Microelectrode Measurements of the Activity Distribution in Nitrifying Bacterial Aggregates

Microelectrodes for ammonium, oxygen, nitrate, and pH were used to study nitrifying aggregates grown in a fluidized-bed reactor. Local reactant fluxes and distribution of microbial activity could be determined from the microprofiles. The interfacial fluxes of the reactants closely reflected the stoichiometry of bacterial nitrification. Both ammonium consumption and nitrate production were localized in the outer shells, with a thickness of approximately 100 to 120 μm, of the aggregates. Under conditions in which ammonium and oxygen penetrated the whole aggregate, nitrification was restricted to this zone; oxygen was consumed in the central parts of the aggregates as well, probably because of oxidation of dead biomass. A sudden increase of the oxygen concentration to saturation (pure oxygen) was inhibitory to nitrification. The pH profiles showed acidification in the aggregates, but not to an inhibitory level. The distribution of activity was determined by the penetration depth of oxygen during aggregate development in the reactor. Mass transfer was significantly limited by the boundary layer surrounding the aggregates. Microelectrode measurements showed that the thickness of this layer was correlated with the diffusion coefficient of the species. Determination of the distribution of nitrifying activity required the use of ammonium or nitrate microelectrodes, whereas the use of oxygen microelectrodes alone would lead to erroneous results.     

Stable coexistence of contrasted nitrification statuses in a wet tropical savanna ecosystem

1. Wet tropical savannas are characterized by strong environmental constraints—particularly low soil nutrient availability—associated with high plant productivity. Nitrogen recycling, and especially nitrification, is supposed to be a strong determinant of the balance between conservation and loss of nutrients at the ecosystem level. Savanna facies dominated by the grass Hyparrhenia diplandra (Andropogoneae) are known to exhibit low levels of nitrification and thus avoid nitrate losses.
2. By comparing two sites in the Lamto area (Côte d'Ivoire, West Africa) with similar soil physico-chemical characteristics and equally dominated by H. diplandra (80% of the grass cover), it was demonstrated that, within this facies, nitrification is highly heterogeneous, with a 240-fold variation in potential nitrification within a specific site.
3. In order to test whether these differences can be considered as permanent in this ecosystem, nitrate reductase activities were compared on H. diplandra plantlets from the two sites, cultivated under identical conditions in the presence of nitrate. The leaves of plants originating from the high nitrification site were always able to reduce nitrate at a significantly higher rate than those from the low nitrification site. This observation indicates a long-term adaptation of the plants and stable nitrification behaviour.
4. Lamto can thus be considered as a contrasted dual ecosystem relative to its nitrogen cycle. The two sites studied therefore constitute useful models to assess the determinism of nitrification in wet savannas and the role of this process on nitrogen retention in such ecosystems.     

Nitrification and nitrogen accumulation in the early stages of primary succession on Mt. Fuji

The relationship between nitrification potential and nitrogen accumulation was studied in an early successional sere on Mt. Fuji. Soil organic nitrogen accumulated with the invasion ofPolygonum cuspidatum and successively withMiscanthus oligostachyus and other species. Laboratory incubation experiments showed a higher nitrification potential at theM. oligostachyus state. The numbers of nitrifying bacteria increased with the progress of succession. No significant difference in nitrate reductase activity was found between pioneer and succeeding species. The soil solution at theM. oligostachyus stage contained a lower level of nitrate than rainwater, while that of the bare ground and theP. cuspidatum stage contained a higher nitrate level than rainwater. It was concluded that the high nitrate levels in the soil solution of the bare ground and theP. cuspidatum stage were due to lower nitrate-absorbing activity, leading to loss of nitrogen with precipitation, while the lower nitrate levels at theM. oligostachyus stage when higher nitrification activity occurred were due to higher nitrate-absorbing activity, preventing net loss of nitrogen from the ecosystem.     

