H.J. Laanbroek

Competition for limiting amounts of oxygen between Nitrosomonas europaea and Nitrobacter winogradskyi grown in mixed continuous cultures

Chemolithotrophic nitrifying bacteria are dependent on the presence of oxygen for the oxidation of ammonium via nitrite to nitrate. The success of nitrification in oxygen-limited environments such as waterlogged soils, will largely depend on the oxygen sequestering abilities of both ammonium- and nitrite-oxidizing bacteria. In this paper the oxygen consumption kinetics of Nitrosomonas europaea and Nitrobacter winogradskyi serotype agilis were determined with cells grown in mixed culture in chemostats at different growth rates and oxygen tensions.Reduction of oxygen tension in the culture repressed the oxidation of nitrite before the oxidation of ammonium was affected and hence nitrite accumulated. Km values found were within the range of 1–15 and 22–166 μM O2 for the ammonium- and nitrite-oxidizing cells, respectively, always with the lowest values for the N. europaea cells. Reduction of the oxygen tension in the culture lowered the half saturation constant Km for oxygen of both species. On the other hand, the maximal oxygen consumption rates were reduced at lower oxygen levels especially at 0 kPa. The specific affinity for oxygen indicated by the Vmax/Km ratio, was higher for cells of N. europaea than for N. winogradskyi under all conditions studied. Possible consequences of the observed differences in specific affinities for oxygen of ammonium-and nitrite-oxidizing bacteria are discussed with respect to the behaviour of these organisms in oxygen-limited environments.

Oxidation of short-chain fatty acids by sulfate-reducing bacteria in freshwater and in marine sediments

Colony counts of acetate-, propionate- and l-lactate-oxidizing sulfate-reducing bacteria in marine sediments were made. The vertical distribution of these organisms were equal for the three types considered. The highest numbers were found just beneath the border of aerobic and anaerobic layers.Anaerobic mineralization of acetate, propionate and l-lactate was studied in the presence and in the absence of sulfate. In freshwater and in marine sediments, acetate and propionate were oxidized completely with concomitant reduction of sulfate. l-Lactate was always fermented. Lactate-oxidizing, sulfate-reducing bacteria, belonging to the species Desulfovibrio desulfuricans, and lactate-fermenting bacteria were found in approximately equal amounts in the sediments. Acetate-oxidizing, sulfate-reducing bacteria could only be isolated from marine sediments, they belonged to the genus Desulfobacter and oxidized only acetate and ethanol by sulfate reduction. Propionate-oxidizing, sulfate-reducing bacteria belonged to the genus Desulfobulbus. They were isolated from freshwater as well as from marine sediments and showed a relatively large range of usable substrates: hydrogen, formate, propionate, l-lactate and ethanol were oxidized with concomitant sulfate reduction. l-Lactate and pyruvate could be fermented by most of the isolated strains.

ALCOHOL CONVERSIONS BY DESULFOBULBUS-PROPIONICUS LINDHORST IN THE PRESENCE AND ABSENCE OF SULFATE AND HYDROGEN

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H2 and CO2, ethanol was quantitatively converted to propionate. H2-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO2 to propionate. Growth was also observed on acetate alone in the presence of H2 and CO2D. propionicus was able to grow mixotrophically on H2 plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.

The chemolithotrophic ammonium-oxidizing community in a nitrogen-saturated acid forest soil in relation to ph-dependent nitrifying activity

Abstract The relationship between pH and nitrification in different layers of a nitrogen-saturated acid Douglas fir forest soil was studied. Nitrification potentials (on basis of dry weight) of overground degrading needles, litter and fermentation layers were much higher than those of the humus and upper mineral layers. In all layers, nitrate production was probably due to chemolithotrophic bacteria as it was inhibited by acetylene. The litter layer contained relatively high numbers (105 g dry soil−1) of acid-sensitive ammonium-oxidizing bacteria, whereas the numbers in the other layers were just above or lower than the detection limit (103 g dry soil−1) of the MPN method used. Measurements of nitrate production in soil suspensions indicated that a pH increase (pH 6 vs pH 4) stimulated ammonium oxidation in the litter layer but not or to a lesser extent in the fermentation and humus layers. It is argued that both acid-sensitive and acid-tolerant ammonium-oxidizing bacteria are contributing to nitrification in the litter layer. In the fermentation and humus layers acid-tolerant or even acidophilic bacteria are thought to be responsible for ammonium oxidation.

Autotrophic nitrification in a fertilized acid heath soil

Abstract The nature of nitrification in fertilized, acid heath soils was studied. Autotrophic ammoniumand nitrite-oxidizing bacteria were enumerated in non-fertilized and fertilized heath soils. Ammoniumoxidizing bacteria were not detected in the non-fertilized soils, whereas nitrite-oxidizing bacteria were only found in the organic layer. Enrichment of acid heath soils with NPK fertilizer increased the number of autotrophic ammonium- and nitrite-oxidizing bacteria in the organic (F + H) layer as well as in the upper part of the mineral (Ah) layer, although the pH of the soil hardly changed with fertilization. In soil suspensions of the upper mineral layer of fertilized heath soils, nitrification was shown to be autotrophic as nitrification was completely inhibited by the addition of nitrapyrin under both neutral and acid conditions. Stimulation of nitrification by addition of peptone appeared to be due to the increase in pH caused by ammonification of peptone. Under acid conditions, nitrification seemed to be coupled with net nitrogen mineralization. The possible influence of vegetation on nitrification is discussed.

