416 Biochemical Society Transactions (2013) Volume 41, part 1

A brief history of the discovery of

hyperthermophilic life
Karl O. Stetter1
University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany

Abstract
Hyperthermophiles, growing optimally at 80◦ C and above were first discovered in 1981. They represent
the upper temperature border of life and are found within water-containing terrestrial and submarine
environments of active volcanism and geothermally heated subterranean rocks. The energy-yielding
www.biochemsoctrans.org

reactions represent mainly anaerobic and aerobic types of respiration rather than fermentation. Within
the ss (single-stranded) rRNA phylogenetic tree, hyperthermophiles occupy all of the short deep branches
closest to the root. Members of the deepest branch-offs are represented by the newly found Nanoarchaeota
and Korarchaeota.

Introduction In the present paper, I give a short overview of the

In 1980, when Wolfram Zillig and I became interested in
thermophilic Archaea, several thermophilic micro-organisms
had already been known which grow optimally (fastest) at
temperatures up to 75◦ C [1]. In following Pasteur’s principle,
similar to mesophiles, they are all killed by incubation
at 100◦ C. At that time, the organism with the highest
growth temperature known was Sulfolobus acidocaldarius
[2]. It thrives aerobically at 75◦ C within acidic hot mud
ponds in Yellowstone National Park. In addition, Tom
Brock had already reported on non-culturable rod-shaped
microbes (now known as Thermocrinis [3]) growing in
boiling (92◦ C) hot springs of neutral pH in Yellowstone
National Park [1]. Sulfolobus had been commonly seen as
a highly derived species, a kind of curiosity (mis-classified)
Biochemical Society Transactions
discovery and properties of hyperthermophiles [8], which
we were able to isolate during the last 32 years. They grow
fastest between 80 and 106◦ C and represent mainly strictly
anaerobic archaea. Some hyperthermophiles grow at 113◦ C
and possibly even higher, and survive autoclaving [9,10].
Discovery and cultivation of the first
hyperthermophiles
On our first trip to Iceland in 1980, using field microscopy,
Wolfram Zillig and I inspected boiling (98–100◦ C) springs
and mud pools in several areas. Surprisingly, a great deal
was teeming with micro-organisms with very unusual

among the Pseudomonads. Its aerobic lifestyle with its
suggested much higher yield of energy appeared essential
to resist thermal destruction [4]. The much lower growth
temperatures observed within the anaerobic thermophilic
methanogens seemed to confirm this prejudice. In sea water,
the high salt concentration was taken as additional stress
preventing an extremely hot lifestyle [4]. Therefore the
possibility of anaerobic extreme thermophiles within boiling
terrestrial and marine environments had never been taken into
consideration. At that time, Carl Woese had just published
his discovery of the Archaea and his revolutionary concept
of a three-domain living world [5]. As Woese had found out,
Sulfolobus, in reality, belonged to the Archaea domain. Within
this domain, it represented the most deeply branching-off
lineage in his dendrogram. The RNA polymerase work with
Wolfram Zillig confirmed Woese’s finding [6,7]. Therefore
this novel view on universal phylogenetic relationships had
convinced me rather early and became essential for my
thinking.
Key words: Archaea, Bacteria, cultivation, evolution, phylogeny, thermophile.
Abbreviation used: ss, single-stranded.
1
email karl.stetter@biologie.uni-regensburg.de
morphology like antler-shaped cells with true branchings
(later to be named Thermoproteales [11]). When I poured the
redox indicator resazurin into such boiling environments,
the blue clouds became reduced immediately, indicating
that they were anaerobic. I took several samples of boiling
water and mud. In order to keep them anaerobic, after
adding resazurin, sodium sulfide and sodium dithionite, I
enclosed them in storage bottles with tightly fitting stoppers.
From a sample taken from a strongly gassed little waterhole
in the Kerlingarfjöll mountains, I was able to isolate
Methanothermus fervidus, a novel rod-shaped methanogen
[12]. For the first time, this organism grew at temperatures
of up to 97◦ C and exhibited its fastest (optimal) growth
at 82◦ C. Therefore, surprisingly, this strictly anaerobic
archaeon grew at much higher temperatures than the aerobic
S. acidocaldarius. It became the key organism of my thinking.
In addition, from the anaerobic samples taken during this
trip, Wolfram Zillig and I were able to isolate the first
members of strictly anaerobic Thermoproteales [11]. Similar
to Methanothermus, the Thermoproteales exhibited growth
temperatures of up to 97◦ C and were unable to grow at 65◦ C
or below. Therefore the novel isolates exhibited a previously
unknown order of extremity of thermophily. Could there


