Acetylene Reduction Assay Research Articles

Spatial and temporal variability in nitrogenase activity and diazotrophic community composition in coastal microbial mats

Coastal microbial mats are highly diverse micro-scale ecosystems in an environment characterized by strongly fluctuating conditions. Nitrogen (N) fixation is an important process in these often N-limited ecosystems. We investigated the characteristics of N2 fixation and the composi- tion of the N2-fixing (diazotrophic) community in 3 microbial mats situated along an intertidal gradi- ent over a period of 3 yr by using the acetylene reduction assay and by analyzing more than 800 envi- ronmental nifH sequences. In 2008, nitrogenase activity (NA) in the 3 mat types differed with respect to the daily pattern and daily integrated rates (ranging from 5 to almost 150 mmol C2H4 m -2 d -1 ). The diazotrophic community composition in the 3 mat types was also dissimilar. The nifH clone libraries at the location that was most influenced by freshwater contained mostly cyanobacterial and alphaproteobacterial sequences, whereas the other 2 locations were dominated by gamma- and deltaproteobacterial diazotrophs. These differences were attributed to the specific environmental conditions at each location resulting from their position along the intertidal gradient and likely caused the differences in the recorded patterns of N2 fixation. Moreover, in each of the mat types, a dramatic shift in the diazotrophic community was observed over a period of 2 to 3 yr. These shifts var- ied greatly between the locations. The characteristics of NA at the same location in different years also varied considerably. N2 fixation in these microbial mats was spatially and temporally variable, which was attributed to the shifts in diazotrophic community composition along the littoral gradient and during the 3 yr of investigation.

Clear increases in acetylene reduction by soil bacteria from an East Siberian Taiga forest bed under conditions mimicking the natural soil environment

In boreal forest ecosystems, nitrogen imbalance has long been discussed as a mystery with regard to its high biomass production. Under conditions widely used for the soybean nodulation bacterium Bradyrhizobium japonicum, microbiota in forest bed soil often do not show any significant acetylene reduction. However, in a recent study, soil bacterial microbiota in an East Siberian Taiga forest bed and bacteria isolated from the soil show remarkable acetylene reduction in nitrogen-poor soft-gel medium solidified with gellan gum instead of agar. Thus, we precisely investigated optimal culture conditions including compositions of the medium; appropriate conditions for the soil microbiota were determined as follows: 0.5 g L−1 D-mannitol as the carbon source, 10–15°C incubation temperature and medium pH 6.2. These culture conditions resembled the physicochemical conditions of the active layer in the East Siberian boreal forest bed on the permafrost. In fact, mannitol, which was the most appropriate carbon source, was obtained as a major sugar component of the cowberry root mats that cover the larch forest bed in this region. The most profitable concentration of D-mannitol (0.5 g L−1) for acetylene reduction was only 1/20 or 1/30 of the amount of the carbon source used in worldwide acetylene reduction assays. In a similar manner, 10–15°C, which was the most profitable incubation temperature range, was close to the reported temperature of the active layer of the bulk soil in summer. us, even in a 3.0 g L−1 gellan gum medium, an acetylene reduction assay under the imitated soil conditions led to clear increases in acetylene reduction of the bacterial communities tested. The N2-fixing potential of boreal forest bed soil had thus ever been evaluated unfairly low, because the world wide assay conditions were too far from the soil environments.

EFFECT OF TEMPERATURE ON THE SENSITIVITY OF NITROGENASE TO OXYGEN IN TWO HETEROCYSTOUS CYANOBACTERIA1

The effect of temperature and oxygen on nitrogenase activity in two heterocystous cyanobacteria, Anabaena variabilis Kütz. ATCC29413 and Nostoc sp. PCC7120, was investigated. The cyanobacteria were grown under a 12:12 light:dark (L:D) cycle at 27°C and were subsequently exposed to different temperatures (27, 36, 39, and 42°C) at different steady‐state O2 concentrations (20, 10, 5, 0%). Light response curves of nitrogenase activity were recorded under each of these conditions using an online acetylene reduction assay combined with a sensitive laser photoacoustic ethylene detection method. The light response curves were fitted with the rectangular hyperbola model from which the model parameters Nm, Nd, and α were derived. In both strains, nitrogenase activity (Ntot = Nm + Nd) was the highest at 39°C–42°C and at 0% O2. The ratio Ntot/Nd was 4.1 and 3.1 for Anabaena and Nostoc, respectively, indicating that respectively 25% and 33% of nitrogenase activity was supported by respiration (Nd). Ntot/Nd increased with decreasing O2 concentration and with increasing temperature. Hence, each of these factors caused a relative increase in the light‐driven nitrogenase activity (Nm). These results demonstrate that photosynthesis and respiration both contribute to nitrogenase activity in Anabaena and Nostoc and that their individual contributions depend on both O2 concentration and temperature as the latter may dynamically alter the flux of O2 into the heterocyst.

