Project description:We developed a laboratory-scale model to improve our understanding and capacity to assess the biological risks of genetically engineered bacteria and their genetic elements in the natural environment. Our hypothetical scenario concerns an industrial bioreactor failure resulting in the introduction of genetically engineered bacteria to a downstream municipal wastewater treatment plant (MWWTP). As the first step towards developing a model for this scenario, we sampled microbial communities from the aeration basin of a MWWTP at three seasonal time points. Having established a baseline for community composition, we investigated how the community changed when propagated in the laboratory, including cell culture media conditions that could provide selective pressure in future studies. Specifically, using PhyloChip 16S rRNA gene-targeting microarrays, we compared the compositions of sampled communities to those of inoculates propagated in the laboratory in simulated wastewater conditionally amended with various carbon sources (glucose, chloroacetate, D-threonine) or the ionic liquid 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl). Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in aeration basin and laboratory-cultured populations. Laboratory-cultured populations were enriched in Gammaproteobacteria. Enterobacteriaceae and Aeromonadaceae were enriched by glucose, Pseudomonadaceae by chloroacetate and D-threonine, and Burkholderiaceae by high (50 mM) concentrations of chloroacetate. Microbial populations cultured with chloroacetate and D-threonine were more similar to sampled populations than thoes cultured with glucose or [C2mim]Cl. Although observed relative richness in operational taxonomic units was lower for laboratory cultures than for sampled populations, both flask and reactor systems cultured phylogenetically diverse communities. These results importantly provide a foundation for laboratory models of industrial bioreactor failure scenarios. 46 samples, flask and reactor experiments were conducted in triplicate with two exceptions: [C2mim]Cl_flask and No-Carbon_flask treatments had only one sample (no replicates).

Project description:Three surface waters in Gainesville, Florida were used in a 48 hour whole effluents exposure to assess gene expression profiles of male fathead minnow liver. Microarray analysis was used to determine changes in gene expression of exposed fish to waters from a site downstream of a wastewater treatment plant (streamwater), a wastewater treatment plant (wastewater), and a lake (stormwater). Differences in gene expression between fish exposed to collected waters and controls were observed. Number of altered genes and biological processes were 1028 and 18 for stormwater; 787 and 19 for streamwater; and: 575 and 12 for wastewater. In general, the effects observed in all exposed fish were related with fatty acid metabolism, DNA repair, oxidation-reduction process, cell wall catabolic process and apoptosis. All exposed fish showed altered expression of genes related with DNA damage repair. In particular fish exposed to stormwater and streamwater showed downregulation of several key intermediates transcripts of cholesterol. The presence and environmental persistence of perfluorinated chemicals (PFCs) in these waters, the resemblance in known effects on transcripts with those found in this study, suggest that the set of genes differentially regulated in fathead minnows after 48 hours of exposure may be attributed to exposure to PFCs. Three surface water sites were chosen for effluent collection in Gainesville, Florida: A lake (stormwater), surface water downstream of a wastewater treatment plant (streamwater), and a wastewater treatment plant effluent used for landscaping irrigation (wastewater). Water from each site was collected two days prior to the fish exposure experiment using Chemfluor ® tubing and a 120 liters steel barrels coated with polyester resin (gel coat) to avoid cross-contamination. Three barrels for each effluent were collected during day 1. Water from the barrel was transported to the laboratory and pumped into four fiberglass cylinders in the aquatic toxicology facility. Water from each cylinder was then pumped into four replicate aquariums per treatment and kept for 1 day without fish (pre-treatment). On day 2, four male fathead minnows from a common tank were transferred to each replicate aquarium and kept for 48 hours, with one 75% water change after first 24 hours. The exposure system consisted of 40 L glass aquaria. Each exposure was conducted in quadruplicate and each aquarium contained the four male fish in 25 L of treatment water . The water used in the control treatment was carbon filtered, dechlorinated tap water. The positions of the treatment tanks were randomized and test initiation times were staggered to ensure an exposure/sampling interval of 48 h. The fish were not fed during the experiment. The temperature range of the water was 24-26 °C with a photoperiod of 16 h light: 8 h dark. Liver was isolate from 4 males indviduals for each treatment except for control group (3 individuals).

Project description:The federally endangered Okaloosa darter (Etheostoma okaloosae) is found almost exclusively on the Eglin Air Force Base in the Choctawhatchee Bay watershed of Florida. Portions of this limited habitat are threatened with erosion of soils, altered hydrology, and impaired water quality. One stream reach in particular, East Turkey Creek, has demonstrated potential water quality problems including poor invertebrate bioassessment scores (IBI), uncharacteristically high conductivity values, and low numbers of Okaloosa darters. General water quality (dissolved oxygen, specific conductance, pH, temperature, and relative turbidity and primary productivity) was characterized in both the potentially impacted East Turkey Creek and a reference stream (Long Creek). Water quality was assessed during a 30 day exposure using passive samplers for both non-polar and polar effluent parameters. Metal loading in the system was assessed via fish tissue burdens in resident Pteronotropis hypseleotris. Additionally, microarray analysis was performed on gonad and liver tissue from fathead minnows, Pimephales promelas, after 48-h exposures to water collected from the two creeks and brought into the laboratory. Gene expression changes were evident at the site below the influence of a wastewater spray field sited along East Turkey Creek, suggesting that anthropogenic compounds in the effluent waters may have affected both liver and testis function and could be related to account the general decrease in populations of the Okaloosa darter. Array hybridizations were performed using a single color design. Four biological replicates consisting of four different individuals were analyzed for each of the treatments (University of Florida control, and each of five sites). Site C was left out of subsequent analysis for publication, due to poor characterization of the site. Two Samples were left out of the normalization/analysis due to QC failure. Therefore, there are only 3 replicates of the "testis, site A water, exposed 48 h." and "liver, site E water, exposed 48 h." groups.

Project description:We developed a laboratory-scale model to improve our understanding and capacity to assess the biological risks of genetically engineered bacteria and their genetic elements in the natural environment. Our hypothetical scenario concerns an industrial bioreactor failure resulting in the introduction of genetically engineered bacteria to a downstream municipal wastewater treatment plant (MWWTP). As the first step towards developing a model for this scenario, we sampled microbial communities from the aeration basin of a MWWTP at three seasonal time points. Having established a baseline for community composition, we investigated how the community changed when propagated in the laboratory, including cell culture media conditions that could provide selective pressure in future studies. Specifically, using PhyloChip 16S rRNA gene-targeting microarrays, we compared the compositions of sampled communities to those of inoculates propagated in the laboratory in simulated wastewater conditionally amended with various carbon sources (glucose, chloroacetate, D-threonine) or the ionic liquid 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl). Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in aeration basin and laboratory-cultured populations. Laboratory-cultured populations were enriched in Gammaproteobacteria. Enterobacteriaceae and Aeromonadaceae were enriched by glucose, Pseudomonadaceae by chloroacetate and D-threonine, and Burkholderiaceae by high (50 mM) concentrations of chloroacetate. Microbial populations cultured with chloroacetate and D-threonine were more similar to sampled populations than thoes cultured with glucose or [C2mim]Cl. Although observed relative richness in operational taxonomic units was lower for laboratory cultures than for sampled populations, both flask and reactor systems cultured phylogenetically diverse communities. These results importantly provide a foundation for laboratory models of industrial bioreactor failure scenarios.

Dataset Information

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure