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.

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