Hoi-Ying Holman and her colleagues Dale Perry, Michael Martin, Wayne McKinney, and Jennie Hunter-Cevera of the Department of Energy's Lawrence Berkeley National Laboratory made the discovery by examining core samples from beneath the Radioactive Waste Management Complex at DOE's Idaho National Engineering and Environmental Laboratory. By using infrared spectromicroscopy at Berkeley Lab's Advanced Light Source (ALS), they were able to follow the reduction of toxic metals among populations of living organisms on minerals for the first time. The researchers will discuss their findings in the forthcoming October/November issue of Geomicrobiology Journal.

SPATIALLY RESOLVED INFRARED SPECTROSCOPY OF A BASALT SURFACE SHOWS A STRONG CORRELATION BETWEEN CONCENTRATIONS OF BIOLOGICAL MOLECULES CHARACTERISTIC OF MICROBES (TOP) AND PEAK CONCENTRATIONS OF REDUCED CHROMIUM (BELOW).

"We have shown that organic vapor may accelerate the transformation of mobile, toxic chromium pollutants into less mobile, less toxic, stable compounds," says Holman, a chemist and engineer with Berkeley Lab's Earth Sciences Division and the Center for Environmental Biotechnology (CEB). "This should help in the design and implementation of new, environmentally benign remediation techniques for cleaning up mixed waste sites."

The Idaho site is polluted with mixtures of hexavalent chromium and other inorganic ions, radionuclides, petroleum hydrocarbons, and volatile organic compounds. Hexavalent chromium (Cr6+) is carcinogenic, mutagenic, and highly toxic to living organisms because it occurs in soluble chromates that readily cross cell membranes. Once inside the cell, these ions are reduced (electrons are added) first to pentavalent chromium (Cr5+), then to trivalent chromium (Cr3+), which disrupts DNA replication.

Outside the cell, however, trivalent chromium is much less toxic, because it is insoluble and can't cross cell membranes. "Polyvalent metal ions at this and other polluted sites are reduced on the surfaces of geologic materials -- that's been known for some time," Holman says, "but there were two views of how this happens."

The dry Columbia River basalts are limited in organic carbon and other nutrients, Holman says, but dense clusters of microorganisms live in the pores and fractures of the rock. "When water infiltrates from rain or melting snow, a burst of nutrients reaches them through fissures in the rock, and they bloom like flowers in the desert" -- suggesting that microbes might play a role in metal-ion reactions.

It was uncertain which microorganisms might be involved in a biological reduction mechanism and what metabolic processes were important, however. An alternate, chemical-mechanism hypothesis proposed that metal oxides such as iron oxides in basalt could help catalyze reductions with no help from living microbes.

To determine which mechanism was at work, Holman and her colleagues obtained basalt core samples beneath the site from the unsaturated rock above the water table. From these, Tamas Torok, a CEB microbiologist with the Lab's Life Sciences Division, isolated and purified 85 strains of microorganisms, many tolerant of hexavalent chromium and able to reduce it -- especially in the presence of toluene (C7H8), another of the site's contaminants, which is a common product of leaking fuel tanks.

These chemical reactions typically proceeded through one or more steps, and many of the organisms encountered bottlenecks that slowed the process. One strain of bacteria, Arthrobacter oxydans, emerged as the most effective.

Arthrobacter oxydans tends to concentrate in areas rich in magnetite, an iron-oxide compound common in basalt; 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.

Arthrobacter oxydans was reintroduced on some of the sterilized magnetite samples. Dilute chromate solution was applied to both the abiotic (barren) and biotic (inhabited) magnetite samples; in a separate set of tests, the samples were also bathed in a tenuous vapor of toluene.

Working on the ALS's infrared beamline over a five-day period, the researchers applied Fourier-transform spectromicroscopy to observe the steps in the reduction process and the precise location of reduced chromium.

"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. You have an extremely complicated spectrum in the ten-micrometer region," which is the dimension of the beam. "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. On samples with living Arthrobacter oxydans, in the absence of toluene, chromium reduction was weak.

But where Arthrobacter oxydans had been exposed to toluene, infrared spectromicroscopy showed that hexavalent chromium and toluene had been replaced by pentavalent chromium and products of hydrocarbon degradation, in association with biomolecules -- right where the bacteria were concentrated.

Was this microbial reduction an accurate reflection of what happened at the waste site? "We now needed to study natural communities in the basalt," Holman says, and to do so she had to devise a unique diamond saw that could cut thin slices from fresh basalt cores, slowly, at cool temperatures, under aseptic conditions.

Over a period of four months the slices of native rock, with their resident communities of microbes still in place, were exposed to solutions of hexavalent chromium and toluene vapor. 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 natural microorganisms were seen to be thriving -- in association with trivalent chromium.

Says Holman, "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."

The Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.

Additional Information:

• Microbial Reduction Of Hexavalent Chromium