Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Convergence in the relationship of CO2 and N2O exchanges between soil and atmosphere within terrestrial ecosystems

Ecosystem CO₂ and N₂O exchanges between soils and the atmosphere play an important role in climate warming and global carbon and nitrogen cycling; however, it is still not clear whether the fluxes of these two greenhouse gases are correlated at the ecosystem scale. We collected 143 pairs of ecosystem CO₂ and N₂O exchanges between soils and the atmosphere measured simultaneously in eight ecosystems around the world and developed relationships between soil CO₂ and N₂O fluxes. Significant linear regressions of soil CO₂ and N₂O fluxes were found for all eight ecosystems; the highest slope occurred in rice paddies and the lowest in temperate grasslands. We also found the dominant role of growing season on the relationship of annual CO₂ and N₂O fluxes. No significant relationship between soil CO₂ and N₂O fluxes was found across all eight ecosystem types. The estimated annual global N₂O emission based on our findings is 13.31 Tg N yr⁻¹ with a range of 8.19–18.43 Tg N yr⁻¹ for 1980–2000, of which cropland contributes nearly 30%. Our findings demonstrated that stoichiometric relationships may work on ecological functions at the ecosystem level. The relationship of soil N₂O and CO₂ fluxes developed here could be helpful in biogeochemical modeling and large-scale estimations of soil CO₂ and N₂O fluxes.     

Nitrogen fixation by Rhodopseudomonas palustris OU 11 with aromatic compounds as carbon source/electron donors

Abstract A purple non-sulfur anoxygenic phototrophic bacterium, Rhodopseudomonas palustris (ATCC 51186; DSM 7375), grew fixing N₂ using aromatic compounds as the sole carbon source/electron donor. Benzoate, cinnamate and benzyl alcohol were used as electron donors for N₂ fixation, while aniline and nitrobenzene supported poor growth under N₂ atmosphere (in the absence of any other combined nitrogen in the medium) but did serve as sole carbon source/e⁻ donor in the presence of ammonium chloride as nitrogen source.     

Protective mechanisms of nitrogenase against oxygen excess and partially-reduced oxygen intermediates

Diazotrophic systems have developed a number of strategies to protect nitrogenase (N₂ase; EC 1.18.6.1) from O₂ excess and active-oxygen species (AOS). Protection against O₂ excess is given by biochemical modifications of N₂ase, increased rates of low-efficiency respiration, temporal segregation of N₂ fixation and photosynthesis, physical barriers to O₂ diffusion, and hemoglobins. On the other hand, AOS may originate from oxidation of N₂ase components, ferredoxins, flavodoxins and hemoglobins; interaction among the AOS themselves, or between H₂O₂ and hemoglobins; and during reactions catalyzed by hydrogenase (EC 1.18.99.1), xanthine oxidase (EC 1.1.3.22) and uricase (EC 1.7.3.3). Active-oxygen species are scavenged enzymatically [superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6). peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11)] or through non-enzymic reaction with low-molecular-weight compounds (ascorbate, α-tocopherol, glutathione).     

Consortial N2 fixation: a strategy for meeting nitrogen requirements of marine and terrestrial cyanobacterial mats

Abstract: Four microbial mat-forming, non-axenic, strains of the non-heterocystous, filamentous, cyanobacterial genus Microcoleus were maintained in culture and examined for the ability to fix atmospheric nitrogen (N₂). Each was tested for nitrogenase activity using the acetylene reduction assay (ARA) and for the presence of the dinitrogenase reductase gene ( nifH ), an essential gene for N₂ fixation, using the polymerase chain reaction (PCR). The Microcoleus spp. cultures were incapable of growth without an exogenous nitrogen source and never exhibited nitrogenase activity. Attempts to amplify a 360-bp segment of the nifH gene using DNA purified from the cyanobacterial cultures did not produce any cyanobacteria-specific nifH sequences. However, several non-cyanobacterial homologous nifH sequences were obtained. Phylogenetic analysis showed these sequences to be most similar to sequences from heterotrophic bacteria isolated from a marine microbial mat in Tomales Bay (California, USA), and bulk DNA extracted from a cryptobiotic soil crust in Moab (Utah, USA). Microcoleus spp. dominated the biomass of both systems. Cyanobacteria-specific 16S rDNA sequences obtained from the cultured cyanobacterial strains demonstrate that the lack of cyanobacteria-specific nifH sequences was not due to inefficiency of extracting Microcoleus DNA. Hence, both the growth and genetic data indicate that, contrary to earlier reports, Microcoleus spp. appear incapable of fixing N₂ because they lack at least one of the requisite genes for this process. Furthermore, our study suggests epiphytic N₂-fixing bacteria form a diazotrophic consortium with these Microcoleus spp. and are likely key sources of fixed N₂ generated within soil crusts and marine microbial mats.     

