University of Groningen
Thermophilic methanol utilization by sulfate reducing bacteria
Goorissen, Helene Petronel
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4
Endospores of thermophilic
sulfate reducing bacteria
survive extreme heat treatments
Heleen P. Goorissen, Manny H. M. Nienhuis-Kuiper,
Alfons J. M. Stams, and Theo A. Hansen
The ability of microorganisms to survive extreme environmental conditions is
partly determined by their capacity to protect DNA from destructive chemical and
physical conditions. Some bacteria form endospores that can resist extreme
conditions — including pressure, extreme heat or cold, drought, starvation, most
poisons, and UV-radiation. Spores may remain dormant for centuries and may even
survive over geological time and through interstellar travel.
Here we report on the extreme heat resistance of spores of the thermophilic
sulfate-reducing bacterium Desulfotomaculum kuznetsovii. About 10% of the spores
of this organism survived a heat treatment at 140 ºC for 15 min., which is
unprecedented. This unusual characteristic is represented by a z-value (thermal
inactivation coefficient) of 16.7 ºC. The decimal reduction value at 120 ºC is 4 hours,
which is highest ever.
Submitted for publication
Chapter 4
Introduction
Heat resistant spores are found among several species, mainly those belonging to the
genera Bacillus and Clostridium (Hyung et al., 1983; Fernandez et al., 2001). Most of the research
into heat resistance of spores has been done using mesophilic species in food industrial
processes. Less attention has been paid to the heat resistance of spores formed by thermophilic
bacteria. Spores from thermophilic bacteria are more heat resistant than spores from mesophilic
species (Warth, 1978). Heat resistant spores from thermophilic anaerobes can be troublesome in
research laboratories and routine autoclaving protocols of culture media and materials might be
insufficient to inactivate bacterial spores. On the contrary, normal autoclaving procedures (20 min.,
121 ºC) may even activate and increase the apparent heat resistance of spores (Hyung et al.,
1983; Byrer et al., 2000).
The first high decimal reduction value DT (the incubation time at temperature T necessary
for a 90% decrease in the viability of the spores) for thermophilic species was reported in 1965 for
Clostridium thermoaceticum, an acetogen with a D124 of more than 72 minutes (Xezones et al.,
1965). More recently, extremely heat resistant spores have been detected in other thermophilic
Clostridium and Moorella species, i.e. Cl. thermohydrosulfuricum (D120 = 11 min.) (Hyung et al.,
1983), Cl. thermoautotrophicum (D120 = 70 min.) (van Rijssel et al., 1992), and Moorella
thermoacetica JW/DB2 and JW/DB4 (D120 = 84 min., 111 min., respectively) (Byrer et al., 2000).
What determines the spore heat resistance is not known with certainty. Multiple factors are
involved, such as the composition of the proteinaceous spore-coat (Henriques & Moran, 2000), the
dipicolinic acid concentration of the spore-cortex (Beaman & Gerhardt, 1986), its thickness, and its
Ca2+ content, the dehydration and mineralization state of the spore (Popham et al., 1999), and
specialized DNA-binding proteins termed α/β type small acid soluble spore proteins (SASP)
(Setlow & Setlow, 1998).
Virtually no studies have been made of the heat resistance of spores of the thermophilic
sulfate-reducing bacteria of the genus Desulfotomaculum, which are widespread in nature. Only
for D. nigrificans, a moderate thermophile that was responsible for sulfur stinker spoilage in canned
food products, a D120 = 5.4 min. was reported (Donnelly & Busta, 1980). We studied the
metabolism of Desulfotomaculum kuznetsovii, a thermophilic, rod-shaped, spore-forming, sulfatereducing bacterium isolated from a geothermal hot spring (Nazina et al., 1987). In continuous
cultures, it appeared that sterilization procedures of even longer than two hours at 120 °C were
insufficient to kill the spores of D. kuznetsovii in our fermentors. This observation led to the
question whether this extremely heat resistant spore production is a typical phenomenon for all
thermophilic Desulfotomaculum strains or is specific to D. kuznetsovii. All strains present in the IC
and ID subclusters of the phylogenetic tree of the Desulfotomaculum main cluster were examined
for the production of heat resistant spores.
64
Heat resistant spores
Methods
Strains. The following strains were purchased from the Deutsche Sammlung von Mikroorganismen
und Zellkulturen (Braunschweig, Germany): Desulfotomaculum kuznetsovii (DSM 6115),
Desulfotomaculum thermoacetoxidans (DSM 5813), Desulfotomaculum thermobenzoicum (DSM
6193), and Desulfotomaculum thermocisternum (DSM 10259). Desulfotomaculum australicum
(AB33) and Desulfotomaculum luciae (SMCC W644) were kindly provided by Bharat Patel (Griffith
University, Nathan, Queensland) and David Boone (Oregon Graduate Institute of Science and
Technology, Portland Oregon) respectively. Strain TPOSR, strain WW1, and strain V21 were
isolated in our laboratories.
