Investigating the mediation of hemoglobin proteins in nitrification at low dissolved oxygen conditions

Due to the high aeration and energy requirements of nitrifying activated sludge processes, there is great interest in developing biological nitrifying processes that operate efficiently under low dissolved oxygen (DO) conditions. In this framework, the present study has investigated the acclimation of ammonia-oxidizing communities (AOC) to low DO concentrations. Under controlled laboratory conditions, two sequencing batch reactors seeded with activated sludge from the same source were operated at high DO (near saturation) and low DO (0.1 mg O2/L) concentrations for a period of 385 days. Stable and complete nitrification at low DO after an acclimation period of approximately 140 days was demonstrated. Modeling of oxygen transfer and uptake behavior demonstrated the low DO reactor to achieve equal performance to the high DO reactor, when the latter is operated at design DO concentration (2 mg O2/L), with approximately 20 % improvement in aeration requirements and oxygen mass transfer efficiency. The effect of DO on AOC dynamics was evaluated using the 16S rRNA gene as the basis for phylogenetic comparisons and organism quantification. Ammonium consumption by ammonia-oxidizing archaea and anaerobic ammonia-oxidizing bacteria was ruled out in both reactors. Even though N. europaea was the dominant AOB lineage in both SBRs at the end of operation, this enrichment could not be linked to acclimation to oxygen-limited conditions. This finding challenges the hypothesis that low DO conditions select for ammonia-oxidizing lineages characterized by high oxygen affinities, and points to the alternative mechanism of a physiological change of a generalist nitrifying community. Acclimation brought about increased specific oxygen uptake rates and enhanced expression of a particular heme protein in the soluble fraction of the biomass in the low DO reactor as compared to the high DO reactor. The heme protein induced was determined not to be any of the enzymes playing a role in ammonia metabolism of ammonia oxidizing bacteria, including a soluble oxidase and soluble peroxidase of unknown function. Further molecular studies are required to verify the hypothesis put forward in this study that the heme protein detected is a hemoglobin.