1. No category

Microbiology (2014), 160, 1–11
Review
DOI 10.1099/mic.0.071696-0
The Geobacillus paradox: why is a thermophilic
bacterial genus so prevalent on a mesophilic
planet?
Daniel R. Zeigler
Correspondence
Daniel R. Zeigler
zeigler.1@osu.edu
Department of Microbiology, Ohio State University, 484 W 12th Ave, Columbus, OH 43210, USA
The genus Geobacillus comprises endospore-forming obligate thermophiles. These bacteria have
been isolated from cool soils and even cold ocean sediments in anomalously high numbers, given
that the ambient temperatures are significantly below their minimum requirement for growth.
Geobacilli are active in environments such as hot plant composts, however, and examination of
their genome sequences reveals that they are endowed with a battery of sensors, transporters and
enzymes dedicated to hydrolysing plant polysaccharides. Although they appear to be relatively
minor members of the plant biomass-degrading microbial community, Geobacillus bacteria have
achieved a significant population with a worldwide distribution, probably in large part due to
adaptive features of their spores. First, their morphology and resistance properties enable them to
be mobilized in the atmosphere and transported long distances. Second, their longevity, which in
theory may be extreme, enables them to lie quiescent but viable for long periods of time,
accumulating gradually over time to achieve surprisingly high population densities.
Introduction: an environmental enigma
Geobacillus bacteria are rod-shaped, aerobic or facultatively
anaerobic, endospore-forming microbes. Depending on the
strain, the temperature range for growth can extend as low as
35 uC or as high as 80 uC. But for most isolates, temperatures
between about 45 and 70 uC are required (Nazina et al.,
2001). These characteristics make Geobacillus bacteria
(geobacilli) attractive to the biotechnology industry as sources
of thermostable enzymes (de Champdore´ et al., 2007;
Karagu¨ler et al., 2007), as platforms for biofuel production
(Cripps et al., 2009; Taylor et al., 2009) and as potential
components of bioremediation strategies (Markossian et al.,
2000; Obojska et al., 2002; Perfumo et al., 2007). Obligate
thermophiles, however, face a significant challenge outside
the lab: they must survive on a planet with an average annual
land surface temperature that has only ranged between 7 and
10 uC over the past three centuries (Rohde et al., 2013). It
would be logical to assume, then, that Geobacillus would only
be found in substantial numbers in the warmest regions of the
planet, such as equatorial deserts or naturally occurring
geothermal and hydrothermal hotspots. The truth, however,
is rather more complicated.
As Fig. 1 illustrates, people have been able to detect
Geobacillus nearly everywhere they have thought to look.
(This figure is based on a survey of 146 mostly recent
references describing the isolation of Geobacillus in natural
or human-made environments; for full details, see Table
Three supplementary tables are available with the online version of this
paper.
071696 G 2014 SGM
Printed in Great Britain
S1, available in Microbiology Online.) One can see at a
glance that these bacteria have been isolated from sources
on all seven continents as well as the Pacific Ocean and the
Mediterranean Sea. What may not be obvious from a twodimensional map is that there has been much diversity in
the third dimension as well. Geobacillus has been isolated
from the Bolivian Andes at an altitude of 3653 m
(Marchant et al., 2002) and has reportedly been detected
by molecular methods in the upper troposphere at
~10 000 m (DeLeon-Rodriguez et al., 2013), although
some aspects of the latter study have been questioned
(Smith & Griffin, 2013). Isolates have also been obtained
from the lower regions of the Earth, including oil wells at
depths of 1700–2150 m (Kato et al., 2001; Wang et al.,
2006), gold mines at depths of 1500–3200 m (DeFlaun
et al., 2007; Rastogi et al., 2009), continental shelf
sediments at 800–2060 m below sea level (Bartholomew
& Paik, 1966), seawater collected at a depth of 3083 m (Liu
et al., 2006) and sediments at 10 897 m below sea level in
the Mariana Trench, the lowest point on the Earth’s surface
(Takami et al., 2004). The ecological range of isolation
sources is equally diverse. The first reported Geobacillus
isolation nearly a century ago was from a can of spoiled
corn (Donk, 1920). It is now recognized that fresh
agricultural produce often carries Geobacillus spores, which
may survive heat treatment and cause ‘flat sour’ spoilage of
processed foods (Sevenier et al., 2012). Members of the
genus are likewise frequent contaminants of dairy production facilities and milk products (Ru¨ckert et al., 2004; Seale
et al., 2012). Hot composts have also been a common site
for isolating Geobacillus strains (Strom, 1985; Takaku et al.,
1
D. R. Zeigler
Isolation sources
Marine thermal
Geothermal
Temperate soil
Dairy
Hot springs
Compost
Other
Fig. 1. Geographical and environmental diversity of Geobacillus isolation. Each circle represents a geographical location
reported to be a source of cultured Geobacillus isolates; colour denotes type of source material. Each circle denotes a
published report that may describe one or many isolated strains. For details, see Table S1.
