A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.     

The importance of autotrophic versus heterotrophic oxidation of atmospheric ammonium in forest ecosystems with acid soil

Abstract The role of autotrophic and heterotrophic nitrifying microorganisms in the oxidation of atmospheric ammonium in two acid and one calcareous location of a Dutch woodland area was investigated. In soil slurries nitrate formation was completely inhibited by acetylene, a specific inhibitor of autotrophic ammonium-oxidizing bacteria. A survey of nitrifiers in the forest soils showed that both autotrophic ammonium- and nitrite-oxidizing bacteria were present in high numbers and evidence was obtained that autotrophic bacteria are able to nitrify below pH 4. These results show that autotrophic nitrifying bacteria may account for most of the nitrification in the examined soils. To assess the contribution of heterotrophic nitrifiers, about 200 strains of heterotrophic bacteria and 21 morphologically distinct fungal strains were isolated from the acid soil locations and tested for their ability to nitrify. Only one Penicillium strain produced nitrate in test media, but its nitrate formation when added to acid soils was poor. These findings indicate that in the investigated soil heterotrophs are of minor importance in the oxidation of atmospheric ammonium.     

Measurements of sediment toxicity of autotrophic and heterotrophic picoplankton by epifluorescence microscopy

Sediment toxicity from Toronto (Ontario) and Toledo (Ohio) harbours to autotrophic and heterotrophic picoplankton was evaluated simultaneously using epifluorescent microscopy. The approach is a simple, fast and effective combination of autofluorescence and fluorescence probes - 1-anilino-8-naphthalene sulfonic acid. The procedure is ideally suited for use with sediment slurries since it can differentiate fluorescent biotic material against a background of abiotic sediment particles and detritus.     

The stoichiometry of nitrogen and phosphorus spiralling in heterotrophic and autotrophic streams

1. Nutrient spiralling provides a conceptual framework and a whole‐system approach to investigate ecosystem responses to environmental changes. We use spiralling metrics to examine how the coupling of nitrogen and phosphorus uptake varies between streams dominated by either heterotrophic (i.e. bacteria‐dominated) or autotrophic (algal‐dominated) microbial communities. 2. Algae generally exhibit greater capacity to store nutrients than bacteria because of differences in cellular structures. These differences led us to hypothesise that the uptake of N and P in heterotrophic ecosystems should have reduced stoichiometric variation in response to changes in supply N : P compared to autotrophic ecosystems when assimilation dominates nutrient uptake. 3. To test this hypothesis, we used an array of serial nutrient additions in several streams in the South Fork Eel River watershed in Northern California. In one set of experiments, N and P were added alone and simultaneously in separate experiments to two small, heterotrophic streams to assess uptake rates and interactions between nutrient cycles. In a second set of experiments, N and P were added simultaneously at a range of N : P in one heterotrophic and one autotrophic stream to assess differences in uptake responses to changes in supply N : P. 4. Results of these experiments suggest two important conclusions. First, increased N supply significantly shortened P uptake lengths, while P addition had little impact on N uptake in both streams, indicating that uptake of non‐limiting nutrients is tightly coupled to the availability of the limiting element. Second, changes in P uptake and uptake ratios (U_N : U_P) with increased supply N : P supported our hypothesis that heterotrophic streams are more homeostatic in their responses to changes in nutrient supply than autotrophic streams, suggesting that physiological controls on nutrient use scale up to influence ecosystem‐scale patterns in nutrient cycling.     

Influence of tree species and ground vegetation on nitrification in an acid forest soil

Soil N transformations were studied at Ironhill, near Liphook, UK as part of a forest fumigation experiment. Nitrification potential was measured in a humoferric podzol soil, of pH 3 (in 0.01 M CaCl₂). An initial experiment into nitrogen mineralisation potential indicated that nitrification was linked strongly to the species of coniferous tree growing in the soil. Transfer of soil solution between soils had no influence on mineralisation potential and allelopathic effects of the trees were not demonstrated. The initial finding was attributed subsequently to the type of ground vegetation and its management. Attempts to reproduce soil conditions, which promoted nitrification, were partially successful.Soil, from the Ironhill site, was incubated with various nitrogenous substrates and other nutrients and sources of carbon to test whether heterotrophs were responsible for nitrification. Organic N (which was ammonified) promoted nitrification, but the addition of ammonium was inhibitory unless supplied with a readily available carbon source such as acetate. Nitrification potential was unaffected when soils were incubated with an inhibitor of autotrophic nitrification. The results of these experiments supported strongly the hypothesis that heterotrophic organisms were responsible for nitrification in this soil.     

