Isotope Tracer Experiments Research Articles

Indole-3-acetic acid synthesized through the indole-3-pyruvate pathway promotes Candida tropicalis biofilm formation.

We previously found that the elevated abundance of the fungus Candida tropicalis is positively correlated with the bacteria Escherichia coli and Serratia marcescens in Crohn's disease patients and the three pathogens, when co-cultured, form a robust mixed-species biofilm. The finding suggests that these three pathogens communicate and promote biofilm formation, possibly through secretion of small signaling molecules. To identify candidate signaling molecules, we carried out a metabolomic analysis of the single-species and triple-species cultures of the three pathogens. This analysis identified 15 metabolites that were highly increased in the triple-species culture. One highly induced metabolite was indole-3-acetic acid (IAA), which has been shown to induce filamentation of certain fungi. We thus tested the effect of IAA on biofilm formation of C. tropicalis and demonstrated that IAA promotes biofilm formation of C. tropicalis. Then, we carried out isotope tracing experiments using 13C-labeled-tryptophan as a precursor to uncover the biosynthesis pathway of IAA in C. tropicalis. The results indicated that C. tropicalis synthesizes IAA through the indole-3-pyruvate pathway. Further studies using inhibitors of the indole-3-pyruvate pathway are warranted to decipher the mechanisms by which IAA influences biofilm formation.

Release of significant fraction of primary production as dissolved organic carbon in the Bay of Bengal

Despite nutrients supply through river discharge, atmospheric deposition and eddy-driven mixing, low primary production in the Bay of Bengal (BoB) is attributed to light limitation and non-availability of nutrients during summer monsoon. We report here the first ever measurements of release of significant fraction of primary production as dissolved organic carbon (DOC) in the BoB through isotopic tracer experiments. Our results suggested that about half of the primary production is released as DOC due to existence of oligotrophic conditions, warm waters and dominance of picophytankton biomass during the summer monsoon. The mean Percentage of Extracellular Release (PER) ranged between 30 and 60% (mean of 45 ± 8%) suggesting that primary production through particulate organic matter measurements were significantly underestimated total primary production in the BoB. Higher DOC release compared to particulate organic carbon (POC) production was noticed in the cyclonic eddy (CE) regions, where high nutrients were found, than anticyclonic eddy (ACE) region, where balanced POC production and DOC release observed associating with warm waters and nutrient deplete conditions. This study suggests that primary production, including the DOC release, is not low than hitherto hypothesized due to lack of measurements on DOC and this study explains the paradox between low production and high sinking carbon fluxes to the deep BoB and underestimation of sequestration of atmospheric carbon dioxide through biological pump in the BoB.

Natural organic matter inhibits Ni stabilization during Fe(II)-catalyzed ferrihydrite transformation

Trace metals, such as nickel (Ni), are often found associated with ferrihydrite (Fh) in soil and sediment and have been shown to redistribute during Fe(II)-catalyzed transformation of Fh. Fe(II)-catalyzed transformation of Fh, however, is often inhibited when natural organic matter (NOM) is associated with Fh. To explore whether NOM affects the behavior of Ni during Fe(II)-catalyzed transformation of Fh, we tracked Ni distribution, Fe atom exchange, and mineral transformation of Fh and Fh coprecipitated with Suwannee River natural organic matter (SRNOM-Fh). As expected, in the absence of Fe(II), Fh and SRNOM-Fh did not transform to secondary Fe minerals after two weeks. We further observed little difference in Ni adsorption on SRNOM-Fh compared to Fh. In the presence of Fe(II), however, we found that Ni associated with SRNOM-Fh was more susceptible to acid extraction than Fh. Specifically, we found almost double the amount of Ni remaining in the Fh after mild extraction compared to SRNOM-Fh. XRD showed that Fh transformed to goethite and magnetite whereas SRNOM-Fh did not transform despite 57Fe isotope tracer experiments confirmed that SRNOM-Fh underwent extensive atom exchange with aqueous Fe(II). Our findings suggest that Fe atom exchange may not be sufficient for obvious Ni stabilization and that transformation to secondary minerals may be necessary for Ni stabilization to occur.

