SYNTHETIC QUIRKINESS
Consider the example of life's hydrocarbons, the molecular family that includes waxes, soaps, oils, and all manner of fuels, from gasoline to Sterno. All cells require a rich variety of these molecules, which incorporate long, chainlike segments of carbon and hydrogen atoms. Hydrocarbons, which we eat in the form of fats and oils, serve many cellular functions, including the production of flexible cell membranes, efficient energy storage, varied internal support structures, and more.
In life and in commerce, long hydrocarbon molecules are usually made by linking smaller pieces end-to-end. When industrial chemists want to synthesize hydrocarbons, or when these molecules arise by natural nonbiological processes, the molecular chains are usually lengthened one carbon group at a time. This process ordinarily yields a suite of molecules with many different lengths, from just a few to many dozens of carbon atoms long, but all formed by the same stepwise mechanism.
Life builds hydrocarbons differently and in a strikingly idiosyncratic way. In each cell an amazing tool kit featuring half-a-dozen different protein catalysts, collectively called the “fatty acid synthase,” facilitates the assembly of hydrocarbon chains by adding units of three carbon atoms to a growing chain and then stripping one away. The net result is carbon addition by pairs. So life's biochemistry is often characterized by a preponderance of hydrocarbon chains with an even number of carbon atoms: chains of 12, 14, or 16 carbon atoms occur in preference to 11, 13, or 15. As a result, given a suite of molecules from some unknown source and a mass spectrometer that can analyze the size distribution of those molecules, it's not too difficult to tell whether the hydrocarbons came from living cells or from nonbiological processes.
Polycyclic compounds, an even more dramatic example of life's molecular idiosyncrasies, include a diverse group of carbon-based molecules with several interlocking 5- and 6-member rings. A variety of cyclic molecules are found everywhere in our environment. Even before Earth was born, they were produced abundantly by chemical reactions in interstellar space and during star formation—processes that littered the cosmos and seeded the primitive Earth with cyclic organic molecules. The PAHs (polycyclic aromatic hydrocarbons) found in the Martian meteorite ALH84001 are examples of these ubiquitous compounds. Cyclic molecules continue to be synthesized on Earth as an inescapable by-product of all sorts of burning: They are found in the soot of fireplaces and candles, the smoke of incinerators and forest fires, and the exhaust of diesel engines. Travel to the remotest places on Earth—the driest deserts of North Africa, deep ocean sediments, even Antarctic ice—and you'll find PAHs.
Every living cell manufactures a variety of polycyclic carbon compounds but, as with hydrocarbon chains, the polycyclic compounds produced by life are much less varied than those produced by inorganic processes. The 4-ring molecules called sterols, including cholesterol, steroids, and a host of other vital biomolecules, underscore this point. Literally hundreds of different 4-ring molecules are possible, yet while the relatively random processes of combustion or interstellar synthesis yield a complex mixture of cyclic compounds, life zeroes in almost exclusively on sterols and their by-products.
Again, cells employ a remarkably quirky synthesis pathway. The first step in forming a sterol is to manufacture lots of isoprene, a 5-carbon branching molecule (which the cell makes from three smaller molecules). Six isoprene molecules line up end-to-end to form squalene, with 30 carbon atoms—24 of them in a chain, with six single carbon atoms branching off at regular intervals. This long molecule then folds up into the 4-ring sterol backbone.
Cells manufacture polycyclic molecules in an idiosyncratic three-step process. First, three small molecules link together to form isoprene (A). Then six isoprene molecules line up end-to-end to make squalene (B). Finally, squalene folds up into the 4-ring cholesterol molecule (C). In these and subsequent drawings of molecules, each short line segment represents a chemical bond between two carbon atoms.
