|Marilyn at Mammoth hot springs, Yellowstone National Park, 1981
On the way to attending a Plant Physiology conference in eastern Washington state in 1980, I made a trip through Yellowstone National Park armed with a newly purchased book authored by Thomas Brock entitled Thermophilic microorganisms and life at high temperatures (1978). The juxtaposition of my earlier work with microalgal cultures and isotope fractionation in comparison to naturally growing algal and bacterial mats struck me as the perfect analogous system with which to study Precambrian stromatolites. I devoured Brock’s book and was determined to set my sights on carrying out an ambitious field-based study in summer of 1981 in Yellowstone.
Most people think of Old Faithful Geyser when they think of Yellowstone’s hot springs. The big geysers, fed by surface groundwater, attract the crowds that stand by to watch periodic eruptions of hot water extending many feet in the air. To me, however, it was the beauty of the living microbes—their vibrant colors and the awe that they thrived in such an extreme environment--that hooked me.
I wrote and submitted a memo to Director Hat Yoder requesting permission to spend four months studying and collecting thermophiles in Yellowstone National Park:
“Carbon isotope ratios of naturally occurring blue-green algae have yet to be measured that show a similar fractionation to the Precambrian carbon. Some of these modern stromatolites consist primarily of photosynthetic, mat-forming bacteria, Chloroflexus. Perhaps the difference between modern blue-green algal carbon isotope patterns and Precambrian carbon isotope patterns is that the photosynthetic bacteria, not the blue-green algae, were responsible for the stromatolites found in the Precambrian.”
Yoder liked to keep a “tight ship” and to know what his staff was doing at all times. Every year in April, Director Yoder sent out a memo to staff members detailing the timeline for writing and producing the Carnegie Institution’s Annual Yearbook Report. We termed this the “rigid adherence” memo because in the memo’s first paragraph Yoder stated “Rigid adherence to the schedule” was required. I had to promise to handle all of my Yearbook submissions prior to departing for the west. Somewhat reluctantly, Hat Yoder approved my request. Armed with a permit from Yellowstone National Park (YNP), my first big field season began.
No one had measured the isotopic compositions of any of these unusual thermophilic (heat loving) microorganisms before. The idea that different carbon assimilation and metabolic pathways might impart different isotope fractionations was an exciting new lead that might explain the measurements of carbon in 2-3 billion year old rocks. It was a hot new field spurred on by Bill Schopf, a paleobiologist at UCLA, who won the National Science Foundation’s Alan T. Waterman award in 1972. He used his prize money to start the Precambrian Paleobiology Research Group (PPRG) in the late 1970s, bringing scientists from around the world to brainstorm on studying and analyzing early Earth rocks. I was aware of this august group—and a bit jealous of them--but had not yet contributed to the field enough to warrant an invitation to participate. My work in Yellowstone was carried out as an independent, individual investigator on a parallel track.
Carnegie scientists had previously worked in Yellowstone National Park. In 1935, E.T. Allen and Arthur Day published a megavolume on the chemistry and location of all of the thermal springs. Allen and Day consulted with a University of California botanist W. A. Setchell to learn about the organisms in the hot springs they were studying:
“Algae are a factor favoring the precipitation of both travertine and silica from these thermal waters. Professor W. A. Setchell concludes from his observation in the Yellowstone Park and elsewhere, that a few species of blue-green algae are probably instrumental in the formation of limited amounts of both travertine and silica deposits, though they are not responsible for anywhere near all of either. Dr. Setchell lays special emphasis on several species of algae, within whose woolly or gelatinous layers masses of travertine, shaped by the growing plant, accumulate. These particular organisms may indeed exercise a more potent influence than the rest; still, from the chemical viewpoint, all photo-synthetic plants, so far as they derive their carbon dioxide from the water, should be regarded as a factor in the formation of carbonate.”
