Succinic acid

All Reagents and standard compounds were of analytical or high performance liquid chromatography (HPLC) grade (≥99%). Acetic acid, acetaldehyde, ethanol, formic acid, d-glucose, mannitol and methanol were obtained from Merck, Darmstadt, Germany; HMF was provided by Acros, Geel, Belgium; succinic acid was sourced from VWR, Darmstadt, Germany; isobutanol, isopentanol and 2-phenylethanol were acquired from Alfa Aesar (ThermoFisher, Karlsruhe, Germany); citric acid, ethyl acetate, ethyl lactate, fructose, 1-propanol, sodium benzoate, sodium propionate, sucrose, and 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (“TSP”, 98 atom% D) and the reagents for the buffer potassium dihydrogen phosphate and potassium hydroxide and sodium azide were bought from Sigma-Aldrich, Steinheim, Germany; deuterium oxide (D₂O, 99.9 atom% D) came from Deutero, Kastellaun, Germany. All aqueous solutions were made using demineralised water.

Distilled sterile water was used for water-soluble molecules, including glutathione (Sigma-Aldrich), galactose (Sigma-Aldrich), and succinic acid (VWR Chemicals). galactose, L-aspartic acid, and acylases I were dissolved in 0.9 M NaCl, 2 M NaOH, and 100 mM potassium phosphate buffer, pH 7, respectively, according to the manufacturer's recommendations (Sigma-Aldrich). Concentrated solutions of the compounds were prepared and applied to ASM cultures to obtain a final concentration of 20 mM of aspartic acid, succinic acid, and galactose, 10 mM of glutathione, and 5 and 15 mg/L of acylases I as described in literature (Xu et al., 2003 (link); Sauer et al., 2004 (link); Klare et al., 2016 (link); Bahamondez-Canas and Smyth, 2018 (link)). All stock solutions were freshly prepared before their application on ASM. Three different concentrations of ciprofloxacin, two subinhibitory concentrations and one inhibitory concentration according to the MIC values obtained for the P. aeruginosa strains, were combined with the compounds.