Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.     

Specific Isolation of Clostridium botulinum Group I Cells by Phage Lysin Cell Wall Binding Domain with the Aid of S-Layer Disruption

Clostridium botulinum is a notorious pathogen that raises health and food safety concerns by producing the potent botulinum neurotoxin and causing botulism, a potentially fatal neuroparalytic disease in humans and animals. Efficient methods for the identification and isolation of C. botulinum are warranted for laboratory diagnostics of botulism and for food safety risk assessment. The cell wall binding domains (CBD) of phage lysins are recognized by their high specificity and affinity to distinct types of bacteria, which makes them promising for the development of diagnostic tools. We previously identified CBO1751, which is the first antibotulinal phage lysin showing a lytic activity against C. botulinum Group I. In this work, we assessed the host specificity of the CBD of CBO1751 and tested its feasibility as a probe for the specific isolation of C. botulinum Group I strains. We show that the CBO1751 CBD specifically binds to C. botulinum Group I sensu lato (including C. sporogenes) strains. We also demonstrate that some C. botulinum Group I strains possess an S-layer, the disruption of which by an acid glycine treatment is required for efficient binding of the CBO1751 CBD to the cells of these strains. We further developed CBO1751 CBD-based methods using flow cytometry and magnetic separation to specifically isolate viable cells of C. botulinum Group I. These methods present potential for applications in diagnostics and risk assessment in order to control the botulism hazard.     

Screening of twelve Clostridium botulinum (group I) spores for high-pressure resistance at elevated-temperatures

Twelve strains of Clostridium botulinum group I spores, suspended in phosphate buffer (0.1 M) at approximately 10⁷ CFU/ml concentration, were subjected to high pressure treatments (800 and 900 MPa; 0.5–15 min) at elevated temperatures (90 and 100 °C). The treatments were chosen to have a range of pressure/temperature severity to be able to discriminate the spore strains for their pressure resistance. An insulated test chamber was used to achieve temperature stability during treatment. Preliminary tests showed the need for an 8 day anaerobic incubation for enumeration. Results showed that strains PA9508B, HO9504A and CK2-A had higher pressure resistance than others among the 12 strains studied. Strain 62A was least resistant and completely inactivated by the treatment. Estimated D values of the more resistant strains were in the 0.66–1.8 min range at 900 MPa at 100 °C treatment. The temperature sensitivity parameter (Z_P value) in the 800–900 MPa pressure range varied between 10 and 16 °C, and pressure sensitivity parameter (Z_T value) in the 90–100 °C temperature range varied between 340 and 760 MPa. The most pressure resistant strain was PA9508B with an estimated Z_P value of 16.0 °C and Z_T value of 470 MPa. Since the pathogenic strains of C. botulinum have different pressure resistance, the most resistance strain should be selected as the basis for process establishment.     

Penicillium commune spore production in solid‐state fermentation of coffee pulp at laboratory scale and in a helical ribbons rotating reactor

Penicillium commune was grown on coffee pulp (CP) by solid‐state fermentation (SSF). The effects of the duration of CP thermal treatment and the effects of incubation temperature on spore production yield were studied at laboratory scale. The effect of mixing during fermentation was assayed at pilot plant scale in a 70 L stainless steel non‐aseptic reactor equipped with helical ribbons for mixing solids. For thermal treatments of CP at 121 °C for 10, 20, 30 and 40 min, no significant difference in spore production yield was observed. Maximum sporulation yield was found at 25 °C; when the incubation temperature was higher than 30 °C, the sporulation yield decreased significantly. A spore production yield of 3.7 × 10⁹ spores g^?1 dry CP was obtained when continuous mixing (0.25 rpm) was used at pilot plant scale; however, a decrease in spore yield (1.4 × 10⁹ spores g^?1 dry CP) was observed under static conditions. Spore production was not affected when a scale factor between 79 and 105 was assayed from laboratory to pilot plant; at this level, the productivity obtained was 3.1 × 10⁷ spores g^?1 dry CP h^?1. This value is similar to that found in other reports using natural substrates but working at a smaller scale. Copyright © 2006 Society of Chemical Industry     

