COMPREH ENS IVE REVIEWS IN FOOD SC IENCE AND FOOD SAFETY Bacillus spore germination at moderate high pressure: A review on underlying mechanisms, influencing factors, and its comparison with nutrient germination

Funding information SwissNational ScienceFoundation, Grant/AwardNumber: 31003A_182273 Abstract Spore-forming bacteria are resistant to stress conditions owing to their ability to form highly resistant dormant spores. These spores can survive adverse environmental conditions in nature, as well as decontamination processes in the food and related industries. Bacterial spores may return to their vegetative state through a process called germination. As spore germination is critical for the loss of resistance, outgrowth, and development of pathogenicity and spoilage potential, the germination pathway has piqued the interest of the scientific community. The inhibition and induction of germination have critical applications in the food industry. Targeted germination can aid in decreasing the resistance of spores and allow the application of milder inactivation procedures. This germination-inactivation strategy allows better maintenance of important food quality attributes. Different stimuli are reported to trigger germination. Among those, isostatic high pressure (HP) has gained increasing attention due to its potential applications in industrial processes. However, pressure-mediated spore germination is extremely heterogeneous as some spores germinate rapidly, while others exhibit slow germination or do not undergo germination at all. The successful and safe implementation of the germination-inactivation strategy, however, depends on the germination of all spores. Therefore, there is a need to elucidate the mechanisms of HP-mediated germination. This work aimed to critically review the current state of knowledge on Bacillus spore germination at a moderate HP of 50–300 MPa. In this review, the germination mechanism, heterogeneity, and influencing factors have been outlined along with knowledge gaps.


INTRODUCTION
Many bacteria belonging to the orders Bacillales and Clostridiales can survive adverse environmental conditions through the formation of spores, a process called sporulation. The dormant spores do not exhibit metabolic activity and are resistant to heat, radiation, desiccation, extreme pH conditions, and toxic chemicals (Reineke & Mathys, 2020;Setlow, 2006). These high resistance properties of spores are attributed to a number of unique features, including their multilayer structure and the presence of characteristic molecules. The inner layer of the spore, which is termed core, contains DNA that is protected by small acid-soluble proteins (SASPs). The water content of the spore core decreases through various processes, including the accumulation of dipicolinic acid (DPA) chelated with Ca 2+ . The core is surrounded by a low permeability inner membrane, a germ cell wall, an additional peptidoglycan layer called the cortex, several proteinaceous coats, and in some cases, an outermost layer called exosporium ( Figure 1) (Leggett et al., 2012;Setlow et al., 2017). The unique structure and properties of dormant spores contribute to their survival for hundreds of years under adverse environmental conditions. Some studies have even claimed the isolation and revival of spores trapped for millions of years in amber or salt crystals (Cano & Borucki, 1995;Vreeland et al., 2000). The success of this unique survival strategy, however, is dependent on the presence of an efficient mechanism to return to the vegetative state under favorable conditions. This process is called germination. Spore germination is an active area of research for the last seven decades. The regulatory systems involved in the maintenance of a dormant state for several years and the subsequent germination within a few minutes have piqued the interest of the scientific community. Additionally, spores can survive various food processing conditions (Reineke & Mathys, 2020) F I G U R E 1 Schematic representation of the layered structure of a dormant bacterial spore. Structures are not drawn to scale. Note that not all species have an exosporium. Based on Setlow et al. (2017) and germinate during storage, which may result in food spoilage or adversely affect food safety. Spore-forming bacteria, such as Bacillus cereus, Clostridium botulinum, and Clostridium perfringens, cause foodborne diseases. Additionally, spore-forming bacteria can cause human diseases, such as tetanus, gas gangrene, pseudomembranous colitis, and anthrax. Spore-former Clostridioides difficile (formerly known as Clostridium difficile) is an emerging public health threat. Bacillus anthracis can be a potential bioweapon. In order to exert their spoilage capacity or pathogenicity, however, spores need to germinate first. Therefore, the understanding of spore germination mechanisms is crucial to control food spoilage and foodborne diseases and develop efficient strategies to induce or prevent spore germination.
A strategy known as germination-inactivation strategy aims to artificially germinate spores to mitigate their resistance to inactivation processes, including physical and chemical treatments, such as heat and antibiotics (Christie & Setlow, 2020;Setlow, 2003Setlow, , 2014Wells-Bennik et al., 2016). In contrast to the state-of-the-art thermal inactivation, which aims at directly inactivating the dormant spore stage (Reineke & Mathys, 2020), the germinationinactivation strategy does less adversely affect the food quality attributes and hence has potentially interesting applications in the food industry (Martínez-Monteagudo et al., 2014;Sevenich & Mathys, 2018). In fact, in order to control bacterial spores in food, the most widespread current practices are to either inactivate spores by intensive wet heat treatment or alternatively, to inactivate vegetative bacteria and control spore outgrowth by the application of additional hurdles, such as low water activity (a w ), low pH (pH <4.5), cooling, and combinations thereof (Lenz & Vogel, 2015). The first approach requires high thermal loads, which adversely affect the food quality attributes, such as vitamin content, color, and taste. The latter approach retains the food quality attributes but has other downsides, for example, cooling chains are a logistical challenge and environmental burden and acidification and lowering of the water activity is not possible for every food product (Heard & Miller, 2016). The development of a mild and an effective spore inactivation technology, which retains the food quality attributes and improves food safety, is a more sustainable long-term solution for the food processing industry. The germination-inactivation strategy can potentially fulfill these requirements. The actual inactivation efficiency that must and can be achieved by such a strategy depends on many factors, including the present organisms, food matrix, chosen process parameters for germination, as well as the chosen technology, and accordingly process parameters for the subsequent inactivation. However, the major limitation for the application of this strategy is heterogeneous spore germination between genera and species, as well as within the same strain. Slow germinating and nongerminating spores are called superdormant (SD) spores (Zhang & Mathys, 2019). The detailed understanding of the germination process and the elucidation of the underlying mechanism involved in superdormancy are critical for the successful implementation of germination-inactivation strategies.
Various stimuli are reported to trigger spore germination. The chronological discovery of these stimuli is outlined in Section 2. This review will focus on high pressure (HP)-mediated germination as it has the highest potential for implementation in germination-inactivation strategies for the food industry. Advantages include homogeneous treatment of food products without the need for chemical addition, simultaneous spore germination and inactivation of vegetative cells, minimal adverse effects on the food quality, and good consumer acceptance Knorr et al., 1998;Martínez-Monteagudo et al., 2014;Sevenich & Mathys, 2018;Zhang & Mathys, 2019). It is worth noting that HP can be applied in continuous dynamic (Dong et al., 2015;Georget, Miller, et al., 2014) and discontinuous isostatic modes (Knorr et al., 2010). Relevant HP-induced germination was mainly observed for discontinuous isostatic treatment (Sevenich & Mathys, 2018).
This review aimed to summarize the findings of the last two decades on isostatic moderate HP (mHP; 50-300 MPa)-mediated Bacillus spore germination. Additionally, this review compared mHP-mediated germination and nutrient-mediated germination as various studies have reported similarities between these two stimuli Doona et al., 2014;Kong et al., 2014). The very limited know-how on the effect of mHP on Clostridiales is shortly summarized. However, as the great majority of research has investigated the effects on Bacillales, this work focuses on this order.

