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

179physiology and the utilization of different carbon sources. In the presentwork, we investigated the glycerol/acetate co-consumption by fission yeast.In contrast to other well-known yeasts like Saccharomyces cerevisiae, S.pombe is not able to use C2-compounds, such as ethanol or acetic acid assole carbon source because the necessary enzymes of the glyoxylat cycleare missing. In 2010, Matsuzawa, et al. reported, that S. pombe is alsounable to use glycerol as sole carbon source, which is in accordance withour results but cannot be explained up to now. In 2011, the simultaneousconsumption of glycerol and acetate by fission yeast has been reported(Klement, et al., 2011). Therefore we composed a minimal mediacontaining glycerol and acetate as sole carbon sources. The specific growthrate of S. pombe was determined as 0.11 h -1 . The biomass yield was 0.48 gCDW g substrate -1 and the respiratory quotient (RQ) 1.05. No ethanol orother typical fermentation products were detected in the culturesupernatant. These findings suggest that glycerol and acetate are coconsumedunder completely respiratory conditions. This is a strikingdifference compared to other yeasts, e.g. S. cerevisiae, where glycerol isused in the fermentative processes for the production of bioethanol.We performed experiments with 13 C-labeld acetate to gain a deeperknowledge of the substrate distribution throughout the entire centralcarbon metabolism. Our results show, that glycerol is used as precursor forglycolysis, gluconeogenesis and the pentose phosphate pathway. Acetate ismetabolized via the tricarboxylic acid cycle (TCA) but glycerol alsocontributes to the acetyl-CoA pool. No transport of mitochondrialoxaloacetate (OAA) into the cytosol was detected. Specific labelingpatterns of proteinogenic amino acids revealed, that amino acids derivedfrom OAA are synthesized exclusively in the cytosol. Further work willconcentrate on the identification of possible regulatory mechanisms tounderstand, why S. pombe does not utilize glycerol as sole carbon source.Klement, T., Dankmeyer, L., Hommes, R., van Solingen, P. and Buchs, J. (2011). Acetate-glycerolcometabolism: Cultivating Schizosaccharomyces pombe on a non-fermentable carbon source in adefined minimal medium.J Biosci Bioeng.112, 20-25.Matsuzawa, T., Ohashi, T., Hosomi, A., Tanaka, N., Tohda, H. and Takegawa, K. (2010). Thegld1+ gene encoding glycerol dehydrogenase is required for glycerol metabolism inSchizosaccharomyces pombe.Appl Microbiol Biotechnol87,715-27.PSP015A miniaturized parallel bioreactor system for continuouscultivation studies on yeastK. Schneider*, T. Klein, E. HeinzleSaarland University, Biochemical Engineering, Saarbruecken, GermanyChemostat cultivation is a powerful tool for physiological studies onmicroorganisms. The cells are kept at a stable physiological steady stateand manipulations of environmental parameters like aeration and substrateavailability are possible. The disadvantages of this system involve a longcultivation time to achieve a steady state and high substrate consumption,which can be problematic if expensive substances are used, e.g.isotopically labeled compounds.We report the construction and application of a set of parallel bioreactorswith 10 ml working volume for continuous cultivation. A similiar systemhas already been described for E. coli (Nanchen, et al., 2006) but has notbeen adapted to yeast cultivation up to now. Hungate tubes are used asculture vessels and placed in a water bath to maintain 30°C cultivationtemperature. The rubber septum is pierced by needles, one connected to amultichannel peristaltic pump for feeding fresh media. A secondmultichannel pump is used for constant removal of culture broth to keepthe culture volume at 10 ml. Since the efflux pump rate is far in excess ofthe feeding rate it is also used to induce aeration by generating underpressure inside the culture vessel. Sterile, water-saturated air is sucked intothe tube via the third needle. A magnetic stirrer bar (9 x 6 mm) at thebottom of the vessel allows proper mixing and boosts oxygen transfercompared to a purely bubbled system. Dissolved oxygen (DO) wasconstantly measured via optical DO sensors to ensure aerobic conditions.In addition the DO-concentration is a powerful indicator of thephysiological state of the cells inside the bioreactor. Off-gas analysis isperformed by means of mass spectrometry.Our system can be applied for continuous cultivation of yeast cells in up to8 parallel bioreactors. DO-concentration profiles clearly indicate theachievement of the steady state. Utilization of magnetic stirrer barsguarantees proper mixing prohibiting sedimentation of cells and permitsthe use of small aeration rates (1 vvm) which is beneficial for accurate offgasanalysis. We used this system to characterize the shift from respiratoryto respiro-fermentative