Valerate

The concentrations of cecal SCFAs were detected by gas chromatography. Briefly, the SCFA external standards (acetic acid, butyrate, propionic acid, isovalerate, isobutyric acid, and valerate) were obtained from Sigma-Aldrich (Shanghai, China). One gram of cecal content was mixed with 6% phosphorous acid (m/v, 1:3). After vibration and centrifugation, the supernatant was injected into the Agilent Technologies GC7890 Network System (Agilent Technologies, USA), with a column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies) and flame ionization.

Methanol (MeOH), acetonitrile and formic acid were of HPLC grade. Analytical grade of NaOH, propan-1-ol, pyridine, hexane and propylchloroformate (PCF) were purchased as well from Sigma-Aldrich (Saint Quentin Fallavier, France). Deionized water comes from a Milli-Q Elix system fitted with a LC-PaK and a MilliPak filter at 0.22 μm (Merck Millipore, Guyancourt, France). The following bile acid standards: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA), lycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), and taurolithocholic acid (TLCA) and ammonium acetate were purchased from Sigma Chemical (St. Louis, MO, USA). All tryptophan reference, isotope labeled metabolites and SCFAs (acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, acetate-D3, butyrate-13C2 and valerate-D9) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The stock solutions of bile acids, tryptophan metabolite and SCFAs were prepared separately in methanol at the concentration of 10 mmol/l.

Fifty-milliliter Methylocystis parvus OBBP cultures were grown to final optical densities (OD₆₀₀) of 0.8–1.2 then centrifuged (3000g) for 15 min. The pellets were resuspended in 30 mL of JM2 medium to create the inoculum for triplicate 160 mL serum bottle cultures. Each culture received 10 mL inoculum plus 40 mL of fresh medium (39.5 mL of medium JM2 plus 0.5 mL of 1.35 M ammonium chloride stock) and was flushed for 5 min with a CH₄/O₂ mixture (molar ratio of 1:1.5). After growth at 30 °C for 24 h, the headspace in each culture was again flushed for 5 min with the CH₄/O₂ mixture then incubated at 30 °C for a second 24 h period of balanced growth.
After 48 h, all cultures were harvested and subjected to nitrogen-limiting conditions. Triplicate samples were centrifuged (3000g) for 15 min and suspended in fresh medium without nitrogen. The headspace of each bottle was flushed with the CH₄:O₂ gas mixture at t = 0 h and t = 24 h. To assess the effects of co-substrate addition of PHA synthesis, the medium was amended with varying concentrations of 4HB, 5HV, and 6HHx monomers. Other organic acid co-substrates including 3HB, butyrate, valerate, hexanoate, and octanoate (Sigma-Aldrich, St Louis, MO, USA) were also tested for PHA copolymer synthesis. After 48 h of incubation, cells were harvested by centrifugation (3000g) and freeze-dried. Preserved samples were assayed for PHA content.