Life in (and on) the Rocks

Life on and in the rocks

Processes on the Earth's surface range from microbial to global and from seconds to eons, writes organic geochemist Kate Freeman.

three shots of microbes in different colors

"It's kind of poetic isn't it," Freeman, a Penn State associate professor of geosciences says now. "I think that's what drew me to the geosciences. The range of scale across space and time. The way things are interconnected."

Freeman's research group studies everything from the earliest life on Earth to methane cycling in modern wetlands and bogs. She is one of 17 faculty members involved in an interdisciplinary program at Penn State called BRIE—Biogeochemical Research Initiative for Education—now in its second year.

Biogeochemists, says Freeman, study "the physical, chemical, and biological processes that influence the distribution of elements on the planet's surface": life-sustaining elements like carbon that play a role in global warming, for instance, as well as nitrogen, hydrogen, and metals like iron and nickel. "We're looking at the source and fate of those elements in the environment from an interdisciplinary perspective," she explains. The term biogeochemistry acknowledges that biology—specifically microorganisms—plays a role in the Earth's chemical cycles.

The program consists of five research teams of faculty, post-doctoral research assistants, graduate students and undergraduates. The researchers come from four colleges—Science, Engineering, Agricultural Sciences, and Earth and Mineral Sciences—and five departments—biochemistry and molecular biology, geosciences, agronomy, civil and environmental engineering, and materials science and engineering.

"Some of the teams have coalesced already and other teams are still evolving," says Freeman. The teams explore an array of interdisciplinary research questions: Can geochemistry improve methods for isolating microorganisms from a wide range of natural environments? What are the specific cell-surface forces by which organisms attach to mineral and engineered surfaces? How do bacteria acquire metals from minerals during soil formation and rock weathering? What are the specific chemical and microbiological controls on the bioavailability and biodegradation of natural soil organic matter? How do sediment oxygen dynamics influence the rates and mechanisms of carbon and nutrient cycling and the release of greenhouse gases?

Freeman is no stranger to interdisciplinary research. "The program is successful because the faculty were already talking to each other," she says. "The research is great," she adds, "but BRIE is primarily a graduate education program."

The Biogeochemical Research Initiative for Education began in 1998 when geosciences professor Sue Brantley, microbiology professor Jean Brenchley, and Freeman garnered a $2.7 million National Science Foundation grant that was matched by Penn State.

"Different fields are so highly specialized, how can a researcher who has training in one discipline break into another?" Brantley asks. "Start with graduate students. That's what the BRIE program aims to do. Train graduate students in the techniques of another discipline to promote interdisciplinary research programs."

All BRIE students must take a survey course their first semester in the program. The course, team-taught by all the BRIE faculty members, is designed to introduce students to the analytical tools necessary to answer interdisciplinary research questions. "You're taking ten students and exposing them to equipment that might take one to six months to learn. And they have one afternoon," says Freeman. "You try to strike a balance between inundating them with information and giving them a broad overview of the instruments available."

This year, the survey course focused on analyzing water, sediment, and mineral samples taken from the Bear Meadows Natural Area. Three of the geochemists on the BRIE faculty, Mike Arthur, Todd Sowers, and Freeman, worked together to make their lab sessions complementary. Arthur had the students measure the flux of methane gases in the Bear Meadows swamp. Sowers had them look at the properties and isotopic composition of the gases. For Freeman, the students checked the isotopic signatures of the organic matter in the wetlands. The students learned how to do a wet chemistry analysis to learn the composition of the water samples. They cultivated and isolated bacteria samples. They used fluorescence microscopy and scanning electron microscopy to image the number of bacterial colonies. And they used mass spectrometry to measure carbon isotopic signatures—the chemical fingerprint—of the materials in the swamp.

"The students are motivated. They help each other," says Freeman. "They figure out each other's strengths and elbow their neighbors when they don't understand a term the instructor has used. As a community, they pool their resources."

