Elevated CO2 stimulates associative N2 fixation in a C3 plant of the Chesapeake Bay wetland

In this study, the response of N₂ fixation to elevated CO₂ was measured in Scirpus olneyi, a C₃ sedge, and Spartina patens, a C₄ grass, using acetylene reduction assay and ¹⁵N₂ gas feeding. Field plants grown in PVC tubes (25 cm long, 10 cm internal diameter) were used. Exposure to elevated CO₂ significantly (P < 0·05) caused a 35% increase in nitrogenase activity and 73% increase in ¹⁵N incorporated by Scirpus olneyi. In Spartina patens, elevated CO₂ (660 ± 1 μ mol mol ^{− 1}) increased nitrogenase activity and ¹⁵N incorporation by 13 and 23%, respectively. Estimates showed that the rate of N₂ fixation in Scirpus olneyi under elevated CO₂ was 611 ± 75 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} compared with 367 ± 46 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} in ambient CO₂ plants. In Spartina patens, however, the rate of N₂ fixation was 12·5 ± 1·1 versus 9·8 ± 1·3 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} for elevated and ambient CO₂, respectively. Heterotrophic non-symbiotic N₂ fixation in plant-free marsh sediment also increased significantly (P < 0·05) with elevated CO₂. The proportional increase in ¹⁵N₂ fixation correlated with the relative stimulation of photosynthesis, in that N₂ fixation was high in the C₃ plant in which photosynthesis was also high, and lower in the C₄ plant in which photosynthesis was relatively less stimulated by growth in elevated CO₂. These results are consistent with the hypothesis that carbon fixation in C₃ species, stimulated by rising CO₂, is likely to provide additional carbon to endophytic and below-ground microbial processes.     

Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

Effect of H2 evolution on 15N2 fixation, C2H2 reduction and relative efficiency of leguminous symbionts

The (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratios of 15 clover- Rhizobium symbionts. soybean, and black medick symbionts were measured. Relative efficiency based on the C2H4 production and on ¹⁵N₂ incorporation were compared, and in most symbionts there was little difference between the two measures of relative efficiency. Total measurable electron flux through nitrogenase during acetylene reduction and ¹⁵N₂ incorporation were nearly equal for most symbionts studied. The relative efficiency and the (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratio showed an inverse correlation. Use of this ratio appears preferable to use of the ratio of C2H2 reduction/N₂ reduction. Some evolution of H₂ was observed in the presence of C₂H₂.     

Drought turns a Central European Norway spruce forest soil from an N2O source to a transient N2O sink

Based on current climate scenarios, a higher frequency of summer drought periods followed by heavy rainfall events is predicted for Central Europe. It is expected that drying/rewetting events induce an increased matter cycling in soils and may contribute considerably to increased emissions of the greenhouse gas N₂O on annual scales. To investigate the influence of drying/rewetting events on N₂O emissions in a mature Norway spruce forest in the Fichtelgebirge area (NE Bavaria, Germany), a summer drought period of 46 days was induced by roof installations on triplicate plots, followed by a rewetting event of 66 mm experimental rainfall in 2 days. Three nonmanipulated plots served as controls. The experimentally induced soil drought was accompanied by a natural drought. During the drought period, the soil of both the throughfall exclusion and control plots served as an N₂O sink. This was accompanied by subambient N₂O concentrations in upper soil horizons. The sink strength of the throughfall exclusion plots was doubled compared with the control plots. We conclude that the soil water status together with the soil nitrate availability was an important driving factor for the N₂O sink strength. Rewetting quickly turned the soil into a source for atmospheric N₂O again, but it took almost 4 months to turn the cumulative soil N₂O fluxes from negative (sink) to positive (source) values. N₂O concentration and isotope analyses along soil profiles revealed that N₂O produced in the subsoil was subsequently consumed during upward diffusion along the soil profile throughout the entire experiment. Our results show that long drought periods can lead to drastic decreases of N₂O fluxes from soils to the atmosphere or may even turn forest soils temporarily to N₂O sinks. Accumulation of more field‐scale data on soil N₂O uptake as well as a better understanding of underlying mechanisms would essentially advance our knowledge of the global N₂O budget.     

