Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I–III. We propose two distinct sporulation strategies used by C. botulinum Groups I–III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.


Introduction
The endospore-forming, bacterial species Clostridium botulinum is divided into phylogenetically and ecologically distinct groups of clostridia that produce the highly potent botulinum neurotoxin (BoNT) [1]. The toxin blocks neurotransmission, causing a potentially lethal, flaccid paralysis known as botulism [2]. While BoNT is the causative agent of botulism, C. botulinum spores play a key role in the survival and dispersion of C. botulinum in the environment, in contamination of food and feed raw materials [3][4][5][6], and in pathogenesis [1,7]. Inadequate storage of contaminated food and feed products may result in spore germination and outgrowth into neurotoxinogenic C. botulinum cultures, posing a botulism risk for humans and animals [8][9][10]. Alternatively, spore germination in toxinogenic culture and colonization in the gut or in deep wounds may result in toxicoinfectious botulism [7,11]. Indeed, infant botulism with toxic gut colonization affecting babies under one year of age is the most common form of human botulism in the United States [12].
Sporulation has been extensively studied in the spore-forming model organisms Bacillus subtilis and Clostridioides difficile [13,14]. Sporulation is a stress response strategy to evade adverse environmental conditions, such as starvation or high cell density [15]. Upon initiation of sporulation, the asymmetrical septation leads to the formation of a mother cell and a forespore [13]. The forespore is then engulfed and surrounded by protective layers by the enveloping mother cell. These layers determine the spore properties, resistance, and how it interacts with its environment [16]. Earlier spore research in C. botulinum focused on the regulation and activation of sporulation [17][18][19][20], spore heat resistance [9,21,22], and germination [23]. Sporulation triggers, the proportion of sporulating cells, and the Table 1. Enumeration of cell types of 10 Clostridium botulinum strains by phase contrast microscopy. The table shows the average and standard deviation of the percentage of cells belonging to three replicates except for UN1/10-7B strain (only two replicates).  Sporulation progressed slowly for Group I strains, and we observed slow accumulation of sporulating cells and only a small number of free spores at the end of the monitoring period. In Group II and III strains, sporulation progressed faster than in Group I strains as they did not display accumulation of sporulating cells but had a dramatic increase in the proportion of free spores (Table 1). However, two Group II strains had low cell density at the assumed mid-logarithmic time point and yielded no cell pellet. Some free spores were phase-dark, indicating rehydration of their cores either due to damage or to germination. In Group II and III strains, we observed free phase-dark spores appearing as early as 48 h and accumulating in these cultures. This cell type was only detected in one strain from Group I, ATCC17841, and in a very low percentage (0.3 ± 0.2%, Table 1).

