Effects of long-term fertilization of forest soils on potential nitrification and on the abundance and community structure of ammonia oxidizers and nitrite oxidizers

Forest fertilization in British Columbia is increasing, to alleviate timber shortfalls resulting from the mountain pine beetle epidemic. However, fertilization effects on soil microbial communities, and consequently ecosystem processes, are poorly understood. Fertilization has contrasting effects on ammonia-oxidizing bacteria and archaea (AOB and AOA) in grassland and agricultural ecosystems, but there are no studies on AOB and AOA in forests. We assessed the effect of periodic (6-yearly application 200 kg N ha?1) and annual (c. 75 kg N ha?1) fertilization of lodgepole pine and spruce stands at five long-term maximum productivity sites on potential nitrification (PN), and the abundance and diversity of AOB, AOA and Nitrobacter and Nitrospira-like nitrite-oxidizing bacteria (NOB). Fertilization increased AOB and Nitrobacter-like NOB abundances at some sites, but did not influence AOA and Nitrospira-like NOB abundances. AOB and Nitrobacter-like NOB abundances were correlated with PN and soil nitrate concentration; no such correlations were observed for AOA and Nitrospira-like NOB. Autotrophic nitrification dominated (55–97%) in these forests and PN rates were enhanced for up to 2 years following periodic fertilization. More changes in community composition between control and fertilized plots were observed for AOB and Nitrobacter-like NOB than AOA. We conclude that fertilization causes rapid shifts in the structure of AOB and Nitrobacter-like NOB communities that dominate nitrification in these forests.     

Community analysis of ammonia and nitrite oxidizers during start-up of nitritation reactors

Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30 degrees C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N. europaea-N. eutropha group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25 degrees C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day(-1) compared to 0.2 day(-1) for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.     

Aquarium nitrification revisited: Thaumarchaeota are the dominant ammonia oxidizers in freshwater aquarium biofilters

Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in many terrestrial and aquatic environments. Although nitrification is the primary function of aquarium biofilters, very few studies have investigated the microorganisms responsible for this process in aquaria. This study used quantitative real-time PCR (qPCR) to quantify the ammonia monooxygenase (amoA) and 16S rRNA genes of Bacteria and Thaumarchaeota in freshwater aquarium biofilters, in addition to assessing the diversity of AOA amoA genes by denaturing gradient gel electrophoresis (DGGE) and clone libraries. AOA were numerically dominant in 23 of 27 freshwater biofilters, and in 12 of these biofilters AOA contributed all detectable amoA genes. Eight saltwater aquaria and two commercial aquarium nitrifier supplements were included for comparison. Both thaumarchaeal and bacterial amoA genes were detected in all saltwater samples, with AOA genes outnumbering AOB genes in five of eight biofilters. Bacterial amoA genes were abundant in both supplements, but thaumarchaeal amoA and 16S rRNA genes could not be detected. For freshwater aquaria, the proportion of amoA genes from AOA relative to AOB was inversely correlated with ammonium concentration. DGGE of AOA amoA genes revealed variable diversity across samples, with nonmetric multidimensional scaling (NMDS) indicating separation of freshwater and saltwater fingerprints. Composite clone libraries of AOA amoA genes revealed distinct freshwater and saltwater clusters, as well as mixed clusters containing both freshwater and saltwater amoA gene sequences. These results reveal insight into commonplace residential biofilters and suggest that aquarium biofilters may represent valuable biofilm microcosms for future studies of AOA ecology.     

Nitrosomonas Nm143-like ammonia oxidizers and Nitrospira marina-like nitrite oxidizers dominate the nitrifier community in a marine aquaculture biofilm

Zero-discharge marine aquaculture systems are an environmentally friendly alternative to conventional aquaculture. In these systems, water is purified and recycled via microbial biofilters. Here, quantitative data on nitrifier community structure of a trickling filter biofilm associated with a recirculating marine aquaculture system are presented. Repeated rounds of the full-cycle rRNA approach were necessary to optimize DNA extraction and the probe set for FISH to obtain a reliable and comprehensive picture of the ammonia-oxidizing community. Analysis of the ammonia monooxygenase gene (amoA) confirmed the results. The most abundant ammonia-oxidizing bacteria (AOB) were members of the Nitrosomonas sp. Nm143-lineage (6.7% of the bacterial biovolume), followed by Nitrosomonas marina-like AOB (2.2% of the bacterial biovolume). Both were outnumbered by nitrite-oxidizing bacteria of the Nitrospira marina-lineage (15.7% of the bacterial biovolume). Although more than eight other nitrifying populations were detected, including Crenarchaeota closely related to the ammonia-oxidizer 'Nitrosopumilus maritimus', their collective abundance was below 1% of the total biofilm volume; their contribution to nitrification in the biofilter is therefore likely to be negligible.     

Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea

The hydrolysis of urea as a source of ammonia has been proposed as a mechanism for the nitrification of ammonia-oxidizing bacteria (AOB) in acidic soil. The growth of Nitrososphaera viennensis on urea suggests that the ureolysis of ammonia-oxidizing archaea (AOA) might occur in natural environments. In this study, ¹⁵N isotope tracing indicates that ammonia oxidation occurred upon the addition of urea at a concentration similar to the in situ ammonium content of tea orchard soil (pH 3.75) and forest soil (pH 5.4) and was inhibited by acetylene. Nitrification activity was significantly stimulated by urea fertilization and coupled well with abundance changes in archaeal amoA genes in acidic soils. Pyrosequencing of 16S rRNA genes at whole microbial community level demonstrates the active growth of AOA in urea-amended soils. Molecular fingerprinting further shows that changes in denaturing gradient gel electrophoresis fingerprint patterns of archaeal amoA genes are paralleled by nitrification activity changes. However, bacterial amoA and 16S rRNA genes of AOB were not detected. The results strongly suggest that archaeal ammonia oxidation is supported by hydrolysis of urea and that AOA, from the marine Group 1.1a-associated lineage, dominate nitrification in two acidic soils tested.     

Heterotrophic nitrification and denitrification are the main sources of nitrous oxide in two paddy soils

Plant and Soil - Fusarium wilt (FW) is the major constraint on cape gooseberry (Physalis peruviana L.) production. Fungicides have been ineffective in disease control and alternative tools are not...     

Links between ammonia oxidizer community structure, abundance, and nitrification potential in acidic soils

Ammonia oxidation is the first and rate-limiting step of nitrification and is performed by both ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the environmental drivers controlling the abundance, composition, and activity of AOA and AOB communities are not well characterized, and the relative importance of these two groups in soil nitrification is still debated. Chinese tea orchard soils provide an excellent system for investigating the long-term effects of low pH and nitrogen fertilization strategies. AOA and AOB abundance and community composition were therefore investigated in tea soils and adjacent pine forest soils, using quantitative PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of respective ammonia monooxygenase (amoA) genes. There was strong evidence that soil pH was an important factor controlling AOB but not AOA abundance, and the ratio of AOA to AOB amoA gene abundance increased with decreasing soil pH in the tea orchard soils. In contrast, T-RFLP analysis suggested that soil pH was a key explanatory variable for both AOA and AOB community structure, but a significant relationship between community abundance and nitrification potential was observed only for AOA. High potential nitrification rates indicated that nitrification was mainly driven by AOA in these acidic soils. Dominant AOA amoA sequences in the highly acidic tea soils were all placed within a specific clade, and one AOA genotype appears to be well adapted to growth in highly acidic soils. Specific AOA and AOB populations dominated in soils at particular pH values and N content, suggesting adaptation to specific niches.     

Links between ammonia oxidizer species composition, functional diversity and nitrification kinetics in grassland soils

Molecular approaches have revealed considerable diversity and uncultured novelty in natural prokaryotic populations, but not direct links between the new genotypes detected and ecosystem processes. Here we describe the influence of the structure of communities of ammonia-oxidizing bacteria on nitrogen cycling in microcosms containing natural and managed grasslands and amended with artificial sheep urine, a major factor determining local ammonia concentrations in these environments. Nitrification kinetics were assessed by analysis of changes in urea, ammonia, nitrite and nitrate concentrations and ammonia oxidizer communities were characterized by analysis of 16S rRNA genes amplified from extracted DNA using ammonia oxidizer-specific primers. In natural soils, ammonia oxidizer community structure determined the delay preceding nitrification, which depended on the relative abundance of two Nitrosospira clusters, termed 3a and 3b. In batch cultures, pure culture and enrichment culture representatives of Nitrosospira 3a were sensitive to high ammonia concentration, while Nitrosospira cluster 3b representatives and Nitrosomonas europaea were tolerant. Delays in nitrification occurred in natural soils dominated by Nitrosospira cluster 3a and resulted from the time required for growth of low concentrations of Nitrosospira cluster 3b. In microcosms dominated by Nitrosospira cluster 3b and Nitrosomonas, no substantial delays were observed. In managed soils, no delays in nitrification were detected, regardless of initial ammonia oxidizer community structure, most probably resulting from higher ammonia oxidizer cell concentrations. The data therefore demonstrate a direct link between bacterial community structure, physiological diversity and ecosystem function.     

