The gene encoding ammonia monooxygenase subunit A exists in three nearly identical copies in Nitrosospira sp. NpAV

Abstract The gene encoding ammonia monooxygenase subunit A (AmoA) was found in three copies in the genome of the chemolithotrophic soil bacterium, Nitrosospira sp. NpAV. The open reading frame and flanking regions of the three copies were isolated from digested and size fractionated genomic DNA using oligodeoxyribonucleotide primers and polymerase chain reaction. The three gene copies of amoA were sequenced and the sequences compared to each other. The open reading frames and the upstream and downstream flanking regions were nearly identical in the three copies. All three copies were expressed in recombinant Escherichia coli strains from the indigenous promoter producing a product of approximately 30 kDa. All amoA copies encode 274 amino acid polypeptides which have similarity to the ammonia monooxygenase acetylene-binding protein from Nitrosomonas europaea .     

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the beta- and the gamma-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2-3) nearly identical copies in the beta-subdivision representatives but in single copies in the gamma-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the beta-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.     

The viable but non-culturable state in the human pathogen Vibrio vulnificus

Abstract Genes encoding paniculate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides ( pmoA and amoA ) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (α- and γ-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (β-and y-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the γ-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.     

Nitrification and anammox with urea as the energy source

Urea is present in many ecosystems and can be used as an energy source by chemolithotrophic aerobic ammonia oxidizing bacteria (AOB). Thus the utilization of urea in comparison to ammonia, by AOB as well as anaerobic ammonia oxidizing (Anammox) bacteria was investigated, using enrichments cultures, inoculated with activated sludge, and molecular ecological methods. In batch enrichment cultures grown with ammonia a population established in 2 weeks, which was dominated by halophilic and halotolerant AOB as determined by fluorescence in situ hybridization (FISH) experiments, with the 16S rRNA targeting oligonucleotide probe NEU. In other batch enrichment cultures using urea, the AOB population was assessed by PCR amplification, cloning and phylogenetic analysis of amoA and ribosomal 16S rRNA genes. While only one of the 48 16S rRNA gene clones could be identified as AOB (Nitrosomonas oligotropha), the amoA approach revealed two more AOB, Nitrosomonas europaea and Nitrosomonas nitrosa to be present in the enrichment. FISH analysis of the enrichment with probe NEU and newly designed probes for a specific detection of N. oligotropha and N. nitrosa related organisms, respectively, showed that N. oligotropha-like AOB formed about 50% of the total bacterial population. Also N. nitrosa (about 15% of the total population) and N. europaea (about 5% of the total population) were relatively abundant. Additionally, continuous enrichments were performed under oxygen limitation. When ammonia was the energy source, the community in this reactor consisted of Anammox bacteria and AOB hybridizing with probe NEU. As the substrate was changed to urea, AOB related to N. oligotropha became the dominant AOB in this oxygen limited consortium. This resulted in a direct conversion of urea to dinitrogen gas, without the addition of organic carbon.     

The small-scale production of [U-14C]acetylene from Ba14CO3: application to labeling of ammonia monooxygenase in autotrophic nitrifying bacteria

A small-scale method has been adapted from an established procedure for the generation of [U-14C]acetylene from inexpensive and commonly available precursors. The method involves the fusing of Ba14CO3 with excess barium metal to produce Ba14C2. The BaC2 is reacted with water to generate acetylene which is then selectively dissolved into dimethyl sulfoxide (DMSO). The results presented demonstrate the effect of Ba:BaCO3 ratio on the concentrations of various gases released during the hydrolysis reaction and quantify the selectivity of the DMSO-trapping process for each gas. [U-14C]Acetylene generated by this method has been used to inactivate ammonia monooxygenase in three species of autotrophic nitrifying bacteria: Nitrosomonas europaea, Nitrosococcus oceanus, and Nitrosolobus multiformis. Our results demonstrate that acetylene inactivation of this enzyme in all three species results in the covalent incorporation of radioactive label into a polypeptide of apparent Mr of 25,000-27,000, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography.     

