Part 1
Prologue
1.1 Prologue: Definition,
Categories, Distribution,
Origin and Evolution,
Pioneering Studies, and
Emerging Fields of
Extremophiles
Koki Horikoshi1 . Alan T. Bull2
1
Japan Agency for Marine-Earth Science and Technology (JAMSTEC),
Yokosuka, Japan
2
University of Kent, Canterbury, Kent, UK
What are Extremophiles? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Categories of Extremophiles and Extremotrophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Distribution of Extremophiles and Extremotrophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Concerning Origins and Evolution of Extremophily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Pioneering Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Thermophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Alkaliphiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Deep-Sea Extremophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Emerging Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
The Deep Biosphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Hyper-Arid Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Purpose and Organization of the Handbook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Koki Horikoshi (ed.), Extremophiles Handbook, DOI 10.1007/978-4-431-53898-1_1.1,
# Springer 2011
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1.1
"
Prologue
I do not know what I may appear to the world, but to myself I seem to have been only like a boy
playing on the sea-shore, and diverting myself in now and then finding a smoother pebble or
a prettier shell than ordinary, whilst the great ocean of truth lay all undiscovered before me.
Isaac Newton
What are Extremophiles?
Extremophiles are organisms that are adapted to grow optimally at or near to the extreme ranges
of environmental variables. Most extremophiles are microorganisms that thrive under conditions
that, from a human perspective, are clearly hostile. RD MacElroy first coined the term ‘‘extremophile’’ in a 1974 paper entitled ‘‘Some comments on the evolution of extremophiles,’’ but
definitions of extreme and extremophile are of course anthropocentric; from the point of view
of the organism per se, its environment is that to which it is adapted and thence is completely
normal. A much larger diversity of organisms are known that can tolerate extreme conditions
and grow, but not necessarily optimally in extreme habitats; these organisms are defined as
extremotrophs (Mueller et al. 2005). This distinction between extremophily and extremotrophy
is not merely a semantic one and it highlights a number of fundamental issues relating to the
experimental study such as (1) inappropriate methods that may have been used to isolate putative
extremophiles, (2) claims of extremophily that may not have been tested rigorously, (3) putative
extremophily that may be compromised by subsequent serial cultivation under laboratory
conditions, and (4) inadequate attempts to determine whether organisms are adaptable to only
small differences in environmental variables (see Bull, > Chap. 12.1 Actinobacteria of the
Extremobiosphere). Note also that many species can survive extreme conditions in a dormant
state but are not capable of growing or reproducing indefinitely under those conditions.
An extremophile is an organism that thrives under extreme conditions; moreover the term
most frequently is used to refer to organisms that are unicellular and prokaryotic. Because many
extremophiles are members of the Domain Archaea and most known archaea are extremophilic,
on occasion, the terms have been used interchangeably. However, this is a very misleading
conception because many organisms belonging to the Bacteria and Eukarya have extremophilic
or extremotrophic life cycles. Additionally, not all extremophiles are unicellular. Most
extremophiles are microorganisms, for example, the presently known upper optimum growth
temperature is 113 C for archaea (upper known maximum is for a black smoker strain at 121 C),
95 C for bacteria, and 62 C for single-celled eukaryotes, in contrast to multi-cellular eukaryotes
that have rarely been shown to grow above 50 C. Members of the Archaea are uniquely
hyperthermophilic, that is, they exhibit optimum growth at 80 C and above, but organisms
having other expressions of hyper-extremophily have evolved in each of the domains.
The study of extremophilic and extremotrophic eukaryotic organisms has been relatively
neglected in comparison to their prokaryotic counterparts. Nevertheless, extremophily is being
increasingly reported among algae and fungi, for example. The case of halophilic green algae such
as Dunaliella species has been known for several decades but more recently discovered
examples of unicellular eukaryotic extremophiles/extremotrophs include Cyanidiales algae
(Toplin et al. 2008) that show obligate acidophily and moderate thermophily. Similar adaptations to extreme environments can be seen among unicellular and mycelial fungi. Members of
the black yeast genus Hortaea include extreme halophiles and extreme acidotrophs and to date
represent the most salt- and acid-tolerant eukaryotic biota on Earth. Moreover, quite a wide
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taxonomic range of yeast-like and mycelial fungi have evolved potent radiation resistance (see
Grishkan, > Chap. 10.2 Ecological Stress: Melanization as a Response in Fungi to Radiation;
Dadachova and Casaadevall, > Chap. 10.3 Melanin and Resistance to Ionizing Radiation
in Fungi). Further discussion of eukaryotes in extreme environments can be found in Weber
et al. (2007).
From the mid-1970s onward, increasing numbers of novel extremophilic and extremotrophic
organisms have been isolated as researchers have acknowledged that extreme environments are
capable of sustaining life. Studies on extremophiles have progressed to the extent that there
are now regular international extremophile symposia, as well as dedicated scientific societies
and journals such as Extremophiles – Microbial Life Under Extreme Conditions and Archaea.