Phylogenetic analysis. Sequences were downloaded from the Genbank Database and aligned
using Clustal W. The 16S rDNA phylogenetic tree was constructed from a distance matrix based
on the neighbor-joining method (Saitou & Nei, 1987) as implemented in the TREECON program
(van de Peer & de Wachter, 1995). A manual correction method was applied and tree topology
was re-examined by using bootstrap analysis (100 replicants, all bootstrap values ≥ 68).
Cultivation techniques. Anaerobic culture techniques were used throughout this study, with all
media prepared as described in the catalog of strains of the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (http://www.gbf.de/dsmz/media/media.htm). For the production
and germination of the spores, substrates methanol (20 mM, filter sterilized) or lactate (20 mM,
heat sterilized) were added from anoxic stock solutions. Incubations were carried out at 60 °C in 1
liter bottles filled with 100 ml medium for the spore-production experiments or in 16 ml tubes filled
with 10 ml medium for the spore-germination experiments and most probable number counts.
Determination of D-values. From a 1 liter culture, cells and spores were harvested and
concentrated 25-fold by centrifugation. Samples (2 ml) of this suspension were transferred to new
anaerobic sterile tubes. Tubes were heated in an oil-bath for different times (1 to 40 hours) and
different temperatures. After heat treatment, the spore suspensions were diluted in fresh anaerobic
medium and incubated at 60 °C for most probable number counts. Decimal reduction values (D)
were calculated from the best linear fit from y = ax + b, in which x is the incubation time (hours) and
y is log N. N is the number of viable spores after heat treatment. Experiments were carried out in
duplo.
Results
Determination of the heat resistance of D. kuznetsovii spores
Heat resistance of the spores of D. kuznetsovii was determined in duplicate culture
suspensions (3·109 viable cells per ml) at incubation temperatures of 120, 130, and 140 °C. The
survival of viable spores after heat treatment was counted using the most probable number
method. Survival curves of D. kuznetsovii spores are depicted in Fig. 4.1.
The decimal reduction value for D. kuznetsovii spores at 140º C is 15 min., an
unprecedented value. At lower temperatures, the D-values are extremely high (D130 = 79.2 min.,
D120 = 240 min.) and this slow decrease of viability of the spores at higher temperatures is also
illustrated by a high z-value i.e. 16.7 ºC (Fig. 4.2).
65
Log decimal reduction values
Chapter 4
12
10
8
6
4
2
0
0
5
10
15
20
25
30
35
40
Incubation time (h)
Figure 4.1.
Survival curve of D. kuznetsovii spores at different temperatures.
Symbols: !, T = 120 ºC; ", T = 130 ºC; #, T = 140 ºC.
Log decimal reduction value
3
y = -0.0655x + 10.392
R2 = 0.988
2.5
2
1.5
1
0.5
0
115
120
125
130
135
140
145
Incubation temperature (ºC)
Figure 4.2.
Thermal inactivation curve. Best fit through log decimal reduction values
of D. kuznetsovii spores
Assuming first order kinetics for the inactivation of spores by heat, extrapolation of the
thermal inactivation curve results in a D100 of approximately 70 hours and a D90 of more than 11
days. Altering the growth conditions could influence the apparent heat resistance of the spores.
Omission of calcium from the sporulation medium reduced the D120 to 87 min. This confirms the
observation that bacterial spores accumulate calcium, which contribute to spore heat resistance,
depending on the concentration in the growth medium (Popham et al., 1999). The assumed
relation between spore cortex/cytoplasm ratio and degree of heat resistance as postulated by
Hyung et al (1983) could not be confirmed in our study. In D. kuznetsovii spores, this ratio is
relatively low (1.7) in view of their extreme heat resistance.
66
Heat resistant spores
Desu
l fo to
De
mac
su
ulum
lfo
therm
tom
oace
ac u
toxid
De
lum
ans
su
the
lfo
rm
ob
to
enz
m
oic
ac
um
ul
um
au
st
ra
lic
um
Phylogenetic analysis of thermophilic Desulfotomaculum species
The complete 16S rDNA sequences of all Desulfotomaculum strains in subclusters IC and ID and
some other strains that form heat resistant spores were compared. In the resulting phylogenetic
tree (Fig. 4.3), species within the Desulfotomaculum subcluster IC are closely related, showing
similarity values of 95-98%. The sequence similarity of the subclusters IC and ID is more than
90%.
ID
Desu
lfoto
Mo
ore
l
ac
te
rs
id
ns
ifica
er
ob
n i gr
er
m
oa
na
u lu m
mac
Th
er
o
ph
ilu
s
la
the
Moo
rm
rella
therm
oa
c
oaut
otro etic
phic a
a
m
to
fo
ul
s
De WW1
Strain
a
lum
cu
e
th
r
um
rn
e
t
ci s
mo
ulum
Desulfotomac
Strain
TPOSR
V2
1
luciae
De
sul
fot
om
acu
lum
kuz
n
ets
o
vii
Ba
cill
us
me
tha
no
licu
s
St
ra
in
IC
Figure 4.3.
