FEATURE
On the Trail of Bioremediating Microbes By tracking bacteria as they "swim" toward contaminants, researchers are improving models for bioremediation. REBECCA RENNER major barrier to designing in situ bioremediation projects has been the lack of a theoretical model that incorporates the basic properties of bacteria, contaminant, and soil, which are necessary for understanding what happens underground. With such a model, remediators could simulate a bioremediation strategy, predict its performance, and evaluate it economically. Using existing mathematical models for contaminant transport phenomena as a starting point, a research group based at the University of Virginia is working on a model that includes a critical piece of information missing from current models: how bacteria move in soil (1, 2). The model, being developed by chemical engineers Roseanne Ford of the University of Virginia and Peter Cummings from the University of Tennessee with support from IBM's Environmental Research Program, is being put to its first real-world test in the design of a new in situ bioremediation technology for contaminated sediments. The technology will have its first field test this summer In a partnership with Environmental Solutions, Inc., a Richmond, Va., remediation and recycling company, the researchers are working on a project to seed creosote-contaminated silts in southern Virginia's Elizabeth River with small ceramic balls permeated with bacteria. The technology has never been tried before, said Bobby Harrell, director of Virginia's Center for Innovative Technology, who set up the demonstration project. "We already know that the bacteria can do away with the creosote," said Harrell. "What we need to know is how many it takes to do the job, how much space they can cover, and how to get the bacteria out of the balls and into the sediments." The project has attracted the attention of the Elizabeth River Project, a nonprofit group working to clean up the river, which empties into the Chesapeake Bay. Ford and Cummings began their research collaboration with the idea that the mathematics to de-
Motility: An overlooked trait The bacteria that interest Ford not only consume contaminants, they also "swim" for their suppers, exhibiting a property called motility. Although no definitive experiment or observation has established how common this trait is, many of the bacteria that have been identified in bioremediation are Pseudomonas strains, and motility is common among this family of bacteria, said Ford. "Most bioremediation models being created assume that motility isn't important, in part because the appropriate mathematical descriptions are just now being developed," she explained. "But many of the bacteria that degrade contaminants are motile, and this characteristic may give them an advantage." Motile bacteria are self-propelling because of the rotating motion of flagella located on their outer surfaces. These bacteria also exhibit chemotaxis: They move in the direction of increasing concentration of the chemicals that they degrade.
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scribe bacterial transport should look something like existing numerical models for molecular transport phenomena, which can be derived from statistical mechanics. Although intermolecular forces tend to dominate the movement of molecules and affect bacteria, the microbes can also move independently. Therefore, to apply this approach to bacteria, the researchers have to understand how bacteria move. Supported by IBM, EPA, the National Science Foundation, and the Department of Energy, the research group combines observations of bacteria on the microscopic scale with computer simulations and quantitative analysis. The laboratory is equipped with a microscope that tracks the movement of an individual bacterium in three dimensions and a stoppedflow diffusion chamber in which populations of bacteria respond to chemical gradients. Computer simulations are enlisted to perform virtual experiments as another tool in pinning down a mathematical description of bacterial movement.
To find out everything about the comings and goings of motile bacteria underground, the group started by studying the detailed movements of an individual bacterium and populations of bacteria. Using the microscope that tracks movement in three dimensions, Ford is studying three things: the unimpeded swimming behavior of bacteria, their reactions near surfaces and in porous media, and their response to a chemical gradient. The experiments are a bit like playing a video game, according to Ford. First, a member of the research group uses a joystick to search for a bacterium swimming in suspension on a glass slide. Once the bacterium is located in the cross-hairs of the tracking microscope, the instrument locks onto the bacterium's movements and records its position every 1/12-second. These traces can then be visualized on one of 13 IBM RS/6000 workstations. According to Ford, unimpeded bacteria exhibit "run and tumble" movement—they move briefly in a random direction, tumble, and then move off again in another direction. It may sound ineffective, but as a result of this running and tumbling, bacteria diffuse through a fluid three orders of magnitude faster than inert particles of the same size. The magnitude of this effect means that mathematical models cannot really ignore bacterial motility, Ford claims. To move toward a chemical gradient, the bacteria tend to run a bit longer when they are going toward the higher concentration. Current experiments are simplifying the interactions of the soil and bacterial movement by analyzing the response of bacteria to glass surfaces and simple porous media such as clean sand of uniform size (3, 4). Devising a way to translate these seemingly erratic movements into an expression suitable for a computer model requires some ingenuity. First, Ford and Cummings analyze the results of enough experiments to generate tumbling frequency and turn angle distributions for when and where bacteria move. Then, to simulate "run and tumble" motion using Monte Carlo methods, the computer program can call up the distributions and "roll the dice" to determine where each bacterium moves after each tumble. Taking on contaminated sediments The Elizabeth River contaminated-sediments project is the first real-world application of the group's work. The models are being used to predict what a mixture of different bacteria encapsulated in ceramic beads will do when the beads are shot into the sediments. Ford evaluates the technology's design by determining whether the bacteria will respond chemotactically and move out of the beads toward the contaminants. After just over a year's work on the project, she said that the results have pushed the group to move from simple laboratory scenarios to more complex real-world applications. To verify the modeling work the group is also testing the technology in aquarium experiments. If the pilot project works, the rewards could be great. Others have found it difficult to use in situ bioremediation on sediments. "Underwater in situ bioremediation has usually proved too difficult. You can't just spread the bacteria on the sediments, because turbulence and currents wash them away," according to
Microscopic observations tracking individual microbes as they move through a solution (top left) and near a surface (top right) are being used by researchers to better understand the role of microbial self-propulsion in remediation. Characteristic "run-and-tumble" behavior is seen in a bacterium moving far (>400 urn) from a surface. The bacterium swims in circles parallel and adjacent to a nearby surface as the surface forces act on the bacterium and the surface. A model developed from these observations produces a simulated track of a bacterium moving in a porous medium (above). (Courtesy National Academy of Sciences, USA (4) and Roseanne Ford, University of Virginia)
Diana Bailey, a member of the Elizabeth River Project and spokesperson for the Army Corps of Engineers. "If it were possible, it would really improve our ability to handle sediment contamination." Initial results of their research have changed Ford's views about what mobile bacteria can do for remediation. "Originally, we were thinking that the bacteria could move from an injection site toward contamination that was about one or two meters away," she said. "Now we think that motility and chemotaxis are more likely to be useful for getting rid of residual contamination trapped in difficult-to-reach areas." References (1) Frymier, P. D.; Ford, R. M.; Cummings, P. T.AIChEJ. 1994, 40, 704-15. (2) Strauss, I.; Frymier, P. D.; Hahn, C. M.; Ford, R. M. AIChE J. 1995, 41, 402-14. (3) Duffy, K. J.j Cummings, P. T.; Ford, R. M. Biophys.J. 1995, 68, 800-6. (4) Frymier, P. D. et al. "Three-dimensional tracking of motile bacteria near a solid planar surface," Proc. Natl. Acad. Sci. USA 1995, 92, 6195-99. Rebecca Renner is a contributing editor of ES&T. VOL. 31, NO. 4, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS • 1 8 9 A