Fe(II)-induced transformation of iron minerals in soil ferromanganese nodules

Recrystallization of iron (hydr)oxides by ferrous iron [Fe(II)] is an important part of the biogeochemical cycling of iron in earth surface environments. Ferromanganese nodules are complex assemblages of natural Fe and Mn minerals, which are widely found in soils and marine environments, and critically impact the geochemical behavior of trace metals and nutrients. However, little is known regarding the Fe(II)-induced recrystallization of ferromanganese nodules, in comparison to pure iron (hydr)oxides. Accordingly, the objective of this study was to elucidate the reaction processes of aqueous Fe(II) with ferromanganese nodules, and to use complementary spectroscopic techniques to characterize the products. Goethite in the nodule that underwent phase transformation to magnetite or Fe(II) oxidation to form magnetite has not been reported in previous studies with respect to pure goethite. 57Fe-enriched isotope tracer experiments confirmed Fe atom exchange between aqueous Fe(II) and structural Fe(III) of the nodule and was enhanced at higher pH conditions. The coexistence of Mn oxides and other minerals possibly make the goethite in the nodule more reactive for phase transformation to magnetite than pure synthetic goethite. In addition, Fe(III) precipitates were formed due to Fe(II) oxidation by Mn(III) of the nodule, and magnetite was likely consequently produced through further Fe(II)-induced reaction. Considering the enrichment of trace metals and nutrients in ferromanganese nodules, the observed Fe(II)-induced recrystallization of nodules in the study is expected to exert important effects on the geochemical behavior of the elements, especially in nodule-enriched soils.

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae).

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm-isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)-like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria-colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high-affinity sucrose-specific proton-dependent importer. Plasma-membrane/tonoplast localization of GeSUT4-GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant-microbe interactions.

Metabolite AutoPlotter - an application to process and visualise metabolite data in the web browser

BackgroundMetabolomics is gaining popularity as a standard tool for the investigation of biological systems. Yet, parsing metabolomics data in the absence of in-house computational scientists can be overwhelming and time-consuming. As a consequence of manual data processing, the results are often not analysed in full depth, so potential novel findings might get lost.MethodsTo tackle this problem, we developed Metabolite AutoPlotter, a tool to process and visualise quantified metabolite data. Other than with bulk data visualisations, such as heat maps, the aim of the tool is to generate single plots for each metabolite. For this purpose, it reads as input pre-processed metabolite-intensity tables and accepts different experimental designs, with respect to the number of metabolites, conditions and replicates. The code was written in the R-scripting language and wrapped into a shiny application that can be run online in a web browser on https://mpietzke.shinyapps.io/autoplotter.ResultsWe demonstrate the main features and the ease of use with two different metabolite datasets, for quantitative experiments and for stable isotope tracing experiments. We show how the plots generated by the tool can be interactively modified with respect to plot type, colours, text labels and the shown statistics. We also demonstrate the application towards 13C-tracing experiments and the seamless integration of natural abundance correction, which facilitates the better interpretation of stable isotope tracing experiments. The output of the tool is a zip-file containing one single plot for each metabolite as well as restructured tables that can be used for further analysis.ConclusionWith the help of Metabolite AutoPlotter, it is now possible to simplify data processing and visualisation for a wide audience. High-quality plots from complex data can be generated in a short time by pressing a few buttons. This offers dramatic improvements over manual analysis. It is significantly faster and allows researchers to spend more time interpreting the results or to perform follow-up experiments. Further, this eliminates potential copy-and-paste errors or tedious repetitions when things need to be changed. We are sure that this tool will help to improve and speed up scientific discoveries.

