Deoxyribonucleic acid homologies among 96 strains of ammonia-oxidizing bacteria

DNA of 96 strains of the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio was isolated and analysed spectrophotometrically. Percentages of guanine plus cytosine (G+C) content, genome sizes, and DNA-DNA homologies were determined. The results indicated the presence of eight Nitrosomonas species, three or four Nitrosococcus species, five Nitrosospira species, and two species of both Nitrosolobus and Nitrosovibrio. DNA homologies between strains of a separate species ranged from 56–100%. Average homologies between strains of different species were 33% in Nitrosococcus, 36% in Nitrosomonas, 37% in Nitrosolobus, 40% in Nitrosospira, and 42% in Nitrosovibrio. Average homologies between species of different genera were 33% and thus not significantly above the background value of 30% detected between DNA of ammonia-oxidizing bacteria and Escherichia coli. Genome sizes ranged from 1.90–2.74×10⁹ dalton in Nitrosomonas, 2.09–2.37×10⁹ dalton in Nitrosococcus, 1.87–2.15×10⁹ dalton in Nitrosospira, 1.92–2.10×10⁹ dalton in Nitrosolobus, and 1.91–2.15×10⁹ dalton in Nitrosovibrio. Differences in genome sizes were in accordance with DNA homologies.     

Inhibitory Effect of Nitrapyrin on Three Genera of Ammonia-Oxidizing Nitrifiers

Five strains of Nitrosomonas and one each of Nitrosospira and Nitrosolobus were examined for sensitivity to the nitrification inhibitor nitrapyrin. Considerable variation in sensitivity was observed, with some strains about five times more resistant than others. Sensitivity to nitrapyrin varied more with strain than with genus.     

Nitrosovibrio spp., the Dominant Ammonia-Oxidizing Bacteria in Building Sandstone

In five historical buildings in the Federal Republic of Germany, ammonia-oxidizing bacteria of the genera Nitrosovibrio, Nitrosospira, and Nitrosomonas were detected in high cell numbers. In building stones, Nitrosovibrio was the most abundant ammonia-oxidizing organism. In the soil at the foot of each building, Nitrosomonas spp. were the most common ammonia oxidizers, whereas Nitrosovibrio spp. were not detected.     

Genera-Specific Immunofluorescence Labeling of Ammonia Oxidizers with Polyclonal Antibodies Recognizing Both Subunits of the Ammonia Monooxygenase

Polyclonal antibodies that recognize the two subunits AmoA and AmoB of the ammonia monooxygenase (AMO) were applied to identify ammonia-oxidizing bacteria by immunofluorescence (IF) labeling in pure, mixed, and enriched cultures. The antibodies against the AmoA were produced using a synthetic peptide of the AmoA of Nitrosomonas eutropha, whereas the antibodies against the AmoB had been developed previously is against the whole B-subunit of the AMO [Pinck et al. (2001) Appl Environ Microbiol 67:118–124]. Using IF labeling, the AmoA antibodies were specific for the detection of all species of the genus Nitrosomonas. In contrast, the antiserum against AmoB labeled all genera of ammonia oxidizers of the -subclass of Proteobacteria (Nitrosomonas, Nitrosospira, Nitrosolobus, and Nitrosovibrio). The fluorescence signals of the AmoA antibodies were spread all over the cells, whereas the signals of the AmoB antibodies were associated with the cytoplasmic membranes. The specificity of the reactions of the antisera with ammonia oxidizers were proven in pure and mixed cultures, and the characteristic IF labeling and the morphology of the cells enabled their identification at the genus level. The genus-specific IF labeling could be used to identify ammonia oxidizers enriched from various habitats. In enrichment cultures of natural sandstone, cells of the genera Nitrosomonas, Nitrosovibrio, and Nitrosospira were detected. Members of the genus Nitrosovibrio and Nitrosolobus were most prominent in enriched garden soil samples, whereas members of the genus Nitrosomonas dominated in enriched activated sludge. The antibodies caused only slight background fluorescence on sandstone and soil particles compared to oligonucleotide probes, which could not be used to detect ammonia oxidizers on these materials because of strong nonspecific fluorescence.     

