1. Biodegradation of Pharmaceuticals and Personal Care Products by Bacteria from Freshwater Biofilms Samantha Retzloff, McNair Scholar, Dr. Kevin Crawford*, Dr. Sabrina Mueller-Spitz University of Wisconsin-Oshkosh, Department of Biology and *Department of Chemistry Abstract Pharmaceuticals and personal care products (PPCPs) are an emerging class of contaminants that potentially impact environment and human health. Wastewater treatment plants are unable to fully remove these compounds from sewage effluent, thereby being released into surface waters. These chemicals may collect in biofilms, which are communities composed of various bacteria and other organisms. Therefore, it is probable that biofilm bacteria are able to metabolize and/or degrade PPCPs. The objective of this research was to isolate and characterize bacteria from freshwater biofilms capable of degrading acetaminophen, caffeine, or triclosan. To do this, biofilm samples were cultured on media containing low nutrients and the PPCP chemicals as carbon sources. Once bacteria grew, individual colonies were isolated and their potential for degradation was measured. A total of 22 cultures were isolated and screened for degradation. The loss of the parent PPCP over time was assessed using High Pressure Liquid Chromatography method. Identities of possible degraders were determined by sequencing the 16S rRNA gene. Nine isolates were classified as possible degraders including four for acetaminophen, four for caffeine, and one for triclosan. Among the collection, three bacterial phyla were represented including Deinococcus-Thermus, Proteobacteria, and Firmicutes. Proteobacteria was the most represented bacterial phyla (n=4) with two members of Rhizobiales isolated from different carbon sources. For three of the isolates that were determined probable degraders, the average percent remaining of the parent chemical over 72 hours was 89.7%. The loss of the parent chemicals over time were similar to the uninoculated control, suggesting the loss may be due to other factors and that further experimentation is required. This research suggests species of bacteria present in freshwater biofilms are capable of growing in the presence and may degrade these chemicals. Therefore, the bacteria that naturally occur in freshwater biofilms may lessen the environmental impact of PPCPs. Research Objective The research objective was to determine if biofilm bacteria can degrade acetaminophen, caffeine, or triclosan as sole carbon sources or grow in the presence of the chemicals with low nutrient levels. Then, we sought to characterize the members of biofilm bacterial consortia responsible for degradation. We hypothesized that bacteria occurring in freshwater biofilms would be capable of degrading various common pharmaceuticals and personal care products that have high logKoc because these molecules can sorb into the extracellular matrix and particles that accumulate in freshwater biofilms. Methods Method for isolation and characterization of bacteria capable of degrading PPCP chemicals were adapted from Zhou et al (2013). Low-nutrient medium; R2A and freshwater base were prepared augmented with 5 g/L concentration of caffeine, triclosan, and acetaminophen. Freshwater biofilms on three substrates at Sunset Park (Figure 1) were sampled on July 1, 2015.. Biofilm samples were inoculated in media supplemented with PPCPs and monitored for growth for 3 days. Multiple colonies per chemical were subcultured and isolated. Degradation was tested using HPLC method after 72 hours of growth. The identity of probable degraders was determined by sequencing the 16S rRNA gene. Degradation experiments were repeated with three isolates to confirm PPCP degradation. Figure 1: Sampling location downstream from Appleton WWTP at Sunset Park in Kimberly, WI. This park is downstream from Appleton’s Wastewater Treatment plant. IsolateChemical Gram Reaction Cell Shape Bacterial Taxonomy based on 16S rRNA Gene Identity (Phylum, Class, Order, Family, Genus) RA1Acetaminophen Gram Negative Bacilli Proteobacteria, Gammaproteobacteria, Xanthomonadales, Xanthomonadaceae, Lysobacter RA2Acetaminophen Gram Negative Cocci Proteobacteria, Alphaproteobacteria, Rhizobiales, Rhizobiaceae, Rhizobium FA3Acetaminophen Gram Negative Bacilli Firmicutes, Bacilli, Bacillales, Bacillaceae, Unclassified Bacillaceae genus FA4Acetaminophen Gram Negative Cocci Proteobacteria, Gammaproteobacteria, Pseudomonadales, Moraxellaceaea, Acinetobacter RC3Caffeine Gram Positive Cocci Proteobacteria, Alphaproteobacteria, Rhizobiales, Methylobacteriaceae, Methylobacterium RC5Caffeine Gram Positive CocciUnidentified FC2Caffeine Gram Positive Cocci Firmicutes, Bacilli, Bacillales, Bacillales-Incertae Sedis XII, Exiguobacterium FC4Caffeine Gram Positive Cocci Deinococcus-Thermus, Deinococci, Deinococcales, Deinococcaceae, Deinococcus RT3Triclosan Gram Negative Bacilli Proteobacteria, Gammaproteobacteria, Chromatiales, Chromatiaceae, Rheinheimera Results 22 bacteria (9 from acetaminophen, 10 from caffeine, and 3 from triclosan) were isolated from low-nutrient media supplemented with the three PCPPs (Figure 2). Nine possible bacterial degraders were identified following the degradation testing. Both Gram positive and Gram negative bacteria were isolated. The majority of isolates belong to phylum Proteobacteria. (Table 1) Three isolates were determined probable degraders because of approximately 10% loss of the parent chemical identified using HPLC method (Figure 3A). RC5 yielded the greatest cell growth, supporting that it was able to utilize caffeine as a carbon and nitrogen source. Table 1: Characterization of nine bacterial isolates that were determined possible degraders. References 1.Zhou, Nicolette A. et al. Cultivation and characterization of bacterial isolates capable of degrading pharmaceutical and personal care products for improved removal in activated sludge wastewater treatment. Biodegradation 2013, 24, 813-827. 2.Wang Q, Garrity GM, Tiedje JM, Cole JR. Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy. Applied and Environmental Microbiology. 2007;73:5261-5267. Figure 3: A. The average percent loss of PPCP parent chemical over 72 hours of isolates RA2, RC5, and RT3 measured by HPLC analysis.. B. The average change (n=3) in optical density over 72 hours of isolates RA2, RC5, and RT3 in liquid culture. Figure 2: Representative bacterial isolates that were able to grow on low-nutrient media augmented with PPCP chemicals. Discussion ●Bacteria capable of growing in the presence of acetaminophen, caffeine, and triclosan were easily isolated from freshwater biofilm samples. ●Probable caffeine, triclosan, and acetaminophen degraders are common bacterial taxa present in freshwater biofilms (Mueller-Spitz, personal communication). ●In order to confirm degradation potential of RA2, RC5, and RT3, these isolates need to be exposed to greater concentrations of each chemical over longer periods of time and determine how the parent chemical is modified. The UW Oshkosh McNair Scholars Program is 100% funded through a TRIO grant from the United States Department of Education PR/Award Number P217A120210. For 2014/2015, the UW Oshkosh McNair Scholars Program will receive $216,000.00 each year in federal funds.