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Early Life and Exolife (split from Exoplanet Stuff)

Swammerdami

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Whenever I see this thread title, I think of Exolife — life on other planets.

I think Nick Lane may well be correct, that our life based on water, peptide chains and some variant of nucleic acid chains is "perfect" and that no other biochemistry is plausible. In this view, if life is ever discovered on another planet the basic building-blocks will be similar to Earth's life.

But, if only to develop into a science fiction novel, it would be interesting to explore the possibility of very different biochemistry. Are there any chemists here? One possibility I see mention of is complex molecules with a silicone backbone ( -Si-O-Si-O- ) in an ocean of sulfuric acid. Although the backbone of the complex molecule might be based on silicon. side chains might be mostly ordinary organics — perhaps similar to amino acids, sugars or nucleotides — but with occasional silicons substituted for carbon.
 
Whenever I see this thread title, I think of Exolife — life on other planets.

I think Nick Lane may well be correct, that our life based on water, peptide chains and some variant of nucleic acid chains is "perfect" and that no other biochemistry is plausible. In this view, if life is ever discovered on another planet the basic building-blocks will be similar to Earth's life.

Considering that Earth life is composed of some of the most common elements in the universe and that the very most basic molecular building blocks can be created in interstellar chemistry, I think that you are probably on to something.
 
Yes. But Silicon is also plentiful on Earth.
One " paradox" is that Phosphorus is rare on Earth, yet is essential in Earth's biochemistry: dna, rna, atp.
 
Yes. But Silicon is also plentiful on Earth.
One " paradox" is that Phosphorus is rare on Earth, yet is essential in Earth's biochemistry: dna, rna, atp.
On earth silicon is locked up in the rocks mostly, yes? And in space silicon is mostly locked up in dust, which aggregates to become the rocks, so it follows.

Elements like phosphorus may just be at low concentration but still common. I don’t know enough about abiogenesis and evolution to comment on how or why these trace elements became important to life.
 
Silcon is often compared with Carbon — the element right above it in the periodic table. But there are huge differences. For starters, CO2 is a gas very convenient as a precursor to organic molecules. SiO2, on the other hand, is ... useless silica rock!

But silica does react with sulfuric acid. And complex molecules can arise if you somehow prevent the silica from re-forming.

But it may be unrealistic to think silicon-based life is likely. I mentioned it mainly as a possibility for mention in science fiction!

Massive presumption that it is "Earth's" biochemistry to begin with. There massive clouds of organic compounds out in space?
All I meant by "Earth's biochemistry" is the chemistry of biological processes on Earth!

To support my claim that phosphorus is anomalous ("paradoxical" was a stupid word — sorry), consider the following table. It shows the eleven commonest elements in the human body, which match fairly closely with the eight commonest elements in sea water. Carbon (CO2) and Nitrogen (N2) are present in sea water, but such dissolved gases might have been omitted from the list I copied off the 'Net.

% in human body% in seawater
Oxygen65.0%85.7%
Carbon18.5%-
Hydrogen9.5%10.8%
Nitrogen3.2%-
Calcium1.5%0.04%
Phosphorus1.0%0.000006%
Potassium0.4%0.04%
Sulfur0.3%0.09%
Sodium0.2%1.1%
Chlorine0.2%1.9%
Magnesium0.1%0.1%

[

Magnesium is one of the commonest dissolved minerals in seawater; and is also a key ingredient — and at the same 0.1% concentration — in many human proteins. (Magnesium gives plants their green color, and may be higher than 0.1% there, but Googling "human composition by elements " is easier.)

Look at the numbers. Do you agree that life's absolute dependence on Phosphorus is interesting?
 

Elemental Composition of Bacteria (%dry wt basis) | Download Table
The elemental composition of modern bacteria 1 (33 elements). | Download Scientific Diagram

Phosphorus has a big bump in bacteria also, to around the same fraction.


Comparing organisms' compositions to the results of prebiotic-chemistry experiments, one finds a lot of differences, even in the smaller molecules, like amino acids.

A simple sort of difference is which of mirror-image asymmetric pairs: chirality or handedness. A carbon atom that bonds to four different groups will be asymmetric. Its mirror image will be different.

