Acetylene hydratase: a non-redox enzyme with tungsten and iron–sulfur centers at the active site

In living systems, tungsten is exclusively found in microbial enzymes coordinated by the pyranopterin cofactor, with additional metal coordination provided by oxygen and/or sulfur, and/or selenium atoms in diverse arrangements. Prominent examples are formate dehydrogenase, formylmethanofuran dehydrogenase, and aldehyde oxidoreductase all of which catalyze redox reactions. The bacterial enzyme acetylene hydratase (AH) stands out of its class as it catalyzes the conversion of acetylene to acetaldehyde, clearly a non-redox reaction and a reaction distinct from the reduction of acetylene to ethylene by nitrogenase. AH harbors two pyranopterins bound to W, and a [4Fe–4S] cluster. W is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. AH activity requires a strong reductant suggesting W(IV) as the active oxidation state. Two different types of reaction pathways have been proposed. The 1.26 Å structure reveals a water molecule coordinated to W which could gain a partially positive net charge by the adjacent protonated Asp-13, enabling a direct attack of C₂H₂. To access the W–Asp site, a substrate channel was evolved distant from where it is found in other members of the DMSOR family. Computational studies of this second shell mechanism led to unrealistically high energy barriers, and alternative pathways were proposed where C₂H₂ binds directly to W. The architecture of the catalytic cavity, the specificity for C₂H₂ and the results from site-directed mutagenesis do not support this first shell mechanism. More investigations including structural information on the binding of C₂H₂ are needed to present a conclusive answer.     

Energetics of syntrophic ethanol oxidation in defined chemostat cocultures

The ethanol-oxidizing, proton-reducing Pelobacter acetylenicus was grown in chemostat cocultures with either Acetobacterium woodii, Methanobacterium bryantii, or Desulfovibrio desulfuricans. Stable steady state conditions with tightly coupled growth were reached at various dilution rates between 0.02 and 0.14 h^-1. Both ethanol and H₂ steady state concentrations increased with growth rate and were lower in cocultures with the sulfate reducer < methanogen < homoacetogen. Due to the higher affinity for H₂, D. desulfuricans outcompeted M. bryantii, and this one A. woodii when inoculated in cocultures with P. acetylenicus. Cocultures with A. woodii had lower H₂ steady state concentrations when bicarbonate reduction was replaced by the energetically more favourable caffeate reduction. Similarly, cocultures with D. desulfuricans had lower H₂ concentrations with nitrate than with sulfate as electron acceptor. The Gibbs free energy (G) available to the H₂-producing P. acetylenicus was independent of growth rate and the H₂-utilizing partner, whereas the G available to the latter increased with growth rate and the energy yielding potential of the H₂ oxidation reaction. The critical Gibbs free energy (G_c), i.e. the minimum energy required for H₂ production and H₂ oxidation, was-5.5 to-8.0 kJ mol^-1 H₂ for P. acetylenicus,-5.1 to-6.3 kJ mol^-1 H₂ for A. woodii,-7.5 to-9.1 kJ mol^-1 H₂ for M. bryantii, and-10.3 to-12.3 kJ mol^-1 H₂ for D. desulfuricans. Obviously, the potentially available energy was used more efficiently by homoacetogens > methanogens > sulfate reducers.     

Differences of cardiac output measurements by open-circuit acetylene uptake in pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension: a cohort study

Background

As differences in gas exchange between pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) have been demonstrated, we asked if cardiac output measurements determined by acetylene (C₂H₂) uptake significantly differed in these diseases when compared to the thermodilution technique.

Method

Single-breath open-circuit C₂H₂ uptake, thermodilution, and cardiopulmonary exercise testing were performed in 72 PAH and 32 CTEPH patients.

Results

In PAH patients the results for cardiac output obtained by the two methods showed an acceptable agreement with a mean difference of -0.16 L/min (95% CI -2.64 to 2.32 L/min). In contrast, the agreement was poorer in the CTEPH group with the difference being -0.56 L/min (95% CI -4.96 to 3.84 L/min). Functional dead space ventilation (44.5 ± 1.6 vs. 32.2 ± 1.4%, p < 0.001) and the mean arterial to end-tidal CO₂ gradient (9.9 ± 0.8 vs. 4.1 ± 0.5 mmHg, p < 0.001) were significantly elevated among CTEPH patients.