2006). The most frequently reported natural sources for
Geobacillus isolates have been hot springs (Canakci et al.,
2007; Derekova et al., 2006; Pinzo´n-Martı´nez et al., 2010),
geothermal soils (Lama et al., 2004; Meintanis et al., 2006),
hot subterranean oilfields and natural gas wells (Nazina
et al., 2001; Struchtemeyer et al., 2011), and hydrothermal
vents (Kimura et al., 2003; Maugeri et al., 2001).
Yet many temperate and even cold places of the Earth have
also yielded these thermophiles. There have been numerous
reports of Geobacillus isolation from cool soils (Marchant
et al., 2002; Rahman et al., 2004; Zeigler, 2005) and
permanently cold ocean sediments (Bartholomew & Paik,
1966; Takami et al., 2004). It is not simply that geobacilli
were found in unexpected locations; it is that they are
found in such large numbers there. Nearly 50 years ago,
researchers at the University of Southern California
examined ocean basin cores collected from the North
American continental slope and discovered up to 900
aerobic thermophiles per gram of wet sediment. All
seven isolates chosen for taxonomic identification were
Geobacillus. The authors recognized an enigma in their
findings, stating that ‘the presence of obligate thermophilic
bacteria in an environment having a constant temperature
of about 4 uC is difficult to explain’ (Bartholomew & Paik,
1966). A decade later, another group examined soil samples
collected in Reykjavik, Iceland, where bare soil temperatures at 5 cm depth averaged only about 14 uC and never
exceeded 27 uC, even on a sunny July day. The researchers
argued that ‘based upon normal recorded weather data, B.
stearothermophilus [now Geobacillus] and other thermophilic bacteria should not be present in the soil at all’. Yet
they cultured 1.66104 aerobic thermophiles per gram of
soil and identified all 33 selected isolates as Geobacillus
(Fields & Chen Lee, 1974). More recently, workers at the
2
University of Ulster, Coleraine, examined soils in Northern
Ireland and cultured aerobic thermophiles at a frequency
of 1.5–8.86104 g21, depending on the site. They could
readily isolate similar bacteria from cool soils collected in
the Andes and the northern USA. Each of five isolates
selected for identification turned out to be geobacilli. The
researchers noted that meteorological records showed that
local soil temperatures in Northern Ireland never reached
the isolates’ minimum growth requirement. ‘We have to
ask’, they concluded, ‘how can these organisms exist in
such large numbers in an environment where they are
unable to grow?’ (Marchant et al., 2002).
Herein lies the Geobacillus paradox. If the soils and
sediments examined in these studies are typical of the
planet as a whole, then Earth’s population of viable,
culturable aerobic thermophiles, as exemplified by the
genus Geobacillus, is enormous. Why have they accumulated in such numbers on a planet like ours? This review
will briefly examine the role of Geobacillus in the
ecosystem, the mechanism of their global dispersal and
the potentially extreme longevity of their spores, in hopes
of framing an answer to that question.
How do geobacilli earn their living?
Lessons from compost
One challenge after isolating a bacterium is determining
whether it was actively growing in that habitat or whether
it merely was transiently present, perhaps in a metabolically
dormant form such as a spore. There are few reports in
the research literature that unambiguously document the
proliferation of geobacilli in the natural world. There is at
least one habitat, however, where these bacteria are
undeniably active participants in a complex, dynamic
Microbiology 160
Geobacillus: why are thermophiles so prevalent?
microbial community: composts. Composting is a biodegradation of organic matter, usually derived from plants.
A consortium of microbes, mostly located in biofilms
clinging to the surface of the organic particles, cooperates
to break down the plant polysaccharides, liberating heat as
the sugar molecules are oxidized. Over a period of weeks or
months, active compost passes from a mesophilic phase
(10–42 uC) to a thermophilic phase (45–70 uC), followed
by a second mesophilic phase and eventual maturation and
stabilization (Ryckeboer et al., 2003). Anyone who has
watched steam pour out of active compost realizes that an
enormous amount of heat can indeed be produced. It is
during that thermophilic phase that geobacilli are active.
