Antifoam 289

Batch fermentations were carried out in a 5-l New Brunswick FS300 baffled fermenter (New Brunswick Scientific Co., New Brunswick, NJ), with two flat-bladed turbines with six blades (Rushton turbine). The fermenter was equipped with controllers for pH, temperature, agitation, and dissolved oxygen concentration and contained 4.0-l of the optimized medium previously determined. Inocula were carried out essentially as described for Erlenmeyer flasks, except that the liquid medium for the inoculum was the same as for the fermenter. Agitation and aeration were varied to maintain dissolved oxygen concentration at 40% air saturation. Foam production was controlled by addition of antifoam (Antifoam 289; Sigma, Saint Louis, MO).

Batch growths were performed in Biostat MD fermenter/Bioreactors (B. Braun Biotech International, Germany) in 2.5 liters. Six batch growths, three with BH500 (pYS5) and three with BH500 (pSW4), were performed under identical conditions to achieve biological triplicates for each strain. Modified FA medium containing (per liter) 35 g Soy peptone E110 (Organotechnie, La Courneuve, France), 5 g yeast extract (Difco Laboratories, Detroit, MI, USA), and 100 ml of 10X salt solution, was used. The 10X salt solution consisted of (grams per liter) 60 g Na₂HPO₄.7H₂O, 10 g KH₂PO₄, 55 g NaCl, 0.4 g L-tryptophan, 0.4 g L-methionine, 0.05 g thiamine, and 0.25 g uracil and was filter sterilized. The pH of the medium was adjusted to 7.5 with 10% phosphoric acid and 2 N NaOH. Starting cultures were inoculated from frozen stocks and grown for 12–14 hr. Fermenters were inoculated with 3% of the overnight grown culture. The medium in the fermenter was supplemented with 0.2 ml/l of antifoam 289 (Sigma, St. Louis, MO). The growth was conducted at 37 °C and 30% oxygen saturation, which was maintained by varying agitation speed and air flow rate through adaptive control^{52 (link)}. Culture pH was monitored and controlled only when it started to rise above the set point of 7.5.