Some chemical and physical factors affecting the rate and dunamics of nitrification in urine-affected soil

The effects of urinary chloride and nitrogen concentration and osmotic pressure on the nitrification of ammonium in a calcareous soil treated with cow urine were examined. Urinary chloride concentrations of up to 7.4 g L^–1 had no effect on the rate of nitrification, as determined by the accumulation of soil nitrate. Osmotic stress, generated using a mixed salt solution, had an inhibitory effect on nitrification at soil osmotic pressures lower than or equal to –1.0 PMa. Nitrification was completely inhibited at a soil osmotic pressure of –2.6 MPa. Accumulation of nitrate after a lag phase of 18 days was noted in the –2.0 MPa soil osmotic pressure treatment, indicating some degree of adaptation or osmo-regulation within the nitrifying population at this stress level. High urine-N concentrations resulted in considerable nitrite accumulations and reduced nitrification activity through the effect of free ammonia. It is concluded that in most temperate grassland soils at near-neutral pH, urinary chloride and nitrogen are unlikely to reduce nitrification rates, except where urine-N concentrations exceed 16 g N L^–1. Inhibition due to osmotic stress will be directly related to soil moisture status and may be particularly severe in dry, light-textured soils.     

Nitrogen (N) Dynamics in the Mineral Soil of a Central Appalachian Hardwood Forest During a Quarter Century of Whole-Watershed N Additions

The structure and function of terrestrial ecosystems are maintained by processes that vary with temporal and spatial scale. This study examined temporal and spatial patterns of net nitrogen (N) mineralization and nitrification in mineral soil of three watersheds at the Fernow Experimental Forest, WV: 2 untreated watersheds and 1 watershed receiving aerial applications of N over a 25-year period. Soil was sampled to 5 cm from each of seven plots per watershed and placed in two polyethylene bags—one bag brought to the laboratory for extraction/analysis, and the other bag incubated in situ at a 5 cm depth monthly during growing seasons of 1993–1995, 2002, 2005, 2007–2014. Spatial patterns of net N mineralization and nitrification changed in all watersheds, but were especially evident in the treated watershed, with spatial variability changing non-monotonically, increasing then decreasing markedly. These results support a prediction of the N homogeneity hypothesis that increasing N loads will increase spatial homogeneity in N processing. Temporal patterns for net N mineralization and nitrification were similar for all watersheds, with rates increasing about 25–30% from 1993 to 1995, decreasing by more than 50% by 2005, and then increasing significantly to 2014. The best predictor of these synchronous temporal patterns across all watersheds was number of degree days below 19°C, a value similar to published temperature maxima for net rates of N mineralization and nitrification for these soils. The lack of persistent, detectable differences in net nitrification between watersheds is surprising because fertilization has maintained higher stream-water nitrate concentrations than in the reference watersheds. Lack of differences in net nitrification among watersheds suggests that N-enhanced stream-water nitrate following N fertilization may be the result of a reduced biotic demand for nitrate following fertilization with ammonium sulfate.     

Nitrification activities and the changes in the populations of nitrifying bacteria in soil perfused at two different H-ion concentrations

Summary The rate of nitrification by soil aggregates at pH 5.5 and 7.5 was examined by a perfusion technique. Nitrification occurred at both levels of H-ion concentration but at the higher pH the rate of nitrification was greater. The population estimates of nitrifying bacteria were correspondingly greater at the high pH. Once the pH was lowered from 7.5 to 5.5, the nitrification rate decreased slowly with a corresponding decrease in the numbers of nitrifying bacteria. The distribution of nitrifying bacteria throughout soil aggregates was homogenous. The lower limit of pH for nitrification was 4.3.     

Is nitrification affected by the diameter and other properties of soil aggregates?

Nitrification was measured in fractions of chernozemic rendzina and lessivē soil differing in aggregate size. In both soils the maximum rates occurred in aggregates between 1 and 3 mm in diameter. The effects of structural and other properties (particle composition, pore-size distribution, surface area, organic C and total N content, ratio of air volume to water volume in aggregates) proved to be insignificant except for the nitrification rate in the lessivē soil, which positively correlated with the fraction of particles between 20 and 50 μm in diameter.     

