MDH, malate dehydrogenase. Citrate, which was part of the simulation medium, was largely metabolized by the lactic acid bacteria. NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. Physiology of A. ghanensis DSM 18895 and A. pasteurianus NCC 316 growth.A. Extracellular fluxes are given in Table 2. The mass isotopomer distributions of proteinogenic amino acids were analyzed by gas chromatography/mass spectrometry (GC/MS) in the selective ion monitoring mode (34). The relative flux intensity is illustrated by the arrow thickness, and the individual contribution of lactate and ethanol to the fluxes is indicated by the arrow color. 1). With a P/O ratio of 0.5 (53), this corresponds to 62 mmol of ATP per 100 mmol of total substrate influx and 53% of total ATP formation. These systems are directly coupled to respiratory chains, located at the cytoplasmic membrane, and allow oxidation of external substrates in a rather simple manner (41). The enzymatic reactions and their corresponding genes and the definitions of the abbreviations for the enzymes are given in Table S1 in the supplemental material. For this purpose, labeling of the latter was quantified from culture supernatants that were derived from cultures with cocoa pulp simulation medium (first exponential growth phase) containing [U-13C]lactate or [U-13C]ethanol as a tracer substrate (see Tables S7 and S8 in the supplemental material). Lactate conversion into acetoin is responsible for 8% of the ATP pool. In addition, Acetobacter ghanensis and Acetobacter senegalensis are found during spontaneous cocoa bean fermentation (9, 13, 17, 18). Additionally, acetoin (18 mM) and pyruvate (13 mM) were produced. Additionally, in some bacteria, carbon flux control through the PEP-pyruvate-oxaloacetate node is complex; e.g., pyruvate carboxylase (PC) and ME are simultaneously active during growth on glucose (62, 63). As a positive control, an isocitrate dehydrogenase assay was used (39). This confirms that carbon from ethanol does not enter this upper part of metabolism. For precultures and the main cultivation of Acetobacter species, cocoa pulp simulation medium for acetic acid bacteria (PSM-AAB) was used (12). Copyright © 2020 American Society for Microbiology | Privacy Policy | Website feedback, Print ISSN: 0099-2240; Online ISSN: 1098-5336, The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions, Sign In to Email Alerts with your Email Address. A. pasteurianus possesses membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenases for oxidation of ethanol into acetate, as well as genes encoding intracellular alcohol dehydrogenase and aldehyde dehydrogenase (see Table S1 in the supplemental material) (21, 40–43). To account for the potential evaporation of volatile compounds, their loss was monitored in control experiments without cells, in which the cultures were incubated under the fermentation conditions described above, and evaporation rates were used to correct formation and consumption rates. Nevertheless, acetoin formation may be advantageous for cellular homoeostasis, as this compound is less toxic than the organic acid. A pathway from lactate to acetate as the final product has been proposed; however, the role of some of its enzymes remains unclear (26). With depletion of lactate and ethanol, the dissolved oxygen level sharply increased. Black circles, 13C-labeled carbon atoms; white circles, unlabeled carbon (12C); PP, pentose phosphate; Rel. Cultivations with L. fermentum NCC 575 and S. cerevisiae NYSC 2 were conducted in nonbaffled 250-ml shake flasks at 37°C and a rotation speed of 50 rpm. However, in recent years, functional drinks have often been contaminated by other acetic acid bacteria belonging to the genus Asaia [4–7]. Their papers give no data for the amount of gas formed or for the per- centage of sugar represented by the end-products.