Ultrastructure of Sporulating Cells and Spores
We used transmission electron microscopy to analyze the ultrastructure of cells in different stages of sporulation and spores in C. botulinum Group I-III cultures ( Figure 1). In all strains, we observed asymmetric septum formation and full engulfment of the forespore before the formation of the coat (Figure 1, Asy and Eng panels), unlike the earlier coat formation in Clostridium tetani and Clostridium histolyticum [42].
In all 10 strains, the coat was assembled from small subunits that surrounded the forespore ( Figure 2) and, apparently, fused together, forming a mature coat. In cells of Group I strains, an exosporium surrounding the forespore was visible while still in the mother cell ( Figure 1, MF panel, black arrows).
Spores of both Group III strains and those of Group II CB11/1-1 and Eklund 17B also harbored an exosporium (Figures 3 and 4); however, this structure was very thin and was not visible in cells carrying a mature spore (Figure 1, MF panel). Both free and purified spores of Group II Beluga did not present an exosporium (Figures 1 and 3). Three spore morphotypes were identified based on TEM images of purified spores ( Figure 3). All Group I strains and Group II Eklund 17B displayed spores surrounded by a loose-fitting exosporium with a lamellar structure ( Figure 3A,D). Spores of both Group I isolates from botulism cases UN1/10-7B and UN5/11-8 appeared to show a multi-layered exosporium. Group II Beluga spores, instead, presented with antenna-like appendages and no exosporia at all ( Figure 3B,D). Spores of both Group III strains showed thin, tight-fitting exosporia ( Figure 3C,D). Unfortunately, we were not able to purify CB11/1-1 spores due to the presence of excess cell debris and free phase-dark spores. However, free CB11/1-1 spores found in sporulating cultures in TEM images displayed a fourth morphotype with appendages typical for Beluga spores and an exosporium typical for Eklund 17B spores ( Figure 4). Free spores of Beluga and Eklund 17B did not present this morphotype ( Figure 1, MCL panel).
A systematic analysis of the structures of purified spores of each strain was performed by taking measurements of 13 key spore structures, as observed in the TEM images of 10 individual spores of each strain (Tables S1 and S4).
Only spores that appeared to be centrally cut by their longest axis were selected for this analysis in order to measure their longest and widest points and the correct thickness of their structures. The absence and poor visibility of Beluga, BKT015925, and Stockholm C exosporia removed their eligibility for related measurements. In all 10 strains, the coat was assembled from small subunits that surrounded the forespore ( Figure 2) and, apparently, fused together, forming a mature coat. In cells of Group I strains, an exosporium surrounding the forespore was visible while still in the mother cell ( Figure 1, MF panel, black arrows). Spores of both Group III strains and those of Group II CB11/1-1 and Eklund 17B also harbored an exosporium (Figures 3 and 4); however, this structure was very thin and was not visible in cells carrying a mature spore ( Figure 1, MF panel). Both free and purified spores of Group II Beluga did not present an exosporium (Figures 1 and 3). Three spore morphotypes were identified based on TEM images of purified spores ( Figure 3). All Group I strains and Group II Eklund 17B displayed spores surrounded by a loose-fitting exosporium with a lamellar structure ( Figure 3A,D). Spores of both Group I isolates from botulism cases UN1/10-7B and UN5/11-8 appeared to show a multi-layered exosporium. Group II Beluga spores, instead, presented with antenna-like appendages and no exosporia at all ( Figure 3B,D). Spores of both Group III strains showed thin, tight-fitting exosporia ( Figure 3C,D). Unfortunately, we were not able to purify CB11/1-1 spores due to the presence of excess cell debris and free phase-dark spores. However, free CB11/1-1 spores found in sporulating cultures in TEM images displayed a fourth morphotype with appendages typical for Beluga spores and an exosporium typical for Eklund 17B spores ( Figure 4). Free spores of Beluga and Eklund 17B did not present this morphotype ( Figure 1, MCL panel).
A systematic analysis of the structures of purified spores of each strain was performed by taking measurements of 13 key spore structures, as observed in the TEM images of 10 individual spores of each strain (Tables S1 and S4). Overall, when considering the exosporium, Group I strains and Group II Eklund 17B had the longest spores, and the Group I strains UN1/10-7B and UN5/11-8 and Group II Eklund 17B had the widest spores ( Figure S4A,B).
Excluding the exosporium, Group II Beluga and Group III Stockholm C had the longest spores ( Figure S4C), and Group III Stockholm C had the widest spores. Group I UN1/10-7B and UN5/11-8 and Group II strains Beluga and Eklund 17B had medium-wide spores, and Group I strains ATCC 3502, ATCC 19397, and ATCC 17841 had the thinnest spores ( Figure S4D).
The Group III strains had the thickest coats ( Figure S4H). We analyzed the exosporia and their position relative to the coats. Group I strains UN1/10-7B and UN5/11-8 and Group II Eklund 17B had the largest space between the coat and the exosporium, while Group III strains had the smallest ( Figure S4I). The spacing from the top of the coat to the exosporium and bottom of the coat to the exosporium were consistent across the strains ( Figure S4J,K). Group I UN1/10-7B had a thicker exosporium than any other strain ( Figures 5A,B and S4L).
Finally, the only strain to present appendages was Beluga. The number of appendages per spore varied in the observed spores from none to nine, and their length varied from 47 to 642 nm ( Figure S4M).     spores ( Figure S4D). The Group III strains had the thickest coats ( Figure S4H). We analyzed the exosporia and their position relative to the coats. Group I strains UN1/10-7B and UN5/11-8 and Group II Eklund 17B had the largest space between the coat and the exosporium, while Group III strains had the smallest ( Figure S4I). The spacing from the top of the coat to the exosporium and bottom of the coat to the exosporium were consistent across the strains ( Figure S4J,K). Group I UN1/10-7B had a thicker exosporium than any other strain (Figures 5A,B and S4L).

