University of Georgia

The Metabolism of Hydrogen by Extremely Thermophilic Bacteria

A Case Study of the R&D Value Mapping Project

Institute for Policy Research and Development

School of Public Policy

Georgia Institute of Technology

Atlanta, GA 30332-0345

This case was written by David Roessner, based on interviews and other materials developed by Hans Klein and Juan Rogers. Comments or questions should be directed to Hans Klein at 404 894 2258 or by email at hans.klein@pubpolicy.gatech.edu. The Research was sponsored by the Department of Energy, Division of Basic Energy Sciences, Contract ER 45562. The views presented here are the case author’s and do not necessarily represent those of the Department of Energy or Georgia Institute of Technology.

I. Project description

            This project seeks to identify and characterize the properties of a peculiar class of anaerobic microorganisms called thermophiles that have the property of growing optimally near and above 100°C. They have been isolated mainly from marine volcanic environments such as deep sea vents. When the project began in 1988, almost nothing was known about these bacteria, but their unique properties made them interesting to study, and they have possible applications in energy technology, pharmaceuticals, and the food industry. The research has focused primarily on these organisms’ enzymes, particularly their hydrogenases, which enable them to metabolize hydrogen. The boundaries of the research are fairly easy to define, because it is distinct from other enzyme research in several respects: thermophilic enzymes contain metal; enzymes that function at lower temperatures are fairly well understood; and the metal involved is tungsten, whereas virtually all other enzymes contain molybdenum.

II. Technical background of the project

            The project initially examined thermophilic hydrogenases. One of the organisms was grown on a large scale (400 liters) and hydrogenase and related proteins were purified, and their molecular and catalytic properties characterized using various biochemical and electron and nuclear resonance spectroscopic techniques. In the early stages, research questions involved identifying the metal centers that activate hydrogen and other gases at 100°C and above, and determining the limits to their stability and catalytic activity.

            The researchers purified hydrogenase and its physiological electron carrier, a novel ferredoxin. Later, rubredoxin, pyruvate ferredoxin oxidoreductase, and a unique tungsten-containing enzyme, aldehyde ferredoxin oxidoreductase, were purified. The latter two enzymes are thought to couple substrate oxidation to hydrogen production in a new type of tungsten-dependent glycolytic pathway. From the most thermophilic eubacterium currently known, a new type of iron-containing hydrogenase, which lacks the catalytic iron-sulfur cluster of lower temperature hydrogenases, was purified.

            Subsequently, the researchers purified two different types of tungsten-containing enzymes that both function as aldehyde ferredoxin oxidoreductases. One type (AOR) is thought to couple substrate oxidation to hydrogen production in a new glycolytic pathway, with the other type (FOR) appears to be involved in peptide catabolism. Crystals of AOR and POR suitable for structural determinations were obtained and their amino acid sequences determined by recombinant DNA techniques. The genes for ferredoxin, AOR and POR were cloned. The complete amino acid sequences of these proteins were determined. Most recently, in collaborative studies with D. Rees at California Institute of Technology, the crystal structure of Pf AOR was determined. This was the first structure for a hyperthermophilic, a tungsten-containing, or a pterin-containing enzyme.

III. Technical Focus and Project History

            In 1987 Michael W. W. Adams, the Principal Investigator for the research on which this case focuses, joined the Department of Biochemistry at the University of Georgia as an Assistant Professor. Previously, he had worked for six years at Exxon Research & Engineering in New Jersey, working on enzymes that metabolize hydrogen and nitrogen gas. These enzymes contain metals that were being used to remove sulfur from oil and coal, so Exxon researchers were asking what could be learned from naturally-occurring enzymes that contained such metals. Exxon was growing 400 liter cultures of gas-metabolizing microorganisms. They wanted to have researchers who knew the biology that would be relevant if the energy industry was going to be revolutionized. Since Adams left, Exxon has downsized and reorganized, with the New Jersey facility’s budget halved. The focus became much more commercially relevant.

