Search for: All records

ABSTRACT Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, the comparability of isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell remains largely unexplored. This knowledge gap creates uncertainty about the consistency of single-cell SIP data obtained independently from each method. Here, we conducted a comparative analysis of 543Escherichia colicells grown in M9 minimal medium in the absence or presence of heavy water (²H₂O) using correlative Raman and NanoSIMS measurements to quantify the results between the two approaches. We demonstrate that Raman and NanoSIMS yield highly comparable measurements of²H incorporation, with varying degrees of similarity based on the mass ratios analyzed using NanoSIMS. The¹²C²H/¹²C¹H and¹²C₂²H/¹²C₂¹H mass ratios provide targeted measurements of C-H bonds but may suffer from biases and background interference, while the²H/¹H ratio captures all hydrogen with lower detection limits, making it suitable for applications requiring comprehensive²H quantification. Importantly, despite its higher mass resolution requirements, the use of C₂²H/C₂¹H may be a viable alternative to the use of C²H/C¹H due to lower background and higher overall count rates. Furthermore, using an empirical approach in determining Raman wavenumber ranges via the second derivative improved the data equivalency of²H quantification between Raman and NanoSIMS, highlighting its potential for enhancing cross-technique comparability. These findings provide a robust framework for leveraging both techniques, enabling informed experimental design and data interpretation. By enhancing cross-technique comparability, this work advances SIP methodologies for investigating microbial metabolism and interactions in diverse systems.IMPORTANCEAccurate and reliable measurements of cellular properties are fundamental to understand the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. Here, we study the relationship of the incorporation of a stable isotope (²H through incorporation of²H₂O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with²H₂O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research. 

Marine dissolved organic matter (DOM) is a major reservoir that links global carbon, nitrogen, and phosphorus. DOM is also important for marine sulfur biogeochemistry as the largest water column reservoir of organic sulfur. Dissolved organic sulfur (DOS) can originate from phytoplankton-derived biomolecules in the surface ocean or from abiotically “sulfurized” organic matter diffusing from sulfidic sediments. These sources differ in 34S/32S isotope ratios (δ34S values), with phytoplankton-produced DOS tracking marine sulfate (21‰) and sulfurized DOS mirroring sedimentary porewater sulfide (∼0 to –10‰). We measured the δ34S values of solid-phase extracted (SPE) DOM from marine water columns and porewater from sulfidic sediments. Marine DOM_SPE δ34S values ranged from 14.9‰ to 19.9‰ and C:S ratios from 153 to 303, with lower δ34S values corresponding to higher C:S ratios. Marine DOM_SPE samples showed consistent trends with depth: δ34S values decreased, C:S ratios increased, and δ13C values were constant. Porewater DOM_SPE was 34S-depleted (∼-0.6‰) and sulfur-rich (C:S ∼37) compared with water column samples. We interpret these trends as reflecting at most 20% (and on average ∼8%) contribution of abiotic sulfurized sources to marine DOS_SPE and conclude that sulfurized porewater is not a main component of oceanic DOS and DOM. We hypothesize that heterogeneity in δ34S values and C:S ratios reflects the combination of sulfurized porewater inputs and preferential microbial scavenging of sulfur relative to carbon without isotope fractionation. Our findings strengthen links between oceanic sulfur and carbon cycling, supporting a realization that organic sulfur, not just sulfate, is important to marine biogeochemistry. 

The hydrogen-isotopic compositions ( 2 H/ 1 H ratios) of lipids in microbial heterotrophs are known to vary enormously, by at least 40% (400‰) relative. This is particularly surprising, given that most C-bound H in their lipids appear to derive from the growth medium water, rather than from organic substrates, implying that the isotopic fractionation between lipids and water is itself highly variable. Changes in the lipid/water fractionation are also strongly correlated with the type of energy metabolism operating in the host. Because lipids are well preserved in the geologic record, there is thus significant potential for using lipid 2 H/ 1 H ratios to decipher the metabolism of uncultured microorganisms in both modern and ancient ecosystems. But despite over a decade of research, the precise mechanisms underlying this isotopic variability remain unclear. Differences in the kinetic isotope effects (KIEs) accompanying NADP + reduction by dehydrogenases and transhydrogenases have been hypothesized as a plausible mechanism. However, this relationship has been difficult to prove because multiple oxidoreductases affect the NADPH pool simultaneously. Here, we cultured five diverse aerobic heterotrophs, plus five Escherichia coli mutants, and used metabolic flux analysis to show that 2 H/ 1 H fractionations are highly correlated with fluxes through NADP + -reducing and NADPH-balancing reactions. Mass-balance calculations indicate that the full range of 2 H/ 1 H variability in the investigated organisms can be quantitatively explained by varying fluxes, i.e., with constant KIEs for each involved oxidoreductase across all species. This proves that lipid 2 H/ 1 H ratios of heterotrophic microbes are quantitatively related to central metabolism and provides a foundation for interpreting 2 H/ 1 H ratios of environmental lipids and sedimentary hydrocarbons.