Spore Autoaggregation, Sedimentation, and Hydrophobicity
We tested spore autoaggregation by allowing the spore suspensions to sediment. While all spore suspensions eventually sedimented at the bottom of the cuvette, spores capable of autoaggregating were expected to attach to each other and sedimented faster. Active autoaggregation was observed in Group I strains UN1/10-7B and UN5/11-8, Group II Beluga, and Group III BKT015925. Spore suspensions from these strains sedimented in less than 6 to 7 h and had high sedimentation values from 96% to 84% ( Figure 6A,B). Using phase contrast microscopy, we observed autoaggregation in these strains ( Figure 6C). Other strains did not display autoaggregation. Instead, their spore suspensions displayed passive sedimentation with long periods to clear the suspensions (Figure 6A), lower sedimentation values (78% to 51%, 37% for Eklund 17B- Figure 6B), and an absence of autoaggregation ( Figure 6C).
We studied the hydrophobicity of C. botulinum spores with the BATH assay [33], measuring the adhesion of purified spores to hexadecane by monitoring the decrease of OD 440nm in aqueous solution. Most strains had hydrophobic spores with hydrophobicity values varying from 86% to 100%, with the only outlier being ATCC 17841 (61%, Figure 7). II Beluga, and Group III BKT015925. Spore suspensions from these strains sedimented in less than 6 to 7 h and had high sedimentation values from 96% to 84% ( Figure 6A,B). Using phase contrast microscopy, we observed autoaggregation in these strains ( Figure 6C). Other strains did not display autoaggregation. Instead, their spore suspensions displayed passive sedimentation with long periods to clear the suspensions (Figure 6A), lower sedimentation values (78% to 51%, 37% for Eklund 17B- Figure 6B), and an absence of autoaggregation ( Figure 6C).  We studied the hydrophobicity of C. botulinum spores with the BATH assay [33], measuring the adhesion of purified spores to hexadecane by monitoring the decrease of OD440nm in aqueous solution. Most strains had hydrophobic spores with hydrophobicity values varying from 86% to 100%, with the only outlier being ATCC 17841 (61%, Figure  7).

Figure 7.
Hydrophobicity of Clostridium botulinum spores. The capacity of the purified spores of each strain to adhere to hexadecane was tested in a BATH assay; bars represent the average of three technical replicates with standard deviation shown by the error bars.

Thermal Destruction of Spores
We tested the heat resistance of purified C. botulinum spores. As expected, Group I spores showed a relatively high heat resistance with D98°C values varying from 1.1 min to Figure 7. Hydrophobicity of Clostridium botulinum spores. The capacity of the purified spores of each strain to adhere to hexadecane was tested in a BATH assay; bars represent the average of three technical replicates with standard deviation shown by the error bars.

Thermal Destruction of Spores
We tested the heat resistance of purified C. botulinum spores. As expected, Group I spores showed a relatively high heat resistance with D 98 • C values varying from 1.1 min to 24.3 min, Group III spores showed a moderate heat resistance with D 90 • C values of 11.5 min and 11.9 min, and Group II spores showed a relatively low resistance with D 75 • C values of 6.3 min and 27.3 min. Within Group I, we observed an interesting pattern: isolates from recent botulism cases (UN1/10-7B and UN5/11-8) showed substantially higher D 98 • C values (11.4-24.3 min) than the laboratory strains (ATCC 3502, ATCC 1784, and ATCC 19397, 1.1-2.1 min) ( Figure 8A). Within Group II, we observed a four-fold difference between the D 75 • C of Eklund 17B (27.3 min) and Beluga (6.3 min) ( Figure 8B). Both Group III strains had similar D 90 • C values (11.9 min and 11.5 min for BKT015925 and Stockholm C, respectively) ( Figure 8C).

