International Journal of Food Microbiology 343 (2021) 109088

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Isolation, stability, and characteristics of high-pressure superdormant

Bacillus subtilis spores
Alessia I. Delbrück a, 1, Yifan Zhang a, 1, Vera Hug a, Clément Trunet b, Alexander Mathys a, *
a
Sustainable Food Processing Laboratory, Institute of Food, Nutrition and Health, Department of Health Science and Technology, ETH Zurich, Zurich, Switzerland
b
Université de Bretagne Occidentale, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, UMT ACTIA 19.03 ALTER’iX, Quimper, France

A R T I C L E I N F O A B S T R A C T

Keywords: Bacterial spores are a major challenge in industrial decontamination processes owing to their extreme resistance.
Bacterial spore High-pressure (HP) of 150 MPa at 37 ◦ C can trigger the germination of spores, making them lose their extreme
Superdormancy resistance. Once their resistance is lost, germinated spores can easily be inactivated by a mild decontamination
Germination
step. The implementation of this gentle germination-inactivation strategy is hindered by the presence of a
Heterogeneous
Flow cytometry
subpopulation of so-called high-pressure superdormant (HPSD) spores, which resist germination or germinate
Mild inactivation only very slowly in response to HP. It is essential to understand the properties of HPSD spores and the underlying
causes of superdormancy to tackle superdormant spores and further develop germination-inactivation strategies
involving HP. This study investigated factors influencing the prevalence of HPSD spores and successfully isolated
them by combining buoyant density centrifugation and fluorescence-activated cell sorting, which allowed further
characterisation of HPSD spores for the first time. The prevalence of HPSD spores was shown to be strongly
dependent on the HP dwell time, with increasing treatment times reducing their prevalence. Spore mutants
lacking major germinant receptors further showed a highly increased prevalence of HPSD spores; 93% of the
spores remained dormant even after a prolonged HP dwell time of 40 min. In contrast to nutrient germination,
sublethal heat treatment of 75 ◦ C for 30 min prior to pressure treatment did not induce spore activation and
increase germination.
The isolated HPSD spores did not show visible structural differences compared to the initial dormant spores
when investigated with transmission electron microscopy. Re-sporulated HPSD spores showed similar germi­
nation capacity compared to the initial dormant spores, indicating that HPSD spores are most likely not
genetically different from the rest of the population. Moreover, the majority of HPSD spores germinated when
exposed a second time to the same germination treatment; however, the germination capacity was lower than
that of the initial population. The fact that the majority of spores lost superdormancy when exposed a second
time to the same trigger makes it unlikely that there is one factor that determines whether a spore germinates
with a certain HP treatment or not. Instead, it seems possible that there are other reversible or cumulative causes.
This study investigated the factors influencing spore HP superdormancy to improve the understanding of HPSD
spores with regard to their stability, germination capacity, and potential underlying causes of spore HP super­
dormancy. This knowledge will contribute to the development of HP-based germination-inactivation strategies
for gentle but effective spore control.

1. Introduction decontamination processes owing to their ability to form extremely

1.1. Germination-inactivation-based strategies for gentle bacterial spore
control
Spore-forming bacteria are a major hurdle in industrial food
resistant spores, which can survive classical preservation methods like
thermal pasteurization (den Besten et al., 2018; Mtimet et al., 2015;
Setlow, 2006). Depending on the post-treatment environment, surviving
bacterial spores can potentially germinate and proliferate, causing food
safety- or spoilage-related issues (Trunet et al., 2018, 2015; Wells-

* Corresponding author at: Schmelzbergstrasse 9, 8092 Zurich, Switzerland.

E-mail address: alexander.mathys@hest.ethz.ch (A. Mathys).
1
Equal first authorship.

