Acetylene Reduction Assay Research Articles

Anthropogenic impacts on nitrogen fixation rates between restored and natural Mediterranean salt marshes

AbstractTo test the effects of site and successional stage on nitrogen fixation rates in salt marshes of the Venice Lagoon, Italy, acetylene reduction assays were performed with Salicornia veneta‐ and Spartina townsendii‐vegetated sediments from three restored (6–14 years) and two natural marshes. Average nitrogen fixation (acetylene reduction) rates ranged from 31 to 343 μmol C2H4·m−2·h−1 among all marshes, with the greatest average rates being from one natural marsh (Tezze Fonde). These high rates are up to six times greater than those reported from Southern California Spartina marshes of similar Mediterranean climate, but substantially lower than those found in moister climates of the Atlantic US coast. Nitrogen fixation rates did not consistently vary between natural and restored marshes within a site (Fossei Est, Tezze Fonde, Cenesa) but were negatively related to assayed plant biomass within the acetylene reduction samples collected among all marshes. Highest nitrogen fixation rates were found at Tezze Fonde, the location closest to the city of Venice, in both natural and restored marshes, suggesting possible site‐specific impacts of anthropogenic stress on marsh succession.

Studies on nitrogenase activity of diazotrophic isolates from different rice production systems

The present study was carried out to evaluate the nitrogen fixing ability of diazotrophs isolated from the rhizosphere soils of rice which were grown in three different rice growing systems. A total of hundred and ten isolates obtained were subjected to Acetylene Reduction Assay (ARA) and ninety eight isolates recorded significant amount of nitrogenase activity in a range of 185.73 to 3794.55 nmoles of ethylene mg of protein-1 h-1. The highest nitrogenase activity was recorded by Derxia (3794.55 nmoles of ethylene mg of protein-1 h-1) isolated from Trichy (lowland). Among the three different rice production systems, isolates obtained from lowland rice (Derxia – 3794.5 nmoles of ethylene mg of protein-1 h-1) recorded higher nitrogenase activity followed by Aerobic (Pseudomonas - 2194.89 nmoles of ethylene mg of protein-1 h-1) and SRI (Azotobacter - 1971.85 nmoles of ethylene mg of protein-1 h-1) rice isolates. The results revealed marked variation in the ARA of the diazotrophic isolates obtained from lowland, SRI and Aerobic rice. The nitrogenase activity of diazotrophs from rice fields have been reported earlier but the nitrogenase activity of diazotrophs from three different rice production systems from various parts of Tamil Nadu is reported for the first time from India.

English

Two “dissimilar”“bacteria” were “isolated” from a “ Barak Valley Tea garden”, both were “poles apart” in their natures. One, “isolated variety” wa s “plucked” from the “surrounding” where there is “soil root interaction section”, it was “growned ” in a “nitrogen”. “deprived”“medium”. The “isolated variety” was then taken upon for its “N-g luing competency” through a well definite “system” of “Acetylene Reduction Assay”. Another “isolate” was “plucked” in same way, from t he same “site” and in similar manner and then the “detached bacteria” was arranged to “incul cate” on another “medium” at which it was preordained to be “infused”. “In the end” the “phys ico-chemical behavior” for that “another” “isolate” was made “reachable”. Alongside, the “bot h of them” were accomplished for “widespread” of “antibiotics”.

Variation in N2 Fixation in Subarctic Tundra in Relation to Landscape Position and Nitrogen Pools and Fluxes

ABSTRACTBiological N2 fixation in high-latitude ecosystems usually exhibits low rates but can significantly contribute to the local N budget. We studied N2 fixation in three habitats of East European subarctic tundra differing in soil N stocks and fluxes: N-limited vegetated peat plateau (PP), frost formations of bare peat called “peat circles” (PC) with high availability of soil N, and vegetated upland tundra (UT) with low to intermediate N-availability. Nitrogen fixation was measured at field conditions twice during summer 2011 by acetylene reduction assay, and N2 fixation rates were verified by 15N2 fixation assay. Response to variation in nutrients, carbon, and temperature was studied in complementary laboratory experiments. Further, we aimed to link N2 fixation rates to N deposition and major N transformation rates (gross and net mineralization, plant N uptake) including high N2O emissions recently found from PC. We hypothesized that N2O emissions in PC were fueled partly by biologically fixed N. Contrary to that hypothesis, N2 fixation was found solely in PP (0.01–0.76 mg N m−2 d−1), where N2 was fixed by moss-associated cyanobacteria and heterotrophic soil bacteria. The low N and high P availability corresponded with the occurrence of N2 fixation in these soils. Nitrogen fixation represented only a small portion of plant N uptake in PP. Conversely, bare PC (as well as vegetated UT) lacked N2 fixation and thus N2O efflux is most likely fueled by release of mineral N to the soil through internal nutrient cycling.

Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer.

Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N2 -fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P.flexilis-AAB association is consistent across years, we re-sampled P.flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N2 , we incubated twigs with (13) N2 -enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using (13) N. We used the acetylene reduction assay to test for nitrogenase activity within P.flexilis twigs four times from June to September. We found evidence for N2 fixation in P.flexilis foliage. N2 diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8-13.6μgNm(-2) d(-1) to P.flexilis stands. AAB dominated the P.flexilis needle endophyte community. We propose that foliar endophytes represent a low-cost, evolutionarily stableN2 -fixing strategy for long-lived conifers. This novel source of biological N2 fixation has fundamental implications for understanding forest N budgets.

Alternative nitrogenase activity in the environment and nitrogen cycle implications

Biological nitrogen fixation, the main natural input of fixed nitrogen into the biosphere, is catalyzed by Mo-, V-, or Fe-only nitrogenase metalloenzymes. Although “alternative” V- and Fe-only nitrogenase genes are found in many environments, the contribution of these isoenzymes to N2 fixation is unknown. Here we present a new method (ISARA, isotopic acetylene reduction assay) that distinguishes canonical Mo and alternative nitrogenase activities based on in vivo 13C fractionation of acetylene reduction to ethylene (13eMo = 13.1–14.7 ‰, 13eV = 7.5–8.8 ‰, 13eFe = 5.8–6.5 ‰). ISARA analyses indicate significant contributions of alternative nitrogen fixation in boreal cyanolichens and salt marshes (~10–40 % acetylene reduction, ~20–55 % N2 fixed). These results affect the quantitative interpretation of natural abundance 15N data or traditional acetylene reduction assays. They also invite a reexamination of the conditions under which the different nitrogenase isozymes are active and suggest significant interactions between the cycles of nitrogen and trace metals.

Cultivable Nitrogen Fixing Bacteria from Extremely Alkaline-Saline Soils

Three soils with different Electrolytic Conductivity (EC) from the former Lake Texcoco (soil with low EC 1.2 dS·m-1, medium with EC 83.1 - 107.8 dS·m-1, and high with EC 137.3 - 152.5 dS•m-1) were used to isolate nitrogen-fixing bacterial strains through enrichment cultures in nitrogen-free media. The medium and high EC in the soil affected negatively the nitrogen-fixing activity, which was generally ten times lower compared to the activity in the soil with low EC. Twenty-one bacterial strains were isolated, identified and characterized for their nitrogen fixation capacity. The diazotrophic genetic potential of all isolates was confirmed by amplification and sequencing of partial nifH and nifD genes and diazotrophic activity quantified by the acetylene reduction assay. Azospirillum brasilense, and several species of Paenibacillus (P. fujiensis, P. durus, P. borealis, P. graminis, P. massiliensis and P. wynnii) were identified. Isolates belonging to the Paenibacillus genus were found in the three soils. Paenibacillus fujiensis and P. durus showed a high nitrogenase activity. The phylograms based on nifH and nifD gene sequences were consistent with 16S rRNA gene phylogeny.

Characterization of N2-fixing plant growth promoting endophytic and epiphytic bacterial community of Indian cultivated and wild rice (Oryza spp.) genotypes

