Friday, January 17, 2020

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 20th 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.

Tuesday, January 14, 2020

Retirement for the Isotope Queen

Wild storm surge, The Sea Ranch, California--our town on the coast

            I am the Isotope Queen. What will I be when I retire? We scientists train for decades on how to succeed at our jobs—we are not mentored nor taught how to slow down, divest, and retire. I’ll be officially retiring June 30, 2020.

            My persona for 40+ years has been associated with being a hard-driving, serious Biogeochemist. I’ve been playing on the Isotope Geochemists Baseball squad at first playing on the second string, in the outfield. For the last couple of decades, I was a starter--pitcher, second baseman, maybe shortstop working tirelessly to get the team through rough games and championships. Now I’m getting off the Team, not even sitting on the bench. Whew—I’ll need to reinvent myself.

            When I turned 60--seven years ago--I decided to give the University of California ten years of good work before retiring at the age of 70. I was determined not to hang on and be thought of as that “Old Bat”, who should move aside for younger scientists to get a chance to show what they were made of. I had decided that I wasn’t going to be the Old Person who shuffled into their office after retirement, dusting off old tomes, and nodding off at my desk.

            Within a year and a half of moving to Mariposa California in the Sierra foothills, Chris and I branched out, purchasing a small house of our dreams on a beautiful stretch of the wild northern Pacific coast 100 miles north of the Golden Gate Bridge. We spent long weekends, spring breaks, and holidays there enjoying the fresh ocean breezes and vistas. Our plan was to transition eventually to full time coastal living—maybe upgrade the house, but certainly keep up an active life hiking, swimming, playing tennis, and drinking fine wines.

            In retirement, my goal was to be the best grandmother a grandchild could ever have. When I grew up, I had two of the best grandmothers you could imagine. There was Polish Grandmom Hencinski, my mother’s mother--a real character. During the Depression to make ends meet, she told peoples’ fortunes by reading their palms, made bootleg whiskey in her basement, and rolled cigars. As a grandmother, she took my brother Fred and me to the Jersey shore by train or bus. She made the best lasagna, spaghetti, and pizza that I had ever eaten. I got into a fight in 4th grade insisting that these were Polish foods—not Italian! Grandmom Hencinski also made a kick-ass vegetable soup, stuffed cabbages, and perogis. Fred and I spent many a Saturday night at our grandparents’ small row house in Camden, New Jersey, while they served as weekend baby sitters. Grandmom Hencinski was outspoken, outrageous, funny, and adventurous.
I AM the Grandmother of a fine dog--Stella, Evan and Meghan's Korean Mutt

            Grandmom Fogel was an opposite. She took us to Sunday school when we visited. She sang Lutheran hymns and never touched a drop of whiskey. She baked several pies and chocolate chip cookies—every week. Once a year, she sewed a patchwork quilt for me, as well as dresses for my dolls and me. Grandmom Fogel kept my grumpy Pop Pop in line, banishing him to the back porch to smoke his cigars. Her cooking was influenced by what was growing in her half-acre garden and included Pennsylvania Dutch specialties. She was sweet and gentle.

            In contrast, when Dana and Evan were babies and young children, their grandmothers were otherwise generally occupied. My sister Barb and her two boys, Chris and Mike, lived with my parents fulltime. Come the weekend, my mother was more interested in adult conversation than grandkids. Chris’s parents lived across the country from us and rarely traveled. Both grandmothers were well meaning, but neither babysat or cooked special dishes for the grandkids. Spoiling was not in their nature.

            I thought maybe by the age of 70 “Grandmom” status was a possibility, but of course this is out of my control. I had visions of providing relief for parents, withholding my opinions unless expressly asked, being there for support, cooking special meals for them, taking them on brief vacations, and of course, some grandmotherly spoiling. Chris and I envisioned traveling to see our children Dana and Evan wherever they lived, helping out, and being pleasant supporters.

            We also expected we’d take a few trips that we never took while we were working. I wanted to take a European trip touring English pubs, hiking on wind swept Scottish isles, taking a barge through British canals then hopping over to the Netherlands to enjoy the Dutch scene. I’d been to both of these countries before, but Chris had not. Further, the family had planned to take more adventurous trips. In 2015, we spent Christmas vacation on a wild trip to Thailand. The list of adventures we wanted to have was pretty long. Chris and I had lived frugally all of our lives—we had the resources to now have fun.

