Ultrastructure and extreme heat resistance of spores from thermophilic Clostridium species

H H Hyun, J G Zeikus, R Longin, J Millet, A Ryter
1983 Journal of Bacteriology  
The heat resistance and ultrastructural features of spore suspensions prepared from Clostridium thermocellum LQRI, Clostridium thermosulfurogenes 4B, and Clostridium thermohydrosulfuricum 39E were compared as a function of decimal reduction time. The decimal reduction times at 121°C for strains LQRI, 4B, and 39E were 0.5, 2.5, and 11 min. The higher degree of spore heat resistance was associated with a spore architecture displaying a thicker cortex layer. Heat resistance of these spores was
more » ... hese spores was proportional to the ratio of spore cortex volume to cytoplasmic volume. These ratios for spores of strains LQRI, 4B, and 39E were 1.4, 1.6, and 6.6, respectively. The extreme heat resistance and autoclavable nature of C. thermohydrosulfuricum spores under routine sterilization procedures is suggested as a common cause of laboratory contamination with pure cultures of thermophilic, saccharide-fermenting anaerobes. The cellular basis for the extreme heat resistance of bacterial endospores remains an active research topic, principally in the genus Bacillus. Previous studies by Murrell and Warth (13) on the mechanism of spore heat resistance reported that Bacillus spores which developed in the presence of cycloserine had normal dipicolinic acid contents but lacked cortex development and were not thermally stable. Millet and Ryter (11) showed that Bacillus sporulation mutants blocked in cortex formation were not heat stable. Gould and Dring (5) proposed that the cortex of an endospore functioned as an osmoregulatory organelle and conferred heat resistance by prevention of cytoplasmic dehydration. Recently, Beaman et. al. (2) examined five types of Bacillus spores and reported that an exponential increase in the heat resistance correlated directly with the wet density and inversely with the water content and with the cytoplasm/cytoplasm plus cortex volume ratio. Less attention has been directed toward understanding the heat resistance of endospores formed by thermophilic bacteria. Previously, Warth (18) demonstrated that spores from thermophilic bacteria were more heat resistant than those from mesophilic or psychrophilic species. Problems associated with extreme heat resistance of spores from thermophilic anaerobes (e.g., Clostridium thermosaccharolyticum) have been reported in the canned food industry (22). t Present address: Department of Bacteriology, University of Wisconsin, Madison, WI 53706. Recently, Donelly and Basta (4) showed that Desulfotomaculum nigrificans, a thermophilic, sulfate-reducing bacterium, produced spores of unusually high heat resistance with a decimal reduction time (D) at 121°C of 5.6 min. This value was higher than that indicated for the aerobic sporeforming thermophile, Bacillus stearothermophilus (1, 4). During the past 8 years, our laboratory has been examining the metabolic properties of different thermophilic bacterial species that transform polysaccharides to ethanol (14, 25). In the past 4 years, our laboratory cultures have been routinely contaminated despite cautious use of rigorous asceptic cultural techniques. Unknowingly, we have sent to other investigators Clostridium thermocellum cultures that were contaminated by pentose-fermenting species. Also, upon receipt of nonsporeforming, thermophilic, ethanol-producing bacteria (3, 20, 24) from other investigators or major culture collections, we have routinely noted the presence of contaminating spores even after modification of our medium autoclaving procedures. These problems led us to examine whether spores of thermophilic anaerobes could withstand normal autoclaving procedures. In view of these facts, the present research report was directed toward comparing the heat resistance and ultrastructural features of three thermophilic ethanologenic species: C. thermocellum LQRI (9), Clostridium thermohydrosulfuricum 39E (23), and Clostridium thermosulfurogenes 4B (16a). The data show 1332 on May 8, 2020 by guest
doi:10.1128/jb.156.3.1332-1337.1983 fatcat:5sjkf6h6nfezrmusxou53wrpmm