A BRIEF HISTORY OF GERMINANT DISCOVERY
Bacterial spores were first studied in 1876 by Cohn and Koch (Cohn, 1876). Already then, Cohn reported that spores in old media do not germinate and that the addition of fresh media promoted spore germination. However, the explanation for this observation was provided after more than seven decades. Various studies have contributed to today's understanding of spore germination, including the different stimuli that trigger spore germination (Setlow et al., 2017).
The elucidation of germination mechanisms began in the 1940s after Hills (1949) observed that amino acids, such as L-alanine, promote the spore germination of var-ious Bacillus species in nutrient-rich media. Levinson and Hyatt (1966) later discovered that certain sugars, such as glucose function as germinants of Bacillus spores and demonstrated their synergy with amino acids and cations (especially K + ). The germination of spores by amino acids and/or sugars is commonly referred to as "nutrient" germination in the scientific literature (Setlow, 2003). Rode and Forster (1960) were the first to provide evidence that some surfactants, especially dodecylamine, strongly induce spore germination and that they were lethal to the germinated spores. Riemann and Ordal (1961) demonstrated that exogenous Ca 2+ -DPA can induce germination. The discovery of lysozyme as germination stimulus once the spore coat has been permeabilized also followed shortly later by Gould and Hitchins (1963).
The ability of HP to induce germination of spores was serendipitously discovered while examining the mechanism of HP-mediated spore inactivation. The effect of HP on the inactivation of vegetative cells was reported more than 120 years ago (Hite, 1899). It was also found that spores are extremely resistant to pressure inactivation and may survive pressures above 1200 MPa (Basset et al., 1932;Larson et al., 1918). Decades later, scientists started to notice that under certain conditions, inactivation of spores occurred more rapidly at lower pressures than at higher pressures. This observation was counterintuitive and difficult to reconcile with the expected effect of a physical treatment on a biological system, where an increase in the intensity is expected to result in enhanced inactivation efficiency (Clouston & Wills, 1969;Sale et al., 1970). Clouston and Willis (1969) and Sale et al. (1970), therefore, investigated the spore inactivation of various Bacillus and Clostridium species by HP up to 810 MPa in detail. They observed that a proportion of spores pressurized under certain conditions became heat sensitive. This and other observations from chemical, phase-contrast microscopy, and electron microscopy analyses suggested that pressure inactivated the spores by first promoting spore germination. The observation that germination precedes inactivation also explained why inactivation did not necessarily increase with increased pressure.

THE GERMINATION PATHWAY AT MODERATE HIGH PRESSURE (MHP)
The elucidation of the germination pathway at mHP has been greatly facilitated by research on nutrient germination. In the 1970s, first genetic studies on the spore germination process were initiated, which led to the identification of proteins associated with the germination process (Smith et al., 1978). In the past 40 years, multiple studies have identified the proteins involved in spore germination. Most proteins associated with spore germination were found to be located in the inner membrane of the spore. Proteins associated with spore germination include (1) germinant receptors (GRs) (in B. subtilis GerA, GerB, and GerK, which are present in a complex termed germinosome), (2) GerD, a protein whose function is not completely elucidated but is reported to be critical for germinosome assembly and hence rapid GR-dependent germination, (3) SpoVA proteins which form a channel to release Ca 2+ -DPA from the spore core during germination, and (4) cortex-lytic enzymes (CLEs) responsible for degrading the cortex, which allows the spore to take up more water, thus completing the germination process (Christie & Setlow, 2020;Pelczar et al., 2007;Setlow, 2014). Wuytack et al. (1998) observed that spores germinated with 100 MPa exhibit changes similar to those during nutrient germination, including Ca 2+ -DPA release, SASP degradation, and ATP generation. They further found that these germinated spores lose their resistance to various stresses to a similar degree as nutrient-germinated spores. In contrast, they found that spores germinated at 600 MPa retain their resistance to some stresses and do not initiate two key enzymatic reactions of nutrient-induced germination: SASP degradation and rapid ATP generation. In a later study, Wuytack et al. (2000) demonstrated that indeed it seemed very likely that the pathways activated in spores exposed to 100 MPa were different from those exposed to 600 MPa. The authors hypothesized that the 100 MPa germination pathway shared one or more steps with the nutrient-induced germination pathway and that germination at 600 MPa likely shortcuts the nutrient germination pathway, at least partly.
The analysis of gene products involved in spore germination led to the construction of various mutants lacking and/or overexpressing germination-related proteins (Black et al., 2005;Cabrera-Martinez et al., 2003;Paidhungat & Setlow, 1999Setlow & Setlow, 1996). These allowed substantiating observations by Wuytack et al. (2000) according to which different germination pathways exist depending on the pressure level. Paidhungat et al. (2002) demonstrated that B. subtilis spores lacking all GRs germinated more than 500-fold slower than wildtype spores under nutrient-rich conditions and that the mutant spores also did not germinate upon exposure to a pressure level of 100 MPa. However, the exposure to a pressure level of 550 MPa induced the germination of spores lacking all GRs. This indicated that exposure to a pressure level of 100 MPa induces spore germination by activating the GRs, whereas exposure to a pressure level of 550 MPa may induce spore germination by directly opening the DPA-channels for the release of Ca 2+ -DPA from the spore core.
To differentiate between these two pathways, the scientific community often classified pressure treatments into moderate HP (mHP) and very HP (vHP). Unfortunately, the terms "moderate" and "very" are not accurate and have been used by different authors for different pressure ranges Lenz & Vogel, 2015;. However, most studies classify pressure levels between 50 and 300 MPa as mHP, and pressure levels between 400 and 800 MPa as vHP . Although this classification might seem arbitrary, it is attributed to the different germination mechanisms that dominate at these pressure levels. mHP and vHP induce GR-dependent and GR-independent germination pathways, respectively. For GR-dependent pressure germination, it has been shown that the dominant range is between 100 and 200 MPa, typically at 30-50 • C . This work focused on mHP-induced germination. For an overview of all different germination and inactivation pathways depending on pressure and temperature level, the reader is referred to  and Lenz and Vogel (2015).
Several studies have investigated various mechanistic aspects of mHP-mediated germination (Aertsen et al., 2005;Black et al., 2005;Black, Wei, et al., 2007;Borch-Pedersen et al., 2017;Delbrück et al., 2021;Doona et al., 2014Doona et al., , 2016Georget, Kapoor, et al., 2014;Kong et al., 2014;Luu et al., 2015;Mathys et al., 2007;Reineke et al., 2012;Reineke, Schlumbach, et al., 2013;Vepachedu et al., 2007;Wuytack et al., 1998Wuytack et al., , 2000Zhang et al., 2020). Most studies have used the model organism B. subtilis due to the wealth of information available as well as the ease with which mutant strains can be constructed. Occasionally, mHP germination has been studied on other Bacillus species, and it appears that general germination mechanisms are shared among Bacillus species . The latest understanding of the mHP germination pathway in B. subtilis and by inference other Bacillus species is depicted in Figure 2. The mHP-mediated direct or indirect GR activation results in the release of Ca 2+ -DPA through an inner membrane channel composed of multiple SpoVA proteins. This process is accompanied by partial water uptake and the activation of CLEs of the spores. The activated CLEs in turn degrade the cortex, which is essential for core swelling and further water uptake and the crucial step for the loss of the majority of resistance Setlow, 2003). The hydrolysis of cortex peptidoglycans promotes water uptake, which results in approximately twofold expansion of the spore core ( Figure 3) (Laue et al., 2018). Following DPA release, spores also degrade their SASPs (Reineke, Ellinger, et al., 2013). This was shown by Wuytack et al. (1998) by means of immunoblot analysis of spore suspensions treated at 100 MPa and 40 • C.

