The methanol tolerance level of the anammox process was investigated in continuous feeding tests. In the conducted pulse methanol addition tests of gel carrier-immobilized anammox bacteria, the nitrogen removal performance was not significantly affected at influent methanol concentrations of up to 30 mg L−1; however, irreversible inhibition occurred at 40 mg L−1. In the absence of methanol acclimation, the anammox process showed low methanol tolerance levels. Moreover, the stepwise addition of 5 mg L−1 of methanol resulted in no anammox inhibition even when the methanol concentration became 100 mg L−1. The effluent total organic carbon concentration did not increase with the increase in the influent methanol concentration; this indicates that methanol was consumed along with the nitrate or nitrite by heterotrophic denitrification. These results show that the increase in the methanol tolerance level was due to the increase in the methanol consumption ability during the anammox process because of the increased denitrifying bacteria and not because the anammox bacteria exhibited resistance to methanol. The microbial community analysis revealed the dominant species in the gel carriers (Candidatus Jettenia asiatica), existence of methylotrophic bacteria, and growth in the methanol-consuming bacterial population.
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