Biochemical textbooks describe dozens of other examples of elaborate synthetic pathways: photosynthesis to make the sugar glucose, glycolysis (splitting glucose) to make the energy-rich molecule ATP (adenosine triphosphate), metabolism via the citric acid cycle, the production of urea, and countless other vital chemical processes. Over and over, we find that cells zero in on a few key molecules. DNA and RNA, which carry the genetic code, rely on ribose and deoxyribose alone, eschewing the dozens of other 5-carbon sugars. Proteins are constructed from only 20 of the hundreds of known amino acids. What's more, sugars and amino acids often come in mirror-image pairs—so-called “right-handed” and “left-handed” variants—but life uses right-handed sugars and left-handed amino acids almost exclusively.
The take-home lesson is that life is exceedingly choosy about its chemistry. Of the millions of known organic molecules with up to a dozen carbon atoms, cells typically employ just a few hundred. This selectivity is perhaps the single most diagnostic characteristic of living versus nonliving systems. If an ancient rock is found to hold a diverse and nondescript suite of organic molecules, then there's little we can conclude, yea or nay, about its biological origins. It may once have held life, or it may simply represent an abiotic accumulation of organic junk. If, on the other hand, an old rock holds a highly selective suite of carbon-based molecules—predominantly even-numbered hydrocarbon chains or left-handed amino acids, for example—then that's strong evidence that life was involved.
A crucial requirement, if this logic is to be implemented in the search for life here or on other worlds, is that biomolecules must be stable over time spans of billions of years. Large protein molecules won't last that long, and neither will the 20 amino acids that comprise the building blocks of proteins. Nor will most carbohydrates or hydrocarbon chains. Over time, water attacks the bonds of these biomolecules, breaking them into smaller fragments of no diagnostic use. But polycyclic compounds, like sterols, degrade more slowly and might survive over geological spans of time. Therein lies a possible top-down path to the discovery of life that is distant in space or time.
THE HOPANE STORY
Once in a very great while, extremely old rocks are found to hold microscopic droplets of a petroleum-like black residue—hydrocarbons that represent the remains of ancient marine algae. When such droplets were first discovered, decades ago, most scientists discounted the possibility that these organic remains were very old; no oil could survive billions of years of geological processing, they said. But subsequent discoveries and improved analytical techniques have convinced the geological community that a hardy breed of organic hydrocarbons can survive in ancient rock provided that temperatures never got too high.
In their quest for life signs, a group of Australian scientists has focused upon what are perhaps the ideal biomarkers—distinctive sterol-derived polycyclic hydrocarbon molecules called hopanes. This group of elegant 5-ring molecules is known in nature only from the biochemical processes of cellular life, where it concentrates in protective cell membranes. Furthermore, different variants of hopanes point to specific groups of microbes with distinctive biochemical lifestyles. If an ancient rock happens to encase and preserve hopane-related molecular fragments with the diagnostic structures of once-living biomolecules, then we have convincing evidence of ancient life.
In 1999, a team of scientists led by Roger Summons (then at the Australian Geological Survey Organisation) presented compelling evidence for the survival of hopanes in a sequence of 2.7-billion-year-old sedimentary rocks called the Pilbara Craton, in Western Australia. The black, carbon-rich shale layers in question came from a section of drill core extracted from a depth of about 700 meters. The mineralogy of the shale revealed that it had never experienced a temperature higher than about 300°C—an unusually benign history for such an ancient deposit.
This 5-ring structure is characteristic of hopane, a distinctive biomolecule whose backbone may be preserved for billions of years in ancient sediments.
Hopanes have been common biomolecules for a l
ong time, so the Australian team's principal challenge was ruling out contamination from more recent life. The rocks might have been contaminated hundreds of millions of years ago by subsurface microbes, or by groundwater carrying biomolecules from the surface, or perhaps even by oil, seeping from some other sedimentary horizon. Summons and his colleagues discounted the last of these possibilities because they found no trace of petroleum in adjacent sediment layers. Modern contamination from living cells, which abound in the lubricants that scientists use to drill their deep holes in the host rock, was also a concern. The team ruled out such contamination, too, because the suite of molecules preserved in the shale was “mature,” containing none of the fragile organic species that would point to recent lubricants and accompanying microbial activity.