https://doi.org/10.1016/j.ijfoodmicro.2021.109088
Received 6 July 2020; Received in revised form 15 November 2020; Accepted 30 January 2021
Available online 11 February 2021
0168-1605/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A.I. Delbrück et al.
Bennik et al., 2016). To control bacterial spores in the food industry,
intensive heat treatment is commonly applied. However, the use of
thermal processing often has a detrimental impact on the nutritional and
sensorial quality of food (Sevenich and Mathys, 2018). While most spore
inactivation strategies try to directly target the resistant dormant stage
of bacterial spores (Kim et al., 2020; Kort et al., 2005; Zhang et al.,
2020b, 2018), a potentially milder approach is to let spores germinate in
the first step, so they lose their extreme resistance. In the second step,
which should follow rather quickly to avoid proliferation and potential
toxin production, the susceptible germinated spores can then easily be
inactivated by a mild inactivation treatment. This follow-up treatment
could be for instance a mild heat treatment or in theory also a treatment
at higher pressures in the range of 600 MPa. This strategy is commonly
known as a germination-inactivation strategy (Abee et al., 2011; Collado
et al., 2004; Lovdal et al., 2011; Zhang and Mathys, 2019). Given the
potential utility of this strategy, the scientific community has been
studying the germination of bacterial spores extensively in the last
decades.
1.2. Bacterial spore germination
It is well established that spore germination can be triggered by
different stimuli, including nutrients, Ca2+- dipicolinic acid (Ca2+-DPA),
cationic surfactants, such as dodecylamine, and isostatic high-pressure
(HP), typically hundreds of megapascals (Setlow, 2013; Setlow et al.,
2017). Most of the knowledge about bacterial spore germination is
established on studies of Bacillus spores and based on germination
triggered by nutrients like L-alanine or a mix of L-asparagine, D-glucose,
D-fructose and K (AGFK). The process of spore germination has been
+
associated with several proteins, which are mainly located in the inner
spore membrane. These proteins include i) germinant receptors (GRs),
namely GerA, GerB, and GerK in Bacillus subtilis, which respond to
nutrient germinants, ii) GerD, a protein that is essential for GR-
dependent germination, iii) the SpoVA proteins, which form a channel
in the inner membrane through which the spore core’s depot of Ca2+-
DPA is released during germination, and iv) cortex-lytic enzymes
responsible for degrading the peptidoglycan cortex, which allows the
spore to take up more water and swell, thus completing the spore
germination (Setlow, 2014). Unlike nutrients, Ca2+-DPA and dodecyl­
amine trigger spore germination through mechanisms that are inde­
pendent of GRs (Paidhungat et al., 2001).
Bacterial spore germination triggered by HP is still not fully under­
stood. However, it is known that different mechanisms dominate within
different pressure and temperature ranges (Reineke et al., 2013). At
lower pressures of 150 MPa and moderate temperatures of approxi­
mately 30–50 ◦ C, HP triggers germination through stimulation of the
GRs and the resultant germination process is believed to be similar to the
nutrient germination process (Aertsen et al., 2005; Black et al., 2005;
Paidhungat et al., 2002; Wuytack et al., 1998). At higher pressures of
500–600 MPa and temperatures below 60 ◦ C, Ca2+-DPA channels are
directly opened (Black et al., 2007b; Paidhungat et al., 2002; Reineke
et al., 2013).
1.3. Heterogeneous bacterial spore germination and the concept of
superdormancy
It is well recognised that the germination response of bacterial spores
is extremely diverse among different genera or species. Even within the
same strain, their germination response is also heterogeneous, inde­
pendent of the germination trigger (Abee et al., 2011; Doona et al.,
2017; Luu et al., 2015; Modugno et al., 2020; Setlow, 2003; Wells-
Bennik et al., 2016; Zhang et al., 2020a; Zhang and Mathys, 2019). For
nutrient germinants or isostatic pressure of 150 MPa, the heterogeneity
in germination rates is mainly determined by the initial stages of
germination and is observed as the time differences between stimulation
(germinant addition or HP application) and commitment, and between
2
International Journal of Food Microbiology 343 (2021) 109088
commitment and initiation of rapid Ca2+-DPA release (Kong et al., 2014;
Setlow et al., 2017; Wang et al., 2015).
While the majority of spores within a population germinate upon
exposure to a germination trigger, there is a small fraction of the spore
population that germinates more slowly or not at all in response to the
germination trigger. This fraction of spores is termed superdormant. It is
important to understand that superdormancy is a relative term. It de­
scribes a subpopulation of spores that is phenotypically different
compared to the rest of a population with respect to germination ca­
pacity. Hence, it is not a static subpopulation but rather a subpopulation
that is largely dependent on the germination and isolation conditions
and the cut-off point chosen in the corresponding experiment. Super­
dormancy has been reported for different germination triggers and
superdormant spores are categorised according to their germination
stimulus, e.g. nutrient superdormant spores or high-pressure super­
dormant (HPSD) spores (Zhang and Mathys, 2019). It is worth pointing
out that being superdormant to one stimulant does not mean being
superdormant to all stimulants.
The presence of a superdormant spore subpopulation hinders the
successful implementation of germination-inactivation-based strategies.
Spores that remain dormant will survive the subsequent mild inactiva­
tion step and can eventually grow out during storage, potentially
causing food safety or spoilage issues. Spore superdormancy is not only a
concern in the food industry but also in the medical, pharmaceutical,
and (bio)chemical sectors, where spore elimination is required (Brook­
meyer et al., 2003; Heine et al., 2007; Setlow, 2012). To understand the
mechanisms of superdormancy and ways to overcome this super­
dormant subpopulation it is important to look more closely at this
subpopulation.
1.4. Current knowledge on superdormant spores
In recent decades, a handful of researchers have put great effort into
the isolation and characterisation of different groups of superdormant
spores to elucidate the cause of their germination deficiency as well as
their germination capacity using other triggers. These studies mainly
focused on nutrient superdormant Bacillus spores (Ghosh et al., 2012;
Ghosh and Setlow, 2010, 2009; Wei et al., 2010; Zhang et al., 2012) and
to a much lesser extent on Ca2+-DPA and dodecylamine superdormancy
(Perez-Valdespino et al., 2013). For nutrient superdormant spores, it has
been shown that the following factors, among others, influence the
prevalence of the subpopulation: i) nutrient concentration; high ger­
minant concentrations decrease the superdormant spore subpopulation,
ii) heat activation; a sublethal heat treatment prior to nutrient germi­
nation reduces the superdormant spore subpopulation; this activation
effect is reversible, and iii) GR levels; with increasing GR levels, the
percentage of superdormant spores decreases drastically. Since the
factors influencing the levels and properties of superdormant Bacillus
cereus, B. subtilis, and Bacillus megaterium spores are reportedly similar, it
is suggested that common mechanisms are involved in the super­
dormancy of most Bacillus species (Ghosh and Setlow, 2010, 2009).
A loss of superdormancy was observed upon extended storage of
isolated nutrient superdormant B. subtilis and B. cereus spores at 4 ◦ C,
giving rise to the hypothesis that at least two factors cause spore
superdormancy, one is transient or reversible and the other is permanent
(Ghosh and Setlow, 2010, 2009). For the transient cause, Ghosh and
Setlow (2010) suggested it might be related to the activation status of
the spores, since heat activation, which has a reversible effect on spore
germination, influences the prevalence of nutrient superdormant spores.
A probable permanent cause of nutrient superdormancy seems to be low
GR levels (Chen et al., 2014; Ghosh et al., 2012; Zhang et al., 2013). The
average number of GRs per spore is relatively low and spore-to-spore
phenotypic variation in GR expression is large, hence difference in GR
levels were shown to at least partially contribute to the heterogeneity in
nutrient germination time (Ghosh et al., 2012; Setlow, 2003; Zhang
et al., 2013). A genetic change, e.g. a defect in a gene coding for a critical
A.I. Delbrück et al.
part of the germination machinery, is most probably excluded as a
permanent cause of nutrient superdormancy (Ghosh and Setlow, 2009).
Ca2+-DPA and dodecylamine superdormant spores have been char­
acterised only to a limited extent (Perez-Valdespino et al., 2013) and
HPSD spores have received the least attention to date. To our best
knowledge, HPSD spores have not yet been systematically characterised
despite their importance in industrial applications. From an applied
perspective, HP shows the highest potential for germination-
inactivation strategies among the different germination stimuli. Its ad­
vantages are as follows: (i) HP can treat food products very homoge­
neously, (ii) no chemicals are required, and (iii) the technology itself
offers the possibility to simultaneously germinate spores and inactivate
vegetative cells (Doona et al., 2016; Gould and Sale, 1970; Knorr et al.,
1998; Sevenich and Mathys, 2018; Zhang and Mathys, 2019). In addi­
tion, HP is known as a technology with little detrimental impact on
important food quality attributes, such as organoleptic and nutritional
value, and is generally well accepted by consumers (Martínez-Mon­
teagudo et al., 2014; Sevenich and Mathys, 2018). To date, however, the
presence of HPSD spores hindered an effective elimination of all spores
by HP alone at mild temperatures. Therefore HP has been combined
with higher temperatures to effectively inactivate resistant bacterial
spores (Ahn et al., 2007; Doona et al., 2017; Margosch et al., 2006,
2004). In order to further develop mild HP-based spore inactivation
methods at mild temperatures, it is important to get a better under­
standing of HPSD spores.
Recently, we reported the successful isolation of HPSD spores using
fluorescence-activated cell sorting (FACS) (Zhang et al., 2020a). The
present study aimed to further improve the isolation throughput of
HPSD spores to yield HPSD spores of high purity and in amounts that are
sufficient for further characterisation. A suitable isolation protocol was
thereby optimised and the important characteristics of HPSD spores,
such as their prevalence, stability, microscopic structure, and germina­
tion capacity, were investigated. These results shed light on the prop­
erties of HPSD spores as well as the potential causes of spore HP
superdormancy at 150 MPa and 37 ◦ C.
2. Material and methods
2.1. B. subtilis strain used and spore preparation
B. subtilis PS533 and an isogenic mutant were used in this study.
PS533 itself is an isogenic mutant of strain PS832, a laboratory deriva­
tive of 168. PS533 (herein defined as wildtype) carries plasmid pUB110
conferring kanamycin resistance (Setlow and Setlow, 1996). FB72
(ΔgerA::spc, ΔgerB::cat, ΔgerK::erm) lacks the three functional GR op­
erons, namely gerA, gerB, and gerK (Paidhungat and Setlow, 2000).
Unless otherwise stated, all experiments were conducted with B. subtilis
PS533. FB72 was used to confirm the role of GRs in HP germination at
150 MPa and 37 ◦ C. Spore preparation was done as previously described
(Zhang et al., 2020a). Spores used in this study were ≥98% phase-bright
dormant spores as determined using phase-contrast microscopy.
2.2. HP spore germination
Prior to HP treatment, spores were diluted to a final spore concen­
tration of 109–1010 spores/mL in 50 mM 0.1 μm filtered (Minisart®,
Germany) N-(2-acetamido)-2-aminoethanesulfonic acid (ACES, Ther­
moFisher, Kandel, Germany) buffer solution at pH 7.0. The initial
dormant spore samples were HP-treated in 1.5 mL cryotube vials and
sealed with a sealing tube (Nunc CryoFlex Tubing, Nunc A/S, Roskilde,
Denmark) to prevent leakage during HP treatment. All samples were
treated at 150 MPa and 37 ◦ C using a dual vessel high-pressure unit
(modified Model U111, Unipress, Warsaw, Poland) as described previ­
ously (Zhang et al., 2020a). Different HP treatment dwell times were
investigated, ranging from 1 to 40 min. If FACS-isolated HPSD spores
were further HP-treated, the procedure was slightly adapted. In this
3
International Journal of Food Microbiology 343 (2021) 109088
