Evaluating the potential of engineering a microbial community of DNRA and anammox bacteria for mainstream treatment

Abstract

Currently, one of the main challenges in anammox-based systems for mainstream treatment is the stable production of nitrite within the system and the possibility that increased organic loading rates would lead to the out-competition of slow-growing anammox bacteria by heterotrophs. To study the potential of engineering a microbial community of DNRA and anammox bacteria for mainstream treatment, an anammox MBBR was started and operated for 180 days with a filling ratio of 40%, an HRT of 2 days, and a Nitrogen Load Rate (NLR) of 140 $g m^{-3} day^{-1}$ (ammonium plus nitrite). At the end of the operation, $NH_4^+$ removal and Total Nitrogen Removal (TNR) reached an average of about 80%. The ratios of nitrite consumed and nitrate produced per ammonium consumed were 1.24 and 0.23, respectively. Metagenomic analysis of the inoculum and the added virgin carriers in the MBBR on days 22, 105, and 145 showed the following relative abundance of C. anammox Brocadia: 82.1%, 41.9%, 3.6%, and 6.4%. From day 23 to 40 and day 53 to 69, a technical trouble in the reactor led to a ratio <0.2 of nitrate produced per ammonium consumed, including periods where nitrate was not measured in the effluent. This event is the most likely reason for the decrease in the relative abundance of anammox bacteria in the MBBR due to the out competition by the heterotroph community present in the anammox carriers. Despite the decreased relative abundance of anammox bacteria, it was observed an anaerobic consumption of ammonium coupled with nitrite consumption and nitrate production in ratios similar to the theoretical anammox stoichiometry during the whole operation of the reactor. Thus, anammox activity was concluded to be present during the 180 days of operation. Parallel to the operation of the anammox MBBR, a total of 9 DNRA-positive bacteria, all identified as the genus Citrobacter, were isolated from the anoxic tank of Daejeon WWTP using two different mediums for the isolation. A mixed DNRA culture was prepared with three Citrobacter isolates. At a high C-to-N ratio of 10, the mixed culture transformed nearly all the nitrate into ammonium, and no nitrite was accumulated in the medium. At a low C-to-N ratio of 1.25, the mixed culture of DNRA accumulated 50% of the nitrate in the form of nitrite, produced 12% ammonium, and the nitrate remaining reached 16%. This study demonstrates the potential of a mixed culture of DNRA bacteria to perform partial DNRA and accumulate nitrite that could be used as a substrate for anammox reaction. We suggest the engineering of a community composed of anammox and DNRA bacteria to solve the anammox-related mainstream challenges. (The author of this thesis is a Global Korea Scholarship scholar sponsored by the Korean Government)