From the Princeton Weekly Bulletin, November 17, 1997

Making bacteria move

$2.8 million grant supports research on subsurface
method of toxic cleanup

By JoAnn Gutin

Every geology student learns there are three classes of rocks: sedimentary, igneous and metamorphic. If Associate Professor of Geosciences T. C. Onstott (pictured with collaborator Envirogen microbiologist Mary de Flaun) were a rock, he would definitely fall into the metamorphic category.

Over the space of only a few years Onstott has changed his research focus several times: from dating ancient rocks, to studying bacteria that live deep underground in those ancient rocks, to studying soil bacteria that live right beneath our feet. In the process, he has shifted from the purest of pure science to science with practical applications in the here and now.

Evidently, metamorphosis has its rewards, among them a grant totalling $2.8 million dollars from the Natural and Accelerated Bioremediation Program of the Department of Energy (DOE). The grant will be administered jointly by Princeton's Department of Geosciences and by Envirogen Inc., a Lawrenceville-based biotechnology company specializing in the cleanup of industrial effluent and hazardous waste. The challenge of the three-year research project will be to find ways to speed up the underground movement of bacteria that degrade toxins in the water-bearing rock formations known as aquifers.

Bacteria three km. deep

If bacteria strike you as a strange focus for a geologist, join the club: Onstott admits that as recently as 1994 he felt the same way. That was the year he was first introduced to the strange micro-organisms called deep sub-surface bacteria.

First discovered less than a decade ago, these one-celled creatures can live within rocks as deep as three kilometers below the earth's surface, in environ-ments where temperatures can reach 75 degrees C. Virtual living fossils, probable descendants of the bacteria that lived when these buried rocks were part of the earth's surface, they were at first studied mostly by biologists. "The first time I went to a meeting about deep subsurface bacteria," says Onstott, "I thought I was in the wrong place. It didn't seem like geology at all."

But when the organizer of that meeting, Frank Wobber, a DOE program manager, implored geochronologists to help date the rocks where the bacteria lived, Onstott took up the challenge. He started learning about microbiology, he says, and "pretty quickly got bitten badly by the sub-surface bug." By 1996, the athletic Onstott was happily squirming through narrow mine shafts 3,500 m. below ground, taking bacteria-laden rock samples and trying not to think about the unimaginable weight of the earth poised just above his head.

However, that same year the roof fell in, metaphorically, when Onstott's funding source -- a different branch of DOE -- shut down its deep subsurface research program. This impelled Onstott to search for other ways to stay involved with the underground bacteria that by now intrigued him. Knowing that DOE was funding projects on bioremediation -- the use of enzymes and bacteria for environ-mental cleanup -- Onstott decided to collaborate with Envirogen microbiologist Mary de Flaun, another veteran of the now defunct deep subsurface bacteria program, on a new venture.

Not yet practical on large scale

For the past few years, scientists at Envirogen and elsewhere have been experimenting with a form of bio-remediation called bioaugmentation, which involves injecting pollution-eating bacteria directly into the ground. The bacteria, specially selected for their appetites for particular organic toxins, either metabolize the contamin-ants or otherwise make them harmless.

The method has shown great promise in small field tests but isn't yet practical on a large scale. Getting bacteria to degrade toxins isn't the problem; the problem is getting them to move through the aquifer. Most bacteria are homebodies: left to their own devices, they tend to adhere to solid particles in the soil and to stay in one place for the whole of their lifespans. This happens because most bugs carry a negative electrostatic charge on their surfaces, which causes them to adhere to the positively charged iron oxides in the soil. In order to clean up a big contaminated site with bacteria, "You'd be drilling a bajillion holes," Onstott says.

This is where the DOE grant comes in. With these funds, Onstott, de Flaun and colleagues will try to see how to get the most remediation from the fewest injections of microbes -- the biggest bang for the bacterial buck.

Microbiologist de Flaun has begun working on breeding "nonadhesive" bacteria, which she does by growing a colony of bacteria and inducing the bugs to migrate through a container of sand. She collects the ones that finish first and uses those peppier, less adhesive bacteria to start another colony. "It's natural selection, only cranked up," she explains.

Onstott, for his part, will be trying to change the environment in which the bacteria live, by altering the geo-chemistry of the aquifer. "Can we get the sediments to be less `sticky,' less attractive to the microbes?" he asks. "This is one of the things we want to learn." One possible approach is to change the pH of the groundwater, in the process altering the electrostatic charge of the soil. "Maybe you could just dump some carbonates in, like you do in a swimming pool."

Bacterial road race

Come this spring, the research team, which includes stratigraphers and hydrologists as well as micro-biologists and geologists, will have a chance to put some of their ideas about improving bioagumentation into practice at a test site in Virginia. There, they'll be staging what Onstott calls "a bacterial road race," pitting various strains of bacteria against one another under various soil conditions.

Ironically, the data on bacterial transport generated by this real-world project may answer some of Onstott's highly academic questions about the first microbes that piqued his interest, those exotic bacteria from the center of the earth. "As it stands now," he says, "we don't know whether the deep subsurface stuff took a thousand years to get there, or a thousand million. This work will help us figure that out."

Onstott, who earned his BS in geophysics at California Institute of Technology in 1976, came to Princeton as a graduate student. He received his PhD in 1980 and was a postdoctoral fellow and research associate for five years before joining the faculty as assistant professor in 1985. He has been associate professor since 1991.