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Figure 1 Disc-shaped cells of Pyrodictium within a network of
ultrathin tubules
Scanning electron micrograph.
be life even growing at 100◦ C and above? Of course, life
in steam would be impossible owing to the lack of life-
supporting components solubilized in liquid water. However,
an increase of 1 bar (100 kPa) above atmospheric pressure
raises the boiling point of water from 100 to 121◦ C and
therefore keeps water liquid up to 121◦ C. This pressure
corresponds to a water depth of only 10 m, which is
easily accessible by scuba diving. In order to hunt for
life above 100◦ C, during my holidays in 1981 at Vulcano
Island (Italy), I took anaerobic samples from a submarine
solfataric field close to the bay of Porto di Levante with
temperatures up to 103◦ C. From these samples, after 3 months
of cultivation experiments, members of the novel strictly
anaerobic Pyrodictium grew up (Figure 1). For the first
time, these organisms were able to grow above 100◦ C in
superheated water with an optimal growth temperature of
Figure 2 ‘Archaea Center’ (fermentation plant to grow hyperthermophiles), University of Regensburg
Partial view, showing two 300-litre fermenters and one 130-litre fermenter.
Molecular Biology of Archaea 3 417
105◦ C and an upper limit at 110◦ C [13]. On the basis of their
volcanism-adapted primitive lifestyle, I raised the hypothesis
that similar hyperthermophilic organisms could have existed
already at the early Earth, 3.9 billon years ago. At those
Hadean times, because of a still very brittle crust and very
active volcanism, the sea had been much hotter than today.
Continued hunting for novel
hyperthermophiles
Based on my new cultivation experience, in order to find
more exciting hyperthermophiles, during the last 32 years, I
visited high-temperature areas all over the world and isolated
high-temperature organisms from there. At present, hot
environments such as terrestrial and submarine heated soils,
sediments and hot springs are mainly found in areas of active
volcanism along tectonic fracture zones and hot spots. I had
visited several of these sites including deep sea hot vents with
their spectacular ‘black smokers’. In addition, I discovered
communities of hyperthermophiles within deep subterranean
(non-volcanic) geothermally heated oil-bearing sandstone
and limestone with in situ temperatures of approximately
100◦ C some 3500 m below the bottom of the North sea and
the surface of the Alaskan North Slope permafrost soil [14].
During that period of time, my laboratory isolated and de-
scribed approximately 50 new species of hyperthermophiles,
among those representatives of the novel bacterial genera
Thermotoga, Thermosipho, Aquifex, Thermocrinis and the
novel archaeal genera Acidianus, Metallosphaera, Stygiolobus,
Thermoproteus, Pyrobaculum, Thermofilum, Desulfuro-
coccus, Staphylothermus, Thermosphaera, Ignicoccus,
Thermodiscus, Pyrodictium, Pyrolobus, Thermococcus, Pyro-
coccus, Archaeoglobus, Ferroglobus, Methanothermus, Meth-
anopyrus, Nanoarchaeum and ‘Candidatus Korarchaeum’

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Figure 3 N. equitans (tiny cocci) attached to I. hospitalis (large cocci)

Left: freeze-etching of a I. hospitalis cell (large) with several cells of N. equitans (small) attached. Scanning electron
micrograph. Right: Ultrathin section of a cell of N. equitans and its attachment site to I. hospitalis. Transmission electron
micrograph.