Removal Capacities and Environmental Constrains of Denitrification and Anammox Processes in Eutrophic Riverine Sediments

Excessive nitrogen (N) loading has had severe consequences in coastal zones around the world. Denitrification and anammox are major microbial pathways for removing N in aquatic environments before it is exported to the coast. To assess two processes in eutrophic riverine systems, the denitrification and anammox and their bacterial participants were investigated in sediments of the Xiaoqing (XQ) River and Jiaolai (JL) River in Northeast China. By combining the evidence from N15 isotope tracing experiment and functional gene-based analysis, it was found that denitrification and anammox are ubiquitous along the investigated riverine sediments. The denitrification varied from 39.38 to 1433.01 nmol N2 m−2 h−1. Moreover, the anammox rates were in the range of 15.91 to 1209.97 nmol N2 m−2 h−1. Quantitative PCR results revealed that the nirK and nirS genes were in the order of 104–106 copies g−1 and 103–105 copies g−1, respectively, in both river sediments, while the hzsA was in the order of 106–105 copies g−1 in XQ at approximately two orders of magnitude compared with JL. The phylogenetic analysis of functional genes revealed the high diversity of the denitrifier and low diversity of anammox bacteria. Variance partitioning analyses verified that the grain particle characteristics were the major factor group determined the N removal efficiency. The denitrification and anammox processes were estimated to have removed 16.1% of the inorganic nitrogen inputs before being exported to Laizhou Bay, which highlights that a more extensive understanding of the regularity of the N removal processes is important in the technical remediation of eutrophication problems.

Community dynamics and activity of nirS-harboring denitrifiers in sediments of the Indus River Estuary

Denitrification is an important pathway for reactive nitrogen removal from aquatic ecosystems. In this study, the biodiversity, abundance, and activity of cytochrome cd1-type nitrate reductase gene (nirS)-harboring denitrifiers in the sediments of the Indus River Estuary were examined by molecular and isotope-tracing techniques. Results showed that the nirS-harboring denitrifier communities showed significant geographical variations along the estuarine salinity gradient. Real-time quantitative PCR showed that the abundance of nirS-harboring denitrifiers ranged from 5.3 × 106 to 2.5 × 108 copies g−1, without significant spatiotemporal variation. The potential rates of denitrification varied from 0.01 to 6.27 μmol N kg−1 h−1 and correlated significantly to TOC and Fe(II) (P < 0.05). On the basis of 15N isotope-tracing experiments, the denitrification process contributed 18.4–99.4% to the total nitrogen loss in the sediments of the Indus River Estuary. This study provides novel insights into the microbial mechanism of nitrogen removal process in estuarine ecosystems.

Survey of dissimilatory nitrate reduction to ammonium microbial community at national wetland of Shanghai, China

Dissimilatory nitrate reduction to ammonia (DNRA) process is an important nitrate reduction pathway in the environment. Numerous studies focused on the DNRA, especially in various natural habitats. However, little is known about the envrionmental parameters driving the DNRA process in anthropogenic ecosystem. Human activities put forward significant influence on nitrogen cycle and bacterial communities of sediment. This study aimed to assess the DNRA potential rates, nrfA gene abundance, DNRA bacterial community’s diversity and influencing factors in a national wetland park near the Yangtze River estuary, Shanghai. The results of 15N isotope tracer experiments showed that DNRA potential rates from 0.13 to 0.44 μmol N/kg/h and contribution of nitrate reduction varied from 1.56% to 7.47%. The quantitative real-time PCR results showed that DNRA functional gene nrfA abundances ranged from 9.87E+10 to 1.98E+11 copies/g dry weight. The results of nrfA gene pyrosequencing analysis showed that Lacunisphaera (10.4–13.4%), Sorangium (7.1–10.7%), Aeromonas (4.2–6.8%), Corallococcus (1.8–6.9%), and Geobacter (3.3–6.6%) showed higher relative abundances in their genus levels. Combined with environmental parameters of sediments, redundancy analysis indicated that the nrfA functional gene was positively correlated with moisture content, the concentration of NO2−-N and NO3−N; the DNRA rates was positively correlated with sediment organic carbon (SOC), C/NO3− ratio and salinity (ranked by explains %). This study is the first simultaneous determination of nitrate reduction pathways including denitrification, anammox and DNRA rates to assess the role of DNRA in a national wetland park and revealed the community abundance, diversity of DNRA bacteria and its relationship with environmental factors.

