Biotechnology Letters 23: 317–324, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 317 Characterization of plant suspension cultures using the focused beam reflectance technique Karen A. McDonald * , Alan P. Jackman & Shaelyn Hurst Department of Chemical Engineering and Materials Science, University of California, Davis, CA 95616, USA * Author for correspondence (Fax: +530 752 1031; E-mail: kamcdonald@ucdavis.edu) Received 2 October 2000; Revisions requested 19 October 2000; Revisions received 1 December 2000; Accepted 2 December 2000 Key words: aggregate size, bioreactor monitoring, plant cell culture Abstract Development of bioreactor systems utilizing plant suspension cultures has been hindered by the lack of on-line sensors for monitoring important process variables such as biomass concentration and aggregate size. An optical technique, the focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA), was used to characterize several plant suspension cultures: rice (Oryza sativa), tobacco (Nicotiana benthamiana) and wild Chinese cucumber (Trichosanthes kirilowii). These cultures differ in a number of respects such as individual cell size and morphology, aggregate shape and size distribution, initial culture density, and color. For plant suspensions comprised of relatively spherical aggregates (rice and cucumber), the area under the cube-weighted FBRM chord length distribution was linearly correlated to biomass concentration (R 2 > 0.99) while the mean of the cube- weighted FBRM chord length distribution was nonlinearly related to aggregate size. Introduction Plant suspension cultures are currently being investi- gated as a source of novel and commercially important natural compounds as well as for production of re- combinant proteins using transgenic plant cell lines (see review by Fischer et al. 1999). Advantages of plant cultures over other host cells include their abil- ity to grow well in suspension in relatively inexpen- sive, chemically defined media, capacity for complex glycosylation, ease of scale-up compared with mam- malian host cells, and safety (plants are not known to propagate human pathogens). However, plant cells typically grow as aggregates rather than single cells. Aggregate size distribution not only plays an impor- tant role in mixing, oxygen and nutrient mass transfer, and ease of product purification, but in many cases it is directly related to productivity. For example, Pépin et al. (1999) and Kinnersley & Dougall (1980) have shown that the production of the pigment anthocyanin from plant suspension cultures is inversely related to aggregate size. In contrast, Jianfeng et al. (1998) have shown that the production of the secondary metabo- lite salidroside is significantly increased in 3–7 mm compact callus aggregates compared with dispersed cells. While there are a number of bioreactor variables that can easily be monitored on-line, sensors for in- situ monitoring of the two most critical variables for plant suspension cultures, biomass concentration and aggregate size are lacking. Although there are sev- eral ways to characterize biomass concentration in plant suspension cultures, dry weight measurements are often considered to be the most meaningful and reliable. However this method requires sampling the bioreactor and takes 1–3 days to obtain a measurement minimizing the measurement’s usefulness for on-line control. Similarly, aggregate size distribution is often measured by gently screening a sample from the biore- actor using a series of standard sieves and obtaining the dry weight of the biomass retained on each screen. In addition to the disadvantages described above, the mechanical sieving process has the potential to al- ter the size distribution. Thus, in-situ techniques to monitor biomass concentration (dry weight l -1 ) and