Estimating the Abundance of Endospores of Sulfate-Reducing Bacteria in Environmental Samples by Inducing Germination and Exponential Growth

Abstract

It is a challenge to quantitatively distinguish active from dormant microbial populations in environmental samples. Here we present an approach for estimating the abundance of dormant sulfate-reducing bacteria (SRB), present as viable endospores in environmental samples. This is achieved by inducing endospores to germinate and grow exponentially. We demonstrate this approach for thermophilic SRB in temperate sediment from Aarhus Bay, Denmark. The approach is based on measuring bulk sulfate reduction rates (SRRs) in pasteurized sediment and calculating associated cell-specific SRRs based on average values for SRB growth yield and cell size known from exponentially growing pure cultures. The method presented is a faster bioassay than most probable number enumerations and has the potential to distinguish between slow-and fast-growing SRB populations in the same sample. This bioassay is attractive given the challenges posed by endospores with respect to cell permeabilization and lysis, which are prerequisite in quantitative microscopy-and nucleic acid extraction-based strategies. These molecular approaches additionally rely on designing target-appropriate oligonucleotide probes, whereas the method presented here considers the trait of interest more broadly. This strategy thus presents a useful complement to other methods in ecological investigations of microbial biogeography and for specific industrial applications such as reservoir souring and corrosion risk assessments in the oil and gas sector.

Identity, Abundance, and Reactivation Kinetics of Thermophilic Fermentative Endospores in Cold Marine Sediment and Seawater

“…In conclusion the time course incubation experiment shows that our incubation procedure was highly efficient in reactivating dormant TFEs from surface layers of cold marine sediments. Our data suggest that TFEs are highly abundant, as our estimates of 6 × 10 6 TFEs mL -1 sediment make TFEs 100-fold more abundant than endospores of thermophilic sulfate reducing bacteria ( de Rezende et al, 2013 , 2016 ). This estimate of TFE abundance in the surface sediment is higher than the estimate obtained by MPN of 7 × 10 3 to 2 × 10 5 TFEs mL -1 ( Figure 2 ).…”

“…Cultivation allows unambiguous discrimination of endospores from vegetative cells and thermophilic endospores from psychro- and mesophilic endospores. Furthermore, it circumvents the need for endospore permeabilization and lysis, which challenge the use of molecular approaches for studies of endospore populations in environmental samples ( de Rezende et al, 2016 ). Our approach, however, depends on the germination and growth of the endospores, which may lead to a minimum-estimate of endospore abundance.…”

“…So far, investigations of the abundance of thermophilic anaerobic endospores in cold marine sediments and estimates of their rates of supply to the seafloor only considered sulfate-reducing members of the endospore community. The abundance of thermophilic sulfate-reducing endospores has been determined in samples from surface sediments from Aarhus Bay and from Svalbard by either most probable number (MPN) enumeration ( de Rezende et al, 2013 ) or by estimations based on measuring bulk rates of sulfate reduction in incubation experiments and relating these to cell-specific sulfate reduction rates ( Hubert et al, 2009 ; de Rezende et al, 2016 ). The endospore abundances ranged from ∼10 4 to 10 5 endospores cm -3 with corresponding rates of supply of ∼10 7 –10 8 endospores m -2 year -1 ( Hubert et al, 2009 ; de Rezende et al, 2013 , 2016 ).…”

“…The abundance of thermophilic sulfate-reducing endospores has been determined in samples from surface sediments from Aarhus Bay and from Svalbard by either most probable number (MPN) enumeration ( de Rezende et al, 2013 ) or by estimations based on measuring bulk rates of sulfate reduction in incubation experiments and relating these to cell-specific sulfate reduction rates ( Hubert et al, 2009 ; de Rezende et al, 2016 ). The endospore abundances ranged from ∼10 4 to 10 5 endospores cm -3 with corresponding rates of supply of ∼10 7 –10 8 endospores m -2 year -1 ( Hubert et al, 2009 ; de Rezende et al, 2013 , 2016 ). The thermophilic sulfate-reducing endospores are affiliated with the genus Desulfotomaculum of the Firmicutes family Peptococcaceae ( Isaksen et al, 1994 ; Hubert et al, 2009 , 2010 ; de Rezende et al, 2013 ; Müller et al, 2014 ; O’Sullivan et al, 2015 ).…”

Historical Factors Associated With Past Environments Influence the Biogeography of Thermophilic Endospores in Arctic Marine Sediments

“…(Tardy-Jacquenod et al, 1998; Vandieken et al, 2006; de Rezende et al, 2013). The enrichment is also necessary in order to effectively target only those cells which are present as endospores because it (a) kills vegetative cells, allowing endospores to be distinguished (Müller et al, 2014) and (b) induces germination of endospores, thus circumventing the problem of endospores being missed in direct sequencing libraries due to their resistance to commonly used DNA extraction methods (Wunderlin et al, 2013; de Rezende et al, 2017). The incubations do not necessarily elicit germination and growth of all thermophilic endospore taxa present in the samples, but are effective at enriching communities that are comparable across samples by employing equivalent incubation conditions and substrate amendments (Müller et al, 2014).…”