All protein-forming amino acids but one have an asymmetric carbon atom: one where their amino group -NH2 and carboxyl group -COOH are attached. Also attached is a hydrogen atom -H and a side chain -R (the usual abbreviation). The one exception, glycine, has H for R, thus making that carbon atom mirror symmetric.

All our planet's biota has one asymmetry of amino acids, with exceptions like in the cell walls of many bacteria. Prebiotic experiments, however, make both.

Other biomolecules have asymmetric carbon atoms, like nucleic acids. Ribose, in RNA, has 4 of them.

The origin of this asymmetry has been much discussed, with a variety of mechanisms proposed. But most of them, like being struck by electrons from beta decay, are very weak.

So it's most likely a case of one or the other being frozen in some early organism's biochemistry.
 
The asymmetry issue aside, there is the issue that only about 10 of the 20 protein-forming amino acids are plausibly prebiotic.

Reduced alphabet of prebiotic amino acids optimally encodes the conformational space of diverse extant protein folds | BMC Ecology and Evolution | Full Text lists them:

A,D,E,G,I,L,P,S,T,V

From  Proteinogenic amino acid, in order of increasing length of side chain, in number of carbon atoms
  • 0: G Gly glycine
  • 1: A Ala alanine, S Ser serine
  • 2: D Asp aspartate, T Thr threonine
  • 3: E Glu glutamate, P Pro proline, V Val valine
  • 4: I Ile isoleucine, L Leu leucine
The remaining ones are
  • 2: N Asn asparagine
  • 3: Q Gln glutamine
  • 4: K Lys lysine
  • Carbon with three aminos: R Arg arginine
  • Sulfur: C Cys cysteine, M Met methionine
  • Benzene: F Phe phenylalanine, Y Tyr tyrosine
  • Imidazole: H His histidine
  • Indole: W Trp tryptophan

Origin and evolution of the genetic code: the universal enigma

The genetic code is a translation table for nucleotide triplets to amino acids and a "stop" or "terminate" (Ter) signal. There are 64 possible triplets or codons and 21 amino acids and signals, so the code is somewhat degenerate.



That article mentions some other work which proposes order of addition 1 G Gly, 2 A Ala, 3 D Asp, 4 V Val, 5 P Pro, 6 S Ser, 7 E Glu, 8 T Thr, 9 L Leu, 10 R Arg, 11 N Asn, 12 I Ile, 13 Q Gln, 14 H His, 15 K Lys, 16 C Cys, 17 F Phe, 18 Y Tyr, 19 M Met, 20 W Trp

The triplet code from first principles - PubMed
G, A, D, V, P, S, E, (L, T), R, (I, Q, N), H, K, C, F, Y, M, W
 
Which Amino Acids Should Be Used in Prebiotic Chemistry Studies? | SpringerLink - Which_Amino_Acids_Should_Be_Used_in_Preb.pdf

Prebiotic amino acids are racemic mixtures, having all mirror-image variants, whether found in meteorites or made in lab experiments. They typically include most or all of the smaller protein-forming amino acids, but they also include a lot of non-protein amino acids like beta-alanine (COOH and NH2 on opposite ends).


One can make various other biomolecules prebiotically, like nucleobases and porphyrins, and nucleobases have been found in meteorites.

Terpenes, fatty acids, and sugars are more difficult, however.
 
Let's look more generally at early evolution.

By cellular architecture, all cellular organisms fall into two categories: prokaryotes and eukaryotes. The ancestral eukaryote most likely originated as some symbiotic association of prokaryotes, a sort of Frankenstein cell. So it's a long way from the origin of life.

Prokaryotes get much closer. The deepest split in them is between the Bacteria (Eubacteria) and the Archaea (Archaebacteria). It was discovered in the mid 1970's, and no deeper split has since been discovered.

But the Last Universal Common Ancestor had a DNA genome and full-scale transcription and translation systems with a full set of protein-forming amino acids. It likely was autotrophic, making all its biomolecules from simple precursors as a plant does, and its energy source was most likely chemical reactions like combining hydrogen and carbon dioxide to make simple organic molecules like methane and acetic acid.

So it had a lot of evolution behind it. Part of it was making non-prebiotic amino acids for its proteins, and part of it was the origin of proteins and DNA.