Phylogenetic tree based on 16S rRNA gene sequence comparisons. The neighbor-joining
tree was reconstructed from distance matrices; bootstrap values are not shown. Cluster
designation according to Stackebrandt et al. (1997) The GenBank accession numbers for
the organisms used in this analysis are Desulfotomaculum kuznetsovii AF009646; Desulfotomaculum luciae AF069293; Desulfotomaculum nigrificans X62176; Desulfotomaculum
thermoacetoxidans Y11573; Desulfotomaculum thermobenzoicum L15628; Desulfotomaculum thermocisternum U33455; Moorella thermoacetica M59121; Moorella thermoautotrophica L09168; Thermoanaerobacter siderophilus AF120479; Strain TPOSR AF442686;
Strain WW1 AF442687. Bacillus methanolicus X64465 S42879 served as outgroup
67
Chapter 4
Determination of the heat resistance of spores of thermophilic Desulfotomaculum species
Among all the studied strains, only strain TPOSR and strain WW1 produced extremely heat
resistant spores, although their spores were considerably less heat resistant than D. kuznetsovii
spores. The D120 value for WW1 is 60 min. and for TPOSR D120 is 114 min (Table 4.1). For both
strains, tubes were positive after a heat treatment of 16 hours and negative after 20 hours. We
were unable to obtain sporulating cultures of D. australicum and D. luciae. For D. luciae, it has
been reported that cells were still viable after a 30 min. boiling procedure (Karnauchow et al.,
1992). All other strains from subcluster IC and ID produced spores with D120-values equal to or
below 3 min. (Table 4.1).
Table 4.1.
Highest reported D120-values (values < 3 min. are not taken into account), combined with
D120-values of Desulfotomaculum strains from the 1C and 1D phylogenetic cluster
Strain
D120 (min)
Sporecortex to
Reference
cytoplasm ration
3
2.8
Aiba & Humphrey, 1978
32.5
nr
Nakamura et al., 1985
Cl. thermoautotrophicum
70
1.4
van Rijssel et al., 1992
Cl. thermohydrosulfuricum 39E
11
6.6
Hyung et al., 1983
Cl. thermosaccharolyticum
72.5
nr
Xezones et al., 1965
Desulfotomaculum nigrificans
5.6
nr
Bacillus stearothermophilus
Clostridium difficile 88
Donnelly & Busta, 1980
a
D. kuznetsovii
240
1.7
This report
D. thermoacetoxidans
<3
nd
This report
D. thermobenzoicum
<3
nd
This report
D. thermocisternum
<3
nd
This report
Strain V21
<3
nd
This report
Strain TPOSR
114
nd
This report
Strain WW1
60
nd
This report
Moorella thermoacetica JW/DB-4
111
5.97
Byrer, et al., 2000
Moorella thermoacetica JW/DB-2
83
3.13
Byrer, et al., 2000
nr, not reported; nd, not determined; a) determined as described previously (van Rijssel et al., 1992)
Discussion
The production of extremely heat resistant spores with a D140 of 15 min. is an exclusive
feature of D. kuznetsovii and makes this strain unique, not only within the genus
Desulfotomaculum, but among all spore-forming bacteria. To our knowledge, D140-values higher
than 7.9 sec. have not been demonstrated previously (Huemer et al., 1998). Highest DT values
reported are D120 values, found for thermophilic low G+C % gram-positive Clostridium and Moorella
species (Table 4.1). On the basis of 16S rDNA sequence comparisons a relative high degree of
relatedness between these species and the thermophilic Desulfotomaculum species exists (8087%). Desulfotomaculum species have been isolated from anaerobic bioreactors and from
environmental samples. In both classes, heat-sensitive spore producers are also present. This
68
Heat resistant spores
indicates that the occurrence of Desulfotomaculum species with extremely heat resistant spores is
not confined to a particular habitat.
The comparison of data published on the heat resistance of spores is complicated because
no standard quantification and incubation protocols are available. Besides, environmental factors
greatly affect microbial heat resistance. Composition of the growth medium (Payot et al., 1999;
Casadei et al., 2001) sporulation temperature (Beaman & Gerhardt, 1986), the heating conditions
and recovery medium (Coroller et al., 2001; Palop et al., 2000), and increased heating time (Byrer
et al., 2000) influence the apparent heat resistance. Often a survival of boiling treatments is
reported to indicate heat resistance, e.g. D. thermoacetoxidans survived 10 min. boiling, but did not
survive 15 min. boiling (Min & Zinder, 1990); Thermoanaerobacter siderophilus survived a 90 min.
boiling treatment (Slobodkin et al., 1999), and methanol utilizing Desulfotomaculum species
survived a heat treatment at 131 ºC for 20 min. (Rosnes et al., 1991). Although these data might
give a prediction of heat resistance, such observations are not comparable with D-values. In all our
experiments, the same conditions have been applied. Therefore it is clear that the differences in
heat resistance among the spores of different Desulfotomaculum species is an intrinsic property of
the thermophilic Desulfotomaculum species
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