D 3 hydroxybutyric acid assay kit

To check enantiopurity of 3-HB, a d-3-hydroxybutyric acid assay kit (Megazyme, Ireland), which is specific for (R)-3-HB, was used. Samples were prepared as recommended by the manufacturer and the results were compared with GC/MS measurements. Each sample was analyzed 5 times and the results meaned to give the final values.

Plasma 3-hydroxybutyric acid (3-HB) concentration was determined using D-3-hydroxybutyric acid assay kit (Megazyme, Ireland) following manufacturer's instructions for use in a microplate. Briefly, 10 μl plasma samples were mixed with 200 μl distilled water, 50 μl buffer mix containing sodium azide, 20 μl NAD⁺ plus iodonitrotetrazolium chloride, and 2 μl diaphorase suspension, mixed, and absorbances read at 492 nm after 3 min (A1 read). Distilled water (210 μl), and 10 μl standard solution (0.06 mg/ml; provided by manufacturer) were used as blank control and internal standard concentration, respectively. Next, 2 μl 3-Hydroxybutyrate dehydrogenase suspension (3-HBDH) was added, mixed, and incubated at 25°C for ~6 min, and absorbances read at 492 nm at the end of the reaction (A2 read). D-3-hygroxybutyric acid concentration was calculated by:
The 96 well plate was read twice with a Synergy HT multimode microplate reader (BioTek, Winooski, VT). Average concentration of individual samples was used with SE.

For quantitative PHB analysis, aliquots of AT and CS cells were collected at the beginning of the main culture and on day 8 of MC1000 cultivation grown under 20 μmol photons m⁻² s⁻¹ with supplemented 15 mM acetate. The PHB quantitation was carried out for four biological replicates for each strain, using a commercial D-3-Hydroxybutyric Acid Assay Kit (Megazyme) according to manufacturer’s protocol. The samples were first adjusted to correspond to OD₇₅₀ = 0.1 in 50 mL total volume. After centrifugation, the pellets were suspended to 400 µL of 0.5 M NaOH and incubated for 1 h at 85 °C in shaking. The samples were then cooled down and neutralized with 100 µl of 1 M HCl, and the generated D-3-hydrozybutyric acid (corresponding to the amount of PHB in the cells) was analysed colourimetrically from the supernatant by recording the absorbance at 492 nm with a PlateReader (Tecan infinite 200 PRO).