Growth and respiration ofBradyrhizobium japonicum USDA 143 with nitrous oxide as the terminal electron acceptor

Bradyrhizobium japonicum USDA 143 grew chemoorganotrophically under anoxic conditions with exogenous N₂O as the sole terminal electron acceptor. Cell growth and dissimilatory N₂O reduction were significantly inhibited by C₂H₂ when either N₂O or N₂O plus NO ₃ ^– served as terminal electron acceptor(s). Reduction of N₂O accounted for 20% of the energy for cell growth in cultures supplied with NO ₃ ^– as the terminal electron acceptor. Nitrous oxide was produced stoichiometrically in cultures containing NO ₃ ^– and C₂H₂, but cell growth was proportionately reduced when compared with cultures supplied with an equal amount of NO ₃ ^– . Exogenous N₂O delayed the reduction of NO ₃ ^– in cultures supplied with both electron acceptors. Direct amperometric monitoring of N₂O respiration showed a specific activity of 0.082±0.004 moles N₂O/min/mg cell protein, and azide inhibited cell respiration.     

Nitrite reduction in Rhizobium “hedysari” strain HCNT 1

Rhizobium hedysari strain HCNT 1 rapidly reduced nitrite to N₂O, only slowly reduced nitrate to nitrite and did not exhibit nitrous oxide reductase activity. Nitrite reduction in this rhizobium strain may be a detoxification mechanism for conversion of nitrite, which inhibits O₂ uptake, to non-toxic N₂O. Concentrations of nitrite as small as 3 M diminished O₂ uptake in whole cells. The bacterium did not couple energy conservation with nitrate or nitrite reduction. Cells neither grew anaerobically at the expense of these nitrogen oxides nor translocated protons during reduction of nitrite. Induction of nitrite reductase activity was not a response to the presence of nitrate or nitrite, but occurred instead when the O₂ concentration in culture atmospheres fell to <16.5% of air saturation. Sensitivity of cytochrome o, which is synthesized only in cells grown under O₂-limited conditions, may account for the toxicity of nitrite in strain HCNT 1.     

The reduction of nitrous oxide to dinitrogen by Escherichia coli

Escherichia coli K12 reduces nitrous oxide stoichiometrically to molecular nitrogen with rates of 1.9 mol/h x mg protein. The activity is induced by anaerobiosis and nitrate. N₂+formation from N₂O is inhibited by C₂H₂ (K _i 0.03 mM in the medium) and nitrite (K _i=0.3 mM) but not by azide. A mutant defective in FNR synthesis is unable to reduce N₂O to N₂. The reaction in the wild type could routinely be followed by gas chromatography and alternatively by mass spectrometry measuring the formation of ¹⁵N₂ from ¹⁵N₂O. The enzyme catalyzing N₂O-reduction in E. coli could not be identified; it is probably neither nitrate reductase nor nitrogenase. E. coli does not grow with N₂O as sole respiratory electron acceptor. N₂O-reduction might not have a physiological role in E. coli, and the enzyme involved might catalyze something else in nature, as it has a low affinity for the substrate N₂O (apparent K _m3.0 mM). The capability for N₂O-reduction to N₂ is not restricted to E. coli but is also demonstrable in Yersinia kristensenii and Buttiauxella agrestis of the Enterobacteriaceae. E. coli is able to produce NO and N₂O from nitrite by nitrate reductase, depending on the assay conditions. In such experiments NO _inf2 ^sup- is not reduced to N₂ because of the high demand for N₂O of N₂O-reduction and the inhibitory effect of NO _inf2 ^sup- on this reaction.Dedicated to Professor L. Jaenicke, Köln, on the occassion of his 70th birthday     

Aspects of nitrogen fixation and denitrification byAzospirillum

Summary Model experiments were performed to investigate the nitrogen fixation (C₂H₂ reduction) and denitrification (N₂O formation) capabilities ofAzospirillum spp. in association with wheat. Plants and bacteria were grown together for a week and then assayed for activities. This association performed C₂H₂ reduction or N₂O formation, depending on the concentrations of nitrate and oxygen in the vessels. Both activities depended on theAzospirillum strains used. The newly isolatedAzospirillum amazonense strains Y1 and Y6 showed significant C₂H₂ reduction and low N₂O formation in association with wheat under the conditions employed and are possibly useful in practice. A cell-free preparation fromAzospirillum brasilense Sp 7 possessed a cytochrome cd type dissimilatory nitrite reductase.     