case, the approximate concentration was 105 spores/mL and further HP
treatments were performed in Gibco™ PBS (1×, pH 7.4, Thermo Fisher
Scientific, Waltham, US). The reason for this is that the isolation pro­
cedure by FACS used PBS as sheath fluid and after isolation the HPSD
spores concentration is around 105 CFU/mL. Furthermore, because of
the limited output volume of FACS isolation, the isolated HPSD spores
were treated in polyethylene pouches (Hyperion heat shrink tubes,
Hyperion, Hong Kong) with smaller volumes of approximately 200 μL.
Notably, experiments that were to be compared with each other were
paired and conducted under the same conditions, i.e. spore concentra­
tion, volume, and buffer. After HP treatment, the samples were imme­
diately removed from the vessel and placed on ice until further analyses
were conducted. In addition, an untreated control was prepared for each
experiment and stored on ice until used for further analysis. Represen­
tative pressure/temperature/time profiles of the HP treatments can be
found in the supplementary material. Experimental replicates were
coming from the same spore batch but were performed on three different
days (n = 3). Results are expressed as mean value ± standard deviation.
2.3. Assessment of germination and determination of the prevalence of
HPSD spores
Germination was assessed using flow cytometry (FCM), which has
previously been extensively validated and described (Mathys et al.,
2007; Zhang et al., 2020a). Briefly, spores were stained with the nucleic
acid stains SYTO16 and propidium iodide (PI); while SYTO16 staining is
indicative for germinated cells, PI staining is indicative of cells with
compromised membranes. Dormant spores are stained neither by
SYTO16 nor PI. Two staining procedures were used; one standard
staining procedure for high spore concentrations and the other for lower
spore concentrations, which was applied for the isolated HPSD spores
and their paired comparison experiments. The staining procedure for
high spore concentrations diluted the spores to a final concentration of
approximately 107 CFU/mL and is described in detail by Zhang et al.
(2020a). For the lower spore concentration of 105 CFU/mL in PBS, the
same staining procedure was applied, except that the concentration of
the stains was 0.05 μM SYTO16 and 0.75 μM PI.
A BD LSR Fortessa™ cytometer (BD Biosciences, Franklin Lakes,
USA) was used to perform FCM analysis and the acquired fcs. files were
analysed in the FlowJo software (FlowJo LLC, Oregon, US) as described
previously (Zhang et al., 2020a). During the FCM analysis, there were
background signals in the HPSD spores gate, which can influence the
accuracy of the prevalence of HPSD spores determined by FCM. How­
ever, these background signals were very weak. Based on the experi­
ments performed in this study, only when the prevalence of HPSD spores
is below 1.5%, the contribution of the background becomes substantial
and influences the results. Therefore, data with HPSD spores prevalence
below 1.5% need to be considered with some suspicion.
2.4. Impact of heat activation on HP germination at 150 MPa
To investigate the influence of heat activation on the prevalence of
HPSD spores, the initial spore population was heat-activated for 30 min
at 75 ◦ C in a 1.5 mL Eppendorf tube in a water bath and then cooled for
15 min in an ice bath prior to HP-treatment at 150 MPa and 37 ◦ C for 2,
4, 6, and 10 min. The germination capacities of the heat-activated and
non-activated spores were compared. The experiments were performed
three times independently (n = 3) and the results are expressed as mean
value ± standard deviation.
2.5. Isolation and stability of HPSD spores
To isolate HPSD spores, 1010 CFU/mL spores were HP-treated in 50
mM ACES at 150 MPa and 37 ◦ C for 6 and 20 min, respectively, to
compare a milder and stronger trigger. The reason for choosing two
different cut-off points is that the cut-off point might influence the
A.I. Delbrück et al.
characteristics of the superdormant spores. By choosing a milder and
stronger germination trigger, one can understand the general trends and
factors influencing the properties of HPSD spores. The 6 min threshold
time was selected because preliminary experiments showed that the
great majority of spores (>95%) germinate within the first 5 min of HP
treatment. In addition, 6 min is within the realistic time frame of po­
tential industrial treatments. In comparison to that, 20 min was chosen
as a stronger germination trigger.
Unless otherwise stated, the HPSD spores were isolated in a two-step
process. Buoyant density centrifugation (Lindsay et al., 1985) was used
in a first step to enrich the HPSD spores fraction. Buoyant density
centrifugation alone, however, was not always sufficient to isolate
superdormant spores with high purity. Therefore, in a second step, FACS
was used to sort out HPSD spores, to reach a final purity of 91–98%. HP
treatment, pre-enrichment, and isolation were conducted within one
day. The protocol for buoyant density centrifugation of spores was
adapted from Ghosh and Setlow (2009). Briefly, 1.4 mL of HP-treated
spores were centrifuged for 2 min at 12,000 ×g and 4 ◦ C (Micro Star
17R, VWR International GmbH, Dietikon, Switzerland) to spin down the
cells and remove the supernatant. The pellets were suspended in 100 μL
20% Nycodenz® (Axis-Shield, Oslo, Norway) and carefully layered on
top of 1.8 mL 50% Nycodenz® in an Eppendorf tube. The tube was then
centrifuged for 45 min at 13,000 ×g and 4 ◦ C. The principle behind
buoyant density centrifugation is that the germinated spores have a
lower density compared to the dormant spores because of the water
uptake. By centrifuging the mixture in a density gradient, the dormant
spores formed pellets, while the germinated spores floated. The super­
natant was carefully removed, and the dormant spores were re-
suspended and washed four times in MilliQ water to remove Nyco­
denz®. A second Nycodenz® treatment did not yield improved purity,
and was, therefore, not applied in this study. To achieve better separa­
tion of the dormant and germinated spores, HP-treated spores were
incubated at 4 ◦ C for 45 min before performing the buoyant density
centrifugation. This allowed germinating spores to take up water, thus
increasing the density difference between the dormant and germinated
spores. To further increase the purity, the enriched HPSD spores were
sorted using FACS as previously described (Zhang et al., 2020a). The
HPSD spores were gated as sorting targets and the gating window was
intentionally set conservatively, i.e. quite narrow, to ensure that no
germinated spores or cells with compromised cell membranes were
sorted out. The isolated HPSD spores were stored in the dark at 4 ◦ C.
The storage stability of the isolated HPSD spores at 4 ◦ C was inves­
tigated by checking their purity using FCM the day after isolation
(defined as day 1), a week later, and a month later. The isolations were
performed three times independently (n = 3) and the results are
expressed as mean value ± standard deviation. Unless otherwise stated,
all further characterisations of the HPSD spores were performed within
one day after isolation.
2.6. Transmission electron microscopy analysis of isolated HPSD spores
Since very little is known about the underlying reasons for HP spore
superdormancy, it was of interest to study the microscopic structure of
the isolated superdormant spores and compare them to the initial
dormant spore population. In theory, it could be possible that the
superdormant spores e.g. have a thicker cortex or another phenotypic
difference in structure. To investigate the potential structural differ­
ences, 1.4 mL of spores with a concentration of 1010 CFU/mL were HP-
treated in 50 mM ACES at 150 MPa and 37 ◦ C for 6 min. Spores were
only isolated using buoyant density centrifugation since an additional
purification step via FACS would dilute them too strongly to allow
microscopic analysis. To increase the purity of the buoyant density
centrifugation, the HP-treated spores were incubated at 37 ◦ C instead of
4 ◦ C for 45 min before centrifugation to speed up the germination of the
triggered spores. With this approach, ≥96% superdormant spores could
be isolated.
4
International Journal of Food Microbiology 343 (2021) 109088
Samples for transmission electron microscopy (TEM) were prepared
as follows: Approx. 2 mL of spore suspensions were centrifuged at 370
×g and the sediment was transferred into Eppendorf tubes. This was
again briefly centrifuged to form a cell pellet; the supernatant was
removed and the cell pellet was re-suspended in a volume of supernatant
equal to the volume of the pellet. The cells were put into cellulose tubes
(Hohenberg et al., 1994) and frozen in triplicate with the supernatant as
the filling medium in 6 mm aluminium planchettes in an HPM 100 high-
pressure freezing system (Bal-Tec, Balzers, Liechtenstein). The samples
were subjected to freeze-substitution in acetone, dried over a molecular
sieve, and supplemented with 0.5% uranyl acetate (Polysciences, War­
rington, USA) added from a 5% stock solution in methanol and 2%
osmium tetroxide (Polysciences, Warrington, USA), kept for 10 h at
− 90 ◦ C, then warmed to − 45 ◦ C at 5 ◦ C/h, left at − 45 ◦ C for 8 h, then
warmed to 0 ◦ C at 7.5 ◦ C/h, left for 8 h at 0 ◦ C, and then kept for 1.5 h at
room temperature. The samples were then rinsed three times in dry
acetone, microwave-assisted (Pelco BioWave, Ted Pella Inc., California,
US). For resin embedding in Epon (Fluka Epoxy Embedding Kit), the
samples were infiltrated in duplicate with 30% Epon in dry acetone
three times, microwave-assisted, followed by three changes of 70%
Epon, microwave-assisted twice, then once overnight at 4 ◦ C, and three
changes of 100% Epon (1, 2, and 3 h at room temperature on a shaker).
The samples were then transferred into moulds with fresh resin and
polymerised for 3 days at 60 ◦ C. Thin sections of 50 nm were obtained
with a diamond knife (Diatome Ltd., Nidau, Switzerland) on a Leica UC6
ultramicrotome (Leica Microsystems, Heerbrugg, Switzerland), placed
on formvar and carbon-coated TEM grids (Quantifoil, Großlöbichau,
Germany), and stained with 2% aqueous uranyl acetate and Reynold’s
lead citrate for 30 s each. The stained sections were then visualised using
a Morgagni 268 TEM at 100 kV (FEI Company, Eindhoven,
Netherlands). The average length (n ≥ 48 spores for each condition) and
width (n = 50 spores for each condition), and the cortex thickness (n =
50 spores for each condition) of dormant and HPSD spores were
measured using the ImageJ software (National Institutes of Health,
USA). For cortex measurement, the cortex width was measured in four
different positions within each cell. The significance of the difference
between the means was determined using a two-tailed, independent
samples t-test in SPSS Statistics after testing for normality and equal
variance with the Shapiro Wilk and Levene’s tests, respectively. When
normality and equal variance were given, a Student’s t-test was used,
while when normality was given but no equal variance, Welch’s t-test
was used.
2.7. Characterisation of re-sporulated HPSD spores
To test whether the germination defect in the HPSD spores is due to a
genetic change, e.g. in a gene coding for a critical part of the germination
machinery, isolated HPSD spores were germinated and re-sporulated. If
a permanent genetic change was responsible for superdormancy, we
would expect the re-sporulated spores to have a decreased germination
capacity. To test this, HPSD spores isolated after a 6 min HP treatment at
150 MPa and 37 ◦ C as described in Section 2.5 were heated for 10 min at
80 ◦ C and plated on tryptic soy agar (TSA) with kanamycin. The applied
heating step ensured that the growing colonies were only derived from
HPSD B. subtilis PS533 spores. It cannot be excluded that some of the
HPSD spores could not germinate on nutrient agar too. However, pre­
vious research has shown that the great majority of HPSD spores can
germinate on nutrient-rich TSA (Zhang et al., 2020a). In this study, we
used the same nutrient-rich agar. Six single colonies on TSA were picked
and re-sporulated independently as described in Section 2.1. The re-
sporulated spores from these six colonies were harvested as described
before and HP-treated for 6 min at 150 MPa in triplicate. A Kruskal-
Wallis-test in the R package was performed to test the differences in
germination capacity between the spores originating from these six
different colonies. In addition, spores originating from one colony were
HP-treated at 150 MPa and 37 ◦ C for various dwell times to investigate
A.I. Delbrück et al. International Journal of Food Microbiology 343 (2021) 109088

