Pilot-Scale Denitrification Bioreactors for Replicated Field Research

Carbon-based denitrification bioreactors are designed to intercept tile drainage and are a promising technology for reducing NO3- export to surface water. While these systems have been tested extensively in the laboratory, the ability to study in-field bioreactors under controlled conditions with statistical replicates has been limited. Nine pilot-scale bioreactors (5.79 x 1.05 x 1.07 m) were designed and installed for systematic field testing, allowing for variation in retention time, fill material, and influent water quality parameters. Each bioreactor is constructed from a concrete trench in-line with influent flow control, dosing port, flow diffusion, and effluent water level control. Sampling ports are installed at two points in each bioreactor for access to water samples or fill materials. A potassium bromide (KBr) tracer study was conducted and Morrill Dispersion Index (MDI) values averaged 2.8 ± 0.3, indicating plug flow characteristics. The average tracer residence time () was 2.3 ± 0.3 h, in close agreement with the estimated hydraulic retention time (HRT) value of 2.1 ± 0.3 h, which was calculated using a porosity value of 0.70. Hydraulic efficiency was good (λ = 0.78 ± 0.03) and there was no evidence of short circuiting (S = 0.73 ± 0.03). This system is expected to provide useful insight regarding design for improved field performance of denitrification bioreactors

Session 2A: \u3cem\u3ePanel Discussion: Developing Post-Incident Risk Communication Guidelines for Intentional Water Contamination Events\u3c/em\u3e

This panel will discuss emerging findings from a US EPA-funded research project intended to improve risk communication for post-incident decontamination and clearance activities associated with intentional contamination of a water system. The session will center around Phase II of the study, which focuses on extending the Phase I case study findings that were presented at last year’s KWRRI Symposium, to identify ways in which disparate stakeholder groups in metropolitan areas differentially perceive risk and subsequent risk communication efforts

The human gastric pathogen Helicobacter pylori has a potential acetone carboxylase that enhances its ability to colonize mice

<p>Abstract</p> <p>Background</p> <p><it>Helicobacter pylori </it>colonizes the human stomach and is the etiological agent of peptic ulcer disease. All three <it>H. pylori </it>strains that have been sequenced to date contain a potential operon whose products share homology with the subunits of acetone carboxylase (encoded by <it>acxABC</it>) from <it>Xanthobacter autotrophicus </it>strain Py2 and <it>Rhodobacter capsulatus </it>strain B10. Acetone carboxylase catalyzes the conversion of acetone to acetoacetate. Genes upstream of the putative <it>acxABC </it>operon encode enzymes that convert acetoacetate to acetoacetyl-CoA, which is metabolized further to generate two molecules of acetyl-CoA.</p> <p>Results</p> <p>To determine if the <it>H. pylori acxABC </it>operon has a role in host colonization the <it>acxB </it>homolog in the mouse-adapted <it>H. pylori </it>SS1 strain was inactivated with a chloramphenicol-resistance (<it>cat</it>) cassette. In mouse colonization studies the numbers of <it>H. pylori </it>recovered from mice inoculated with the <it>acxB:cat </it>mutant were generally one to two orders of magnitude lower than those recovered from mice inoculated with the parental strain. A statistical analysis of the data using a Wilcoxin Rank test indicated the differences in the numbers of <it>H. pylori </it>isolated from mice inoculated with the two strains were significant at the 99% confidence level. Levels of acetone associated with gastric tissue removed from uninfected mice were measured and found to range from 10–110 μmols per gram wet weight tissue.</p> <p>Conclusion</p> <p>The colonization defect of the <it>acxB:cat </it>mutant suggests a role for the <it>acxABC </it>operon in survival of the bacterium in the stomach. Products of the <it>H. pylori acxABC </it>operon may function primarily in acetone utilization or may catalyze a related reaction that is important for survival or growth in the host. <it>H. pylori </it>encounters significant levels of acetone in the stomach which it could use as a potential electron donor for microaerobic respiration.</p

Session 2A: \u3cem\u3eDeveloping Post-Incident Risk Communication Guidelines for Intentional Water Contamination Events\u3c/em\u3e

This panel will discuss a US EPA-funded research project intended to improve risk communication for post-incident decontamination and clearance activities associated with intentional contamination of a water system. The study incorporates two complementary methods conducted in successive phases. The recently-completed first phase included robust case study analyses of risk communication related to recent and significant contamination incidents. The second phase, which is currently underway, will identify ways in which disparate stakeholder groups in a metropolitan area differentially perceive risk and subsequent risk communication efforts

A New Approach to Measuring Estrogen Exposure and Metabolism in Epidemiologic Studies

