Sabbatical in California: Global Oxygen Cycle

The Oxygen cycle and Dole Effect--Guy, Berry and Fogel

         Everyone knows about the global carbon cycle. Humans in the past couple of hundred years have changed it due to the massive combustion of fossil fuels. Since I was a grad student in 1975 to now (2020) the concentration of carbon dioxide (CO₂) in the atmosphere has gone from 340 parts per million (ppm) to over 410 ppm and is now increasing about 10 ppm per year! It may be that we’re not going to be able to reverse this trend in our lifetimes—maybe not even in the next 100 years. But, the Earth has gone through many boom and bust periods of high and low concentrations of carbon dioxide in the atmosphere before, in fact many times. Now, however, we’ve made substantial changes to carbon dioxide in the air at a much more rapid pace than ever before. 

         While increased CO₂ in the air causes global temperatures to rise and sea levels to flood low lying areas, plants are pretty happy. More CO₂ is like more food for plants, which means more photosynthesis. More photosynthesis means they produce more oxygen into the atmosphere as well. Currently, oxygen (O₂) is about 20% of all the gases in air. If there were much more O₂ in the air, we’d have a significant shift in many of the biogeochemical cycles, not just with oxygen, but nitrogen and sulfur as well. The interactions between the carbon and the oxygen cycles—now and in the past—are major drivers of how organisms manage to grow and reproduce on Earth.

         With a PhD in botany, one of the more intriguing intersections, to me, between botany and biogeochemistry was the “Dole Effect.” Malcolm Dole, a chemist at Northwestern University, had been doing experiments with oxygen isotopes in O₂ and water in the atmosphere for over 15 years starting in the 1930s. He and others, including Harold Urey—a Nobel Prize winner--noted that the O₂ in the atmosphere had an excess of the heavy isotope of oxygen (¹⁸O) relative to what it should be if atmospheric O₂ was in equilibrium with Earth’s surface water (Dole, 1935). This observation was important because the anomaly in the oxygen isotopes in air was caused by a balancing act in the Earth’s carbon cycle between photosynthesis, which produces oxygen, and respiration, which consumes it. 

         In 1956, Dole and his colleagues published experiments that measured oxygen isotope changes (i.e., fractionation) during respiration by various organisms. As expected, organisms used the lighter isotope (¹⁶O) faster than they did the heavier isotope (¹⁸O). As a result, the O₂ that hadn’t been consumed by respiration had an isotope composition with an increased ¹⁸O concentration. The problem was that the magnitude of isotope changes Dole and his colleagues measured didn’t easily explain the isotope values of atmospheric O₂. Something didn’t match up. Studies by Michael Bender, then at the University of Rhode Island, showed once again that oxygen isotope fractionation during respiration by marine phytoplankton and microbes in oceans was considerably less than what had been measured in the oxygen of the atmosphere (Bender and Grande, 1987). There still was no explanation for the strange isotope values in the atmosphere. To me it meant that we didn’t understand all the parts and pieces of Earth’s oxygen cycle. 

         In 1984, I was looking for additional challenges to my work as an isotope biogeochemist. I had spent eight years at the Geophysical Laboratory in the company of geochemists and earth scientists, far removed from biologists and botanists at the start of my career. I met Joe Berry at Carnegie’s Plant Biology Department at a Carnegie Institution of Washington meeting, and we immediately struck up a conversation about how we could weave together experiments on modern plant biology to tackle the potential factors that cause the Dole Effect. Berry, Chris Field, and Olle Bjorkman, all staff members at Carnegie’s Department of Plant Biology, were studying O₂ uptake reactions (i.e., photorespiration) in leaves, as well as conducting in situ work with Rubisco. If conditions are right, Rubisco has the potential to fix O₂, rather than CO₂. This can occur when temperatures are high, and the plant has made an excess of oxygen that might damage the plant via free radical production.

         At that time, we could not account for the factors that caused the Dole effect, which had implications for how the most important biological processes—photosynthesis and respiration--were balanced globally. Clearly, the studies to explain the global oxygen cycle using stable isotopes could not resolve the problem. Thus, I started on a major discovery endeavor to describe the oxygen isotope changes that occur from living organisms that deal with oxygen. Photosynthesis had been measured. Earlier experiments had shown that O₂ created from the splitting of water during photosynthesis had the same isotopic composition as that of the water in the plant (Dole and Jenks, 1944). Nevertheless, we wanted to make sure we understood it perfectly. Photorespiration, the uptake of photosynthetic O₂ by plants in hot, dry environments, had not been measured. Plants also take up and use oxygen in many other reactions that the geochemists who’d been thinking about the Dole effect, did not consider. There was plenty of work to do to figure this out.

