Journal of General Microbiology (1980), 116, 225-232. Printed in Great Britain Growth of Mixed Cultures of Bacteria on Methanol By J. S. ROKEM, I. GOLDBERG AND R. I. MATELES Laboratory of Applied Microbiology, Institute of Microbiology, Hebrew University-Hadassah Medical School, P. 0. Box 1 172, Jerusalem, Israel (Received 5 April 1979; revised 10 July 1979) 225 The bacterium Pseudomonas C was grown in a chemostat on methanol as sole source of carbon and energy. At a dilution rate of 0.1 h-I, other methanol-utilizing bacteria (Pseudo- monas 1 and Pseudomonas 135), when added separately at a steady state, became dominant in the fermenter and Pseudomonas C was excluded. At a dilution rate of 0.3 h-l, however, Pseudomonas C dominated and the other bacteria were excluded. When various bacteria unable to utilize methanol were added to the chemostat during a steady state growth of Pseudomonas C, they remained in the fermenter independent of the dilution rate, but as a very low percentage of the total population (about 1%). When pathogenic bacteria (Staphylococcus aureus and SalmoneEEa typhimurium), which are unable to utilize methanol as a sole carbon source, were added separately to a pure culture of Pseudomonas C in a chemostat, they too remained in the fermenter independent of the dilution rate. However, they constituted less than 1 yo of the population in the culture broth but a high percentage of the population on the fermenter wall. When added to a mixture of Pseudomonas C and bacteria unable to utilize methanol, the pathogenic bacteria could not be found in the fermenter after a few medium changes. The results suggest that operation of a continuous culture of Pseudomonas C at high dilution rates serves to prevent contamination with other methylotrophs that may have lower yields. A mixture of Pseudornonas C and heterotrophs from soil is relatively resistant to invasion by pathogens. INTRODUCTION Although the production of single-cellprotein (SCP) by continuous culture using methanol as a substrate is usually based on pure cultures of micro-organisms (Terui et al., 1973; MacLennan et al., 1974 ; Mateles et al., 1976 ; Haggstrom, 1977 ; Chen et al., 1977), a number of reports have suggested the desirability of working with mixed cultures (Snedecor & Cooney, 1974; Haggstrom, 1969; Harrison et af., 1976; Cremieux et al., 1977; Ballerini et al., 1977), and a review of mixed cultures has appeared recently (Harrison, 1978). The mixed culture may offer several possible advantages compared with a pure culture, e.g. (1) the mixed culture may grow at a faster rate or give a higher yield than the pure culture, or (2) the mixed culture may be able to maintain itself in the face of accidental contamination more easily than the pure culture, thus permitting substantial savings in capital and operating costs associated with sterilization. The mixed culture may consist of organisms competing for growth on methanol itself or on C1 compounds derived from the oxidation of methanol, or may involve a single methanol- utilizer and one or more other organisms growing on compounds (other than C,) excreted by the methanol-utilizer or arising from its partial lysis. The purpose of this work was to investigate the relationship of Pseudomonas C grown in continuous culture to various types of competing organisms, both methanol-utilizers and 0022-1 287/8O/OooO-8700 $02.00 43 1980 SGM 15-2