May 22, 2001 (Blacksburg, Va., May 18, 2001) -- For decades, people have been interested in how microbes attach and release from mineral surfaces. The interaction is fundamental to the way microorganisms are transported through water treatment facilities, the effective use of agrochemicals, or the movement of toxic metals in ground water, for instance. But the forces between the molecules at the surfaces of microbes and minerals had never been measured.
Now, a Virginia Tech graduate student has invented a technique called biological force microscopy and he and his major professor have used it to determine what happens when Shewanella, a microorganism found in most soils, meets goethite, the most important iron oxide in soils worldwide. The research provides some of the first evidence of recognition between a living organism and an inanimate object such as a mineral.
The research will be featured in Science on May 18, 2001, in the article, "Bacterial Recognition of Mineral Surfaces: Nanoscale Interactions Between Shewanella and a-FeOOH," by Steven Lower, Michael F. Hochella Jr., and Terry J. Beveridge. Lower, soon to begin work as an assistant professor at the University of Maryland, College Park, invented the technique while a Ph.D. student in geology at Virginia Tech. Hochella is professor of geochemistry and mineralogy at Tech, and Beveridge is with the microbiology department at the University of Guelph.
Respiration is the process by which living things, including humans, break down carbohydrates to produce energy. Shewanella use oxygen to breakdown carbohydrates (breath); but, if there is no oxygen, the bacteria use iron3 (Fe III) to breath. This ability has a significant impact on the way minerals dissolve and the movement of iron in the environment.
So the researchers looked at Shewanella and goethite, the iron oxide known for turning soil yellowish. "The bacteria use the mineral as a terminal electron acceptor," Hochella says. "That is, the bacteria transfer (carbohydrate) electrons to the mineral. The addition of each electron breaks a bond between atoms on the mineral's surface and allows the goethite to shed iron. Its surface molecules convert from iron3 (Fe III) to iron2 (Fe II). The result can be iron shed into ground water. Or, if a toxic metal, such as lead or arsenic, is bound to the surface of the goethite, these metals are also shed."
Complex biomolecules on the surface of Shewanella bacteria facilitate the electron transfer. The researchers have determined that Shewanella have a much greater affinity for Fe III-containing minerals when oxygen is absent. "Upon a more detailed analysis of our data, we see that Shewanella make a special protein that interacts specifically with the surface of the Fe III mineral," says Lower. "It is as if Shewanella recognize the mineral as beneficial to life," he says. "This protein appears to be made for the expressed purpose of transferring electrons from Shewanella to goethite." The Virginia Tech researchers have been studying the nanoscale interactions between bacteria cells and mineral surfaces for about five years, since receiving "ASPIRE" funds from the university's research division to launch the new, interdisciplinary area. The way microbes and minerals effect each other is of fundamental importance in many sciences, including geochemistry, agronomy, and environmental sciences, Hochella explains. Researchers from all these areas are members of the minerals-microbe group.
"Working at the atomic level is not new," says Hochella. "But working at the nanometer level is." One nanometer equals 10 angstroms, or about 10 atoms. "We're working with pieces of matter the size of molecules that have unusual properties. We are just beginning to understand what the properties are and how we can put them to use," he says.
To study nanoscale interactions, Lower modified an atomic force microscope so he could attach a living bacterial cell to an arm and move the cell toward a mineral surface to observe and measure the interaction. "A cell is about one micron, or one-millionth (10-6) of a meter," says Hochella. "Under this new microscope, it looks like a blimp attached to the bottom of a huge pole."
Before the cell touches the mineral, there is an interaction -- attraction or repulsion. It is a weak force in terms of measurement, but it determines whether or not the microbe attaches to the mineral, Hochella explains. Lower's microscope measures the attraction in "nanoNewtons" -- a force that is between one millionth and one billionth of the force between a record player's stylus and a vinyl record when it is being played.
"By measuring the forces of the attraction, we think we can tell which biomolecules are facilitating the process. Some of these complex biomolecules haven't even been identified," Hochella says. Why is knowing about attraction or repulsion between microbes and minerals important?
"Identification of this protein is the first step in our complete understanding of how a bacteria moves electrons from itself to minerals," says Lower. "By understanding this process, we may then be able to control related processes, such as the release of metals or other contaminants from a mineral surface, which often go hand-in-hand with the electron transfer reactions. Also, Shewanella can couple the metabolism (i.e., breakdown) of organic contaminants, many of which are found in oxygen-poor environments, to their electron transfer process. By giving Shewanella minerals, such as like goethite, to live and breath in oxygen-poor environments, we may be able to remediate harmful pollutants," Lower says.
Hochella adds, "If you have a pathogenic bacteria in the soil, you want to know if it's going to travel into and within the ground water and get into drinking water. Or will it attach to a mineral surface and be removed from the water? So, we hope to be able to predict microbial movement," Hochella says.
Many scientists are also interested from the perspective of basic research, to answer fundamental questions of how things work.
"But for the article in Science we wanted to demonstrate the capability of the new microscope with a question of microbe-mineral interaction," Hochella says.
Probing the unknown properties of single biomolecules is the subject of the Virginia Tech group's continued research, for which they have just received a $1.1 million grant from the National Science Foundations newest initiative, nanoscale science and engineering.
Since the nanoscale interactions are mediated by biomolecules and the inorganic complements of the mineral surface, another Virginia Tech graduate student, Treavor Kendall, figured out how to attach a single biomolecule to the arm under the atomic force microscope. "You can't even see it under the microscope. It's very tricky work," says Hochella.
But the interaction can definitely be measured. "When you use the microscope to pull the biomolecule away from the mineral, it's like stepping on chewing gum then pulling your foot away from the pavement. You stretch the biomolecule until it snaps off. By measuring the stretching characteristics, we think we can tell which biomolecule is there. We are literally looking at their nano-mechanical characteristics."
Co-investigators on the NSF grant are Lower at the University of Maryland and Virginia Tech researchers Susan Eriksson, associate professor of geological sciences; Maddy Schreiber, assistant professor of hydrology, and Chris Tadanier, research scientist with the microbe-mineral group. In addition to microbe-mineral interactions, the group is studying protein folding and biofilms. "We will continue to study and attempt to mimic the natural specificity between biomolecules on bacteria and mineral surfaces," Lower says. "We are also trying to probe the elementary forces that cause a protein to fold and unfold because it is the folding of a protein that ultimately determines its function and activity. We are applying biological force microscopy to questions pertaining to medical science. For example, biofilm formation affects issues such as the decay of teeth and the function of artificial implants in humans. Biofilms form (or don't form) because of attractive (or repulsive) forces between two bacterial cells. We are using biological force microscopy to study these fundamental forces between living microorganisms."
The NSF grant is the second award in five years stemming from the $25,000 investment by Virginia Tech's research division (the ASPIRES program to launch new areas of research). The first external award was $386,000 from the U.S. Department of Energy for research by the new interdisciplinary group into mineral-microbe interactions. The ASPIRES grant was matched by funds from the College of Arts and Sciences and elsewhere and resulted in the hiring of Tadanier as a post doc. Tadanier's background is environmental science and engineering. Lower joined the program as a graduate student because of his interest in the interdisciplinary opportunities.
The microbe-minerals group's youngest graduate student, Andrew Madden, also received an NSF Graduate Student Fellowship, one of only 30 such fellowships awarded to earth scientists in the country this year. Tracy Cail, another recent addition to the mineral-microbe team, was awarded a prestigious graduate fellowship from the Department of Education.
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