The diversity of endophytic and epiphytic diazotrophs in different parts of rice plants has specificity to the niche (i.e. leaf, stem and root) of different genotypes and nutrient availability of the organ. Inoculation of the indigenous, polyvalent diazotrophs can facilitate and sustain production of non-leguminous crops like rice. Therefore, N2-fixing plant growth promoting bacteria (PGPB) were isolated from different parts of three Indian cultivated [Oryza sativa L. var. Sabita (semi deep/deep water)/Swarna (rain fed shallow lowland)/Swarna-Sub1(submergence tolerant)] and a wild (O. eichingeri) rice genotypes which respond differentially to nitrogenous fertilizers. Thirty-five isolates from four rice genotypes were categorized based on acetylene reduction assay on nitrogenase activity, biochemical tests, BIOLOG and 16S rRNA gene sequencing. The bacteria produced 9.36-155.83 nmole C2H4 mg(-1) dry bacteria h(-1) and among them nitrogenase activity of 11 potent isolates was complemented by nifH-sequence analysis. Phylogenetic analysis based on 16S rDNA sequencing divided them into five groups (shared 95-100 % sequence homology with type strains) belonging to five classes-alpha (Ancylobacter, Azorhizobium, Azospirillum, Rhizobium, Bradyrhizobium, Sinorhizobium, Novosphingobium, spp.), beta (Burkholderia sp.), gamma (Acinetobacter, Aeromonas, Azotobacter, Enterobacter, Klebsiella, Pantoea, Pseudomonas, Stenotrophomonas spp.) Proteobacteria, Bacilli (Bacillus, Paenibacillus spp.) and Actinobacteria (Microbacterium sp.). Besides, all bacterial strains possessed the intrinsic PGP traits of like indole (0.44-7.4 µg ml(-1)), ammonia (0.18-6 mmol ml(-1)), nitrite (0.01-3.4 mol ml(-1)), and siderophore (from 0.16-0.57 μmol ml(-1)) production. Inoculation of rice (cv. Swarna) seedlings with selected isolates had a positive impact on plant growth parameters like shoot and root elongation which was correlated with in vitro PGP attributes. The results indicated that the diverse polyvalent phytonic PGP bacteria, which may be exploited as bio-inoculants to improve rice production.

Phylogeny and nitrogen fixation potential of Bradyrhizobium species isolated from the legume cover crop Pueraria phaseoloides (Roxb.) Benth. in Eastern Cameroon

Pueraria is an herbaceous, perennial legume crop originating in Asia. Pueraria phaseoloides (Roxb.) Benth. (tropical kudzu) is frequently introduced into production systems and is used as green manure, a cover crop and a forage plant, making it important economically. We used P. phaseoloides as a trap crop to study and characterize soil rhizobia in Eastern Cameroon. Bacteria were isolated from fresh nodules collected from field-grown P. phaseoloides roots. The 16S-23S rRNA internal transcribed spacer (ITS) sequences from 30 bacterial isolates were amplified by polymerase chain reaction (PCR) and the reaction products were sequenced. Phylogenetic analysis revealed that all isolates were ascribed to the genus Bradyrhizobium and were grouped into three clusters of Bradyrhizobium sp. strains, one cluster of B. yuanmingense strains, and one cluster of B. elkanii strains. Acetylene reduction assay (ARA) results indicated that the B. yuanmingense strains had significantly higher nitrogen fixation potential and that they could be used as inoculants to enhance nitrogen fixation in Pueraria grown in Eastern Cameroon.

Degradation of kerosene hydrocarbon by indigenous diazotropic bacteria isolated from crude oil contaminated soil

Hydrocarbon contamination significantly increases supply of carbon, but the availability of nitrogen and phosphorus generally become the growth limiting factors for indigenous oil degraders. This nitrogen imbalance selectively favors the development of the soil asymbiotic dinitrogen fixing microorganisms. The present study was undertaken to isolate such diazotrophic bacteria capable of degrading petroleum hydrocarbons. Two bacterial strains were isolated, and identified by using their 16SrDNA as Achromobacter sp. and Arthrobacter sp . Both the isolates could degrade almost all major components of kerosene hydrocarbon within 60 days. Hydrocarbon degradation by the isolates was studied under Gas chromatography-Mass spectroscopy (GC-MS) and the nitrogenase activity of the isolates was measured by Acetylene Reduction Assay. Results of the present investigation revealed the potentiality of free living indigenous nitrogen fixing bacteria to degrade petroleum hydrocarbon and thereby contribute to bioremediation and bio-fertilization of crude oil contaminated soil.

