Acetylene Reduction Rates Research Articles

Nitrogen fixation and nifH gene diversity in cyanobacteria living on feather mosses in a subalpine forest of Mt. Fuji

In the boreal forests, feather mosses such as Hylocomium splendens and Pleurozium schreberi are colonized by cyanobacteria, which provide large amounts of nitrogen to forest ecosystems through nitrogen fixation. Although these feather mosses are also ubiquitous in subalpine forests of East Asia, little is known regarding their associated cyanobacteria and their ability to fix nitrogen. In this study, we investigated (1) whether cyanobacteria co-exist and fix nitrogen in the two species of feather mosses that cover the ground surface in a subalpine forest of Mt. Fuji, (2) whether cyanobacteria belonging to a common cluster with boreal forests are found in feather mosses in Mt. Fuji, and (3) whether moss-associated nitrogen fixation rates differed among moss growing substrates, canopy openness, and moss nitrogen concentrations in the same forest area. Our results showed that cyanobacteria colonized feather mosses in the subalpine forests of Mt. Fuji and acetylene reduction rates as an index of nitrogen fixation tended to be higher in H. splendens than in P. schreberi. Based on analysis of the nifH gene, 43 bacterial operational taxonomic units (OTUs) were identified, 28 of which represented cyanobacteria. Among the five clusters of cyanobacteria classified based on their nifH gene and identified in northern Europe, four (Nostoc cluster I, Nostoc cluster II, Stigonema cluster, and nifH2 cluster) were also found at Mt. Fuji. The acetylene reduction rate differed depending on the moss growing substrate and the total nitrogen concentration of moss shoots, and a strong negative correlation was observed with the total nitrogen concentration.

Influence of environmental factors and exogenous nitrogen on nitrogen fixation by intact thallus and excised cephalodia of Peltigera aphthosa

The present study investigates the effects of various abiotic environmental factors: air humidity, moisture content, oxygen and exogenously supplied nitrogen on acetylene reduction by intact thallus and excised cephalodia of Peltgera aphthosa. Intact thallus and excised cephalodia of Peltigera aphthosa were incubated at various conditions of air humidity, moisture contents, oxygen tensions, and addition of exogenous nitrogen, and comparative nitrogen fixation by the intact thallus and excised cephalodia was used as a method for assessment. Acetylene reduction (nitrogen fixation) was enhanced at conditions of lowered oxygen tension and at initial addition of nitrogen to the medium. However, prolonged incubation of Peltigera aphthosa in the medium with combined nitrogen addition resulted in fluctuation of nitrogenase synthesis. Acetylene reduction rates were stimulated in an atmosphere of 100% relative humidity (RH) and moisture content range of 570-620% of dry weight of Peltigera aphthosa. The decrease of nitrogenase activity measurable by acetylene reduction of thallus after prolonged incubation in the medium with combined nitrogen addition shows susceptibility of the symbiosis (lichen) since the thallus showed signs of disintegration at this time. Furthermore, air humidity and moisture content of the thallus influenced nitrogenase synthesis of Peltigera aphthosa considerably on separation of cephalodia such approach decreased nitrogenase activity and also elicited differences in their response to the various treatments. Applicability of this technology would enhance plant sustainability and yield in agricultural farms.

Immobilized heterocysts as microbial factories for sustainable nitrogen fixation

A novel thin-layer biocatalyst for photosynthetic N2 fixation and H2 photoproduction was assembled using a Ca2+-alginate matrix and heterocysts isolated from wild-type Anabaena sp. PCC 7120 filaments. Compared to suspension heterocysts, heterocysts entrapped in Ca2+-alginate films showed improved stability of the nitrogenase system. While suspension heterocysts lost nitrogenase activity within 24 h, immobilized heterocysts supported nitrogenase activity for up to 125 h. The maximum specific rate of acetylene reduction was the same in both cases (∼0.4 μmol C2H2 mg Chl−1 h−1), but the catalyst with entrapped heterocysts required a much longer time to achieve the maximum rate (60 h instead of 3 h in suspension). Simultaneously with acetylene reduction, the immobilized heterocysts were able to photoproduce H2 for 125 h, yielding up to 1.1 mmol H2 mg Chl−1. The absence of acetylene increased the H2 photoproduction rate to a maximum of 25–30 μmol H2 mg Chl−1 h−1, and the catalyst was capable of H2 photoproduction for 190 h, yielding up to 2.5 mmol H2 mg Chl−1. The recovery of the catalyst with entrapped heterocysts was achieved through placing the cells in a N2 atmosphere for 24 h. This engaged a second cycle of H2 photoproduction, which lasted for another 240 h (10 days), thus yielding ∼3 mmol H2 mg Chl−1 in total after 454 h. Together, these findings demonstrate great potential for a heterocyst-based thin-layer platform for the sustainable production of chemicals and biofuels.

Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum.

Symbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) in Sinorhizobium meliloti Rm1021 dct mutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF with Medicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-type S. melilotiRhizobium leguminosarum bv. viciae 3841 dct mutants unable to transport C4-dicarboxylates but expressing the maeP transporter had strong symbiotic properties, with Pisum sativum plants inoculated with these strains appearing similar to plants inoculated with wild-type R. leguminosarum This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combined P. sativum-R. leguminosarum nodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses, l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCE Symbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

Nitrogenase activity by biological soil crusts in cold sagebrush steppe ecosystems

In drylands worldwide, biological soil crusts (BSC) form a thin photosynthetic cover across landscapes, and provide vital benefits in terms of stabilizing soil and fixing nitrogen (N) and carbon (C). Numerous studies have examined the effects of climate and disturbance on BSC functions; however, few have characterized these responses in rolling BSCs typical of northern ecosystems in the Intermountain West, US. With temperature increases and shifts in precipitation projected, it is unclear how BSCs in this region will respond to climate change, and how the response could affect their capacity to perform key ecosystem functions, such as providing ‘new’ N through biological N2 fixation. To address this important knowledge gap, we examined nitrogenase activity (NA) associated with rolling BSCs along a climatic gradient in southwestern Idaho, US, and quantified how acetylene reduction rates changed as a function of climate, grazing (using exclosures), and shrub-canopy association. Results show that warmer, drier climates at lower elevations hosted greater cover of late successional BSC communities (e.g., mosses and lichens), and higher NA compared with colder, wetter climates at higher elevations. Highest NA (0.5–29.3 µmol C2H4 m−2 h−1) occurred during the early summer/spring, when water was more available than in late summer/autumn. Activity was strongly associated with soil characteristics including pH and ammonium concentrations suggesting these characteristics as potentially strong controls on NA in BSCs. The relationship between grazing and NA varied with elevation. Specifically, lower elevation sites had lower NA at grazed locations, whereas higher elevation sites had higher NA with grazing. At both low and high ends of the elevation gradient, shrub-canopy associated BSCs maintained two to three times higher NA compared to BSCs in the interspace among shrubs. Taken together, our findings indicate that the controls and rates of NA in BSCs vary seasonally and strongly with climate in the Intermountain West, and that drier springs are likely to influence rates of NA more than warmer summers.

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.

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.

Effect of sulfur dioxide (SO2) on growth and physiological activity in Alnus sieboldiana at Miyakejima Island in Japan

AbstractWe used a physiological approach to investigate the effect of sulfur dioxide (SO2) on Alnus sieboldiana Matsum. (A. sieboldiana) and symbiotic Frankia spp. in Miyakejima Island, which was devastated by volcanic action in July of 2000. In April of 2008, three study sites were chosen in the forest of Miyakejima Island, and were categorized as high, medium, or low in relation to their sulfur dioxide concentration (volcanic gas). Nine A. sieboldiana trees were selected from naturally regenerated forest at each site. The higher the SO2 concentration at the study site, the lower was the photosynthetic rate and the maximum photochemical efficiency of PSII (Fv/Fm) of A. sieboldiana and its apparent quantum yield. The acetylene reduction rate of the Frankia spp. symbiotic with A. sieboldiana was significantly less at the high SO2 concentration (p<0.05). The nitrogen concentration in their leaves remained high, however. There was no significant difference in the chlorophyll concentration (Chl a + b) in leaves from the three sites. The photosynthetic nitrogen use efficiency decreased significantly in sites with a high SO2 concentration, however. As a result, A. sieboldiana regenerated at the sites with high or medium SO2 concentration underwent approximately 50–70 % less growth than at the site with a low SO2 concentration.

