Aerobic Ammonium-oxidizing Bacteria Research Articles

Autotrophic nitrogen removal in sequencing batch biofilm reactors at different oxygen supply modes

Conventional nitrification-denitrification treatment is a common way to treat nitrogen in wastewater, but this process is costly for low COD/N wastewaters due to the addition of air and external carbon-source. However, ammonia may alternatively be converted to dinitrogen gas by autotrophic bacteria utilizing aerobically autotrophically produced nitrite as an electron acceptor under anoxic conditions. Lab-scale sequencing batch biofilm reactors (SBBRs) inoculated with normal nitrifying sludge were employed to study the potential of an oxygen-limited autotrophic nitrification-denitrification process initiated with typical nitrifying sludge for treating a synthetic ammonia wastewater devoid of organic carbon in one step. The ring-laced fibrous carrier (length 0.32 m, surface area 3.4 m2/m) was fixed vertically in a 3 L reactor. Two different air supply modes were applied:continuous aeration to control dissolved oxygen at 1.5 mg/L and intermittent aeration. High nitrogen removals of more than 50% were obtained in both SBBRs. At an ammonia loading of 0.882 gm N/m2-day [hydraulic retention time (HRT) of 24 hr], the SBBR continuously aerated to 1.5 mg DO/L had slightly higher nitrogen removal (64%) than the intermittently alternated SBBR (55%). The main form of residual nitrogen in the effluent was ammonia, at concentrations of 25 mg/L and 37 mg N/L in continuous and intermittent aeration SBBRs, respectively. Ammonia was completely consumed when ammonia loading was reduced to 0.441 gm N/m2-day [HRT extended to 48 hr]. The competitive use of nitrite by aerobic nitrite oxidizing bacteria (ANOB) with anaerobic ammonia-oxidizing bacteria (anammox bacteria) during the expanded aeration period under low remaining ammonia concentration resulted in higher nitrate production and lower nitrogen loss in the continuous aeration SBBR than in the intermittent aeration SBBR. The nitrogen removal efficiencies in SBBRs with continuous and alternating aerated were 80% and 86% respectively. Specific microorganisms in the biofilm were characterized using fluorescence in situ hybridization. Aerobic ammonia-oxidizing bacteria (AAOB) occurred side by side with putative anammox bacteria (cells hybridizing with probe AMX820) throughout the biofilm, though ANOB were rarely detected.

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'.

Environmental detection of octahaem cytochrome c hydroxylamine/hydrazine oxidoreductase genes of aerobic and anaerobic ammonium‐oxidizing bacteria

Bacterial aerobic ammonium oxidation and anaerobic ammonium oxidation (anammox) are important processes in the global nitrogen cycle. Key enzymes in both processes are the octahaem cytochrome c (OCC) proteins, hydroxylamine oxidoreductase (HAO) of aerobic ammonium-oxidizing bacteria (AOB), which catalyses the oxidation of hydroxylamine to nitrite, and hydrazine oxidoreductase (HZO) of anammox bacteria, which converts hydrazine to N(2). While the genomes of AOB encode up to three nearly identical copies of hao operons, genome analysis of Candidatus'Kuenenia stuttgartiensis' showed eight highly divergent octahaem protein coding regions as possible candidates for the HZO. Based on their phylogenetic relationship and biochemical characteristics, the sequences of these eight gene products grouped in three clusters. Degenerate primers were designed on the basis of available gene sequences with the aim to detect hao and hzo genes in various ecosystems. The hao primer pairs amplified gene fragments from 738 to 1172 bp and the hzo primer pairs amplified gene fragments from 289 to 876 bp in length, when tested on genomic DNA isolated from a variety of AOB and anammox bacteria. A selection of these primer pairs was also used successfully to amplify and analyse the hao and hzo genes in community DNA isolated from different ecosystems harbouring both AOB and anammox bacteria. We propose that OCC protein-encoding genes are suitable targets for molecular ecological studies on both aerobic and anaerobic ammonium-oxidizing bacteria.