(overview in [15]). In Woese’s small subunit rRNA-based follow inorganic redox reactions (chemolithotrophic), and
phylogenetic tree [16], hyperthermophiles exclusively CO2 is the only carbon source required to build up organic
represent all extremely short and deeply branching-off cell material (autotrophic). Molecular hydrogen serves as an
lineages within the Archaea and Bacteria, indicating a low important electron donor. Other electron donors may be
rate of evolution of their ss (single-stranded) rRNAs. sulfide, sulfur and ferrous iron. Whereas chemolithoauto-
trophic hyperthermophiles produce organic matter, there
are some obligate heterotrophic hyperthermophiles which
depend on organic material as energy and carbon sources. In
Enrichment and cultivation
addition, several chemolithoautotrophic hyperthermophiles
Enrichment cultures can be obtained by simulating the
are opportunistic heterotrophs.
varying geochemical and geophysical composition of the en-
Hyperthermophiles are adapted to distinct environmental
vironments. Various plausible electron donors and acceptors
factors including composition of minerals and gasses,
may be used under anaerobic, microaerophilic or (rarely)
pH, redox potential, salinity and temperature. Similarly
aerobic culture conditions. Depending on the (unknown)
to mesophiles, they grow within a temperature range
initial cell concentration and the doubling time of the
of approximately 25–30◦ C (in between the minimal and
organism, positive enrichment cultures of hyperthermophiles
maximal growth temperature). Reproduction of cells at and
can be identified by microscopy within 1–7 days. For a deeper
even above 110◦ C (maximal growth temperature) has been
understanding of the organisms, the study of pure cultures
observed in members of Pyrodictium (110◦ C), Methanopyrus
is required. Owing to the high-incubation temperatures,
(110◦ C) and Pyrolobus (113◦ C) [9,13,19]. A recent new
the traditional way of cloning by plating does not work
Japanese isolate of Methanopyrus had been reported to
well. Therefore we developed a new procedure to clone
grow, slowly, even at 122◦ C [10]. Hyperthermophiles grow
single cells anaerobically under the laser microscope by
fastest (optimal) at temperatures between 80 and 106◦ C,
employing optical tweezers [17,18]. Large cell masses are
depending on the strains. At the low-temperature end, as
required for biochemical and biophysical investigations. For
a rule, hyperthermophiles do not propagate at 50◦ C or
mass culturing of hyperthermophiles, in collaboration with
below, some not even below 80 or 90◦ C. Although unable
an engineering company, a new type of high-temperature
to grow at ambient temperatures (and space temperatures of
fermenter was developed, which became the basis of the
− 140◦ C, of course!), they are able to survive there for many
University of Regensburg Archaea Center (Figure 2). The
years. On the basis of their simple growth requirements,
steel casing of these fermenters is enamel-protected to resist
hyperthermophiles could grow in any hot-water-containing
the highly corrosive culture conditions. Sharp-edged parts
site, even on other planets and moons.
such as stirrers, gassing and sampling pipes and condensers
are made of titanium. The cell yield of a 300-litre fermentation
may vary from approximately 3 g to 2 kg (wet weight),
depending on the hyperthermophilic isolate. My ultimate discoveries of
hyperthermophiles
Finally, I want to introduce two members of novel groups
Energy sources and lifestyle of hyperthermophiles discovered in my laboratory recently.
Usually, the energy sources of hyperthermophiles can be The first one is a hyperthermophilic virus-sized archaeon.
simple: most species exhibit a chemolithoautotrophic mode In ecological studies based on PCR, it had been completely
of nutrition. Anaerobic and aerobic types of respiration overlooked, so far. We isolated this novel organism from a


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 Molecular Biology of Archaea 3 419

Figure 4 Localization of the ATP synthase in I. hospitalis Figure 5 Cell of ‘Ca. Korarchaeum cryptofilum’ showing its
Left: ultrathin section. Scale bar as indicated. Right: ultrathin section S-layer composed of uniquely tiny subunits
treated with immunogold-labelled antibodies against A1 Ao ATP Scanning electron micrograph (courtesy of Gerhard Wanner, LMU
synthase. Scale bar, 1 μm. C, cytoplasm; IM, inner membrane; V, vesicles Munich).
in the periplasm; OM, outer membrane. Reproduced with permission
from Küper, U., Meyer, C., Müller, V., Rachel, R. and Huber, H. (2010)
Energized outer membrane and spatial separation of metabolic processes
in the hyperthermophilic archaeon Ignicoccus hospitalis. Proc. Natl. Acad.
Sci. U.S.A. 107, 3152–3156.

