Numbers of nitrite-oxidizing bacteria in the root zone of grassland plants

Abstract The results of Most Probable Number determinations applying low and high concentrations of nitrite reveal the presence of at least two different communities of potential nitrite-oxidizing bacteria in a number of soil types. The effect of plant roots on these two communities was studied in pot experiments with soil from natural grassland in the presence or absence of either Festuca rubra or Plantago lanceolata . Both plant species are dominant on the grassland soil used in this study. Plant roots had a stimulating effect on the numbers of nitrite-oxidizing bacteria determined with 0.05 mM nitrite in the enumeration medium as well as on the potential nitrite-oxidizing activity. On the other hand, plants roots, especially in younger plants, repressed the numbers of nitrite-oxidizing bacteria enumerated with 5.0 mM nitrite in the counting medium. Pure culture studies with organotrophically grown Nitrobacter species clearly showed that this type of potential nitrite-oxidizing bacteria could not have been responsible for the relatively high Most Probable Numbers observed in the root zones when applying 0.05 mM nitrite in the enumeration medium.     

Most Probable Numbers of chemolitho-autotrophic nitrite-oxidizing bacteria in well drained grassland soils: Stimulation by high nitrite concentrations

Abstract The results of Most Probable Number (MPN) enumerations of chemolitho-autotrophic nitrite oxidizers are very much dependent on the nitrite concentration applied in the incubation medium. In order to explain this dependency, the influence of pH, nitrite and resultant nitrous acid concentration of the incubation medium on the MPN-enumeration was investigated. It appeared that none of these factors were exclusively responsible for the result of the enumeration. In samples from a well drained grassland soil, highest number have been obtained with a combination of a low pH and a low nitrite concentration in the counting medium. The relation between the MPN-counting results and the nitrous acid concentration showed an optimum with the same soil samples. It was hypothesized that the relatively high numbers of nitrite-oxidizing cells determined in soil samples at a high nitrite concentration and pH 7.3 was due to the presence of dormant cells. However, this hypothesis could not be confirmed with enumerations of aged cell suspensions of different Nitrobacter species. In contrast to the field observations, these resting cells were always enumerated more efficiently at a low nitrite concentration. The importance of the use of more than one incubation medium for the enumeration of nitrite-oxidizing bacteria is emphasized.     

Enumeration of nitrite-oxidizing bacteria in grassland soils using a Most Probable Number technique: Effect of nitrite concentration and sampling procedure

Abstract Numbers of nitrite-oxidizing bacteria were determined in grassland soils using a Most Probable Number technique. Two concentrations of nitrite were used in the incubation medium, i.e. 0.05 and 5.0 mM. The results of the enumerations were highly dependent on the nitrite concentration as well as on the grassland soil sampled. In the one soil the highest numbers were counted with 5.0 mM, whereas in other soils highest numbers were obtained in media with 0.05 mM nitrite. The spatial distribution of nitrite-oxidizing bacteria was determined i two field plots. Variation between individual samples was low in one plot and high in the other. The implications of the observed differences for the sampling procedure in each sampling plot are discussed. In the waterlogged, oxygen-limited soils, numbers of nitrite-oxidizing bacteria were as high as in the drained soils. The chemolitho-autotrophic nature of these nitrifiers has been confirmed.     