Categories of Extremophiles and Extremotrophs
The literature contains several terms that are used to describe extremophiles and extremotrophs
and sub-definitions exist for organisms that present moderate, extreme, hyper-extreme,
and/or obligate extremophily. Among the terms that are frequently used, and to be found in
this book, are the following (further values for minimum, optimum, and maximum growth
characteristics can be found in Bull, > Chap. 12.1 Actinobacteria of the Extremobiosphere):
Acidophile: an organism with a pH optimum for growth at, or below 3–4
Alkaliphile: an organism with optimal growth at pH values above 10
Endolith: an organism that lives inside rocks
Halophile: an organism requiring at least 1 M salt for growth
Hyperthermophile: an organism having a growth temperature optimum of 80 C or higher
Hypolith: an organism that lives inside rocks in cold deserts
Metalotolerant: organisms capable of tolerating high levels of heavy metals, such as copper,
cadmium, arsenic, and zinc
Oligotroph: an organism capable of growth in nutritionally deplete habitats
Piezophile: an organism that lives optimally at hydrostatic pressures of 40 MPa or higher
Psychrophile: an organism having a growth temperature optimum of 10 C or lower, and
a maximum temperature of 20 C
Radioresistant: organisms resistant to high levels of ionizing radiation
Thermophile: an organism that can thrive at temperatures between 60 C and 85 C
Toxitolerant: organisms able to withstand high levels of damaging agents, such as organic
solvents
Xerophile: an organism capable of growth at low water activity and resistant to high desiccation
These anthropocentric definitions that we make of extremophily and extremotrophy focus
on a single environmental extreme but many extremophiles may fall into multiple categories,
for example, organisms living inside hot rocks deep under the Earth’s surface. The phenomena
of polyextremophily and polyextremotrophy refer to organisms adapted to more than two
extreme conditions but have received comparatively little detailed study. Examples of adaptation to multiple extremes can be found throughout this book and, again, while most attention
is given to prokaryotic organisms, there are dramatic instances of polyextremophily among
eukaryotes. A case in point is the unicellular eukaryotic red alga Cyanidioschyzon (order
Cyanidiales), a strain of which is acidophilic (pH 0.2–3.5), moderately thermophilic
(38–57 C), has a high tolerance of arsenic, and the capacity for its biotransformation
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Prologue
(Qin et al. 2009); these authors consider that algae play a significant role in arsenic cycling in
marine and freshwater geothermal environments.
Distribution of Extremophiles and Extremotrophs
At the wider scale, extreme environments on Earth have arisen, and continue to arise as
a consequence of plate tectonic activity, the dynamic nature of the cryosphere, and the
formation of endorheic basins. Plate boundaries occur wherever two tectonic plates collide
and result in the formation of mid-ocean ridges, mountains, deep-ocean trenches, and
volcanoes and other geothermal phenomena such as marine hydrothermal vent systems. The
latter, for example, are distributed globally and consist of very contrasting black smokers and
carbonate chimneys. Such tectonic manifestations variously produce extreme heat, pH,
dissolved gasses, and metals.
A high proportion of the Earth’s surface contains water in solid form (sea ice, ice caps and
sheets, glaciers, snowfields, permafrost) forming the cryosphere, the longevity of which may be
thousands or even a few million years. Cryosphere-climate dynamics are complex and influence
precipitation, hydrology, and ocean circulation. In regions where precipitation is very low (or
zero) and also unpredictable, deserts develop, the aridity of which is defined as hyper- (annual
precipitation to annual evaporation <0.05) or extreme hyper-arid (<0.002). Highly saline lakes
and pans often develop under these circumstances; they also arise more frequently in endorheic
basins that are drainage basins with no outflow of water. Given that the average depth of the
world’s oceans is ca. 3,800 m, high pressure generates yet another extreme environment.
Oligotrophic environments are defined as those presenting very low nutrient concentrations;
they include oceans deplete in iron, nitrate, phosphate, tropical laterite soils, and white sands.
Finally, a range of environments are deemed to be extreme by virtue of chemically and/or
physically caused toxicity (e.g., soils high in arsenic, lakes exposed to high incident radiation).
The foregoing descriptions include most of the world’s dominant ecosystems, all of which
have evolved as the results of natural processes over geological time scales. In more recent
times, similar or significantly different extreme conditions have been imposed as a consequence
of human insult of the environment, for example, soil salinity as a result of deep well drilling for
irrigation water, radioactivity contamination from power plant failure, persistently high
xenobiotic chemical challenge as a result of industrial pollution, and agrochemical use. Thus
the totality of these global and local, natural and anthropogenic ‘‘extreme’’ environments has
provided a remarkable panoply of opportunities for the evolution of the organisms that are
treated in this handbook.
A few additional points need to be made before we leave this topic. First, new extreme
ecosystems continue to be discovered and investigated including the deep biosphere that exists
at great depths in sub-seafloor sediments and in subterranean rock formations, and the
carbonate chimney vent system (Kelley et al. 2001). Such is the combination of extreme
conditions that characterize the marine deep biosphere that Sass and Parkes (see > Chap. 9.1
Sub-seafloor Sediments - An Extreme but Globally Significant Prokaryotic Habitat (Taxonomy, Diversity, Ecology)) prompt the thought that only organisms found therein might be
extremophiles sensu stricto! Second, as we have stated several times, extreme environments
almost invariably are affected by two or more extreme conditions. Third, in describing
extremophilic and extremotrophic organisms, care must be taken to discriminate between
the mere presence of an organism (or its phylogenetic signature) and those that are growing or
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metabolically active in an extreme environment. Fourth, certain extremophilic organisms can
be recovered from ‘‘normal’’ environments. Fifth, is our current knowledge of extremophile
diversity comprehensive? The results of recent analyses made by Pikuta et al. (2007) point to
a lack of evidence for the existence of acidopsychrophiles, acidohalophiles, and thermohalophiles – a challenge that should not be neglected by microbiologists! And finally, the question
of conservation of what might be termed extremophile ‘‘hot spots.’’ Priority-setting systems
were established originally for defining and conserving regions or localities that possessed
unusually high levels of animal and plant diversity. A similar strategy seems desirable for
conserving representative habitats that are dominated by extremophilic and extremotrophic
organisms, and paradoxically, would include heavily degraded environments that are providing
conditions for ongoing extremophile evolution.
