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Origin of first cells at terrestrial, anoxic
geothermal fields
Armen Y. Mulkidjaniana,b,1, Andrew Yu. Bychkovc, Daria V. Dibrovaa,d, Michael Y. Galperine, and Eugene V. Koonine,1
a
School of Physics, University of Osnabrück, D-49069 Osnabrück, Germany; bA. N. Belozersky Institute of Physico-Chemical Biology and Schools of cGeology
and dBioengineering and Bioinformatics, Moscow State University, Moscow 119992, Russia; and eNational Center for Biotechnology Information, National
Library of Medicine, National Institutes of Health, Bethesda, MD 20894
Edited* by Norman H. Sleep, Stanford University, Stanford, CA, and approved January 17, 2012 (received for review October 28, 2011)
All cells contain much more potassium, phosphate, and transition
metals than modern (or reconstructed primeval) oceans, lakes, or
rivers. Cells maintain ion gradients by using sophisticated, energy-
dependent membrane enzymes (membrane pumps) that are
embedded in elaborate ion-tight membranes. The first cells could
possess neither ion-tight membranes nor membrane pumps, so the
concentrations of small inorganic molecules and ions within proto-
cells and in their environment would equilibrate. Hence, the ion
composition of modern cells might reflect the inorganic ion
composition of the habitats of protocells. We attempted to re-
construct the “hatcheries” of the first cells by combining geochem-
ical analysis with phylogenomic scrutiny of the inorganic ion
requirements of universal components of modern cells. These ubiq-
uitous, and by inference primordial, proteins and functional systems
show affinity to and functional requirement for K+, Zn2+, Mn2+, and
phosphate. Thus, protocells must have evolved in habitats with
a high K+/Na+ ratio and relatively high concentrations of Zn, Mn,
and phosphorous compounds. Geochemical reconstruction shows
that the ionic composition conducive to the origin of cells could not
have existed in marine settings but is compatible with emissions of
vapor-dominated zones of inland geothermal systems. Under the
anoxic, CO2-dominated primordial atmosphere, the chemistry of
basins at geothermal fields would resemble the internal milieu of
modern cells. The precellular stages of evolution might have tran-
spired in shallow ponds of condensed and cooled geothermal vapor
that were lined with porous silicate minerals mixed with metal sul-
fides and enriched in K+, Zn2+, and phosphorous compounds.
prebiotic chemistry abiotic photosynthesis
origin of life Na+/K+ gradient
|
|
| hydrothermal alteration |
he utility of the geological record for reconstruction of the
habitats of the earliest life forms is limited. Because of the
heavy impact bombardment, the Earth surface underwent major
changes approximately 3.8 to 3.9 Gigayears (Gyr) ago, so that
only few rock samples are older than 4.0 Gyr (1, 2). Diverse
recent data indicate that life might be older than the oldest
known rocks (2). If life originated in the Hadean, finding any
geological traces of the first life forms is unlikely.
In 1926, Archibald Macallum noted that, although similarities
between seawater and organismal fluids, such as blood and
lymph, indicate that the first animals emerged in the sea, the
inorganic composition of the cell cytosol dramatically differs
from that of modern sea water (3). Macallum insightfully pointed
out that “the cell. . . has endowments transmitted from a past
almost as remote as the origin of life on earth.” Thus, in our
inference of the features of the primordial organisms and their
environment, we are left with the biological record which, given
the evolutionary continuity, is as old as life itself. The ideas of
Macallum (3) can be generalized in a “chemistry conservation
principle” (4): the chemical traits of organisms are more con-
servative than the changing environment and hence retain in-
formation about ancient environmental conditions. Chemistry
conservation is manifest, for example, in the highly reduced state
of the cell interior even in those organisms that dwell in oxy-
www.pnas.org/cgi/doi/10.1073/pnas.1117774109T
genated habitats (4). The reduced state of the cytoplasm indi-
cates that the major biochemical pathways were fixed before the
atmosphere became oxygenated as a result of the activity of
cyanobacteria approximately 2.4 Gyr ago (5), so that substantial
modification of these pathways in response to the oxygenation of
the atmosphere was impossible. Instead, cellular life forms have
evolved numerous energy-requiring membrane transport systems
to sustain redox and (electro)chemical gradients between their
interior and the environment.
It stands to reason that simultaneous consideration of various
boundary conditions has the potential to eliminate most of the
vast number of scenarios for the early evolution of life that ap-
pear possible in principle (4). Under this premise, we have
previously addressed diverse facets of the early life problem from
the viewpoint of photochemistry (6), comparative genomics (7–
9), and energetics (10, 11). The principle of chemistry conser-
vation can be used as an additional major constraint for recon-
structing primordial environmental conditions in the absence of
reliable geological record. For example, ancient, ubiquitous
proteins often use Zn and Mn, but not Fe, as transition metal
cofactors; this preference is retained across the three domains of
life (12). The abundance of Zn- and Mn-dependent enzymes
during the earliest steps of evolution and the later recruitment
of Fe has been inferred also from a global phylogenomic re-
construction (13). The prevalence of Zn-dependent ancestral
enzymes is particularly remarkable given the low estimated
concentration of Zn in the anoxic ocean of 10−12 to 10−16 M (14,
15) and indicates that the first organisms might have dwelled in
specific, Zn-enriched habitats (12, 16).
Here we combine geochemical evidence with the data on the
overall ionic composition of the modern cells, with a particular
emphasis on their universal preference for K+ ions over Na+
ions. Geochemical analysis shows that, contrary to the common
belief that associates the origin of life with marine environments,
the first cells could have emerged at inland geothermal fields
within ponds of condensed and cooled geothermal vapor. Con-
ceptually, this scenario of early evolution resembles Darwin’s
“warm little pond” vision (17).† Under this scenario, the ocean
Author contributions: A.Y.M. designed research; A.Y.M., A.Y.B., D.V.D., M.Y.G., and E.V.K.
performed research; A.Y.M., A.Y.B., and E.V.K. contributed new reagents/analytic tools;
A.Y.M., A.Y.B., D.V.D., M.Y.G., and E.V.K. analyzed data; and A.Y.M., A.Y.B., D.V.D., M.Y.G.,
and E.V.K. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
*This Direct Submission article had a prearranged editor.
