Shawn A. Hawkins

Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

ABSTRACT The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

Changes in the relative expression pattern of multiple vitellogenin genes in adult male and larval zebrafish exposed to exogenous estrogens

Production of the lipoprotein vitellogenin (Vg) is induced in fish upon exposure to estrogens and is a biomarker of endocrine disruption in fish. In some fish, three types of Vg (VgA, VgB, and VgC) are recognized and transcribed from at least three distinct Vg genes (vtg). We investigated expression of vtg coding for Vg1A/B, Vg2A/B, and VgC in adult male and larval zebrafish exposed to various estrogenic substances. Quantitative PCR was conducted for transcripts of each vtg and a control gene (beta-actin). Male fish were exposed to 17beta-estradiol (E2) and 17alpha-ethinylestradiol, total RNA was extracted from excised liver, and histopathology of liver, trunk kidney, and gonads was conducted. Larval fish were exposed to 10 different estrogenic substances and total RNA was extracted from groups of whole larvae. In adult male fish, the relative fold change varied, but pattern of expression change (i.e., Vg1A/B > Vg2A/B > VgC) was consistent. Larger males exposed to E2 had significantly higher induction of each vtg. In larval zebrafish, the relative fold change in vtg expression varied according to specific estrogenic substance tested, but the pattern of change (i.e., Vg2A/B > Vg1A/B > VgC) was consistent for each substance that induced vtg.

Limited impact of free ammonia on Nitrobacter spp. inhibition assessed by chemical and molecular techniques.

Free ammonia has long been identified as a nitrite oxidation inhibitor. However, past attempts to use this compound to eliminate nitrite oxidation and thereby promote more efficient nitrogen removal strategies during biological wastewater treatment have often failed. Additionally, contradictory results exist in the literature where direct measurements of free ammonia inhibition of nitrite oxidation have been reported. In this study, suspended biomass samples (nitrifier enriched activated sludge) were collected from a bench scale nitrification reactor with Nitrobacter spp. as the dominant nitrite oxidizer and subjected to batch respirometric experiments designed to quantify free ammonia inhibition of nitrite oxidization. A variety of data including ammonia, nitrite, and nitrate conversion rates, oxygen consumption rates, and Nitrobacter ribosomal RNA transcript abundance, a molecular indicator of growth activity, were used to assess nitrite oxidation and growth activity. Both the traditional and molecular activity assessments indicated that free ammonia had a limited inhibitory effect on Nitrobacter spp. In fact, the pH changes necessary to induce high free ammonia concentrations (>10mg-N/L) had a demonstrably more important inhibiting effect on nitrite oxidation than free ammonia. In contrast, during high ammonia oxidizing activity (5.3mg-N/L/h), low nitrite oxidation rates (0.2mg-N/L/h) and severely impaired Nitrobacter spp. growth activity, indicated by a low abundance of the Nitrobacter spp. ribosomal gene transcript relative to the ribosomal gene (0.08), were measured. The findings suggest that pH changes and ammonia oxidizing bacteria activity are more important factors limiting Nitrobacter spp. mediated nitrite oxidation, rather than the free ammonia concentration.

Genome sequence of the Bacteroides fragilis phage ATCC 51477-B1

The genome of a fecal pollution indicator phage, Bacteroides fragilis ATCC 51477-B1, was sequenced and consisted of 44,929 bases with a G+C content of 38.7%. Forty-six putative open reading frames were identified and genes were organized into functional clusters for host specificity, lysis, replication and regulation, and packaging and structural proteins.