Design and evaluation of a new device for fungal spore aerosolization for laboratory applications

Fungi have significant health effects varying from serious allergic reactions from inhalation to toxic effects and even fatal infections. Indoor and outdoor sampling may identify potentially contaminated environments, but sampling techniques need refining to set risk levels regarding occupational exposure or to assess and analyse sudden outbreaks of fungi. As little is known of the sporulation and dispersal dynamics of fungi, we have experimented with aerosolizing fungal spores from cultures grown in standard 9 cm Petri dishes for 3–10 h using a new device that incorporates vibration. We evaluated the effect of the air flow and frequency on a quantity and pattern of spores aerosolized. Frequency had positive, negative or no effect. Positive quantitative effects were noted in Aspergillus versicolor at 50 Hz, in Aspergillus niger at 400 Hz and at 100–400 Hz in Rhizopus. Aerosolization of Aspergillus species dropped significantly in the first 20 min and then stabilised for 2.5–3 h. After this time period, the generation process became highly unstable with large quantities of small particles produced. It could be explained by appearance of cracks on the nutrient surface due to desiccation with corresponding release of particles of nutrient to the air carrier. For Rhizopus, the initial steep decrease in particle generation for the first 20 min of the process was followed by quite stable spore production for the following 10 h of process operation. There were no cracks observed on the nutrient surface for the entire 10 h operation of the device. The air flow also affects the efficiency of the spore release. It was found that increase in the air flow was associated with more efficient sporulation from the nutrient dish. On the other hand, the size distribution curve of the airborne spores produced at higher air flows was correspondingly slightly shifted towards larger sizes, which is related to less efficient loosening of spores and corresponding increase in concentration of clumps consisting of more than one spore. Also, it was found that incubation of fungal cultures for extended periods (2–6 weeks) led towards production of larger numbers of airborne fungal spores.     

Dynamics of Germinosome Formation and FRET-Based Analysis of Interactions between GerD and Germinant Receptor Subunits in Bacillus cereus Spores

Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores’ extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor’s (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)–SGFP2 and GerD–mScarlet-I, but there was no FRET between GerD–mScarlet-I and either GerRA–SGFP2 or GerRC–SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)–SGFP2 and GerD–mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)–SGFP2 and GerD–mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.     

Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.     

Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.     

Characterization of Two Highly Arsenic-Resistant Caulobacteraceae Strains of Brevundimonas nasdae: Discovery of a New Arsenic Resistance Determinant

Arsenic (As), distributed widely in the natural environment, is a toxic substance which can severely impair the normal functions in living cells. Research on the genetic determinants conferring functions in arsenic resistance and metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. More and more new arsenic resistance (ars) determinants have been identified to be conferring resistance to diverse arsenic compounds and encoded in ars operons. There is a hazard in mobilizing arsenic during gold-mining activities due to gold- and arsenic-bearing minerals coexisting. In this study, we isolated 8 gold enrichment strains from the Zijin gold and copper mine (Longyan, Fujian Province, China) wastewater treatment site soil, at an altitude of 192 m. We identified two Brevundimonas nasdae strains, Au-Bre29 and Au-Bre30, among these eight strains, having a high minimum inhibitory concentration (MIC) for As(III). These two strains contained the same ars operons but displayed differences regarding secretion of extra-polymeric substances (EPS) upon arsenite (As(III)) stress. B. nasdae Au-Bre29 contained one extra plasmid but without harboring any additional ars genes compared to B. nasdae Au-Bre30. We optimized the growth conditions for strains Au-Bre29 and Au-Bre30. Au-Bre30 was able to tolerate both a lower pH and slightly higher concentrations of NaCl. We also identified folE, a folate synthesis gene, in the ars operon of these two strains. In most organisms, folate synthesis begins with a FolE (GTP-Cyclohydrolase I)-type enzyme, and the corresponding gene is typically designated folE (in bacteria) or gch1 (in mammals). Heterologous expression of folE, cloned from B. nasdae Au-Bre30, in the arsenic-hypersensitive strain Escherichia coli AW3110, conferred resistance to As(III), arsenate (As(V)), trivalent roxarsone (Rox(III)), pentavalent roxarsone (Rox(V)), trivalent antimonite (Sb(III)), and pentavalent antimonate (Sb(V)), indicating that folate biosynthesis is a target of arsenite toxicity and increased production of folate confers increased resistance to oxyanions. Genes encoding Acr3 and ArsH were shown to confer resistance to As(III), Rox(III), Sb(III), and Sb(V), and ArsH also conferred resistance to As(V). Acr3 did not confer resistance to As(V) and Rox(V), while ArsH did not confer resistance to Rox(V).     

