FEMS MicrobiologyRex-:ews75 0999) 155-170
Publishedby Elsevier
155
FEMSRE00136
Temperature spans for growth: hypothesis and discussion *
Jtirgen Wiegel
Department of MicrabiologF. Unioersity of Georgia, Athens. GA, U.S.A.
Key words: Thermophiles; Psychrophilas; Anaerobic cryptic thermophiles; Anaerobic extreme
thermophiles; Temperature tolerance; Temperature span
1. SUMMARY
In recent years the upper and lower temperature limits for growth of pure cultures of microorganisms have been extended at least to l l 0 ° C
and - 1 4 ° C, respectively. There are no organisms
which grow at both 0 ° C and 100°C and, therefore, organisms are grouped according to their
ranges of growth temperatures. Thus, the questions of importance are: (1) What is the widest
temperature range (temperature span) over w~ch
a single organism can grow? and (2) How much
can one alter the temperature spans of an
organism.'? The concept of 'cryptic thermophiles' is
used to explain some of the published data on the
latter question. A wider temperature range can be
very important for organisms in various ways,
since it makes an organism more versatile with
regard to changes in the environment. Also, it
enables the organism to utilize a wider range of
ecological niches. Some aerobic and anaerobic extreme thermophiles will grow within a span of
more than 40°C. Furthemtore, such organisms
are regarded suitable for biotechnological applications as well. The following hypothesis is presented and discussed: these organisms have two
* Dedicated to J.L lngraham, at the occasion of his 65th
birthday, who has authored more than 30 publicationson
temperature effects and minimalgrowthtemperatures.
Correspondence to: J. Wiegel, Department of Microbiology,
Universityof Georgia,Athens,GA 30602, U.S.A.
sets of key enzymes, and their synthesis is regulated by temperature. Such organisms are capable
of growing in two different ranges, such as the
mesophilie and thermophilie ranges. The hypothesis is based on the fact that these bacteria exhibit
broken Arrbenius plots (growth temperature versus
doubling time as parameters), and is illustrated
with 'temperature tolerant extreme thermophiles'
as the major example. However, the hypothesis is
not restricted to this group, but is also applicable
to the 'temperature tolerant tbermophiles and
mesophiles'.
2. GROWTH TEMPERA'I ORES
2.1. Upper and lower temperature limits of living
organisms
The records for the upper and lower temperature limits of growth are held by microorganisms
[11. Microorganisms growing at elevated temperatures, such as 60 ° C and above, or at temperatures
below O°C, were regarded as curiosities for many
years. Aerobic thermophilic microorganisms were
described at the end of the last century, and these
were mainly species of Bacillus. The first anaerobic
thermophile described, still validly published [2],
is Clostridium thermocellum [3]. A recent list of
thermophiles with a Topt above 6 0 ° C is given by
Wiegel and Ljungdahl [4]. The list contains 48
aerobic and 32 anaerobic thennophilie eu- and
archaebacteria, demonstrating the versatility of
0168-6445/90/$03.50 © 1990Federationof European MicrobiologicalSocieties
156
Table 1
Definitior~ of organisms according to their cardinal growth temperatures (in °C)
Psychrophiles (kryophiles)
Temperature tolerant mesophiles (psychrotrophs ')
Mesophiles
Thermotolerants (thermodurie 2)
Temperature tolerant 2 thermophiles
Thcrmophiles
Temperature tolerant extreme thermophiles
Extreme thermophiles
Barodiermotolerants 3
Barothermophiles ~
rm,,
<0
<5
>5
-
< 25
> 25
< 45
> 45
?
?
Top,
< 15
> 15
< 45
< 45
> 45
> 45
> 65
> 65
< 104)
> 104)
rm~,
< 20
> 20
< 50
> 50
> 50
> 50
> 70
> 70
> IIKI
> 100
z l~finilion according to Morita [17] based on the Dictionary for Microbiology {18]. Morita defines psych, otrophie organisms as
t ~ which grow at low temperatures, but do not meet the strict requirements given by the above cardinal temperatures of
psyehrophiles; see also Gow and Mills [191 for pragmatic criteria. The symbol ' <' indicates that the given temperature or
temperatures below are fulfilling the definition.
z BrOCk,in his recent book [11 suggests using the older terms (201aurythormal, for organisms with a wide grc~th temperature range.
and nenothermal for those with a nari~w range. Thus. one would replace psyehrotrophic, an unfitting term (cold-eating)
introduced by Eddy [2t], with eurytherrnal mesophiles, and temperature tolerant with eurodiermal and eurythermal extreme
thermophile&
Sletter recently introduced for both the simpler term hyperthermophilic.
thermephilic organisms. Several new, extreme
thermophiles in particular, have been described
since then (see review by Stetter in this issue).
Only a few species of anaerobic, thermophilic and
extreme thermophilic bacteria have been studied
extensively. T h e same is true for psyehrophilie
organisms. For most of these organisms, little is
known about their distribution, physiology, and
extremes o f their individual growth temperatures.
Life is possible as long as there is water. This
assumption is true with some limitation. M o r e
than 25 years ago, the upper temperature for
growth was thought to be around the boiling point
o f water based on the now famous studies of T . D .
Brock and co-workers [5] in Yellowstone N a t i o n a l
Park. Recently, Heinen and Lauwers [6] and esped a l l y Stetter and his co-workers ([7] and this
issue), extended the upper temperature limit for
microbial growth beyond the boiling point of
water. Presently, the highest growth temperature
for an isolated organism is approximately 110 ° C,
reported for the anaerobic archaebacterium, Pyrodietium oceultum [8]. The organism exhibits unusual morphology which may be linked to its
energy metabolism, involving sulfur, at extremely
high temperatures [8,91. Bacterial growth at 120 ° C
has been reported [10], but the characterization of
pure organisms growing at this temperature has
not yet been published. Whether there are
organisms that can grow at 1 5 0 ° C or higher is
subject to discussion and further discoveries.
However, the thermostability of molecules such as
A T P and most a m i n o acids suggests that the upper temperature limit is somewhere between 125
and 1 5 0 ° C ([8,11] and literature cited therein).
As with the boiling point of water, the freezing
point of water is also not the limit for microbial
growth. G r o w t h for microorganisms (unspecified
strains) has been recorded at temperatures as low
as - 1 2 ° C for bacteria [12], - 2 0 ° C for fungi
[13], and - 3 4 0 C for yeast [14]. However, the
growth temperatures below - 1 2 ° C are presently
regarded as unconfirmed [15]. Nevertheless, the
' p r e s e n t record' for growth at low temperatures is
held by eukaryotes and not by arehaebaeteria as
found for the upper temperature limit. Whether a
valid conclusion can be d r a w n from this about the
evolution of life is not clear [16]. Theoretically, the
lowest temperature which might permit growth is
around - 1 3 0 ° C .
At this temperature water
changes to the crystalline or glassy state, and no
'free' water (i.e., no water activity) is measurable.
157
However, usually bacteria require an aw of 0.9,
fungi of 0.8, and the lowest value found was 0.61.
This matter is discussed in detail by Mazur [15].
Thus, the temperatures at which bacterial
growth occurs range from at least - 1 2 ° C to at
least I I 0 ° C . Because no organisms are known to
grow over the entire range, organisms are best
classified according to the temperature range in
which they grow (Table 1).
Although the given boundaries are somewhat
artificial, they still serve their purpose. So far, no
microorganism has been described that has a T,,pt
in the mesophilic range (Topt ~ 40 ° C), and is able
to grow at temperatures of the extreme thermo9bilie range (T,~x >/70°C). Nor has any microorganism been described that has a T,,,pt in the
psyehrophilic range (T~,pt< 15°C) and is able to
grow above dO o C.
