|Mammoth Hot Springs, Yellowstone National Park, 1982|
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 δ13C and Precambrian δ13C is that the photosynthetic bacteria, not the blue-green algae, were responsible for the stromatolites found in the Precambrian.”
No one had measured the isotopic compositions of any of these organisms before. The idea that different carbon assimilation and metabolic pathways might impart different isotope fractionations was an exciting new lead. 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.
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:
“Accepting the facts of biological research, the writers concede that 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.”
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 km 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.
There were three major photosynthetic organisms in the neutral pH, silica depositing springs: Synochocccus lividus (coccoid, yellow cyanobacteria); Chloroflexus sp. (a phototrophic eubacteria); and Phormidium sp. (orange, filamentous conophyton-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). The pink filaments from Octopus Spring, were identified in the 1990s as Aquifex species (Reysenbach, 2012 ).
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 were discovered. Chloroflexus, for example, uses the 3-hydroxyproprionate cycle, whereas Aquifex sp. uses the reductive TCA cycle. Cyanobacteria all use Rubisco and the pentose phosphate pathway for CO2 fixation. Unwittingly, I had been measuring microbes with three different CO2 fixation pathways with attendant carbon isotope signatures. House et al. (2003) published a paper summarizing literature data from cultures, along with original data from culture experiments. Organisms using the 3-hydroxyproprionate pathway had the smallest carbon isotope fractionations averaging around; those associated with the reverse TCA cycle were intermediate; and organisms using the pentose phosphate pathway, had the largest fractionation patterns. Without knowing the biochemistry behind these different pathways, I discovered them through measuring their carbon isotope signatures. The work applies directly to understanding the carbon isotope patterns that we find in the Earth’s oldest rocks.
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.
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 more greater isotope differences because Rubsico would have an unlimited supply of carbon for fixation. Organisms collected in acidic springs (pH 4-5) plotted on a different line, probably because the inorganic carbon source was CO2 rather than the bicarbonate ion at this pH. Around the same time, Farquhar, O’Leary, and Berry (1982) published their equations describing how intra- and extracellular partial pressures of CO2 influence carbon isotope fractionation in higher plants. Subsequently, such relationships have proven to be important for interpreting paleo-CO2 conditions by measuring compounds from living organisms found in marine sediments (see Freeman, 2001, for a review).
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