VAAM-Jahrestagung 2012 18.–21. März in Tübingen

135Union’s 7 th Framework Programme FP7/2007-2013 under grant agreementN° 266025.[1] Liu, C. et al. (2011): Biomacromolecules 12: 3291-3298[2] Yang, Y.X. et al. (2010): Biomacromolecules 11: 259-268[3] Craft, D.L. et al. (2003): Appl Environ Microbiol 69: 5983-5991[4] van Beilen, J.B. et al. (2006): Appl Environ Microbiol 72: 59-65[5] Fujii, T. et al. (2006): Biosci Biotechnol Biochem 70: 1379-1385[6] Scheps, D. et al. (2011): Org Biomol Chem 9: 6727-6733OTV020Using yeast and fungi to produce electricity- Towards a self-regenerating enzymatic biofuel cell cathodeS. Sané* 1 , S. Rubenwolf 1 , C. Jolivalt 2 , S. Kerzenmacher 11 University Freiburg, MEMS application, Freiburg, Germany2 Chimie ParisTech, Paris, FranceBiofuel cells (BFCs) directly transform chemical energy into electricity foras long as fuel and oxidant are supplied. To catalyze the electrode reactionin biofuel cells, for instance biochemical pathways of completemicroorganisms or enzymatic biocatalysts can be used [1].The aim of our research is to improve the long-term stability of efficient,but currently short-lived enzymatic biofuel cell electrodes [2]. We aim tocontinually supply catalytically-active enzymes at the electrode usingliving microorganisms that grow in an electrode-integrated microbioreactor.In the present work, we demonstrate the feasibility of using the crudeculture supernatant of the fungus Trametes versicolor and the recombinantyeast Yarrowia lipolytica [3] to supply the biocatalyst laccase to a biofuelcell cathode. Both T. versicolor and Y. lipolytica were grown in a syntheticdeficient (SD) medium. At approximately the highest enzyme activity,which was 3.6 U/ml for T. versicolor and 0.02 U/ml for Y. lipolytica,culture supernatant was transferred into a biofuel cell cathodecompartment [4]. To record the loadcurve, current was incrementallyincreased (steps of 5.6 A/(cm 2 *h)) and the cathode potential wasmeasured against a saturated calomel electrode (SCE). At a cathodepotential of 0.4 V vs. SCE, we obtained a current density of 134 A/cm 2for T. versicolor. The same enzyme activity of commercial T. versicolorlaccase (Sigma) in SD medium yielded a current density of only 75A/cm 2 and in citrate buffer a current density of 87 A/cm 2 . For Y.lipolytica, a current density of 4 A/cm 2 was measured. The same amountof purified laccase from Y. lipolytica in SD medium and in citrate bufferresulted in a current density of 8 A/cm 2 and 12 A/cm 2 respectively.Our results are a first step towards constructing a self-regeneratingenzymatic biofuel cell with extended lifetime. Furthermore, we haveshown that the choice of microorganism, has an influence on the obtainedcurrent density, because it has a large influence on the composition of theculture supernatant as well as on laccase activity. Important topics forfuture work will be the clarification of the secreted byproducts and theintegration of the laccase-producing microorganisms in the electrodecompartment.[1] R.A. Bullen et al., Biosens. Bioelectron., (2006), pp. 2015-2045[2] S. Rubenwolf et al., Appl. Microbiol. Biotechnol., (2011), pp. 1315-132[3] C. Jolivalt et al.,Appl Microbiol Biotechnol., (2005) pp. 450-456[4] A. Kloke et al., Biosens. Bioelectron. (2010), pp. 2559-2565OTV021Biofilms - a new Chapter in BiocatalysisK. Bühler*, R. Karande, B. Halan, A. SchmidTU Dortmund, BCI / Biotechnik, Dortmund, GermanyIn biocatalysis, the traditional bottlenecks like low biocatalyst stability,toxicity problems, and difficulties in running continuous processes are stillprevailing. A most promising approach to counteract such shortfalls is theexploitation of biofilms for producing industrially relevant compounds.Biofilm formation is a common feature of microbes. Under certainconditions, they attach to various kinds of surfaces and form a sort ofsessile community at aqueous solid interfaces [1] . Advantages of biofilmgrowing organisms as compared to their planktonic counterparts are theirphysical robustness, the ability to self-immobilize, and their long-termstability. In the recent years, we developed a number of different biofilmreactors and characterized the biofilm catalyst under reaction conditions.In this presentation, we will introduce three different biofilm reactorconcepts and point out advantages and disadvantages. The basicconfiguration of a membrane attached biofilm reactor (MABR) turned outto be severely oxygen limited and difficult to scale up [2] . This shortcomingwas circumvented by introducing a dual purpose ceramic membrane intothe reactor system, which simultaneously served as growth surface for thebiocatalyst and as aeration device [3] . This system is currently underevaluation for scale up.In a novel approach, we combined a capillary three phase (aqueousorganic-gas)segmented-flow reactor with catalytic biofilms [4] . Based onthe internal shear forces within such micro-capillary reactor systems, thisset-up takes advantage of high mass transfer rates. In addition, these shearforces prevent the system from clogging and control the biofilm thickness.We will present data regarding the conversions of octane and styrene tooctanol and (S)-styrene oxide, respectively, in these different set-ups anddiscuss the pros and cons of these approaches for biofilm based catalysis.