Concerning Origins and Evolution of Extremophily
It may appear overambitious to introduce the evolution of extremophiles into this chapter
given the uncertainty and disparate hypotheses and opinions attending discussions of the
origin of life, the last common ancestor, and the origin of eukaryotes. It is not our intention to
examine these issues in detail but rather to attempt a realistic framework – incomplete and
speculative as parts of it may be – that might encourage discussion and further researches.
A pertinent starting point for this topic is the stimulating article published recently by
Martin and his colleagues (Martin et al. 2008). These authors consider the case for hydrothermal vents as systems where life might have originated and, ipso facto, where extremophiles
could be expected to have had their genesis. Such vent systems are found in abundance
worldwide and are presumed to have existed as soon as liquid water accumulated on Earth
(ca. 4.2 Ga). Martin et al. are at pains to differentiate black smoker and carbonate chimney
vents: black smokers arise at diverging plate boundaries above magma chambers, they are
highly acidic (pH 2–3), very hot (up to 405 C), with vent fluids rich in Fe and Mn, and CO2,
H2S, H2, and CH4; carbonate chimneys in contrast are found off-axis (away from diverging
boundaries), are highly alkaline (pH 9–11), moderately hot (up to 90 C) and rich in H2,
CH4, and low-molecular-weight hydrocarbons. The process of serpentinization (see Takai,
> Chap. 9.2 Physiology) results in the geochemical production of hydrogen at both types of
vent systems; at the carbonate chimneys hydrogen can reduce CO2 to methane, also geochemically. The conditions that arose in the Hadean oceans could have been conducive for supporting
chemolithotrophic life prior to the much later generation of photosynthetic carbon. The
microbiota of present-day, actively venting carbonate chimneys is dominated by anaerobic
methanogens that are replaced by anaerobic methanotrophs in cooler, less active vents. Present-day black smoker communities contain a variety of hyperthermophilic archaea and bacteria,
some of which also grow chemoautotrophically by gaining energy from the reduction of sulfate
or CO2 by H2. Thus, the intriguing possibility posed by Martin et al. is that the contemporary
microbiota of marine thermal vents ‘‘harbour relict physiological characteristics that resemble
the earliest microbial ecosystem on Earth.’’ Several authors have cautioned that present-day
hyperthermophiles may reflect later evolutionary adaptations to altered conditions on Earth
and that inferences made about distant ancestral life from contemporary hyperthermophiles
may be inappropriate (see Glansdorff et al. 2008, for example).
Another conceptual step relevant to ideas about evolution and the physiological divergence
of extremophilic and extremotrophic life has been made by Battistuzzi and Hedges (2009).
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Prologue
By combining data from phylogenetic (core protein, and small- and large-subunit rRNA genes)
surveys, cytology, and environmental surveys, they have proposed that a large clade of
prokaryotic organisms evolved on land during the mid-Arcaean era (ca. 3.18 Ga). This clade,
termed the Terrabacteria, comprises two-thirds of all recognized species of prokaryotes and
includes the Actinobacteria, Chloroflexi, Cyanobacteria, and Firmicutes. This evolutionary
hypothesis claims to be consistent with a number of geological and biomarker calibration
points and posits that members of the Terrabacteria include those showing salient adaptations
to a wide spectrum of extreme environments shaped by factors such as desiccation, high
salinity, and radiation exposure. Some lineages of the Terrabacteria are proposed to have
subsequently re-invaded marine environments. A further informative approach to extremophile biology has been to plot the occurrence of known species in 2-dimensional matrices,
pH versus temperature, pH versus salinity, etc. (Pikuta et al. 2007). While we have referred
above to some of the results of these analyses, Pikuta and her colleagues, in scrutinizing their
matrices, pose the interesting question as to the direction in which biological (and particularly
extreme biological) changes to organisms may have occurred during the geochemicalgeophysical evolution of Earth. Assuming a thermophilic beginning, acidophily probably
arose at an early stage, while alkaliphily evolved only after certain mineral precipitation and
sufficient buffer concentration of CO2 was established in the atmosphere. Moreover, halophily
could have developed only after an arid climate was imposed on land, and psychrophily only
after a major temperature fall. From the analysis of a large and environmentally diverse
collection of 16S rRNA gene sequences, Lozupone and Knight (2007) concluded that salinity
was the major determinant of microbial community composition rather than extreme temperature, pH, or other environmental factor(s). It might be the case, therefore, that despite the
powerful selective pressure of extreme temperatures and pH, the more general properties of
such environments exemplified by salinity are the primary determinants of which lineages
adapt and evolve.
It is clear that a cautious approach needs to be taken to the topic raised in this section and
excellent research and scholarship notwithstanding, conjecture inevitably is a significant element in attempts to unravel questions of extremophile evolution. What is apparent is the
stimulus that work and discussions of this kind will provide for further investigations. In
addition to the work referred to above, the interested reader will discover a large relevant
literature among which the papers of Sheridan et al. (2003), Cox et al. (2008), Glansdorff et al.
(2008), and Cavalier-Smith (2010) are thought provoking.
