Thursday, August 8, 2019

Astrobiology! Are we alone? How did we get here? My start...

Rus Hemley, Nabil Boctor, George Cody, Jen Blank, Hat Yoder, Jay Brandes, Bob Hazen, and Marilyn, Hat Yoder's Lab circa 1999

“The probability for the chance of formation of the smallest, simplest form of living organism known is 1 to 10,340,000,000.... The size of this figure is truly staggering, since there are only supposed to be approximately 1080 electrons in the whole universe!” Harold Morowitz, in Energy Flow in Biology (1968).

         In 1996, NASA announced that it had found evidence of life contained in the Martian meteorite Alan Hills 84001 (McKay et al., 1996). They held press conferences and presented evidence for what they termed bacterial cell structures in the meteorites, along with some amino acid profiles. News spread like wildfire among Mars aficionados and skeptics alike. The search for life on Mars stimulated several lander and orbital missions to Mars. At the same time, NASA under the advice of Wesley T. Huntress, Associate Administrator for Space Science, had the idea to form the NASA Astrobiology Institute (NAI), a virtual institute in which scientists in disciplines as far-ranging as astronomy and astrophysics would regularly engage with molecular biologists and geochemists. Objectives central to this effort was to understand how life arose on planet Earth, to determine when it arose, and to devise a set of criteria everyone could agree on that constituted evidence of life.
         This task has been more difficult than it sounds. To this day, NASA and NAI scientists still are discussing how they will determine which chemical or physical line(s) of evidence constitute proof that there is life on another planetary body. I was part of two original NAI teams that took very different approaches to search for life. The Jet Propulsion Lab (JPL) team focused on biosignatures. Biosignatures are fingerprints of life such as isotope fractionation patterns or elemental ratios. Ken Nealson, who specifically moved from University of Wisconsin to JPL, led the effort. Our work was carried out in both modern extreme environments as well as in ancient rocks. Being on the JPL campus, this NAI team used several new techniques that were being specifically designed for space flight. The goal of this group was to develop biosignatures in tandem with new analytical methods.
         Working with postdoc Sue Ziegler, we started with a simple mixed culture of microorganisms and fed them a single protein, Rubisco the protein I studied for my Ph.D. work. We then analyzed the chemical isotope signature in the microbes and found that bacteria totally scrambled the isotopic patterns in amino acids of the Rubisco protein. The process was rapid and completed overturned in a matter of a day or two. Bacterial proteins were decomposed and recycled as well. Our data shows that on very short time scales, hours to days to months, microbial products are formed and degraded. On a geological time scale of hundreds, thousands, or millions of years, biologically-resistant material gradually obtains an isotopic and chemical composition that has little resemblance to original biochemical compounds. If we were to find evidence of life in martian rocks billions of years, we needed to broaden our thinking.
         The approach by the Carnegie team, which I also working with, was completely different. Geophysical Laboratory staff member Bob Hazen and Harold Morowitz (George Mason University) had the idea that we might be able to study some of the most basic reactions of the TCA cycle using hydrothermal techniques rather than biological enzymes. Hazen wrote a book, Genesis, about how this project evolved. Geophysical Laboratory staff member George Cody, former Geophysical Laboratory Director Hat Yoder, and myself as well as Bob and Harold, postdocs Mark Teece, Jay Brandes, Tim Filley, Jennifer Blank, and Nabil Boctor discussed how this could be carried out. Bob and postdocs would seal organic reactants into gold tubes that served as reaction vessels; Hat Yoder would subject them to high temperatures and pressures in his internally heated, gas media pressure bombs; George and postdocs would analyze the products of the reaction; Mark Teece and I would measure the isotopic patterns in the products; Harold and the rest of us would interpret the results. Experiments were more complicated than we ever anticipated. 
Hat Yoder's Internally heated high pressure "rig"
         Bob Hazen is an accomplished writer of popular science books, textbooks, as well as scientific articles in his discipline. Over the years, I watched him transform from a high-pressure mineralogist to an astrobiologist to a big picture thinker on the nature of carbon in the deep earth. He was never a “lab guy” like the rest of us at the Geophysical Lab. Instead, he partnered with bright people like Larry Finger, George Cody, Robert Downs, and others to anchor the data collecting. Often, we sparred with one another, at unpredictable times. He saw my work as incremental, which is what most scientific advances truly are. I saw his work as broad brush, not getting at the heart of problems. Either way, Hazen sustained having novel scientific ideas which he supported with his encyclopedic ability to read articles, understand them, and write about them with style.
         Harold Morowitz was a real original thinker and intellect. Working at a second tier university in northern Virginia, he struck me as a person who could work anywhere that welcomed complex thoughts. Harold knew the biochemical pathways and enzymes thoroughly, which we appreciated. When he made visits to discuss experiments, with time, there was a tension when hypotheses did not match results and findings. Both he and Bob Hazen were scholars, while the rest of us were soldiers in the laboratory figuring out difficult analytical problems. Fortunately, George Cody and Harold worked on the same plane and were able to match their talents to produce the detailed work on abiotic organic synthesis.
