Microbial Population Dynamics and Community Structure during the Formation of Nitrifying Granules to Treat Ammonia-Rich Inorganic Wastewater

Abstract

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...

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