Returning to Darwin's "Warm Little Pond" with the Terrestrial Origin of Life Hypothesis. Presented at the Australasian Astrobiology Meeting, Perth, July 2016. Presented by Dr. Bruce Damer, U.C. Santa Cruz.

1. An Origin of Life in Salt Water or Fresh Water?
Returning to Darwin’s “Warm Little Pond”
-> The Terrestrial Origin of Life Hypothesis
Australasian Astrobiology 2016, Curtin University
Bruce Damer, University of California, Santa Cruz
David Deamer, Martin Van Kranendonk, Malcolm Walter, Tara Djokic

2. “Strong Inference, John Platt in Science-1964
“Strong inference consists of applying the following steps to
every problem in science, formally and explicitly and regularly
1.Devising alternative hypotheses
2.Devising a crucial experiment...with alternative possible
outcomes, each of which will, as nearly as possible, exclude
one or more of the hypotheses
3.Carrying out the experiment so as to get a clean result”

3. Charles Darwin’s Intuition-1871
"But if (and oh what a big if) we could conceive in
some warm little pond with all sorts of ammonia
and phosphoric salts, light, heat, electricity etcetera
present, that a protein compound was chemically
formed, ready to undergo still more complex
changes [..] " ~Charles Darwin, in a letter to Joseph
Hooker (1871)
“...some warm little pond“ or perhaps
“...a hot, fluctuating little pool?”

4. Working Model: Steps to an Origin of Cellular Life
1. There must be a non-enzymatic process by which potentially
functional, catalytic and replicating polymers of sufficient length are
continuously synthesized and re-synthesized within a cycling system
which drives Darwin’s “still more complex changes”.
2. The polymers must also be encapsulated by self-assembly within
membranous compartments to prevent dispersion and allow selective
transport, thereby forming vast numbers of protocells.
3. The protocells represent microscopic experiments in a natural
version of combinatorial chemistry. A rare few happen to contain
systems of polymers that stabilize their encapsulating protocell and
catalyze their own reproduction.
4. These populations of functional systems are then selected by
competition and interaction to be inherited by subsequent generations of
protocells and evolve toward the first cellular life.

5. Platt’s Alternative Hypotheses and Crucial Experiment
Life must have begun in an aqueous medium which implies two
alternatives: salt water (99% of water on early Earth: hydrothermal vents or
tidal pools) or fresh water (distilled water from precipitation: pools in
hydrothermal fields found on volcanic landmasses)
Chemical reactions require concentration and a source of energy ->
eliminates dilute environments (open ocean, lakes, rivers).
Alternative hypothesess:
I. Hydrothermal vents (submarine-salt water)
II. Hydrothermal fields (land surface-fresh water)
Crucial experiment:
1. There must be a non-enzymatic process by which potentially
functional, catalytic and replicating polymers of sufficient length are
continuously synthesized and re-synthesized within a cycling system
which drives Darwin’s “still more complex changes”.

6. I. Hydrothermal vents
Proposed by Corliss, Baross, Hoffman 1980s
Extended by Mike Russell and Bill Martin, 1990s
Laboratory simulations by Herschey et al., 2014
and Burcar et al. 2015
Lost City - an
alkaline
hydrothermal
vent

7. An origin of life at hydrothermal vents?
Vents would have existed on the early Earth
Chemical energy & some chemical inventory available
Microbial life exists today in vent structures
Q: How do hydrothermal vent environments satisfy the Crucial
Experiment (synthesis and open ended complexification of
systems of long chain polymers)?
A: Thermondynamic barriers (hydrolysis) prevent the formation
of biopolymers & salt water prevents the formation of stable
membranous compartments (products are dispersed, no
combinatorial selection). 30 years of laboratory work has so far
failed to achieve this suggesting falsification of this approach.