Chain Transfer to Toluene in Styrene Coordination Polymerization

AbstractThe chain‐transfer reaction is rather important in coordination polymerization regarding catalytic efficiency, adjustment of molecular weight, and control of chain structure. To date, chain transfer to H2 and Al, Mg, and Zn alkyl compounds and β‐H elimination are the commonly encountered modes. Now a novel chain transfer to toluene is reported. By introducing fluorine atoms into the β‐diketimine ligands, an inert catalytic system for styrene (St) coordination polymerization was transferred into the highly active one. The activity increased with an increase in the number of fluorines in the ligands. Surprisingly, the molecular weights of resultant polystyrenes are very low (Mn=2000–6600 Da) despite of St loadings, corresponding up to 121 chains per active species. The mechanisms were investigated by DFT simulation, MALDI‐TOF MS, isotope tracing experiment and 2D NMR spectrum analyses, which revealed that the fluorine activated the polymerization and directed chain transfer to toluene.

Chain Transfer to Toluene in Styrene Coordination Polymerization.

The chain-transfer reaction is rather important in coordination polymerization regarding catalytic efficiency, adjustment of molecular weight, and control of chain structure. To date, chain transfer to H2 and Al, Mg, and Zn alkyl compounds and β-H elimination are the commonly encountered modes. Now a novel chain transfer to toluene is reported. By introducing fluorine atoms into the β-diketimine ligands, an inert catalytic system for styrene (St) coordination polymerization was transferred into the highly active one. The activity increased with an increase in the number of fluorines in the ligands. Surprisingly, the molecular weights of resultant polystyrenes are very low (Mn =2000-6600 Da) despite of St loadings, corresponding up to 121 chains per active species. The mechanisms were investigated by DFT simulation, MALDI-TOF MS, isotope tracing experiment and 2D NMR spectrum analyses, which revealed that the fluorine activated the polymerization and directed chain transfer to toluene.

P165 INFLUENCE OF THE GUT MICROBIOTA ON COLONIC HISTONE MODIFICATIONS THROUGH BUTYRATE METABOLISM UNDER HEALTHY AND INFLAMMATORY CONDITIONS

Abstract Inflammatory bowel disease (IBD) is often associated with a disruption in the composition and activity of the gut microbiota, referred to as dysbiosis. Since the microbiota has the potential to interact with host epithelial cells through small molecules generated from microbial metabolism, knowledge of how inflammation alters the microbial metabolome and how epithelial cells react is important for a better understanding of how IBD develops and persists. Butyrate, a short chain fatty acid produced through fermentation of dietary polysaccharides, has long been known to inhibit histone deacetylases (HDACs), which represent one of the many types of enzymes responsible for the epigenetic control of gene expression through the post-translational modification of histone proteins. We and others have observed that colonic epithelial cells from germ-free mice have reduced levels of acetylation on histone H4, which appears to be distributed throughout the genome based on sequencing analysis. The decreased levels of H4 acetylation may stem from a lack of butyrate, and therefore uninhibited HDAC activity, in germ-free mice. However, since colonic epithelial cells utilize short chain fatty acids as an energy source, an alternative explanation is that the germ-free condition results in less oxidation of butyrate to acetyl-CoA, which is the donor substrate for histone acetylation reactions. Isotope tracing experiments, in which cultured cells were incubated with labeled butyrate, demonstrated that the acetyl groups of histones contained carbon derived from butyrate. We have also performed isotope tracing experiments in mice using labeled inulin, a plant polysaccharide that presumably undergoes fermentation into short chain fatty acids. In this more physiologically relevant model, we detected isotope incorporation into the acetylated histones of colonic epithelial cells at rates of 5–20%, which appears dependent on the microbiota since labeling is sensitive to antibiotic treatment. To identify the metabolic pathways that link inulin to histone acetylation, we are investigating which metabolites become isotopically labeled using untargeted metabolomics. We will apply the same approach to the DSS-induced model of colitis to investigate how inflammation modulates the gut metabolome as well as the metabolic connections between the microbiota and the host. Our studies may uncover metabolic pathways that become dysregulated during inflammation, which may contribute to the pathogenesis of diseases such as IBD.