Methane oxidation in soil profiles of Dutch and Finnish coniferous forests with different soil texture and atmospheric nitrogen deposition

Abstract We studied methane oxidation capacity in soil profiles of Dutch and Finnish coniferous forests. The Finnish sites ( n = 9) had nitrogen depositions from 3 to 36 kg N ha −1 a −1 . The deposition of N on the Dutch sites ( n = 13) was higher ranging from 50 to 92 kg N ha −1 a −1 . The Dutch sites had also limed counterparts. Methane oxidation rates were determined by incubating soil samples in the laboratory at + 15°C with 10 μl CH 4 l −1 (10 ppmv CH 4 ). In general, CH 4 oxidation rates were highest in the uppermost mineral layers. The average CH 4 oxidation rate in the Finnish mineral soils was three times higher than that in the Dutch soils. The litter layers did not oxidize CH 4 . In the Netherlands all organic horizons had a negligible capacity to oxidize CH 4 . However, some Finnish organic horizons showed high CH 4 oxidation capacity. In the Netherlands, in contrast to Finland, there were some soil profiles lacking CH 4 oxidation. Higher contents of nitrate and ammonium, as well as greater production of nitrous oxide (N 2 O) and lower production of carbon dioxide in the Dutch than in the Finnish forest soils reflected the high N deposition rate in the Netherlands. Not only the N deposition, but also the highly sorted soil texture (fine sand) with low amounts of both coarse and fine particles is an important reason for the low CH 4 oxidation in the Dutch soils. The proportions of fine and coarse particles, both well represented in moraine soils typical in northern Europe, correlated positively with the CH 4 oxidation. Fine particles provide a good surface for microbial growth. Coarse particles, on the other hand, enhance diffusion of CH 4 and oxygen into the soil. Methane oxidation in the Dutch mineral soils was slightly enhanced by liming.

THE BIOENERGETICS OF AMMONIA AND HYDROXYLAMINE OXIDATION IN NITROSOMONAS-EUROPAEA AT ACID AND ALKALINE PH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a ΔΨ. Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a ΔΨ. Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.

UREA STIMULATED AUTOTROPHIC NITRIFICATION IN SUSPENSIONS OF FERTILIZED, ACID HEATH SOIL

Abstract The effect of various organic N-compounds on nitrate production in suspensions of fertilized heath soil was studied. Urea increased nitrate production the most. Nitrification in these suspensions was stimulated specifically by urea and not by an increase in pH due to urea hydrolysis, as the addition of equivalent amounts of ammonium and carbonate or ammonium and hydroxide resulted in a similar increase in the pH but had only limited effect on the production of nitrate. Numbers of autotrophic nitrifiers increased significantly in urea-enriched suspensions although these suspensions remained acidic (pH

Contribution of nitrification and denitrification to the NO and N2O emissions of an acid forest soil, a river sediment and a fertilized grassland soil

Abstract Most studies determining the contribution of nitrification and denitrification to NO and N2O emissions from soils have been performed in agricultural systems, often with homogenized soil samples. More information about the nitrifier and denitrifier contribution in non-agricultural systems nitrification and denitrification to NO and N2O emissions from three different ecosystems: an acid forest soil; a river sediment in the intertidal zone; and a fertilized peat grassland, using intact soil cores. Samples were taken in the spring of 1993 and the autumn of 1994. Intact soil cores (5 cm deep) were incubated at field temperature in the laboratory and the accumulation of NO and N2O during 24 h was measured. The nitrification and denitrification contribution was determined by specific inhibition of nitrification. The highest mean N2O production was in the same range for all sites. Nitrification dominated N2O production in spring at all sites. In contrast, denitrification was the main source of N2O in the acid forest soil and grassland soil in the autumn. However, the tight coupling of nitrification and denitrification in the river sediment could have resulted in an over-estimation of the contribution of nitrification to N2O and NO production. A large part of denitrified N in the acid forest soil was emitted as N2O, whereas in the river sediment, except for the autumn, the denitrification N2O-to-N2 ratio was low, which coincided with a low nitrate content. Nitrification was the dominant NO source in spring at all sites. In autumn, high contributions of both nitrification and denitrification were observed.

Two types of chemolithotrophic nitrification in acid heathland humus

The nature of nitrification in the organic horizons (FH) of two heathland soils was studied. Two types of chemolithotrophic nitrification but no heterotrophic nitrification were detected in the acid heathland humus. One type was predominant in slow nitrate producing humus. It was acid-sensitive but could be stimulated by urea at low pH. The other type was acid-tolerant, it was not stimulated by urea. The latter type was predominant in fast nitrate producing humus. The occurrence of acid-tolerant chemolithotrophic nitrification provides indirect evidence for the existence of previously unknown acid-tolerant, chemolithotrophic, ammonium-oxidizing bacteria. Nitrification by these micro-organisms may be an important source of soil acidification in acid soils that are exposed to high levels of ammonium deposition.