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PubMed1976

Growth of nitrobacter in the presence of organic matter. II. Chemoorganotrophic growth of Nitrobacter agilis.

1. After a resting period of up to 6 months cells of Nitrobacter agilis grow with acetate, formate, and pyruvate as carbon and energy source. Yeast extract and peptone were added to supply the organism with nitrogen and to meet possible vitamin requirements. 2. The length of the growth period depends on the substrate; it increases according to the following sequence: pyruvate, formate, acetate. The highest growth yield is observed with pyruvate, the lowest with formate. 3. O2 consumption is increased in the presence of substrates as compared to endogenous respiration. With pyruvate and acetate twice as much O2 is consumed, with formate 7 times, with yeast extractpeptone 10 times as much. 4. The ability of nitrite oxidation is largely preserved, except in cells grown with acetate or pyruvate in the presence of 0.015% yeast extract and peptone. Such cells have nearly no cytochrome a1. Accordingly. the cytochrome spectra of nitrite oxidizers grown under chemoorganotrophic and lithoautotrophic conditions coincide qualitatively. 5. The nitrite oxidizing system is inducible. It is induced by nitrite but also by substances present in yeast extract and peptone. Cells grown on acetate and yeast extract and peptone (0.015%) require 3--4 weeks before they regain the ability to grow with nitrite. Cells grown chemoorganotrophically with the same substrates and yeast extract and peptone (0.15%) start growing and nitrite as energy source without a lag. 6. Cell size and form, distribution of storage materials, order and fine structure of double membranes are correlated with growth conditions.

Acetates↗
PubMed2006

Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes.

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2.

Bioreactors↗
PubMed2006

Competition between Nitrospira spp. and Nitrobacter spp. in nitrite-oxidizing bioreactors.

In this work the question was addressed if in nitrite-oxidizing activated sludge systems the environmental competition between Nitrobacter spp. and Nitrospira spp., which only recently has been discovered to play a role in these systems, is affected by the nitrite concentrations. Two parallel chemostats were inoculated with nitrifying-activated sludge containing Nitrospira and operated under identical conditions. After addition of Nitrobacter to both chemostats, the nitrite concentration in the influent of one of the chemostats was increased such that nitrite peaks in the bulk liquid of this reactor were detected. The other chemostat served as control reactor, which always had a constant nitrite influent concentration. The relative cellular area (RCA) of Nitrospira and Nitrobacter was determined by quantitative fluorescence in situ hybridization (FISH). The nitrite perturbation stimulated the growth of Nitrobacter while in the undisturbed control chemostat Nitrospira dominated. Overall, the results of this experimental study support the hypothesis that Nitrobacter is a superior competitor when resources are abundant, while Nitrospira thrive under conditions of resource scarcity. Interestingly, the dominance of Nitrobacter over Nitrospira, caused by the elevated nitrite concentrations, could not be reverted by lowering the available nitrite concentration to the original level. One possible explanation for this result is that when Nitrobacter is present at a certain cell density it is able to inhibit the growth of Nitrospira. An alternative explanation would be that the length of the experimental period was not long enough to observe an increase of the Nitrospira population.

Bioreactors↗
PubMed1976

Growth of Nitrobacter in the presence of organic matter. I. Mixotrophic growth.

1. Culture filtrates of heterotrophic bacteria were tested for their stimulatory effect on nitrification of three strains of Nitrobacter. 2. Yeast extract-peptone solution, in which Pseudomonas fluorescens had grown, after removal of the cells was added to autotrophically growing cultures of Nitrobacter agilis; it caused a stimulated nitrite oxidation and growth of Nitrobacter agilis. 3. The degree of stimulation depended on: a) the proportion of the culture filtrate to the autotrophic medium; b) the composition of the complex medium in which Pseudomonas fluorescens had been grown; c) the time the heterotrophic bacterium had been grown in the complex medium. 4. The stimulatory effect was highest with Nitrobacter agilis, less with Nitrobacter winogradskyi and negligible with Nitrobacter K4. 5. It was possible to adapt nutrifying cells of Nitrobacter agilis to higher concentrations of yeast extract and peptone. After the nitrite had been completely oxidized the cell-N still increased up to 30% before growth stopped.

Culture Media↗
PubMed2007

Effect of free ammonia on the respiration and growth processes of an enriched Nitrobacter culture.

The inhibitory effect of free ammonia (FA;NH(3)) on the metabolism of Nitrobacter is investigated using a method that allows decoupling energy generation from growth processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of Nitrobacter. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population in the reactor was Nitrobacter, while no Nitrospira was detected. Batch tests were carried out to measure the oxygen uptake rate (OUR) by the culture at various FA levels, in the presence (OUR with CO(2)) or absence (OUR without CO(2)) of inorganic carbon (CO(2), HCO(3)(-) and CO(3)(2-)). The FA inhibition on the respiration initiated at below 1mgNH(3)-NL(-1) in both cases. OUR without CO(2) gradually decreased by 12% when the FA concentration increased from 0 to approximately 4mgNH(3)-NL(-1) and remained at the same level till an FA level of 9mgNH(3)-NL(-1) (the highest FA concentration applied in this study). This indicates that FA has a limited inhibitory effect on the respiratory capability of Nitrobacter. Starting from a level that is 15% higher than OUR without CO(2) when no FA was present, OUR with CO(2)decreased more rapidly than OUR without CO(2) reaching the same level as OUR without CO(2) when FA was between 6-9mgNH(3)-NL(-1). This implies that in this range of FA the presence of inorganic carbon did not cause any increase in the respiration activity of Nitrobacter. The results suggest that, while still oxidizing nitrite at approximately 75% of the non-inhibited rate, Nitrobacter likely ceased to grow at an FA level of above 6mgNH(3)-NL(-1). While the real mechanisms remain to be identified, this study indicates that the FA inhibition on Nitrobacter is likely much more serious than suggested by previous studies where OUR with CO(2) (or the equivalent nitrite oxidation rate) was used as the sole measure of the inhibitory effects.

Ammonia↗
PubMed2006

The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched nitrobacter culture.

The inhibitory effects of nitrite (NO2-)/free nitrous acid (HNO2-FNA) on the metabolism of Nitrobacter were investigated using a method allowing the decoupling of the growth and energy generation processes. A lab-scale sequencing batch reactor was operated forthe enrichment of a Nitrobacter culture. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population was Nitrobacter. Batch tests were carried out to assess the oxygen and nitrite consumption rates of the enriched culture at low and high nitrite levels, in the presence or absence of inorganic carbon. It was observed that in the absence of CO2, the Nitrobacter culture was able to oxidize nitrite at a rate that is 76% of that in the presence of CO2, with an oxygen consumption rate that is 85% of that measured in the presence of CO2. This enabled the impacts of nitrite/FNA on the catabolic and anabolic processes of Nitrobacter to be assessed separately. FNA rather than nitrite was likely the actual inhibitor to the Nitrobacter metabolism. It was revealed that FNA of up to 0.05 mg HNO2-N x L(-1) (3.4 microM), which was the highest FNA concentration used in this study, did not have any inhibitory effect on the catabolic processes of Nitrobacter. However, FNA initiated its inhibition to the anabolic processes of Nitrobacterat approximately 0.011 mg HNO2-N x L(-1) (0.8 microM), and completely stopped biomass synthesis at a concentration of approximately 0.023 mg HNO2-N x L(-1) (1.6 microM). The inhibitory effect could be described by an empirical inhibitory model proposed in this paper, but the underlying mechanisms remain to be revealed.

Energy Metabolism↗
PubMed1994

Molecular phylogenetic analysis of Nitrobacter spp.

The phylogeny of bacteria belonging to the genus Nitrobacter was investigated by sequencing the whole 16S rRNA gene. The average level of similarity for the three Nitrobacter strains examined was high (99.2%), and the similarity level between Nitrobacter winogradskyi and Nitrobacter sp. strain LL, which represent two different genomic species, was even higher (99.6%). When all of the Nitrobacter strains and their phylogenetic neighbors Bradyrhizobium and Rhodopseudomonas species were considered, the average similarity level was 98.1%. When complete sequences were used, Nitrobacter hamburgensis clustered with the two other Nitrobacter strains, while this was not the case when partial sequences were used. The two Rhodopseudomonas palustris strains examined exhibited a low similarity level (97.6%) and were not clustered.