Denitrification rate determined by nitrate disappearance is higher than determined by nitrous oxide production with acetylene blockage

A mixed beech and spruce forest soil was incubated under potential denitrification assay (PDA) condition with 10% acetylene (C₂H₂) in the headspace of soil slurry bottles. Nitrous oxide (N₂O) concentration in the headspace, as well as nitrate, nitrite and ammonium concentrations in the soil slurries were monitored during the incubation. Results show that nitrate disappearance rate was higher than N₂O production rate with C₂H₂ blockage during the incubation. Sum of nitrate, nitrite, and N₂O with C₂H₂ blockage could not recover the original soil nitrate content, showing an N imbalance in such a closed incubation system. Changes in nitrite and ammonium concentration during the incubation could not account for the observed faster nitrate disappearance rate and the N imbalance. Non-determined nitric oxide (NO) and N₂ production could be the major cause, and the associated mechanisms could vary for different treatments. Commonly applied PDA measurement likely underestimates the nitrate removal capacity of a system. Incubation time and organic matter/nitrate ratio are the most critical factors to consider using C₂H₂ inhibition technique to quantify denitrification. By comparing the treatments with and without an antibiotic, the results suggest that microbial N uptake probably played a minor role in N balance, and other denitrifying enzymes but nitrate reductase could be substantially synthesized during the incubation.     

Nitrous oxide production by nitrogen-fixing,fast-growing Rhizobia

Rhizobium trifolii, R. leguminosarum, andR. hedysarum, grownex planta under anoxic conditions in a chemically defined medium, evolve N₂O from NO₃ ^–, NO₂ ^–, and (NH₄)₂NO₃. The amount of nitrous oxide formed after 96 hours is about 0.2M×mg^–1 cells d.w. Large availability of organic matter enhances the production of N₂O from nitrate by free-livingR. trifolii in peat/sand mixtures. Denitrification of the above species andR. meliloti was detected also in planta. Nitrous oxide production increases almost linearly from 10–45M×mg^–1 nodules d.w. when nitrogen-fixing plants are exposed to increasing concentrations of nitrate (1–12M).     

Evolution of nitrous oxide in a nitrate-treated soil at a low partial pressure of acetylene

Summary Soil cores incubated under air atmosphere with C₂H₂ at partial pressure of 0.1 kPa, reduced NO₃ ^– to N₂O. Glucose and nitrate at higher concentration stimulated the N₂O production. Further reduction of N₂O to N₂ seemed to be temporarely inhibited. However after 12 h N₂O concentrations decreased, irrespective of soil treatment.     

N2 fixation by red alder (Alnus rubra) and scotch broom (Cytisus scoparius) planted under precommerically thinned Douglas-fir (Pseudotsuga menziesii)

Summary From acetylene reduction assays over a 10-month period starting in April 1979, nodule activities averaged 18.78 (se 4.67) moles C₂H₄ g nodule dw^–1 h^–1 forAlnus rubra and 59.95 (se 12.14) moles C₂H₄ g nodule dw^–1 h^–1 forCytisus scorparius. Plant rates were 1.91 (se. 47) moles C₂H₄ plant^–1 h^–1 forA. rubra and 0.55 (se. 17) moles C₂H₄ plant^–1 h^–1 forC. Scoparius. Plant activity and total leaf N were strongly correlated with the dw of other plant parts, but nodule activity and percent leaf N were not. Plant and nodule activities were not associated with temperature, moisture stress, precipitation events or percent light for either species over the growing season nor for 54A. rubra sampled in mid-season 1979 on one replication. After 5 to 6 growing seasons, 14A. rubra on the same site ranged from 30 to 332 cm in height and showed strong correlation between nodule dw, leaf dw, plant size and total leaf N. Results from this study and others indicate logistic equations may be modified to predict the effect of adding a N₂ fixing plant to a population of non N₂ fixing trees.     