the germination kinetics among a range of HP treatment times. The
germination capacity of the re-sporulated HPSD spores was compared to
that of the normal dormant spores that were sporulated in parallel under
the same conditions. The experimental design of this investigation is
shown in Fig. 1A.
2.8. Germination capacity of isolated HPSD spores
An interesting question about HPSD spores is whether they would
stay dormant or germinate when treated with a second HP trigger (re-
HP). To test this, the initial dormant spores were HP-treated at 150 MPa
and 37 ◦ C for 6 or 20 min, isolated with buoyant density centrifugation,
purified with FACS as described in Section 2.5, and re-HP-treated either
6 or 20 min at 150 MPa and 37 ◦ C. The germination capacity of HPSD
spores, reflected by the percentage of the germinated and dormant
spores after the re-HP treatment, was compared to the germination ca­
pacity of the initial dormant spore population. To take into account a
potential change in the germination capacity over time, re-HP treat­
ments were performed the day after isolation and a week later. All ex­
periments were performed three times independently (n = 3) and the
results are expressed as mean value ± standard deviation. The experi­
mental design of this investigation is shown in Fig. 1B.

Fig. 1. Visualisation of the experimental design of A) Resporulating high-pressure superdormant (HPSD) Bacillus subtilis spores to investigate whether super­
dormancy is due to a genetic change (Section 2.7). For the resporulation experiment, the germination capacity of re-sporulated spores originating from HPSD spores
was compared to that of the normal dormant spores that were sporulated in parallel to the spores originating from HPSD spores. B) Investigating the germination
capacity of HPSD B. subtilis spores (Section 2.8). HPSD spores isolated after a 6 and 20 min treatment at 150 MPa and 37 ◦ C are referred to as HPSD6min and
HPSD20min, respectively. Dormant spores are represented in white, germinated spores in black.

