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PubMed1983

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 . h) 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.

Journal Article↗

PubMed1989

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.

Journal Article↗

PubMed1996

Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria.

Three nucleic acid probes, two for autotrophic ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria and one for alpha subdivision nitrite-oxidizing bacteria, were developed and used to study nitrifying bacterial phylotypes associated with various freshwater and seawater aquarium biofilters. Nitrosomonas europaea and related species were detected in all nitrifying seawater systems and accounted for as much as 20% of the total eubacterial rRNA. In contrast, nitrifying bacteria belonging to the beta-proteobacterial subdivision were detected in only two samples from freshwater aquaria showing vigorous nitrification rates. rRNA originating from nitrite-oxidizing alpha subdivision proteobacteria was not detected in samples from either aquarium environment. The data obtained indicate that chemolithotrophic ammonia oxidation in the freshwater aquaria was not due to beta-proteobacterial phylotypes related to members of the genus Nitrosomonas and their close relatives, the organisms usually implicated in freshwater nitrification. It is likely that nitrification in natural environments is even more complex than nitrification in these simple systems and is less well characterized with regard to the microorganisms responsible.

Ammonia↗

PubMed1999

Nitrogen cycling and community structure of proteobacterial beta-subgroup ammonia-oxidizing bacteria within polluted marine fish farm sediments.

A multidisciplinary approach was used to study the effects of pollution from a marine fish farm on nitrification rates and on the community structure of ammonia-oxidizing bacteria in the underlying sediment. Organic content, ammonium concentrations, nitrification rates, and ammonia oxidizer most-probable-number counts were determined in samples of sediment collected from beneath a fish cage and on a transect at 20 and 40 m from the cage. The data suggest that nitrogen cycling was significantly disrupted directly beneath the fish cage, with inhibition of nitrification and denitrification. Although visual examination indicated some slight changes in sediment appearance at 20 m, all other measurements were similar to those obtained at 40 m, where the sediment was considered pristine. The community structures of proteobacterial beta-subgroup ammonia-oxidizing bacteria at the sampling sites were compared by PCR amplification of 16S ribosomal DNA (rDNA), using primers which target this group. PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) and with oligonucleotide hybridization probes specific for different ammonia oxidizers. A DGGE doublet observed in PCR products from the highly polluted fish cage sediment sample was present at a lower intensity in the 20-m sample but was absent from the pristine 40-m sample station. Band migration, hybridization, and sequencing demonstrated that the doublet corresponded to a marine Nitrosomonas group which was originally observed in 16S rDNA clone libraries prepared from the same sediment samples but with different PCR primers. Our data suggest that this novel Nitrosomonas subgroup was selected for within polluted fish farm sediments and that the relative abundance of this group was influenced by the extent of pollution.

Ammonia↗

PubMed2001

Dissimilatory nitrite reductase genes from autotrophic ammonia-oxidizing bacteria.

The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of beta-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria.

Amino Acid Sequence↗

PubMed2004

Denaturing gradient gel electrophoretic analysis of ammonia-oxidizing bacterial community structure in the lower Seine River: impact of Paris wastewater effluents.

The Seine River is strongly affected by the effluents from the Acheres wastewater treatment plant (WWTP) downstream of the city of Paris. We have shown that the effluents introduce large amounts of ammonia and inoculate the receiving medium with nitrifying bacteria. The aim of the present study was to investigate the diversity of the ammonia-oxidizing bacterial population by identifying autochthonous bacteria from upstream and/or allochthonous ammonia-oxidizing bacteria from the WWTP effluents. Measurements of potential nitrifying activity, competitive PCR, and denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments specific to ammonia-oxidizing bacteria (AOB) were used to explore the succession and shifts of the ammonia-oxidizing community in the lower Seine River and to analyze the temporal and spatial functioning of the system at several different sampling dates. A major revelation was the stability of the patterns. The CTO primers used in this study (G. A. Kowalchuk, J. R. Stephen, W. D. Boer, J. I. Prosser, T. M. Embley, and J. W. Woldendorp, Appl. Environ. Microbiol. 63:1489-1497, 1997) were shown not to be completely specific to AOB of the beta subclass of Proteobacteria. We further demonstrated that when DGGE patterns are interpreted, all the different bands must be sequenced, as one major DGGE band proved to be affiliated with a group of non-AOB in the beta subclass of Proteobacteria. The majority of AOB (75 to 90%) present in the lower Seine river downstream of the effluent output belong to lineage 6a, represented by Nitrosomonas oligotropha- and Nitrosomonas ureae-like bacteria. This dominant lineage was represented by three bands on the DGGE gel. The major lineage-6a AOB species, introduced by the WWTP effluents, survived and might have grown in the receiving medium far downstream, in the estuary; it represented about 40% of the whole AOB population. The other two species belonging to lineage 6a seem to be autochthonous bacteria. One of them developed a few kilometers downstream of the WWTP effluent input in an ammonia-enriched environment, and the other appeared in the freshwater part of the estuary and was apparently more adapted to estuarine conditions, i.e., an increase in the amount of suspended matter, a low ammonia concentration, and high turnover of organic matter. The rest of the AOB population was represented in equal proportions by Nitrosospira- and Nitrosococcus mobilis-like species.