Key Parameters for Control of DEMON Deammonification Process

Key Parameters for Control of DEMON Deammonification ProcessA suspended growth deammonification process has been in full-scale operation for over two years in Austria. Three US utilities have embarked on piloting this process at two locations: New York City and Alexandria, Virginia. Deammonification is a two-part autotrophic reaction involving two distinct biomass populations. In the first step aerobic ammonia oxidizing bacteria (AOB) nitrify partially...Author(s)B. WettS. MurthyI. TakácsM. HellG. BowdenA. DeurM. O'ShaughnessySourceWater PracticeSubjectArticlesDocument typeConference PaperPublisherWater Environment FederationPrint publication date Nov, 2007ISSN1933-1770SICI1933-1770(20071112)1:5L.1;1-DOI10.2175/193317707X257017Volume / Issue1 / 5Content sourceWater PracticeFirst / last page(s)1 - 11Copyright2007Word count150Subject keywordsDEAMMONIFICATIONREJECT WATERSLUDGE LIQUORSIDE-STREAMNITRITE TOXICITYDEMONANAMMOX

Characterization of functional microbial community in a membrane-aerated biofilm reactor operated for completely autotrophic nitrogen removal

The 16S rDNA-based molecular technique was applied to investigate the functional microbial community of a membrane-aerated biofilm (MAB) that was used for completely autotrophic nitrogen removal over nitrite (CANON). The relationships among two kinds of key bacteria responsible for CANON: aerobic ammonia-oxidizing bacteria (AOB) and Anammox bacteria, and their possible distributions in the MAB were discussed based on the microbial community analysis. FISH analysis showed the existence of two visible active layers in experimental MAB. One is the partial nitrifying layer located in the region of oxygen-rich membrane–biofilm interface, dominated by NSO190-positive AOB. The other is the Anammox active layer located in the region of anoxic liquid–biofilm interface, dominated by PLA46 and AMX820-positive Anammox microorganisms. As a result of this study, the AOB as well as Anammox bacteria were present and active in experimental MABR, and the cooperation between AOB and Anammox bacteria was considered to be responsible for CANON.

Whole‐genome analysis of the ammonia‐oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation

Analysis of the structure and inventory of the genome of Nitrosomonas eutropha C91 revealed distinctive features that may explain the adaptation of N. eutropha-like bacteria to N-saturated ecosystems. Multiple gene-shuffling events are apparent, including mobilized and replicated transposition, as well as plasmid or phage integration events into the 2.66 Mbp chromosome and two plasmids (65 and 56 kbp) of N. eutropha C91. A 117 kbp genomic island encodes multiple genes for heavy metal resistance, including clusters for copper and mercury transport, which are absent from the genomes of other ammonia-oxidizing bacteria (AOB). Whereas the sequences of the two ammonia monooxygenase and three hydroxylamine oxidoreductase gene clusters in N. eutropha C91 are highly similar to those of Nitrosomonas europaea ATCC 19718, a break of synteny in the regions flanking these clusters in each genome is evident. Nitrosomonas eutropha C91 encodes four gene clusters for distinct classes of haem-copper oxidases, two of which are not found in other aerobic AOB. This diversity of terminal oxidases may explain the adaptation of N. eutropha to environments with variable O(2) concentrations and/or high concentrations of nitrogen oxides. As with N. europaea, the N. eutropha genome lacks genes for urease metabolism, likely disadvantaging nitrosomonads in low-nitrogen or acidic ecosystems. Taken together, this analysis revealed significant genomic variation between N. eutropha C91 and other AOB, even the closely related N. europaea, and several distinctive properties of the N. eutropha genome that are supportive of niche specialization.

