Citrate and Sugar Cofermentation in Leuconostoc oenos, a (sup13)C Nuclear Magnetic Resonance Study

(sup13)C nuclear magnetic resonance spectroscopy was used to investigate citrate-glucose cometabolism in nongrowing cell suspensions of the wine lactic acid bacterium Leuconostoc oenos. The use of isotopically enriched substrates allowed us to identify and quantify in the end products the carbon atoms derived from each of the substrates supplied; furthermore, it was possible to differentiate between products derived from the metabolism of endogenous carbon reserves and those derived from external substrates. Citrate-sugar cometabolism was also monitored in dilute cell suspensions for comparison with the nuclear magnetic resonance results. A clear metabolic shift of the end products from glucose metabolism was observed when citrate was provided along with glucose: ethanol was replaced by acetate, and 2,3-butanediol was produced. Reciprocally, the production of lactate and 2,3-butanediol from citrate was increased in the presence of glucose. When citrate was cometabolized with glucose, a 10-fold reduction in the intracellular concentration of glucose-6-phosphate was observed, a result in line with the observed citrate-induced stimulation of glucose consumption. The presence of citrate provided additional pathways for NADP(sup+) regeneration and allowed the diversion of sugar carbon to reactions in which ATP was synthesized. The increased growth rates and maximal biomass yields of L. oenos growing on citrate-glucose mixtures resulted from increased ATP synthesis both by substrate-level phosphorylation and by a chemiosmotic mechanism.