P165 INFLUENCE OF THE GUT MICROBIOTA ON COLONIC HISTONE MODIFICATIONS THROUGH BUTYRATE METABOLISM UNDER HEALTHY AND INFLAMMATORY CONDITIONS

Inflammatory bowel disease (IBD) is often associated with a disruption in the composition and activity of the gut microbiota, referred to as dysbiosis. Since the microbiota has the potential to interact with host epithelial cells through small molecules generated from microbial metabolism, knowledge of how inflammation alters the microbial metabolome and how epithelial cells react is important for a better understanding of how IBD develops and persists. Butyrate, a short chain fatty acid produced through fermentation of dietary polysaccharides, has long been known to inhibit histone deacetylases (HDACs), which represent one of the many types of enzymes responsible for the epigenetic control of gene expression through the post-translational modification of histone proteins. We and others have observed that colonic epithelial cells from germ-free mice have reduced levels of acetylation on histone H4, which appears to be distributed throughout the genome based on sequencing analysis. The decreased levels of H4 acetylation may stem from a lack of butyrate, and therefore uninhibited HDAC activity, in germ-free mice. However, since colonic epithelial cells utilize short chain fatty acids as an energy source, an alternative explanation is that the germ-free condition results in less oxidation of butyrate to acetyl-CoA, which is the donor substrate for histone acetylation reactions. Isotope tracing experiments, in which cultured cells were incubated with labeled butyrate, demonstrated that the acetyl groups of histones contained carbon derived from butyrate. We have also performed isotope tracing experiments in mice using labeled inulin, a plant polysaccharide that presumably undergoes fermentation into short chain fatty acids. In this more physiologically relevant model, we detected isotope incorporation into the acetylated histones of colonic epithelial cells at rates of 5-20%, which appears dependent on the microbiota since labeling is sensitive to antibiotic treatment. To identify the metabolic pathways that link inulin to histone acetylation, we are investigating which metabolites become isotopically labeled using untargeted metabolomics. We will apply the same approach to the DSS-induced model of colitis to investigate how inflammation modulates the gut metabolome as well as the metabolic connections between the microbiota and the host. Our studies may uncover metabolic pathways that become dysregulated during inflammation, which may contribute to the pathogenesis of diseases such as IBD.

Benthic carbon fixation and cycling in diffuse hydrothermal and background sediments in the Bransfield Strait, Antarctica

Abstract. Sedimented hydrothermal vents are likely to be widespread compared to hard substrate hot vents. They host chemosynthetic microbial communities which fix inorganic carbon (C) at the seafloor, as well as a wide range of macroinfauna, including vent-obligate and background non-vent taxa. There are no previous direct observations of carbon cycling at a sedimented hydrothermal vent. We conducted 13C isotope tracing experiments at three sedimented sites in the Bransfield Strait, Antarctica, which showed different degrees of hydrothermalism. Two experimental treatments were applied, with 13C added as either algal detritus (photosynthetic C), or as bicarbonate (substrate for benthic C fixation). Algal 13C was taken up by both bacteria and metazoan macrofaunal, but its dominant fate was respiration, as observed at deeper and more food-limited sites elsewhere. Rates of 13C uptake and respiration suggested that the diffuse hydrothermal site was not the hot spot of benthic C cycling that we hypothesised it would be. Fixation of inorganic C into bacterial biomass was observed at all sites, and was measurable at two out of three sites. At all sites, newly fixed C was transferred to metazoan macrofauna. Fixation rates were relatively low compared with similar experiments elsewhere; thus, C fixed at the seafloor was a minor C source for the benthic ecosystem. However, as the greatest amount of benthic C fixation occurred at the “Off Vent” (non-hydrothermal) site (0.077±0.034 mg C m−2 fixed during 60 h), we suggest that benthic fixation of inorganic C is more widespread than previously thought, and warrants further study.

Labile Fe(III) from sorbed Fe(II) oxidation is the key intermediate in Fe(II)-catalyzed ferrihydrite transformation

