Differences in cell sizes were observed in recombinant Bacillus subtilis studied in cyclic batch culture. It was observed that under this mode of operation both plasmid-containing and plasmid-free cells were present in the culture broth fur an extended period of time when compared to batch culture. In cyclic batch culture, plasmid-containing cells decreased from 100% to 0% over a period of 75 h, compared with batch culture where plasmid-containing cells decreased to 0 over a period of 20 h. Plasmid copy number was found to be similar in cyclic batch culture to that found in batch culture. Copy number increased from 90 copies up to a maximum of 190 copies in cyclic batch culture after the drop in plasmid-containing cells below 40%. Tn batch culture, copy number ranged fram 90 to 140. Cell size distributions by using image analysis and particle-size equipment indicated the existence of two populations in cyclic batch culture. Average cell size decreased with decreasing percentage of plasmid-containing cells, whereas the average plasmid-containing cell size increased with increasing plasmid copy number. (C) 2000 Elsevier Science Inc. All rights reserved.