Modification of the pII protein in response to carbon and nitrogen availability in filamentous heterocystous cyanobacteria

Abstract In the filamentous cyanobacterium Calothrix PCC 7504, which fixes N₂ aerobically, the modification state of the regulatory P_II protein (GlnB) was shown to depend on nitrogen and carbon availability, as observed in the unicellular non-fixing strain Synechococcus PCC 7942. However, the conditions for modifications, the time dependence of the process and the electrophoretic behavior of the native P_II isoforms differed somewhat between the two strains. In another strain, Calothrix PCC 7601, which has lost the capability to fix N₂, P_II was modified only if ammonia plus an inhibitor of glutamine synthetase were present. It is proposed that: (i) the behavior of the P_II proteins depends upon the physiological properties of the strains; and (ii) the modification system of P_II per se may differ between the two cyanobacterial genera.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

Snow depth, soil freezing, and fluxes of carbon dioxide, nitrous oxide and methane in a northern hardwood forest

Soil–atmosphere fluxes of trace gases (especially nitrous oxide (N₂O)) can be significant during winter and at snowmelt. We investigated the effects of decreases in snow cover on soil freezing and trace gas fluxes at the Hubbard Brook Experimental Forest, a northern hardwood forest in New Hampshire, USA. We manipulated snow depth by shoveling to induce soil freezing, and measured fluxes of N₂O, methane (CH₄) and carbon dioxide (CO₂) in field chambers monthly (bi-weekly at snowmelt) in stands dominated by sugar maple or yellow birch. The snow manipulation and measurements were carried out in two winters (1997/1998 and 1998/1999) and measurements continued through 2000. Fluxes of CO₂ and CH₄ showed a strong seasonal pattern, with low rates in winter, but N₂O fluxes did not show strong seasonal variation. The snow manipulation induced soil freezing, increased N₂O flux and decreased CH₄ uptake in both treatment years, especially during winter. Annual N₂O fluxes in sugar maple treatment plots were 207 and 99 mg N m⁻² yr⁻¹ in 1998 and 1999 vs. 105 and 42 in reference plots. Tree species had no effect on N₂O or CO₂ fluxes, but CH₄ uptake was higher in plots dominated by yellow birch than in plots dominated by sugar maple. Our results suggest that winter fluxes of N₂O are important and that winter climate change that decreases snow cover will increase soil:atmosphere N₂O fluxes from northern hardwood forests.     

Nitrogen use efficiency. 3. Nitrogen fixation: genes and costs

The nitrogen use efficiencies (NUE) of N₂ fixation, primary NH ₄⁺ assimilation and NO ₃⁻ assimilation are compared. The photon and water costs of the various biochemical and transport processes involved in plant growth, N-assimilation, pH regulation and osmolarity generation, per unit N assimilated are respectively likely to be around 5 and 7% greater for N₂ fixation than for a combination of NH ₄⁺ and root and shoot NO ₃⁻ assimilation as occurs with most crops. Studies on plant and rhizobial genes involved in nodulation and N₂ fixation may lead to more rapid nodulation, production of more stress-tolerant N₂ fixing systems and transfer of the hydrogenase system to rhizobium/legume symbioses which currently do not have this ability. The activity of an uptake hydrogenase is predicted to decrease the photon cost of diazotrophic plant growth by about 1%.     

Plant regulated aspects of nodulation and N2 fixation

Abstract. Root nodule organogenesis is described. Plant regulated aspects of nodulation and N₂ fixation are reviewed and discussed. Since the effective N₂ fixing symbiosis requires the interaction of the host plant and bacterium in an appropriate environment (the rhizosphere and the root nodule) it is essential that research aimed at improving N₂ fixation involve a knowledge and understanding of the plant genes that affect nodule development, growth, and function. Current knowledge of host plant genes involved in N₂ fixation is summarized. Various experimental approaches to the study of the host plant's contribution to nodulation are noted. The functions of nodule specific proteins (nodulins) in symbiosis are delineated. Future areas of research are suggested.     