The students must take a certain number of courses outside of their discipline. Microbiology students can take geochemistry or civil engineering courses. Geoscience students can take microbiology or soil science courses. They must also choose research advisers from two different fields. The challenge of venturing outside of their home department is exciting for most of them.

man digs in soil

Andres Marin, a first-year BRIE student in the geosciences department, for example, had earned a bachelor's in geology at Humboldt State University in California. When he was deciding on graduate programs, he wanted to focus on aqueous geochemistry, but he was also interested in applied aspects of the field, such as soil remediation. He "didn't want to be stuck in a certain discipline," he says. He liked the BRIE program because he was able to explore areas outside of the geosciences department. "We have support from the faculty in other departments," says Marin, and an invitation to use the instruments in their labs.

"One of the difficulties of interdisciplinary programs is that students get lonely," says Freeman. "They fall through the cracks between disciplines and often they feel like they don't have a home department. But the BRIE program gives them a home, both scientifically and socially.

"One of the challenges is that the students are coming from different departments that have different requirements and different cultures. The executive committee tries to navigate through those cultures." Brantley, Freeman, Brenchley, environmental engineer Bruce Logan, and soil scientist Jon Chorover, make up the executive committee. The faculty "interact at different levels," says Freeman. "The research carried on by BRIE students serves as a bridge. It spawns interaction between the faculty and can lead to collaboration in terms of teaching and research."

"At Penn State, we're lucky," adds Brantley. "The infrastructure is such that it's easy to work between colleges—easier, at least, than it is for some of my colleagues around the country. It's pretty forward-thinking."

As a geosciences graduate student at Indiana University, Freeman had taken a course in microbiology. "I was thinking this week about how grateful I am for that class. It has given me a foundation for the research I'm working on now."

BRIE is a program that would have appealed to her as a graduate student, she adds. "I would have been attracted to the interconnectedness, and to the independence that these students have. It gives me great pleasure to offer the BRIE program to students."

BRIE students are given a $4,000 research "credit card" to carry out their thesis work. The money can be used for research-related expenses, or to subsidize their living expenses if they take an internship outside of the University. The students are also given an $1,800 teaching credit card to develop a teaching module for an undergraduate or a graduate course. Mark Strynar, a BRIE doctoral student in soil science, developed a teaching module for an undergraduate course on techniques in environmental chemistry that Brantley and Freeman teach.

"I showed the students how to isolate natural organic material from the waters and sediments at Bear Meadows," says Strynar. "You can't study the material if you can't isolate it." The course credit card allowed Strynar to purchase the specialized glassware and resins that are needed to carry out the techniques that he demonstrated.

woman stands with arms crossed with man experimenting in background

In his own research, Strynar studies ways to clean up soil, specifically soil contaminated with TNT and other xenobiotics—manmade substances. "Soils scientists are good at understanding the interaction between natural organic material, microbes, and inorganic material in the soil," he says. Microbes are abundant in soil: More than a million per gram. Natural organic material is what's left over after the decay of plants and animals. Inorganic material could be anything from a rock buried in the ground, to heavy metal contaminants. "In normal conditions in soil, microbes cannot metabolize TNT, but they can partially transform the compound," Strynar explains. Under the right conditions, TNT can be transformed to a reactive metabolite. Through covalent binding, the TNT becomes strongly attached to humic material in the soil ("humus is the stuff that makes soil brown," Strynar explains). The compound is then immobilized and cannot leach into the groundwater.

When a xenobiotic substance is in the soil, Strynar explains, the microbes in the soil will try to degrade it. "The microbes may not have the right enzymatic pathway to fully degrade the xenobiotic. Sometimes, the xenobiotic is similar to a compound that the microbe recognizes. The microbe may then partially degrade it," Strynar explains. "It's not always one microbe doing the work. Microbe A might partially transform the contaminant, and then microbe B might come in and further transform it.