Conclusion

Cardiac output evaluation by the C₂H₂ technique should be interpreted with caution in CTEPH, as ventilation to perfusion mismatching might be more relevant than in PAH.     

Exploring the active site of the tungsten, iron-sulfur enzyme acetylene hydratase

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H₂O to acetylene (H—C☰C—H) to form acetaldehyde (CH₃CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme.Molybdenum and tungsten are the only transition metals of the second (Mo) and third (W) row of the periodic table of elements with known biological functions (7). In their biologically active form, both metals are bound to the cofactor molybdopterin (Moco), which is present in all molybdenum and tungsten enzymes with the exception of nitrogenase, where molybdenum is coordinated to a large iron-sulfur cluster, MoFe₇S₉ (9). Virtually all organisms including plants and mammals use either molybdenum or tungsten proteins in important metabolic pathways (35). Microorganisms carry a wide variety of molybdenum enzymes, such as nitrate reductase (NAR), formate dehydrogenase (FDH), dimethyl sulfoxide reductase (DMSOR), or trimethylamine N-oxide reductase (TMAOR) (7). These enzymes are involved in either oxygen atom transfer reactions or in reductive hydroxylations. By this means, the metal shuttles between the oxidation states +IV and +VI (16). Notably, the tungsten, iron-sulfur enzyme acetylene hydratase ([AH] EC 4.2.1.112), isolated from the soluble fraction of the mesophilic anaerobe Pelobacter acetylenicus, is an exception (26). It catalyzes the hydration of acetylene to acetaldehyde via an enol intermediate as an initial step for the fermentation of acetylene by P. acetylenicus, clearly a nonredox reaction (equation 1): Except for nitrogenase, which reduces acetylene to ethylene (H₂C=CH₂), AH is the only enzyme known to accept acetylene as a substrate. However, acetylene is well known to act as an inhibitor for numerous metal-dependent enzymes (10). AH is a member of the DMSOR family and carries one [4Fe-4S] cluster and two molybdopterin-guanosine-dinucleotide (referred to as P- and Q-MGD) ligands that coordinate the tungsten atom (Fig. ​(Fig.1)1) (18). The enzyme is sensitive toward dioxygen, and its [4Fe-4S] cluster is converted to a truncated [3Fe-4S] cluster upon exposure to air, as shown by electron paramagnetic resonance (EPR) (18). In AH prepared under the exclusion of dioxygen, the EPR signal of the [3Fe-4S] cluster was absent, and reaction with sodium dithionite led to a rhombic EPR signal (g_z of 2.048, g_y of 1.939, and g_x of 1.920) originating from a [4Fe-4S]⁺ cluster. Upon oxidation with hexacyanoferrate(III), a new signal appeared (g_x of 2.007, g_y of 2.019, and g_z of 2.048; average g value [g_av] of 2.022), which was assigned to a W(V) center (18).Open in a separate windowFIG. 1.Acetylene hydratase from P. acetylenicus. (Left) Overall structure, with the [4Fe-4S] cluster and W(MGD)₂ buried inside the protein; the peptide backbone is shown in dark blue at the N-terminal end, and continues as light blue, cyan, green, and yellow to orange at the C-terminal end. (Right) [4Fe-4S] cluster and W(MGD)₂ center. C is shown in gray, N in blue, O in red, P in orange, S in yellow, Fe in brown, and W in cyan (Protein Data Bank [PDB] code 2E7Z).For catalytic activity, AH requires a strong reductant, such as sodium dithionite or titanium(III) citrate (18). Recently, the X-ray structure of AH in the reduced state could be solved at 1.26-Å resolution (28) which gave a first view of its active site: W(IV) is coordinated by four sulfur atoms delivered by the two dithiolene ligands (MGD), one cysteinyl sulfur (Cys141), and one oxygen ligand at a distance of 2.04 Å (Fig. ​(Fig.1).1). Mechanistically, the nature of this oxygen ligand is critical. The observed W-O distance of 2.04 Å is right between the values expected for a hydroxide ligand (1.9 to 2.1 Å) and a coordinated water (2.0 to 2.3 Å), thus not allowing an unequivocal assignment of the sixth ligand of the WS₅O core. Two different reaction mechanisms have been proposed: (i) nucleophilic attack of the hydroxide group and (ii) electrophilic attack of a polarized water molecule, on the C,C triple bond of acetylene (28). As a consequence of theoretical calculations, and in agreement with the observed bond distances, the active W(IV) state should favor a water ligand and therefore an electrophilic addition mechanism (28). Acetylene can access the tungsten ion through a well-defined channel close to the N-terminal domain that harbors the [4Fe-4S] cluster. One residue, Asp13, interacts with the oxygen ligand bound to the W ion, forming a short hydrogen bond of 2.4 Å. Above the W ion and the coordinated H₂O molecule, the substrate channel ends in a ring of six hydrophobic residues. These residues build a cavity with dimensions perfect for accommodating acetylene. Experiments to bind the substrate acetylene, ethylene, the inhibitor propargyl alcohol (H—C☰C—CH₂OH), and dinitrogen or carbon monoxide have failed thus far to produce a complex in the crystal. However, computer docking of one acetylene molecule at this position led to a reasonable fit, positioning the two carbon atoms of the substrate exactly above the H₂O molecule coordinated to tungsten (28).To gain further information about the reaction mechanism of AH, we initiated a study by site-directed mutagenesis and exchanged several amino acids which have been suggested to be important for catalysis at the active site cavity. To achieve this goal, we had to develop a suitable procedure for the heterologous expression of AH in Escherichia coli. Notably, E. coli uses a chaperone system for the insertion of Moco into its enzymes (6). These chaperones of the TorD superfamily act in two ways. First, they bind at the N-terminal signal sequence, similar to the sequence of the TAT export system, thereby delaying the folding of the newly synthesized molybdenum enzyme until Moco has been properly inserted (25). Second, they actively facilitate the incorporation of the molybdopterin cofactor by binding to a second, yet unknown site (13). To improve the assembly of the metal sites as well as to increase the enzymatic activity of the recombinant AH, the N-terminal chaperone binding site of the nitrate reductase, NarG, from E. coli was fused to the AH gene in the expression vector.     

