M br 034p shaker

Single colonies of positive transformants were grown aerobically in nutrient-rich CGTG15 medium supplemented with kanamycin sulfate (50 mg/L). The composition of CGTG15 medium was designed with reference to CGXII medium [51 –53 (link)]. Transformants of KC551 were cultured at 30 °C and 800 rpm in 750 µL of the medium in deep-well plates sealed with Axygen® Breathable Sealing Film (Corning Inc., Corning, NY, USA) using M·BR-034P shaker (TAITEC Corporation, Saitama, Japan). To produce 4,3-AHBA in fed-batch culture, the strains were precultured in 6 mL of the medium in a test tube for 24 h at 30 °C. Thereafter, 3 mL of the precultured cells were used to inoculate 60 mL of the medium containing 250 mg/L antifoam FERMOL1000 (Kao Corporation, Tokyo, Japan) in a small-scale multi-channel fermenter (Bio Jr.8; ABLE, Tokyo, Japan), and cultured at 32 °C (aeration, 60 mL/min). Agitation was automatically controlled to maintain the dissolved O₂ concentration constant at 0.2 ppm. The pH was maintained at 7.2 by automatic addition of 10% ammonia solution, and glucose in the culture medium was supplemented with 60% (w/w) glucose solution as needed to prevent depletion. Aliquots of culture medium were taken to calculate cell density, followed by quantification of products and glucose. Cell density was calculated by monitoring the OD₆₀₀ using a spectrophotometer.

SP was inoculated into 802 medium and cultured at 30 °C overnight to serve as an inoculum. After dispensing 0.6 mL of 802 medium containing each sugar alcohol (erythritol, xylitol, sorbitol, and maltitol) at 0, 5, 10, or 15% (w/w) into 96 Deep Well Plates (AxyGen Scientific, CA, USA), each well was inoculated with 4 µL of each bacterium and cultured at 30 °C using an MBR-034P shaker (Taitec, Aichi, Japan) with shaking at 1,000 rpm for 16 h. Twenty microliters of these cultures were suspended in 180 µL of water in the 96-well flat-bottomed plates (4845-96 F; Watson Bio Lab, CA, USA), and turbidity (OD₆₆₀) was measured using a microplate reader (SpectraMax M2; Molecular Devices, CA, USA) (n = 4). To assess the significance of differences, comparisons between the control (0%) group and each sugar-added group were performed by Tukey’s method using SPSS @ Statistics Version 26.0, with the probabilistic significance p < 0.05 indicating a significant difference.