            The University of Georgia seemed to be the obvious place for Adams to come, because they wanted to expand work on metal-containing enzymes. Adams wanted to get into a new field, but did not want to start from scratch. At UGA, Professor Harry Peck was trying to set up a center on metalloenzymes. He hired six young professors, of whom Adams was one. Peck was funded by DOE/BES, and knew several people there. Indeed, a number of researchers at UGA had been funded by BES for many years. Adams had never written a grant before, but BES seemed to fit his research interests, so he submitted a proposal in 1986/87. His description of the proposal preparation process is interesting:

"I was politically inept, and still am. I didn’t know what was going on. My knees tremble even now when I talk with these [DOE] guys. I didn’t know whether I had a good chance or not. But I knew we had done good work and just scratched the surface. And we were pretty productive in that first grant. The second submission, you have a lot of preliminary data, history, background, etc. The amount of effort for the first grants was night and day compared to the later ones."

            Adams became interested in organisms that grow at high temperature: thermophilic organisms. They metabolize hydrogen, and so were an interest of his because of the link to energy. He wanted to look at hydrogen, nitrogen, and methane products from metabolism. At the time his work began, very little was known about these organisms; it was virgin territory. It was also odd that they were anaerobic. There were very few labs in 1988 that had any expertise in this field, and Adams surmises that may have been one reason he was funded. The thermophilic microorganisms contained some of the enzymes Adams had been working on, and there were in addition several intriguing questions: how do they grow at 100°C? How do they live in deep sea vents at enormous pressures? Further, they were among the most slowly evolving, the most primitive, of organisms, and thus were remnants of life when the earth was very young and much hotter.

            The Energy Department came back with good reviews on the Adams proposal, but recommended that he do less--focus just on hydrogen. The research was funded in April 1988 for three years. At the same time, the group at UGA put together a large center proposal to NSF that was funded in 1989; almost 20 faculty were interested in metals in biology. The center grant (for a Center for Metalloenzyme Studies) was funded for five years and was renewed a couple of years ago. The Center proposal required a lot of interaction among faculty to get the grant. Each student had to have two major professors in different departments to meet NSF’s requirement for interdisciplinarity. Adams stated that this fostered a lot of interaction that probably would not have occurred otherwise. Every summer the Center sponsors a workshop on metalloenzymes with 60-70 students and experts from outside. According to Adams, this was part of Peck’s grand plan. He was head of biochemistry, but subsequently also head of chemistry, where he was asked to revitalize the department. He brought in younger, more aggressive faculty to accomplish this.

            The fermentation facility at UGA, which was the basis for an ongoing series of enzyme studies and probably helped Adams get his first grant, was set up by Harry Peck in the 1970s. It was state funded and run by a full time faculty person. At present UGA has a 600 liter and a 400 liter facility that are unique in the country for an academic institution. For a fee they supply cells to researchers and industry, while those in the university system get cells free. The product is the organism, usually frozen. The facility puts UGA at the center of this area. Adams wanted to use these facilities to grow large quantities of thermophilic organisms, extract their enzymes, and work with them.

            The year after Adams wrote his first proposal to BES and was awarded the grant, he applied to NSF to continue what he was doing at Exxon, studying the low temperature organisms. They are very distinct, different types of organisms. Almost nothing was known about the thermophilic organisms ("hot bugs") whereas the low-temperature ones were well characterized. Adams had two postdocs working on the DOE grant at first, and nine have worked on the grant from 1988 to the present.

            Since 1988 the field of research has expanded dramatically. However, UGA remains the only place that is doing this work on a large scale. The only other group working exclusively in this area is headed by Robert Kelly at North Carolina State, with whom Adams collaborates. Kelly’s interests are much more applied than Adams’; as a chemical engineer, Kelly is interested in enzymes that have more obvious commercial applications. The two are not in competition, but have compatible interests and got together. The two of them joined with John Baross at the University of Washington, who actually goes down in deep sea submersibles to capture thermophilic and other microorganisms. The three of them applied for a grant in 1991 or 92 to the bioengineering program at NSF to look at the properties of some of the enzymes studied in the DOE-funded work from a more applied, engineering perspective. That work was very successful and got renewed last year. The low temperature work funded by NSF has tapered off. When Adams renewed his NSF grant the main focus was on the enzyme his team discovered through the DOE work. Adams finds it easy to separate the various streams of scientific work funded from different agencies. Ferrodoxin work, for example, came under NIH.

