Wastewater contains pharmaceuticals, endocrine disruptors, personal care products, and emerging contaminants originating from human waste and other activities. Endocrine disruptors encompass a wide array of chemicals, including human hormones, phytoestrogens, and flame retardants, among others. Once these chemicals enter wastewater treatment facilities, their fates vary depending on the treatment techniques present. Standard wastewater treatment has not been designed to treat such contaminants, as human and synthetic hormones are only partially degraded during secondary (biological) treatment. The bacteria present in wastewater treatment are exposed to all classes of emerging contaminants, including antibiotics. Antibiotic resistant bacteria populations are becoming more common in wastewater treatment. Recently, hormones have been shown to be substrates for major classes of antibiotic pumps, leading to hormone resistance. Here, it is hypothesized that hormones are inefficiently degraded by bacteria because they are exported out of the cell by antibiotic resistance proteins before encountering oxidizing proteins. The suspected proteins involved in hormone oxidation are oxygenases, which insert a hydroxyl moiety onto a carbon chain. This BRIGE award will investigate whether the lack of degradation of endocrine disrupting compounds in secondary biological treatment is due to hormone resistance. The proposed work encompasses three main tasks: (1) Various endocrine disrupting compounds, both estrogenic and androgenic, will be tested as substrates for the major Resistance Nodulation Division (RND) antibiotic pump AcrAB-TolC in e. coli; (2) Numerous classes of monooxygenase and dioxygenase genes will be cloned into e. coli; and (3) endocrine disrupting compound degradation will be tested in e. coli strains expressing and lacking oxygenase genes.
Intellectual Merit: If the outlined activities are successful, bacteria populations may be engineered to efficiently degrade hormone and other endocrine disruptors. Pure culture batch reactors will be constructed, studied, and re-designed to incorporate other bacteria populations required for adequate wastewater treatment. These results have significant implications for the reduction of endocrine disrupting compounds released into the environment.
Broader Impacts: The anticipated results will explain why endocrine disrupting compounds survive wastewater treatment. To determine if this hypothesis is valid, bacteria populations and hormone concentrations will be evaluated at local wastewater treatment facilities. Sample collection will occur in the metropolitan Salt Lake City area, rural treatment works, and from tribal lands. Working in an environmental capacity with Tribal entities requires special permission from their respective leaders. A research protocol will be developed on behalf of the University of Utah which will outline the steps required to pursue such research. Sampling on tribal lands will incorporate educational and research opportunities for interested community members, including high-school students.
Endocrine disrupting chemicals (EDCs) such as estrogenic hormones, plasticizers, and detergents are problematic to aquatic animals because they mimic the body’s natural hormone system. Such chemicals are responsible for causing fish to acquire both male and female physiological haracteristics. EDCs enter the environment primarily from wastewater treatment facilities, which were not designed to remove specific emerging contaminants at low, biologically active concentrations. This project examines biological mechanisms responsible for attenuation of EDCs during wastewater treatment. The overall research objective of this project is to determine if antibiotic resistance in bacteria contributes to environmental endocrine disruption by competing with biological degradation. First, we determined that wastewater pollutants 17a-ethynylestradiol (synthetic hormone in the birth control pill), bisphenol-A (BPA, a plasticizer), nonylphenol and octylphenol (detergent by-products) are exported by antibiotic-resistant proteins in E. coli and Pseudomonas aeruginosa. Other bacteria, designated as EDC-resistant, include Acinetobacter sp., Pseudomonas putida, and Salmonella enterica. To determine if efflux competes with biodegradation, we tested growth and chemical degradation in bacteria strains with and without efflux proteins and oxidizing enzymes. If efflux competes with biodegradation, the EDC will not be removed during wastewater treatment. This was verified with the chemical nonylphenol. Two bacteria strains, one with an antibiotic-resistant protein, and one without, both containing oxidizing enzymes were exposed to nonylphenol. Results showed that the strain with the antibiotic pump grew slower than the strain without the antibiotic resistant protein. The strain with the efflux pump exported nonylphenol from the cell, rendering it non-bioavailable as a carbon source. Nonylphenol remained in the cell when the efflux pump was deleted, and was available as a carbon source and for growth. These results demonstrated that antibiotic-resistant proteins contribute to lack of biodegradation of EDCs and also supports findings that a longer contact time between bacteria and EDCs is needed for better removal during wastewater treatment.
