Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 3/13/2013
Publication Date: 3/13/2013
Citation: Nishiyama, T., Fujii, T., Vanotti, M.B., Martinez, J., Kunz, A., Furukawa, K. 2013. A bacterial community analysis using reverse transcription (RT) PCR which detects the bacteria with high activity in a wastewater treatment reactor. In: Proceedings of the 47th Annual Conference of Japan Society on Water Environment, Osaka Institute of Technology (OIT), March 13, 2013, Osaka, Japan. Interpretive Summary:
Technical Abstract: This research used reverse transcription polymerase chain reaction (RT-PCR) method to help detect active bacteria in a single-tank deammonification reactor combining partial nitritation and anammox. The single-tank aerobic deammonification reactor effectively removed the ammonia in anaerobically digested swine wastewater. Because of the stoichiometry and rate of reaction, it was predicted that ammonium oxidizing bacteria (AOB) and anammox bacteria (Planctomycetes bacteria) will show high activities in the single-tank. However, if the upstream reactor is operated in the anaerobic conditions, the discharged bacteria might die or rest their activities in the downstream aerobic reactor. Because the working and resting bacteria could not be discriminated by the ordinal bacterium community analyses with 16S rRNA gene sequences of bacteria, other methods are needed to reflect the activities of the bacterial community in wastewater treatment. It is reported that the number of ribosomes per cell is well correlated with the growth rate of purified bacterial cell and that the number of ribosomes in an actively growing cell is about tenfold over that in a resting cell. Thus, to know the community activities, we analyzed reverse transcription product of 16S rRNA in addition of the RNA gene of the sludge in an aerobic single-tank nitritation-anammox reactor where ammonium in anaerobic digestion effluent was treated. The numbers of clones detected by RNA-RT analyses shows the relative quantities of ribosomes, which correspond to physiological activities in the reactor. Many bacterial groups (23 operational taxonomic units, OTUs) were detected in the single-tank. Ammonia oxidizing bacteria (AOB) (OTU1-3), especially Nitrosococcus mobilis (OTU 1), had the most abundant ribosomes (51%), indicating flourishing growth and physiologically high activity. Candidatus Brocadia caroliniensis (anammox bacteria) was secondarily abundant (4.4% of ribosomes), being definitely working in the reactor. OTU 17 and 18 (Chlorobi) occupied only 2.2%, showing minor activity. The analyses of the influent (from anaerobic digestion) detected bacteria belonging to phyla Bacteroidetes, Firmicutes, Chloroflexi and Synergistetes, which are strictly or facultative anaerobes. Almost all clones in the anaerobically digested influent were not detected in the single-tank deammonification reactor. This indicated that most of bacteria in the inflow sludge had disappeared in the single-tank reactor and had little effect on the bacterial community in the reactor. Our results show that the microbial community analysis using RT-PCR method could detect the bacteria that were supposed to grow vigorously and be active in the reactor. This method was considered better method than the bacterium community analyses with 16S rRNA gene sequences for the analysis of wastewater treatment.