Now that we’re getting closer to analyzing the atmospheres of terrestrial-size exoplanets, it’s worth remembering how difficult the call on the existence of life is going to be. Long-time Centauri Dreams contributor Alex Tolley takes on the issue in his essay for today, pointing out along the way just how easy it is to see what we want to see in our data. While we can learn much from terrestrial biology, new approaches looking at ‘pathway complexity’ may offer useful indications of biology and a set of markers not constrained by our own unique sample of life on Earth. A lecturer in biology at the University of California, Alex brings us up to speed with extending our methods of life detection in ways that are ‘biology agnostic.’ Expect controversy ahead — will we know life when we see it, and how can we be sure?
by Alex Tolley
Manuel Werner, CC BY-SA 2.5, https://commons.wikimedia.org/w/index.php?curid=633977
Life: [noun] The condition that distinguishes animals and plants from inorganic matter, including the capacity for growth, reproduction, functional activity, and continual change preceding death. – Oxford Living Dictionary 
Life, like pornography, is notoriously hard to define, but we mostly recognize it when we see it. Life, as we know it, is identified by a set of features, which individually, may be shown by non-living systems. A classic example is “fire”, that can exhibit a simple metabolism (combustion), growth (size and spread), and even “reproduction” (sparks ignite new fires). Fire, however, fails the test of life, as all terrestrial life has the cell as a basic unit, which is not a feature of fire, nor can fire evolve.
Fossils are clearly not living, yet they show the order that life exhibits which indicates that they are a remnant of an organism that was living. For example, the fossilized skull of a dinosaur shows considerable order with features that indicate it was from a living animal and very similar to other fossil skulls of its type. Fossil bone fragments are far harder to identify and experts can detect these when a layperson would see only a piece of rock. Microfossils are even harder, as the controversial objects in the meteorite ALH84001 indicate . Are they natural formations or organisms?
When we consider how to recognize extraterrestrial life, we are largely constrained by the single sample we have. That should not stop us looking for Earth-type life as the low hanging fruit, as Earth-type life is an existence proof and well worth searching for signs of, whether with telescopes or probes.
Recent focus has shifted to spectroscopic analysis of exoplanet atmospheres. The logic is largely that of James Lovelock’s Gaia hypothesis, where the production of certain gases is a proxy for their generation by life. For a terrestrial-like world in the habitable zone (HZ), the existence of both oxygen (O2) and methane (CH4) implies life as these are primarily produced by life on Earth in the ratios required to prevent equilibrium. For a world more like the Archaean Era, an atmosphere rich in methane but excluding other gases like carbon monoxide (CO) indicates bacterial methanogens, as the geological serpentinization of ultramafic rocks like olivine is insufficient to maintain the CH4 levels.
It is this geological reaction that makes the presence of CH4 in the Martian atmosphere so ambiguous, as the masses are small enough to be produced by geology as well as subsurface pockets of life.
Where we have extraterrestrial samples, such as carbonaceous meteorites, asteroids and the recent confirmation of organic material on Mars, there is a need to differentiate abiotic from biotic processes. The classic examples of biotic processes based on our Earthly sample include the chirality of amino acids and sugars, the isotopic changes of elements due to favored selection in biological processes, such as the reduced carbon-13/carbon-12 ratios, and the odd number of carbon atoms in many lipids.
As our planetary probes increase in sophistication, and the idea that subsurface icy moons might be hospitable for life, there is a need to include the instruments to test for possible biosignatures to try to reduce ambiguity.
Biology as a System
Returning to the question of recognizing life, a key point is that it exhibits a number of features that need to be present so that we can distinguish it from inanimate objects. For terrestrial life, the basic unit is the cell, which encapsulates all the components needed to exhibit the features we identify with life, maintaining order and fighting entropy, by interacting with the external world. With the evolution of photosynthesis, that order is maintained by the capture of a tiny amount of the energy emitted by our sun. This is now the dominant source of energy for the terrestrial biosphere. Even the simplest unicellular organisms require hundred of genes, and therefore unique functional protein molecules to maintain themselves. Higher organisms require tens of thousands of genes, producing hundreds of thousands of unique proteins to maintain their more complex structures and life cycles.
We can again see the problem of detecting life from limited features with the three Viking experiments that proved ambiguous. Had there been a microscope to view a culture, the presence of cells, their growth and reproduction over time would have clinched the presence of life.
While this approach can work for samples in our solar system, for exoplanets, we must rely on proxies that are primarily measurable using spectrographic techniques. Conceivably, a telescope could image a world, detecting seasonal changes in photosynthetic organisms, providing direct evidence. For worlds with life still only in its prokaryotic state, remote direct imaging of life may prove impossible.