Whether in controlled laboratory settings or in waste
recycling centres, Geobacillus species can become the
dominant culturable bacterial taxon in composts at 60–
69 uC (Blanc et al., 1997; Ronimus et al., 2003; Strom,
1985; Takaku et al., 2006). When culture-independent
methods are employed, however, a richer and much more
nuanced picture emerges in which Geobacillus and other
thermophilic members of class Bacilli still play a part,
although a numerically minor one (Martins et al., 2013;
Michel et al., 2002; Partanen et al., 2010; Peters et al.,
2000). Yet the prevalence of geobacilli in active, hot
composts gives us a place to start in assessing the likely
role of Geobacillus in the natural environment. Earth’s
biosphere produces an estimated 100 gigatonnes of plant
biomass each year, about 90 % of which escapes consumption by herbivores and enters the dead organic matter pool,
an abundant carbon source that could certainly fuel the
production of huge numbers of bacteria (Cebrian, 1999).
We can hypothesize, then, that geobacilli are likely
opportunistic decomposers of plant-derived organic matter, capable of rapid growth under transient thermophilic
conditions, but endowed with mechanisms to survive long
periods of time when growth is impossible. This picture is
admittedly developed from circumstantial evidence and
therefore requires corroboration. For this it is necessary to
take a detour into the realm of comparative genomics, in
the hope that these organisms’ secret lives are recorded in
their genes.
At the time of writing, there were ten publicly available
complete genome sequences for Geobacillus (see Table S2).
It is thus possible to bring the powerful tools of
comparative genomics to bear on this question. A key
concept in the field is that of the core genome, the set of
orthologous genes shared by all members of a bacterial
taxon. Additional genes found in a given bacterium that
may provide increased functionality are called its dispensable genome. The sum of the core genome, together with all
the dispensable genes found in at least one member of the
taxon, is termed the pan-genome (Tettelin et al., 2005).
1665 genes. Significantly, 1409 of them (85 %) are also
found in the genome of Bacillus subtilis 168, a mesophilic
model organism that has been intensively investigated by
classical genetics since 1958 and by ‘omics’ technologies
since 1997. It is therefore possible to use the thoroughly
annotated and carefully curated B. subtilis 168 genome
sequence (Belda et al., 2013; Ma¨der et al., 2012) to create an
annotated Geobacillus core genome (Table S2). The
Geobacillus genome includes orthologues of 236 of the
278 genes known to be essential for viability in B. subtilis
(Commichau et al., 2013) and 67 of the 75 genes deemed
essential for endospore production (Galperin et al., 2012).
(Presumably, some essential functions are carried out by
non-orthologous proteins in Geobacillus.) The Geobacillus
core genome includes orthologues of the majority of B.
subtilis genes involved in key cellular processes such as
DNA replication, repair, recombination, condensation
and segregation; transcription and translation; cell division
and maintenance of cell shape; membrane dynamics
and protein secretion; carbon core metabolism and ATP
synthesis; biosynthesis and acquisition of nucleotides,
amino acids and cofactors; and RNA processing. The
Geobacillus core genome also features orthologues of many
genes involved in the B. subtilis post-exponential growth
lifestyle. Besides genes encoding alternative sigma factors
and other proteins participating in sporulation and
germination, it includes genes involved in chemotaxis and
motility, biofilm formation, DNA uptake, and (at least
potentially) genetic competence. These observations imply
strongly that Geobacillus and B. subtilis share an overarching
survival strategy, inherited from a common ancestor,
featuring rapid growth when metabolizable nutrients are
readily available, followed by concerted efforts to create,
scavenge or move towards additional sources when nutrients
are exhausted, with spore formation as a last resort if these
efforts fail (Fujita et al., 2005; Fujita & Losick, 2005). Some
Geobacillus isolates lyse following nutrient exhaustion in
batch culture, a potential boon for certain biotechnological
applications (Pavlostathis et al., 2006). However, most
geobacilli in my own collection of environmental isolates
(Zeigler, 2005) do indeed form endospores on solid media
under laboratory conditions, and many show not only
motility but the ability to swarm on agar surfaces, as one
would predict from genomic analysis. But for Geobacillus,
what specialized adaptations have been overlaid on this basic
lifestyle? The remaining 15 % of the core genome is of little
help in answering this question; it seems mostly to encode
additional transporters and proteins of unknown function.
Instead we must turn our attention to the dispensable
genome for this genus. Does it feature genes devoted to the
utilization of plant biomass? The enzymology of plant
polysaccharide decomposition has been very thoroughly
studied (Gilbert, 2010). So, what is necessary is to scan the
Geobacillus dispensable genomes for genes encoding known
enzymes for hydrolysis, uptake, and utilization of these
polysaccharides and their components.
The Geobacillus core genome, determined with the help of
the EDGAR application (Blom et al., 2009), consists of about
To this end we are greatly assisted by the work of Y.
