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A comparative survey of several bacterial aa3-type cytochrome c oxidases.

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show alpha and gamma absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 microM KCN, while 50% inhibition of the activity requires 100 microM KCN in the case of the N. europaea enzyme.

Azides↗

PubMed1977

A comparison between Nitrosomas europaea and Thiobacillus novellus on the basis of their oxidation systems of inorganic compounds.

Nitrosomonas europaea and Thiobacillus novellus were compared with each other on the basis of the biochemical properties of their inorganic compound-oxidizing systems. Cytochromes c of the two organisms differ considerably from each other; N. europaea cytochrome c-552 belongs to the "bacterial-type" cytochrome c, while T. nouellus cytochrome c-550 resembles eucaryolic cytochrome c. The specificity of cytochrome oxidase for cytochrome c as the electron donor is different between the two organisms; T novellus oxidase reacts rapidly with cytochromes c of the organisms which seem to be higher than the organisms whose cytochromes c react rapidly with N. europaea oxidase. On the basis of these facts, N. europaea seems to be older organism than T. novellus in terms of evolution.

Biological Evolution↗

PubMed1995

Sequence of an ammonia monooxygenase subunit A-encoding gene from Nitrosospira sp. NpAV.

One of three gene copies encoding ammonia monooxygenase subunit A (AmoA) and flanking sequences was isolated from genomic DNA of the chemolithotrophic soil bacterium Nitrosospira sp. NpAV using oligodeoxyribonucleotide primers and the polymerase chain reaction (PCR). The gene (amoA) encodes a 274-amino-acid polypeptide which has similarity to the ammonia monooxygenase acetylene-binding protein from Nitrosomonas europaea.

Amino Acid Sequence↗

PubMed1995

Generation of polymerase chain reaction-specific probes for library screening using single degenerate primers.

Degenerate oligonucleotide primers were made to peptide sequences from hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea. The primers were used singly in PCR reactions to amplify portions of the gene for HAO from genomic DNA. Southern hybridizations using fragments amplified with each primer showed that they labeled the same genomic DNA fragments. The PCR-amplified fragments were successfully used to screen a gene library for clones containing the HAO gene. The method of isolating genes by PCR with single primers has general utility.

Base Sequence↗

PubMed2006

Heme crevice disorder after sixth ligand displacement in the cytochrome c-551 family.

1H NMR and visible absorption spectroscopy were used to monitor sixth ligand methionine displacement reactions in four members of the ferricytochrome c-551 family from Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas stutzeri substrain ZoBell, and Nitrosomonas europae. Potassium cyanide displaces the methionine ligand with very modest changes in the visible spectra, but profound changes in the NMR spectra. The initial product formed kinetically, designated complex I, changes with time and/or heating to a more thermodynamically favored product termed complex II. Spectra indicate that both I and II are actually a family of closely related conformational isomers. Low temperature NMR spectra of complex II indicate that some of the isomers are in chemical exchange on the NMR time scale. High pH also displaces the methionine ligand in a manner similar to the well-known alkaline transition of mitochondrial cytochrome c. However, the reaction occurs at higher pH values and over a narrower pH range for the c-551 family, and the transition pH range is different for the different proteins studied. The final alkaline forms also show peak widths and a number of peaks indicative of multiple conformational isomers.

Bacterial Proteins↗

PubMed2005

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH.

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3)d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L(-1). Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosomonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662.

Ammonia↗

PubMed2005

Simulation modeling for nitrogen removal and experimental estimation of mass fractions of microbial groups in single-sludge system.