Pioneering Studies
Breakthroughs in the discovery and physiological characterization of extremophilic organisms
occurred in a number of laboratories throughout the world during the early and middle
decades of the twentieth century. Here we consider a selection of major achievements that
changed the course of much subsequent research with microorganisms.
Thermophiles
In June 1965, Thomas Brock, a microbiologist at Indiana University (he moved later to the
University of Wisconsin), discovered a bacterium, Thermus aquaticus, in the thermal vents of
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Yellowstone National Park that could survive at near-boiling temperatures (Brock and Freeze
1969). At that time, the upper temperature for life was thought to be 73 C. At one particular
site, Octopus Spring, Brock had discovered large amounts of pink filamentous bacteria at
temperatures of 82–88 C; here were organisms living at temperatures above the then ‘‘upper
temperature for life.’’ Attempts to cultivate these pink bacteria were initially unsuccessful, but
during the next several years, as he continued broader work on the ecology of the Yellowstone
springs, Brock isolated and collected many microbes from geothermal areas. Strain YT-1 of
T. aquaticus collected from Mushroom Spring on September 5, 1966 was the first to be
developed as the source of Taq polymerase that would become universally and routinely
used in molecular biology. His group showed that T. aquaticus was widespread in hot-water
environments and that enzymes from T. aquaticus were temperature tolerant in boiling water.
During various travels to study thermal areas in other parts of the world, Brock isolated
a number of other cultures of T. aquaticus, one interesting strain being recovered from the
hot-water system on the Indiana University campus. Subsequently, he could show that
T. aquaticus was widespread in artificial hot-water environments, and other workers have
isolated it from hot tap water in other parts of the world.
Brock commented that for many years his Yellowstone work had seemed somewhat
‘‘exotic’’ to many microbiologists, perhaps because of the presumed restricted distribution of
hot springs on Earth. This attitude changed after the discovery of the deep-sea vents, with their
very high temperatures and their associated diverse and flourishing life forms. Now deep-sea
thermal vents are known to be widespread in the oceans, just as the overall range of geothermal
environments supporting life has been revealed. Seventeen years after Brock’s discovery, Karl
Stetter isolated, from a shallow marine vent, the first organisms that could grow optimally at
temperatures greater than 100 C and so began the era of hyperthermophilic microbiology.
Although the upper temperature for life remains an open question, there is now a great
opportunity to pursue it by studying microorganisms living in the deep seas, the deep marine
and terrestrial biosphere, and the myriad of other extremely hot locations.
Alkaliphiles
In 1956, Koki Horikoshi first encountered moderate alkaliphilic bacteria when working as
a graduate student in the Department of Agricultural Chemistry, University of Tokyo on the
lysis of Aspergillus oryzae. One day in November he found one cultivation flask in which the
mycelia of A. oryzae had completely disappeared; the flask smelt of ammonia and the pH of its
contents had increased to nine. The lytic microorganism isolated from the flask was Bacillus
circulans, and strong endo-1,3-b-glucanase activity was detected in the culture fluid. However,
this bacterium showed very poor growth in conventional media but on the addition of 0.5%
sodium bicarbonate, good growth and enzyme production occurred.
Later, in 1968, during a visit to Florence and looking at Renaissance buildings so different
from Japanese architecture, a voice whispered in his ear, ‘‘There might be a whole new world of
microorganisms in different unexplored cultures’’ and memories of those experiments with B.
circulans flashed into his mind. Could there be an entirely unknown world of microorganisms
at alkaline pH? Hardly any microbiological work had been done in the alkaline region
principally because alkaline foods are uncommon, except a few Chinese types. Upon his return
to Japan, Horikoshi prepared an alkaline medium and inoculated it with small amounts of soil
collected from various sites on campus of The Institute of Physical and Chemical Research
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(RIKEN). To his surprise, after overnight incubation at 37 C, various microorganisms
flourished in all test tubes. Horikoshi named these microorganisms that grow well in alkaline
environments ‘‘alkaliphiles,’’ and found subsequently that they were widely distributed
throughout the earth (even at ca.10,900 m depth in the Mariana Trench in Pacific Ocean)
and that they produced new products (Horikoshi 1971). Over the past four decades, studies of
alkaliphiles have been comprehensive with one big question being ‘‘how do alkaliphiles tolerate
extreme alkaline environments?’’ Alkaliphiles can keep the intracellular pH at about 7–8 in
environments of pH 10–13. How pH homeostasis is maintained is one of the most fascinating
aspects of alkaliphiles and is discussed in > Chap. 2.2 Distribution and Diversity of Soda Lake
Alkaliphiles of this handbook.
Studies of alkaliphiles have led to the discovery of many types of enzymes that exhibit
unique properties; about 35 new kinds of enzymes have been isolated and purified by
Horikoshi’s group and some produced on the industrial scale (see > Chap. 2.10 BetaCyclomaltodextrin Glucanotransferase of a Species of Alkaliphilic Bacillus for the Production
of Beta-Cyclodextrin and > Chap. 2.11 Alkaline Enzymes in Current Detergency).
Deep-Sea Extremophiles
Claude ZoBell was instrumental in laying the foundations for modern marine microbiology,
and during his long career at the Scripps Institution of Oceanography provided the first
convincing evidence for the existence of indigenous marine bacteria, among them being
ones that were growing in the deep seas. In a seminal paper of 1949, Zobell and Johnson
reported bacteria in sediment sampled at 5,800 m off Bermuda that could grow under high
hydrostatic pressures (500–600 atmospheres 50–60 MPa) that were equivalent to the in situ
pressures of the deep-sea environment; they coined the term barophile to ‘‘characterize
microbes which grow preferentially or exclusively at high hydrostatic pressures.’’ Note that
the term barophile has been replaced in more recent times by piezophile (Gk piezein, to press).
