Dichloromethane

The PHA content was measured using the spectrometric method as suggested in Law and Slepecky (1960a (link)). The PHAs of the samples were extracted from the cells with dichloromethane (Carl Roth, Karlsruhe, Germany). Two milliliters of the chlorinated hydrocarbon is added to each previously lyophilized sample and the mixture was boiled in a water bath for 10 min. After cooling down to room temperature, cell pellets were centrifuged for 10 min at 3200×g and 4 °C (Allegra X-12R, Beckman Coulter, Brea, USA) and the supernatants were transferred to a 10-mL glass tube. Afterwards, the extraction step was repeated two times. The supernatants were then evaporated to obtain the extracted PHAs. After complete evaporation of the dichloromethane, samples were boiled with 2 mL of concentrated sulfuric acid (98%, Carl Roth, Kalrsruhe, Germany) in a water bath for 30 min. After cooling down to room temperature, 200 μL of the sample was transferred in a microtiter plate and measured in a spectrometer (Eon, BioTek, Winooksi, USA). Thereby, a spectrum was recorded between 200 and 800 nm using 5 nm slit. The lamp of the device provides monochromatic light in the visual and UV range. The maximum absorbance at 235 nm can be used to determine the amount of crotonic acid in the sample.

Bacteria were cultivated in 6 ml NB II overnight at 30°C and 170 rpm, and once OD₆₀₀ = 0.5–1 was reached transferred into a 100 ml modified Erlenmeyer flask of the VOC-collection system (Kai et al., 2010 (link)). Volatile compounds of the headspace of three 24 h intervals (air flow 1.1 ml/min) were trapped on the absorbent SuperQ (50 mg, brand new SuperQ to avoid cross-contamination; Alltech, IL, USA). The trapped VOCs were eluted with dichloromethane (Carl Roth, Karlsruhe, Germany). Nonyl acetate (Carl Roth, Karlsruhe, Germany) was added as internal standard (stock solution 5 ng/10 μg), peak in the chromatogram corresponds to 5 ng.

CBN (1 mg/mL in methanol, certified reference material), CBN-d₃ (100 μg/mL in methanol, certified reference material), CBD (1 mg/mL in methanol, certified reference material), and CBD-d₃ (100 μg/mL in methanol, certified reference material) were bought from Cerilliant; acetonitrile (HPLC gradient grade), methanol (HPLC gradient grade), n-hexane (HPLC grade), n-heptane (HPLC grade), and formic acid (98–100%) were obtained from VWR Chemicals; dichloromethane (HPLC grade) from Carl Roth, triethylamine from ACROS Organics, Fast Blue Salt B (FBS, dye content ~ 95%) from Sigma-Aldrich, Chromabond SiOH (1 ml/100 mg), Chromabond C₁₈ ec (1 ml/ 100 mg) as well as TLC (thin-layer chromatography) plates (silica gel 60, ALUGRAM Xtra SIL G UV₂₅₄ and octadecyl-modified silica, ALUGRAM RP-18 W/UV₂₅₄) from Macherey-Nagel, TLC plates (silica gel 60 without fluorescent indicator on aluminum sheets), and HPTLC (high-performance thin-layer chromatography) plates (silica gel 60 F₂₅₄ MS-grade for matrix-assisted laser desorption/ionization (MALDI) and silica gel 60 F₂₅₄ on glass plates) were purchased from Merck KGaA and analytical sea sand from Grüssig GmbH. Distilled diethyl ether and acetone were produced with a rotary evaporator from BÜCHI.