Ammonia↗

PubMed1985

Nitrite and nitrous oxide production by Methylosinus trichosporium.

Conditions for the production of nitrite and nitrous oxide by an obligate methanotroph, Methylosinus trichosporium (OB 3b), were studied. The rate of nitrite production (V NO2-) was correlated with the concentration of ammonia up to 20 mM in the presence of sufficient amounts of oxygen and inversely correlated with the amounts of methane in the system. The rate of nitrous oxide (N2O) production (V N2O) was correlated positively with V NO2- and the amount of nitrite produced and inversely with the oxygen concentration in the system. Nitrite started to disappear when either oxygen or methane or both were depleted, but only a part of the loss could be accounted for by an increase in N2O. Maximum rates of nitrite and N2O production by Ms. trichosporium were 6.9 X 10(-16) and 2.2 X 10(-17) mol . cell-1 X day-1, respectively. These values are about 0.2 and 1.6% of the values for Nitrosomonas europaea. Therefore, production of nitrite and N2O by methanotrophs in aquatic environments may not be as significant as that of Nitrosomonas.

Ammonia↗

PubMed2001

Purification and comparison of triosephosphate isomerases from ammonia-oxidizing bacteria isolated from terrestrial and marine environments.

Triosephosphate isomerases [TIMs, EC 5.3.1.1] were purified from two ammonia-oxidizing bacteria: Nitrosomonas sp. K1 (K1), Nitrosomonas sp. TNO632 (TNO). The molecular masses of the native enzymes were estimated to be about 53.6 (K1-TIM) and 51.9 kDa (TNO-TIM) by gel filtration, whereas SDS-PAGE produced one band for each enzyme with M(r) values of 27.1 (K1-TIM) and 26.4 kDa (TNO-TIM), respectively, suggesting that the enzymes consist of identical subunits. The apparent K(m) for d-glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) were about 1.19 and 4.78 mM (K1-TIM), and 0.41 and 6.01 mM (TNO-TIM), respectively. The two TIMs had different pH-activity curves with an optimum pH range of 6.5 (K1-TIM) and 8.0 (TNO-TIM). The temperature optima of K1-TIM and TNO-TIM were 50-60 and 60-65 degrees C, respectively. Both enzymes were strongly inhibited by 5,5'-ditiobis at 1.0 mM. The N-terminal amino acid sequences of K1-TIM and TNO-TIM were MRAGFVAGNWKMHG (K1-TIM) and MVRTGLVAGNWKMNG (TNO-TIM). A homology of 74.1% was observed between K1-TIM and TNO-TIM.

Journal Article↗

PubMed2006

Quantitative structure-activity relationships for toxicity of nonpolar narcotic chemicals to Pseudokirchneriella subcapitata.

This study presents data for 27 nonpolar narcotic compounds regarding toxicity to Pseudokirchneriella subcapitata as evaluated using a closed-system algal toxicity test with an exposure time of 48 h. Two test endpoints, dissolved oxygen production and algal growth rate, were used to assess the toxicity of nonpolar narcotic chemicals on algae. Hydrophobicity (1-octanol-water partition coefficient [K(OW)]) provided satisfactory descriptions for the toxicity of nonpolar narcotic compounds, and quantitative structure-activity relationships based on log K(OW) were established. The relative sensitivity of various aquatic organisms to nonpolar chemicals was as follows: P. subcapitata > Vibriofischeri > or = Nitrosomonas sp. > fathead minnow > Daphnia magna > polytox > activated sludge. In addition, linear relationships were found between the toxicity observed in P. subcapitata and other aquatic organisms, except in the case of Nitrosomonas sp. Therefore, for nonpolar toxicants, the closed-system technique applied in the present study can be an ideal surrogate for other tests, such as fathead minnow and D. magna, that are either time-consuming or labor-intensive. However, because the current toxicity database is based primarily on the conventional batch tests, it cannot provide adequate assessment regarding the effects of various organic toxicants. Therefore, more extensive research is needed to revise the database for the toxicity of organic compounds on phytoplankton using the closed-system technique.

