Thiosphaera pantotropha: a sulphur bacterium capable of simultaneous heterotrophic nitrification and aerobic denitrification

Thiosphaera pantotropha, a facultative anaerobe is capable of mixotrophic and heterotrophic growth on a wide range of substrates. It can oxidize reduced sulfur compounds, nitrify ammonia heterotrophically to nitrite, and reduce nitrate or nitrite to nitrogen gas irrespective of the ambient dissolved oxygen concentration.¹ The ammonia oxygenase has similarities with that of autotrophic nitrifiers (such as, light sensitivity, Mg²⁺ requirement, and NAD(P)H utilization), so has hydroxylamine oxidoreductase (cytochrome C oxidation, hydrazine inhibition) but there are some differences too (e.g., hydroxylamine inhibition of ammonia oxidation).² It is the denitrifying enzyme system expression and operation under aerobic conditions, however, which is shrouded with controversy. The typical enzyme system of the bacterium throws open interesting possibilities of its applications for wastewater treatment. T. pantotropha has been tested in mixed bacterial cultures in suspended as well as fixed film systems to treat simulated industrial and domestic wastewaters. It has also been used in a flocculating algal-bacterial system to treat synthetic fertilizer wastewater. Fixed film systems have yielded better results. High rates of simultaneous removal of organics and nitrogen have been achieved. This indicates a vast improvement over conventional treatment strategies.     

Utilization and leaching of nitrate from two Deschampsia-dominated heathland sites: a lysimeter study using intact soil columns*

High deposition levels of atmospheric ammonia in the Netherlands have led to the major replacement of dwarf shrubs by grasses and to elevated nitrification rates in acid heathland soils. In order to study the efficacy of a naturally established grass-heath of Deschampsia flexuosa at capturing NO^- ₃, an outdoor 'mesocosm' lysimeter experiment was set up with relatively large and undisturbed soil columns from two Deschampsia-dominated heathland sites. One of the sites (Ede) had a relatively high rate of nitrate production, whereas the other (Hoorneboeg [HB]) showed practically no nitrate formation. For part of the Ede columns, the fate of labeled nitrate, split-applied at two rates (30 or 150 kg ha^-1 yr^-1) during two seasons, was studied. D. flexuosa was highly effective in acquiring fertilizer nitrate, as demonstrated by (1) distinct rises in foliar NRA, especially at high N; (2) increased ¹⁵N enrichments in all plant components with N rate; (3) significant increases in organic-N and carboxylate concentration in several plant compartments; and (4) clear shifts in biomass allocation in favour of the aboveground tissues.After 18 months at low N, an average 39 and 23% of the applied N was immobilized in the plant and soil compartments, respectively; at high N rate, corresponding recoveries were 33 and 20%. Total leaching of nitrate (beyond a depth of 35 cm) from the unfertilized Ede columns corresponded to an annual loss of 1.9 kmol N ha^-1, whereas leaching was virtually zero from HB columns. Relatively high amounts of N leached from the fertilized columns with apparent fertilizer recovery in the leachate reaching an average 60% at high N. However,¹⁵ N analyses revealed only recoveries of 2.0% (low N) and 7.2% (high N) of the applied N in the leachate. From columns where the plant cover had been removed, apparent and real leaching losses reached values of >100 and 10% of the applied N, respectively. Hence, soil-derived N appeared by far the major source of leaching. Unplanted and unfertilized HB columns displayed high rates of nitrification and leached high amounts of nitrate, suggesting a plant-induced repression of the in situ nitrification at this site.On average, planted columns had lost 37% (low N) and 40% (high N) of the applied N, whereas unplanted lysimeters had lost 89% (for both low and high N). The N not recovered was presumed lost by denitrification due to favourable conditions with respect to nitrate concentration, moisture, carbon supply, and temperature.     