DNA is a modification of RNA that has only one known function: carrying master copies of genetic information. RNA has several functions, and DNA building blocks are made from RNA ones. This suggests that RNA was the first nucleic acid and DNA came later.

Proteins are assembled on structures called ribosomes from information carried in messenger RNA, with their amino acids being carried in transfer RNA. Ribosomes' main working parts are their RNA.

RNA can also act as an enzyme, a ribozyme, thus doing the work of protein enzymes.

You might have noticed a pattern: RNA, RNA, RNA, RNA, ... this is what leads to the hypothesis of the RNA world.
 
Cofactors are Remnants of Life’s Origin and Early Evolution | SpringerLink

Mentioning:
  • ATP - adenosine triphosphate (nucleotide with extra phosphates) - transfers phosphates
  • SAM - S-adenosylmethionine (from methionine, an amino acid) - transfers methyl groups
  • NAD - nicotinamide adenine dinucleotide (with niacin, vitamin B3, acting as a nucleobase) - does electron transfer
  • Coenzyme A (CoA, with adenosine and pantothenate, vitamin B5) - transfers acyl (acetic acid) groups
  • FAD - flavin adenine dinucleotide - FMN - flavin mononucleotide (with riboflavin, vitamin B2) - does electron transfer
  • thiamine (vitamin B1), with a pyrimidine part
  • histidine, an amino acid, with an imidazole (half-purine) part
  • porphyrins - in heme, chlorophyll, and vitamin B12
  • pyridoxal - vitamin B6
The remaining  B vitamins are biotin: B7, and folate: B9.

The origin of the genetic code: amino acids as cofactors in an RNA world - PubMed

So some RNA-world organisms ended up making multi-amino-acid cofactors and these cofactors ended up taking over, as proteins.
 
The Origins of the RNA World - although there is strong evidence for the existence of its former existence, its origin is another story.
It might be imagined that all of the components of RNA were available in some prebiotic pool, and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this “RNA-first” view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth.
While nucleobases can be made prebiotically without much trouble, that is very difficult for ribose.
Perhaps these concerns will eventually be resolved, and recent experimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteins were preceded by RNA.
 
Some comments about early evolution.

While attaching amino acids into a polypeptide chain is trivial — a single water molecule is expelled to create each junction — weaving nucleotides together to form RNA is non-trivial: you need a phosphate backbone, and ribose molecules linking the backbone to the nucleotides; ribose is not a sugar that is built easily. For this reason some theorists guess that the earliest life used a simpler chain of nucleotides. (Polyamide nucleic acid, glycol nucleic acid, threose nucleic acid, and peptide nucleic acid have been explored in labs and are mentioned as possibilities; one of these is synthesized naturally by a certain bacteria IIRC.)

But let's ignore that complication and just write 'RNA' to denote the nucleotide chain which was used in the earliest proto-life.

Some hypothesize a 'RNA World' followed only later by a world of RNA and Protein. But a theory which seems more likely to many researchers is that RNA and protein co-evolved. A bath of very short peptide chains and very short nucleotide chains evolved to catalyze the production of other short RNA and protein chains; slightly longer chains emerged and then longer still. In other words the evolution was a "boot-strap" process with proteins of growing complexity helping to synthesize larger RNA's, and vice versa. In this view the Genetic Code would be extremely ancient. The earliest mappings (nucleotide pair or triplet --> amino acid) would rely on the intrinsic structure of those units, but at some point transfer RNA would be "invented" to improve and expand the mappings. (In a similar way, some of the earliest enzymes incorporate a FeS catalytic core which emulated the FeS (iron pyrite) rock, micropores of which provided proto-life's earliest venue.

The ribosome is by far the most complex "machine" present in early life (i.e. LUCA). In addition to the small and large subunits (each composed of RNA and proteins, there are 100+ species of tRNA (even though logically 64 should be "enough"), a large number of specific enzymes needed to attach amino acids to tRNA, and several other proteins for "housekeeping." This unique complexity suggests to me that the Genetic Code (or its predecessors) must be very ancient, which in turn implies that there was never an "RNA World," but rather that RNA and Proteins co-evolved as described above.

Although just a single molecule, in contrast to a ribosome, ATP synthase is another elegant "machine" present in LUCA and essential to all known life.