Discussion
Sporulation is a survival and pathogenesis strategy in bacteria. Producing a resistant cell type allows spore-forming pathogens to disperse and infect new hosts. The spores are dormant, which incurs a loss of individuals actively contributing to the population growth and pathogenesis. Therefore, each sporulating organism needs to adopt a sporulation strategy that ensures a balance between survival and growth in prevailing conditions. We studied the population-level sporulation strategies of C. botulinum strains by following their sporulation with phase contrast microscopy. Overall, the studied strains displayed two main strategies for sporulation. While the times when sporulating cells were first observed were similar for most strains, Group I strains showed an accumulation of cells in the late stages of sporulation, delaying the release of mature spores into cultures. By contrast, Group II and III cultures had small sporulating populations that released mature spores as soon as 48 h after inoculation. C. botulinum Groups I, II, and III are diverse species; thus, it is tempting to speculate that the different population-level sporulation strategies have evolved in adaptation to different metabolisms and natural environments. The proteolytic metabolism of Group I strains allows them to obtain more nutrients via proteolysis than non-proteolytic strains, which might afford them a longer sporulation time and a larger proportion of sporulating individuals. For the non-proteolytic, Group II and some Group III strains, it appears to be beneficial to promptly guarantee survival of a small part of the population.
We observed discontinuous spore coat formation in all strains studied. This is a common mechanism in clostridia; however, its timing during sporulation varies between species [29,[43][44][45]. For example, C. tetani and C. histolyticum show spore coat fragments during engulfment [42]. In all our C. botulinum Group I, II, and III strains, we observed the coat fragments appearing after completed engulfment when a cortex was already visible. This suggests that, in C. botulinum, activation of coat gene expression is temporally regulated, as is the case in B. subtilis [46].
We identified four spore morphotypes through TEM. C. botulinum Groups I and III showed their own consistent morphotypes. Group I spores had thick and loose-fitting exosporia, while Group III strains displayed spores with thin and tight-fitting exosporia.
The three Group II strains showed three different morphotypes. Eklund 17B spores were similar to the spores of Group I strains, Beluga spores presented with antennae protruding from the spore coat and no exosporium, and CB11/1-1 spores showed both antennae and exosporium. This last morphotype has previously been reported as the spore morphology of Group II, type E strains, including Beluga, using spores obtained with lytic enzymes and not by natural mother cell lysis [30]. The apparently contradictory findings on the presence of an exosporium in Beluga can be explained by the loss or destruction of an exosporium during mother cell lysis. Alternatively, the structure observed in [30] was an artefact of enzymatic cell lysis. This assumption might be supported by the lack of csxA encoding CsxA, an essential protein for the assembly of the exosporium in C. sporogenes [47,48], in the Beluga genome, inferring that a proper exosporium is likely not formed. It is of note that csxA homologs can be found in the genomes of several C. botulinum Group I strains and Eklund 17B [48]. Interestingly, other exosporium-related genes identified in C. sporogenes were not found in the genomes of the Eklund 17B strain [48], suggesting that Group II exosporia composition differs from C. sporogenes and C. botulinum Group I strains.
Previous studies have shown moderate hydrophobicity of spores of C. botulinum Group I strain 213B [49]. All our purified C. botulinum Group I, II, and III spore suspensions displayed high or very high hydrophobicity, regardless of the spore morphotypes. Similarly, regarding autoaggregation, we did not observe differences between spore morphotypes. It is possible that spores with similar morphology have similar structural spore proteins, but adhesion proteins on their surface differ. Interestingly, all strains that displayed active autoaggregation were originally isolated from botulism cases, while strains with no or little autoaggregation were from environmental sources. While aggregation might assist in infectious processes, such as being toxicoinfectious or wound botulism, its possible relevance in the aetiology of foodborne botulism, which is an intoxication, remains unclear.
In line with previous knowledge, spores of each C. botulinum group displayed different levels of heat resistance, with Group I spores being the most heat resistant, Group II being the least heat resistant [22], and Group III showing moderate heat resistance [50]. The observed D-values, however, were mostly lower than those previously reported, which is likely due to the spore purification used. Spores for thermal destruction assays used for food safety testing were often prepared by using sonication and washes of the spore stock without further density gradient purification of spores from culture debris. Density gradient-purified spore suspensions lack the heat protection effects that cell debris and sugar, present in culture media, confer to the spores.
In both C. botulinum Group I and II spore suspensions, we observed two different patterns of heat resistance, with high and low D-values for strains within the same group. Spores of strains with the lower D-values within a group possessed no (Group II) or a very thin exosporium (Group I). Additionally, the strains with higher D-values within a group had larger spores (total spore size) than strains with lower D-values. Meanwhile, spores of both Group III strains had a similar size, exosporium thickness, and D-value. It appears that the exosporium contributes to heat resistance, probably by presenting an additional barrier to protect the spore core from heat, as well as by enlarging the spore size, which can potentially affect the heat distribution. However, further studies encompassing more strains and exosporium gene knockout mutants would bring insights into the role of the exosporium in the heat resistance of C. botulinum spores.
While the heat resistance of Groups I and II C. botulinum spores has been widely studied for food safety purposes, studies on the heat resistance of Group III spores unrelated to human disease are scarce. An early study demonstrated different heat resistance patterns for Group III spores of terrestrial and marine origin, with terrestrial spores showing higher heat resistance than marine spores [50]. Both of our Group III strains could be characterized as terrestrial and showed resistance patterns similar to [50]. To better understand the diverse spore heat resistance and its mechanisms in C. botulinum strains, a genome-wide association analysis of a large number of strains would be invaluable.
To conclude, we provide a systematic characterization of sporulation and spores of C. botulinum Groups I, II, and III strains. C. botulinum appears to have two distinct sporulation strategies, where the proteolytic, Group I strains take longer to sporulate and reach larger numbers of free spores than the non-proteolytic, Group II and III strains. The morphological stages of sporulation appeared to be similar in all strains. We observed discontinuous coat formation after engulfment and initiation of cortex formation, which suggests tight temporal regulation of coat gene expression. Four spore morphotypes were identified: one for Group I strains, one for Group III strains, and two for Group II strains. Each Group II strain had its own morphotype with one strain sharing the Group I morphotype. The differences in morphotypes did not appear to play a role in spore hydrophobicity or autoaggregation, with autoaggregation displayed only by strains isolated from botulism cases. Finally, the exosporium appeared to play a role in spore heat resistance as both the presence of an exosporium and its thickness seemed to confer heat resistance.
Our work expands on the knowledge of C. botulinum sporulation and spore biology and highlights the heterogeneity of spore heat resistance within each group, providing novel insights in the fields of food and feed safety and epidemiology.

