, which are absent in minimal media but readily-available for direct uptake and incorporation from complex media.…”

Raman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges

“…The metabolic profile of bacteria changes due to different environmental conditions [1,2]. Understanding the dynamics of bacterial metabolism is the key to elucidating biological processes [1][2][3][4].…”

“…The metabolic profile of bacteria changes due to different environmental conditions [1,2]. Understanding the dynamics of bacterial metabolism is the key to elucidating biological processes [1][2][3][4]. The identification of metabolically active bacterial cells is of high relevance in clinical diagnosis [5][6][7][8], industrial biotechnology, and microbial ecology [1,[9][10][11][12].…”

“…Understanding the dynamics of bacterial metabolism is the key to elucidating biological processes [1][2][3][4]. The identification of metabolically active bacterial cells is of high relevance in clinical diagnosis [5][6][7][8], industrial biotechnology, and microbial ecology [1,[9][10][11][12]. The standard method for identifying metabolically active cells in biomedical samples involves the use of a gas sensor to monitor the increase in CO 2 concentration in bacterial cultures [13,14].…”