Refractomax 520

Substrate and metabolite concentrations were measured by HPLC with an Ultimate 3000 system (Thermo Scientific, Waltham/MA, USA) using an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules/CA, USA). The column was operated with 4 mM H₂SO₄ as mobile phase at 60 °C and a flow of 0.6 ml/min for 30 min. A refractive index detector (Refractomax 520, Thermo Scientific, Waltham/MA, USA) and an UV detector (Ultimate 3000, Thermo Scientific, Waltham/MA, USA) were used for peak detection. Controlling, monitoring and quantification of the HPLC run was performed with Chromeleon 7.2.6 Chromatography Data System (Thermo Scientific, Waltham/MA, USA). Samples and calibration standards were prepared by mixing 450 µl cell-free supernatant with 50 µl 40 mM H₂SO₄. 10 µl sample was injected for analysis and 5-point calibration curves were used for quantification.

Organic acids, alcohols and amino acids were determined using an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules/CA, USA) in an Ultimate 3000 system (Thermo Scientific, Waltham/MA, USA). The mobile phase was 4 mM H₂SO₄ and the column was operated at 60 °C and a flow of 0.6 ml min⁻¹ for 30 min. The injection volume was 10 µl. Detection was performed using a refractive index (Refractomax 520, Thermo Scientific, Waltham/MA, USA) and a DAD detector (Ultimate 3000, Thermo Scientific, Waltham/MA, USA). Chromeleon 7.2.6 Chromatography Data System (Thermo Scientific, Waltham/MA, USA) was used for control, monitoring and evaluation of the analysis.
For the measurement of organic acids and alcohols, 450 µl of culture supernatant were mixed with 50 µl of 40 mM H₂SO₄ and centrifuged for 5 min at 14,000 rpm (21,913g) at 4 °C. The remaining supernatant was used for further analysis.
For the measurement of amino acids and particularly aspartic acid, 250 µl of culture supernatant was mixed with 50 µl of 1 M sodium nitrite and 10 µl of 12 M HCl. The solution was heated to 45 °C for 90 min and the reaction was stopped by adding 50 µl 2 M NaOH [52 (link)]. The derivatized amino acid solution was directly transferred to an HPLC tube and measured at the conditions described above.
Standards were treated like samples and a 5-point calibration was used for quantification.

Sugars, organic acids, and alcohols were determined using an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules/CA, USA) in an Ultimate 3000 system (Thermo Scientific, Waltham/MA, USA). 4 mM H₂SO₄ was used as a mobile phase at 60 °C and a flow of 0.6 ml min⁻¹ for 40 min and the injection volume was 10 µl. Metabolites were detected using a refractive index (Refractomax 520, Thermo Scientific, Waltham/MA, USA) and a DAD detector (Ultimate 3000, Thermo Scientific, Waltham/MA, USA). Chromeleon 7.2.6 Chromatography Data System (Thermo Scientific, Waltham/MA, USA) was used for control, monitoring and evaluation of the analysis.
For sample preparation, 450 µl of cell-free culture supernatant was mixed with 50 µl of 40 mM H₂SO₄ and centrifuged for 5 min at 14 000 rpm at 4 °C. The supernatant was used for analysis and standards were treated the same way. A 5-point calibration was used for substrate and metabolite concentrations in the samples.

D-glucose and trehalose concentration in the clarified cell broth and in the feed was determined with an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules, CA, United States) using a Vanquish Core HPLC system (Thermo Fisher Scientific, Waltham, MA, United States) and a refractive index detector (RefractoMax 520, Thermo Fisher Scientific, Waltham, MA, United States). The mobile phase consisted of 4 mM H₂SO₄ with a constant flow rate of 0.6 mL/min. The system was run isocratically at 60 °C. Chromatograms were analyzed using Chromeleon 7.2.6 Chromatography Data System (Thermo Fisher Scientific, Waltham, MA, United States). The concentration of glutamic acid was assessed photometrically with a Cedex Bio HT Analyzer (Roche, Basel, Switzerland). The glutamic acid concentration was then multiplied by 1.15 to obtain the MSG content in the supernatant.

Lactate was quantified in the supernatant of the cell suspension. Samples of cell suspension of 0.5 to 1.0 mL were centrifuged for 5 min at 17,000 × g and 4°C (Heraeus Fresco 17; Thermo Scientific, Waltham, MA, USA). The supernatant was stored at −20°C until further analysis. The lactate concentration was determined either by high-performance liquid chromatography (HPLC) (Ultimate 3000 series; Dionex, Thermo Scientific) equipped with a refractive index (RI) detector (Refractomax 520; Thermo Scientific) or an assay kit according to the supplier’s instructions (MAK065; Sigma-Aldrich). For quantification via HPLC, a HyperREZ XP carbohydrate H⁺ column was used with 5% sulfuric acid as mobile phase at 40°C and a flow rate of 1.2 mL min⁻¹. The specific lactate production rate (mmol g_CDW⁻¹ day⁻¹) was calculated based on the lactate concentration in the supernatant (c_Lac) (mmol L⁻¹), the biomass concentration (CDW in g L⁻¹), and the dilution rate (D) (day⁻¹) with the equation
$$r_{\text{Lac}}=\frac{c_{\text{Lac}}\times D}{\text{CDW}}$$
For the calculation of carbon partitioning into lactate, r_Lac was converted into a carbon-based production rate (mmol C g_CDW⁻¹ day⁻¹).

Optical density of broth was measured daily at 660 nm (OD₆₆₀). When constant, the biomass concentration was measured (at least in triplicate) by determination of the volatile suspended solids (VSS) concentration in the broth (Clesceri et al. 1999 ), from 150 mL broth samples collected continuously and anaerobically from the effluent of the bioreactor.
Acetate, ethanol, 2,3-butanediol, and formate concentrations in filtered broth samples (0.22-µm pore size, Millipore, Millex-GV, MA, USA) were determined using ultra high-performance liquid chromatography (UPLC) with an Aminex HPX-87 H column (BioRad, CA, USA) and 1.5 mmol L⁻¹ phosphoric acid as eluent at 50 °C with RI detection (RefractoMax 520, Thermo Fisher Scientific, MA, USA).
The bioreactor exhaust gas was continuously diluted 1:10 (v/v) with pure nitrogen gas to obtain the minimum flow required for gas analysis (Rosemount™ X-STREAM XEGP, Emerson, MO, USA). This custom-built analyser was equipped with a nondispersive infrared (NDIR) sensors for CO and CO₂ measurement and a thermal conductivity detector (TCD) for H₂ measurement.

At each sampling point, 1 mL of culture broth was centrifuged at 10,000 g for 10 min at 4 °C. The obtained supernatant was analyzed for its substrate and metabolite composition. D-glucose and trehalose concentrations in the supernatant were determined with an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) employing an Ultimate 3000 high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific, Waltham, MA, USA). 10 µL sample were analyzed at a flow rate of 0.6 mL/min and a column temperature of 60 °C. 4 mM H₂SO₄ served as mobile phase. For quantitative determination a RI detector (RefractoMax 520, Thermo Fisher Scientific, Waltham, MA, USA) and a UV detector (VH-D10-A, Thermo Fisher Scientific, Waltham, MA, USA) at 210 nm were used. Chromeleon 7.2.6 Chromatography Data System (Thermo Fisher Scientific, Waltham, MA, USA) was used for control and data analysis. Glutamic acid was determined via a photometric assay using a Cedex Bio HT Analyzer (Roche, Basel, Switzerland). The obtained concentration of glutamic acid was converted to the used substrate MSG by multiplication with the factor 1.15.