A final big breakthrough in the evolution leading up to LUCA was the "invention" of DNA. RNA and DNA are very similar molecules, but a very minor difference leads to important functional differences. RNA is very flexible: it can easily fold into the intricate shapes of rRNA and tRNA and mRNA can easily uncoil itself to be read by a ribosome. But this flexibility is also its weakness; to preserve and transcribe a genome reliably, the less flexible DNA is much more suitable. So already by LUCA, both nucleic acids were in use: DNA to preserve a genome reliably with very few mutations, and RNA's flexibility needed on the "factory floor" where proteins are churned out.

Since RNA came first, the chronological order in which four replication mechanisms developed is clear. Here they are, along with the name of the facilitating enzyme:
(1) RNA --> RNA. RNA replicase.
(2) RNA --> DNA. Reverse transcriptase
(3) DNA --> RNA. Transcriptase.
(4) DNA --> DNA. DNA replicase.
Once DNA genome became the standard, mechanisms (1) and (2) became obsolete, and these mechanisms (and their associated enzymes) are no longer present in living organisms. (1) RNA replicase and (2) reverse transcriptase are present in some viruses; these viral enzymes MIGHT have an ancestry tracing back to the counterparts known to exist in pre-LUCA proto-life!

It seems fascinating how RNA "invented" the (RNA,DNA) system. Similarly the earliest life might have used some more primitive nucleic acid (call it PNA), with the genetic code developing at that stage and the (PNA,Protein) system bootstrapping into the (RNA,Protein) world.

The mechanisms for (4) above (DNA replication) are very different between archaeotes and bacteria, suggesting that this mechanism was still evolving when LUCA split into the first two domains of life.

Finally, a comment about the energy source for the earliest life. A HUGE amount of evolution was required to turn the simple assembly of raw ingredients like nucleotides into the huge complexity of the genetic code and other machines in LUCA. Evolution requires huge amounts of growth and reproduction, which in turn require huge amounts of energy.

This raises a "chicken and egg" problem. Photosynthesis is an amazingly complex "machine" which harnesses energy from sunlight VERY efficiently; this energy can drive further growth and evolution. But where did the energy to evolve the machinery of photosynthesis come from? Answer: The energy needed by the very earliest life (or proto-life) could not have come from complex living processes like photosynthesis; it must have been FREE energy.

ATP synthase is a main step in the energy development of all living organisms. That enzyme positions itself between the two sides of a membrane, and harnesses energy when a proton pushes its way through the enzyme to get from one side of the membrane to the other. If one side of the membrane has a positive charge and the other side a negative charge, this is just the same as harnessing energy from a capacitor as it discharges. (A proton H+ is the acid counterpart to the alkali OH- and sometimes it is the acid/alkali gradient that yields energy rather than simple electric gradient.)

Pumping a proton through a membrane seems irrelevant to the mechanism of photosynthesis — for one thing, you have to expend the same amount of energy to move the proton to the other side of the membrane in the first place, just to watch it flow back in. And yet that happens as a part of photosynthesis. And indeed the same thing happens in cellular respiration and every method of energy harnessing used in life today. All these mechanisms are mimicking the energy harnessing of their most ancient ancestors. Those ancient life-forms (including LUCA?) had no way to generate energy themselves (or to push protons against an electric gradient); they were able to operate their ATP synthase (and thereby get the energy needed for growth and reproduction) with protons that were already on the other side of the membrane! These early creatures simply located themselves at the boundary between alkaline water gushing upward from beneath the ocean floor into "white smoker" vents, and the relatively acidic sea water. The boundary between subterranean water and ocean was a rocky (iron pyrite?) vent full of micropores and thin mineral membranes. This boundary had a fractal-like shape, so there were micropores and thin membranes with just the right "Goldilocks" shape for these proto-cells to thrive.
 
Some comments about ATP. It's adenosine triphosphate, a RNA nucleotide with two extra phosphates, with structure
A-P-P-P

Its energy resides in its P-P (pyrophosphate) bonds. A loose phosphate ion is Pi, for inorganic phosphate.

