Two common quantification methods for subseafloor microorganisms are catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and quantitative PCR (qPCR). Using these methods, we quantified Bacteria and Archaea in Baltic Sea basin sediments (IODP Exp. 347) down to 90 mbsf, testing the following hypotheses in an inter-laboratory comparison: 1) proteinase K permeabilization of Archaeal cell walls increases CARD-FISH accuracy, and 2) qPCR varies by more than an order of magnitude between laboratories using similar protocols. CARD-FISH counts did not differ between permeabilization treatments, demonstrating that proteinase K did not increase accuracy of CARD-FISH counts. However, 91% of these counts were below the quantification limit of 1.3 × 107 cells cm−3. For qPCR, data varied between laboratories, but were largely within the same order of magnitude if the same primers were used, with 88% of samples being above the quantification limit. Copy number values were elevated by preparing a sediment slurry before DNA extraction: 3.88 ×106 to 2.34 ×109 16S rRNA gene copies cm−3 vs. 1.39 × 107 to 1.87 × 109 total cells cm−3. By qPCR, Bacteria were more abundant than Archaea, although they usually were within the same order of magnitude. Overall, qPCR is more sensitive than CARD-FISH, but both require optimization to consistently achieve both precision and accuracy.
Deep subsurface microbiology is a rising field in geomicrobiology, environmental microbiology and microbial ecology that focuses on the molecular detection and quantification, cultivation, biogeographic examination, and distribution of bacteria, archaea, and eukarya that permeate the subsurface biosphere. The deep biosphere includes a variety of subsurface habitats, such as terrestrial deep aquifer systems or mines, deeply buried hydrocarbon reservoirs, marine sediments and the basaltic ocean crust. The deep subsurface biosphere abounds with uncultured, only recently discovered and—at best—incompletely understood microbial populations. So far, microbial cells and DNA remain detectable at sediment depths of more than 1 km and life appears limited mostly by heat in the deep subsurface. Severe energy limitation, either as electron acceptor or donor shortage, and scarcity of microbially degradable organic carbon sources are among the evolutionary pressures that may shape the genomic and physiological repertoire of the deep subsurface biosphere. Its biogeochemical importance in long-term carbon sequestration, subsurface elemental cycling and crustal aging, is a major focus of current research at the interface of microbiology, geochemistry, and biosphere/geosphere evolution. The papers of this Frontiers e-volume bear evidence of the rapid advances in deep subsurface microbiology.