Improvement of the Reliability of the Anaerobic Ammonium Oxidation (Anammox) Process: Mechanisms of Nitrite Inhibition and Recovery Strategies

Abstract

Anaerobic ammonium oxidizing (Anammox) bacteria are known to utilize ammonium and nitrite as electron donor and acceptor, respectively, to produce nitrogen gas as their main final product with by-product formation of nitrate. Anammox bacteria provide the advantages of significant saving in aeration, no requirement for external electron donor, reduction of greenhouse gas emission, lowered sludge production, and higher specific nitrogen-removing activity compared to the conventional nitrification-denitrification process used in nutritent-N removal. Therefore, the anammox process has recently been widely studied and applied as a state-of-the-art biotechnology to remove nutrient nitrogen from ammonium-rich wastewater. However, the inhibitory impact of nitrite (one of the two main substrates) on the anammox process has been reported in both lab- and full-scale anammox systems, which limits the application of anammox process. Based on the current knowledge, a wide range of nitrite concentrations causing anammmox inhibition was reported to be correlated to the pH and energy status of anammox bacteria, and the understanding of the mechanisms of nitrite inhibition to anammox bacteria is still not clear. Therefore, the purpose of this work is to investigate the mechanism of nitrite inhibition and develop a strategy for recovering nitrite inhibited anammox processes. The effects of pre-exposing anammox bacteria to nitrite alone on their subsequent activity and metabolism after ammonium has been added was evaluated in batch bioassays. The results showed that pre-exposure of anammox bacteria to nitrite without ammonium caused dramatic inhibition with observed 50% inhibition concentration (IC₅₀) of 52 mg NO₂⁻-N L⁻¹, compared to an IC₅₀ of 384 mg NO₂⁻-N L⁻¹ obtained in the control group with ammonium and nitrite added simultaneously. The accumulated nitric oxide (NO) found in the group with anammox bacteria pre-inhibited by nitrite indicated that pre-exposure to nitrite most likely caused disruption of the anammox biochemistry by interrupting the hydrazine synthesis step. Meanwhile, active metabolic status of anammox bacteria fueled by a strong proton gradient maintained by controlling pH in the optimal range of 7.2-7.8 enhanced the ability of anammox bacteria to tolerate nitrite inhibition. This was evaluated by depleting the proton gradient by utilizing two uncouplers of respiration, 2,4 dinitrophenol (24DNP) and carbonyl cyanide m-chlorophenyl hydrazine (CCCP). The results showed that presence of 0.28 mg CCCP L⁻¹ caused enhancement of nitrite inhibition to anammox bacteria, with a calculated IC₅₀ of 18.7 mg NO₂⁻-N L⁻¹ compared to an IC₅₀ greater than 150 mg NO₂⁻-N L⁻¹ in the control group lacking CCCP. Meanwhile, the sensitivity to NO₂⁻ was 3 times in anammox bacteria pre-exposed to 100 mg NO₂⁻ L⁻¹ for 24 h than in treatments lacking 37.8 mg 24DNP L⁻¹. A potential strategy of detoxifying the nitrite inhibition to anammox bacteria was proposed by using nitrate due to the finding of the presence of NarK, with potential function of NO₃⁻/NO₂⁻ antiporter, encoded in the anammox genome. Both batch- and continuous-experiments were carried out to test this hypothesis. The relative contribution of nitrate to nitrite detoxification was found to be pH dependent but the attenuation of nitrite inhibition is independent of the proton motive force which is supported by the result that nitrate caused almost complete attenuation of nitrite toxicity in cells exposed to the proton gradient disruptor, CCCP, at pH 7.5. Increase in nitrate concentration also improved the attenuation of nitrite inhibition to anammox process, with the maximum recovery being achieved at 0.85 mM in batch experiment and 2.0 mM for 3 days in continuous-fed bioreactor. Moreover, the timing of nitrate addition is significant because long-term nitrite inhibition of anammox biomass results in irreversible damage of the cells, under which condition addition of nitrate showed no positive impact on recovery of nitrite inhibition. This study also investigated the inhibitory effects of six metals (Cu²⁺, Cd²⁺, Ni²⁺, Zn²⁺, Pb²⁺, and molybdate) commonly found in landfill leachate on anammox activity. Results from batch bioassays indicated that precipitation reactions decreased considerably the soluble concentration of the cationic metals. Cu, Zn, Cd, and Ni were the most toxic metals with 50% inhibiting soluble concentrations of 4.2, 7.6, 11.2, and 48.6 mg L⁻¹, respectively. Molybdate and Pb²⁺ were not or only moderately inhibitory at the highest soluble concentrations tested (22.7 mg Mo L-1 and 6.0 mg Pb L⁻¹, respectively). Microbial inhibition was strongly correlated with both the added- and the dissolved metal concentration. These relationships could be described by a noncompetitive inhibition model for all inhibitory metals except for Pb. The results of this dissertation indicate that the resistance of anammox bacteria to nitrite inhibition could be enhanced by maintaining either an active metabolism in simultaneous presence of ammonium and nitrite, or sufficient proton gradient to enable relieving nitrite accumulation in sensitive regions of the anammox cells through an active nitrite transport system. An alternative nitrite detoxification mechanism was also demonstrated which relied on a secondary transport system facilitated by exogenous nitrate to avoid the accumulation of toxic intraorganelle nitrite concentration. Moreover, the results obtained in the study investigating the impact of heavy metals on anammox process provides new insights on the sensitivity of anammox bacteria to common metals and can be used to devise strategies to minimize inhibition of the anammox process when treating wastewater containing heavy metals.