It's assembled with
A-P + Pi -> A-P-P
A-P-P + Pi -> A-P-P-P

It works like this: to assemble X and Y, it does something like
A-P-P-P + X -> A-P-P + X-P
X-P + Y -> X-Y + Pi

ATP energy does mechanical work in similar ways, operating membrane pumps, making flagella and cilia move, and making muscle fibers move relative to each other.


Now to energy metabolism. Where did the first organisms get their energy from? Not from photosynthesis. Its distribution is very patchy, and it looks like it was added on to earlier energy-metabolism systems. I will be discussing what these earlier systems are, systems that many organisms continue to use.
 
There are two major parts of organisms' energy metabolism,  Chemiosmosis and  Electron transport chain

First, chemiosmosis. It is pumping protons (hydrogen ions) across a cell membrane, then letting them back across in ATP-synthase protein complexes, with their return helping to assemble ATP.

In most prokaryotes, the protons are pumped to outside of the cell, something carried over into mitochondria, while in cyanobacteria, the protons are pumped into flattened vesicles called thylakoids, something carried over into chloroplasts. The interiors of thylakoids are topologically equivalent to the outsides of their cells.

There is a suspected connection between chemiosmotic energy metabolism and the origin of life.
An alkaline hydrothermal vent's fluids would have a higher pH than the surrounding ocean, and that pH difference can drive some chemistry. It would eventually be carried over into chemiosmotic energy metabolism.
 
That has the nice feature of suggesting a place to look for organism origination, so one can ask if some planet or moon can have alkaline hydrothermal vents in liquid water. One needs a combination of liquid water and geological activity.

Let's see where one might find such a combination in the Solar System.

Venus might have had this combination very early in its history, but it clearly does not have it in the present day, and it will be hard to search its surface for possible fossils.

Mars likely had this combination early in its history, since there is plenty of evidence of both geological activity and liquid water in the planet's history. But there is very little of both at present, though there might be hydrothermal systems some distance down in the planet's crust.

Some of the outer-planet moons may have both, since some of those moons have icy surfaces and tidal heating, moons like Jupiter's moon Europa. Though that moon's surface is far too cold for liquid water, there is some evidence of a subsurface ocean of liquid water beneath it.

List of outer-Solar-System minor planets and moons with possible subsurface oceans (underlined: strong evidence, bold: confirmed)
  • Jupiter: Europa, Ganymede, Callisto
  • Saturn: Enceladus, Dione, Rhea, Titan
  • Uranus: Titania, Oberon
  • Neptune: Triton
  • (minor planets): Pluto, Eris, Orcus, Sedna
This means that some exoplanets and exomoons may also have subsurface oceans.

Some exoplanets have evidence of oceans, though in the form of average density. Oceans that show up in that way are oceans that are very deep, enough to form high-pressure ice phases. If any organisms originate there, they would likely be stuck on the bottoms of those oceans.
 
I now get to the second part,  Electron transport chain - this works by making a set of chained redox reactions that transmit electrons. Some of the protein complexes that make these reactions also transmit protons to outside of the cell, by consuming them inside the cell and releasing them outside the cell. Likewise for thylakoids, with inside and outside reversed.

In general:

Donor -> dehydrogenase (proton pump) -> quinone
Donor -> quinone -> oxidase (reductase) -> acceptor
Quinone -> bc1 (proton pump) -> cytochrome
Donor -> cytochrome -> oxidase (reductase) -> acceptor

The donors may release protons and the acceptors may accept protons, thus being additional parts of membrane proton pumps.

The first donor may involve NAD, which contains niacin, and the second donor may involve FAD or FMN, which contain riboflavin. Both niacin and riboflavin go back to the RNA world.

-

Donors: organic molecules and a variety of inorganic ones: hydrogen, carbon monoxide, ammonia, nitrite, sulfur, sulfide, manganese oxide, ferrous iron, ...

Acceptors: organic molecules and a variety of inorganic ones: oxygen, nitrate, nitrite, ferric iron, sulfate, carbon dioxide, ...
 
I now get to photosynthesis. In cyanobacteria and chloroplasts, it involves two light-capture protein-pigment complexes, Photosystems I and II, and it is incorporated into the aforementioned electron-transport chain, with the photosystems supplying energy to electrons.