Alcohols↗

PubMed2003

A comparitive study of ammonia-oxidizing bacteria in lab-scale industrial wastewater treatment reactors.

The diversity and community structure of the beta-proteobacterial ammonia oxidising bacteria (AOB) in a range of different lab-scale industrial wastewater treatment reactors were compared. Three of the reactors treat waste from mixed domestic and industrial sources whereas the other reactor treats waste solely of industrial origin. PCR with AOB selective primers was combined with denaturing gradient ge electrophoresis to allow comparative analysis of the dominant AOB populations and the phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing or direct sequencing of bands excised from DGGE gels. Different AOB were found within and between different reactors. All AOB sequences identified were grouped within the genus Nitrosomonas. Within the lab-scale reactors there appeared to be selection for a low diversity of AOB and predominance of a single AOB population. Furthermore, the industrial input in both effluents apparently selected for salt tolerant AOB, most closely related to Nitrosococcus mobilis and Nitrosomonas halophila.

Ammonia↗

PubMed2006

Low-temperature EPR and Mössbauer spectroscopy of two cytochromes with His-Met axial coordination exhibiting HALS signals.

C-type cytochromes with histidine-methionine (His-Met) iron coordination play important roles in electron-transfer reactions and in enzymes. Low-temperature electron paramagnetic resonance (EPR) spectra of low-spin ferric cytochromes c can be divided into two groups, depending on the spread of g values: the normal rhombic ones with small g anisotropy and g(max) below 3.2, and those featuring large g anisotropy with g(max) between 3.3 and 3.8, also denoted as highly axial low spin (HALS) species. Herein we present the detailed magnetic properties of cytochrome c(553) from Bacillus pasteurii (g(max) 3.36) and cytochrome c(552) from Nitrosomonas europaea (g(max) 3.34) over the pH range 6.2 to 8.2. Besides being structurally very similar, cytochrome c(553) shows the presence of a minor rhombic species at pH 6.2 (6 %), whereas cytochrome c(552) has about 25 % rhombic species over pH 7.5. The detailed Mössbauer analysis of cytochrome c(552) confirms the presence of these two low-spin ferric species (HALS and rhombic) together with an 8 % ferrous form with parameters comparable to the horse cytochrome c. Both EPR and Mössbauer data of axial cytochromes c with His-Met iron coordination are consistent with an electronic (d(xy))(2) (d(xz))(2) (d(yz))(1) ground state, which is typical for Type I model hemes.

Anisotropy↗

PubMedDate not supplied

The use of a hollow fiber membrane module in sample conditioning prior to electrophoresis.

A new method to continuously change the buffer conditions in samples prior to electrophoresis by counter-current dialysis has been developed using a hollow fiber membrane module (VariPerm M, bitop, Witten). It is demonstrated that the collected fractions of a column eluate after hydrophobic interaction chromatography cannot be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) due to the high content of ammonium sulfate. In contrast, when the hollow fiber membrane module is used for on-line desalting of the eluate during chromatography, it is possible to detect distinct bands on SDS-gels even after sample concentration by evaporation. Moreover, adding polyethyleneglycol 20,000 to the counter-current dialysis buffer the hollow fiber membrane module is suitable for combined on-line sample desalting and sample concentration of living organisms during free flow electrophoresis (FFE): a culture of Nitrosomonas eutropha which could not significantly be detected by FFE without conditioning was successfully analyzed when concentrated and desalted on-line during sample application.

Ammonium Sulfate↗

PubMed1982

Incorporation of organic compounds into cell protein by lithotrophic, ammonia-oxidizing bacteria.