Summons and his co-workers had to develop meticulous procedures to expose and clean unadulterated fresh rock surfaces: Break the rock, wash the surface, and measure the wash for contamination. They resorted to smaller and smaller rock fragments to avoid the inevitable impurities that had seeped in along cracks. Summons found that properly prepared powdered shale contained a distinct suite of ancient hydrocarbon molecules at hundreds of times higher concentrations than in adjacent chert and basalt layers from the same drill core. Nevertheless, the amount of hopanes was minuscule: of all the carbon-rich material extracted from the rock, no more than a precious few hundred parts per million were hopanes and related polycyclic molecules. Still, the very presence of hopanes provided evidence for ancient microbial life.
Having overcome daunting hurdles, Summons and his colleagues announced their finding in August 1999, in two remarkable papers, one in Science and the other in Nature. The Science article detailed extraction of hopanes from 2.7-billion-year-old shale—results that broke the previous record for the oldest molecular biomarker by about a billion years. The Nature article described the discovery of hopanes from 2.5-billion-year-old Australian black shale—a younger nearby formation, but with a twist. That formation included 2-methylhopanoid, a hopane variant known to occur in cyanobacteria, which are the primitive photosynthetic microbes responsible for generating Earth's oxygen-rich atmosphere. The Australian team had found suggestive evidence that cyanobacteria were thriving long before 2 billion years ago, when Earth's atmosphere is thought to have achieved modern levels of oxygen.
By extracting and identifying unambiguous biomarkers in ancient rocks, Summons and colleagues had made a major advance in detecting and characterizing ancient life. They also helped close the gap in our ignorance of life's emergence by embellishing the top-down story and pushing it just a little bit further back in time.
BIOSIGNATURES AND ABIOSIGNATURES
The quest for unambiguous “biosignatures,” including hopanes and other distinctive molecules, represents an effective strategy in the search for ancient life on Earth and other worlds. However, the identification of “abiosignatures”—chemical evidence that life was never present in a particular environment—might also prove important in constraining models of life's emergence.
Abiosignatures hold special significance to astrobiologists, who search for life in Martian meteorites and other exotic specimens. Are there physical or chemical tests that might preclude the presence of past life in those specimens? “NO LIFE ON MARS!” would be a bummer of a headline, but would nevertheless carry great scientific, not to mention philosophical, implications about the frequency of life's emergence.
Hopanes not only represent biosignatures for ancient life on Earth, but they also point to a search strategy for other worlds, especially our nearest neighbor, Mars. Based on what we now know about life and its fossil preservation, we are unlikely to find unambiguous Martian fossils of single cells, much less animals or plants, at least not any time soon. A Mars sample return mission won't happen for at least a dozen years, while human exploration of the red planet is many decades away. And even with such hands-on exploration, we'd be incredibly lucky to find a convincing fossil. We're much more likely to find local concentrations of carbon-based molecules, from which we can determine the carbon-isotopic composition. However, as the Greenland incident reveals, a simple isotopic ratio may not be sufficient to distinguish nonbiological chemical systems from those that were once living.
Suites of carbon-based molecules, if we can find them, hold much greater promise. An array of molecular fragments derived from a colony of cells, if not too degraded, will differ fundamentally from a geochemical suite synthesized in the absence of life. For now, molecules represent our best hope of finding proof of life both here and elsewhere in our solar system.
The ideal molecular biosignatures—and abiosignatures as well—must display three key characteristics. First, biosignatures should consist of distinctive molecules or their diagnostic fragments that are essential to cellular processes. Similarly, abiosignatures should consist of molecules that clearly point to nonbiological processes.
