Friday, January 10, 2020

Getting Started on Nitrogen Isotope Biogeochemistry

Steve Macko attending his handmade nitrogen isotope line, 1982
         In September 1982, my first postdoctoral fellow, Steve Macko, arrived from my old alma mater, the University of Texas Marine Science Institute. Macko, as he is affectionately called, sported a thick mustache and a shock of brown hair along with coke-bottle lens glasses. His preferred outfit was a blue and red zippered sweatshirt with a pair of slightly rumpled jeans. He is known the world over for his thrifty ways—with food, his clothing, his hairstyles, and his lab’s gear. Almost 40 years later, Macko has held on to a perpetually youthful appearance. 
Steve (right) and Sue Ziegler, Carnegie, 2016

         We shared an office during his time as a postdoc. Steve brought with him to the Geophysical Lab, not only boundless enthusiasm and a plethora of ideas, but the technical wherewithal to measure nitrogen isotopes in organic matter. He came with a host of marine science projects, collaborators from around the globe, as well as a zeal to learn as much as he could.
         Often, after reading a new paper at his desk, he would turn to me and say, “Ya’ wanna know something interesting?” He’d have a devilish little smile on his face and follow this with a soft chuckle, then follow that question up with “I have an idea!”
         At the Geophysical Lab, Macko quickly set up the methods he’d worked out as a PhD student for analyzing isotopes in organic matter. He prided himself on his glassblowing talents. Tom Hoering had been the Geophysical Lab’s only glass blower. The two of them had a bit of a rivalry in terms of prowess at making connections and curves with the glass tubing used to build vacuum extraction lines. Eventually they reached a détente and managed to support each other’s skills.
         Steve’s nitrogen isotope technique became what is popularly known as the Sealed Tube method in which 5 milligrams of a sample—plant or animal tissue-- was placed into a quartz tube with copper reagents and sealed off after it had been evacuated on our homemade vacuum extraction line. We used an oxygen-natural gas torch (at 2770°C!) to carefully melt the quartz and seal the tubes shut. That was a white-knuckle operation that took patience and care to do properly.
         The next step required heating the whole tube, with the sample sealed inside, in a furnace at 950°Ç, cooling it carefully back to room temperature. We then cracked open the tube within a finicky metal device that usually worked, but failed about 10% of the time. Once cracked open, the gases from the combustion of the sample—carbon dioxide, nitrogen, and water vapor—were distilled into a glass trap using liquid nitrogen (-196°C) to freeze out the gases.
         After several more steps, nitrogen gas was isolated in a sample bulb, which was then taken to the isotope ratio mass spectrometer for isotope analysis. Macko was a whiz at this and could process 15-16 samples in a day. I was slower, maxing out at around a dozen. It was not unusual to have one or two of these samples be contaminated with air that had somehow leaked into the vacuum extraction line. I used Steve’s method from 1982 until 1999, when I installed a new automated instrument used by all labs today. During those 17 years, I trained multiple students and postdocs how to do this procedure. 
Nitrogen isotope line, Carnegie 1991

         Macko’s methods spread to many other laboratories like wildfire. During my career, I published 120+ papers using nitrogen isotope measurements to study everything from humans to extraterrestrial organic matter. Nitrogen isotope measurements now form the backbone of stable isotope ecology research along with carbon isotopes. Who is eating whom can be figured out with nitrogen isotopes. One of my first projects with nitrogen isotopes was the study of the hot springs microbes, figuring out how they were surviving in nutrient poor environments. In a subsequent chapter, I describe how nitrogen isotopes allowed Noreen Tuross and me to figure out how long women nursed their babies during prehistoric times. I used nitrogen isotopes to study pollution in the Delaware Estuary, the Atlantic Ocean, and coastal coral reefs. Ocean scientists use nitrogen isotopes to learn about ocean circulation and primary production.
Univ. of South Carolina's lab early 1980s with lots of glass 'lines', David Mucciarone (left)