1
To whom correspondence may be addressed. E-mail: amulkid@uos.de or koonin@ncbi.
nlm.nih.gov.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1117774109/-/DCSupplemental.
†
“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 &c. present, that a protein com-
pound was chemically formed, ready to undergo still more complex changes. . ..” —from
Darwin’s 1871 letter to Joseph Hooker (17).
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EVOLUTION
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was invaded by life at a later stage, following the emergence of
ion-tight phospholipid membranes.
Results and Discussion
Inorganic Ion Requirements of Ubiquitous Cellular Systems. The total
intracellular content of an ion reflects the ability of the cell to
accumulate this ion against the concentration gradient. In par-
ticular, Table 1 shows that concentrations of K+, Zn2+, phos-
phate, and several other inorganic ions in all cells are orders of
magnitude higher than the levels of these ions in modern sea
water, as well as in the primordial, anoxic ocean. Conversely, the
content of Na+ ions in the cells is much lower than it is in the sea
water. Many halophiles that can tolerate high external levels of
NaCl increase the internal K+ concentration up to approxi-
mately 1.0 M, to keep the internal K+/Na+ ratio high (18).
Apparently, it is not so much the actual concentrations of K+
and Na+ but the K+/Na+ ratio of at least 1 that is critical for the
proper functioning of the cell.
Modern cells can maintain the ionic disequilibria because their
membranes are ion-tight and contain a plethora of membrane-
embedded, energy-dependent ion-translocating protein com-
plexes (i.e., ion pumps). Accordingly, cells invest large amounts of
energy into sustaining the respective ion gradients. For example,
neurons, even in the resting state, use approximately 20% of their
ATP to maintain the K+/Na+ gradient across the membrane (19).
Under the chemistry conservation principle, the striking dif-
ference between the intracellular inorganic chemistry and the
composition of sea water suggests that the first cellular organisms
dwelled in specific habitats that were enriched for the elements
that are prevalent in modern cells (3, 4, 12, 16, 20). A potential
alternative to this explanation is that the chemical differences
between the intracellular milieu and the environment are un-
related to the conditions under which the first cells evolved (21).
Then, the dramatic enrichment of modern cells for K+, Zn2+,
and phosphate could be viewed as a relatively late shift that
came after the emergence of powerful ion-translocating mem-
brane pumps and was driven by the growing demand of the
newly evolving enzymes for particular inorganic ions as catalysts
or substrates.
To distinguish between these two explanations, we turned to
the proteins that are shared by (nearly) all cellular organisms
with sequenced genomes and by inference originate from the so-
called last universal cellular (or common) ancestor (LUCA) or
an even earlier stage of evolution (7, 22–27). The ion preferences
of the ubiquitous, ancient proteins are expected to provide in-
formation about the habitats of the first cells. Indeed, the ion-
tight membranes of modern cells are extremely complex energy
conversion and transport systems that obviously are products of
long evolution and could not possibly exist in the first protocells.
According to the available reconstructions, the first lipids were
simple and single-tailed (28–31). The experiments with such
lipids compounds have shown that vesicles made of fatty acids
(28, 32) or of phosphorylated isoprenoids (33) can reliably en-
trap polynucleotides and proteins. Such membranes, however,
are leaky to small molecules (30, 32). Hence, the membranes of
first cells probably could occlude biological polymers and even
facilitate their transmembrane translocation but could not pre-
vent (almost) free exchange of small molecules and ions with the
environment. Furthermore, before the emergence of diverse
membrane translocators, the exchange of small molecules via
leaky membranes should have been of vital importance for the
first cells, which also implies that their interior was equilibrated
with the surroundings, at least with respect to small molecules
and ions (30, 32, 34–38).
SI Appendix, Table S1, lists the ion requirements and affinities
of the ubiquitous proteins that represent the heritage of the
LUCA and probably of protocells (7, 27). Besides the preference
for Zn and Mn, which has been discussed previously (12, 16),
several proteins and functional systems that can be traced back
to the LUCA—and probably beyond—require K+, whereas none
of the surveyed ancestral proteins specifically requires Na+. The
majority of the (nearly) universal proteins that can be confidently
traced to the LUCA are involved in translation, which is potas-
sium-dependent both in bacteria (39) and in archaea (40, 41).
Potassium seems to be required for at least two essential ribo-
somal reactions. First, K+ ions are needed for the peptidyl
transferase center to assume its functional conformation (42).
Second, our sequence and structure comparisons indicate that
the key translation factors are K+-dependent GTPases (SI Ap-
pendix, Figs. S1–S4 and Table S2 provide further details).
Phylogenetic analysis of GTPases shows that extensive di-
versification of GTPase domains antedated the LUCA (43). The
K+-binding sites are highly conserved in diverse GTPases, in-
dicating that they were already present in the primordial GTPase
domains (SI Appendix). Perhaps even more telling are recon-
structions showing that the peptidyl transferase center is the core,
ancestral part of the ribosome (44, 45). Thus, the K+-dependent
components of the translation system appear to stem from the
protocell (or even earlier) stage of evolution. Apparently, the
dominance of K+ over Na+ in modern cells, which is reverse to
the case in sea water, was important also for the protocells.
The concentration of phosphate in the cytosol is at least four
orders of magnitude greater than in the sea water (Table 1). Not
surprisingly, the energetics of the protocells, which can be
inferred from the inspection of the ubiquitous protein set, must
have been based on phosphate transfer reactions and specifically
Table 1. Approximate concentrations of key ions in various environments
Ion, mol/L
Na
K+
Ca2+
Mg2+
Fe
Mn2+
Zn2+
Cu
Cl−
PO43−
+
Modern sea water
0.4
0.01
0.01
0.05
10−8 (mostly Fe3+)
10−8
10−9
10−9 (Cu2+)
0.5
10−6 to 10−9
Anoxic water of primordial ocean
>0.4
∼0.01
∼0.01
∼0.01
10−5
10−6 to 10−8
<10−12
<10−20 (Cu1+)
>0.1
<10−5
Cell cytoplasm
0.01
0.1
0.001
0.01
10−3 to 10−4
10−6
−3
10 to 10−4
10−5
0.1
∼10−2 (mostly bound)
The intracellular concentration is defined here as the total content of a particular element divided by the cell
volume and should be discriminated from the much lower free ion concentration, which does not account for the
ions that are bound to biological molecules. The reconstructed chemical composition of the anoxic ocean
includes data from refs. 14, 15, 58, 141. The data on intracellular concentrations of different chemical elements
are based on refs. 14, 142–145.