Inducible nitrate reductase of rice plants as a possible indicator for nitrification in water-logged paddy soils

When following the pattern of the disappearance of NH ₄ ⁺ –N from ammonium sulfate applied to the flooded soil-rice plant system (field and greenhouse experiments) during a growing season, it was observed that the lowest NH ₄ ⁺ –N level coincided with the highest value of NR activity in the leaves. Nitrate was detected in both the root and shoot systems of the rice plants and autotrophic nitrifiers (Nitrosomonas and Nitrobacter) were particularly abundant. Since it was also demonstrated in this work that the NR activity of rice plants grown with nitrate fertilization (growth chamber culture experiments) was inducible by its substrate, it can be assumed that NH ₄ ⁺ –N oxidation takes place in the water-logged soil studied. Therefore, the occurrence of the nitrification process following NH ₄ ⁺ –N fertilizer application can be predicted by thein vitro orin situ evaluation of the NR activity of the rice leaf as an indicator.     

Applicability of two-step models in estimating nitrification kinetics from batch respirograms under different relative dynamics of ammonia and nitrite oxidation

A mechanistically based nitrification model was formulated to facilitate determination of both NH(4)(+)-N to NO(2)(-)-N and NO(2)(-)-N to NO(3)(-)-N oxidation kinetics from a single NH(4)(+)-N to NO(3)(-)-N batch-oxidation profile by explicitly considering the kinetics of each oxidation step. The developed model incorporated a novel convention for expressing the concentrations of nitrogen species in terms of their nitrogenous oxygen demand (NOD). Stoichiometric coefficients relating nitrogen removal, oxygen uptake, and biomass synthesis were derived from an electron-balanced equation.%A parameter identifiability analysis of the developed two-step model revealed a decrease in correlation and an increase in the precision of the kinetic parameter estimates when NO(2)(-)-N oxidation kinetics became increasingly rate-limiting. These findings demonstrate that two-step models describe nitrification kinetics adequately only when NH(4)(+)-N to NO(3)(-)-N oxidation profiles contain sufficient information pertaining to both nitrification steps. Thus, the rate-determining step in overall nitrification must be identified before applying conventionally used models to describe batch nitrification respirograms.     

Impacts of reduced sulfur components on active and resting ammonia oxidizers

While there has been significant research on the nature and extent of the impact of inhibitory reduced sulfur with respect to anaerobic (e.g., methanogenic and sulfidogenic) microbial systems, only limited study has yet been conducted on the comparable effects of soluble sulfides which might occur within aerobic wastewater treatment systems. Admittedly, aerobic reactors would not normally be considered conducive to the presence of reduced sulfur constituents, but there do appear to be a number of processing scenarios under which related impacts could develop, particularly for sensitive reactions like nitrification. Indeed, the following scenarios might well involve elevated levels of reduced sulfur within an aerobic reactor environment: (1) mixed liquor recycle back through sulfide-generating anaerobic zones (e.g., in conjunction with biological nutrient removal processes, etc.), (2) high-level side-stream sulfide recycle via sludge digestion, etc., back to aerobic reactors, and (3) high-level influent sulfide inputs to wastewater treatment facilities via specific industrial, septage, etc., streams. The objective of this study was, therefore, to determine the subsequent metabolic impact of soluble sulfide under aerated and unaerated conditions, focusing in particular on ammonia-oxidizing bacteria due to their critical first-step role with nitrification. The obtained results indicated that, under catabolically active conditions, cultures of ammonia oxidizers were extremely sensitive to the presence of sulfide. At total soluble sulfide concentrations of 0.25 mg l^–1 S, active ammonia oxidation was completely inhibited. However, immediately following the removal of this soluble sulfide presence, ammonia oxidation started to recover; and it continued to improve over the next 24 h. Similar sulfide impact tests conducted with inactive ammonia oxidizers exposed during anaerobic conditions, albeit at higher dosage levels, also revealed that their subsequent aerobic activity would correspondingly be retarded. These results indicated that, after sulfide exposure under unaerated conditions, subsequent aerobic oxidative activity rates rapidly decreased as the soluble sulfide exposure was increased from 0.5 gm l^–1 S to 5 mg l^–1 S and that further reductions in this activity progressively developed as the concentration was increased to 200 mg l^–1 S. The recovery following unaerated exposure to sulfide was significantly higher at pH 7, as compared with pH 8, and although the specific nature of this variation was not established, a hypothetical explanation appeared warranted.     

