Acetylene Reduction Method Research Articles

Temporal variations of biological nitrogen fixation and diazotrophic communities associated with artificial seaweed farms

Diazotrophic communities contribute inorganic nitrogen for the primary productivity of the marine environment by biological nitrogen fixation (BNF). They play a vital role in the biogeochemical cycle of nitrogen in the marine ecological environment. However, there is still an incomplete understanding of BNF and diazotrophs in artificial seaweed farms. Therefore, this study comprehensively investigated the temporal variations of BNF associated with Gracilariopsis lemaneiformis, as well as the diazotrophic communities associated with macroalgae and its surrounding seawater. Our results revealed that a total of 13 strains belonging to Proteobacteria and Bacteroidetes were identified as N2-fixing bacteria using azotobacter selective solid medium and nifH gene cloning. Subsequently, BNF and diazotrophic communities were characterized using the acetylene reduction method and high-throughput sequencing of the nifH gene, respectively. The results showed that nitrogenase activity and nifH gene abundance of epiphytic bacteria on G. lemaneiformis varied significantly among four different cultivation periods, i.e., Cultivation Jan. (CJ), Cultivation Feb. (CF), Cultivation Mar. (CM), Cultivation Apr. (CA). Among them, the nitrogenase activity and nifH gene abundance of epiphytic bacteria on G. lemaneiformis in CM were significantly higher than those in CJ, CF, and CA, indicating that the BNF of eiphytic bacteria on G. lemaneiformis was markedly enhanced. Combined with the data on environmental factors, it was found that the low concentration of nitrogen and phosphorus in CM might considerably boost the BNF of epiphytic bacteria in G. lemaneiformis. The sequencing results of the nifH gene showed that the α-diversity of diazotrophic communities associated with G. lemaneiformis and seawater in CM was higher than that in other cultivation periods. In addition, the diazotrophic communities on G. lemaneiformis were significantly different in CJ, CF, CM, and CA, and they were significantly diverse from diazotrophic communities in seawater. LEfSe analysis indicated that Rhodobacterales, Hyphomonadaceae, Robiginitomaculum, and Robiginitomaculum antarcticum within α-proteobacteria played a remarkable role in BNF in response to nitrogen nutrient deficiency. Taken together, these results provide a unique insight into the interaction between macroalgae and its epiphytic bacteria and lay a foundation for further research on the mechanism of action of nitrogen-cycling microorganisms associated with macroalgae.

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity

BackgroundBiological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166.ResultsTwenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain.ConclusionsThe endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Cyanobacteria and Algae in Biological Soil Crusts of Frost Boils in the Mountain Tundra of the Urals

Diversity of cyanobacteria and eukaryotic algae was recovered from biological soil crusts formed on bare spots in tundras of the Ural mountains. The research was carried out in two typical tundra types, dominated by shrub-moss-lichen and dwarf birch-lichen-moss communities. In total, 46 species belonging to five divisions were identified: Cyanobacteria (19), Ochrophyta (1), Bacillariophyta (2), Chlorophyta (22), Charophyta (2). Core species of the biological crusts come from Cyanobacteria (Stigonema minutum, Gloeocapsopsis magma, Schizothrix fuscescens, Dasygloea cf. lamyi, Fischerella muscicola, Nostoc commune, Scytonema hofmannii) and Chlorophyta (Sporotetras polydermatica, Coccomyxa simplex, Elliptochloris bilobata, E. subsphaerica, Lobochlamys culleus, Pleurastrum terricola). The low diversity of algae and cyanobacteria results from harsh environmental conditions of their habitat: extreme fluctuation of upper ground temperatures, acidic soils with low base content, and reduced levels of nitrogen, phosphorus and other crucial biogenic elements. The total number of cyanobacterial and algal cells per g of soil was estimated at 0.03 to 34.19 million. Based on the acetylene reduction method, the average nitrogen fixation rates in biological crusts ranged from 0.009 to 0.015 mg С2Н4 m–2h–1. The activity varied between soil crust types from 1.48 to 2.25 mg С2Н4 m–2h–1. In future, regular observations are planned on the studied sites to reconstruct and predict succession processes in cryogenic landscapes under mountain tundra conditions.

Effect of Phosphorus Supply Levels on Nodule Nitrogen Fixation and Nitrogen Accumulation in Soybean (Glycine max L.)