F I G U R E 2
Moderate high pressure-mediated germination pathway of Bacillus subtilis spores. Schematic representation of the germination steps induced by moderate high pressure (mHP). Germinant receptors (GR) in the inner membrane (IM) are activated, followed by release of ions and Ca 2+ -dipicolinic acid (Ca 2+ -DPA) from the spore core. Partial core hydration is followed by full core hydration after degradation of the cortex (Cx) by cortex lytic enzymes (CLEs). Small acid-soluble proteins (SASP) binding to DNA are degraded. Note that the temporal sequence of cortex and SASP degradation under moderate HP is not fully clear yet. Structures are not drawn to scale F I G U R E 3 Transmission electron micrographs of dormant spores (a) (reproduced from Delbrück et al., 2021) and spores germinated at 150 MPa and 37 • C for 6 min (b) (unpublished data Delbrück et al., 2021) of Bacillus subtilis. Typical spore structures are depicted. Outer spore coat (OSC), inner spore coat (ISC), outer membrane (OM), cortex (Cx), cell wall (Cw), inner membrane (IM) (additionally highlighted in yellow), and core (Co). Note the expansion of the inner membrane and core with germination Consistently, spores germinated at 100 MPa were sensitive to both hydrogen peroxide and UV light. α/β-type SASPs protect the spores against UV light and hydrogen peroxide-induced DNA damage. Hence, the increased UV and hydrogen peroxide sensitivity of the spores indicated the degradation of SASPs (Mason & Setlow, 1986;Setlow & Setlow, 1993;Setlow, 2007). A proof of SASP and cortex degradation in mHP germination is the ability of germinated spores to be stained with the nucleic acid stain SYTO16. Cortex degradation plays a critical role in the uptake of SYTO16 in the spore core and SASP degradation is required for DNA-SYTO16 binding (Black et al., 2005;Kong et al., 2010;Zhang et al., 2020).
Although the broad mHP-mediated germination pathway has been elucidated, the first step of GR activation requires further investigation. Even though there is a consensus that mHP is considered to activate GRs, how this activation works at the molecular level is still not understood. Two hypotheses have been proposed for the GR activation. mHP may alter membrane properties, such as membrane fluidity and/or configuration, which in turn affect GRs. Alternatively, mHP may directly promote structural changes in the GRs, thereby initiating the germination cascade Doona et al., 2014Doona et al., , 2016Kong et al., 2014;Paidhungat et al., 2002;Setlow et al., 2017;Wuytack et al., 1998). Certainly, HP can cause structural changes in both, proteins or membranes, and possibly, also both components might be altered. However, the nature of these changes and importance during mHP-mediated germination have not been elucidated.
Although the inner membrane has a critical role in mHP-mediated germination, there are limited studies on the effect of mHP on the inner membrane, which can be attributed to the challenges associated with in situ membrane analysis. The inner membrane of dormant spores is reported to exhibit extremely low permeability and low mobility. Most lipids (almost 70%) in the inner membrane of dormant B. subtilis are immobile, which likely results in a highly ordered gel-like lipid phase. However, the inner membrane comprises some fraction of disordered and mobile lipids (Cowan et al., 2004;Georget, Kapoor, et al., 2014;Hofstetter et al., 2012;Setlow, 2006;Sunde et al., 2009).  investigated Geobacillus stearothermophilus spore HP germination at 200 MPa by in situ infrared spectroscopy and fluorometry. The lipid packing and order of the inner membrane were analyzed using fluorospectroscopy with the polarity-sensitive fluorophore Laurdan. The secondary protein structural changes were recorded using Fourier-transform infrared spectroscopy. The findings of this study suggested that mHP promotes reversible minor changes that affect the lipid phase and protein conformation. Changes under mHP include the induction of a different ordered gel-like lipid phase in the inner membrane.
Further insights into inner membrane modifications and protein conformational changes would likely aid in the understanding of mHP-mediated germination and GR activation.

METHODS TO STUDY SPORE GERMINATION
Several methods have been developed to monitor spore germination (Table 1). Different methods measure different physiological changes that occur during germination. Many methods measure the average germination rate of a population. However, with increased awareness for heterogeneity in germination rates, the need for technologies that allow single-cell studies increased (Zhang et al., 2020). In its most instructive way, spore germination is measured in real-time at the single-cell level. However, the implementation of real-time measurements is difficult for HP-exposed cells. Kong et al. (2014) developed a protocol for continuous monitoring of individual HP-germinated spores with phase-contrast microscopy. The pressure-mediated germination of multiple individual spores could be monitored in real-time in a diamond anvil cell. This study provided valuable insights into the germination kinetics of single cells, which will be outlined in the next section.