While temperature tolerant mesophiles and
tbermotolerant organisms both have a Topt in the
mesopbilic range, the former grows at lower temperatures and the latter grows at higher temperatures than the mesophilic boundary.
2.2. What is the temperature span for growth of the
average organism?
Temperature spans vary and tend to be more
narrow at the upper and lower end of the temperature scale for growth. Spans of 2 0 - 3 0 ° C for
extreme thermophiles and for strict psyehrophiles
[21], and around 3 0 - 3 5 ° C for mesophilic
organisms, are common. Some psyehrophiles do
not grow above ] 0 ° C [22] indicating an even
smaller temperature span. However, unpublished
data from Wiebe (personal communication) indicate the existence of bacteria that are able to grow
at - 3 o C and also at 4 0 ° C (Top~ above 30°C).
These organisms are an example of temperature
tolerant mesophiles (see Table 1). Escherichia colt,
a very versatile organism, has evolved as an
organism able to adapt fast to various stress conditions, and grows between 43.5 [23] and 7 - 9 ° C
[241.
I found (unpublished data) that Methanobacterium thermoautotrophicum
can grow in the
mesophilic temperature range (above 22°C), but
has a Topt = 6 8 / 6 9 ° C and a Tma~ = 78°C, clearly
in the lower range of extreme thermophiles. Thus,
it grows over a temperature span of about 55°C.
This finding might also explain why this extreme
thermophilic species is so widespread in mesobiotic
[251 environments, as well as in mud samples from
lakes of North America. which do not reach temperatures of 25°C and above [26]. Strain AH
(isolated by Zeikus from sewage sludge) does not
exhibit as wide a range, but is able to grow at
30°C (28°C when transferred from 4 0 ° C or
above, but not when subcultured from 28 ° C). The
presence of sterile anaerobic sediments helps the
organism to stay alive during incubations at lower
temperatures (unpublished results). Even at 15°C,
strain JW 501 produces methane at a linear rate
over several months. However, no growth could be
detected in laboratory mineral medium at 18°C or
below, in the presence or absence of yeast extract.
Based on these observations, 1 ask the following questions: Is this range exceptionally large and
is a span of 55 ° C the widest for growth of a single
organism? Can other organisms grow in the same
span or even larger spans if provided with the
right growth conditions (i.e., given the correct
supplements, which they cannot pi'oduce at the
lower or higher temperature)?
At the present time one may only speculate,
since no further data are available on extended
temperature spans. However, to evaluate the possibilities for a larger span, one must look at the
parameters which determine the Tram and T ~ , for
an organism.
2.3. What determines the cardinal temperatures Tin,.
and T~,,?
An excellent and detailed discussion of the
chemical and physieo-ehemical basis of what determines the Tm~n and Tr,~, for thermophiles is
given by Sundaram [27] and by Frommel and
Sander [28]. Further discussion emphasizing various aspects of lipids and membranes are summarized by Russell [29,30]. Therefore a detailed
discussion is not given in this article. Many of the
parameters discussed for thermophiles are also
valid for the cardinal temperatures of mesophiles
and psyehrophiles. One has to keep in mind that
the limiting factors for those two cardinal temperatures are independent. While changing some biochemical parameters may ehat~ge both tempera-
158
tures, in general the changes of those temperatures
will occur independently. This is especially true if
the property of one of the compounds determining
T=~n or Tm~ is changed. Usually, it is not the same
compor.ent that is responsible for both temperature limits. Although it is possible that temperature-induced conformational changes regulate an
enzyme to function at different temperature
ranges.
The important parameters governing T,,~ and
Tmin include (i) the fluidity of lipids and membrane components (ratios of saturated and unsaturated fatty adds, polarity of phospholipids,
etc.); (ii) the forces which affect tertiary and
quaternary structures of enzymes, their capability
to undergo conformational changes and, to stay
flexible to facilitate catalysis; These forces include
the hydrophobie/hydrophilie interactions of the
protein side chains governing the stability of the
tertiary structure, as well as the stabilizing associations with other proteins or membrane components; (iii) temperature dependent interactions of
regulatory compounds at the enzyme, ribosome,
R N A or D N A level, causing a breakdown in regulation such as a total derepression or repression
(this type of event can lead to an accumulation of
poisoning metabolites, or drain off energy-rich
compounds by favoring one reaction at the expense of other essential reactions); and (iv) changes
in transport of substrates, ions or products.
From this general list it is evident that factors
such as changes in solutes and in ionic strength
[31] and pH in the cell can have a drastic effect on
the cardinal data, since they are influenced by
most of the above mentioned forces. As stressed
by Sundaram [27], the energy required for stabilization of most thermostable enzymes is low, less
than 13 kJ, thus minor changes in the above
parameters can have an effect on Tmln and Tmax.
It is clear that the choice of medium will frequently have a strong influence on cardinal temperatures. In instances such as those described by
lngraham and Marr [24] or Run and Shani [321,
where the synthesis of one or two amino acids
became impaired at T~n and Tmax, respectively,
the addition of the non-synthesized amino acid
will extend the temperature range l'or growth until
another limitation becomes effective. We assume
that this is also the explanation for the enhanced
growth of M. thermoautotrophicum below 3 5 ° C
down to 2 2 " C by addition of yeast extract (0.1%)
or sterilized anaerobic sedimen:s (unpublished
data). Although methane was formed below 15 o C,
growth 0nerease in cell number) was not observed
below 20°C, indicating that the limitation is not
in the enzymes or membrane functions required
for methane formation, but that it is in the biosynthetic capability for some other biomolecules required for growth and multiplication. Below 15 o C,
other limitations than that are effective. Much
more work is needed to clarify this situation.
Similar observations were made by Miller et al.
[331. They found that by increased helium pressure
(250 atm), methanogenesis could be extended
above the Tmax of Methanococcus jannaschii, to
98 o C, but they could not raise the Tm~ for growth.
Sometimes other circumstances may be limiting
factors. The observed upper temperature for
growth of Pyrodictium may not be caused by die
instability of components of the organism at higher
temperatures, but may be due to the melting of
the sulfur, which in that form may no longer be
utilizable by the organism [7]. At 114.5°C, which
is the 'natural melting point' of the beta(monokline)- form of sulfur, the Ss-rings convert to the
liquid lambda sulfur, consisting of high molecular
chains or rings of 1000 or more sulfur atoms.
The decline in growth rates with increasing
temperature above optimal temperature is usually
sharp. Although the range varies between 5 and
12°C, the sharp decline allows the derivation of a
relatively exact ( + 1 - 2 ° C ) Tm~, in contrast to the
Tmi,. The difference between Train and Topt is
frequently 15-25 ° C. The determination of T~n is
often less aeenrate due to the slow growth rates
over a wider temperature range. Not all organisms
show clearly an abrupt cessation of growth, so
that Tmln may not be easily obtained from the
Arrbenius graph. In some instances, growth may
even stop more abruptly than the growth temperature curve would suggest. Ratkowsky et al. [34]
investigated a variety of organisms. From their
curves they calculate the cardinal data, which as
first approximation values are useful, but still may
be inconsistent because the effect of inte.anediary
plateaus was not considered, nor was any possible
159
sudden change in the rate limiting step at lower
temperature ranges. This is especially important
when low growth rates (e.g., t o of 100-200 h) are
not determined experimentally. Broken Arrhenius
graphs are not characteristic of thermophiles but
occur with organisms from all temperature ranges.
3. CHANGES I N RANGES OF G R O W T H
TEMPERATURES
3.1. Gain and loss of the property "thermophilic"
Presently, being thermophilic or extremely thermophilic or being psychrophilic is regarded by
mozt investigators as an intrinsic property of the
corresponding bacteria. Thus, a tree mesophile
cannot gain thermophily by adaptation or single
step mutations [4,35-39], nor ;.s there abundant
evidence for the existence of 'thermal adaptor
genes', as suggested by Lindsay and Creaser [40].