"I don't really care what microbe is doing it," he adds. "My main interest is in the contaminant's interaction with the organic material in the soil. As a soil scientist, from a bioremediation perspective, I'm interested in stimulating microbes already in the soil that are capable of detoxifying the target contaminant. The hope is that the contaminant is transformed to carbon dioxide, water, and other innocuous nutrients.

"Soil is such a complex system, but soil scientists don't see it as a black box, they understand much of what's happening in the system."

Amy Barnes, a third-year graduate student in materials science, came to Penn State on a National Science Foundation fellowship to work with Carlo Pantano, a world-renowned glass expert. Barnes makes a batch of glass about every other week. She mixes simple raw materials—silica, alumina, and other oxides—and heats them to a high temperature. For color, she might add transition metal oxides—chromium or iron for green glass, cobalt for blue. She then cools the mixture before it crystallizes. "Making glass is simple, but it often seems mysterious to people," she says.

Most of the time, her glass-making is part of an undergraduate laboratory class that she helps Pantano teach. But she also makes phosphate glass for her doctoral research. "Phosphate glass is different from the glass that's in windows, which is built around sand, or silica. It's a different network, it's made from different building blocks," says Barnes.

Barnes works with both Pantano and Brantley studying the interaction between microbes and minerals. She is particularly interested in how microbes obtain phosphorous from their surroundings. "Biological systems require phosphorous to survive and it's in low availability in the environment," says Barnes. She wants to know how microbes bind to—or "attack"—minerals to get the nutrients they need.

Barnes' research model is a simplified version of the mineral-microbe interaction. She substitutes phosphate glass for minerals found in nature. "Minerals are very heterogeneous," says Barnes. "When you're trying to understand fundamental interactions at the surface, it's difficult to take materials from nature. You have no control over how they're made. There's a lot of variability. With minerals, you take what you get. If you make a glass in the lab, you can control what goes into it," she says. Neither does Barnes use microbes. "Living organisms are complex. I'm starting by looking at simple organic molecules and how they bind to phosphate glass surfaces, then I'll extrapolate," says Barnes. "Organic molecules are organic molecules whether they're natural or synthetic."

woman pours hot lava-like substance out of beaker

Barnes takes a close look at the interface between the glass and the organic material using an atomic force microscope (AFM), a powerful tool that gives her an image with a resolution on the order of a micron. The AFM operates in two modes. In contact mode, the tip of the microscope drags along the surface of the material. A laser detects how the tip is displaced as it moves over the surface. In the tapping mode—the one that Barnes usually uses—the tip resonates over the surface of the material. Changes in the amplitude of the resonance indicate whether there are pits or peaks on the surface. "It's like making a topographical map of the material, on a very small scale," says Barnes.

Barnes also uses x-ray photoelectron spectroscopy (XPS) to determine the chemical composition of the surfaces of materials. The instrument shoots electrons at a material and measures the photoelectrons that are emitted from the outermost ten nanometers of the material's surface. The energy and intensity of these electrons reveal the identity and concentration of the atoms in those ten nanometers.

Barnes learned about the BRIE program after she had already been at Penn State for a year. At first, she was wary of expanding her research to include biological systems. "I figured out that I hadn't taken a biology class since 1989, my first year of high school. I started out taking a 400-level biochemistry course at Penn State and I thought—whoa—this isn't going to happen. So I've been sitting in on some introductory-level courses for a little more background information."

As a veteran BRIE student, Barnes agreed to help with the biogeochemical analysis survey course last fall. Pantano asked her to bury glass samples at Bear Meadows so that the first-year BRIE class could learn how to use surface techniques to examine them. Fellow graduate student Mark Strynar gave her a pair of chest waders for the job. "I got in the water and I started sinking," says Barnes. "The bottom of the meadow is anaerobic," she adds. "Every time you take a step, a cloud of gas comes to the surface and it stinks."

But Barnes was able to bury the glass, at several different depths, in both water and sediment. Two months later, she dug up the samples for the BRIE students to study.