Nitrospira-like bacteria associated with nitrite oxidation in freshwater aquaria

Oxidation of nitrite to nitrate in aquaria is typically attributed to bacteria belonging to the genus Nitrobacter which are members of the alpha subdivision of the class Proteobacteria. In order to identify bacteria responsible for nitrite oxidation in aquaria, clone libraries of rRNA genes were developed from biofilms of several freshwater aquaria. Analysis of the rDNA libraries, along with results from denaturing gradient gel electrophoresis (DGGE) on frequently sampled biofilms, indicated the presence of putative nitrite-oxidizing bacteria closely related to other members of the genus Nitrospira. Nucleic acid hybridization experiments with rRNA from biofilms of freshwater aquaria demonstrated that Nitrospira-like rRNA comprised nearly 5% of the rRNA extracted from the biofilms during the establishment of nitrification. Nitrite-oxidizing bacteria belonging to the alpha subdivision of the class Proteobacteria (e.g., Nitrobacter spp.) were not detected in these samples. Aquaria which received a commercial preparation containing Nitrobacter species did not show evidence of Nitrobacter growth and development but did develop substantial populations of Nitrospira-like species. Time series analysis of rDNA phylotypes on aquaria biofilms by DGGE, combined with nitrite and nitrate analysis, showed a correspondence between the appearance of Nitrospira-like bacterial ribosomal DNA and the initiation of nitrite oxidation. In total, the data suggest that Nitrobacter winogradskyi and close relatives were not the dominant nitrite-oxidizing bacteria in freshwater aquaria. Instead, nitrite oxidation in freshwater aquaria appeared to be mediated by bacteria closely related to Nitrospira moscoviensis and Nitrospira marina.     

Oxygen uptake activity by Nitrobacter spp. in the presence of metal ions and sulfooxyanions

Abstract Several metal ions inhibited the oxygen uptake activity of Nitrobacter agilis , but their effects on the kinetic parameters of nitrite oxidation were mixed. Growth of Nitrobacter winogradskyi was inhibited by persulfate (>0.1 mM), tetrathionate (>0.5 mM), and trithionate (>5 mM). Oxygen uptake activity was, however, relatively insensitive to persulfate and tetrathionate ions.     

Temporal and spatial variation in the nitrite-oxidizing bacterial community of a grassland soil

Abstract The temporal and spatial distribution of the nitrite-oxidizing community of a non-fertilized, semi-natural grassland soil was studied to obtain more insight into the possible variation in nitrate production in this soil throughout the year. Data describing the size, potential nitrite-oxidizing activity and serotype composition of the nitrite-oxidizing community are reported. In addition, several abiotic soil parameters potentially related to the activity of this community were measured. Whereas numbers and potential activities largely varied with time and place, the specific affinity for nitrite oxidation, defined as the ratio V _max/ K _m, was relatively constant. The serotypes Nitrobacter agilis, N. winogradskyi and N. hamburgensis were all present in the top 5-cm soil in every 500-g sample examined, showing that these species co-exist in this soil.     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Continuously Percolated Soil Columns

Although the absence of nitrate formation in grassland soils rich in organic matter has often been reported, low numbers of nitrifying bacteria are still found in these soils. To obtain more insight into these observations, we studied the competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi with soil columns containing calcareous sandy soil. The soil columns were percolated continuously at a dilution rate of 0.007 h^-1, based on liquid volumes, with medium containing 5 mM ammonium and different amounts of glucose ranging from 0 to 12 mM.A. globiformis was the most competitive organism for limiting amounts of ammonium. The numbers of N. europaea and N. winogradskyi cells were lower at higher glucose concentrations, and the potential ammonium-oxidizing activities in the uppermost 3 cm of the soil columns were nonexistent when at least 10 mM glucose was present in the reservoir, although 10⁷ nitrifying cells per g of dry soil were still present. This result demonstrated that there was no correlation between the numbers of nitrifying bacteria and their activities. The numbers and activities of N. winogradskyi cells decreased less than those of N. europaea cells in all layers of the soil columns, probably because of heterotrophic growth of the nitrite-oxidizing bacteria on organic substrates excreted by the heterotrophic bacteria or because of nitrate reduction at reduced oxygen concentrations by the nitrite-oxidizing bacteria. Our conclusion was that the nitrifying bacteria were less competitive than the heterotrophic bacteria for ammonium in soil columns but that they survived as viable inactive cells. Inactive nitrifying bacteria may also be found in the rhizosphere of grassland plants, which is rich in organic carbon. They are possibly reactivated during periods of net mineralization.     