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on hydrolysis of nucleoside triphosphates (SI Appendix, Table
S1). That phosphate-based metabolism is ancestral in cellular life
follows also from the results of the recent global phylogenomic
analysis (13). Given that the backbones of nucleic acids contain
phosphate groups, there is no doubt that phosphate was a central
component of life from its inception.
However, the concentration of phosphate ions in natural
aqueous systems, such as lakes or seas, could never be as high as
it is inside cells because of the poor solubility of Ca and Mg
phosphates. Thus, although the requirement for a high phos-
phate concentration in the protocells is indisputable, it remains
unclear how the protocells could accumulate phosphate without
tight membranes and phosphate-scavenging pumps. It has been
argued that more reduced phosphorous compounds such as
hypophosphite (PO23−) and/or phosphite (PO33−), which are ap-
proximately 1,000 times more soluble than phosphate, could have
been abundant under primordial reduced conditions (46–49).
Hence a major conundrum:
a) Intracellular concentrations of key ions, in particular K+,
Zn2+, and phosphate, are several orders of magnitude higher
compared with sea water, both extant and that of Hadean
ocean (according to the available reconstruction; Table 1);
b) (Nearly) universal, and by inference primordial, proteins and
functional systems show affinity to and functional requirement
for K+, Mg2+, Zn2+, Mn2+, and phosphate, but not Na+ (SI
Appendix, Table S1); and
c) It is extremely unlikely that protocells possessed ion-tight
membranes with built-in ion pumps.
Given these observations and inferences, it appears most likely
that protocells evolved in habitats characterized by a high K+/
Na+ ratio and relatively high concentrations of Zn2+, Mn2+ and
phosphorous compounds.
Vapor-Dominated Zones of Terrestrial Geothermal Systems as
Possible Hatcheries of First Cells. Is it possible to envision any
Meteoric
water
Cl- and Na+ enriched
thermal waters
natural habitats with high levels of transition metals and phos-
phorous compounds, as well as a K+/Na+ ratio substantially
greater than 1?
As argued previously (10–12), high concentrations of transi-
tion metals, such as Zn and Mn, are found only where extremely
hot hydrothermal fluids leach metal ions from the crust and bring
them to the surface. Such thermal systems operate either on the
sea floor (50, 51), or at sites of continental (i.e., terrestrial)
geothermal activity where the metal ions are carried not only by
hot fluids, but also by steam (52, 53).
Phosphate concentrations are low both in the sea water (Table
1) and in the fluids of the deep sea hydrothermal vents (∼0.5
μM) (50). The content of phosphorous compounds is higher in
terrestrial thermal springs, where it varies within a broad range,
reaching 60 to 70 μM in some Yellowstone springs (54) and as
much as 1 mM in the acidic mud pots of Kamchatka (55). In an
attempt to discriminate phosphite from phosphate in field sam-
ples, Pech et al. have found comparable amounts of phosphate
and phosphite in a pristine geothermal pool at Hot Creek Gorge
near Mammoth Lakes, CA, which is fed by hot, bicarbonate-rich
geothermal waters (56). The discovery of highly soluble phos-
phite in a modern geothermal pool can at least partly account for
high amounts of phosphorus in the discharges of terrestrial
geothermal systems. Furthermore, this finding could explain why
diverse prokaryotes possess systems of hypophosphite and phos-
phite oxidation (57).
The high K+/Na+ ratio should be taken as the key search
criterion because accumulation of transitional metals or phos-
phorous compounds is conceivable in primordial evaporating
water basins; evaporation, however, cannot affect the K+/Na+
ratio. No marine environment with a K+/Na+ ratio greater than
Mulkidjanian et al.
Vapor
dominated
zone
Cl -
Liquid
dominated
zone
Leaching of metals
from the rock by
hot fluids
Meteoric
water
Magma
chamber
Fig. 1. A terrestrial geothermal system (scheme based on refs. 52, 53, 62,
138) that is fed mostly by water from rain and snow (meteoric water) which,
when it is deep underground, mixes with cation- and anion-enriched mag-
matic fluids and becomes heated to 300 to 500 °C; such hot fluids can leach
diverse ions from the hot rock. Upon heating, the water becomes lighter
and, being enriched in metal cations and such anions as Cl−, HS−, and CO32−,
ascends toward the surface. At shallower depths, the rising hot water starts
to boil because of lower pressure. The vapor phase usually separates from
the liquid phase, which leads to the typical zoning (53, 62). The separation is
not only physical but also chemical; e.g., whereas Cl− anions mostly stay in
the liquid phase, the gaseous compounds, such as CO2, NH3, and H2S, re-
distribute into vapor. The flow route of the liquid phase and the exact point
of its discharge are determined by the crevices within the rock; the ejected
fluids are characterized by slightly alkaline pH and high content of chloride
and sodium, which both can be traced to the contribution of magmatic
waters. The vapor rises upward and spreads within the rock; the subsurface
area that is filled by steam and gas is called the vapor-dominated zone. Part
of the steam condenses near the surface and is ejected by the thermal
springs, and the rest of the steam reaches the surface through fissures of
the rock to form fumaroles (i.e., steam vents). Metal cations are carried both
by the liquid and by the vapor phases (52, 53), although the K+/Na+ ratio is
higher in the vapor phase (Table 2).
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EARTH, ATMOSPHERIC,
ANDPLANETARYSCIENCES
H2S, NH3, CO2 and K+-enriched
exhalations
EVOLUTION
1 has ever been described or reconstructed to our knowledge. In
trapped samples of Archaean seawater, the K+/Na+ ratio is
approximately 0.025 and is similar to that in modern oceans (58).
Arguably, this low K+/Na+ ratio was established in the ocean
shortly after its formation, when it was still too hot to be com-
patible with life (2, 58). The K+/Na+ ratio is similarly low in
hydrothermal fluids of marine hot vents because these vents are
fed predominantly by sea water (50).