ISOLATION OF CALOTHRIX RHIZOSOLENIAE (CYANOBACTERIA) STRAIN SC01 FROM CHAETOCEROS (BACILLARIOPHYTA) SPP. DIATOMS OF THE SUBTROPICAL NORTH PACIFIC OCEAN1

In November 2004, Chaetoceros spp. (diatom) cells were collected from 5 m at Station ALOHA (22º45′ N, 158º0′ W) in the subtropical North Pacific Ocean. Attached to the spines of several Chaetoceros spp. were symbiotic heterocystous cyanobacterial cells, identified as Calothrix rhizosoleniae Lemmerm. The symbiotic diatom cells were handpicked and placed in N-deplete media. The host cells have been absent since isolation, and the cyanobacterial symbiont has persisted in a free-living state in two different liquid and solid media: one made from artificial seawater, and another with natural seawater. All media are devoid of fixed inorganic nitrogen. Trichome length varied from three to four vegetative cells when growing colonially, to >10 vegetative cells when growing as single trichomes. The DNA and amino acid sequences of the narB, rbcL, and rnpB genes are most similar to those from other heterocystous cyanobacteria (Anabaena and Fischerella). Acetylene reduction (AR) assays were run in conjunction with multiplexing quantitative reverse transcription–PCR (qRT–PCR) assays. Gene transcription for rbcL and nifH was high, coincident with maximum AR, and occurred in the middle of the photoperiod. Eight irradiance curves of nitrogenase activity at varying biomass concentrations showed evidence of photoinhibition at high light intensities. Here, we report on the genetic identification and photophysiology for the first symbiotic isolate, Ca. rhizosoleniae SC01, of an open-ocean diatom (Chaetoceros).

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5–7.5 μmol C 2H 4 h −1 g −1, comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.

Methodological Underestimation of Oceanic Nitrogen Fixation Rates

The two commonly applied methods to assess dinitrogen (N2) fixation rates are the 15N2-tracer addition and the acetylene reduction assay (ARA). Discrepancies between the two methods as well as inconsistencies between N2 fixation rates and biomass/growth rates in culture experiments have been attributed to variable excretion of recently fixed N2. Here we demonstrate that the 15N2-tracer addition method underestimates N2 fixation rates significantly when the 15N2 tracer is introduced as a gas bubble. The injected 15N2 gas bubble does not attain equilibrium with the surrounding water leading to a 15N2 concentration lower than assumed by the method used to calculate 15N2-fixation rates. The resulting magnitude of underestimation varies with the incubation time, to a lesser extent on the amount of injected gas and is sensitive to the timing of the bubble injection relative to diel N2 fixation patterns. Here, we propose and test a modified 15N2 tracer method based on the addition of 15N2-enriched seawater that provides an instantaneous, constant enrichment and allows more accurate calculation of N2 fixation rates for both field and laboratory studies. We hypothesise that application of N2 fixation measurements using this modified method will significantly reduce the apparent imbalances in the oceanic fixed-nitrogen budget.

