Roger Knowles

Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria

Acetylene (0.1 atm) caused complete or almost complete inhibition of reduction of N/sub 2/O by whole cell suspensions of Pseudomonas perfectomarinus, P. aeruginosa and Micrococcus denitrificans. Acetylene did not inhibit reduction of NO/sub 3//sup -/ or NO/sub 2//sup -/ by these organisms. In the presence of acetylene there was stoichiometric conversion of NO/sub 3//sup -/ or NO/sub 2//sup -/ to N/sub 2/O with negligible subsequent reduction of the latter. In the absence of acetylene there was no or only transient accumulation of N/sub 2/O. The data are consistent with the view that N/sub 2/O is an obligatory intermediate in the reduction of NO/sub 2//sup -/ to N/sub 2/ in all of the three organisms studied.

Methane production and consumption in temperate and subarctic peat soils: Response to temperature and pH

Abstract Rates of methane (CH4) production under anaerobic conditions and CH4 consumption under aerobic conditions were studied in slurries of peat samples kept at different temperatures (0–35°C) and pH values (buffered at pH 3.5–8). Apparent Xn, CH4 for consumption was 1 μm. Optimum temperatures for both processes were about 25°C but CH4 production showed much more temperature-dependence (activation energies 123–271 kJ mol−1, Q10 values 5.3–16) than did CH4 consumption (activation energies 202−80 kJ mol−1 values 1.4–2.1). In the 0–10°C range, CH4 production was negligible but CH4 consumption was 13–38% of maximum. Both processes showed optimum pH values which were about 2 pH units higher than the native peat pH in acidic peats and only 0–1 pH unit higher in the more alkaline peats. We conclude that the microflora involved in CH4 metabolism is not well adapted to either low temperatures or low pH values.

Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil

Reduction of N2O in moist soil was inhibited completely by 10−2 atm C2H2 and partially by 10−5 atm C2H2. The effect of C2H4 was 104 times less than that of C2H2. Denitrification of NO−3 occurred in anaerobically or aerobically incubated waterlogged soil and in anaerobic but not in aerobic moist soil. In the absence of C2H2 there was transient accumulation of N2O. In the presence of C2H2 there was stoichiometric conversion of NO−3 to N2O. Some kinetics of the reduction of N2O and of NO−3 to N2O are presented. Denitrification of 1 μg added NO−3-N.g− could be measured within 1 h. Stoichiometries of production of N2O from NO−2 and NO−3, respectively, and production of CO2 attributable to denitrification were consistent with reported energy yields. Reduction of C2H2 to C2H4 occurred immediately following complete denitrification of added NO−3. The incubation of soil in the presence and in the absence of C2H2 thus permits assay of both denitrification and N2 fixation and provides information on the mole fraction of N2O in the products of denitrification.

Acetylene Inhibition Technique: Development, Advantages, and Potential Problems

The discovery in 1966 of the significant interaction of C2H2 with nitrogenase (reviewed in Burns and Hardy, 1975) was rapidly followed by the recognition that many other small double-or triple-bonded molecules such as N2O could act as substrates (Hardy and Knight, 1966). Interactions between C2H2, N2O, and nitrogenase have since been studied in more detail and N2O is in fact a poor substrate, with apparent Km values of 24 kPa for Klebsiella pneumoniae (Jensen and Burris, 1986) and 50 kPa for Azotobacter vinelandii (Liang and Burris, 1988) components in vitro.

Effect of nitrogen fertilizers and moisture content on CH4 and N2O fluxes in a humisol: Measurements in the field and intact soil cores

Field and laboratory studies were conducted to determine effects of nitrogen fertilizers and soil water content on N2O and CH4 fluxes in a humisol located on the Central Experimental Farm of Agriculture Canada, Ottawa. Addition of 100 kg N ha−1 as either urea or NaNO3 had no significant effect on soil CH4 flux measured using chambers. Fertilization with NaNO3 resulted in a significant but transitory stimulation of N2O production. Inorganic soil N profiles and the potential nitrification rate suggested that much of the NH4+ from urea hydrolysis was rapidly nitrified. CH4 fluxes measured using capped soil cores agreed well with fluxes measured using field chambers, and with fluxes calculated from soil gas concentration gradients using Ficks diffusion law. This humisol presents an ideal, unstructured, vertically homogeneous system in which to study gas diffusion, and the influence of gas-filled porosity on CH4 uptake. In soil cores gradually saturated with H2O, the relationship of CH4 flux to gas-filled porosity was an exponential rise to a maximum. Steepening CH4 concentration gradients partially compensated for the decreasing diffusion coefficient of CH4 in soil matrix air as water content increased, and diffusion limitation of CH4 oxidation occurred only at water contents > 130% (dry weight), or gas-filled porosities < 0.2.

Methane production and consumption in a cultivated humisol

SummaryLaboratory studies were conducted on a cultivated humisol containing populations of both methanotrophs and methanogens. The molar ratio CO2 produced : O2 consumed :CH4 consumed was 0.27:1.0:1.0. Methane oxidation showed typical Michaelis-Menten kinetics with apparent Km values for CH4 and O2 of 66.2 μM and 37.0 μM, respectively. The low CO2 yields and the effects of low dissolved oxygen indicated the presence of aerobic obligate methanotrophs. It is suggested that the methanotrophs in this soil are not entirely dependent on atmospheric CH4 for growth and survival in situ.