Bacterial Strains and Growth Conditions
The present work included 10 C. botulinum strains belonging to Group I, Group II, and Group III ( Table 2). The strain selection included historical and representative strains from each metabolic group, as well as more recent isolates from human botulism cases. We also selected strains producing different toxin type within each group. All strains were cultured in anaerobic conditions (atmosphere of 85% N 2 , 10% CO 2 , and 5% H 2 ) in an anaerobic workstation (MACS MG-1000, Don Whitley, Bingley, UK). Group I strains were propagated in tryptone-peptone-glucose-yeast extract (TPGY) broth at 37 • C. Group II strains were grown in TPGY broth and a bi-phasic sporulation medium (Cooked Meat Medium Agar + TPGY) at 30 • C [26]. Finally, Group III strains were grown at 37 • C in TPGY supplemented with 0.2% (w/v) L-cysteine [51].

Phase Contrast Microscopy
Sporulation of C. botulinum cultures was monitored by phase contrast microscopy. One milliliter of each culture was pelleted, washed, and re-suspended in 100 µL of anaerobic phosphate buffered saline buffer (PBS, Sigma-Aldrich, St. Louis, MO, USA). Three microliters of washed bacterial preparation was spotted onto an agarose slide and mounted samples were observed using a Leica DMi8 microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a 100X objective and an Orca-Flash4.0LT camera (Hamamatsu Photonics K. K., Iwata City, Japan). Acquired images were further processed and analyzed with MetaMorph (version 7.10.0.119, Molecular Devices, San Jose, CA, USA). Cells were classified and counted according to their morphology as follows: non-sporulating vegetative cells, sporulating cells, free phase-bright spores, and free phase-dark spores. Bacterial cells that were out of focus, overlapped, or not entirely located within the boundaries of the image field were excluded from the analysis.

Spore Purification
For each strain, 50 mL of the appropriate sporulation medium was inoculated from an overnight culture to an initial optical density at 600 nm (OD 600nm ) of~0.05 (AU), and the OD 600nm was recorded over time ( Figure S5). This was performed in triplicate (biological replicates).
At the mid-logarithmic growth phase (5 h for Group I strains and 8 h for Group II and III strains) and at 24 h, 48 h, and 72 h post inoculation, the cultures were observed by phase contrast microscopy and sampled for further transmission electron microscopy analyses.
The total duration of sporulation was estimated to be 3 days for Group II and III strains and 10 days for Group I strains, according to phase contrast microscopy observations ( Figure S6). Once the sporulation was completed, free spores were harvested by centrifugation (10,000× g, 5 min, 4 • C) and washed twice with cold 0.1% (v/v) Triton X-100 (ICN Biomedicals Inc., Irvine, CA, USA). Washed spore pellets were then resuspended in 20% (v/v) solution of Gastrografin ® (Bayer AG, Leverkusen, Germany) and layered over 50% (v/v) Gastrografin ® solution in round-bottom tubes to create a density gradient. The gradient tubes were then centrifuged (10,000× g, 30 min, 4 • C) and the layer of debris and extra Gastrografin solution were removed from the tubes. The pellets were resuspended in cold 0.1% (v/v) Triton X-100 and transferred to a clean Eppendorf tube. Finally, the spore suspensions were washed five times with cold 0.1% (v/v) Triton X-100 (5000× g, 2 min, room temperature), and purity was assessed by phase contrast microscopy until >90% of the cells were free phase-bright spores and the amount of cell debris was minimal/absent. If needed, the gradient centrifugation and subsequent washes were repeated, as described above.
The purification process was affected by high numbers of free phase-dark spores, indicating re-germination or damage of newly formed spores. Three strains had a particularly high number of free phase-dark spores already at 72 h (Beluga 17.2 ± 7.4%, CB11/1-1 14.9 ± 7.8%, BKT015925 7.5 ± 6.2%; Table 1). This led us to set 72 h as the endpoint of the sporulation experiments for Group II and III cultures.