Nitrification-denitrification in a single-sludge nitrogen removal system (SSNRS; with a sufficient carbon source for denitrification) was performed. With an increase in the mixed liquor recycle ratio (R(m)) from 1 to 2, the total nitrogen (TN) removal efficiency at a lower volumetric loading rate (VLR=0.21 NH(4)(+)-N m(-3) d(-1)) increased, but the TN removal efficiency at a higher VLR (0.35 kg NH(4)(+)-N m(-3) d(-1)) decreased. A kinetic model that accounts for the mass fractions of Nitrosomonas, Nitrobacter, nitrate reducer and nitrite reducer (f(n1), f(n2), f(dn1), and f(dn2)) in the SSNRS and an experimental approach for the estimation of the mass fractions of nitrogen-related microbial groups are also proposed. The estimated f(dn1) plus f(dn2) (0.65-0.83) was significantly larger than the f(n1) plus f(n2) (0.28-0.32); the f(n1) (0.21-0.26) was larger than the f(n2) (0.05-0.07); and the f(dn1) (0.32-0.45) varied slightly with the f(dn2) (0.33-0.38). At the lower VLR, the f(dn1) plus f(dn2) increased with increasing R(m); however at the higher VLR, the f(dn1) plus f(dn2) did not increase with increasing R(m). By using the kinetic model, the calculated residual NH(4)(+)-N and NO(2)(-)-N in the anoxic reactor and NO(2)(-)-N and NO(3)(-)-N in the aerobic reactor were in fairly good agreement with the experimental data; the calculated NO(3)(-)-N in the anoxic reactor was over-estimated and the calculated NH(4)(+)-N in the aerobic reactor was under-estimated.

Aerobiosis↗

PubMed2005

Moderately thermophilic nitrifying bacteria from a hot spring of the Baikal rift zone.

Samples from three hot springs (Alla, Seya and Garga) located in the northeastern part of Baikal rift zone (Buryat Republic, Russia) were screened for the presence of thermophilic nitrifying bacteria. Enrichment cultures were obtained solely from the Garga spring characterized by slightly alkaline water (pH 7.9) and an outlet temperature of 75 degrees C. The enrichment cultures of the ammonia- and nitrite oxidizers grew at temperature ranges of 27-55 and 40-60 degrees C, respectively. The temperature optimum was approximately 50 degrees C for both groups and thus they can be designated as moderate thermophiles. Ammonia oxidizers were identified with classical and immunological techniques. Representatives of the genus Nitrosomonas and Nitrosospira-like bacteria with characteristic vibroid morphology were detected. The latter were characterized by an enlarged periplasmic space, which has not been previously observed in ammonia oxidizers. Electron microscopy, denaturing gradient gel electrophoresis analyses and partial 16S rRNA gene sequencing provided evidence that the nitrite oxidizers were members of the genus Nitrospira.

Ammonia↗

PubMed2005

A new soluble 10kDa monoheme cytochrome c-552 from the anammox bacterium Candidatus "Kuenenia stuttgartiensis".

The chemolithoautotrophic anammox bacterium Candidatus "Kuenenia stuttgartiensis" grows anaerobically using ammonium as electron donor for nitrite reduction. More than 10% of the proteins in cell extracts of "K. stuttgartiensis" consist of c-type heme proteins. A 10kDa soluble cytochrome c was purified from cell extracts using ultracentrifugation and anion exchange chromatography. The UV/Vis spectrum of the reduced cytochrome showed the gamma, beta and alpha absorption maxima at 419, 522 and 552nm, respectively. The N-terminal amino acid sequence and peptide fragments of the tryptic digest of the protein were used to identify the corresponding gene. Analysis of the gene product showed that the protein was preceded by a 30 amino acids long leader sequence and that it belonged to the low-spin class ID cytochrome c. The CXXCH motive was located at the N-terminal site of the protein. The gene organization of the cytochrome showed some resemblance to cytochrome c clusters of unknown function in the genome of Nitrosomonas europaea and Geobacter sulfurreducens PCA.

Amino Acid Sequence↗

PubMed2005

DNA microarray detection of nitrifying bacterial 16S rRNA in wastewater treatment plant samples.

A small scale DNA microarray containing a set of oligonucleotide probes targeting the 16S rRNAs of several groups of nitrifying bacteria was developed for the monitoring of wastewater treatment plant samples. The microarray was tested using reference rRNAs from pure cultures of nitrifying bacteria. Characterization of samples collected from an industrial wastewater treatment facility demonstrated that nitrifying bacteria could be detected directly by microarray hybridization without the need for PCR amplification. Specifically, the microarray detected Nitrosomonas spp. but did not detect Nitrobacter. The specificity and sensitivity of direct detection was evaluated using on-chip dissociation analysis, and by two independent analyses--an established membrane hybridization format and terminal restriction fragment length polymorphism fingerprinting (T-RFLP). The latter two analyses also revealed Nitrospira and Nitrobacter to be contributing populations in the treatment plant samples. The application of DNA microarrays to wastewater treatment systems, which has been demonstrated in the current work, should offer improved monitoring capabilities and process control for treatment systems, which are susceptible to periodic failures.