Apparent and Measured Rates of Nitrification in the Hypolimnion of a Mesotrophic Lake

Three distinct phases were observed in the change of dissolved inorganic nitrogen concentrations in the hypolimnion of Grasmere. The second phase of decreasing ammonia and increasing nitrate concentrations was typical of the nitrification process. Observations on nitrate concentration gradients between surface sediments and the water column and experiments using the nitrification inhibitor N-Serve indicated the in situ activity of chemolithotrophic nitrifying organisms. Nitrification rates were estimated throughout the period of stratification by using the N-Serve and [¹⁴C]bicarbonate uptake method. Comparison of the field nitrate concentrations with the predicted nitrate concentrations (from estimates of the nitrification rate) indicated that the method underestimated the true rate of nitrification. Possible reasons for this are discussed.     

Aggregate Size Distribution of Ammonia-Oxidizing Bacteria and Archaea at Different Landscape Positions

To quantify the spatial distribution of ammonia-oxidizing bacteria (AOB) and archaea (AOA) and to determine nitrification activity in soil aggregates along a landscape, soil samples were collected from three landscape positions (shoulder, backslope, and toeslope) at two pasture sites with contrasting climatic conditions. The abundance of AOB and AOA was estimated by quantifying their respective bacterial and archaeal amoA gene copies using real-time polymerase chain reaction. Soil organic C (SOC), total N (TN), and the potential nitrification rate (PNR) were measured in aggregate size ranges (4–1, 1–0.25, and 0.25–0.05 mm). At site 1, a decreasing trend in PNR was observed as the size of aggregates decreased. Both bacterial and archaeal amoA genes were higher in the macroaggregates (4–1 and 1–0.25 mm) than in the microaggregates (0.25–0.05 mm) along the landscape. At site 2, PNR was higher in the smallest size of aggregates. In the 0.25–0.05-mm fraction, the abundance of bacterial and archaeal amoA genes was equal to, or greater than, those found in larger aggregate sizes. The relative abundance of archaeal amoA gene and the PNR correlated with relative SOC and TN contents along the landscapes. The positive relationship between relative archaeal amoA gene abundance and PNR suggests that nitrification in the studied pastures is probably driven by ammonia-oxidizing Thaumarchaeota.     

Acetylene reduction (dinitrogen fixation) and nitrification in soil as affected by the structural aggregate size

The effect of size of structural aggregates on the intensity of nitrification and nitrogenase (nitrogen: acetylene oxidoreductase) activity was investigated in three soils. In two of them the nitrogenase activity was limited by addition of glucose. Aggregates of a larger diameter (2-4 mm) exhibited a considerably higher nitrogenase activity than those with a diameter smaller than 2 mm. This effect was even more pronounced when the soil samples were repeatedly intensively aerated. On the contrary, smaller aggregates (0.5-2 mm) exhibited more intensive nitrification.     

Measurement of nitrification rates in lake sediments: Comparison of the nitrification inhibitors nitrapyrin and allylthiourea

A method for measuring rates of nitrification in intact marine sediment cores has been modified and adapted for use in freshwater sediments. The technique involves subsampling a sediment core into minicores. Half of these cores are treated with an inhibitor of chemolithotrophic nitrification and, after incubation, differences in ammonia and nitrate concentration between inhibited and uninhibited systems are calculated. The within-treatment variability of ammonia and nitrate concentrations could be reduced by storing the cores overnight prior to subsampling. Estimates of the nitrification rate using the difference in ammonia concentrations between the inhibited and uninhibited mini-cores were always greater than the rate estimate using the difference in nitrate concentrations. Comparison between the results using the nitrification inhibitors allylthiourea (ATU) and nitrapyrin (N-Serve) indicated that the former appeared to give larger values for the nitrification rate than did the latter. Differences in the efficiency of these inhibitors in the control of nitrification under the conditions used partly explain these results. Data are also presented on the effect of N-Serve and ATU on some other nitrogen transformations affecting ammonia and nitrate concentrations.     

Microscale distribution of populations and activities of Nitrosospira and Nitrospira spp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors.

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 microm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.     

