Paula Krauter

Removal of Cr(VI) from ground water by Saccharomyces cerevisiae

Chromium can be removed from ground water by the unicellular yeast, Saccharomyces cerevisiae. Local ground water maintains chromium as CrO42- because of bicarbonate buffering and pH and Eh conditions (8.2 and +343 mV, respectively). In laboratory studies, we used commercially available, nonpathogenic S. cerevisiae to remove hexavalent chromium [Cr(VI)] from ground water. The influence of parameters such as temperature, pH, and glucose concentration on Cr(VI) removal by yeast were also examined. S. cerevisiae removed Cr(VI) under aerobic and anaerobic conditions, with a slightly greater rate occurring under anaerobic conditions. Our kinetic studies reveal a reaction rate (Vmax) of 0.227 mg h-1 (g dry wt biomass)-1 and a Michaelis constant (Km) of 145 mg/l in natural ground water using mature S. cerevisiae cultures. We found a rapid (within 2 minutes) initial removal of Cr(VI) with freshly hydrated cells [55–67 mg h-1 (g dry wt biomass)-1] followed by a much slower uptake [0.6–1.1 mg h-1 (g dry wt biomass)-1] that diminished with time. A materials-balance for a batch reactor over 24 hours resulted in an overall shift in redox potential from +321 to +90 mV, an increase in the bicarbonate concentration (150–3400 mg/l) and a decrease in the Cr(VI) concentration in the effluent (1.9-0 mg/l).

A new direct microscopy based method for evaluating in-situ bioremediation

A new epifluorescent microscopy based method using 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) and 5-(4,6-dichlorotriazinyl) aminofluoroscein (DTAF) was developed for quantifying total microbial biomass and evaluating levels of microbial activity. CTC is a tetrazolium dye that forms fluorescent intracellular formazan when biologically reduced by components of the electron transport system and/or dehydrogenases of metabolically active bacteria. DTAF is a fluorescein-based fluorochrome that selectively stains bacterial cell walls thereby enabling quantification of total bacterial biomass. CTC can be used in conjunction with DTAF to provide the optical resolution necessary to differentiate metabolically active cells from inactive cells in microbial populations associated with subsurface soils. The CTC/DTAF staining method has been shown to be effective for quantifying the metabolic activity of not only aerobic bacteria, but also diverse groups of anaerobic bacteria. This method allows for the rapid quantification of total and active bacterial numbers in complex soil samples without enrichment or cell elution. In this study, CTC/DTAF staining was applied to evaluate in-situ microbial activity in petroleum hydrocarbon contaminated subsurface soils from Sites 3 and 13 at Alameda Point, CA. At each site, subsurface microbial activity at two locations within contaminated plumes were examined and compared to activity at two geologically similar but uncontaminated background locations. Significant bacterial populations were detected in all soils examined, and the biomass estimates were several orders of magnitude higher than those obtained by conventional culture-based techniques. Both the total bacterial concentrations and the numbers of active bacteria in soils from contaminated areas were substantially higher than those observed in soils from background locations. Additionally, the percentages of metabolically active bacteria in the contaminated areas were consistently higher than those detected in background areas, suggesting that the enhanced microbial activity was due to microbial contaminant degradation. Although conventional heterotrophic plate counts failed to show significant microbial activity at either of the sites, soil gas carbon dioxide and methane measurements confirmed that hydrocarbon contaminant degradation was occurring in both areas. The CTC/DTAF staining protocol proved to be a rapid, reliable, and inexpensive method to evaluate the progress of in-situ bioremediation.

Reaerosolization of Fluidized Spores in Ventilation Systems

ABSTRACT This project examined dry, fluidized spore reaerosolization in a heating, ventilating, and air conditioning duct system. Experiments using spores of Bacillus atrophaeus, a nonpathogenic surrogate for Bacillus anthracis, were conducted to delineate the extent of spore reaerosolization behavior under normal indoor airflow conditions. Short-term (five air-volume exchanges), long-term (up to 21,000 air-volume exchanges), and cycled (on-off) reaerosolization tests were conducted using two common duct materials. Spores were released into the test apparatus in turbulent airflow (Reynolds number, 26,000). After the initial pulse of spores (approximately 1010 to 1011 viable spores) was released, high-efficiency particulate air filters were added to the air intake. Airflow was again used to perturb the spores that had previously deposited onto the duct. Resuspension rates on both steel and plastic duct materials were between 10−3 and 10−5 per second, which decreased to 10 times less than initial rates within 30 min. Pulsed flow caused an initial spike in spore resuspension concentration that rapidly decreased. The resuspension rates were greater than those predicted by resuspension models for contamination in the environment, a result attributed to surface roughness differences. There was no difference between spore reaerosolization from metal and that from plastic duct surfaces over 5 hours of constant airflow. The spores that deposited onto the duct remained a persistent source of contamination over a period of several hours.