Imaging Mass Spectrometry of a Coral Microbe Interaction with Fungi

Fungal infections are increasing worldwide, including in the aquatic environment. Microbiota that coexist with marine life can provide protection against fungal infections by secretion of metabolites with antifungal properties. Our laboratory has developed mass spectrometric methodologies with the goal of improving our functional understanding of microbial metabolites and guiding the discovery process of anti-infective agents from natural sources. GA40, a Bacillus amyloliquefaciens strain isolated from an octocoral in Panama, displayed antifungal activity against various terrestrial and marine fungal strains. Using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), the molecular species produced by this microbe were visualized in a side-by-side interaction with two representative fungal strains, Aspergillus fumigatus and Aspergillus niger. The visualization was performed directly on the agar without the need for extraction. By evaluating the spatial distributions, relative intensities and m/z values of GA40 secreted metabolites in the fungal interactions and singly grown control colonies, we obtained insight into the antifungal activity of secreted metabolites. Annotation of GA40 metabolites observed in MALDI-IMS was facilitated by MS/MS networking analysis, a mass spectrometric technique that clusters metabolites with similar MS/MS fragmentation patterns. This analysis established that the predominant GA40 metabolites belong to the iturin family. In a fungal inhibition assay of A. fumigatus, the GA40 iturin metabolites were found to be responsible for the antifungal properties of this Bacillus strain.     

Catalase (CAT) Gene Family in Rapeseed (Brassica napus L.): Genome-Wide Analysis,Identification, and Expression Pattern in Response to Multiple Hormones and Abiotic Stress Conditions

Catalase (CAT) is an antioxidant enzyme expressed by the CAT gene family and exists in almost all aerobic organisms. Environmental stresses induce the generation of reactive oxygen species (ROS) that eventually hinder plant growth and development. The CAT enzyme translates the hydrogen peroxide (H₂O₂) to water (H₂O) and reduce the ROS levels to shelter the cells’ death. So far, the CAT gene family has not been reported in rapeseed (Brassica napus L.). Therefore, a genome-wide comprehensive analysis was conducted to classify the CAT genes in the rapeseed genome. The current study identified 14 BnCAT genes in the rapeseed genome. Based on phylogenetic and synteny analysis, the BnCATs belong to four groups (Groups I–IV). A gene structure and conserved motif analysis showed that Group I, Group II, and Group IV possess almost the same intron/exon pattern, and an equal number of motifs, while Group III contains diverse structures and contain 15 motifs. By analyzing the cis-elements in the promoters, we identified five hormone-correlated responsive elements and four stress-related responsive elements. Further, six putative bna-miRNAs were also identified, targeting three genes (BnCAT4, BnCAT6, and BnCAT8). Gene ontology (GO) enrichment analysis showed that the BnCAT genes were largely related to cellular organelles, ROS response, stimulus response, stress response, and antioxidant enzymes. Almost 10 BnCAT genes showed higher expression levels in different tissues, i.e., root, leaf, stem, and silique. The expression analysis showed that BnCAT1–BnCAT3 and BnCAT11–BnCAT13 were significantly upregulated by cold, salinity, abscisic acid (ABA), and gibberellic acid (GA) treatment, but not by drought and methyl jasmonate (MeJA). Notably, most of the genes were upregulated by waterlogging stress, except BnCAT6, BnCAT9, and BnCAT10. Our results opened new windows for future investigations and provided insights into the CAT family genes in rapeseed.     