The second criterion is stability: biosignatures—and abiosignatures as well—must be molecules able to survive through geological time. Even the least altered ancient sediments have been subjected to billions of years of temperatures greater than the boiling point of water—conditions that significantly alter the chemical characteristics of any suite of organic molecules, whether biological or not. This criterion of stability, consequently, focuses our attention on unusually stable molecules.
Finally, the molecules must occur commonly and in reasonable abundance. A molecular biosignature or abiosignature is of no use unless it can be detected by mass spectrometry or other standard analytical techniques.
Hopanes, and the related sterols, are unquestionably excellent diagnostic biosignatures from the standpoint of stability, and they're reasonably easy to analyze. Many ancient deposits yield traces of these molecules, and they will continue to be a tempting target for analysis, as well as a model for finding other biosignatures. But hopanes are probably not the ultimate answer in the search for signs of life: They seldom occur in abundance, and their absence cannot be taken as a reliable abiosignature.
An alternative to the search for reliable biosignatures and abiosignatures might be to identify diagnostic ratios of molecular fragments, akin to the carbon-12/carbon-13 isotopic ratio. However, we're confronted with a vast multitude of possible molecule pairs. Which pair of molecules should we study?
My first foray into the search for biomarkers occurred in the summer of 2004. Preliminary studies by George Cody on organic compounds in meteorites prompted us to look at the ratio of two of the commonest PAHs: anthracene and phenanthrene. These 3-ring polycyclic molecules, both made up of 14 carbon atoms and 10 hydrogen atoms (C14H10), differ only in the arrangement of the rings: In anthracene the rings form a line, in phenanthrene a dogleg. We realized that the ratio of these two molecules might fulfill the essential biomarker requirements: Both are distinctive, relatively stable, common in the geological record, and easy to detect in trace amounts.
Phenanthrene and anthracene form in abundance through a variety of nonbiological processes, including any burning process that produces soot. These cyclic compounds are also synthesized in deep space, where they contribute to the molecular inventory of the carbon-rich meteorites called carbonaceous chondrites. The most celebrated of these is the Murchison meteorite, which fell to Earth in a cow field outside the small town of Murchison, about 100 miles north of Melbourne, Australia, on September 28, 1969. Meteorites hit Earth all the time, but the Murchison fall was special. For one thing, it was big—several kilograms of rock. For another, it was fresh and relatively uncontaminated—a number of pieces were collected while they were still warm. But, most important, the Murchison was a carbonaceous chondrite, containing more than 3 percent by weight of organic molecules. That black, resinous matter, formed billions of years ago in dense molecular clouds and protoplanetary disks, held a treasure trove of the molecules that could have accumulated on the prebiotic Earth.
Phenathrene (top) and anthracene are 3-ring polycyclic molecules tha
t differ only in their shape. The ratio of these two molecules differs in abiotic and biological systems.
George Cody had found that such meteorites often display about a 1:1 ratio of phenanthrene to anthracene. But biochemical processes seem to produce a different ratio. Many polycyclic biomolecules—including sterols and the varied hopanes—incorporate a 3-ring dogleg, so phenanthrene is a common and expected biomolecular fragment, and it should persist in rocks and soils, even when larger molecules break down. But for some reason, life almost never uses anthracene's linear arrangement of three rings. Anthracene would thus seem to be correspondingly rare as a biomolecular fragment. Cody had found that biogenic coals typically hold 10 times more phenanthrene than anthracene.
Is the ratio of phenanthrene to anthracene a useful biomarker? Testing this idea required measuring the ratios of cyclic compounds in lots of samples, so that was the task I gave to Rachel Dunham, a bright and energetic undergraduate summer intern from Amherst College. Over the course of her 10-week stay in Washington, Rachel assembled dozens of natural and synthetic PAH-containing samples from around the world, analyzed them with our gas chromatograph/mass spectrometer, and managed to track down many more analyses from the vast coal and petroleum literature, since it turns out that PAHs are especially abundant in some fossil fuels.
Genesis: The Scientific Quest for Life's Origin Page 9