HETEROGENEITY IN MHP-MEDIATED GERMINATION AND SUPERDORMANCY
Bacterial spores are notorious for their unpredictable germination responses and it is well known that the germination of individual spores in populations is heterogeneous. This heterogeneity in spore germination is not only observed between different species and strains, but even within isogenic populations. Germination heterogeneity has been observed for all germination stimuli including mHP (Zhang & Mathys, 2019).
The kinetics of spore germination can be described based on the following time points: T 0 (application of the germination trigger), T c (time of commitment), T lag (initiation of rapid Ca 2+ -DPA release), T release (completion of Ca 2+ -DPA release), and T lys (completion of cortex lysis) (Setlow et al., 2017). The heterogeneity of nutrient germination is mainly due to the variability in T lag. In contrast, ∆T release (T release -T lag ) is relatively constant for spores of any particular strain or species (Chen et al., 2006;Stringer et al., 2005Stringer et al., , 2011 Kong et al. (2014) reported that the kinetics of HP-mediated germination of individual B. subtilis spores at approximately 150 MPa are similar to those of nutrient-induced germination with a variable T lag prior to a period of rapid Ca 2+ -DPA release (∆T release ) ( Figure 4). Hence, heterogeneity in both nutrient and ∼150 MPa pressure germination is attributed to variability in T lag (Kong et al., 2014). This suggests that events after T lag , such as cortex lysis, likely play no critical role in germination heterogeneity.
Although most spores germinate rapidly, a small fraction of spores germinates extremely slowly or not at all. Consistent with their apparently increased dormancy compared to the rest of the dormant population, this fraction is termed superdormant (SD) spores (Kong et al., 2014;Luu et al., 2015;Setlow, 2003Setlow, , 2013Setlow et al., 2017;Wells-Bennik et al., 2016;Zhang et al., 2020;Zhang & Mathys, 2019). This heterogeneous germination behavior is most likely a population survival strategy. The maintenance of SD spores decreases the risk of complete eradication of the population under adverse environmental conditions (Setlow et al., 2017). However, this evolutionary advantageous strategy is a challenge for designing germinationinactivation-based strategies. In food products, SD spores might survive subsequent inactivation steps and germinate at a later point during storage and adversely affect food safety or quality (Zhang & Mathys, 2019).
It is important to understand that superdormancy is a relative term. It describes a subpopulation of spores which behaves phenotypically different in their germina-tion capacity in comparison to the rest of the dormant population. This subpopulation is not static, instead its size depends on the germination and isolation conditions and analysis timepoints (Zhang & Mathys, 2019). Thus, the number of SD spores is the difference between the total number of spores and the number of germinated spores. Increased HP dwell times for instance will decrease the prevalence of SD spores (Delbrück et al., 2021;Zhang et al., 2020). Therefore, generalizing statements about properties of SD spores should be avoided. Instead, statements about properties of SD spores should always be accompanied by stating the isolation conditions chosen to define superdormancy.
The isolation of SD spores can aid in determining their properties and providing insights into the mechanisms of superdormancy. The comparison of SD spores with the initial dormant spore population, generally just termed dormant, can give valuable information on what characteristics lead to superdormancy. While nutrient SD spores have been isolated and readily characterized Ghosh et al., 2012;Ghosh & Setlow, 2009Zhang et al., 2012), it is only very recently that attempts have been made to isolate and characterize mHP SD spores. Accordingly, the literature is still very scarce concerning the properties of mHP SD spores. Zhang et al. (2020) provided a proof of concept and a detailed methodology for the use of fluorescence-activated cell sorting (FACS) to isolate the SD spores. This sophisticated method can isolate highly pure SD spores at a single-cell level. However, the equipment is expensive and requires skilled training. Additionally, the method yields a low concentration of isolated spores in the range of 10 5 spores/ml. Combined with the low throughput where the sorting of 1 ml of SD spores can take hours if the SD subpopulation is small, this technology is not suitable for isolating large numbers of SD spores. However, FACS-based spore isolation technology can aid single-cell analysis. An alternate spore isolation method is buoyant density centrifugation, which takes advantage of density differences between (super)dormant and germinated spores (Ghosh & Setlow, 2009). Buoyant density centrifugation is a simple method that requires minimal resources or technical skills. However, the purity of SD spores isolated using buoyant density centrifugation is typically lower than that isolated using FACS. Depending on the required purity and further analysis, buoyant density centrifugation alone, as a pre-enrichment step in combination with FACS or FACS alone, may be used for the isolation of SD spores (Delbrück et al., 2021). There are ongoing studies for the characterization of isolated mHP SD spores. The overall goal would be to fully understand the underlying reasons of mHP superdormancy in order to potentially design strategies to target this subpopulation.
One identified reason for nutrient superdormancy is significantly lower GR levels Zhang et al., 2012). Other authors have suggested that nutrient germination is slowed down by the presence of the SpoVA 2mob operon (Krawczyk et al., , 2017. The SpoVA 2mob operon encodes seven proteins, of whom three, SpoVAC 2mob , SpoVAD 2mob , and SpoVAEb 2mob , are homologous to the conserved SpoVAC, SpoVAD, and SpoVAEb proteins that form Ca 2+ -DPA channels in the inner membrane Krawczyk et al., 2016;Velásquez et al., 2014). It was also found that the copy number of the SpoVA 2mob operon correlated with the resistance of Bacillus spores to pressure treatments conducted at vHP, that is, 600 MPa and 80 • C (Li et al., 2019). The role of the SpoVA 2mob operon in mHP germination has not been investigated so far. However, while the presence of the SpoVA 2mob operon may influence interstrain variability in nutrient and potentially mHPinduced germination, it cannot explain the heterogeneity in germination within an isogenic spore population. mHP superdormancy and its underlying reasons are still poorly understood. What is clear is that GR levels also seem to play a role in mHP superdormancy, which is outlined in Section 6.7. There is limited understanding of other factors contributing to mHP superdormancy and the current knowledge is mainly based on the principle of exclusion. Delbrück et al. (2021) studied potential quantitative and qualitative structural differences between mHP SD spores (isolated after exposure to 150 MPa at 37 • C for 6 min) and the initial dormant spore population using transmission electron microscopy. The size, cortex thickness, and structural properties were not significantly different among the SD and the initial dormant spore population. A potential other reason for superdormancy could have been genetic changes. However, already the fact that superdormancy is not a static subpopulation makes it unlikely that the underlying reason is a genetic defect, for example, in a gene coding for a critical part of the germination machinery (Zhang & Mathys, 2019). Indeed, Delbrück et al. (2021) demonstrated that genetic factors are most likely not involved in mHP superdormancy. The mHP SD spores were isolated after exposure to 150 MPa at 37 • C for 6 min and subjected to resporulation to gain a new generation of dormant spores. The resporulated spores were descendants of former SD spores. If a permanent genetic defect promoted superdormancy, the resporulated population must exhibit decreased germination capacity when compared with a normal dormant spore population. However, the resporulated population did not exhibit decreased germination. The same observation was made by Ghosh and Setlow (2009) for nutrient superdormancy. The conclusion that a genetic change is unlikely to cause mHP superdormancy was reinforced by the finding that the majority of isolated mHP SD spores could germinate upon exposure to the same treatment at 150 MPa at 37 • C. This finding further suggested that GR levels alone cannot fully explain superdormancy, which is discussed in further detail in Section 6.8.
Further studies are needed to elucidate the mechanism underlying mHP superdormancy. Recent studies indicate that the spore size, cortex thickness, and genetic changes are not involved in mHP superdormancy. Although genetic changes may not contribute to the SD phenotype, stochastic variations in gene expression can influence the phenotype. Current evidence indicates that GR levels are one but not the only factor that determines mHP superdormancy and that other cumulative causes or transient factors may contribute to superdormancy (Delbrück et al., 2021).

IMPACT OF VARIOUS FACTORS ON MHP-MEDIATED BACILLUS SPORE GERMINATION
The analysis of various factors involved in mHP-mediated germination has improved the mechanistic understanding of mHP germination and can enhance germination in mHP applications. In the following, different factors and their influence on mHP are discussed.

6.1
Pressure level, temperature, and pressure dwell time The most straight forward parameters that can be controlled during mHP treatments are the pressure level, temperature, and pressure dwell time. In the following section, the current knowledge on these parameters is outlined. Most studies were performed using B. subtilis spores. However, some findings may vary among different species.

6.1.1
Pressure level Kong et al. (2014) demonstrated that exposure to HP <10 MPa does not induce B. subtilis spore germination. However, there seems to be a consensus that pressure levels as low as 50 MPa can promote Bacillus spore germination (Heinz & Knorr, 2001). Heinz and Knorr (1998) investigated the effect of HP levels between 50 and 300 MPa on B. subtilis spore germination using in situ scanning of the optical density during pressure treatment. The maximum germination rate was observed at a pressure range of 100-200 MPa. Consistently, most mechanistic studies on mHP-mediated germination used pressure levels between 100 and 200 MPa. Kong et al. (2014) demonstrated that the average T lag values of B. subtilis spore germination at 50 MPa were longer than those at 150 MPa (an average of 29 min at 50 MPa and 13 min at 150 MPa). However, the ∆T release values were identical at the two pressure levels. Very interestingly, the germination of B. subtilis spores exposed to a 150 MPa HP pulse as short as 30 s, followed by exposure to a constant pressure of 1 MPa at 37 • C, was identical to that of spores exposed to a constant pressure of 150 MPa. This indicated that a short HP pulse of 150 MPa is sufficient to potentiate the germination when the spores were subsequently incubated at 37 • C and 1 MPa (a pressure level that cannot independently induce spore germination). However, the germination of spores incubated at 0.1 MPa (ambient pressure) stopped 5-10 min after the application of a short pressure pulse. This indicates that exposure to a short HP pulse of 150 MPa is sufficient to convert spores into a state in which they are committed to germinate. This may be mediated through the activation of spore GRs, which remain activated at 1 MPa but can deactivate at ambient pressure. This commitment concept has been reported for nutrient-induced germination during which the spores become committed to germinate once they encounter nutrients and the germination proceeds even after the removal of the nutrient germinant (Setlow, 2003). However, this deactivation phenomenon observed with mHP has not been previously reported. Clearly, the molecular mechanisms of nutrientmediated and mHP-mediated activation in this committed state have not been fully elucidated. However, the committed state may involve the conversion of GRs into an activated state (Kong et al., 2014). 6.1.2 Temperature  investigated the B. subtilis germination rate in Tris-HCl buffer (pH 7.5) at a pressure level of 150 MPa and a temperature range of 20-80 • C. Nearly, no germination was observed at 20 • C. The germination of mesophilic spores increased with temperature but only up to 40 • C. Similar findings have been observed in other studies (Black et al., 2008;. It was suggested that temperatures above 40 • C may decrease the efficiency of GRs and/or the Ca 2+ -DPA channel proteins, which leads to decreased germination rates at high temperatures . Heinz and Knorr (1998) hypothesized that the increased germination up to 40 • C could be related to the accelerated mass transfer from the core of the spore into the medium. Generally, slight heating and thereby increased germination could also be related to property changes of the inner membrane and/or structural changes of proteins involved in germination.
It is important to note that thermophilic spores likely have higher temperature requirements for optimal germination.  demonstrated that the germination of G. stearothermophilus spores at 30 • C was lower than that at high temperatures. The minimum temperature required to achieve significant germination of G. stearothermophilus spores at 200 MPa was 55 • C, which may be related to the optimal growth temperature of this strain.

