New view of bacteria-mineral interface to advance bioremediation

December 11, 2001

Researchers studying the feasibility of in situ bioremediation have a new tool for their analytical arsenal. The Department of Energy's Idaho National Engineering and Environmental Laboratory (INEEL) can now precisely map mineral crystals and bacterial growth on basalt using a customized laser imaging Fourier transform mass spectrometer.

In the first reported application of imaging Fourier transform mass spectrometry (FTMS) to the field of biogeochemistry, INEEL researchers created high-resolution pseudo-images showing the arrangement of minerals within basalt and bacterial growth on the rock surface.

Researchers need to better understand why some microbes are attracted to specific minerals before they can effectively harness microbial populations for bioremediation.

The technique creates highly reproducible two-dimensional maps of the bacterial-mineral interface, providing critical information about bacterial metabolism. Researchers can also create three-dimensional images (depth profiles) by stacking the maps. Geomicrobiologist Mary Kauffman will present a poster on this research at the American Geophysical Union meeting in San Francisco, Dec. 11, 2001.

"To exploit microbes for bioremediation, we need to understand the complex relationships between microbes, minerals and contaminants to determine if the microbes can degrade, mobilize or sequester contaminants," said INEEL chemist Jill Scott, lead researcher for the project. Understanding what environments subsurface bacterial populations prefer is a defining piece of the puzzle for researchers developing environmental remediation techniques.

If microbes are getting enough energy from specific minerals on the basalt, they may be less available to researchers for bioremediation activities. "Our imaging FTMS can spatially resolve the location of microbes on heterogeneous minerals, allowing us to answer the questions of which microbes are present, where are they located on the minerals, and what are they doing," said Scott.

"Bacteria are extremely energy efficient organisms, "Kauffman explains. They do what's easiest to survive and put the least amount of energy into getting what they need. So if they are more active in one location than another, there's a reason."

The research team chose to evaluate microbial activity on basalt because the INEEL subsurface is dominated by layers of basalt. "The composition of the basalt is so complex it presents a unique problem for those of us doing geomicrobiology here. The fine-grained heterogeneity is very difficult to work with when you're looking at microbial interactions with the minerals," said Kauffman.

The scanning technique works in the same way a person reads text. The laser starts at the top left of a sample, scans across various locations in a row, and then returns to the left to start again. The FTMS detects the spectrum of the minerals and bacteria present, and stores that data in memory along with spatial information about the locations of each sample spot. At the end of the scan, researchers can assemble the data into a kind of paragraph of information they can decipher.

The ability to take multiple data points and retain the spatial context of the information is a major advance for geomicrobiology research. "In the past, we could find out if a specific microbe was present, but not where it was and why," said Kauffman.

The team creates dime-sized (19 mm), sterilized disks of INEEL basalt and characterizes the surface of the minerals with the FTMS. Then they inoculate the disks with a lean culture of a single form of bacteria found in INEEL soils and allow the bacteria to form a biofilm. The samples are rinsed to remove any bacteria that are not actually attached to the mineral surfaces and then analyzed in the FTMS.

Once the information about microbial growth has been collected, the team can blast through the microbial layer to evaluate the surface of the minerals. "Changes in the spectra of the minerals tell us what the bacteria were metabolizing," explains Kauffman.

Because INEEL researchers want to successively scan the sample layer by layer and yet still be able to correlate the information spatially, they needed to keep the sample stationary and re-measure the same locations. INEEL's Paul Tremblay, a certified Naval nuclear engineer, created what he calls a Virtual Source for the FTMS.

"The Virtual Source makes it look as though the source of the laser is coming from different locations," he said. "But actually, we've designed a complex arrangement of prisms to redirect the laser beam at precise angles to reach the locations we need."

The size of the sample spots being analyzed can be adjusted depending on the researcher's needs. "The smallest, most precise spot size we've been able to achieve is around 2 micrometers, about the size of an individual bacteria cell," said Tremblay. "It's more common to sample spot sizes between 10 to 12 micrometers, which is about one-tenth the thickness of a human hair."

In the past, researchers have created simulated basalt material for analysis, but best efforts still generate simulated materials that are just too uniform and don't adequately represent natural basalt samples.

"We may find out that the microbes we're interested in prefer the boundaries between two minerals. It's hard to say. But a major benefit of this technique is that we can analyze real basalt samples," explains Scott.

In the future, the team plans to study how bacteria change the mineral surfaces over time. "We'll create a batch of inoculated samples and analyze the bacteria-mineral interactions over time," said Kauffman.

As the team grows increasingly sophisticated in understanding the capabilities of this new application of FTMS and interpreting the data they collect, they will progress to multiple strains of bacteria to fully mimic the real world environment under the INEEL site. "Right now we're keeping the number of variables manageable as we're learning," said Kauffman.

The instrument development and testing is funded through the INEEL's discretionary Laboratory Directed Research and Development Program. The LDRD program provides seed money to develop instrument capabilities at the INEEL to address experimental needs related to the subsurface research projects.

The geoscience portion of this research is funded by the INEEL's Environmental Systems Research and Analysis (ESRA) program, authorized and supported by the Department of Energy's Environmental Management Office EM-50.

The ESRA program supports research designed to better understand the interplay of biogeochemical processes in the subsurface environment. Improving our understanding of such processes will help researchers develop more effective detection, characterization, remediation, and monitoring technologies.

ESRA and LDRD research support the objectives of INEEL's Subsurface Science Initiative and the DOE's environmental management mission.
-end-
The INEEL is a science-based, applied engineering national laboratory dedicated to supporting the U.S. Department of Energy's mission in environment, energy, science and national defense. The INEEL is operated for the DOE by Bechtel BWXT Idaho, LLC.

Technical contacts: Lead researcher Jill Scott, 208-526-0429, or scotjr@inel.gov; Mary Kauffman, 208-526-2684, or kaufme@inel.gov; Paul Tremblay, 208-526-9664, or plt@inel.gov

Note to journalists: Biogeochemist Mary Kauffman will be presenting this research at the AGU Poster Session in the Moscone Center, San Francisco on Tuesday Dec. 11, 2001, at 1:30 p.m. in Hall D.

DOE/Idaho National Laboratory

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