Base Sequence↗
PubMed1983

Proton electrochemical gradients in washed cells of Nitrosomonas europaea and Nitrobacter agilis.

The components of the proton motive force (Deltap), namely, membrane potential (Deltapsi) and transmembrane pH gradient (DeltapH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis. In these bacteria both Deltapsi and DeltapH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Deltapsi values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [(3)H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, (14)C-benzoic and (14)C-acetyl salicylic, and the weak base [(14)C]methylamine. Nitrosomonas europaea accumulated (14)C-benzoic acid and (14)C-acetyl salicylic acid when the external pH was below 7.0 and [(14)C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [(14)C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a DeltapH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis, Deltapsi only contributed to Deltap. In Nitrosomonas europaea the total Deltap remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis, Deltap decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea, Deltap and its components (Deltapsi and DeltapH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Deltap. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on DeltapH and Deltapsi in the two nitrifying bacteria are considered.

Hydrogen-Ion Concentration↗
PubMed1995

Detection and counting of Nitrobacter populations in soil by PCR.

Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient.

Colony Count, Microbial↗
PubMed1965

MOLYBDENUM AS A MICRONUTRIENT FOR NITROBACTER.

Finstein, M. S. (University of California, Davis), and C. C. Delwiche. Molybdenum as a micronutrient for Nitrobacter. J. Bacteriol. 89:123-128. 1965.-The requirement of Nitrobacter for molybdenum was investigated by use of a medium purified by chemical means, as well as by depletion by the prior growth of this autotrophic bacterium. During 5 days of growth, added molybdenum induced an approximately 11-fold increase in both nitrite utilization and cell-mass development. In short-term studies, maximal response was obtained with concentrations as low as 10(-9)m. No other metal tested substituted for molybdenum. It is estimated that a minimum of 2,000 atoms of molybdenum is required for the synthesis of one Nitrobacter cell. Molybdenum did not directly influence the thermodynamic efficiency of Nitrobacter. The efficiency of molybdenum-deficient cultures and of cultures supplied with the micronutrient declined equally during the early stage of development, despite large differences in the amount of nitrite consumed. After the utilization of about 100 mumoles of nitrite per ml, cultures supplied with molybdenum became relatively less efficient. A procedure is described whereby large masses of Nitrobacter cells can be obtained with relative ease.

Bacteria↗
PubMed1998

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 (> or = 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.

Adaptation, Physiological↗
PubMed1975

Autoradiography and immunofluorescence combined for autecological study of single cell activity with Nitrobacter as a model system.

Specific detection of a particular bacterium by immunofluorescence was combined with estimation of its metabolic activity by autoradiography. The nitrifying bacteria Nitrobacter agilis and N. winogradskyi were used as a model system. Nitrobacter were incubated with NaH14CO3 and 14CO2 prior to study. The same preparations made for autoradiograms were stained with fluorescent antibodies specific for the Nitrobacter species. Examination by epifluorescence and transmitted dark-field microscopy revealed Nitrobacter cells with and without associated silver grains. Direct detection and simultaneous evaluation of metabolic activity of Nitrobacter was demonstrated in pure cultures, in a simple mixed culture, and in a natural soil.

Autoradiography↗
PubMed1992

Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer.

DNA sequences from the intergenic spacer (IGS) region of the ribosomal operon were amplified by the polymerase chain reaction (PCR) technique using two primers derived from 16S and 23S rRNA conserved sequences. The PCR products, cleaved by 4 base cutting restriction enzymes, were used to differentiate Nitrobacter strains. This method offered a convenient alternative to serological testing for characterization of Nitrobacter isolates and enabled a large number of strains to be genotypically characterized easily and rapidly. This method was successfully used to characterize natural populations of Nitrobacter from various soils and a lake. A diversity was demonstrated in various soils, and in a lake both in freshwater and in sediments. Strains closely related to both WL and LL were found in these ecosystems. It seems that the diversity of Nitrobacter populations was not associated with global environments but may be related to the presence of locally coexisting niches.