The specific mechanism by which phosphorus affects nodule nitrogen fixation and nitrogen absorption in soybeans remains inconclusive. To further quantitatively analyze the effect of phosphorus on nodule nitrogen fixation and nitrogen accumulation in soybeans, this experiment was carried out under sand culture conditions. The experiment consisted of six phosphorus supply levels (1 mg/L, 11 mg/L, 21 mg/L, 31 mg/L, 41 mg/L, 51 mg/L). The acetylene reduction method and 15N tracer method (50 mg/L (NH4)2SO4) were used to determine and analyze the nodule growth status, nodule nitrogenase activity, nitrogen content, and nodule nitrogen fixation rate at initial flowering (R1 stage), initial pod (R3 stage), seed filling (R5 stage) and maturity stages (R8 stage). The results are described as follows: 1. The nitrogen fixation of soybean nodules at different growth stages has different requirements for phosphorus supply levels. The initial flowering stage and seed-filling stage were 31 mg/L–41 mg/L, and the initial pod stage was 51 mg/L. 2. The nitrogen source in different parts of soybean showed different trends with different growth periods and phosphorus supply concentrations. Among them, from the initial flowering stage to the seed filling stage, the main body of the nitrogen supply of soybean shoots in the low phosphorus treatment (1 mg/L–31 mg/L) gradually changed from fertilizer nitrogen to nodule nitrogen fixation, while the main body of the nitrogen supply of soybean shoots in the high phosphorus treatment (41 mg/L–51 mg/L) always showed nodule nitrogen fixation and was transformed into fertilizer nitrogen at the mature stage. The main nitrogen supply to the roots of soybean at different levels of phosphorus supply from the initial flowering to the initial pods and maturity stage was fertilizer nitrogen, and the main nitrogen supply at the seed filling stage was nodule nitrogen fixation. The nitrogen supply to the main body of soybean nodules was constantly nodule nitrogen fixation. 3. Different phosphorus supply levels significantly affected the nitrogen fixation of soybean nodules (R2 ≥ 0.803), and both the acetylene reduction method and the 15N tracer method could be used to determine the nitrogen fixation capacity of soybean nodules. This study indicated the optimal phosphorus supply level of nodules in different growth stages of soybean and clarified the main body of phosphorus supply in different parts of soybean at different growth stages, which pointed out the direction for further improving the utilization efficiency of soybean nitrogen and phosphorus fertilizer.

Dried Nostoc commune exhibits nitrogen-fixing activity using glucose under dark conditions after rehydration

ABSTRACT Nostoc commune is an edible cyanobacterium that produces a massive gelatinous polysaccharide matrix around the filamentous cells. The polysaccharides, more than 70% of which comprise glucose, are essential for resistance to environmental stresses. In the present study, we collected naturally growing N. commune colonies, dried them for preservation, rehydrated them, and then examined their nitrogen-fixing activity using the acetylene reduction method. As expected, the rehydrated N. commune performed nitrogen fixation after illumination with white light. Notably, under dark, aerobic conditions, the rehydrated N. commune exhibited nitrogen fixation in the presence of glucose. In contrast, under dark, anaerobic conditions, nitrogen fixation was low. Because the natural habitats of N. commune are aerobic but lack carbohydrates, N. commune cells may exhibit glucose utilization activity constitutively.

Composition and Activity of N2-Fixing Microorganisms in Mangrove Forest Soils

Mangrove forests are considered to be a highly productive ecosystem, but they are also generally nitrogen (N)-limited. Thus, soil N2 fixation can be important for the stability of both mangrove ecosystem functions and upland N supply. This study evaluates the N2 fixation activity and composition of relevant microorganisms in two coastal mangrove forests—the Guandu mangrove in an upstream estuary and the Bali mangrove in a downstream estuary—using the acetylene reduction method, real-time polymerase chain reaction, and next-generation sequencing. The results demonstrated that ambient nitrogenase activity was higher in downstream mangrove forests (13.2–15.6 nmol h−1 g−1 soil) than in upstream mangrove forests (0.2–1.4 nmol h−1 g−1 soil). However, both the maximum potential nitrogenase activity and nitrogenase gene (nifH gene) copy number were found to be higher in the upstream than in the downstream mangrove forests, implying that the nitrogenase activity and diazotrophic abundance may not necessarily be positively correlated. In addition, amended MoO4 (which inhibits the activity of sulfate-reducing bacteria in N2-fixation) yielded low nitrogenase activity, and sulfate-reducing bacteria made up 20–50% of the relative diazotrophic abundance in the mangrove forests, indicating that these bacteria might be the major active diazotrophs in this environment.