The Tmax and Topt values can be raised to some
extent by manipulating growth c~nditinns, and by
increasing the availability of gaseous substrates,
by applying higher gas pressure (only for psychrophiles [41-43]), providing suitable surface for
adsorption, optimal ionic strength, etc~ However,
these changes are relatively small.
The route of genetic engineering of mesophiles
into extreme thermophiles is possible in theory but
not yet in practice, because many unknown properties may need to he transferred in order to
change the organism into an extreme thermophile.
Several genetic approaches which appear to be
successful ?,ave been reported [44,451. These inctade the engineering of a disulfide bond into T4
lysozyme, rendering it a thermostable enzyme. A
chemical oxidation to the disulfide increased the
thermostability drastically, whereas the reduction
~tto SH-groups made the enzyme as labile as the
wild-type enzyrc.~ [46]. Droffner and Yamamoto
(45] concluded from their Bacillus mutants that
t-~v~ genes are responsible for the conversion into
thermophiles. The requirement of two mutations
explains why a low frequency of mutation to
thermophilic growth is observed. Generally, one
can except that even more than just two mutations
are required.
3.2. Cryptic thermophiles, another view on the conversion of mesophiles into thermophiles
Thus it has been claimed in the fiterature that
conversion of organisms from one group to the
other, such as psychrophiles into mesophiles (e.g.
[47]), and mesophiles into ff~ermophiles or extreme
thermophiles (e.g., [6,26,35-39]) is possible
through simple mutations a n d / o r adaptations. In
my opinion, these conversions are only possible if
cryptic thermophiles or cryptic extreme thermophiles are used. Cryptic thermophiles are
organisms which (I) possess mainly ~hermostable
enzymes, lipids, membrane components, and
means to stabilize their D N A at the elevated temperatures; or (2) organisms that have the information to synthesize thermostable isoenzymes a n d / o r
alter their lipids into the thermostable form but
apparently are not using it (e.g., Tsien et at. [48]).
Both classes of bacteria can not grow at elevated
temperatures because one or two of the enzymes
lack thermostability, or one of the lipids causes
too much flnidi:y of the membrane with increasing temperatures. If one adds to these organisms
the gene product or th~ miz:,ing component not
formed by the impaired enzymes, engineers the
proper corresponding enzymes into the organism,
or changes the lipid composition in the thermostable direction, the barrier may be overcome and the
organism may grow at the higher temperature. In
these instances, remarkable increases in the Tma~
are possible. In all these instances, the new information can be located on a plasmid, so that "thermophily could be cured" with acridine orange, as
described by Stahl and Olssen [44]. Similar corresponding arguments can he made for lowering the
T~, by overcoming the limitations just mentioned.
The possibility that a greater num~."r of
mesophilic organisms, especially those helongin 3
to the genera Bacillus and Clostridium, indeed are
cryptic therrnophiles increases if one assumes that
thermophiles are the origin of marly of our present
day microorganisms [16]. However, this view is
controversial as expressed by Zeikas [26]. The
existence of cryptic enzymes, (enzymes which are
not expressed at all under normal growth conditions) has been demonstrated recently for
'fermentative enzymes: in bacteria, so far regarded
as strict aerobes [49].
To prove or disprove this explanation, one needs
an extensive knowledge about the genetic information of the organism for which such changes in the
growth range have been described~ Without this
information, caution mast be taken regarding
claims of being able to change a mesophile into a
thermophil¢ etc. For industrial purposes and applications, this discussion is academic. There is
always the possibility that the microorganism of
industrial interest may have cryptic thermostalite
traits. Therefore, if a higher growth/fermentation
temperature is advantageous, the use of undirected mutations and selection by using increasing
temperatures may he the route to unlocking these
traits.
In contrast to expression of thermophilic enzymes in mesophJles (e.g., Eseherichia coil or
Bacillus subtilis), little is known about the expression of mesophillc gene products in thermophiles.
Enzymes from thermophiles, when expressed in
mesophiles, usually maintain their thermostable
properties. However, little is known of how stable
mesophilic (thermolabile) enzymes will be when
expressed in thermophiles. Such an example is the
transformation of 11. stearothermophilus reported
by Koizumi et al. [50], in which the transformants
gained the penicillioase activity from Bacillus
licheniformis. However, although B. Iieheniformis
is regarded as a mesophile, it contains other thermostable enzymes (used in the brewing industry)
and the stability and activity of the pcuicillinas¢ is
dependent on the stability and on the rate of
retaining the plasmid in use.
The fate of mesophilic genetic information in
thetmophiles is of interest with respect to biomass
conversion processes using various waste materials. Presently it is generally assumed that there are
no true thermophilic pathogens. If one uses a
thermophdic fermeutation (e.g., 60oC), most
vegetative pathogenic organisms will he killed, but
the number of spores of pathogenic clostridia may
at best be reduced if long retention time3 are
involved in the fermentation process. However, in
reality this may work out quite differently. Let us
assume that genetic information of mesophilic
origin can be maintained in a thermophile, espe-
cially in those bacteria termed temperature tolerant
thermophiles (Table 1). Efficient biomass conversion, especially for solvent or chemical production, requires high productivity and thus high biocatalyst concentrations (high cell density). If cell
recycling is employed, cell densities of more than
1012 cells/ml can be reached. In such a system,
free D N A or phages can occur. Although relatively high DNAse activity can be expected, an
uptake or a transf~ via phages may occur. In a
large waste fermentor with thousands of liters of
cultures containing 10 Is cells, very rare ovents can
occur. If slaughter house waste or agricultural
waste containing pathogens is used, there may
well be a slight though real possibility that a
thermophile will incorporate the genetic information from a mesophili¢ pathogen for producing a
toxic protein. If the thermophile can keep the
information and can passs it along to its daughter
cells, severe impfications have to 5e envisaged
even if the toxic gene product is not formed at the
elevated temperatures. Thus, if the cells are discharged, or pass through a stage of reduced temperatures during the process, the gene product
could be formed and causing a health hazard. This
becomes even more likely if temperature tolerant
thermophiles (see below) and thermotolerant
mesophiles are involved. These organisms can grow
both in mesobiotic and (extreme) thermobintic
[25] environments. We have started to investigate
this question.
4. CONCEPT O F "TEMPERATURE TOLERA N T EXTREME THERMOPHILES', A N ATTEMPT T O EXPLAIN EXTENDED TEMPERA T U R E SPAN A N D BROKEN A R R H E N I U S
PLOTS
The following hypothesis was developed based
on results obtained with the extreme thermophilic
anaerobic eubacteria, ~ermoanaerobium et,hanolicus [51] and Clostridium thermohydrosulfuricum
[52], C. thermobutyricum [53] and with the
archaebact~ium M. thermoautotrophicum, as well
as on data published about the properties of enzymes from Bacillus stearothermophilus [35]. This
hypothesis attempts to explain what determines
161
~
-tl
-o5
~"
~
, o
-io
o
.~.0
31×10 ~
~.9 1/T (*K-11
Fig. 1. Arrheniusplol (natural log of 1/ta; ra = doublingtime
versus 1/growth temperature in °K) for Cl~tridium therm~:hydmsulfurieura strainJW102{O)and finalopticaldensityat 600
nm in relationto growthtemperature ( c, C) (zx).
2.9
the growth temperature span for a single organism,
and what steps are necessary to extend the span.
4.1. The hypothesis
Bacteria which grow within an extended temperature span of 4 0 ° C or more should exhibit a
relatively pronounced biphasic curve in the
Arrhenius graph. This effect should be due to the
fact that these organisms contain two sets of
otherwise rate or range limiting enzymes, one set
for the lower range (in the ease of C. thermohydrosulfuricum, about 35-68°C; Fig. 1) and one for
the higher range (50-78* C).
