Effects of triclosan exposure on nitrification in activated sludge, biofilms, and pure cultures of nitrifying bacteria

Emerging contaminants, such as pharmaceuticals or personal care products, have the potential to impact many wastewater treatment processes due to their antimicrobial properties. Nitrifying bacteria initiate the nitrogen removal process in wastewater treatment, and are particularly sensitive to inhibition by these and other contaminants. The impacts of the emerging contaminant triclosan (TCS) on two common nitrifying bacteria were evaluated under multiple growth conditions. The resilience of biofilms and suspended cell cultures of the ammonia oxidizing bacterium (AOB) Nitrosomonas europaea was compared during TCS exposure. Impacts of TCS on Nitrobacter winogradskyi, a common nitrite oxidizing bacterium (NOB), were also considered. Lastly, activated sludge biofilms and suspended cells were also exposed to TCS to further evaluate impacts on nitrification. Triclosan at part per million levels was found to reduce respiration in nitrifying biofilms, and NOB were much more impacted by TCS than AOB. Interestingly, biofilms of N. europaea were just as impacted by TCS as suspended cells. Triclosan adsorbed strongly to cellular material and degradation was only observed in activated sludge at low concentrations. Altogether, TCS was found to reduce nitrification by AOB and NOB, and the results suggest that its presence at high levels in wastewater treatment is likely to have negative consequences. 1 CHAPTER ONE – INTRODUCTION Effective wastewater treatment is necessary for protection of both human health and natural water systems. Nitrogen removal is particularly important in wastewater treatment, as its release can cause eutrophication of water systems, leading to algal blooms, oxygen depletion, and fish die off (1). Microbial activity is used in wastewater treatment plants to remove nitrogen and other nutrients from wastewater. Nitrification, the first step in the nitrogen removal process, consists of the microbial conversion of ammonia (NH3) to nitrite (NO2) by ammonia oxidizing bacteria (AOB). Nitrite oxidizing bacteria (NOB) then convert NO2 to nitrate (NO3). Nitrate is eventually converted to harmless dinitrogen gas by denitrifying bacteria, thus completing the nitrogen removal cycle. Of specific interest here are the first steps in the nitrogen removal process, or the conversion of ammonia to nitrite by AOB, followed by the conversion of nitrite to nitrate by NOB. The first step is significant because it is catalyzed in wastewater in large part by Nitrosomonas europaea, a well-studied microorganism on which most knowledge of AOB is based (2). N. europaea is ubiquitous in wastewater treatment, and is a fairly slow-growing microorganism that is sensitive to various changes in its growth environment, such as the introduction of chemical contaminants (3-8). In turn, this makes the ammonia oxidation step vulnerable to contaminants in wastewater, thereby jeopardizing the success of the entire nitrogen removal process—if ammonia oxidation cannot proceed, nitrification will not occur, and neither will denitrification. 2 In particular, the enzyme used for ammonia oxidation (ammonia monooxygenase or AMO) is non-specific and capable of oxidizing a wide variety of substrates other than ammonia (3-5, 9). Cometabolism by this enzyme does not support growth of N. europaea, and therefore can inhibit successful nitrification. AMO can oxidize multiple aliphatic and aromatic compounds, many of which are found in municipal and industrial wastewater (10-12). Consequently, wastewater contamination by these groups of compounds can be a barrier to successful nitrification due to the non-specificity of the AMO enzyme. Another ubiquitous wastewater bacterium, Nitrobacter winogradskyi, can catalyze the second step in nitrification—the conversion of nitrite to nitrate. N. winogradskyi is less well-studied than N. europaea, but is still typically used as the model organism for nitrite oxidation (13). The nitrite oxidoreductase (NXR) enzyme is used by N. winogradskyi for nitrite consumption. Little is known about inhibition of this enzyme, but NXR belongs to a family of non-specific enzymes, and therefore potentially may cometabolize non-growth-supporting compounds in a similar manner as AMO, leading again to inhibition of the nitrification process (13, 14). Thus, AOB and NOB are sensitive microorganisms that use enzymes that can interact with or cometabolize many different aliphatic or aromatic compounds. Emerging contaminants (ECs) such as pharmaceuticals and