Competition for Ammonia Influences the Structure of Chemotrophic Communities in Geothermal Springs

Source waters sampled from Perpetual Spouter hot spring (pH 7.03, 86.4°C), Yellowstone National Park, WY, have low concentrations of total ammonia, nitrite, and nitrate, suggesting nitrogen (N) limitation and/or tight coupling of N cycling processes. Dominant small-subunit rRNA sequences in Perpetual Spouter source sediments are closely affiliated with the ammonia-oxidizing archaeon "Candidatus Nitrosocaldus yellowstonii" and the putatively nitrogen-fixing (diazotrophic) bacterium Thermocrinis albus, respectively, suggesting that these populations may interact at the level of the bioavailable N pool, specifically, ammonia. This hypothesis was evaluated by using a combination of geochemical, physiological, and transcriptomic analyses of sediment microcosms. Amendment of microcosms with allylthiourea, an inhibitor of ammonia oxidation, decreased rates of acetylene reduction (a proxy for N2 fixation) and nitrite production (a proxy for ammonia oxidation) and decreased transcript levels of structural genes involved in both nitrogen fixation (nifH) and ammonia oxidation (amoA). In contrast, amendment of microcosms with ammonia stimulated nitrite production and increased amoA transcript levels while it suppressed rates of acetylene reduction and decreased nifH transcript levels. Sequencing of amplified nifH and amoA transcripts from native sediments, as well as microcosms, at 2 and 4 h postamendment, indicates that the dominant and responsive populations involved in ammonia oxidation and N2 fixation are closely affiliated with Ca. Nitrosocaldus yellowstonii and T. albus, respectively. Collectively, these results suggest that ammonia-oxidizing archaea, such as Ca. Nitrosocaldus yellowstonii, have an apparent affinity for ammonia that is higher than that of the diazotrophs present in this ecosystem. Depletion of the bioavailable N pool through the activity of ammonia-oxidizing archaea likely represents a strong selective pressure for the inclusion of organisms capable of nitrogen fixation in geothermal communities. These observations help to explain the strong pattern in the codistribution of ammonia-oxidizing archaea and diazotrophs in circumneutral-to-alkaline geothermal springs.

Seasonal patterns of nitrogen fixation in biological soil crusts from British Columbia’s Chilcotin grasslands

This study provides estimates of N2 fixation in biological soil crusts (BSCs) from shrub–steppe grasslands in British Columbia’s Chilcotin plateau. We identify key seasonal periods and environmental conditions during episodes of BSC nitrogen-fixation activity. Predicted nitrogen-fixation activity showed two peak periods: one during late winter and early spring snowmelt events, and the second during summer precipitation events. BSC nitrogen fixation, especially summer activity, was quite variable from year to year. The magnitude of predicted nitrogen fixation at the landscape level was dependent upon the ratio used to convert rates of acetylene reduction to nitrogen fixed. Using a theoretical ratio of 3:1, estimates of average annual BSC nitrogen fixation were 1.0 kg N·ha−1. However, using a conversion ratio of 0.06, obtained from side-by-side measurements of acetylene reduction and 15N uptake, annual estimates are 52 kg N·ha−1. These estimates are based on assessments of the aerial extent of dark BSC communities, which at the Farwell Canyon study site exceeded 50%. The fate of fixed nitrogen, especially during the late winter period, when underlying soils are frozen, remains unclear.

Effect of the acidity and chemical nature of the protonating agent on the rate of acetylene reduction catalyzed by the nitrogenase active site isolated from the enzyme

The effect of the acidity (pK a) of the source of protons on the rate and selectivity of acetylene reduction has been investigated in order to elucidate the mechanism of protonation of substrate molecules coordinated to the reduced FeMoco cluster. A number of compounds whose pK a in DMF varies between 6 and 20 have been examined as protonating agents. The rate of the reaction is almost independent of the acidity of the proton donor in a wide pK a range. This can be explained in terms of the specific features of substrate protonation catalyzed by iron-sulfur clusters. Active protonating agents in the system are those which react with the catalyst to form hydrogen-bonded association species or those which are ligands reversibly binding to the cluster and are capable of donating protons, likely with simultaneous electron transfer.

