[Isolation, identification and characterization of nitroso-bacteria in one-step completely nitrogen removal process].

OBJECTIVE: To study the characteristics of the Nitroso-bacteria in one-step completely autotrophic nitrogen removal process. METHODS: We took samples from activated sludge of the one-step completely autotrophic nitrogen removal reactor. Through four times of enrichments and isolations, one Nitroso-bacteria named N1 was obtained. We identified this strain by microscope and 16S rDNA analysis and studied the effect of pH, temperature as well as ammonium concentration on the metabolism of N1. RESULTS: Of the N1 nucleotides, 97%, 96% and 96% were identical with the conserved fragment of Nitrosomonas sp. NL7 (AY958677), Nitrosomonas AS1 (EF016119) and Nitrosomonas sp. Is32 (AJ621027) respectively. The optimal temperature and pH of N1 were 8.0 and 30 degrees C and sufficient dissolved oxygen was demanded. In addition, no restraint effect was turned up in the ammonium concentration of 80-800 mg/L. CONCLUSION: N1 was identified as Nitrosomonas sp. Compared with the relative references, this strain was ammonium-tolerant.

Electron transfer during the oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas europaea.

The combined action of ammonia monooxygenase, AMO, (NH(3)+2e(-)+O(2)-->NH(2)OH) and hydroxylamine oxidoreductase, HAO, (NH(2)OH+H(2)O-->HNO(2)+4e(-)+4H(+)) accounts for ammonia oxidation in Nitrosomonas europaea. Pathways for electrons from HAO to O(2), nitrite, NO, H(2)O(2) or AMO are reviewed and some recent advances described. The membrane cytochrome c(M)552 is proposed to participate in the path between HAO and ubiquinone. A bc(1) complex is shown to mediate between ubiquinol and the terminal oxidase and is shown to be downstream of HAO. A novel, red, low-potential, periplasmic copper protein, nitrosocyanin, is introduced. Possible mechanisms for the inhibition of ammonia oxidation in cells by protonophores are summarized. Genes for nitrite- and NO-reductase but not N(2)O or nitrate reductase are present in the genome of Nitrosomonas. Nitrite reductase is not repressed by growth on O(2); the flux of nitrite reduction is controlled at the substrate level.

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.

[Preliminary study of characteristics of a special Nitrosomonas].

A special obligately-autotrophic Nitrosomonas was isolated from high temperature and hypersaline drainage water obtained off a wastewater treatment plant. It was Gram-negative, nonsporulating, ellipsoidal or short-rod shaped bacterium that singly arranged or arranged in a circle or in a row. Cells were 0.7 to 0.9 microm wide and 1.2 to 1.98 microm long. Electron-microscopic scanning of these cells indicated that they were wrapped by unknown fluff-like matter. They oxidized ammonium to nitrite and caused the removal of nitrogen from the system. But the decrease of NH4+ -N did not accord with the increase of NO2 - -N and NO3- -N was almost not detected. After 12 d of cultivation at 50 degrees C, about 10% of the NH4+ -N was converted to NO2- -N, 15% remained as NH4+ -N, and the other 75% was removed from the system, including 17% of NH4+ -N volatilized.

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.

Immunocytochemical localization of membrane-bound ammonia monooxygenase in cells of ammonia oxidizing bacteria.

The intracellular location of the membrane-bound ammonia monooxygenase (AMO) in all genera of ammonia oxidizing bacteria (Nitrosomonas, Nitrosococcus and Nitrosospira) was determined by electron microscopic immunocytochemistry. Polyclonal antibodies recognizing the two subunits, AmoA- and AmoB-proteins, were used for post-embedding labeling. Ultrathin sections revealed that the AmoB-protein was located in all genera on the cytoplasmic membrane. In cells of Nitrosomonas and Nitrosococus additional but less AmoB-labeling was found on the intracytoplasmic membrane (ICM). In contrast to the detection of AmoB-protein, the AmoA-antibodies failed to detect the AmoA-protein. Based on quantitative immunoblots the extent of ICM in Nitrosomonas eutropha was correlated with the amount of AmoA in the cells. The highest extent of ICM and amount of AmoA was found at low ammonium substrate concentrations.

Polyhedral inclusion bodies in cells of Nitrosomonas spec.

Polyhedral inclusion bodies were observed in cells of a Nitrosomonas species. They were present in growing cells as well as in resting cells. In thin sections their size was about 130 nm in growing cells and about 185 nm in diameter in resting cells. The bodies were commonly located in the nucleoplasm. They appeared to be bounded by a nonunit membrane and had a granular substructure. In thin sections about 70% of the exponentially grown cells and about 20% of the resting cells of the investigated strain showed 1-7 respectively 1-3 inclusion bodies.

In situ morphology of nitrifying-like bacteria in aquaculture systems.

The in situ microbiota from several aquaculture facilities with active nitrification was examined by transmission electron microscopy of thin sections for the presence of bacteria that contained intracytoplasmic membranes characteristic of the nitrifying bacteria. Colonies of bacteria with the cellular morphology of a species of Nitrosomonas were found to be present in both the culture water and in the biological filter slime of a freshwater chinook salmon (Oncorhynchus tshawytscha) culture system. bacteria in the water possessed the normal nitrosomonas type of ultrastructure, whereas similar bacteria in the slime had an aberrant morphology due to multiple invaginations of the cell wall and cyto-membranes and a significantly greater number of ribosomes. These nitrosomonas-like bacteria lysed during enrichment in commonly used media. Bacteria with the morphology of species of Nitrosomonas and Nitrosococcus were also observed in colonies in the surface slimes of marine culture systems for striped bass (Morone saxatilis) and quahaug (Mercenaria mercenaria).

Effects of gaseous NO2 on cells of Nitrosomonas eutropha previously incapable of using ammonia as an energy source.

Cells of Nitrosomonas eutropha grown under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were initially unable to oxidize ammonia (ammonium) and hydroxylamine when transferred to oxic conditions. Recovery of ammonia and hydroxylamine oxidation activity was dependent on the presence of NO2. Under oxic conditions, without addition of NO2, ammonia consumption started after 8 - 9 days, and small amounts of NO and NO2 were detectable in the gas atmosphere. Removing these nitrogen oxides by intensive aeration, ammonia oxidation activity decreased and broke off after 15 days. Addition of gaseous NO2 (25 ppm1) led to a fast recovery of ammonia oxidation (3 days). Simultaneously, the arrangement of intracytoplasmic membranes (ICM) changed from circular to flattened vesicles, the protein pattern revealed an increase in the concentration of a 27 and a 30 kDa polypeptide, and the cytochrome c content increased significantly.