submarine hydrothermal system at the Kolbeinsey Ridge,

north of Iceland. It is coccoid in shape. With a cell diameter
of only 0.4 μm, it is among the smallest living organisms
known. We named it Nanoarchaeum equitans. Most likely,
it represents a novel kingdom of Archaea [20]. Cells of N.
equitans grow only attached to the surface of a specific
crenarchaeal host, Ignicoccus hospitalis (Figure 3). With
490 885 bp, the genome of N. equitans is among the smallest
microbial genomes known to date [21]. It encodes the of N. equitans suggests that its symbiotic relationship to
complete machinery for information processing and repair, its Ignicoccus host is mandatory. The genome of the host
but lacks genes for lipid, cofactor, amino acid and nucleotide organism, I. hospitalis, has also been analysed [22]. Although
biosynthesis. The limited biosynthetic and catabolic capacity able to live free of its symbiont, I. hospitalis exhibits the

Table 1 Examples of genes throught to be characteristic of either the Crenarchaeota or the Euryarchaeota both occurring in ‘Ca.
Korarchaeum cryptofilum’

arCOG COG functional category∗ Function Eur† Cr‡

04447 L DNA polymerase II, large subunit 27 0

04455 L DNA polymerase II, small subunit 26 0
00872 L ERCC4-like helicase 26 0
02610 L Rec8/ScpA/Scc1-like protein 24 0
02258 L Subunit of RPA complex 20 0
00371 D Chromosome segregation ATPase, SMC 24 0
02201 D Cell division GTPase FtsZ 26 0
01013 R Protein with L13E-like domain 0 11
04327 R Ribosomal protein S25 0 13
04293 R Ribosomal protein S30 0 13
04305 R Ribosomal protein S26 0 13
04271 T RNA polymerase, subunit RPB8 0 12
00393 T Membrane-associated transcriptional regulator 0 9
∗ COG (Clusters of Orthologous Groups) functional categories: L, Replication, recombination and repair; D, Cell cycle control, cell division, chromosome
partitioning; R, Translation, ribosomal structure and biogenesis; T, Transcription.
†Number of euryarchaeal (Eur) genomes containing that arCOG (of a total of 27).
‡Number of crenarchaeal (Cr) genomes containing that arCOG (of a total of 13).


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420 Biochemical Society Transactions (2013) Volume 41, part 1
smallest genome among all free-living Bacteria and Archaea.
This organism exhibits a cellular organization unique to
Archaea and all other living organisms [23]: two membranes;
the outer membrane harbours the energy conservation (sulfur
reductase and A1 A0 ATP synthase), and the inner membrane
comprises the DNA and the ribosomes (Figure 4). At
present, we are still far away from a deeper understanding
of the Nanoarchaeum–Ignicoccus relationship and further
investigations are required.
On the basis of a unique environmental ss rRNA gene
sequence within the hot Obsidian Pool in Yellowstone
National Park [24], the second novel organism was obtained
by a continuous enrichment culture from there. This
hyperthermophile represents one of the deepest-branching
lineages among the Archaea in the tree of life, tentatively
named the Korarchaeota. We were able to physically enrich
the Korarchaeota cells to approximately 99.4% purity.
They consist of ultrathin rod-shaped cells, 10–100 μm long
and only 0.16 μm in diameter, below the resolution of a
regular light microscope. Owing to its difficult detection
and unusual morphology, we named the first cultivated
strain ‘Korarchaeum cryptofilum’ (‘Candidatus Korarchaeum
cryptofilum’). Under the electron microscope, cells of ‘Ca.
Korarchaeum cryptofilum’ can be easily recognized by their
tiny cell diameter and their unique S-layer consisting of
very tiny subunits (Figure 5). In collaboration with the JGI
(Joint Genome Institute), the genome of ‘Ca. Korarchaeum
cryptofilum’ was sequenced [25]. With a total of 1 590 757 bp,
it is pretty small. Analyses of the genes revealed that the korar-
chaeal genome harboured an unprecedented combination of
genes thought to be characteristic of either the Euryarchaeota
or the Crenarchaeota (Table 1). The heterogeneous gene
complement suggests that the Korarchaeota may have
diverged very early from those two major lineages. Further
comparisons may illuminate the early evolution of Archaea
and the origins of life.
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Received 26 October 2012
doi:10.1042/BST20120284