Three-Dimensional Stratification of Bacterial Biofilm Populations in a Moving Bed Biofilm Reactor for Nitritation-Anammox

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.

Dynamics of biofilm formation on microscopic slides submerged in an anammox bioreactor

“…This suggestion was confirmed by microsensor mea surements [10]. In order to provide conditions for immobilization, bioreactors for the cultivation of anammox bacteria are equipped with special carriers, usually polymeric [7,8] or made of nonwoven fabric [11,12]. The shape and size of the biofilm depend on the size and localization of the carriers and on the cul tivation conditions.…”

“…The shape and size of the biofilm depend on the size and localization of the carriers and on the cul tivation conditions. Biofilms can be three dimen sional, spherical, or thin and flat [7,8,11]. In OLAND and SBR type reactors, which are not equipped with 1 Corresponding author; e mail: botchkovaekat@gmail.com carriers and involve media stirring during cultivation, anammox bacteria form granules and flocs 1-2 and 0.5 mm in diameter, respectively [13,14].…”

“…It is suggested that nitrifiers are the first to develop during biofilm formation de novo, forming the initial biofilm with anaerobic microniches, which are subsequently occupied by anammox bacteria. This process can last for up to five months [8]. In some cases, a biofilm has one more layer, the deepest one, localized in the middle part of the granule.…”

“…It is applied in wastewater treatment bioreactors of different scale, from laboratory to industrial, including the reactors for nitrogen rich wastewater treatment. In all habitats anammox bacteria exhibit a strong tendency for attached growth and biofilm for mation [7,8]. This tendency is especially important in bioreactors, since anammox bacteria have an extremely low growth rate (doubling time is 11 days, according to Strous et al) [9], and attached growth decreases the undesirable washout of anammox cells from the bioreactor.…”

“…Another group of filamentous prokaryotes, members of the phylum Bacteroidetes, are also found in biofilms. Their cells are distributed evenly along biofilms, suggesting the structural func tion of these bacteria in biofilms [8]. Anammox bacte ria were cultivated in an anaerobic laboratory upflow bioreactor with working volume of 1 L and cell immo bilization on flexible carriers "ersh."…”