The effect of acetylene on N transformations in an acid oak-beech soil

The effectiveness of acetylene (C₂H₂) as inhibitor of nitrification was studied in relation to the decomposition of C₂H₂. This was done by examining the effects of single and multiple additions of different C₂H₂ concentrations (10, 100, 1000 Pa) on mineral N and NO₃ ^–-N production in samples of the organic (FH) and upper mineral (Ah) layer of an acid oak-beech forest soil. The decomposition of C₂H₂ was much faster in Ah samples than in FH samples. A single addition of 10 Pa C₂H₂ was not sufficient for complete inhibition of nitrification in the Ah samples. Nitrification was blocked completely by all other C₂H₂ treatments in both FH and Ah samples. Addition of C₂H₂ decreased net mineral N production in Ah samples but not in FH samples. Addition of carboxymethyl-cellulose and chitin to Ah soil had no affect on the rate of decomposition of C₂H₂. Chitin had a negative effect on net NO₃ ^–-N production.     

In situ determination of nitrogen fixation in Antarctica using a high sensitivity portable gas chromatograph

A portable gas chromatograph was employed in the Vestfold Hills, Antarctica, during the austral summer of 1979-80 for determining nitrogenase activity of the blue-green alga Nostoc commune Vaucher by the acetylene reduction assay. Acetylene reduction was measured in samples taken along a transect where the vegetation changed with respect to differing topography and water availability. Submerged colonies of Nostoc recorded the highest fixation rates (6.89 nmol C₂H₄. cm^-2 h^-1). Damp mosscyanophyte associations growing on shallow slopes showed moderate rates of acetylene reduction (1.99 nmol C₂H₄. cm^-2 h^-1) whilst the drier vegetation of the steeper terrain was the least active (0.19 nmol C₂H₄. cm^-2 h^-1. The employment of a high sensitivity portable gas chromatograph provided an accurate and reliable method of measuring acetylene reduction.     