Comparative ecophysiology of three Placopsis species, pioneer lichens in recently exposed Chilean glacial forelands

Lichen species belonging to the genus Placopsis are early colonisers on snow free moraines of exposed land surfaces in the subantarctic region of Tierra de Fuego, South Chile. The physiological performance of three co-occurring species, P. pycnotheca, (terricolous), and P. perrugosa, and P. stenophylla (both saxicolous) was studied. All, possess green algal photobionts but have cyanobacteria in cephalodia. It was found that there was (i) a strong positive correlation between the acetylene reduction rate (AR) and the maximum photosynthetic rate (Amax), between the N content and the AR rate, and between the N and P contents, and (ii) the relationship between the CO2-exchange rates and the responses obtained in the laboratory reflected the ecology of these three lichens in the field. The results provide new information about the dynamics of some of the fastest growing crustose lichens. We hypothesize that the performance of these three species may have developed as a response to growing in an unstable environment that resulted from frequent glacial fluctuations.

Cyanolichens: a link between the phosphorus and nitrogen cycles in a Hawaiian montane forest

Abstract:Low phosphorus (P) supply frequently has been shown to limit the abundance and activity of nitrogen (N)-fixing organisms, potentially constraining N inputs to ecosystems. Previous research in a montane Hawaiian forest has shown that ground-level P-fertilization led to significant increases in the population size of epiphytic N-fixing lichens (cyanolichens), as well as a shift in community composition from crustose to leafy species. In this study, we ask whether these changes in the cyanolichen community have resulted in increased N inputs to the forest, and also whether the very high levels of P in the canopy of P-fertilized forest stimulate individual lichen fixation rates over those of lichens from a nearby unfertilized reference forest. We used acetylene reduction (AR) assays to measure the fixation rates of 14 cyanolichen species from P-fertilized forest, and calibrated these rates by measuring15N2fixation incorporation in four species. We found that the ratio of acetylene reduced to N fixed ranged from 2.4 ± 0.4 inPseudocyphellaria crocatato 9.3 ± 2.4 inLeptogium denticulatum. Nitrogen fixation rates in the P-fertilized forest ranged from 0.64 ± 0.05 nmol N cm−2h−1inNephroma helveticumto 3.97 ± 1.48 nmol N cm−2h−1inParmeliella nigrocincta. Fixation rates did not vary greatly among species from P-fertilized forest. We compared these P-fertilized rates to those of 10 species from the reference forest, and found that mass-based fixation rates of P-fertilized lichens were not greater than those of lichens from the unfertilized forest. Using the measured AR rates, we estimate that the P additions increase cyanolichen N inputs to the forest 30-fold, from ~0.3 kg N ha−1y−1to ~9 kg N ha−1y−1. These results suggest that P additions to this ecosystem increase N inputs primarily by increasing the abundance of cyanolichens, and that shifts in cyanolichen community composition and changes in individual fixation rate were of lesser importance in determining ecosystem N inputs.

Peanut plant growth and yield as influenced by co-inoculation with Bradyrhizobium and some rhizo-microorganisms under sandy loam soil conditions