(2H2O -> O2 + 4H+ + 4e)
Photosystem II (photons -> electron energy)
Quinone (proton pump)
Photosystem I (photons -> electron energy)
Electron acceptors like NAD for biosynthesis

Photosystem I and quinone can transfer electrons in a closed-loop cycle, supplying energy without doing biosynthesis.

There are some bacteria with only one of the reaction centers:  Photosynthetic reaction centre They use a variety of electron donors other than oxygen: organic molecules, sulfur, ...

Early Evolution of Photosynthesis

By phylogeny,
  • Terrabacteria:
    • Cyanobacteria: PS I, PS II, Calvin
    • Firmicutes > Clostridia > Heliobacteria: PS I, no C fixation
    • Chloroflexi > (filamentous green nonsulfur): PS II, 3-hydroxypropionate cycle
  • Gracilicutes:
    • Proteobacteria > Alpha- (purple nonsulfur), Gamma- (purple sulfur): PS II, Calvin cycle
    • FCB > Chlorobi (green sulfur): PS I, reverse tricarboxylic cycle
This suggests either lots of losses of photosynthetic capability or else lots of lateral gene transfer -- or both.
 
There is another kind of photosynthesis, one not very well-known. This kind is done by some oddball organisms,  Haloarchaea named after their salt tolerance. Though most closely related to methanogens, these organisms have acquired a large number of genes from organisms in Bacteria, enough to make their metabolism heterotrophic.

These organisms photosynthesize using  Bacteriorhodopsin a protein that contains  Retinal - related to carotenoids and Vitamin A. But their photosynthesis is very limited, pumping protons through the cell membrane as part of their chemiosmotic metabolism.


 Purple Earth hypothesis
The Purple Earth hypothesis is an astrobiological hypothesis that photosynthetic life forms of early Earth were retinal-based rather than chlorophyll-based, making Earth appear purple rather than green.[1][2] An example of retinal-based organisms that exist today are the photosynthetic microbes collectively called Haloarchaea.[3] That time would date somewhere between 2.4 and 3.5 billion years ago, prior to the Great Oxygenation Event.[4] Many Haloarchaea contain the retinal protein, bacteriorhodopsin, in their purple membrane which carries out light-driven proton pumping, generating a proton-motive gradient across the cell membrane and driving ATP synthesis. The haloarchaeal purple membrane constitutes one of the simplest known bioenergetic systems for harvesting light energy.

Autotrophic organisms, like plants, must get their carbon with  Carbon fixation There are six known ways that organisms are known to do that.
The W-L pathway is interesting.

The first part of it uses tetrahydrofolate, a modification of folic acid. CO2 is added to THF, then does reduction, adding electrons and protons, until the added CO2 becomes a methyl group, that is, -CH3

It is then transferred to a cobalamin (vitamin B12) and then combined with carbon monoxide and Coenzyme A to make acetyl-CoA. Carbon monoxide is made by reducing carbon dioxide, and Coenzyme A contains pantothenic acid.

Methanogens do  Methanogenesis which uses the first half of the W-L pathway and does a final reduction of the methyl group to make methane.

Some organisms are "acetogens", doing the W-L pathway, and then removing and excreting the resulting acetic acid.

Both methanogens and acetogens do their metabolism to extract energy from H2 and CO2.

CO2 + 4H2 -> CH4 + 2H2O
2CO2 + 4H2 -> CH3COOH + 2H2O
 
The Emergence and Early Evolution of Biological Carbon-Fixation

The Last Universal Common Ancestor used both the reductive Krebs cycle and the Wood-Ljungdahl pathway for biosynthesis and carbon fixation. It also had the pentose phosphate pathway for biosynthesis, a relative of the Calvin cycle.


The physiology and habitat of the last universal common ancestor | Nature Microbiology

That organism was anaerobic, and it got its energy from combining H2 and CO2. It also fixed nitrogen and it was thermophilic. Its enzymes had lots of iron-sulfur clusters and it had lots of cofactors.

"The 355 phylogenies identify clostridia and methanogens, whose modern lifestyles resemble that of LUCA, as basal among their respective domains. LUCA inhabited a geochemically active environment rich in H2, CO2 and iron. The data support the theory of an autotrophic origin of life involving the Wood–Ljungdahl pathway in a hydrothermal setting."

In effect, acetogens that lived in hydrothermal vents.
 
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