Nitrification and denitrification by Thiosphaera pantotropha in aerobic chemostat cultures

Abstract: Thiosphaera pantotropha has been reported to denitrify aerobically and nitrify heterotrophically. However, recent evidence has indicated that these properties (particularly aerobic denitrification) have been lost. The occurrence and levels of aerobic denitrification and heterotrophic nitrification by T. pantotropha in chemostat cultures have therefore been re-evaluated. Only low nitrate reduction rates were observed: the apparent nitrogen loss was of the same order of magnitude as the combined error in the calculated nitrogen consumption. However, ¹⁵N mass spectrometry revealed low aerobic denitrification rates (about 10% of the rates originally published by this group). Heterotrophic nitrification rates were about a third of previous observations. N₂ and N₂O were both produced from NH₄⁺, NO₃⁻ and NO₂⁻. Periplasmic nitrate reductase was present in aerobically grown cells.     

Urea production by Thiosphaera pantotropha and by anaerobic enrichment cultures from marine sediments

Abstract The production of urea by Thiosphaera pantotropha was studied. Batch cultures were grown on acetate as energy source and with NO₃⁻ or O₂ as terminal electron acceptor. Urea accumulated in the media during exponential growth in aerobic and anaerobic cultures of T. pantotropha . Urea production continued after the cells had entered the stationary growth phase. Bacterial ability to produce urea was supported by studies of cultures enriched for denitrifying, sulphate-reducing and fermenting bacteria. The results implied that urea production was common among bacteria normally considered to be important in marine sediments.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Nitrification and nitrous oxide production potentials in aerobic soil samples from the soil profile of a Finnish coniferous site receiving high ammonium deposition

Abstract Using aerobic soil slurry technique nitrification and nitrous oxide production were studied in samples from a pine site in Western Finland. The site received atmospheric ammonium deposition of 7–33 kg N ha⁻¹ a⁻¹ from a mink farm. The experiments with soil slurries showed that the nitrification potential in the litter layer was higher at pH 6 than at pH 4. However, the nitrification potentials in the samples from the organic and mineral horizons at pH 6 and 4 were almost equal. Also N₂O was produced at a higher rate at pH 6 than at pH 4 in slurries of the litter layer samples. The reverse was true for samples from the organic and mineral horizons. The highest N₂O production and nitrification rates were measured in the suspensions of litter layer samples. Nitrification activity in field-moist soil samples was lower than the activity in the slurries indicating that the availability of ammonium limited nitrification in these soils. Acetylene (2.5 kPa) retarded nitrification activity (70-–100%) and N₂O production (40 – 90%) in soil slurries. Acetylene inhibited the N₂O production by 40–60% during the first 3 days after its addition to field-moist samples incubated in aerobic atmosphere. After 3 days the inhibition became much lower (4–5%). The results indicate that, in soil profiles of boreal coniferous forests receiving ammonium deposition, chemolithotrophic nitrification may have importance in the N₂O production, and that changes in soil pH affect differently nitrification as well as N₂O production in litter and deeper soil layers.     

Reduction of nitrous oxide by a soil Cytophaga in the presence of acetylene and sulfide

Abstract A denitrifying Cytophaga was isolated from soil enriched by anaerobic incubation with glucose, sulfide (S²⁻), nitrous oxide (N₂O), and acetylene (C₂H₂). Such soil enrichments and pure cultures of the isolated Cytophaga reduced N₂O rapidly even in the presence of a normally inhibitory concentration of C₂H₂ (4 kPa) providing S²⁻ was present (8 μmol/g soil or 0.4 μmol/ml culture). Since C₂H₂ inhibition of the reduction of N₂O is used as a tool in the assay of denitrification, the presence in large numbers of such a Cytophaga may influence the effectiveness of this assay especially in sulfidic environments.     

Denitrification and nitrate-reducing bacterial populations in abandoned and reclaimed minesoils

Abstract Microbial populations, nitrogen mineralization potentials, and denitrification enzyme activities were examined in two abandoned carbolithic minesoils. Numbers and activities of bacteria and fungi were lower in nonamended than in lime and/or fly ash amended sites. Rates of aerobic NO₃⁻ production (3 to 38 μg-N kg⁻¹ h⁻¹) and anaerobic NO₃⁻ reduction to N₂O (5 to 68 μg-N kg⁻¹ h⁻¹) were measured. Organisms capable of N₂O production under anaerobic soil conditions were present in low numbers, and their activity was restricted in part by low soil pH. Nondenitrifying nitrate-reducing bacteria were more diverse and in greater numbers than respiratory denitrifiers and may have been responsible for N₂O production in assays measuring denitrification enzyme activity.     