Spore Measurements in Thin Section Samples
For each strain, we selected ten individual spores cut in their center following their longest axis. Their ultrastructures were visualized and measured using Adobe Photoshop CS6 version 13.0 (Adobe Systems Inc., Mountain View, CA, USA). Thirteen different measurements (Table S1) were collected for each spore. Length and width were measured at a single site per spore, while thickness was measured at three different sites per structural component per spore and averaged. The length of every single appendage present in each C. botulinum Group II Beluga spore was measured ( Figure S4). One-way ANOVA (Post-Hoc test: Tukey HSD) was performed using IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA) on the spore measurements, using the single measurements and averages thickness which were measured three times. All original measurements used for the analysis are presented in Table S2.

Spore Autoaggregation
The capacity of purified spores to autoaggregate and sediment was tested for each strain in triplicate [32]. Purified spore suspensions were adjusted to OD 600nm~1 . Initial OD 600nm was measured, and the suspensions were allowed to settle in OD cuvettes for 24 h without agitation. Estimations of the sedimentation velocity were achieved by acquiring still images of the spore suspensions every half hour with a Canon EOS 40D digital camera (Canon Inc., Tokyo, Japan). After 24 h, the upper fraction (top 80%) of the suspension was sampled, and the OD 600nm was measured. The percentage of sedimentation was calculated as Sedimentation (%) = 100 × [1 − (OD f /OD i )], where OD f is the final OD 600nm and OD i is the initial OD 600nm . Additionally, in one of the replicate experiments, phase contrast images were acquired to monitor the changes in the number and dispersion of cells before and after allowing the suspensions to settle.

Spore Hydrophobicity
Hydrophobicity of the purified spores was tested by measuring the bacterial adherence to hydrocarbons (BATH) using a modified protocol [33]. Pure spore suspensions in sterile ddH 2 O were adjusted to OD 440nm~0 .5, and 100 µL of hexadecane (Sigma-Aldrich) was added to the suspension. Each cuvette was vortexed at maximum speed for 1 min and allowed to settle for 10 min to separate the aqueous and hexadecane phases. Using thin pipette tips, the aqueous phase was transferred to a clean cuvette, allowed to rest for an additional 10 min, and OD 440nm was measured. The percentage of spores captured by hexadecane was calculated as Hydrophobicity (%) = [100 × (OD i − OD f )]/OD i , where OD f is the final OD 440nm and OD i is the initial OD 440nm .

Spore Heat Resistance
Purified spore suspensions were adjusted to OD 600nm~1 in sterile ddH 2 O, which, when using purified spore suspensions, translates to an approximate 10 8 spore titer. To determine the initial spore titer, 150 µL of the spore suspensions was transferred into a sterile tube. The remaining suspension was aliquoted in replicate tubes to a volume of 150 µL. Samples were heat-treated in a water bath at 80 • C, 90 • C, or 98 • C for a duration ranging from 1 min to 120 min. After heat treatment, the tubes were cooled to room temperature and transferred to an anaerobic chamber. Most probable number (MPN) assays were performed as previously described to determine the initial and heat-treated spore titers [20]. Results were read after two days of incubation and D-values were calculated. Several time-temperature combinations were tested until we achieved at least a 3-log kill, a time/temperature combination where at least 10 3 spores were inactivated. D-values were calculated by plotting the log 10 (N t /N 0 ) values from each individual replicate against time (min), where N t and N 0 were the MPN values after and before heating, respectively. Regression lines were fitted to each set of data, and the D-values were calculated using D T = −1/a, where a is the slope of the regression line.