Study sites and sampling procedures
Samples were collected from redoxclines in the anoxic basins of the central Baltic Sea, specifically, from the Landsort Deep in 2009 and the Gotland Deep in 2005. Water was retrieved via free-flow bottles attached to a conductivity–temperature–depth (CTD) probe that also recorded turbidity. The Landsort Deep pelagic redoxcline (station 284: 57°19.2′N, 20°03.0′E) was sampled in three characteristic depths with distinct redox conditions: the suboxic zone (78 m), the oxic–anoxic interface layer (86 m), and the sulfidic zone (110 m). The sulfidic zone of the Gotland Deep redoxcline (station 271: 58°34.88′N, 18°14.11′E) was sampled at a depth of 160 m. Concentrations of oxygen, nitrate, nitrite, ammonium, and hydrogen sulfide were analyzed according to Grasshoff et al. (1983) on board the research vessel directly after sampling.
Landsort Deep redoxcline 2009: 13C-acetate incorporation and 16S rRNA SIP analyses
To identify acetate-incorporating prokaryotes present in pelagic redoxclines of the Landsort Deep in 2009, a 13C incorporation assay was carried out as described in Glaubitz et al. (2009). Water retrieved from the suboxic zone, the oxic–anoxic interface layer, and the sulfidic zone was carefully filled into 1-L glass bottles with overflow, closed gas-tight with polytetrafluoroethylene (PTFE) septum stoppers, and then supplemented with 12C- or 13C-acetate (1,2-13C2-sodium acetate, 99%, Eurisotop, France) to a final concentration of 100 μM. The bottles were incubated for 72 h in the dark at the in situ temperature. Cells were harvested via filtration onto 0.2-μm polycarbonate filters (47 mm) (GE Water & Process Technologies). The filters were shock-frozen at −196 °C and stored at −80 °C until further processing. Subsampling for flow cytometric enumeration of total prokaryotic cell numbers was conducted as described by Gasol et al. (2004) and Labrenz et al. (2007).
RNA was extracted with an acidic extraction protocol as described by Glaubitz et al. (2009). Residual DNA was removed with DNase I (Ambion) and the RNA was purified using phenol/chloroform. A maximum of 500 ng RNA was loaded in a cesium trifluoroacetate density gradient (illustra CsTFA, GE Healthcare Lifesciences) and then subjected to isopycnic centrifugation for at least 68 h under vacuum at 111 544 rcf in a Beckman Coulter Ultima L-100 Xp centrifuge with a VTi 65.2 vertical rotor. The RNA was thereby separated according to its buoyant density, in turn a function of the amount of 13C labeling. The resulting gradient was collected in 14 fractions of equal volumes, and the RNA within each fraction was purified.
Bacterial or archaeal 16S rRNA copy numbers within the gradient fractions were quantified in a one-step reverse transcriptional quantitative polymerase chain reaction (RT-qPCR) using the Access One-Step-RT-PCR kit (Promega). 13C incorporation into 16S rRNA during the incubation was assessed by comparing the buoyant densities of the 12C and 13C gradients yielding the maximum 16S rRNA copy numbers.
The PCR assay mixture contained 1 mmol MgSO4 L−1, 0.1 mmol of each dNTP L−1, 0.2 μg BSA μL−1, 0.1× SybrGreen™, 100 μmol fluorescein L−1, 0.26 μmol of each primer L−1 (Bacteria: Ba519f/Ba907r, Stubner (2002); Archaea: Ar109f/Ar912rt, Lueders & Friedrich (2003), see Supporting Information, Table S1), and 1.5 units of both AMV reverse transcriptase and Tfl DNA polymerase. Thermal cycling conditions were as follows: 30 min at 45 °C, 5 min at 95 °C, and 35 cycles of 95 °C for 30 s, 52 °C for 30 s, and 84 °C for 10 s fluorescence measurement, with a final elongation at 68 °C for 5 min. After amplification, a melting curve to exclude unspecific amplicons from the analysis was generated as follows: 1 min at 95 °C, 30 s at 50 °C, and 10 s at 50 °C with +0.5 °C intervals of increasing temperature for 84 repeats.
For single-strand conformation polymorphism (SSCP) fingerprinting analysis, RNA of the gradient fractions was reverse-transcribed and amplified using the same procedure in 50-μL volumes but with a phosphorylated reverse primer; thermal cycling conditions were 45 °C for 30 min, 95 °C for 5 min, and 35 cycles of 95 °C for 1 min, 50 °C for 1 min, and 68 °C for 1 min with a 5-min final elongation at 68 °C. SSCP electrophoresis was carried out according to Schwieger & Tebbe (1998), and the gel was silver-stained as described in Lee et al. (1996). Gels were digitalized and relative band intensities quantified based on densitometric curves using Applied Maths GelCompar v 4.5. Selected bands were excised and reamplified according to Labrenz et al. (2005). PCR products were purified using the NucleoSpin Extract II kit (Macherey-Nagel) and sequenced by Qiagen (Hilden, Germany) with forward and reverse primers. The excised and eluted bands of interest were reamplified via touch-down PCR (Don et al., 1991) using the primers com1f and com2r (Schwieger & Tebbe, 1998) and the following thermal cycling conditions were as follows: 95 °C for 3 min, 25 cycles of 94 °C for 1 min, initially at 53 °C for 1 min and 72 °C for 1:30 min but then lowering the annealing temperature by 0.1 °C with each cycle; final elongation was carried out at 72 °C for 5 min.