Terrestrial aqueous systems, which are mostly fed by water
from rain and snow, are more variable with respect to the K+/
Na+ ratios. Generally, the concentrations of K+ and Na+ ions in
rivers and lakes are much less than 1 mM, and the K+/Na+ ratio
is in the range of 0.1 to 1.0, although in streams that interact with
potassium-rich igneous rocks, this ratio can reach 2 or 3 (59, 60).
At sites of inland geothermal activity, the levels of K+ and Na+
are higher as a result of extensive leaching of metals from rocks
by hot, carbonate-enriched waters, and the K+/Na+ ratio varies
within a broad range (54, 55) owing to the intrinsic heterogeneity
of such systems. The heterogeneity is a result of the boiling of the
ascending hot hydrothermal fluids at shallower depths followed
by separation of the vapor phase from the liquid phase (Fig. 1).
Upon separation, gaseous compounds, such as H2S, CO2, and
NH3, redistribute into vapor that rises upward toward the sur-
face. The subsurface area in which steam and gas prevail in open
fractures is called the vapor-dominated zone (Fig. 1). The ex-
halations from vapor-dominated zones, which are enriched in
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H2S, CO2, NH3 and metal cations, discharge as steam (i.e.,
fumaroles) or, after condensation, as mud pots (SI Appendix, Fig.
S5) because of the silica that is also carried by the vapor (52–55,
61, 62). Numerous fumaroles and mud pots overlaying a vapor-
dominated zone make a geothermal field.
The emissions from the vapor-dominated zones of inland
geothermal systems are K+-enriched, unlike the discharges from
the liquid-dominated zones, which contain much more Na+ than
K+ (54, 55). To our knowledge, the causes of this enrichment
have not been explicitly addressed. Comparison of the concen-
trations of some essential elements in the fluids of thermal
springs and in the vapor of the same springs (Table 2 shows data
from Kamchatka volcanic system) sheds light on the probable
mechanisms of K+ enrichment. As follows from the data in
Table 2, the K+/Na+ ratio is, on average, higher in the vapor
condensate than in the liquid. A similar dependence can be
inferred from data on the two largest vapor-dominated geo-
thermal fields of modern Earth: at the Larderello geothermal
field in Italy, the K+/Na+ ratio reached 32 in the steam con-
densate (63), whereas the steam condensates at The Geysers
geothermal field in California showed a K+/Na+ ratio as high as
75 (64). Thus, the high K+/Na+ ratios in the exhalations from the
vapor-dominated zones of inland hydrothermal systems could be
a result of the higher volatility of K+ ions within the vapor phase;
the larger K+ ions are expected to more readily form complexes
with such molecules as H2O, H2S, or CO2 and anions.
Thus, among the well characterized environments on Earth,
only emissions from vapor-dominated zones of inland geothermal
systems simultaneously show K+/Na+ ratios much greater than 1,
a high content of transition metals, and substantial levels of phos-
phorous compounds (Table 2) (55, 62, 63, 65). Although terres-
trial geothermal systems have been occasionally suggested as
potential habitats of the early life (37, 61, 66), the unique role of
their vapor-dominated zones as natural chemical separators, to
our knowledge, has not been specifically addressed. The principal
reason why the vapor-dominated fields were not considered as
suitable hatcheries for the protocells is that the fluids at such
fields are highly acidic [with pH values reaching −0.5 (54, 55);
Table 2] and hence inhospitable to life. However, acidity appears
to be a characteristic of modern geothermal fields but not the
primordial ones. Indeed, the ascending vapor carries large
amounts of hydrogen sulfide, which, when it reaches the surface,
is oxidized by atmospheric oxygen to strong sulfuric acid. In
the absence of oxygen on the primordial Earth, the geochemistry
of vapor-dominated geothermal fields should have been quite
different:
a) The pH of the discharges from the vapor-dominated zones
should have been closer to neutral because both H2S and CO2,
which ascend with the vapor, are weak acids, and their acidity is
usually compensated by the interaction with basic rocks;
b) At neutral pH, silica would precipitate at the outlets of ther-
mal springs and around them not as amorphous kaolinite/
mud, as it does now (61), but as porous, ordered silicate min-
erals. Thus, the formation of clays such as smectite/montmoril-
lonite and illite, as well as zeolites such as laumontite and
natrolite, should be expected;
c) In the absence of oxygen, sulfide ions would cause precipita-
tion of metal sulfides, as is the case at modern deep-sea hy-
drothermal systems, where slowly precipitating ZnS particles
form halos around the vent throats which are built of fast-
precipitating sulfides of iron and copper (50, 51). At ancient
geothermal fields, because of the high silica content in the
Table 2. Concentration of some essential elements in the water of thermal springs and in the
condensate of the same springs
Spring Number
Element
t, °C
pH
B
Ca
Fe
K
Mg
Mn
Na
Ni
P
Ti
Zn
pH
B
Ca
Fe
K
Mg
Mn
Na
Ni
P
Ti
Zn
S6–14
94.00
0.50
95,109
279,893
384,075
89,606
168,491
7,355
128,609
140
7,399
9,170
657
2.29
2,635.0
566.7
760.4
15,787.2
141.0
9.0
5,427.1
16.2
18.0
18.7
19.0
S6–15
S6–16
S6–17
S6–18
S6–19
96.00
−0.30
133,910
168,640
250,982
155,190
98,071
4,325
121,597
67
9,163
7,874
439
2.03
4,295.5
288.9
99.4
8,398.6
24.5
2.3
3,082.5
0.7
4.3
4.1
10.8
Water composition, parts per billion
93.00
89.00
93.00
96.00
−0.28
0.25
−0.58
−0.09
54,142
35,927
72,639
83,813
121,911
455,703
213,657
334,430
174,308
245,163
258,688
446,416
138,879
22,881
882,720
86,835
68,883
118,968
78,648
202,059
2,909
3,358
3,942
9,424
100,599
79,224
479,027
143,699
89
82
96
593
8,615
6,434
33,689
7,568
2,345
2,300
3,106
8,533
324
734
471
830
Condensate composition, parts per billion
2.19
2.54
2.03
1.05
84.4
1,092.3
184.6
214.6
219.2
424.4
30.0
90.0
216.3
798.5
10.7
154.6
45.5
2,317.2
22.6
37.6
48.7
138.9
2.5
15.5
2.3
7.0
0.1
1.9
127.8
797.6
14.9
50.7
0.4
9.2
0.2
1.3
5.2
11.8
2.0
6.6
16.6
8.3
0.5
2.6
3.4
12.8
6.0
6.9
For Mutnovsky volcano, Kamchatka peninsula, see Methods and refs. 62, 95.