Inhibition of Ammonia Oxidation in Nitrosomonas europaea by Sulfur Compounds: Thioethers Are Oxidized to Sulfoxides by Ammonia Monooxygenase

Organic sulfur compounds are well-known nitrification inhibitors. The inhibitory effects of dimethylsulfide, dimethyldisulfide, and ethanethiol on ammonia oxidation by Nitrosomonas europaea were examined. Both dimethylsulfide and dimethyldisulfide were weak inhibitors of ammonia oxidation and exhibited inhibitory characteristics typical of substrates for ammonia monooxygenase (AMO). Depletion of dimethylsulfide required O(2) and was prevented with either acetylene or allylthiourea, two inhibitors of AMO. The inhibition of ammonia oxidation by dimethylsulfide was examined in detail. Cell suspensions incubated in the presence of ammonia oxidized dimethylsulfide to dimethyl sulfoxide. Depletion of six other thioethers was also prevented by treating cell suspensions with either allylthiourea or acetylene. The oxidative products of three thioethers were identified as the corresponding sulfoxides. The amount of sulfoxide formed accounted for a majority of the amount of sulfide depleted. By using gas chromatography coupled with mass spectrometry, allylmethylsulfide was shown to be oxidized to allylmethylsulfoxide by N. europaea with the incorporation of a single atom of O derived from O(2) into the sulfide. This result supported our conclusion that a monooxygenase was involved in the oxidation of allylmethylsulfide. The thioethers are concluded to be a new class of substrates for AMO. This is the first report of the oxidation of the sulfur atom by AMO in whole cells of N. europaea. The ability of N. europaea to oxidize dimethylsulfide is not unique among the ammonia-oxidizing bacteria. Nitrosococcus oceanus, a marine nitrifier, was also demonstrated to oxidize dimethylsulfide to dimethyl sulfoxide.     

Detection of Nitrosococcus oceanus in a Mediterranean Iagoon by immunofluorescence

The autotrophic ammonium-oxidizing bacterium Nitrosococcus oceanus was studied in the brackish lake of Ganzirri by cultural and immunofluorescent methods. The preparation of the specific antiserum in rabbits is reported. The polyclonal antiserum for Nitrosococcus oceanus cross-reacted with other ammonia oxidizing strains, but not with other marine bacteria. Temporal changes were determined by taking monthly water samples from a station 6 m deep and the distribution in sediments was investigated in four stations. Isolation of a strain of Nitrosococcus sp. was obtained from a sediment sample collected in December. The abundance of Nitrosococcus spp. bacteria correlated positively with particulate organic carbon (POC), particulate organic nitrogen (PON), temperature and total bacteria, whereas there was a negative relationship with oxygen tension. No correlation was found between immunofluorescent and MPN counts of Nitrosococcus spp. bacteria.     

Analysis of ammonia-oxidizing bacteria from hypersaline Mono Lake, California, on the basis of 16S rRNA sequences

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the beta subdivision of the division Proteobacteria (beta-subdivision Proteobacteria) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different beta-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured Nitrosomonas strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the Nitrosomonas europaea group that are acclimated to alkaline, high-salinity environments. Primers specific for Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium in the gamma subdivision of the Proteobacteria, did not amplify target from any samples.     

Susceptibility of ammonia-oxidizing bacteria to nitrification inhibitors

Activity of nitrification inhibitors to several typical ammonia-oxidizing bacteria isolated recently, i. e. Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira and Nitrosovibrio species was assayed using 2-amino-4-methyl-trichloromethyl-1,3,5-triazine (MAST), 2-amino-4-tribromomethyl-6-trichloromethyl-1,3,5-triazine (Br-MAST), 2-chloro-6-trichloromethylpyridine (nitrapyrin) and others, and compared to confirm the adequate control of ammonia-oxidizing bacteria by the inhibitors. The order of activity of the inhibitors to 13 species of ammonia-oxidizing bacteria examined was approximately summarized as Br-MAST > or = nitrapyrin > or = MAST > other inhibitors. Two Nitrosomonas strains, N. europaea ATCC25978 and N. sp. B2, were extremely susceptible to Br-MAST, exhibiting a pI50 > or = 6.40. These values are the position logarithms of the molar half-inhibition concentration. The 16S rRNA gene sequence similarity for the highly susceptible 4 strains of genus Nitrosomonas was 94% to 100% of Nitrosomonas europaea, although those of the less susceptible 3 strains of ammonia-oxidizing bacteria, Nitrosococcus oceanus C-107 ATCC19707, Nitrosolobus sp. PJA1 and Nitrosolobus multiformis ATCC25196, were 77.85, 91.53 and 90.29, respectively. However, no clear correlation has been found yet between pI50-values and percent similarity of 16S rRNA gene sequence among ammonia-oxidizing bacteria.     

Diversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor

The ammonium-oxidizing microbial community was investigated in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor that was operated for about 1 year with high anaerobic ammonium oxidation activity (up to 0.8 kg NH(4)(+)-N m(-3) day(-1)). A Planctomycetales-specific 16S rRNA gene library was constructed to analyse the diversity of the anaerobic ammonium-oxidizing bacteria (AnAOB). Most of the specifically amplified sequences (15/16) were similar to each other (> 99%) but were distantly related to all of the previously recognized sequences (< 94%), with the exception of an unclassified anammox-related clone, KSU-1 (98%). An ammonia monooxygenase (amoA) gene library was also analysed to investigate the diversity of 'aerobic' ammonium-oxidizing bacteria (AAOB) from the beta-Proteobacteria. Most of the amoA gene fragments (53/55) clustered in the Nitrosomonas europaea-Nitrosococcus mobilis group which has been reported to prevail under oxygen-limiting conditions. The quantitative results from real-time polymerase chain reaction (PCR) amplification showed that the dominant AnAOB comprised approximately 50% of the total bacterial 16S rRNA genes in the reactor, whereas the AAOB of beta-Proteobacteria represented only about 3%. A large fragment (4008 bp) of the rRNA gene cluster of the dominant AnAOB (AS-1) in this reactor sludge was sequenced and compared with sequences of other Planctomycetales including four anammox-related candidate genera. The partial sequence of hydrazine-oxidizing enzyme (hzo) of dominant AnAOB was also identified using new designed primers. Based on this analysis, we propose to tentatively name this new AnAOB Candidatus'Jettenia asiatica'.     

Linking Diversity and Stable Isotope Fractionation in Ammonia-Oxidizing Bacteria

The link between similarity in amino acid sequence for ammonia monooxygenase (AMO) and isotopic discrimination for ammonia oxidation ( l AMO ) was investigated in g -subdivision ammonia-oxidizing bacteria. The isotope effects for ammonia oxidation in pure cultures of the nitrifying strains Nitrosomonas marina , Nitrosomonas C-113a, Nitrosospira tenuis , Nitrosomonas europaea , and Nitrosomonas eutropha ranged from 14.2 to 38.2. The differences in isotope effects could not be readily explained by differential rates of ammonia oxidation, transport of NH 4 + , or accumulation of NH 2 OH or N 2 O among the strains. The major similarities and differences observed in l AMO are, however, paralleled by similarities and differences in amino acid sequences for the f -subunit of AMO (AmoA). Robust differences in l AMO among nitrifying bacteria may be expected to influence the stable isotopic signatures of nitrous oxide (N 2 O) produced in various environments.     

Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys

The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.     

Primers containing universal bases reduce multiple amoA gene specific DGGE band patterns when analysing the diversity of beta-ammonia oxidizers in the environment

The gene encoding the active site of the ammonia monooxygenase (amoA) has been exploited as molecular marker for studying ammonia-oxidizing bacteria (AOB) diversity in the environment. Primers amplifying functional genes are often degenerated and therefore produce multiple band patterns, when analysed with the Denaturing gradient gel electrophoresis (DGGE) approach. To improve the DGGE band patterns we have designed new primer sets which contain inosine residues and are specific for the amoA gene. Primers were evaluated analysing pure AOB cultures and two habitats (wastewater treatment plant, soda pools). We found that the application of inosine primers helped to reduce the apparent complexity of the DGGE band pattern. Comparison of sequences from environmental samples using either degenerated or inosine containing amoA primers retrieved both identical and additional sequences. Both primer sets seem to be limited in their ability to detect the presence of all AOB by DGGE analyses.     