I was excited to be following in the footsteps of Allen and Day. My laboratory at the Geophysical Lab was in the same space as that of E.T. Allen. I prepared for the trip by assembling a plan for collection of samples, measuring chemical parameters in the springs, carrying out growth experiments, examining the samples via microscopy, and preparing them for analysis back in my lab in Washington. My husband (then) Jack and I drove two vehicles out to Montana in early June. Jack had won a small, old, beat-up travel trailer in a poker game in April. It fit the bill for a place to live and I thought it would work as a mobile laboratory. We also owned an older VW bus that I drove across country somehow managing to figure out how to use a manual transmission, a driving skill I have rarely used since. The trailer was parked in Corwin Springs at a small “resort” that included a post office, general store selling candy bars and soda, and a place for parking a dozen or so RVs. It looked out over the Yellowstone River valley and had a sweeping view of the mountains just north of the national park.
My field plans included sampling a series of hot springs with very different water chemistries. I highlighted carbonate springs vs. silica depositing springs vs. highly acidic springs. Brock and others had shown that even in nearly boiling water at pH 2.0, organisms could survive and even thrive (e.g., Sulfolobus). For my first year of fieldwork, I had to choose hot springs that were within 1 to 5 kilometers of a road as well as relatively hidden from Park visitors. At this time, park regulations stipulated that the public should not view researchers in action, a policy that has changed since then. The silica depositing springs I chose were in the Lower Geyser Basin: Fairy Creek, Queen’s Laundry, Wiegert’s Channel, and Octopus Spring. As part of this work, I incubated glass slides in the spring to sample freshly grown material that matched with the environmental conditions that I was measuring at the same time (e.g., temperature, pH, CO2 concentration). Once sufficient growth was noted on the glass slides, biomass was scraped off and dried for isotopic analysis.
|Mounds of precipitated carbonate were enhanced by microbes
A typical week in the field started off with a long day’s trip into the Park. Usually, I needed to hike in a mile or more to the spring I was examining. I carried a daypack with my sampling gear, a portable pH and temperature probe, lunch, water, my notebook, and camera. Attached to the outside of my pack were a pair of “bear bells”, noisy jingle bells that were supposed to warn a grizzly bear I was in the neighborhood and hopefully signal it to go elsewhere.
The next day was spent in the “mobile lab” examining specimens under a microscope I’d brought with me. I set up a chemistry lab on the kitchen table to analyze carbon dioxide concentrations using an electrode especially designed for the task. I’d brought standards with me, kept them in the trailer’s small refrigerator, and calibrated the instrument each time before making measurements. In subsequent years, I had a portable hand held instrument that measured light absorption (a spectrophotometer) for measuring nitrate, sulfate, and sulfide concentrations. I poured cadmium columns for reducing nitrate to nitrate, a method that is both tedious and finicky. Today, there are much more sophisticated methods for these analyses, but in 1981, I had to use the technology of the day.
Wednesday was often used to go to “town”--Livingston Montana, about an hour away, for groceries and supplies and to visit the bank for cash. There was no bank (or no ATM machines) in Corwin Springs, and I didn’t have a single credit or debit card. Thursday and Friday were repeats of Monday and Tuesday with field work and sample prep.
I soon learned the “Ins and Outs” of my favorite hot springs and could identify most of the organisms inhabiting them. There were three major photosynthetic microbes in the neutral pH, silica depositing springs: Synochocccus lividus (a coccoid, yellow cyanobacteria); Chloroflexus sp. (a phototrophic eubacteria); and Phormidium sp. (an orange, filamentous stromatolite-forming cyanobacteria). Rarely were these organisms found in isolated forms, however, temperature segregated the populations. S. lividus grew at the highest temperatures (60-75°C); Chloroflexus slightly lower (48-65°C); Phormidium at the lowest temperatures (40-48°C). Pink filaments from Octopus Spring, were identified in the 1990s as Aquifex species (Reysenbach, 2012).
To me, the array of colors and the arrangement of subtle shapes created by these organisms was the height of beauty. After hiking through conifer forests and along meadows to get to the springs, the sight of the raised carbonate terraces and mounds never failed to excite me. Faced with so many potential organisms to collect, it required thought and meditation to choose the right samples. Much of my time was spent measuring distances and temperatures from the hot spring’s source. Typically, the water in source pools was boiling with gas bubbles emanating with great force. Collecting water from areas like this required patience and care. One time at the Norris Geyser Basin, my foot broke through a thin crust covering boiling, acidic water. It was a lesson for me to always have a clear, stable path when sampling boiling pools.