Incorporation of organic compounds into cell protein by the obligate chemolithotrophs Nitrosomonas spec., Nitrosococcus oceanus, Nitrosococcus mobilis, Nitrosovibrio tenuis, Nitrosolobus spec., and Nitrosospira spec. was studied. In the presence of ammonia as energy source organic substrates were supplied. Distribution of 14C into cell amino acids arising from 14C-labelled glucose, Na-pyruvate, and Na-acetate was investigated. While carbon from glucose was distributed unrestricted, carbon from pyruvate preferably entered into the amino acids of the pyruvate and glutamate family and from acetate mainly into leucine and the glutamate family. Among the strains examined, slight differences were observed, but all should be included under group A of the scheme of Smith and Hoare (1977).

Acetates↗

PubMed2001

Isolation and properties of obligately chemolithoautotrophic and extremely alkali-tolerant ammonia-oxidizing bacteria from Mongolian soda lakes.

Five mixed samples prepared from the surface sediments of 20 north-east Mongolian soda lakes with total salt contents from 5 to 360 g/l and pH values from 9.7 to 10.5 were used to enrich for alkaliphilic ammonia-oxidizing bacteria. Successful enrichments at pH 10 were achieved on carbonate mineral medium containing 0.6 M total Na(+) and < or =4 mM NH(4)Cl. Five isolates (ANs1-ANs5) of ammonia-oxidizing bacteria capable of growth at pH 10 were obtained from the colonies developed on bilayered gradient plates. The cells were motile and coccoid, with well-developed intracytoplasmic membranes (ICPM) and carboxysomes. At pH 10.0, ammonia was toxic for growth at concentrations higher than 5 mM NH(4)Cl. The bacteria were able to grow within the salinity range of 0.1-1.0 M of total Na+ (optimum 0.3 M). In media containing 0.3-0.6 M total Na(+), optimal growth in batch cultures occurred in the presence of a bicarbonate/carbonate buffer system within the pH range 8.5-9.5, with the highest pH limit at pH 10.5. At pH lower than 8.0, growth was slower, most probably due to decreasing free ammonia. The pH profile of the respiratory activity was broader, with limits at 6.5-7.0 and 11.0 and an optimum at 9.5-10.0. In pH-controlled, NH(3)-limited continuous culture, isolate ANs5 grew up to pH 11.3, which is the highest pH limit known for ammonia-oxidizing bacteria so far. This showed the existence of extremely alkali-tolerant ammonia-oxidizing bacteria in the soda lakes. Comparative 16S rDNA sequence analysis of the five isolates demonstrated that they possess identical 16S rDNA genes and that they are closely related to Nitrosomonas halophila (sequence similarity 99.3%), a member of the beta-subclass of the Proteobacteria. This affiliation was confirmed by comparative sequence analysis of the amoA gene, encoding the active-site subunit of the ammonia-monoxygenase, of one of the isolates. DNA-DNA hybridization data further supported that the soda lake isolates are very similar to each other and represent an alkali-tolerant subpopulation of N. halophila whose species description is herewith amended.

Ammonia↗

PubMed2002

Biodegradation of monohalogenated alkanes by soil NH(3)-oxidizing bacteria.

Although cooxidative biodegradation of monohalogenated hydrocarbons has been well studied in the model NH(3)-oxidizing bacterium, Nitrosomonas europaea, virtually no information exists about cooxidation of these compounds by native populations of NH(3)-oxidizing bacteria. To address this subject, nitrifying activity was stimulated to 125-400 nmol NO(3)(-) produced g(-1) soil h(-1) by first incubating a Ca(OH)(2)-amended, silt loam soil (pH 7.0+/-0.2) at field capacity (270 g H(2)O kg(-1) soil) with 10 micro mol NH(4)(+) g(-1) soil for 14 days, followed by another 10 days of incubation in a shaken slurry (2:1 water:soil, v/w) with periodic pH adjustment and maintenance of 10 mM NH(4)(+). These slurries actively degraded both methyl bromide (MeBr) and ethyl chloride (EtCl) at maximum rates of 20-30 nmol ml(-1) h(-1) that could be sustained for approximately 12 h. Although the MeBr degradation rates were linear for the first 10-12 h of incubation, they could not be sustained regardless of NH(4)(+) level and declined to zero over 20 h of incubation. The transformation capacity of the slurry enrichments (~1 micro mol MeBr ml(-1) soil slurry) was similar to the value measured previously in cell suspensions of N. europaea with similar NH(3)-oxidizing activity. Several MeBr-degrading characteristics of the nitrifying enrichments were found to be similar to those documented in the literature for MeBr-degrading methanotrophs and facultatively methylotrophic bacteria.