Serological studies on lithotrophic,ammonia oxidizing bacteria

Rabbit antisera were prepared against living cells of six different ammonia oxidizing nitrifying bacteria. They were examined as to cross-reactivity in the agglutination test (Microtiter-system) with 24 nitrifier strains, including members of all known genera.Usually distinct cross-reactions were obtained only within the genera, but some exceptions were noticed. There was stated a clear cross-reaction between the two anti-Nitrosospira-antisera and the four tested Nitrosolobus strains. In some cases cross-reactions between cells of the Nitrosovibrio strains and the anti-Nitrosospira- as well as the anti-Nitrosococcus-antisera could be observed. The interpretation of the results obtained with the Nitrosomonas group was complicated by the fact that all strains showed positive zero titers with the control sera. In seven cases lipopolysaccharides were isolated and tested in the passive hemagglutination test to their cross-reactivity with the above mentioned antisera. hemagglutination could only be observed in the homologous system, cross-reactivity was never expressed.Abbreviations LPS Lipopolysaccharide(s) - G+C Guanine + cytosine - PBS Phosphate-buffered saline - SRBC Sheep red blood cells     

Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently Aerated Reactors

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of thereactor's performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.     

Application of Molecular Biological Techniques to a Seasonal Study of Ammonia Oxidation in a Eutrophic Freshwater Lake

The autotrophic ammonia-oxidizing bacteria in a eutrophic freshwater lake were studied over a 12-month period. Numbers of ammonia oxidisers in the lakewater were small throughout the year, and tangential-flow concentration was required to obtain meaningful estimates of most probable numbers. Sediments from littoral and profundal sites supported comparatively large populations of these bacteria, and the nitrification potential was high, particularly in summer samples from the littoral sediment surface. In enrichment cultures, lakewater samples nitrified at low (0.67 mM) ammonium concentrations only whereas sediment samples exhibited nitrification at high (12.5 mM) ammonium concentrations also. Enrichments at low ammonium concentration did not nitrify when inoculated into high-ammonium medium, but the converse was not true. This suggests that the water column contains a population of ammonia oxidizers that is sensitive to high ammonium concentrations. The observation of nitrification at high ammonium concentration by isolates from some winter lakewater samples, identified as nitrosospiras by 16S rRNA probing, is consistent with the hypothesis that sediment ammonia oxidizers enter the water column at overturn. With only one exception, nested PCR amplification enabled the detection of Nitrosospira 16S rDNA in all samples, but Nitrosomonas (N. europaea-eutropha lineage) 16S rDNA was never obtained. However, the latter were part of the sediment and water column communities, because their 16S rRNA could be detected by specific oligonucleotide probing of enrichment cultures. Furthermore, a specific PCR amplification regime for the Nitrosomonas europaea ammonia monooxygenase gene (amoA) yielded positive results when applied directly to sediment and lakewater samples. Patterns of Nitrosospira and Nitrosomonas detection by 16S rRNA oligonucleotide probing of sediment enrichment cultures were complex, but lakewater enrichments at low ammonium concentration were positive for nitrosomonads and not nitrosospiras. Analysis of enrichment cultures has therefore provided evidence for the existence of subpopulations within the lake ammonia-oxidizing community distinguishable on the basis of ammonium tolerance and possibly showing a seasonal distribution between the sediment and water column.     

Abundance and Diversity of Ammonia-Oxidizing Microorganisms in the Sediments of Jinshan Lake

Community structures of ammonia-oxidizing microorganisms were investigated using PCR primers designed to specifically target the ammonia monooxygenase α-subunit (amoA) gene in the sediment of Jinshan Lake. Relationships between the abundance and diversity of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), and physicochemical parameters were also explored. The AOA abundance decreased sharply from west to east; however, the AOB abundance changed slightly with AOB outnumbering AOA in two of the four sediment samples (JS), JS3 and JS4. The AOA abundance was significantly correlated with the NH₄–N, NO₃–N, and TP. No significant correlations were observed between the AOB abundance and environmental variables. AOB had a higher diversity and richness of amoA genes than AOA. Among the 76 archaeal amoA sequences retrieved, 57.89, 38.16, and 3.95 % fell within the Nitrosopumilus, Nitrososphaera, and Nitrososphaera sister clusters, respectively. The 130 bacterial amoA gene sequences obtained in this study were grouped with known AOB sequences in the Nitrosomonas and Nitrosospira genera, which occupied 72.31 % and 27.69 % of the AOB group, respectively. Compared to the other three sample sites, the AOA and AOB community compositions at JS4 showed a large difference. This work could enhance our understanding of the roles of ammonia-oxidizing microorganisms in freshwater lake environment.     

