AEM Accepts, published online ahead of print on 16 May 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.01048-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved. 1 2 The key to acetate: Metabolic fluxes of acetic acid bacteria under cocoa pulp 3 fermentation simulating conditions 4 5 Philipp Adlera, Lasse Jannis Freya, Antje Bergera, Christoph Josef Boltenb, Carl Erik 6 Hansenb and Christoph Wittmanna,c* 7 8 9 a Institute of Biochemical Engineering, Technische Universität Braunschweig, 38106 10 Braunschweig, Germany 11 b Nestlé Research Center, Vers-Chez-Les-Blanc, 1000 Lausanne 26 Switzerland 12 c Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, 13 Germany 14 15 16 17 *Corresponding address: Christoph Wittmann, Institute of Systems Biotechnology, 18 Saarland University, Campus A 1.5, 66123 Saarbrücken, Germany, phone 0049-681- 19 30271971, FAX: 0049-681-30272972, Email: [email protected] 20 21 1 22 Abstract 23 Acetic acid bacteria (AAB) play an important role during cocoa fermentation, as their 24 main product acetate is a major driver for development of desired cocoa flavors. Here, 25 we investigated the specialized metabolism of these bacteria under cocoa pulp 26 fermentation simulating conditions. A carefully designed combination of parallel 13C 27 isotope experiments allowed the elucidation of intracellular fluxes in the complex 28 environment of cocoa pulp, including lactate and ethanol as primary substrates, 29 among undefined ingredients. We demonstrate that AAB exhibit a functionally 30 separated metabolism during co-consumption of carbon-two and carbon-three 31 substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for 32 formation of acetoin and biomass building blocks. Although suboptimal from cellular 33 energetics, this allows maximized growth and conversion rates. The functional 34 separation results from lack of phosphoenolpyruvate carboxykinase and malic 35 enzymes, typically present in bacteria to interconnect metabolism. In fact, 36 gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced 37 ratio of lactate and ethanol is important for optimum performance of AAB. As lactate 38 and ethanol are individually supplied by lactic acid bacteria and yeasts, during the 39 initial phase of cocoa fermentation, respectively, this underlines the importance of a 40 well-balanced microbial consortium for a successful fermentation process. Indeed, 41 AAB performed best and produced the largest amounts of acetate in mixed culture 42 experiments, when lactic acid bacteria and yeasts were both present. 43 44 45 Key words 46 Metabolic flux, 13C, fluxome, systems biology, acetate, cocoa 47 2 48 Introduction 49 Acetic acid bacteria (AAB) play an important role in cocoa fermentation (1). During 50 fermentation of pulp, surrounding the cocoa beans, they form acetate. Acetate then 51 diffuses into the beans (2–4), where it initiates a cascade of chemical and biochemical 52 reactions leading to precursor molecules for cocoa flavor (2, 5, 6). Potential 53 substrates for AAB are lactate and ethanol, which are individually produced by lactic 54 acid bacteria (LAB) (mainly Lactobacillus fermentum) and yeasts (diverse yeasts such 55 as Saccharomyces cerevisiae, Hanseniaspora oppuntiae, and Candida crusei) during 56 the fermentation process (6–12). Hereby, degradation of lactate by AAB is desired, 57 since remaining lactate may provide off-flavor in the final cocoa product (11, 13, 14). 58 In recent years, AAB were extensively analyzed for their contribution to cocoa 59 fermentation. Obviously, the most prevalent AAB species is Acetobacter pasteurianus 60 (13, 15–17). In addition, A. ghanensis and A. senegalensis are found during 61 spontaneous cocoa bean fermentation (9, 13, 17, 18). Further studies provided first 62 insights into basic microbiological properties of such strains and macroscopic 63 dynamics during cocoa pulp fermentation (12, 15, 18–20). At this point, it appears 64 relevant to resolve the metabolic contribution of AAB to a greater detail. Basic 65 knowledge that we share indicates a unique metabolism among members of the 66 genus Acetobacter, including a non-functional Embden-Meyerhof-Parnas (EMP) 67 pathway and the generation of energy from incomplete oxidation of ethanol into 68 acetate (21, 22). A few species are able to catabolize lactate (23–26). A pathway from 69 lactate to acetate as final product has been proposed, however the role of some of its 70 enzymes remains unclear (26). In general, little is known about the in vivo contribution 71 of these carbon core pathways to cellular metabolism of AAB. Particularly, their life 72 style in complex environments such as cocoa pulp is largely unknown. In the past 73 decade, 13C metabolic flux analysis has emerged as a routine approach to study 3 74 individual microbes grown on single-carbon substrates on the level of molecular 75 carbon fluxes (in vivo reaction rates) (27, 28). Without resolving details, advanced 76 experimental designs meanwhile provide fluxes for complex microbial systems, 77 including mixed populations (29) and nutrient mixtures (30). 