The effect of thiosulphate and other inhibitors of autotrophic nitrification on heterotrophic nitrifiers

It has been found that heterotrophic nitrification by Thiosphaera pantotropha can be inhibited by thiosulphate in batch and chemostat cultures. Allythiourea and nitrapyrin, both classically considered to be specific inhibitors of autotrophic nitrification, inhibited nitrification by Tsa. pantotropha in short-term experiments with resting cell suspensions. Hydroxylamine inhibited ammonia oxidation in chemostat cultures, but was itself fully oxidized. Thus the total nitrification rate for the culture remained the same.Heterotrophic nitrification by another organism, a strain of Pseudomonas denitrificans has also been shown to be inhibited by thiosulphate in short term experiments and in the chemostat. During these experiments it became evident that this strain is able to grow mixotrophically (with acetate) and autotrophically in a chemostat with thiosulphate as the energy source.     

Seasonal determinations of extracellular hydrolytic activities in heterotrophic and mixed heterotrophic/autotrophic biofilms from two contrasting rivers

The temporal changes in extracellular enzyme activities in freshwater microbial biofilms were examined in two contrasting river sites in North Wales over a 12 month period. Sites were a first order, unshaded oligotrophic upland stream (Nant Waen) and a fourth order, mildly eutrophic river with riparian tree cover (River Clywedog). When algal populations were low, biofilms of the more eutrophic site supported greater enzyme activities and higher population densities than the oligotrophic site. Composition, concentration and origin of substrates available to the respective biofilm communities influenced extracellular processing patterns. Reduction in algal populations depressed total and extracellular activities in biofilms from the first order site, suggesting that biofilm communities here were maintained by in situ primary production. Biofilms from Nant Waen were often found to contain higher extracellular activities per cell than the more eutrophic River Clywedog biofilms, which might represent the enhanced ability of an oligotrophic biofilm to accumulate extracellular enzymes. In contrast, light and darkgrown River Clywedog biofilms were not enzymatically distinct, inferring a less important role for biofilm phototrophs. Some evidence was found for increased reliance on allochthonous substrates in the River Clywedog for biofilm maintenance.     

Investigation of nitrification in forest soils with soil percolation columns

Soil percolation columns in which a pF of 2 could be maintained were developed to study nitrification in soils and litter of an acid and a calcareous forest soil location. High nitrification rates were observed in the calcareous soil. In the acid soil nitrification was much slower. A column filled with leaf litter gave a low nitrification rate at the start of the experiment, but a high rate was found after 60 days of percolation with an ammonium-containing medium of pH 4. In this leaf litter high numbers of autotrophic bacteria were just present at the beginning of the experiment, whereas at the end only low numbers were detected. Results indicate that autotrophic bacteria from acid soils are sensitive to a pH increase.     

Fertilization of boreal forest reduces both autotrophic and heterotrophic soil respiration

The boreal forest is expected to experience the greatest warming of all forest biomes, raising concerns that some of the large quantities of soil carbon in these systems may be added to the atmosphere as CO₂. However, nitrogen deposition or fertilization has the potential to increase boreal forest production and retard the decomposition of soil organic matter, hence increasing both tree stand and soil C storage. The major contributors to soil‐surface CO₂ effluxes are autotrophic and heterotrophic respiration. To evaluate the effect of nutrient additions on the relative contributions from autotrophic and heterotrophic respiration, a large‐scale girdling experiment was performed in a long‐term nutrient optimization experiment in a 40‐year‐old stand of Norway spruce in northern Sweden. Trees on three nonfertilized plots and three fertilized plots were girdled in early summer 2002, and three nonfertilized and three fertilized plots were used as control plots. Each plot was 0.1 ha and contained around 230 trees. Soil‐surface CO₂ fluxes, soil moisture, and soil temperature were monitored in both girdled and nongirdled plots. In late July, the time of the seasonal maximum in soil‐surface CO₂ efflux, the total soil‐CO₂ efflux in nongirdled plots was 40% lower in the fertilized than in the nonfertilized plots, while the efflux in girdled fertilized and nonfertilized plots was 50% and 60% lower, respectively, than in the corresponding nongirdled controls. We attribute these reductions to losses of the autotrophic component of the total soil‐surface CO₂ efflux. The estimates of autotrophic respiration are conservative as root starch reserves were depleted more rapidly in roots of girdled than in nongirdled trees. Thus, heterotrophic activity was overestimated. Calculated on a unit area basis, both the heterotrophic and autotrophic soil respiration was significantly lower in fertilized plots, which is especially noteworthy given that aboveground production was around three times higher in fertilized than in nonfertilized plots.     