The ability of tested rhizomicrobial isolates (Serratiamarcescens and Trichoderma harzianum) along with a strain of root nodule bacteria (Bradyrhizobium spp.) to exhibit some PGP-properties was evaluated in vitro conditions. The main PGP-properties, namely the ability to solubilize-P and production of IAA, as well as production of siderophores and HCN were examined. Additionally, field trials were conducted on sandy loam soil at El-Tahrir Province during two successive summer seasons to study the effect of co-inoculation with Bradyrhizobium either individually or together with S.marcescens and/or T. harzianum on nodulation, some plant growth characters, peanut yield and its yield components. The in vitro experiment revealed that all of the tested microorganisms were apparently able to trigger PGP-properties. Phosphate solubilization was the common feature of the employed microorganisms. However, T. harzianum appeared to be superior to other microorganisms, and Bradyrhizobium displayed the lowest capacity. The ability of the microorganisms to produce indole compounds showed that S.marcescens was more effective in IAA production and followed by Bradyrhizobium. Capacity of S.marcescens and T. harzianum to excrete ferric-specific ligands (siderophores) and HCN was detected, while Bradyrhizobium failed to produce such compounds. Results of field trials showed that the uninoculated peanut had the least nodulation status, N2-ase activity and all vegetative growth characters in both studied seasons. Bacterization of peanut seeds with bradyrhizobia exerted considerable improvement in number and mass of root nodules, increased the rate of acetylene reduction and all growth characters in comparison to the uninoculated control. The synergy inoculation between bradyrhizobia and any of the tested microorganisms led to further increases of all mentioned characters and strengthened the stimulating effect of the bacterial inoculation. However, the promotive action on peanut nodulation, N2-fixation performance and vegetative characters was obvious with the dual inoculation with Bradyrhizobium plus S.marcescens. The other tested treatments, Bradyrhizobium conjugated with Trichoderma or with a mixture of the examined microorganisms, occupied the second rank. Additionally, peanut yield (pods and straw) and it’s components (seed and straw protein contents, hundred seed weight, as well as pod and seed weight/plant and shelling%) along the two consecutive seasons followed a similar pattern to that of the vegetative growth stage.

Short-term effects of flooding on bacterial ­community structure and nitrogenase activity in microbial mats from a desert stream

Flood-induced disturbance causes major shifts in the diversity and function of microbial communities in desert streams. We compared bacterial community structure (using automated rRNA intergenic spacer analysis, ARISA), pigment composition and nitrogen fixation rates in benthic micro- bial mats from stone surfaces collected from 6 sites along a desert stream in the Sultanate of Oman before and 2 wk after an intense flood. Flooding caused partial flushing out of old mats, and new mats re-established on the surfaces of rocks. Visually, the filamentous green algae Spirogyra spp. in - creased in abundance in the water column at most sites after the flood, and direct microscopy revealed that the newly developed mats were dominated by the heterocystous Calothrix spp. Multi- variate analyses of ARISA profiles revealed that, in each mat, the structure of the bacterial commu- nity was different after the flood (analysis of similarities (ANOSIM), R = 0.49, p < 0.001), and pairwise comparison of the presence/absence of operational taxonomic units (OTUs) showed that only between 18 and 26% shared OTUs. The OTU richness in the newly developed microbial communi- ties decreased in 6 mats after the flood, but remained similar or slightly increased in the other 6 mats. Out of the 9 detected pigments, chlorophyll a, scytonemin, fucoxanthin, diadinoxanthin and β- carotene showed site- specific changes with flooding. The average acetylene reduction rates increased after the flood at all sites; however, this increase was statistically significant at only 2 sites due to the high variance among samples. In conclusion, flooding resulted in the replacement of over 74% of the microbial communities within mats, while nitrogen fixation rates remained stable or increased.

Biological nitrogen fixation in acidic high‐temperature geothermal springs in Yellowstone National Park, Wyoming

The near ubiquitous distribution of nifH genes in sediments sampled from 14 high-temperature (48.0-89.0°C) and acidic (pH 1.90-5.02) geothermal springs in Yellowstone National Park suggested a role for the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) (e.g. nitrogen fixation or diazotrophy) in these environments. nifH genes from these environments formed three unique phylotypes that were distantly related to acidiphilic, mesophilic diazotrophs. Acetylene reduction assays and (15) N(2) tracer studies in microcosms containing sediments sampled from acidic and high-temperature environments where nifH genes were detected confirmed the potential for biological N(2) reduction in these environments. Rates of acetylene reduction by sediment-associated populations were positively correlated with the concentration of NH(4)(+), suggesting a potential relationship between NH(4)(+) consumption and N(2) fixation activity. Amendment of microcosms with NH(4)(+) resulted in increased lag times in acetylene reduction assays. Manipulation of incubation temperature and pH in acetylene reduction assays indicated that diazotrophic populations are specifically adapted to local conditions. Incubation of sediments in the presence of a N(2) headspace yielded a highly enriched culture containing a single nifH phylotype. This phylotype was detected in all 14 geothermal spring sediments examined and its abundance ranged from ≈ 780 to ≈ 6800 copies (g dry weight sediment)(-1), suggesting that this organism may contribute N to the ecosystems. Collectively, these results for the first time demonstrate thermoacidiphilic N(2) fixation in the natural environment and extend the upper temperature for biological N(2) fixation in terrestrial systems.