Ferrihydrite (Fh) is a major Fe(III)-(oxyhydr)oxide nanomineral distinguished by its poor crystallinity and thermodynamic metastability. While it is well known that in suboxic conditions aqueous Fe(II) rapidly catalyzes Fh transformation to more stable crystalline Fe(III) phases such as lepidocrocite (Lp) and goethite (Gt), because of the low solubility of Fe(III) the mass transfer pathways enabling these rapid transformations have remained unclear for decades. Here, using a selective extractant, we isolated and quantified a critical labile Fe(III) species, one that is more reactive than Fe(III) in Fh, formed by the oxidation of aqueous Fe(II) on the Fh surface. Experiments that compared time-dependent concentrations of solid-associated Fe(II) and this labile Fe(III) against the kinetics of phase transformation showed that its accumulation is directly related to Lp/Gt formation in a manner consistent with the classical nucleation theory. 57Fe isotope tracer experiments confirm the oxidized Fe(II) origin of labile Fe(III). The transformation pathway as well as the accelerating effect of Fe(II) can now all be explained on a unified basis of the kinetics of Fe(III) olation and oxolation reactions necessary to nucleate and sustain growth of Lp/Gt products, rates of which are greatly accelerated by labile Fe(III).

Multiplexed Profiling and Data Processing Methods to Identify Temperature-Regulated Primary Metabolites Using Gas Chromatography Coupled to Mass Spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of plant material that was exposed to temperature stress. The method can be combined with stable isotope labeling and tracing experiments and is equally applicable to preparations of plant material and microbial photosynthetic organisms. The described methods are modular and can be multiplexed, that is, the same sample or a paired identical backup sample can be analyzed sequentially by more than one of the described procedures. The modules include rapid sampling and metabolic inactivation protocols for samples in a wide weight range, sample extraction procedures, chemical derivatization steps that are required to make the metabolite fraction amenable to gas chromatographic analysis, routine GC-MS methods, and procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The methods have two applications: (1) The rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification by exact quantification using GC-MS protocols that are extended by internal and external standardization.

Productivity and carbon fluxes depend on species and symbiont density in soft coral symbioses

Soft corals often constitute one of the major benthic groups of coral reefs. Although they have been documented to outcompete reef-building corals following environmental disturbances, their physiological performance and thus their functional importance in reefs are still poorly understood. In particular, the acclimatization to depth of soft corals harboring dinoflagellate symbionts and the metabolic interactions between these two partners have received little attention. We performed stable isotope tracer experiments on two soft coral species (Litophyton sp. and Rhytisma fulvum fulvum) from shallow and upper mesophotic Red Sea coral reefs to quantify the acquisition and allocation of autotrophic carbon within the symbiotic association. Carbon acquisition and respiration measurements distinguish Litophyton sp. as mainly autotrophic and Rhytisma fulvum fulvum as rather heterotrophic species. In both species, carbon acquisition was constant at the two investigated depths. This is a major difference from scleractinian corals, whose carbon acquisition decreases with depth. In addition, carbon acquisition and photosynthate translocation to the host decreased with an increase in symbiont density, suggesting that nutrient provision to octocoral symbionts can quickly become a limiting factor of their productivity. These findings improve our understanding of the biology of soft corals at the organism-scale and further highlight the need to investigate how their nutrition will be affected under changing environmental conditions.

An Alkylpyrazine Synthesis Mechanism Involving l-Threonine-3-Dehydrogenase Describes the Production of 2,5-Dimethylpyrazine and 2,3,5-Trimethylpyrazine by Bacillus subtilis.

Alkylpyrazines are important contributors to the flavor of traditional fermented foods. Here, we studied the synthesis mechanisms of 2,5-dimethylpyrazine (2,5-DMP) and 2,3,5-trimethylpyrazine (TMP). Substrate addition, whole-cell catalysis, stable isotope tracing experiments, and gene manipulation revealed that l-threonine is the starting point involving l-threonine-3-dehydrogenase (TDH) and three uncatalyzed reactions to form 2,5-DMP. TDH catalyzes the oxidation of l-threonine. The product of this reaction is l-2-amino-acetoacetate, which is known to be unstable and can decarboxylate to form aminoacetone. It is proposed that aminoacetone spontaneously converts to 2,5-DMP in a pH-dependent reaction, via 3,6-dihydro-2,5-DMP. 2-Amino-3-ketobutyrate coenzyme A (CoA) ligase (KBL) catalyzes the cleavage of l-2-amino-acetoacetate, the product of TDH, into glycine and acetyl-CoA in the presence of CoA. Inactivation of KBL could improve the production of 2,5-DMP. Besides 2,5-DMP, TMP can also be generated by Bacillus subtilis 168 by using l-threonine and d-glucose as the substrates and TDH as the catalytic enzyme.IMPORTANCE Despite alkylpyrazines' contribution to flavor and their commercial value, the synthesis mechanisms of alkylpyrazines by microorganisms remain poorly understood. This study revealed the substrate, intermediates, and related enzymes for the synthesis of 2,5-dimethylpyrazine (2,5-DMP), which differ from the previous reports about the synthesis of 2,3,5,6-tetramethylpyrazine (TTMP). The synthesis mechanism described here can also explain the production of 2,3,5-trimethylpyrazine (TMP). The results provide insights into an alkylpyrazine's synthesis pathway involving l-threonine-3-dehydrogenase as the catalytic enzyme and l-threonine as the substrate.