            Adams’ research group also collaborates with researchers at Oak Ridge. This collaboration arose because the UGA facility sent them some hydrogenase. (The group gets requests about every two weeks for samples of organisms; they want cells or DNA. Sometimes they want purified enzymes.) In the Oak Ridge case, they got an inquiry in 91-92 about purchasing hydrogenases from Oak Ridge, where researchers were using high temp hydrogenase to try to take sulfur out of coal. Adams said he did not understand what they were trying to do, but when the original researcher at Oak Ridge retired and Eric Kaufman took over, Adams found communication easy and formed a relationship. Currently they have a joint grant, funded by DOE, in which most of the money goes to Oak Ridge. Internally, Adams collaborates with Robert Scott, Michael Johnson, and Donald Kurtz, all of whom are in the Chemistry Department. This work, funded by NIH, focuses on gene modification. Adams has approximately ten current collaborators who are working on various aspects of the DOE grant or on research stemming from that grant.

            The Adams research group has two competitors, both at the same university in the Netherlands. One group is headed by De Voos at the University of Wagingen, the other by Fred Haagen. De Voos is interested in physiology and metabolism, while Haagen is interested in the metal-containing enzymes. But Adams pointed out that one of his current postdocs if from Haagen’s lab, and a student working on the DOE project got his master’s degree from De Voos. Adams regards the competition as healthy.

            Many of the collaborations are a product of relationships initiated when the UGA group supplies enzymes to people who are experts in spectroscopic techniques. Michael Johnson is one such expert. Metal-containing enzymes are amenable to many studies in which other proteins cannot be used. These relationships are more than just sending researchers a sample. There is professional interaction, and students are encouraged to become involved. Other collaborators come to UGA to work on enzymes that do not interest Adams and his colleagues.

            Adams describes the major output of the DOE-funded research as publications. There has been no patenting. Kelly and Adams tried to start their own company about three years ago, but Adams quickly became convinced that he did not want to be involved in commercial activities. Kelly knew two former vice presidents from major chemical firms, now retired, who thought enzymes from these organisms would make tremendous contributions, and they knew that the necessary basic research was far removed from the commercial world. In their retirement they wanted to build bridges, and Kelly and Adams would be the academic partners for them. Both Kelly and Adams are now on the board of a company, Recombinant Biocatalysis, that is seeking to commercialize exotic enzymes, including thermophiles. There is an enormous potential market for enzymes in general; the thermophilic ones might be replacements for existing ones. There might be new applications because of their bizarre properties.

            By 1994 Adams’ lab had gotten quite large, with 20 active researchers. This includes 12 students and nine postdocs. In addition, typically 5-6 undergraduates are involved most of the time. The two prime enzyme systems they have been working on for DOE have "mushroomed." They found that the high temperature "bugs" were dependent on tungsten, whereas virtually everything else that lives has molybdenum. In 1995 the group published the first work on the crystal structure of these enzymes, including both molybdenum and tungsten enzyme structures. Much of this work was due to Douglas Rees at Caltech, a collaborator, and to DOE. The big difference in the group’s new proposal to BES is that they have acquired experience in molecular biology by hiring postdocs. The new focus is on how these enzymes work at the atomic level.

IV. Outputs and Impacts

            The primary outputs from this project have been publications by the UGA group and its collaborators. Adams lists 56 publications spanning 1988-1997 that resulted from DOE funding. In addition, Adams has edited two books on high-temperature microorganisms, one published in 1992 (Biocatalysis at Extreme Temperatures: Enzyme Systems Near and Above 100°C; American Chemical Society) and one in 1996 (Enzymes and Proteins from Hyperthermophilic Microorganisms; Academic Press). The list of publications is appended to this case. The DOE grants have enabled Adams and his colleagues to develop considerable funding for related work from NSF, NIH, and DOE’s Environmental Management Science Program. Adams and Johnson (Chemistry, UGA) received two NIH grants during 1991-98 totaling over $600K; Adams alone received grants from NSF during 1991-99 totaling over $800K for work on iron-sulfur clusters of hydrogenase; Kaufman (Oak Ridge) and Adams received $1.4 million from DOE; Kurtz, Scott, and Adams received a 4-year grant from NIH with the first year for $132K; and Kelly, Baross, and Adams have received $1.6 million from NSF for the period 1993-99 for work on biocatalysis at 100°C. Details are appended to this case.

