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To improve the cell protocol for Gymnema sylvestre, we investigated the influence of initial sucrose concentration, inoculum density, and optimal concentrations of auxins (IBA and NAA) in flask cultures, as well as the role of aeration volume in bioreactor cultures. Cell growth was enhanced 9-fold when the medium was supplemented with 3% sucrose versus a sucrose-free environment. Increasing the inoculum density to 60 g (wet weight) $L^{-1}$, but no further, greatly improved the growth of these cultures. All concentrations of IBA proved inhibitory while supplementation with $5mg\;L^{-1}$ NAA was associated with significantly higher dry-cell weights. In our bioreactor cultures, a step-wise increase in aeration volume from 0.05 to 0.40 vvm was optimal for cell growth. Although biomass (i.e., fresh weight) accumulated in the bioreactor up until Day 20, the dry-cell weights increased 10-fold, but only through Day 15. The internal dynamics of our culture media indicated that sucrose was preferentially utilized and that its concentration steeply decreased at the log phase. In contrast, both glucose and fructose supplies were exhausted only at the beginning of the declining phase. Our findings suggest that a 15-d culture period is optimal for G. sylvestre cell growth in a bioreactor.
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