         Macko’s main project was to culture microorganisms, then separate and purify amino acids from them for isotopic analysis, similar to the original work that Phil Abelson and Tom Hoering published in 1961. We were interested in learning about the isotope patterns originating from biochemical synthesis. Analyses of this type are used for understanding unique biochemical pathways, sources of food for animal diets, and the trophic position (e.g., primary consumer vs. top carnivore) for animals in which their diets are unknown.
         Our work in 1982 involved all three Geophysical Lab staff members in biogeochemistry, as well as Macko: me for culturing the organisms and interpreting the biochemistry learned from the isotope patterns; Ed Hare for separation of the amino acids; and Tom Hoering for IRMS and other isotope support.  To carry out these experiments, I needed to ramp up my culturing ability in order to grow the substantial amounts of material needed for amino acid isotope analyses. Ed Hare needed to produce grams of the special ion exchange resin required to separate milligram quantities of amino acids. Hare and Macko worked hard in order to have the amino acid analyzers going full time to assay compounds as they eluted from the separation column. We used two IRMS instruments for the isotope measurements: a newer Nuclide 6” IRMS for carbon and for nitrogen a much older IRMS constructed at the Geophysical Laboratory by Hoering and others [the one previously sandblasted…].
         It took several weeks to grow up the microbial cultures. We analyzed an aliquot of the microbes using the Sealed Tube method, then hydrolyzed the remaining sample to produce hydrochloride salts of amino acids.  Individual amino acids in the hydrolysate, the solution after acid heating, were separated using column chromatography that took 24 to 48 hours to complete.  The fact that amino acids do not separate at regular or specific time intervals was a problem. The trick was to use a separate liquid chromatography system to determine when each amino acid started and finished eluting. 
         With this information, we could isolate and capture each amino acid in its entirety but separate it from other amino acids in the sample. Fractions were collected from the eluant every few minutes. After analysis via liquid chromatography, fractions were pooled to capture the entire amino acid eluting from the column.  This resulted in 13-15 different amino acid samples, each containing the hydrochloride salts of a single amino acid. The samples were then dried by evaporation in the fume hood. The amino acids were then analyzed by the Sealed Tube method described above.
         This was an elaborate and slow process where each sample took a total of about 6 weeks from start to finish, depending on whether everything was working in tip-top order (Macko et al., 1987). We were the only research group in the world doing these detailed, complicated analyses and in the early 1980s were twenty years ahead of our time. Today, experiments of this sort can be accomplished with much less material, and by automated analysis, where five to ten samples can be measured in a week. The framework we established in the 1980s through this laborious work was important for subsequent measurements of nitrogen isotopes in amino acids from animals, a critical method for studying the mechanisms that are now used by laboratories globally to study ecological trophic levels.
         Another pivotal postdoctoral fellow from this time was Michael Engel. Engel and Macko became fast friends and colleagues, a relationship that has lasted over 30 years. Engel came to work with Ed Hare to learn as much as he could about amino acid analytical techniques. Engel is the opposite of Macko in many ways. He’s a flashy dresser, loves to eat at the finest restaurants, spends money without guilt, and sometimes seems a bit down in the dumps. 
Dr. Engel and Macko, Hoeringfest, 1995