Shoham and colleagues with a xylanase-secreting strain
Lessons from genomes
http://mic.sgmjournals.org
3
D. R. Zeigler
with potential industrial applications, Geobacillus stearothermophilus strain T-6 (Khasin et al., 1993). The genome
of strain T-6 has a hemicellulose utilization island of 76 kb,
divided equally between two subclusters providing functionality for utilizing xylan/xylose and arabinan/arabinose
(Shulami et al., 1999, 2011). The region also contains
several mobile elements, suggesting that the island could have
been assembled by horizontal gene transfer. Both the xylan
and the arabinan subclusters contain sensing systems that can
alert the cell to the presence of the units that make up these
hemicelluloses; glycohydrolases that can remove side chains
from them and cleave them into oligosaccharides; ABC
transporters that can import the oligo- or monosaccharides
into the cell; suites of sugar metabolism genes; and master
transcriptional regulators. Nine of the ten completed
genomes contain at least some portion of this island, and
seven contain nearly all of it (see Table 1 for a summary and
Table S3 for details). Recently, a galactan utilization operon
has also been identified in strain T-6, enabling the bacterium
to break down a key component of pectin, type I galactan
(Tabachnikov & Shoham, 2013). The entire 9.4 kb cluster is
also found in orthologous form in the genome of Geobacillus
thermoleovorans CCB_US3_UF5. Examination of the finished genomes also reveals other likely genes and clusters
devoted to plant degradation. Orthologues of a well-studied
neopullulanase (Hondoh et al., 2003) are encoded within an
apparent pullulan or starch utilization cluster found in eight
of the ten genomes (Table 1 and Table S3). A previously
studied system (Lai & Ingram, 1993) for the uptake and
utilization of cellobiose, a disaccharide produced from partial
cellulose hydrolysis, is found in four of the genomes, while a
five-gene cluster highly similar to a well-characterized B.
subtilis glucomannan utilization operon (Sadaie et al., 2008)
is found in nine of the genomes.
In summary, most Geobacillus isolates are equipped with a
battery of sensors, transporters and hydrolytic enzymes
specifically adapted to extract energy and metabolic building
blocks from plant biomass. Interestingly, this generalization
holds for Geobacillus isolates taken from seemingly unrelated
sources: Geobacillus thermodenitrificans NG80-2 from a hot
deep-subsurface oil reservoir in China (Wang et al., 2006),
G. thermoleovorans CCB_US3_UF5 from a hot spring in
Malaysia (Muhd Sakaff et al., 2012), Geobacillus sp. GHH01
from cool garden soil in Germany (Wiegand et al., 2013) and
Geobacillus kaustophilus HTA426 from cold sediment at the
bottom of the Mariana Trench (Takami et al., 2004). While
some Geobacillus strains have been isolated from oilcontaminated soils and deep oil reservoirs, and some have
been shown to metabolize long-chain hydrocarbons, it is
also true that nearly all of these environments have been
disturbed by human activity, and at least some of these
isolates have been introduced in subterranean oil and
gas fields by the drilling and extraction processes
(Struchtemeyer et al., 2011). Further, as mentioned above,
the oil reservoir isolate NG80-2 possesses many clusters of
genes involved in plant degradation. For these reasons,
further work will be required to establish whether
subterranean hydrocarbon deposits compose a significant
ecological niche for Geobacillus.
An emerging picture
From these considerations, a coherent picture begins to
emerge, although one still lacking in many important
details. Geobacillus has evolved to tap into the vast global
carbon cycle, helping decompose complex polysaccharides
found in dead plant biomass. It is a bit player in the drama,
joining with a large community of other microbes when
thermophilic conditions persist. When conditions are no
longer favourable for growth, geobacilli form endospores
to ensure their long-term survival. This genetic toolkit
allows opportunistic Geobacillus to decompose hot polysaccharides in human-created environments, such as in
compost piles or food processing plants. This picture does
not, of course, preclude geobacilli from occupying more
specialized niches in nature, but it is consistent with the
bulk of what we know about these bacteria. Yet the
question at this point remains unanswered: why is
Geobacillus found in high numbers in environments where
the organism cannot grow?
Table 1. Gene clusters predicted to function in plant polysaccharide degradation, as detected in the dispensable genomes of
Geobacillus isolates
Target compound
Cellulose
Starch/pullulan
Pullulan
Glucomannan
Xylan
Xylan
Arabinan
Galacturonan
Galactan, type I
4
Gene cluster
Named genes
Length (kb)
ORFs
Genomes
celRABCD
5.3
¡17.8
0.7
4.3
35.2
3.0
¡38.3
0.8
9.4
5
4–9
1
5
25
2
11–21
1
6
4
8
8
9
6
9
7
4
1
gmuRDCAB
uxu, kdg, agu, xyn, axe operons
xylAB
ara, abf, abn operons
ganREFGBA
Microbiology 160
Geobacillus: why are thermophiles so prevalent?