Similar temperature spans for growth of 4 0 ° C
or more, and concomitant Arrhenius plots with an
intermediary plateau, are not unique to the extreme thermoph~,lie anaerobes. Other groups of
bacteria arc expected to contain species with those
properties. Organisms with extended temperature
spans will then be called 'temperature tolerant
organisms of the mesophilic, thermophilic or extreme thermophilic growth range', respectively, depending on the temperature range in which they
exhibit their TopI,
4.2, Critical evaluation of the working hypothesis
4.2.1. Supportive arguments
(1) The hypothesis assumes that some organisms
contain enzymes and structural proteins, as well as
lipid components, that are more stable than the
upper temperature limit of the organisms indicate.
However, a few temperamre-sensilive gone products limit the growth range. These gene products
can include one or more enzymes for catabolic
reactions, for energy generation, or for the formation or modification of lipids and/or other cell
components which exhibit increased temperature
stability or which allow the cell to grow at the
higher temperature. In the course of evolution, the
temperature tolerant organisms have acquired the
necessary information for producing a second set
of limiting enzyme(s) with a higher temperatur,~
stability or cell components with a higher t~m:,,,erature stability. Another explanation ,nay be that
these organisms were originally thetmophilic, and
then acquired the information to synthesize enzymes with a lower T~i.. In this manner, they
could overcome the limitation set by the enzymes
(lipid components) which became unfunctional
('freezing') at lower temperatures. With the additional enzymes, the organisms could survive at
lower environmental temperatures, as the global
temperature decreased.
(2) Many known anaerobic and aerobic thermophiles have a Tm,~ around 65-69°C and a T,,,
around 35-38°C [4]. Several anaerobic organisms
and several Bacillus species have been described
that have about the same Tram bat have their Topt
around 6 7 - 6 9 ° C and their T ~ at 75-78°C.
Thus, these organisms grow within a temperature
range of more than 40 ° C. Some of these bacteria
are listed in Table 2, including the anaerobic
eubaeteria C. thermohydrosulfuricum, T. ethanolicus, the aerobic eubacterium B. stearothermophilus, and the anaerobic archaebacterium M. therraoaulolrophic'um. In contrast, most bacteria ineluding bacteria such as the very extremely thermophilic anaerobic archaebacteria P. occultum
(Tmax) about l l 0 ° C ) , T. tenax and M. fervidus
(Tm~x 100°C), and the moderate thermophilic
eubacteria C. thermocellum, Desulfotomaculum
nigrificans and C. thermosaccharolyticum ( T ~
67-69°C) grow within much narrower temperature spans of less than 35°C. An extended list of
growth temperature ranges has been compiled by
Wiegel and Ljungdahl [41. Except that the upper
temperature limit is held by an arehacbacteriam,
the known kryophiles are euhacteria, there are no
162
obvious differences in the temperature spans found
with eubacteria and archaebacteria. Whether or
not there are really distinct j u m p s in the 7ma~ as is
indicated ~rom the above list (e.g., 65--70 and then
7 5 - 8 0 ° C ) , or whether this is due to limited precision of the data and a low number and variety of
isolated organisms, needs to be seen. Unfortunately, for a great number of organisms, the
cardinal temperatures and the temperature responses over the whole temperature range have
not been determined in detail, especially for the
minimal growth temperature, Tmin. T h i s is partly
doe to the fact that more interest was placed on
other features of the isolates, e.g., their arcbaebacterial nature or other special properties. Thus,
the Tm~ and T~i. values reported in the literature
have not always been investigated thoroughly in
small temperature increments; in several cases
temperature intervals of more than 5 (up to 1 0 ) ° C
have been used. Such intervals are too far apart to
obtain the correct cardinal temperature data, T~i .
and Tm~x, needed to answer this question.
(3) O f the organisms listed in T a b l e 2 and for
which we have determined an accurate temperature curve, we observed that the relationship between the growth temperature and the doubling
time led to a biphasic curve. This was d e a r l y
shown using an Arrhenius plot (Fig. 1; [52]). The
remaining bacteria have yet to be investigated.
N o n e of the investigated organisms with a significantly smaller temperature range (e.g., C. thermocellum, C. thermoautotrophicum) exhibited an intermediary plateau. T h e tested organisms included
M. thermoautotrophicum strain J W S l 0 . with r.
lower Tmax of only 6 9 - 7 0 ° C and a temperature
span of 3 5 ° C . T h i s strain lacks a main component
of the typical M. thermoautotrophicum lipid spect r u m (Wiegel and Mnkula, unpublished results).
Strain JW510 is otherwise a typical M. thermoautotrophicum strain with respect to RNA-poly-
Table 2
Examples of thermophilie organisms wilh extended temperalure (°C) spans that exhibit intermediary plateaus in the Arrbenius
graphs
Organism
Desulfuroc¢occus mobilis
1). mueosus
Methanoeocctt-vjanm~dlii
Thermodesulfotobacterium
commune
Methanobacte~ium
thermoautrotrophicum
CIostridium fervidur
Bacillus schlegelii
Bacill~ stearothermophilus
Thertnoanaerobacter ethanolic~
Clostridium
thermohydrosulfuricum
Thermoanaerobium brockii
Clostridium
thermosulfurogenes
Acetogeniur~l kivui
Methanococcus
thermolithotrophicus
Thermobacteroides
aceroethylict~s
T~ax
tO0
tO0
95
85
75-78
< 80
< 80
70-77
78
Tmin
Span I
55
55
(50)
45
45
(45) z
40
45
22 (30) 3
37
35
35
35
55
4-40
+ 40
42
43
Intermediary plat~u in Arrhenius
graph demonstrated
n.d,
n.d.
n.d.
38-43
55-60 (weak)
n.d.
n.d.
55-60
55-60
78
77
35
35
43
42
55-60
55-60
75
75
35
35
40
40
53-58
57-60
> 70
30
> 40
n.d.
75
36
39
n.d.
Only the greatest span is given, for references to cardinal lemperatures see Wiegal and Ljungdald [4].
Approximated values.
3 Strain AH did not grow below 30°C.
163
/
i/
f'~
Temperature
Fig. 2. Temperature dependence of the cellulose dcgradanon
by growing cultures of C therm~ellum with a Tmax 69DC (@),
Clostridum spec. with a T~a, 76°C (El) and the coculture
(---). The relativecellulose degradation rate reflects growth
of the cultures as indicated by an increase in cell number.
merase (Stetter, personal communication). As predicted by the hypothesis, this special strain does
not yield a biphasie Arrhenius graph. The same is
true for the temperature-sensitive mutant isolated
in our lab, and listed below.
(4) With C thermohydrosulfuricum JW102 and
the type strain E100-69, it has been demonstrated
convincingly that the broken Arrhenius plots were
obtained with pure cultures [52]. The same is true
for M. thermoautotrophicum and T. ethanolicus,
and for C. thermobutyricum, a moderate thermophile growing between 61.5 and 2 6 ° C [53]. However, the proposed concept can be illustrated by
using two bacterial strains with different temperatare optima and comparing the temperature curve
of the pure cultures and of the co-culture (Fig. 2).