Disruption of narH,narJ, and moaE inhibits heterotrophic nitrification in Pseudomonas strain M19

Interruptions in three nitrate reductase-related genes, narH, narJ, and moaE, inhibited heterotrophic nitrification in Pseudomonas strain M19. No nitrate was detected in the medium, and nitrification proceeded in the presence of a nitrate reductase inhibitor. Heterotrophic nitrification was greatly stimulated by the addition of nitrate.     

Leaching of nitrogen from subtropical soils as affected by nitrification potential and base cations

A soil column method was used to determine the effect of nitrification on leaching of nitrate and ammonium from three acid subtropical soils after application of ammonium bicarbonate. Three soils, designated QF, GB and SU, derived from Quaternary red earth, granite and tertiary red sandstone, were collected from forest land, brush land and upland field, ranged in nitrification potential and cation exchange capacity. The results indicated that nitrate leaching increased with the soil nitrification potential. The soils with higher nitrification potential had a higher nitrate peak concentration and required a shorter time to reach it. In soils QF and GB with low cation exchange capacity, and a low content of exchangeable base cations, there were not sufficient base cations to accompany the nitrate leached with the result that ammonium and hydrogen ions were leached from the soil, and pH changes occurred in different layers of the soil column.     

温度、水分及不同氮源对土壤硝化作用的影响

选用陕西省 3个自然生态区 3种主要耕作土壤土样 ,在实验室培养条件下 ,研究温度、水分及不同氮肥品种对其硝化作用的影响 ,并用 d N/ dt=b N(B-N) / B方程描述硝化作用过程中硝态氮含量随时间的累积变化 ,获得定量描述硝化作用强弱的两个指标 (Kmax和 td)。结果表明 :不同土壤水分含量对硝化作用的影响在不同土壤间差异明显 ;但不同土壤在田间持水量 (F H C)的 60时 ,硝化作用的最大速率 (Kmax)及硝化率最高。土壤温度不仅显著影响硝化作用的最大速率 (Kmax)和硝化率 ,而且迟缓期 (td)也有明显变化。不同氮肥品种对硝化作用的影响主要表现在硝化率不同 ,3种土壤硝化率均为硫酸铵 >尿素和碳铵 >氯化铵 ,显示硫酸根离子的促进作用和氯离子的强烈抑制作用。但氮肥品种对硝化作用的最大速率 (Kmax)和迟缓期 (td)的影响不规律。     

硝化基质和产物对发光细菌的急性毒性

摘要：【目的】对硝化基质和产物对硝化过程的影响进行初步研究。【方法】采用发光细菌法,在pH=7.0的条件下,测定了氨、羟胺、亚硝酸和硝酸对发光细菌的急性毒性（15min-半抑制浓度（the half inhibitory concentration,IC50））。【结果】单一物质的毒性试验结果表明,硝化基质和产物对发光细菌的毒性随浓度的升高而增大,且具有较好的线性关系;氨、羟胺、亚硝酸和硝酸的IC50分别为2180.2 mg/L、6.2740 mg/L、1207.2 mg/L和3140.3 mg/L;其毒性大小顺序为：羟胺 >亚硝酸 >氨 >硝酸。按等效浓度混合法测定硝化基质和产物的联合毒性,结果表明：氨与羟胺、氨与亚硝酸、羟胺与亚硝酸对发光细菌的联合毒性呈相加作用;氨与硝酸、羟胺与硝酸、亚硝酸与硝酸对发光细菌的联合毒性呈独立作用;氨、羟胺、亚硝酸、硝酸四元混合物的联合毒性也呈相加作用。【结论】根据硝化基质和产物对发光细菌和硝化细菌抑制浓度的相关性,可用发光细菌发光强度的变化指示硝化基质和产物的抑制作用。     

