The absence of pyruvate decarboxylase activity (low activity) likely seems to be responsible for the low contribution of lactate to acetate formation. 8). A few species are able to catabolize lactate (23–26). Additionally, the glyoxylate shunt is missing in A. pasteurianus (21, 40) and A. ghanensis (31). The OD600 was monitored to determine cell growth (Libra S11; Biochrome, Cambridge, United Kingdom). Based on 13C labeling, our results clearly show that lactate is poorly oxidized into acetate and, vice versa, that ethanol does not contribute to acetoin formation. (D) Elucidation of specific metabolic routes of [3-13C]lactate utilization. Ethanol is almost completely converted into acetate. Indeed, AAB performed the best and produced the largest amounts of acetate in mixed culture experiments when lactic acid bacteria and yeasts were both present. Metabolic origin of proteinogenic amino acids. 3A). Obviously, aspartate serves as a source of oxaloacetate and replenishes the TCA cycle, replacing anaplerosis via PEPC. We conclude that the flux through PEPCK is zero. As an exception to the otherwise strongly separated functional modules of lactate and ethanol metabolism, a minor fraction of lactate-derived carbon replenishes the TCA cycle. A proton-translocating nicotinamide nucleotide transhydrogenase might be involved in NADPH oxidation (21). In this constellation, the main role of ethanol is to generate metabolic energy via acetate production. A pathway from lactate to acetate as the final product has been proposed; however, the role of some of its enzymes remains unclear (26). Violet circles, nodes between the modules of lactate and ethanol metabolism. Metabolic model for flux calculations.The metabolic model for flux estimations was derived from the genomic repertoire (Fig. Here, glucose is an energy source, while fructose is entirely used as an electron acceptor for NADH + H+. The glyoxylate pathway and the Entner-Doudoroff (ED) pathway are not encoded in the genomes of A. pasteurianus IFO 3283 and A. pasteurianus 386B, respectively (21, 40). A deeper inspection of the labeling patterns provides direct evidence of their in vivo significance. (A) Theoretical 13C-labeled mass isotopomer distribution of alanine obtained by de novo synthesis from the labeled substrates ([U-13C]lactate and [U-13C]ethanol) or amino acid uptake (12C). 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. Disruption was performed mechanically (with 100-μm-diameter silica glass beads and disruption 2 times for 20 s each time at 6.0 m s−1; FastPrep-24; MP Biomedicals, Santa Ana, CA). In this regard, Acetobacter species can make use of pyruvate phosphate dikinase, which converts pyruvate directly into PEP (58, 59). 6A). The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. As a positive control, an isocitrate dehydrogenase assay was used (39). Taken together, metabolic flux analysis of the acetic acid bacteria integrated labeling information from four parallel tracer studies (Fig. Similarly, Acetobacter aceti lacks oxaloacetate decarboxylation during growth on three-carbon substrates, while pyruvate phosphate dikinase is active (58, 60). PEP carboxykinase and ME, enzymes of gluconeogenesis, however, were not expressed (Table 3). In contrast, channeling of ethanol or acetate into the central metabolism via acetyl-CoA synthetase costs two extra moles of ATP (22, 52, 53). melanogenum bymeansof the methodol-ogy and system of clasification of Frateur (1950). 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 basic knowledge that we share indicates a unique metabolism among members of the genus Acetobacter, including a nonfunctional Embden-Meyerhof-Parnas (EMP) pathway and the generation of energy from the incomplete oxidation of ethanol into acetate (21, 22). (A) Carbon transition in the reaction of phosphoenolpyruvate carboxykinase and experimental labeling profiles of oxaloacetate and phosphoenolpyruvate resulting from growth on a mixture of [12C3]lactate and [13C2]ethanol. PDF; ABSTRACT. The resulting metabolic flux distribution is shown in Fig. As a central finding, our data demonstrate a strict separation of two-carbon (ethanol, acetaldehyde, acetate) and three-carbon (lactate, pyruvate, phosphoenolpyruvate) metabolism in AAB. 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). Simulated and experimental mass isotopomer distributions are presented in Fig. Computer-based experimental design predicts optimum combinations of 13C-labeled lactate and ethanol to resolve metabolic fluxes in Acetobacter.In the medium that represented cocoa pulp, lactate and ethanol were the primary substrates among complex ingredients. Copyright © 2014, American Society for Microbiology. The mean squared error between experimental and simulated mass isotopomer distributions was 4.2 × 10−4, indicating an excellent fit. 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. Samples were diluted 1:10 with deionized water prior to analysis. Analogously, it was previously shown that A. ghanensis DSM 18895 and A. pasteurianus NCC 316 did not grow on ethanol with ammonium as the only nitrogen source (31). In particular, their lifestyle in complex environments, such as cocoa pulp, is largely unknown. Acetic acid bacteria exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Our long cooperation with Polish drinks factories shows that Asaia spp. Notably, this reaction requires 2 moles of ATP. Consequently, a balanced ratio of lactate and ethanol is important for the success of the fermentation process. Species interactions determine the quality of cocoa fermentation. Ethanol contributed to only a small set of biosynthetic pathways. Energetic efficiency of different metabolic modes for consumption of lactate, ethanol, acetate, and acetoin. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. Briefly, 500 μl of culture supernatant was mixed with 50 μl of a 0.5% (w/vol) aqueous O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) solution, and the mixture was incubated for 30 min at 80°C. Con-version of ethanol to acetic acid in the presence of a small concentration of oxygen is the main form of spoilage caused by these bacteria (Greenshields 1978; Drysdale and Fleet 1988). In this set of reactions, 2 moles of ATP is consumed in order to produce 1 mole of PEP.