Response of the floating aquatic fern Azolla filiculoides to elevated CO2, temperature, and phosphorus levels

Azolla filiculoides is a floating aquatic fern growing in tropical and temperate freshwater ecosystems. As A. filiculoides has symbiotic nitrogen-fixing cyanobacteria (Anabaena azollae) within its leaf cavities, it is cultivated in rice paddies to improve N availability and suppress other wetland weeds. To understand how C assimilation and N accumulation in A. filiculoides respond to elevated atmospheric carbon dioxide concentration (CO2) in combination with P addition and higher temperatures, we conducted pot experiments during the summer of 2007 and 2008. In 2007, we grew A. filiculoides in pots at two treatment levels of added P fertilizer and at two levels of [CO2] (380 ppm for ambient and 680 ppm for elevated [CO2]) in controlled-environment chambers. In 2008, we grew A. filiculoides in four controlled-environment chambers at two [CO2] levels and two temperature levels (34/26°C (day/night) and 29/21°C). We found that biomass and C assimilation by A. filiculoides were significantly increased by elevated [CO2], temperature, and P level (all P < 0.01), with a significant interaction between elevated [CO2] and added P (P < 0.01). Tissue N content was decreased by elevated [CO2] and increased by higher temperature and P level (all P < 0.01). The acetylene reduction assay showed that the N-fixation activity of A. filiculoides was not significantly different under ambient and elevated [CO2] but was significantly stimulated by P addition. N-fixation activity decreased at higher temperatures (34/26°C), indicating that 29/21°C was more suitable for A. azollae growth. Therefore, we conclude that the N accumulation potential of A. filiculoides under future climate warming depends primarily on the temperature change and P availability, and C assimilation should be increased by elevated [CO2].

Wetland response to sedimentation and nitrogen loading: diversification and inhibition of nitrogen‐fixing microbes

Anthropogenic inputs of nutrients and sediment simultaneously impact coastal ecosystems, such as wetlands, especially during storms. Independent and combined effects of sediment and ammonium nitrate loading on nitrogen fixation rates and diversity of microbes that fix nitrogen (diazotrophs) were tested via field manipulations in Spartina foliosa and unvegetated zones at Tijuana Estuary (California, USA). This estuary is subject to episodic nitrogen enrichment and sedimentation associated with rain-driven flooding and slope instabilities, the latter of which may worsen as the Triple Border Fence is constructed along the U.S.-Mexico border. Responses of diazotrophs were assessed over 17 days using acetylene reduction assays and genetic fingerprinting (terminal restriction fragment length polymorphism [T-RFLP]) of nifH, which codes for dinitrogenase reductase. Sulfate-reducing bacteria performed approximately 70% of nitrogen fixation in Spartina foliosa rhizospheres in the absence of nitrogen loading, based on sodium molybdate inhibitions in the laboratory. Following nutrient additions, richness (number of T-RFs [terminal restriction fragments]) and evenness (relative T-RF fluorescence) of diazotrophs in surface sediments increased, but nitrogen fixation rates decreased significantly within 17 days. These responses illustrate, within a microbial community, conformance to a more general ecological pattern of high function among assemblages of low diversity. Diazotroph community composition (T-RF profiles) and rhizosphere diversity were not affected. Pore water ammonium concentrations were higher and more persistent for 17 days in plots receiving sediment additions (1 cm deep), suggesting that recovery of diazotroph functions may be delayed by the combination of sediment and nutrient inputs. Nitrogen fixation constitutes a mechanism for rapid transfer of fixed N to S. foliosa roots and a variety of primary consumers (within 3 and 8 days, respectively), as determined via 15N2 enrichment studies with in situ microcosms of intact marsh sediment. Thus, long-term declines in nitrogen fixation rates in response to increasingly frequent nutrient loading and sedimentation may potentially alter nitrogen sources for vascular plants as well as trophic pathways in wetland ecosystems.

Biological dinitrogen fixation by selected soil cyanobacteria as affected by strain origin, morphotype, and light conditions

The potential for N(2) fixation by heterocystous cyanobacteria isolated from soils of different geographical areas was determined as nitrogenase activity (NA) using the acetylene reduction assay. Morphology of cyanobacteria had the largest influence on NA determined under light conditions. NA was generally higher in species lacking thick slime sheaths. The highest value (1446 nmol/h C(2)H(4) per g fresh biomass) was found in the strain of branched cyanobacterium Hassalia (A Has1) from the polar region. A quadratic relationship between NA and biomass was detected in the Tolypothrix group under light conditions. The decline of NA in dark relative to light conditions ranged from 37 to 100 % and differed among strains from distinct geographical areas. Unlike the NA of temperate and tropical strains, whose decline in dark relative to light was 24 and 17 %, respectively, the NA of polar strains declined to 1 % in the dark. This difference was explained by adaptation to different light conditions in temperate, tropical, and polar habitats. NA was not related to the frequency of heterocysts in strains of the colony-forming cyanobacterium Nostoc. Colony morphology and life cycle are therefore more important for NA then heterocyst frequency. NA values probably reflect the environmental conditions where the cyanobacterium was isolated and the physiological and morphological state of the strain.