Pantano's main role in the BRIE program is to bring to the BRIE table the tools of materials science—powerful microscopes, ion-guns, x-ray guns, and lasers—that can be used to analyze the surfaces of materials that the geoscientists pull out of the ground—rocks, soil, minerals. Pantano works with Brantley, for instance, to study the effects of biological species on the weathering and corrosion of minerals in the environment.

"We try to use non-destructive methods," says Pantano. "We don't have to dissolve anything in solution to analyze it. These tools allow us to look as shallow as one to two atomic layers, and as deep as ten to 20 microns—that's 'billions and billions' of layers, as Carl Sagan might say," Pantano explains. "These tools were developed for the analysis of computer chips," he adds. "And now geochemists are using them."

Brantley and Pantano started working together long before the BRIE program was in place. "We have been studying the surface chemistry of natural oxide minerals, using synthesized glasses as laboratory models," Pantano says. "She wanted to know how to analyze the surface of the materials. We used to clean the dirt and bugs off the natural mineral surfaces, but now we're interested in the bugs, too."

The surface of a material, Pantano explains, can reveal how the material has been altered over time. Pantano holds up a smooth piece of glass that he made in lab. "If I bury this in the ground and dig it up later and clean off the dirt, you'll see a crusty white layer where water penetrated into the material. The structure of the layers tells us about the conditions under which the layers formed. You can use the thickness of the layer to calculate the time over which the material has changed. Water is our favorite tracer," he adds. "It's everywhere."

He is also interested in whether the presence of bacteria enhances how fast glass degrades in the environment. "Do bugs get on the surface and slow it down? Or do the bugs find something in the material that they can get energy out of?" If so, the bugs will accelerate the rate of degradation. The BRIE program is so interdisciplinary, Pantano says, you can ask these questions from diverse points of view.

Bruce Logan works at a very small scale. Like Strynar, Logan, a professor of environmental engineering, is also interested in remediating soils, using bacteria to clean up chemical pollutants. "But soils filter out bacteria," he says. "The bacteria go about five centimeters and stop." They stick to the soil and are washed away. "How do we make bacteria non-sticky?" he asks. "We realized that we didn't really understand the molecular properties that determine the adhesive nature of bacteria."

woman in red sweater posed near chemicals

Logan and his research group use an atomic force microscope to understand adhesion. The tip of the AFM is two to ten nanometers across. Bacteria are 1000 nanometers by 500 nanometers. "We can probe the surface of the bacteria and look at the interaction between the tip and the surface of the bacteria." They can also measure the force of attraction: usually on the scale of pico- and nano-Newtons.

"The BRIE program affords graduate students the opportunity to learn chemical techniques, surface techniques, transport of chemicals in the environment," says Logan. "The big picture is to understand biogeochemical cycling—minerals, organic matter, bacteria are all a part of that."

"Geochemistry is the chemistry of the Earth. Add biology onto it and you're looking at how life interacts with the chemical cycles of the Earth, how microbes influence the cycles of nutrients and minerals," says Pete Sheridan, a BRIE post-doc in microbiology.

Microbes shape the environment as much as they are shaped by it. There's an incredible diversity of bacteria in the soil, yet scientists are able to culture less than one percent of those bacteria in the laboratory. "Most of the organisms in the environment can't be cultured in the lab because we don't have the right media to grow them on," says Sheridan. Which means, he adds, that "what we're growing in the lab is not really representative of what is in the soil." The repercussions for biogeochemistry are great: "We might not be studying the right bugs.

"I'm interested in improving methods to culture what's out there. To go back and try to cultivate some of this great phylogenetic diversity that we're missing." To do so, working with an interdisciplinary team is key. Engineers using an atomic force microscope "can tell us what a surface looks like. Do organisms need a certain type of surface on which to grow? How does the rock surface determine which microorganisms can grow there? We can get a lot of information from each other. Geochemists can give me a detailed profile of the environment that these bugs are found in. That can help me better design a medium to grow the bugs."

