Labile Carbon Triggers Microbial Priming of Deep Peat Carbon Breakdown Unveiled by Coupled DNA SIP-Metabolomics : NMR raw data

METHODS:

180 µL of each DOM sample (two depths, 3 treatments (control, labeled glucose, unlabeled glucose,) and 6 times points (7, 14, 28, 42, 56, and 70 days); n=36) were combined with 2,2-dimethyl-2-silapentane- 5-sulfonate-d6 (DSS-d6) in D2O (20 µL, 5 mM) and thoroughly mixed prior to transfer to 3mm NMR tubes.  NMR spectra were acquired on a Varian 600 MHz VNMRS spectrometer equipped with a 5-mm triple-resonance (HCN) cold probe at a regulated temperature of 298K. The 90° 1H pulse was calibrated prior to the measurement of each sample. The one-dimensional (1D) 1H spectra were acquired using a nuclear Overhauser effect spectroscopy (NOESY) pulse sequence with a spectral width of 12 ppm and 512 transients. The NOESY mixing time was 100ms, and the acquisition time was 4s, followed by a relaxation delay of 1.5s during which pre-saturation of the water signal was applied. Time-domain free induction decays (57,472 total points) were zero filled to 131,072 total points prior to Fourier transform. Chemical shifts were referenced to the 1H methyl signal in DSS-d6 at 0 ppm. The 1D 1H spectra were manually processed, assigned metabolite identification, and quantified using Chenomx NMR Suite 8.3. Metabolite identification was based on matching the chemical shift, J-coupling, and the intensity of experimental signals to compound signals in the Chenomx and custom in-house databases. Quantification was based on fitted metabolite signals relative to the internal standard (DSS-d6). Signal-to-noise ratios (S/N) were measured using MestReNova 14 with the limit of quantification equal to an S/N of 10 and the limit of detection equal to an S/N of 3. 13C labeling was assessed by 13C satellite analysis from the 1D spectra described above or from a 1D-(13C-edited) HSQC experiment. In several cases further corroboration of metabolite identity was made using standard 2-D experiments such as 1H / 13C - heteronuclear correlation (HSQC) experiments or 2-D 1H/ 1H Total Correlation spectroscopy (TOCSY).

FUNDING:

This research was supported by U.S. Department of Energy Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0023297.