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Rationale Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling. Methods We used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases. Results All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27–0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41–0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing a more complete picture of the reactions catalyzed by hydrogenases. Conclusions The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities. Copyright © 2015 John Wiley & Sons, Ltd.

Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling.We used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases.All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27–0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41–0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing a more complete picture of the reactions catalyzed by hydrogenases.The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities.

Acid scavengers are frequently used as stabilizer compounds in a variety of applications. When used to stabilize volatile compounds such as nerve agents, the lower volatility and higher stability of acid scavengers make them more persistent in a post-event forensic setting. We are employing compound-specific stable isotope analysis of the carbon, nitrogen, and hydrogen components of three acid scavenging compounds (N,N-diethylaniline, tributylamine, and triethylamine) as a tool for distinguishing between different samples of the stabilizers. Combined analysis of three stable isotopes in these samples improves the technique’s resolving potential, enhancing sample matching capabilities. The compound specific methods developed here can be applied to instances where these compounds are not pure, such as when mixed with an agent or when found as a residue at an event site. Effective sample matching can be crucial for linking compounds at multiple event sites or linking a supply inventory to an event.

This paper presents design, fabrication, and testing of a microscale ceramic reactor for the pyrolysis of organic compounds. One application for this pyrolysis reactor is to convert the oxygen and hydrogen atoms in organic compounds to CO and H2 for isotope ratio measurements in a continuous flow mode. Existing commercial pyrolyzers use high carrier gas flow rates (typically 80−100 mL/min) such that >95% of the CO and H2 produced from a given sample is vented before introduction into the mass spectrometer. We describe here the fabrication and testing of a microscale pyrolysis reactor designed to be compatible with existing isotope ratio mass spectrometers. The microreactor uses carrier gas flow rates of 3−5 mL/min, decreasing the proportion of the CO and H2 lost in venting and permitting analysis of samples 20−50 times smaller than can be analyzed with conventional pyrolysis reactors. Results have shown that organic compounds, such as 1-butanol, ethanol, and ethanolamine, can be fully decomposed to desire...

Stable isotope ratios of hydrogen and oxygen in microbes have been shown to be functions of the corresponding isotope ratios of the water with which the culture medium was prepared, and thus to contain a potential geographic signal. Water can evaporate from agar (solid) media during culturing, changing its isotope ratios. Here we describe the effect of drying on the isotope ratios of water extracted from agar media and the H and O stable isotope ratios ratios of Bacillus subtilis spores cultured on agar. The δ 2 Hv sδ 18 O relationship of water in Petri dish agar was surprisingly constant during evaporation regardless of the ambient relative humidity, making it possible to calculate the approximate isotope ratios of the original water, even in significantly evaporated agar. The H stable isotope ratios of spores cultured on agar remained relatively unchanged as the agar dried, but the O ratio became significantly enriched.

The stable isotope ratios of a seized pathogen culture could potentially reveal information about the environment in which the agent was produced. In this paper we describe general relationships between stable isotopes of carbon, nitrogen, and hydrogen in bacteriological culture media and spores of Bacillus subtilis, an endospore-forming soil bacterium. In numerous media that varied both in nutrient composition and water stable isotope ratios, medium to spore enrichment in carbon isotopes was 0.3 ± 2.0‰ (parts per thousand), and in nitrogen, 4.5 ± 0.7‰. We achieved mass balance for the contribution of hydrogen isotopes from nutrients (70%) and water (30%) to spores in independent experiments by varying the isotope ratios of nutrients or water. A model was derived for predicting the isotope ratio values of spores from those in nutrients and water.

We successfully tested the viability of using synchrotron-based full-field infrared imaging to study biochemical processes inside living cells. As a model system, we studied fibroblast cells exposed to a medium highly enriched with D2O. We could show that the experimental technique allows us to reproduce at the cellular level measurements that are normally performed on purified biological molecules. We can obtain information about lipid conformation and distribution, kinetics of hydrogen/deuterium exchange, and the formation of concentration gradients of H and O isotopes in water that are associated with cell metabolism. The implementation of the full field technique in a sequential imaging format gives a description of cellular biochemistry and biophysics that contains both spatial and temporal information., Biophysical Chemistry, 189, ISSN:0301-4622, ISSN:1873-4200

Shewanella oneidensis MR-1 encodes both a [NiFe]- and an [FeFe]-hydrogenase. While the output of these proteins has been characterized in mutant strains expressing only one of the enzymes, the contribution of each to H2 synthesis in the wild-type organism is not clear. Here, we use stable isotope analysis of H2 in the culture headspace, along with transcription data and measurements of the concentrations of gases in the headspace, to characterize H2 production in the wild-type strain. After most of the O2 in the headspace had been consumed, H2 was produced and then consumed by the bidirectional [NiFe]-hydrogenase. Once the cultures were completely anaerobic, a new burst of H2 synthesis catalyzed by both enzymes took place. Our data are consistent with the hypothesis that at this point in the culture cycle, a pool of electrons is shunted toward both hydrogenases in the wild-type organisms, but that in the absence of one of the hydrogenases, the flux is redirected to the available enzyme. To our knowledge, this is the first use of natural-abundance stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.