Nitrogen mineralization and denitrification as influenced by crop residue particle size

Managing the crop residue particle size has the potential to affect N conservation in agricultural systems. We investigated the influence of barley (Hordeum vulgare) and pea (Pisum sativum) crop residue particle size on N mineralization and denitrification in two laboratory experiments. Experiment 1: ¹⁵N-labelled ground (3 mm) and cut (25 mm) barley residue, and microcrystalline cellulose+glucose were mixed into a sandy loam soil with additional inorganic N. Experiment 2: inorganic¹⁵ N and C₂H₂ were added to soils with barley and pea material after 3, 26, and 109 days for measuring gross N mineralization and denitrification.Net N immobilization over 60 days in Experiment 1 cumulated to 63 mg N kg^-1 soil (ground barley), 42 (cut barley), and 122 (cellulose+glucose). More N was seemingly net mineralized from ground barley (3.3 mg N kg^-1 soil) than from cut barley (2.7 mg N kg^-1 soil). Microbial biomass peaked at day 4 with the barley treatments and at day 14 with the cellulose+glucose whereafter the biomass leveled out at values 79 mg C kg^-1 (ground), 104 (cut), and 242 (cellulose+glucose) higher than for the control soil. Microbial growth yields were similar for the two barley treatments, ca. 60 mg C g^-1 substrate C added, which was lower than the 142 mg C g^-1 C added with cellulose+glucose. This suggests that the 75% (w/w) holocelluloses and sugars contained with the barley material remained physically protected despite grinding. In Experiment 2 gross mineralization on day 3 was 4.8 mg N kg^-1 d^-1 with ground pea, twice as much as for all other treatments. On day 26 the treatment with ground barley had the greatest gross N mineralization. In static cores ground barley denitrified 11-fold more than did cut barley, whereas denitrification was similar for the two pea treatments. In suspensions denitrification was similar for the two treatments both with barley and pea residue.We conclude that the higher microbial activity associated with the initial decomposition of ground plant material is due to a more intimate plant residue-soil contact. On the long term, grinding the plant residues has no significant effect on N dynamics.     

Effect of oxygen on denitrification in continuous chemostat culture withComamonas sp SGLY2

Continuous cultures ofComamonas sp SGLY2 were grown anaerobically prior to establishing steady states at different oxygen flow rates. At a low oxygen transfer rate, no dissolved oxygen accumulated in the medium and all nitrate was reduced to dinitrogen. Concurrently with the increase of dissolved oxygen concentration in the liquid phase, the rate of denitrification decreased. However, at a dissolved oxygen concentration near saturation (33 mg L^–1), a part of the electron flow always diverted to nitrate with production of dinitrogen: the aerobic denitrification rate was equivalent to 35% of that calculated under anaerobic conditions. These experiments reflected the co-utilization of oxygen and N-oxides and the production of dinitrogen, up to saturated conditions, which implied synthesis and activity of the four denitrifying enzymes under various aeration conditions.     

Long-term effects of nitrate on lucerne (Medicago sativa L.) nitrogen fixation is not influenced by the denitrification status of the microsymbiont

Studies on the inhibitory effects of combined nitrogen on biological nitrogen fixation in legume crops have been usually carried out after short-term nitrate treatments at high concentrations. As these treatments are quite different from field conditions, a study was conducted to evaluate the effects of the continuous presence of nitrate (0, 1, 5 and 10 mM) throughout three months on lucerne (Medicago sativa L.). Plants were grown in a greenhouse with perlite as substrate and were inoculated with a denitrifying Sinorhizobium meliloti strain (102-F-51) and a non-denitrifying strain (102-F-65). During the first 60 days of growth, the highest nitrate treatment resulted in a complete inhibition of the main symbiotic parameters (nodule initiation and development and specific nitrogen fixation) in plants inoculated with either strain. However, after 3 months of growth in the presence of nitrate, this inhibition was partly abolished, with a high number of new functioning nodules being formed. Acetylene reduction activity (ARA) of these plants was 70% of the control plants. As this process was observed in plants nodulated with either strain, it is concluded that this was not related to the denitrifying ability of the strain, but is an intrinsic property of the lucerne nitrogen fixing system. As legume plants usually grow under natural field conditions in the continuous presence of nitrate, the ability to use simultaneously nitrate and atmospheric nitrogen could be of adaptive and agronomic importance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bacterial oxidation of sulphide under denitrifying conditions

Anoxic H2S oxidation under denitrifying conditions produced sulphur and sulphate in almost equal proportions by an isolated Thiobacillus denitrificans. Under nitrate reducing conditions the rate of sulphide oxidation was approximately 0.9 g sulphide/g biomass h. Nitrate was reduced to nitrite and accumulated during sulphide oxidation. Above 100 mg nitrite/l, the sulphide oxidation rate declined and at 500 mg/l it was totally arrested. The optimum pH for the anoxic sulphide oxidation was around 7.5. Concentrations of sulphate 1500 mg/l and acetate 400 mg/l had no effect on anoxic sulphide oxidation.     

