Microbial Composition and Structure of a Rotating Biological Contactor Biofilm Treating Ammonium-Rich Wastewater without Organic Carbon

Abstract

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in Kölliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.

Anaerobic ammonium oxidation in a bioreactor treating slaughterhouse wastewater

“…The light area represents hybridization of the biomass sample with the Amx 820 fluorescently labeled probe; stained cells in dense aggregates of coccoid morphology can be seen. The cells showed an inner area with a lower signal intensity, as had been described previously for the anammox-like microorganisms (Egli et al, 2003).…”

“…To confirm this hypothesis a fluorescence in situ hybridization analysis was carried out with a specific probe for the group of anammox-like bacteria (Amx 820) described in the literature (Egli et al, 2003). Hybridization of the enriched biomass with the specific probe can be observed in Figure 6.…”

“…For enumeration, the biomass w as resuspended in sodium pyrophospate (2.8gL -1 ) and homogenized for 1 min with an ultrasonic probe. Five µl of fixed samples were spotted onto microscopic slides and sequentially dehydrated and hybridized with a Cy3-labeled probe (Amx820) in accordance with Egli et al (2003). Slides were incubated in the washing buffer with 0.1 m gL -1 of 4,6-diamidino-2-phenylindole (DAPI) for 10 min at 48°C, dried and observed with an Olympus BX41 microscope equipped with MWU (DAPI) and MWIG (Cy3) filters.…”

Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

“…In addition, the concentrations of Nitrospira in reactors with different SRTs are of interest because Nitrospira is considered the primary organism responsible for nitrite oxidation in activated sludge. Reported percentages for Nitrospira by in situ hybridization in a sludge from an industrial plant connected to a rendering factory were 9 and 12% (26, 27), 2.5% for a combined activated sludgerotating biological contactor process (36), and less than 5% for another rotating biological contactor (15). For comparison purposes, the percentages of Nitrospira for these current reactors under study were calculated assuming 1 ribosomal operon copy per Nitrospira cell and 3.6 ribosomal operon copies per general bacterial cell.…”

Bacterial Communities and Antibiotic Resistance Communities in a Full-Scale Hospital Wastewater Treatment Plant by High-Throughput Pyrosequencing

“…Sphingobacteria is known to possess a tetracycline resistance gene which harbors a functional tet (X) gene for the degradation of tetracycline [33,34]. In addition, Sphingobacteria could form a biofilm and contribute to the treatment of ammonium-rich wastewater [35]. Interestingly, Cytophagia (14.8%) and Anaerolineae (7.6%) were predominant classes in the HWW, compared to the DWW (1.4% Cytophagia and 1.2% Anaerolineae).…”