N2(C2H2)-fixation in early stages of a primary succession on a reclaimed salt marsh

Nitrogen fixation activity was determined for Lotus tenuis. Medicago lupulina and Trifolium pratense . The three species grew in clones in grassland in an area reclaimed from brackish water in the 1940s. The N₂[C₂H₂]-fixation was measured in soil cores throughout 1974 and 1975. From cores taken in dense and uniform stands of the species, the yearly N₂[C₂H₂]-fixation at maximum cover was estimated. L. tenuis fixed about 4 g N m⁻² yr⁻¹ (area with max. cover 130%), i.e. 30–56% of its requirement. Both M. lupulina and T. pratense fixed about 7 g N m⁻² yr⁻¹ (maximum cover 37% and 80%) i.e. 67% of their N-requirement. Average N₂[C₂H₂]-fixation for the whole area was 0.4 g N m⁻² yr⁻¹, considerably less than the N-addition through rainfall.     

Sources of nitrous oxide production following wetting of dry soil

Abstract Production of N₂O was detected within 30 min of adding water to very dry soil (matric water potential < −9 MPa) sampled at the end of the dry season from an annual grassland of California, U.S.A. Using C₂H₂ to inhibit nitrification, we demonstrate that nitrification was a modest source of N₂O in sieved soil wetted to a water content below field capacity, but that denitrification was the major source of N₂O in sieved soils wetted to a water content above field capacity and in intact cores wetted either below or above field capacity. Significant abiological sources of N₂O were not detected. De novo enzyme synthesis began within 4–8 h of wetting, and denitrifying enzyme activity doubled within 26 h, indicating that denitrifying bacteria can quickly transform their metabolic state from adaptation to severe drought stress to rapid exploitation of changing resources.     

Interactions between respiration and denitrification during growth of Thiosphaera pantotropha in continuous culture

Abstract The effects of oxygen on the use of nitrate as an electron acceptor by the denitrifying bacterium Thiosphaera pantotropha were investigated during growth on acetate. In batch cultures under aerobic conditions nitrate was not utilised and the growth rate constant was 0.55 h⁻¹. The corresponding value for growth on nitrate under anoxic conditions was 0.37 h⁻¹. In acetate-limited continuous cultures with feedback control of the dissolved oxygen concentration, nitrate utilisation was totally inhibited by the lowest concentration of oxygen tested (22 μM). Carbon conversion efficiencies with acetate increased from 0.28 with nitrate to 0.44 with oxygen. The rates of nitrification calculated from nitrogen balance studies were not greater than 1.5% of the rate of anoxic denitrification.     

Symbiotic N2 fixation in a high Alpine grassland: effects of four growing seasons of elevated CO2

1. Increasing carbon dioxide concentration (E: 680 μl CO₂ litre^–1 vs ambient, A: 355 μl CO₂ litre^–1) around late-successional Alpine sedge communities of the Swiss Central Alps (2450 m) for four growing seasons (1992–1995) had no detectable effect on symbiotic N₂ fixation in Trifolium alpinum —the sole N₂-fixing plant species in these communities (74 ± 30 mg N m^–2 year^–1, A and E plots pooled).
2. This result is based on data collected in the fourth growing season showing that elevated CO₂ had no effect on Trifolium above-ground biomass (4·4 ± 1·7 g m^–2, A and E plots pooled, n = 24) or N content per unit land area (124 ± 51 mg N m^–2, A and E pooled), or on the percentage of N Trifolium derived from the atmosphere through symbiotic N₂ fixation (%Ndfa: 61·0 ± 4·1 across A and E plots) estimated using the ¹⁵N dilution method.
3. Thus, it appears that N inputs to this ecosystem via symbiotic N₂ fixation will not be dramatically affected in the foreseeable future even as atmospheric CO₂ continues to rise.     

