David T. Tan

Tertiary-treated municipal wastewater is a significant point source of antibiotic resistance genes into Duluth-Superior Harbor.

In this study, the impact of tertiary-treated municipal wastewater on the quantity of several antibiotic resistance determinants in Duluth-Superior Harbor was investigated by collecting surface water and sediment samples from 13 locations in Duluth-Superior Harbor, the St. Louis River, and Lake Superior. Quantitative PCR (qPCR) was used to target three different genes encoding resistance to tetracycline (tet(A), tet(X), and tet(W)), the gene encoding the integrase of class 1 integrons (intI1), and total bacterial abundance (16S rRNA genes) as well as total and human fecal contamination levels (16S rRNA genes specific to the genus Bacteroides ). The quantities of tet(A), tet(X), tet(W), intI1, total Bacteroides , and human-specific Bacteroides were typically 20-fold higher in the tertiary-treated wastewater than in nearby surface water samples. In contrast, the quantities of these genes in the St. Louis River and Lake Superior were typically below detection. Analysis of sequences of tet(W) gene fragments from four different samples collected throughout the study site supported the conclusion that tertiary-treated municipal wastewater is a point source of resistance genes into Duluth-Superior Harbor. This study demonstrates that the discharge of exceptionally treated municipal wastewater can have a statistically significant effect on the quantities of antibiotic resistance genes in otherwise pristine surface waters.

Direct and indirect photolysis of sulfamethoxazole and trimethoprim in wastewater treatment plant effluent

The photolysis of two antibacterial compounds, sulfamethoxazole and trimethoprim, was studied in wastewater effluent. The rate of loss of sulfamethoxazole was enhanced in wastewater effluent due to indirect photolysis reactions, specifically reactions with hydroxyl radicals and triplet excited state effluent organic matter. Photolysis in the presence of natural organic matter, however, did not lead to enhanced degradation of sulfamethoxazole. Trimethoprim was also found to be susceptible to indirect photolysis in wastewater effluents, with hydroxyl radical and triplet excited effluent organic matter being the responsible species. Deoxygenation of solutions led to more rapid direct photolysis of sulfamethoxazole and trimethoprim, indicating that direct photolysis proceeds through a triplet excited state, which was verified by demonstrating that trimethoprim is a singlet oxygen sensitizer. In the wastewater effluents tested, photolysis could be apportioned into direct photolysis (48% for sulfamethoxazole, 18% for trimethoprim), reaction with hydroxyl radicals (36% and 62%, respectively) and reaction with triplet excited effluent organic matter (16% and 20%, respectively). These results indicate that allowing photolysis in wastewater stabilization ponds or wastewater treatment wetlands may lead to enhanced pharmaceutical removal prior to discharge and that effluent organic matter has different photoreactivity than natural organic matter.

Impact of Organic Carbon on the Biodegradation of Estrone in Mixed Culture Systems

The effects of organic carbon concentrations and loading on the degradation of estrone (E1) were examined under various conditions in batch reactors and membrane-coupled bioreactors (MBRs). Experiments examined effects on individual microorganisms (substrate competition and growth) and on the whole community (selection). Substrate competition with organic carbon (competitive inhibition and catabolic repression) was not a factor in E1 degradation (P = 0.19 and 0.29 for two different analyses). Conversely, addition of organic carbon increased E1 degradation rates, attributable to biomass growth in feast-famine reactors over a five-day period (P = 0.016). Subsequently, however, community dynamics controlled E1 degradation rates, with other organisms outcompeting E1 degraders. More moderate but sustained increases in E1 degradation rates were observed under starvation conditions. Low influent organic carbon strength was detrimental to E1 degradation in MBRs, where organic carbon concentration and loading were decoupled (P = 0.018). These results point to the importance of multiple substrate utilizers in E1 degradation. They also suggest that while initial growth of biomass depends on the presence of sufficient organic carbon, further enrichment under starvation conditions may improve E1 degradation capability via the growth and/or stimulation of multiple substrate utilizers rather than heterotrophs characterized by an r-strategist growth regime.

