|Marilyn as a young postdoctoral scholar, 1977-1979|
Every summer, the esteemed Dr. Tom Hoering from the Carnegie Institution of Washington’s Geophysical Laboratory would come to the Marine Science Institute in Port Aransas on “vacation” which generally consisted of him hanging around the lab and chatting with the graduate students and postdocs. Hoering was the major professor of one of my major professors, Pat Parker, making me Hoering’s academic granddaughter. Tom had a thick head of grey hair, sharp blue eyes, and facial expressions that let you easily know what he was thinking about you. He was one of the earliest pioneers in the field of stable isotopes carrying out studies as varied as measuring nitrogen-fixing bacteria to ammonia formed by lightening strikes. Not only was he carrying out studies on his own, but he also built his own mass spectrometers, one reportedly out of used automobile parts. When he came to Port Aransas, we grad students were on our best behavior.
The summer before I graduated, he stood in the hallway and quizzed me informally about my work and my findings. At the end of the conversation, he remarked that the Geophysical Laboratory might be an excellent place for me to consider for a postdoc. I was thrilled! As the year went by and I wrote up my work, I applied to two places: University of Georgia to work with Clanton Black, a noted plant physiologist who dabbled in stable isotopes, and the Geophysical Laboratory of the Carnegie Institution of Washington. I received offers from both, and it was a fairly easy choice to go to Washington DC and work on hydrogen isotopes with Tom Hoering.
Many people are not familiar with the Carnegie Institution of Washington. The financier, railroad man, and founder of US Steel, Andrew Carnegie, donated $22 million dollars in 1901 to found a basic research institution dedicated to a small, select group of scientists who were provided a full salary and ample support to study questions that they felt were most important. Carnegie’s Presidents were all distinguished men; for most of its history, the senior staff scientists, called Staff Members, were also only men. The Geophysical Laboratory was formed some years later (1906) and the first building constructed on a hilltop in upper Northwest Washington DC one block off of Connecticut Avenue on Upton Street. The US Geological Survey at that time was on a campus directly across the street on Connecticut Ave. In the early 1900s, the nation’s capital was the center of geological research in the United States.
Several leading scientists and Nobel Prize winners did their awarding winning research at the Carnegie including Edwin Hubble (discovered the expanding universe); Charles Richter (developed the earthquake measurement scale), and Barbara McClintock (work on genetic inheritance). A postdoctoral fellowship at the Carnegie meant that you were running with the Big Dogs, particularly in the field of earth science and geochemistry. It was well known that a postdoc at the Carnegie could land you a prestigious position at a good university after you finished.
The Geophysical Laboratory was the polar opposite of the Port Aransas Marine Lab in almost every way. In Texas, I came to work in shorts, a ragged T-shirt; shoes were optional. At the Geophysical Laboratory, shorts were out of the question. I needed to purchase a week’s worth of “work” clothes—khaki pants, blouses, and decent shoes. The staff scientists at that time were all men, who came to work wearing ties and white shirts. The halls of the lab were quiet; the average age of people I saw on a regular basis was about 50. They were all geochemists; I was the only biologist. I was assigned a desk that was once occupied by “Mrs. Navrotsky,” who I later learned was Dr. Navrotsky, a never married and very distinguished geochemist. I required a sterile environment to do culturing work, so a small plywood “hood” was constructed for me in Tom Hoering’s back lab, which I shared with Doug Rumble and an oxygen isotope extraction line laced with explosive fluorine compounds.
My project for the next two years was to bring my expertise in biochemistry and physiology to the study of hydrogen isotopes in living organisms, which at this point in time had been studied primarily by geochemists with little knowledge about biology. This information is important because scientists are interested in using hydrogen isotopes to learn about past climate. Hydrogen has two stable isotopes: the “light” isotope--protium (1H) and “heavy” isotope--deuterium (2H), which has twice the mass of 1H. As these isotopes move through the environment, the “light” isotope moves faster than the “heavy” one--just like you’d expect.
Everyone knows that water is essential for life, and it made perfect sense that the hydrogen in the water molecule (H2O) was the source of hydrogen for plants, animals, and microbes. The global water cycle is learned by every 5th grader in elementary school. It is responsible for changing the hydrogen isotope signatures in precipitation, groundwater, rivers, and subsequently plants.
Isotopically “light” water with a preponderance of 1H evaporates faster than isotopically “heavy” water that has more 2H. Conversely, isotopically “light” water likes to remain as happy-go-lucky water vapor. It condenses slower than isotopically “heavy” water. Therefore, as air masses laden with moisture pass over continents, the isotope composition of precipitation from those air masses changes. The heavy isotopes rain out and the light isotopes remain as water vapor.
