Stabilwax column

The total fatty acid content was measured according to a modified version of the method described by Kang and Wang (2005). To 30 mg of freeze dried cells in a capped test tube with screw cap (VWR art. no. SCERE5100160011G1 and SCERKSSR15415BY100) 1 ml BF₃/methanol reagent (Merck art. no. 8.01663.0500) and 1 ml heptane (Acros organics art. no. 120340025) was added. After overnight incubation at 70 °C 2 ml water was added and mixed. Subsequently, the tubes were centrifuged at 2000 g for 5 min, and the upper (heptane) layer was transferred to a GC vial and analysed on methylated fatty acids by using a Focus-GC (Interscience) equipped with FID. GC was equipped with a Stabilwax Column (Restek art. no. 10624) and uses hydrogen as carrier gas. The sum of the methylated fatty acids was quantified using methylheptadocanic acid as a standard.

Aldehydes and alcohols were separated and quantified by gas chromatography using Trace GC Ultra (Thermo Scientific, Braunschweig, Germany), equipped with Headspace Tri Plus autosampler, an agitator and FID. All compounds were separated via a Stabilwax column (length 30 m, 0.25 mm internal diameter, 0.25 μm film thickness; Restek, Bad Homburg, Germany), with helium (1.2 mL min⁻¹) as carrier gas. The oven temperature was programed to be held at 50°C for 2 min, raised with a ramp of 10 to 200°C min⁻¹ and held for 1 min. Injector and detector were kept at 200°C. Samples were incubated prior to injection at 40°C for 15 min. For the analysis, 700 μL of the headspace were injected (headspace syringe 100°C) in the split mode with a flow of 10 mL min⁻¹.
Pyruvate was separated and quantified via HPLC, using an Ultimate-3000 HPLC-system (Thermo Scientific, Braunschweig, Germany), equipped with an autosampler, a thermostatic column compartment, and a diode-array detector. The separation was achieved on a Metrosep Supp A16 column (25 mm, particle size 4.6 μm; Metrohm, Filderstadt, Germany) at 65°C by isocratic elution with 12 mM ammonium bicarbonate (pH 10), followed by a washing step with 30 mM sodium carbonate (pH 10.4). Mobile phase flow was adjusted to 0.2 mL min⁻¹.

The GC-MS analysis of PHBV was performed using a modified procedure based on the findings of Riis and Mai (1988 (link)). Five to ten milligrams of dried biomass was simultaneously hydrolyzed and transesterified in sealed headspace vials using 4 mL 1,2-dichloroethane, 2 mL 4:1 (vol/vol) propanol sulfuric acid mixture, and 100 μL of 20 g/L benzoic acid as internal standard. Acidic propanolysis was carried out for 4 h at 120 °C. Then, 4 mL water was added, mixed, and waited until phase reseparation. One millilitre of the lower phase was transferred for GC analysis. Separation took place on a Stabilwax column (Restek, Bad Homburg, Germany) using Helium as carrier at a flow rate of 1.44 mL/min, and a gradient of 120 °C (3 min), 140 °C at 3 °C/min, 230 °C at 50 °C/min, and 240 °C at 10 °C/min. A PHBV standard curve (12% 3HV, Sigma-Aldrich/Merck, Darmstadt, Germany) was used for quantification and qualification of the propionyl-3HB and propionyl-3HV.

Volatile compounds were analyzed using an Agilent 7890B gas chromatograph (GC) connected to a 5977A mass detector (Agilent Technologies, Santa Clara, CA, USA). A Stabilwax column (30 m length × 0.25 mm internal diameter × 0.25 μm film thickness, Restek Corporation, Bellefonte, PA, USA) was used. The oven temperature was initiated at 40 °C (5 min), increased to 220 °C at a rate of 4 °C/min and then held at 220 °C (10 min). Helium was used as carrier gas at a constant flow rate of 0.8 mL/min. The mass spectrum was obtained in EI (electron ionization) mode at 70 eV, mass scan rate of 4.5 scans/sec, and mass scan range of 35–350 m/z. In addition, the analysis of some volatile compounds, which could be derived from phenylalanine, were conducted using selective ion monitoring (SIM) mode. The list of volatile compounds measured and the SIM qualifying ions are presented in Table 4.

A sample volume of 2 μl of the hexane extract was injected in splitless mode into a Thermo Scientific ISQ single quadrupole GC/MS (Waltham, MA) with Xcalibur software (ThermoElectron Corp.) for separation and analysis of the deposited hydrocarbons. The GC/MS was equipped with a Stabilwax column, 30 m × 0.32 mm with 1.0 µm film thickness (Restek Corp., Bellefonte, PA). The injection port was set at 230°C. The oven temperature was set to 60°C, raised to 180°C at 6°C min⁻¹, held at 180°C for 20 min, and then raised to 220°C at 6°C min⁻¹ where it was maintained for an additional 20 min. Helium carrier gas constantly flowed at 2.5 mL min⁻¹. The mass spectrometer was operated at an ionizing energy of 70 eV with a 2 scan/s rate over a scan range of m/z 40–400 and an ion source temperature of 200°C. Identification of structures/compounds was performed using the National Institute of Standards and Technology library, as well as comparisons with known literature compounds and commercially available standards. Relative retention times were obtained by comparison of sample hydrocarbons to authentic standards. All standards were purchased from Sigma or Cayman Chemical Company at the highest available purity.