208 Chapter 7 sulfate reduction inhibitor to incubations supplemented with MnO2 (10 mM) and sulfate (Figure 1B). Finally, we purged the mixed slurries with He before injecting 20 mL 13C-CH4 (99.8 atom %, Campro Scientific). All incubation bottles were kept in the dark in an anoxic chamber at 25 °C for 36 months. Chemical characterization of slurry samples At the end of the incubation, we collected the liquid phase of the slurries and filtered it through 0.45-µm filters for nutrient analysis. Sulfate was quantified by ion-chromatography (940 Professional IC Vario, Metrohm, Switzerland), while Mn2+, Fe2+, and H 2S were determined by ICP and photometry, as described previously (Cojean et al., 2020). DIC concentrations were quantified on a TOC analyzer (Shimadzu, Corp., Kyoto, Japan). The carbon isotopic composition of DIC was determined by acidifying 1 mL filtered supernatant with 200 µL 85% H3PO4 in 12 mL He-flushed exetainers. The δ13C of the released CO 2 was determined in the headspace after overnight equilibration via a gas-bench coupled to a Delta V Plus IRMS. The solid phase of incubation slurries was centrifuged and lyophilized prior to elemental analysis. Total carbon (TC) and total organic carbon (TOC, after acidification of the samples) as well as their δ13C values were determined by EA-IRMS using a Delta V Plus IRMS and a ConFlow IV interface (Thermo Fisher Scientific, Bremen Germany). Stable carbon isotope values are reported in the conventional δ-notation (in ‰) relative to the Vienna Pee Dee Belemnite standard (V-PDB). DNA extraction, amplicon sequencing and metagenomics We extracted DNA from the inocula sediments and the incubation slurries using the FastDNATM Spin Kit for Soil (MP Biomedicals, city, country) and performed amplicon sequencing of 16S rRNA genes following the two-step PCR approach (https://support. illumina.com/documents/documentation/chemistry_documentation/16s/16smetagenomic-library-prep-guide-15044223-b.pdf) with primers 515F-Y (5′-GTGYCAGCMGCCGCGGTAA) and 926R (5′-CCGYCAATTYMTTTRAGTTT-3”) (Parada et al., 2016), targeting the V4 and V5 regions of the 16S rRNA gene. Amplicons were sequenced at the Genomics Facility Basel (University of Basel/ETHZ). Details of library preparation, sequencing, quality control, and bioinformatical processing are described in (Su et al., 2023). We used SINTAX (v11.0.667_i86linux64, Edgar,
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