         He’d come into my lab sometimes and shake his head, “I don’t know…I can’t figure something out.” Then, he’d proceed to argue with himself, while I listened, and figure things out for him self. I call him “Dr. Engel”, rarely Mike, and today when we see each other, we laugh and giggle about the Olden Days like a bunch of teenagers.
         Engel and his major professor Bart Nagy at the University of Arizona studied the organic chemistry of one of the most famous meteorites—the Murchison, which fell in Australia several decades ago (Engel and Nagy, 1982). This particular meteorite contains abundant organic matter that has been studied by scientists around the world. Dr. Engel developed detailed procedures to extract and identify amino acids found in the meteorite. Many of these amino acids have similar structures to those found in the proteins of living organisms on Earth.
         All living things use proteins to do the metabolic work of a cell. These proteins are composed of building blocks known as amino acids. There are about 20 different amino acids that are found in proteins. Anywhere from 50 to several hundred amino acids are linked together to form a long chain-like protein. Almost every amino acid comes in two forms, levorotary (left-L) and dextrorotary (right-D). These forms of amino acids are mirror images of each other but cannot be overlaid onto each other, just like right and left hands. The words come from the fact that if you shine a light on left-handed molecules; they bend light to the left. The same is true for the right-handed variety. However, most living things only use the L amino acids. When a creature dies the L amino acids begin to convert to D amino acids through a process called amino acid racemization in which one mirror image molecule turns into another.
         Other geochemists had found amino acids in the Murchison meteorite before, but they did not report the ratio of L- to D- amino acids. Engel and Nagy found a slight enrichment in the L-form of the amino acids and argued that this enrichment might have been important in influencing the ultimate form (L-) that was “chosen” by living organisms. Other geochemists denounced their findings as contamination from minute quantities of terrestrial amino acids, all of which would have had just the L- form.
         This plagued Engel, then a postdoc looking for a permanent faculty position, for many years. He was subsequently proved right by other researchers and by developing a new stable isotope method that I’ve used for nearly thirty years. Collaborating with Steve Macko, they published their first paper in 1991 using continuous flow methods in which amino acids are separated by gas chromatography then swept one-by-one into the mass spectrometer without all the messy chemistry. Engel and Macko published multiple papers in the coming decade on isotopic compositions of amino acids from meteorites, which confirmed Engel’s earlier assertion that the enantiomeric excess he measured and published with Nagy in 1982 was in fact correct. 
            While Engel and Macko were at the Geophysical Laboratory, we conducted experiments measuring nitrogen isotopic fractionation by the enzyme that adds the amino (NH2) group to acid carbon compounds, thereby forming amino acids. This enzyme, glutamate-aspartate transaminase, was the second enzyme system that I used to measure isotope fractionation. It was much easier to do these experiments than working with Rubisco during my PhD. First, we didn’t have to grow organisms, could purchase the enzyme directly from Sigma Chemical Company rather than purify it ourselves. The chemicals needed to carry out the reaction (i.e., reactants--aspartate, glutamate, α-ketoglutarate and oxaloacetate) were all inexpensive and readily obtainable. We determined the nitrogen isotope fractionation for the reaction going in one direction, then the other. This is one of the most basic and important enzymatic reactions for producing amino acids in all organisms. The paper has been cited hundreds of times but never repeated. 

            Having the capability for measuring nitrogen, carbon, and hydrogen isotopes in my laboratory opened up the entire world of isotope biogeochemistry for me. Every experiment that we could think of was new, and the outcomes unknown. At that time, the literature on stable isotopes was so small that you knew each publication that came out, memorized its contents, and thought about it long and hard. The work on amino acids quickly progressed to investigating fossil collagen samples and lab grown animals. The amino acid isotope field plodded long incrementally for a decade before resurging as advances in instrumentation permitted reliable automated measurements.

Tuesday, January 7, 2020

Failure: am I an "imposter?"