Retention of nitrogen in small streams artificially polluted with nitrate

A simple method was developed to test hypotheses on nitrogen retention in first-order streams in an agricultural region near Oslo, SE Norway. A gravity-operated system added a nitrate solution to the streams continuously at a constant rate. Water samples were collected at fixed intervals downstream to follow the rate of decline in streamwater nitrate. Repeated sampling allowed calculation of regression lines from experiments with different levels of additions of nitrate.The experiments showed that removal of nitrate generally increased with higher initial nitrate concentration, regardless of temperature (range 8–16 °C). Higher nitrate removal rates were found in a stream polluted by easily degradable organic matter than in a similar stream fed by groundwater.Experiments conducted in indoor channels lined with a layer of stream sediment gave reproducible, exponential rates of nitrate decrease in the recirculated water.The results are discussed in the framework of first-order streams as protective ecotones between agricultural areas and higher-order parts of the watersheds.     

Nitrate limitation of N2O production and denitrification from tropical pasture and rain forest soils

Nitrous oxide production was measured in intact cores taken from active pasture and old-growth forest Inceptisols in the Atlantic Lowlands of Costa Rica. Following additions of aqueous KNO₃ or glucose, or the two combined amendments, the cores were incubated in the laboratory to determine if N₂O production rates were either N-limited or C-limited in the two land use types. Differences in rates of denitrification (N2₂O + N₂ production) among amended forest and pasture soils were determined by addition of 10% C₂H₂.The forest soils were relatively insensitive to all amendment additions, including the acetylene block. Forest N₂O production rates among the treatments did not differ from the controls, and were consistently lower than those of the pasture soils. With the addition of glucose plus nitrate to the forest soils, production of N₂O was three times greater than the controls, although this increase was not statistically significant. On the other hand, the pasture soils were definitely nitrogen-limited since N₂O production rates were increased substantially beyond controls by all the amendments which contained nitrate, despite the very low N level (5 mg N kg^–1 soil) relative to typical fertilizer applications. With respect to the nitrate plus glucose plus acetylene treatment, denitrification was high in the pasture soils; N₂O production in the presence of C₂H₂ was 150% of the rate of N₂O production measured in the absence of the acetylene block. The results are discussed in relation to the effects of agricultural land use practices and subsequent impacts of disturbance on N₂O release.     

Denitrification measurements in aquatic sediments: A comparison of three methods

Measurements of denitrification using the acetylene inhibition,¹⁵N isotope tracer, and N₂ flux methods were carried out concurrently using sediment cores from Vilhelmsborg sø, Denmark, in an attempt to clarify some of the limitations of each technique. Three experimental treatments of overlying water were used: control, nitrate enriched, and ammonia enriched water. The N₂ flux and¹⁵N tracer experiments showed high rates of coupled nitrification/denitrification in the sediments. The acetylene inhibition method did not capture any coupled nitrification/denitrification. This could be explained by acetylene inhibition of nitrification. A combined¹⁵N tracer/acetylene inhibition experiment demonstrated that acetylene inhibition of N₂O reduction was incomplete and the method, therefore, only measured approximately 50% of the denitrification due to nitrate from the overlying water. Similar rates of denitrification due to nitrate in the overlying water were measured by the N₂ flux method and the acetylene inhibition method, after correcting for the 50% efficiency of acetylene inhibition. Rates of denitrification due to nitrate from the overlying water measured by the¹⁵N tracer method, however, were only approximately 35% or less of those measured by the acetylene inhibition or N₂ flux methods.     

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 10³ to 10⁵ bacteria ml^–1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.Abbreviations BTEX benzene, toluene, ethylbenzene, andp-xylene - HPLC high performance liquid chromatography - GAC granular activated carbon