Dionex ultimate 3000

LGP32 was cultured in 3 L of Marine Broth (Difco, Le pont de Claix, France) at 25 °C under shaking (100 RPM) for 24 h (representing a late exponential phase, pH 7.5). A liquid-liquid extraction of the culture was performed with ethyl acetate (1/3 v:v) in a separatory funnel. The organic phase was evaporated to dryness and the extract was re-suspended in 1 mL of HPLC grade DMSO. The extract was fractionated using a separative HPLC system with two Varian Prep Star pumps, a manual injector, a Dionex Ultimate 3000 RS variable wavelength detector and a Dionex Ultimate 3000 fraction collector (Thermo Scientific, Courtaboeuf, France). The column was a Phenomenex Luna C18 (21.2 mm × 250 mm), with 5 μm particle size, and the flow rate was set to 20 mL·min⁻¹. The mobile phase consisted of HPLC grade H₂O and CH₃CN at different proportions starting at 70:30 for 3 min, followed by a 12 min linear gradient from 70:30 to 0:100, followed by 100% CH₃CN for 10 min. 22 fractions were collected every minute between 3 and 25 min. The solvent was removed with a HT-4X system (Genevac, Biopharma Technologies France, Lyon, France), each fraction was dissolved in 100 μL DMSO and diluted at 1/4 with LB medium (v/v) to perform the biosensor tests. Positive fractions were further analyzed by UHPLC-HRMS/MS. pH was controlled at each step of our experimental process and maintained in between 6 and 7.

We isolated chondrocytes from each cartilage sample as described^{113 (link)} and detailed in full in Supplementary Methods (see Supplementary Information). Assays and analyses were performed as described in Supplementary Methods (see Supplementary Information), and RNA sequencing was performed on the Illumina HiSeq2000 or Hiseq4000 (75 bp paired-ends), with quality control including FastQC 0.11.5 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). For raw RNA sequencing data details see Data Availability Statement. For protein extracts, we carried out digestion, 6-plex or 10-plex tandem mass tag labeling and peptide fractionation. For samples from 12 knee osteoarthritis patients, we applied a liquid chromatography mass spectrometry (LC-MS) analysis using the Dionex Ultimate 3000 ultra-high-performance liquid chromatography (UHPLC) system coupled with the high-resolution LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific GmbH, Dreieich, Germany). For all remaining samples, LC-MS analysis was performed on the Dionex Ultimate 3000 UHPLC system coupled with the Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific). For proteomics data details see Data Availability Statement.

Dried iTRAQ-labeled peptides were resuspended in buffer A [3% (v/v) acetonitrile and 0.1% (v/v) trifluroacetic acid (TFA) in HPLC water] and off line fractionated using an Hypercarb Porous Graphite column (Thermo Fisher Scientific, United Kingdom), with 3 μm particle size, 50 mm length, 2.1 mm diameter and 250 Å pore size. Peptides were reverse-phase separated using buffer A [3% (v/v) acetonitrile and 0.1% TFA in water] and buffer B [97% (v/v) acetonitrile and 0.1% TFA in water]. The Hypercarb separation was performed on a Dionex UltiMate 3000 Autosampler linked to Dionex UltiMate 3000 Flow Manager and Pump system (Thermo Scientific, United Kingdom). Gradient elution was performed at a flow rate of 30 μL min^–1 as follows: 3% B -10% B for 10 min, 10% B – 50% B for 75 min, 50% B – 90% B for 1 min, 90% B for 10 min, 3% B for 14 min. The fractions were collected every 2 min from 10 to 120 min, and dried by vacuum centrifugation (Scanvac Labogene, Denmark) ready for reverse-phase LC-MS/MS.

SEC traces were determined
after passing through a size-exclusion chromatography column (5000–5000000
g/mol) Superose 6 Increase 10/300 GL (GE Healthcare) in a mobile phase
of PBS containing 300 ppm of sodium azide at a flow rate of 0.75 mL/min
(Dionex Ultimate 3000 pump, degasser, and autosampler, Thermo Fisher
Scientific). Detection consisted of an Optilab T-rEX refractive index
detector operating at 658 nm and a diode array detector operating
at 280 nm (Dionex Ultimate 3000, Thermo Fischer Scientific). Before
injection, samples at a concentration of 1 mg/mL were filtered through
a 0.22 μm PVDF membrane.

To evaluate the concentration of short-chain VFA (scVFA) in the formation waters collected from the four reservoirs and their content after batch cultures, an HPLC methodology developed for the detection of acetic, propionic, n-butyric, iso-butyric, n-valeric, and iso-valeric acid adapted from Ricci et al. (2021) (link) was applied in the present study. Quantification of organic acids was performed by HPLC using a Thermo-Fischer Dionex Ultimate 3,000 system (Thermo-Fischer, USA) coupled with a Thermo-Fischer Dionex Ultimate 3,000 Variable wavelength detector operating at 210 nm. For organic acids, the column (Metab AAC, ISERA GmbH, Düren, Germany) was eluted isocratically with 9 mM H₂SO₄ at a flow rate of 0.6 mL min-1 and an oven temperature of 40°C. Calibration was performed by generating individual stock solutions (100 mM) of the above-mentioned scVFA species by solubilizing their relative weight/volume in MilliQ water. Stock solutions were then used to prepare calibration standards containing all the acid species for concentrations between 10 and 1 mM. Data collected were screened against the VFA levels contained in the media and in the inactivated formation waters used as blanks allowing to differentiate between the degradation/generation of VFAs naturally occurring in the culture media from that caused by microbial activity.

Optical rotations were measured on a JASCO P-2000 spectropolarimeter (Easton, MD, USA) at 20 °C in MeOH with Spectramanager software.
HPLC used configuration of analytical system by Agilent 1100 Series (Degasser G1322A, Quaternary Pump G1311A, Autosampler ALS G1313A, Column Compartment G1316, DAD G1315B, Loop 20 µL, UV spectrum 200–900 nm) with column Kinetex^® PFP 100 A, 250 mm × 4.6 mm I.D., 5 µm (Phenomenex, CA, USA), and flow rate of 1 mL/min. Semi-preparative HPLC was carried out using Dionex UltiMate 3000 system (Pump Dionex UltiMate 3000 UPLC+ Focused, Dionex UltiMate 3000 RS Variable Wavelength Detector, fraction collector Dionex UltiMate 3000 with 6 positions, LCO 101 ECOM column oven, constant temperature 40 °C, autosampler Dionex UltiMate 3000, loop 100 µL), column Ascentis^® RP-AMIDE, 250 mm × 10 mm, 5 µm (Supelco, PA, USA), and flow rate of 5 mL/min. TLC was carried out on precoated Silica gel plates (Supelco Kieselgel G, F254, 60, Merck, Darmstadt, Germany) with the solvent systems EtOAc:MeOH:H₂O (100:13.5:10, v/v/v). Spots were visualized under UV light (365 nm) after spraying with NTS/PEG reagent. Column chromatography (CC) was performed using Diaion HP-20 (Supelco, PA, USA), Ø = 80 mm, height 70 cm ~ 700 g and Silica gel (40−63 μm, Sigma-Aldrich^®, St. Louis, MO, USA) Ø = 35 mm, height 60 cm.