Marilyn and the ill-fated oxygen line, 1981

         As a new staff member, it was exciting to move into my own dedicated laboratory space. The original Geophysical Laboratory on Upton Street in Washington DC was built in 1908, had 18-inch walls and big hallways. Director Yoder assigned me a laboratory to share with retiring staff scientist, Gordon Davis. Davis’ career focused on radiogenic dating of rocks. His laboratory was primarily a preparatory, “semi-clean” lab where he purified lead and other radiogenic isotopes for dating. For about a year, we “shared” the space with my culturing equipment on benches in the clean lab. It was an arrangement that would never really work, because I did not have full control over my lab space. Only many years later did I realize that this arrangement put the start of my career at a moderate disadvantage. Fortunately for me, after one year Gordon fully retired and I was able to renovate the laboratory, move in two isotope mass spectrometers, and build my first original vacuum line.
         After investigating hydrogen isotopes for several years, my interests turned to oxygen in organic matter with an experimental plan similar to what I had carried out for hydrogen isotopes. Oxygen has three stable isotopes: 16O, roughly 99.6% of all oxygen, with six protons and six neutrons; 17O, at <0.1% the rarest isotope, has six protons and seven neutrons; 18O, 0.3% of all of the oxygen isotopes, has six protons and eight neutrons. Our isotope ratio mass spectrometers (IRMS), then, were set to measure 16O and 18O, and were not tuned to measure 17O.
         Oxygen enters in the biological realm from three sources: water that is taken up directly into cells, oxygen in food for animals and heterotrophic microbes, and oxygen in the air for aerobic organisms. Oxygen in air is metabolized to water during aerobic respiration in animals and microbes and during photorespiration in plants. Because the analytical methods were limited to the measurement of carbohydrates, almost nothing was known about how oxygen and its isotopes were incorporated into other important biological molecules such as collagen, the protein in bones and teeth, and keratin, the protein in feathers.
         Oxygen isotopes in water have very different compositions relative to the oxygen isotope composition of oxygen in air. I took advantage of these natural differences in designing my first experiments. Using microalgae growing in cultures, I wanted to know whether any of the oxygen in air was permanently incorporated into the cell’s organic substances. If it did, I should have a way of determining the oxygen isotope composition of atmospheric oxygen over geological time by analyzing kerogen or fossil proteins. The ultimate goal was to understand long-term global oxygen cycles, which operate in tandem with global carbon cycles that are linked through living organisms’ respiration processes.
         I had modified a method for my hydrogen studies that enabled me to separate lipids from carbohydrates and proteins in cells. I cultured cyanobacteria and green algae in liquid broth under several conditions. I varied the oxygen isotope composition of the water, an easy thing to accomplish by adding “heavy” water composed of H218O, while keeping the isotopic composition of air constant. I modified the microalgae’s ability to carry out photorespiration by varying carbon dioxide concentrations. High concentrations of carbon dioxide inhibit photorespiration. I thought I was particularly clever.
         The analysis of organic oxygen was, at this time, limited to carbohydrates. The Caltech group, Sam Epstein and his students, was analyzing cellulose in plants and tree rings. John Hayes and his student Kim Wedeking at Indiana University were revising a method, originally described by Rittenberg and Ponticorvo (1956), for analyzing proteins and kerogen—the intractable molecule that forms the bulk of organic matter preserved in ancient sediments and rocks. These compounds contain nitrogen, sulfur, and other elements besides carbon and hydrogen. Tom Hoering and I followed their work closely. Their method, published only in Kim Wedeking’s dissertation, was based on a reaction of organic matter with mercuric chloride (HgCl2) to form a mixture of carbon monoxide (CO) and carbon dioxide (CO2). Tom Hoering and I were working on a similar method. Organic matter was heated in an evacuated sealed glass tube with HgCl2 at 500°C.  The reaction of this compound with the organic matter resulted in products that included both CO and CO2, along with hydrochloric acid and other impurities.
         Hoering and I built a system—a gas chromatograph—to separate the CO from the CO2 and the other impurities (Hoering and Estep, 1981). Gas chromatography is an analytical method designed to separate mixtures of compounds based on their chemical and physical characteristics, e.g., boiling point and reactivity. No one builds their own gas chromatograph these days! We started out with a 5 meter long ¼” copper tube, a funnel, and beaker full of molecular sieve, a compound known to separate simple gases like CO2. The copper tube was suspended in the stairwell of the Geophysical Lab. Hoering stood at the top with the funnel and beaker of molecular sieve. I stood halfway down tapping the side of the tube to promote even packing. Once filled, we rolled the tubing around a 2” diameter pipe to form a neatly wound “column” that was then fastened to our analytical system.
         We connected a series of valves to move the gases from our reactions through the gas chromatograph. We had a simple detector attached to a paper, strip-chart recorder, that I used to measure the yields of CO and CO2. As the gases were pushed through the column with helium gas under pressure, I manually flipped the valves to capture the CO and CO2, while letting the impurities exit.
         CO was converted to CO2 by reacting it in a high-voltage discharge chamber where excess carbon is plated out on platinum electrodes. The method was never robust, and as a byproduct of the high-voltage discharge in the reaction chamber, nitric oxide (NO2) was formed from the nitrogen from proteins in the organic matter. Multiple analyses of biological samples introduced NO2 unwittingly into the metal guts of our homemade IRMS. NO2 is notoriously sticky on metal surfaces. The vacuum pumps on the IRMS couldn’t get rid of it.
         Eventually the IRMS would not reach the level of vacuum needed to operate and measure isotope compositions. This was a real disaster. Tom Hoering removed the metal guts of the mass spectrometer called the flight tube. He took it out onto the lawn of the Geophysical Laboratory and sandblasted it to remove the contaminating nitric oxide. In the environment of the Geophysical Lab, where everyone seemed to have research successes all the time, I felt deflated, maybe even an “imposter.” I didn’t know anyone else who caused such damage to an isotope ratio mass spectrometer. [With time, I did hear of equally awful things--blowing up expensive metal “bellows” like balloons; dripping strong acid onto expensive electronics, for example.] I immediately, and sheepishly, moved on to other projects while Hoering reassembled the IRMS.
         The oxygen isotope vacuum and extraction line I designed for this project was my first. As a “girl”, I wasn’t taught how to use tools, really. I figured things out on my own—sometimes not until I did something stupid, did I learn to do it correctly. Parts of my line were constructed using a type of metal compression fitting composed of four important parts. The fittings, called Swagelock fittings, had a small cone, a ring called a ferrule, a nut that screwed on, and the body of the fitting made out of brass. Assembled correctly, the fittings made a water and gas tight seal. The cone went on first, then the ferrule, then the body, and finally the nut. I used these fittings to construct a water line designed to cool parts of the reaction line. When the water was first turned on, the line dripped at every connection. I did not know how to assemble a Swagelock fitting properly. Tom Hoering strolled into my lab and snorted: “You have these in ass-backward.” I had reversed the cone and the ferrule during assembly.
A lot of work- for not much, 1982

