Waters alliance e2695 hplc

Pretreatment and/or enzymatic hydrolysis was performed in a high throughput pretreatment and enzymatic hydrolysis (HTPH) system [19 (link)-21 (link)], using a customized 96-well plate reactor. Dry biomass weighing 4.5 mg was added to each well using an automated solid and liquid dispensing robotics platform (Core Module II, Freeslate Inc., Sunnyvale, CA, United States) followed by 445 μL of deionized (DI) water. The well plates were then clamped together and placed in a custom-built steam chamber for pretreatment, as described in detail elsewhere [19 (link)]. Following pretreatment, 30.5 μL of a mixture of citric acid buffer (1 M, pH 4.8), sodium azide (10 g/L), and dilute enzyme mixture was added to each well, and the plates were incubated at 50°C in a Multitron shaker (Multitron Infors-HT, ATR Biotech, MD) at 150 rpm for 72 hours. The well-plates were then centrifuged at 2700 rpm for 30 minutes and the liquid hydrolyzate was transferred to HPLC vials for analysis. All enzymatic hydrolysis experiments were performed in quadruplicate. Sugar concentrations were determined by a Waters Alliance e2695 HPLC with a 2414 refractive index (RI) detector (Waters Corporation, Milford, MA, United States) and a BioRad Aminex HPX-87H column (Bio-Rad Life Science, Hercules, CA, United States).

The amount of lutein in the samples was quantified using Waters Alliance e2695 HPLC (Waters Corporation, Milford, MA, USA) equipped with 2996 photodiode array (PDA) detector and Empower 2.0 software. The analysis was performed using reverse-phase Waters^® C-18 column (5 µm; 250 mm × 4.6 mm) under isocratic conditions (flow rate of 1 mL/min at 25 °C). Mobile phase was a mixture (90:10) of methanol and tetrahydrofuran. The analyte was monitored at 450 nm. Sample injection volume was 20 µL and the run time was 10 min. Retention time of lutein was 3.85 min [43 (link)]. All the samples injected were filtered through 0.45 µm membrane filter. Stock solution (1 mg/mL) of lutein was prepared in the mobile phase, and calibration standards (n = 3) ranging from (1 µg/mL to 100 µg/mL) were serially diluted in the mobile phase. Similar standard curve was also prepared by dissolving lutein in DMSO. Calibration curves were obtained by plotting peak area against the concentration of lutein. The lutein content in the samples was determined quantitatively using the linear regression equations from the calibration curves (R² > 0.99). The HPLC method provided rapid and reproducible results without a significant difference in intra and inter-day analysis.

For fast and efficient growth evaluation, Bioscreen C (Bioscreen, Finland) was used. When strains grew to exponential phase after overnight cultivation, cells were collected and inoculated into fermentation medium with an initial OD₆₀₀ of 0.1 in a microtiter plate. The growth (OD₆₀₀) of strains was measured and recorded by Bioscreen C within an interval of 0.5 h at 30°C and medium-speed shaking. We added 5.0 g/L acetic acid, 10 mM H₂O₂, 5.0 g/L furfural, or 1.0 M NaCl into the medium to evaluate stress tolerance.
Batch fermentation evaluation was performed in a 250-mL flask containing 100 mL fermentation medium with or without acetic acid stress. The inoculation method was consistent with the processes mentioned before. In contrast, the cells of flocculating strain SPSC01 had to be deflocculated and resuspended by 0.1 M sodium citrate buffer (pH 4.5) (46 (link)). The fermentation conditions were controlled at 30°C and 150 rpm without pH adjustment. For analysis of fermentation performance, samples were collected every 12 h until glucose was consumed completely. The concentrations of glucose and ethanol in fermentation broth at each time point were detected via a high-performance liquid chromatography (HPLC) system (Waters Alliance e2695 HPLC; Waters, USA) with a Bio-Rad Aminex HPX-87H column. The column was eluted with 4 mM sulfuric acid at a 0.6-mL/min flow rate at 50°C.