Effect of Environmental Conditions, Wastewater Constituents and Contaminants on the Anaerobic Ammonium Oxidation (Anammox) Process

Abstract

The anaerobic ammonium oxidation (anammox) is a microbial process that plays an important role in the global nitrogen cycle. Due to their unique metabolism, the chemolithoautotrophic anammox bacteria can convert ammonium (NH4+) and nitrite (NO2-) to produce dinitrogen gas (N2) under anaerobic conditions. Since the discovery of the anammox bacteria in the early 1990s, there has been a huge interest in studying the metabolism of the anammox bacteria, particularly aimed towards the application of the anammox process for nitrogen removal during wastewater treatment. Successful implementation of the anammox process for wastewater treatment depends on understanding the factors that can potentially affect the process stability. Thus, our study was aimed towards studying the effect of environmental conditions, wastewater constituents and contaminants on the anammox process. Since the anammox bacteria are slow growing in nature, the effect of such factors has been traditionally evaluated based on anammox bacteria activity rather than bacterial growth. Long term bioreactor studies provide the opportunity to study the growth of the anammox bacteria. However, due to extensive time and equipment required for such testing, of a wide variety of conditions is not feasible. Thus, a novel batch bioassay was developed such that the growth of the anammox bacteria would be measured in a short period of time using simple apparatus and simple analytical measurements such as N2 gas generation. Using this novel bioassay, the effect of environmental conditions such as temperature and pH as well as the effect of common wastewater constituents such as NH4+ on the anammox growth was evaluated. In addition, the effect of trace elements and iron on the anammox growth was also measured. Following the investigation of the effects of various factors on the anammox growth, the effect of a ubiquitous component of wastewater, organic carbon, on the anammox process was investigated in detail. A comparative study was carried out between two anammox moving bed biofilm reactor (MBBR), a control reactor (CR) receiving no organic substrates and a treatment reactor (TR) receiving variable loadings of acetate used as a model for organic substrates. The effect of acetate on the reactor performance and microbial community were evaluated. The results showed that the total nitrogen removal (TNR) improved by 8 to 13% when a low concentration of acetate of 58.7 mg L-1 chemical oxygen demand (COD), was introduced in the TR. This was due to complete removal of the anammox substrates, NO2- and NH4+, combined with the removal of NO3- (a byproduct of the anammox process) potentially consumed by the heterotrophic/denitrifying bacteria enriched in the culture. However, increasing the acetate concentration, up to 153 mg L-1 COD, led to a 37% decrease in the TNR in the TR. The loss in the TNR was attributed to the decline in the NH4+ removal since high organic substrate loading promoted denitritation, thereby removing NO2- that would be needed to achieve NH4+ removal by the anammox bacteria. Wastewater is a complex mixture containing inorganic aqueous species, natural organic matter, and may also contain organic contaminants. The presence of organic contaminants is of concern as some of these compounds can be particularly toxic towards specific microbial communities. One of the prominent examples is the severe toxicity of nitrogen containing heterocyclic aromatic compounds, or the azole compounds towards nitrification (aerobic microbial oxidation of NH4+). Since nitrifying bacteria are a crucial part of the biological wastewater treatment process, evaluation of the effect of organic contaminants on other biotechnologies used in wastewater treatment is essential. Thus, the acute toxicity of azole compounds towards the anammox bacteria was evaluated. The results showed that most of the azole compounds tested were either nontoxic or at best mildly toxic towards the anammox bacteria, except for 1H-benzotriazole and 5-methyl-1H-benzotriazole causing 50% decrease in anammox activity (IC50) at concentrations of 19.6 and 17.8 mg L-1, respectively. Overall, the anammox bacteria were found to be far less sensitive to azoles compared to nitrifying bacteria. The evaluation of the toxicity of the azole compounds led to the investigation of the fate of the azole compounds in the presence of the anammox enrichment culture. This investigation led to the discovery of the biotransformation of the azole compounds 1H-pyrazole and 1H-1,2,4-triazole by the anammox enrichment culture. 1H-pyrazole and 1H-1,2,4-triazole were biotransformed yielding major biotransformation products, 3-amino-1H-pyrazole and 3-amino-1H-1,2,4-triazole, respectively. NO3- and glucose greatly stimulated the biotransformation. 80.7% of the 1H-pyrazole and 16.4% of the 1H-1,2,4-triazole were biotransformed in an incubation period of 6 days, when provided with 1 mmol L-1 of the respective azole compounds, and of 2 mmol L-1 each of glucose and NO3-. Overall, this investigation provided insights into the effects of environmental conditions such as pH and temperature, as well as common wastewater constituents such as NH4+, organic carbon on the anammox process. The study of toxicity of contaminants such as the azole compounds as well as the discovery of the novel biotransformation capabilities of the anammox enrichment culture have expanded our understanding of the anammox process.