         Joe Berry and I submitted a proposal to the Department of Energy to fund our work. We received the following reviews:

“The investigators underestimate the pitfalls of their experimental technique. The trapping of oxygen “on a molecular sieve column” (page 8) and the application of an oxygen electrode (pages 8,9) shift the ratio of the two oxygen isotopes...The hypothesis that photorespiration can account for part of the photosynthetic fractionation is interesting. Is there any reason (literature, preliminary data) to expect it to be so?”

Perhaps we hadn’t accounted for all of those supposed pitfalls, but both of us were experimental isotope biological chemists. I was confident that we could figure this out. Amazingly, the proposal was funded and on we went. 

         Oxygen is not the only gas in air. The bulk is nitrogen (about 80%). There are trace amounts of carbon dioxide and water vapor in air. Another 1% of air is the inert gas argon. To measure the stable isotopes of O₂, we needed to purify it from all of the other components. We knew how to separate carbon dioxide, water vapor, and nitrogen from O₂, but it was a bigger challenge to remove the argon. To purify atmospheric oxygen from argon in air samples, Tom Hoering and I designed and constructed an isotope vacuum line at the Geophysical Laboratory in Washington, DC. This particular line targeted O₂ gas, a procedure distinct from my first oxygen experiments that did not work out. 

         We built another homemade gas chromatograph to separate out the argon. It was a time-consuming process, but it worked. After oxygen was separated from argon, it was chemically converted to CO2 in which the oxygen atoms in CO₂ came from the oxygen atoms in air. In late February 1985, we deconstructed the line piece by piece and shipped it to the Department of Plant Biology in Palo Alto, California. Tom Hoering and I flew out to California and pieced it back together. Then, working closely with postdoctoral fellow Robert Guy, we designed and built the “biological” part of the extraction line where enzyme reactions and cells would be incubated for experiments. Experiments began in earnest in 1986, when I spent a year’s sabbatical leave at Plant Biology.  

Our vacuum line, 1985-1987

         Working at another Carnegie department was an interesting experience for me because I could see there were many things the same and some things quite different. Both the Geophysical Lab and Plant Biology had strong leaders. Winslow Briggs, Plant Biology’s Director, made me feel right at home. I was assigned a comfortable desk space and introduced to the staff. Winslow had an easy way of checking up on everyone at his laboratory, including me. The support staff were similar to those at the Geophysical Lab as well—a building manager, a business officer (Mary Smith), men in the instrument shop, and grounds keepers. Scientific staff members were quiet, nose-to-their-work folks who livened up at seminar time. Because Plant Biology was located on the edge of the Stanford University campus, there were many more students around than I was used to. In fact, being on a campus give a larger feel to the small department because you could walk to any number of seminars, enjoy a big library, and shop at a superb bookstore. 

         Joe Berry, my host staff member, kept a certain distance after I arrived in 1986. A tall, lanky man, Berry would come into the isotope lab maybe every couple of weeks, then we’d regale him about our successes and failures. He listened carefully, thought over what we’d said, and provided very measured responses. Sometimes, he even laughed a bit. I was somewhat disappointed that we didn’t interact more. I was used to seeing my Geophysical Lab colleagues every day and checking in with a brief “hello, how ya’ doing?” and “see you later” at the end of the day. Eventually, I didn’t pay much attention to this and enjoyed working with postdoc Rob Guy and chatting mostly to Olle Bjorkman, whose lab was next door to ours. Years later, I learned from Winslow Briggs that Joe really enjoyed having me in residence, which I couldn’t figure out at the time. 

         Rob Guy, a Canadian to the hilt, and I worked hard on the oxygen isotope experiments with long days filled with hard work operating the vacuum line and preparing samples. Short in stature, but long in thought and level of detail, Rob was a great colleague for me to work with. I often had outlandish ideas, whereas Rob was a pragmatist. Sometimes, we would spend several hours going over an experimental design or an outcome, followed by a compromise.  It was fortunate for me, looking back, that Rob worked with me on this project, because his input was critical for its ultimate success. 