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exhalations of the vapor-dominated systems, the formation of
metal-sulfide–contaminated clays and zeolites rather than pure
metal-sulfide precipitates should be expected.
It is generally believed that the primordial atmosphere was
CO2-dominated and that the atmospheric pressure was higher
than it is now (67, 68). Both these factors would boost the
transportation of diverse ions by the ascending vapor. The high
CO2 concentration would enhance the leaching from the rock by
carbonate ions, whereas the high atmospheric pressure would
bring the boiling isotherm (Fig. 1) closer to the surface, shorten
the distance that had to be covered by the ascending vapor, and
thereby increase the amount of transported inorganic ions.
In summary, the operation of geothermal systems under an-
oxic, CO2-dominated atmosphere would result in vigorous dis-
charge of neutral geothermal fluids and steam from their vapor-
dominated zones; the discharges would have a K+/Na+ ratio
greater than 1 and would be enriched in NH3, H2S, CO2, phos-
phorous compounds, and transition metals. These terrestrial
geothermal fields appear to provide the best environment on the
primordial Earth for the origin of protocells.
Evolution of Protocells at Anoxic Geothermal Fields. Fig. 2 shows
a scenario for the origin of protocells at anoxic geothermal fields
overlaying the vapor-dominated zone of a primordial geothermal
system (as detailed in the legend to Fig. 2). Such systems should
have been typical of the first Earth continent(s) that are believed
to have formed from Mg-, K-rich ultramafic rocks (2, 69). The
analysis of the 4.02- to 4.19-Gyr–old inclusion-bearing zircons
indicates an early presence of subduction zones and, hence, the
overlying geothermal fields (70). In the absence of oxygen, the
transition metals would precipitate mostly as sulfides. While ZnS
and MnS precipitate slowly, Cu2S, PbS, and FeS2 are promptly
Inflow of
abiotically
synthesized
substrates
Zone of abiotic
photosynthesis
Abiotic
condensation reactions,
the catchment area
A
Ponds of condensed vapor
B
UV-protected habitat of protocells
Fig. 2. Evolution of protocells at a primordial anoxic geothermal field. (A)
Anoxic geothermal field over a terrestrial geothermal system; the figure
corresponds to the boxed section in Fig. 1. A primordial geothermal system
could form over a “hot spot,” similar to modern Island (139) or a primitive
subduction zone (52, 69, 70, 140). The cooling of the ascending, H2S-enriched
vapor causes precipitation of metal sulfides, particularly pyrite, which starts
beyond the surface. At the point of water/vapor discharge, H2S starts to
escape into the atmosphere, thus increasing the pH of the discharging fluids.
By analogy with modern geothermal fields, the geothermal fluids and
condensed vapor are expected to run down the slope, cool down and loose
transition metals through sulfide precipitation. At neutral pH, Cu2S, PbS, and
FeS2, shown by dark colors, should have precipitated first (71–73), leaving
Mn and Zn ions in the liquid phase. The relief depressions gave rise to lakes,
ponds or puddles; at a certain distance from the thermal springs, after the
cooling of geothermal fluids and the fall-out of Cu2S, PbS, and FeS2, these
basins should have became particularly enriched in Zn2+ and Mn2+ ions, with
their beds covered by ZnS and MnS-containing silicate minerals (shown by
yellow color). (B) An anoxic geothermal pond as a sink for diverse (organic)
substrates delivered by geothermal fluids and abiotically (photo)synthesized
at minerals. These substrates could be consumed by protocells that are
shown dwelling in the deeper, UV protected layers of the pond bed, within
inorganic compartments build of silica minerals and metal sulfide particles.
removed by precipitation at neutral pH and at temperatures
lower than 300 °C (71–73). Therefore, Cu2S, PbS, and FeS2 could
not spread far away from points of discharge, especially taking
into account the cooling of the geothermal fluids to the ambient
temperatures. In addition, Zn is much more volatile than Fe, as
could be judged from the analyses of geothermal springs (Table
2) and volcanic vapor (74). Hence, far-off ponds and puddles, fed
by cooled geothermal fluids and condensed vapor, would have
been particularly enriched in slowly precipitating Zn2+ and
Mn2+ ions, with their beds covered by clays and zeolites con-
taminated by sulfides and carbonates of Zn and Mn (Fig. 2A).
We hypothesize that such loose, Zn- and Mn-enriched sedi-
ments served as the cradles for protocells (Fig. 2B). The affinity
of many ubiquitous proteins for Zn2+ and, to a lesser extent,
Mn2+ (SI Appendix, Table S1) implies that these proteins might
have evolved in such environments.
The absence of any enzymes related to autotrophy in the
ubiquitous protein set (SI Appendix, Table S1) suggests that the
protocells were heterotrophs, i.e., their growth depended on
the supply of abiotically produced organic compounds (32, 75–
77). At least two continuous, abiotic sources of such compounds
would exist in the described geothermal systems. First, even in
modern vapor-dominated geothermal systems, exhalations carry
organic molecules that are believed to be formed, at least partly,
in the process of hydrothermal alteration of ultramafic rocks (78,
79). Hydrothermal alteration occurs when iron-containing rocks
interact with water at temperatures of approximately 300 °C,
which is typical of terrestrial geothermal systems. Under these
conditions, part of the Fe2+ in the rock is oxidized to Fe3+,
yielding magnetite (Fe3O4). The electrons released through this
reaction are accepted by protons of water yielding H2; in the
presence of water-dissolved CO2, diverse hydrocarbons are ul-
timately produced (78). It could be argued that the hydrothermal
rock alteration might also account for the reduction of insoluble
apatite to soluble phosphite (47), explaining the presence of
phosphite in the geothermal fluids (56). Similar reactions could
lead to the ammonia formation (80), which might account for the
high ammonia content in the exhalations of geothermal fields [as
much as 130 mg/L in the mud pot solutions of Kamchatka (55)].