DNA Fingerprinting↗

PubMed2006

Cometabolism of trihalomethanes by mixed culture nitrifiers.

Three mixed-culture nitrifier sources degraded low concentrations (25-450 microg/L) of four trihalomethanes (THMs) (trichloromethane (TCM) or chloroform, bromodichloromethane (BDCM), dibromochloromethane (DBCM), tribromomethane (TBM) or bromoform) commonly found in treated drinking water. Individual THM rate constants (k1THM) increased with increasing THM bromine-substitution with TBM>DBCM>BDCM>TCM and were comparable to previous studies with the pure culture nitrifier, Nitrosomonas europaea. A decrease in temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. The significant effect of temperature indicates that seasonal variations in water temperature should be a consideration for technology implementation. Product toxicity, measured by transformation capacity (T(c)), was similar to that observed with N. europaea. Because both rate constants and product toxicities increase with increasing THM bromine-substitution, a water's THM speciation is an important consideration for process implementation during drinking water treatment. Even though a given water is kinetically favored, the resulting THM product toxicity may not allow stable treatment process performance.

Ammonia↗

PubMed2002

Distribution and activity of ammonia oxidizing bacteria in a large full-scale trickling filter.

The biofilm in a full-scale nitrifying trickling filter (NTF) treating municipal wastewater has been investigated with microbiological methods using fluorescence in situ hybridization (FISH) with 16S rRNA oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM) and mathematical modeling using a dynamic multi-species biofilm reactor model. Ammonia oxidizing bacteria (AOB) were found to belong to the genus Nitrosomonas at different depths in the NTF at every sampling occasion, corresponding to different long-term operational conditions for the NTF. Both the measurements and the corresponding simulated predictions showed the same general trend of a decrease with filter depth of the amount of biofilm, the proportion of AOB to all bacteria and the total amount of AOB. The latter decreased by several times from top to bottom of the NTF. Measurements and simulations of potential ammonium oxidizing activity in the biofilm also showed a decreasing activity with depth in the NTF, which generally was operating at close to complete nitrification. However, no difference was observed when the activity was normalized to the amount of biofilm, despite decreasing proportions of AOB to all bacteria with depth in the NTF. This could be explained by diffusion limitations in the biofilm from the upper parts of the NTF according to the biofilm reactor model. The relatively good agreement between the simulations and the measurements shows that the kind of biofilm reactor model used can qualitatively describe an averaged behavior and averaged composition of the biofilm in the reactor.

Ammonia↗

PubMed2003

Nitrification efficiency and nitrifying bacteria abundance in combined AS-RBC and A2O systems.

This study makes a comparison between the nitrification performance of TNCU-I (a combined activated sludge-rotating biological contactor process) and A2O systems by the use of a pilot plant and batch experiments. The nitrifier abundance in both systems was determined, using cloning-denaturing gradient gel electrophoresis (DGGE) and fluorescent in-situ hybridization (FISH), to investigate the role of rotating biological contactor in the TNCU-I process. The stability of the nitrification performance and the specific nitrification rate were found to be greater in TNCU-I system than in the A2O system. RBC biofilm promoted nitrifying activity that contributed to the nitrification performance, especially at a low SRT. By using the cloning-DGGE method, the genera Nitrosospira and Nitrospira were found to be present in all the samples, while the genus Nitrosomonas was observed only in the TNCU-I RBC biofilm. In addition, the proportions of ammonia oxidizer in the TNCU-I RBC biofilm, the TNCU-I activated sludge and the A2O activated sludge were 11.4%, 13.2%, and 4.1%, respectively, higher than the nitrite oxidizer fractions of 3.3%, 5.7% and 2.1%, respectively, according to the cloning-DGGE method. On the other hand, the proportions of ammonia oxidizers in the afore-mention materials were 10.3%, 13.7%, and 5.2%, higher than the nitrite oxidizer fractions of 2.5%, 3.6% and 2.3%, according to the FISH experiments. This implies that the proportion of ammonia oxidizer in the TNCU-I process was 3.2 and 2.6 times that in the A2O process, determined by the cloning-DGGE and FISH methods, respectively. These amounts are also close to the ammonia oxidization rate of 2.9 times. All the data show that RBC added to the aerobic zone of TNCU-I process would increase the nitrifier abundance and enhance the nitrification performance of the system.