Short-term increases in relative nitrification rates due to trenching in forest floor and mineral soil horizons of different forest types

The representation of NO₃ ^– dynamics within forest growth simulation models could improve forest management. An extensive literature review revealed an 88% probability of measuring a higher relative nitrification index (i.e. RNI = [NO₃ ^–] ÷ [NO₃ ^– + NH₄ ⁺]) in mineral soil horizons than in forest floors, across a wide range of conifer and hardwood ecosystems. We then hypothesised that humus form and fine root density could be used as two crude variables to predict changes in in situ, potential and relative nitrification rates. Twenty-seven trench plots were established in 1999, across nine contrasting hardwood and coniferous stands in the Eastern Townships of Québec. Forest floor and mineral soil samples were collected from each plot, and from a 1 m radius surrounding each plot, on three dates during summer 2000. In situRNI values increased significantly in trench plots as the season progressed. Potential nitrification rates (i.e. NO₃ ^– concentrations following incubation) were two orders of magnitude higher in forest floor than in mineral soil samples. RNI was significantly higher in mineral soil than in forest floor samples after incubations, but the relative increase in RNI due to trenching was higher in forest floor samples. Indices of available C were significantly higher in forest floor than mineral soil samples, and decreased only in forest floor samples during incubations. Likewise, trenching significantly reduced available C in forest floor samples only. Seasonal changes in soil temperature and fine root growth were the most plausible explanations for seasonal changes in NO₃ ^– dynamics, whereas other factors such soil acidity and moisture appeared less important in determining NO₃ ^– dynamics in this study. We conclude that crude assessments of humus form and fine root density have the potential to be used as calibration parameters for the simulation of NO₃ ^– dynamics in forest growth and yield models.     

Inhibition of nitrification in forest soil by monoterpenes

Nitrate production was detected in untreated soil of a Norway spruce (Picea abies L.) stand only after clear-cutting the stand. The aim of this study was to determine whether allelochemical inhibition of nitrification by monoterpenes played any role in inhibiting nitrification in the stand. Therefore, soils from a clear-cut plot and from a forest plot were studied. In the field, monoterpenes (mostly - and -pinenes), measured by soil microair diffusive samplers, were intensively produced in the forest plot, but not in the clear-cut plot. In the laboratory, soil samples taken from the forest plot produced only small amounts of monoterpenes, indicating that monoterpenes were mainly produced by the roots and not to great extent by the soil microbial population. The effect of a mixture of monoterpenes (seven major monoterpenes detected in the field) on net nitrification, net N mineralization and denitrification activities of soil from the clear cut plot, and on carbon mineralization of soils from both the forest and clear-cut plots, was studied in the laboratory. In both aerobic incubation experiments and in soil suspensions with excess NH4-N, nitrification was inhibited by exposure to the vapours of monoterpenes at similar concentrations at which they had been detected in forest plot. This indicates direct inhibition of nitrification by monoterpenes. Exposure to monoterpenes did not affect denitrification. However, it increased respiration activity of both soils. This could also indicate indirect inhibition of nitrification by monoterpenes, due to immobilization of mineral N. Thus it seems that monoterpenes could play a role in inhibiting nitrification in the forest soil.     

The use of mean pool abundances to interpret 15N tracer experiments

A pulse dilution ¹⁵N technique was used in the field to determine the effect of the ammonium to nitrate ratio in a fertilizer application on the uptake of ammonium and nitrate by ryegrass and on gross rates of mineralization and nitrification. Two experiments were performed, corresponding approximately to the first and second cuts of grass. Where no substantial recent immobilization of inorganic nitrogen had occurred, mineralization was insensitive to the form of nitrogen applied, ranging from 2.1–2.6 kg N ha^-1 d^-1. The immobilization of ammonium increased as the proportion of ammonium in the application increased. In the second experiment there was evidence that high rates of immobilization in the first experiment were associated with high rates of mineralization in the second. The implication was that some nitrogen immobilized in the first experiment was re-mineralized during the second. Whether this was nitrogen taken up, stored in roots and released following defoliation was not clear. Nitrification rates in this soil were low (0.1–0.63 kg N ha^-1 d^-1), and as a result, varying the ratio of ammonium to nitrate applied markedly altered the relative uptake of ammonium and nitrate. In the first experiment, where temperatures were low, preferential uptake of ammonium occurred, but where >90% of the uptake was as ammonium, a reduction in yield and nitrogen uptake was observed. In the second experiment, where temperatures and growth rates were higher, the proportion of ammonium to nitrate taken up had no effect on yield or nitrogen uptake.     