         I gave myself a grade of “C-“—sloppy work. On the science side, it was learned a few years later that all of the oxygen in a living organism is in equilibrium with cellular water. My original hypotheses wouldn’t have produced much. But, I did learn how to build a gas chromatograph, a skill needed for later work on oxygen isotopes in a project with molecular O2 that did work out. Even later, I used this knowledge to understand much more complex continuous flow gas chromatography-combustion-IRMS (GC-C-IRMS) techniques that I still use today. 
         The analysis of oxygen isotopes in organic matter was my first failed project, but I learned a lot during the process. The most important lesson was learning to deal with failure. As a high school student, I got a couple “C”s in history classes to scuff up my reputation as a nerd. In college, I breezed through with only a couple more “C”s in genetics and statistics. In the early 1970s, Penn State taught genetics through its Ag departments. The class concentrated heavily on traits of chickens and corn. I was not at all interested and sloughed off. In grad school, with the exception of a difficult biochemistry class on Intermediary Metabolism in which I earned a “B”, I was a straight A student. I needed to learn about real failure.
         A scientist’s life is filled with a certain amount of rejection: manuscripts, proposals, and ideas. Only rarely does one get affirmation and accolades. Learning to not give up, but to keep searching for the next promising idea, is key to a successful long-term career. As a University professor, I counsel undergraduates to take difficult courses—quantum physics, climate modeling, or Bayesian statistics—and figure out how to learn difficult concepts and accept that you aren’t perfect. For those straight A students, this can be frightening! What better time to know that part of yourself as to how you deal with small failures.

Winter in the "Olden Days"

  Greenvale Raiders: Marilyn, Albert Stein, Freddy, David Fuhrman, 1960 My mother claimed, and rightly so, that she walk...