C. thermocellum JW20 and CIostridium spec.
JW51 are both cellulolytic anaerobes with Top~ of
59 and 69 ° C, respectively. Their growth temperature ranges are relatively narrow when compared
to those of the extreme thermophiles with a broken
Arrhenius plot, but similar to those of other thermophiles without a broken Arrhenius plot. In
accordance with the observation that the more
thermophilic organisms frequently have shorter
doubling times [26,27,54,55], strain JWSI grew
faster. The mixed culture yields a broken Arrhenius
graph similar to those seen by the above-mentioned organisms (Fig. 1). Obviously, the two cellulolytic bacteria have enzymes with different temperature characteristics. The distance between their
temperature optima, and the differences in shape
of the two temperature curves, determines how
strongly pronounced the intermediary plateau will
be in the graph of the mLxed culture. The same
holds true for an organism containing different
enzymes, as suggested by the hypothesis. In some
organisms (e.g., M. thermoautotrophicum strain
H and JW501) the intermediary plateau is minor,
while in others ((7. thermohydrosulfuricum JW102),
the intermediary plateau is very distinct.
(5) With C. thermohydrosulfuricum and M. thermoautotrophicam, the presence of two different
sets of enzymes has been indicated by temperature-dependent changes in the protein pattern obtained in two-dimensional gel electrophoresis [56].
Other than changes in the intensity (indicative for
the concentration) of some stained proteins, several
proteins which are present in the protein map of
the cells grown at 5 0 ° C (below the intermediary
plateau) are absent in the map of the cells grown
at 7 0 ° C (above the intermediary plateau), and
vice versa. However, more data have to be collected, especially for the 'temperature range'
mutants of M. thermoautotrophicum described below.
These mutants differ from other mutants we
have obtained which display changes in their temperature range, but were not isolated as temperature sensitive mutants. The ethanol-producing
mutants (high ethanol and high glucose/starch
resistant) of T ethanolicus exhibit the same biphasic response to temperature as the wild type,
although their protein patterns were significantly
modified. The Tm~. shifted from 35 (for the wild
type) to 3 9 - 4 0 ° C and the maximal growth temperature dropped from 77-78 (wild type) to 747 5 ° C for the mutant (Carreira and Ljungdahl,
unpublished results).
During our studies (unpublished results), we
also compared the morphological features of our
strains, to help detect changes in the temperaturesensitive mutants. All tested strains (AH, JWS00,
JW501, JW510) contained two types of up to 7
Poili, mainly monopolar inserted pill of the 60
Angstrom type, 3 - 4 p m long, and 4-10 p m thin
(about 30 Angstrom thick) fibrils which were peritrichously inserted. No changes at low or high
temperature were encountered. Contrary to an
164
earlier report [57], we could not correlate drastic
changes in morphology of the cells with changes
in growth temperature. The strongly twirled cells
are more an indication of the physiological state
and the age of the cells than of temperature. We
obtained even below 300C straight cells when the
cells were growing in the logarithmic growth phase.
A strong emphasis was put on the purity of the
investigated strain; repeatedly single cell colonies
were isolated at several temperatures before experiments were carried out.
(6) The observed response of M. thermoautotrophicum to various antibiotics differs with the
growth temperature. The temperatures for changes
in the susceptibility of M. thermoautotrophicum
antibiotics coincided with the appearance of the
intermediary plateau, thus supporting the hypothesis of major changes when growing at the
extremely thermophilic temperature range. For example, 5', 5', 5'-trifluoro leucine (leucinc antagonist) kills cells growing above 700C, but has very
little effect at temperatures below 55°C. Unfortunately, this compound is presently unavailable
for further experiments; we therefore examined
several common antibiotics (Sharma and Wiegel,
Ann. Meeting Am. Soe., 1987). Neomycin,
kanamycin and amphotericin B displayed effects
similar to the leucine antagonist at a level of 250
jtg/ml and a temperature of 72°C, but had no
effect at 5 7 ° C and 48°C. Metronidazole, streptomycin and trimethoprim killed at 1, 100 and 100
/~g/rnl respectively at 72°C, but required 25, 250,
and 250 F g / m l respectively, at 48*C and 57°C.
Bacitracin, ampicillin, ehloramphenieol, tetracycline monensin, novobiocin and other antibiotics showed no variation in the bactericidal concentration required at those temperatures. These
facts were applied to develop methods for isolating temperature sensitive mutants, which may also
be usable as a general enrichment method for
anxotrophie mutants of M. thermoautotrophicum
in analogy to the penicillin technique of eubacteria.
The technique was first tested with a co.culture of
strain JWS01 (Tm~~ 78) and JW510 (T~a~ 69"C).
After incubation at 7 2 ° C in the presence of
streptomycin and trimethoprim, the subsequent
subcultures did not grow at 72°C, indicating that
all cells of JWS01 were killed. Growth occurred at
60°C, indicating that cells of JW510 survived.
Consequently, mutants of strain JW501 not able
to grow at 7 2 ° C were isolated. From 10 mutants
isolated independently, two reverted and grew at
72°C. Two mutants were checked in the temperature gradient incubator. The Topt for the two
mutants dropped from 690C to 5 5 ° C and 50°C,
and T ~ shifted from 7 7 - 7 8 ° C to 68-69°C, both
as predicted by the hypothesis. Both mutants also
grew at 25°C, indicating that the lower temperature range was not affected to a great extent, if at
all (manuscript in prep.). The isolation of mutants
not able to grow within the lower temperature
range has not been done yet, but this should be
possible using lasalocid or other antibiotics. The
detailed characterization of the mutants in respect
to the protein pattern is required. Furthermore, we
are in the process of analysing whether the mutants
have a changed lipid profile similar to the isolated
natural mutant from Yellowstone National Park
(strain .l'W510). The recently isolated strain JWS10
of M. thermoautotrophieum from hot springs of
Yellowstone National Park lacks the intermediary
plateau in the Arrhenius plot and a major component of the glucoproteins found with the other
tested M. thermoautotrophicum strains AH, JW500
and 501. This strain has only a Tm~~ of 700C and
a temperature span for growth of about 350C.
The other strains of M. thermoaatotrophicum described all have a Tm~x of 7 7 - 7 8 ° C and a span of
about 4 3 ° C for growth.
(7) C. thermohydrosulfuricum I51] exhibits a distinct temperature dependence in spore formation.
If a growing culture is cooled from temperatures
above the intermediary plateau to temperatures
below the plateau, the culture will sporulate up to
30% in liquid culture. At other temperatures the
sporulation in liquid medium is less than 1%.
(8) C. thermohydrosulfuricum (Wiegel and
Ljungdahl, Ann. Meet. Am. Soc., 1979) and T.
ethanolicus [51] each have two different ferredoxins which vary in amino acid composition and in
the temperature stability of the protein-(Fe-S)duster association. The four iron ferredoxin is
stable for 2 h at 80°C, whereas the eight-iron
ferredoxin is labile and disintegrates at similar
rates as the ferredoxin from the mesophilic C.
pasteurianum.
165
(9) B. stearothermophilus only produces ethanol
anaerobically above 5 0 ° C [58] concomitant with
changes observed in the enzyme patterns [59,601.
For example, Brown et al. [61] also noticed that
the enzyme pyrophosphatase (EC 3.6.1.1.) has different thermal stabilities depending on the growth
temperature of B. stearothermophilus. Amelunxen
and Murdock [37] summarize the effect of growth
temperatures on the formation of the three
aminepeptides, at dear dependence was observed.
Several enzymes of B. stearothermophilus (see review by Ljungdahl [35]) have their optimum either
at 5 0 - 6 0 ° C or above 70°C, or exhibit changes in
their properties at 55-60°C. These include
changes in the mode of feed-back inhibition or
changes in the substrate saturation curves from
hyperbolic to sigmoidal. In those cases that two
enzymes were found, they had different thermostability. Unfortunately, there exist only a few
precise experiments on the temperature dependence of the synthesis of those enzymes; in must
cases, only the differences between cells grown at
37 and 55°C were studied. Until now no one has
made a connection between temperature effects on
enzymes of B. stearothermophilis and the occurrence of the intermediary plateau in the Arrhenius
plot. It is of interest that B. stearothermophilus has
only one extremely thermostable (4Fe-4S)-ferredoxin. One could speculate that this organism
needs only a ferredoxin for its anaerobic growth
occurring above 50 ° C (or vice-versa). On the other
hand, ferredoxin can be regarded as a protein,
which does not undergo the eonformational
changes of most enzymes in functioning as effective catalysts.