A field study on the fate of 15N-ammonium to demonstrate nitrification of atmospheric ammonium in an acid forest soil

To demonstrate the contribution of atmospheric ammonium to soil acidification in acid forest soils, a field study with¹³N-ammonium as tracer was performed in an oak-birch forest soil. Monitoring and analysis of soil solutions from various depths on the¹³N-ammonium and¹⁵N-nitrate contents, showed that about 54% of the applied¹⁵N-ammonium was oxidized to nitrate in the forest floor. Over a period of one year about 20% of the¹⁵N remained as organic nitrogen in this layer. The percentage¹⁵N enrichment in ammonium and nitrate were in the same range in all the forest floor percolates, indicating that even in extremely acid forest soils (pH < 4) nitrate formation from ammonium can occur. Clearly, atmospheric ammonium can contribute to soil acidification even at low soil pH.     

Dynamics of viable nitrifier community and nutrient availability in dry tropical forest habitat as affected by cultivation and soil texture

Seasonal dynamics of N-mineralization and the size of the viable community of nitrifying bacteria were studied for a forest site and an adjoining cropland site. The forest site was dominated by Boswellia serrata and Acacia catechu in the tree layer, and by Nyctanthes arbortristis and Zizyphus glaberrima in the shrub layer. Crop sequence on the cropland site was Oryza sativa/Lens culinaris. The soil type in both the sites was ultisol (USDA). The cropland soil had significantly higher bulk density, and clay content but lower organic C, total N and total P than forest soil. The soil moisture content, numbers of ammonia-and nitrite oxidizing bacteria and N-mineralization rates were highest in the wet season and lowest in the dry season, while the size of mineral N and P pools showed a reverse trend in both sites. The numbers of free-living cells of ammonia-and nitrite oxidizing bacteria were significantly related with each other as well as with the soil moisture content and N-mineralization rates. In N-mineralization, NO₃^– was the dominating form in the forest site during rainy season, while in other seasons in this site and in all the seasons in the cropland site, NH₄⁺ -N was predominant. The N-mineralization rate and the number of viable nitrifying cells were consistently higher for the forest soil compared to the clay-rich cropland soil. The combination of low soil organic matter and high clay content suppressed the number of free-living cells of nitrifying bacteria and N-mineralization rates in the cropland site.     

Fine root mass in relation to soil N supply in a cool temperate forest

Soil inorganic nitrogen supply and fine root mass in the top layers of mineral soil (0–5 and 5–10cm) were investigated at upper and lower sites of a cool temperate forest where Fagus crenata and Quercus crispula dominate. At both sites, soil inorganic nitrogen supply was greatest in the 0–5cm layer. The predominant forms of soil inorganic nitrogen supply were NH₄⁺-N at the upper site and NO₃^–-N at the lower site. Fine roots were concentrated in the 0–5cm layer at the upper site, but not at the lower site. The form of supplied soil inorganic nitrogen supply can be important in determining the vertical distribution of fine roots.     

The influence of sulphite mill effluents on heterotrophic activity as measured by glucose assimilation

The influence of sulphite mill effluents on the heterotrophic activity of natural populations in the recipient water was studied during the summer of 1979. A single concentration method (3-H-glucose, 0.05 µg 1^–1) was used to measure the heterotrophic activity. The influence of the effluent was studied by adding it in concentrations varying from 0.001 to 10% to water samples derived from different sections of the watercourse. Adaptation to rather high concentrations of the effluent was observed near to the point of discharge. Low concentrations of the effluent had a stimulatory effect on heterotrophic activity. Short turnover times (5–20 h) of glucose and inhibition of primary productivity demonstrated the influence of effluents on planktonic populations.The single concentration method allows rapid and simple detection of horizontal and vertical effects of effluents on natural bacterioplankton in the recipient water.     