Characteristics of the H2 oxidizing system in soybean nodule bacteroids

A derivative of Rhizobium japonicum (strain 122 DES) has been isolated which forms nodules on soybeans that evolve little or no H₂ in air and efficiently fixes N₂. Bacteroids isolated from nodules formed by strain 122 DES took up H₂ with O₂ as the physiological acceptor and appeared to be typical of those R. japonicum strains that possess the H₂ uptake system. The hydrogenase system in soybean nodules is located within the bacteroids and activity in macerated bacteroids is concentrated in a particulate fraction. The pH optimum for the reaction is near 8.0 and apparent K _m values for H₂ and O₂ are 2 M and 1 M, respectively. The H₂ oxidizing activity of a suspension of 122 DES bacteroids was stable at 4°C for at least 4 weeks and was not particularly sensitive to O₂. Neither C₂H₂ nor CO inhibited O₂ dependent H₂ uptake activity.Non-physiological electron acceptors of positive oxidation reduction potential also supported H₂ uptake by bacteroids. The rate of H₂ uptake with phenazine methosulfate as the acceptor was greater than that with O₂. When methylene blue, triphenyltetrazolium, potassium ferricyanide or dichlorophenolindophenol were added to bacteriod suspensions, without preincubation, rates of H₂ uptake were supported that were lower than those in the presence of O₂. Preincubation of the bacteroids with acceptors increased the rates of H₂ uptake. No H₂ evolution was observed from reaction mixtures containing bacteroid suspensions and reduced methyl or benzyl viologens. Of a series of carbon substrates added to bacteroid suspensions only acetate, formate or succinate at concentrations of 50 mM resulted in 20% or greater inhibition of H₂ oxidation.The H₂ uptake capacity of isolated 122 DES bacteroids (expressed on a dry bacteroid basis) was at least 10-fold higher than the rate of the nitrogenase reaction in nodules expressed on a comparable basis. Since about 1 mol of H₂ is evolved for every mol of N₂ reduced during the N₂ fixation reaction, these observations explain why soybean nodules formed by strain 122 DES and other strains with high H₂ uptake activities have a capacity for recycling all the H₂ produced from the nitrogenase reaction.Abbreviations PMS PHenazine methosulfate - MB Methylene blue     

Denitrification, Acetylene Reduction, and Methane Metabolism in Lake Sediment Exposed to Acetylene

Samples of sediment from Lake St. George, Ontario, Canada, were incubated in the laboratory under an initially aerobic gas phase and under anaerobic conditions. In the absence of added nitrate (NO₃⁻) there was O₂-dependent production of nitrous oxide (N₂O), which was inhibited by acetylene (C₂H₂) and by nitrapyrin, suggesting that coupled nitrification-denitrification was responsible. Denitrification of added NO₃⁻ was almost as rapid under an aerobic gas phase as under anaerobic conditions. The N₂O that accumulated persisted in the presence of 0.4 atm of C₂H₂, but was gradually reduced by some sediment samples at lower C₂H₂ concentrations. Low rates of C₂H₂ reduction were observed in the dark, were maximal at 0.2 atm of C₂H₂, and were decreased in the presence of O₂, NO₃⁻, or both. High rates of light-dependent C₂H₂ reduction occurred under anaerobic conditions. Predictably, methane (CH₄) production, which occurred only under anaerobiosis, was delayed by added NO₃⁻ and inhibited by C₂H₂. Consumption of added CH₄ occurred only under aerobic conditions and was inhibited by C₂H₂.     

Proton translocation during denitrification by a nitrifying-denitrifying Alcaligenes sp.

A heterotrophic nitrifying Alcaligenes sp. from soil was grown as a denitrifier on nitrate and subjected to oxidant pulse experiments to ascertain the apparent effeciencies of proton translocations during O₂ and nitrogen-oxide respirations. With endogenous substrate as the reducing agent the H⁺/2e^– ratios, extrapolated to zero amount of oxidant per pulse, were 9.4, 3.7, 4.3 and 3.5 for O₂, nitrate, nitrite and N₂O, respectively. The value for O₂ and those for the N-oxides are, respectively, somewhat larger and smaller than corresponding values for Paracoccus denitrificans. None of the three permeant ions employed with the Alcaligenes sp. (valinomycin-K⁺, thiocyanate and triphenylmethylphosphonium) was ideal for all purposes. Thiocyanate provided highest ratios for O₂ but abolished the oxidant pulse response for nitrate and N₂O. Valinomycin was slow to penetrate to the cytoplasmic membrane and relatively high concentrations were required for optimal performance. Triphenylmethylphosphonium enhanced passive proton permeability and diminished proton translocation at concentrations required to realize the maximal oxidant pulse response.     

