Case Study: Fermentative Production Of RL

1. Materials and methods
1.1. Microorganism
The P. aeruginosa 15GR is a RL hyperproducer mutant obtained by gamma radiation of P. aeruginosa isolate P6 in our previous study (in press). This isolate was stored in Luria Bertani broth (Lab M, Topley house, England) containing 20% glycerol at -80 °C.
1.2. Culture media
The mineral salts medium [9] containing glycerol 2 % v/v as the carbon source (GMSM) was prepared and used in this study.
1.3. Fermentative production of RL
1.3.1. Seed culture preparation
A loopful from a fresh culture grown onto nutrient agar was inoculated into a 250 ml Erlenmeyer flask containing 25 ml trypticase soy broth, incubated at 250 rpm and 30°C for 15-16 h. After that, the culture obtained was centrifuged at 10,000
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1.6. Studying the different factors affecting RL production by P. aeruginosa 15GR using SSF
1.6.1. Studying the time course of RL production using the selected substrate (sugarcane bagasse and sunflower meal)
Six Erlenmeyer flasks containing sugarcane bagasse and sunflower meal (total mass 10 g) were prepared. Twenty milliliters impregnating solution (GMSM) was inoculated with 0.4 ml of seed culture (2% v/v), and this was mixed into the solid substrate. The inoculated flasks were incubated at 30°C. Over an incubation period of 12 days, one flask was removed at specific time intervals for extraction and determination of RL concentration. One flask was left uninoculated and served as a control.
1.6.2. Effect of agitation rate
In these experiments, two flasks were prepared as described above; one was incubated at 30°C without agitation and the other incubated at 30°C with an agitation rate of 250 rpm. After incubation, RLs were extracted as described. Control uninoculated flasks were prepared and treated similarly.
1.6.3. Effect of using variable concentrations of glycerol in impregnating
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Twenty milliliters aliquots of MSM containing different concentrations of glycerol (2%, 5%, 10% v/v) were inoculated with seed culture (2% v/v), mixed with the solis substrate and incubated at 30°C. After incubation, RLs were extracted as described above. Control uninoculated flasks were prepared and treated similarly.
1.6.4. Response surface methodology (RSM) for the optimization of RL production under SSF
Factors such as inoculum size (represented by the code A), temperature (represented by the code B) and pH (represented by the code C) were optimized by RSM. Experimental Box-Behnken design (BBD) was employed and the factors and levels used for these experiments were: inoculum size of 1, 2 or 5 % v/v; temperature of 30, 33.5 or 37°C; and pH of 6, 7 or 8. In total, 13 runs of experiments were carried out with 1 centerpoint. Each treatment had a control without inoculum. One response value, the RL concentration (RL, g/L) was employed. After 10 days incubation, the response value was measured accordingly. The design of experiments was performed using Design Expert® v. 7.0 (DesignExpert ® Software, Stat-Ease Inc., Statistics Made Easy, Minneapolis, MN,

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