Performance assessment of UVAPS: Influence of fungal spore age and air exposure

This work focused on two main outcomes. The first was the assessment of the response of the Ultraviolet Aerodynamic Particle Sizer Spectrometer (UVAPS) for two different fungal spore species. The UVAPS response was investigated as a function of fungal age and the frequency of air current that their colonies exposure to. This outcome was achieved through the measurement of fungal spore fluorescent percentage and fluorescent intensity throughout a period of culturing time (three weeks), and the study of their fluorescent percentage as a function of exposure to air currents. The second objective was to investigate the change of fungal spore size during this period, which may be of use as a co-factor in this differentiation. Fungal spores were released by blowing the surface of the culture colonies with continuous filtered flow air. The UVAPS was used to detect and measure auto-fluorescing biomolecules such as riboflavin and nicotinamide adenine dinucleotide phosphate (NAD(P)H) present in the released fungal spores.The study demonstrated an increase in aerodynamic diameter for fungal spores under investigation (Aspergillus niger and Penicillium species) over a period of time. The fluorescent percentage of spores was found to decrease for both fungal genera as they aged. It was also found that the fluorescent percentage for tested fungi decreased with frequency of air exposure. The results showed that, while the UVAPS could discriminate between Aspergillus and Penicillium species under well-controlled laboratory conditions, it is unlikely to be able to do so in the field.     

The Distribution of Several Genomic Virulence Determinants Does Not Corroborate the Established Serotyping Classification of Bacillus thuringiensis

Bacillus thuringiensis, commonly referred to as Bt, is an object of the lasting interest of microbiologists due to its highly effective insecticidal properties, which make Bt a prominent source of biologicals. To categorize the exuberance of Bt strains discovered, serotyping assays are utilized in which flagellin serves as a primary seroreactive molecule. Despite its convenience, this approach is not indicative of Bt strains’ phenotypes, neither it reflects actual phylogenetic relationships within the species. In this respect, comparative genomic and proteomic techniques appear more informative, but their use in Bt strain classification remains limited. In the present work, we used a bottom-up proteomic approach based on fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) coupled with liquid chromatography/tandem mass spectrometry(LC-MS/MS) protein identification to assess which stage of Bt culture, vegetative or spore, would be more informative for strain characterization. To this end, the proteomic differences for the israelensis-attributed strains were assessed to compare sporulating cultures of the virulent derivative to the avirulent one as well as to the vegetative stage virulent bacteria. Using the same approach, virulent spores of the israelensis strain were also compared to the spores of strains belonging to two other major Bt serovars, namely darmstadiensis and thuringiensis. The identified proteins were analyzed regarding the presence of the respective genes in the 104 Bt genome assemblies available at open access with serovar attributions specified. Of 21 proteins identified, 15 were found to be encoded in all the present assemblies at 67% identity threshold, including several virulence factors. Notable, individual phylogenies of these core genes conferred neither the serotyping nor the flagellin-based phylogeny but corroborated the reconstruction based on phylogenomics approaches in terms of tree topology similarity. In its turn, the distribution of accessory protein genes was not confined to the existing serovars. The obtained results indicate that neither gene presence nor the core gene sequence may serve as distinctive bases for the serovar attribution, undermining the notion that the serotyping system reflects strains’ phenotypic or genetic similarity. We also provide a set of loci, which fit in with the phylogenomics data plausibly and thus may serve for draft phylogeny estimation of the novel strains.     

Association Between Increased DNA Mutational Frequency and Thermal Inactivation of Aerosolized Bacillus Spores Exposed to Dry Heat