In the early 1980s, the carbon fixation pathways in many of these organisms were not known. In the ensuing decades, detailed pathways for the diversity of microbial carbon fixation pathways were discovered. Three of the four species of microbes described above use different photosynthetic pathways! Chloroflexus, for example, uses the 3-hydroxyproprionate pathway, whereas Aquifex sp. uses the reductive TCA cycle. Cyanobacteria all use Rubisco and the pentose phosphate pathway for CO2 fixation. Without knowing the biochemistry behind these different pathways, I discovered them through measuring their carbon isotope signatures. Organisms using the 3-hydroxyproprionate pathway had the smallest carbon isotope fractionations; those associated with the reverse TCA cycle were intermediate; and organisms using the pentose phosphate pathway, had the largest fractionation patterns.
I was thoroughly familiar with Thomas Brock’s studies on the microbes in thermal springs, but I hadn’t yet read some of the recent papers coming out of Carl Woese’s lab at the University of Illinois. In 1981, only a handful of scientists realized that there was a third form of life—the Archaea—a branch of microorganisms that included many of those I collected in Yellowstone. Sulfolobus, a microbe growing at 100°C in acid water is now known to be an Archaea. The gelatinous filaments growing near the boiling pools were mostly Archaea; the most famous, Thermus aquaticus, produced an enzyme that revolutionized molecular biology (Taq polymerase). It was not until almost a decade later that the scientific community embraced this branch of life as the third kingdom.
|Roaring Mountain where I collected Sulfolobus, the acid lover
My research applied directly to understanding the carbon isotope patterns that we find in the Earth’s oldest rocks—one of the studies carried out by UCLA’s Precambrian Paleobiology Research Group. All of these modern organisms have deep roots in the tree of life. Their ancestors were the organisms thriving billions of years ago. One of the major findings of my studies at Yellowstone was discovering the relationship between the concentration of inorganic CO2 with the carbon isotope patterns of organic carbon fixed by cyanobacteria and other photosynthetic microorganisms (Estep, 1984). The relationship between the carbon isotopes and carbon dioxide concentration was not well known at the time. I hypothesized that high concentrations of CO2 in the Precambrian would have resulted in greater isotope differences because Rubsico would have an unlimited supply of carbon for fixation. While I was making empirical measurements, Graham Farquhar, Marion O’Leary, and Joe Berry (1982) were developing a set of equations describing how concentrations of CO2 influence carbon isotope fractionation in higher plants. Subsequently, such relationships have proven to be important for interpreting what the Earth’s carbon dioxide levels were over geologic time (see Freeman, 2001, for a review).
In 1982, I collected organisms related to nitrogen cycling and pathways, a new avenue of research opened up for me by postdoc Steve Macko. The following year, I concentrated on sulfur cycling. Methods for analyzing the isotopic compositions for nitrogen and sulfur were just being developed at the Carnegie. I was not able to analyze sulfur isotopes then—something I regret to this day. The timing was just not right. At the end of the 1983 field season, I returned to Washington DC by myself with my small dog Sputnik. I boarded a plane to attend the International Organic Geochemistry Meeting in The Hague in Netherlands. I had decided to move on in life, separate from Jack, and call it quits in Yellowstone.
The work I’d accomplished studying microbes in Yellowstone had a far greater impact on my scientific career than the two papers I published on the subject. From this project, I learned how to carry out fieldwork in a remote, distant location where one had to be resourceful and calculating. There were grizzly bears in the Park, bison and elk that commonly charged tourists, and of course, boiling hot springs with fragile crusts. The work required permitting, regulations, and reporting. The park supervisor required that researchers do their work out of the public view, something very difficult in crowded and popular areas. I chose sample locations off the beaten track. Taking samples and setting up biogeochemical measurements in the field, preserving the samples properly for subsequent isotopic analysis, and creating experimental protocols were all skills learned during the three summers in Yellowstone and carried through my years as a field savvy biogeochemist.