Agilent 1200 series system

Following exposure to acetate, LX2 cells were removed the supernatant and washed three times with ice-cold PBS, and then added 3 mL of ice-cold aqueous acetonitrile (50%, v/v) (VWR) to break cells to extracte intracellular nucleotides. The resulting extract was maintained on ice for 10 min, followed by centrifugation at 14,000 × g for 1 min at 0°C. The extract of cell contents was collected and dried using a refrigerated SpeedVac (Savant). The dried extract was resuspended in 240 μL of deionized water and filtered using a 0.22-μm syringe filter unit for high-performance liquid chromatography (HPLC) analysis (30 (link)). The chromatographic separation and analysis were performed on an Agilent system (1,200 series), equipped with a diode-array detector and a C18 reverse-phase column (Kromasil, 5 μm, 100 Å; 4.6 × 150 mm) at a flow rate of 1 mL/min and a linear gradient of acetonitrile (0–7%) in 10 mM triethylammonium acetate buffer (Glen Research) over 20 min. AMP and ATP were identified based on their retention times.

HPLC–UV system: Agilent Series 1200 system (Agilent Technologies, Palo Alto, CA, USA), equipped with a quaternary pump, an automatic injection system, a diode array detector (DAD), and Agilent ChemStation software for data analysis. Ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC–HRMS) system: Accela (Thermo Scientific, Hemel Hempstead, UK) equipped with a quaternary pump, a thermostatic autosampler, a DAD, and coupled to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, Hemel Hempstead, UK) with an ESI source and Xcalibur Qual Browser software for HRMS data handling. Spectrophotometer: Double beam Perkin Elmer UV/Vis/NIR Lambda 19 (Waltham, MA, USA) with QS quartz glass high performance cuvettes (10 mm optical path) from Hellma Analytics (Jena, Germany). UAE system: Ultrasonic bath (Branson 5510, Danbury CT, USA), with a frequency of 42 kHz and power of 135 W. MAE system: Milestone Microwave Labstation (Ethos E, Milestone, Shelton, CT, USA). PLE system: Accelerated solvent extractor Dionex ASE 350 (Dionex Corp., Sunnyvale, CA, USA) equipped with 5 mL stainless steel extraction cells. Other: Labofuge 400 centrifuge (Heraeus, Hanau, Germany), Vibra mix R agitator (OVAN, Badalona, Spain).

To quantify the production of astaxanthin, zeaxanthin and canthaxanthin, samples comprising 1 mL of culture broth were centrifuged at 16,200 × g for 3 min, and the cell pellet was washed with sterile water. Subsequently, 750 μL of extraction solution (acetonitrile/methanol/dichloromethane, 21:21:8, v/v/v) was added to the pellet and ultrasonicated in an ice bath for 30 min, after which the resulting lysate was centrifuged at 16,200 × g for 3 min. The resulting supernatant was collected and another 750μL of extraction solution was added to repeat the extraction [27 (link)]. The supernatants of both extraction steps were combined, centrifuged at 16,200 × g for 3 min, and filtered through a 0.22 μm organic nylon filter before analysis by high-performance liquid chromatography (HPLC) using an Agilent Series 1200 system with a variable wavelength detector set at 476 nm and a Symmetry C18 column (250 mm × 4.6 mm, 5 μm, Waters, Ireland). The carotenoid detection method was the same as reported before [11 (link), 28 (link)]. The results represent the means of three independent experiments. The reference standards of the indicated compounds were purchased from Sigma (Sigma-Aldrich, USA). Dry cell weight (DCW) was calculated according to the empirical formula: 1 OD6₀₀ = 0.323 g DCW/L.

Corrinoid extractions were performed as previously described [16 (link)]. For corrinoids extracted from 1 L cultures of C. sporogenes, C. scindens, and T. primitia, high-performance liquid chromatography (HPLC) analysis was performed with an Agilent Series 1200 system (Agilent Technologies, Santa Clara, CA) equipped with a diode array detector with detection wavelengths set at 362 and 525 nm. Samples were injected onto an Agilent Eclipse XDB C18 column (5 µm, 4.6 × 150 mm) at 35 °C, with 0.5 mL/min flow rate. Compounds in the samples were separated using acidified water and methanol (0.1% formic acid) with a linear gradient of 18 to 30% acidified methanol over 20 min.
For all other bacteria excluding B. hydrogenotrophica, extracted corrinoids were analyzed as above, except with a 1.5 mL/min flow rate and a 40 °C column. Corrinoids were eluted with the following method: 2% acidified (0.1% formic acid) methanol for 2 min, 2 to 10% acidified methanol in 0.1 min, and 10 to 40% acidified methanol over 9 min.
For B. hydrogenotrophica, corrinoids were analyzed as above with the following changes. Samples were injected onto an Agilent Zorbax SB-Aq column (5 µm, 4.6 × 150 mm) with 1 mL/min flow rate at 30 °C. The samples were separated with a gradient of 25 to 34% acidified (0.1% formic acid) methanol over 11 min, followed by 34 to 50% over 2 min, and 50 to 75% over 9 min.

Liquid chromatography was performed on an Agilent Series 1200 system (Agilent Technologies, Santa Clara, CA, USA) equipped with a degasser, binary pump, autosampler and thermostatted column compartment. Chromatographic separation was performed on an Agilent Extend C18 column (2.1 mm × 100 mm, 1.8 μm) at 30 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B). A gradient elution of 40–50% B at 0–10 min, 50–80% B at 10–15 min, 80–95% B at 15–20 min, and 95–95% B at 20–25 min was used for the diterpenoid and triterpenoid analysis. An alternative gradient elution of 40–50% B at 0–10 min, 50–68% B at 10–13 min, and 95–95% B at 13–20 min was used for the sesquiterpene alkaloid analysis. The flow rate was set at 0.3 mL/min. The injection volume was 2 μL.
All MS experiments were conducted on a 6410B triple quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an ESI source. The analytes were determined in positive ionization mode, with the SIM method used for diterpenoids and triterpenoids and the MRM method for sesquiterpene alkaloids. Data acquisition was performed using a Mass Hunter Workstation. Capillary voltage was set to 4000 V. Desolvation gas (nitrogen) was delivered at 540 L/h and 350 °C. Nebulizer pressure was set to 0.2 MPa.