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PubMed2006

Effect of nitrite concentration on the distribution and competition of nitrite-oxidizing bacteria in nitratation reactor systems and their kinetic characteristics.

Genus Nitrospira and Nitrobacter species are the key nitrite-oxidizing bacteria (NOB) in nitrifying wastewater treatment plants. It has been hypothesized that genus Nitrospira are K-strategists and can exploit low amounts of nitrite more efficiently than Nitrobacter. In contrast, Nitrobacter species are r-strategists that can grow faster than Nitrospira. To elucidate the K/r hypothesis and to analyze the effect of substrate (nitrite) concentration on the competition and distribution of the two NOB, two different reactor types were employed for nitrite oxidation (nitratation) and NOB growth. The continuous biofilm airlift reactor (CBAR) maintained low nitrite concentration due to the complete oxidation of nitrite in continuous operation while the sequencing batch reactor (SBR) was kept in a relatively high nitrite concentration environment due to a cyclic substrate concentration profile. Fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) showed that both Nitrobacter species and genus Nitrospira were present in the CBAR and the SBR. Quantitative FISH analyses of the CBAR showed that Nitrospira occupied 59% of the total bacteria while Nitrobacter occupied only 5%. On the other hand, Nitrobacter, occupying 64%, was the dominant NOB in the SBR, and only 3% of total bacteria belonged to genus Nitrospira. Nitrite oxidation kinetics and quantitative FISH analyses revealed that the specific nitrite oxidation activities of Nitrobacter and Nitrospira are 93.8 and 10.5 mg/g NOB h, respectively, and the specific activity of Nitrobacter is about 9 times higher than that of Nitrospira. In conclusion, the results confirm the K/r hypothesis and the distribution of Nitrobacter and Nitrospira is likely to depend mainly on nitrite concentration. It seems that nitrite load and starvation conditions do not give a direct effect on the distribution of NOB.

Bacteria↗
PubMed1998

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.

Journal Article↗
PubMed1999

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.

Antibodies, Bacterial↗
PubMed2001

Factors affecting nitrite build-up in nitrifying biofilm reactor.

Nitrite build-up in a nitrifying biofilm reactor for direct denitrification from the nitrite stage was investigated. At least three factors were found to influence the nitrite build-up: (i) the relative specific growth rates of Nitrosomonas to Nitrobacter, microNs/microNb in the biofilm; (ii) the relative initial ratio between Nitrosomonas and Nitrobacter on the support surface, (Mao)Ns/(Mao)Nb; (iii) the level of free ammonia, particularly at greater than 0.1 mg N/l that can be inhibitory to Nitrobacter. Results showed that without free ammonia inhibition, the first two factors determined the degree of nitrite build-up. The effect of (Mao)Ns and (Mao)Nb on nitrification kinetics were more significant than the specific growth rates of Nitrosomonas and Nitrobacter. By regulating effects of these three factors, extremely high concentrations of nitrite build-up could occur in a biofilm reactor. This offers a potential for achieving direct denitrification through a nitrate shunt.

Ammonia↗
PubMed1968

Properties of some reductase enzymes in the nitrifying bacteria and their relationship to the oxidase systems.

The reductase enzymes in Nitrosomonas and Nitrobacter were studied under anaerobic conditions when the oxidase enzymes were inactive. The most effective electron-donor systems for nitrate reductase in Nitrobacter were reduced benzyl viologen alone, phenazine methosulphate with either NADH or NADPH, and FMN or FAD with NADH. Nitrite and hydroxylamine reductases were found in both nitrifying bacteria, and optimum activity for each enzyme was obtained with NADH or NADPH with either FMN or FAD. The product of both these enzymes was identified as ammonia. In extracts of Nitrosomonas the ammonia was further utilized by an NADPH-specific glutamate dehydrogenase. (15)N-labelled nitrite, hydroxylamine and ammonia were rapidly incorporated into cell protein by Nitrosomonas, and Nitrobacter in addition incorporated [(15)N]nitrate. Relatively gentle methods of cell disruption were compared with ultrasonic treatment, to enable a more exact study to be undertaken of the intracellular distribution of the oxidase and reductase enzymes. The functional relationship of these opposing enzyme systems in the nitrifying bacteria is considered.

Ammonia↗
PubMed2000

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.

Ammonia↗
PubMed2005

Influence of inorganic nitrogen management regime on the diversity of nitrite-oxidizing bacteria in agricultural grassland soils.

To assess links between the diversity of nitrite-oxidizing bacteria (NOB) in agricultural grassland soils and inorganic N fertilizer management, NOB communities in fertilized and unfertilized soils were characterized by analysis of clone libraries and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Previously uncharacterized Nitrospira-like sequences were isolated from both long-term-fertilized and unfertilized soils, but DGGE migration patterns indicated the presence of additional sequence types in the fertilized soils. Detailed phylogenetic analysis of Nitrospira-like sequences suggests the existence of one newly described evolutionary group and of subclusters within previously described sublineages, potentially representing different ecotypes; the new group may represent a lineage of noncharacterized Nitrospira species. Clone libraries of Nitrobacter-like sequences generated from soils under different long-term N management regimes were dominated by sequences with high similarity to the rhizoplane isolate Nitrobacter sp. strain PJN1. However, the diversity of Nitrobacter communities did not differ significantly between the two soil types. This is the first cultivation-independent study of nitrite-oxidizing bacteria in soil demonstrating that nitrogen management practices influence the diversity of this bacterial functional group.