Last spring, Sheridan taught a graduate level microbial biogeochemistry with Kate Freeman. Freeman lectured for the first ten weeks, and Sheridan taught a lab component for the last five weeks.

Sheridan asked the students in the course, mostly geoscientists with limited backgrounds in biology, to collect soil samples from outside of Deike Building. They put the dirt in a growth medium. Sheridan incubated the samples to give the microbes a chance to grow in the soil. Later, the students spread the growth media on plates and grew bacterial colonies. Working in small groups, the students selected individual bacterial colonies to study.

All the colonies were white on the plate and they all grew on the same media, making it almost impossible to distinguish them. "But when you get to the molecular level, what a difference," says Sheridan. The students had isolated several different strains of bacteria: Bacillus anthracis, Bacillus mycoides, Aureobacterium, Acinetobactor.

The lab component of the course focused on three major techniques: how to grow and isolate bacterial colonies, how to isolate DNA ("I had to explain DNA to the geologists," says Sheridan), and how to use PCR to amplify and sequence subunit of the DNA. "It takes a lot of expertise to do this and I was surprised at how well it worked in a class setting," says Sheridan.

As a graduate student at the University of Cincinnati, Sheridan did two tours in Antarctica. The first was six weeks, the second four months. At Penn State he works with microbiologist Jean Brenchley, whose lab specializes in extremophiles, or bacteria that thrive in extremes of temperature, pH, pressure, and salinity. "Often the bacteria are extremophilic for more than one condition. They may like to live where it's very salty, and very cold," says Sheridan.

"A lot of extremophiles are anaerobes"—meaning they don't need oxygen to survive. "Bacteria can exist anywhere. They change the environment and the environment changes them. If you killed everything with a nucleus, life would go along its merry way. If you killed all the prokaryotes—the bacteria—life would come crashing down very quickly."

Bacteria oxygenated the atmosphere over a billion and a half years ago. "Really it was the first global pollution event," says Sue Brantley. Oxygen made it possible for life to explode into multicellular organisms and complex plants and animals.

"Geologists like to go to funky places," Sheridan had noted, "and just about wherever they go, they find life." In Yellowstone's hot springs. Under three thousand meters of ice covering a lake in Antarctica. Nestled inside ice cores from Greenland. On the barrels of giant drills that plunge deep into the sea floor.

crowd in golden meadow

Brantley has been to some funky places, everywhere from the glaciers of Iceland to the deserts of Peru. As an aqueous geochemist, she studies natural waters and water-rock interactions—the slow process by which water, oxygen, and microbes break down rocks into soil. Brantley added the microbial realm to her research about five years ago. "It fits right into the theme of what I've been doing my whole career," she says. "Some people call it biogeochemistry. Others call it geomicrobiology."

Brantley has spent a good deal of her career talking to researchers in other fields. Her curiosity has led her in many different directions: She's worked with microbiologists "who help with classical techniques and with modern molecular techniques" and with engineers "who really understand the upper 100 angstroms of a mineral."

What gets the microbiologists and the geologists and the soil scientists and the engineers talking? Realizing that they could help each other shed some light inside those black boxes. It isn't easy to do. Brantley has written, "Neither faculty nor students cross disciplinary boundaries because of the extra time and resources necessary to learn the fundamentals of a second discipline."

But a broad scientific background is necessary to answer questions of biogeochemical importance.

When Brantley first began including biological systems in her study of water-mineral interactions, she asked microbiologist Greg Ferry for help. Ferry specializes in a class of bacteria called anaerobes—organisms that live in the absence of oxygen. "They're a special group of bugs and they have special nutritional requirements," says Ferry. That makes growing them a challenge.

But for Ferry's research group, growing anaerobes is "a basic laboratory chore," he adds. Nutrients are delivered to the anaerobes in temperature-controlled vats. "In nature, nutrients are limited, while in the lab, we can supply unlimited nutrients," says Ferry. The liquid slurry is then taken from the vats and the bacteria are "harvested" with a centrifuge—a machine that separates particles by their density. The resulting paste looks like peanut butter. While bacterial densities in soil or water are usually one million per cubic centimeter, the cell density in Ferry's paste can be as high as one billion per cubic centimeter.