Differential responses of auto‐ and heterotrophic soil respiration to water and nitrogen addition in a semiarid temperate steppe

Evaluating how autotrophic (SR_A), heterotrophic (SR_H) and total soil respiration (SR_TOT) respond differently to changes of environmental factors is critical to get an understanding of ecosystem carbon (C) cycling and its feedback processes to climate change. A field experiment was conducted to examine the responses of SR_A and SR_H to water and nitrogen (N) addition in a temperate steppe in northern China during two hydrologically contrasting growing seasons. Water addition stimulated SR_A and SR_H in both years, and their increases were significantly greater in a dry year (2007) than in a wet year (2006). N addition increased SR_A in 2006 but not in 2007, while it decreased SR_H in both years, leading to a positive response of SR_TOT in 2006 but a negative one in 2007. The different responses of SR_A and SR_H indicate that it will be uncertain to predict soil C storage if SR_TOT is used instead of SR_H to estimate variations in soil C storage. Overall, N addition is likely to enhance soil C storage, while the impacts of water addition are determined by its relative effects on carbon input (plant growth) and SR_H. Antecedent water conditions played an important role in controlling responses of SR_A, SR_H and the consequent SR_TOT to water and N addition. Our findings highlight the predominance of hydrological conditions in regulating the responses of C cycling to global change in the semiarid temperate steppe of northern China.     

Nitrification and occurrence of Nitrobacter by MPN-PCR in low and high nitrifying coniferous forest soils

The nitrification of three coniferous forest soils was investigated: a podzol (Fontainebleau, France) with low N deposition and no nitrate accumulation, an acid mull (Haldon, UK) with low N deposition and moderate nitrate accumulation, and a podzol (Wekerom, The Netherlands) with high N deposition and high nitrate accumulation. Twenty-one months in situ lysimeter experiments and short-term and long-term in vitro incubations were performed to respectively establish the status of NO ₃ ^- accumulation and potential nitrification of these soils. These complementary approaches allowed to conclude that the absence of NO ₃ ^- accumulation in Fontainebleau soil was effectively due to a lack of nitrifing activity in the whole profile while the high NO ₃ ^- accumulation observed in the Wekerom soil essentially resulted from an active nitrifying activity in the Oh horizon of this soil. For the Haldon soil, the inadequacy between the lysimeter and the short-term in vitro experiment was discussed.Surprisingly, relatively high densities of the nitrite-oxidiser Nitrobacter (enumerated by PCR-MPN technique) were found in all the studied horizons of the three soils. Moreover, a long-term incubation experiment showed that inhibition of nitrification in the Oh Fontainebleau soil could be removed after submitting the soil samples to constant conditions during 82 days. This demonstrates that in these soils, ammonium-oxidisers (as well as nitrite-oxidisers) are present and that extrinsic factors were involved in the inhibition of nitrification.     

N uptake and N status in ponderosa pine as affected by soil compaction and forest floor removal

Soil compaction and forest floor removal influence fundamental soil processes that control forest productivity and sustainability. We investigated effects of soil compaction and forest floor removal on tree growth, N uptake and N status in ponderosa pine. Factorial combinations of soil compaction (non-compacted and compacted) and forest floor removal (forest floor present and no forest floor) were applied to three different surface soil textures. For studying N uptake, four trees from every treatment were ¹⁵N labeled with 130.6 mg m^–2 of ¹⁵N. Tree responses to compaction were dependent on the forest floor removal level. In loam and clay soils, non-compacted+no forest floor was beneficial to tree growth. Tree growth was depressed with compaction+no forest floor in clay soil. In sandy loam soil, compaction+no forest floor showed the best tree growth. No N deficiency was found in any soil type but a graphical method suggested correlation between N status and tree growth. In loam and clay soils, compaction+forest floor present increased N uptake. Nitrogen uptake was explained significantly by potential N mineralization in loam and clay soils. In sandy loam soil, the effects of compaction and forest floor removal were more complex, with the N uptake improved in the compaction+no forest floor treatment and reduced under compaction+forest floor present. Soil compaction may have influenced N tracer uptake because of improved unsaturated flow and root-soil contact. However, N immobilization may have restricted N uptake in compaction+forest floor present in the sandy loam soil. The study illustrates how soil properties and site preparation can potentially interact to affect N dynamics and forest productivity.