Base Sequence↗
PubMed2000

Application of an amperometric immunosensor for the enumeration of Nitrobacter in activated sludge.

A competitive immunosensor using a monoclonal antibody has been developed for the enumeration of Nitrobacter in activated sludge and other environmental samples. Its cross-reactivity was tested against a number of bacterial strains and isolates. All strains of the nitrite-oxidising genera Nitrobacter and Nitrococcus reacted strongly with the monoclonal antibody. The nitrite-oxidising Nitrospira moscoviensis, as well as the ammonia oxidising bacteria and the heterotrophic bacteria tested, did not show any affinity towards the antibody in the immunosensor. The numbers of Nitrobacter were analysed in sludge samples from several wastewater treatment plants in Sweden. Detectable amounts were found in all samples. This study shows the adequacy of using this immunosensor for the enumeration of Nitrobacter in natural environments.

Antibodies, Bacterial↗
PubMed2001

Development of nitrification inhibition assays using pure cultures of Nitrosomonas and Nitrobacter.

Restricted requirements for nitrogen reduction at wastewater treatment plants have increased the need for assays determining the inhibition of nitrification. In this paper, two new assays studying ammonia oxidation and nitrite oxidation, respectively, are presented. As test organisms, pure cultures of Nitrosomonas and Nitrobacter isolated from activated sludge are used. The assays are performed in test tubes where the bacteria are incubated with the compound or wastewater to be tested. The nitrification rate is measured during 4 h and compared with reference samples. The test organisms were characterised with respect to temperature, pH and cell activity. Optimum temperature was 35 degrees C for Nitrosomonas and 38 degrees C for Nitrobacter; optimum pH was 8.1 for Nitrosomonas and 7.9 for Nitrobacter. There was a linear relationship between the nitrification rate and the cell concentration in the studied interval. The cell activity decreased slightly with storage time. A significant level of inhibition was calculated to 11% for the Nitrosomonas assay, and to 9% for the Nitrobacter assay. The assays are applicable to determination of nitrification inhibition in samples of industrial waste waters or influents of treatment plants, or chemical substances likely to be found in wastewater.

Ammonia↗
PubMed1977

Phosphoribulokinase from Nitrobacter winogradskyi: activation by reduced nicotinamide adenine dinucleotide and inhibition by pyridoxal phosphate.

CO2 fixation by particle-free extracts from Nitrobacter winogradskyi increased by addition of reduced nicotinamide adenine dinucleotide (NADH). Ribulose-1,5-diphosphate, however, increased CO2 fixation, even in the absence of NADH. Phosphoribulokinase (EC 2.7.1.19) was the enzyme of Nitrobacter extracts that was activated specifically by NADH. Pyridoxal-5-phosphate inhibited both CO2 fixation and NADH-activated phosphoribulokinase from Nitrobacter. However, it did not affect phosphoribulokinase from spinach leaves. Since the spinach enzyme had also no requirement for reduced pyridine nucleotides, it appears that pyridoxal phosphate interferes only with the binding of NADH and not with the binding of ribulose-5-phosphate and adenosine-5'-triphosphate. The regulation of phosphoribulokinase activity by NADH provided Nitrobacter with an energy-dependent control mechanism of CO2 assimilation.

Carbon Dioxide↗
PubMed1977

Immunofluorescence studies of Nitrobacter populations in soils.

Certain steps of a protocol to enumerate a bacterium directly in soil by immunofluorescence were studied with respect to the enumeration of Nitrobacter in soils of diverse properties. Maximal counts of Nitrobacter were obtained by varying factors involved in the release of bacteria from the soil. Differences with respect to these factors were related to soil-colloidal properties. Enumeration protocols modified with regard to soil properties were used with strain-specific fluorescent antibodies (FA) to enumerate Nitrobacter populations (a) in soil during storage, (b) in comparison with most probable number (MPN) enumeration, and (c) occurring in a spectrum of soil samples. FA counting was rapid and precise and gave counts generally 10- to 100-fold higher than obtained by MPN. Nitrobacter cells of the two serotypes studied, designated as agilis and Winogradsky, occurred in each soil at levels of 10(4)-10(5) per gram.

Bacteriological Techniques↗