3 l bioreactor

Fed-batch cultivation was performed at 37 °C for 24 h in a 3-L bioreactor (Infors HT, Bottmingen, Switzerland) [30 (link)] containing 1.2 L of optimized medium. Seed culture (100 mL LB medium containing 30 μg/mL kanamycin) was cultivated in 500-mL shake flasks and grown at 37 °C for 8 h with shaking at 200 rpm before inoculation into the bioreactor.
Dissolved oxygen (DO) and temperature were controlled at 30% and 33 °C, respectively, and phosphoric acid or ammonia solution was added, as needed, to maintain the pH at 7.0. DO, pH, temperature, and flow rates (200–700 rpm) were regulated automatically by the Infors fermentation device (Infors HT). Upon reduction of the carbon source and increase in DO in the primary medium, the feed medium was added to maintain the B. subtilis growth rate, thereby initiating the exponential-feeding phase of fed-batch cultivation. After fermentation the quantity of SPase was determined by scanning the area of each band on reduced SDS-PAGE gels, and then calculating with Image-Master TotalLab software (Amersham Biosciences) using purified SPase as a reference.

The media included Luria–Bertani (LB) medium, Yeast Extract Peptone Dextrose (YPD) medium, Buffered Methanol-complex (BMMY) medium and Basal Salts (BSM) medium [13 (link)]. The yeast cells were pre-cultured in YPD medium for 24 h, at 30 °C and 220 rpm. Then the pellets were resuspended in BMMY medium for 144 h cultivation with the same condition. Also, 1% methanol (v/v) was added into medium every 24 h for inducing the pAOX1 promoter. The scale-up cultivation was carried out in 3-L bio-reactor (INFORS, Switzerland), with 800 mL BSM medium. The cultivation process was divided into three phases. During the glycerol batch cultivation, the yeast cells were cultured under pH 5.5, 30 °C, and the dissolved oxygen (DO) controlled over 30% by constant agitation speed. Glycerol fed-batch cultivation was carried out when the glycerol was depleted with DO over 50%. And the feeding solution (50% glycerol with 1.2% PTM1 solution) was gradually added into medium to confer high-density cultivation with increased DO level by increased agitation speed (800 rpm). In the methanol fed-batch phase, the trypsin was expressed by methanol induced promoter pAOX1. In order to avoid the repression effect of pAOX1 by the glycerol, the inducer methanol was added 2 h later when DO was over 60%. And the methanol was gradually fed, according to the method developed by Wang et al. [46 (link)].

A final preculture of 150 ml was used to inoculate 1.2 l of deuterated Enfors minimal medium in a 3 l bioreactor (Infors, Switzerland). The pD of the culture medium was regulated at 7.2 by addition of 4% NaOD (Eurisotop, France). The temperature was maintained at 30 °C. After consumption of the deuterated glycerol-d₈ from the culture medium, the fed-batch phase was initiated by continuous feeding with the additional 30 g of the deuterated glycerol-d₈. The protein expression was induced overnight (17 h) with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at the OD₆₀₀ of 13 at 30 °C. After the fermentation, the cells were recovered by centrifugation (10,500 × g for 1 h at 6 °C) and the wet cell paste was frozen at −80 °C for long-term storage. The final yield was 51 g of the deuterated cell paste from a final volume of 1.6 l culture medium.