Influence of organic fertilization on the number of culturable diazotrophic endophytic bacteria isolated from sugarcane

The numbers of culturable diazotrophic endophytic bacteria (CDEB) from roots, stems and leaves of sugarcane submitted to organic, inorganic or no fertilization were compared. In order to determine the size of the N 2 fixing populations, the Most Probable Number technique (MPN) was used. The quantification of diazotrophic bacteria by using the acetylene reduction assay (ARA) was more accurate than observing the bacterial growth in the vials; to confirm N 2 fixing capability, the detection of gene nifH was performed on a sample of 105 isolated bacteria. The production of extracellular enzymes involved in the penetration of the plants by the bacteria was also studied. The results showed that organic fertilization enhances the number of CDEB when compared with conventional fertilization used throughout the growing season. The maximum number of bacteria was detected in the roots. Roots and stems presented the greatest number of CDEB in the middle of the cropping season and in leaves numbers varied according to the treatment. Using two pairs of primers and two different methods, the nifH gene was found in 104 of the 105 tested isolates. Larger amounts of pectinase were released by isolates from sugarcane treated with conventional fertilizers (66%), whereas larger amounts of cellulase were released by strains isolated from sugarcane treated with organic fertilizers (80%).

Rhizobacteria as bioenhancer and biofertilizer for growth and yield of banana ( Musa spp. cv. ‘Berangan’)

Banana, an important fruit crop, requires high amounts of fertilizers for commercial cultivation, which is costly and can be hazardous to the environment when used excessively. Inoculations of plant growth promoting rhizobacteria (PGPR) have been shown to produce beneficial effects through growth stimulation in legumes and cereals, and an attempt has been made to use rhizobacteria in bananas. Three experiments were conducted to observe the effects of plant growth promoting rhizobacteria (PGPR) inoculation on root stimulation and colonization, nutrient absorption, growth and yield of bananas ( Musa spp. cv. ‘Berangan’, AA type). The results showed that inoculation together with 33% N improved the bioenhancing activity by increasing root and shoot growth, and photosynthetic rate. The PGPR inoculation with 33% N-fertilizer also increased the Ca uptake capacity resulting in higher Ca concentration in root, corm and pulp but increased the Mg concentration in the root only. In addition, the total accumulation of nutrients was heavily influenced by PGPR inoculation due to enhanced root proliferation. The PGPR inoculation greatly increased the bunch yield (35–51%). The strains Sp7 and UPMB10 were evaluated for their N 2 fixing capacities in association with banana roots by 15N isotopic dilution technique and acetylene reduction assay (ARA). The results conclusively showed that roots of PGPR-inoculated plants fixed N 2 and produced higher ARA values and inoculated plants together with the least N-fertilizer supply showed the highest amount of nitrogen derived from atmosphere (Ndfa). The findings from the above studies demonstrated that PGPR strains (Sp7 and UPMB 10) inoculation with minimal N-fertilizer supply are effective as a bioenhancer for increasing plant growth, nutrient uptake and yield of bananas.

Ectopic expression of hetP can partially bypass the need for hetR in heterocyst differentiation by Anabaena sp. strain PCC 7120

Differentiation of nitrogen-fixing cells, called heterocysts, by the cyanobacterium Anabaena sp. strain PCC 7120 requires HetR, which is considered the master regulator of heterocyst differentiation. In this study, ectopic expression of hetP from an inducible promoter was shown to partially bypass the need for hetR in heterocyst differentiation. A hetR-deletion strain with hetP expressed ectopically produced cells morphologically similar to heterocysts that produced exopolysaccharide and glycolipids specific to heterocysts, and nitrogenase activity was present under anaerobic conditions, as indicated by an acetylene reduction assay. Five potential transcription start points or transcript processing sites located at positions -727, -545, -208, -177 and -12 bp relative to the putative translational start site were identified in the promoter region of hetP. Relative levels of transcription and their location in filaments resembled that previously reported for HetR protein in various genetic backgrounds, and an inverted repeat sequence in the promoter of hetP was necessary for binding of HetR. Two of the three identified homologues of hetP found in PCC 7120 partially complemented a hetP-null mutant. Taken together, these results suggest that HetP functions directly downstream of HetR in the regulatory network responsible for heterocyst differentiation.

