title: Interlaboratory quantification of Bacteria and Archaea in deeply buried sediments of the Baltic Sea (IODP Expedition 347).
abstract: 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 interlaboratory 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 × 106-2.34 × 109 16S rRNA gene copies cm-3 vs. 1.39 × 107-1.87 × 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.
authors.affiliation: Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: Joy
authors.initials: J
authors.lastname: Buongiorno
authors.affiliation: Geomicrobiology, Federal Institute for Geosciences and Natural Resources (BGR), Hannover 30655, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Turner
authors.affiliation: Cardiff School of Biosciences, Sir Martin Evans Building, Cardiff University, Cardiff CF10 3AX, UK.
authors.forename: Gordon
authors.initials: G
authors.lastname: Webster
authors.affiliation: Cardiff School of Biosciences, Sir Martin Evans Building, Cardiff University, Cardiff CF10 3AX, UK.
authors.forename: Masanori
authors.initials: M
authors.lastname: Asai
authors.affiliation: Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: Alexander K
authors.initials: AK
authors.lastname: Shumaker
authors.affiliation: Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: Taylor
authors.initials: T
authors.lastname: Roy
authors.affiliation: Cardiff School of Biosciences, Sir Martin Evans Building, Cardiff University, Cardiff CF10 3AX, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Weightman
authors.affiliation: Geomicrobiology, Federal Institute for Geosciences and Natural Resources (BGR), Hannover 30655, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Schippers
authors.affiliation: Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: Karen G
authors.initials: KG
authors.lastname: Lloyd
mesh_terms: D001105:Archaea / Q000235:genetics* / Q000254:growth & development; D001419:Bacteria / Q000235:genetics* / Q000254:growth & development; D015169:Colony Count, Microbial; D017931:DNA Primers; D005101:Expeditions; D019015:Geologic Sediments / Q000382:microbiology*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012623:Seawater / Q000382:microbiology*; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S
keywords: Archaea; Bacteria; CARD-FISH; qPCR; quantification; subsurface