NMR spectra from photogranule metabolome

This repository contains 532 NMR spectra and associated metadata used to study the metabolome of oxygenic photogranules (OPGs). Photogranules are phototrophic microbial aggregates typically used for wastewater treatment (Milferstedt et al., 2017). Photogranules were grown in a sequencing batch photobioreactor fed with synthetic wastewater, as described in Galea-Outón et al., 2024. RMN spectra come from round-shape photogranules of 0.6-1 mm in diameter. Photogranules were exposed to light in absence or in presence of available carbon (acetate). RMN spectra were obtained at different time-points.

Three carbon conditions were tested: “early carbon series”, “late carbon series”, “no carbon series”. At t0, the early carbon series received 500 µL of an aqueous solution containing 400 mg COD·L-1 (acetate) and 21 mg NH4+·L-1. For the late carbon series, this solution was added after 90 minutes, while for the no carbon series, OPGs received no solution. OPGs were exposed to 70 µmol·m-2·s-1 of photosynthetically active radiation between 0 and 180 minutes, then the light was switched off between 180 and 240 minutes. A sacrificial approach was applied and 8 replicates were used per condition and time point. To stop all metabolic activity, tubes after sampling were immediately immersed in liquid nitrogen and stored at -70°C until metabolites were extracted.

For OPGs metabolome extraction, a citrate buffer was added to frozen samples that were then grinded, centrifuged and filtered. After the addition of methanol to the eluate, the aqueous phase was extracted with dichloromethane. The remaining aqueous phase and combined organic layers were evaporated separately to give crude solids. Before NMR analysis, solids were re-suspended in a deuterated buffer phosphate solution (for the aqueous phase) and deuterated chloroform/methanol (2/1, V/V) solution (for the lipid phase), both containing 0.03% of tetramethylsilane (TMS).

1H-NMR spectra were obtained from a 300 K Bruker Advance III HD 600 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany), operating at 600.13 MHz for 1H resonance frequency, equipped with a 5 mm 1H-13C-15N-31P cryoprobe attached to a Cryoplatform (the preamplifier unit). Probe tuning and matching, locking, shims tuning, pulse (90Å) and gain computation were automatically performed for each sample. For aqueous samples, 1H-NMR spectra were acquired using the noesy-presat 1H experiment (noesypr1d: sequence used suppression of the water signal) whereas for organic phase, 1H-NMR spectra were acquired using the zg 1H experiment. A total of 256 transients were collected into 64k data points using a spectral width of 20 ppm, a relaxation delay of 5 s and an acquisition time of 2.72 s. Free induction decay (FID) data were obtained from the measurements processed using Topspin software. Prior to Fourier transform, an exponential line broadening function of 0.3 Hz was applied to the FID. Then, all NMR spectra were phase- and baseline-corrected and referenced to the chemical shift of TMS (0 ppm).

NMR spectra were imported into the software environment R, version 4.4.0 (R Core Team, 2022), using ASICS package (version 2.21.2, Lefort et al., 2019). An automatic process was performed for baseline correction, normalisation and alignment. Then two tables were generated for the lipid phase (“NMR_spectra_photogranules_lipid_phase”) and the aqueous phase (“NMR_spectra_photogranules_lipid_phase”), containing chemical shifts in ppm (first column) associated with each spectrum, for each sample.

Metadata describing the samples are available in tables “metadata_photogranules_lipid_phase” and “metadata_photogranules_aqueous_phase”. For each sample is indicated the sample name, the sample type (OPG/quality control or blank), the experimental time (in minutes), light condition (on or off), if acetate was supplied, the metabolome extraction date and metabolome analysis date.