Diversity and abundance of aerobic and anaerobic ammonium‐oxidizing bacteria in freshwater sediments of the Xinyi River (China)

Here we report on the biodiversity and abundance of aerobic and anaerobic ammonium-oxidizing bacteria in sediment samples from the Xinyi River, Jinagsu Province (China). The biodiversity of aerobic ammonium-oxidizing bacteria in the sediment was assessed using the amoA gene as functional marker. The retrieved amoA clones were affiliated to environmental sequences from freshwater habitats. The closest cultivated relative was Nitrosomonas urea. Anaerobic ammonium-oxidizing (anammox) bacteria were studied using anammox and planctomycete-specific 16S rRNA gene primers. The sediments contained 16S rRNA genes and bacterial cells closely related to the known anammox bacterium Candidatus'Brocadia anammoxidans'. Anaerobic continuous flow reactors were set up to enrich anammox organisms from the sediments. After an adaptation period of about 25 days the reactors started to consume ammonium and nitrite, indicating that the anammox reaction was occurring with a rate of 41-58 nmol cm(-3) h(-1). Community analysis of the enrichments by quantitative fluorescence in situ hybridization showed an increase in the abundance of anammox bacteria from < 1% to 6 +/- 2% of the total population. Analysis of the 16S rRNA genes showed that the enriched anammox organisms were related to the Candidatus'Scalindua' genus.

A potential new process for improving nitrogen removal in constructed wetlands—Promoting coexistence of partial-nitrification and ANAMMOX

Conventional nitrification by vertical flow constructed wetlands beds and denitrification by horizontal flow constructed wetlands beds were considered as the main ways to remove nitrogen in constructed wetlands. However, recent evidences showed that there are other microbial pathways for nitrogen removal which are based on partial-nitrification of ammonium to nitrite with combining anaerobic ammonium oxidation (ANAMMOX). The full-scale experiments, lasting 100 days in Evieu, east of France, showed that with appropriate design and modification, for example dividing the depth of 80 cm conventional vertical flow beds into a 25 cm unsaturated layer and a 55 cm saturated layer, this new model cannot only get a better performance in removal efficiency of total nitrogen, but also achieved a promoted growth of ANAMMOX bacteria which will function to further remove the ammonium in horizontal flow beds. With fluorescent in situ hybridization (FISH) analysis, aerobic ammonia oxidizing bacteria (AOB) in conventional vertical flow CWs and ANAMMOX bacteria in the interface between unsaturated–saturated layer of amelioration vertical flow CWs and the followed horizontal flow CWs were tested. This paper described the design, the performance and the results. Comparison experiments with conventional process of nitrification–denitrification beds were also done at the same times; results indicated that the two processes carried on are completely different pathways. Results of experiments, nitrogen balance and FISH analysis proved that the process of partial-nitrification with ANAMMOX can be realized in constructed wetlands and can obtain higher removal efficiency of total nitrogen than conventional methods.

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria.

Vertical migration of aggregated aerobic and anaerobic ammonium oxidizers enhances oxygen uptake in a stagnant water layer

Ammonium can be removed as dinitrogen gas by cooperating aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). The goal of this study was to verify putative mutual benefits for aggregated AerAOB and AnAOB in a stagnant freshwater environment. In an ammonium fed water column, the biological oxygen consumption rate was, on average, 76 kg O(2) ha(-1) day(-1). As the oxygen transfer rate of an abiotic control column was only 17 kg O(2) ha(-1) day(-1), biomass activity enhanced the oxygen transfer. Increasing the AnAOB gas production increased the oxygen consumption rate with more than 50% as a result of enhanced vertical movement of the biomass. The coupled decrease in dissolved oxygen concentration increased the diffusional oxygen transfer from the atmosphere in the water. Physically preventing the biomass from rising to the upper water layer instantaneously decreased oxygen and ammonium consumption and even led to the occurrence of some sulfate reduction. Floating of the biomass was further confirmed to be beneficial, as this allowed for the development of a higher AerAOB and AnAOB activity, compared to settled biomass. Overall, the results support mutual benefits for aggregated AerAOB and AnAOB, derived from the biomass uplifting effect of AnAOB gas production.

Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors

To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-N m −3 day −1 was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH 4 + to NO 2 - molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH 4 + / NO 2 - molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria). They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH 4 + and NO 2 - ) of 4.45 g-N of ( NH 4 + + NO 2 - ) m −2 day −1 was detected in the upper 800 μm of the biofilm, which was consistent with the spatial distribution of anammox bacteria.