Fungal endophyte symbiosis alters nitrogen source of tall fescue host, but not nitrogen fixation in co-occurring red clover

Infection of tall fescue with the common toxic fungal endophyte Epichloe coenophiala harms livestock via toxic alkaloid production; therefore, non-toxic ‘novel’ strains of the endophyte have been developed and released. How different endophyte strains impact biological nitrogen fixation (BNF) in mixed species pastures is unknown. We asked whether novel endophyte or common toxic endophyte-infected (NE+; CTE+) tall fescue affects symbiotic and non-symbiotic BNF, and utilization of biologically-fixed nitrogen in tall fescue. Tall fescue was planted either endophyte-free (E-), infected with CTE, two non-toxic strains AR542 NE, AR584 NE, or a blend of endophyte treatments. We measured natural abundance of 15 N in plant and soil samples, and conducted soil acetylene reduction assays. Endophyte presence and strain significantly affected the δ15N of tall fescue. Near red clover, CTE+ and AR584 NE+ tall fescue were most 15 N-depleted; but away, E- tall fescue was most 15 N-depleted. Endophyte strain significantly influenced N concentration in red clover, but not symbiotic or non-symbiotic BNF. Endophyte strains produce different effects on tall fescue’s competitive ability and nitrogen utilization. In mixed pastures, deployment of NE strains for decreased alkaloid toxicity will differentially impact use of biologically fixed nitrogen in tall fescue and nitrogen concentration in red clover.

Examining the impact of acetylene on N-fixation and the active sediment microbial community.

Here we examined the impact of a commonly employed method used to measure nitrogen fixation, the acetylene reduction assay (ARA), on a marine sediment community. Historically, the ARA technique has been broadly employed for its ease of use, in spite of numerous known artifacts. To gauge the severity of these effects in a natural environment, we employed high-throughput 16S rRNA gene sequencing to detect differences in acetylene-treated sediments vs. non-treated control sediments after a 7 h incubation. Within this short time period, significant differences were seen across all activity of microbes identified in the sediment, implying that the changes induced by acetylene occur quickly. The results have important implications for our understanding of marine nitrogen budgets. Moreover, because the ARA technique has been widely used in terrestrial and freshwater habitats, these results may be applicable to other ecosystems.

Nitrogenase Activity and Nodule Biomass of Cowpea (Vigna unguiculata L. Walp.) Decrease in Cover Crop Mixtures

Legume cover crops are often used to build soil nitrogen (N) fertility and there is increasing interest in cover crop mixtures. The objective of this mechanistic greenhouse study was to determine the effect of cover crop community diversity and soil fertility on nitrogenase activity and nodule biomass of cowpea. Cover crops were grown for 42–53 days, aboveground biomass was harvested, and nitrogenase activity was estimated with the acetylene reduction assay. Roots were then excavated to determine nodule and root biomass. Nitrogenase activity and nodule biomass per plant were greatest in cowpea monoculture and reduced by 71–98 percent in four-species mixtures. Reduced capacity for N2 fixation was partially driven by lower cowpea biomass in mixtures. The ratio of root nodule / shoot biomass increased by 81–297 percent in low-fertility relative to high-fertility soils, which contributed to increased nitrogenase activity. Results suggest cowpea monocultures in low-fertility soils have the greatest potential for N2 fixation.

Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus.

The Amazonian catfish, Panaque nigrolineatus, consume large amounts of wood in their diets. The nitrogen-fixing community within the gastrointestinal (GI) tract of these catfish was found to include nifH phylotypes that are closely related to Clostridium sp., Alpha and Gammaproteobacteria, and sequences associated with GI tracts of lower termites. Fish fed a diet of sterilized palm wood were found to contain nifH messenger RNA within their GI tracts, displaying high sequence similarity to the nitrogen-fixing Bradyrhizobium group. Nitrogenase activity, measured by acetylene reduction assays, could be detected in freshly dissected GI tract material and also from anaerobic enrichment cultures propagated in nitrogen-free enrichment media; nifH sequences retrieved from these cultures were dominated by Klebsiella- and Clostridium-like sequences. Microscopic examination using catalyzed reporter deposition-enhanced immunofluorescence revealed high densities of nitrogenase-containing cells colonizing the woody digesta within the GI tract, as well as cells residing within the intestinal mucous layer. Our findings suggest that the P. nigrolineatus GI tract provides a suitable environment for nitrogen fixation that may facilitate production of reduced nitrogen by the resident microbial population under nitrogen limiting conditions. Whether this community is providing reduced nitrogen to the host in an active or passive manner and whether it is present in a permanent or transient relationship remains to be determined. The intake of a cellulose rich diet and the presence of a suitable environment for nitrogen fixation suggest that the GI tract microbial community may allow a unique trophic niche for P. nigrolineatus among fish.