Nutrient enrichment in tropical wetlands: shifts from autotrophic to heterotrophic nitrogen fixation

We used established long-term experimental P-amended plots in freshwater marshes of northern Belize to determine the impact of P input on nitrogen (N) fixation. Marshes with different conductivities and sulfate concentrations were selected to elucidate the effect of salinity and the contribution of sulfur reducing bacteria to the overall N fixation. Rates of N fixation in sediment, roots, and cyanobacterial mats was measured in laboratory incubation experiments (acetylene reduction assay calibrated by 15N2 reduction assay) with and without the addition of sodium molybdate (sulfur reducing bacteria inhibitor). P has increased macrophyte primary production significantly, which led to the rapid elimination of cyanobacterial mats and the elimination of autotrophic N fixation. P addition enhanced heterotrophic N fixation in both the sediments and rhizosphere due primarily to increased C supply to the sediment. When expressed on a dry weight basis, root associated N fixation was higher than sediment N fixation, but the contribution of the root associated fixation to the total N fixation was small when expressed per square meter. Sulfur reducing bacteria were an important component of N fixation, contributing from 20 to 53% to the overall N fixation. A simple N budget was created to determine if N demands are met following P addition. The heterotrophic N fixation substituted in part for autotrophic cyanobacterial N fixation when P limitation was alleviated.

Protection of free-living nitrogen-fixing bacteria within the soil matrix

Habitat based protection gives soil microbial communities the ability to withstand unfavourable environmental periods. Soils differ in their ability to support and maintain microbial functions through their variation in physical and chemical properties. This paper explores the use of biocidal treatments to assess the ability of soils to protect N 2-fixing communities from adverse conditions. The acetylene reduction assay was used to measure nitrogenase activity by free-living bacteria in soil exposed to additional C, and biocidal treatments (steam or CHCl 3). N 2-fixing microbial communities in soils with higher clay contents (∼50%) withstood fumigation treatment, demonstrated through acetylene reduction activity and a nifH PCR response, whereas those in sandy soils failed to survive. Based on this study and other research on soil aggregates, it was concluded that microbial survival in different soils was related to the ability of aggregates to provide protection against biocidal exposure within the soil matrix. This has consequences for the amount and duration N 2-fixing activities in soils exposed to adverse environmental conditions.

Nitrogen fixation by a marine non‐heterocystous cyanobacterium requires a heterotrophic bacterial consort

Cultures of the non-heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro-oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark-mediated nitrogenase in the absence of DCMU nor light-mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non-axenic cultures of L. nodulosa. RT-PCR demonstrated that this gene was expressed only in non-axenic cultures. Western blotting showed that the Fe-protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.

Nitrogen fixation by the verrucomicrobial methanotroph ‘Methylacidiphilum fumariolicum’ SolV

The ability to utilize atmospheric nitrogen (N(2)) as a sole nitrogen source is an important trait for prokaryotes. Knowledge of N(2) fixation by methanotrophs is needed to understand their role in nitrogen cycling in different environments. The verrucomicrobial methanotroph 'Methylacidiphilum fumariolicum' strain SolV was investigated for its ability to fix N(2). Physiological studies were combined with nitrogenase activity measurements and phylogenetic analysis of the nifDHK genes, encoding the subunits of the nitrogenase. 'M. fumariolicum' SolV was able to fix N(2) at low oxygen (O(2)) concentration (0.5 %, v/v) in chemostat cultures. This low oxygen concentration was also required for an optimal nitrogenase activity [47.4 nmol ethylene h(-1) (mg cell dry weight)(-1)]. Based on acetylene reduction assay and growth experiments, the nitrogenase of strain SolV seems to be extremely oxygen sensitive compared to most proteobacterial methanotrophs. The activity of the nitrogenase was not inhibited by ammonium concentrations up to 94 mM. This is believed to be the first report on the physiology of N(2) fixation within the phylum Verrucomicrobia.

