Alkaloid production by hairy root cultures

Alkaloids are a valuable source of pharmaceuticals. As alkaloid producers, hairy roots show rapid growth in hormone-free medium and synthesis of alkaloids whose biosynthesis requires differentiated root cell types. Oxygen mass transfer is a limiting factor in hairy root cultures because of oxygen's low solubility in water and the mass transfer resistance caused by entangled hairy roots. In addition to limited oxygen supply, alkaloids accumulating primarily in the root tissue present a bioprocessing challenge because the root tissue must be homogenized and the phytochemical extracted and purified from a chemically complex solution. The present research focused on improving oxygen mass transfer with microbubbles and enhancing alkaloid release in hairy root cultures. Hairy roots are heterotrophic and require sucrose and oxygen for respiration. The tobacco hairy roots were able to grow with glucose and/or fructose, but the growth was only 10% of that with sucrose as the carbon source. Oxygen was a limiting nutrient for tobacco hairy root growth. In a 1-liter fermentor, microbubbles stabilized by 50 mg l -1 Triton X-100 increased oxygen mass transfer, hairy root growth, and nicotine production in medium. Basification of the culture medium associated with root growth resulted in a dramatic reduction in nicotine accumulation in the medium, which was reversed by adjusting the medium pH from 8 to 6. Confocal microscope observation showed that the H. niger hairy root cells with less aeration were much wider than the cells with sufficient aeration. The combination of microbubble generator and a novel ground-joint column bioreactor improved the oxygen mass transfer and consequently the growth of H. niger hairy roots. When the medium pH was adjusted from 6 to 3, the concentration of hyoscyamine in hairy root culture medium increased from zero (undetectable) to 42 mg l-1 , whereas the scopolamine in medium increased from 0.1 to 11 mg l-1 . Medium pH 3 inhibited H. niger hairy root growth but the root branches appeared to survive short time medium pH 3 treatment. In comparison to the wild-type H. niger hairy root line, the transgenic line showed about 10-fold increase in total tropane alkaloid released at the medium pH3.