Hairy root culture in a liquid-dispersed bioreactor: Characterization of spatial heterogeneity

A liquid-dispersed reactor equipped with a vertical mesh cylinder for inocu lum support was developed for culture of Atropa belladonna hairy roots. The working volume of the culture vessel was 4.4 L with an aspect ratio of 1.7 . Medium was dispersed as a spray onto the top of the root bed, and the roo ts grew radially outward from the central mesh cylinder to the vessel wall. Significant benefits in terms of liquid drainage and reduced interstitial liquid holdup were obtained using a vertical rather than horizontal support structure for the biomass and by operating the reactor with cocurrent air and liquid flow. With root growth, a pattern of spatial heterogeneity devel oped in the vessel. Higher local biomass densities, lower volumes of inters titial liquid, lower sugar concentrations, and higher root atropine content s were found in the upper sections of the root bed compared with the lower sections, suggesting a greater level of metabolic activity toward the top o f the reactor. Although gas-liquid oxygen transfer to the spray droplets wa s very rapid, there was evidence of significant oxygen limitations in the r eactor. Substantial volumes of non-free-draining interstitial liquid accumu lated in the root bed. Roots near the bottom of the vessel. trapped up to 3 -4 times their own weight in liquid, thus eliminating the advantages of imp roved contact with the gas phase offered by liquid-dispersed culture system s. Local nutrient and product concentrations in the non-free-draining liqui d were significantly different from those in the bulk medium, indicating po or liquid mixing within the root bed. Oxygen enrichment of the gas phase im proved neither growth nor atropine production, highlighting the greater imp ortance of liquid-solid compared with gas-liquid oxygen transfer resistance . The absence of mechanical or pneumatic agitation and the tendency of the root bed to accumulate liquid and impede drainage were identified as the ma jor limitations to reactor performance. Improved reactor operating strategi es and selection or development of root lines offering minimal resistance t o liquid flow and low liquid retention characteristics are possible solutio ns to these problems.