Hydrogenases catalyze the reversible formation of H(2), and they are key enzymes in the biological cycling of H(2). H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H(2) production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H(2) evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom-designed high-throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H(2). Using our new approach, we determined that the fractionation factor for H(2) production by the [NiFe]-hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H(2) evolution. Potential applications of this newly developed method are discussed.

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.

Our understanding of the δ2H and δ18O of plant water and tissues ranges from model descriptions of multiple fractionating processes and mechanistic drivers, to primarily observational relationships often described by simple regression relationships. Plant hydrogen and oxygen isoscapes are therefore produced with a range of model sophistications and demonstrate some of the diversity of approaches to producing and utilizing isoscapes in general. Leaf water is central to much of plant H & O isoscape modeling. It affects the atmosphere at large scales by influencing atmospheric CO2 and O2 δ18O, and the biosphere at a range of scales through its influence on the isotopic composition of plant organic compounds. Leaf water isoscapes have therefore been produced as part of efforts to understand atmospheric gas isotopic composition and also have the potential to contribute to other areas where spatial variation in plant-derived organic compound isotope ratios is of interest. For example, improving our understanding of the isotope ratios of cellulose or n-alkanes improves their utility for paleoreconstructions. Spatially-explicit models of food isotope ratios may improve the use of animal isotope ratios to infer migration patterns. Finally, plant hydrogen and oxygen isoscapes can yield forensic information for a variety of products, including food, drugs, or other plant-derived materials. Given this wide range of applications, we discuss approaches to producing plant H & O isoscapes that vary in their complexity depending on the level of mechanistic understanding, data availability, and the question being pursued.

It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.

Stable isotope ratios of hydrogen and oxygen in microbes have been shown to be functions of the corresponding isotope ratios of the water with which the culture medium was prepared, and thus to contain a potential geographic signal. Water can evaporate from agar (solid) media during culturing, changing its isotope ratios. Here we describe the effect of drying on the isotope ratios of water extracted from agar media and the H and O stable isotope ratios ratios of Bacillus subtilis spores cultured on agar. The delta2H vs delta18O relationship of water in Petri dish agar was surprisingly constant during evaporation regardless of the ambient relative humidity, making it possible to calculate the approximate isotope ratios of the original water, even in significantly evaporated agar. The H stable isotope ratios of spores cultured on agar remained relatively unchanged as the agar dried, but the O ratio became significantly enriched.

The stable isotope ratios of a seized pathogen culture could potentially reveal information about the environment in which the agent was produced. In this paper we describe general relationships between stable isotopes of carbon, nitrogen, and hydrogen in bacteriological culture media and spores of Bacillus subtilis, an endospore-forming soil bacterium. In numerous media that varied both in nutrient composition and water stable isotope ratios, medium to spore enrichment in carbon isotopes was 0.3 +/- 2.0% per thousand (parts per thousand), and in nitrogen, 4.5 +/- 0.7% per thousand. We achieved mass balance for the contribution of hydrogen isotopes from nutrients (70%) and water (30%) to spores in independent experiments by varying the isotope ratios of nutrients or water. A model was derived for predicting the isotope ratio values of spores from those in nutrients and water.

Since the anthrax attacks of 2001 the need for methods to trace the origins of microbial agents has become urgent. The stable isotope ratios of bacteria record information from both the nutrients and the water used to make their culture media and could potentially be used to provide information about their growth environment. We present a survey of carbon (C), nitrogen (N), and hydrogen (H) stable isotope ratios in 516 samples of bacteriological culture media. The observed variation was consistent with expected isotopic variation in the plant and animal products upon which the media are based. The variation is sufficient to translate into substantial isotope variation in cultures grown on different batches of media, and thus to allow investigators to determine whether seized media could have been used to produce seized bioweapons agents.

Bacillus subtilis , a Gram-positive, endospore-forming soil bacterium, was grown in media made with water of varying oxygen (δ 18 O) and hydrogen (δD) stable isotope ratios. Logarithmically growing cells and spores were each harvested from the cultures and their δ 18 O and δD values determined. Oxygen and hydrogen stable isotope ratios of organic matter were linearly related with those of the media water. We used the relationships determined in these experiments to calculate the effective whole-cell fractionation factors between water and organic matter for B. subtilis . We then predicted the δ 18 O and δD values of spores produced in nutritionally identical media and local water sources for five different locations around the United States. Each of the measured δ 18 O and δD values of the spores matched the predicted values within a 95% confidence interval, indicating that stable isotope ratio analyses may be a powerful tool for tracing the geographic point-of-origin for microbial products.