Pressure dwell time
It is not surprising that, independently of pressure level and temperature, germination increases with increasing pressure dwell times (Delbrück et al., 2021;Zhang et al., 2020). However, as stated above, Kong et al. (2014) showed that a short pressure pulse at mHP followed by a constant pressure of 1 MPa can be as effective in triggering germination as a constant mHP. Not only the effect of the total treatment time but also the effect when the treatment time is split into cycles of pressurization, holding time, and decompression has been investigated (López et al., 2003;Zhang & Mittal, 2008). Pressure cycling has not only been shown to promote spore inactivation and injury but also to increase mHP germination. Furukawa et al. (2003) exposed B. subtilis spores to six 5-min-cycles at 200 and 300 MPa combined with several temperatures (25, 35, 45, and 55 • C) and found a significantly increased Ca 2+ -DPA release compared to corresponding continuous pressure treatments. While pressure cycling may increase germination, its practicability in industrial applications may be limited due to the increased energy consumption, wearing of the equipment and associated costs. Generally, it is important to be aware that mHPmediated germination does not follow log 10 -linear kinetics (Heinz & Knorr, 2001). The choice of optimal pressure dwell times depends on the application, involved matrices, and species. Prolonged pressure dwell times are not economically feasible and may not be beneficial if germinated spores can potentially multiply in nutrient-rich environments and produce toxins. However, multiplication and toxin production are unlikely to occur under pressure. Though, to the best of our knowledge, this has not been studied systematically.

6.2.1
Dissociation equilibrium, pKa, and pH Several authors have examined the effect of pH on B. subtilis and B. cereus mHP germination by using different buffers. All found that highest germination was observed at pH 7 Raso et al., 1998;Wuytack & Michiels, 2001). However, the optimum pH range was much broader than for nutrient-mediated germination of spores at ambient pressure . Wuytack and Michiels (2001) further investigated the effect on B. subtilis spore inactivation considering spore inactivation as a two-step process of germination followed by inactivation. An acidic environment did not enhance spore inactivation. However, the exposure of spores to mHP at neutral pH, followed by exposure to low pH conditions for 1 h, increased the levels of inactivation. This indicated that optimal spore inactivation in acid products can be achieved by acidifying the product after the pressure treatment. The rationale for this observation is that a neutral pH maximizes the germination of spores exposed to mHP and the exposure to acidic conditions afterward enhances the inactivation of germinated spores as they are more sensitive to low pH conditions.
It is worth highlighting that the consideration of pH alone in experiments involving pressure can be misleading. Generally, pressure and temperature can change the dissociation equilibrium, pKa, and pH of solutions with the sign and magnitude of the change depending on the composition of the solution (Kitamura & Itoh, 1987;Mathys et al., 2008;Reineke et al., 2011). The pH value is defined as the negative decadic logarithm of the oxonium concentration (H 3 O + ). A decreased pH value under pressure (a higher H 3 O + concentration) does not necessarily indicate acidic conditions, that is, the H 3 O + concentration increased relatively to the hydroxide (OH − ) concentration. Instead, pressure can also increase the H 3 O + concentration by promoting the dissociation of an electrolyte. For example, pressure shifts the dissociation equilibrium of H 2 O toward H 3 O + and OH − , which results in an equal increase in H 3 O + and OH − concentrations. Although the pH value decreases due to increased H 3 O + concentration, the conditions can still be "neutral." The pKa value reflects the dissociation equilibrium and should, therefore, additionally be considered to the pH. A change in the dissociation equilibrium influences the ion strength of a solution which in turn might influence cellular processes, such as germination (Mathys et al., 2008).

Suspending medium composition
The effects of suspending medium factors, such as the presence of nutrients, a w , and fat concentration, on mHPmediated germination efficiency were examined (Black et al., 2008;Raso et al., 1998;Van Opstal et al., 2004). Several studies have comparatively analyzed mHPinduced germination and nutrient/mHP combinationinduced germination. Van Opstal et al. (2004) demonstrated that the germination of B. cereus spores at 100 and 300 MPa in milk was significantly higher than that in potassium phosphate buffer (pH 6.7). Pressure and germination-inducing components exerted synergistic activities in the milk. Similarly, Black et al. (2008) demonstrated that the germination levels of B. subtilis spores after 5 min at 100 MPa and 40 • C in milk were two-fold higher than those of spores suspended in PBS (>4 log 10 vs. 2 log 10 ). Raso et al. (1998) reported that the combination of 250 MPa and 100 mM L-alanine in McIlvaine citrate phosphate buffer at pH 7 exerted an additive effect to promote B. cereus spore germination. Raso et al. (1998) examined the germination of B. cereus spores at 250 MPa and 25 • C in various solutions of sucrose with known a w values. High concentrations of sucrose inhibited the germination of spores at a w of 0.92 and no germination was detected after 15 min. Rao et al. (2018) investigated the effect of various humectants on the mHP germination of Bacillus spores with different germinants. They also found that glycerol and sucrose addition decreased HP germination of B. subtilis and B. cereus at 150 MPa. A w reduction by sucrose was further found to be more effective in slowing down mHP germination than glycerol. Even though humectant inhibition for mHP germination was observed, the authors found that the effect was less pronounced than for germination by nutrient germinants with both B. cereus and B. subtilis spores. Further investigations on the effect of dietary humectants on germination would be important, as this is of high industrial relevance. Little data are available about the effect of other food constituents. The fat concentration in milk was shown to not affect HP-mediated B. cereus spore germination at 250 MPa (Raso et al., 1998). However, further research would be beneficial here.  Gould and Sale (1970) a Concentrations that inhibit nutrient germination were used. b AGFK is a mixture of L-asparagine, glucose, fructose, and K + .