         George Cody is a one of a kind, often self-absorbed and oblivious to humans, other times thinking about the long-term vision for the Geophysical Laboratory and his colleagues. Cody is most comfortable at the console of his custom built NMR spectrometer. As he evolved from being a coal chemist to an organic geochemist to an astrobiologist to a generalist, his skills in analyzing complex data sets thoroughly distinguish his work. At parties, George often can’t leave his work behind. After a couple glasses of red wine, he can regale his friends about hamiltonians (an obscure parameter measured by his NMR spectrometer), while his wife looks patiently on. When we worked together, I could always rely on George to huddle together with me and figure out gnarly lab problems, personnel issues, and weird data.
         The team learned that the anticipated products were unstable at high temperatures (e.g., 170°C). Hundreds, if not thousands, of reactions were sealed in gold tubing, heated, and chemically analyzed. After almost two years, Cody and Hazen realized that they were examining a different type of reaction than they had originally thought. Rather than studying synthesis, the work slowly evolved to studying decomposition. Further, although the starting reactants were often a single molecule (e.g., citrate), the products were very complex. We never made it to the point of measuring stable isotopes. Bob and George shifted to investigating metal-sulfide catalysts and iron-nickel catalysts, searching for the Holy Grail of recreating the biological tricarboxylic acid (TCA) cycle via hydrothermal reactions.
         Eventually, Cody and team members found a potential entry point into the reductive TCA cycle utilizing metal sulfides and reduced carbon bearing fluids. This pathway, named the hydrothermal redox pathway, is not used in any extant organisms but may have been the ignition point for primary metabolism during the early evolution of living organisms. The potentially critical catalytic role of such metallic catalysts may be a crucial link between geochemistry and biochemistry at the point of life’s emergence. Integrating all of this, they discovered that they were within one reaction of demonstrating a purely geochemical carbon fixation pathway that closely mimics the TCA pathways.
Hiroshi Ohmoto giving astrobiology lecture with Boz Wing to left, Canada
         Origin of life studies were being carried out across the NAI and internationally at this time (2000s). The RNA (ribonucleic acid, one of the molecules carrying the genetic code) world hypothesis (Shostack et al) had been a favored pathway for jump-starting life. RNA can catalyze simple reactions, directs the construction of more complicated molecules like proteins, and holds heritable information. The problem with this model was figuring out how to make RNA molecules in the first place. One hypothesis was that meteorites that bombarded early Earth brought these molecules with them.
         Zita Martins and the organic geochemists at Goddard Space Flight Center were searching for the building blocks of DNA and RNA, nucleobases, in organic extracts of Murchison meteorite, one of the most famous carbonaceous chondrites of all time. Zita was working with Danny Glavin and Jason Dworkin at Goddard, and her colleagues and Ph.D. advisor in the Netherlands. Using a sophisticated type of mass spectrometry, she could detect nucleobases in some of the Murchison extracts. She was able to zero in on these unusual and complicated structures, essentially picking the “needles out of the haystack” of other compounds. We began a collarboration to measure the stable isotope patterns in these particular molecules. If they really came from outer space they should have an isotope pattern similar to other meteorite organic molecules others had measured. Alternatively, if these were terrestrial contaminants, they should resemble isotope patterns of biological molecules.
         ZIta arrived with extracts in hand at the Geophysical Laboratory, and we were confident that we could readily measure the carbon isotope patterns based on the results she measured at Goddard Space Flight Center. Unfortunately, the analytical system I was using, a combustion-GC-IRMS system, combusts all carbon compounds in the chromatogram, which was loaded with a multitude of other compounds, mostly organic acids, as well as with trace amounts of nucleobases (Martins et al., 2008).
         We were able to devise a clever analytical scheme for our instrument that separated the acids from the nucleobases without any serious overlap. During chromatography, the isotopic composition of the head of the peak can differ quite a bit from the tail of the peak. The carbon isotope patterns of the organic, dicarboxylic acids were very positive and similar to measurements others had made. We measured a similar carbon isotope pattern for uracil, a component in RNA, in the Murchison, relative to a much different carbon isotope pattern for uracil from the soil surrounding the location in Australia where it was found. Xanthine, a potential compound for forming nucleic acids, from Murchison had a carbon isotope pattern matching with uracil and the organic acids (Martins et al., 2008), confirming that these nucleobases originated from meteoritic organic carbon. This work supports one of the many theories about the origins of life. One of these theories holds that meteorites delivered organic molecules to Earth during its formation and the period when Earth was continually bombarded by incoming asteroids. Although it was known that amino acids (e.g., Engel et al., 1980; Martins et al., 2007) were common monomers in some chondrites, no one had ever found nucleobases.
         Eventually, the stable isotope biogeochemistry studies of the Carnegie team shifted in emphasis. In phase two of the NAI project at the Geophysical Laboratory, we began to study the isotopic compositions of ancient sedimentary rocks and stromatolites and began an ambitious field campaign in Svalbard, an archipelago high above the Arctic Circle in a project termed AMASE: Arctic Mars Analogue Svalbard Expedition. Both the NAI and AMASE endeavors required field trips to far flung places to collect samples from ecosystems of interest to astrobiologists.

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