Abstract
Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers.
Highlights
Nitrogen removal with anaerobic ammonium oxidizing bacteria is an emerging technology for treatment of high strength wastewater
The nitritation-anammox Moving bed biofilm reactors (MBBRs) was operated for three years
Since only trace amounts of organic carbon was added to the synthetic wastewater, and even though ammonia oxidizing bacteria (AOB) in the biofilm could be a source for organic carbon per se [21,22], it is unlikely that denitrification would account for any significant nitrogen removal
Summary
Nitrogen removal with anaerobic ammonium oxidizing (anammox) bacteria is an emerging technology for treatment of high strength wastewater. For moving bed biofilm reactors (MBBRs), which are used today in full-scale nitritation-anammox [15,16], a recent investigation has indicated a more orderly biofilm structure [17], but detailed analysis of biofilm establishment and stratification is still lacking for these systems. We have utilized a cryosectioning approach for the retrieval and study of intact biofilm of different age from protected compartments of carriers residing in a nitritation-anammox MBBR. By combining FISH-CLSM with a newly developed digital image analysis method [20], the three-dimensional stratification of AOB and anammox bacteria in intact biofilms was determined and quantified. Image analysis of total carrier compartment i.e., “wall-to-wall” biofilms, allowed an assessment of problems with biofilm retrieval and shrinkage from dehydration and cryo-embedding, which has often been suggested as a serious draw-back of these biofilm research methods
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