Acetylene Metabolism and Stimulation of Denitrification in an Agricultural Soil

The effects of C₂H₂ metabolism on N₂O production were examined in soil slurries. Enrichment of C₂H₂ consumption activity occurred only in aerobic incubations. Rapid disappearance of subsequent C₂H₂ additions, stimulation of CO₂ production, and most-probable-number enumerations of C₂H₂ utilizers indicated enrichment of the population responsible. During C₂H₂ consumption in slurries incubated statically under air, maximal rates of N₂O evolution were 19 times higher than those in anaerobic incubations. After 20 days of enrichment with C₂H₂, the production of N₂O by slurries supplemented with C₂H₂ and nitrate was 10 times higher than that in the unenriched controls. A Nocardia- or Arthrobacter-like bacterium was isolated that grew on C₂H₂ but did not denitrify. The behavior of soil inoculated with this bacterium became similar to that of C₂H₂-enriched soil incubated aerobically. Ethanol, acetate, and acetaldehyde were identified in enrichment experiments, and denitrification in soil slurries was stimulated by addition of the supernatant from a pure culture grown on mineral medium with C₂H₂. These results indicate that denitrification can be stimulated by the actions of an aerobic, nondenitrifying C₂H₂-metabolizing population. Utilization of intermediate metabolites by denitrifiers and enhanced O₂ consumption are two possible mechanisms for this stimulation.     

Interaction of Cytokinins and Abscisic Acid During Regulation of Stomatal Opening in Bean Leaves

Effects of benzyladenine (BA) and abscisic acid (ABA) applied separately or simultaneously on parameters of gas exchange of Phaseolus vulgaris L. leaves were studied. In the first two experimental sets) 100 M ABA and 10 M BA were applied to plants sufficiently supplied with water. Spraying of leaves with ABA decreased stomatal conductance (g _s) and in consequence transpiration rate (E) and net photosynthetic rate (P _N) already 1 h after application, but 24 h after application the effect almost disappeared. 10 M BA slightly decreased gas exchange parameters, but in simultaneous application with ABA reversed the effect of ABA. Immersion of roots into the same solutions markedly decreased gas exchange parameters and 24 h after ABA application the stomata were completely closed. The effect of ABA was ameliorated by simultaneous BA application, particularly after 1-h treatment. In the third experimental set, plants were pre-treated by immersing roots into water, 1 M BA, or 100 M ABA for 24 h and then the halves of split root system were dipped into different combinations of 1 M BA, 100 M ABA, and water. In plants pre-treated with ABA all gas exchange parameters were small and they did not differ in plants treated with H₂O+H₂O, H₂O+BA, or BA+BA. In plants pre-treated with BA or H₂O, markedly lower values of P _N were found when both halves of roots were immersed in ABA. Further, the effects of pre-treatment of plants with water, 1 M BA, 100 M ABA, or ABA+BA on the development of water stress induced by cessation of watering and on the recovery after rehydration were followed. ABA markedly decreased gas exchange parameters at the beginning of the experiment, but in its later phase the effect was compensated by delay in development of water stress. BA also delayed development of water stress and increased P _N in water-stressed leaves. BA reversed the effect of ABA at mild water stress. Positive effects of BA and ABA pre-treatments were observed also after rehydration.     