5
A.I. Delbrück et al.
3. Results and discussion
3.1. Factors influencing the prevalence of HPSD spores after 150 MPa HP
germination
3.1.1. Pressure dwell time
Several factors may influence the germination capacity of spores at
150 MPa and 37 ◦ C in ACES. As expected, a strong influencing factor is
the pressure dwell time. With increasing pressure dwell time, the frac­
tion of HPSD spores decreases (Fig. 2). Within 5 min, >95% of spores
germinated. The germination of the majority of B. subtilis spores within
less than 5 min of HP treatment at 150 MPa and 37 ◦ C was also observed
by other researchers using other buffer systems like Tris-HCl and K-
HEPES buffer (Black et al., 2005; Luu et al., 2015). After 6 min, 2.6 ±
0.3% of spores remained dormant and the dormant spore fraction kept
decreasing with increasing treatment time. The shortest investigated HP
treatment consisted only of a 3 s dwell time and therefore, mainly of
compression and decompression. No germination was observed in this
sample (98.9 ± 0.5% dormant spores before HP treatment and 98.2 ±
0.6% dormant spores after 3 s HP treatment), suggesting that the pres­
sure build-up and release used in these experiments did not significantly
contribute to the extent of germination.
3.1.2. Germinant receptors
It is generally understood that germination at 150 MPa and 37 ◦ C is
via the GRs (Black et al., 2007a, 2005; Paidhungat et al., 2002; Reineke
et al., 2012; Wuytack et al., 2000, 1998). Accordingly, this research also
reinforced this conclusion as it found that the germination of B. subtilis
FB72, an isogenic mutant of PS533 with deletion of the three main GRs,
is strongly deficient to germinate. Even after a long holding time of 40
min, 93.1 ± 0.6% of spores remained dormant (data not shown).
3.1.3. Heat activation
A sublethal heat treatment prior to germination is known to strongly
increase nutrient germination (Setlow et al., 2017). Since nutrient
germination and HP germination at 150 MPa seem to share similarities,
especially concerning their dependency on GRs, it was of interest to also
Fig. 2. Prevalence of high-pressure superdormant (HPSD) spores in depen­
dence of the high-pressure dwell time. Bacillus subtilis spores were high-
pressure-treated at 150 MPa and 37 ◦ C in 50 mM ACES, pH 7 for different
dwell times. The percentage of HPSD spores within the total population was
determined using flow cytometry. With increasing pressure dwell time, HPSD
spores prevalence decreased. Error bars represent standard deviations of three
independent experiments (n = 3). Data points below 20% are additionally
plotted in the top right corner for better readability.
6
International Journal of Food Microbiology 343 (2021) 109088
investigate the effect of heat activation on HP germination. Interest­
ingly, no increase was observed in germination under HP when prior
heat activation was applied. On the contrary, for all tested pressure
dwell times, the prevalence of HPSD spores was even higher when spores
were heat-activated. Heat-activated spores showed up to 23% more
HPSD spores compared to HP-treated spores without prior heat activa­
tion (Fig. 3). These results were rather unexpected, and no plausible
explanation can be found for the observed decrease in germination after
heat activation.
However, the result that heat activation did not increase germination
at 150 MPa is mostly aligned with findings of previous research (Kong
et al., 2014; Luu et al., 2015; Wei et al., 2010). Some research found that
prior heat activation of B. subtilis spores had no significant effect on
germination at 150 MPa (Kong et al., 2014; Wei et al., 2010). Since GRs
have been hypothesised as potential direct targets of heat activation
(Setlow et al., 2017), it was rather unexpected that heat activation
would have a positive effect on nutrient germination but not on HP
germination at 150 MPa. However, the exact mechanism of heat acti­
vation is too poorly understood to draw any conclusions here. Luu et al.
(2015) investigated this aspect in greater detail on isogenic mutants of
B. subtilis PS533 lacking certain GRs. They found that heat activation
stimulated the germination at 150 MPa via GerB and GerK, and to a
lesser extent via GerA. The fact that germination at 150 MPa is pre­
dominantly triggered through the activation of GerA (Black et al., 2005)
could explain why the effect of heat activation was not seen in the
wildtype in this study. However, even though this might explain why
increased germination was not seen, it remains unclear why germination
at 150 MPa was even reduced after heat activation.
3.2. Characteristics of isolated HPSD spores
3.2.1. Isolation and stability of HPSD spores
HPSD spores were isolated after 6 and 20 min HP treatment at 150
MPa and 37 ◦ C. To facilitate the comprehension of the following results
and discussion, HPSD spores isolated after 6 and 20 min treatment are
referred to as HPSD6min and HPSD20min, respectively. Buoyant density
centrifugation has been used by other researchers for the isolation of
nutrient, Ca2+-DPA, and dodecylamine superdormant spores (Ghosh and
Setlow, 2010, 2009; Perez-Valdespino et al., 2013). However, in our
case, the purity of the HPSD spores was only approximately 80% for 6
min HP-treated spores after the first run of buoyant density
Fig. 3. Prevalence of high-pressure superdormant (HPSD) Bacillus subtilis
spores after high-pressure (HP) germination with and without prior heat acti­
vation. Spores were or were not heat-activated at 75 ◦ C for 30 min before HP
germination at 150 MPa and 37 ◦ C in 50 mM ACES, pH 7. The percentage of
HPSD spores within the total population was determined using flow cytometry.
Heat activation slowed down germination. Error bars represent standard de­
viations of three independent experiments (n = 3).
A.I. Delbrück et al.
centrifugation and a second cycle did not increase the purity. Therefore,
FACS was applied as a further step to increase the purity and by
combining these two methods, we reached a purity of 98.3 ± 0.8%
HPSD6min spores the day after isolation. These isolated spores remained
dormant over the entire test period of 28 days at 4 ◦ C (Fig. 4). For HPSD
spores left after 20 min HP treatment, buoyant density centrifugation
only reached an isolation purity of approximately 20% HPSD20min
spores. The reason for this reduced purity achieved using buoyant
density centrifugation is most probably that the percentage of HPSD
spores became so low after this strong germination trigger that impu­
rities left from imperfect removal of floating germinating cells propor­
tionally had a higher impact than in the 6 min HP-treated sample.
Subsequent FACS isolation, however, increased the purity of the
HPSD20min spores to 91.8 ± 1.8% the day after isolation. Contrary to the
HPSD6min spores, the HPSD20min spores showed an average of approxi­
mately 10% germination over the monitored period of 28 days at 4 ◦ C
(Fig. 4).
It is not entirely clear why HPSD20min spores would start to partially
germinate during storage at 4 ◦ C, while the HPSD6min spores do not. One
hypothesis could be that the strong germination trigger of 20 min
stimulated some difficult germinators, i.e. spores that, for instance, need
a higher threshold as a signal to elicit germination, while the milder 6
min treatment only triggered the germination of the easy germinators.
These difficult germinators germinated extremely slowly, and within the
3–5 h between HP treatment and isolation with FACS they did not fully
degrade the cortex; therefore, they were categorised as dormant spores
based on their fluorescent signal and sorted out together with the HPSD
spores fraction. After sorting, germination proceeded and these slow
germinators fully germinated. Certain compounds released by the
germinating cells, such as Ca2+-DPA, in turn, might have triggered the
germination of other spores potentially explaining the decreased
stability.
3.2.2. The microscopic structure of HPSD spores
To investigate the potential structural differences in the super­
dormant and normal dormant spores, the isolated HPSD spores were
compared to the initial dormant population using phase-contrast
Fig. 4. Storage stability of isolated high-pressure superdormant (HPSD) Bacillus
subtilis spores at 4 ◦ C. HPSD spores were isolated by a combination of buoyant
density centrifugation and fluorescence-activated cell sorting after an initial
high-pressure treatment of 6 and 20 min at 150 MPa and 37 ◦ C in 50 mM ACES,
pH 7. Dormant spores were quantified using flow cytometry. HPSD spores
isolated after 6 min showed very good stability, while HPSD spores isolated
after 20 min showed partial germination during one-month storage. Error bars
represent standard deviations of three independent experiments (n = 3). To
allow comparison between the different replicates, the displayed data has been
normalised to 100% dormant spores on day 1.
7
International Journal of Food Microbiology 343 (2021) 109088
microscopy and TEM (Fig. 5). As expected, there was no difference be­
tween the initial dormant spores and the isolated HPSD spores under the
phase-contrast microscope. In phase-contrast microscopy, both the
dormant and HPSD spores appeared phase-bright owing to their high
core refractive index as a consequence of the very low water content
(Hashimoto et al., 1969; Kong et al., 2011). A closer look was enabled by
TEM where the different layers of the dormant spores were visible.
However, no obvious difference was observed between the initial
dormant spore population and the isolated HPSD spores via visual in­
spection. Not to overlook any potential differences, the average total
spore length and width, and the cortex thickness of the dormant and
HPSD spores were quantitatively compared. Quantitative comparison of
the average spore length of the dormant (M = 1322 nm, SD = 80 nm)
and the HPSD spores (M = 1340 nm, SD = 112 nm) showed no signif­
icant difference (n ≥ 48, p = 0.382). Quantitative comparison of the
average spore width of the dormant (M = 670 nm, SD = 59 nm) and the
HPSD spores (M = 682 nm, SD = 49 nm) also showed no significant
difference (n = 50, p = 0.272). Similarly, systematic quantification of
the spore cortex thickness showed no significant difference between the
dormant spores (M = 105 nm, SD = 21 nm) and the HPSD spores (M =
107 nm, SD = 20 nm) (n = 50, p = 0.231) (data visualisation can be
found in the supplementary material). It, therefore, seems unlikely that
spore length, width, and cortex thickness are a determining factor in
spore HP superdormancy.
3.2.3. Spore HP superdormancy is most likely not due to a genetic change
To test whether superdormancy might be traced back to a genetic
change, e.g. a defect in a gene coding for a critical component of the
germination machinery, the isolated HPSD spores were germinated and
re-sporulated to a new generation of dormant spores, which were all
descendants of the former HPSD spores. If a permanent genetic change
was responsible for superdormancy, we would expect the re-sporulated
population to have an increased germination deficiency compared to the
initial dormant spores. A similar approach was used previously by Ghosh
and Setlow (2009) and Chen et al. (2014) to investigate whether
nutrient superdormant spores are genetically different from normal
dormant spores.
In this study, six single colonies of the germinated HPSD spores were
re-sporulated independently. In the first step, the resulting six spore
populations were tested for similarity among each other by treating
them individually at 150 MPa and 37 ◦ C for 6 min. All six spore pop­
ulations were able to germinate well under these conditions and no
significant difference was observed in the germination capacity between
the six populations (n = 3, p = 0.63). In a second step, one of the six
descendant spore populations derived from the HPSD spores was
randomly chosen to compare its germination capacity under HP at
different dwell times with the normal spores that were sporulated in
parallel (experimental design shown in Fig. 1A). The results showed that
there is no difference in the germination capacity between the de­
scendants of the HPSD spores and the normal dormant spores (Fig. 6).
Thus, even though the HPSD spores behaved phenotypically different
from the rest of the population, it seems unlikely that they are geneti­
cally different. These findings suggest that the germination defect of the
HPSD spores is most probably not due to genetic alteration. This
conclusion is also supported by results presented in Section 3.2.4
showing that the majority of the HPSD spores germinated when exposed
a second time to the same trigger. If a genetic alteration of a component
vital for germination was responsible for superdormancy, we would not
expect that spores that were superdormant in a first HP treatment,
germinate in a second one. Similarly, Ghosh and Setlow (2009) and
Chen et al. (2014) found that the germination defect in nutrient super­
dormant spores was not due to a genetic change.
3.2.4. Germination capacity of HPSD spores
To investigate the extent of the superdormancy of the isolated HPSD
spores and to gain some mechanistic understanding of superdormancy,
A.I. Delbrück et al.
Fig. 6. Germination capacity of the re-sporulated high-pressure superdormant
(HPSD) and normal Bacillus subtilis spores. Spores were high-pressure-treated at
150 MPa and 37 ◦ C in 50 mM ACES, pH 7 for various dwell times. The per­
centage of the HPSD spores within the total population has been determined
using flow cytometry. The HPSD spores were isolated after 6 min HP treatment,
re-sporulated, and their germination capacity was compared to that of the
normal spores that were sporulated in parallel. The germination capacities of
the re-sporulated and normal spores were very similar. The graphs show the
means and standard deviations of n = 3.
it was of interest to see whether they would also be defective to
germinate in a second HP treatment of 150 MPa at 37 ◦ C. To test this, the
initial dormant spores were HP-treated at 150 MPa and 37 ◦ C for 6 or 20
min representing a milder and stronger trigger, respectively. The HPSD
spores were isolated and re-HP-treated either for 6 or 20 min at 150 MPa
and 37 ◦ C. The re-HP treatments were performed one day after isolation
and a week after isolation. As no difference was observed in germination
capacity (data not shown), only results obtained from re-HP the day
after isolation are presented below (Fig. 7). The results revealed that the
majority of isolated HPSD spores could germinate in a second HP
treatment with the same treatment parameters used for their isolation
(Fig. 7). However, their germination capacity decreased compared to
that of the initial dormant spore population.
More than 98% of the initial dormant spore population germinated
when exposed to 6 or 20 min HP treatment. Compared to that, only
approximately 85% of the HPSD6min germinated when re-HP-treated for
another 6 min (15.4 ± 0.5% remain dormant) and approximately 57% of
the HPSD20min germinated when exposed a second time to 20 min HP
treatment (43 ± 3.5% remain dormant) (Fig. 7). Comparing the
germination capacities of the HPSD6min and HPSD20min spores re-treated
8
International Journal of Food Microbiology 343 (2021) 109088
Fig. 5. Transmission electron microscopy micro­
graphs of a dormant spore and a high-pressure
superdormant (HPSD) Bacillus subtilis spore. The
microscopic appearance of the initial dormant spore
population was compared to the HPSD spores isolated
with buoyant density centrifugation after 6 min high-
pressure treatment at 150 MPa and 37 ◦ C. Repre­
sentative images of the initial and HPSD spore pop­
ulation are depicted (>150 spores visually
inspected). A) Initial dormant spore population, B)
HPSD spores. Typical spore structures are depicted,
with OSC: outer spore coat, ISC: inner spore coat,
OM: outer membrane, Cx: cortex, Cw: cell wall, IM:
inner membrane, Co: core. No qualitative difference
in microscopic appearance was observed between the
dormant and superdormant spores.
with the same intensity (6 min), it is evident that the HPSD20min spores
showed increased HP germination deficiency compared to the HPSD6min
spores (Fig. 7). Only 15.4 ± 0.5% of HPSD6min remained dormant in a
second HP of 6 min, while 71.3 ± 0.7% of HPSD20 min remained dormant
when re-treated for 6 min.
The finding that HPSD20min spores showed increased germination
deficiency compared to HPSD6min spores, was expected because
increasing trigger intensity increases the selection pressure for
dormancy. It is, however, interesting that the majority of HPSD spores
can germinate when exposed a second time to the same trigger intensity.
Considering the similarities in the GR-dependent germination pathway
between HP at 150 MPa and nutrients (Black et al., 2005; Paidhungat
et al., 2002; Wuytack et al., 1998), it is worthwhile putting the char­
acteristics observed in the context of what is known about nutrient
superdormancy. However, given the poor understanding of HP germi­
nation via GRs, the comparison needs to be done cautiously.
Similar to this study, Ghosh and Setlow (2009) investigated the
germination capacity of nutrient superdormant B. subtilis and
B. megaterium spores when re-exposed to the same trigger intensity used
for their isolation. They observed that nutrient superdormant spores
remained dormant or germinated very poorly when re-exposed. This
observation was made for both moderate and high nutrient concentra­
tions. This is different from our observation with the HPSD spores,
where the majority of the HPSD spores germinated when re-exposed to
the trigger. However, care must be taken when comparing the charac­
teristics of superdormant spores, because they are strongly influenced by
isolation conditions. Ghosh and Setlow (2009) used a more stringent
isolation procedure with two germination and isolation cycles in a row.
Ghosh and Setlow (2010) also observed that a substantial fraction of the
superdormant B. cereus spores isolated after one germination cycle
germinated when exposed a second time to the same nutrient trigger.
Therefore, it is possible that the HPSD spores isolated after two HP and
isolation cycles could have also shown very little germination in a
(third) re-HP. In addition, our results suggest that with increasing
pressure dwell time before isolation, the HPSD spores show decreased
germination capacity in a second exposure. It is, therefore, possible that
HPSD spores isolated after an even longer pressure dwell time than 20
min might have germinated very poorly.
The observed partial loss in superdormancy when re-exposed to the
HP germination trigger, might also give some first hints on the under­
lying mechanism of HP superdormancy. Given the common dependency
of nutrient and HP germinations at 150 MPa on GRs, it seems reasonable
to hypothesise that low GR expression levels might also contribute to HP
superdormancy as they have been shown to play a major role in nutrient
superdormancy (Ghosh and Setlow, 2010, 2009). Findings by Black
et al. (2005), which showed that the levels of GRs influenced the rates of
pressure germination at 150 MPa and that elevated levels of individual
receptors increased spore germination, further substantiate this hy­
pothesis. On one hand, the findings made in this study seem not to
A.I. Delbrück et al. International Journal of Food Microbiology 343 (2021) 109088