Why are geobacilli found worldwide?
Bridges in the sky
In our search for planetary mechanisms for redistributing
geobacilli, we need to give special consideration to the
atmosphere. It has been said that air is ‘as alive as soil or
water’ (Womack et al., 2010). And for good reason:
according to current estimates, every square metre of the
Earth’s surface emits on average 50–220 bacteria s21
(Burrows et al., 2009b). In total, Earth’s atmosphere
includes 7.661023 to 3.561024 particles that contain
bacteria (Burrows et al., 2009a). This distribution is not
completely homogeneous. The near-surface atmosphere
over the oceans has about 104 bacteria m–3, with similar
levels over deserts and tundra. Bacterial loads in air over
grasslands, wetlands and crops are about an order of
magnitude higher. The highest levels are found over
shrubs, where bacteria may exceed 36105 m23, and urban
environments, where they may approach concentrations of
106 m23 (Burrows et al., 2009b). Models based on global
wind patterns suggest that most bacterium-sized particles
reside in the atmosphere for 2–15 days. They are usually
removed from the air, or ‘scavenged’, by water, and icephase scavenging is much more efficient than liquid-phase.
Bacteria from tropical grasslands, shrubs and deserts are
more likely to participate in long-distance travel, as they
can be caught up in tropical convective systems and
potentially carried all the way to the upper troposphere.
Interestingly, smaller particles of about 1 mm diameter fall
into a ‘scavenging gap’ and reside in the atmosphere longer
than particles of 3 mm diameter. The atmosphere thus
regularly redistributes bacteria around the surface of the
planet, although the patterns of transport are by no means
completely uniform. The air can even serve as a bridge
allowing intercontinental transit of microbes (Burrows
et al., 2009a; Smith et al., 2012).
There have been many published studies documenting the
isolation of viable bacteria from the air, beginning with
the work of Louis Pasteur (Ariatti & Comtois, 1993).
Unfortunately, most of these studies provide no specific
data about Geobacillus. With very few exceptions, researchers using culture-dependent methods to study atmospheric
microbes have incubated their samples under mesophilic
conditions that preclude the growth of any aerobic
thermophiles. Those who have employed DNA sequencebased methods have very rarely identified short sample
sequences beyond the family taxonomic level, and there
are many genera besides Geobacillus within the family
Bacillaceae. Nevertheless, reports of mesophilic bacilli in
the atmosphere are probably broadly applicable to
morphologically similar thermophilic geobacilli as well.
In general, culture-based studies have usually reported
Bacillus as a major mesophilic component of bioaerosols.
In air samples taken from various locations in Oregon, for
example, the frequency of Bacillus among all cultured
bacteria was 41.7 % from forest air samples, 13.6 % from
coastal samples, 45.2 % from urban samples and 25.8–
41.7 % from rural samples (Shaffer & Lighthart, 1997).
http://mic.sgmjournals.org
Bacillus was also a dominant taxon among cultured
airborne bacteria sampled from urban Beijing (Tong et al.,
1993), from a border market in Thailand (Reanprayoon
& Yoonaiwong, 2012) and from a North American
mountain observatory 2.7 km above sea level (Smith
et al., 2012). Culture-independent methods report a much
higher bacterial diversity. A study of urban aerosols
collected from four cities in the US Midwest reported the
presence of 200–300 bacterial phylotypes, among which the
order Bacillales represented a minority component (Bowers
et al., 2011). A similar study of urban aerosols from two
cities of the south-western US detected large numbers of
Firmicutes, including members of the family Bacillaceae
(Brodie et al., 2007). One interesting study that does bear
directly on Geobacillus aerobiology examined the copious
bioaerosols generated when hot compost piles are turned.
Using a culture-independent approach, the researchers
reported that Firmicutes and Actinobacteria were the most
frequently detected bacterial phyla, and that Geobacillus
was the most common phylotype among Firmicutes (Le
Goff et al., 2010). In a study with direct bearing on the
paradox that forms a theme for this review, Geobacillus has
also been cultured from air samples collected above cool
soils in Northern Ireland. The isolation frequency was
relatively low, 1.55 c.f.u. per 1000 litres of air. The
frequency of culturable Geobacillus in rainwater collected
in the location was measured at 1.1 cells per 100 ml of
water. These numbers are not large, but based on Northern
Ireland climate records, they imply that each year about
1.46105 Geobacillus cells or spores are deposited on 1 m2
of soil surface (Marchant et al., 2008). In summary, there
is a dynamic but largely invisible interaction between
microbes and Earth’s atmosphere, and Bacillus sensu lato is
a minor but not insignificant part of this process. What is
true for mesophilic bacilli is likely true for their thermophilic relatives, the geobacilli, as well.