Gas Exchange of Carrot Leaves in Response to Elevated CO2 Concentration

Short-term responses of four carrot (Daucus carota) cultivars: Cascade, Caro Choice (CC), Oranza, and Red Core Chantenay (RCC) to CO₂ concentrations (C _a) were studied in a controlled environment. Leaf net photosynthetic rate (P _N), intercellular CO₂ (C _i), stomatal conductance (g _s), and transpiration rate (E) were measured at C _a from 50 to 1 050 mol mol^–1. The cultivars responded similarly to C _a and did not differ in all the variables measured. The P _N increased with C _a until saturation at 650 mol mol^–1 (C _i= 350–400 mol mol^–1), thereafter P _N increased slightly. On average, increasing C _a from 350 to 650 and from 350 to 1 050 mol mol^–1 increased P _N by 43 and 52 %, respectively. The P _N vs. C _i curves were fitted to a non-rectangular hyperbola model. The cultivars did not differ in the parameters estimated from the model. Carboxylation efficiencies ranged from 68 to 91 mol m^–2 s^–1 and maximum P _N were 15.50, 13.52, 13.31, and 14.96 mol m^–2 s^–1 for Cascade, CC, Oranza, and RCC, respectively. Dark respiration rate varied from 2.80 mol m^–2 s^–1 for Oranza to 3.96 mol m^–2 s^–1 for Cascade and the CO₂ compensation concentration was between 42 and 46 mol mol^–1. The g _s and E increased to a peak at C _a= 350 mol mol^–1 and then decreased by 17 and 15 %, respectively when C _a was increased to 650 mol mol^–1. An increase from 350 to 1 050 mol mol^–1 reduced g _s and E by 53 and 47 %, respectively. Changes in g _s and P _N maintained the C _i:C _a ratio. The water use efficiency increased linearly with C _a due to increases in P _N in addition to the decline in E at high C _a. Hence CO₂ enrichment increases P _N and decreases g _s, and can improve carrot productivity and water conservation.     

Physiology and kinetics of autotrophic denitrification by Thiobacillus denitrificans

Summary A strain of Thiobacillus denitrificans was isolated after enrichment under anaerobic conditions by the continuous culture technique using thiosulfate as energy source and nitrate as electron acceptor and nitrogen source. The isolate was an active denitrifyer, the optimal conditions being 30°C and pH 7.5–8.0. Denitrification was inhibited by sulfate (the reaction product) above 5 g SO ₄ ⁼ /l, whereas high concentrations of the substrates nitrate and thiosulfate were less harmful; nitrite affected denitrification above 0.2 g NO ₂ ^– /l. During the time course of denitrification in a batch culture growth and substrate consumption slowed down already after only half the substrate was utilized due to product inhibition. The following parameters were determined in continuous culture under nitrate limitation: _max=0.11 h^–1, K _S=0.2 mg NO ₃ ^– /l, maximum denitrification rate=0.78 g NO ₃ ^– /g cells·h, g cells/g NO ₃ ^– , g cells/g S₂O ₃ ⁼ . Nitrite did not accumulate during steady state denitrification; the denitrification gas was almost pure N₂. The concentrations of N₂O and NO were below 1 ppm.     

Utilization of H2O2 as the Oxygen Source by Bacteria of the Genera Pseudomonas and Rhodococcus

The growth of bacteria of the genera Pseudomonas and Rhodococcus in the presence of hydrogen peroxide as the sole source of oxygen was studied. The toxic effect of H₂O₂ in the concentration range of 100–200 g/ml was shown to extend the lag phase by two to three days. Apart from the peroxide toxicity, the bacterial growth was inhibited by the toxic effect of dissolved oxygen in concentrations over 100 g O₂/ml; in the presence of a liquid hydrocarbon phase, this effect was alleviated. Under decreased partial pressure of oxygen in the presence of hydrocarbons (12–15 vol %), culture growth was initiated at high initial concentrations of H₂O₂ (300 g/ml). When hydrogen peroxide concentrations exceeded 320 g/ml, no growth occurred, regardless of how much hydrocarbon was added.     

Production of N2O in soil during decomposition of dead yeast cells with different spatial distributions

Production and sources of N₂O were determined in soil columns amended with autoclaved yeast cells either mixed into or added as 0.5 cm³ lumps to the soil in combination with no or 200 g NO₃ ^--N g^-1. At four occasions over a two-week study period, subsets of cores were measured for N₂O production during 4-hour incubations under atmospheres of ambient air, 10 Pa of C₂H₂, and N₂, respectively. Denitrification enzyme activity (DEA) was assessed in subsamples of cores that had been incubated continuously under air.Autoclaved yeast provided a C-source readily available for denitrifying bacteria in the soil. Nitrous oxide production was negligible in unamended columns whereas accumulated N₂O losses in the presence of yeast material were substantial, varying between 15 to 49 ng N₂O-N g^-1 h^-1. Mixing yeast into the soil caused the highest production of N₂O followed by the yeast lump and no yeast treatments. Incubation in the presence of 10 Pa C₂H₂ indicated that denitrification was the sole source of N₂O, in accordance with an increase in DEA. Nitrous oxide production and DEA peaked after 4–7 days of incubation, and both were unaffected by additional NO₃ ^-. Two-to four-fold responses to anaerobiosis and accumulation of NO₃ ^- and NH₄ ⁺ in proximity of the lumps indicated that N₂O production here was limited by relatively low C-availability. In contrast, 10- to 12-fold responses to anaerobiosis and no accumulation of inorganic N suggested a higher C-availability where yeast was mixed into the soil.     

