The present study investigated the phylogenetic affiliation and physiological characteristics of bacteria responsible for anaerobic ammonium oxidization (anammox); these bacteria were enriched in an anammox reactor with a nitrogen removal rate of 26.0 kg N m "3 day "1 . The anammox bacteria were identified as representing 'Candidatus Brocadia sinica' on the basis of phylogenetic analysis of rRNA operon sequences. Physiological characteristics examined were growth rate, kinetics of ammonium oxidation and nitrite reduction, temperature, pH and inhibition of anammox. The maximum specific growth rate (m max ) was 0.0041 h "1 , corresponding to a doubling time of 7 days. The half-saturation constants (K s ) for ammonium and nitrite of 'Ca. B. sinica' were 28±4 and 86±4 mM, respectively, higher than those of 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis'. The temperature and pH ranges of anammox activity were 25-45 6C and pH 6.5-8.8, respectively. Anammox activity was inhibited in the presence of nitrite (50 % inhibition at 16 mM), ethanol (91 % at 1 mM) and methanol (86 % at 1 mM). Anammox activities were 80 and 70 % of baseline in the presence of 20 mM phosphorus and 3 % salinity, respectively. The yield of biomass and dissolved organic carbon production in the culture supernatant were 0.062 and 0.005 mol C (mol NH þ 4 ) "1 , respectively.This study compared physiological differences between three anammox bacterial enrichment cultures to provide a better understanding of anammox niche specificity in natural and man-made ecosystems.

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 m) 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 mol cm ؊2 h ؊1 at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 mol 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.Anaerobic ammonium oxidation (anammox) is a microbiological oxidation of ammonium, with nitrite as the electron acceptor and dinitrogen gas as the main product, and is mediated by a group of deep-branching Planctomycete-like bacteria (12, 39). Anammox bacteria have been detected in different wastewater treatment facilities and environments in the world (for a review, see reference 34). Currently, four genera of anammox bacteria have been reported and named, including Brocadia, Kuenenia, Scalindua, and Anammoxoglobus (14,34).The anammox process is a new and promising alternative to conventional nitrogen removal processes. A better understanding of the ecophysiology (i.e., microbial community structure and in situ activity) of anammox bacteria in complex biofilms is essential for implementing the anammox process as a manageable and reliable nitrogen removal process in wastewater treatment. However, related information is limited because anammox bacteria are strict anaerobic autotrophs and thus have not yet been isolate...

The anaerobic ammonium oxidation (Anammox) process is a new efficient and cost effective method of ammonium removal from wastewater. Under strictly anoxic condition, ammonium is directly oxidised with nitrite as electron acceptor to dinitrogen gas. However, it is extremely difficult to cultivate Anammox bacteria due to their low growth rate. This suggests that a rapid and efficient start-up of Anammox process is the key to practical applications. To screen appropriate seeding sludge with high Anammox potential, a real-time quantitative PCR assay with newly designed primers has been developed. Thereafter, the seeding sludge with high abundance of Anammox bacteria (1.7 x 10(8) copies/mg-dry weight) was selected and inoculated into an upflow anaerobic biofilters (UABs). The UABs were operated for more than 1 year and the highest nitrogen removal rate of 24.0 kg-N m-3 day(-1) was attained. In addition, the ecophysiology of Anammox bacteria (spatial distribution and in situ activity) in biofilms was analysed by combining a full-cycle 16S rRNA approach and microelectrodes. The microelectrode measurement clearly revealed that a successive vertical zonation of the partial nitrification (NH4+ to NO2-), Anammox reaction and denitrification was developed in the biofilm in the UAB. This result agreed with the spatial distribution of corresponding bacterial populations in the biofilm. We linked the micro-scale information (i.e. single cell and/or biofilm levels) with the macro-scale information (i.e. the reactor level) to understand the details of Anammox reaction occurring in the UABs.

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