Changes in the biological N2-fixation rates and diazotrophic community as vegetation recovers on abandoned farmland in a karst region of China

The successful regeneration of karst landscapes that have been severely degraded by intensive agriculture relies on the recovery of biological nitrogen fixation (BNF) by diazotrophic communities in soils after nitrogen (N) derived from synthetic fertilizers dissipates through the soil. It is important to understand the mechanisms that drive BNF because there must be enough bioavailable N in soils to support the recovery of native vegetation communities that are best adapted to erosion-prone karst landscapes. In this study, we quantified the response of the diazotrophic population in topsoil (0–10 cm deep) along a vegetation restoration gradient (sloping cropland > abandoned sloping cropland > secondary forest > primary forest) in the Puding Karst Critical Zone Observatory, Guizhou Province, southwest China from the absolute and specific BNF rates, determined using the acetylene reduction method and qPCR and MiSeq sequencing of nifH genes. The nifH phylogenetic diversities increased as the vegetation increased in maturity and were positively correlated with soil moisture and the N to phosphorus ratio (p < 0.05). The diazotrophic community structure changed with the vegetation recovery time (p < 0.05). Rhizobiales accounted for between 38% and 45% of the total nifH gene sequences and were the most abundant order in the vegetation that had been recovering for the longest time. We found that the soil bulk density was a strong control on the diazotroph community structure and was negatively correlated with Rhizobiales. The absolute and specific BNF rates were the highest in primary forest and abandoned sloping cropland, respectively (p < 0.05). The results from stepwise multiple regression showed that 50% of the variation in the absolute BNF rates was explained by SWC, and 55% of the variation in the nifH specific rates was explained by the available N contents and the diazotrophic community structure. We suggest that the recovery of degraded karst could be accelerated by managing the soil physical structure so that the environment is suitable for diazotrophic communities, which would then improve the BNF efficiency.

Assessing the Systematic Effects of the Concentration of Nitrogen Supplied to Dual-Root Systems of Soybean Plants on Nodulation and Nitrogen Fixation

The specific mechanisms by which nitrogen affects the nodulation and nitrogen fixation of soybean plants are unclear. Investigating the relationship between nitrogen, nodulation and nitrogen fixation can provide new insights for the rational and proper use of nitrogen fertilizer in soybean plants. In this study, we grafted soybean roots to construct a dual-root system with a single nodulated side. Experiment I was performed at the third trifoliate leaf to initial seed filling (V3-R3) growth stages (for 30 days) for long-term nitrogen supply, and Experiment II was performed at the third trifoliate leaf to fourth trifoliate leaf (V3-V4) growth stages (for 5 days) for short-term nitrogen supply. For the two experiments, a nutrient solution providing 15NH415NO3 or NH4NO3 as the nitrogen source was added to the non-nodulated side, while a nitrogen-free nutrient solution was added to the nodulated side. The concentrations of nitrogen supplied were 0 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, and 100 mg/L. The results showed the following: (1) Short-term nitrogen supply systematically regulated the specific nitrogenase activity (SNA), thereby inhibiting the acetylene reduction activity (ARA). Under long-term nitrogen supply, the recovery of SNA was generally consistent across treatments, and the concentration of nitrogen supplied systematically regulated the growth of root nodules, thereby inhibiting the ARA. (2) Using the 15N tracer method, the concentration of fertilizer nitrogen was positively correlated with the amount of nitrogen redistributed to other organs. Although the percentage of nitrogen derived from the atmosphere (Ndfa%) decreased significantly with increasing concentrations of nitrogen supplied, the effect on the accumulation of nitrogen fixed by nodules (Naccumulation of nodules) was not significant. By establishing the relationships between the ARA (measured by the acetylene reduction method), Ndfa% (based on 15N calculations), and Naccumulation nodules (based on 15N calculations), it was found that the ARA reliably reflected the Ndfa% but not the Naccumulation of nodules.