Desert dust
One special focus of aerobiology has been the study of desert
dust storms. These massive events mobilize on the order of a
billion tonnes of soil each year, most of it originating from
the Sahara and Sahel in Africa, with significant contributions
from the Gobi, Takla Makan and Badain Jaran deserts in
Asia. Dust storms can be very precisely monitored from
Earth observation satellites. The storms can be observed
crossing the entire Atlantic westward from Africa in 3–
5 days and crossing the entire Pacific eastward from Asia in
7–10 days (Griffin, 2007; Kellogg & Griffin, 2006; WeissPenzias et al., 2006). When microbes have been cultured
from mobilized African or Asian desert dust, Bacillus usually
have constituted a significant proportion of the population
(Griffin et al., 2001, 2003, 2006; Hua et al., 2007). As
expected, when culture-independent methods are employed,
a greater diversity is observed in aerosolized desert dust.
Nevertheless, the Bacillaceae can still constitute one of the
dominant taxa detected in DNA-based studies (Smith et al.,
2013; Yamaguchi et al., 2012).
5
D. R. Zeigler
Could the geobacilli found in unexpectedly high levels in
cool European soils have their ultimate origin in Saharan
dust storms? Desert dust deposited in Mediterranean
Turkey and Greece yielded several culturable Geobacillus
isolates in a recent study. On a phylogram of 16S rRNA
gene sequences, these isolates were interspersed with
Northern Ireland cool soil isolates (Perfumo & Marchant,
2010). However, one should be cautious not to overinterpret these data. For one thing, they are more
qualitative than quantitative; no estimate of Geobacillus
loads in Saharan dust are possible from this study, because
neither the air volume that contained the dust, the mass of
the dust itself nor the titre of aerobic thermophiles in
the dust was measured. For another, there are a priori
reasons to question whether Saharan dust carries enough
Geobacillus spores to account for what has been measured
in Icelandic soils or in Irish soils or rainwater. Although
Geobacillus strains have occasionally been isolated from
desert soils (Abdel-Fattah & Gaballa, 2008; Al-Hassan et al.,
2011), careful quantitative studies are generally lacking.
One notable exception is a report of aerobic thermophiles
isolated from Saudi Arabian soils (Abu-Zinada et al.,
1981). As expected, the desert soil was found to be very
poor in nutrients and moisture; it consisted of 91 % sand
and contained only 0.3 % water and 0.2 % organic matter.
It was also very low in culturable thermophiles, with only
760 c.f.u. g21 of soil. In contrast, Arabian loams and sandy
clays yielded thermophiles at 20–40 times higher frequencies, roughly equivalent to what is seen in European cool
temperature soils. Ten isolates were analysed further, and
all were classified as Geobacillus (then named Bacillus
stearothermophilus). We should be cautious about extrapolating from a single study, and research is needed to
confirm these results and extend them to other desert
regions. Yet it would hardly be surprising if Saudi Arabian
desert microbial communities are broadly similar to those
found in the Sahara and Gobi deserts. It therefore seems
likely that while desert dusts may be important vehicles for
redistributing Geobacillus spores, the deserts themselves
should not be thought of as huge microbial incubators
generating the high levels of Geobacillus observed in soils
and sediments worldwide. If Geobacillus is primarily a
decomposer of plant biomass under thermophilic conditions, it may be that shrubs and grasslands are the major
contributors to the atmospheric load of geobacilli. This
question remains open.
Are spores of (Geo)bacillus adapted for atmospheric
transport?
There is an intriguing correspondence between the size of
the ‘scavenging gap’ and the size of Bacillus and Geobacillus
spores. Particles with a diameter of about 1 mm have the
longest residence in the atmosphere and so can presumably
be transported the greatest distances before falling back to
earth (Burrows et al., 2009a). Strain descriptions of novel
Geobacillus species rarely report spore size. But it is
apparent, from both my unpublished observations and
6
others’ published micrographs, that most Geobacillus
spores are slightly ovoid, with an average diameter close
to the size required to fit into the scavenging gap (Fortina
et al., 2001; Nazina et al., 2001). Further, Bacillus and
Geobacillus endospores are notorious for their extreme
resistance to UV light, temperature changes and desiccation (Driks, 2002; Nicholson et al., 2000; Setlow, 2006;
Takamatsu & Watabe, 2002), the very environmental
challenges faced by airborne bacteria (Griffin, 2007).