Membrane inlet mass spectrometric analysis of N-isotope labelling for aquatic denitrification studies

Abstract: Techniques are described for measuring the isotope distribution in dissolved nitrate and N₂ using membrane inlet mass spectrometry, which allows several gases to be measured in a water sample without the need for any separation steps. The isotope distribution in dissolved nitrate was measured using denitrifying Pseudomonas nautica to reduce the nitrate to N₂ which was then measured by mass spectrometry. Pseudomonas nautica NCIMB 1967 was easily grown in nitrate-limited continuous culture minimising intra- or extracellular nitrate or nitrite pools, and the bioassay was tolerant of a range of salinities. The precision of the bioassay when measuring samples with high ¹⁵NO₃⁻ contents (0.5 μmol) was 0.05 atom%; with 0.1 μmol ¹⁵NO₃⁻, the precision was around 0.2 atom%. Differences in labelling of N₂ in preserved samples obtained from ¹⁵NO₃⁻ incubations of water-covered sediment cores were measured on parallel samples with membrane inlet MS and GC-MS. The membrane inlet technique was accurate but the precision on ratio measurements was lower than by GC-MS.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Convergence in the relationship of CO2 and N2O exchanges between soil and atmosphere within terrestrial ecosystems

Ecosystem CO₂ and N₂O exchanges between soils and the atmosphere play an important role in climate warming and global carbon and nitrogen cycling; however, it is still not clear whether the fluxes of these two greenhouse gases are correlated at the ecosystem scale. We collected 143 pairs of ecosystem CO₂ and N₂O exchanges between soils and the atmosphere measured simultaneously in eight ecosystems around the world and developed relationships between soil CO₂ and N₂O fluxes. Significant linear regressions of soil CO₂ and N₂O fluxes were found for all eight ecosystems; the highest slope occurred in rice paddies and the lowest in temperate grasslands. We also found the dominant role of growing season on the relationship of annual CO₂ and N₂O fluxes. No significant relationship between soil CO₂ and N₂O fluxes was found across all eight ecosystem types. The estimated annual global N₂O emission based on our findings is 13.31 Tg N yr⁻¹ with a range of 8.19–18.43 Tg N yr⁻¹ for 1980–2000, of which cropland contributes nearly 30%. Our findings demonstrated that stoichiometric relationships may work on ecological functions at the ecosystem level. The relationship of soil N₂O and CO₂ fluxes developed here could be helpful in biogeochemical modeling and large-scale estimations of soil CO₂ and N₂O fluxes.     

The microbial flora of smoked pork loin and frankfurter sausage stored in different gas atmospheres at 4°C

The development of the microflora of smoked pork loin and frankfurter sausage was followed during storage in vacuum, N₂ and CO₂ atmospheres at 4°C. The total aerobic count on the smoked pork loin reached 10⁷ organisms/g after 37 d in vacuum, 43 d in N₂ and 49 d in CO₂. The corresponding value for the sausage was 77 d in vacuum, while the growth stopped at 6 times 10⁴ organisms/g after 98 d in N₂, and at 4 times 10² organisms/g after 48 d in CO².
The predominant organisms on the fresh products were Bacillus spp., coryneform bacteria, Flavobacterium spp. and Pseudomonas spp.
At the end of the storage time the microflora on both products in the three gas atmospheres, consisted mainly of Lactobacillus spp. and two large groups of organisms that could not be identified as any described genus. Some of the unidentified strains could be classified as a Lactobacillus sp. after subsequent subculturing on laboratory media.
The numbers of Lactobacillus spp. at the end of storage decreased in the order, CO₂ > N₂ > vacuum. Lactobacillus viridescens generally constituted a substantial part of the Lactobacillus flora (5–72%). On the sausages two large uniform groups of unidentifiable homofermentative Lactobacillus spp. were also found.     

Seasonal patterns of nitrogen fixation in termites

1. Termite nitrogenase activity was highest in autumn and spring (≈ 3 μg N₂ fixed termite fresh mass (g)^–1 day^–1) and lowest in winter and summer (≈ 0·8 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
2. The nitrogenase activity of worker termites was significantly higher than all other castes (1·58 ± 0·27 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
3. Worker termites constituted the largest proportion of all the castes throughout the study period (≈ 90%).
4. The localized input of fixed nitrogen by termites may reach 15·3 mg N log^–1 day^–1 and 5·6 g N log^–1 year^–1.     