Alkanes↗

PubMed2004

Removal of carbonaceous and nitrogenous pollutants from a synthetic wastewater using a membrane-coupled bioreactor.

Two modified Ludzack-Ettinger (MLE)-type membrane-coupled bioreactors (MBRs) were investigated in this study for the purpose of removing both nitrogenous and carbonaceous pollutants from a synthetic wastewater. During the first MBR experiment, removal efficiencies were high (>90%) for chemical oxygen demand (COD) and ammonia, but total nitrogenous pollutant removal efficiency was poor (approximately 25%). Bacterial community analysis of ammonia oxidizing bacteria (AOB) by a nested PCR-DGGE approach detected two Nitrosomonas-like populations and one Nitrosospira-like population. During the initial portion of the second MBR experiment, COD and ammonia removal efficiencies were similar to the first MBR experiment until the COD of the influent wastewater was increased to provide additional electron donors to support denitrification. Total nitrogen removal efficiencies eventually exceeded 90%, with a hydraulic residence time (HRT) of 24 h and a recirculation ratio of 8. When the HRT of the MBR experiment was decreased to 12 h, however, ammonia removal efficiency was adversely affected. A subsequent increase in the HRT to 18 h helped improve removal efficiencies for both ammonia (>85%) and total nitrogenous compounds (approximately 70%). Our research demonstrates that MBRs can be effectively designed to remove both carbonaceous and nitrogenous pollutants. The ability of the microbial community to switch between anoxic (denitrifying) and oxic (nitrifying) conditions, however, represents a critical process constraint for the application of MLE-type MBR systems, such that little benefit is gained compared to conventional designs.

Aerobiosis↗

PubMed2006

Characterization and performance of constructed nitrifying biofilms during nitrogen bioremediation of a wastewater effluent.

Constructed ammonium oxidizing biofilms (CAOB) and constructed nitrite oxidizing biofilms (CNOB) were characterized during the bioremediation of a wastewater effluent. The maximum ammonium removal rate and removal efficiency in CAOB was 322 mg N-NH4+ m(-3) d(-1) and 96%, respectively, while in CNOB a maximum removal rate of 255 mg N-NH4+ m(-3) d(-1) and a removal efficiency of 76% was achieved. Both constructed biofilms on low-density polyester Dacron support achieved removal efficiencies higher than that of the concentrations normally present in reactors without constructed biofilms (P < 0.05). Nitrifying bacteria from the constructed biofilms cultures were typed by sequencing 16S rRNA genes that had been amplified by PCR from genomic DNA. Analysis of enrichment biofilms has therefore provided evidence of high removal of ammonium and the presence of Nitrosomonas eutropha, N. halophila and N. europaea in CAOB, while in CNOB Nitrobacter hamburgensis, N. winogradskyi and N. alkalicus were identified according to 16S rRNA gene sequences comparison. The biofilm reactors were nitrifying over the whole experimental period (15 days), showing a definite advantage of constructed biofilms for enhancing a high biomass concentration as evidenced by environmental electron microscopic analysis (ESEM). Our research demonstrates that low-density polyester Dacron can be effectively used for the construction of nitrifying biofilms obtaining high removal efficiencies of nitrogen in a relatively short time from municipal effluents from wastewater treatment plants. CAOB and CNOB are potentially promissory for the treatment of industrial wastewaters that otherwise requires very large and expensive reactors for efficient bioremediation of effluents.

Bacteria↗

PubMed1984

3,3',5,5'-tetramethylbenzidine/H2O2 staining is not specific for heme proteins separated by gel electrophoresis.

Staining of sodium dodecyl sulfate or lithium dodecyl sulfate gels with 3,3',5,5'-tetramethylbenzidine (TMBZ)/H2O2 after electrophoresis has frequently been used as a specific method of detecting heme proteins. That TMBZ is an electron donor for O2 reduction by the nonheme-soluble cytochrome oxidase/nitrite reductase from Nitrosomonas europaea is now shown; this protein is detected by the TMBZ/H2O2 method. A method for the determination of TMBZ oxidase activity is given; hence, the detection of artifactual staining due to proteins of this type is possible.

Benzidines↗