Fig. 7. Germination capacity of isolated HPSD spores at 150 MPa and 37 ◦ C. Bacillus subtilis spores were high-pressure (HP)-treated for 6 or 20 min at 150 MPa and
37 ◦ C. The HPSD spores were isolated and re-HP-treated the day after isolation for 6 and/or 20 min at 150 MPa and 37 ◦ C. The displayed data has been normalised to
100% dormant spores after the isolation. The majority of the isolated HPSD spores were able to germinate when exposed a second time to an HP trigger; however,
their germination capacity was reduced compared to that of the initial dormant spore population. The graph shows the rounded means of n = 3; the exact means and
standard deviations can be found in the text.

strongly support this hypothesis. The GR levels of the individual spores
should not change between the first and second HP treatments. Still, the
majority of spores that remained dormant in the first HP treatment were
able to germinate in the second treatment. Hence, the GR level alone
would not explain why they germinated in the second HP treatment. In
contrast, it is difficult to draw a concrete conclusion here because the
mechanism of HP germination via GRs is very poorly understood. There
seems to be a consensus that an HP of 150 MPa somehow activates GRs.
How this activation works at the molecular level, however, is not un­
derstood. It has been suggested that HP might alter membrane proper­
ties directly or cause structural changes in the GRs itself, thereby
initiating the germination cascade (Black et al., 2007a; Doona et al.,
2016, 2014; Kong et al., 2014; Paidhungat et al., 2002; Setlow et al.,
2017). In theory, it could, for instance, be possible that the first HP
trigger leads to a slight, but (at least temporary) stable conformational
change in the GRs, which is not enough to trigger germination in the first
treatment. However, the additional, cumulative, conformational change
caused by a second HP treatment might be strong enough to trigger
germination for some of the spores. In more general terms, spores might
get partially activated in the first HP treatment and full activation takes
place only in the second HP treatment. While this might be an intriguing
explanation, this is at most a hypothesis at this point and further in­
vestigations are needed.
To conclude, there are still too many unknowns concerning the
mechanistic effect of pressure activation via GRs to conclude on the role
of GR levels in HP superdormancy. Based on previous studies showing
that GR levels play a major role in the 150 MPa germination rate (Black
et al., 2005; Doona et al., 2014), it is possible that they also play a role in
superdormancy, as superdormancy is a direct consequence of poor
germination. However, the results of this study strongly suggest that this
is not the only determining factor and that other factor(s) might play a
role in HP superdormancy. This is also in alignment with the results of
Black et al. (2005) and Doona et al. (2014), who both found that GR
levels are a major factor in determining the germination rate of 150 MPa
germination but that there must be one or several other factors that
determine the rate of spore germination. For example, based on a study
with mutant strains, Doona et al. (2014) suggested the levels of a small
protein that appears to be a D subunit of some GRs and perhaps GerD as
possible factors that influence the germination rate.
4. Conclusion
HPSD spores are the major hurdle in the successful implementation
of a germination-inactivation strategy for mild bacterial spore inacti­
vation in industrial applications. Thorough characterisation of HPSD
spores is therefore needed to better understand spore HP
superdormancy.
Therefore, this study isolated HPSD spores and reported for the first
time some relevant properties thereof. Systematic characterisation of
HPSD spores helped improve the understanding of HP superdormancy
and gave some first hints of the potential underlying reasons for HP
superdormancy. The following main conclusions can be drawn from this
study:
(i) The prevalence of HPSD spores is strongly influenced by the
pressure dwell time. Increasing pressure dwell times reduce the
amount of HPSD spores. A sublethal heat activation did not
decrease the prevalence of HPSD spores; it even increased the
amount of HPSD spores. In addition, GRs are important in 150
MPa germination as confirmed by the extremely poor germina­
tion of a mutant strain lacking the three functional GRs GerA,
GerB, and GerK.

