c Acetic acid bacteria (AAB) play an important role during cocoa fermentation, as their main product, acetate, is a major driver for the development of the desired cocoa flavors. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. A carefully designed combination of parallel 13 C isotope labeling experiments allowed the elucidation of intracellular fluxes in the complex environment of cocoa pulp, when lactate and ethanol were included as primary substrates among undefined ingredients. We demonstrate that AAB exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for the formation of acetoin and biomass building blocks. Although this is suboptimal for cellular energetics, this allows maximized growth and conversion rates. The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. In fact, gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. As lactate and ethanol are individually supplied by lactic acid bacteria and yeasts during the initial phase of cocoa fermentation, respectively, this underlines the importance of a well-balanced microbial consortium for a successful fermentation process. 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.A cetic acid bacteria (AAB) play an important role in cocoa fermentation (1). During fermentation of the pulp that surrounds the cocoa beans, they form acetate. Acetate then diffuses into the beans (2-4), where it initiates a cascade of chemical and biochemical reactions leading to precursor molecules for cocoa flavor (2, 5, 6). Potential substrates for AAB are lactate and ethanol, which are individually produced by lactic acid bacteria (LAB; mainly Lactobacillus fermentum) and yeasts (diverse yeasts such as Saccharomyces cerevisiae, Hanseniaspora opuntiae, and Candida krusei), respectively, during the fermentation process (6-12). Hereby, the degradation of lactate by AAB is desired, since the remaining lactate may provide an off flavor in the final cocoa product (11,13,14). In recent years, AAB have been extensively analyzed for their contribution to cocoa fermentation. Obviously, the most prevalent AAB species is Acetobacter pasteurianus (13,(15)(16)(17). In addition, Acetobacter ghanensis and Acetobacter senegalensis are found during spontaneous cocoa bean fermentation (9,13,17,18). Further studies provided first insights into the basic microbiological properties of such strains and macroscopic dynamics during cocoa pulp fermentation (12,15,(18)(19)(20). At this point, it appears to be relevant to resolve the metabolic contribution of AAB in greater detail. The basic know...

Pseudomonas aeruginosa is a human pathogen that frequently causes urinary tract and catheter-associated urinary tract infections. Here, using 13C-metabolic flux analysis, we conducted quantitative analysis of metabolic fluxes in the model strain P. aeruginosa PAO1 and 17 clinical isolates. All P. aeruginosa strains catabolized glucose through the Entner-Doudoroff pathway with fully respiratory metabolism and no overflow. Together with other NADPH supplying reactions, this high-flux pathway provided by far more NADPH than needed for anabolism: a benefit for the pathogen to counteract oxidative stress imposed by the host. P. aeruginosa recruited the pentose phosphate pathway exclusively for biosynthesis. In contrast to glycolytic metabolism, which was conserved among all isolates, the flux through pyruvate metabolism, the tricarboxylic acid cycle, and the glyoxylate shunt was highly variable, likely caused by adaptive processes in individual strains during infection. This aspect of metabolism was niche-specific with respect to the corresponding flux because strains isolated from the urinary tract clustered separately from those originating from catheter-associated infections. Interestingly, most glucose-grown strains exhibited significant flux through the glyoxylate shunt. Projection into the theoretical flux space, which was computed using elementary flux-mode analysis, indicated that P. aeruginosa metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response.

BackgroundSingle cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.ResultsHere different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied.ConclusionsIt was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites. To investigate the expression rates of P. aeruginosa proteins commonly expressed by different clinical isolates, absolute protein quantities were determined employing a gel-free and data-independent LC-IMS(E) approach. Moreover, the metabolic diversity of these isolates was investigated by (13) C-metabolic flux analyses. 812 proteins were reproducibly identified and absolutely quantified for the reference strain P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all isolates. Whilst the majority of these proteins were expressed in constant amounts, expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in burned wounds isolates compared to lung/urinary tract isolates. Moreover, proteins involved in iron/amino acids uptake were found to be highly abundant in urinary tract isolates. The fluxome data revealed a conserved glycolysis, and a niche-specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated proteome/fluxome analysis did not indicate straightforward correlation between the protein amount and flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments. All MS data have been deposited in the ProteomeXchange with identifier PXD002373 (http://proteomecentral.proteomexchange.org/dataset/PXD002373).