Agilent 1100 apparatus

The biomass production of JW15 was determined by measuring the dry cell weights [20 (link)]. An aliquot (10 ml) of the culture broth was transferred to pre-weighed plastic conical tubes and centrifuged (5,000 ×g, 10 min, 4°C). The cell-free supernatant was separated to determine the concentration of organic acids. Pellets were washed thrice with ice-cold distilled water, dried at 80°C and left in a vacuum oven to obtain a constant weight.
The lactic and acetic acid production of JW15 was assessed using HPLC analysis, as described previously [16 (link)]. The obtained supernatant was passed through a 0.22-μm membrane filter and used for the HPLC analysis. Analysis was conducted using an Agilent 1100 apparatus (Agilent Technologies, USA), which comprises a column oven, auto-sampler, UV detector, and Aminex HPX-87H column (300 mm × 7.8 mm, 5 μm) (Bio-Rad, USA). The injection volume was 20 μl. Isocratic elution was performed using a 5 mM H₂SO₄ aqueous solution at 50°C. The flow rate was 0.6 ml/min. The chromatogram were detected at 210 nm using a UV detector. The concentration of each organic acid was evaluated from the external regression curve, which was constructed using standard mixtures at five concentrations.

The supernatants of FCW, NPCW, and SC7-PCW were filtered and derivatized using an Agilent automatic on-line derivatization method for the analysis of amino acids according to the previous study [19 (link),20 (link)]. The primary and secondary amino acids were reacted with O-phthalaldehyde (OPA) and fluorene methoxycarbonyl chloride (FMOC), respectively.
High-performance liquid chromatography (HPLC) was used to determine the amino acids with an Agilent 1100 apparatus (Agilent, Santa Clara, CA, USA) and a ZORBAX Eclipse AAA column (4.6 mm × 150 mm, 3.5 μm, Agilent, Santa Clara, CA, USA). 40 mM sodium dihydrogen phosphate (pH7.8) was used as the mobile phase A. The mobile phase B contained acetonitrile, methanol, and water (45:45:10, v/v/v). The gradient was 0% B (0 min), 0% B (1 min), 57% B (23 min), 100% B (27 min), 100% B (34 min), 0% B (40 min), and 0% B (41 min). The mixed standard of 17 kinds of amino acids (Sigma, Saint Louis, MO, USA), including aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline, and the tryptophan standard (Sigma, Saint Louis, MO, USA) were used in the identification and quantification. Experiments were conducted in triplicate.

We used for specific rotation a Perkin-Elmer 343 Polarimeter (Perkin-Elmer, Waltham, MA, USA); for NMR, a Bruker Avance III 700 Bruker FT-NMR (Bruker BioSpin GmbH, Rheinstetten, Germany) (700.00/176.03 MHz) (¹H/¹³C) spectrometer; for ESI MS (positive and negative ion modes), an Agilent 6510 Q-TOF apparatus (Agilent Technology, Santa Clara, CA, USA), sample concentration of 0.01 mg/mL; and for HPLC, an Agilent 1100 apparatus with a differential refractometer (Agilent Technology, Santa Clara, CA, USA). The column was a Supelco Discovery HS F5-5 (10 × 250 mm, 5 μm) (Supelco, inc., Bellefonte, PA, USA).