Sample collection and quantitative stable-isotope probing (qSIP)

Homogenized carbonate surface sediment (top ~10 cm; ~4 g; 74 °C) and spring water (4 mL) collected from Gongxiaoshe Hot Spring in Tengchong County (Fig. 1A; GPS location N25.44012°; E98.44081°) during the winter dry season (Jan 5, 2016) was supplemented with sterile 95 atom% ¹³C-acetate (n = 3, 9.6 µmol C g^-1 sediment; ~3.2 mM) or 95 atom% ¹³C-aspartate (n = 4, 9.6 µmol C g^-1 sediment; ~1.6 mM) in 50 mL Falcon tubes, covered with aluminum foil, and incubated in the spring for 48 hours. Identical replicates were amended with acetate with natural abundance ¹³C (n = 4, 1.6 µmol C g^-1 sediment) or with no substrate addition (n = 1) to increase the number of ASVs with natural C isotope abundance for the AFE calculation. Following incubation, sediments were transferred aseptically to sterile polypropylene tubes, immediately frozen on dry ice, and transported to the lab. DNA was extracted using the Fast DNA Spin Kit for Soil (MP Biomedicals), separated by isopycnic ultracentrifugation on a CsTFA density gradient, fractionated, and used as templates for 16S rRNA gene PCR and Illumina tag sequencing in parallel with a sample of the homogenized, unincubated sediment (n = 4). For each sample, 13-15 fractions were collected and each was sequenced. Primers 515 F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to target the V4 region of the bacterial and archaeal 16S rRNA gene [42, 43]. Additionally, a broad-coverage quantitative PCR was used to measure the microbial 16S rRNA gene copy number in each fraction as described previously using the same primers [26].

A Location of Gongxiaoshe Hot Spring (red star). B Bar plots showing the relative abundance of microbial phyla before incubation (initial), after incubation without added substrate (control), or with ¹³C-acetate (n = 3) or ¹³C-aspartate (n = 4). C Effect of incubation on beta diversity. Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity index of ASVs followed by ANOSIM significance test. The five groups are: initial (purple), control (brown), natural abundance acetate (green), ¹³C-acetate (orange), and ¹³C-aspartate (blue). Dotted ellipses represent 95% confidence intervals. D 16S rRNA gene copies per gram (wet weight) determined by qPCR. Different letters represent significant differences as determined by Kruskal–Wallis test. (p < 0.05).