ROLES OF MOSS SPECIES AND HABITAT IN METHANE CONSUMPTION POTENTIAL IN A NORTHERN PEATLAND

In northern peatlands with water tables at or near the surface, the Sphagnum moss layer is potentially the only aerobie region where CH4 oxidation can occur. We hypothesized that mosses with varying physiologies would create different conditions for methane-oxidizing bacteria and, in turn, affect rates of CH4 consumption. We measured in-vitro CH4 consumption potential of Sphagnum magellanicum and Sphagnum capillifolium taken from the same habitat and S. magellanicum and Sphagnum majus across habitats to compare and contrast species and environmental effects. In certain cases, S. capillifolium consumed CH4 more rapidly than S. magellanicum taken from identical habitats, although the greatest difference in consumption rates between species was only 29 μg CH4 g−1 dry moss d−1, compared to a maximum difference of 126 and 415 μg CH4 g−1 dry moss d−1 in S. magellanicum and S. majus sampled from different habitats. In most cases, CH4 was consumed most rapidly in the lower, non-photosynthetic portions of the Sphagnum mosses, and consumption potential increased with an increase in the concentration of CH4 in the habitat. We hypothesize that CH4 consumption occurred internally, likely in the hyaline cells, as external surface sterilization did not significantly alter CH4 consumption rates. This work provides evidence that different Sphagnum moss species have variable ability to oxidize CH4, although inter-species differences are small compared to differences across habitats.

Inhibition of methane consumption in forest soils and pure cultures of methanotrophs by aqueous forest soil extracts

Aqueous extracts of forest soils inhibited atmospheric methane consumption by boreal and temperate forest soils by up to 100%. Extracts from the upper soil layers (0–5 cm) were generally inhibitory, while those from deeper in the soil column (5–12 cm) were not. The inhibitory effect was concentration-dependent and transient, becoming insignificant after as little as 3 d of exposure of soil to the aqueous extracts. Ammonium concentration alone could not explain the inhibitory effect and treatment with activated charcoal suggested involvement of an organic component. Methane oxidation by pure cultures of the methanotrophs Methylosinus trichosporium OB3b and Methylobacter luteus was significantly inhibited by the aqueous extracts of upper, but not lower soil layers. Removal of phenolic compounds by polyvinylpolypyrrolidone alleviated inhibition in the pure cultures but not in a forest soil. The results support the hypothesis that naturally occurring substances may prevent methane consumption in the upper layers of forest soils.

Effect of ammonium-, nitrite- and nitrate nitrogen on anaerobic nitrogenase activity in soil

Abstract Sandy loam soil, with added glucose, was incubated anaerobically under N2 and subjected to repeated 1-h C2H2 reduction assays. In the presence of 1% glucose the addition of 50 μg NH4+ −N/g or of 20 μg NO−3 N/g (untreated soil contained 1.2 μg NH+4−N and 7.10 μg NO−3-N/g) caused at least some suppression of nitrogenase activity. Activity developed when the KCl-extractable soil inorganic nitrogen concentration dropped below 35 μg/g. In the presence of 0.1 or 0.05% glucose the addition of 5 μg NH+4−N/g caused some suppression of nitrogenase activity. However, activity developed when the soil NH4+-N concentration dropped below about 4 μg/g. With 0.1% glucose and 5 μg added NO−2 N/g, activity did not develop until the soil NO−2 -N concentration dropped to zero. Added NO−3 N was rapidly reduced and denitrified to NO−2- N, N2O-N and NH+4 N and furthermore caused some inhibition of CO2 evolution. The data from NH4−-addition experiments are consistent with a nitrogenase repression/ derepression threshold of 4 and 35μg NH+4-N/g at 0.05 and 1% glucose concentrations, respectively. The data from NO−2- and NO−3-addition experiments suggest a combination of repression and toxicity effects in the presence of added NO−3 N.

Efficiency of acetylene reduction (nitrogen fixation) in soil: Effect of type and concentration of available carbohydrate

Abstract Sandy loam field soil and Acer saccharum (maple) forest soil were amended with different concentrations of glucose and mannitol and incubated at different pO 2 levels. Nitrogenase activity was determined by repeated 1-h C 2 H 2 reduction assays performed at the ambient pO 2 of incubation. Calculated efficiencies of N 2 fixation increased with increasing anaerobiosis and with decreasing added carbohydrate concentration. Efficiencies up to 30 mg N 2 fixed per gram of glucose consumed were obtained under anaerobic conditions in the presence of 0.25% (w/w) glucose. Evidence suggested that low aerobic efficiencies were caused by intense competition for carbohydrate and by lower pH values attained. High concentrations (up to 3.0% w/w) of glucose under aerobic conditions suppressed the development of N 2 ase activity. Mannitol supported N 2 ase activity the development of which was very much delayed under aerobic conditions but little delayed under anaerobic conditions.