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Hydrogen isotope ratios (δ 2 H) represent an important natural tracer of metabolic processes, but quantitative models of processes controlling H-fractionation in aquatic photosynthetic organisms are lacking. Here, we elucidate the underlying physiological controls of 2 H/ 1 H fractionation in algal lipids by systematically manipulating temperature, light, and CO 2 (aq) in continuous cultures of the haptophyte Gephyrocapsa oceanica . We analyze the hydrogen isotope fractionation in alkenones (α alkenone ), a class of acyl lipids specific to this species and other haptophyte algae. We find a strong decrease in the α alkenone with increasing CO 2 (aq) and confirm α alkenone correlates with temperature and light. Based on the known biosynthesis pathways, we develop a cellular model of the δ 2 H of algal acyl lipids to evaluate processes contributing to these controls on fractionation. Simulations show that longer residence times of NADPH in the chloroplast favor a greater exchange of NADPH with 2 H-richer intracellular water, increasing α alkenone . Higher chloroplast CO 2 (aq) and temperature shorten NADPH residence time by enhancing the carbon fixation and lipid synthesis rates. The inverse correlation of α alkenone to CO 2 (aq) in our cultures suggests that carbon concentrating mechanisms (CCM) do not achieve a constant saturation of CO 2 at the Rubisco site, but rather that chloroplast CO 2 varies with external CO 2 (aq). The pervasive inverse correlation of α alkenone with CO 2 (aq) in the modern and preindustrial ocean also suggests that natural populations may not attain a constant saturation of Rubisco with the CCM. Rather than reconstructing growth water, α alkenone may be a powerful tool to elucidate the carbon limitation of photosynthesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

The hydrogen isotope ratios (δ 2 H AA values) of amino acids in all organisms are substantially fractionated relative to growth water. In addition, they exhibit large variations within microbial biomass, animals, and human tissues, hinting at rich biochemical information encoded in such signals. In lipids, such δ 2 H variations are thought to primarily reflect NADPH metabolism. Analogous biochemical controls for amino acids remain largely unknown, but must be elucidated to inform the interpretation of these measurements. Here, we measured the δ 2 H values of amino acids from five aerobic, heterotrophic microbes grown on different carbon substrates, as well as five Escherichia coli mutant organisms with perturbed NADPH metabolisms. We observed similar δ 2 H AA patterns across all organisms and growth conditions, which-consistent with previous hypotheses-suggests a first-order control by biosynthetic pathways. Moreover, δ 2 H AA values varied systematically with the catabolic pathways activated for substrate degradation, with variations explainable by the isotopic compositions of important cellular metabolites, including pyruvate and NADPH, during growth on each substrate. As such, amino acid δ 2 H values may be useful for interrogating organismal physiology and metabolism in the environment, provided we can further elucidate the mechanisms underpinning these signals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Silverman, Wijker and Sessions.)

Mono Lake is a closed-basin, hypersaline, alkaline lake located in Eastern Sierra Nevada, California, that is dominated by microbial life. This unique ecosystem offers a natural laboratory for probing microbial community responses to environmental change. In 2017, a heavy snowpack and subsequent runoff led Mono Lake to transition from annually mixed (monomictic) to indefinitely stratified (meromictic). We followed microbial succession during this limnological shift, establishing a two-year (2017-2018) water-column time series of geochemical and microbiological data. Following meromictic conditions, anoxia persisted below the chemocline and reduced compounds such as sulfide and ammonium increased in concentration from near 0 to ~400 and ~150 µM, respectively, throughout 2018. We observed significant microbial succession, with trends varying by water depth. In the epilimnion (above the chemocline), aerobic heterotrophs were displaced by phototrophic genera when a large bloom of cyanobacteria appeared in fall 2018. Bacteria in the hypolimnion (below the chemocline) had a delayed, but systematic, response reflecting colonization by sediment "seed bank" communities. Phototrophic sulfide-oxidizing bacteria appeared first in summer 2017, followed by microbes associated with anaerobic fermentation in spring 2018, and eventually sulfate-reducing taxa by fall 2018. This slow shift indicated that multi-year meromixis was required to establish a sulfate-reducing community in Mono Lake, although sulfide oxidizers thrive throughout mixing regimes. The abundant green alga Picocystis remained the dominant primary producer during the meromixis event, abundant throughout the water column including in the hypolimnion despite the absence of light and prevalence of sulfide. Our study adds to the growing literature describing microbial resistance and resilience during lake mixing events related to climatic events and environmental change., (© 2021 The Authors. Geobiology published by John Wiley & Sons Ltd.)