8. II. Hydrothermal fields
Example: Bumpass Hell, Mount Lassen California
3 interfaces:
mineral/water
mineral/atmosphere
atmosphere/water.
Only mineral/water
in hydrothermal vents.
Collection and concentration
of large molecular inventory:
extraterrestrial infall (lipid,
amino acid, nucleobases,
nutrients) + hydrothermal
sources. Dilution in oceans.
Access to 3 energy sources:
heat activation, dehydration
and sunlight.
Regular cycling to drive
reactions.
Clays 90 C, pH~3
Wet-dry cycles

9. Archaean hydrothermal field

10. “Hot Little Pool” Mt. Mutnovski, Kamchatka
4 amino acids
4 nucleobases (A,U,G,C)
Amphiphilic molecule
(14 carbon fatty acid)
Glycerol
Phosphate
pH 3 (mildly acidic)
Testing the hypothesis in the field
Membraneous structures form.
Polymerization of RNA-like polymers has also been
observed in exposure to hydrothermal field conditions.

11. Simulation chamber: many “hot little fluctuating pools”
CO2 atmosphere (anaerobic)
Elevated temperature 85 C
Acidic pH range (pH 3)
Testing the hypothesis in the lab
-> RNA-like polymers are produced through a few cycles of
4-24 hours. Evidence will be presented next.
Hydration-dehydration cycles
with a range of reageants:
AMP, UMP, lipid, etc.
H2O rehydrationCO2 dehydration

12. Gel elecrophoresis, nanopore sequencing, X-ray diffraction
Gel elecrophoresis
(Rajamani et al. 2008)
Evidence
-> RNA-like polymers of AMP and UMP are produced in the
50-100mer range
Nanopore sequencing (De
Guzman et al. 2014)
Polymer in presence of
different lipids
Polymer product
through cycles
-> Using hydration-dehydration other groups are
polymerizing amino acids producing peptides (Hud et al.,
Cronin et al.).
X-ray diffraction reveals
AMP order in bilayer
(Toppozini et al. 2013)

13. When lipid vesicles are dried in the presence of solute molecules,
the solutes are captured between layers of a multilamellar matrix,
ordered and polymerized through condensation reactions as water
leaves.
How it works – synthesis of polymers
o
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oooo
oo oo
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DRY
FUSE
MULTILAMELLAR
LIPID MATRIX
2 µm 20 nm
Phospholipid vesicles in water Dry multilamellar lipid phase

14. Lipid: decanoic acid+
decanoyl monoglyceride
Monomers: dAMP+TMP
Fluorescent stain: DAPI
When dried lamellae are exposed to rehydration, large numbers of
vesicles “bud off” some containing random polymers. Each provides
a natural experiment in a natural combinatorial selection process.
How it works – encapsulations of polymers
We will now introduce the more
speculative aspects of our work to
satisfy steps 2-4 of the Working
Model.

15. Protocells cycle through three coupled phases
Polymers carried by stable protocells cycle between hydrated (top)
and dehydrated (bottom) phases. If the rate of synthesis exceeds the
rate of hydrolysis a kinetic trap is created permitting continuous re-
synthesis and combinatorial selection of functional polymers. A
growing population of stable protocells causes a third “gel phase”
(center) to form. Micrographs shown to right.

16. Chemical Framework of the Terrestrial Origins of Life Model

17. The three coupled phases cycle polymers through the kinetic trap toward
increasing complexity through continuous synthesis (dry), selection (wet) and
interaction (gel). Competition and cooperation drive molecular evolution to
gradually replace self-assembly processes with the functions of biology.

18. These functions emerge as an evolutionary process that involves the following steps:
•An S-polymer arises by chance that stabilizes protocell membranes allowing them
to survive to return their contents for re-synthesis in the anhydrous phase.
• Protocells acquire P-polymers which enables access to nutrients through pores.
• Metabolism generates products that support replication and systems of R-
polymers that catalyze their own replication. Feedback through F-
polymers provides controls for the rates of the above processes.
• Access to nutrients supports metabolism through catalyzed M-polymers.
Predictions on the Stepwise Emergence of Protocell Functions
• Templating and coding through I-polymers provides inheritance of functional
expression through cycles and initiates Darwinian selection.