Diversity in the Ammonia-Oxidizing Nitrifier Population of a Soil

Multiple genera of ammonia-oxidizing chemoautotrophic nitrifiers in a soil were detected, isolated, and studied by means of modified most-probable-number (MPN) techniques. The soil examined was a Waukegon silt loam treated with ammonium nitrate or sewage effluent. The genera Nitrosomonas and Nitrosospira were found to occur more commonly than the genus Nitrosolobus. Three different MPN media gave approximately the same overall ammonia oxidizer counts within statistical error after prolonged incubation but differed markedly in ratios of Nitrosomonas to Nitrosospira. Selectivity and counting efficiency of MPN media were studied by observing the growth response of representative pure cultures isolated from the soil. Selectivity was evident in each medium with respect to all strains tested, and the media differed greatly in incubation times required to obtain maximum counts.     

Diversity,Abundance, and Spatial Distribution of Ammonia-Oxidizing β-Proteobacteria in Sediments from Changjiang Estuary and Its Adjacent Area in East China Sea

Changjiang Estuary, the largest estuary in China, encompasses a wide range of nutrient loading and trophic levels from the rivers to the sea, providing an ideal natural environment to explore relationships between functional diversity, physical/chemical complexity, and ecosystem function. In this study, molecular biological techniques were used to analyze the community structure and diversity of ammonia-oxidizing bacteria (AOB) in the sediments of Changjiang Estuary and its adjacent waters in East China Sea. The amoA gene (encoding ammonia monooxygenase subunit A) libraries analysis revealed extensive diversity within the β-Proteobacteria group of AOB, which were grouped into Nitrosospira-like and Nitrosomonas-like lineages. The majority of amoA gene sequences fell within Nitrosospira-like clade, and only a few sequences were clustered with the Nitrosomonas-like clade, indicating that Nitrosospira-like lineage may be more adaptable than Nitrosomonas-like lineage in this area. Multivariate statistical analysis indicated that the spatial distribution of the sedimentary β-Proteobacterial amoA genotype assemblages correlated significantly with nitrate, nitrite, and salinity. The vertical profile of amoA gene copies in gravity cores showed that intense sediment resuspension led to a deeper mixing layer. The horizontal distribution pattern of amoA gene copies was nearly correlated with the clayey mud belt in Changjiang Estuary and its adjacent area in East China Sea, where higher β-Proteobacteria phylogenetic diversity was observed. Meanwhile, those areas with high amoA copies in the surface sediments nearly matched those with low concentrations of dissolved oxygen and ammonium in the bottom water.     

Production of Nitrous Oxide by Ammonia-Oxidizing Chemoautotrophic Microorganisms in Soil

Gas chromatographic studies showed that nitrous oxide was produced in each instance when sterilized (autoclaved) soil was incubated after treatment with ammonium sulfate and inoculation with pure cultures of ammonia-oxidizing chemoautotrophic microorganisms (strains of Nitrosomonas, Nitrosospira, and Nitrosolobus). Production of N₂O in ammonium-treated sterilized soil inoculated with Nitrosomonas europaea increased with the concentration of ammonium and the moisture content of the soil and was completely inhibited by both nitrapyrin and acetylene. Similar effects of nitrapyrin, acetylene, ammonium concentration, and soil moisture content were observed in studies of factors affecting N₂O production in nonsterile soil treated with ammonium sulfate. These observations support the conclusion that, at least under some conditions, most of the N₂O evolved from soils treated with ammonium or ammonium-producing fertilizers is generated by chemoautotrophic nitrifying microorganisms during oxidation of ammonium to nitrite.     

Community composition of ammonia-oxidizing bacteria and archaea in rice field soil as affected by nitrogen fertilization

Little information is available on the ecology of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in flooded rice soils. Consequently, a microcosm experiment was conducted to determine the effect of nitrogen fertilizer on the composition of AOB and AOA communities in rice soil by using molecular analyses of ammonia monooxygenase gene (amoA) fragments. Experimental treatments included three levels of N (urea) fertilizer, i.e. 50, 100 and 150 mg N kg⁻¹ soil. Soil samples were operationally divided into four fractions: surface soil, bulk soil deep layer, rhizosphere and washed root material. NH₄⁺-N was the dominant form of N in soil porewater and increased with N fertilization. Cloning and sequencing of amoA gene fragments showed that the AOB community in the rice soil consisted of three major groups, i.e. Nitrosomonas communis cluster, Nitrosospira cluster 3a and cluster 3b. The sequences related to Nitrosomonas were predominant. There was a clear effect of N fertilizer and soil depth on AOB community composition based on terminal restriction fragment length polymorphism fingerprinting. Nitrosomonas appeared to be more abundant in the potentially oxic or micro-oxic fractions, including surface soil, rhizosphere and washed root material, than the deep layer of anoxic bulk soil. Furthermore, Nitrosomonas increased relatively in the partially oxic fractions and that of Nitrosospira decreased with the increasing application of N fertilizer. However, AOA community composition remained unchanged according to the denaturing gradient gel electrophoresis analyses.     