Non-invasively probing metabolites within single live cells is highly desired but challenging. Here we utilize Raman spectro-microscopy for spatial mapping of metabolites within single cells, with the specific goal of identifying druggable metabolic susceptibilities from a series of patient-derived melanoma cell lines. Each cell line represents a different characteristic level of cancer cell de-differentiation. First, with Raman spectroscopy, followed by stimulated Raman scattering (SRS) microscopy and transcriptomics analysis, we identify the fatty acid synthesis pathway as a druggable susceptibility for differentiated melanocytic cells. We then utilize hyperspectral-SRS imaging of intracellular lipid droplets to identify a previously unknown susceptibility of lipid mono-unsaturation within de-differentiated mesenchymal cells with innate resistance to BRAF inhibition. Drugging this target leads to cellular apoptosis accompanied by the formation of phase-separated intracellular membrane domains. The integration of subcellular Raman spectro-microscopy with lipidomics and transcriptomics suggests possible lipid regulatory mechanisms underlying this pharmacological treatment. Our method should provide a general approach in spatially-resolved single cell metabolomics studies.

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., Competing Interests: The authors declare no conflict of interest.

Quantifying the extent of biodegradation of nitroaromatic compounds (NACs) in contaminated soils and sediments is challenging because of competing oxidative and reductive reaction pathways. We have previously shown that the stable isotope fractionation of NACs reveals the routes of degradation even if it is simultaneously caused by different bacteria. However, it is unclear whether compound-specific isotope analysis (CSIA) can be applied in situations where multiple pollutants are biodegraded by only one microorganism under multi-substrate conditions. Here we examined the C and N isotope fractionation of 2-nitrophenol (2-NP) and 3-nitrophenol (3-NP) during biodegradation by Pseudomonas putida B2 through monooxygenation and partial reductive pathways, respectively, in the presence of single substrates vs. binary substrate mixtures. Laboratory experiments showed that the reduction of 3-NP by Pseudomonas putida B2 is associated with large N and minor C isotope fractionation with C and N isotope enrichment factors, ε C and ε N , of -0.3 ± 0.1‰ and -22 ± 0.2‰, respectively. The opposite isotope fractionation trends were found for 2-NP monooxygenation. In the simultaneous presence of 2-NP and 3-NP, 2-NP is biodegraded at identical rate constants and ε C and ε N values (-1.0 ± 0.1‰ and -1.3 ± 0.2‰) to those found for the monooxygenation of 2-NP in single substrate experiments. While the pathway and N isotope fractionation of 3-NP reduction (ε N = -24 ± 1.1‰) are independent of the presence of 2-NP, intermediates of 2-NP monooxygenation interfere with 3-NP reduction. Because neither pH, substrate uptake, nor aromatic substituents affected the kinetic isotope effects of nitrophenol biodegradation, our study illustrates that CSIA provides robust scientific evidence for the assessment of natural attenuation processes.

Assessing the pathways and rates of organic pollutant transformation in the environment is a major challenge due to co-occurring transport and degradation processes. Measuring changes of stable isotope ratios (e.g. (13)C/(12)C, (2)H/(1)H, (15)N/(14)N) in individual organic compounds by compound-specific isotope analysis (CSIA) makes it possible to identify degradation pathways without the explicit need to quantify pollutant concentration dynamics. The so-called isotope fractionation observed in an organic pollutant is related to isotope effects of (bio)chemical reactions and enables one to characterize pollutant degradation even if multiple processes take place simultaneously. Here, we illustrate some principles of CSIA using benzotriazole, a frequently observed aquatic micropollutant, as example. We show subsequently how the combined C and N isotope fractionation analysis of nitroaromatic compounds reveals kinetics and mechanisms of reductive and oxidative reactions as well as their (bio)degradation pathways in the environment.

Calculations of alternative oxidation pathways of toluene and its ortho-substituted nitro derivatives by permanganate anion have been performed. The competition between methyl group and ring oxidation has been addressed. Acceptable results have been obtained using IEFPCM/B3LYP/6-31+G(d,p) calculations with zero-point (ZPC) and thermal corrections, as validated by comparison with the experimental data. It has been shown that ring oxidation reactions proceed via relatively early transition states that become quite unsymmetrical for reactions involving ortho-nitrosubstituted derivatives. Transition states for the hydrogen atom abstraction reactions, on the other hand, are late. All favored reactions are characterized by the Gibbs free energy of activation, ΔG(≠), of about 25 kcal mol(-1). Methyl group oxidations are exothermic by about 20 kcal mol(-1) while ring oxidations are around thermoneutrality.