Geochemists in the 1960s measured the hydrogen isotope composition of water from around the globe. Seawater—in most of the world’s oceans—has a uniform isotope composition. Freshwater and rainwater, on the other hand, have great variations in their hydrogen isotope compositions. The global variations are caused by evaporation and condensation of water in the global water cycle. Earth’s temperature and elevation are the major drivers of evaporation and condensation. For example, rain falling over cold, high mountains has much less of the heavy isotope than rain in warmer, lower elevation coastal areas.
Plants use water that has a hydrogen isotope composition directly related to where they grow and indirectly related to the temperature of their environment. Those growing in the tropics have more 2H, while plants from the tundra have more 1H. Scientists exploit this information in fossil plant compounds found in sediments in order to study temperature fluctuations in Earth’s climate over geological time.
With this background in mind, my postdoctoral research was to learn more about what happened inside of plants with respect to hydrogen isotopes. No one knew which biochemical reactions in plants were responsible for hydrogen values in any of the biochemicals—lipids, proteins, or sugars. I had studied carbon isotope signatures associated with the Rubisco enzyme for my PhD research. I was uniquely qualified to begin to unravel the many biochemical reactions in living organisms that used hydrogen isotopes with the ultimate goal of supporting climate science.
I arrived at the Geophysical Lab with small test tubes of carefully cultured microalgae—simple unicellular plants--from Van Baalen’s lab. Within a few weeks, I learned how to measure hydrogen isotopes in water, oils, and plants. I purchased supplies for growing algal cultures and obtained an old autoclave from our sister institution, the Department of Terrestrial Magnetism. In my office, I set up an aquarium I’d purchased at a yard sale as a water bath, adding gas mixing lines and a temperature controller for growing pure, axenic cultures of microbes. The field of hydrogen isotopes in plants and algae was wide open at this point in time. After six months, I gave Tom Hoering a report of my work: investigating hydrogen isotope ratios in plants. I attempted to survey the hydrogen isotope values of various terrestrial plants, including lichens, liverworts, mosses, a fern, and angiosperm leaves collected on the Lab’s extensive grounds. Leaves on different branches of the same tree have basically the same isotope ratio although one leaf sample during the three-week period showed a considerable variation. Similar experiments with three species of mosses showed similar results, although one moss species was consistently different.
Today viewed from a 40-year perspective, these findings on isotopic variation in plants are both simplistic and profound because they highlight biological variations that capture the big trends. In fact, data like these are fundamental to understanding isotope biogeochemistry of hydrogen isotopes in plants. My report also included data on microalgal culture experiments and some phytoplankton samples collected in Chesapeake Bay. After reading my two-page report, I met with Tom who criticized my approach and told me “I wouldn’t have done things this way.” I answered that his opinion wasn’t my own, and I was proceeding just as I had planned. I am not entirely sure what Tom’s approach would have been, but given his strong talent in chemistry, I think he would have moved more quickly to studying plant lipids.
Biologists typically replicate their experiments. I needed to confirm that I wasn’t making “one off” measurements with just “5 well chosen” natural samples. Before proceeding further, I needed to know if different taxonomic groups fractionated hydrogen isotopes in the same way or in different ways. These fundamental questions obviously did not appeal to a geochemist, but they appealed to me and were important for me to follow my own ideas to completion.
In the 1970s, the method for measuring hydrogen isotopes in organic compounds was a three-step process requiring the exact procedure to be followed every time. Glass pumps filled with mercury and hot uranium metal could explode if you opened the wrong valve. The combination of 100 kg of mercury and hot uranium (750°C) made this a particularly dangerous analysis. Samples, after thorough grinding, were weighed into a ceramic combustion boat and inserted into a vacuum line where oxygen was introduced. The sample was then pushed into a 900°C furnace. A pump circulated the gases from combustion including carbon dioxide, water, and nitrogen. The water was frozen into a 6 mm Pyrex tube, which was sealed off with a torch. For every single measurement I made, the sample was analyzed about 5 to 6 times. The tubes with water samples were then attached to a vacuum line in which water was converted to hydrogen gas by reaction with hot uranium at 750°C. This vacuum line used a Toepler pump, a frightening, fragile glass contraption that cycled mercury up and down a glass column to move gases around. Hydrogen gas was collected in a sample bulb with a glass stopcock, which was then taken to a Nuclide 3” mass spectrometer especially designed for measuring the two isotopes of hydrogen. The inlet systems of mass spectrometers in the 1960s and 1970s had mercury pistons to compress the gas samples to match the sample and standard pressures. Because I was on top of things and paid attention, I could analyze 10-12 individual aliquots in two days, which resulted in two publishable data points.