Analysis of β-Subgroup Proteobacterial Ammonia Oxidizer Populations in Soil by Denaturing Gradient Gel Electrophoresis Analysis and Hierarchical Phylogenetic Probing

A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic β-subgroup proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DNA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE. Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers. A sequence specific to all β-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147–4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters. DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results were ambiguous because of overlapping banding patterns. Unambiguous band identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes. The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol 62:4147–4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor. The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of β-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.     

Community shift of ammonia-oxidizing bacteria along an anthropogenic pollution gradient from the Pearl River Delta to the South China Sea

The phylogenetic diversity and abundance of ammonia-oxidizing beta-proteobacteria (beta-AOB) was analyzed along an anthropogenic pollution gradient from the coastal Pearl River Delta to the South China Sea using the ammonia monooxygenase subunit A (amoA) gene. Along the gradient from coastal to the open ocean, the phylogenetic diversity of the dominant genus changed from Nitrosomonas to Nitrosospira, indicating the niche specificity by these two genera as both salinity and anthropogenic influence were major factors involved. The diversity of bacterial amoA gene was also variable along the gradient, with the highest in the deep-sea sediments, followed by the marshes sediments and the lowest in the coastal areas. Within the Nitrosomonas-related clade, four distinct lineages were identified including a putative new one (A5-16) from the different sites over the large geographical area. In the Nitrosospira-related clade, the habitat-specific lineages to the deep-sea and coastal sediments were identified. This study also provides strong support that Nitrosomonas genus, especially Nitrosomonas oligotropha lineage (6a) could be a potential bio-indicator species for pollution or freshwater/wastewater input into coastal environments. A suite of statistical analyses used showed that water depth and temperature were major factors shaping the community structure of beta-AOB in this study area.     

Response of Nitrosospira sp. Strain AF-Like Ammonia Oxidizers to Changes in Temperature, Soil Moisture Content, and Fertilizer Concentration

Very little is known regarding the ecology of Nitrosospira sp. strain AF-like bacteria, a unique group of ammonia oxidizers within the Betaproteobacteria. We studied the response of Nitrosospira sp. strain AF-like ammonia oxidizers to changing environmental conditions by applying molecular methods and physiological measurements to Californian grassland soil manipulated in the laboratory. This soil is naturally high in Nitrosospira sp. strain AF-like bacteria relative to the much-better-studied Nitrosospira multiformis-like ammonia-oxidizing bacteria. Increases in temperature, soil moisture, and fertilizer interacted to reduce the relative abundance of Nitrosospira sp. strain AF-like bacteria, although they remained numerically dominant. The overall abundance of ammonia-oxidizing bacteria increased with increasing soil moisture and decreased with increasing temperature. Potential nitrification activity was altered by interactions among temperature, soil moisture, and fertilizer, with activity tending to be higher when soil moisture and temperature were increased. The increase in potential nitrification activity with increased temperature was surprising, given that the overall abundance of ammonia-oxidizing bacteria decreased significantly under these conditions. This observation suggests that (i) Nitrosospira sp. strain AF-like bacteria may respond to increased temperature with an increase in activity, despite a decrease in abundance, or (ii) that potential nitrification activity in these soils may be due to organisms other than bacteria (e.g., archaeal ammonia oxidizers), at least under conditions of increased temperature.     

Characterization of Microbial Communities and Composition in Constructed Dairy Wetland Wastewater Effluent

Constructed wetlands have been recognized as a removal treatment option for high concentrations of contaminants in agricultural waste before land application. The goal of this study was to characterize microbial composition in two constructed wetlands designed to remove contaminants from dairy washwater. Water samples were collected weekly for 11 months from two wetlands to determine the efficiency of the treatment system in removal of chemical contaminants and total and fecal coliforms. The reduction by the treatment was greatest for biological oxygen demand, suspended solids, chemical oxygen demand, nitrate, and coliforms. There was only moderate removal of total nitrogen and phosphorus. Changes in the total bacterial community and ammonia-oxidizing bacterial composition were examined by using denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified fragments of the gene carrying the α subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and DGGE bands. DGGE analysis of wetlands and manure samples revealed that the total bacterial community composition was dominated by bacteria from phylogenetic clusters related to Bacillus, Clostridium, Mycoplasma, Eubacterium, and Proteobacteria originally retrieved from the gastrointestinal tracts of mammals. The population of ammonia-oxidizing bacteria showed a higher percentage of Nitrosospira-like sequences from the wetland samples, while a higher percentage of Nitrosomonas-like sequences from manure, feces, raw washwater, and facultative pond was found. These results show that the wetland system is a natural process dependent upon the development of healthy microbial communities for optimal wastewater treatment.     