Monooxygenation is an important route of nitroaromatic compound (NAC) biodegradation and it is widely found for cometabolic transformations of NACs and other aromatic pollutants. We investigated the C and N isotope fractionation of nitrophenol monooxygenation to complement the characterization of NAC (bio)degradation pathways by compound-specific isotope analysis (CSIA). Because of the large diversity of enzymes catalyzing monooxygenations, we studied the combined C and N isotope fractionation and the corresponding (13)C- and (15)N-apparent kinetic isotope effects (AKIEs) of four nitrophenol-biodegrading microorganisms (Bacillus spharericus JS905, Pseudomonas sp. 1A, Arthrobacter sp. JS443, Pseudomonas putida B2) in the pH range 6.1-8.6 with resting cells and crude cell extracts. While the extent of C and N isotope fractionation and the AKIE-values varied considerably for the different organisms, the correlated C and N isotope signatures (δ(15)N vs δ(13)C) revealed trends, indicative of two distinct monooxygenation pathways involving hydroxy-1,4-benzoquinone or 1,2- and 1,4-benzoquinone intermediates, respectively. The distinction was possible based on larger secondary (15)N-AKIEs associated with the benzoquinone pathway. Isotope fractionation was neither masked substantially by nitrophenol speciation nor transport across cell membranes. Only when 4-nitrophenol was biodegraded by Pseudomonas sp. 1A did isotope fractionation become negligible, presumably due to rate-limiting substrate binding steps pertinent to the catalytic cycle of flavin-dependent monooxygenases.

Assessing the fate of nitroaromatic explosives in the subsurface is challenging because contaminants are present in different phases (e.g., bound to soil or sediment matrix or as solid-phase residues) and transformation takes place via several potentially competing pathways over time-scales of decades. We developed a procedure for compound-specific analysis of stable C, N, and H isotopes in nitroaromatic compounds (NACs) and characterized biodegradation of 2,4,6-trinitrotoluene (TNT) and two dinitrotoluene isomers (2,4-DNT and 2,6-DNT) in subsurface material of a contaminated site. The type and relative contribution of reductive and oxidative pathways to the degradation of the three contaminants was inferred from the combined evaluation of C, N, and H isotope fractionation. Indicative trends of Δδ(15)N vs Δδ(13)C and Δδ(2)H vs Δδ(13)C were obtained from laboratory model systems for biodegradation pathways initiated via (i) dioxygenation, (ii) reduction, and (iii) CH3-group oxidation. The combined evaluation of NAC isotope fractionation in subsurface materials and in laboratory experiments suggests that in the field, 86-89% of 2,4-DNT transformation was due to dioxygenation while TNT was mostly reduced and 2,6-DNT reacted via a combination of reduction and CH3-group oxidation. Based on historic information on site operation, our data imply biodegradation of 2,4-DNT with half-lives of up to 9-17 years compared to 18-34 years for cometabolic transformation of TNT and 2,6-DNT.

Oxidation of aromatic rings and its alkyl substituents are often competing initial steps of organic pollutant transformation. The use of compound-specific isotope analysis (CSIA) to distinguish between these two pathways quantitatively, however, can be hampered by large H isotope fractionation that precludes calculation of apparent (2)H-kinetic isotope effects (KIE) as well as the process identification in multi-element isotope fractionation analysis. Here, we investigated the C and H isotope fractionation associated with the transformation of toluene, nitrobenzene, and four substituted nitrotoluenes by permanganate, MnO4(-), to propose a refined evaluation procedure for the quantitative distinction of CH3-group oxidation and dioxygenation. On the basis of batch experiments, an isotopomer-specific kinetic model, and density functional theory (DFT) calculations, we successfully derived the large apparent (2)H-KIE of 4.033 ± 0.20 for the CH3-group oxidation of toluene from H isotope fractionation exceeding >1300‰ as well as the corresponding (13)C-KIE (1.0324 ± 0.0011). Experiment and theory also agreed well for the dioxygenation of nitrobenzene, which was associated with (2)H- and (13)C-KIEs of 0.9410 ± 0.0030 (0.9228 obtained by DFT) and 1.0289 ± 0.0003 (1.025). Consistent branching ratios for the competing CH3-group oxidation and dioxygenation of nitrotoluenes by MnO4(-) were obtained from the combined modeling of concentration as well as C and H isotope signature trends. Our approach offers improved estimates for the identification of contaminant microbial and abiotic oxidation pathways by CSIA.