Nitrification at Low pH by Aggregated Chemolithotrophic Bacteria

A study was performed to gain insight into the mechanism of acid-tolerant, chemolithotrophic nitrification. Microorganisms that nitrified at pH 4 were enriched from two Dutch acid soils. Nitrate production in the enrichment cultures was indicated to be of a chemolithoautotrophic nature as it was (i) completely inhibited by acetylene at a concentration as low as 1 μmol/liter and (ii) strongly retarded under conditions of carbon dioxide limitation. Electron microscopy of the enrichment cultures showed the presence of bacteria that were morphologically similar to strains of known chemolithotrophic nitrifying genera. Many of the enriched bacteria, in particular those that were identified as ammonium oxidizers, were aggregated. Filtration experiments indicated that aggregated cells were able to nitrify at low pH, whereas single cells were not. It is hypothesized that cells inside the aggregates are protected against the toxicity of nitrous acid. Nitrification by aggregated chemolithoautotrophic bacteria may be the dominating process of nitrate formation in many acid soils as it does not appear to depend on the existence of microsites of high pH (acid-sensitive autotrophic nitrification) or on the availability of organic carbon (heterotrophic nitrification).     

Watershed liming effects on the forest floor N cycle

The forest floor was expected to play a major role in determining the total ecosystem response to watershed liming because of its high concentration of nutrients and its high level of activity. Net N mineralization and net nitrification were estimated in a field survey using the buried-bag approach. In a laboratory incubation experiment, forest floor humus was mixed with 6 doses of lime to determine the sensitivity of N mineralization and nitrification to lime dose. Forest floor microcosms with and without live tree roots were used to calculate a N budget for the system.The pH of the forest floor increased from 3.6 to 4.9 in the Oe and to 4.0 in the Oa two years after liming. The extractable ammonium pool in both the field survey and microcosm study was substantially smaller after liming and was probably a result of the 36% to 55% lower net N mineralization rate in limed plots than in reference plots. The laboratory incubation results agreed with the field survey results and further demonstrated that at higher lime doses (pH 5 to 6), N mineralization increased above controls. Net nitrification in limed humus in both the buried bags and laboratory incubation was as much as three times higher than controls, which could explain why nitrate leaching in limed microcosms was greater than in control microcosms. However, nitrate leaching from microcosms with live. roots was not affected by liming, suggesting that roots in the forest floor may prevent excess nitrate leaching. Reductions in N mineralization had no effect on N leaching or N uptake, but reduced the extractable ammonium pool.     

Comparison of nitrification rates in three branches of the lower river Rhine

The nitrogen cycle in the lower river Rhine was analysed, using data on concentrations of ammonium, nitrite and nitrate, measured in the period from 1972 to 1986. The massive discharge of ammonium in densely populated areas in the Federal Republic of Germany led to microbial nitrification, detectable as decreases in ammonium and nitrite concentrations in the lower river Rhine over reaches 85–133 km long. The distribution of the nitrogen-rich Rhine waters over three different branches in the Netherlands permits some of the factors governing microbial nitrification in the river bed to be discriminated. In the fast-flowing main channel, intensively used by ships, nitrification is more important than in the smaller branches, despite the short residence time of the water in the main channel. Differences in the flow rate of water, in grain size distribution of sediments, and in intensity of shipping (aeration, turbulence) seemed to be responsible for the different rates of nitrification.     

The conversion of amino acids in soils

Summary In an investigation on the conversion of amino acids in percolated soils, it was found that during the breakdown of glutamic acid to ammonia micro-organisms developed in the soil capable of denitrifying nitrite and nitrate to gaseous nitrogen. The enrichment of a soil with these micro-organisms was studied.Drying of the enriched soil had a deleterious effect on the activity of these micro-organisms.The interaction between denitrification and soil nitrification processes was studied in soil subjected to various percolation treatments. When the denitrifying micro-organisms and their metabolites were present in the soil the amount of nitrogen lost by denitrification depended on the availability of nitrite and nitrate. When this was supplied externally, in glutamate—nitrite or glutamate—nitrate mixtures, considerable reduction occurred. Losses were less severe where nitrite and nitrate entered the system internall y by nitrification of the ammonia produced from the breakdown of the amino acid. In fresh soils there were indications that the amount of nitrification occurring during amino-acid breakdown was the important factor.All the data appeared to be consistent with the hypothesis that during the conversion of amino acids in soils a delicate balance is established between nitrification and denitrification reactions by different types of soil micro-organisms.