Endogenous estrogen plays an integral role in the etiology of breast and endometrial cancer, and conceivably ovarian cancer. However, the underlying mechanisms and the importance of patterns of estrogen metabolism and specific estrogen metabolites have not been adequately explored. Long-standing hypotheses, derived from laboratory experiments, have not been tested in epidemiologic research because of the lack of robust, rapid, accurate measurement techniques appropriate for large-scale studies. We have developed a stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS(2)) method that can measure concurrently all 15 estrogens and estrogen metabolites (EM) in urine and serum with high sensitivity (level of detection=2.5-3.0fmol EM/mL serum), specificity, accuracy, and precision [laboratory coefficients of variation (CV\u27s) \u3c or =5% for nearly all EM]. The assay requires only extraction, a single chemical derivatization, and less than 0.5mL of serum or urine. By incorporating enzymatic hydrolysis, the assay measures total (glucuronidated+sulfated+unconjugated) EM. If the hydrolysis step is omitted, the assay measures unconjugated EM. Interindividual differences in urinary EM concentrations (pg/mL creatinine), which reflect total EM production, were consistently large, with a range of 10-100-fold for nearly all EM in premenopausal and postmenopausal women and men. Correlational analyses indicated that urinary estrone and estradiol, the most commonly measured EM, do not accurately represent levels of total urinary EM or of the other EM. In serum, all 15 EM were detected as conjugates, but only 5 were detected in unconjugated form. When we compared our assay methods with indirect radioimmunoassays for estrone, estradiol, and estriol and enzyme-linked immunosorbent assays for 2-hydroxyestrone and 16alpha-hydroxyestrone, ranking of individuals agreed well for premenopausal women [Spearman r (r(s))=0.8-0.9], but only moderately for postmenopausal women (r(s)=0.4-0.8). Our absolute readings were consistently lower, especially at the low concentrations characteristic of postmenopausal women, possibly because of improved specificity. We are currently applying our EM measurement techniques in several epidemiologic studies of premenopausal and postmenopausal breast cancer

Survival in Nuclear Waste, Extreme Resistance, and Potential Applications Gleaned from the Genome Sequence of Kineococcus radiotolerans SRS30216

Kineococcus radiotolerans SRS30216 was isolated from a high-level radioactive environment at the Savannah River Site (SRS) and exhibits γ-radiation resistance approaching that of Deinococcus radiodurans. The genome was sequenced by the U.S. Department of Energy's Joint Genome Institute which suggested the existence of three replicons, a 4.76 Mb linear chromosome, a 0.18 Mb linear plasmid, and a 12.92 Kb circular plasmid. Southern hybridization confirmed that the chromosome is linear. The K. radiotolerans genome sequence was examined to learn about the physiology of the organism with regard to ionizing radiation resistance, the potential for bioremediation of nuclear waste, and the dimorphic life cycle. K. radiotolerans may have a unique genetic toolbox for radiation protection as it lacks many of the genes known to confer radiation resistance in D. radiodurans. Additionally, genes involved in the detoxification of reactive oxygen species and the excision repair pathway are overrepresented. K. radiotolerans appears to lack degradation pathways for pervasive soil and groundwater pollutants. However, it can respire on two organic acids found in SRS high-level nuclear waste, formate and oxalate, which promote the survival of cells during prolonged periods of starvation. The dimorphic life cycle involves the production of motile zoospores. The flagellar biosynthesis genes are located on a motility island, though its regulation could not be fully discerned. These results highlight the remarkable ability of K radiotolerans to withstand environmental extremes and suggest that in situ bioremediation of organic complexants from high level radioactive waste may be feasible

Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA

Insights into the complex regulation of rpoS in Borrelia burgdorferi

Co-ordinated regulation of gene expression is required for the transmission and survival of Borrelia burgdorferi in different hosts. The sigma factor RpoS (σS), as regulated by RpoN (σ54), has been shown to regulate key virulence factors (e.g. OspC) required for these processes. As important, multiple signals (e.g. temperature, pH, cell density, oxygen) have been shown to increase the expression of σS-dependent genes; however, little is known about the signal transduction mechanisms that modulate the expression of rpoS. In this report we show that: (i) rpoS has a σ54-dependent promoter that requires Rrp2 to activate transcription; (ii) Rrp2Δ123, a constitutively active form of Rrp2, activated σ54-dependent transcription of rpoS/P-lacZ reporter constructs in Escherichia coli; (iii) quantitative reverse transcription polymerase chain reaction (QRT-PCR) experiments with reporter cat constructs in B. burgdorferi indicated that Rrp2 activated transcription of rpoS in an enhancer-independent fashion; and finally, (iv) rpoN is required for cell density- and temperature-dependent expression of rpoS in B. burgdorferi, but histidine kinase Hk2, encoded by the gene immediately upstream of rrp2, is not essential. Based on these findings, a model for regulation of rpoS has been proposed which provides mechanisms for multiple signalling pathways to modulate the expression of the σS regulon in B. burgdorferi