9
A.I. Delbrück et al.
(ii) A TEM structural analysis of normal dormant and HPSD spores
did not show any visible difference. Furthermore, a quantitative
comparison of the spore length and width, and the cortex thick­
ness did not show any significant difference suggesting that these
features are not the underlying causes of superdormancy.
(iii) Re-sporulated HPSD spores yielded a spore population that
exhibited similar germination characteristics compared to the
initial dormant spore population, which indicates that HPSD
spores are not genetically different from the rest of the popula­
tion. Hence, an underlying genetic reason for superdormancy is
most likely ruled out. This conclusion was further reinforced by
the finding that the majority of the HPSD spores germinated after
a second, same HP treatment.
(iv) A second HP treatment of isolated HPSD spores showed that the
germination capacity of HPSD spores markedly decreased
compared to that of the initial spore population, but the majority
of the HPSD spores germinated when exposed a second time to
the same trigger. This finding suggests that it is rather unlikely
that there is one “deal-or-no-deal” type of permanent cause that is
solely responsible for superdormancy. Instead, it is possible there
are other cumulative factors or factors of transient or reversible
nature, that influence superdormancy.
Therefore, if general spore size, cortex thickness, an underlying ge­
netic alteration, and GR levels alone are unlikely to explain super­
dormancy, what then is causing some spores to remain dormant while
others germinate? While this study is the first step towards a better
understanding of HP superdormancy, further research is needed to
answer this question. It is also unclear to what extent the underlying
causes of HP superdormancy at 150 MPa and nutrient superdormancy
are similar. The potential roles of GRs, GerD, and other germination-
related proteins could be investigated using proteome analysis as well
as the comparison between dormant and HPSD spores. The reason for
some spores remaining superdormant might be because of stochastic
variations in the expression of certain proteins. In addition, the study of
potential transient or reversible causes might clarify HP superdormancy.
Notably, understanding what the activation of spores by 150 MPa means
in molecular terms would greatly benefit the understanding of HP
superdormancy and most probably also nutrient superdormancy. To
further explain the potential superdormancy mechanisms, it would also
be of interest to investigate and compare the germination capacity of
HPSD spores with other germination triggers including nutrients,
dodecylamine, and Ca2+-DPA. Apart from potentially giving mecha­
nistic insights, these findings are also of applied interest. Of special in­
terest is the investigation of the germination capacity of HPSD spores
with nutrients, given their natural presence in food. Further, it is
important to keep in mind that the germination pathway of moderate HP
around 150 MPa and very HP around 500–600 MPa, is different and
hence a combination of moderate and very HP might trigger more
bacterial spores. Finally, it is important to investigate whether the in­
sights gained from the model organism B. subtilis can be transferred to
other Bacillus species and it will be of particular importance to also
thoroughly study superdormancy in spores of the genus Clostridium.
In conclusion, we are only at the beginning of understanding
superdormancy and further studies are required to unravel its underly­
ing cause. However, once these causes are known, potential strategies to
target superdormant spores can be developed, allowing the germination
of all bacterial spores and enabling safe and gentle germination-
inactivation-based spore control strategies.
Declaration of competing interest
The authors declare that this research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
10
International Journal of Food Microbiology 343 (2021) 109088
Acknowledgment
This study was supported by the Swiss National Science Foundation
SNF (Grant number: 31003A_182273). We further thank the Scientific
Center for Optical and Electron Microscopy (ScopeM) at ETH Zurich for
their support with the transmission electron microscopy pictures as well
as the Cytometry Facility of the University of Zurich for their technical
support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijfoodmicro.2021.109088.
References
Abee, T., Groot, M.N., Tempelaars, M., Zwietering, M., Moezelaar, R., van der Voort, M.,
2011. Germination and outgrowth of spores of Bacillus cereus group members:
diversity and role of germinant receptors. Food Microbiol. https://doi.org/10.1016/
j.fm.2010.03.015.
Aertsen, A., Van Opstal, I., Vanmuysen, S.C., Wuytack, E.Y., Michiels, C.W., 2005.
Screening for Bacillus subtilis mutants deficient in pressure induced spore
germination: identification of ykvU as a novel germination gene. FEMS Microbiol.
Lett. https://doi.org/10.1016/j.femsle.2004.12.029.
Ahn, J., Balasubramaniam, V.M., Yousef, A.E., 2007. Inactivation kinetics of selected
aerobic and anaerobic bacterial spores by pressure-assisted thermal processing. Int.
J. Food Microbiol. 113, 321–329. https://doi.org/10.1016/J.
IJFOODMICRO.2006.08.012.
den Besten, H.M.W., Wells-Bennik, M.H.J., Zwietering, M.H., 2018. Natural diversity in
heat resistance of bacteria and bacterial spores: impact on food safety and quality.
Annu. Rev. Food Sci. Technol. https://doi.org/10.1146/annurev-food-030117-
012808.
Black, E.P., Koziol-Dube, K., Guan, D., Wei, J., Setlow, B., Cortezzo, D.E., Hoover, D.G.,
Setlow, P., 2005. Factors influencing germination of Bacillus subtilis spores via
activation of nutrient receptors by high pressure. Appl. Environ. Microbiol. 71,
5879–5887. https://doi.org/10.1128/AEM.71.10.5879-5887.2005.
Black, E.P., Setlow, P., Hocking, A.D., Stewart, C.M., Kelly, A.L., Hoover, D.G., 2007a.
Response of spores to high-pressure processing. Compr. Rev. Food Sci. Food Saf. 6,
103–119. https://doi.org/10.1111/j.1541-4337.2007.00021.x.
Black, E.P., Wei, J., Atluri, S., Cortezzo, D.E., Koziol-Dube, K., Hoover, D.G., Setlow, P.,
2007b. Analysis of factors influencing the rate of germination of spores of Bacillus
subtilis by very high pressure. J. Appl. Microbiol. 102, 65–76. https://doi.org/
10.1111/j.1365-2672.2006.03062.x.
Brookmeyer, R., Johnson, E., Bollinger, R., 2003. Modeling the optimum duration of
antibiotic prophylaxis in an anthrax outbreak. Proc. Natl. Acad. Sci. U. S. A. https://
doi.org/10.1073/pnas.1631983100.
Chen, Y., Ray, W.K., Helm, R.F., Melville, S.B., Popham, D.L., 2014. Levels of
germination proteins in Bacillus subtilis dormant, superdormant, and germinating
spores. PLoS One 9, e95781. https://doi.org/10.1371/journal.pone.0095781.
Collado, J., Fernández, A., Rodrigo, M., Martínez, A., 2004. Variation of the spore
population of a natural source strain of Bacillus cereus in the presence of inosine.
J. Food Prot. https://doi.org/10.4315/0362-028X-67.5.934.
Doona, C.J., Ghosh, S., Feeherry, F.F., Ramirez-Peralta, A., Huang, Y., Chen, H.,
Setlow, P., 2014. High pressure germination of Bacillus subtilis spores with alterations
in levels and types of germination proteins. J. Appl. Microbiol. 117, 711–720.
https://doi.org/10.1111/jam.12557.
Doona, C.J., Feeherry, F.E., Setlow, B., Wang, S., Li, W., Nichols, F.C., Talukdar, P.K.,
Sarker, M.R., Li, Y.-Q., Shen, A., Setlow, P., 2016. Effects of high-pressure treatment
on spores of Clostridium species. Appl. Environ. Microbiol. 82, 5287–5297. https://
doi.org/10.1128/AEM.01363-16.
Doona, C.J., Feeherry, F.E., Kustin, K., Chen, H., Huang, R., Philip Ye, X., Setlow, P.,
2017. A quasi-chemical model for bacterial spore germination kinetics by high
pressure. Food Eng. Rev. https://doi.org/10.1007/s12393-016-9155-1.
Ghosh, S., Setlow, P., 2009. Isolation and characterization of superdormant spores of
Bacillus species. J. Bacteriol. 191, 1787–1797. https://doi.org/10.1128/JB.01668-
08.
Ghosh, S., Setlow, P., 2010. The preparation, germination properties and stability of
superdormant spores of Bacillus cereus. J. Appl. Microbiol. 108, 582–590. https://
doi.org/10.1111/j.1365-2672.2009.04442.x.
Ghosh, S., Scotland, M., Setlow, P., 2012. Levels of germination proteins in dormant and
superdormant spores of Bacillus subtilis. J. Bacteriol. 194, 2221–2227. https://doi.
org/10.1128/JB.00151-12.
Gould, G.W., Sale, A.J., 1970. Initiation of germination of bacterial spores by hydrostatic
pressure. J. Gen. Microbiol. https://doi.org/10.1099/00221287-60-3-335.
Hashimoto, T., Frieben, W.R., Conti, S.F., 1969. Germination of single bacterial spores.
J. Bacteriol. https://doi.org/10.1128/JB.98.3.1011-1020.1969.
Heine, H.S., Bassett, J., Miller, L., Hartings, J.M., Ivins, B.E., Pitt, M.L., Fritz, D.,
Norris, S.L., Byrne, W.R., 2007. Determination of antibiotic efficacy against Bacillus
anthracis in a mouse aerosol challenge model. Antimicrob. Agents Chemother.
https://doi.org/10.1128/AAC.01050-06.
A.I. Delbrück et al.
Hohenberg, H., Mannweiler, K., Müller, M., 1994. High-pressure freezing of cell
suspensions in cellulose capillary tubes. J. Microsc. https://doi.org/10.1111/j.1365-
2818.1994.tb04785.x.
Kim, S.S., Kim, S.H., Park, S.H., Kang, D.H., 2020. Inactivation of Bacillus cereus spores on
stainless steel by combined superheated steam and UV-C irradiation treatment.
J. Food Prot. https://doi.org/10.4315/0362-028X.JFP-19-133.
Knorr, D., Heinz, H., Schlüter, O., Zenker, M., 1998. The potential impact of high
pressure as unit operation for food processing. In: Isaacs, N.S. (Ed.), High Pressure
Food Science. Bioscience and Chemistry. The Royal Society of Chemistry,
Cambridge, pp. 227–235. https://doi.org/10.1533/9781845698379.3.227.