Sublethal heat treatment prior to mHP-mediated germination
It is well known that GR-dependent nutrient-induced germination can be reversibly increased if the dormant spores are subjected to a sublethal heat treatment (commonly in the range of 60-75 • C for 30 min depending on the organism) (Luu et al., 2015;Setlow et al., 2017). Given the similarities of the nutrient-induced and mHP-induced germination pathways, the effect of a sublethal heat treatment on mHP-induced germination was examined. From the available literature, we found that the answer to this question is not straightforward, even though one might expect a positive effect of heat activation on mHP germination. Some studies have reported that heat activation does not markedly affect the mHP-mediated germination of B. subtilis spores (Kong et al., 2014;Wei et al., 2010). Delbrück et al. (2021) even reported that heat activation decreased the subsequent mHP-mediated germination of B. subtilis spores. Luu et al. (2015) investigated this aspect in great detail and reported that heat activation did not affect the mHP-mediated germination of wildtype B. subtilis spores. However, heat activation promoted the mHPmediated germination of spores from mutants lacking certain GRs at 150 MPa. Heat activation stimulated the germination at 150 MPa through GerB and GerK, but not so much through GerA. As GerA showed little stimulation, the effect of heat activation was most likely not seen in the wildtype because germination at 150 MPa is predominantly induced through the activation of GerA (Black et al., 2005;Luu et al., 2015). This is discussed in greater detail in Section 6.7.
Given the fact that no significant increase in mHP germination is achieved, recent studies on mHP-mediated germination have applied HP treatments without prior sublethal heat treatment .

Potential chemical inhibitors
Several substances are known to inhibit nutrient-induced germination (Cortezzo et al., 2004). Multiple studies have investigated the effect of nutrient-induced germination inhibitors on mHP-mediated germination (Black et al., 2005;Wuytack et al., 2000) (Table 2). Nutrient-induced germination inhibitors, such as HgCl 2 , o-chlorosresol, tert-amyl methyl ether (TAME), and phenol, can at least partially inhibit mHP-induced spore germination. However, treatment with other nutrientinduced germination inhibitors, such as ethanol, octanol, and amiloride, does not inhibit mHP-mediated spore germination at concentrations that inhibit nutrient-induced germination (Black et al., 2005;Wuytack et al., 2000). Gould and Sale (1970) reported that D-alanine, which is a strong nutrient-induced germination inhibitor at atmospheric pressure (Setlow, 2013;Zhang et al., 2014), potentiated mHP-mediated germination of B. cereus spores. This suggested that pressure promotes the racemization of the inhibitory D-enantiomorph to the stimulating L-form.

Sporulation conditions
6.5.1 Nutrient richness of sporulation medium Spores prepared in nutrient-poor sporulation medium germinated approximately four-fold slower than those prepared on nutrient-rich medium . This is likely because the spores prepared in rich, complex media exhibit higher GR levels than those prepared in nutrient-poor medium (Hornstra et al., 2006;Ramirez-Peralta, Stewart, et al., 2012).

6.5.2
Sporulation temperature and inner membrane unsaturated fatty acid content Multiple studies have investigated the effect of sporulation temperature on the heat resistance of bacterial spores. Most of these studies have reported that spores obtained at high temperatures exhibit enhanced heat resistance (Beaman & Gerhardt, 1986;Condon et al., 1992;Melly et al., 2002;Palop et al., 1999). Melly et al. (2002) demonstrated that the core water content was lower in spores prepared at high temperatures, which can explain the increased heat resistance. In contrast, spores generated at low temperatures exhibited enhanced pressure resistance. Igura et al. (2003) reported that the B. subtilis spores sporulated at low temperatures exhibited higher resistance to HP treatments at 100-300 MPa and 55 • C than those prepared at high temperatures. As inactivation is preceded by germination, this observation is consistent with the results of previous studies, which reported that B. cereus and B. subtilis spores sporulated at low temperatures (20-23 • C) were more resistant to the initiation of germination at 150-250 MPa than those prepared at high temperatures (37-44 • C) (Black et al., 2005;Raso et al., 1998). At low sporulation temperatures, the spores exhibited decreased germination capacity. Thus, low sporulation temperatures decrease the efficiency of mHP-mediated inactivation.
The underlying mechanistic explanation for this observation is still unclear. Based on the current understanding, high sporulation temperatures decrease the amount of unsaturated fatty acids, which decreases the fluidity and permeability of the inner membrane . However, how a decreased fluidity of the inner membrane should enhance mHP-induced germination is not clear. Indeed, Black et al. (2005) demonstrated that changes in the levels of inner membrane unsaturated fatty acids have minimal effect on mHP-mediated germination. Therefore, another explanation than the altered unsaturated fatty acid level can be the reason for the increased mHP-induced germination rates of spores prepared at high temperatures. Generally, it is likely that higher sporulation temperatures either influence (1) certain inner membrane properties, (2) the GRs, or (3) other components involved in germination. In theory, also combinations thereof are conceivable. It seems intuitive that changes in the membrane fluidity may influence the responsiveness of GRs to pressure. However, since so little is known about the structure of the inner membrane and the exact mode of action of the GRs, it is very difficult to formulate hypotheses.

Salt concentration in sporulation
The preparation of spores with salt (NaCl) concentrations ranging from 0 to 1 mol/L demonstrated that the salt concentrations were inversely proportional to mHP-mediated germination (Black, Wei, et al., 2007). The cell membrane properties of Bacillus species, especially the levels of individual fatty acids and phospholipids, can be influenced by the ionic strength of the growth medium (López et al., 1998(López et al., , 2002Machado et al., 2004). Therefore, the decreased mHP-mediated germination at high salt concentrations could be attributed to changes in the cell membranes. However, the underlying mechanisms are poorly understood.

Lipid addition to GRs
The covalent addition of diacylglycerol to a cysteine residue in the N-terminal region of C proteins of some nutrient receptors is essential for receptor function in nutrient-induced germination (Igarashi et al., 2004). Black et al. (2005) investigated the effect of receptor diacylglycerylation on the pressure-induced germination of B. subtilis spores at 150 MPa and 37 • C. A B. subtilis strain that lacked the only lipoprotein diacylglycerol transferase was used to examine the role of receptor diacylglycerylation. Diacylglycerylation of the receptors exhibited similar roles in the pressure-induced and nutrient-induced activation of these receptors. The rate of pressure-induced germination of spores lacking diacylglycerol transferase was 8% of that of wildtype spores. This suggested that diacylglycerylation is critical for nutrient receptors to respond to mHP. In particular, GerA requires diacylglycerylation to respond to nutrients and pressure, whereas GerK does not require diacylglycerylation to respond to nutrients or pressure (Black et al., 2005).