In vitro creation of artificial nitrogen fixing Cyanobacterium (Nostoc muscorum) association with wheat

Using sonication technique and addition of 2,4 dichlorophenoxyacetic acid to wheat roots, increasing colonization of Nostoc muscorum on roots and enhancement of nitrogen fixation as novel associations between nitrogen fixing Nostoc muscorum and nonlegume wheat, had been occurred. Sonication of roots (10 and 20 s) enhanced abundance of N. muscorum within / on the root tissue.The N. muscorum penetrated the roots in the form of motile filaments (hormogonia) and once inside, they divided and transformed into aseriate packages. Sonication technique does not have significant effect on root growth, and there are no significant differences in shoot growth. Treatment of wheat roots with low concentrations of 2,4 dichlorophenoxyacetic acid (0.5, 1.0 and 3.0 ppm) developed nodule like structures (para-nodules) mainly along primary roots. N. muscorum colonized the paranodules externally at the junction of the paranodules. Addition of 2,4 dichlorophenoxyacetic acid significantly enhanced the rate of acetylene reduction of inoculated plants, and also abundance of N. muscorum on 2,4 dichlorophenoxyacetic acid induced wheat root compared to untreated wheat roots. Reduction of the growth of both root and shoot occured at a tested concentrations of 2,4 dichlorophenoxyacetic acid. Nitrogenase activity of N. muscorum co-cultivated with wheat roots was increased up to 1.9 times in the absence of combined nitrogen (nitrates) compared with nitrate treated plants, and also abundance of N. muscorum on roots increased in absence of combined nitrogen (nitrates). Results of this study revealed that N. muscorum is a promising organism for achieving efficient association between N2 fixing cyanobacteria and nonlegumes by using sonication technique or 2,4 dichlorophenoxyacetic acid at low concentrations. Key words: Nostoc muscorum, sonication, 2,4 dichlorophenoxyacetic acid (2,4-D), nonlegume.

The effect of abiotic factors on asymbiotic nitrogen fixation in dead roots from the Pacific Northwest

High rates of asymbiotic nitrogen fixation have been observed in dead roots. However, little is known about how biotic and abiotic factors influence asymbiotic nitrogen fixation in dead roots. The objective of this study was to examine how nitrogen fixation in dead roots responded to three abiotic factors: temperature, oxygen concentration, and moisture content by measuring acetylene reduction rates in dead roots. For measuring the response to temperature and oxygen, we collected dead roots of Douglas-fir (Pseudotsuga menziesii) from one site in western Oregon, while the effect of moisture content was tested with samples from three species including Douglas-fir, western hemlock (Tsuga heterophylla), and Ponderosa pine (Pinus ponderosa) from three sites in western Oregon. We measured acetylene reduction rates at 5, 13, 20, 30, and 40°C and at oxygen concentrations of 0, 1, 2, 4, 8, and 21% in dead root samples that had been previously wetted. Unaltered root samples with different moisture contents were used to measure the effect of moisture content on acetylene reduction. The optimal temperature for acetylene reduction in roots was 20°C with an acetylene reduction rate of 9.6 nmol/g/d and a standard error of 3.2 nmol/g/d. Asymbiotic nitrogen fixation was greatly reduced above and below this temperature with acetylene reduction rates of 1.2 ± 0.6 nmol/g/d at 5°C and 0.2 ± 0.2 nmol/g/d at 40°C. The highest acetylene reduction rates (13.8 ± 4.2 nmol/g/d) occurred at an oxygen concentration of 2%. Rates dropped sharply under no oxygen condition (3.0 ± 2.8 nmol/g/d) and at an oxygen concentration of 21% (3.0 ± 0.8 nmol/g/d). Optimal acetylene reduction activity occurred above 60% root moisture content and activity was near zero below 60%. These abiotic factors have a similar influence on asymbiotic nitrogen fixation activity in other substrates like dead wood. In the Pacific Northwest, annual nitrogen fixation activity in dead roots tends to be primarily controlled by temperature while above-ground substrates like dead wood can have reduced activity during the dry summer months when lower moisture contents and higher oxygen concentrations can reduce activity.