For samples in our solar system, we can expect a search based on terrestrial life analogs, so the usual suspects will be searched – proteins with chiral amino acids, DNA, lipids with odd-numbered carbon atoms, as well as more subtle signs such as carbon-13/carbon-12 ratios. But we should also look for evidence that is terrestrial biology agnostic, especially if we are hoping to discover very different life forms from unique geneses.
Sara Seager: Going Beyond the Presence of a Molecule
Sara Seager’s team has been at the forefront of considering biosignatures beyond the usual proxies of atmospheric gas mixing ratios. Her paper  (see also CD post Ambiguity in Life Detection, October 31, 2017) collated the range of small organic molecules that exist and their source whether biotic or abiotic or both. At the 2018 Breakthrough Discuss conference, she noted that biology does have some apparent constraints and explained the paucity of biotic molecules with nitrogen-sulfur (N-S) bonds, even though these compounds abound in industrial chemistry because of their usefulness. Terrestrial biology is rich with thiol reactions and has evolved replication and metabolisms that generally eschew molecules with such N-S bonds. This phenomenon constitutes a possible biosignature. While this is one specific example, there are likely many others. However, constraining our ideas to terrestrial biology may result in us missing non-terrestrial biologies that are different, providing false negatives. What is needed is a more general approach that is biology agnostic.
Lee Cronin: A Generalized Approach
Lee Cronin’s group has been formulating a more biology agnostic approach, one that is based on living organisms being homeostatic systems . His approach is to assume molecules can be constructed by assembling sub-units and that this confers a minimal construction pathway, which he calls “pathway complexity”. One can consider this as a tree of all possible molecules composed of the building blocks with the number of construction steps needed to build the molecule. This is an indication of the non-randomness of the molecule. If a molecule in a sample is highly enriched compared to the possible random set of molecules that could be constructed at random, this is indicative of a construction pathway that in turn is indicative of life.
Figure 1 below shows the concept of construction of a specific molecule from building blocks.
Figure 1. Illustration of a complexity pathway in blocks, with the target shown by the yellow box. A combinatorial explosion in structures is illustrated by the other faded structures shown, which are just a small set of the many alternative structures that could be constructed. (Online version in color.) 
Figure 2. An illustrative graph of complexity against size of the state space. Orange regions are impossible as they are above or below the bounds of the measure. The green region is where living systems may be most probable, where structures are neither too simple to be definitively biological, nor too complex to exist at all. 
Cronin states :
“We can extend the basic complexity measure above to cope with assessing the complexity of a group of objects that contain identical connection motifs (figure 5). In this case, we examine a population of objects and abstract out a common graph based on connected subunits that share features. For example, if examining a set of cups or mugs, then we can create a common graph of ‘handle connected to body’, regardless of potential variations in size/colour etc. If examining a set of human beings, then we could create a common graph of bone connectivity, ignoring variations in size/shape of individual bones, or any material in the body other than bones.”
“It is clear that biological and biologically derived systems have an ability to create complex structures, whether proteins or iPhones, that is not found elsewhere in nature. Assessing the complexity of such artefacts will be instrumental in searching for undiscovered biospheres, either on Earth  or elsewhere in the Solar System, and would make no assumptions about the details of the biology found. We propose Pathway Complexity as the natural measure of complexity for the production of artefacts. In this context, we argue that there is a critical value of Pathway Complexity above which all artefacts must be biologically derived. This approach provides a probabilistic context to extending the physical basis for life detection proposed by Lovelock . In further work, we will show how this applies to a range of other systems, and propose a series of experimental approaches to the detection of objects and data that could be investigated as a possible biosignature. In the laboratory, we are interested in using this approach to develop a system that can explore the threshold between a non-living and living system. Pathway Complexity may also allow us to develop a new theory for biology. This might inform anew way to search for life in the laboratory in terms of the complex products a system produces and if they could have arisen in any abundance by chance, rather than trying to measure the intrinsic complexity of the living system itself.”
Kauffman: Self Organization Theory of Life
In 1993, Stuart Kauffman published The Origins of Order: Self Organization and Selection in Evolution . I consider it a tour de force in theoretical biology. Of relevance to this post are chapters 7 and 8 on the concept of autocatalytic sets, and the crystallization of metabolisms.
Autocatalytic sets are best thought of as a linked set of components, e,g, catalytic RNA, that can build each component from others in the set. The effect of which is to rapidly increase the RNA species included in the set. From an origin of life perspective, Kauffman showed that the probability of autocatalytic sets arising increases to unity as the number of RNA species increases. Metabolisms similarly crystalize when there are enough reactants so that a complete, self-contained metabolism can be sustained. Again, the probability of such a complete metabolism will increase as the number of reactants increase.