Direct detection by in situ PCR of the amoA gene in biofilm resulting from a nitrogen removal process.

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.     

Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.     

Differential inhibition in vivo of ammonia monooxygenase, soluble methane monooxygenase and membrane-associated methane monoxygenase by phenylacetylene

Phenylacetylene was investigated as a differential inhibitor of ammonia monooxygenase (AMO), soluble methane monooxygenase (sMMO) and membrane-associated or particulate methane monooxygenase (pMMO) in vivo. At phenylacetylene concentrations > 1 microM, whole-cell AMO activity in Nitrosomonas europaea was completely inhibited. Phenylacetylene concentrations above 100 microM inhibited more than 90% of sMMO activity in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b. In contrast, activity of pMMO in M. trichosporium OB3b, M. capsulatus Bath, Methylomicrobium album BG8, Methylobacter marinus A45 and Methylomonas strain MN was still measurable at phenylacetylene concentrations up to 1,000 microM. AMO of Nitrosococcus oceanus has more sequence similarity to pMMO than to AMO of N. europaea. Correspondingly, AMO in N. oceanus was also measurable in the presence of 1,000 microM phenylacetylene. Measurement of oxygen uptake indicated that phenylacetylene acted as a specific and mechanistic-based inhibitor of whole-cell sMMO activity; inactivation of sMMO was irreversible, time dependent, first order and required catalytic turnover. Corresponding measurement of oxygen uptake in whole cells of methanotrophs expressing pMMO showed that pMMO activity was inhibited by phenylacetylene, but only if methane was already being oxidized, and then only at much higher concentrations of phenylacetylene and at lower rates compared with sMMO. As phenylacetylene has a high solubility and low volatility, it may prove to be useful for monitoring methanotrophic and nitrifying activity as well as identifying the form of MMO predominantly expressed in situ.     

Effects of nitrite on ammonia-oxidizing activity and gene regulation in three ammonia-oxidizing bacteria

Nitrite is the highly toxic end product of ammonia oxidation that accumulates in the absence of a nitrite-consuming process and is inhibitory to nitrifying and other bacteria. The effects of nitrite on ammonia oxidation rates and regulation of a common gene set were compared in three ammonia-oxidizing bacteria (AOB) to determine whether responses to this toxic metabolite were uniform. Mid-exponential-phase cells of Nitrosomonas europaea ATCC 19718, Nitrosospira multiformis ATCC 25196, and Nitrosomonas eutropha C-91 were incubated for 6 h in mineral medium supplemented with 0, 10, or 20 mM NaNO(2) . The rates of ammonia oxidation (nitrite production) decreased significantly only in NaNO(2) -supplemented incubations of N. eutropha; no significant effect on the rates was observed for N. europaea or N. multiformis. The levels of norB (nitric oxide reductases), cytL (cytochrome P460), and cytS (cytochrome c'-β) mRNA were unaffected by nitrite in all strains. The levels of nirK (nitrite reductase) mRNA increased only in N. europaea in response to nitrite (10 and 20 mM). Nitrite (20 mM) significantly reduced the mRNA levels of amoA (ammonia monooxygenase) in N. multiformis and norS (nitric oxide reductase) in the two Nitrosomonas spp. Differences in response to nitrite indicated nonuniform adaptive and regulatory strategies of AOB, even between closely related species.     

Grassland management regimens reduce small-scale heterogeneity and species diversity of beta-proteobacterial ammonia pxidizer populations.

