Inhibitory Impact of Nitrite on the Anaerobic Ammonium Oxidizing (Anammox) Bacteria: Inhibition Mechanisms and Strategies to Improve the Reliability of the Anammox Process as a N-Removal Technology

Abstract

The anaerobic oxidation of ammonium (anammox) with nitrite as electron acceptor is a microbial process that generates nitrogen gas as main final product. After being discovered in the Netherlands in the 1990s, anammox has been applied in state-of-the-art biotechnologies for the removal of N pollution from ammonium rich wastewaters. The anammox process offers significant advantages over traditional nitrification-denitrification based processes. Since anammox does not need elemental oxygen, it allows for important savings in aeration. Furthermore, due to the autotrophic nature of the bacteria, anammox does not require external addition of electron donor, often needed in systems with post-denitrification. Although the anammox bacteria have high specific activity, they are slow growing, with doubling times that can range from 10 to 25 d. Therefore, in case of a toxic event causing the death of the biomass, a long recovery period will be required to reestablish full treatment capacity. The purpose of this work is to investigate the inhibition of anammox bacteria by compounds commonly found in wastewaters, including substrates, intermediates and products of the anammox reaction. Among common wastewater constituents, sulfide was shown to be especially harmful, causing complete inhibition of anammox activity at concentrations as low as 11 mg H₂S L⁻¹. Dissolved oxygen was moderately toxic with a 50% inhibiting concentration of 2.3 and 3.8 mg L⁻¹ to granular and suspended anammox cultures, respectively. Among the various compounds involved in the anammox reaction, special attention was paid to nitrite. Numerous literature reports have indicated inhibition of anammox bacteria by its terminal electron acceptor. However to date, there is no consensus explanation as to the mechanism of nitrite inhibition nor on how the inhibition is impacted by variations in the physiological status of anammox cells. The mechanisms of anammox inhibition by nitrite were thoroughly investigated in batch and continuous experiments of this dissertation. The results of this work demonstrate that conditions hindering generation of metabolic energy have a detrimental effect on the tolerance of anammox cells to toxic levels of nitrite. The absence of ammonium during events of nitrite exposure was shown to exacerbate its toxic effect. As a result of nitrite inhibition, nitric oxide, an intermediate of the anammox reaction, accumulated in the head space of the batch experiments. Moreover, nitrite inhibition was enhanced at the lowest range of pH tested (6.4-7.2), while same nitrite concentrations caused no inhibition under mildly alkaline conditions (7.5-7.8). Although other authors have relied on the classic concept that undissociated nitrous acid is the species responsible for the inhibition, the results in this work indicate that the pH affects the inhibitory effect of nitrite, irrespective of the free nitrous acid concentration. Nitrite stress triggered an active response of the anammox bacteria, which temporarily increased their ATP content to mitigate the inhibition. Additionally, starvation of anammox microorganisms, caused during storage or by sustained underloading of bioreactors, was found to limit the capacity of the bacteria to tolerate exposure to nitrite. The results of this dissertation indicate that the tolerance of anammox bacteria to NO₂⁻ inhibition relies on limiting its accumulation in sensitive regions of the cell. Active metabolism in presence of NH₄⁺ allows for active consumption of NO₂⁻, avoiding accumulation of toxic intracellular NO₂⁻ concentrations. Furthermore, secondary active transport proteins may be used by anammox bacteria to translocate nitrite to non-sensitive compartments. Nitrite active transport relies on a proton motive force. Therefore, conditions such as low pH (below 7.4) or absence of energy sources, which may disturb the maintenance of the intracellular proton gradient, will increase the sensitivity of anammox cells to NO₂⁻ inhibition. Strategies for the operation and control of anammox bioreactors must be designed to avoid exposure of the biomass to nitrite under the absence of ammonium, low pH or after periods of starvation.