A Review on Marine N2 Fixation: Mechanism, Evolution of Methodologies, Rates, and Future Concerns

Investigations on marine N2 fixation have gained momentum since 1960s with eventual establishments of relevant methodologies to identify species involved and quantify the rates. The evolution of various methodologies to understand N2 fixation and to estimate its rates were underpinned by the constant efforts of pioneers in the ocean biogeochemical research field. Those efforts succeeded in introducing various methodologies that include experimental (15N2 bubble method and acetylene reduction method), geochemical (N* and P* method), mathematical modelling, and remote sensing techniques. However, the construction of an accurate N budget is still under progress due to inseparable issues associated with each method and difficulties in conducting the experiments onboard on a larger scale. Nevertheless, the contributions by each of the methodologies are significant and helped in forming basic ideas about N2 fixation activities on a global scale. It is not only important to recognize the contributions made by the formation of various methodologies by marine research pioneers, but also vital to summarize what we have achieved in the marine N2 fixation research area so far. Hence, this review is an attempt to brief on the various milestones achieved in research on the N2 fixation mechanism, species involved, evolution of methodologies to estimate N2 fixation rates, species identification, budgets, and future concerns.

Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation

To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for use with soil/rhizosphere samples. We tested the impact of different carbon (C) sources, oxygen concentrations (O2), and incubation times on FLNF rates of two low-fertility Michigan soils via ARA and 15N2. FLNF rates were greatest with addition of a C cocktail, at low O2, and with 7-day incubations for both methods. FLNF via ARA was 1700x greater with a C cocktail versus glucose only and via 15N2 was 17x greater with a C cocktail compared to other C sources and no-C controls. Specific O2 optimum varied by method and site. A 7-day incubation was needed for the ARA, but a 3-day incubation was suitable for 15N2. Lastly, we confirm previously identified issues with the ARA of acetylene-independent ethylene production/consumption resulting in potential FLNF measurement error of 1.3–52.3 μg N g−1 day−1. We present an optimized method for measuring potential FLNF in soil/rhizosphere samples which will allow for consistent and comparable FLNF rate measurements. Researchers should account for C source, O2, and incubation time when assessing FLNF and use the ARA method with caution.

Diazotrophic Macroalgal Associations With Living and Decomposing Sargassum.

Despite several studies reporting diazotrophic macroalgal associations (DMAs), biological nitrogen fixation (BNF) is still largely overlooked as a potential source of nitrogen (N) for macroalgal productivity. We investigated the role of BNF, via the acetylene reduction method, throughout different life stages of the invasive macroalga, Sargassum horneri, in its non-native Southern California coastal ecosystem. Throughout most of its life cycle, BNF rates were not detectable or yielded insignificant amounts of fixed N to support S. horneri productivity. However, during late summer when nutrient concentrations are usually at their minimum, BNF associated with juvenile S. horneri contributed ∼3–36% of its required N, potentially providing it with a competitive advantage. As DMAs remain poorly understood within macroalgal detrital systems, long term (15–28 days) laboratory decomposition time series were carried out to investigate the role of BNF throughout decomposition of the endemic macroalga, S. palmeri, and the invasive S. horneri. Nitrogenase activity increased drastically during the second phase of decomposition, when increasing microbial populations are typically thought to drive macroalgal degradation, with BNF rates associated with S. palmeri and S. horneri reaching up to 65 and 247 nmol N × g-1(dw) × h-1, respectively. Stimulation of BNF rates by glucose and mannitol additions, up to 42× higher rates observed with S. palmeri, suggest that labile carbon may be limiting at varying degrees in these detrital systems. Comparable, if not higher, dark BNF rates relative to light incubations during S. horneri decomposition suggest an important contribution from heterotrophic N fixers. Inhibition of nitrogenase activity, up to 98%, by sodium molybdate additions also suggest that sulfate reducers may be an important constituent of the detrital diazotrophic community. As labile N can become limiting to the microbial community during macroalgal decomposition, our results suggest that BNF may provide a source of new N, alleviating this limitation. Additionally, while BNF is rarely considered as a source for N enrichment with aging macroalgal detritus, we found it to account for ∼1–11% of N immobilized with decaying S. horneri. Our investigations suggest that DMAs may be globally important with Sargassum and potentially occur within other macroalgal detrital systems.

