1. Early Life on Earth

2. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Early Life on earth The evidence for the earliest life is highly controversial and still debated. Much of the debate surrounds the oldest fossils. The timespan that considers the biological and geological context for the emergence of life on the Earth includes the Hadean and the early Archean. The Hadean eon ran from 4.56 to 4.0 Ga ago. Archean eon ran from 4.0 to 2.5 Ga ago.

3. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Early metabolisms Some of the habitats available to early life could been similar today and would have included the open oceans, hydrothermal vent-like environments in the oceans, the surface of early volcanic land-masses, the margins or shallow-waters around the continents (inter-tidal regions), freshwater pools of water formed on continental crust from precipitation, the deep subsurface (both in ocean sediments and in the crust) and so on. Some researchers favour the suggestion that the earliest organisms on the Earth were autotrophic. The reductive citric acid cycle or acetyl-CoA pathways, which are widespread in the domains bacteria and archaea, and ubiquitous in the deepest branches, may have been an early means by which CO 2 from the atmosphere was assimilated.

4. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Early metabolisms It is also supposed that H 2 could have played an important role in early metabolisms. Hydrogen would have been exhaled at hydrothermal vents and produced by serpentinization reactions whereby water reacts with minerals such as olivine, resulting in the formation of serpentine and hydrogen. Hydrogen concentrations in the atmosphere in a mildly reducing state could have been ~0.1% or greater. The evolution of the capacity to use hydrogen as an electron donor would have opened up the possibility of chemoautotrophic iron and sulfate reduction (using oxidised iron and sulphate ions as the electron acceptors, respectively). It would also have made possible methanogenesis, whereby hydrogen is coupled to CO 2 as the electron acceptor, to generate energy.

5. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Early metabolisms Sulfur-based metabolisms seem to have an ancient heritage and may reflect a time in the early history of the Earth with more hydrothermal activity. However, low concentrations of sulfate inferred for the Archean oceans (perhaps less than 2.5 μM) may have limited the possibilities for sulfate-reduction in the ancient oceans. Many of the organisms that oxidise sulfur compounds prefer to use sulphite (HSO 3 - ) as the electron acceptor, a compound readily formed from the reaction of SO 2 (from volcanic gases) with water. Sulphate requires initial ATP investment to activate into a form in which it can be used. Thus, sulphite reduction, which does not require this step, is more energetically favourable and could have been the precursor metabolism.

6. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Early metabolisms Organisms capable of using organic compounds on the early Earth may have existed. Many deep branching bacteria and archaea are capable of fermentation, suggesting that this could have been an early trait. These early fermenters would have used dead chemoautotrophs as a source of food or even organic material produced in non-biological syntheses or delivered from space. An enduring question in astrobiology is: which came first, heterotrophy or autotrophy, or did they emerge at roughly the same time? As the evidence described above shows, this is an unresolved question. However, the availability of inorganic redox couples and the presence of organic carbon, both from chemoautotrophs and exogenously delivered organics, would suggest that early Earth was habitable with respect to both modes of metabolism.

7. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Early metabolisms One major metabolic conundrum is the evolution of photosynthesis. It seems plausible to imagine that the earliest photosystems may have resembled the simple light driven pumps in organisms like the Halobacterium. These halophiles use bacteriorhodopsin, a simple light-driven pump, to create a proton motive force that can do work. The later evolution of bacteriochlorophylls would have allowed for simple cyclic electron transport chains in early anoxygenic photosynthesisers. These organisms would have used cyclic phosphorylation to make ATP. At some momentous time, organisms developed the ability to couple photosystems to the use of water as an electron donor. The two photosystems in cyanobacteria are thought to have evolved from an ancestral photosystem I, which bears similarities to the photosystem in green sulfur bacteria and other anoxygenic photosynthesisers, followed by the evolution of photosystem II, perhaps by gene duplication.

8. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Isotopic fractionation One way in which we can search for evidence of this past life that might have lived in early habitats is to look for isotopic evidence. Isotopes are atoms in two or more forms of the same element that contain equal numbers of protons, but different numbers of neutrons in their nuclei. Many elements of low atomic weight (including those used extensively in life) have two or more stable isotopes. Some examples are shown below with their atomic mass numbers shown as superscripts. Hydrogen - 1 H, 2 H (sometimes written as D) (deuterium) Carbon - 12 C, 13 C Nitrogen - 14 N, 15 N Oxygen - 16 O, 17 O, 18 O Sulfur - 32 S, 33 S, 34 S, 36 S

9. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Isotopic fractionation The different numbers of neutrons confer on elements different chemical properties. The lighter isotope has a higher molecular vibrational frequency, thus it tends to form a weaker bond and so it is slightly more reactive. As molecules with the lighter isotope react faster they tend to become concentrated in the products of reactions. This chemical process whereby isotopes are separated or enriched in products is called isotopic fractionation. For most processes (we will encounter an exception later in the book), this fractionation is proportional to the mass of the isotopes and so it is sometimes referred to as mass dependent isotope fractionation. The principle of this is shown for 12 C and 13 C.

10. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Measuring isotope fractionation: The delta notation We need some way to express isotope fractionation which creates an internationally recognised norm and an easy way of comparing different samples. The ‘delta’ notation is the most important way of specifying the degree of fractionation. For carbon isotopes, for instance, the degree of fractionation is defined as:

11. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Measuring isotope fractionation: The delta notation There are several things to notice about this equation. We first measure a ratio between the two carbon isotopes ( 13 C/ 12 C) for our sample of interest. It is the ratio of the heavier isotope over the lighter one. The ratio for a standard (std) is subtracted from this value and this value is expressed as the fraction of the same standard ratio. For any given element the standard is a rock or material that has been internationally agreed by the scientific community to be the standard of choice. Name of standard Isotope ratio it provides Value of standard Standard Mean Ocean Water (SMOW) 2 H/ 1 H0.00015575 18 O/ 16 O0.0020052 Pee Dee Belemnite (PDB) 13 C/ 12 C0.0112372 18 O/ 16 O0.0020672 Canyon Diablo Troilite(CDT) 34 S/ 32 S0.045005 Air 15 N/ 14 N0.003676 Table 1. Typical isotope standards.

12. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Measuring isotope fraction: The delta notation The delta notation means that a sample containing less of the heavy isotope compared to a lighter one will yield a lower isotope ratio and a lower delta value. If the ratio is lower than the standard, then the isotopic value will be negative. For carbon, typical values of  13 C range from ~0 +/- 2 ‰ for seawater and limestones to –31 ‰ in petroleum (which is essentially the remains of dead organisms) to -70 ‰ for methane produced by microorganisms, illustrating the general principle that as biological fractionation increases the content of 12 C, so the  13 C becomes more negative (lower).

13. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Measuring isotope fractionation: The delta notation

14. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Sulfur isotope fractionation Another commonly studied set of isotopes in the rock record are sulfur isotopes. The stable sulfur isotopes are 32 S, 33 S, 34 S, 36 S. The fractionation between 32 S and 34 S is the most studied. In analogy to carbon, biology prefers the lighter isotope, 32 S, as it reacts slightly faster than the heavier isotope 34 S. Sulfur isotope fractionation in the natural environment is largely due to sulfate-reducing bacteria (Chapter 5), which you’ll remember are anaerobic bacteria that oxidize organic matter (or hydrogen) using sulfate, SO 4 2-, as the electron acceptor. When organisms are limited in sulphate, fractionation from the source is quite small, but when they have plenty of it (greater than 1 mmol) then fractionation values as great as -45‰ have been measured. Fractionation values also vary between species. SO 4 2- is reduced to H 2 S during the microbial activity, which becomes enriched in 32 S because of isotopic fractionation. When the H 2 S reacts with minerals to form sulfides (such as iron sulfide), then the signature of sulfur fractionation is preserved in the minerals of the rock and it can be detected later. Thus, low values of  34 S in sulfidic rocks imply the presence of life.

15. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Using ancient isotopes to look for life We can look at ancient carbon in rocks laid down through Earth history and see what the isotopic fractionation patterns and values are. In the next figure you can see a whole range of carbon isotope measurements that have been made for rocks of different ages. The Figure shows two values, the values at the top (C carbonate ) are the isotopic values for non-biological carbonate rocks, precipitated in watery environments. Unsurprisingly, without biology, we see no significant carbon isotope fractionation. The values in the bottom part of the graph (C organic ) are the values obtained from carbon that is presumed to have once been organisms (for example, kerogen).These materials do show isotopic fractionation (a depletion of 13 C) throughout time, which is taken as evidence that organisms (autotrophs) have been at work through Earth history, taking up CO 2, preferentially using 12 C and thereby causing a 13 C depletion and a negative δ 13 C value.

16. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Using ancient isotopes to look for life Ranges of carbon isotope fractionation values through time from the present day to ~3.5 Ga ago in the inorganic fraction (C carbonate ) and organic fraction (C organic ) of rocks showing evidence for biological fractionation through time.

17. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Using ancient isotopes to look for life Evidence for early life on Earth has been presented from the ancient Akilia rocks from Greenland (which are greater than 3.8 Ga old). The carbon (graphite) within them was found to be depleted in 13 C, which was reported as evidence that organisms had preferentially taken up 12 C and left a signature of their presence. This was presented as evidence for life just over 3.85 Gyr ago. Other data, which involved analysing graphite within phosphate minerals in the rocks (apatite) showed remarkable fractionation values with a mean value -37‰, which, given losses of light carbon through processes such as metamorphism, would give isotopic values that could be interpreted to be caused by organisms such as methanotrophs that use methane as an electron donor. The rocks were interpreted to be sedimentary, supposedly formed by chemical precipitation and settling out of particles from seawater. They were critical indicators of early life because they were thought to establish the existence of a liquid hydrosphere in a habitable temperature range.

18. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Using ancient isotopes to look for life However, re-mapping and new petrologic and geochemical analyses do not support a sedimentary origin for some of the rocks. They appear instead to be igneous (volcanic) rocks such as lavas and it is therefore improbable that they hosted life at the time of formation. The apatite may not be as old as originally thought, which has further called into question the evidence for life and underlined the potential problems with using isotope data in isolation as evidence for life. Evidence has also been sought in another outcrop of Greenland rocks, the Isua rocks. These rocks, about 3.7-3.8 Ga have been claimed to have light carbon isotope values in carbonates and ancient sedimentary rocks (with a mean of ~ -15‰), indicative of life. However, the carbonates are thought to have been produced more recently during the metamorphism of the ancient volcanic rock. Isotopic studies of carbon from rocks in the Pilbara of Australia (3.5-3.2 Ga old) and the Fig Tree groups in South Africa (~3.3 Ga old) similarly show evidence of isotopic excursions (δ 13 C) of up to -32‰ that some have claimed to be biological and by others to have been caused by alteration processes since the rocks were laid down.

19. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Using ancient isotopes to look for life Evidence for life has been sought in other isotopic fractionations. For example, fractionation in sulfur isotopes caused by biological sulfate reduction is a favoured means to attempt to infer a biological process. Such signatures are reported in 3.47 Ga ago rocks from the North Pole region of Western Australia (it has nothing to do with the geographical north pole!). Sulfur isotope fractionation values in pyrite grains of up to -21.1‰ and a mean of -11.6‰ suggest that biological sulfate-reduction occurred in these sediments when they were being formed and could be evidence of life at that time. The sulfate used by these microbes is thought to have been gypsum (CaSO 4 ), which forms at low temperatures (less than ~60°C), suggesting that the microbes grew in a clement, mesophilic environment. The evidence, if correct, further corroborates the idea that sulphate reduction is an ancient metabolism. Nitrogen isotope fractionations (negative δ 15 N values) in Archean rocks have been variously interpreted as evidence of nitrogen fixation on the early Earth or for the activity of chemoautotrophic organisms using fixed nitrogen.

20. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Morphological evidence for life Another way to look for evidence of ancient life is to search for shapes that resemble fossilised cells, the basis of life. We can look for tell-tale signs of spirals, filaments and other shapes that suggest cellular structures consistent with the sorts of morphologies we find associated with cells. William Schopf famously presented photo-montages of inferred microfossils from rocks ranging in age from 0.7-3.5 Ga. Some of them are from the Warrawoona Group of the Pilbara Craton in West Australia and are ~ 3.5 Ga old. They were interpreted to be early cells with striking resemblance to the filamentous, branched structures of modern-day cyanobacteria with segmentation (septation). However, the morphological evidence has since been questioned by Martin Brasier (1947-2014) and colleagues. Rather than being in an ancient sedimentary rock, the microfossils were claimed to be associated with a hydrothermal vein created by the interaction of hot water in the ancient rock that was emplaced more recently. Temperatures within the vein could have been over 200°C, above the known upper temperature limit for life.

21. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Morphological evidence for life Some of the most convincing evidence from the Archean (although not the early Archean when life first arose) are microfossils in the 2.55 Ga cherts and carbonate formations in the Transvaal Supergroup in South Africa. These are made of tubes and sphere-shaped structures of about 2-3 μm size or less. Moving back further in time, microfossils that include long filaments from a hydrothermal set of rocks ~3.23 Ga old have been reported from the Sulphur Springs Group of rocks in the Pilbara Craton, Australia. The filaments, which are made of pyrite are narrow (~2 μm or less) and very long (~200-300 μm). They are not hollow, but have carbon coatings.

22. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Morphological evidence for life Morphological evidence poses some similar challenges to investigating isotopic evidence including:  The problem of dealing with ancient samples that have long since been pressurised and heated during rock deformation, making the signature of morphology difficult to discern.  The problem of ensuring that the fossils are associated with a plausible geological context for life.  Ensuring that the fossils are not abiotic.

23. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Morphological evidence for life One problem with morphology is that you can create microfossil-looking structures (so called biomorphs) by chemical means. Morphology on its own is at best an ambiguous indicator of biogenicity. Filament-looking structures have been produced by some researchers in the laboratory using only chemical solutions such as BaCl 2, Na 2 SiO 3, NaOH. When the solutions are added together, inorganic aggregates precipitate. In silica-carbonate solutions, filamentous materials are precipitated which appear very much like the ancient fossils reported from the rock record. To add to the complexity of this situation, these inorganic precipitates can even be shown to bind organic carbon, so that when they are lithified or preserved in hydrothermal fluid by silicification, they could easily appear to be the organic remains of organisms.

24. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Morphological evidence for life These complications have led some in the community to propose criteria by which all suggested microfossils should be judged - a sort of minimum set of criteria to accept that they could be of biological origin. One set of criteria is that putative ancient fossils should: 1)Be in rocks that are shown to be sedimentary and have undergone very low levels of geological alteration (metamorphism). 2)Be made of kerogen. 3)Exist with other fossils (and not just be an isolated occurrence). 4)Be of a size at least as great as the minimum known size for viable cells. 5)Be hollow (suggesting a cellular origin).

25. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Morphological evidence for life A variety of abiotic precipitates produced by chemical reactions that look like fossil life. (A to D) Scanning electron microscopy images of filaments. (A), (B), and (D) Filaments containing silica and barium carbonate. (C) Barium carbonate crystal aggregate after dissolution of silica in mild alkaline solution. (D) Silica skin coating the exterior of the aggregates. (E and F) Filamentous structures found in the Warrawoona chert, Australia for comparison. (G to I) Optical micrographs of synthetic filaments, showing the progressive dissolution of the solid interior of the filaments in dilute ethanoic acid, leaving a hollow silica membrane whose morphology is that of the original filament. Scale bars in (A) and (B), 40 μm; in (C), 1 μm; in (D), 4 μm; in (F) and (H), 40 μm [(G), (H) and (I) are at the same magnification]. (from Garcia-Ruiz et al., 2003) (image: Science magazine).

26. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Stromatolites Life could potentially manifest itself at microscopic scale as microfossils as we have just seen. Another type of morphological feature of life is stromatolites. These are features that can be observed at the macroscopic scale and they are found in some locations on the present-day Earth. Shark Bay stromatolites in Australia, for instance, result from the interaction between microbes and the physical and chemical environment. Present-day stromatolites growing in the Shark Bay, Australia. Each one is about half a meter across.

27. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Stromatolites Although one might think that these large structures would be easier to identify than the microfossils discussed earlier, their remains in the rock record nevertheless remain disputed. Some early putative stromatolites are from the Strelley Pool Formation, near Nullagine, Pilbara, Australia. The putative stromatolites are about 3.43 Ga old. Stromatolites are also observed in the Fig Tree Group rocks in South Africa which are about 3.3 Ga old. Putative early stromatolites (seen here as wavy rock textures) from the Strelley Pool Formation, Pilbara, Australia.

28. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. Biomarkers Some microorganisms produce compounds that are quite specific to biology and even particular metabolisms. For example, hopanoids are lipid compounds that are thought to be involved in the rigidification of cell membranes. When they degrade they leave hard-to-degrade carbon structures. One class of compounds, the 2α- methyl-hopanes, are associated with cyanobacteria and have been found in 2.6-2.5 Ga old shales and iron formations in the Hamersley Group of rocks in Western Australia. Biomarkers offer excellent potential since they can allow for specific metabolisms to be identified. One of their weaknesses is their tendency to be destroyed by the high temperatures (greater than ~300°C) encountered during metamorphism.

29. Astrobiology: Understanding Life in the Universe, First Edition. Charles S. Cockell. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. What have we learned?  Various methods can be used to search for ancient life in the rock record.  The three most common methods of searching for ancient life are: - Morphology - Isotope composition - Left over organic remnants  All of these lines of evidence are open to intense debate and questioning.  Major problems include: - Alteration of rocks over such long time scales - Contamination - Production of signatures from non-biological processes.  Overcoming these problems is key to searching for life elsewhere.