The impact of soil management practices on ammonia oxidizer diversity and spatial heterogeneity was determined in improved (addition of N fertilizer), unimproved (no additions), and semi-improved (intermediate management) grassland pastures at the Sourhope Research Station in Scotland. Ammonia oxidizer diversity within each grassland soil was assessed by PCR amplification of microbial community DNA with both ammonia oxidizer-specific, 16S rRNA gene (rDNA) and functional, amoA, gene primers. PCR products were analysed by denaturing gradient gel electrophoresis, phylogenetic analysis of partial 16S rDNA and amoA sequences, and hybridization with ammonia oxidizer-specific oligonucleotide probes. Ammonia oxidizer populations in unimproved soils were more diverse than those in improved soils and were dominated by organisms representing Nitrosospira clusters 1 and 3 and Nitrosomonas cluster 7 (closely related phylogenetically to Nitrosomonas europaea). Improved soils were only dominated by Nitrosospira cluster 3 and Nitrosomonas cluster 7. These differences were also reflected in functional gene (amoA) diversity, with amoA gene sequences of both Nitrosomonas and Nitrosospira species detected. Replicate 0.5-g samples of unimproved soil demonstrated significant spatial heterogeneity in 16S rDNA-defined ammonia oxidizer clusters, which was reflected in heterogeneity in ammonium concentration and pH. Heterogeneity in soil characteristics and ammonia oxidizer diversity were lower in improved soils. The results therefore demonstrate significant effects of soil management on diversity and heterogeneity of ammonia oxidizer populations that are related to similar changes in relevant soil characteristics.     

Characteristics of aerobic and anaerobic ammonium-oxidizing bacteria in the hyporheic zone of a contaminated river

Both β-proteobacterial aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (ANAMMOX) bacteria were investigated in the hyporheic zone of a contaminated river in China containing high ammonium levels and low chemical oxygen demand. Fluorescence in-situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and cloning-sequencing were employed in this study. FISH analysis illustrated that AOB (average population of 3.5?%) coexisted with ANAMMOX bacteria (0.7?%). The DGGE profile revealed a high abundance and diversity of bacteria at the water-air-soil interface rather than at the water-soil interface. The redundancy analysis correlated analysis showed that the diversity of ANAMMOX bacteria was positively related to the redox potential. The newly detected sequences of ANAMMOX organisms principally belonged to the genus Candidatus "Brocadia", while most ammonia monooxygenase subunit-A gene amoA sequences were affiliated with Nitrosospira and Nitrosomonas. These results suggest that the water-air-soil interface performs an important function in the nitrogen removal process and that the bioresources of AOB and ANAMMOX bacteria can potentially be utilized for the eutrophication of rivers.     

Physiological and phylogenetic study of an ammonium-oxidizing culture at high nitrite concentrations

Oxidation of high-strength ammonium wastewater can lead to exceptionally high nitrite concentrations; therefore, the effect of high nitrite concentration (> 400 mM) was studied using an ammonium-oxidizing enrichment culture in a batch reactor. Ammonium was fed to the reactor in portions of 40-150 mM until ammonium oxidation rates decreased and finally stopped. Activity was restored by replacing half of the medium, while biomass was retained by a membrane. The ammonium-oxidizing population obtained was able to oxidize ammonium at nitrite concentrations of up to 500 mM. The maximum specific oxidation activity of the culture in batch test was about 0.040 mmol O(2)g(-1)proteinmin(-1) and the K(s) value was 1.5 mM ammonium. In these tests, half of the maximum oxidation activity was still present at a concentration of 600 mM nitrite and approximately 10% residual activity could still be measured at 1200 mM nitrite (pH 7.4), or as a free nitrous acid (FNA) concentration of 6.6 mg l(-1). Additional experiments showed that the inhibition was caused by nitrite and not by the high sodium chloride concentration of the medium. The added ammonium was mainly converted into nitrite and no nitrite oxidation was observed. In addition, gaseous nitrogen compounds were detected and mass balance calculations revealed a nitrogen loss of approximately 20% using this system. Phylogenetic analyses of 16S rRNA and ammonium monooxygenase (amoA) genes of the obtained enrichment culture showed that ammonium-oxidizing bacteria of the Nitrosomonas europaea/Nitrosococcus mobilis cluster dominated the two clone libraries. Approximately 25% of the 16S rRNA clones showed a similarity of 92% to Deinococcus-like organisms. Specific fluorescence in situ hybridization (FISH) probes confirmed that these microbes comprised 10-20% of the microbial community in the enrichment. The Deinococcus-like organisms were located around the Nitrosomonas clusters, but their role in the community is currently unresolved.