Nitrogen fixation by Bacillus strains isolated from the rhizosphere of Ammophila arenaria

Summary Several Bacillus strains, from the rhizosphere of Ammophila arenaria, appeared on ‘nitrogen-free’ agar plates. They were able to grow in nitrogen-poor medium to which 0.1% yeast extract was added. Three of these bacilli were tested for their ability to fix nitrogen using the acetylene reduction assay. The C₂H₂-reducing activity was determined at 8-hour intervals during their growth cycle. C₂H₂ reduction (and accordingly N₂ fixation) was greater under anaerobic than aerobic conditions. Additions of 0.1% CaCO₃ significantly increased the C₂H₂-reducing activity under both conditions. Characterisation suggests that these strains are new nitrogen-fixing Bacillus species. re]19740121     

Isolation and characterization of heterocysts from Anabaena sp. strain CA

A comparative study has been made on the pigment composition and nitrogenase activity of whole filaments and isolated beterocysts from a mutant strain of Anabaena CA. The whole cell absorption spectra of intact filaments and isolated heterocysts showed close resemblance especially between 550–700 nm region. On a quantitative basis the chlorophyll a content was found almost equal between the vegetative cell and heterocyst but the c-phycocyanin content in the heterocyst was about 1/2 that of the vegetative cell. The purification of the phycobiliprotein on DEAE-cellulose showed the presence of c-phycocyanin (_max 615 nm) and allophycocyanin (_max 645 nm, shoulder 620 nm). Isolated heterocysts under H₂ showed acetylene reduction rates of 57 nmol C₂H₄/mg dry wt·min (342 mol C₂H₄/mg chl a·h), whereas intact filaments reduced at the rate of 18 nmol C₂H₄/mg dry wt·min (108 mol C₂H₄/mg chl a·h). This rate accounts for 30% recovery of nitrogenase activity in isolated heterocysts compared to whole filaments. The activity was strictly light dependent and was linear under H₂ for more than 3 h. Addition of as little as 5% H₂ under argon stimulated the C₂H₂ reductionseveral fold. The acetylene reduction (nitrogenase activity) also showed tolerance to 5% added O₂ either under H₂ or argon. The results suggest that the heterocyst of Anabaena CA-V is different in some characteristics (viz., higher endogenous C₂H₂ reduction rate, prolonged activity and higher levels of phycobiliproteins) than those reported in other Anabaena species.     

Utility of Environmental Primers Targeting Ancient Enzymes: Methylotroph Detection in Lake Washington

Methods have been explored for detection of methylotrophs in natural samples, using environmental primers based on genes involved in the tetrahydromethanopterin (H₄MPT)-linked C₁ transfer pathway. The underlying hypotheses were that the H₄MPT-linked pathway is an ancient methylotrophy pathway, based on gene divergence, and that primers targeting more divergent genes will detect a broader variety of methylotrophs compared to the variety uncovered using probes and primers targeting highly conserved genes. Three groups of novel primer sets were developed targeting mch, mtdB, and fae, key genes in the H₄MPT-linked pathway, and these were used to assess the variety of microorganisms possessing these genes in sediments from Lake Washington in Seattle, WA. Environmental clone libraries were constructed for each of the genes and were analyzed by RFLP, and representatives of different RFLP groups were sequenced and subjected to phylogenetic analysis. A combination of all three sets of novel primers allowed detection of the two previously characterized groups of methylotrophs in the site: methanotrophs of the (- and the ó-proteobacterial groups, belonghg to genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, Methylomicrobium, and Methylococcus. In addition to the genes belonging to known methanotroph populations, novel genes were identified, suggesting existence of previously undetected microbial groups possessing C₁ transfer functions in this site. These included sequences clustering with the well-characterized methylotrophic phyla, Methylobacterium, Hyphomicrobium, and Xanthobacter. In addition, sequences divergent from those known for any groups of methylotrophs or methanogens were obtained, suggesting the presence of a yet unidentified microbial group possessing this H₄MPT-linked C₁ transfer pathway.     

