Understanding the biodegradation products and pathways of selected pharmaceuticals atenolol and acyclovir by an enriched nitrifying sludge through experiments and modeling

Abstract

Pharmaceuticals could potentially pose detrimental effects on aquatic ecosystems and human health, with wastewater treatment being one of the major pathways for pharmaceuticals to enter into the environment. Enhanced removal of pharmaceuticals has been widely observed by ammonia oxidizing bacteria (AOB). However, the degradation mechanisms involved in pharmaceutical biotransformation were still ambiguous. In addition, pharmaceutical biotransformation models have not yet considered transformation products associated with the metabolic type of microorganisms. The overall objective of this thesis is to understand the contribution of different metabolisms by relevant microorganisms to the biotransformation of selected pharmaceuticals (i.e., atenolol and acyclovir) accompanied with the formation of their transformation products in an enriched nitrifying sludge, in terms of product identification, influencing factor assessment and mathematical modeling. Biodegradation of atenolol in an enriched nitrifying sludge was studied under different metabolic conditions. The positive link was observed between atenolol biodegradation and the cometabolic activity of AOB in the presence of ammonium, likely due to a broad substrate spectrum of ammonia monooxygenase (AMO). In the presence of ammonium, atenolol was transformed into P267 (atenolol acid) and three new products including P117 (1-isopropylamino-2-propanol), P167 (1-amino-3-phenoxy-2-propanol), and an unknown product P227. However, atenolol was only transformed to P267 and P227 in the absence of ammonium. The formation of P117, P167 and P227 was further confirmed from follow-up atenolol acid biodegradation experiments in the presence of ammonium. Therefore, a tentative biodegradation pathway of atenolol is proposed in the enriched nitrifying sludge, consisting of two steps regardless of the presence of ammonium: i) microbial amide-bond hydrolysis to carboxyl group, producing P267 and ii) a possible formation of P227 and other two cometabolically induced reactions: iii) breakage of ether bond in the alkyl side chain to produce P117 and iv) a minor pathway through N-dealkylation and loss of acetamide moiety from the aromatic ring, yielding P167. An important insight was herein provided regarding the biotransformation pathways of pharmaceuticals under different metabolic conditions. To further assess the influence of the growth substrate on atenolol biotransformation in enriched nitrifying culture, different ammonium concentrations were applied constantly to study atenolol degradation kinetics and the biotransformation product formation dynamics. Higher ammonium concentrations led to the lower atenolol removal efficiencies probably due to the substrate competition between ammonium and atenolol. The formation of biotransformation product atenolol acid was positively related to the ammonium oxidation activity, resulting in a higher amount of atenolol acid at the end of experiments at higher ammonium concentrations. Positive correlations between ammonia oxidation rate and atenolol degradation rate at ammonium levels of both 25 and 50 mg-N L-1, suggested the cometabolism of atenolol by AOB in the presence of ammonium. The revealed biotransformation reaction, i.e., hydroxylation on amide group to carboxylic group, could be catalyzed by the non-specific enzyme AMO. It was also demonstrated the formation of atenolol acid was independent on the ammonium availability. Biotransformation of acyclovir by the enriched nitrifying culture was evaluated under different metabolic conditions at different initial levels of acyclovir (15 mg L-1 and 15 mg L-1). Higher degradation rates of acyclovir were observed under higher ammonia oxidation rates in the presence of ammonium than those constant degradation rates in the absence of ammonium. The positive correlation between acyclovir degradation rate and ammonia oxidation rate further confirmed the cometabolic biodegradation of acyclovir by AOB in the presence of ammonium. Carboxy-acyclovir (P239) was produced from acyclovir biodegradation. The main biotransformation pathway was aerobic oxidation of the terminal hydroxyl group, which was independent on the metabolic type (i.e. cometabolism or metabolism). This enzyme-linked reaction might be catalyzed by monooxygenase from AOB or heterotrophs (HET). The formation of carboxy-acyclovir was irrelevant to the acyclovir concentrations applied, indicating the revealed biotransformation pathway might be dominant in acyclovir removal during wastewater treatment processes. A comprehensive mathematical model was developed therein to describe and evaluate the biodegradation of pharmaceuticals accompanied with the formation of biotransformation products by enriched nitrifying culture. Microbial processes including cometabolism induced by AOB growth, metabolism by AOB, cometabolism by HET growth and metabolism by HET were involved. Model calibration and validation were accomplished using pharmaceutical biodegradation experimental data at environmentally-relevant initial concentrations, demonstrating a good prediction performance of the developed model under different metabolic conditions and the reliability of the established model in predicting different pharmaceuticals biotransformation. The linear positive relationship between ammonia oxidation rate and pharmaceutical degradation rate confirmed the potential role of cometabolism induced by AOB in pharmaceutical removal. Dissolved oxygen (DO) was able to regulate the pharmaceutical biotransformation cometabolically and the substrate competition between ammonium and pharmaceuticals existed especially at higher ammonium concentrations. The outcomes of this thesis improve our understanding of the microbially induced metabolic types involved in the pharmaceutical biotransformation in enriched nitrifying sludge. Potential application of these insights into the fate of pharmaceuticals in engineered systems could help optimize their removal during wastewater treatment processes.