Kong, L., Zhang, P., Wang, G., Yu, J., Setlow, P., Li, Y.Q., 2011. Characterization of
bacterial spore germination using phase-contrast and fluorescence microscopy,
Raman spectroscopy and optical tweezers. Nat. Protoc. 6, 625–639. https://doi.org/
10.1038/nprot.2011.307.
Kong, L., Doona, C.J., Setlow, P., Li, Y., 2014. Monitoring rates and heterogeneity of
high-pressure germination of Bacillus spores by phase-contrast microscopy of
individual spores. Appl. Environ. Microbiol. 80, 345–353. https://doi.org/10.1128/
AEM.03043-13.
Kort, R., O’Brien, A.C., Van Stokkum, I.H.M., Oomes, S.J.C.M., Crielaard, W.,
Hellingwerf, K.J., Brul, S., 2005. Assessment of heat resistance of bacterial spores
from food product isolates by fluorescence monitoring of dipicolinic acid release.
Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.71.7.3556-3564.2005.
Lindsay, J.A., Beaman, T.C., Gerhardt, P., 1985. Protoplast water content of bacterial
spores determined by buoyant density sedimentation. J. Bacteriol. 163, 735–737.
Lovdal, I.S., Hovda, M.B., Granum, P.E., Rosnes, J.T., 2011. Promoting Bacillus cereus
spore germination for subsequent inactivation by mild heat treatment. J. Food Prot.
74, 2079–2089. https://doi.org/10.4315/0362-028x.Jfp-11-292.
Luu, S., Cruz-Mora, J., Setlow, B., Feeherry, F.E., Doona, C.J., Setlow, P., 2015. The
effects of heat activation on Bacillus spore germination, with nutrients or under high
pressure, with or without various germination proteins. Appl. Environ. Microbiol.
81, 2927–2938. https://doi.org/10.1128/AEM.00193-15.
Margosch, D., Gänzle, M.G., Ehrmann, M.A., Vogel, R.F., 2004. Pressure inactivation of
Bacillus endospores. Appl. Environ. Microbiol. 70, 1564–1569. https://doi.org/
10.1128/aem.70.12.7321-7328.2004.
Margosch, D., Ehrmann, M.A., Buckow, R., Heinz, V., Vogel, R.F., Gänzle, M.G., 2006.
High-pressure-mediated survival of Clostridium botulinum and Bacillus
amyloliquefaciens endospores at high temperature. Appl. Environ. Microbiol. 72,
3476–3481. https://doi.org/10.1128/AEM.72.5.3476-3481.2006.
Martínez-Monteagudo, S.I., Gänzle, M.G., Saldaña, M.D.A., 2014. High-pressure and
temperature effects on the inactivation of Bacillus amyloliquefaciens, alkaline
phosphatase and storage stability of conjugated linoleic acid in milk. Innov. Food
Sci. Emerg. Technol. https://doi.org/10.1016/j.ifset.2014.05.003.
Mathys, A., Chapman, B., Bull, M., Heinz, V., Knorr, D., 2007. Flow cytometric
assessment of Bacillus spore response to high pressure and heat. Innov. Food Sci.
Emerg. Technol. 8, 519–527. https://doi.org/10.1016/J.IFSET.2007.06.010.
Modugno, C., Peltier, C., Simonin, H., Dujourdy, L., Capitani, F., Sandt, C., Perrier-
Cornet, J.M., 2020. Understanding the effects of high pressure on bacterial spores
using synchrotron infrared spectroscopy. Front. Microbiol. https://doi.org/10.3389/
fmicb.2019.03122.
Mtimet, N., Trunet, C., Mathot, A.G., Venaille, L., Leguérinel, I., Coroller, L., Couvert, O.,
2015. Modeling the behavior of Geobacillus stearothermophilus ATCC 12980
throughout its life cycle as vegetative cells or spores using growth boundaries. Food
Microbiol. https://doi.org/10.1016/j.fm.2014.10.013.
Paidhungat, M., Setlow, P., 2000. Role of Ger proteins in nutrient and nonnutrient
triggering of spore germination in Bacillus subtilis. J. Bacteriol. 182, 2513–2519.
https://doi.org/10.1128/Jb.182.9.2513-2519.2000.
Paidhungat, M., Ragkousi, K., Setlow, P., 2001. Genetic requirements for induction of
germination of spores of Bacillus subtilis by Ca2+-dipicolinate. J. Bacteriol. 183,
4886–4893. https://doi.org/10.1128/jb.183.16.4886-4893.2001.
Paidhungat, M., Setlow, B., Daniels, W.B., Hoover, D., Papafragkou, E., Setlow, P., 2002.
Mechanisms of induction of germination of Bacillus subtilis spores by high pressure.
Appl. Environ. Microbiol. 68, 3172–3175. https://doi.org/10.1128/AEM.68.6.3172-
3175.2002.
Perez-Valdespino, A., Ghosh, S., Cammett, E.P., Kong, L., Li, Y.-q., Setlow, P., 2013.
Isolation and characterization of Bacillus subtilis spores that are superdormant for
germination with dodecylamine or Ca2+ -dipicolinic acid. J. Appl. Microbiol. 114,
1109–1119. https://doi.org/10.1111/jam.12125.
Reineke, K., Doehner, I., Schlumbach, K., Baier, D., Mathys, A., Knorr, D., 2012. The
different pathways of spore germination and inactivation in dependence of pressure
and temperature. Innov. Food Sci. Emerg. Technol. 13, 31–41. https://doi.org/
10.1016/J.IFSET.2011.09.006.
Reineke, K., Mathys, A., Heinz, V., Knorr, D., 2013. Mechanisms of endospore
inactivation under high pressure. Trends Microbiol. 21, 296–304. https://doi.org/
10.1016/J.TIM.2013.03.001.
11
International Journal of Food Microbiology 343 (2021) 109088
Setlow, B., Setlow, P., 1996. Role of DNA repair in Bacillus subtilis spore resistance.
J. Bacteriol. 178, 3486–3495.
Setlow, P., 2003. Spore germination. Curr. Opin. Microbiol. 6, 550–556. https://doi.org/
10.1016/J.MIB.2003.10.001.
Setlow, P., 2006. Spores of Bacillus subtilis: their resistance to and killing by radiation,
heat and chemicals. J. Appl. Microbiol. 101, 514–525. https://doi.org/10.1111/
j.1365-2672.2005.02736.x.
Setlow, P., 2012. Dynamics of the assembly of a complex macromolecular structure - the
coat of spores of the bacterium Bacillus subtilis. Mol. Microbiol. 83, 241–244. https://
doi.org/10.1111/j.1365-2958.2011.07948.x.
Setlow, P., 2013. Summer meeting 2013 - when the sleepers wake: the germination of
spores of Bacillus species. J. Appl. Microbiol. https://doi.org/10.1111/jam.12343.
Setlow, P., 2014. Germination of spores of Bacillus species: what we know and do not
know. J. Bacteriol. 196, 1297–1305. https://doi.org/10.1128/JB.01455-13.
Setlow, P., Wang, S., Li, Y.-Q., 2017. Germination of spores of the orders Bacillales and
Clostridiales. Annu. Rev. Microbiol. 71, 459–477. https://doi.org/10.1146/annurev-
micro-090816-093558.
Sevenich, R., Mathys, A., 2018. Continuous versus discontinuous ultra-high-pressure
systems for food sterilization with focus on ultra-high-pressure homogenization and
high-pressure thermal sterilization: a review. Compr. Rev. Food Sci. Food Saf. 17,
646–662. https://doi.org/10.1111/1541-4337.12348.
Trunet, C., Mtimet, N., Mathot, A.G., Postollec, F., Leguerinel, I., Sohier, D., Couvert, O.,
Carlin, F., Coroller, L., 2015. Modeling the recovery of heat-treated Bacillus
licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 spores at suboptimal
temperature and pH using growth limits. Appl. Environ. Microbiol. https://doi.org/
10.1128/AEM.02520-14.
Trunet, C., Mtimet, N., Mathot, A.G., Postollec, F., Leguérinel, I., Couvert, O., Carlin, F.,
Coroller, L., 2018. Effect of incubation temperature and pH on the recovery of
Bacillus weihenstephanensis spores after exposure to a peracetic acid-based
disinfectant or to pulsed light. Int. J. Food Microbiol. https://doi.org/10.1016/j.
ijfoodmicro.2018.04.014.
Wang, S., Setlow, P., Li, Y.Q., 2015. Slow leakage of ca-dipicolinic acid from individual
Bacillus spores during initiation of spore germination. J. Bacteriol. https://doi.org/
10.1128/JB.02490-14.
Wei, J., Shah, I.M., Ghosh, S., Dworkin, J., Hoover, D.G., Setlow, P., 2010. Superdormant
spores of Bacillus species germinate normally with high pressure, peptidoglycan
fragments, and bryostatin. J. Bacteriol. 192, 1455–1458. https://doi.org/10.1128/
JB.01497-09.
Wells-Bennik, M.H.J., Eijlander, R.T., den Besten, H.M.W., Berendsen, E.M., Warda, A.K.,
Krawczyk, A.O., Nierop Groot, M.N., Xiao, Y., Zwietering, M.H., Kuipers, O.P.,
Abee, T., 2016. Bacterial spores in food: survival, emergence, and outgrowth. Annu.
Rev. Food Sci. Technol. 7, 457–482. https://doi.org/10.1146/annurev-food-041715-
033144.
Wuytack, E.Y., Boven, S., Michiels, C.W., 1998. Comparative study of pressure-induced
germination of Bacillus subtilis spores at low and high pressures. Appl. Environ.
Microbiol. 64, 3220–3224.
Wuytack, E.Y., Soons, J., Poschet, F., Michiels, C.W., 2000. Comparative study of
pressure- and nutrient-induced germination of Bacillus subtilis spores. Appl. Environ.
Microbiol. https://doi.org/10.1128/AEM.66.1.257-261.2000.
Zhang, J. qiao, Griffiths, K.K., Cowan, A., Setlow, P., Yu, J., 2013. Expression level of
Bacillus subtilis germinant receptors determines the average rate but not the
heterogeneity of spore germination. J. Bacteriol. https://doi.org/10.1128/JB.02212-
12.
Zhang, P., Kong, L., Wang, G., Scotland, M., Ghosh, S., Setlow, B., Setlow, P., Li, Y.-Q.,
2012. Analysis of the slow germination of multiple individual superdormant Bacillus
subtilis spores using multifocus Raman microspectroscopy and differential
interference contrast microscopy. J. Appl. Microbiol. 112, 526–536. https://doi.org/
10.1111/j.1365-2672.2011.05230.x.
Zhang, Y., Mathys, A., 2019. Superdormant spores as a hurdle for gentle germination-
inactivation based spore control strategies. Front. Microbiol. 9, 3163. https://doi.
org/10.3389/fmicb.2018.03163.
Zhang, Y., Moeller, R., Tran, S., Dubovcova, B., Akepsimaidis, G., Meneses, N.,
Drissner, D., Mathys, A., 2018. Geobacillus and Bacillus spore inactivation by low
energy electron beam technology: resistance and influencing factors. Front.
Microbiol. https://doi.org/10.3389/fmicb.2018.02720.
Zhang, Y., Delbrück, A.I., Off, C.L., Benke, S., Mathys, A., 2020a. Flow cytometry
combined with single cell sorting to study heterogeneous germination of Bacillus
spores under high pressure. Front. Microbiol. https://doi.org/10.3389/
fmicb.2019.03118.
Zhang, Y., Huber, N., Moeller, R., Stülke, J., Dubovcova, B., Akepsimaidis, G.,
Meneses, N., Drissner, D., Mathys, A., 2020b. Role of DNA repair in Bacillus subtilis
spore resistance to high energy and low energy electron beam treatments. Food
Microbiol. https://doi.org/10.1016/j.fm.2019.103353.