Hydrogen production by nitrogenase as a potential crop rotation benefit

Hydrogen production by nitrogenase as a potential crop rotation benefit

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well-defined levels of oxygen to which the organisms are exposed, using an online and near real-time acetylene reduction assay combined with sensitive laser-based photoacoustic ethylene detection. The cultures were grown under an alternating 12-12 h light-dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 mumol m(-2) s(-1) provided at the same light-dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5-7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29 degrees C. At 31 degrees C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41 degrees C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.

Wetland response to sedimentation and nitrogen loading: diversification and inhibition of nitrogen-fixing microbes

Anthropogenic inputs of nutrients and sediment simultaneously impact coastal ecosystems, such as wetlands, especially during storms. Independent and combined effects of sediment and ammonium nitrate loading on nitrogen fixation rates and diversity of microbes that fix nitrogen (diazotrophs) were tested via field manipulations in Spartina foliosa and unvegetated zones at Tijuana Estuary (California, USA). This estuary is subject to episodic nitrogen enrichment and sedimentation associated with rain-driven flooding and slope instabilities, the latter of which may worsen as the Triple Border Fence is constructed along the U.S.–Mexico border. Responses of diazotrophs were assessed over 17 days using acetylene reduction assays and genetic fingerprinting (terminal restriction fragment length polymorphism [T-RFLP]) of nifH, which codes for dinitrogenase reductase. Sulfate-reducing bacteria performed ~70% of nitrogen fixation in Spartina foliosa rhizospheres in the absence of nitrogen loading, based on sodium molybdate inhibitions in the laboratory. Following nutrient additions, richness (number of T-RFs [terminal restriction fragments]) and evenness (relative T-RF fluorescence) of diazotrophs in surface sediments increased, but nitrogen fixation rates decreased significantly within 17 days. These responses illustrate, within a microbial community, conformance to a more general ecological pattern of high function among assemblages of low diversity. Diazotroph community composition (T-RF profiles) and rhizosphere diversity were not affected. Pore water ammonium concentrations were higher and more persistent for 17 days in plots receiving sediment additions (1 cm deep), suggesting that recovery of diazotroph functions may be delayed by the combination of sediment and nutrient inputs. Nitrogen fixation constitutes a mechanism for rapid transfer of fixed N to S. foliosa roots and a variety of primary consumers (within 3 and 8 days, respectively), as determined via 15N2 enrichment studies with in situ microcosms of intact marsh sediment. Thus, long-term declines in nitrogen fixation rates in response to increasingly frequent nutrient loading and sedimentation may potentially alter nitrogen sources for vascular plants as well as trophic pathways in wetland ecosystems.

Comparison of Nitrogen Fixation for North- and South-facing Robinia pseudoacacia Stands in Central Korea

The nitrogenase activity, root nodule biomass, and rates of nitrogen (N) fixation were measured in 25-year-old pure north- and south-facing Robinia pseudoacacia stands in an urban forest of Seoul (Kkachisan Mountain) in central Korea. The nitrogenase activity was estimated using an acetylene reduction (AR) assay, which showed an increasing trend during the early growing season, with sustained high rates from June through to September with a decrease thereafter. July had the highest nitrogenase activity rate (micromoles C2H4 per gram dry nodule per hour), averaging 95.8 and 115.1 for the north- and south-facing stands, respectively. The maximum root nodule biomass (kilograms per hectare) was 45.7 and 9.1 for the north- and south-facing stands in July, respectively. The AR rate appeared to be strongly correlated to the soil temperature (r 2 = 0.68, P < 0.001) and soil pH (r 2 = 0.59, P < 0.001) while root nodule biomass was correlated to the soil temperature (r 2 = 0.36, P < 0.01) and water content (r 2 = 0.35, P < 0.05). The soil temperature showed clear differences between seasons, while there was a significant difference in soil pH, organic matter, total N concentrations, and available phosphorus between the north- and south-facing stands. The N2 fixation rates during the growing season varied from 0.1 to 37.5 kg N ha−1 month−1 depending on the sampling location and time. The annual N2 fixation rate (kg N per hectare per year) was 112.3 and 23.2 for the north- and south-facing stands, respectively. The differences in N2 fixation rate between the two stands were due mainly to the differences in total nodule biomass.