In addition, diverse organic molecules could be produced by
abiotic photosynthesis catalyzed by ZnS and MnS particles (81–
84). Such crystals are semiconductors, which can trap quanta
with a λ of less than 320 nm and transiently store their energy in
a form of charge-separated states, capable of reducing diverse
compounds at the surface (81). Thereby, crystals of ‡ZnS are the
most powerful photocatalysts known in nature (10). Particles of
ZnS can catalyze photopolymerization reactions (85) and pho-
toreduce carbonaceous compounds to diverse organic molecules,
including intermediates of the tricarboxylic acid cycle (83, 84);
the highest quantum yield of 80% was observed upon reduction
of CO2 to formate (81).
Generally, two types of environments relevant for the early
stages of evolution can be discriminated at primordial geo-
thermal fields: (i) periodically wetted and illuminated mineral
surfaces that could serve as templates and catalysts for diverse
abiotic syntheses and (ii) geothermal pools that could serve as
hatcheries of first replicating life forms (Fig. 2). At mineral
surfaces of primordial geothermal fields, ammonia, sulfide,
phosphite, and phosphate ions would react with carbonaceous
compounds, yielding aminated, sulfurated, and phosphorylated
molecules (48, 49), which could provide nourishment and fuel
‡
ZnS, broadly known as phosphor (from “phosphorescence”), shows a unique ability to
convert diverse kinds of energy, including that of light quanta, X-rays, electrons (as in
displays), α-particles (ZnS was introduced as the first inorganic scintillator by Sir William
Crookes in 1903), into (electro)chemical energy of separated electric charges (reviewed in
ref. 10).
Mulkidjanian et al.
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ANDPLANETARYSCIENCES
EVOLUTION
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for the protocells within the geothermal ponds. Each such pool
would “harvest,” with the help of geothermal streams and rain
water, substrates from its catchment area. Only water-soluble
compounds or compounds that could be carried by water (e.g., as
micelles of amphiphilic molecules) could reach such ponds. This
harvesting mechanism essentially excludes the interference of
“tar,” which would inevitably form under conditions of abiotic
syntheses (4), with the chemistry within geothermal ponds.
In the absence of an ozone shield, the protocells would need
protection from the UV component of solar light (86). Both ZnS
and MnS crystals efficiently scavenge UV up to approximately
320 nm (81, 87). The molar absorption coefficient of ZnS par-
ticles is approximately 2 mM cm−1 at 260 nm, at which nucleo-
tides absorb (88). It is easy to estimate that a thin, 5-μm layer of
ZnS would attenuate the UV light by a factor of 1010. Thus, even
conservatively assuming a 90% porosity of ZnS-containing
sediments and a 1% ZnS content in the sediments, a 5-mm layer
of ZnS-containing precipitates would give the same UV pro-
tection as a greater than 100 m water column (cf. ref. 86). This is
a low bound estimate because other mineral constituents of si-
liceous sediments would also absorb UV and protect the pri-
mordial life forms (89). Hence, a stratified system could be
established within geothermal ponds, where the illuminated
upper layers would be involved in the “harvesting” and pro-
duction of reduced organic compounds, whereas the deeper, less
productive but better protected layers could provide shelter for
the protocells (Fig. 2B). The porosity of the silica minerals would
enable metabolite transport between the layers. Both the light
gradient and the interlayer metabolite exchange are typical of
modern stratified phototrophic microbial communities (90).
Thus, Hadean anoxic geothermal fields would provide:
(a) Water basins with ionic composition compatible with that of
modern cells, meeting the chemistry conservation criterion;
(b) A supply of organic molecules that could fuel biosynthetic
reactions;
(c) Abundant, efficient, and versatile (photo)catalysts, above all
ZnS and Zn2+ ions;
(d) Microcompartments within porous, siliceous ZnS- and MnS-
containing masses.
The proposed scenario is robust because its critical parame-
ters, such as the K+/Na+ ratio greater than 1 and the continuous
supply of reduced compounds, are sustained by multiple com-
plementary mechanisms. In particular, the high K+ levels and the
K+/Na+ ratio greater than 1 would have been maintained by the
K-enrichment of the primordial igneous rocks (2), by the higher
mobility of K+ ions in the vapor phase (Table 2), and by ability of
2:1 clay minerals, such as smectites and illite, to select potassium
over sodium (91). The only vital parameter for the model is the
absence of atmospheric oxygen, which is not disputed when it
comes to the first eons of Earth history (5, 67).
Furthermore, geothermal fields have autonomous heat sour-
ces and good thermal isolation provided by the air, so the tem-
perature and chemical composition of water basins in these
habitats are defined primarily by the geothermal activity and are
effectively independent of the climate, potentially allowing pro-
tocells to endure climate changes or even periods of early gla-
ciations (67). Taken together, these considerations seem to make
inland anoxic geothermal fields the best incubators for the pro-
tocells among all currently known habitats on Earth.
Terrestrial Anoxic Geothermal Fields as Cradles for Earliest Life
Forms? So far, we have focused on the conditions under which
analysis of those protein families that were represented in the
LUCA by multiple paralogues such as GTPases or aminoacyl-
tRNA synthetases (92) (SI Appendix, Table S1). Most likely, the
ancestors of these protein families shared the ionic requirements
of the extant family members, such as those for K+ and Zn2+.
A similar preference for Zn2+, Mn2+, and ATP as substrate is
shown by viral hallmark genes (SI Appendix, Table S3). These
genes encode proteins which are present in many viral families
but are absent from cellular organisms and could stem from
organisms that preceded the LUCA (9, 93). Thus, extending the
chemistry conservation principle, we hypothesize that terrestrial
geothermal fields, similar to those illustrated in Fig. 2A, might
have also served as the cradles of life itself, sheltering the first,
precellular life forms up to the stage of the LUCA. This scenario
seems to be compatible with several lines of evidence:
a) Remaining almost independent of the ambient climate, inland
geothermal fields could exist for millions of years, long enough
to serve as incubators not only for the protocells but also for
the preceding life forms.
b) The major biochemical building blocks are derivatives of those
molecules that preferably partition to the vapor phase upon
the geothermal separation, namely simple carbonaceous and
phosphorous compounds, ammonia, and sulfide. In addition,
the vapor phase of geothermal systems is particularly enriched
in borate, the concentration of which can reach 10 mM (Table
2) (54, 94, 95) and which seems to be important for the sta-
bilization of ribose (96, 97).
c) Geothermal fields should have offered ample opportunity for
the reagents to concentrate and interact upon evaporation.