Subsequently, ZoBell joined the famous Danish Galathea round the world deep-sea expedition,
and for four months in 1951 he was on board during the Manila (Philippines) to Port Moresby
(Papua New Guinea) leg of the cruise. Sediments taken from the Philippine Trench
(10,120–10,190 m depth) were found to contain viable bacteria representing a variety of
physiological types (ZoBell 1952) showing thereby that life existed in the deepest parts of the
ocean. These pioneering studies, together with essential developments in marine engineering
(notably submersibles, deep drilling, sampling equipment) have inspired successive generations of microbiologists and organizations to invest in deep-sea research, the results of much of
which are described in this handbook.
Emerging Fields
Since the early pioneering days of extremophile discovery, a growing number of individuals
and organizations have been drawn into this compelling field of research and new vistas
continue to be opened up. In Japan, for example, a 15-year research program called DEEP
STAR (Deep-sea Environment Exploration Program, Science and Technology for Advanced
Research) was launched in October 1990 and directed by Horikoshi with a mission to expand
the sources of microorganisms for study and application from the surface of the Earth to the
deep sea. We illustrate these new developments by reference to just two examples but recognize
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that several others deserve mention; in addition, opportunities for innovative biotechnological
applications of extremophiles and extremotrophs are briefly introduced.
The Deep Biosphere
ZoBell also found microorganisms in sub-seafloor sediments, but culturable organisms were
not recovered at depths greater than ca. 7 m. It was only after applying a portfolio of culturedependent, culture-independent, and biogeochemcal techniques several decades later that the
existence of deep submarine and subterranean microbial communities was established.
The modern era of deep biosphere studies began in the 1980s and was catalyzed particularly by
concerns for the contamination of ground waters and by the launch of the Ocean Drilling
Program. Early explorations of coastal sediments by the Savannah River Laboratory in South
Carolina revealed a diversity and abundance of microorganisms at depths of 850 m. The first
intensive study of deep submarine sediments, from the Peru Margin, at about the same time,
confirmed the existence of prokaryotic communities several hundred meters below the sea floor.
The extent of these deep sub-seafloor populations led Parkes and his colleagues to propose that
approximately 10% of global biomass carbon might exist as prokaryotic organisms in these
sediments down to a depth of 500 m (Parkes et al. 1994). The deepest submarine sediments
proven to sustain prokaryotic organisms are greater than 1,600 m. The physiology and ecology
of these organisms are discussed in > Chap. 9.1 Sub-seafloor Sediments - An Extreme but
Globally Significant Prokaryotic Habitat (Taxonomy, Diversity, Ecology) of this handbook.
Some of the more recent investigations of the deep subterranean biosphere are ongoing in Sweden
and South Africa. Research at the Äspö Hard Rock Laboratory, for example, has led to the proposal
that a H2-driven deep biosphere has developed in crystalline bedrocks at depths greater than
1,200 m (Pedersen 1997). Subsequently, the study of a fracture zone 2,825 m below land surface
(Mponeng gold mine, S. Africa) has produced clear evidence for a microbial community sustained
by geochemically derived H2 and sulfate (Lin et al. 2006) and dominated by one Firmicutesrelated phylotype. Here the remarkable conclusion is that these organisms might have been
sustained by lithotrophic metabolism for millions of years without inputs from photosynthesis.
Hyper-Arid Environments
Approximately 15% of the land surface of Earth is desert, a biome that is found in all of the
geographic realms. Deserts are classified as subtropical (e.g., Sahara, the largest non-polar
desert), cool coastal (e.g., Namibian), cold winter (e.g., Gobi), and polar. Hyper-aridity and
extreme hyper-aridity were defined above (see ‘‘> Distribution of Extremophiles and
Extremotrophs’’) on the basis of precipitation: evaporation ratios and deserts of this type,
exemplified by the world’s driest (the cool coastal Atacama Desert of northern Chile), also may
experience high salinity, intense atmospheric radiation, and very low nutrient availability. Such
is the nature of the extreme arid region of the Atacama that its soils have been proposed as
a model for those of Mars, and taken to be the dry limit for microbial survival in the
extremobiosphere (Navarro-Gonzalez et al. 2003). However, subsequent investigations made
in the same hyper-arid core of this Desert have shown that amplifiable DNA and a variety of
culturable bacteria can be recovered from this harsh environment (see > Chap. 12.1
Actinobacteria of the Extremobiosphere, this handbook for details).
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The microbiology of high-elevation (>5,000 m) hyper-arid and periglacial landscapes in
Peru and Chile/Argentina, some of which could be appropriate analogs of Martian polar
regions, have been explored recently by Steve Schmidt’s group (Schmidt et al. 2009; Costello
et al. 2009). These multi-extreme environments harbor diverse populations of bacteria, fungi,
and micro-metazoans, many members of which are novel taxa; in addition, metabolic activities
are sustained even under conditions of extreme diurnal fluctuations of soil temperature
( 12 C to 27 C).
Biotechnology
The exploration of the extremobiosphere and the surge of interest in biotechnology occurred
during roughly the same period; so it is not surprising that extremophiles and extremotrophs
became prime targets in search and discovery programs aimed at new natural products, new
biocatalysts, and other goods and services. The application of these organisms opened up an
exciting phase of biotechnology innovation.