Assessing N2 fixation in estuarine mangrove soils

Nitrogen (N) limited mangrove forest may have a high potential for microbial N2 fixation. Previous research has focused on soil nitrogenase activity in pristine mangrove forests with little anthropogenic impact. This research was designed to evaluate the magnitude of nitrogenase activity of mangrove soils in a high anthropogenic N-loading environment and the way in which soil N2 fixation in mangrove forest may be related to organic carbon and salinity. The test involved an acetylene reduction method under controlled laboratory conditions. The mangrove forests with high anthropogenic N loading may have high nitrogenase activity in the soils. The diazotrophs in these mangrove soils were mostly heterotrophs and the sulfate-reducing bacteria were the major N2-fixing bacteria. The nitrogenase activity was little affected by the soil salinity, which suggests that these groups of N2 fixation bacteria adapted well to saline conditions in the estuary.

Incompatible Nodulation of &amp;lt;i&amp;gt;Bradyrhizobium elkanii&amp;lt;/i&amp;gt; Strains BLY3-8 and BLY6-1 with &amp;lt;i&amp;gt;Rj&amp;lt;/i&amp;gt;3 Gene-Harboring Soybean Cultivars

Bradyrhizobia are known symbiotic partners of soybean. However, some soybean cultivars restrict nodulation by some Bradyrhizobium bacterial strains. These restrictions are related to compatibility between the Rj genes of soybean cultivars and nodulation types of inoculated bacteria. The objective of this study was to determine nodulation incompatibility of Type B strains with Rj3 soybean cultivars. Newly isolated B. elkanii strains BLY3-8 and BLY6-1 from Myanmar and specific strain Bradyrhizobium elkanii USDA33, which are incompatible with Rj3 soybean cultivars, and B. japonicum USDA110 were used as inoculants to check compatibility or incompatibility with Rj3 soybean cultivars. Nitrogen fixation activity was measured by the acetylene reduction method. Ethylene concentration (reduction of acetylene) was determined by flame ionization gas chromatography. According to the inoculation test results, USDA110 was compatible with all soybean cultivars because it formed effective nodules (Figure S1 in Appendix) and possessed nitrogenase activity. Similarly, B. elkanii strains BLY3-8, BLY6-1, and USDA33 were highly compatible with non-Rj and Rj4-gene harboring soybean cultivars because they had the ability to form functional nodules and possessed nitrogenase activity. Inversely, BLY3-8, BLY6-1, and USDA33 were incompatible with Rj3 soybean cultivars because they produced ineffective nodules. Consequently, the ratio of ineffective nodule number to total nodule number was >0.5. Therefore, nodule formation by the newly isolated B. elkanii strains BLY3-8 and BLY6-1 was restricted by the Rj3 soybean cultivars potentially making them useful as specific strains to detect the Rj3 gene in soybean cultivars.

Environmental factors associated with heterotrophic nitrogen-fixing bacteria in water, sediment, and riparian soil of Suquía River

In this study we investigated the environmental factors associated with biological nitrogen (N) fixation (BNF) in water, sediments, and riparian soil along a polluted river (Suquia River of Cordoba, Argentina). Here, we screened heterotrophic nitrogen-fixing bacteria and assessed the magnitude of BNF at different sites of Suquia River. To this aim, samples of the three habitats (riparian soil, water, and sediment) were collected from five polluted sites and one reference site during low and high flow water periods. In all samples the abundance of N-fixing bacteria was evaluated in solid nitrogen-free medium and the biological N fixation was measured by nitrogenase (Nase) enzyme activity using the acetylene reduction method. To identify the heterotrophic N-fixing taxa DNA of nine cultures isolated from sites with different Nase enzyme activity was extracted and the 16S rRNA gene was amplified and sequenced. In addition, the ammonia and organic carbon (C) content in all samples, the dissolved O2 concentration in water, and the water content in riparian soil were measured. The N-fixing bacteria were detected in all study sites and habitats. The abundance of them correlated significantly with organic C content in sediment, and with water and organic C contents in riparian soil, whereas in water a negative correlation with dissolved O2 was observed. In addition, the water and sediment Nase enzyme activity varied among sites during low flow period presenting significant correlation with ammonia and organic C contents in sediment. The identified taxonomic groups in the Suquia River are related to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Actinobacteria although the N-fixing capacity of them was not established. Altogether these findings demonstrate that BNF occurs in all habitats of Suquia River, being in sediments influenced mainly by the higher organic C present in the most polluted sites, while in riparian soil the organic C and water contents were the major abiotic factors that control the abundance of N-fixing bacteria. In Suquia River water the density of N-fixing bacteria were associated with low dissolved O2 concentration. These data suggest that the BNF in Suquia River is a complex process that depends on numerous environmental factors that act together.