The next step must take place in a glove bag—a plastic bubble filled nitrogen gas stretched over a lab bench. "Anaerobic microbes are difficult to culture because they're poisoned by oxygen," says Ferry. Working inside a glove bag, researchers like Ferry can break open bacterial cells and release the enzymes within them for study.

One third of all enzymes in nature contain a single atom of a metal at the active site. The active site is where chemical transformations take place, where one compound changes to another. "Metals are very reactive atoms," Ferry says. "They join with other atoms, or attack other atoms, you could say. Metals are good at breaking bonds and creating new ones." Thus heavy metals are an essential part of the nutritional requirements of anaerobic bacteria, which catalyze reactions in nature. "Getting the metals out of the environment so that the cells can grow and proliferate is one of the jobs of the cell," says Ferry. Heavy metals are found in the soil and they are bound within the structure of minerals.

Together Brantley and Ferry have elucidated the mechanism by which bacteria extract iron from rocks. Other heavy metals used in bacterial enzymes include nickel, cobalt, zinc, selenium, molybdenum, copper, and cadmium.

"It's a very sophisticated interaction between the microbe and the mineral," Ferry adds. "Bugs are extremely intelligent. They have to find the metal—sense that it is near. They have to dissolve the mineral to release the metal and know how to get the metal into the cell in the right amount while excluding metals that are toxic."

Ferry teaches a unit on growing anaerobic bacteria in the BRIE survey course; he particularly enjoys teaching grad students with no background in microbiology. "The field of microbiology is so enormously fascinating," says Ferry.

Growing up on a dairy farm, he says, he had "an intuitive idea about the role of microbes in nature." He'd always been interested in chemistry and in college he took a microbiology course. "I loved the way that microbes manipulate chemistry and how that can be harnessed for applied uses," says Ferry. "I was amazed at how such tiny living creatures could be so complex—in both their chemistry and their architecture.

"And think about the impact they've had—these organisms have completely transformed the Earth geochemically."

Sue Brantley, Ph. D., is professor of geosciences in the College of Earth and Mineral Sciences, director of the Center for Environmental Chemistry and Geochemistry, and director of the Biogeochemical Research Initiative for Education, 239 Deike Building, University Park, PA 16802; 814-863-1739; Kate Freeman, Ph.D., is associate professor of geosciences,242 Deike Bldg.; 863-8177; J. Greg Ferry, Ph.D., is the Stanley Person professor of biochemistry and molecular biology in the Eberly College of Science, 205 South Frear Bldg.; 863-5721; Carlo Pantano, Ph.D., is distinguished professor of materials science in the College of Earth and Mineral Sciences and director of the Materials Research Institute, 198 Materials Research Institute; 863-8561; Bruce Logan, Ph.D., is Kappe Professor of Environmental Engineering in the College of Engineering, 231Q Sackett Bldg.; 863-7908; Jean Brenchley, Ph.D., is professor of microbiology and biotechnology in the Eberly College of Science; 209 S. Frear; 863-7794; Jon Chorover, Ph.D., is assistant professor of environmental soil chemistry in the College of Agricultural Sciences, 418 Agricultural Sciences and Industry Bldg.; 863-5394; Andres Marin is a BRIE graduate student in the geosciences department, 213 Deike Building; 865-1215; Amy Barnes is an NSF Fellow in materials science and engineering, 102 Materials Research Institute; 863-0540; Mark Strynar is a BRIE graduate student in the agronomy department; 129 Land and Water Bldg.; 863-7787; Peter Sheridan, Ph.D., is a BRIE postdoctoral scholar in microbiology; 865-3330; The BRIE program is funded by the National Science Foundation and the University.

Last Updated May 01, 2001