The information summarized in > Table 1.1.1 provides a snapshot of biotechnologically
interesting compounds and activities produced by extremophilic and extremotrophic microorganisms. This is not an exhaustive compilation and it is also important to view these
products as a potential resource since only relatively few of them have been brought to market
at this stage. From a commercial perspective, enzymes (extremozymes) from extremophiles
have made the most impact so far. As an example, alkaline proteases, derived from alkaliphilic
species, constitute an important group of enzymes that find applications primarily as proteindegrading additives in detergents. Given the robust nature of alkaliphiles, these enzymes
can be subjected to harsh operational environments, including elevated temperature, high
pH, surfactants, bleach chemicals, and chelating agents, where applications of many other
enzymes are limited because of their low activity or stability. Enzyme production for detergents is a huge market constituting approximately 40% of the total enzymes produced
worldwide. In addition to detergent enzymes, the application of extremophile products has
been wide, ranging from large-scale processing (e.g., metal recovery, coal desulphurization,
waste treatment, and paper bleaching) to smaller-scale, high-value-added products (e.g., food
additives, optical switches, and photocurrent generators). Research on anti-infective agents
from extremophiles/extremotrophs has been slower to develop but the potential in this area
is now being appreciated with the discovery of new chemical entities and first-in-a-class
modes of action as illustrated by the abyssomicins. A recent review by Wilson and Brimble
(2009) describes the large chemical diversity found in extremophiles that probably have
evolved in part ‘‘as unique defences against their environment, leading to the biosynthesis
of novel molecules ranging from simple osmolytes and lipids to complex secondary
metabolites.’’
The cost and effort involved in collecting samples from extreme environments both for
fundamental research and for screening campaigns is far from being trivial. The search for
extremophiles is an expedition in itself; researchers are found deep-sea diving in Hawaii and
Japan, sending submersibles to the ocean depths, foraging in Yellowstone National Park and
other regions of geothermal activity, ascending high mountains and volcanoes, and collecting
ice core samples in polar and similar cryoenvironments. Although this matter is rarely
commented upon by contributors to the Extremophiles Handbook, it is one that the reader
might usefully ponder as she or he is brought close to the extremobiosphere.
✓
✓
✓
✓
✓
High salinity
High alkalinity
High acidity
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
Carbohydrases
Biocatalysts
✓
✓
✓
✓
Proteases
✓
✓
✓
✓
Lipases
✓
✓
Other
Antifoulant, antifreeze, anti-inflammatory, antioxidant, bacteriorhodopsin, biopolymer, cyclodextrin, osmolyte, pigment, polyunsaturated fatty acid, receptor agonist, surfactant.
a
✓
✓
✓
✓
✓
✓
High pressure
✓
Low temperature
✓
✓
Miscellaneousa
✓
Antitumor
High temperature
Antiparasitic
Antifungal
Antibacterial
Environmental extreme
Therapeutics
. Table 1.1.1
Products of extremophilic microorganisms
Prologue
1.1
13
14
1.1
Prologue
Purpose and Organization of the Handbook
The environmental limits to life on Earth are defined by the distribution of microorganisms as
primary colonizers, and it is this marvel of evolutionary history that is the principal theme of
this book. Environmental limits in this context describe the outermost boundaries of the
physicochemical world as we know it, and, by extension, those organisms that grow under such
extreme conditions are known as extremophiles.
Until quite recently, temperature, pH, and pressure extremes of the order of about 18 C
to 121 C, 0–13, and up to 100 MPa respectively would have been thought as inimical to life.
However, the progressive discoveries of recent years, of life in environments controlled by these
and other extreme conditions such as radiation, hyper acidity and salinity, intense pollution, or
very low nutrient availability, has revealed that extremophilic organisms are both abundant
and diverse. It is important to distinguish between true extremophiles that flourish only in
extreme environments and those organisms that, for various reasons, can merely tolerate or
survive at environmental extremes. This distinction is not simply a matter of semantics but has
major implications, for example, in understanding the behavior of extreme ecosystems and for
the biotechnological exploitation of these organisms. The relevance of molecular biological
approaches in revealing hidden organisms and metabolic potential within the communities of
extreme ecosystems is undisputable, but in this post-genomics era, the importance of developing innovative isolation and cultivation methods for extremophiles remains an important
objective in gaining a comprehensive picture of their physiology and ecology.
The purpose of the Extremophiles Handbook is to bring together the rapidly growing
and often scattered information on microbial life in the whole range of extreme environments
and to evaluate it in relation not only to the biodiversity, biochemistry, physiology, and
ecology that it comprises, but also to assess how we can gain clues to the origin of life and
the search for astrobiology, and finally to explore the biotechnological potential of these
fascinating organisms. In some cases, the reader will find little or no information on certain
aspects of extremophile biology; ecology, for example, often stands out as a void in our
understanding. We have asked contributors to mention such information gaps in their
accounts of particular extremophile groups as a means of directing further research into
extremophily.
References
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Mol Biol Evol 26:335–343
Brock TD, Freeze H (1969) Thermus aquaticus gen. n. and
sp. n., a nonsporulating extreme thermophile.
J Bacteriol 98:289–297
Cavalier-Smith T (2010) Deep phylogeny, ancestral
groups and the four ages of life. Philos Trans R Soc
Lond B 365:111–132
Costello EK, Halloy SRP, Reed SC, Sowell P, Schmidt SK
(2009) Fumarole- supported islands of biodiversity
within a hyperarid, high-elevation landscape on
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Environ Microbiol 75:735–747
Cox CJ, Foster PG, Hirt RP, Harris SR, Embley TM
(2008) The archaebacterial origin of eukaryotes.