Oxygen exchange between nitrate molecules during nitrite oxidation by Nitrobacter

During oxidation of nitrite, cells of Nitrobacter winogradskyi are shown to catalyze the active exchange of oxygen atoms between exogenous nitrate molecules (production of 15N16/18O3- during incubation of 14N16/18O3-, 15N16O3-, and 15N16O2- in H216O). Little, if any, exchange of oxygens between nitrate and water also occurs (production of 15N16/18O3- during incubation of 15N16O3- and 14N16O2- in H218O). 15N species of nitrate were assayed by 18O-isotope shift in 15N NMR. Taking into account the O-exchange reactions which occur during nitrite oxidation, H2O is seen to be the source of O in nitrate produced by oxidation of nitrite by N. winogradskyi. The data do not establish whether the nitrate-nitrate O exchange is catalyzed by nitrite oxidase (H2O + HNO2----HNO3 + 2H+ + 2e-) or nitrate reductase (HNO3 + 2H+ + 2e-----HNO2 + H2O) or both enzymes in consort. The nitrate-nitrate exchange reaction suggests the existence of an oxygen derivative of a H2O-utilizing oxidoreductase.     

Maintenance energy demand and starvation recovery dynamics of Nitrosomonas europaea and Nitrobacter winogradskyi cultivated in a retentostat with complete biomass retention.

Nitrosomonas europaea and Nitrobacter winogradskyi (strain "Engel") were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25 degrees C and pH 8. Fitting the retentostat biomass and oxygen consumption data of N. europaea and N. winogradskyi to the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 micromol of O2 mg-1 of biomass-C x h-1 for both N. europaea and N. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster for N. winogradskyi than for N. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable. N. europaea more readily recovers from starvation than N. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 microg x ml-1) inhibition experiments with N. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514-1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.     

A new obligately chemolithoautotrophic,nitrite-oxidizing bacterium,Nitrospira moscoviensis sp. nov. and its phylogenetic relationship

A gram-negative, non-motile, non-marine, nitrite-oxidizing bacterium was isolated from an enrichment culture initiated with a sample from a partially corroded area of an iron pipe of a heating system in Moscow, Russia. The cells were 0.9–2.2 μm × 0.2–0.4 μm in size. They were helical- to vibroid-shaped and often formed spirals with up to three turns 0.8–1.0 μm in width. The organism possessed an enlarged periplasmic space and lacked intracytoplasmic membranes and carboxysomes. The cells tended to excrete extracellular polymers, forming aggregates. The bacterium grew optimally at 39°C and pH 7.6– 8.0 in a mineral medium with nitrite as sole energy source and carbon dioxide as sole carbon source. The optimal nitrite concentration was 0.35 mM. Nitrite was oxidized to nitrate stoichiometrically. The doubling time was 12 h in a mineral medium with 7.5 mM nitrite. The cell yield was low; only 0.9 mg protein/l was formed during oxidation of 7.5 mM nitrite. Under anoxic conditions, hydrogen was used as electron donor with nitrate as electron acceptor. Organic matter (yeast extract, meat extract, peptone) supported neither mixotrophic nor heterotrophic growth. At concentrations as low as 0.75 g organic matter/l or higher, growth of nitrite-oxidizing cells was inhibited. The cells contained cytochromes of the b- and c-type. The G+C content of DNA was 56.9 ± 0.4 mol%. The chemolithoautotrophic nitrite-oxidizer differed from the terrestrial members of the genus Nitrobacter with regard to morphology and substrate range and equaled Nitrospira marina in both characteristics. The isolated bacterium is designated as a new species of the genus Nitrospira. Comparative analysis of 16S rRNA gene sequences revealed a moderate phylogenetic relationship to Nitrospira marina, leptospirilla, Thermodesulfovibrio yellowstonii, "Magnetobacterium bavaricum," and the isolate OPI-2. Initial evidence is given that these organisms represent a new phylum of the domain bacteria. Received: 17 February 1995 / Accepted: 18 April 1995     

Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm

The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium. Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas europaea -like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1–2 orders of magnitude lower than those of N. europaea . Nitrospira sp. were virtually absent in this part of the biofilm, whereas they were most abundant at the oxic–anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.     

Lipopolysaccharide of Paracoccus denitrificans ATCC13543

Abstract Nitrobacter hamburgensis was shown to synthesize at least two distinct membrane-bound b -type cytochromes. One of these, a minor component detected during nitrite oxidation, was also found in the obligately autotrophic species Nitrobacter winogradskyi . During heterotrophic growth of N. hamburgensis a second (major) cytochrome b was detected, which we assume functions as an alternative terminal oxidase.     

