Diazotrophic macroalgal associations (DMAs) can contribute fixed nitrogen (N) to the host macroalgae. Biological nitrogen fixation (BNF) rates investigated using acetylene reduction assays with living macroalgae surrounding Santa Catalina Island were low (maximum: 36 nmol N × g−1(dw) × h−1) and probably insufficient towards helping meet macroalgal N demand. However, DMAs were observed during periods of low nitrate availability in Southern California coastal waters, highlighting the potential importance of diazotrophs during N depleted conditions. Eleven long‐term (16–32 days) litter bag decomposition experiments with various macroalgae, especially those with high (> 10) C:N ratios, resulted in much higher BNF rates (maximum: 693 nmol N × g−1(dw) × h−1) than observed with living macroalgae. BNF rates were lower at the beginning of macroalgal decomposition but rapidly increased during the second phase before declining towards the end of decomposition. Labile carbon availability is likely influencing BNF rates throughout macroalgal degradation and limits BNF in the final decomposition stage. Comparable dark and light BNF rates with most macroalgae surveyed suggest macroalgal detrital systems are an overlooked, potentially global, niche for heterotrophic N2fixation. Lastly, suppressed BNF rates with sodium molybdate additions highlight the prevalence of sulfate reducing diazotrophs.
Biological nitrogen fixation (BNF) by canonical molybdenum and complementary vanadium and iron-only nitrogenase isoforms is the primary natural source of newly fixed nitrogen. Understanding controls on global nitrogen cycling requires knowledge of the isoform responsible for environmental BNF. The isotopic acetylene reduction assay (ISARA), which measures carbon stable isotope (13C/12C) fractionation between ethylene and acetylene in acetylene reduction assays, is one of the few methods that can quantify isoform-specific BNF fluxes. Application of classical ISARA has been challenging because environmental BNF activity is often too low to generate sufficient ethylene for isotopic analyses. Here we describe a high sensitivity method to measure ethylene δ13C by in-line coupling of ethylene preconcentration to gas chromatography-combustion-isotope ratio mass spectrometry (EPCon-GC-C-IRMS). Ethylene requirements in samples with 10% v/v acetylene are reduced from > 500 to ~ 20 ppmv (~ 2 ppmv with prior offline acetylene removal). To increase robustness by reducing calibration error, single nitrogenase-isoform
- NSF-PAR ID:
- 10386749
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 12
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Symbiotic nitrogen fixation (SNF) by higher plants and their bacterial symbionts is a globally important input of nitrogen. Our understanding of the mechanisms that control SNF and the time‐scales over which they operate has been constrained by the limitations of the existing methods for measuring SNF. One method, Acetylene Reduction Assays by Cavity ring‐down laser Absorption Spectroscopy (ARACAS), seems promising, as it is highly sensitive and gives rapid, continuous, repeatable and real‐time measurements of nitrogenase activity. ARACAS has been used to study nitrogen fixation in lichens, mosses and asymbiotic bacteria, but adapting it to higher plants poses challenges because acetylene and ethylene can influence plant function.
Here, we report modifications to ARACAS that allow it to be used on higher plants in an environmentally controlled incubation chamber. The modifications include lower concentrations of acetylene (2%) and ethylene and concurrent measurements of whole‐chamber CO2exchange, H2O exchange and nitrogenase activity, linking nitrogenase activity to whole‐plant rates of photosynthesis and respiration.
After propagating the error terms from all sources, we establish the following parameters of the method: (a) The detection limit of our method was 2–3 ppbv C2H4per hour, although it rose substantially when we used tank‐derived acetylene, which has much higher ethylene contamination; (b) Repeated measures at a frequency of 3 days or longer did not diminish nitrogenase activity or photosynthesis, although daily measurements diminished nitrogenase activity; (c) This method can detect changes at time‐scales as short as seconds; (d) Continuous measurement of nitrogenase activity is maintained above 90% of the maximum rate for 7.0 ± 1.3 (
M ±SD ) hours.This method has the potential to improve our understanding of the controls over SNF, and therefore, how SNF and global nitrogen and carbon cycling are likely to be affected by global change.
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Glass, Jennifer B. (Ed.)Nitrogen gas (N2) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH4+) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (−0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N2 fixation genes. At −0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH4+ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at −0.15 V. N2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at −0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at −0.15 V, suggesting that these mechanisms may be involved in N2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2 fixation rates. We hypothesize that at −0.15 V, they increase N2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use.more » « less
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Summary Cu+‐chaperones are a diverse group of proteins that allocate Cu+ions to specific copper proteins, creating different copper pools targeted to specific physiological processes.
Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required.
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Abstract High rates of biological nitrogen fixation (BNF) are commonly reported for tropical forests, but most studies have been conducted in regions that receive substantial inputs of molybdenum (Mo) from atmospheric dust and sea‐salt aerosols. Even in these regions, the low availability of Mo can constrain free‐living BNF catalyzed by heterotrophic bacteria and archaea. We hypothesized that in regions where atmospheric inputs of Mo are low and soils are highly weathered, such as the southeastern Amazon, Mo would constrain BNF. We also hypothesized that the high soil acidity, characteristic of the Amazon Basin, would further constrain Mo availability and therefore soil BNF. We conducted two field experiments across the wet and dry seasons, adding Mo, phosphorus (P), and lime alone and in combination to the forest floor in the southeastern Amazon. We sampled soils and litter immediately, and then weeks and months after the applications, and measured Mo and P availability through resin extractions and BNF with the acetylene reduction assay. The experimental additions of Mo and P increased their availability and the lime increased soil pH. While the combination of Mo and P increased BNF at some time points, BNF rates did not increase strongly or consistently across the study as a whole, suggesting that Mo, P, and soil pH are not the dominant controls over BNF. In a separate short‐term laboratory experiment, BNF did not respond strongly to Mo and P even when labile carbon was added. We postulate that high nitrogen (N) availability in this area of the Amazon, as indicated by the stoichiometry of soils and vegetation and the high nitrate soil stocks, likely suppresses BNF at this site. These patterns may also extend across highly weathered soils with high N availability in other topographically stable regions of the tropics.