Nitrogen Fixation Associated with Sand Grain Root Sheaths (Rhizosheaths) of Certain Xeric Grasses

Nitrogen fixation (acetylene reduction) was found to be associated with sand grain root sheaths (rhizoseaths) occurring on the following xeric grasses: Oryzopsis hymenoides (Roem. and Shult.) Ricker, Agropyron dasystachyum (Hook.) Scrib., Stipa comata Trin. and Rupr., and Aristida purpurea Nutt. Acetylene reduction rates associated with whole plant specimens of these species varied from 515 to 920 nmol C₂H₄/(g dry wt.) × (6 days). Nitrogenase activity was shown to be associated with the rhizosheaths. Bacillus polymyxa-like nitrogen fixers were isolated from the rhizosheaths of each grass. The isolates reduced acetylene and assimilated ¹⁵N₂.     

Biological Dinitrogen Fixation (Acetylene Reduction) Associated with Florida Mangroves

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C₂H₄/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C₂H₂) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction.     

Pyrene Biodegradation by Bacillus spp. Isolated from Coal Tar–Contaminated Soil

Aerobic, mesophilic bacteria from coal tar–contaminated soil were analyzed for pyrene utilization capacity and identified by 16S ribosomal DNA sequencing as members of three genera: Bacillus spp., Pseudomonas sp., and Rhodococcus sp. The soil contained nine different hazardous polyaromatic hydrocarbons (PAHs): benzo[g, h, i]perylene, dibenzo[a, h]anthracene, indeno[1,2,3-c,d]pyrene, pyrene, acenaphthylene, fluorene, phenanthrene, benzo[k]fluoranthene, and benzo[b]fluoranthene. Bacillus spp. (PK-6) MTCC 1005 showed 56.4% utilization of pyrene (C₁₆H₁₀) (50 μg ml^?1) in 4 days, with growth associated biosurfactant activity and resulted in the formation of five new intermediates: phenanthrene (C₁₄H₁₀), 9,10-diphenylphenanthrene (C₂₆H₁₈), 9-methoxyphenanthrene (C₁₅H₁₂O), 5,6,7,8-tetrahydro-1-naphthoic acid (C₁₁H₁₂O₂), and 1,6,7-trimethylnaphthalene (C₁₃H₁₄). The results suggested that Bacillus spp. could be found suitable for practical field application for effective in situ PAH bioremediation.     

Effect of oxygen partial pressure on nitrogen fixation and acetylene reduction in a sandy loam soil amended with glucose

Summary Small samples of soil amended with 2% (w/w) of glucose were preincubated either aerobically or anaerobically and then assayed (N₂ ¹⁵ and C₂H₂-C₂H₄) either aerobically or anaerobically for different time periods. One-hour C₂H₂-C₂H₄ assays showed greatest activity when anaerobic assay followed anaerobic preincubation. During the anaerobic preincubation a lag of 12–24 h occurred before rapid increase in one-hour assay activity was observed. When aerobic assay followed aerobic preincubation a longer lag was observed and lower activities were obtained. When anaerobic assay followed aerobic preincubation (orvice versa) negligible activities were observed in short assays, and longer assays showed increasing activity related to changes in atmosphere and/or microbial population in the closed system. Preincubation of soil on a diffusion gradient at a series of different partial pressures of oxygen confirmed the above pattern and showed that as preincubation pO₂ increased, the anaerobic assay activity rapidly decreased. As preincubation pO₂ decreased from 0.2 atm the aerobic assay activity decreased but less rapidly. The activities observed were related to the sizes of the Azotobacter and Clostridium populations. There was no evidence of aerobic or anaerobic C₂H₂ reduction in any cultures of ‘oligonitrophiles’ isolated. Incorporation of N₂ ¹⁵ was related to C₂H₂ reduction activity in the soil system studied. However, observed C₂H₄/N₂ molar ratios ranged from 10 to 22 and appeared to be highest in samples which were preincubated anaerobically. Issued as Macdonald College Journal Series No.618 and as Canadian IBP contribution No.84.     

Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Differences in Sediment Organic Matter Composition and PAH Weathering between Non-Vegetated and Recently Vegetated Fuel Oiled Sediments

We examined polycyclic aromatic hydrocarbon (PAH) attenuation in contaminated field sediments after only 2 years of plant growth. We collected sediments from vegetated and non-vegetated areas at the Indiana Harbor Canal (IHC), an industrialized area with historic petroleum contamination of soils and sediments. PAH concentrations, PAH weathering indices, and organic matter composition in sediments colonized by Phragmites, cattails, or willow trees were compared to the same indices for non-vegetated sediments. We hypothesized that bulk sediment and humin fractions with measurable increases in plant organic matter content would show measurable changes to PAH attenuation as indicated by more weathered PAH diagnostic ratios or reduced PAH concentrations. Carbon-normalized PAH concentrations were lower in vegetated bulk sediments but higher in vegetated humin fractions relative to non-vegetated sediment fractions. Total organic carbon content was not indicative of more weathered N₃/P₂ ratios or reduced PAH concentrations in vegetated sediment fractions. More weathered N₃/P₂ ratios were observed with increased modern carbon (plant carbon) content of vegetated sediment fractions. Phragmites sediments contained more modern carbon (plant carbon) and more weathered PAH ratios [C₃-naphthalenes and C₂-phenanthrenes (N₃/P₂)] than willow, cattail, and non-vegetated sediments.     

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.     

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.     

Acetylene Reduction (Nitrogen Fixation) by Pulp and Paper Mill Effluents and by Klebsiella Isolated from Effluents and Environmental Situations

High rates of acetylene (C₂H₂) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C₂H₄ produced per ml per h) and under anaerobic (up to 135 nmol of C₂H₄ produced per ml per h) conditions. Pasteurized effluent developed C₂H₂ reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N₂-fixing Azotobacter and Klebsiella were abundant, and N₂-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.     

Dinitrogen fixation (C2H2 reduction) by bacterial strains at various temperatures

Acetylene-reducing activities (ARA) of strains ofEnterobacter agglomerans, Azospirillum brasilense, Azotobacter chroococcum, and Bacillus, isolated from temperate or tropical soils, were compared at different temperatures to study temperature adaptability. All Enterobacter strains and Bacillus strain C-11-25 reduced C₂H₂ at temperatures as low as 5°C. ARA by Enterobacter strains declined sharply above 30°C but ARA by Bacillus strain C-11-25 continued to increase with an increase in temperature.A. brasilense strain sp 245, isolated from wheat roots in Brazil, reduced more C₂H₂ at lower temperatures than strain Cd, isolated from a Californian soil. Similarly, the temperate strain ofA. chroococcum was a better N₂ fixer than the tropicalA. chroococcum strain at lower temperatures. Tropical strains ofA. brasilense andA. chroococcum reduced more C₂H₂ than temperate strains at higher temperatures. Therefore, it appears that temperate and tropical N₂-fixing organisms adapt themselves to their particular environment and should have more potential to benefit crops grown at the particular temperatures favorable to them. Only Bacillus strain C-11-25 has potential to benefit both temperate and tropical crops because it reduced significant acetylene over a wide temperature range.     

Nitrate requirement for acetylene inhibition of nitrous oxide reduction in marine sediments

The inhibition of nitrous oxide (N₂O) reduction by acetylene (C₂H₂) in saltmarsh sediment was temporary; we investigated this phenomenon and possible causes. The reduction of N₂O in the presence of C₂H₂ was biological. N₂O consumption in the presence of C₂H₂ began when nitrate concentration became very low. The time course of N₂O consumption after periods of N₂O accumulation was unaffected by initial nitrate concentrations between 16 and 200M, or C₂H₂ concentrations between 10 and 100% of the gas phase. Sulfide had no effect on the kinetics of N₂O reduction in the presence of C₂H₂. In more dilute slurries of saltmarsh sediments and in estuarine sediment, N₂O persisted in the presence of C₂H₂ unless sufficient organic carbon was added to deplete nitrate. In saltmarsh sediments, the rate of N₂O consumption in the presence of C₂H₂ was not changed by preincubation with C₂H₂. Initial positive rates of N₂O production in the presence of C₂H₂ occurred only when the block was apparently effective (i.e., at nitrate concentrations greater than about 5–10M) and appeared to represent a valid estimate of denitrification. Conversely, and in agreement with previous studies, concentrations of NO₃ ^– below these levels resulted in reduced efficiency of C₂H₂ blockage of N₂O reductase.