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| 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.
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| 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.
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| 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.
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