Freezing Tolerance of Thermophilic Bacterial Endospores in Marine Sediments

Abstract

Dormant endospores of anaerobic, thermophilic bacteria found in cold marine sediments offer a useful model for studying microbial biogeography, dispersal, and survival. The dormant endospore phenotype confers resistance to unfavorable environmental conditions, allowing dispersal to be isolated and studied independently of other factors such as environmental selection. To study the resilience of thermospores to conditions relevant for survival in extreme cold conditions, their viability following different freezing treatments was tested. Marine sediment was frozen at either −80°C or −20°C for 10 days prior to pasteurization and incubation at +50°C for 21 days to assess thermospore viability. Sulfate reduction commenced at +50°C following both freezing pretreatments indicating persistence of thermophilic endospores of sulfate-reducing bacteria. The onset of sulfate reduction at +50°C was delayed in −80°C pretreated microcosms, which exhibited more variability between triplicates, compared to −20°C pretreated microcosms and parallel controls that were not frozen in advance. Microbial communities were evaluated by 16S rRNA gene amplicon sequencing, revealing an increase in the relative sequence abundance of thermophilic endospore-forming Firmicutes in all microcosms. Different freezing pretreatments (−80°C and −20°C) did not appreciably influence the shift in overall bacterial community composition that occurred during the +50°C incubations. Communities that had been frozen prior to +50°C incubation showed an increase in the relative sequence abundance of operational taxonomic units (OTUs) affiliated with the class Bacilli , relative to unfrozen controls. These results show that freezing impacts but does not obliterate thermospore populations and their ability to germinate and grow under appropriate conditions. Indeed the majority of the thermospore OTUs detected in this study (21 of 22) could be observed following one or both freezing treatments. These results are important for assessing thermospore viability in frozen samples and following cold exposure such as the very low temperatures that would be encountered during panspermia.

Metabolic responses of thermophilic endospores to sudden heat-induced perturbation in marine sediment samples

“…To investigate microbial community response in laboratory incubations, triplicate anoxic slurries consisting of cold Arctic marine sediment supplemented with and without six volatile fatty acids (VFAs) were incubated for 216 h in the dark at 50 • C. VFA amendment provides electron donors for sporeforming SRB and has been used similarly in previous studies (Hubert et al, 2010;de Rezende et al, 2013;Müller et al, 2014;Bell et al, 2018;Chakraborty et al, 2018;Cramm et al, 2019;Hanson et al, 2019;Gittins et al, In Press). Changes in VFA and sulfate concentrations were observed in both VFA-amended and unamended sediment slurries (Figure 1).…”

“…DNA-based biodiversity surveys generally do not include the portion of the microbiome that exists as endospores, most likely due to endospore resistance to physical or chemical lysis steps employed during community DNA extraction from environmental samples (Bueche et al, 2013;Wunderlin et al, 2014). Laboratory incubations have been successfully implemented for investigating the physiology, diversity, abundance, and distribution of endospore-forming bacteria in seawater and marine sediments (Hubert et al, 2010;de Rezende et al, 2013de Rezende et al, , 2017Nielsen et al, 2017;Volpi et al, 2017;Bell et al, 2018;Chakraborty et al, 2018;Cramm et al, 2019). Some of these studies have showcased the metabolic versatility displayed by these thermophilic populations, including organic matter mineralization by obligately fermentative bacteria (Hubert et al, 2010;Volpi et al, 2017) and sulfate reduction by members of the Desulfotomaculia (Hubert et al, 2010;Cramm et al, 2019;Bell et al, 2020).…”

“…Incubation-based spore investigations depend on a multitude of factors such as the temperature, pressure and nutrients, spore abundance in the sample inoculum, sample storage conditions, and sediment heterogeneities (Hubert et al, 2010;Cramm et al, 2019;Bell et al, 2020). Furthermore, sporulation and germination are complex stepwise processes that can be temporally variable between diverse spore-forming taxa (Setlow, 2003;Paredes-Sabja et al, 2011;Tocheva et al, 2016).…”

Cultivation With Powdered Meteorite (NWA 1172) as the Substrate Enhances Low-Temperature Preservation of the Extreme Thermoacidophile Metallosphaera sedula

“…How these thermophilic species, abundantly represented in permanently cold habitats, can tolerate low temperatures significantly below their minimum requirement for growth is an intriguing subject of current investigations. Thermophilic spore-forming bacteria in the marine sediments of Svalbard have been constantly reported (Vandieken et al, 2006;Hubert et al, 2009Hubert et al, , 2010Cramm et al, 2019). Their endospores with specialized cellular features that protect cells from extreme harsh environmental factors contribute to the survival of these thermophilic bacteria in Arctic sediments.…”

Antifreeze Water-Rich Dormant Cysts of the Terrestrial Ciliate Colpoda cucullus Nag-1 at −65 ℃: Possible Involvement of Ultra-Antifreeze Polysaccharides

“…In dormant forms (resting cysts of protists and endospores of bacteria) and vegetative forms of microorganisms, tolerance to harsh environments including desiccation (Corliss and Esser 1974), UV rays (Nicholson and Galeano 2003, Lonnen et al 2014, Matsuoka et al 2017, Yamane et al 2020) and freezing (Müller et al 2010, Anderson 2016, Cramm et al 2019) is an adaptive strategy for survival in terrestrial environments. To survive on the soil surface where puddles appear temporarily and dry out rapidly, soil unicellular eukaryotes such as the ciliate Colpoda promptly transform into resting cysts when they detect approaching desiccation.…”