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A quantitative model of nitrogen fixation in the presence of ammonium.

ABSTRACT: Nitrogen fixation provides bioavailable nitrogen, supporting global ecosystems and influencing global cycles of other elements. It provides an additional source of nitrogen to organisms at a cost of lower growth efficiency, largely due to respiratory control of intra-cellular oxygen. Nitrogen-fixing bacteria can, however, utilize both dinitrogen gas and fixed nitrogen, decreasing energetic costs. Here we present an idealized metabolic model of the heterotrophic nitrogen fixer Azotobacter vinelandii which, constrained by laboratory data, provides quantitative predictions for conditions under which the organism uses either ammonium or nitrogen fixation, or both, as a function of the relative supply rates of carbohydrate, fixed nitrogen as well as the ambient oxygen concentration. The model reveals that the organism respires carbohydrate in excess of energetic requirements even when nitrogen fixation is inhibited and respiratory protection is not essential. The use of multiple nitrogen source expands the potential niche and range for nitrogen fixation. The model provides a quantitative framework which can be employed in ecosystem and biogeochemistry models.

SUBMITTER: Inomura K

PROVIDER: S-EPMC6264846 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A quantitative model of nitrogen fixation in the presence of ammonium.

Inomura Keisuke K Bragg Jason J Riemann Lasse L Follows Michael J MJ

PloS one 20181129 11

Nitrogen fixation provides bioavailable nitrogen, supporting global ecosystems and influencing global cycles of other elements. It provides an additional source of nitrogen to organisms at a cost of lower growth efficiency, largely due to respiratory control of intra-cellular oxygen. Nitrogen-fixing bacteria can, however, utilize both dinitrogen gas and fixed nitrogen, decreasing energetic costs. Here we present an idealized metabolic model of the heterotrophic nitrogen fixer Azotobacter vinelan ...[more]

PMID: 30496286

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Project description:Diazotrophs can produce bioavailable nitrogen from inert N2 gas by bioelectrochemical nitrogen fixation (e-BNF), which is emerging as an energy-saving and highly selective strategy for agriculture and industry. However, current e-BNF technology is impeded by requirements for NH4 +-assimilation inhibitors to facilitate intracellular ammonia secretion and precious metal catalysts to generate H2 as the energy-carrying intermediate. Herein, we initially demonstrate inhibitor- and catalyst-less extracellular NH4 + production by the diazotroph Pseudomonas stutzeri A1501 using an electrode as the sole electron donor. Multiple lines of evidence revealed that P. stutzeri produced 2.32±0.25 mg/L of extracellular NH4 + at a poised potential of -0.3 V (vs. standard hydrogen electrode (SHE)) without the addition of inhibitors or expensive catalysts. The electron uptake mechanism was attributed to the endogenous electron shuttle phenazine-1-carboxylic acid, which was excreted by P. stutzeri and mediated electron transfer from electrodes into cells to directly drive N2 fixation. The faradaic efficiency was 20%±3% which was 2-4 times that of previous e-BNF using the H2-mediated pathway. This study reports a diazotroph capable of producing secretable NH4 + via extracellular electron uptake, which has important implications for optimizing the performance of e-BNF systems and exploring the novel nitrogen-fixing mode of syntrophic microbial communities in the natural environment.IMPORTANCE Ammonia greatly affects the global ecology, agriculture and the food industry. Diazotrophs with an enhanced capacity of extracellular NH4 + excretion have been proven to be more beneficial to the growth of microalgae and plants, whereas most previously reported diazotrophs produce intracellular organic nitrogen in the absence of chemical suppression and genetic manipulation. Here, we demonstrate that Pseudomonas stutzeri A1501 is capable of extracellular NH4 + production without chemical suppression or genetic manipulation when the extracellular electrode is used as the sole electron donor. We also reveal the electron uptake pathway from the extracellular electron-donating partner to P. stutzeri A1501 via redox electron shuttle phenazines. Since both P. stutzeri A1501 and potential electron-donating partners (such as electroactive microbes and natural semiconductor minerals) are abundant in diverse soils and sediments, P. stutzeri A1501 has broader implications on the improvement of nitrogen fertilization in the natural environment.

Project description:Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium.IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

Project description:BACKGROUND: Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. In many diazotrophs, addition of excess ammonium in the growth medium results in immediate repression of nif gene transcription. Although the regulatory cascades that control the transcription of the nif genes in proteobacteria have been well investigated, there are limited data on the kinetics of ammonium-dependent repression of nitrogen fixation. RESULTS: Here we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501, a root-associated and nitrogen-fixing bacterium. A total of 166 genes, including those coding for the global nitrogen regulation (Ntr) and Nif-specific regulatory proteins, were upregulated under nitrogen fixation conditions but rapidly downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in each of Azoarcus sp. BH72 and Azotobacter vinelandii AvoP. In particular, a 49-kb expression island containing nif and other associated genes was markedly downregulated by ammonium shock. Further functional characterization of pnfA, a new NifA-sigma54-dependent gene chromosomally linked to nifHDK, is reported. This gene encodes a protein product with an amino acid sequence similar to that of five hypothetical proteins found only in diazotrophic strains. No noticeable differences in the transcription of nifHDK were detected between the wild type strain and pnfA mutant. However, the mutant strain exhibited a significant decrease in nitrogenase activity under microaerobic conditions and lost its ability to use nitrate as a terminal electron acceptor for the support of nitrogen fixation under anaerobic conditions. CONCLUSIONS: Based on our results, we conclude that transcriptional regulation of nif gene expression in A1501 is mediated by the nif-specific and ntr gene regulatory systems. Furthermore, microarray and mutational analyses revealed that many genes of unknown function may play some essential roles in controlling the expression or activity of nitrogenase. The findings presented here establish the foundation for further studies on the physiological function of nitrogen fixation-inducible genes.

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A quantitative model of nitrogen fixation in the presence of ammonium.

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A quantitative model of nitrogen fixation in the presence of ammonium.

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