Carbohydrate metabolism by Actinomyces viscosus growing in continuous culture

Abstract

A human oral strain of Actinomyces viscosus, GN431/75, was grown anaerobically in a defined medium in continuous culture with a glucose limitation at dilution rates (D) between 0.025 and 0.2 h-1 and with a nitrogen limitation at D = 0.005 and 0.1 h-1. With 5 mg of glucose per ml, the culture was limited for carbon at D = 0.025 and 0.05 h-1, but became nitrogen limited (asparagine) at D = 0.1 and 0.2 h-1. The molar growth yield (Y(glucose)) decreased from 50.0 to 40.9 g of cells per mol of glucose as the dilution rate was increased from 0.025 to 0.2 h-1, reflecting the limitation of asparagine. With high glucose and low amino acid concentrations (nitrogen limited), the cell yields at D = 0.05 and 0.1 h-1 were 37 to 33% lower than in the glucose-limited culture. The major products of metabolism were succinic and lactic acids with less amounts of acetic and formic acids and ethanol. The rate of glucose fermentation by resting cells removed from the glucose-limited culture and assayed in a pH state increased with the dilution rate and was always higher than that for the fermentation of sucrose (60%) and fructose (40%). Activity for the glucose-P-enolpyruvate phosphotransferase system was observed in whole homogenates, with the highest activity evident at D = 0.1 h-1 with the glucose-limited culture. The observed activity was significantly lower than the rate of glucose metabolism at each dilution rate, suggesting that glucose-P-enolpyruvate phosphotransferase system was underestimated or that an additional transport system exists in the organism. The glucose-limited culture showed considerable ability to synthesize glycogen during the transition from carbon to nitrogen limitation, when 35% of the cell mass was present at this polymer. The organism was shown to possess the glycogen synthetic enzymes ADP glucose synthase and ADP glucose transferase, as well as the degradative enzyme maltodextrin phosphorlase. Washed cells of A. viscosus GN431/75 were shown to be relatively insensitive to the inhibiting actions of NaF in pH-fall and constant-pH experiments at all dilution rates. At pH 7.0, 25 mM NaF was required to completely inhibit glycolysis by glucose-limited cells at D = 0.05 h-1, whereas a concentration of only 11 mM Naf was required with cells of Streptococcus mutans grown and incubated under identical conditions. An interesting feature of the growth of A. viscosus GN431/75 in the chemostat was the shift from individual nonadherent cells at the low dilution rates to the appearance at D = 0.2 h-1 of large cell aggregates forming tenacious adherent films reminiscent of its characteristics in the oral cavity.