Elevated, but highly variable, acetylene reduction in soils associated with the invasive shrub Rhamnus cathartica in a Midwestern oak forest

Common buckthorn (Rhamnus cathartica) is an important invasive shrub in North American forests, where it is thought that the plant’s retention of green leaves during autumn canopy senescence helps it succeed in deciduous understory habitat. This trait results in loss of nitrogen (N) in N-rich leaf litter, which has led some workers to suggest buckthorn may foster associative N-fixation in soil. We examined this possibility in an oak woodland understory in eastern Minnesota using the acetylene reduction assay to compare apparent nitrogenase activity in soils collected from beneath buckthorn individuals with soils collected from a canopy species (Quercus spp.), an important understory shrub (Prunus serotina), and non-vegetated areas. Buckthorn and non-buckthorn soils differed in variability of acetylene reduction (AR) rates, with buckthorn values covering a range 10× the range of non-buckthorn soils. Mean AR also differed between buckthorn and non-buckthorn soils, but the direction and magnitude of the difference varied with sampling location. Estimates of N inputs calculated from our data suggest that AR values at the high end of the buckthorn-associated range are biologically significant. Our results represent the first measurement of AR activity associated with common buckthorn, and are consistent with the hypothesis that this plant supports associative N fixation under some conditions. Suggestions for follow-up studies are provided.

Isolation and identification of indigenous plant growth promoting rhizobacteria from Himalayan region of Kashmir and their effect on improving growth and nutrient contents of maize (Zea mays L.).

Introduction and exploitation of plant growth promoting rhizobacteria (PGPR) in agro-ecosystems enhance plant–microbes interactions that may affect ecosystems sustainability, agricultural productivity, and environmental quality. The present study was conducted to isolate and identify PGPRs associated with maize (Zea mays L.) from twenty sites of Himalayan region of Hajira-Rawalakot, Azad Jammu and Kashmir (AJK), Pakistan. A total of 100 isolates were isolated from these sites, out of which eight (HJR1, HJR2, HJR3, HJR4, HJR5, MR6, HJR7, HJR8) were selected in vitro for their plant growth promoting ability (PGPA) including phosphorus solubilization, indole-3-acetic acid (IAA) production and N2 fixation. The 16S rRNA gene sequencing technique was used for molecular identity and authentication. Isolates were then further tested for their effects on growth and nutrient contents of maize (Z. mays L.) under pouch and pot conditions. The 16S rRNA gene sequencing and phylogenetic analysis identified these isolates belong to Pseudomonas and Bacillus genera. The isolates promoted plant growth by solubilizing soil P which ranged between 19.2 and 35.6 μg mL-1. The isolates HJR1, HJR2, HJR3, and HJR5 showed positive activity in acetylene reduction assay showing their N2-fixation potential. All eight isolates showed the potential to produce IAA in the range of 0.9–5.39 μg mL-1 and promote plant growth. Results from a subsequent pot experiment indicated PGPRs distinctly increased maize shoot and root length, shoot and root dry weight, root surface area, leaf surface area, shoot and root N and P contents. Among the eight isolates, HR3 showed a marked P-solubilizing activity, plant growth-promoting attributes, and the potential to be developed as a biofertilizers for integrated nutrient management strategies.

Significant nonsymbiotic nitrogen fixation in Patagonian ombrotrophic bogs.

Nitrogen (N) nutrition in pristine peatlands relies on the natural input of inorganic N through atmospheric deposition or biological dinitrogen (N2 ) fixation. However, N2 fixation and its significance for N cycling, plant productivity, and peat buildup are mostly associated with the presence of Sphagnum mosses. Here, we report high nonsymbiotic N2 -fixation rates in two pristine Patagonian bogs with diversified vegetation and natural N deposition. Nonsymbiotic N2 fixation was measured in samples from 0 to 10, 10 to 20, and 40 to 50cm depth using the (15) N2 assay as well as theacetylene reduction assay (ARA). The ARA considerably underestimated N2 fixation and can thus not be recommended for peatland studies. Based on the (15) N2 assay, high nonsymbiotic N2 -fixation rates of 0.3-1.4 μmolN2 g(-1) day(-1) were found down to 50cm under micro-oxic conditions (2 vol.%) in samples from plots covered by Sphagnum magellanicum or by vascular cushion plants, latter characterized by dense and deep aerenchyma roots. Peat N concentrations point to greater potential of nonsymbiotic N2 fixation under cushion plants, likely because of the availability of easily decomposable organic compounds and oxic conditions in the rhizosphere. In the Sphagnum plots, high N2 fixation below 10cm depth rather reflects the potential during dry periods or low water level when oxygen penetrates the top peat layer and triggers peat mineralization. Natural abundance of the (15) N isotope of live Sphagnum (5.6 δ‰) from 0 to 10cm points to solely N uptake from atmospheric deposition and nonsymbiotic N2 fixation. A mean (15) N signature of -0.7 δ‰ of peat from the cushion plant plots indicates additional N supply from N mineralization. Our findings suggest that nonsymbiotic N2 fixation overcomes N deficiency in different vegetation communities and has great significance for N cycling and peat accumulation in pristine peatlands.