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Glansdorff N, Xu Y, Labedan B (2008) The last universal
common ancestor: emergence, constitution and genetic
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Horikoshi K (1971) Production of alkaline enzymes by
alkalophilic microorganisms. Part I. Agr Biol Chem
36:1407–1414
Kelley DS, Karson JA, Blackman DK, Fruh-Green GL,
Butterfield DA, Lilley MD, Olson EJ, Schrenk MO,
Roe KK, Lebon GT, Rivizzigno P (2001) An off-axis
hydrothermal vent field near the Mid-Atlantic Ridge
at 30 N. Nature 412:145–149
Prologue
Lin L-H et al (2006) Long-term sustainability of a highenergy, low-diversity crustal biome. Science 314:
479–482
Lozupone CA, Knight R (2007) Global patterns in bacterial diversity. Proc Nat Acad Sci USA 104:
11436–11440
MacElroy RD (1974) Some comments on evolution of
extremophiles. Biosystems 6:74–75
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Microbiol 6:805–814
Mueller DR, Vincent WF, Bonilla S, Laurion I (2005)
Extremotrophs, extremophiles and broadband pigmentation strategies in a high arctic ice shelf ecosystem. FEMS Microbiol Ecol 53:73–87
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Hollen BJ, de la Rosa J, Small AM, Quinn RC,
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FEMS Microbiol Rev 20(399):414
Pikuta EV, Hoover RB, Tang J (2007) Microbial
extremophiles at the limits of life. Crit Rev Microbiol
33:183–209
1.1
Qin J, Lehr CR, Yuan CG, Le XC, McDermott TR, Rosen
BP (2009) Biotransformation of arsenic by
a Yellowstone thermoacidophilic eukaryotic alga.
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King AJ, Seimon A (2009) Microbial activity and
diversity during extreme freeze-thaw cycles in
periglacial soil, 5400 m elevation, Cordillera Vilcanota,
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Sheridan PP, Freeman KH, Brenchley JE (2003) Estimated
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Weber APM, Horst RJ, Barbier GG, Oesterhelt C (2007)
Metabolism and metabolomics of eukaryotes living
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Wilson ZE, Brimble MA (2009) Molecules derived from
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15
Part 2
Extremophiles:
Alkaliphiles
2.1 Introduction and History of
Alkaliphiles
Koki Horikoshi
Japan Agency for Marine-Earth Science and Technology (JAMSTEC),
Yokohama, Japan
Introduction and History of Alkaliphiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Why Did I Study Alkaliphiles? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
History of Alkaliphiles Before 1968 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Koki Horikoshi (ed.), Extremophiles Handbook, DOI 10.1007/978-4-431-53898-1_2.1,
# Springer 2011
20
2.1
Introduction and History of Alkaliphiles
Introduction and History of Alkaliphiles
Why Did I Study Alkaliphiles?
In 1956, I encountered an alkaliphilic bacterium, although not alkaliphilic in the true sense of
the word. I was a graduate student in the Department of Agricultural Chemistry, University of
Tokyo, working on autolysis of Aspergillus oryzae, which was the research theme for my
doctoral thesis.
One day in November, I found one cultivation flask in which mycelia of Asp. oryzae was
completely disappeared. Last night when I saw flasks, the mold flourished in all culture flasks.
I still remembered spectacular pictures of how bacteria thrived and vividly moved. No
mycelium could be seen under microscope.
The microorganism isolated from the flask was Bacillus circulans and strong endo-1,3-bglucanase activity was detected in the culture fluid. This enzyme lyzed Asp. oryzae. It was
the first time that mold cells had been found to be lyzed by bacteria and these results
were published in Nature (Horikoshi and Iida 1958). However, this bacterium showed
very poor growth in the absence of mycelia of Asp. oryzae and production of endo-1,3-bglucanse was very low. Therefore, purification of endo-1,3-b-glucanase could be done only
in culture fluid in the presence of mycelia of Asp. oryzae. I did not realize at the time that
the culture fluid had alkaline pH value. A few years later, I attempted production of endo1,3-b-glucanase in conventional media. I tested many cultivation media containing various
nutrients. An addition of 0.5% sodium bicarbonate to conventional nutrient culture broth gave
good growth and production of the enzyme. Autolysis of Asp. oryzae changed the culture
medium from weakly acidic to alkaline pH. In this way, I discovered that such a change in pH
value accelerated bacterial growth and enzyme production.
In 1968, I visited Florence, Italy, and saw the Renaissance buildings, which are so very
different from Japanese architecture. No Japanese could have imagined this Renaissance culture,
although both cultures date back almost to the same time: between fourteenth century and
fifteenth century (> Fig. 2.1.1). Then suddenly I heard a voice whispering in my ear,
"
There could be a whole new world of microorganisms in different unexplored cultures.
Memories of experiments on B. circulans done almost 10 years ago flashed back into my mind.
Could there be an entirely unknown domain of microorganisms existing at alkaline pH? The
acidic environment was being studied, probably because most food is acidic. Very little work
had been done in the alkaline region. Almost all biologists believed that life could survive only
within a very narrow range of temperature, pressure, acidity, alkalinity, salinity, and so on, in
so-called moderate environments. Therefore, when microbiologists looked around for interesting bacteria and other life-forms, they attempted to isolate microorganisms only from
moderate environments.