The effects of antibiotic cocktails at environmentally relevant concentrations on the community composition and acetate biodegradation kinetics of bacterial biofilms

Antibiotics and antibacterials are present in water bodies worldwide but little is known about their effects on the biological processes often used to treat water. In this research, the effect of antibiotics on bacterial activity and community structure was investigated by growing biofilms in the presence and absence of a mixture of three compounds (sulfamethoxazole, erythromycin, and ciprofloxacin) in a continuous-flow rotating annular bioreactor fed acetate as a carbon and energy source. Steady-state, surface area-normalized substrate utilization rates for all antibiotic treatments (all at 0.33 μg L(-1), all at 3.33 μg L(-1), and 1 at 3.33 μg L(-1) with the other 2 at 0.33 μg L(-1)) were similar to the control experiments. Higher attached biomass levels in the experiments with ciprofloxacin at 3.33 μg L(-1) resulted in lower steady-state biomass-normalized substrate utilization rates in comparison to other runs. Microbial community analyses via automated ribosomal intergenic spacer analysis revealed significant shifts in community structure for the experiments dosed with the highest concentrations of ciprofloxacin, suggesting that the antibiotic selected for more resistant bacterial strains. The results of this research also suggest that mixtures of antibiotics at the sub-μg L(-1) concentrations typically observed in surface waters are unlikely to affect biological process performance, at least in terms of the degradation of easily assimilable compounds. Conversely, changes to community structure and biofilm quantity might be expected with ciprofloxacin at μg L(-1) concentrations.

Estrone degradation: does organic matter (quality), matter?

Understanding the parameters that drive E1 degradation is necessary to improve existing wastewater treatment systems and evaluate potential treatment options. Organic matter quality could be an important parameter. Microbial communities grown from activated sludge seeds using different dissolved organic matter sources were tested for E1 degradation rates. Synthetic wastewater was aged, filter-sterilized, and used as a carbon and energy source to determine if recalcitrant organic carbon enhances E1 degradation. Higher E1 degradation was observed by biomass grown on 8 d old synthetic wastewater compared to biomass grown on fresh synthetic wastewater (P = 0.033) despite much lower concentrations of bacteria. Minimal or no E1 degradation was observed in biomass grown on 2 d old synthetic wastewater. Organic carbon analyses suggest that products of cell lysis or microbial products released under starvation stress stimulate E1 degradation. Additional water sources were also tested: lake water, river water, and effluents from a municipal wastewater treatement plant and a treatment wetland. E1 degradation was only observed in biomass grown in treatment effluent. Nitrogen, dissolved organic carbon, and trace element concentrations were not causative factors for E1 degradation. In both experiments, spectrophotometric analyses reveal degradation of E1 is associated with microbially derived organic carbon but not general recalcitrance.

Research highlights: functions of the drinking water microbiome - from treatment to tap

Maintaining drinking water safety from treatment to point-of use is a critical health priority. Growth and proliferation of opportunistic pathogens in premise plumbing is a well-known concern that can be mitigated by controlling water heater temperatures and water stagnation patterns. However, there is growing evidence that upstream processes, beginning with choice of treatment methods, have significant influences on premise plumbing microbial communities. Here, we highlight four papers that explore the roles of microbial communities in drinking water quality, and how design and treatment choices shape these roles.

Temperature modulates estrone degradation and biological effects of exposure in fathead minnows

Environmental pollutants, including estrogens, are widespread in aquatic environments frequently as a result of treated wastewater effluent discharged. Exposure to estrogens has been correlated with disruption of the normal physiological and reproductive function in aquatic organisms, which could impair the sustainability of exposed populations. However, assessing the effects of estrogen exposure on individuals is complicated by the fact that rates of chemical uptake and environmental degradation are temperature dependent. Because annual temperature regimes often coincide with critical periods of biological activity, temperature-dependent changes in estrogen degradation efficacy during wastewater treatment could modulate biological effects. We examined the interactions between ambient water temperature and degradation of estrone (E1) during wastewater treatment. In addition, we exposed mature fathead minnows (Pimephales promelas) to three environmentally relevant concentrations of E1 at four different water temperatures (15°C, 18°C, 21°C, and 24°C) to reflect natural seasonal variation. E1 degradation occurred with and without the support of robust nitrification at all temperatures; however, the onset of E1 degradation was delayed at cooler water temperatures. In addition, we observed significant interactive effects between temperature and E1 exposure. Female morphometric endpoints were more susceptible to temperature-modulating effects while physiological endpoints were more strongly affected in males. Collectively, the data demonstrate that natural seasonal fluctuations in temperature are sufficient to affect E1 degradation during wastewater treatment and induce sex-dependent physiological and anatomical changes in exposed fish.

