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.     

p-Cresol biodegradation under denitrifying conditions: isolation of a bacterial coculture

Abstract From polluted river sediment, two bacterial species were isolated which utilized p -cresol as the sole source of carbon when grown in coculture under nitrate-reducing conditions. One species, PC-07, metabolized p -cresol (pCr) anaerobically to p -hydroxybenzoate (pOHB), which in turn was further metabolized by the second isolate, PB-04. The PC-07 isolate was unable to degrade and utilize pOHB, and PB-04 was unable to utilize pCr, thereby demonstrating a syntrophic relationship for pCr utilization under anaerobic conditions. Nitrate served as external electron acceptor for both microorganisms under anaerobic conditions and was reduced via NO₂⁻ and N₂O to N₂. pCr, therefore, appears to be metabolized to ring fission products via the formation of pOHB under nitrate reducing conditions, with the metabolism being mediated by a 2-member microbiol food chain.     

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.     

Symbiotic N2 fixation in a high Alpine grassland: effects of four growing seasons of elevated CO2

1. Increasing carbon dioxide concentration (E: 680 μl CO₂ litre^–1 vs ambient, A: 355 μl CO₂ litre^–1) around late-successional Alpine sedge communities of the Swiss Central Alps (2450 m) for four growing seasons (1992–1995) had no detectable effect on symbiotic N₂ fixation in Trifolium alpinum —the sole N₂-fixing plant species in these communities (74 ± 30 mg N m^–2 year^–1, A and E plots pooled).
2. This result is based on data collected in the fourth growing season showing that elevated CO₂ had no effect on Trifolium above-ground biomass (4·4 ± 1·7 g m^–2, A and E plots pooled, n = 24) or N content per unit land area (124 ± 51 mg N m^–2, A and E pooled), or on the percentage of N Trifolium derived from the atmosphere through symbiotic N₂ fixation (%Ndfa: 61·0 ± 4·1 across A and E plots) estimated using the ¹⁵N dilution method.
3. Thus, it appears that N inputs to this ecosystem via symbiotic N₂ fixation will not be dramatically affected in the foreseeable future even as atmospheric CO₂ continues to rise.     

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.     

Physiological interactions between a mesophilic cellulolytic Clostridium and a non-cellulolytic bacterium

Abstract A mesophilic cellulolytic bacterium ( Clostridium strain C7) capable of N₂ fixation and a non-cellulolytic bacterium ( Klebsiella strain W1), both isolated from freshwater environments rich in decaying plant material, were co-cultured in a chemically defined, vitamin-deficient medium containing cellulose as the carbon and energy source. In the co-culture, an extracellular cellulase complex produced by the Clostridium hydrolyzed cellulose to soluble sugars that served as fermentable substrates for the Klebsiella . In turn, the Klebsiella excreted growth factors, identified as biotin and p -aminobenzoic acid, which were required by the Clostridium . Furthermore, demonstration of NH₄⁺-repressible acetylene reduction by co-cultures growing in medium lacking combined nitrogen showed that the Clostridium fixed N₂, thus allowing growth of the Klebsiella , which was not a nitrogen fixer. The mutualistic relationships observed in the co-cultures may be representative of interactions that take place in natural environments in which cellulose-containing plant materials are biodegraded.     

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.     

N2-dependent growth and nitrogenase activity in the metal-metabolizing bacteria, Geobacter and Magnetospirillum species

Cells of Geobacter metallireducens , Magnetospirillum strain AMB-1, Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense showed N₂-dependent growth, the first anaerobically with Fe(III) as the electron acceptor, and the latter three species microaerobically in semi-solid oxygen gradient cultures. Cells of the Magnetospirillum species grown with N₂ under microaerobic conditions were magnetotactic and therefore produced magnetosomes. Cells of Geobacter metallireducens reduced acetylene to ethylene (11.5 ± 5.9 nmol C₂H₄ produced min⁻¹ mg⁻¹ cell protein) while growing with Fe(III) as the electron acceptor in anaerobic growth medium lacking a fixed nitrogen source. Cells of the Magnetospirillum species, grown in a semi-solid oxygen gradient medium, also reduced acetylene at comparable rates. Uncut chromosomal and fragments from endonuclease-digested chromosomal DNA from these species, as well as Geobacter sulphurreducens organisms, hybridized with a nifHDK probe from Rhodospirillum rubrum , indicating the presence of these nitrogenase structural genes in these organisms. The evidence presented here shows that members of the metal-metabolizing genera, Geobacter and Magnetospirillum , fix atmospheric dinitrogen.     

Changes in nitrogen source modify distribution of excitation energy in the cyanobacterium Phormidium laminosum

In an attempt to clarify the interactions between the available nitrogen source and the photosystems in cyanobacteria, O₂ exchange and fluorescence emission were monitored in spheroplasts and intact cells of the non N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) growing on different nitrogen sources or in the absence of nitrogen. Short-term (time scale of seconds to minutes), NH⁺₄ addition to NO⁻₃-growing or N-starved cells and, to a minor extent, NO⁻₃addition to N-starved cells, induced state 2 transitions both in light and dark. Long term (time scale of days), the fluorescence yield of PSII relative to that of PSII at 77 K was higher in NO⁻₃- than in NH⁺₄ growing cells, and even higher in N-starved cells. In the dark, the plastoquinone pool was more reduced in NH⁺₄- than in NO⁻₃-growing cells. Both PSII and PSI activities and the degree of linking between both photosystems were affected in the long term, so that non-cyclic electron transport decreased in parallel to the ferredoxin requirement to assimilate each nitrogen source. Results indicate that nitrogen metabolism exerts short- and long-term control over the photosynthetic apparatus, which acclimates to the energy requirement of the available nitrogen source.     