GR levels and responsiveness of individual GRs
B. subtilis is reported to express three functional GRs (GerA, GerB, and GerK). Each GR comprises at least three subunits termed A (e.g., GerAA, GerBA, or GerKA), B, and C subunits in a 1:1:1 stoichiometry. Some GRs have an additional D subunit (e.g., GerKD), and the loss of D subunit decreases the function of GRs (Christie & Setlow, 2020). This protein is not to be confused with the GerD protein, which is another protein important for GR-dependent germination (Pelczar et al., 2007).
The mutant strain of B. subtilis lacking all three functional GRs (GerA, GerB, and GerK) germinate poorly upon exposure to mHP (Black et al., 2005;Delbrück et al., 2021;Paidhungat et al., 2002). Black et al. (2005) investigated the contribution of individual GRs to 150 MPa-mediated germination using B. subtilis mutants lacking certain GRs and demonstrated that it is the same as to a complex mixture of nutrients. The order of sensitivity was as follows: GerA > GerB > GerK (Black et al., 2005;. The GerA receptor contributed the most to mHP-mediated germination as evidenced by the mutant lacking GerB and GerK germinating at a similar rate as the wildtype spores. The rate of mHP-mediated spore germination lacking the GerA receptor was identical to that of spores lacking GerA and GerK. These findings suggested that GerK has minimal contribution to HP-mediated germination. Consistently, Doona et al. (2014) reported that a loss of GerA decreased the 150 MPa-induced spore germination more than that expected by the <50% decrease in GRs in GerA-lacking spores. One potential explanation for the different responses of different GRs to mHP involves mHP inducing spore germination by promoting conformational changes that activate GRs. Thus, it is conceivable that some GRs may undergo such conformational changes in response to mHP . Generally, it seems that the differences in the rates of pressure-mediated germination with different GRs can be attributed to the following two reasons: (1) the abundancy/levels of the receptors and (2) intrinsic differences in the sensitivities of various receptors to pressure (Black et al., 2005;Doona et al., 2014).
The reasons for the heterogeneity in GR levels in individual spores are not fully understood. However, some factors have been identified to influence GR levels. For example, it was found that alterations in the levels of at least two transcription factors SpoVT and YlyA that modulate the transcription of genes encoding GRs influence GR levels and hence modulate mHP-induced germination rates Ramirez-Peralta, Stewart, et al., 2012;Traag et al., 2013). The other factors include the properties of sporulation media. In particular, spores prepared in nutrient-rich complex media exhibit higher GR levels than those prepared in nutrient-poor media (Hornstra et al., 2006;Ramirez-Peralta, Zhang, et al., 2012). Hence, spores prepared on nutrient-poor sporulation medium germinate slower upon exposure to mHP than those prepared in a nutrient-rich medium .

Other factors modulating mHP-mediated germination rates
Although GR levels are major factors determining mHPmediated germination rates, other factors also modulate mHP-mediated germination and the GR levels alone cannot account for all the heterogeneity in mHP-mediated germination rates. Some scientific findings established with B. subtilis that support the hypothesis that factors other than GR levels may influence mHP germination are discussed below.
I. GerD promotes rapid mHP-mediated spore germination: Li et al. (2014) identified a dominant negative gerD mutation (gerD F87C ) in B. subtilis, which did not affect GR levels. Although the spores of this gerD variant strain exhibited normal GR subunit levels, they germinated slower at mHP than those lacking GerD altogether . Interestingly, and in line with much of scientific evidence available indicating that mHP-mediated germination and nutrientmediated germination are very similar, the presence of the GerD F87C variant inhibited nutrient-mediated germination of these spores . II. D subunits influence mHP-mediated spore germination: The levels of small proteins termed D subunits that are associated with at least some GRs are reported to modulate GR function and alter the rates of mHP-mediated germination independent of GR levels Ramirez-Peralta et al., 2013). For example, the spores of a B. subtilis strain overexpressing gerKD exhibited approximately five-fold slower mHP-mediated germination rates than wildtype spores . However, the effect of D subunit on mHP-mediated spore germination is not well understood. III. No linear relationship between GR levels and mHP germination: Doona et al. (2014) compared the GR levels of different mutants with mHP-mediated germination rates. Even though germination rates were generally reflected in the spores' total GR levels, they clearly found that there is no linear relationship. Different GRs may exert synergistic activities and GRs appear to have different responsiveness to mHP (Black et al., 2005;Doona et al., 2014). The correlation between GR levels and rates of mHP-mediated germination is complex. However, Doona et al. (2014) suggested that the rates of mHP-mediated germination are not directly proportional to GR levels even when the differential responsiveness is considered. Additionally, mHP-mediated germination may not be directly correlated with the rates of nutrient-mediated germination. IV. Most isolated SD spores germinate by a second, identical mHP treatment: Delbrück et al. (2021) isolated the nongerminated spore fraction (SD spores) after exposure to 150 MPa at 37 • C for 6 and 20 min. These SD spores were exposed to the same HP treatment. The germination capacity of SD spores was markedly reduced when compared with the initial dormant spores. However, most SD spores germinated upon re-exposure to the same trigger. As the GR levels did not increase between the first and second HP treatments, the GR level alone cannot account for the germination of most spores after re-exposure. However, the findings must be carefully analyzed as the effect of mHP on GR-dependent germination has not been fully elucidated. Theoretically, spores may be partially activated in the first HP treatment and undergo complete activation only after the second HP treatment. This activation may be due to a conformational change in GRs.
In conclusion, these findings indicate that factors other than GR levels may influence mHP-mediated spore germination. The levels of GR D subunits and GerD are potential factors that determine the mHP-mediated germination rates .

PROPERTIES OF MHP-MEDIATED GERMINATED SPORES
The spores exposed to mHP can germinate and lose most of their resistance to stress. However, it is important to be aware that the suspending medium strongly influences the properties and further fate of germinated spores. For instance, spores germinated using mHP do not outgrow in the absence of nutrients. During outgrowth, the germinated spore emerges from the remaining spore structure (i.e., the coat layers) and transforms into an immature cell. Amino acids derived from SASP degradation can support some metabolism during the early stages of outgrowth and multiplication; however, eventually exogeneous nutrients are required. Nutrients may be absent in buffers used for HP-mediated germination research; however, they are typically present in food products Lenz & Vogel, 2015;Sinai et al., 2015).
Spores germinated by mHP typically have a degraded cortex, degraded SASPs, and are rehydrated . Hence, these spores are susceptible to hydrogen peroxide, UV light, and heat (Heinz & Knorr, 2001;Wuytack et al., 1998;Zhang et al., 2020). Although the spores lose most of their resistance to stress conditions, the application of mHP in the range of 50-300 MPa is not sufficient to lead to significant inactivation of the germinated cells at a moderate temperature of approximately 40 • C. Previous studies have investigated the physiological state of B. subtilis spores germinated at 150 MPa and 37 • C in ACES buffer. HP treatment generates the following four subpopulations with different physiological states: (1) heat-resistant (80 • C, 10 min) and mostly cultivable SD spores, (2) heat-sensitive and cultivable germinated spores, (3) heat-sensitive and partially cultivable germinated spores, and (4) membrane-compromised cells with minimal cultivability. Within a very short pressure dwell time of 3 min, approx. 90% of the spores germinated. However, even after a long pressure dwell time of 40 min, less than 3% of B. subtilis (germinated) spores were membrane compromised and by inference inactivated. Instead, most spores ended up in a presumably sublethally damaged state (Zhang et al., 2020). These cells were still partially able to grow in nutrient-rich environments, such as food. Therefore, mild follow-up treatments, such as mild heating or increased pressure levels, must be applied to completely inactivate the germinated spores. Additionally, germination may continue in the spores committed to germination after the pressure is released (Kong et al., 2014;Zhang et al., 2020). Therefore, the timing of the follow-up treatment is a crucial factor for inactivation. The follow-up treatment should be applied only after germination and the loss of resistance to stress. However, excessive duration between the HP treatment and the follow-up treatment may allow proliferation and potential production of heat-stable toxins. Hence, the timing of germination-inactivation strategies must be optimized and future studies are needed to thoroughly investigate this. Potential follow-up inactivation treatments could include a mild heat treatment or a pressure treatment at higher pressures in the range of 400-600 MPa. TA B L E 3 Comparison of nutrient-mediated and moderate high pressure (mHP)-mediated germination of Bacillus spores Similarities between nutrient and mHP germination Differences between nutrient and mHP germination • Both trigger Ca 2+ -dipicolinic acid (DPA) release, small acid-soluble protein (SASP) degradation, and rapid ATP generation (Wuytack et al., 1998). • Both are dependent on germinant receptors (GRs) for germination (Black et al., 2005;Paidhungat et al., 2002).
• The hierarchy of receptor responsiveness to pressure in B. subtilis is identical to that to a mixture of nutrients (GerA > GerB > GerK) (Black et al., 2005;. • Both are similarly affected by overexpression of various receptors (Black et al., 2005).
• Both exhibit similar germination kinetic parameters of individual spores. Heterogeneity is mainly due to variability in T lag (Kong et al., 2014).
• Both are strongly reduced or essentially eliminated in the presence of the GerD F87C variant Li et al., 2014).
• Both decrease with decreasing a w values due to the presence of humectants (Rao et al., 2018).
• A sublethal heat treatment increases nutrient-mediated germination but not mHP-mediated germination of B. subtilis spores (Luu et al., 2015).
• Both nutrient-mediated and mHP-mediated germination are associated with a commitment stage in which a short germination stimulus is sufficient to commit the spores to germinate even if the stimulus is removed. However, for mHP, it has been observed that these activated spores can decay to the inactivated dormant spore state depending on the incubation conditions. This deactivation phenomenon has not been reported for nutrient-mediated germination (Kong et al., 2014).
Note: This list is not exhaustive but provides an overview.