Assessment of in-situ bioremediation at a refinery waste-contaminated site and an aviation gasoline contaminated site

A combination of geochemical, microbiological and isotopic methods were used to evaluate in-situ bioremediation of petroleum hydrocarbons at one site contaminated with refinery waste and a second site contaminated with aviation gasoline at Alameda Point, California. At each site, geochemical and microbiological characteristics from four locations in the contaminated zone were compared to those from two uncontaminated background locations. At both sites, the geochemical indicators of in-situbiodegradation includeddepleted soil gas and groundwater oxygen, elevated groundwater alkalinity, and elevated soil gas carbon dioxide and methane in the contaminated zone relative to the background. Radiocarbon content of methane and carbon dioxide measured in soil gas at both sites indicated that they were derived from hydrocarbon contaminant degradation. Direct microscopy of soil core samples using cell wall stains and activity stains, revealed elevated microbial numbers and enhanced microbial activities in contaminated areas relative to background areas, corroborating geochemical findings. While microbial plate counts and microcosm studies using soil core samples provided laboratory evidence for the presence of some microbial activity and contaminant degradation abilities, they did not correlate well with either contaminant location, geochemical, isotopic, or direct microscopy data.

Perchlorate and Nitrate Remediation Efficiency and Microbial Diversity in a Containerized Wetland Bioreactor

We have developed a method to remove perchlorate (14–27 μg/L) and nitrate (48 mg/L) from contaminated groundwater using a wetland bioreactor. The bioreactor has operated continuously in a remote field location for more than 2 yr with a stable ecosystem of indigenous organisms. This study assesses the bioreactor for long-term perchlorate and nitrate remediation by evaluating influent and effluent groundwater for oxidation-reduction conditions and nitrate and perchlorate concentrations. Total community DNA was extracted and purified from 10-g sediment samples retrieved from vertical coring of the bioreactor during winter. Analysis by denaturing gradient gel electrophoresis of short, 16S rDNA, polymerase-chain-reaction products was used to identify dominant microorganisms. Bacteria genera identified were closely affiliated with bacteria widely distributed in soils, mud layers, and fresh water. Of the 17 dominant bands sequenced, most were gram negative and capable of aerobic or anaerobic respiration with nitrate as the terminal electron acceptor (Pseudomonas, Acinetobacter, Halomonas, and Nitrospira). Several identified genera (Rhizobium, Acinetobactor, and Xanthomonas) are capable of fixing atmospheric nitrogen into a combined form (ammonia) usable by host plants. Isolates were identified from the Proteobacteria class, known for the ability to reduce perchlorate. Initial bacterial assessments of sediments confirm the prevalence of facultative anaerobic bacteria capable of reducing perchlorate and nitrate insitu.

A Systematic Methodology for Selecting Decontamination Strategies Following a Biocontamination Event

Decontamination and recovery of a facility or outdoor area after a wide-area biological incident involving a highly persistent agent (eg, Bacillus anthracis spores) is a complex process that requires extensive information and significant resources, which are likely to be limited, particularly if multiple facilities or areas are affected. This article proposes a systematic methodology for evaluating information to select the decontamination or alternative treatments that optimize use of resources if decontamination is required for the facility or area. The methodology covers a wide range of approaches, including volumetric and surface decontamination, monitored natural attenuation, and seal and abandon strategies. A proposed trade-off analysis can help decision makers understand the relative appropriateness, efficacy, and labor, skill, and cost requirements of the various decontamination methods for the particular facility or area needing treatment--whether alone or as part of a larger decontamination effort. Because the state of decontamination knowledge and technology continues to evolve rapidly, the methodology presented here is designed to accommodate new strategies and materials and changing information.