These correspondences suggest the following hypothesis:
atmospheric transport has played an important role in the
selective pressures that shaped the morphology and
resistance properties of spores from Bacillus and related
genera. Other more subtle properties of spores, such as
their tendency to clump or stimulate condensation, might
likewise have been shaped by this selection. Or to put it
another way, Bacillus spores have evolved to remain viable
during long residence times in the atmosphere, allowing
them to travel great distances, perhaps to more favourable
locations for growth.
Why have geobacilli accumulated in such high
numbers?
In attempting to explain the paradoxically high populations of thermophilic geobacilli in cool soils and sediments,
I have proposed first that these bacteria probably play
minor roles in the global carbon cycle as decomposers of
plant biomass, and second that geobacilli and their spores
are redistributed widely, although not necessarily uniformly, around the planet through atmospheric transport.
One major component of the question still needs to be
addressed: how have these bacteria accumulated in such
high numbers? It appears that the genus Geobacillus plays
only a modest role in a large microbial community, even in
environments where it is clear that geobacilli are growing
and reproducing, such as hot compost. Perhaps there is
some as yet poorly characterized ecological niche that
serves as a Geobacillus production factory, continuously
releasing enormous numbers of these organisms into the
environment. But another solution is possible, one that
may be more logically economical. Geobacillus could be
produced by the environment only sporadically in
restricted geographical locations and small numbers and
then widely redistributed as spores, provided that these
spores exhibit significant longevity. If so, the high
populations observed in modern environments could have
gradually accumulated over long periods of time. Perhaps
the global population of Geobacillus has reached equilibrium, and the rate of reproduction is matched by the
death rate. Or perhaps the Geobacillus population is still
increasing, slowly but inexorably dusting large swathes of
the planet with viable spores.
Is there evidence for such extreme longevity? There have
been numerous reports of the recovery of viable bacteria
from ancient preserved or fossilized materials (Kennedy
et al., 1994). These reports have historically tended to be
Microbiology 160
Geobacillus: why are thermophiles so prevalent?
greeted with scepticism, although recent publications
demonstrate that researchers have been developing increasingly rigorous methods for mitigating sample contamination concerns (Johnson et al., 2007; Sankaranarayanan
et al., 2011). For Geobacillus, data regarding longevity are
especially sparse. However, in the previously mentioned
study of Pacific Ocean basin cores from the North
American continental slope, aerobic thermophiles identified as Geobacillus were repeatedly isolated from sediments dated up to 7800 years BP in such substantial
numbers that contamination is an unlikely explanation
(Bartholomew & Paik, 1966). It would be fascinating to
learn whether similar organisms could be isolated from
much deeper, older cores, but such studies do not seem to
have looked for thermophiles.
It has been persuasively argued that chemical instability of
DNA is a limiting factor for the longevity of bacteria
(Lindahl, 1993). There is a hint in the literature that
Geobacillus cells may be highly resistant to ionizing
radiation, comparing favourably with cells of Deinococcus
radiodurans in that regard (Saffary et al., 2002). This
observation needs to be confirmed by repetition, and other
Geobacillus isolates should be similarly tested. However,
there is already a considerable body of evidence documenting a bacterial structure that clearly protects genomic
DNA not only from ionizing radiation (Moeller et al., 2008)
but also from many forms of environmental damage
(Nicholson et al., 2000; Setlow, 2006): the Bacillus spore.
It may be that the same mechanisms that protect spores
from these sudden environmental crises could also
dramatically extend their lifespans under ordinary conditions by protecting them from more gradual damage over
time. For once we have an abundance of data for
Geobacillus spores. Because they are a standard biological
indicator for sterilization, their inactivation kinetics when
challenged by steam, dry heat, plasma and various
chemicals have been carefully documented (Kla¨mpfl et
al., 2012; Lambert, 2003; Marquis & Bender, 1985; Mosley
et al., 2005). A convenient concept in sterilization is the
decimal reduction value, or D-value. It denotes the time
increment for a particular treatment, such as incubation
at a given temperature, to reduce the number of viable
spores by one order of magnitude. When samples of the
same spore crop are tested at different temperatures, an
interesting phenomenon is observed: the plot of the log10
of the calculated D-values versus incubation temperature is
linear over all tested temperatures. As incubation temperature decreases, therefore, the time required to inactivate
spores increases rapidly but predictably. Nicholson (2003)
made clever use of these data to estimate the longevity of
spores under temperature conditions typical for a warm
terrestrial climate (25–40 uC). Thermal inactivation curves
for the spores of B. subtilis, a mesophile, required no
extrapolation, because D-values had already been experimentally determined for temperatures in this range. But
Geobacillus spores require much longer incubation times for
thermal inactivation to occur, so Nicholson was forced to
http://mic.sgmjournals.org
extrapolate the available thermal inactivation curves to
obtain an estimate for spore longevity at moderate
temperatures. Yet the result was startling: based on this
extrapolation, the longevity of Geobacillus spores at 40 uC
would be 1.9 billion years, and the longevity at 25 uC even
longer (Nicholson, 2003)! Of course, this estimate of
extreme longevity cannot simply be accepted uncritically,
as Nicholson himself discussed. Other factors besides
thermal inactivation could be at work under natural
conditions, and extrapolating curves is an inherently risky
business. Still, this analysis at least suggests that real-world
longevity of Geobacillus spores could be surprisingly high.