Isolation and characterization of a novel facultatively alkaliphilic Nitrobacter species, N. alkalicus sp. nov.

Five strains of lithotrophic, nitrite-oxidizing bacteria (AN1-AN5) were isolated from sediments of three soda lakes (Kunkur Steppe, Siberia; Crater Lake and Lake Nakuru, Kenya) and from a soda soil (Kunkur Steppe, Siberia) after enrichment at pH 10 with nitrite as sole electron source. Morphologically, the isolates resembled representatives of the genus Nitrobacter. However, they differed from recognized species of this genus by the presence of an additional S-layer in their cell wall and by their unique capacity to grow and oxidize nitrite under highly alkaline conditions. The influence of pH on growth of one of the strains (AN1) was investigated in detail by using nitrite-limited continuous cultivation. Under such conditions, strain AN1 was able to grow at a broad pH range from 6.5 to 10.2, with an optimum at 9.5. Cells grown at pH higher than 9 exhibited a clear shift in the optimal operation of the nitrite-oxidizing system towards the alkaline pH region with respect to both reaction rates and the affinity. Cells grown at neutral pH values behaved more like neutrophilic Nitrobacter species. These data demonstrated the remarkable potential of the new nitrite-oxidizing bacteria for adaptation to varying alkaline conditions. The 16S rRNA gene sequences of isolates AN1, AN2, and AN4 showed high similarity (≥ 99.8%) to each other, and to sequences of Nitrobacter strain R6 and of Nitrobacter winogradskyi. However, the DNA-DNA homology in hybridization studies was too low to consider these isolates as new strains. Therefore, the new isolates from the alkaline habitats are described as a new species of the genus Nitrobacter, N. alkalicus, on the basis of their substantial morphological, physiological, and genetic differences from the recognized neutrophilic representatives of this genus. Received: 3 April 1998 / Accepted: 2 July 1998     

Autecological Study of the Chemoautotroph Nitrobacter by Immunofluorescence

Fluorescent antibodies (FA) prepared for Nitrobacter agilis and N. winogradskyi were highly reactive in homologous staining. Low-level cross-reactions between the two species were removed by adsorption. All 15 pure-culture isolates of Nitrobacter tested reacted strongly with either N. agilis FA or N. winogradskyi FA. All pure-culture isolates from soils were determined to be N. winogradskyi; those from Mammoth Cave sediments and a cattle waste oxidation ditch were N. agilis. No cross-reaction was found in extensive tests that included five isolates of Nitrosomonas europaea and 668 heterotrophic aerobic and anaerobic bacteria isolated from soil, sewage, and cave sites. The FA preparations were used to detect Nitrobacter species in Mammoth Cave sediments, in a cattle waste oxidation ditch, and in surface waters and sediments of a river and to observe that N. winogradskyi can outgrow N. agilis in enrichment culture.     

Identification of nitrite-oxidizing bacteria with monoclonal antibodies recognizing the nitrite oxidoreductase.

Immunoblot analyses performed with three monoclonal antibodies (MAbs) that recognized the nitrite oxidoreductase (NOR) of the genus Nitrobacter were used for taxonomic investigations of nitrite oxidizers. We found that these MAbs were able to detect the nitrite-oxidizing systems (NOS) of the genera Nitrospira, Nitrococcus, and Nitrospina. The MAb designated Hyb 153-2, which recognized the alpha subunit of the NOR (alpha-NOR), was specific for species belonging to the genus Nitrobacter. In contrast, Hyb 153-3, which recognized the beta-NOR, reacted with nitrite oxidizers of the four genera. Hyb 153-1, which also recognized the beta-NOR, bound to members of the genera Nitrobacter and Nitrococcus. The molecular masses of the beta-NOR of the genus Nitrobacter and the beta subunit of the NOS (beta-NOS) of the genus Nitrococcus were identical (65 kDa). In contrast, the molecular masses of the beta-NOS of the genera Nitrospina and Nitrospira were different (48 and 46 kDa). When the genus-specific reactions of the MAbs were correlated with 16S rRNA sequences, they reflected the phylogenetic relationships among the nitrite oxidizers. The specific reactions of the MAbs allowed us to classify novel isolates and nitrite oxidizers in enrichment cultures at the genus level. In ecological studies the immunoblot analyses demonstrated that Nitrobacter or Nitrospira cells could be enriched from activated sludge by using various substrate concentrations. Fluorescence in situ hybridization and electron microscopic analyses confirmed these results. Permeated cells of pure cultures of members of the four genera were suitable for immunofluorescence labeling; these cells exhibited fluorescence signals that were consistent with the location of the NOS.     