A Biological Decontamination Process for Small, Privately Owned Buildings

An urban wide-area recovery and restoration effort following a large-scale biological release will require extensive resources and tax the capabilities of government authorities. Further, the number of private decontamination contractors available may not be sufficient to respond to the needs. These resource limitations could create the need for decontamination by the building owner/occupant. This article provides owners/occupants with a simple method to decontaminate a building or area following a wide-area release of Bacillus anthracis using liquid sporicidal decontamination materials, such as pH-amended bleach or activated peroxide; simple application devices; and high-efficiency particulate air-filtered vacuums. Owner/occupant decontamination would be recommended only after those charged with overseeing decontamination-the Unified Command/Incident Command-identify buildings and areas appropriate for owner/occupant decontamination based on modeling and environmental sampling and conduct health and safety training for cleanup workers.

False negative rate and other performance measures of a sponge-wipe surface sampling method for low contaminant concentrations.

Recovery of spores from environmental surfaces is known to vary due to sampling methodology, techniques, spore size and characteristics, surface materials, and environmental conditions. A series of tests were performed to evaluate a new, validated sponge-wipe method. Specific factors evaluated were the effects of contaminant concentrations and surface materials on recovery efficiency (RE), false negative rate (FNR), limit of detection (LOD) - and the uncertainties of these quantities. Ceramic tile and stainless steel had the highest mean RE values (48.9 and 48.1%, respectively). Faux leather, vinyl tile, and painted wood had mean RE values of 30.3, 25.6, and 25.5, respectively, while plastic had the lowest mean RE (9.8%). Results show a roughly linear dependence of surface roughness on RE, where the smoothest surfaces have the highest mean RE values. REs were not influenced by the low spore concentrations tested (3 x 10{sup -3} to 1.86 CFU/cm{sup 2}). The FNR data were consistent with RE data, showing a trend of smoother surfaces resulting in higher REs and lower FNRs. Stainless steel generally had the lowest mean FNR (0.123) and plastic had the highest mean FNR (0.479). The LOD{sub 90} varied with surface material, from 0.015 CFU/cm{sup 2} on stainless steel up to 0.039 on plastic. Selecting sampling locations on the basis of surface roughness and using roughness to interpret spore recovery data can improve sampling. Further, FNR values, calculated as a function of concentration and surface material, can be used pre-sampling to calculate the numbers of samples for statistical sampling plans with desired performance, and post-sampling to calculate the confidence in characterization and clearance decisions.

Inhibiting the Transport of Hazardous Spores Using Polymer-Based Solutions

A series of polymer solutions were developed for the purpose of immobilizing aerosolized 1–10 μ m sized hazardous biological particles. The polymer solutions were designed as tools for emergency response and remediation personnel. The inhibition of secondary aerosolization and migration of biothreat particles has important implications for public health protection and contamination cleanup. Limiting further dispersion of particles such as Bacillus anthracis spores may reduce inhalation hazards and enhance remediation efficiencies. This study evaluated film-forming polymers that have multiple functional groups capable of attracting and binding particles; these included acrylates, cellulosics, vinyl polymers, and polyurethanes. The selected polymers were combined with appropriate solvents to design solutions that met specific performance objectives. The polymer solutions were then evaluated for key characteristics, such as high adhesion, high elasticity, low density, short drying time, low viscosity, and low surface tension. These solutions were also evaluated for their adhesion to biothreat agent in a series of wind tunnel experiments using highly refined aerosolized Bacillus atrophaeus spores (a simulant for anthrax, 1–3 μ m). Results demonstrated that a polymer solution, an amphoteric acrylate identified as NS-2, was the best candidate for attaching to spores and inhibiting reaerosolization. This polymer solution was anionic, thus providing the electrostatic (coulombic) attraction to cationic spores, had low surface tension, and performed well in wind tunnel tests.

Enhancing activated-peroxide formulations for porous materials: Test methods and results

During an urban wide-area incident involving the release of a biological warfare agent, the recovery/restoration effort will require extensive resources and will tax the current capabilities of the government and private contractors. In fact, resources may be so limited that decontamination by facility owners/occupants may become necessary and a simple decontamination process and material should be available for this use. One potential process for use by facility owners/occupants would be a liquid sporicidal decontaminant, such as pHamended bleach or activated-peroxide, and simple application devices. While pH-amended bleach is currently the recommended low-tech decontamination solution, a less corrosive and toxic decontaminant is desirable. The objective of this project is to provide an operational assessment of an alternative to chlorine bleach for low-tech decontamination applications activated hydrogen peroxide. This report provides the methods and results for activatedperoxide evaluation experiments. The results suggest that the efficacy of an activated-peroxide decontaminant is similar to pH-amended bleach on many common materials.