Appl Microbiol Biotechnol (2002) 60:300–305 DOI 10.1007/s00253-002-1117-2 ORIGINAL PAPER K. Ueda · H. Saka · Yoshiyuki Ishikawa · T. Kato · Y. Takeshita · H. Shiratori · M. Ohno · K. Hosono · M. Wada · Yohichi Ishikawa · T. Beppu Development of a membrane dialysis bioreactor and its application to a large-scale culture of a symbiotic bacterium, Symbiobacterium thermophilum Received: 11 June 2002 / Revised: 19 July 2002 / Accepted: 27 August 2002 / Published online: 12 October 2002  Springer-Verlag 2002 Abstract A simple membrane dialysis bioreactor was developed for a large-scale axenic culture of Symbiobac- terium thermophilum, a symbiotic thermophile that requires co-cultivation with an associating thermophilic Bacillus strain S for normal growth. The bioreactor consisted of an outer- and an inner-coaxial cylindrical compartment bordered across a dialyzing membrane, which enabled a 1 l-scale dialysis culture with exchange of low molecular metabolites between the two compart- ments to be performed. Using the bioreactor, growth characteristics of S. thermophilum and Bacillus strain S were assessed under two medium conditions. The growth of S. thermophilum was measured by quantitative PCR because the bacterium formed no visible colonies and gave abnormally low turbidity. In medium containing 2% tryptone peptone, S. thermophilum proliferated up to 410 7 cells/ml, and strict dependence on the co-culture with Bacillus strain S was observed. On the other hand, medium containing 0.5% yeast extract not only facilitated the growth of S. thermophilum in the co-culture (610 7 cells/ml), but also allowed limited pure growth indepen- dent of Bacillus strain S (110 7 cells/ml), implying that some component of yeast extract can partially replace the growth requirement of S. thermophilum supplied by Bacillus strain S. Both the oxidative redox potential values and the cell morphology in the independently growing culture suggested the occurrence of marked unbalanced growth possibly caused by significant meta- bolic changes. The bioreactor is applicable to the analyses of culturing characteristics in symbiotic systems between free-living microorganisms. Introduction Symbiobacterium thermophilum is a symbiotic and ther- mophilic bacterium, which was discovered by screening for thermostable tryptophanases. Although the bacterium propagates up to 210 9 cells/ml in a mixed culture with an associating thermophilic Bacillus strain S at 60C, and produces a thermostable tryptophanase, attempts to establish it in pure culture by single-colony-isolation on solid media have failed (Ohno et al. 1999; Suzuki et al. 1988). Our study has revealed that these two free-living bacteria comprise a system of commensal symbiosis, in which the growth of S. thermophilum is strictly dependent on co-existence with Bacillus strain S. The 16S rDNA- based phylogeny revealed that S. thermophilum belongs to a Gram-positive group creating a novel phylogenetic branch at an outermost position in the group without clustering with any other genus (Ohno et al. 2000). Despite this novel phylogenetic feature, the ubiquitous distribution of S. thermophilum and its relatives in the natural environment was revealed by extensive screening using specific PCR, which frequently detected identical or homologous 16S rDNA sequences in thermophilic cul- tures obtained from compost, soil, animal intestinal contents and feces, etc (Ueda et al. 2001). The potential of such a symbiotic microbe as a novel genetic resource was demonstrated by the analyses of the genes encoding thermostable tryptophanase and b-tyrosinase, both of which are useful for the enzymatic synthesis of l- tryptophan and l-dihydroxyphenylalanine (l-DOPA), respectively (Hirahara et al. 1992, 1993; Kudo et al. 1999). To analyze the growth requirements of this symbiotic bacterium, we previously developed a dialysis glass vessel, which consisted of two compartments partitioned with a dialysis membrane (see Fig. 2A) (Ohno et al. K. Ueda · H. Saka · Yoshiyuki Ishikawa · T. Kato · Y. Takeshita · H. Shiratori · M. Ohno · K. Hosono · T. Beppu ( ) ) Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252–8510, Japan e-mail: beppu@brs.nihon-u.ac.jp Tel.: +81-466-843931 Fax: +81-466-843935 M. Wada · Yohichi Ishikawa ABLE Corporation, 6–10 Nishigokencho Shinjuku-ku, Tokyo 162-0812, Japan