The occurrence of chemolitho-autotrophic nitrifiers in water-saturated grassland soils

Relatively high most probable number (MPN) counts of chemolithotrophic nitrite oxidizers were present in water-saturated soils compared with MPNs and activity of ammonia oxidizers. These high numbers of nitrite oxidizers were confirmed by fluorescent antibody counts and potential activity measurements. Application of different nitrite concentrations in the MPN procedure discriminated within the community of nitrite oxidizers and revealed a large number of nitrite-sensitive nitrite oxidizers and a subcommunity of nitrite-insensitive nitrite oxidizers. The size of this subcommunity was small but corresponded with the low numbers of ammonium oxidizers. Numbers of nitrite-sensitive nitrite oxidizers outnumbered the ammonia oxidizing bacteria by 2–4 orders of magnitude in these soils. The possibility is discussed that the fraction of the nitrite-insensitive cells was active as aerobic nitrite oxidizers, whereas the nitrite-sensitive cells represented an inactive group of nitrite oxidizers growing as heterotrophs or as anaerobes reducing nitrite. In this situation, both MPN enumerations at a low nitrite concentration and activity measurements could give false information about the size of the in situ nitrite-oxidizing community.     

The effect of the incubation period on the result of MPN enumerations of nitrite-oxidizing bacteria: theoretical considerations

Abstract A computer model based on Monod- and Haldane-kinetics was used to estimate the minimum incubation period required for MPN enumerations of nitrite-oxidizing bacteria. The minimum incubation period was defined as the time needed for one cell, present in the tubes inoculated with the highest dilutions, to grow into a population that oxidized all the nitrite present at the start of the incubation. Kinetic parameters used in the model were derived from literature data and applied in different combinations. The results show that the minimum incubation period may increase with decreasing initial nitrite concentrations in the incubation medium. They also show that the opposite trend, i.e. increasing minimum incubation periods with increasing nitrite concentration periods with increasing nitrite concentration, can be explained by introducing a term for substrate inhibition in the model. A MPN enumeration result obtained with samples from a waterlogged peat bog soil could only partly be explained by the model if only one set of parameters was used. This indicates that the community of nitrite-oxidizing bacteria in this soil is composed of at least two types of nitrite-oxidizing bacteria, with different kinetic parameters of nitrite oxidation and growth.     

First exploration of Nitrobacter diversity in soils by a PCR cloning-sequencing approach targeting functional gene nxrA

Nitrite oxidoreductase (NXR) is the key enzyme responsible for the oxidation of NO(2)(-) to NO(3)(-) in nitrite-oxidizing bacteria. For the first time a molecular approach for targeting the nxrA gene was developed, encoding the catalytic subunit of the NXR, to study diversity of Nitrobacter-like organisms based on the phylogeny of nxrA gene sequences in soils. NxrA sequences of the Nitrobacter strains analysed (Nitrobacter hamburgensis, Nitrobacter vulgaris, Nitrobacter winogradskyi, Nitrobacter alkalicus) by PCR, cloning and sequencing revealed the occurrence of multiple copies of nxrA genes in these strains. The copy number and similarity varied among strains. The diversity of Nitrobacter-like nxrA sequences was explored in three soils (a French permanent pasture soil, a French fallow soil, and an African savannah soil) using a cloning and sequencing approach. Most nxrA sequences found in these soils (84%) differed from nxrA sequences obtained from Nitrobacter strains. Moreover, the phylogenetic distribution and richness of nxrA-like sequences was extremely variable depending on soil type. This nxrA tool extends the panel of functional genes available for studying bacteria involved in the nitrogen cycle.