The effect of abiotic factors on asymbiotic nitrogen fixation in dead roots from the Pacific Northwest

AbstractHigh rates of asymbiotic nitrogen fixation have been observed in dead roots. However, little is known about how biotic and abiotic factors influence asymbiotic nitrogen fixation in dead roots. The objective of this study was to examine how nitrogen fixation in dead roots responded to three abiotic factors: temperature, oxygen concentration, and moisture content by measuring acetylene reduction rates in dead roots. For measuring the response to temperature and oxygen, we collected dead roots of Douglas-fir (Pseudotsuga menziesii) from one site in western Oregon, while the effect of moisture content was tested with samples from three species including Douglas-fir, western hemlock (Tsuga heterophylla), and Ponderosa pine (Pinus ponderosa) from three sites in western Oregon. We measured acetylene reduction rates at 5, 13, 20, 30, and 40°C and at oxygen concentrations of 0, 1, 2, 4, 8, and 21% in dead root samples that had been previously wetted. Unaltered root samples with different moisture contents were used to measure the effect of moisture content on acetylene reduction. The optimal temperature for acetylene reduction in roots was 20°C with an acetylene reduction rate of 9.6 nmol/g/d and a standard error of 3.2 nmol/g/d. Asymbiotic nitrogen fixation was greatly reduced above and below this temperature with acetylene reduction rates of 1.2 ± 0.6 nmol/g/d at 5°C and 0.2 ± 0.2 nmol/g/d at 40°C. The highest acetylene reduction rates (13.8 ± 4.2 nmol/g/d) occurred at an oxygen concentration of 2%. Rates dropped sharply under no oxygen condition (3.0 ± 2.8 nmol/g/d) and at an oxygen concentration of 21% (3.0 ± 0.8 nmol/g/d). Optimal acetylene reduction activity occurred above 60% root moisture content and activity was near zero below 60%. These abiotic factors have a similar influence on asymbiotic nitrogen fixation activity in other substrates like dead wood. In the Pacific Northwest, annual nitrogen fixation activity in dead roots tends to be primarily controlled by temperature while above-ground substrates like dead wood can have reduced activity during the dry summer months when lower moisture contents and higher oxygen concentrations can reduce activity.

Hydrogen cycling by the unicellular marine diazotroph Crocosphaera watsonii strain WH8501.

The hydrogen (H₂) cycle associated with the dinitrogen (N₂) fixation process was studied in laboratory cultures of the marine cyanobacterium Crocosphaera watsonii. The rates of H₂ production and acetylene (C₂H₂) reduction were continuously measured over the diel cycle with simultaneous measurements of fast repetition rate fluorometry and dissolved oxygen. The maximum rate of H₂ production was coincident with the maximum rates of C₂H₂ reduction. Theoretical stoichiometry for N₂ fixation predicts an equimolar ratio of H₂ produced to N₂ fixed. However, the maximum rate of net H₂ production observed was 0.09 nmol H₂ μg chlorophyll a (chl a)⁻¹ h⁻¹) compared to the N₂ fixation rate of 5.5 nmol N₂ μg chl a⁻¹ h⁻¹, with an H₂ production/N₂ fixation ratio of 0.02. The 50-fold discrepancy between expected and observed rates of H₂ production was hypothesized to be a result of H₂ reassimilation by uptake hydrogenase. This was confirmed by the addition of carbon monoxide (CO), a potent inhibitor of hydrogenase, which increased net H₂ production rates ∼40-fold to a maximum rate of 3.5 nmol H₂ μg chl a⁻¹ h⁻¹. We conclude that the reassimilation of H₂ by C. watsonii is highly efficient (> 98%) and hypothesize that the tight coupling between H₂ production and consumption is a consequence of fixing N₂ at nighttime using a finite pool of respiratory carbon and electrons acquired from daytime solar energy capture. The H₂ cycle provides unique insight into N₂ fixation and associated metabolic processes in C. watsonii.