N2 fixation by Acacia species increases under elevated atmospheric CO2

In the present study the effect of elevated CO₂ on growth and nitrogen fixation of seven Australian Acacia species was investigated. Two species from semi‐arid environments in central Australia (Acacia aneura and A. tetragonophylla) and five species from temperate south‐eastern Australia (Acacia irrorata, A. mearnsii, A. dealbata, A. implexa and A. melanoxylon) were grown for up to 148 d in controlled greenhouse conditions at either ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ concentrations. After establishment of nodules, the plants were completely dependent on symbiotic nitrogen fixation. Six out of seven species had greater relative growth rates and lower whole plant nitrogen concentrations under elevated versus normal CO₂. Enhanced growth resulted in an increase in the amount of nitrogen fixed symbiotically for five of the species. In general, this was the consequence of lower whole‐plant nitrogen concentrations, which equate to a larger plant and greater nodule mass for a given amount of nitrogen. Since the average amount of nitrogen fixed per unit nodule mass was unaltered by atmospheric CO₂, more nitrogen could be fixed for a given amount of plant nitrogen. For three of the species, elevated CO₂ increased the rate of nitrogen fixation per unit nodule mass and time, but this was completely offset by a reduction in nodule mass per unit plant mass.     

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.     

Initial cultivation of a temperate-region soil immediately accelerates aggregate turnover and CO2 and N2O fluxes

The immediate effects of tillage on protected soil C and N pools and on trace gas emissions from soils at precultivation levels of native C remain largely unknown. We measured the response to cultivation of CO₂ and N₂O emissions and associated environmental factors in a previously uncultivated U.S. Midwest Alfisol with C concentrations that were indistinguishable from those in adjacent late successional forests on the same soil type (3.2%). Within 2 days of initial cultivation in 2002, tillage significantly (P=0.001, n=4) increased CO₂ fluxes from 91 to 196 mg CO₂‐C m^?2 h^?1 and within the first 30 days higher fluxes because of cultivation were responsible for losses of 85 g CO₂‐C m^?2. Additional daily C losses were sustained during a second and third year of cultivation of the same plots at rates of 1.9 and 1.0 g C m^?2 day^?1, respectively. Associated with the CO₂ responses were increased soil temperature, substantially reduced soil aggregate size (mean weight diameter decreased 35% within 60 days), and a reduction in the proportion of intraaggregate, physically protected light fraction organic matter. Nitrous oxide fluxes in cultivated plots increased 7.7‐fold in 2002, 3.1‐fold in 2003, and 6.7‐fold in 2004 and were associated with increased soil NO₃^? concentrations, which approached 15 μg N g^?1. Decreased plant N uptake immediately after tillage, plus increased mineralization rates and fivefold greater nitrifier enzyme activity, likely contributed to increased NO₃^? concentrations. Our results demonstrate that initial cultivation of a soil at precultivation levels of native soil C immediately destabilizes physical and microbial processes related to C and N retention in soils and accelerates trace gas fluxes. Policies designed to promote long‐term C sequestration may thus need to protect soils from even occasional cultivation in order to preserve sequestered C.     

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.     

Leaf gas exchange and nitrogen dynamics of N2-fixing, field-grown Alnus glutinosa under elevated atmospheric CO2

Few studies have investigated the effects of elevated CO₂ on the physiology of symbiotic N₂-fixing trees. Tree species grown in low N soils at elevated CO₂ generally show a decline in photosynthetic capacity over time relative to ambient CO₂ controls. This negative adjustment may be due to a reallocation of leaf N away from the photosynthetic apparatus, allowing for more efficient use of limiting N. We investigated the effect of twice ambient CO₂ on net CO₂ assimilation (A), photosynthetic capacity, leaf dark respiration, and leaf N content of N2-fixing Alnus glutinosa (black alder) grown in field open top chambers in a low N soil for 160 d. At growth CO₂, A was always greater in elevated compared to ambient CO₂ plants. Late season A vs. internal leaf p(CO₂) response curves indicated no negative adjustment of photosynthesis in elevated CO₂ plants. Rather, elevated CO₂ plants had 16% greater maximum rate of CO₂ fixation by Rubisco. Leaf dark respiration was greater at elevated CO₂ on an area basis, but unaffected by CO₂ on a mass or N basis. In elevated CO₂ plants, leaf N content (μg N cm^?2) increased 50% between Julian Date 208 and 264. Leaf N content showed little seasonal change in ambient CO₂ plants. A single point acetylene reduction assay of detached, nodulated root segments indicated a 46% increase in specific nitrogenase activity in elevated compared to ambient CO₂ plants. Our results suggest that N₂-fixing trees will be able to maintain high A with minimal negative adjustment of photosynthetic capacity following prolonged exposure to elevated CO₂ on N-poor soils.     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

Exploitation of N2-fixation capacity for enrichment of anoxygenic photosynthetic bacteria in ecological studies

Abstract A procedure is described for the selective enrichment of anoxygenic photosynthetic bacteria from diverse natural sources containing numerous types of microorganisms. The enrichment medium contains a mixture of organic acid carbon sources, and the conditions used favor the growth of organisms that can multiply relatively rapidly anaerobically with N₂ as the nitrogen source and light as the source of growth energy; the development of oxygenic cyanobacteria is effectively excluded.