Working with uranium metal is a challenge that probably exceeds safety guidelines in universities today. To prepare the uranium metal to convert water to hydrogen gas, I needed to react it with strong acid--a nerve-wracking experience. Uranium is so reactive, it can spontaneously combust. Uranium metal was placed in a glass beaker. Concentrated nitric acid was slowly poured over top, releasing a dark orange gas that wafted out of the beaker. I had to watch carefully and douse the reaction with water when the uranium showed imminent signs of combustion.
No wonder very few people measured hydrogen isotopes at that time! Today, analyses of this type are accomplished automatically. Instead of a 2-day process to make a few measurements, 150 samples can, in theory, be measured in a day without mercury or uranium. Because methods in the 1970s required such drastic measures, the amount of data and the number of people who could carry out those reactions safely was less than a dozen laboratories worldwide.
Other than the research Tom Hoering and I were carrying out at Carnegie during my postdoc, Sam Epstein and his students at Caltech were turning out the majority of work on organic hydrogen isotopes. Epstein’s work focused on measuring the isotopic composition of non-exchangeable hydrogen in cellulose purified from tree rings. He and his colleagues were using hydrogen isotope patterns in cellulose for studying paleoclimate in the Holocene (Epstein et al., 1977). The basis of their work centered on the assumption that temperature affects the hydrogen isotopes of environmental water, and that the hydrogen in environmental water equilibrates with glucose monomers in cellulose during synthesis, often in a matter of hours. The premise of this work was based primarily on temperature-based isotope exchange of water with cellulose. My work showed that the hydrogen isotope patterns of plants were determined by biosynthetic reactions, not simple exchange. The experiments that I conducted with microalgae revealed that the water used in actively growing plant cells is not environmental water—there is a considerable lag phase between the time when water enters a cell and when it is used in biosynthetic reactions. Based on changing light conditions and using photosynthetic inhibitors, I learned that NADPH producing reactions in photosynthesis are key to providing most of the organic hydrogen in plant tissue (Estep and Hoering, 1980, 1981).
Reviews of my first manuscript, submitted in 1979, on hydrogen isotope fractionation by microalgae were harsh. One reviewer remarked with sharpness: “This paper suffers from a fundamental flaw in experimental design.” The review primarily criticized the measurements on the bulk or total hydrogen in the algal cultures or plant leaves. The reviewer assumed that in living organisms C-bound hydrogen was not exchangeable with water, but hydrogen bonded to nitrogen, oxygen or sulfur, readily exchanged with any contact with water. The exchangeability “controversy” persisted for over 20 years, until finally a number of researchers repeatedly showed that only about 10-15% of hydrogen in molecules like keratin (e.g., feathers) is exchangeable, whereas the remainder of the organic H, if bonded by covalent or ionic bonds is not (e.g., DeBond et al., 2013).
During my PhD studies, fellow graduate student Brian Fry (the son of isotope chemist, Arthur Fry) worked with Pat Parker showing that “you are what you eat” in an elegant grasshopper and plant community study using carbon isotopes (Fry et al., 1978). This study included detailed diet measurements and mass balance equations using the mixtures of two different types of plants found in his study area. Brian’s paper with the grasshoppers and carbon isotope signatures remains a classic. When I teach Stable Isotope Ecology today, we begin the class learning everything about Brian’s study. At the same time, Michael DeNiro and Sam Epstein were growing mice in the laboratory and finding similar carbon isotope results (DeNiro and Epstein, 1977).
In 1980, I was intrigued by these studies and set out to determine if hydrogen isotopes could also be used as a tracer for animal diet. My study included a laboratory-based experiment with mice, as well as with marine snails and their potential algal food sources from a natural environment study on the intertidal coast of Maine (Estep and Dabrowski, 1981). Essentially, I determined that the primary source of hydrogen in the organic tissues of animals originated from their food, rather than their drinking water. The paper, published in Science, attracted the wrath of DeNiro and Epstein, who submitted a technical comment to Science arguing that my findings were invalid owing to hydrogen exchange (DeNiro and Epstein, 1981).
For a quiet, shy young woman from New Jersey and coastal Texas to be challenged by Caltech folks was unsettling. Tom Hoering supported me in writing my rebuttal: “At present, there is some uncertainty about whether the isotopic composition of the hydrogen in prey and predators can be used to follow food chains, but similar criticism may also be applied to the use of carbon or nitrogen isotopes in analogous studies” (Estep, 1981). DeNiro and Epstein’s technical comment included data on hydrogen exchange experiments with mouse tissue that had been freeze-dried, ground, steamed at 100°C, then analyzed. It was no surprise that their experiment showed considerable isotopic exchange with the hydrogen in steam. I argued that because tissues were treated carefully and at room temperature in my studies, exchange was not the major controlling factor in the hydrogen isotopic composition of animal tissues. With time, it has been shown that about 20-30% of the hydrogen in animal tissues comes from drinking water---not by random exchange, but by direct incorporation. I was correct all along.