Nitrogen use efficiency. 3. Nitrogen fixation: genes and costs

The nitrogen use efficiencies (NUE) of N₂ fixation, primary NH ₄⁺ assimilation and NO ₃⁻ assimilation are compared. The photon and water costs of the various biochemical and transport processes involved in plant growth, N-assimilation, pH regulation and osmolarity generation, per unit N assimilated are respectively likely to be around 5 and 7% greater for N₂ fixation than for a combination of NH ₄⁺ and root and shoot NO ₃⁻ assimilation as occurs with most crops. Studies on plant and rhizobial genes involved in nodulation and N₂ fixation may lead to more rapid nodulation, production of more stress-tolerant N₂ fixing systems and transfer of the hydrogenase system to rhizobium/legume symbioses which currently do not have this ability. The activity of an uptake hydrogenase is predicted to decrease the photon cost of diazotrophic plant growth by about 1%.     

The relationship between the in situ reduction level of the cytochrome c pool of Azorhizobium caulinodans growing in a chemostat with NH4+ or N2 as the N source and the total activity of cytochrome c oxidases

Abstract The in situ method for determination of reduction levels of cytochromes b and c pools during steady-state growth (Pronk et al., Anal. Biochem. 214, 149–155, 1993) was applied to chemostat cultures of the wild-type, a cytochrome aa₃ single mutant and a cytochrome aa₃/d double mutant of Azorhizobium caulinodans . For growth with NH₄⁺ as the N source, the results indicate that (i) the aa₃ mutant strains growing at a dissolved O₂ tension of 0.5% possess an active alternative cytochrome c oxidase, which is hardly present during fully aerobic growth, and assuming that (i) also pertains to the wild-type, (ii) the wild-type uses cytochrome aa₃ under fully aerobic conditions. For growth with N₂ as the N source, it was found that the aa₃ mutant strains growing at dissolved O₂ tensions ranging from 0.5 to 3.0% also contain an active alternative cytochrome c oxidase.     

Field determination of denitrification in water-logged forest soils

Abstract Denitrification rates were measured as N₂O production in two water-logged forest soils at monthly intervals. The effect of acetylene inhibition and the addition of nitrate, glucose, acetate and celloboise in field incubations was examined.
N₂O release from the two soils was very low, 26 mg N₂m⁻²y⁻¹ in ash and 178 mg N₂ in alder. In acetylene inhibited incubations N₂O production was higher, 296 and 486 mg N₂m⁻² y⁻¹ in ash and alder respectively. After addition of nitrate and C-sources to a 10 mM concentration, denitrification rates increased to 5–15 times higher values.
The denitrification rates below 4°C were low and most N₂O was produced in late spring and summer.
The highest rate of denitrification during a 50 h incubation experiment occurred between 3 and 23 h.     

The effect of electron acceptor variations on the behaviour of Thiosphaera pantotropha and Paracoccus denitrificans in pure and mixed cultures

Abstract The competitive advantages provided by a capacity for aerobic denitrification have been tested by comparing Thiosphaera pantotropha (which denitrifies aerobically and anaerobically), with a strain of Paracoccus denitrificans (which only denitrifies under anaerobic conditions) in acetate-limited chemostats. A comparison of μ -C_s curves based on K _s and μ _max measurements indicated that Pa. denitrificans could be expected to dominate mixtures of the two species at high growth rates when the dissolved oxygen was above 80% of air saturation and NH₃ was the sole source of nitrogen. The comparison also suggested t that at lower growth rates, lower dissolved oxygen tensions, and/or in the presence of nitrate, Tsa. pantotropha should have the competitive advantage. Chemostat experiments with mixtures of the two species showed that Tsa. pantotropha did, indeed, dominate the population when expected. However, when Pa. denitrificans was expected to dominate, only a small increase in the Pa. denitrificans numbers was possible before Tsa. pantotropha formed a biofilm on the walls of the chemostat instead of washing out, and was again able to out-compete Pa. denitrificans for acetate. Experiments with axenic chemostat cultures subjected to aerobic/anaerobic switches showed that Tsa. pantotropha , with its constitutive denitrifying system, was able to adjust smoothly to the changing environmental conditions and thus continued to grow. Pa. denitrificans does not have constitutive denitrifying enzymes, and could consequently not adjust its metabolism to the lack of oxygen rapidly enough. It therefore washed out at a rate equivalent to the dilution rate.