            This plan made a lot of sense then.

            Things changed—of course. Anyone can have their life alter with just one doctor’s visit. My life took several sharp turns. First, the ALS diagnosis was an extreme left turn. Then, we steered sharply right leaving UC Merced and moving to SoCal and working at UC Riverside. At the end of June, I’ll be veering to the left again and retiring officially. I’ve been taking the last six months getting accustomed to the idea. Chris and I spend time with each other almost 24/7/365. Fortunately, we are pretty happy looking at birds, the weather, and the PBS News Hour together.

            Heck, retirement is something you’re supposed to look forward to! My colleagues and friends are retiring in droves. The science folks are cleaning out their labs, shipping their precious samples to others who might value them, and getting their final studies published. The non-science folks are lining up volunteer activities, traveling to Nepal and Italy, and helping with grandchildren. The transitions are varied, but as far as I can tell working out well for everyone.

            My retirement now requires further planning. I’m a bit anxious about it. As my physical abilities diminish, traveling is nearly impossible. Forget trying to get on an airplane with tiny bathrooms I can no longer use. Hotel rooms are even a challenge that Chris and I eventually figure out, but they take a lot of work to be remotely comfortable. Our house on the coast requires renovation because I can no longer fit into the small bathroom on the first floor. [Last week, I washed my hair out on the deck with a bucket of warm water and a cup.] We’re working with architects and a contractor to retrofit the bathroom creating an accessible space for me. We’ve started looking at wheelchair accessible vans with lifts or ramps. One is in our future. I still love to see new places and meet new people.

            Fortunately, my mental facilities remain as much as they normally are in any 67-year old. I’m still able to talk without any problem; so visiting with people is a major source of fun and stimulation for me. Obviously, I’m still writing and working on several research projects that require reading, thinking, and writing. I’m starting a project on the Salton Sea with UC Riverside colleagues in a couple of weeks. I’ll be writing a biographical article for the National Academy about Phillip Abelson, former Carnegie President. This week, I helped revise a manuscript on hydrogen isotopes that is destined to be a classic. What a blessing it is to have the ability to continue intellectual engagement through retirement. Because academics build strong social networks with their colleagues, I expect to keep in touch with many of my scientific colleagues.

            My responsibilities at the University will diminish. I stopped teaching this year. I taught 18 classes while at the University of California—it’s enough. I’m still in contact with UC Merced students and serving on committees at UC Riverside. No more writing grant proposals! Soon, no more faculty meetings. I don’t mind them, personally, but I’ll no longer have the ability to vote on hiring and promotions. I have two funded projects that will require me to continue to pay attention. I have one student remaining: Mr. POM Bobby Nakamoto, who I need to push towards graduation. Two postdocs, Kaycee Morra and Jon Nye, are doing well, but will need full time positions. We continue to press on with first class work, publishing, presenting, and job hunting. I believe I will have the stamina to do this.

            So, what will retirement look like for me?

            I hope to spend more time aimlessly tinkering, sitting in the sunshine,  painting abstract works of art, and reading more novels. Perhaps, Chris and I will buy a big motorhome complete with accessible bathroom and comfortable bed, then we’ll set out across North America. We might park in your driveway, roll into your living room, and eat your food. We dream of seeing the national parks in the western states again. I’d love to see Chincoteague Virginia, Moorestown New Jersey, and St. John’s Newfoundland again. One more time.
A recent painting--our garden Riverside, 2019

            Chris and I will figure this retirement thing out. He’s writing a book about his famous grandfather Harry Swarth, an ornithologist. I’m working on my memoir. We tap-tap-tap on our laptops, exchange chapters and comment. It’s not climbing the mountains of Nepal, but it’s something. Our homes are Bed and Breakfasts for friends. Rarely does a week go by without sharing our hospitality with special people.

            Together we plan and plot.

            And I can dream.

Rounding Third Base and Heading Home

Cards from Franny and Flowers the Rumbles   My daughter Dana is marrying George Goryan on June 25 at our home in Mariposa...