Dynamics of periphyton nitrogen fixation in short-hydroperiod wetlands revealed by high-resolution seasonal sampling

Periphyton N2 fixation plays a key role in N cycling of aquatic systems, but temporal studies of this process are often lacking, especially in systems with only seasonal flooding. We used seven samplings to characterize nitrogenase activity (acetylene reduction method) of periphyton in short-hydroperiod marl prairies and two wetlands restored from agricultural disturbance in Everglades National Park, USA. We hypothesized that the seasonal drying and rewetting would increase the temporal dynamics of the process. All sites showed significant periphyton N2 fixation, but in restored areas highest rates were observed only in the early wet season (July), while in reference sites fixation was spread throughout the summer. Most N2 fixation in the restored areas was confined to a 3-month-period resulting in large underestimates of annual fixation in previous studies with few seasonal measurements. N2 fixation rates correlated with total P, N and TN:TP ratio, and periphyton moisture content in the dry season. N stable isotopic signature was a good indicator of N2 fixation rates between sites, but did not correctly indicate seasonal patterns. These findings improve our understanding of N cycling in wetlands like the Everglades and indicate a need for more detailed measurements of processes in seasonally flooded systems.

Growth and N2 fixation in an Alnus hirsuta (Turcz.) var. sibirica stand in Japan

To estimate the N2 fixation ability of the alder (Alnus hirsuta (Turcz.) var. sibirica), we examined the seasonal variation in nitrogenase activity of nodules using the acetylene reduction method in an 18-year-old stand naturally regenerated after disturbance by road construction in Japan. To evaluate the contribution of N2 fixation to the nitrogen (N) economy in this alder stand, we also measured the phenology of the alder, the litterfall, the decomposition rate of the leaf litter, and N accumulation in the soil. The acetylene reduction activity per unit nodule mass (ARA) under field conditions appeared after bud break, peaked the maximum in midsummer after full expansion of the leaves, and disappeared after all leaves had fallen. There was no consistent correlation between ARA and tree size (dbh). The amount of N2 fixed in this alder stand was estimated at 56.4 kg ha-1 year-1 when a theoretical molar ratio of 3 was used to convert the amount of reduced acetylene to the amount of fixed N2. This amount of N2 fixation corresponded to the 66.4 percent of N in the leaf litter produced in a year. These results suggested that N2 fixation still contributed to the large portion of N economy in this alder stand.

Effects of Mo(VI) on phototrophic hydrogen production by Rhodobacter sphaeroides

Effects of Mo6+ concentration on phototrophic hydrogen production of Rhodobacter sphaeroides were investigated using lactate as the sole carbon source. Results showed that an increase of Mo6+ from nil to 1000 µg l−1 led to increases in hydrogen yield, maximum production rate, conversion efficiency, biomass yield and lactate removal. At 100 µg‐Mo l−1, the maximum rate was 12.0 ml h−1 l−1 with a conversion efficiency of 36.1%, the cell yields were 1.11 g‐cell g−1‐lactate and 2.4 g‐cell g−1‐TOC removed. Further increase of Mo6+ improved hydrogen production only marginally. Degradation of lactate by R. sphaeroides produced not just hydrogen but also acetate, butyrate, i‐valerate, i‐caproate, hexanoate and some unidentified organic intermediates, but did not produce propionate and alcohols. Nitrogenase activity, as measured by the acetylene reduction method, had no clear correlation with either Mo6+ concentration or hydrogen yield.

Mutational approach for N2-fixing and P-solubilizing mutant strains of Klebsiella pneumoniae RSN19 by microwave mutagenesis.

