Effect of Elicitation on Growth, Respiration, and Nutrient Uptake of Root and Cell Suspension Cultures of Hyoscyamus muticus Edgard B. Carvalho and Wayne R. Curtis* Department of Chemical Engineering, Fenske Laboratory, Life Science Consortium, Wartik Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802 The elicitation of Hyoscyamus muticus root and cell suspension cultures by fungal elicitor from Rhizoctonia solani causes dramatic changes in respiration, nutrient yields, and growth. Cells and mature root tissues have similar specific oxygen uptake rates (SOUR) before and after the onset of the elicitation process. Cell suspension SOUR were 11 and 18 μmol O 2 /g FW‚h for non-elicited control and elicited cultures, respectively. Mature root SOUR were 11 and 24 μmol O 2 /g FW‚h for control and elicited tissue, respectively. Tissue growth is significantly reduced upon the addition of elicitor to these cultures. Inorganic yield remains fairly constant, whereas yield on sugar is reduced from 0.532 to 0.352 g dry biomass per g sugar for roots and 0.614 to 0.440 g dry biomass per g sugar for cells. This reduction in yield results from increased energy requirements for the defense response. Growth reduction is reflected in a reduction in root meristem (tip) SOUR, which decreased from 189 to 70 μmol O 2 /g FW‚h upon elicitation. Therefore, despite the increase in total respiration, the maximum local oxygen fluxes are reduced as a result of the reduction in metabolic activity at the meristem. This distribution of oxygen uptake throughout the mature tissue could reduce mass transfer requirements during elicited production. However, this was not found to be the case for sesquiterpene elicitation, where production of lubimin and solavetivone were found to increase linearly up to oxygen partial pressures of 40% O 2 in air. SOUR is shown to similarly increase in both bubble column and tubular reactors despite severe mass transfer limitations, suggesting the possibility of metabolically induced increases in tissue convective transport during elicitation. Introduction Plants are the source of a wide array of compounds used as food additives, dyes, pharmaceuticals, fragrances, pesticides, and cosmetics. A substantial number of drugs are derived from plants. As of the end of the past decade, 47 major drugs have come from tropical plants (1). Currently several screening projects are being conducted in partnership between American pharmaceutical com- panies and developing countries (2) to identify potential drug candidates. Commercial exploitation of the potential represented by plants could be greatly improved by the ability to economically grow plant tissue culture in bioreactors for the production of secondary metabolites. Some attempts have been taken in this direction. Among them, the development of a plant cell culture process for the production of Taxol in a large-scale (75 m 3 ) bioreactor is being conducted by Phyton Catalytic (3). The biosynthetic pathways for the production of phy- toalexins are commonly inoperative in plant tissues under normal growth conditions. However, upon the addition of an inducer, such secondary metabolite path- ways are turned on and the flux of carbon is diverted from growth metabolism toward these secondary me- tabolite pathways. The model system used in this work is the production of sesquiterpene phytoalexins in the solanaceous plant Hyoscyamus muticus after contact with Rhizoctonia solani fungal extract. Fungal elicitation turns on the branch-point sesquiterpene cyclase enzyme, diverting the flux of carbon from the isoprenoid to the sesquiterpene pathway (4). The defense response causes a profound change in the metabolic state of the cultures. Such changes are similar to those caused by the incom- patible (nonpathogenic) interaction between plants and fungi, which includes a series of cellular processes such as de novo biosynthesis of defense proteins and a vigorous consumption of ATP accompanied by an increase in specific oxygen uptake rate (SOUR) (5). The shift of resource allocation away from growth to secondary metabolism and the associated shifts in respiration are both of interest in bioreactors for phytochemical produc- tion. This report compares the response of H. muticus root and cell suspension cultures after fungal elicitation. Materials and Methods Plant Root and Cell Cultures. “Hairy”-root culture of H. muticus line HM90T (Egyptian henbane) was used in this work. This root culture was initiated in 1990 through an Agrobacterium rhizogenes infection. Roots were maintained since 1990 in B5 medium (6) containing 20 g/L sucrose by a biweekly subculture using 125-mL Erlenmeyer flasks filled with 50 mL of B5 medium. Flasks were cultured in the dark on a gyratory shaker with a 1-in. stroke at 125 rpm at a temperature of 25 °C. The H. muticus cell suspension was obtained by dedifferentiating the hairy-root culture described above * To whom correspondence should be addressed. Ph: (814) 863- 4805. Fax: (814) 865-7946. Email: wrc2@psu.edu. 282 Biotechnol. Prog. 2002, 18, 282-289 10.1021/bp0202970 CCC: $22.00 © 2002 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/15/2002