To test the hypothesis that the microbes play a role in reducing toxic chromium, Holman and her colleagues obtained basalt core samples, from which microbiologist Tamas Torok of the Life Sciences Division isolated and purified 85 strains of microorganisms. They found that many of these microorganisms were tolerant of hexavalent chromium and able to reduce it—especially in the presence of organic vapors such as toluene, a common product of leaking fuel tanks.

HOI-YING HOLMAN       MICHAEL MARTIN         WAYNE McKINNEY Hoi-Ying Holman credits microbiologist Jennie Hunter-Cevera, founder of the Center for Environmental Biotechno- logy, and chemist Dale Perry for broadening her initial focus on toxins to include interactions involving microorganisms, animals, and human cells. Since coming to Berkeley Lab from Brookhaven, IR beamline spokesman Michael Martin has not only become fascinated with anything that fits under an infrared beam, he’s found time to develop an award-winning travel and language website and to coauthor a textbook in solid-state physics. Wayne McKinney credits Malcolm Howell for "pulling me back to pure science" at the Center for X-Ray Optics, after eight years as a manager of research at Bausch & Lomb. McKinney’s optical wizardry has benefited numerous Berkeley Lab divisions.

Most of the microbes eventually encountered bottlenecks that slowed the reaction process, but one strain of bacteria, Arthrobacter oxydans, performed consistently. A. oxydans tends to concentrate in areas rich in magnetite, however, an iron-oxide compound common in basalt. Because a competing "chemical method" hypothesis proposed that iron oxides help catalyze reductions with no help from living microbes, the researchers had to eliminate the possibility that the magnetite itself was responsible for the reduction.

They tested to see if reactions would proceed on sterilized magnetite under realistic environmental conditions: in an aerobic atmosphere, at room temperature, and in the dark.

In other tests, A. oxydans was introduced on sterilized magnetite samples, and dilute chromate solutions were applied to both abiotic (barren) and biotic (inhabited) samples. The bacteria were also introduced on sterilized samples that had been bathed in a tenuous vapor of toluene.

For five days the researchers studied the samples with Fourier-transform spectromicroscopy, working at the ALS’s infrared beamline 1.4.3, designed and operated by Wayne McKinney and Michael Martin.

"The infrared is the end of the spectrum not usually associated with synchrotrons," says Holman, "but for us it’s perfect—and not only because it’s nondestructive of organisms." The bright infrared beam can focus on a region just 10 micrometers in diameter. "We identified markers in this spectral region that tracked the key compounds that undergo changes. We could resolve the spectrum in time, to follow the different steps of the reduction, and also in space, to see exactly where the reactions were happening."

On the samples with no living bacteria, no changes were evident, and even on samples with living A. oxydans, chromium reduction was weak in the absence of toluene. But where the bacteria had been exposed to toluene, infrared spectromicroscopy showed that hexavalent chromium was reduced and toluene was degraded—exactly where the bacteria were concentrated.

To find out if the same thing could happen at the waste site "we now needed to study natural communities in the basalt," Holman says.

With the help of Andre Mei, a machinist in the Engineering Division’s Technical Services Department, she devised a unique diamond saw that could cut thin slices from unaltered basalt cores, slowly, at cool temperatures, and under aseptic conditions.

 The slices of native rock, with their resident communities of microbes still in place, were exposed to hexavalent chromium and toluene vapor over a period of four months.

At first, infrared spectromicroscopy showed no evidence of reduction, and it appeared that many of the organisms were dying. But after four months, chromium-tolerant and chromium-reducing microorganisms were seen to be thriving in association with trivalent chromium.

"As far as we know, this is the first time that infrared synchrotron studies have been used to follow the steps in the transformation of toxic chromium on mineral surfaces," Holman says. Moreover, "we have shown that organic vapor may accelerate the transformation. This should help in the design and implementation of new, environmentally benign remediation techniques for cleaning up mixed waste sites."

Dale Perry of Earth Sciences and Geraldine Lamble of the ALS, along with Jennie Hunter-Cevera, then head of the Center for Environmental Biotechnology and now president of the University of Maryland Biotechnology Institute, were members of the team that studied the reduction of chromium by microbes.

Recently Holman, McKinney, and Martin, working with colleagues from other institutions and other divisions within Berkeley Lab, have extended infrared spectromicroscopy to living human cells. The 10-micrometer focus of the bright beam from the synchrotron is roughly the diameter of a mammalian cell, so the beamline can produce unique spectral signatures of chemical reactions and physical changes within single cells, as well as distinguishing among different phases in the cell cycle and different cell lines.

Using synchrotron-based infrared spectromicroscopy to follow subtle chemical and molecular changes, Holman says, "we can study individual cells in real time without killing them or introducing extraneous factors." 

A technique with spectacular success in establishing the role of microbes in detoxifying carcinogenic pollutants has now been applied to the study of environmental effects, disease mechanisms, and therapies in living human cells— only the beginning for a uniquely useful tool of environmental, biological, and biomedical research.  — Paul Preuss