Molecular indicators of Nitrobacter spp. population and growth activity during an induced inhibition event in a bench scale nitrification reactor

The Nitrobacter spp. ribosomal RNA gene (rDNA) and transcript (rRNAt) abundance were quantified in a bench scale nitrification reactor during baseline periods of high nitrification efficiency and an intervening staged inhibition event. The transcript to gene ratio (rRNAt/rDNA) was highly sensitive to changes in the reactor nitrite oxidation rate. During high nitrification efficiency, the rRNAt/rDNA metric displayed a range from 0.68 to 2.01 with one-sided (α=0.10) lower and upper prediction intervals of 0.70 and 1.78, respectively. When nitrification was inhibited by disabling the reactor pH control system, this activity metric declined an order of magnitude to ≈ 0.05, well below the lower prediction interval reflecting high nitrification efficiency. The decline was rapid (2h) and preceded a significant drop in reactor nitrification performance, which occurred as ammonia accumulated. The rRNAt/rDNA ratio remained low (≈ 0.05) for several days after the pH control system was re-enabled at a setpoint of 8.0, which otherwise induced rapid oxidation of accumulated ammonia and produced high free ammonia concentrations. The timing of a subsequent increase in the rRNAt/rDNA ratio, which transiently exceeded the upper prediction interval established during the baseline period of high nitrification efficiency, was not coincidental with resumption of pH control at 7.2 that lowered free ammonia concentrations to non-inhibitory levels. Rather, nitrite oxidation resumed and the rRNAt/rDNA ratio increased only after oxidation of accumulated ammonia was complete, which was coincidental with reduced reactor oxygen demand. In summary, the Nitrobacter rRNAt/rDNA activity metric reflected timely and easily recognizable changes in nitrite oxidation activity, illustrating that molecular data can be used to diagnose poor biological wastewater treatment performance.

Fate and Transport of 17β-Estradiol beneath Animal Waste Holding Ponds.

Concentrated animal feeding operations typically store livestock waste in clay-lined ponds. Although these ponds are regulated to include a liner with a small hydraulic conductivity to limit leaching, previous studies have traced surface and groundwater contamination from such regulated animal waste ponds. This research examined the transport of 17β-estradiol (E2) and its primary metabolite, estrone (E1), through soil liners using field- and laboratory-based studies. Additionally, a potential engineering solution to limit hormone transport-applying biochar to new pond liners to act as a retardant-was studied. Soil cores 80 cm in length were collected beneath a mature dairy waste pond and analyzed for moisture content and hormone concentrations. Unsaturated conditions and E2 concentrations of 4 to 250 ng g were detected beneath the waste pond. In the laboratory portion of the study, hand-packed columns of sand or clay were subjected to infiltration by a 2.3-m head of dairy waste. A subset of the hand-packed sand columns was amended with powdered biochar to test its ability to retard E2 and E1. For 3 mo, column leachate was analyzed for hormone concentrations, and at the conclusion of the study E2 and E1 concentrations in the soil were measured. In the 44 d after sealing, the clay, sand, sand with a thin layer of biochar, and sand mixed with a biochar amendment leached a total of 0.54, 1.3, 0.09, and 0.45 μg of E2, respectively. The biochar amendments to the hand-packed columns considerably minimized E2 in the leachate.

A study of cattle producer preferences for best management practices in an East Tennessee watershed

Best Management Practices (BMPs) contribute to a broader range of efforts to improve the environmental performance of the livestock sector and its impact on water quality. This research evaluates a survey of cattle producers in an East Tennessee watershed and parts of five surrounding watersheds, along with and the factors correlated with preferences for BMPs designed to reduce sediment, nutrient, and fecal coliform contamination of surface waters by limiting cattle access to streams. The objective of the survey was to gather behavioral information about producer interest in specific BMPs in an effort to supplement a long-term biophysical modeling project. Structures and BMPs analyzed include stream crossings, rotational grazing, pasture improvement, and cattle water tanks. The physical and economic constraints faced by producers and the incentives provided by state and federal programs influence the decision to adopt a set of practices or structures that impact pathogen loading into streams. There was a clear preference for a suite of BMPs that did not include stream crossings, reinforcing anecdotal evidence that the maintenance associated with frequent high flow events may reduce willingness to install stream crossings. Cattle producers were more willing to implement rotational grazing and pasture improvement BMPs, which were associated with cattle health and productivity. The extensive distribution of pastureland in the region analyzed, the relatively inexpensive costs of adopting practices supporting pasture improvement, and higher quality forage correlated with improved pastures suggest a win-win outcome for cattle owners and efforts to enhance water quality.