SIMILARITIES AND DIFFERENCES BETWEEN MHP-MEDIATED AND NUTRIENT-MEDIATED GERMINATION
The nutrient-induced spore germination pathway, which is the predominant pathway in nature, has been widely studied in the last 40 years. The wealth of knowledge on nutrient-mediated germination has aided the understanding of mHP-mediated germination. However, despite remarkable achievements in gaining additional understanding on nutrient-mediated germination, efforts to fully explain this process have remained unsuccessful. In particular, the role and mechanisms of GRs and the effects of activation treatments (e.g., sublethal heat activation) on germination are not well understood. Reversible protein conformational changes are hypothesized to play a major role in germination. However, this hypothesis has not been verified (Setlow, 2014;Setlow et al., 2017). Christie and Setlow (2020) have comprehensively reviewed the unexplored areas related to GRs and GR function. Novel insights into mHP-mediated germination can improve our understanding of these research questions. Thus, the elucidation of the nutrient-mediated and mHP-mediated germination pathways can aid in the identification of common pathways and mechanisms. Multiple studies have compared nutrient-mediated and mHP-mediated germination. Even though most available evidence indicates that underlying germination mechanisms between nutrient-induced and mHP-mediated germination are extremely similar, some aspects seem to be different (Table 3).

UN(DER)EXPLORED AREAS OF MHP-MEDIATED GERMINATION
Since the discovery of HP-induced spore germination more than 50 years ago, several studies have attempted to elucidate the underlying mechanisms. Although the general mechanism has been elucidated, some unanswered fundamental questions that must be addressed remain and are highlighted below.
• The biggest leap forward in understanding mHP germination would probably be made by getting a better mechanistic understanding of the mode of action of mHP on the GRs. In particular, the molecular mechanisms involved in the activation of the GRs and the direct or indirect role of the inner membrane in GR activation must be elucidated. Insights into pressure-induced inner membrane modifications and in situ analysis of protein conformation changes could markedly contribute to the mechanistic understanding. Potentially related to this are also unanswered questions like: (1) Why does a sublethal heat treatment enhance nutrient germination, but not mHP germination?, (2) What does spore commitment with mHP mean?, (3) What role does GerD play in mHP-mediated germination?, (4) Which role does the D subunit of some GRs play?, and (5) what is the exact downstream signaling cascade after GR activation? • Why do some spores within an (isogenic) population germinate significantly slower than other spores or not germinate at all after exposure to mHP? What are the underlying reasons for this observed superdormancy? What proteins, other than GRs, may play a critical role in superdormancy? And ultimately, how could we tackle SD spores in industrial applications? • The general features of spore germination appear to be identical in all species of the order Bacillales investigated so far Christie & Setlow, 2020). However, there is limited information on spore germination in species belonging to the order Clostridiales. Some studies have suggested that the germination behavior of Clostridiales spores can be significantly different from that of spores of the order Bacillales. For example, some species, such as C. difficile and C. perfringens, first hydrolyze the cortex and only release DPA afterward. Other species, such as C. sporogenes, however, have a similar germination pathway to Bacillus spp. where DPA release proceeds cortex hydrolysis (Setlow et al., 2017). Similar to Bacillus species, C. botulinum, C. perfringens, and Clostridium sporogenes all have inner membrane GRs, which are able to sense physiological germinants. In contrast, C. difficile and closely related species encode no such proteins. Instead, they sense bile salt germinants by their binding to a CspC pseudoprotease in the outer layers of the spores (Bhattacharjee et al., 2016;Olguín-Araneda et al., 2015;Setlow et al., 2017). The reader is referred to Bhattacharjee et al. (2016) for a comprehensive overview of germinants and germination receptors of Clostridiales species. Another notable difference is that Clostridiales have no GerD homolog, and a paralog has not been identified. As stated above, GerD and presumably germinosome formation increase rates of GR-dependent spore germination (Setlow et al., 2017). Literature reporting the specific germination behavior of Clostridiales under mHP, however, is very scarce. It was shown that C. perfrin-gens spores germinate upon exposure to mHP. However, other species, such as C. difficile spores that lack the inner membrane GRs, do not germinate upon exposure to mHP (Doona et al., 2016;Paredes-Sabja et al., 2011). Interestingly, Doona et al. (2016) found that mHPinduced C. perfringens germination was much faster and more complete when spores were heat activated prior to the HP treatment. Except for the here reported findings, however, very little is known about mHP germination of Clostridiales. The Clostridiales order comprises multiple pathogenic species. Hence, the effect of mHP on the germination of spores in this order must be elucidated. Ultimately, an inactivation strategy based on prior germination is only as effective as its weakest link, hence, weakest germinator and that might be of either of the two orders.

CONCLUSIONS
mHP represents a promising technology to trigger germination of spores in order to increase their susceptibility to subsequent mild inactivation technologies. Similar to nutrient germination, mHP triggers spore germination via GRs. However, the molecular mechanisms of mHP-mediated GR activation have not been elucidated. Various factors, including temperature, pressure dwell time, pH, suspending medium, and sporulation conditions, are reported to influence mHP germination. For a practical implementation of mHP-induced germination of spores, more in-depth studies investigating the effect of typical food parameters like water activity as well as the effect of food constituents like salt, fat, and sugars would be beneficial. Even though the general mechanisms of mHP germination are fairly well understood, multiple other aspects need further research in order to allow for a successful industrial implementation of an mHPinduced germination-inactivation strategy. A challenge is represented by the heterogeneity with which spores of an isogenic population germinate in response to the trigger. The major limitation for the successful implementation of germination-inactivation strategies is SD spores, which exhibit slow germination or do not germinate. The presence of SD spores makes it currently impossible to guarantee commercial sterility. Heterogenous germination of Bacillus spores exposed to mHP can be partially explained by different GR levels. However, other factors seem to play a role. Further studies are needed to examine the effect of mHP on GRs and other factors that may influence mHP-mediated germination and superdormancy. Finally, and importantly, the effect of mHP on the germination of spores from species of the order Clostridiales must be examined in the future.

A C K N O W L E D G M E N T S
The authors gratefully acknowledge the financial support of the Swiss National Science Foundation SNF (Grant number: 31003A_182273, Title: Isolation and characterization of high pressure superdormant spores). The authors would like to thank Corinne Bieri for valuable comments on the manuscript.

C O N F L I C T O F I N T E R E S T
The authors declare that this review was written in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.