Inactivation of viable bioaerosol particles, especially stress-resistant microorganisms, has important implications for biodefense and air quality control. It has earlier been shown that the loss of viability of bacterial endospores due to exposure to dry heat is associated with mutational damage. Previous studies, however, used non-aerosolized spores, long exposure times, and moderately elevated temperatures. This study was designed to investigate the mechanism of inactivation of aerosolized Bacillus endospores exposed to high temperatures for sub-second time periods. Bioaerosol was tested in a continuous air flow chamber under two flow rates, 18 L/min and 36 L/min. The chamber had a cylindrical electric heating element installed along its axis. The estimated characteristic exposure temperature (T_exposure ) ranged from 164°C to 277°C (with an uncertainty of 21–26°C). To quantify mutational frequency, spores were cultivated after dry heat exposure on tryptic-soy agar and on antibiotic nalidixic acid media. Increases in the exposure temperature caused viability loss and increase in mutational frequency of the spore DNA. Significant association was found between the inactivation factor and the mutational frequency ratio (heat exposed versus non-exposed) with R² of 0.985 for both flow rates combined. The results suggest that mutational damage is involved in the causal chain of events leading to inactivation of aerosolized endospores exposed to heat for sub-second time periods.     

In vitro fracture resistance of endodontically treated premolar teeth restored with a direct layered fiber-reinforced composite post and core

Methods for restoring endodontically treated teeth fall short of restoring the physiologic mechanical properties. Fracture of endodontically treated teeth is a common type of clinical failure. New treatment modalities or perfected versions of existing treatment concepts need to be tested to find a biomimetic solution. A novel method of restoring endodontically treated teeth is presented and compared in vitro with currently accepted restorative methods. Seventy-two extracted and endodontically treated maxillary premolar teeth were divided into six groups (n = 12) depending on restorative technique (Groups 1–6). Group 1: fiber-reinforced composite post (FRC), Group 2: direct layered short FRC post and core, Group 3: short fiber-reinforced obliquely layered composite restoration, Group 4: microhybrid composite restoration, Group 5: fiber-reinforced box, Group 6: control. Specimens were submitted to static fracture resistance test. Fracture thresholds and fracture patterns were evaluated. Group 6 exhibited the highest fracture resistance. Group 2 yielded the highest fracture resistance among the restored groups. The fracture resistance of Group 2 did not differ significantly from Group 6. Groups 1, 3, 4, 5 proved to be significantly different from the control group. There was no statistically significant difference among restored groups. Fracture patterns of tested groups were dominantly non repairable opposed to control groups mostly repairable fractures. Application of direct-layered short FRC post and core in endodontically treated premolars performed statistically similarly in the studied conditions as natural teeth. Therefore, it seems a promising alternative to current endo-restorative solutions. However, further testing is required.     

Thermal inactivation of airborne viable Bacillus subtilis spores by short-term exposure in axially heated air flow

In this investigation, an experimental facility was developed for quantifying the inactivation of viable bioaerosol particles in a controlled axially heated air flow. The tests were conducted with Bacillus subtilis var. niger endospores. The thermal inactivation of aerosolized spores was measured based on the loss of their culturability that resulted from a short-term exposure to air temperatures ranging from ～150 to >1000 °C. The cross-sectional and longitudinal temperature profiles in the test chamber were determined for different heating and flow conditions. The characteristic exposure temperature (T_e) was defined using a conservative approach to assessing the spore inactivation. Experimentally determined inactivation factors (IF) were corrected to account for the temperature profiles in the axially heated air flow. The reported IF-values serve as the lower approximation of the actual inactivation. Two data sets obtained at different flow rates, Q=18 and 36 L min^?1, represent different exposure conditions. In both cases, the thermal exposure of aerosolized spores produced no effect or only a moderate inactivation when the T_e remained below ～200 °C for 18 L min^?1 and ～250^oC for 36 L min^?1. The IF-values increased exponentially by about four orders of magnitude as the temperature rose by 150 °C. Depending on the flow rate, IF exceeded ～10⁴ at T_e>320 °C (Q=18 L min^?1) or >360 °C (Q=36 L min^?1). At T_e≈375–400 °C, the spore inactivation obtained at both flow rates reached the limit of quantification established in this study protocol, which translates to approximately 99.999% viability loss. The findings were attributed primarily to the heat-induced damage of DNA and denaturation of essential proteins. Up to a certain level of the thermal exposure, these damages are repairable; however, the self-repair capability diminishes as the heat rises and then the damage becomes totally irreversible. The data generated in this study provide an important reference point for thermal inactivation of stress-resistant spores in various biodefense/counterterrorism and air quality control applications.