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

Abstract

Cold marine sediments harbor endospores of fermentative and sulfate-reducing, thermophilic bacteria. These dormant populations of endospores are believed to accumulate in the seabed via passive dispersal by ocean currents followed by sedimentation from the water column. However, the magnitude of this process is poorly understood because the endospores present in seawater were so far not identified, and only the abundance of thermophilic sulfate-reducing endospores in the seabed has been quantified. We investigated the distribution of thermophilic fermentative endospores (TFEs) in water column and sediment of Aarhus Bay, Denmark, to test the role of suspended dispersal and determine the rate of endospore deposition and the endospore abundance in the sediment. We furthermore aimed to determine the time course of reactivation of the germinating TFEs. TFEs were induced to germinate and grow by incubating pasteurized sediment and water samples anaerobically at 50°C. We observed a sudden release of the endospore component dipicolinic acid immediately upon incubation suggesting fast endospore reactivation in response to heating. Volatile fatty acids (VFAs) and H2 began to accumulate exponentially after 3.5 h of incubation showing that reactivation was followed by a short phase of outgrowth before germinated cells began to divide. Thermophilic fermenters were mainly present in the sediment as endospores because the rate of VFA accumulation was identical in pasteurized and non-pasteurized samples. Germinating TFEs were identified taxonomically by reverse transcription, PCR amplification and sequencing of 16S rRNA. The water column and sediment shared the same phylotypes, thereby confirming the potential for seawater dispersal. The abundance of TFEs was estimated by most probable number enumeration, rates of VFA production, and released amounts of dipicolinic acid during germination. The surface sediment contained ∼105–106 inducible TFEs cm-3. TFEs thus outnumber thermophilic sulfate-reducing endospores by an order of magnitude. The abundance of cultivable TFEs decreased exponentially with sediment depth with a half-life of 350 years. We estimate that 6 × 109 anaerobic thermophilic endospores are deposited on the seafloor per m2 per year in Aarhus Bay, and that these thermophiles represent >10% of the total endospore community in the surface sediment.

Contrasting community composition of endospores and vegetative Firmicutes in a marine sediment suggests both endogenous and exogenous sources of endospore accumulation

“…S5). Among the 100 most abundant endospore OTUs, six were identical (>97% sequence identity) to sequences recently reported to represent endospores of thermophilic fermentative bacteria present in the Aarhus Bay sediment (Volpi et al ., ; Fig. ).…”

“…*: OTU present in sequence libraries of endospore‐enriched DNA extracts of biomass collected from the water column of Aarhus Bay, Site M5. #: OTU represents a thermophilic endospore phylotype identified by Volpi and colleagues (), see Supporting Information text for details. The thermophilic endospore OTUs collectively constitute 27% of the reads in the endospore enriched sequence libraries from 1 to 85 cmbsf.…”

“…Based on measurements of the endospore‐specific compound dipicolinic acid (DPA) endospores are estimated to be as abundant as vegetative cells in subsurface sediments (Langerhuus et al ., ; Lomstein et al ., ; Braun et al . ; Fichtel et al ., ) and their presence is also documented by cultivation dependent enumeration (Volpi et al ., ; de Rezende et al ., ; Hubert et al ., ; Köpke et al ., ).…”

Distribution of thermophilic endospores in a temperate estuary indicate that dispersal history structures sediment microbial communities

“…The presence of anaerobic thermophilic bacteria in the cold seabed was first reported for sediments from Aarhus Bay, Denmark, where despite in situ temperatures of 0–15°C, a thermophilic strain of Desulfotomaculum was found to grow by reducing sulfate when samples were heated to 60°C (Isaksen et al ., ). Since this discovery, several studies have shown that thermophilic bacteria constitute an exogenous, low abundance, dormant component of microbial communities in cold marine sediments throughout the world (Hubert et al ., ; de Rezende et al ., ; Müller et al ., , Volpi et al ., ). These misplaced thermophiles belong to the phylum Firmicutes , which encompasses all known endospore‐forming bacteria.…”

“…Their physiology with respect to temperature (growth above 40°C; Hubert et al ., ) indicates that they must be being delivered to cold sediments from an external source or sources, while spore‐formation confers a survival strategy that enables persistence at temperatures much below their growth range. Large numbers of endospores of anaerobic thermophilic bacteria are delivered to cold marine sediments each year, with 6 × 10 9 spores m −2 y −1 reported in Aarhus Bay, representing > 10% of the total endospore population (Volpi et al ., ). The detection of the same 16S rRNA gene phylotypes in both the water column and the underlying seabed in Aarhus Bay supports previous assertions that seawater acts as a vector for the dispersal of thermophilic endospores in the marine environment (de Rezende et al ., ; Volpi et al ., ).…”