Vitamin standards

Methanol, formic acid, water, acetonitrile (all HPLC-grade purity), sodium dihydrogen phosphate, disodium hydrogen phosphate, trans-caftaric acid, caffeic acid, syringic acid, quercetin hydrate, quercetin-3-glucoside trans-piceid, and vitamin standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid, protocatechuic acid, p-coumaric acid, ferulic acid, and taxifolin were purchased from Fluka (Buchs, Switzerland). Quercetin-3-glucuronide, procyanidins, (+)-catechin, (−)-epicatechin, piceatannol, and resveratrol were purchased from Extrasyn-these (Genay, France); p-hydroxybenzoic acid and myricetin were purchased from Acros Organics (Geel, Belgium). The cis-isomer of caftaric acid was obtained by UV illumination of a Methanol solution containing the trans-isomer for four hours [30 (link)]. Anthocyanins (monoglucoside chlorides) were purchased from Biosynth Carbosynth (Bratislava, Slovakia). Ultrapure water and all the reagents (60% HNO₃) and standards for analysis using inductively coupled plasma optical emission spectroscopy (ICP-OES) were obtained from Merck (Darmstadt, Germany).

To measure water-soluble vitamins, an Agilent 1200 Infinity HPLC system coupled to a diode array detector was used with an Agilent Poroshell 120 EC-C18 column at 4.6 ×100 mm with particle size of 2.7 µm. The HPLC method was developed by the UC Davis Department of Viticulture and Enology analytical laboratory based on manufacturer suggestions. Briefly, mobile phases consisted of 25 mM HK₂PO₄ (pH 7.0) (A) and acetonitrile (B). The 20.1 min run time was set up with a constant flow rate of 0.5 mL/min. The flow consisted of 1% B at 0 to 5 mins, a gradient increase to 30% B from 5 to 15 mins, 30% B from 15 to 20 mins, a gradient decrease to 1% B from 20 to 20.1 mins, followed by 5 mins post run time at 1% B. Data were acquired at 205, 214, 232, 266, and 280 nm. The column was kept at 35 °C, and injection volume was 20 µL. Vitamin standards were obtained from Sigma and were prepared fresh and shielded from light prior to analysis.

The quantity of thiamine produced by Variovorax was quantified during the growth in 75% fresh minimal media and 25% filter-sterilized spent medium from the final passage of the co-culture experiment as well as during the growth of Variovorax in M9 minimal media with varying concentrations of pantothenate. Thiamine was quantified on an Agilent 1200 liquid chromatograph equipped with a Agilent ZORBAX Eclipse Plus C-18 column with a 5 um particle size, maintained at 35 °C. Using a two-phase mobile phase of (A) 25 mM NaH₂PO₄ (pH = 2.5), and (B) pure methanol. The flow rate was 1.0 mL/min. The mixture of these mobile phases were as follows: 0% mobile phase B from time of injection, 50% mobile phase B from 1.0 min to 75% mobile phase B from 10 to 25 min. We identified vitamins using a G1362A Refractive Index Detector at 220 nm. Vitamin standards were obtained from Sigma-Aldrich®.