Nitrogen-fixing bacillus species from Egyptian soils: Acetylene reduction and cultural conditions

Summary Among 390 isolates from Egytiian soils initially grown on Brown's N-free agar, 15 facultative Bacillus isolates were able to reduce acetylene in Stanier's N-poor broth under both aerobic and anaerobic (N₂ atmosphere) conditions. Some of these isolates were Gram-positive, with unswollen sporangia and thin-walled endospores. Other strains were with slightly or definitely bulged sporangia. Yeast extract (0.01%) was essential for growth stimulation and N₂[C₂H₂] fixation by these isolates. Replacing yeast extract with 20 g/ml (NH₄)₂SO₄ or biotin, thiamine and amino acids (singly or in combination) resulted in stimulation of growth and N₂[C₂H₂] fixation, though at lower rates than in yeast extract.One isolate was able to grow and reduce C₂H₂ in Stanier's N-free liquid medium. Nitrogenase [C₂H₂] activity of the anaerobically grown and incubated cultures was greater than aerobic cultures. Addition of 0.1% CaCO₃ to the culture media significantly increased and O₂ partially inhibited, N₂[C₂H₂] fixation by these Bacillus isolates.Studies of the characteristics and N₂[C₂H₂] fixing activities of these isolates indicate that at least some of them are new nitrogen-fixingBacillus species.     

Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It was found to be able to grow in the presence of micromolar molecular oxygen (O₂). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O₂ to hydrogen peroxide (H₂O₂), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent V_max value of 37 mol NADH oxidized min^–1 mg^–1 protein. Apparent K_m values for NADH and O₂ were determined to be 7.5 M and 85 M, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85°C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H₂O₂ produced by the NADH oxidase to H₂O. It seems possible that O₂ can be reduced to H₂O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O₂.     

Growth, denitrification and nitrate ammonification of the rhizobial strain TNAU 14 in presence and absence of C2H4 and C2H2

Soil-N (NO₃ ^?) initiates as far as a threshold concentration is surpassed manifold physiological reactions on N₂-fixation. Organic N and ammonium oxidised to NO₃ ^? means oxygen depletion. Plants suffering under O₂ or infection stress start to excrete ethylene (C₂H₄). C₂H₄ widens the root intercellulars that O₂-respiration will continue. Now microbes may more easily enter the plant interior by transforming the reached methionine into C₂H₄. Surplus nitrate and C₂H₄ inhibit nodulation of leguminous plants. Excess NO₃ ^? in the nodulesphere could be diminished by N₂-fixing bacteria which in addition can denitrify or ammonify nitrate. Consequently, it was asked whether C₂H₄ interferes with the potential of N₂-fixing bacteria to reduce nitrate. The groundnut-nodule isolate TNAU 14, from which it was known that it denitrifies and ammonifies nitrate, served as inoculum of a KNO₃-mannitol-medium that was incubated under N₂-, 1% (v/v) N₂?C₂H₄-, and 1% (v/v) N₂?C₂H₂-atmosphere in the laboratory. C₂H₂ was included into the experiments because it is frequently used to quantify N₂-fixing potentials (acetylene reduction array, ARA). Gene-16S rDNA-sequencing and physiological tests revealed a high affiliation of strain TNAU 14 toRhizobium radiobacter andRhizobium tumefaciens. Strain TNAU 14 released N₂O into the bottle headspace in all treatments, surprisingly significantly less in presence of C₂H₂. Nitrate-ammonification was even completely blocked by C₂H₂. C₂H₄, in contrast rather stimulated growth, denitrification, and nitrate-ammonification of strain TNAU 14 which consumed the released NH₄ ⁺ during continuing incubation.