As Kaufmann states:
“Thus we arrive at a new point of view. The emergence of a connected metabolism as a supracritical web requires a sufficient complexity of organic molecules and a sufficient complexity of potential catalysts. At that point, such a connected web is an inevitable emergent collective property of the chemical system.”  p348.
The relevance to Cronin’s work should be clear. Once a self-sustaining set of components appears, the components in that set will increase rapidly compared to others in the vast space of possible components. Cronin’s metrics, such as “pathway complexity” naturally emerge when considering the number of components compared to the possible components due to random reactions.
While Kauffman’s work is theoretical, Cronin has shown that lab experiments  support this basic concept. In terms of biology, they are agnostic in origin, therefore freeing us from focusing on terrestrial biology as our single sample of life, and informing us of possible biosignatures.
Sampling the compound space is not something that is likely to be possible anytime soon, if ever, using spectral analysis of [exo]planet atmospheres. Even Seager’s list of possible biosignature compounds are effectively trace compounds, and there is no way to determine whether her N-S bonds hypothesis works for an exoplanet from telescopic observations.
However, the solar system is another matter, and targets such as Mars, the plumes of Enceladus, the organics on the [sub]surface of comets, Ceres and the Europan sub-surface ocean are ripe for this sort of systems analysis using mass spectrometers and IR spectroscopy on probes to determine the mix of compounds in a physical sample.
While future telescopic observations that can image worlds directly may show up life as lush, boreal zones on exoplanets, nearer to home we may be able to sample the biological detritus of such worlds through wanderers like ‘Oumuamua that may have captured bacterial life from living worlds. If exoplanet life is largely bacterial, then probes sampling the upper atmosphere or even the surface can use this technique to determine if life exists without the difficulty of trying to cultivate bacterial colonies and observing the results. While interstellar probes that could sample such worlds are a relatively distant prospect, they are possible in the centuries to come using propulsion technologies that do not require new physics.
Confirmation bias involves seeing the data supporting what you are expecting. The lack of artificial objects in the heavens that is the context for the Fermi Question elicits polar views of “we are the only life” to “technological life is there, we don’t recognize it”. Similarly, the lack of unambiguous signals found by SETI results in a similar dichotomy. As we noted, the ambiguous objects in the ALH84001 meteorite that came from Mars have proponents for either proposition — life and non-life. Early searches for “missing links” in the evolution of humans that found a few bones and partial skulls also resulted in polar views of whether modern humans had evolved from an apelike ancestor or been created in his present form. We can be sure that any spectroscopic evidence of proxies for life – biosignatures – will be similarly interpreted.
So far, chemical analysis of samples in our solar system have been teasers, hinting at possible life, but no more. While a video of a living animal in a sample tube would be unambiguous (although there will no doubt be claims of “it is a hoax”), the most compelling approaches would be confirmation of DNA or proteins, preferably with no known terrestrial copies. This, however, assumes life is very similar to terrestrial life, and techniques used to find such molecules will miss life that may be very different from terrestrial life. Starting from the model that living systems are complex systems, yet not so complex as to be random, chemical analyses within the scope of that with existing analyzers may well be able to indicate life with far less ambiguity than the focus upon a few proxy molecules. In this regard, the theoretical bases described by Kaufmann and Cronin, and confirmed with experiments on terrestrial living organisms, offers perhaps the best approach for sampling probes that we can envisage in the near future, although the mass penalty of a microscope would be very much appreciated.
1. Petkowski J et al “Natural Products Containing a Nitrogen−Sulfur Bond” J. Nat. Prod. 2018, 81, 423−446
2. Kauffman S. “The Origin of a Connected Metabolism” ch 10, p343 in The Origins of Order, 1993
3. Domagal-Goldman S et al “Life Beyond the Solar System: Remotely Detectable Biosignatures” 2018, arXiv:1801.06714 [astro-ph.EP]
4. Cronin Lee “A probabilistic framework for identifying biosignatures using Pathway Complexity” 2017, Philos Trans A Math Phys Eng Sci. 2017 Dec 28;375(2109). pii: 20160342. doi: 10.1098/rsta.2016.0342.
5. Doran D et al “A recursive microfluidic platform to explore
the emergence of chemical evolution” 2017, Beilstein J Org Chem. 2017 Aug 17;13:1702-1709. doi: 10.3762/bjoc.13.164. eCollection 2017.
8. Seager, Bains and Petkowski, “Toward a List of Molecules as Potential Biosignature Gases for the Search for Life on Exoplanets and Applications to Terrestrial Biochemistry,” Astrobiology 16(6) (June 201), 465-485