Effects of temperature and fertilization on nitrogen cycling and community composition of an urban lawn

We examined the influence of temperature and management practices on the nitrogen (N) cycling of turfgrass, the largest irrigated crop in the United States. We measured nitrous oxide (N₂O) fluxes, and plant and soil N content and isotopic composition with a manipulative experiment of temperature and fertilizer application. Infrared lamps were used to increase surface temperature by 3.5±1.3 °C on average and control and heated plots were split into high and low fertilizer treatments. The N₂O fluxes increased following fertilizer application and were also directly related to soil moisture. There was a positive effect of warming on N₂O fluxes. Soils in the heated plots were enriched in nitrogen isotope ratio ( δ ¹⁵N) relative to control plots, consistent with greater gaseous losses of N. For all treatments, C₄ plant C/N ratio was negatively correlated with plant δ ¹⁵N, suggesting that low leaf N was associated with the use of isotopically depleted N sources such as mineralized organic matter. A significant and unexpected result was a large, rapid increase in the proportion of C₄ plants in the heated plots relative to control plots, as measured by the carbon isotope ratio ( δ ¹³C) of total harvested aboveground biomass. The C₄ plant biomass was dominated by crabgrass, a common weed in C₃ fescue lawns. Our results suggest that an increase in temperature caused by climate change as well as the urban heat island effect may result in increases in N₂O emissions from fertilized urban lawns. In addition, warming may exacerbate weed invasions, which may require more intensive management, e.g. herbicide application, to manage species composition.     

Carbon Costs Associated with N2 Fixation in Vicia faba L and Pisum sativum 1. over a 14-Day Period

Abstract: Long-term (14 days) carbon costs of N₂ fixation were studied in pot trials. For this purpose the CO₂ release from the root space of nodulated and non-nodulated (urea nourished) Vicia faba L. and Pisum sativum L. plants was compared and related to the amount of fixed or assimilated N. Additional measurements of shoot CO₂ exchange and dry matter increment were carried out in order to calculate the overall carbon balance. The carbon costs for N₂ fixation in Vicia faba 1. (2.87 mg C/mg N_fiX) were higher than in Pisum sativum L. (2.03 mg C/mg N_fix). However, the better carbon efficiency in Pisum sativum 1. did not lead to a better growth performance compared to Vicia faba L. Vicia faba L. compensated for the carbon and energy expenditure by more intensive photosynthesis in the N₂-fixing treatment. This was not the case with Pisum sativum L., where the carbon balance indicates that the carbon costs of N₂ fixation restricted root growth. It is proposed that low carbon costs for N₂ fixation indicate an adaptation to a critical carbon supply of roots and nodules, e.g., during the pod-filling of grain legumes.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Anatomical and hnctional differences and nyctinastic leaf movements in Chenopodium album L. and Chenopodium hircinum Schrad. (Chenopodiaceae)

The anatomical structure of the leaves of Chenopodium album L. and Chenopodium hircinum Schrad. has been examined. Leaf chlorophyll content (Chl), specific leaf area (SLA), specific leaf weight (SLA^-1) and nitrogen content (N₂) were estimated in both species. Ch1, SLA and N₂ were greater in C. album than in C. hircinum. SLA^-1 data showed that C. album is more efficient than C. hircinum , because the former invests smaller quantities of dry weight to achieve a square centimetre of leaf area. The possible effects of Chl, N₂ and leaf anatomy on efficient gas exchange are discussed. The presence of nyctinastic leaf nocturnal movements was detected in both species. The meaning of this is discussed in relation to the prevention of the loss of pollen grains.     

Elevated atmospheric CO2 does not affect per se the preference for symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.

The objective of this investigation was to examine the effect of an elevated atmospheric CO₂ partial pressure ( p CO₂) on the N-sink strength and performance of symbiotic N₂ fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient p CO₂ in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated p CO₂ (35 and 60 Pa) and two levels of mineral N (N-free and 7·5 mol m^–3 N) for 36 d in single pots filled with silica sand in growth chambers. Elevated p CO₂ evoked a significant increase in biomass production from day 12 after the start of CO₂ enrichment. For plants supplied with 7·5 mol m^–3 N, the relative contribution of symbiotically fixed N (%N_sym) as opposed to N assimilated from mineral sources (¹⁵N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N_sym was unaffected by atmospheric p CO₂ over the entire experimental period. In plants fully dependent on N₂ fixation, the increase in N yield reflects a stimulation of symbiotic N₂ fixation that was the result of the formation of more nodules rather than of higher specific N₂ fixation. These results are discussed with regard to physiological processes governing symbiotic N₂ fixation and to the response of symbiotic N₂ fixation to elevated p CO₂ in field-grown T. repens .     

Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste

Aims:  To monitor emissions of NH₃ and N₂O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB).
Methods and Results:  A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH₃ emissions peaked when pH started to increase. Small amounts of N₂O and CH₄ were also produced. In total, 16% and less than 1% of the initial N was lost as NH₃-N and N₂O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase.
Conclusions:  Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH₃ emissions. Small but significant amounts of N₂O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear.
Significance and Impact of Study:  This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH₃ emissions.     

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Abstract Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C₂H₂ reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C₂H₂ reduction had a K _i value of 16 μM. Both ammonia and glutamine inhibited N₂ fixation (C₂H₂ reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.     

Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary

Summary Anaerobic ammonium oxidation (anammox) specific PCR method was developed to examine diversity and distribution of anammox bacteria in sediments collected from three different sites at Cape Fear River Estuary, North Carolina, where environmental parameters vary greatly over the year. Abundance and activities of anammox bacteria in these sediments were measured using the quantitative PCR (Q-PCR) method and ¹⁵N isotope tracer incubations. Different anammox bacterial communities composed with Brocadia , Kuenenia , Jettenia or Scalindua were found among sites along the estuarine gradient. Seasonal variations of anammox community structures were observed along the estuary based on terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. Correlation analysis suggested that salinity variation influenced the diversity and distribution of different anammox bacteria in the estuary. Q-PCR assays of anammox bacteria showed temporal and spatial variations of their abundances, which were highly correlated to salinity variation. ¹⁵N isotope tracer incubations measured different anammox rates and its per cent contribution to total N₂ production among sites. The highest anammox rate was found at the site where Scalindua organisms dominated with the highest anammox bacterial abundance. Thus, we demonstrated a biogeographical distribution of diverse anammox bacteria influenced by salinity, and provide evidence to link anammox abundance and activities in estuarine sediments.     

Measurement of denitrification in estuarine sediment using membrane inlet mass spectrometry

Abstract Denitrification was measured in intact sediment cores and in homogenised slurries using membrane inlet mass spectrometry. Dissolved concentrations of O₂, N₂, N₂O and CO₂ were simultaneously monitored. Using a 0.8 mm diameter needle probe, a comparison was made of the gas profiles of intact cores obtained under different conditions, i.e. with air or argon as the headspace gas and after the addition of nitrate and/or a carbon source to the sediment surface. O₂ was detectable to a depth of 1 cm under a headspace of air and the depth at which the maxima of denitrification products occurred was 1.5–2 cm. Denitrification products (N₂O, N₂) occurred in the surface layers where O₂ was above the minimum level of detectability (> 0.25 μM): diffusion of N₂ and N₂O upwards from the anoxic zone, local anaerobic microenvironments or aerobic denitrification are alternative explanations for this observation. The addition of nitrate and/or acetate increased the concentrations of N₂, N₂O and CO₂ in the sediment core. In sediment slurries, the pH, nitrate concentration, carbon source and the depth from which the sample was taken affected the rate of denitrification. Nitrogen was the sole detectable end product. Maximum denitrification occurred at pH 7.5 and at 20 mM nitrate. Denitrification was at a maximum in those slurries prepared from sections of core at 1–2 cm depth.     

The metabolism of sugar and malic acid by Leuconostoc oenos: effect of malic acid, pH and aeration conditions

The co-metabolism of sugars by Leuconostoc oenos was studied under different environmental conditions. Under aerobic conditions, growth and sugar metabolism were poorer than under CO₂ or N₂ atmosphere and acetic acid accumulated to a larger extent. Glycerol was found in the aerobic cultures while erythritol was detected under N₂ or CO₂. When medium conditions make growth difficult (low pH, aerobic conditions, low nutrients), sugars were only slightly metabolized and growth was very slow while malic acid was rapidly and completely degraded, leading to an increase in the y _ATP. Aeration effects on the malic acid degradation rate depended on the nutrients and carbon source in the medium. Malic acid clearly stimulated bacterial growth, allowing an increase in the molar growth yields and ATP production. The results suggest that under adverse conditions cells are not able to grow and malic degradation supplies additional energy production.