Roa organic acid h 8

The concentration of glycerol in the samples was analyzed with Agilent 1100 HPLC system (Santa Clara, CA, USA) with RI-detector system (G1362A RI Detector). Separation was carried out using LC column (Rezex TM ROA-organic acid H+ (8 %), 300 × 7.8 mm, with pre-column ROA-organic acid H+ (8 %) (50 × 7.8 mm), both Phenomenex, Germany) and a mobile phase consisting of 5 mM H₂SO₄ at a flow rate of 0.50 mL/min at 50 °C. The injection volume was set to 5 µL.

To verify the specific amount of PHB produced by genetically modified E. coli cells during each test, every time a new culture was set up a sample corresponding to 5 mL of final culture broth was kept and stored at -20 °C for successive analysis of the cell dry weight and of the correspondent PHB amount. Cell dry weight was obtained gravimetrically. The sample collected was centrifuged at 18,816×g for 5 min, washed two times and dried at 80 °C for 12 h in an oven. Once estimated the correspondent biomass concentration (g L⁻¹), 5 mg of biomass were hydrolysed by 1 mL of 96% H₂SO₄ under stirring of 800 rpm, into an agitated oil bath at 90 °C for one hour. At the end of the reaction, a dilution of 1:1000 was done, and the samples were analysed by HPLC equipped with a column ROA-organic acid H+ (8%) (Phenomenex) and a photodiode array detectors (PDA) under an isocratic flux of 0.7 mL min⁻¹ of 5 mM of H₂SO₄ and a temperature of 50 °C. The PHB-related peak was revealed at 210 nm with an elution time of 23.09 min, which corresponds to the crotonic acid (the monomer coming from the PHB hydrolysation) used as a reference standard [54 (link)]. The PHB yield was estimated on the base of Eq. 1: $$\text{PHB extraction yield}\left(\%\right)=\frac{\text{purified PHB}\left(\text{g}\right)}{\text{PHB present in the treated biomass}\left(\text{g}\right)}.$$

Extracellular concentrations of metabolites were determined on a Dionex Ultimate 3,000 HPLC system (Thermo Scientific, France) equipped with a RI detector (RID-10A, Shimadzu, Japan) and UV/Vis detector (SPD-20A, Shimadzu). The sample injection volume was 20 μL. Glucose and (D/L)-DHB were measured with the RI detector by using a cation-exchange column (Rezex RoA-organic acid H⁺ 8%) preceded by a SecurityGuard guard cartridge (Phenomenex, USA). The separation was performed at 80 °C with 0.5 mM H₂SO₄ at 0.5 mL min⁻¹ as mobile phase. PDO concentrations (from RI detector) were measured using an Aminex HPX-87H column protected by a Micro-Guard Cation H⁺ pre-column (BioRad, USA). The separation was performed at 35 °C with 1.25 mM H₂SO₄ at 0.5 mL min⁻¹. Amounts of D-DHB were estimated on the UV detector (at 254 nm) by using a Chirex 3126 column (Phenomenex, USA) with an aqueous mobile phase containing 2 mM CuSO₄ and 15% methanol. Flow rate was 1 mL/min, and the column was held at 22 °C. Concentrations of (L)-DHB were estimated by subtracting estimated amounts of the (D)-form to total (D/L)-DHB. All samples were centrifuged (2 min at 13,000 rpm) and syringe-filtered (0.2 µm), and the resulting supernatant stored at −20 °C before analysis. A standard calibration curve was obtained by injecting standards.