Science, just as much as the arts, relies upon a sense of romance and intuition. Upon my
return to Japan, I prepared two alkaline media containing 1% sodium carbonate ‘‘Horikoshi-I
and Horikoshi-II’’ (as shown in > Table 2.1.1) put small amounts of soil collected from various
area of the Institute of Physical and Chemical Research (RIKEN), Wako, Japan, into 30 test
tubes and incubated them overnight at 37 C.
To my surprise, various microorganisms flourished in all 30 test tubes. I isolated a great
number of alkaliphilic microorganisms and purified many alkaline enzymes. Here was a new
Introduction and History of Alkaliphiles
2.1
Kinkakuji temple in Kyoto
and Duomo in Firenze
. Fig. 2.1.1
Two photos of Kinkauji (Golden Temple) in Kyoto, Japan and Duomo in Firenze, Italy. The Golden
Temple was built at the end of the fourteenth century and The Duomo was built at the middle of
the fifteenth century
alkaline world that was utterly different from the neutral world discovered by Pasteur. This was
my first encounter with alkaliphiles. The first paper concerning an alkaline protease was
published in 1971(Horikoshi 1971).
Then in 1972, I was talking with my father-in-law, Shigeo Hamada about alkaliphilic
microorganisms. He had been in London after World War 1 as a businessman and quidnunc
about everything. He showed interest in alkaliphiles. These microorganisms were unique,
required high alkalinity, and they could produce alkaline enzymes such as alkaline proteases
and alkaline amylases. As I was speaking, he said
"
Koki wait a minute, I have an interesting present for you.
21
22
2.1
Introduction and History of Alkaliphiles
. Table 2.1.1
Basal media for alkaliphilic microorganisms
Horikoshi-I (g/l)
Glucose
Horikoshi-II (g/l)
10
–
Soluble starch
–
10
Polypeptone
5
5
Yeast extract
5
5
KH2PO4
1
1
Mg2SO4 7H2O
0.2
0.2
Na2CO3
10
10
Agar for plats
20
20
He brought out a sheet of old newspaper, Nikkei Shimbun, dated June 11, 1958. A short column
with one electron micrograph was like a punch to my head. As I had been in Purdue University,
Indiana, USA as a graduate student, I could not see this newspaper at all!
The article stated:
"
In Japan, since ancient times, indigo has been naturally reduced in the presence of sodium
carbonate. Indigo from indigo leaves can be reduced by bacteria that grow under high alkaline
conditions. Indigo reduction was controlled only by the skill of the craftsman. Takahara and his
colleagues isolated the indigo-reducing bacterium from a indigo vat.
I carefully checked scientific papers from Chemical Abstracts in the library of RIKEN. Only
16 scientific papers were discovered (Johnson 1923; Downie and Cruickshank 1928; Vedder
1934; Jenkin 1936; Bornside and Kallio 1956; Chesbro and Evans 1959; Kushner and Lisson
1959; Takahara and Tanabe 1960; Chislett and Kushner 1961a,b; Takahara et al. 1961; Takahara
and Tanabe 1962; Wiley and Stokes 1962; Wiely and Stokes 1963; Barghoorn and Tyler 1965;
Siegel and Giumarro 1966).
I felt a sense relief glancing at these scientific papers, because alkaline-loving microorganisms remained little more than interesting biological curiosities. No industrial application of
these microorganisms was attempted, until my papers were published in 1988. I named these
microorganisms that grow well in alkaline environments ‘‘alkaliphiles’’ and conducted systematic microbial physiological studies on them. It was very surprising that these microorganisms,
which are completely different from any previously reported, were widely distributed throughout the globe even in the deepest point of the Mariana Trench in the Pacific Ocean as shown in
> Fig. 2.1.2. Here was a new alkaline world that was utterly different from the neutral world.
Using such simple media, I found thousands of new microorganisms (alkaliphiles) that grow
optimally well at pH values of 10, but cannot grow at neutral pH value of 6.5 (> Fig. 2.1.3).
Many different kinds of alkaliphilic microorganisms have been isolated including bacteria
belonging to the genera Bacillus, Micrococcus, Pseudomonas, Actinobacteria, and eukaryotes,
such as yeasts and filamentous fungi.
Then over the past 4 decades, my coworkers and I have focused on the enzymology,
physiology, ecology, taxonomy, molecular biology, and genetics of alkaliphilic microorganisms
Introduction and History of Alkaliphiles
2.1
Counts of alkaliphilic bacteria/gram of soil
106
105
104
103
102
10
1
2
3
4
5
6 7 8
pH of soil
9
10 11 12
. Fig. 2.1.2
Distribution of alkaliphilic bacteria in soil
120
100
Growth
80
60
40
20
0
3
5
7
9
11
13
pH in media
. Fig. 2.1.3
pH dependence of microorganisms. The typical dependence of the growth of neutrophilic
bacteria (Bacillus subtilis), obligate alkaliphilic bacteria (Bacillus pseudofirmus 2b-2), and
facultative alkaliphilic bacteria (Bacillus halodurans C-125) are shown by solid squares, solid circles,
and solid triangles, respectively
to establish a new microbiology of alkaliphilic microorganisms. Another big question arises:
‘‘Why do alkaliphiles require alkaline environments?’’ The cell surface of alkaliphiles can keep
the neutral intracellular pH values in alkaline environments of pH 10–13. How the pH
homeostasis is maintained is one of the most fascinating aspects of alkaliphiles. In order to
23