Pyruvic acid

All chemicals used in experiments were at analytical grade and all solutions were prepared using de-ionized water. Microcrystalline cellulose (MCC) was purchased from Sigma Aldrich. The standards of reagents used in HPLC analysis are fructose (≥99%), levulinic acid (98%), lactic acid (98%), glycerolaldehyde (99%), glycolaldehyde (99%), 5-HMF (99%), glycolic acid (99%), and pyruvic acid (98%) and they were purchased from Merck. Sulfuric acid (96–98%) was also obtained from Merck. Phosphoric acid (85–90%) were purchased from Fluka. Biomass feedstock, hazelnut shell, was supplied from Ordu, Turkey. Hazelnut shells were dried in an oven at 60°C and then, they ground into small pieces (~1 mm) using a laboratory type grinder.

The culture medium consisted of StemPro‐34 SFM with Stem Pro supplement (Kanatsu‐Shinohara, Ogonuki, et al., 2003; Wu et al., 2015; Zhang et al., 2014; Gibco, #10639–011), 25 μg/mL insulin (Sigma, #I1882), 100 μg/mL transferrin (Sigma, #T1428), 60 μM putrescine (Sigma, #P5780), 30 nM sodium selenite (Sigma, #S5261), 6 mg/mL D‐(+)‐glucose (Sigma, #G7021), 30 μg/mL pyruvic acid (Sigma, #P4562), 1 μL/mL DL‐lactic acid (Sigma, #L7900), 5 mg/mL bovine serum albumin (Calbiochem, #126609), 2 mM L‐glutamine (Millipore, #TMS‐002‐C), 100x β‐mercaptoethanol (Specialty Media, #ES‐007‐E), minimal essential medium vitamin solution (Gibco, #11120–052), 100x nonessential amino acid solution (Gibco, #11140–035), 1% penicillin/streptomycin (Specialty Media, #TMS‐AB2‐C), 0.1 mM ascorbic acid (Sigma, #A4034), 10 μg/mL d‐biotin (Sigma, #B4639), 20 ng/mL recombinant human epidermal growth factor (Gibco, #PMG8041), 10 ng/mL recombinant human basic fibroblast growth factor (PeproTech, #AF‐100‐18B‐250), 10 ng/mL recombinant human GDNF (PeproTech, #450–10‐250), 1 IU/mL Leukemia Inhibitory Factor (Millipore, #ESG1107), and 1% fetal bovine serum (HyClone, #SH30071.03). Cells were maintained at 37°C with 5% CO₂.

Mitochondrial and glycolytic stress test analysis in fibroblasts were performed on an XF24 Seahorse bioanalyser as previously described (Allen et al., 2014 (link), 2015 (link), 2019a (link); Raman et al., 2015 (link)). Specifically for metabolic substitution assays, fibroblast media was supplemented with 0.3 mM glutamine and 5 mM glucose or either 5 mM pyruvic acid, butyric acid, alpha ketoglutaric acid, succinamic acid or adenosine (Sigma) for 40 h prior to metabolic flux analysis. Flux analysis was performed using XF basal media (Agilent) supplemented with the combination of metabolic substrates listed above. Metabolic flux analysis under physiological and stress conditions were assessed following sequential addition of the mitochondrial inhibitors oligomycin, FCCP and antimycin/rotenone (Sigma) as previously described (Allen et al., 2014 (link), 2015 (link)). Flux data were normalized to CyQUANT fluorescence in each well as a proxy for cell number following the manufacturer's instructions as previously described (Allen et al., 2019a (link)).

Bacteria cells were prepared as described above and resuspended in LB media, adjusted OD₆₀₀ to 0.5. Colistin (2 mg/L) or/and a metabolite (1.2, 2.5, 5, and 10 mM) were added and incubated at 37°C for 6 h or 12 h. After incubation, CFU were determined by the 10-fold serial broth microdilution method. The metabolites, including pyruvic acid, citrate, α-ketoglutaric acid, cis-aconitic acid, and (s)-malate and agmatine sulfate, were all purchased from Sigma-Aldrich and dissolved in water.