19. An Archaean volcanic landmass supports a hydrothermal field giving rise to
a sufficiently robust gel phase progenote (Woese, Fox 1977). Progenote
distribution to additional aqueous environments permits adaptation to a
gradient of conditions leading to the more challenging marine shorelines.
Geological Framework of the Terrestrial Origins of Life Model

20. Downhill distribution and
adaptive radiation…
…ultimately gives rise to LUCA
and marine stromatolites

21. Back to Darwin: from a “little warm pond” to the
“roots of the tree of life”?

22. The Terrestrial Origin of Life Model & Hypotheses
is offered to the community as a alternative
hypothesis and template for experimentation. The
full hypothesis is in publication as of May 2016
(Life)
Commentary, teams and publications, testable
hypotheses, experimental designs, and related
work are being prepared at the Terrestrial Origins
Wiki at:
Origins.Biota.org
Collaborators and Supporters
UC Santa Cruz
Australian Centre for Astrobiology
McMaster University
Center for Chemical Evolution,
Georgia Tech
Cronin Lab, University of Glasgow
Resources and Acknowledgements
University of Paris
Harry Lonsdale Research Award
NASA
DigitalSpace Research
Contact: Bruce Damer bdamer@ucsc.edu

23. New work:
1.Archaean Simulation Chamber (McMaster University)
2.Fieldwork: Bumpass Hell (August 2016)
3.Combining species: oligonucleotides & peptides
4.Conceptual work on the first genetic function: oligomers
of an informational polymer & the “genetic cloud”
5.Definition of “rickety early life” and the candidate
progenote.
6.Communal unit beginnings & extending evolutionary
theory (Odling-Smee/Laland).
7.Scientific American article (Martin et al.)

24. Discussion:
Platt’s strong inference applied to an origin of life at
Hydrothermal Field vs. Hydrothermal Vent Sites
Using analog laboratory and field experiments of hydrothermal
field conditions we are capturing the physical energy of
evaporation and concentration with the consequent reduction of
water activity and changing it into the chemical energy of ester
bond synthesis. We and our colleagues have demonstrated
continuous non-enzymatic synthesis and re-synthesis of
oligonucleotides and oligopeptides in lengths necessary to support
the emergence of functions in a stepwise evolution of populations
of protocells toward a form of pre-life capable of growth and
adaptation
In other words, the terrestrial, fresh water hydrothermal field
hypothesis has satisfied our application of Platt’s crucial
experiment.

25. Discussion: Limitations of Hydrothermal Vent Sites
- Vents lack an effective concentrating mechanism and energy
source to form sufficiently long polymers through diester bonds in
nucleic acids and peptide bonds in proteins. In addition,
polymers degrade through hydrolysis in the continuous presence
of water so no kinetic trap mechanism can operate at these sites
meaning that systems of polymers cannot move away from
equilibrium.
Condensation reaction to form ester bond, water must be a leaving group
Consequence: there is a thermodynamic barrier against the
formation of necessary polymers in the vent setting.

26. Discussion: Limitations of Hydrothermal Vent Sites
- Salt and calcium ions (and additional divalent cations in the ancient oceans)
inhibit assembly of membrane-bounded compartments. Life today has evolved
complex homeostatic mechanisms to regulate cell volume by actively pumping
osmotically active ions and thereby maintain transmembrane sodium and
potassium gradients. This suggests that life adapted to marine
environments later in its evolution (Mulkidjanian et al. 2012, Deamer and
Georgiou 2015).
- Mineral compartments may provide temporary localized concentration but are
not selectively permeable or replicable and therefore cannot be a basis
for combinatorial selection of large numbers of compartmentalized
molecular systems. The necessary cycling through synthesis and testing
required for polymers to undergo selection and molecular evolution is therefore
not possible under these conditions. Any products of chemistry will be trapped
indefinitely with the compartments or eventually lost to dispersion and
degraded through hydrolysis.
- Consequence: without effective compartmentalization there are dispersion
and kinetic barriers to the evolution of systems of functional polymers.

27. Discussion: Limitations of Hydrothermal Vent Sites
Conclusions: these thermodynamic and kinetic barriers would be
present in hydrothermal vent environments on Enceladus (and
Europa) as they are on the Earth. No vent analog experiment has
demonstrated polymerization of sufficiently long polymers to exhibit
function required by biology. No model for a kinetic trap supporting
polymerization and repeated combinatorial selection of
encapsulated systems of polymers has been observed at or
proposed for a vent environment.
In other words, the marine, salt water hydrothermal vent sites
proposed as a hypothesis for an origin of life have yet to satisfy
Platt’s crucial experiment and due to these barriers they may never
be able to satisfy that requirement.
Life as we know it, as it originated on Earth or could have
originated on Mars could not have originated on Enceladus