Strategies of aerobic ammonia-oxidizing bacteria for coping with nutrient and oxygen fluctuations

In most natural environments as well as in engineered environments, such as wastewater treatment plants, ammonia-oxidizing bacteria (AOB) experience fluctuating substrate concentrations. Several physiological traits, such as low maintenance energy demand and decay rate, cell-to-cell communication, cell mobility, stable enzymes and RNAs, could allow AOB to maintain themselves under unfavourable circumstances. This review examines whether AOB possess such traits and how these traits might offer advantages over competing organisms such as heterotrophic bacteria during periods of starvation. In addition, within the AOB groups, differences exist in adaptation to and competitiveness under conditions of high or low ammonia or oxygen concentrations. Because these findings are of importance with regard to the ecology and activity of AOB in natural and engineered environments, concluding remarks are directed towards future research objectives that may clarify unanswered questions, thereby contributing to the general knowledge of the ecology and activity of ammonia oxidizers.

Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic ammonia-oxidizing bacteria

Nitric oxide (NO) and nitrous oxide (N 2O) are climatically important trace gases that are produced by both nitrifying and denitrifying bacteria. In the denitrification pathway, N 2O is produced from nitric oxide (NO) by the enzyme nitric oxide reductase (NOR). The ammonia-oxidizing bacterium Nitrosomonas europaea also possesses a functional nitric oxide reductase, which was shown recently to serve a unique function. In this study, sequences homologous to the large subunit of nitric oxide reductase ( norB) were obtained from eight additional strains of ammonia-oxidizing bacteria, including Nitrosomonas and Nitrosococcus species (i.e., both β- and γ-Proteobacterial ammonia oxidizers), showing widespread occurrence of a norB homologue in ammonia-oxidizing bacteria. However, despite efforts to detect norB homologues from Nitrosospira strains, sequences have not yet been obtained. Phylogenetic analysis placed nitrifier norB homologues in a subcluster, distinct from denitrifier sequences. The similarities and differences of these sequences highlight the need to understand the variety of metabolisms represented within a “functional group” defined by the presence of a single homologous gene. These results expand the database of norB homologue sequences in nitrifying bacteria.

Application, eco-physiology and biodiversity of anaerobic ammonium-oxidizing bacteria

The demand for new and sustainable systems for nitrogen removal has increased dramatically in the last decade. It is clear that the conventional systems cannot deal with the increasing nitrogen loads in a cost effective way. As an alternative, the implementation of the anammox (anaerobic ammonium oxidation) process in the treatment of wastewater with high ammonium concentrations has been started. The compact anammox reactors can sustain high nitrogen loads without any problems. The highest observed anammox capacity is 8.9 kg N removed m-3 reactor day-1. The first 75 m3 anammox reactor is operating in Rotterdam, the Netherlands, combined with the partial nitrification process Single reaction system for High Ammonium Removal Over Nitrite (SHARON). Partial nitrification and anammox can also be combined in one reactor systems like Completely Autotrophic Nitrogen removal Over Nitrite (CANON) or Oxygen Limited Ammonium removal via Nitrification Denitrification (OLAND) where aerobic ammonium-oxidizing bacteria (AOB) and anammox bacteria cooperate under oxygen-limitation. These systems remove about 1.5 kg N m-3 reactor day-1. In addition to ammonium, urea can also be converted in the CANON system after a two-week adaptation period. The ecophysiological properties of the anammox bacteria make them very well suited to convert ammonium and nitrite. The Ks values for ammonium and nitrite are below 5 μM. However, nitrite above 10 mM is detrimental for the anammox process, and oxygen reversibly inhibits the process at concentrations as low as 1 μM. Acetate and propionate can be used by the anammox bacteria to convert nitrite and nitrate, whereas methanol and ethanol severely inhibit the anammox reaction. The enzyme hydroxylamine/hydrazine oxidoreductase (HAO), one of the key enzymes, is located in the anammoxosome, which is a membrane bound organelle. The membranes of the anammox bacteria contain unique ladderane lipids and hopanoids. The bacteria responsible for the anammox reaction are related to the Planctomycetes. The first anammox bacteria were isolated via Percoll centrifugation and characterized as Candidatus “Brocadia anammoxidans”. Survey of different wastewater treatment plants using anammox specific 16S rRNA gene primers and anammox specific oligonucleotide probes has revealed the presence of at least three other anammox bacteria, which have been tentatively named Candidatus “Kuenenia stuttgartiensis”, Candidatus “Scalindua wagneri” and Candidatus “Scalindua brodae”. A close relative of the latter, Candidatus “Scalindua sorokinii” was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the Black Sea, making the anammox bacteria an important player in the oceanic nitrogen cycle.

