This study evaluates the community structure in nitrifying granules (average diameter of 1600 mum) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluorescence in situ hybridization revealed that ammonia-oxidizing bacteria dominated within the first 200 mum below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 mum, while heterotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Microprofiles of NH(4)(+), NO(2)(-), NO(3)(-) and O(2) concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria-substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the interaction between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone.

Microbial population dynamics were investigated during the formation of nitrifying granules in an aerobic upflow fluidized bed (AUFB) reactor fed ammonia as a sole energy source. Analyses of clone libraries of 16S rRNA gene and the ammonia monooxygenase subunit A gene (amoA) revealed that although the clones obtained from the seed sludge were widely distributed among the ammonia-oxidizing bacteria (AOB) isolates, the community structure of AOB shifted towards the Nitrosomonas mobilis lineage as granulation proceeded. Quantitative fluorescence in situ hybridization showed that changes in the bacterial population occurred concomitantly with changes in nitrification performance and the size of granules. AOB associated with the N. mobilis lineage were predominant in the early stages as nitrifying granules formed (average diameter, 126 µm). In mature granules (average diameter, 270 µm), at least three types of AOB, N. mobilis, Nitrosomonas oligotropha, and Nitrosomonas europaea, formed different niches and coexisted. Nitrite-oxidizing bacteria (NOB) affiliated with Nitrospira spp. were detected in the start-up period, but were replaced by NOB affiliated with Nitrobacter spp. after granules formed.Key words: community structure, population dynamics, nitrifying bacteriaThe removal of biological nitrogen from various types of wastewater is becoming increasingly important owing to the implementation of strict regulations concerning nitrogen discharge. The process of nitrification involves the conversion of ammonia (NH3) or ammonium ions (NH4. Two types of nitrifying bacteria, namely, lithoautotrophic ammonia-oxidizing bacteria (AOB) and lithoautotrophic nitrite-oxidizing bacteria (NOB), play important roles in this process. AOB are classified into two phylogenetic lineages based on their 16S rRNA gene sequences; Nitrosomonas sp. and Nitrosospira sp. belonging to Betaproteobacteria, and Nitrosococcus oceani and Nitrosococcus halophilus belonging to Gammaproteobacteria (19). NOB are mainly classified into two phylogenetic lineages based on the cell morphology of isolated organisms and on 16S rRNA gene sequences. The most intensively studied NOB, Nitrobacter and Nitrospira, fall within the Alphaproeobacteria and separate phyla in Bacteria, respectively (2).It is generally accepted that nitrifying bacteria have a low growth rate and tend to be washed out from reactors. Thus, it is difficult to retain large numbers of them within a reactor. Therefore, there is a need for a simple yet effective method of immobilizing nitrifying bacteria within a reactor. Nitrifying granule production is one method of immobilization used to preserve a large population of nitrifying bacteria within a reactor (12,(40)(41)(42)(43). Tsuneda et al. (41) produced nitrifying granules in inorganic wastewater using an aerobic upflow fluidized bed (AUFB) reactor. These granules exhibited a good settling ability and a high rate of ammonia removal (1.5 kg-N m −3 d −1 ). Therefore, nitrifying granule production is a promising method for the effective remov...

In this study, the effectiveness of aerobic granular sludge as seed sludge for rapid start-up of nitrifying processes was investigated using a laboratory-scale continuous stirred-tank reactor (CSTR) fed with completely inorganic wastewater which contained a high concentration of ammonia. Even when a large amount of granular biomass was inoculated in the reactor, and the characteristics of influent wastewater were abruptly changed, excess biomass washout was not observed, and biomass concentration was kept high at the start-up period due to high settling ability of the aerobic granular sludge. As a result, an ammonia removal rate immediately increased and reached more than 1.0 kg N/m(3)/d within 20 days and up to 1.8 kg N/m(3)/d on day 39. Subsequently, high rate nitritation was stably attained during 100 days. However, nitrite accumulation had been observed for 140 days before attaining complete nitrification to nitrate. Fluorescence in situ hybridization analysis revealed the increase in amount of ammonia-oxidizing bacteria which existed in the outer edge of the granular sludge during the start-up period. This microbial ecological change would make it possible to attain high rate ammonia removal.