         Our first experiments were with Anacystis nidulans, an easy-to-culture cyanobacterium. Cells were grown in the lab, then transferred to a collapsible bag that was bubbled with helium to remove all traces of atmospheric oxygen. We had an oxygen electrode mounted in the bottom of the apparatus, which was constantly stirred. Light was turned on, and oxygen evolved through photosynthesis. We confirmed earlier studies that showed there was little to no oxygen isotope fractionation during photosynthesis (e.g., Stevens et al., 1975). These first photosynthetic O₂ measurements allowed us to refine our techniques and understand various pitfalls. Typically, we would start on Monday to grow cells, get the chamber and vacuum line in good shape on Tuesday, run the first set of experiments on Wednesday, then repeat Thursday and Friday. Samples of CO₂ were sealed into Pyrex ampules. Every two weeks or so, I traveled down to NASA’s Ames Center at Mountain View to work in David DesMarais’s lab, who kindly allowed me to use his isotope ratio mass spectrometer. 

         Rubisco, which stands for ribulose 1,5 bisphosphate (Rubis) carboxylase (c) oxygenase (o), is an unusual enzyme in that it catalyzes the uptake of both CO₂ and O₂. Our second set of experiments was a “revisit” to my PhD dissertation and the re-measurement of both carbon and oxygen isotope fractionation by Rubisco. Times had changed. Rubisco from spinach, cyanobacteria, and R. rubrum could now be expressed in E. coli; no need for growing massive amounts of cells or purifying enzymes. For these experiments, rather than the time-consuming purification and crystallization of the enzyme’s product, PGA, we simply measured the isotopic composition of the remaining CO₂. It turns out that the values I measured for my PhD were slightly different from those I measured in California, because the carbon isotope ratio of RuBP in my earlier experiments was influenced by unknown contamination. By measuring only CO₂, we avoided that problem. This time I nailed it. We measured an isotope fractionation for carbon in CO₂ nearly identical to recent experiments by Roeske and O’Leary (1984). 

         Oxygen isotope fractionations were calculated using specialized equations (Rayleigh equations) that compare the oxygen isotope compositions of the O₂ in our reaction chamber at the start of the experiment to the oxygen isotope compositions of O₂ when we took a sample. Using our oxygen electrode, we could calculate the fraction of O₂ that was consumed (Guy et al., 1993). We determined the isotope fractionation from the oxygenation part of the Rubisco reaction to be very similar to what was required to explain the Dole Effect. Approximately 30% of all O₂ produced by photosynthesis is “fixed” by Rubisco prior to its being released. Another 20% is taken up by reactions in which oxygen radicals are converted to peroxide (H₂O₂), then converted via another enzyme, catalase, to form H₂O. The isotope fractionation during these reactions yielded further isotope fractionation. 

         We followed with measurements of oxygen isotope fractionations by even more plant enzymes that take up oxygen including cytochrome oxidase, which is involved in respiration for all plants and animals. Experiments with cytochrome oxidase were difficult. To perform these experiments, we needed large quantities of the enzyme cytochrome oxidase, which we could obtain from Sigma Chemical Company. Cytochrome c, the compound that the enzyme requires to make it work, was expensive--about $500 per experiment---and it was destroyed at the end of each experiment. We managed to only have one or two successful runs, measuring an isotope fractionation that was way too small--hardly enough to explain the Dole Effect. 

            We measured oxygen isotope fractionation by almost all of the enzymes that could possibly take up molecular oxygen. We concluded that if all of the plant enzymes that took up oxygen were considered, the Dole effect could be explained. Aerobic respiration is important, but the other five enzymes we’d measured were also key players in Earth’s carbon and oxygen cycles. The work remains important—and the only studies to date—for understanding the biogeochemical cycles of oxygen on Earth. 

            At the same time I was working at Plant Biology, two major events in my life and career were unfolding. First, and most important, Chris proposed in spring of 1986. I returned to the east coast in July by myself with little dog Sputnik to plan our wedding in mid-September. We married on September 20^th in New Jersey then traveled across the country on our honeymoon. Unlike many newly weds of that era, we eschewed going to Florida or Europe and instead went canoeing in Minnesota’s Boundary Waters Park on the border with Canada. It was absolute heaven! We stopped off at Yellowstone National Park, where we checked out my former field areas in the hot springs, then on to California.

Marilyn and Chris, 1986, Geophysical Lab

            The other not-so-pleasant event was fighting for the sovereignty of the Geophysical Laboratory. President James D. Ebert discussed with me the possibility of moving fulltime to Plant Biology. While I would have enjoyed living in California, limiting myself to research only plants—and not microbes, animals, old rocks, and sediments—wasn’t appealing to me. After a Visiting Committee assessed the Geophysical Lab’s science in 1985, they strongly supported keeping the lab the way that it was. The time I spent at Plant Biology strengthened my resolve to continue in biogeochemistry and be as interdisciplinary as I wanted to be.