(10) B. caldotenax grow~ over a temperature
span of about 40 o C and thus a biphasic Arrhenius
plot is expected. It has two glucose 6-phosphate
isomerases: one is produced at growth temperatures between 30 and 5 0 ° C and exhibits low
thermostability, and the second is produced between 60 and 70 * C, and exhibits high thermostabUity.
4.2.2. Critical arguments
(1) It should be stressed that I do not propose
any relationship between the occurrence of the
two temperature optima or the intermediary
plateaus and the structural changes and anomalies
of water, as they were suggested 25 years ago [62].
Those effects have not been reaffirmed.
(2) The forwarded hypothesis neither implies
that only organisms which can grow over extended
temperature spans have broken Arrhenius plots
(growth parameter versus growth temperature), nor
that all these organisms with biphasie Arrhenius
plots have to have two sets of enzymes, There are
several organisms with reported temperature spans
for growth of about 3 0 " C that exhibit broken
Arrhenius plots. In many cases, they have been
drawn as broken lines. The break point is called
Tom (critical point; [63]). However, in several cases
the experimental data clearly indicate an intermediary plateau a~d not the existence of a sharp
breaking point. These organisms should be
analysed further.
(3) It should be mentioned that there are other
possible explanations for the broken Arrhenius
plots of growth temperature versus growth parameter: (i) they might also be due to temperaturedependent activity changes of enzymes responsible
for the rate-limiting step of the bacterial growth.
A temperature-dependent conformation change of
the rate-limiting enzyme could cause higher activity or higher temperature stability, or another enzyme in the bacterium could become rate limiting.
(ii) The broken graphs might be the result of
temperature-dependent changes in the structure of
membranes and lipid layers. (iii) Changes in the
membrane structure and thus changes in the thermostability of membrane-associated or membrane-bound enzymes could be responsible for the
observed effect. There is also the possibility that
in some bacteria all enzymes are stable over the
entire extended temperature span of 40 o C or more
(for examples of such enzymes see review of
Ljungdahl [35]). In this case the enzymes would be
synthesized in amounts needed at the particular
growth temperature and the corresponding growth
rate (e.g.B. stearothermophilus, [59]). This would
lead to a temperature span for growth of more
than 4 0 ° C but without intermediary plateaus in
the Arrhenius plot, and it could also lead to a
wave-type graph as has been found with several
enzymes [64,651.
There is another important aspect which can be
166
discussed here only very briefly. Enzymes are proteins with at least one catalytic- center, but often
contain additional binding sites for effeetors (regulating molecules). Allosterie enzymes are characterized by their property to undergo conformational changes. However, 'simple' enzymes are
somewhat flexible molecules, too. Smaller structural changes are involved in substrate binding (e.g.
induced fit theory of Koshland and co-workers)
and during catalysis. The flexibility of dm protein
chain or the subunit structure often triggers the
limited thermostability at higher temperatures. A
decreased flexibility at lower temperatures is
thought to be the reason for the Tmln of an enzyme. Through temperature-induced conformational changes, an enzyme can exhibit a relatively
broad temperature range. In one conformation,
the enzyme is flexible enough to function at the
low temperature, while in another conformation, it
is rigid enough to be thermostable at the
higher temperature. Thus, substrate-induced
changes coupled with temperature-induced effects
could lead to biphasic Arthenins plots without
involving two sets of enzymes.
(4) Two or more phenomena can be responsible
for the extended temperature span of some
organisms. No uniform biochemical property has
been found for the thermostability of proteins but
organisms have developed various ways to deal
with the individual problems of thermostability
[35,38,39,60,66-68]. Thus, for one reaction an
organism may have two enzymes with different
temperature stabilities, for another reaction it may
use conformational changes, and for yet another
reaction it may possess an enzyme which is thermostable beyond the observed temperature range
for growth.
(5) As indicated earlier, broken Arrhenius
graphs for growth are not restricted to extreme
thermophiles. Similar graphs have been observed
with mesophilie and psychrophilic bacteria [69,70].
These authors have drawn different conclusions
from their experiments than are expressed in the
present hypothesis, but their results are not necessarily contradictory, as discussed above. Several of
the mesophJlic organisms grow with temperature
spans of more than 40°C; for others the cardinal
temperatures Tmjn and T=a~ have not been fully
determined.
Differences in the cellular content of proteins
that affect growth temperature have been demonstrated with the 'common mesophile' Escherichia
eoli. The shift from 28 to 4 2 ° C elicits a rapid
change in the protein pattern (as shown with the
two-dimensionai gel electropfioresis) leading to the
elucidation of heat shock and stress proteins. The
arguments of heat shock response and its regulation can be used against as well as in support of
the above theory. The Arrhenius plot for E. coil,
as shown by Mohr and Krawiee [69], exhibits
obvious deviations from a straight fine; the authors
chose to draw a broken plot with a T~t around
290C. The data indicate one or two plateaus (or
depreviations) in the curve around 2 5 ° C and
40°C. The second optimum (above 4 0 ° C ) is not
as visible as for (7. thermohydrosulfuricum or T
ethanolicus. The temperature span for growth of
E. coil is about 40 o C, depending on the published
data of Tmin. Unfortunately, Molar and Krawiec
[69] did not study the lower temperature ranges of
the mesophiles they investigated. Thus, for
mesophiles there are fewer data available to discuss in relation to the maximal temperature spans
and the hypothesis presented here.
The heat shock or stress proteins have not been
studied in detail in organisms with intermediary
plateaus. The study of heat shock proteins could
help to prove the forwarded hypothesis. The formation of heat shock proteins (temperatureinduced stress proteins) is mainly a temporary
response to a temperature shift and thus a different process from the one forwarded in the hypothesis. The 'induction' of those gene products
which are synthesized to overcome the limitation
are more or less expected to stay at a constant
level similar to the other already temperature-stable gene products of the organism. If the above
hypothesis is correct, the formation of temperature-induced stress proteins should occur above
the intermediary plateau (e.g,, for M. thermoautotrophh:um around 67-70°C), if the organism
'evolved' from a mesophile by adding some genes
to grow at the higher temperature (69-78"C). On
the other hand, if a thermophile is aicered for
167
growth at lower temperatures, stress proteins are
not expected to be formed at or above the intermediary plateau region. However, no studies in
this direction have been reported.
ble that microorganisms would grow at 110 ° C (or
maybe even above), as we now k n o w to be true.
ACKNOWLEDGEMENT
5. C O N C L U S I O N S
The observation that some M. thermoautotrophicum strains grow between 22 and 7 8 ° C , and
thus in a temperature span of about 5 5 ° C , led to
the question: W h a t is the maximal span over
which a single organism can grow?. T o the knowledge of the author, 5 5 0 C (reported here) is currently the most extended range. T h e ability to
grow over such extended temperature ranges was
connected with the observed broken Arrhenius
plots and a hypothesis was forwarded. T h e hypothesis suggests that the organism with an extended temperature range may possess two different enzyme sets for the otherwise limiting reactions: one set for the lower and one for the higher
temperature range. The temperature ranges are
indicated by the temperatures below and above
the intermediary plateau in the Arrhenius graph.