personal care products are typically of an aliphatic or aromatic nature and readily enter wastewater due to widespread use (15, 16). ECs therefore pose a large threat to successful nitrogen removal due to the vulnerability of AOB and NOB. Typically present in ng to μg per liter concentrations in 3 wastewater treatment plant (WWTP) influent and effluent, ECs have the potential to adversely impact ecology and human health, and are generally not monitored or regulated in surface waters (17). Their impacts on surface waters and wastewater treatment efficacy are poorly understood. Triclosan is one such contaminant that is used for household applications and is prevalent in wastewater. Triclosan (TCS) is a broad-spectrum antimicrobial agent that has been incorporated into various personal care products. It is widespread in the environment—in the US, WWTP influent can contain up to 86 μg/l TCS, surface river water up to 35 ng/l, and groundwater up to 2.1 μg/l (15, 18, 19). At low concentrations, TCS is bacteriostatic, and at higher levels it is bactericidal (20). TCS is known to inhibit bacterial reproduction via inhibition of the enzymes responsible for cell membrane synthesis and repair (18, 21), and its aromatic structure suggests that it could also be a potent inhibitor of AMO. Previous studies have indicated that TCS significantly inhibits nitrification in activated sludge, with as little as 1 mg/l causing nearly 20% inhibition (22). Inhibition of both AOB and NOB activity and growth has been observed (23, 24). However, detailed studies of the impacts of triclosan on specific bacterial species present in activated sludge are largely absent. In particular, the effects of TCS on pure cultures of N. europaea and N. winogradskyi are not well characterized. No studies have been conducted on the impacts of TCS on nitrifying biofilms. The lack of information available on TCS impacts on nitrifying biofilms is particularly glaring, since biofilms can act as a major bacterial defense against antimicrobial compounds. Biofilms are dense communities of cells that are attached to a 4 surface and enclosed in an extracellular polymeric matrix (3). Both slower growth of cells within the biofilm and contaminant diffusion limitations increase bacterial resilience in the face of growth-inhibiting conditions (25). Wastewater treatment plants frequently use biofilm-based treatment methods for nitrification, and therefore it is important to determine if the effects of TCS on biofilm-grown cells will differ from those in planktonic treatment systems. Studies have shown that biofilms of N. europaea were less inhibited by the aromatic compounds nitrapyrin, toluene, and phenol than their planktonic counterparts (4, 5, 26). Since TCS is also aromatic, these studies suggest that inhibition by TCS may be reduced when cells are grown as a biofilm. The prevalence of TCS in the environment and the lack of information on its impacts on wastewater treatment efficacy make investigating its effects important. Knowledge of its effects on two ubiquitous wastewater bacteria—N. europaea and N. winogradskyi—is largely undetermined, and therefore one objective of this study was to investigate the effect of triclosan on ammonia and nitrite oxidation by pure cultures of both of the aforementioned bacteria. The inhibition mechanism of triclosan with the AMO enzyme was also investigated. Thirdly, impacts of TCS on ammonia oxidation by biofilms and suspended cell cultures of N. europaea were compared to determine if attached growth had any impact on bacterial resistance. Nitrification efficacy by planktonic activated sludge cells and biofilms during TCS exposure was also investigated in order to compare the response of mixed cultures to that of pure cultures, and oxygen consumption by nitrifying biofilms during triclosan exposure was considered. Finally, triclosan fate in all cultures and growth modes was 5 measured to determine if degradation was possible by any of the bacterial species of interest. This thesis consists first of a literature review that explores current information on nitrifying biofilms and their role in wastewater treatment. Nitrification microbiology and inhibition mechanisms relevant to AOB and NOB are also examined. Finally, characteristics of triclosan and current research on its impacts are summarized, with particular attention paid to studies of its effects on nitrification in WWTPs. Experimental methods are then described, followed by results and a discussion of the results’ relevance and applicability. Lastly, conclusions are presented. 6 CHAPTER TWO – LITERATURE REVIEW