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.Sustainable wastewater treatment systems are being developed that minimize energy consumption, CO 2 emission, and sludge production. However, these systems typically yield effluents rich in ammonium-nitrogen (NH 4 ϩ -N) and poor in biodegradable organic carbon, thereby making them less suitable for biological N removal through the conventional nitrification-denitrification sequence.Different N removal processes that could be successfully integrated in a sustainable wastewater treatment system are being studied. The Sharon process (single-reactor high-activity ammonium removal over nitrite) (15) uses the principle that at higher temperatures (30 to 35°C), pH 7 to 8, and a cell residence time of 1 day, aerobic ammonia-oxidizing bacteria (AAOB) are able to maintain themselves in the system while nitrite-oxidizing bacteria (NOB) are washed out. Given the reaction stoichiometry of the two groups of nitrifying bacteria (equations 1 and 2), this proces...

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, NH 4 + is directly converted to N 2 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 NH 4 + -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 NH 4 + with NO 2 -as electron acceptor (maximal 434 mg of NH 4 + -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.

A membrane-assisted bioreactor (MBR) for sustained nitrite accumulation is presented, treating a synthetic wastewater with total ammonium nitrogen (TAN) concentrations of 1 kg N m À3 at a hydraulic retention time down to 1 day. Complete biomass retention was obtained by microfiltration with submerged hollow fibre membranes. A membrane flux up to 189.5 dm 3 day À1 m À2 could be maintained at a suction pressure below 100 kPa. Nitrification was effectively blocked at the nitrite stage (nitritation), and nitrate concentration was less than 29 g N m À3 . The rate of aeration was reduced to obtain a mixture of ammonium and nitrite, and after adjusting this rate the TAN/NO 2 -N ratio in the reactor effluent was kept around unity, making it suitable for further treatment by anaerobic oxidation of ammonium with nitrite. After increasing again the rate of aeration, complete nitrification to nitrate recovered after 11 days. It is suggested that nitrite accumulation resulted from a combination of factors. First, the dissolved oxygen (DO) concentration in the reactor was always limited with concentrations below 0.1 g DO m À3, thereby limiting nitrification and preventing significant nitrate formation. The latter is attributed to the fact that ammonium-oxidising bacteria cope better with low DO concentrations than nitrite oxidisers. Second, the MBR was operated at a high ammonia concentration of 7-54 g N m À3, resulting in ammonia inhibition of the nitrite-oxidising microorganisms. Third, a temperature of 35°C was reported to yield a higher maximum growth rate for ammonium-oxidising bacteria than for nitrite-oxidising bacteria. Nitrite oxidisers were always present in the MBR but were out-competed under the indicated process conditions, which is reflected in low concentrations of nitrate. Oxygen limitation was shown to be the most important factor to sustain nitrite accumulation. Nevertheless, nitritation was possible at ambient temperature (22-24°C), lower ammonia concentration (<7 g N m À3) and when using raw nitrogenous wastewater containing some biodegradable carbon. Overall, application of the MBR for nitritation was shown to be a reliable technology.

Nitrogen removal from sludge reject water was obtained by oxygen-limited partial nitritation resulting in nitrite accumulation in a first stage, followed by autotrophic denitrification of nitrite with ammonium as electron donor (similar to anaerobic ammonium oxidation) in a second stage. Two membrane-assisted bioreactors (MBRs) were used in series to operate with high sludge ages and subsequent high volumetric loading rates, achieving 1.45 kg N m(-3) day(-1) for the partial nitritation MBR and 1.1 kg N m(-3) day(-1) for the anaerobic ammonium oxidation MBR. Biomass retention in the nitritation stage ensured flexibility towards loading rate and operating temperature. Nitrite oxidisers were out-competed at low oxygen and high free ammonia concentration. Biomass retention in the second MBR prevented wash-out of the slowly growing bacteria. Nitrite and ammonium were converted to dinitrogen gas in a reaction ratio of 1.05, thereby maintaining nitrite limitation to assure process stability. The anoxic consortium catalysing the autotrophic denitrification process consisted of Nitrosomonas-like aerobic ammonium oxidizers and anaerobic ammonium oxidizing bacteria closely related to Kuenenia stuttgartiensis. The overall removal efficiency of the combined process was 82% of the incoming ammonium according to a total nitrogen removal rate of 0.55 kg N m(-3) day(-1), without adding extra carbon source.

A lab-scale Rotating Biological Contactor (RBC) was operated with the purpose of oxygen-limited (autotrophic) nitrification-denitrification of an ammonium-rich synthetic wastewater without Chemical Oxygen Demand (COD). Based on the field observations that RBCs receiving anaerobic effluents come to anoxic ammonium removal, the RBC was inoculated with methanogenic sludge. Some 100 days after the addition of the anaerobic sludge to the reactor as a possible means of a rapid initiation of the nitrogen (N) removal process, a maximum ammonium removal of 1,550 mg N m(-2) d(-1) was achieved. Batch tests with 15N labeled ammonium and nitrite indicated that a large part of that N was removed via oxygen-limited oxidation of ammonium with nitrite as the electron acceptor. The other part was removed via conventional denitrification, presumably with COD released from lysis of cells. Species identification of the most abundant microorganisms revealed that Nitrosomonas spp. were the dominant ammonium-oxidizers in the sludge. Thus far, the molecular characterization of the sludge could not show the presence of Planctomycetes among the most dominant species. Overall this experiment confirms the property of the RBC system to remove ammonium to nitrogen gas without the use of heterotrophic carbon source.