78 79 Here, we applied 13C based metabolic flux analysis to study the metabolism of 80 Acetobacter during cocoa pulp fermentation. Acetobacter pasteurianus NCC 316 and 81 A. ghanensis DSM 18895 were selected as representative strains of two major 82 species of AAB, naturally occurring in cocoa pulp (23, 31). A validated set-up (12) 83 unraveled metabolic fluxes in AAB under cocoa pulp simulating conditions. As central 84 finding, concerted use of lactate and ethanol enables optimum fluxes with regard to 85 growth and energy metabolism. Considering recent insights into extracellular and 86 molecular fluxes of LAB during cocoa fermentation (12, 20, 23, 30) this suggests that 87 the efficiency of AAB depends on compositional traits provided by LAB and yeasts 88 during the initial fermentation process so that the overall success of cocoa 89 fermentation requires a well-balanced microbial consortium. 90 91 Material and Methods 92 Strains and maintenance. Acetobacter pasteurianus NCC 316, Lactobacillus 93 fermentum NCC 575 and Saccharomyces cerevisiae NYSC 2 were obtained from the 94 Nestlé Culture Collection (Lausanne, Switzerland). A. pasteurianus NCC 316 and L. 95 fermentum NCC 575 were previously isolated from cheese (1978) and from coffee 96 (1984), respectively. S. cerevisiae NYSC 2 is the commonly used laboratory strain 97 X2180-1B (MATα gal2 SUC2 mal CUPJ). Acetobacter ghanensis DSM 18895 was 98 obtained from the German Culture Collection (DSMZ, Braunschweig, Germany). 99 Escherichia coli K-12 was used as a reference strain for enzyme activity assays and 4 100 was supplied by the Coli Genetic Stock Center (CGSC, New Haven, CT, USA). A. 101 pasteurianus NCC 316 and A. ghanensis DSM 18895 were stored in mannitol – yeast 102 extract – peptone (MYP) medium (32). L. fermentum NCC 575 was maintained in 103 Lactobacilli MRS (deMan-Rogosa-Sharpe) broth (32). S. cerevisiae NYSC 2 was 104 stored in yeast malt (YM) medium (32). E. coli K-12 was maintained in LB medium 105 (synonymously called lysogeny broth or Luria Bertani broth) (33). All frozen stocks 106 were stored at -80°C and contained 15% (vol/vol) glycerol. 107 108 Cultivation. Cells of A. pasteurianus NCC 316 and A. ghanensis DSM 18895 were 109 re-activated on MYP agar at 30°C for 48 h before use. For pre-cultures and main 110 cultivation of Acetobacter species, the cocoa pulp simulation medium for acetic acid 111 bacteria was used (PSM-AAB) (12). It contained per liter: 10 g ethanol, 7.2 g sodium 112 lactate, 10 g yeast extract (Roth, Karlsruhe, Germany), 5 g soy peptone (Roth), and 1 113 ml Tween 80 (Sigma-Aldrich, Taufkirchen, Germany). The initial pH of the medium 114 was 4.5. In 13C tracer experiments, lactate and ethanol were replaced by equimolar 115 amounts of 98% [U-13C] sodium lactate (Cambridge Isotopes, Andover, MA, USA), 116 98% [3-13C] sodium lactate (Cambridge Isotopes), and 99% [U-13C] ethanol (Sigma- 117 Aldrich), respectively. Pre-cultures and main cultures of L. fermentum NCC 575 and 118 S. cerevisiae NYSC 2 were performed in cocoa pulp simulation medium for lactic acid 119 bacteria (PSM-LAB) (12), which was prepared as described previously (30). For all 120 experiments, cells from agar plates, previously obtained from frozen stocks 121 (Acetobacter), or frozen stocks (L. fermentum and S. cerevisiae) were used to 122 inoculate the first pre-culture (37°C, 12-24 h). Cells were transferred to the second 123 pre-culture, which was incubated at 37°C. Cells were then harvested in the mid- 124 exponential growth phase, washed (6000 × g, 5 min; 4°C) with a peptone mixture (10 125 g liter−1 yeast extract [Roth], 5 g liter−1 soy peptone [Roth]), and used to inoculate the 5 126 main culture to an initial optical density (OD600) of 0.01. All cultivations of A. 127 pasteurianus NCC 316 and A. ghanensis DSM 18895 were performed in disposable 128 baffled shake flasks (250 ml, PreSens Precision Sensing GmbH, Regensburg, 129 Germany) on a rotary shaker at 280 rpm (Infors Multitron II, Infors, Bottmingen, 130 Switzerland). Dissolved oxygen was monitored using a shake-flask reader (SFR, 131 PreSens Precision Sensing GmbH). To account for potential evaporation of volatiles, 132 their loss was monitored in control experiments without cells, incubated under the 133 fermentation conditions described above and evaporation rates were used to correct 134 formation and consumption rates. Cultivations with L. fermentum NCC 575 and S. 135 cerevisiae NYSC 2 were conducted in non-baffled 250-ml shake flaks at 37°C and a 136 rotation speed of 50 rpm. 137 138 In order to study the entire cocoa pulp fermentation, PSM-LAB was inoculated with 139 either L. fermentum, S. cerevisiae or a mixture of both microorganisms and then 140 incubated for 24 h. Subsequently, the fermentation broth was filtered (filter top 250, 141 0.22 µm, TPP Techno Plastic Products, Trasadingen, Switzerland), the pH was 142 adjusted to 4.5, and the medium was further incubated with A.