Start-up of autotrophic nitrogen removal reactors via sequential biocatalyst addition.

A procedure for start-up of oxygen-limited autotrophic nitrification-denitrification (OLAND) in a lab-scale rotating biological contactor (RBC) is presented. In this one-step process, NH4+ is directly converted to N2 without the need for an organic carbon source. The approach is based on a sequential addition of two types of easily available biocatalyst to the reactor during start-up: aerobic nitrifying and anaerobic, granular methanogenic sludge. The first is added as a source of aerobic ammonia-oxidizing bacteria (AAOB), the second as a possible source of planctomycetes including anaerobic ammonia-oxidizing bacteria (AnAOB). The initial nitrifying biofilm serves as a matrix for anaerobic cell incorporation. By subsequently imposing oxygen limitation, one can create an optimal environment for autotrophic N removal. In this way, N removal of about 250 mg of N L(-1) d(-1) was achieved after 100 d treating a synthetic NH4+-rich wastewater. By gradually imposing higher loads on the reactor, the N elimination could be increased to about 1.8 g of N L(-1) d(-1) at 250 d. The resulting microbial community was compared with that of the inocula using general bacterial and AAOB- and planctomycete-specific PCR primers. Subsequently, the RBC reactor was shown to treat a sludge digestor effluent under suboptimal and strongly varying conditions. The RBC biocatalyst was also submitted to complete absence of oxygen in a fixed-film bioreactor (FFBR) and proved able to remove NH4+ with NO2- as electron acceptor (maximal 434 mg of NH4+-N (g of VSS)(-1) d(-1) on day 136). DGGE and real-time PCR analysis demonstrated that the RBC biofilm was dominated by members of the genus Nitrosomonas and close relatives of Kuenenia stuttgartiensis, a known AnAOB. The latter was enriched during FFBR operation, but AAOB were still present and the ratio planctomycetes/AAOB rRNA gene copies was about 4.3 after 136 d of reactor operation. Whether this relates to an active role of AAOB in the anoxic N removal process remains to be solved.

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.

Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab-scale rotating biological contactor.

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH(4)(+) wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% +/- 5% of the influent N at the highest surface load of approximately 8.3 g of N m(-2) day(-1), with N(2) as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 +/- 7.6 and 76.5 +/- 6.4 mg of NH(4)(+)-N g of volatile suspended solids [VSS](-1) day(-1), respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS(-1) day(-1)). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH(4)(+) wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.

The SHARON®-Anammox® process for treatment of ammonium rich wastewater

The treatment of ammonium rich wastewater, like sludge digester effluent, can be significantly improved when new biotechnological processes are introduced. In this paper, the combination of a partial nitrification process (SHARON) and anoxic ammonium oxidation (Anammox) process for the treatment of ammonia rich influents is evaluated. Herein the combined process has been studied with sludge recycle liquor from the WWTP Rotterdam-Dokhaven. The SHARON process was operated stably for more than 2 years in a 10ICSTR under continuous aeration with a HRT of 1 day. The ammonia in the sludge liquor was converted by 53% to nitrite only. During the test period no formation of nitrate was observed. The effluent of the SHARON process was ideally suited as influent for the Anammox reactor. The Anammox process was operated as a granular sludge SBR-process. More than 80% of the ammonia was converted into dinitrogen gas at a load of 1.2 kgN/m3 per day. Planctomycete-like bacteria dominated the mixed community of the Anammox reactor, and only a small percentage of the population consisted of aerobic ammonium-oxidizing bacteria. This showed that the ammonium-oxidizers in the effluent of the SHARON process did not accumulate in the SBR. The test period showed that the combined SHARON-Anammox system can work stably over long periods and the process is ready for full-scale implementation.