            DOE funds supported, in whole or part, nine postdocs during 1988 to the present. Six students have received Ph.D.s on the project and two are currently working on their degrees. The current positions of the six doctoral graduates are:

	NIH
	Staff Scientist at Inual, Inc., in Chile
	Staff Scientist at 3D Pharmaceuticals, Inc., in PA
	Postdoc at Harvard Medical School
	Medical residency program, Emory University
	Postdoc at Caltech.

            The UGA group has hosted ten visiting researchers from Italy, Argonne National Labs, Germany the UK, Hungary Converse College, and Caltech. Collaborators number ten, including one at UGA and others from Northwestern, North Carolina State (Kelly), University of Washington (Baross), Stanford Synchrotron Radiation Lab, Caltech (Rees), University of Utah, Carnegie Mellon, Oak Ridge (Woodward and Kaufman), University of Minnesota, the University of Maryland, University of Cape Town, Argonne, and the University of Puerto Rico. The only significant industrial interaction is the previously-mentioned connection with Recombinant Biocatalysis, Inc., wherein Adams and Kelly serve on the company’s Scientific Advisory Board.

            Although probably not directly related to the DOE funding, Adams has received several honors and awards, including:

	Leo G. Friend Award, American Chemical Society (1993)
	Chemist of the Year, NE Georgia Section of American Chemical Society (1993)
	Creative Research Medal, University of Georgia (1995)
	Research Professor, University of Georgia (1996).

V. Impact Maps

            The "impact map" in this case seems straightforward: DOE support led to an expanding field of investigation and provided the basis for awards from other agencies for related work. A modest number of collaborations and interactions resulted, and postdocs and doctoral students have been supported. There is no record of major impacts such as national or international awards for excellence, patents, or secondary impacts resulting from doctoral or postdoctoral work done subsequent to work at UGA.

VI. Conclusions

            This appears to be a textbook example of successful mission-oriented basic research. The principal investigator is driven primarily by curiosity but is aware that the research might at some point have practical application. The initial award from DOE would appear to be the result of several factors working together: the PI, while new to the university grants game, was known in a network of scientists working on metalloenzymes; he was housed in an institution with a major facility for growing microorganisms of the type being studied; the work proposed was in an area that had just been discovered, had a number of interesting and unique features, and held long-term potential for application to energy-related problems. The initial DOE funded what was clearly good science. The research produced results of sufficient quality and interest to generate, in part at least, related work supported by NSF and NIH. The primary outputs of the DOE-funded work were publications and students. It is difficult to assess the scholarly impact of these knowledge outputs. They are published in respectable journals, but there is little evidence of major breakthroughs or contributions in the materials made available on the case.

            Interaction between the PI and BES has been minimal. He writes the proposal, he gets the grants, he writes the progress reports. Although the work has led to support of related work funded by other agencies, there has been no record of other DOE funding or other agency funding of the hyperthermophilic work itself (the original and continuing subject of the DOE awards). The PI flirted briefly with possible commercial applications of his work, but it was short-lived and, apparently, not particularly promising. If commercial payoff from the DOE-sponsored work is to occur, it is unlikely to occur for many years, and, given the proclivities of the PI, it is unlikely to emerge directly from the UGA project, at least under Adams.

            The six students who received their Ph.D.s under Adams and his colleagues have scattered widely. None seems to be continuing work on thermophilic bacteria. A modest network of collaborators exists, primarily at North Carolina State, Oak Ridge, and recently at Caltech. If commercial applications result from this work, they are likely to come from one of these collaborators rather than from Adams himself at UGA.