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.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

A nitrogen-fixing consortia associated with the bacterial decay of a wooden pipeline

Investigation of superficial decay of 2·0 m diameter wooden pipeline carrying water for hydroelectric power generation, revealed the presence of a wood-decaying, nitrogen-fixing bacterial consortia associated with the attack. The most severe attack occurred in pools of semi-permanent water on the top of the pipeline. Associated N₂-fixing biota were identified as Xanthobacter autotrophicus or X. flavus , Pseudomonas acidovorans , Ps. alcaligenes , Ps. aureofaciens and an Alcaligenes sp. The Alcaligenes and Pseudomonas isolates (but not the Xanthobacter isolates) showed growth with creosote as the sole source of carbon, with gas chromatography/mass spectrometry analysis indicating utilization of the aliphatic components in the C₂₂–C₃₅ range.     

Microaerophilic growth of Wolinella recta ATCC 33238

Abstract The influence of oxygen on growth and fumarate-dependent respiration of Wolinella recta ATCC 33238 was studied in continuous culture. Steady states were obtained with formate-limited cultures grown at a specific growth rate of 0.1 h⁻¹ with different levels of oxygenation. The extent of aeration was regulated by means of a redox control system permitting reproducible cultivation at oxygen levels below the detection limit of conventional lead-silver probes. The ratio of succinate produced to that of formate consumed (Suc/For) decreased from 0.99 in strictly anaerobic cultures to 0.06–0.10 in aerated cultures. The growth yield did not change significantly with increasing redox readings: 4.9–5.2 g cell carbon/mol formate. The ability to use O₂ as the sole electron acceptor was demonstrated in a chemostat culture with formate as electron donor and succinate as carbon source. Washed cells from all chemostat cultures comsumed O₂ with formate as electron donor at a high rate (2.1–3.7 μmol/min per mg protein) and possessed b - and c -type cytochromes and CO-binding pigments. These results clearly indicated the microaerophilic nature of W. recta .     

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.     

A review of nitrogen enrichment effects on three biogenic GHGs: the CO2 sink may be largely offset by stimulated N2O and CH4 emission

Anthropogenic nitrogen (N) enrichment of ecosystems, mainly from fuel combustion and fertilizer application, alters biogeochemical cycling of ecosystems in a way that leads to altered flux of biogenic greenhouse gases (GHGs). Our meta-analysis of 313 observations across 109 studies evaluated the effect of N addition on the flux of three major GHGs: CO₂, CH₄ and N₂O. The objective was to quantitatively synthesize data from agricultural and non-agricultural terrestrial ecosystems across the globe and examine whether factors, such as ecosystem type, N addition level and chemical form of N addition influence the direction and magnitude of GHG fluxes. Results indicate that N addition increased ecosystem carbon content of forests by 6%, marginally increased soil organic carbon of agricultural systems by 2%, but had no significant effect on net ecosystem CO₂ exchange for non-forest natural ecosystems. Across all ecosystems, N addition increased CH₄ emission by 97%, reduced CH₄ uptake by 38% and increased N₂O emission by 216%. The net effect of N on the global GHG budget is calculated and this topic is reviewed. Most often N addition is considered to increase forest C sequestration without consideration of N stimulation of GHG production in other ecosystems. However, our study indicated that although N addition increased the global terrestrial C sink, the CO₂ reduction could be largely offset (53–76%) by N stimulation of global CH₄ and N₂O emission from multiple ecosystems.     

Genetic factors in Rhizobium affecting the symbiotic carbon costs of N2 fixation and host plant biomass production

The effect of genetic factors in Rhizobium on host plant biomass production and on the carbon costs of N₂ fixation in pea root nodules was studied. Nine strains of Rhizobium leguminosarum were constructed, each containing one of three symbiotic plasmids in combination with one of three different genomic backgrounds. The resulting strains were tested in symbiosis with plants of Pisum sativum using a flow-through apparatus in which nodule nitrogenase activity and respiration were measured simultaneously under steady state conditions. Nodules formed by strains containing the background of JI6015 had the lowest carbon costs of N₂ fixation (7.10–8.10 μmol C/μmol N₂), but shoot dry weight of those plants was also smaller than that of plants nodulated by strains with the background of B151 or JI8400. Nodules formed by these two strain types had carbon costs of N₂ fixation varying between 11.26 and 13.95 μmol C/μmol N₂. The effect of symbiotic plasmids on the carbon costs was relatively small. A time-course experiment demonstrated that nodules formed by a strain derived from JI6015 were delayed in the onset of nitrogenase activity and had a lower rate of activity compared to nodules induced by a strain with the background of B151. The relationship between nitrogenase activity, carbon costs of N₂ fixation and host plant biomass production is discussed.     

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%.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

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.