Biotechnology Letters 25: 61–65, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 61 Biomass segregation in sage cell suspension culture Žiga Bolta, Dea Bariˇ ceviˇ c & Peter Raspor ∗ Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia ∗ Author for correspondence (Fax: +386 1 5274092; E-mail: peter.raspor@bf.uni-lj.si) Received 27 May 2002; Revisions requested 17 June/27 August 2002; Revisions received 5 August 2002/28 October 2002; Accepted 29 October 2002 Key words: aggregation, bioreactor, particle size distribution, plant cell suspension, Salvia officinalis L., ursolic acid Abstract The biomass of sage (Salvia officinalis L.) cell suspension culture was composed of single cells and cell aggre- gates. The development of aggregated cell culture from a single-cell suspension was monitored by particle size distribution for four particle size classes. Particle size distribution was compared between the biomass grown in bioreactor and shake flasks. The size of the particles had a strong influence on content of secondary metabolite, ursolic acid (UA). The single cell biomass fraction accumulated up to 7.7 mg UA g -1 DW which was up to 50 times higher compared to aggregated biomass fractions. Introduction Plant cells have the capacity to proliferate as undiffer- entiated tissue. A considerable effort has already been invested in exploiting this property for the production of valuable compounds when growing the biomass in a bioreactor. One major obstacle is the low productivity of the cultures (Verpoorte et al. 1999). Many plant species from genus Salvia have long been recognised as medicinal herbs and their deriv- atives continue to be important components of con- temporary phytopharmaceuticals. Quantity and com- position of bioactive compounds in the plants vary with respect to the plant source and environmen- tal conditions. Bioreactor cultivation enables climate independent cultivation. We have focused our atten- tion to the production of UA, a triterpenoid with an anti-inflammatory and anti-tumour activity (Hsu et al. 1997, Bariˇ ceviˇ c et al. 2001). Growth of a sage cell cul- ture and UA accumulation in the biomass was already reported (Bolta et al. 2000) to be affected by the origin of plant material and its morphological form. Plant cell suspension is a particulate system com- posed of single cells and cell aggregates. Particles forming the plant cell suspension range from a sin- gle cell of 20 μm to an aggregated particle with a diameter of over 20 mm (Hibino & Ushijama 1999) and thus constitute a very heterogeneous system. The conditions of the culture, hydrodynamic shear stress and mass transfer limitations can vary considerably in this range (Hulst et al. 1989, Balica & Ryu 1993, Mei- jer et al. 1993). Hulst et al. (1989), Maddhusudhan & Ravishankar (1996) and Keßler et al. (1999) re- port of aggregate size dependant secondary metabolite production. Plant cell aggregates can also cause rheo- logical problems and can block pipes and openings in a bioreactor (Mavituna & Park 1987). The characterisa- tion of the productive biomass is therefore prerequisite for setting up a secondary metabolite production strat- egy. This present communication demonstrates the development of segregated cell suspension from a sin- gle cell and establishes the importance of particle size distribution on production of biologically active compound, UA. Materials and methods Cell culture Sage shoot (donor-plants originated from Salvia of- ficinalis L., accession 25/89, Gatersleben Gene-bank,