Alongside with the 13C incubations, the incorporation rates of 14C-labeled sodium bicarbonate or 3H-labeled acetate were determined for the same depths in separate individual vials following the methods of Jost et al. (2008) and Jost & Pollehne (1998). From the CTD, water was transferred directly into 10-mL test tubes with glass stoppers (OMNILAB). Labeled sodium bicarbonate (40–60 μL) or acetate (25 μL; final concentration 28.5 nM) was added with a gas-tight syringe (Hamilton) from an anoxic stock solution (9.25 MBq mL−1 for sodium bicarbonate, 8.51 MBq mL−1 for acetate). The samples were incubated at the in situ temperature (6–10 °C) in the dark: the bicarbonate incubations for 24 h, while the acetate incubations varied between 22 h and 29 h. Negative controls were stopped with 100 μL formaldehyde (37%) 10 min prior to substrate addition and subsequently treated as described above. The incubation period was terminated by adding 100 μL formaldehyde. To determine the total activity added, 50 μL was withdrawn prior to filtration and merged with 50 μL of ethanolamine and 5 mL of scintillation cocktail (UltimaGold XR). The remaining volume was filtered onto 0.2-μm cellulose acetate membrane filters (25 mm), which were exposed to HCl fumes for 30 min (sodium bicarbonate incubations only) and subsequently mixed with scintillation cocktail for counting in a scintillation counter (TriCarb 2560 Tr/X). For acetate, incorporation rates are expressed as percentage of the recovered radioactivity in biomass compared with the added radioactivity because the in situ concentrations of acetate were unknown. CO2 incorporation was calculated based on a total dissolved inorganic carbon concentration of 2 mM, which is the concentration typically found for Baltic Sea pelagic redoxclines (Grote et al., 2008).
Gotland Deep redoxcline 2005: growth on organic carbon and metal oxides as electron acceptors
For the sulfidic zone samples of the Gotland Deep, obtained in 2005, heterotrophic and metal-oxide-reducing bacteria were stimulated with a mixture of organic substrates (Table S2), manganese(IV) (Merck), or iron(III) (AppliChem). These were added in different combinations to the sample water to final concentrations of 4 μM (each organic substrate) and 100 μM (metal oxides). The samples and controls (no additions) were incubated at the in situ temperature in the dark for 48 and 96 h until subsampling and harvesting of the cells as described above.
Manganese concentrations were determined as described in Labrenz et al. (2005) using the formaldoxime method (Brewer & Spencer 1971). Dissolved iron(II) concentrations were determined photometrically using ferrozine according to the method described by Stookey (1970). For rinsing the filter, 10-mL sample were filtered through a 0.4-μm polycarbonate filter and the filtrate was then discarded. Afterward, 20 mL of sample water was filtered, followed by the addition of ferrozine, incubation in the dark, and the measurement of absorbance at 562 nm against a ddH2O blank. FeCl2 standards between 0 and 50 μM were prepared anoxically in preboiled ddH2O, which was purged with N2 while the solution cooled to room temperature. Particulate iron(III) concentrations were calculated by subtracting the concentrations of the filtered from the unfiltered fraction.
RNA and DNA were extracted according to Weinbauer et al. (2002), followed by the digestion of co-precipitated DNA with DNAse I (Ambion). Then, cDNA was synthesized from RNA using the iScript cDNA synthesis kit (Bio-Rad) as described in Labrenz et al. (2005) and the universal primer 1492r (Lane, 1991). The reaction started at 25 °C for 5 min, followed by reverse transcription at 42 °C for 30 min and terminal elongation at 85 °C for 5 min. Amplification of the cDNA with the primers com1f/com2rpH (Schwieger & Tebbe, 1998) and gel electrophoresis were carried out as described above.
Sequencing data were quality-revised with DNAStar SeqMan II v5.06, and forward and reverse sequences were assembled into a combined contig. Sequences were aligned using the arb 5.1 software package (Ludwig et al., 2004), and phylogenetic trees were reconstructed based on the PHYML maximum-likelihood (ML), neighbor joining (NJ), and maximum parsimony (MP) algorithms with a specific filter. 16S rRNA partial sequences of the excised fingerprinting bands were integrated into the constructed tree using the quick add marked function in arb.