[1] Rosche, B. et al., 2009, Trends in Biotechnol.27: p. 636-43.[2] Gross, R. et al., 2010, Biotechnol Bioeng.105: p. 705-17[3] Halan, B. et al., 2010, Biotechnol Bioeng,106: p. 516-27[4] Schmid A., et al. 2011, PCT/EP2011/057724, FiledOTV022Growth-decoupled, anaerobic succinate production fromglycerol with pyruvate-kinase deficient E. coli mutantsS. Söllner* 1 , M. Rahnert 2 , M. Siemann-Herzberg 2 , R. Takors 2 , J. Altenbuchner 11 Institut für Industrielle Genetik, Universität Stuttgart, Stuttgart, Germany2 Institute of Biochemical Engineering, University of Stuttgart, Stuttgart,GermanyWe constructed E. coli strains for succinate production from glycerol by arational combination of gene deletions and a concomitant evolutionarydesign. Based on elementary mode calculations the formation of 1 molsuccinate from 1 mol glycerol with simultaneous fixation of 1 mol of CO 2represents the theoretical maximum yield. This can be realized if succinateis exclusively formed by PEP carboxylation followed by the reductivebranch of the tricarboxylic acid cycle. Therefore the genes pykF and pykA,both encoding pyruvate kinases, were deleted. Otherwise, the pyruvatekinases would catalyze the direct conversion of PEP into pyruvate. Theresulting strains could, however, barely grow on glycerol, presumablycaused by a certain pyruvate shortage. Only after a selection procedure forfaster growth, the evolved mutants revealed growth rates in the range of0.3 h -1 . In these strains, pyruvate was most likely formed through a novelpathway. It is proposed to be based on a complete ‘rerouting’ ofmetabolism which includes the following steps: PEP carboxylation tooxaloacetate, conversion of oxaloacetate to malate, and decarboxylation ofmalate to pyruvate, further termed POMP. Evidence for the POMPpathwaycomes from the deleterious effects on growth after furtherdeletion of genes coding for malic enzymes. The strain ss279 (pykApykF gldA ldhA poxB pflB tdcE) was finally used in a ‘zerogrowthcultivation’ setup (direct biotransformation from glycerol tosuccinate) at a cell concentration of 0.4 g/L (DCW). To implement this, thegeneration of cell mass was aerobically assured, prior to the ultimateproduction phase, since E. coli is not able to grow anaerobically onglycerol, as long as a necessary external electron acceptor is absent.Consequently, succinate was produced from glycerol and carbon dioxide(or bicarbonate) in an adjacent anaerobic production phase at non-growingconditions. At initial lab-scale, we observed continuous succinateproduction over a period of 6 days. Herein, 58 mM glycerol was consumedand 48 mM succinate was produced, which corresponded to an averagemolar yield of 82 %. This indicated a net fixation of CO 2 in the productionphase, which was further confirmed by stable isotope labelling assays,proving the incorporation of 13 C-labeled bicarbonate into the producedsuccinate.OTV023Gradual insight into Corynebacterium glutamicum`s centralmetabolism for the increase of L-lysine productionJ. van Ooyen*, S. Noack, M. Bott, L. EggelingForschungszentrum Jülich GmbH, IBG-1: Biotechnologie, Jülich, GermanyCorynebacterium glutamicum is used for the large production of aminoacids like L-glutamate, L-valine or L-lysine, the latter made in a scale of8x10 5 annual metric tons. We applied a stoichiometric model andidentified citrate synthase (CS) as most promising target to increase L-lysine production. We therefore replaced the two promoters which weidentified in front of the CS gene gltA of a lysine producer by ninepromoters of decreasing strength. The resulting set of strains wassubsequently analysed with respect to CS activity, growth, and L-lysineyield. The decrease of CS-activity below 30% led to an increase in L-lysine yield accompanied by a decrease in growth rate. A reduced CSactivityof 6% produced an increase in L-lysine yield from 0.17 g/g to 0.32g/g. As a further step the global consequences at the transcriptome,metabolome, and fluxome level were monitored within the strain series.Reduced CS activity results in altered expression of genes controlled byRamA and RamB, and increased cytosolic concentrations of aspartate andaspartate-derived amino acids. The fluxome study revealed that reducedCS-activity surprisingly has only a marginal influence on CS flux itself,but increases the internal concentration of its substrates oxaloacetate andacetyl-CoA, thus showing that the observed systemwide macroscopiceffects are due to locally bordered differences.This systemic approach opens an exciting new view on the system C.glutamicum as an excellent and robust producer of bulk compounds andraises new challenges for stoichiometric models applied to the living cell.BIOspektrum | Tagungsband 2012