Specifically, the wetted surfaces would undergo continuous
drying resulting in selective accumulation of the least volatile
compounds, which, in this case, would be simple amides, with
boiling points of approximately 200 °C due to their ability to
form strong hydrogen bonds. Formamide, the likely key build-
ing block for abiotic synthesis of nucleotides and amino acids
(98–108), could form via hydrolysis of hydrogen cyanide,
which is found in volcanic gases and in exhalations of geo-
thermal fields (109). In addition, elimination of a water mol-
ecule from ammonia salts of carboxylic acids could also yield
amides, in particular, formamide from ammonia formate. As
noted earlier, exhalations of geothermal fields contain high
amounts of ammonia (55); part of this ammonia is of non-
sedimentary origin (110) and could have been present already
in the primordial geothermal vapor. Formate and other car-
boxylic acids would also have been produced at anoxic geo-
thermal fields (as detailed earlier). Hence, anoxic geothermal
fields could selectively accumulate simple amides, primarily
formamide, most likely mixed with water and other simple
molecules in different ratios. The yield of photochemical
and thermal syntheses in amide-containing solutions could
be further enhanced by catalytic action of mineral surfaces.
Specifically, it has been shown that silica minerals catalyze the
formation of adenine and cytosine from formamide (103, 111)
and that TiO2, the main component of the mineral rutile,
could catalyze the formation not only of purine derivatives
but also of thymine, 5-hydroxymethyluracil, and even acyclo-
nucleosides (112). Even widespread iron oxides have been
shown to catalyze the synthesis of nucleobases from formam-
ide (113).
d) Spontaneous polymerization events, which are thermodynam-
ically unfavorable in the bulk water, would be favored at geo-
thermal fields. Strikingly, a thermodynamic “window” at
concentrations of formamide of greater than 30% has been
identified, at which polynucleotides were more stable than
mononucleotides (114, 115). In addition, condensation reac-
tions would be favored by the wet/dry cycles driven by the
intrinsic pulsation of thermal springs (66), daily oscillations
Mulkidjanian et al.
the protocells might have evolved, without addressing the earlier
steps of evolution. Comparison of extant genomes does not di-
rectly yield information on pre-LUCA life forms. However, fea-
tures of these primordial organisms can be gleaned from the
6 of 10 |
www.pnas.org/cgi/doi/10.1073/pnas.1117774109vide a K+/Na+ ratio of greater than 1 or concentrate phosphate
up to its level in the cells. Thus, our analysis argues against the
widespread belief that the first cells evolved in marine habitats.
Although early evolutionary scenarios usually considered shallow
seawaters where solar light was available as an energy source
(116, 126), deep-sea environments have been invoked later, ini-
tially because of the protection against the hazards of the solar
UV that the water column would provide to the primordial life
forms. In particular, it has been estimated that the UV compo-
nent should have been attenuated by a factor as high as 109 to
avoid irreparable damage to the first organisms (86). Russell and
coworkers have noticed that FeS/FeS2 precipitates around hy-
drothermal vents form expansive honeycomb-like structures and
suggested that such iron-sulfide “bubbles” could encase and
protect the first life forms before the emergence of cells with
modern-type membranes (127, 128). Subsequently, attention has
been drawn to low-temperature vents where the hydrothermal
fluids are enriched in diverse organic compounds that are formed
through serpentinization, a hydrothermal alteration process that
is typical of the basaltic oceanic crust (129).
The terrestrial scenario outlined here incorporates all the
features of the hydrothermal vents that favor the origin and
early evolution of life, and adds more (Table P1 in Summary).
Our scenario includes production of organic molecules from
CO2 not only in reactions of hydrothermal alteration within the
rocks but also via abiotic photosynthesis at the surface. The UV
protection by ZnS, MnS, and silicate minerals is much more
efficient than the protection by a water column. Continental
geothermal fields are even more compartmentalized than ma-
rine hydrothermal systems. Not only do they include micro-
compartments, such as variably hydrated pores within ZnS and
MnS-containing silicate minerals, but in addition, each pond or
puddle can be itself considered a separately evolving macro-
compartment; occasional exchange of genetic material between
Mulkidjanian et al.
Protocells Could Not Emerge in Marine Habitat: Late Escape of Life to
the Ocean. Apparently, no marine environment could ever pro-
Conclusions
Building on the geochemical data and the results of phyloge-
nomic analysis, we argue here that anoxic geothermal fields
overlaying the vapor-dominated zones of terrestrial hydrother-
mal/volcanic systems could be the most suitable hatcheries for
the protocells and, most likely, the preceding replicator systems.
These putative cradles of life share all of the advantages of the
deep sea hydrothermal vents that have been previously proposed
in the same capacity (127–129), including the presence of in-
organic compartments, versatile catalysts, and sources of organic
matter (Table P1 in Summary). In addition, and in contrast to
deep sea vents, terrestrial geothermal fields are conducive to
condensation reactions and enable the involvement of solar light
as an energy source and a selective factor that would favor the
accumulation of nucleotides, which are particularly photostable
(6, 121, 124). Also in contrast to deep sea vents, the geothermal
vapor is enriched in phosphorous and boron compounds (Table
2) that could be essential for the emergence of first RNA-like
oligomers (96, 97).
Reconstruction of conditions under which the first life forms
might have emerged is important for experimental modeling of
the origin of life (32, 37). Some of the most successful attempts
to simulate primitive abiogenic reactions have been conducted
under conditions that are compatible with reconstructed con-
ditions at the geothermal fields of the anoxic Earth. These
promising experiments include syntheses of biologically relevant
compounds in formamide solutions (98–108, 111–115), photo-
synthesis/photoselection of natural nucleotides (120–122, 133),
montmorillonite-catalyzed formation of long RNA oligomers
(118) and membrane vesicles (134), RNA polymerization in the
eutectic phase in water–ice (135), abiotic UV photosynthesis of
the tricarboxylic acid cycle intermediates at ZnS (83, 84) and
TiO2 crystals (136), as well as UV-triggered recharging of ADP
to ATP (137). Further experimental exploration of models that
mimic the conditions at anoxic geothermal fields are expected to
shed more light on precellular evolution.