Nitrogen (N) fixing Klebsiella pneumoniae RSN19 has high inorganic phosphorus (P) solubilizing capability, but its N2-fixing capability is limited. In order to acquire a P-solubilizing mutant strain with high efficiency N-fixing capability, different microwave irradiation intensities and durations were tested on RSN19 in an attempt to produce mutants with improved N2-fixation and P-solubilization capabilities. The effect of microwave irradiation power and time were studied and the microwave mutagenesis parameters were optimized. Nitrogenase activity was tested on the mutant strains by acetylene reduction method; and their P-solubilizing capability and genetic stability were determined. The results indicated that the best conditions for microwave mutagenesis that produced better performed mutant strains were 250W, 36s. Under these conditions a maximum positive mutation rate of 1.66% was obtained, resulting in five genetically stable strains with promoted nitrogenase activity which was designated as RSM-219, RSM-206, RSM-224, RSM-225 and RSM-275. Subculture tests showed that RSM-219 and RSM-206 were genetically stable mutant strains with higher nitrogenase activity and phosphate solubilizing capabilities than the original strain. Both RSM-219 and RSM-206 performed better than the original strain under N-free conditions when supplied with calcium phosphate only, and produced greater increases in the biomass of alfalfa seedlings.

Dinitrogen fixation by biological soil crusts in an Inner Mongolian steppe

Eurasian steppe ecosystems are nitrogen-limited and suffer additionally from high grazing intensities in many areas. Soil surface-bound cyanobacteria are able to fix nitrogen and can be the major source of plant available nitrogen in such ecosystems. In this study, the abundance and dinitrogen fixation capacity of the most common soil surface-bound microbial and lichen species were determined at an ungrazed, a winter-grazed, and a heavily grazed steppe site in the Xilin River catchment, Inner Mongolia, People’s Republic of China. The microorganisms were identified as Nostoc spec. and the lichen species as Xanthoparmelia camtschadalis (Ach.) Hale by a combination of classical light microscopy, confocal laser scanning microscopy and molecular analysis of the internal transcribed spacer (ITS1) region of ribosomal RNA. Both species were found exclusively at grazed steppe sites, with a clear difference in abundance depending on the grazing intensity. At the winter-grazed site, Nostoc was more abundant than Xanthoparmelia; for the heavily grazed site, the opposite was found. N2 fixation was quantified with both the acetylene reduction method and 15N2 incubation. Cyanobacterial colonies of Nostoc fixed N2 vigorously, whereas X. camtschadalis did not at all. The fraction of nitrogen derived from the fixation of molecular nitrogen in Nostoc was 73%, calculated from 15N natural abundance measurements of Nostoc with X. camtschadalis as reference. The conservatively calculated N2 uptake by Nostoc was 0.030–0.033 kg N ha−1 for the heavily grazed site and 0.080–0.087 kg N ha−1 for the winter-grazed site for the growing seasons of 2004 and 2005, respectively. Together with previous findings, this study demonstrates that N2 fixation by Nostoc can potentially replace significant amounts, if not all, of the nitrogen lost in the form of N2O and NO soil emissions in this steppe ecosystem.

Occurrence and diversity of nitrogen-fixingSphingomonasbacteria associated with rice plants grown in Brazil

So far, the occurrence of nitrogen-fixing Sphingomonas bacteria has been restricted to three strains of Sphingomonas azotifigens. In this work, a group of 46 Sphingomonas-like isolates, which originated from two rice varieties grown in two soils in Brazil, were characterized based on morphological, physiological and genetic analyses. The PCR genus specifically applied indicated that all 46 isolates belonged to the Sphingomonas genus and confirmed the results based on the yellow pigment of the colonies grown on potato agar medium and the BIOLOG data. It was also observed that 22 isolates are nitrogen-fixing bacteria as determined by the acetylene reduction method and confirmed by nifH gene detection. The genetic diversity based on the 16S rRNA analysis (amplified rDNA restriction analysis) showed that the isolates formed two distinct groups at a similarity value of 60%. Furthermore, five clusters at 60% similarity were observed with the 16S-23S intergenic space (ribosomal intergenic space analysis) analysis. Sequencing of the 16S rRNA gene and nifH fragments showed that most of the 22 nitrogen-fixing isolates formed clusters apart from that of the S. azotifigens. This is the first report on the occurrence of nitrogen-fixing Sphingomonas bacteria associated with rice grown in Brazil.