Heterotrophic nitrification in add forest soils

Ninety cultures of heterotrophic organisms were isolated from soils of four acid Norwegian forest sites, which were active in nitrifying. The isolates were tested for ability to form nitrite in a glucose-ammonium-inorganic salts medium. Eleven cultures were found capable of oxidizing ammonium to nitrite. The nitrifying organisms consist of 4 bacteria and 7 fungi.     

Abundance and diversity of archaeal ammonia oxidizers in a coastal groundwater system

Nitrification, the microbially catalyzed oxidation of ammonia to nitrate, is a key process in the nitrogen cycle. Archaea have been implicated in the first part of the nitrification pathway (oxidation of ammonia to nitrite), but the ecology and physiology of these organisms remain largely unknown. This work describes two different populations of sediment-associated ammonia-oxidizing archaea (AOA) in a coastal groundwater system in Cape Cod, MA. Sequence analysis of the ammonia monooxygenase subunit A gene (amoA) shows that one population of putative AOA inhabits the upper meter of the sediment, where they may experience frequent ventilation, with tidally driven overtopping and infiltration of bay water supplying dissolved oxygen, ammonium, and perhaps organic carbon. A genetically distinct population occurs deeper in the sediment, in a mixing zone between a nitrate- and oxygen-rich freshwater zone and a reduced, ammonium-bearing saltwater wedge. Both of these AOA populations are coincident with increases in the abundance of group I crenarchaeota 16S rRNA gene copies.     

Estimation of nitrification rates in flooded soils

Three techniques for estimating nitrification rates in flooded soils were evaluated in short-term incubation experiments using three soils. The techniques were based on inhibition of either ammonium or nitrite oxidation and ¹³N isotope dilution. Of four inhibitors of ammonium oxidation evaluated, one (allylthiourea) was ineffective and two (2-ethynylpyridine or phenyl acetylene dissolved in ethanol) promoted immobilization of ammonium. Emulsified 2-ethynylpyridine and acetylene were equally effective inhibitors of ammonium oxidation and had little or no effect on gross rates of N mineralization and immobilization. Four inhibitors of nitrite oxidation were evaluated, but this approach was compromised by the nonspecificity of three of the compounds—potassium cyanide, 2-ethylamino-4-isopropylamino-6-methylthio-s-triazine (ametryne) and 3-(3,4-dichlorophenyl)-1-methylurea (DMU)—and by the partial effectiveness of another (potassium chlorate). Two methods based on isotope dilution gave similar estimates of nitrification rates. These rates were similar to those estimated by inhibition of ammonium oxidation in one soil but were lower in the other two soils. In the latter two soils, nitrification of labeled ammonium derived from dissimilatory nitrate reduction resulted in underestimation of nitrification rates by isotope dilution.     

Use of polyclonal antibodies to detect and quantify the NOR protein of nitrite oxidizers in complex environments

In the approaches or models which aim to understand and/or predict how the functioning of ecosystems may be affected by perturbations or disturbances, little attention is generally given to microorganisms. Even when they are taken into account as indicators, variables which are poorly informative about the changes in the microbial functioning (microbial biomass or diversity or total number of microorganisms) are often used. To be able to estimate, in complex environments, the quantity of enzymes involved in key ecosystem processes may constitute a useful complementary tool. Here, we describe an immunological method for detecting and quantifying, in complex environments, the nitrite oxidoreductase (NOR), responsible for the oxidation of nitrite to nitrate. The alpha-catalytic subunit of the enzyme was purified from Nitrobacter hamburgensis and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NOR by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. They recognized the alpha-NOR of all the Nitrobacter species described to date, but no reaction was observed with members of other nitrite-oxidizing genera. The detection threshold and reproducibility of the proposed method were evaluated. The feasibility of its use to quantify NOR in a soil was tested.