Estrone biodegradation in laboratory-scale systems designed for total nitrogen removal from wastewater

Changes in regional regulations are causing a shift towards the implementation of total nitrogen removal technologies. Conventional nitrification systems do not remove total nitrogen, instead only oxidizing ammonia and ammonium in the influent to nitrate. Conventional nitrification does, however, result in degradation of estrone (E1), a major contributor to the estrogenicity of wastewater treatment plant (WWTP) effluent. The objective of this research was to provide guidance on the impact that changes in wastewater treatment practices could have on E1 degradation. This was accomplished by comparing E1 removal in a laboratory-scale conventional nitrification system with that in a range of idealized laboratory-scale systems designed to remove total nitrogen from wastewater: the modified Ludzack-Ettinger (MLE) system (a two-stage anaerobic–aerobic system with recycle), a granular activated sludge system (cycled anaerobic–aerobic), a sequencing batch reactor (cycled anaerobic–aerobic), and an anaerobic ammonia oxidation (anammox) system. As anticipated, E1 removal was excellent when fed to the nitrification, MLE, and sequencing batch reactors, at >96% mean E1 loss. The granular activated sludge system operated in our laboratory failed to remove E1, which was perhaps not unexpected given the high COD loading under which our system was operated. Despite the anaerobic nature of anammox, it also resulted in excellent E1 removal (95% mean E1 loss) without concomitant 17β-estradiol production. This work demonstrates that the choice of nitrogen removal technology used by a treatment plant could have an impact on the estrogenicity of WWTP effluent, but low energy total nitrogen removal systems do exist that are capable of excellent E1 removal.

Effects of estrone and organic carbon exposure on the transformation of estrone

Exposure of biomass to estrone (E1) and alternate organic substrates was studied to determine whether cometabolism or multiple substrate utilization is an operating mechanism for the transformation of E1 and if feeding intervals affect the selection of E1 degrading bacteria. Biomass generated in membrane bioreactors (MBRs) was capable of degrading E1 regardless of E1 exposure. Nevertheless, pre-exposed biomass had higher E1 transformation rates (P = 0.05) and un-exposed biomass showed a clear lag phase (6 h) prior to E1 tranformation. These results are consistent with and strongly suggest metabolic transformation of E1 via multiple substrate utilization. In the feeding interval study, longer intervals between feeding periods selected for E1 degraders at high organic carbon loads (100 mg COD L−1 d−1; P = 0.018), but had no effect at low organic carbon loads (30 mg COD L−1 d−1; P = 0.32). A lag phase was observed in E1 transformation during famine periods but was absent during feast periods. This result indicates that the presence of other organic carbon substrates speeds the transformation of E1. This research is the first to demonstrate evidence for the role of multiple substrate utilization in the transformation of E1 and suggests operating conditions to improve selection for and activity of E1 degrading bacteria.

Research highlights: applications of atomic force microscopy in natural and engineered water systems

The mechanistic understanding of the fate, transport, and transformation of contaminants in natural and engineered water systems is of great importance for environmental remediation. Recently, atomic force microscopy (AFM) has emerged as a promising tool to provide insights on the properties and interactions of materials, chemicals, and microorganisms. This article highlights three studies using AFM to understand the molecular mechanism of membrane fouling for water and wastewater treatment, to characterize biofilm properties that influence the accumulation and release of pathogens in drinking water distribution systems, and to evaluate the nucleation and growth of manganese (Mn) (hydr)oxide for remediating Mn-contaminated environmental systems.