Surface sediment microbial communities remain viable, culturable, and metabolically active during sequential heating.

Marine sediments harbour a vast and diverse microbial biomass, yet it remains unclear how temperate surface sedimentary microbial communities transition to deep hot subsurface conditions. To simulate the effects of increasing temperature stress associated with deep burial, an estuarine surface sediment was sequentially heated from 15 °C to 90 °C over 434 days. Total cell counts increased during the first 210 days (42 °C), while FISH-detectable cells remained relatively constant for the first 56 days (15 °C). Overall culturability increased by one order of magnitude for heterotrophs and sulfate reducers, and by three orders of magnitude for methanogens. Subsequent heating from 42 °C to 90 °C resulted in a progressive decline in cell numbers, viability and culturability; however, culturable cells were detected throughout the experiment, including at the highest temperature (90 °C). Surprisingly, cells sampled at 90 °C were culturable across all incubation temperatures (15-90 °C) suggesting different members of the community were capable to grow across this large temperature range. Radiolabelled substrates were rapidly metabolised at 90 °C (≥1 day). Microbial community analysis demonstrated that with increasing temperature, members of the bacterial class Clostridia, mainly the Caldicoprobacteraceae (22.1%) and Peptococcaceae (10.5%) dominated the community. This study demonstrates that a phylogenetically diverse microbial community, originally adapted to temperate near-surface physicochemical conditions, can undergo functional and compositional restructuring and that certain members can become metabolically active under deep, thermally elevated sedimentary environments. This transition is likely mediated by the activation and selective enrichment of a cryptic thermophilic 'seed bank' present within the community and therefore may represent a mechanism of how deep sediments are inoculated with microbes and how the deep hot biosphere is sustained.