Interestingly, although my original paper was the first to show the utility of hydrogen for tracing diets, it has only been cited about 129 times since it was published in 1981. This work had little impact until almost twenty years later when the methods for measuring hydrogen isotopes became easier using a thermo-chemolysis procedure that is fully automated. Although methodology was an important factor in delaying the impact for my work, another reason why this paper was largely ignored by the ecological community studying animal migration is that they did not, and still don’t, care about hydrogen in food. Those isotope biogeochemists in the know realize that the hydrogen isotope composition of food reflects local precipitation, as water is the only source of hydrogen available to primary producers to build organic tissues. Broadening the ecological community’s perspective on the influence of hydrogen from all of the major biochemical sources available to an animal would provide them with a nifty source of information that could enhance their studies.
During my two-year postdoctoral fellowship funded by the Carnegie Corporation of New York, I started to apply for permanent positions in academia. I had produced solid work on the enzymology and isotope fractionation by Rubisco. I had transitioned to a new isotope system at the Geophysical Lab and showed I had become independent from my three Texas advisors and from Tom Hoering. My undergraduate degree was in Biology and my PhD was in Botany (Marine Science), and now I was completing a postdoc at a prestigious earth science laboratory. With several papers and presentations under my belt, I began sending out applications for faculty positions. At that time (1978-1979), it was not an advantage to be a woman in almost any scientific discipline. Today, women scientists with talent often compete well for positions. Not so in the 1970s. Further, I didn’t fit easily into either a regular biology department or an earth science department. Marine science positions were relatively rare, and if they did exist, many did not have the type of analytical support (i.e. mass spectrometers) that I needed to do my work. At that time, it was beginning to dawn on me that being an interdisciplinary scientist might have its drawbacks.
After receiving over 20 letters of rejection, I finally had one offer for a second postdoc with a promise of a faculty position in two or three years at a marine science laboratory in South Carolina. At the same time, the Geophysical Lab was searching for a biogeochemist to fill a vacant staff member position. There was no position description and applications were by invitation only. Initially, I was not asked to apply. A couple of men, at my level of experience, interviewed. Things changed for the better when I gave a talk on my hydrogen isotope work at the Geological Society of America meeting in 1979. I was scheduled to speak just prior to an awards session honoring Harmon Craig, the most famous isotope geochemist. The lecture hall was packed with over 400 people in anticipation of hearing the great man speak. With good fortune, as I gave my talk, I noticed the President of the Carnegie Institution, Dr. Philip Abelson, directly in front of me in the audience. As I made my points, he nodded his head in agreement. I felt as though I was talking directly to him, and perhaps I was. The following week, I was called up to the Geophysical Laboratory Director’s office to meet with Dr. Hatten S. Yoder to “discuss my future plans.”
Phil Abelson was also editor in chief of the journal Science, in which he wrote a weekly editorial on controversial topics in science at that time. His scientific accomplishments included discovering nuclear fission (one week after the Germans did), figuring out the biosynthetic pathway that is the central cycle of almost all organisms (but it was named after a German scientist Hans Krebs who again was working at the same time), and finding amino acids in ancient fossil shells thereby jump starting the field of organic geochemistry. For a public figure, he was an exceedingly shy man, largely bald with Roosevelt like glasses, and a toothy smile—if you could get him to smile. At Carnegie functions over the years, I was frequently seated next to him, because I could get him to talk, even joke, and we had good conversations about my work. I always marveled that he remembered not only who I was, but details about my research.
Apparently President Abelson returned from the GSA conference and phoned Yoder directly asking him, “Why haven’t I heard about this postdoc, Marilyn?” I was invited up to talk with the Geophysical Lab’s Director Hatten S. Yoder, who asked me to write a summary of what I might work on if I were to remain at the lab as a new staff scientist. After this conversation I floated out of Dr. Yoder's office elated and headed immediately to Tom Hoering’s office. He encouraged me to write up my ideas. My proposal had three major projects, but I did not carry out these projects exactly as I’d written, but carved out studies with oxygen isotope fractionation of molecular O2 during biological reactions, work on thermophilic microbes, as well as investigations into bacterial manganese mineral formation (see below).
Director Hat Yoder wrote to then Carnegie President James Ebert the following:
“The present staff has the expertise to measure the principal stable isotopes and characterize the complex amino acids and mineral structures, but they do not have the knowledge to maintain the primitive living organisms needed in the proposed studies. After a thorough search, the staff organic geochemists and I have concluded that we already have a most talented, highly motivated, and successful potential staff member among our Postdoctoral Fellows.”
Within a week or so, I had an offer for a Temporary Staff Member position starting July 1, 1979. I was off to a solid career in Biogeochemistry.
|A new staff member--front row, second from right|