Continuous Cultivation of Glucose-Limited Bean Cells (Phaseolus vulgarisL.) in a Modified Bacterial Fermentor

Abstract

A commercially available bacterial fermentor was modified to allow the continuous cultivation of glucose-limited bean cells (Phaseolus vulgaris L.) at a defined pH. A steady state was reached using a dilution rate of 0-004 h_1 and was maintained for 4 months; during this period the dry weight and packed cell volume of the culture remained constant; the yield for glucose was 0-34 (dry wt./dry wt.) and for carbon 0-27 (g atom/g atom). Glucose-limited cells had lower C/N ratios both for cytoplasm and cell walls than cells in the exponential phase of growth in batch cultures. INTRODUCTION Continuous cultures of substrate-limited plant cells are useful for studying meta bolism, because the cells grow in a solution of constant composition at a constant rate. The cells can thus be kept indefinitely in particular metabolic states. However, most studies have been performed with sycamore cells and only a few substrate limitations have so far been investigated (nitrogen limitation: Young, 1973; King, 1976, 1977; phosphate limitation: Wilson, 1976; glucose limitation: King, 1977). One of the difficulties arising from this type of work is the lack of a cheap, commercially available fermentor suitable for the cultivation of plant cells. This paper describes the continuous cultivation of glucose-limited bean cells at a defined pH in a relatively cheap bacterial fermentor that we have adapted to this purpose. MATERIALS AND METHODS Cell suspension culture The culture was initiated from seedling stem expiants of Phaseolus vulgaris L., var. Prelude (Klis and Eeltink, 1979) and was maintained by subculturing every 2 weeks. Cells were grown in 500 ml Erlenmeyer flasks containing 200 ml defined medium (Table 1). The flasks were placed on a rotary shaker (125 rev. min-1) and kept at 27 °C in continuous light (2000 lx). Culture vessel The continuous culture system is shown in Fig. 1. Cells were cultured in a 2 1 glass vessel from a Biolafitte laboratory fermentor. Two glass outlet tubes (i.d. 6 mm) were inserted in the vessel This content downloaded from 207.46.13.111 on Tue, 09 Aug 2016 06:21:12 UTC All use subject to http://about.jstor.org/terms 1224 Bertola and Klis—Glucose-Limited Bean Cells Table 1. Composition of synthetic medium for bean suspension cultures o ^ o o 9 00 g> ^00° C .roo—' <N <N U .9 ^ i «i a ■§ 1 •§ «1 •3.S o « H .S •« 2 I o a .a ■£ o « .s 8 12 -a £,0 .2 4 .9 S3 h2^fcfflri^<0