Even if their lifespans are measured in millennia, a very
modest global production of Geobacillus spores coupled
with atmospheric dispersal could lead to the high populations observed ‘in an environment where they cannot grow’.
What can geobacilli teach us?
The genus Geobacillus is a taxon of humble stature.
Although these bacteria may well achieve some level of
importance in biotechnology, in any given environment
they probably serve a minor and redundant function. If the
entire genus were to become extinct in an instant, one
wonders whether Earth’s biosphere would even notice. Yet
consideration of their paradoxically high populations in
some cool and cold environments has perhaps highlighted
some important points. First, there is a need for studies
surveying microbial diversity in a given environment to
take extremophiles into account, especially when culturedependent methods are used. Just because a given category
of microbe would not be expected to thrive in an
environment does not mean that they are not present.
Second, there is a need for a broad interchange of ideas
between aerobiology and other branches of microbiology.
Most of the environments that bacteria occupy can interact
freely with the atmosphere, and this fact needs to be taken
into account if we are to have a complete and nuanced
understanding of bacterial ecology and evolution. Third,
the importance of the bacterial endospore in environmental microbiology deserves more focused study.
Bacillus sporulation has been primarily approached from
a developmental biology and genetics point of view, a focus
that has yielded some impressive accomplishments. But it
is likely that these organisms interact with their environment primarily as spores, rather than as vegetative cells.
This phenomenon has undoubtedly had a significant
impact on the evolution of these bacteria and their
roles in Earth’s ecosystems. Finally, the distribution of
Geobacillus in the environment requires us to regard the
concept of natural selection from a somewhat unconventional angle. The accumulation of large reservoirs of viable
spores in places where they are unlikely to germinate and
grow suggests that ‘reproductive fitness’ sometimes can
mean ensuring that reproduction happens only rarely. In
this regard, the longevity of spores in the natural
environment is a topic that begs for more attention and
careful analysis. One might expect that if this model is
7
D. R. Zeigler
valid, it should also be possible to find other kinds of
‘misplaced’ spores in the natural environment: strict
anaerobes in aerobic environments, halophiles in mesophilic environments, and the like. These possibilities are
largely unexamined. ‘Everything is everywhere; but the
environment selects’ is a well-worn maxim that provided
the conceptual framework for much of microbial biogeography in the 20th century but has been vigorously debated
in recent years (O’Malley, 2008). I feel unqualified to weigh
in on that discussion. But I hope that the continued study
of Geobacillus will provide fruitful insights into the
complex relationship between ecological variation and
geographical distance.
Burrows, S. M., Butler, T., Jo¨ckel, P., Tost, H., Kerkweg, A., Po¨schl, U.
& Lawrence, M. G. (2009a). Bacteria in the global atmosphere – part
2: modeling of emissions and transport between different ecosystems.
Atmos Chem Phys 9, 9281–9297.
Burrows, S. M., Elbert, W., Lawrence, M. G. & Po¨schl, U. (2009b).
Bacteria in the global atmosphere: part 1 – review and synthesis of
literature data for different ecosystems. Atmos Chem Phys 9, 9263–
9280.
Canakci, S., Inan, K., Kacagan, M. & Belduz, A. O. (2007). Evaluation
of arabinofuranosidase and xylanase activities of Geobacillus spp.
isolated from some hot springs in Turkey. J Microbiol Biotechnol 17,
1262–1270.
Cebrian, J. (1999). Patterns in the fate of production in plant
communities. Am Nat 154, 449–468.
Commichau, F. M., Pietack, N. & Stu¨lke, J. (2013). Essential genes in
Acknowledgements
I thank Wayne L. Nicholson, David J. Smith and Frederick Michel for
helping me understand concepts that fall outside my realm of
specialization, with special thanks to D. J. S. for suggesting that the
surface properties of spores could be subject to the selective pressures
of atmospheric transport. Any errors or misunderstandings that
persist in the paper are of course my own responsibility. The Bacillus
Genetic Stock Center is supported in part by a grant from the
National Science Foundation (0234214).
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