Insights into the role of cation vacancy for significantly enhanced electrochemical nitrogen reduction

Electrochemical nitrogen reduction reaction (NRR) at ambient conditions offers a promising picture for renewable production of ammonia (NH3), but its practical implementation is strictly restricted by lacking efficient catalysts. Defect engineering as an effective strategy has been applied to promote the electrochemical NRR activity. Unfortunately, in contrast to anion vacancy, the study of cation vacancy for insight into the relationship between the activity and structure of NRR electrocatalysts is still very scarce. Herein, we designed a superior cation-vacancy NRR electrocatalyst by fabrication of MoN nanocrystals with abundant Mo vacancies embedded in N-doped hierarchical porous carbon framework for the first time. Compared with the pre-reported anion-vacancy electrocatalysts, it exhibits an impressive improvement for NRR activity (NH3 yield: 76.9 μg h−1 mg−1cat.). We identified Mars-van Krevelen pathway during NRR process by joint 15N2 isotopic tracer experiments with nuclear magnetic resonance spectroscopy. First-principles calculations revealed the critical role of Mo vacancy in regulating the electronic properties of MoN and shifting the rate-limiting step of NRR that significantly reduces the reaction barrier. Our findings of creating cation defects in nitrides to catalyze N2 fixation pave a new avenue for synthesizing realistic and high-efficiency NRR electrocatalysts.

A method for Si isotope tracer kinetics experiments: Using Q-ICP-MS to obtain 29Si/28Si ratios in aqueous solutions

Here we show that silicon isotope tracer kinetics experiments can be economically conducted with measurements of 29Si/28Si ratios in the experimental aqueous solutions using Quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS) with sufficient precision. Although the isotope tracer method promises orders of magnitude improved detection sensitivity of reactions, its application has been hampered by the limited availability of multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) for Si isotope analysis. However, in Si isotope tracer kinetics experiments, dissolution rates are often derived from isotope fractional abundance increments by as much as 0.5 unit (equivalent to 50%) and the method essentially relies on the difference in isotope ratios over a period of time, which render uncertainties in < ±0.01 fraction (or ±1%) in Si isotope abundances tolerable. Laboratory-measured silicate dissolution rates typically carry uncertainties of more than ±25% and with even poorer intra- and inter-laboratory replications. These special situations of kinetics experiments circumvent the conventional concerns of using Q-ICP-MS to measure isotope ratios.To test this hypothesis, we conducted two experiments. The first used Q-ICP-MS to measure a set of mixing solutions or reference solutions with known isotope compositions. The results show that the difference is an average of ±0.009 in f29. A second test used both MC-ICP-MS and Q-ICP-MS to measure the 29Si/28Si ratios in experimental solutions of albite dissolution. Ten albite dissolution experiments were conducted at 50°C and pH 8.8. Student t-test showed that the rates calculated from the two analytical data sets have no statistically significant differences at the 95% confidence level. These results demonstrate that the Q-ICP-MS measurements of 29Si/28Si ratios produced tolerable uncertainties for determining silicate dissolution rates. However, the cost is significantly reduced and more significantly, the reduced wait time makes isotope tracer experiments more feasible. Silicate minerals make up 90% of the Earth’s crust. The kinetics of silicate-water reactions is of paramount significance to many society’s mega-environmental enterprises. This method to conduct Si isotope tracer experiments, as well as the possibility to use this approach for other non-traditional stable isotopes, will have significant impact on geochemical kinetics studies.