Bacteria, Aerobic↗

PubMed2000

Biotreatment of H2S- and NH3-containing waste gases by co-immobilized cells biofilter.

Gas mixture of H2S and NH3 in this study has been the focus in the research area concerning gases generated from the animal husbandry and the anaerobic wastewater lagoons used for their treatment. A specific microflora (mixture of Thiobacillus thioparus CH11 for H2S and Nitrosomonas europaea for NH3) was immobilized with Ca-alginate and packed inside a glass column to decompose H2S and NH3. The biofilter packed with co-immobilized cells was continuously supplied with H2S and NH3 gas mixtures of various ratios, and the removal efficiency, removal kinetics, and pressure drop in the biofilter was monitored. The results showed that the efficiency remained above 95% regardless of the ratios of H2S and NH3 used. The NH3 concentration has little effect on H2S removal efficiency, however, both high NH3 and H2S concentrations significantly suppress the NH3 removal. Through product analysis, we found that controlling the inlet ratio of the H2S/NH3 could prevent the biofilter from acidification, and, therefore, enhance the operational stability. Conclusions from bioaerosol analysis and pressure drop in the biofilter suggest that the immobilized cell technique creates less environmental impact and improves pure culture operational stability. The criteria for the biofilter operation to meet the current H2S and NH3 emission standards were also established. To reach Taiwan's current ambient air standards of H2S and NH3 (0.1 and 1 ppm, respectively), the maximum inlet concentrations should not exceed 58 ppm for H2S and 164 ppm for NH3, and the residence time be kept at 72 s.

Air Pollutants↗

PubMed1999

An evaluated improvement of the extinction dilution method for isolation of ammonia-oxidizing bacteria.

An improved method for isolation of ammonia-oxidizing bacteria (AOB) by the extinction dilution technique is described. It is important to prevent the growth of heterotrophic organisms, which may easily outnumber the AOB in mixed cultures. This was achieved by careful elimination of C sources in the medium and by sealing the cultures from contact with the atmosphere, thus excluding air-borne, volatile compounds which support growth of heterotrophs. The sealing of the cultures reduced the number of heterotrophs by a factor of 10, thus grossly increasing the chances of obtaining pure AOB cultures. Another important factor is to use actively growing 'late log' cultures during the final isolation step. This was achieved by adjusting the buffer capacity to ensure a clearly visible pH indicator shift at a stage when one-third to one-half of the ammonia had been oxidized. By this improved isolation procedure, AOB were isolated from three different locations: an arable soil, a lead-contaminated soil and an animal house. For an unknown reason, several attempts to isolate pure cultures from a forest soil were unsuccessful, despite the presence of AOB in the primary extinction dilution cultures. The isolates from soils were all Nitrosospira spp. For isolation of AOB from the animal house, two growth media were used, one containing ammonium sulfate, and one containing urea. From the cultures with ammonium sulfate, Nitrosomonas spp. were isolated, whereas Nitrosospira spp. were isolated from the cultures with urea as the main ammonia source. The identifications of all isolates are based on morphology and 16S rDNA sequences.

Ammonia↗

PubMed1999

Feasibility of bioremediation of trichloroethylene contaminated sites by nitrifying bacteria through cometabolism with ammonia.