Ammonia- and Nitrite-Oxidizing Bacterial Communities in a Pilot-Scale Chloraminated Drinking Water Distribution System

Nitrification in drinking water distribution systems is a common operational problem for many utilities that use chloramines for secondary disinfection. The diversity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the distribution systems of a pilot-scale chloraminated drinking water treatment system was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene (ribosomal DNA [rDNA]) cloning and sequencing. For ammonia oxidizers, 16S rDNA-targeted T-RFLP indicated the presence of Nitrosomonas in each of the distribution systems, with a considerably smaller peak attributable to Nitrosospira-like AOB. Sequences of AOB amplification products aligned within the Nitrosomonas oligotropha cluster and were closely related to N. oligotropha and Nitrosomonas ureae. The nitrite-oxidizing communities were comprised primarily of Nitrospira, although Nitrobacter was detected in some samples. These results suggest a possible selection of AOB related to N. oligotropha and N. ureae in chloraminated systems and demonstrate the presence of NOB, indicating a biological mechanism for nitrite loss that contributes to a reduction in nitrite-associated chloramine decay.     

Effect of organic matter on growth and cell yield of ammonia-oxidizing bacteria

The effect of various organic compounds on the growth of ammonia-oxidizing bacteria was examined.Nitrosococcus oceanus, a strongly halophilic bacterium, had a very low tolerance to organic matter compared with other organisms tested. Organic compounds scarcely affected the growth of theNitrosomonas strains whereas nitrite formation by bothNitrosococcus mobilis strains was inhibited by nearly all of the substances tested. The growth ofNitrosospira strain Nsp₁ was enhanced more than 30% by acetate and formate, but not growth was detectable in the presence of pyruvate. On the contrary,Nitrosospira strain Nsp₅ was stimulated only by pyruvate. Nitrite formation by the twoNitrosovibrio tenuis strains tested was similar. The growth of both strains was enhanced considerably by formate and glucose; acetate and, to a greater extent, pyruvate inhibited these bacteria.In batch culture, the energy efficiency of autotrophically grown ammonia-oxidizing bacteria varied from strain to strain. The cell yield of mixotrophically grown cultures, per unit of ammonia oxidized, was increased in comparison with autotrophic ones. No heterotrophic growth was detected.     

Toxic Effects of Linear Alkylbenzene Sulfonate on Metabolic Activity, Growth Rate, and Microcolony Formation of Nitrosomonas and Nitrosospira Strains

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC₅₀], 6 to 38 mg liter⁻¹) was affected much less by LAS than the growth rate and viability (EC₅₀, 3 to 14 mg liter⁻¹) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter⁻¹); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH₄⁺ oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.     

Population Ecology of Nitrifiers in a Stream Receiving Geothermal Inputs of Ammonium

The distribution, activity, and generic diversity of nitrifying bacteria in a stream receiving geothermal inputs of ammonium were studied. The high estimated rates of benthic nitrate flux (33 to 75 mg of N · m⁻² · h⁻¹) were a result of the activity of nitrifiers located in the sediment. Nitrifying potentials and ammonium oxidizer most probable numbers in the sediments were at least one order of magnitude higher than those in the waters. Nitrifiers in the oxygenated surface (0 to 2 cm) sediments were limited by suboptimal temperature, pH, and substrate level. Nitrifiers in deep (nonsurface) oxygenated sediments did not contribute significantly to the changes measured in the levels of inorganic nitrogen species in the overlying waters and presumably derived their ammonium supply from ammonification within the sediment. Ammonium-oxidizing isolates obtained by a most-probable number nonenrichment procedure were species of either Nitrosospira or Nitrosomonas, whereas all those obtained by an enrichment procedure (i.e., selective culture) were Nitrosomonas spp. The efficiency of the most-probable-number method for enumerating ammonium oxidizers was calculated to be between 0.05 and 2.0%, suggesting that measurements of nitrifying potentials provide a better estimate of nitrifying populations.