Center for Environmental Biotechnology at UTennessee

The Hydrogeology Research Program at the University of Tennessee, Knoxville was established by Larry McKay in 1993. When founded, the main focus of the program was to examine groundwater flow and contaminant transport in clay-rich materials, including fractured saprolite, shale and till. Although topics of inquiry continue to expand as other researchers join the group, studying groundwater flow and contaminant transport in clay rich materials is still a central focus of the program.

Various types of contaminants (solutes, colloids and immiscible fluids) all behave differently in fractured soil or rock when compared with their behavior in sands and gravels. Migration of dissolved contaminants is strongly retarded by their diffusion from the fast flowing water in the fractures into the relatively immobile pore water of the matrix between fractures. Diffusion has much less of an effect on colloids (such as bacteria) or immiscible fluids (like gasoline or dry cleaning solvent). The goal of the research is to develop and test conceptual models which can describe the behavior of all three of these contaminant types. Studies often involve field or laboratory experiments with two or more contaminant types. Once data is obtained, it is then evaluated using computer models. These studies have great significance for assessing and remediating contamination in groundwater on the Oak Ridge Reservation. Because fractured clay-rich materials cover large areas of the eastern US, these studies can also apply to locations outside the confines of the Oak Ridge Reservation.

      Zoogloeal Polymer Production and Dewatering in Activated Sludge (contact Gary Sayler)
Municipal and industrial wastewater treatment plants have persistent problems with sludge quality that affect plant performance and discharge. To maintain effluent discharge criteria, plant operators spend hundreds of thousand of dollars for physical remedies to maintain water quality. Over the past few years, CEB researchers have made significant progress in determining how microbial interactions affect wastewater plant upsets. They successfully isolated an organism, Thauera sp. MZ1T, whose overabundance has been positively correlated to episodes of poor dewatering of activated sludge. Isolation and analysis of the exopolysaccharide (EPS) obtained from pure cultures of this strain revealed an unusual composition that possesses functional groups in common with Zoogloea ramigera EPS. Furthermore, the EPS appears to exhibit interesting metal-binding properties. Future research on wastewater treatment will be directed towards the identification of the genes involved in EPS biosynthesis and their regulation. The analysis and subsequent regulation of these genes may stimulate continued development of potential biotechnological applications of this unusual polymer, as well as help researchers determine what factors influence viscous bulking and poor dewaterability in activated sludge systems.

      Natural Attenuation of Hydrocarbon Contaminants (contact Gary Sayler)
Petroleum hydrocarbons, including gasoline, diesel fuel and jet fuel, are common groundwater contaminants. Bacteria capable of metabolizing these compounds are ubiquitously distributed in subsurface environments. In order to gain insight into microbial interactions with petroleum hydrocarbons in groundwater, a multidisciplinary field scale study is currently underway at Columbus AFB, MS. A groundwater system has been purposely contaminated with residual concentrations of a model jet fuel. Instead of using an active scheme of site remediation, a passive form known as natural attenuation was chosen. Natural attenuation involves an initial site characterization to assess the potential for biodegradation at the site, followed by intensive monitoring of the native microbiota. Using molecular probes targeting genes commonly associated with the degradation of components of jet fuel, we are monitoring natural attenuation of the contaminants by measuring changes in the microbial community. These molecular diagnostics have provided a direct means for monitoring the effects of hydrocarbon perturbation on the groundwater microbial community.

      Toxicity Screening Using Bioluminescent Reporter Technology (contact Paul D. Frymier)

The activated sludge process has been widely used in publicly owned treatment works (POTWs). Toxic loadings can cause upsets in the operations of the activated sludge process. Such upsets include decreased BOD removal, reduction of solids separation efficiency, and modification of sludge compacting properties. In the most severe circumstances, the activated sludge microorganisms may be completely inhibited and the treatment plant has to be shut down. Such toxic effects can be avoided if influent wastewater is screened for toxicity and protective actions are taken.

Whole-cell marine luminous bacteria have been employed in assays for wastewater toxicity assessment, e.g., the Microtox assay. Luminescent bacteria-based assays have a quick response to toxicants and an easily measured signal. However, because of their high sensitivity to toxicants, marine luminous bacteria are not representative of the activated sludge microorganisms.

A new strain of bioluminescent bacteria was created at the University of Tennessee's Center for Environmental Technology and was designated Shk1. The host strain of Shk1 was a Pseudomonad isolated from the aeration basin in an industrial wastewater treatment plant. This project involves the development and validation of influent toxicity screening methods based on Shk1. To date, the following tasks have been completed

Validation of Shk1 as an accurate surrogate for toxicity effects to the respiration rate of activated sludge microoranisms
Development of a lab-scale continuous toxicity monitoring system
Validation of Shk1 as an accurate and rapid screening sensor for toxicity associated with heavy metals and organic and phenolic compounds
Construction of QSAR models for prediciting Shk1 EC50 values
Development of a kinetic model of heavy metal toxicity to Shk1
Development and validation of a new protocol for batch-wise screening of wastewater treatment plant influent
Field-scale implementation of a continuous toxicity monitoring system for the influent to the Kuwahee wastewater treatment plant of the Knoxville Utility Board.

We are currently gathering data with the field installation in order to determine if the continuous monitoring system we are developing is an adequate predictor of plant performance degradation as a result of influent toxicity. The degree to which the response of Shk1 can be used to predict overall measures of plant performance (effluent BOD and ammonia, sludge volume index, etc.) is being evaluated by simultaneously considering several factors relevant to treatment efficiency, such as influent BOD, rainfall amount, etc. Principal component analysis (PCA) will be performed to analyze the field data.