The autotrophic ammonia-oxidizing bacteria (Nitrosomonas sp.) are able to dechlorinate trichloroethylene (TCE) through cometabolism using ammonia (NH(3)) as a growth substrate. Cometabolic kinetics models suggest that TCE is a potent competitive inhibitor of NH(3) oxidation because it competes with NH(3) for oxidation by the enzyme of ammonia monooxygenase (AMO). In this study, an enriched culture of nitrifying bacteria was used to investigate the efficiencies of cometabolism of TCE by AMO. In addition, the relationships among specific growth substrate (NH(3)) utilization rate (qNH(3)), specific nongrowth substrate (TCE) cometabolic rate (qTCE), NH(3) and TCE concentrations, and NH(3)/TCE and TCE/NH(3) ratios were also analyzed. We found that the relationships between qNH(3) and NH(3) for the systems with and without TCE followed the Alvarez-Cohen competitive inhibition model and Monod model, respectively. Our results demonstrate that TCE could be cometabolized in a nitrification system when sufficient oxygen and NH(3)200 microg/l) were also found to show inhibitory effects towards NH(3) oxidation in enriched nitrifying culture. We also found that the NH(3)/TCE ratio rather than TCE concentrations alone exhibited strong correlation with qNH(3), much the same as the Ely activity recovery model presented. Our results suggest that the relationship between qTCE and TCE concentrations followed the Oldenhuis enzyme inactivation model for systems without NH(3).

Ammonia↗

PubMed2002

Spectroscopic characterization of the NO adduct of hydroxylamine oxidoreductase.

Hydroxylamine oxidoreductase (HAO) from the autotrophic nitrifying bacterium Nitrosomonas europaea catalyzes the oxidation of NH2OH to NO2-. The enzyme contains eight hemes per subunit which participate in catalysis and electron transport. NO is found to bind to the enzyme and inhibit electron flow to the acceptor protein, cytochrome c554. NO is found to oxidize either partially or fully reduced HAO, but NO will not reduce ferric HAO. Since NO can be reduced but not oxidized to product by HAO, NO is not considered to be a long-lived intermediate in the catalytic mechanism. Substrate oxidation occurs in the presence of bound NO or cyanide, suggesting a second interaction site for substrate with HAO and providing a means for recovery of the NO-inhibited form of the enzyme. Upon addition of NO to oxidized HAO, the integer-spin EPR signal from the active site vanishes, an IR band from NO appears at 1920 cm(-1), and a diamagnetic quadrupole iron doublet appears in Mössbauer spectroscopy with delta = 0.06 mm/s and DeltaEq = 2.1 mm/s. The NO stretching frequency and Mössbauer parameters are characteristic of an [FeNO]6 heme complex. New Mössbauer data on ferric myoglobin-NO are also presented for comparison. The results indicate that NO binds to heme P460 and that the loss of the integer-spin EPR signal is due to the conversion of heme P460 to a diamagnetic S = 0 state and concomitant loss of magnetic interaction with neighboring heme 6. In previous studies where the heme P460-heme 6 interaction was affected by substrate or cyanide binding, a signal attributable to heme 6 was not observable. In contrast, in this work, the NO-induced loss of the signal is accompanied by the appearance of a previously unobserved large g(max) (or HALS) low-spin EPR signal from heme 6.

Electron Spin Resonance Spectroscopy↗

PubMed2000

Involvement of a novel hydroxylamine oxidoreductase in anaerobic ammonium oxidation.

In this study a novel hydroxylamine oxidoreductase (HAO) was purified and characterized from an anaerobic ammonium-oxidizing (Anammox) enrichment culture. The enzyme, which constituted about 9% of the protein mass in the soluble fraction of the cell extract, was able to oxidize hydroxylamine and hydrazine. When phenazine methosulfate and methylthiazolyltetrazolium bromide were used as electron acceptors, a V(max) [21 and 1.1 micromol min(-)(1) (mg of protein)(-)(1)] and K(m) (26 and 18 microM) for hydroxylamine and hydrazine were determined, respectively. The hydroxylamine oxidoreductase is a trimer and contains about 26 hemes per 183 kDa. As deduced from UV/vis spectra, hydroxylamine reduced more and different cytochromes than hydrazine. The dithionite-reduced spectrum showed an unusual 468 nm peak. Inhibition experiments with H(2)O(2) showed that hydroxylamine bound to this P-468 cytochrome, which is assumed to be the putative substrate binding site. Cyanide and hydrazine inhibited the oxidation of hydroxylamine. The amino acid sequences of several peptide fragments of HAO from Anammox showed a clear difference with the deduced amino acid sequence of HAO from the aerobic ammonia-oxidizing bacterium Nitrosomonas europaea. In EPR spectra of the Anammox HAO, two g-values (g(z)() = 2.37 and 2.42) were observed, which were not present in HAO of N. europaea.

Bacterial Proteins↗