If the assumptions are correct, more organisms
should be found growing over a wide temperature
range. Furthermore, it should b e possible to extend the growth temperature spans of some
organisms in both directions, especially using the
cryptic extreme thermophiles. Cryptic thermophiles are those organisms which could grow at
higher temperatures than their Tm~, if it were not
for having one or two gene products that are not
thermostable. However, the different requirements
to maintain protein flexibility and stability, and
the correct fluidity of membranes and their components at low temperatures (e.g. below 5 o C) and
at higher temperatures (e.g. above 7 5 ° C ) , m a k e it
very unlikely that organisms exist that could grow
over a temperature span of 7 5 ° C or more. Such
organisms would need to have the information to
synthesize cell components for psychrobiotic as
well as extreme thermobiotie temperature ranges,
and perhaps even for some between. T h e presence
of so much information (temperature isoenzymes)
in one organism is unlikely, but we cannot know
for certain. Ten years ago, it was thought impossi-
T h i s research was supported by a grant from
the U.S. D e p a r t m e n t of Energy ( D E - F G 0 9 - 8 6
ER13614).
REFERENCES
[1] Brock, T.D. (1986) Introduction: an ovendew of the thermophiles, in Tbermophiles: General, Moleodar, and APplied Microbiology, (T.D. Brock, Ed.), John Wiley and
Sons. New York.
[2] Skennan, V.B.D., McGowan, v. and Soeath, P.M. (1980).
Approved list of bactelial names. ASM, Washington, DC.
[3l Viljcen, J.A., Fred, E.B. and Peterson, W.H. (1926). The
fermentation of cellulose by thennophilic bacteria, J. Agri.
Sci., 16,1-17.
[4] WiegeLJ. and Ljungdahl, L.G. (1986). The importance o1
thermophilic bacteria in biotechnohigy. CRC-Crit. Rev.
BiotechnoL 3, 39-107.
|5l Brock, T.D. (19"/8)Thermophilic Mioroorganimmsmad Life
at High Temperature. St~tingerVerlag, New YoA..
[6] Heinen, W. and Lanwers, A.M. (1981) Growth of bacteria
at 100°C and beyond. Arch. Mierobiol. 129,127-128.
[7] Steuer, K.O. (1982) Uhrathin mycelia-forming organisms
from submarine volcanic areas having an optimum growth
temverature of 105°(?. Nature 300, 258.
{8] Stetter, i/ O. (1986) Diversity of extremely thermophili¢
archaebacteria, in Tbermophiles: General, Molecular, and
Applied Microbiology ('r.D. Brock, Ed.), pp. 75-106. Wiley & Sons, New York.
[91 K0nig. H., Messner. P. and Stette't, K.O. (198g) The fine
structure of the fibers of Pyrodictium occultum. FEMS
Microbiol. Len. 49, 207-212.
[10] Doming, J.W. and Baross, J.A. (1986) Solid medium for
culturing black smoker bacteria at t ~ t u r e s
to 120o C.
AppL Environ. MierobioL 51, 238-243.
White,
R.H.
(1984)
Hydrolytic
stability
of
blomolecules
at
[111
high temperatures and its implication for life at 250°C.
Nature 310, 430-432.
[12] lngraham, J.L, (1969) Factors which preclude growth of
bacteria at low temperature. Cr.cobiolos~ 6,188-193.
[13l Hawker, L.E.. Linton. A.H.. Folkes, B.F. and Carlile, M./.
(1960) An Introduction to the Biology of Microorganisms.
Edward Arnold, London.
[14] Michener, H.D. and Ellion, R.P. (1964) Minimum growth
temperatures for tonal-poisoning, fecal indicators, and
psychrophili¢ microorganisms. Adv. Food Res. 13. 349396 and lit. cited therein.
168
[15] Mazur, P. (19801 Limits to life at low temperatures and at
reduced water contents and water activities. Orig. Life 10,
137-159.
[16] Achenbach-Richter, L., Geupta, IL, Stetter, K.O. ahd
Woese, CR. 0987) Were the original eubaeterla theemophiles? Syst. AppL Microbiol. 9, 34-39.
[17] Morila. R.Y. (19751 Psychrophilic bacteria. Bactcriol. Roy.
39,144-167.
[18] Jacobs, M.B.. G~stein, M.J. and Walter, W.G. (1957)
Dictionary of Microbiology. Van Nostrand, New York.
[19] Gow, J.A, and Mills, F.H.J. (19841 Pragmatic c r i t ~ a to
distinguish psychrophiles and psychrotrophs in ecological
systems. Appl. Environ. Mierobiol. 47, 213-215.
[20] Williams, R.A.D. (19751 Caldoac0ve and thermopifflic
bactccia and their thermos[able proteins. Sci, Progr. Oxford 62, 373-383.
[21] Eddy, B.F. (1960) The use and meaning of the term
'psyehrophilic'. J. Appl. Bacterial. 23,189-190.
[22] Christian, R.R. and Wiebe, W.I. (19741 The effect of
temperature upon the reproduction and respiration of a
marine obligate psychrophile. Can. J. Microiffol. 20,
1341-1345.
[23] Hoffmm~ H. and Frank, M.E. (1963). Temperature limits,
genealogical origin, developmental course, and ultimate
fate of heabindueed filaments in Escherichia eoli microcullures. J. Bacteriol. 85,1221-1234,
[24] lngrah~an, J.L. and Marr, A.G. (1963) Control of enzyme
biosynthesis at temperatures near the minimum for growth
of Escherichia coil Col. Int. Ctr. Natl. Rech. Sci. 124,
319-328.
[25] Aragno, M. (19811 Responses of microorganisms to temperature, in Encyclopedia of Plant Physiology, New Series,
Vol. 12a. (O.L. Lunge, P.S. Nobel, C.B. Osmand and H.
Ziegler, Eds.), pp. 339-369. Springer Verlag, Heidalberg,
[26] Zeikus. J.G. (19791 Thermopifflic bacteria: ecology, physiology and technology. Enz. Microbiol. Tech. L 243-252.
[27[ Sundaram, T.K. (19861 Physiology and growth of thermophilie bacteria, in Thermophits*: General, Molecular and
Applied Microbiology (T.D. Brock. Ed.), John Wiley &
Sons. New York.
[28] Fr'dmmeL C. and Sander, C. (1989) Thennitase, a thermostable subtilisin: Comparison of predicted and experimental structures and the molecular cause of thermos[ability,
Proteins St~et., Funct. Oenet. 5, 22-37.
[291 Russel, N J . 0984) Mechanisms of thermal adaptation in
bacteria: Iffueprinls for survival. TIBS 9,108-112.
[30] RusseL N.J. (19881 Thermoadaptation of bacterial membranes. J. Chem. TechnoL Biotechnol. 42, 312-315.
131] Hensel, R. and K~nig, H. (19881 Thermoadaptation of
methanogenic bacteria by intracellular ion concentration.
FEMS Misroiffal. Lets. 49. 75-79.
[32] l~.on, E.Z. and Shunt, M. (1971) Growth rate of Escherlchia
colt at elevaled ,c;nperutures: reversible inhibition of hoa~oserine trans-succinylase, J. Bacteriol. 107, 397-400.
[33] Miller, J.F., Shah, N.N., Nelson, C.M., Ludlow, J.M. and
Clark, D.S. (19881 Pressure and temperature effects on
growth and methane production of the extreme thormo-
piffle Methan~coccus jannaschiL Environ. Mierobiol. 54,
3039-3042.
[34] Ratkowshy, P.A., Lowry, R.R., MeMeekin, T.A., Stokes,
AM. and Chandler, R.F- (1983) Model for bacterial culthre growth rate throughout the entire biokinetic temperalure range. J. Bacteriol. 154,1222-1226.
[35] Ljungdaiff, L.G. (19791 Physiology of thermophibe
hacteria. Adv. Mierob. Physiol. 19,149-243.
[3fi] Tansey, M.IL and Brock, T.D. (19781 Microbial life at
high temperatures: e~ological aspects, in Microbial Life in
Extreme Environmollts (D, Kushner, Ed.), pp. 159-216.