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of temperature and light, and the capacity of silicate minerals
to serve as apt templates (116–118).
e) The exceptional photostability of biological nucleotides sug-
gests that they could have been selected under solar UV ra-
diation from a plethora of diverse abiotically (photo)
synthesized organic compounds (6, 119–122). Analogously,
photoselection might have facilitated the transition from com-
plex mixtures of small organic molecules to the “RNA world”
(123) by favoring photostable RNA-like polymers with exci-
tonically coupled, stacked nucleotides forming Watson–Crick
pairs (6, 119, 124). In addition, solar UV radiation could
support primeval syntheses not only by catalyzing photopoly-
merization, but also by breaking the less photostable organic
molecules and thus supplying building blocks for new syn-
thetic cycles (10).
f) Under the low luminosity of the young sun (67), the daily
temperature oscillations could lead to periodic freezing
events, favoring the concentration of reactants, the endurance
of RNA-like oligomers, and their pairing (37).
g) The Zn2+ and Mn2+ ions could shape the primeval biochem-
istry as selective catalysts and as stabilizers of nascent biopol-
ymers (10, 12). It has been shown that Zn2+, to a much greater
extent than any other transition metal ion, favored the forma-
tion of naturally occurring 3′–5′ phosphodiester bonds during
abiotic polymerization of activated nucleotides (125).
h) Last but not least, evolution of life from the very first RNA-
like molecules to the stage of protocells in the same habitats is
the most parsimonious scenario: otherwise, one would have to
envision mechanisms for relocation of the first precellular
organisms to geothermal fields from some other location
and their accommodation in new habitats.
these macrocompartments could be triggered by rains or over-
flowing of the geothermal fields.
Detailed analysis of the transition from the first biomolecules to
the first cells is beyond the scope of this work; it is nevertheless
clear that this transition should have been accompanied by selec-
tion for increasingly tighter cellular envelopes (36–38). Increasing
sequestering of primordial life forms should have followed the
evolution of their metabolic pathways (36, 130) and also would
protect the informational systems from external hazards (10, 12).
The dramatic difference between the ionic compositions of the
cytosol and seawater (Table 1) implies that cellular organisms
could invade the ocean only after the emergence of ion-tight
membranes. These membranes and the appropriate ion pumps
were required to maintain the intracellular chemical environ-
ment similar to that in which the protocells evolved. Being
encased by ion-tight membranes and endowed with ion pumps,
the first cells could invade terrestrial water basins with low K+/
Na+ ratios and then, via rivers, reach the ocean, where they
would have been severely challenged by the high sodium levels.
Therefore, they would require ion pumps capable of ejecting Na+
ions out of the cell against large concentration backpressure. As
argued previously on the basis of phylogenomic analysis of rotary
ATPases, the interplay between several Na+ pumps might have
led to the emergence of membrane bioenergetics, initially in its
ancestral, Na+-using form (38, 131, 132).
The proposed terrestrial origin of the first cells implies that life
started not as a planetary but as a local event, confined to a long-
lasting inland geothermal field or to a network of such fields at
a continental volcanic system. Only the invasion of the ocean by
membrane-encased organisms transformed life into a planetary
phenomenon.
EVOLUTION
PNAS PLUS
Methods
Steam samples were collected by using a specially constructed condensing
device that aimed to minimize the possible contamination from the drops of
liquid phase or incomplete condensation of vapors. The thermal spring (i.e.,
mud pot) was covered by a vapor collector that contained a refractor to
prevent the eventual contamination by drops of liquid (SI Appendix, Fig. S6)
(95). The temperature was controlled by a temperature sensor; the differ-
ence between the temperature in the vent and at the wall of the collector
did not exceed 1 °C. The collector was connected to a glass Allihn condenser
(i.e., bulb condenser). The condenser was continuously cooled by cold water
from a tank. The vapor flow was regulated by changing the placement of
the vapor collector. The sampling conditions were chosen in such a way that
the temperature of the condensate outflow did not exceed 30 °C. Ac-
cordingly, if the vapor flow was too strong, the condenser was elevated
so part of the steam could escape around the edges of the collector (SI
Appendix, Fig. S6). After installation at a steam vent, the collector was
equilibrated for 10 min. After that, the samples were gathered in several
50-mL vials (at least two per spring) during 2 h to ensure the reproducibility
of results. When checked afterward, the concentration difference between
samples obtained from the same spring did not exceed 10%, whereas the
concentration differences between the samples taken from different
springs could vary by orders of magnitude (Table 2). The samples of the
liquid phase of the same thermal springs were filtered at the spot by using
0.45-μM membrane filters. All samples were preserved by the addition of
HNO3 up to a final concentration of 3%. The samples were later analyzed
by inductively coupled plasma MS by using an Element2 (Finnegan) mass
spectrometer.
ACKNOWLEDGMENTS. Valuable discussions with Drs. D. A. Cherepanov,
M. Eigen, R. M. Hazen, G. F. Joyce, M. J. van Kranendonk, V. N. Kompaninchenko,
D.-H. Lankenau, D. L. Pinti, M. J. Russell, V. P. Skulachev, H.-J. Steinhoff,
J. Szostak, N. E. Voskoboynikova, R. J. P. Williams, Y. I. Wolf and A. Yonath are
greatly appreciated. The authors are thankful to Drs. A. S. Karyagina and I. Y.
Nikolaeva for providing photographs of boiling mud pots. This study was
supported by Deutsche Forschungsgemeinschaft (DFG) Grants DFG-Mu-1285/1-
10 and DFG-436-RUS 113/963/0-1 (to A.Y.M.), Russian Government Grant
02.740.11.5228 (to A.Y.M.), the Volkswagen Foundation (A.Y.M.), EU COST
CM0902 Action (A.Y.M.), Deutscher Akademischer Austausch Dienst (D.V.D.),
Russian Foundation for Basic Research Grants 10-05-00320 (to A.Y.B.) and 0-04-
91331 (to D.V.D.), and the Intramural Research Program of the National Library
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[*/quote*]
So, Ihr Analphabeten von der Fake-Universität "Viadrina", das ist Forschung.
.