Characterization and crop production efficiency of diazotrophic isolates from the rhizosphere of semi-arid tropical grasses of India

The search for diverse plant growth-promoting diazotrophic bacteria is gaining momentum as efforts are made to exploit them as bioinoculants for various crops. In particular, the use of strains with multiple plant growth promoting properties would help to increase crop productivity on a sustainable basis. This study investigated the effects of plant growth promoting potential of diazotrophs isolated from rhizosphere of semi-arid tropical grasses and evaluated their inoculation effects on the growth of rice plants under in vitro and in vivo conditions. The diazotrophic isolates from grass species were characterized for nitrogenase activity by acetylene reduction assay (ARA) and 16S rRNA gene sequencing. The ARA activity of the isolates ranged from 50.83 to 172.25nmolethylene/mgprotein/h and the putative diazotrophs from rhizosphere of grass species were identified by nifH gene amplification. The 16S rRNA gene sequence analysis identified the isolates as belonging to class of alpha Proteobacteria and Firmicutes. Plant growth promoting traits of all the selected diazotrophic isolates were analysed and results revealed that the diazotrophs were found to produce phytohormone, siderophores, HCN, solubilized minerals such as P, K and Zn. Diazotrophs also produced enzyme such as ACC deaminase that can modulate plant growth and development. Based on the presence of multiple plant growth promoting traits, the isolates were selected for inoculation studies. In gnotobiotic experiment, inoculation of diazotrophic isolates significantly improves the growth of rice. In the field experiment, Serratia sp. (CB2) and K. pneumoniae (CR3) treated plots, grain yields were recorded more by 31 and 28%, respectively, over yield obtained using full doses of fertilizers. This trait of improving growth parameters and yield of rice indicates that the diazotrophs isolated from grass species can be utilized as bioinoculant for rice.

Revisiting N₂ fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach.

Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N2 fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N2 fixation, whereas 15N2 tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of 15N2-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in 15N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% 15N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N2 fixation in the intertidal mats, whereas support for significant N2 fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.

Free-living nitrogen fixation responds to elevated nutrient inputs in tropical montane forest floor and canopy soils of southern Ecuador

Although often overlooked in forest research, the canopy can play an important role in forest nutrient cycling. Since the canopy is spatially isolated from the forest floor, nutrient cycling in the two areas may differ as terrestrial nutrients accumulate. We measured rates of free-living N2 fixation along an elevation gradient (1,000, 2,000 and 3,000 m) of tropical montane canopy soils, compared these to rates measured in the top 5 cm of forest floor soils (excluding fresh litter), and assessed the effects of elevated nutrient inputs to the forest floor. N2 fixation was measured using the acetylene reduction assay. Measurements occurred in the field, in the wet and dry seasons, using intact cores of soil. The forest floor had been fertilized biannually with moderate amounts of nitrogen (N) and phosphorus (P) for 4 years; treatments included control, N, P and N + P. N2 fixation rates exhibited little variation with elevation but were higher in the dry season than the wet season. Fixation was inhibited in forest floor N plots compared to control and P plots, and stimulated in canopy P plots compared to control. At 2,000 m, the canopy contributed 12 % of measured canopy and forest floor N2 fixation (1.2 kg N ha−1 year−1). Results suggest that N2 fixation is an active process in canopy soils, which is variable across seasons and sensitive to changes in terrestrial nutrient availability. Long-term terrestrial accumulation of N and/or P has the potential to significantly change the dynamics of soil N cycling in these canopies.