Bacteria↗
PubMed2001

Energy model and metabolic flux analysis for autotrophic nitrifiers.

The behavior of pure cultures of nitrifying microorganisms under autotrophic growth operating conditions was investigated and the relations between their energy metabolism and their anabolism analyzed by means of metabolic network computation. The description of the metabolism of the nitrifiers is extended to their energy metabolism by introducing compartmentalization (cytoplasmic and periplasmic sides) and studying coupling between the electron transport chain and the proton gradient generation. The energy model of Nitrosomonas and Nitrobacter was developed based on the oxidoreduction reactions known to be involved. The electron transport chains and the associated proton translocation for these models are described. Several possible hypotheses are analyzed and discussed concerning the thermodynamic consistency of all the oxidoreduction reactions. For Nitrosomonas, the most delicate point is the second step of hydroxylamine oxidation. For Nitrobacter a new energy model is proposed in which NO plays an important role as node in the distribution of electrons from NO(2)(-) oxidation to the membrane electron transport chain. The compartmentalization enables us to consider a proton gradient dissipation flux as the expression of the overall energy loss in metabolic analysis (the so-called maintenance phenomena). The energy model (electron transport chain, proton gradient) is associated with an overall description of the metabolism of Nitrosomonas and Nitrobacter in terms of metabolic flux calculation. This representation demonstrates that a maintenance in nitrifiers expressed as a proton leak is no higher than for other aerobes. The yields calculated from the energy models integrated with the metabolic models of nitrifiers are consistent with the experimental yields in the literature.

Ammonia↗
PubMed2002

Use of natural zeolite to enhance nitrification in biofilter.

To enhance nitrification, natural zeolite and activated carbon were tested as a media in a biofilter to treat wastewater containing relatively high concentrations of TKN. Using those media, the adsorption isotherms for ammonium ion were compared; the K values for natural zeolite and activated carbon were found to be 0.5117 and 0.0006, respectively. In comparison of the performance of the two media, two identical, lab-scale biofilters were then operated for 4 months. The effect of NH3-N loading rates on the performance was investigated. The results showed that higher NH3-N removal efficiency and faster nitrification were achieved in the biofilter with natural zeolite throughout the experimental period. Nitrosomonas and Nitrobacter, two principal nitrifiers, in biofilm grown on two different media were counted and compared. Nitrobacter which is the more fragile of the two principal nitrifiers was outgrown in the biofilm on natural zeolite media. The reason for this may be due to the ammonium ion exchanging capacity of natural zeolite which provided the favorable environment for Nitrobacter.

Biodegradation, Environmental↗
PubMedDate not supplied

Unusual lipid A types in phototrophic bacteria and related species.

Photosynthetic bacteria of the Rhodospirillaceae family (sulfur-free purple bacteria) possess lipopolysaccharides (LPS) that deviate markedly from the Salmonella lipopolysaccharides in the chemical makeup of the lipid A component and in their biologic properties. LPS of Rhodopseudomonas gelatinosa is highly toxic and pyrogenic, while that of Rhodospirillum tenue shows cryptic toxicity. Two LPS types are completely non-toxic. The Rhodopseudomonas sphaeroides lipid A has the same backbone as that of Salmonella, but a part of the amide-linked fatty acids has the unusual 3-oxo structure (3-oxo-14:0). The lipid A's of Rhodopseudomonas viridis and Rhodopseudomonas palustris have 2,3-diamino-2,3-dideoxy-D-glucose as the backbone sugar. This is the first demonstration of this sugar in nature. In recent studies using 16S rRNA sequencing, the nonphotosynthetic Nitrobacter strains were shown to be phylogenetically closely related to R. palustris. The R. palustris lipid A type has been identified in three Nitrobacter species, including Nitrobacter winogradskyi, the type strain. The data demonstrate the taxonomic significance of lipid A constituents and structures.

Bacteria↗
PubMed2006

Impact of prehybridization PCR amplification on microarray detection of nitrifying bacteria in wastewater treatment plant samples.

A gel-based microarray that included a set of 26 oligonucleotide probes targeting all nitrifying bacteria at varying levels of specificity suggested the presence of targeted microorganisms when hybridized to RNA isolated from a wastewater treatment plant, but could not discriminate between perfectly matched and mismatched sequences due in part to low signal intensity. To enhance sensitivity and improve discrimination, polymerase chain reaction was used to selectively amplify the 16S rRNA genes of specific nitrifier groups. RNA transcribed from these DNA templates was hybridized to the microarray and thermal dissociation analysis was used to characterize the specificity of hybridization. Amplification with Nitrospira-specific primers resulted in the selective amplification of this target group, confirmed by both a significant increase in signal intensity and a melting profile identical to the reference RNA. In contrast, Nitrobacter was not detected in the environmental samples with probe Nbac1000 despite pre-amplification with Nitrobacter-specific primers, indicating the absence of strains containing this Nitrobacter-specific sequence. Pre-amplification using primers specific for beta-Proteobacterial ammonia-oxidizing bacteria resulted in a significant increase in signal intensity for probe Nso190, but melting profiles for probe Nso190 showed a slight deviation between amplified RNA and the reference microorganism, suggesting that the amplification products contained some sequences that varied by a single nucleotide difference in the probe target region.

Bacteria↗