Academic Press, London.
[37] Amelunxen, R.E. and Murdock, A.L. (19781 Microbial life
at high temperatures: mechanisms and molecular aspecLs.
in Microbial Life in Extreme Environments (D.J. Kushner,
Ed.) pp. 216-278. Academic Press, London.
[38] Somero, G.N. (19781 Temperature adaptation of enzymes:
Biological optimization through structure-function compromises. Ann. Rcv. Ecol. Syst. 9,1-29.
[39] Kogut, M. (19801 Are there strategies of microbial adaptation to exlreme environments? TIBS 5, 15-18.
[40] Liodsay, J.A. and Creaser, E.H. (19751 Enzyme thermos[ability is a transformable property between Bacill~ spp.
Nature (Lond.) 255, 650-652.
[4ll Bernhardt, G., Jaenicke, R., L0demann, H D . , Kibnig, H.
and Stetter, K.O. (1988) High pressure ~nhances the
growth rate of the thermophilic archaebacterium
Methanococcus thermolirholrophicus without extending its
temperature range. AppL Environ. Microbiol. 54, 1258
1261.
[42] Yayanos. A.A.. van Boxtel, R. and Die[z, A.S. (1983)
Reproduction of Bacillus stearothermophilus as a function
of temperature and pressure. AppL Environ. Microbial.
46,1357-1363.
[43] Yayanos, A.A. (19861 Evolutional and ec:!ogieal implications of the properties of deep-sea baropifflic bacteria.
Proc. Natl. Acad. SCi. USA 83, 9542-9546.
[44] Stahl, S, alld Olsson, O. (1977) Temperature range variants
of Bacillum megateeium. Arch. Microbiok 113, 221-229.
[45] Droffner, M.L. and Yamamoto, N. (19851 halation of
ihermopifflic mutants of Bacillus sub[ills and Bacillus
pumi/us and transformation of thermophiiie trait to
mesopifflic strains. J. Gen. MicrobioL 131, 2789-2794.
[46] perry, LJ. and Wetzal, R. (1984) Disulfide botld ellgineered into T4 lysozyme: Stabilization of the protein
toward tliermal inactivation. Science 226. 555-557.
[47] Kim, K.J. and Larkin, J.M, (19731 Pr~uction of
mesophilic mutants from a psychropifflic Bacillus. Can. J.
MicrobioL 19,1452-1454 and lit. cited therein.
[48) Tsien, H., Panos, Ch., Shockman, G.D. and Higgins, M.L.
(19801 Evidence that Streptococcus mutans constructs its
membranes with excess fluidity for suwival al suboptimal
temperatures. J. Gea. Microbio[. 121,105-111.
[49] Schlegel, H.G. and Vollbrecht, D. (19801 Formation of ihe
dehydrogenases for lactate, ethanol, and butanediol in the
strictly aerobic bacterium Aicaligenes eulrophus. J. Gen.
Microbial. 117, 475-480.
169
[50] Koizumi, J., Monden, Y. and Aiba, S. 0985) Effects of
temperature and dilution rate on the copy number of
recombinant pIasmld in continuous culture of Bacillus
stearothermophil~ (pLPlI). Biotechnol. Bioenf~ 27, 721728.
[51] Wiesel, J. and Ljunsdehl, L.G. (1981) Thermoanaerobacter
ethanOh~ gen. nov., spec. nov., a new, extreme thermophillc, anaerobic bacterium. Arch. Mierobiol. 128, 343MS.
[52] Wiegel, J., Ljungdahl, L.G. and Rawson, J.R. 0979) I,'oialion from soil aad propcrti©s of th~ extrem~ thgrmophilic
Closlridium ihermohydrosulf~tcum. J. Bacterlol. 139, 800810.
[53] Wiegel, J., Kuk, S.-U. and Kohrlng, G.W. (1989)
CIostridiwn thermobutyrzcum, spec. nov., a moderate thermophile isolated from a cellulolytic culture producing
butyrate as major product. Int. J. Syst. Bacteriol. 39,
199-206.
[54] Brock, T.D. (1967) Life at high temperatures. Science 158,
1012.
[55] Sonnl©itner, B. and Fiechler, A. (1983) Advantages of
usin$ thermophiles in bloteclmolo~ical processes: expectations and reality. Trends BiotechnoL I, 74-80.
[56] Ljungdahl, L.G., Bryant, F., Carreira. L., Saiki, T. and
Wiegel, J. (1981) Some aspects of thermophilic and ex.
Ireme thermophilic anaerobic mieroorganisms, in Trends
in the Biology of Fermentations for Fuels and Chemicals
(A. Hollaender, Gen. PAl.), pp. 397-419, Plenum Press,
New York.
[57] Zeikus, J.t'~. and Wolfe, ~S. (19"/2) Methanobacterimn
therm~awolrokfiicum sp. n., an anaerobic, autotrophic,
extreme thermophil©. J. BactcrioL 109, 707-713.
[58] At kinson, A., Elwood, D.C., Evans, G.G.T. and Yeo, R.G.
(197,4) Production of alcohol by Bacillus srenrother~ph&
/us. Biotechnol. Biting. 17.1375-1377.
[59] Jung, L., Jost, R., Stall, E. and Zuber, H. (1974) Metabolic
differences in Bacillus stearothermoph~lus grown at 55°C
and 37°C. Arc6. Microbial. 95,125-138.
[60] Haberstick, H.-U. and Zuber, H. (1974) Thermoadaptatlon of enzymes in t h e ~ h i l l c and mesophitlc cultures
of Bacillus stearothermophilu~ and Bacillus caldotenax.
Arch. Microhial. 98, 275-287.
[61] Brown, D.K., Militzer, W. and Georgi, C.E. (1957) The
effect of growth Igmpetattlfc on the heal stability of
bacterial pyropho~phatas~. Arch. Bi~help_ Biophys. 70,
248-256.
[62] Oppenheimer, C.H. and Drost-Hansem W. (1960) The
relationship between multiple temperature optima for biolo,~icul systems and the properties of water. J. Baeteriol.
80, 2i-24.
[63] Lamam~a, C. and Malette, M.F. (1973) Basic Bac~riokigy.
4th edn., Williams and Wilkins. Baltimore.
[641 Talsky, G. (1971) Zur anomalen TemperatarahhItnsigkeit
enzymkatalysierter Reaktionen. Angew. Chem. 83, 553594.
[65] Han, M.H. (1972) Non-||near Atrhenius plots in ~mperature-dependent kinetic studies of enzyme reactions. L
Single transition processe~ J. Thcor. Biol. 35, 543-568.
[66] [jungduld, L.G. and Shero.i, D. (1976) Proteins from
thermophilic mlcrccrganlsms, in Extreme Environments:
Mechanisms of Microbiad Adaptation (M.R. Halnrich,
Ed.), pp. 167-187. Academic Press, New York.
[67] Welker, N. (1978) Physiological and genetic factors affecting transfection and transformation in Bacillus stearcihermophilus in Biochemistry of Thermophily (S.M. Friedman, Ed.). pp. 127-147. Ac~tdemi¢ Press, New York.
]68] Zuber. H. (1981) Thermophily, in Trends in the Biolosy of
Fermentations for Fuels and Chemieuls (A. Hollaender,
Gen. Ed.), pp. 499-512. Plenum Press, New York.
[69] Mohr, P.W. and Krawim:, S. Temperatures characteristics
and Arrhenius plots for nominal psychrophiles, mesophiles
and thermophiles. J. Gram Microhiol. 121,311-317.
[70] Reichardt, W. and Morita, R.Y. (1982)Temperature characteristics of psychrotrophic and psychrophilic bacteria. I.
Coon. MicrobioL 128, 565-568.