growth for Schizosaccharomyces pombe andperformed cultivations with 13 C-labeled substrate to determine intracellularfluxes through the central carbon metabolism.Nanchen, A., Schicker, A. and Sauer, U. (2006). Nonlinear dependency of intracellular fluxes ongrowth rate in miniaturized continuous cultures of Escherichia coli.Appl EnvironMicrobiol72,1164-72.PSP016Biochemical and kinetic analysis of the acidophilic c-typecytochrome thiosulfate dehydrogenase from differentProteobacteriaK. Denkmann* 1 , A. Siemens 1 , J. Bergmann 1 , R. Zigann 1 , F. Grein 2 ,I. Pereira 2 , C. Dahl 11 Universität Bonn, Institut für Mikrobiologie und Biotechnologie, Bonn,Germany2 Universidade Nova de Lisboa, Instituto de Tecnologia Química eBiológica, Oeiras, PortugalThe acidophilic tetrathionate-forming enzyme thiosulfate dehydrogenasewas isolated from the purple sulfur bacterium Allochromatium vinosum[1]and the corresponding gene (tsdA, YP_003442093) was identified on themain A. vinosum chromosome (NC_013851) on the basis of the previouslydetermined N-terminal amino acid sequence. Thiosulfate dehydrogenase isa periplasmic, monomeric 25.8 kDa c-type cytochrome with an enzymeactivity optimum at pH 4.3. UV-Vis and EPR spectroscopy indicatemethionine (strictly conserved M 222 or M 236) and cysteine (strictlyconserved C 123) as probable sixth distal axial ligands of the two heme ironsin TsdA. In addition UV-Vis spectroscopy revealed a minor peak at 635nm which was assigned to the iron high-spin state. The low intensity ofthis high-spin-marker indicates that only a small portion of hemes exists in5-coordination. An EPR spectrum of TsdA supplemented with its naturalelectron donor thiosulfate showed that the high spin heme is completelyreduced at pH 5.0 but not at pH 8.0, which corresponds with the enzymesoptimum pH for activity. Furthermore we determined the redox potentialof the hemes.Genes homologous to tsdA are present in a number of -, -, - and -proteobacteria. The wide-spread occurrence of tsdA agrees with reports oftetrathionate formation not only by specialized sulfur oxidizers but also bymany chemoorganoheterotrophs that use thiosulfate as a supplemental butnot as the sole energy source. For further analysis of TsdA we chose thefacultative chemolithoautotrophic well-established sulfur oxidizerThiomonas intermedia[2], the chemoorganoheterotrophic Pseudomonasstutzeri, for which tetrathionate formation from thiosulfate had previouslybeen reported [3] and the psychro and halotolerant heterotrophicPsychrobacter arcticus[4], for which sulfur-oxidizing capabilities havenever been investigated. All three proteins were produced in E. coli andproven to be c-type cytochromes which exhibited high specific thiosulfatedehydrogenase activities.[1] Hensen et al. (2006) Mol. Microbiol.62, 794-810[2] Moreira and Amils (1997) Int. J. Syst. Bacteriol.47,522-528[3] Sorokin et al. (1999) FEMS Microbiol. Ecol.30, 113-123[4] Bakermans et al. (2006) Int. J. Syst. Evol. Microbiol.56, 1285-1291PSP017Effects of High CO 2 Concentrations on Typical AquiferMicroorganismsA. Schulz*, C. Vogt, H.H. RichnowHelmholtz Centre for Environmental Research - UFZ, IsotopeBiogeochemistry, Leipzig, GermanyThe sequestration of carbon dioxide into the deep underground isconsidered as one option to reduce the emission of carbon dioxide into theatmosphere. A leakage of carbon dioxide from a deep storage site into ashallow aquifer is one of the main concerns connected to the CarbonCapture and Storage (CCS) technology. For a proper risk assessment it isnecessary to study the influence of high CO 2 concentrations, as a result ofleakage, on microorganisms, occurring in shallow aquifers. Therefore,growth curves and survival rates for four ecophysiologically distinct modelorganisms, ubiquitous in shallow aquifers, were determined. CO 2concentrations in the gas phase varied between approximately 0 (refers tono amendment of CO 2) to 80% for the aerobic strains Pseudomonas putidaF1 and Bacillus subtilis 168 and roughly 0 to 100% CO 2 for the nitratereducingstrain Thauera aromatica K172 and the sulfate-reducing strainDesulfovibrio vulgaris Hildenborough. Carbon dioxide that infiltrates afreshwater aquifer under oxidizing conditions and under atmosphericpressure will have an immediate impact on water chemistry, leading to areduction in pH. In our experiments, the pH of the growth mediumdecreased for about one unit from seven to six after the addition of CO 2.To distinguish between effects caused by carbon dioxide and the influenceof decreasing pH-values, parallel experiments without CO 2 addition anddecreased pH were performed. The results showed that growth andviability of all four strains were reduced at high CO 2 concentrations (>50%), however, the aerobic strains are more sensitive to CO 2 stresscompared to the anaerobic strains. After experiments at ambient pressure,growth experiments with increasing CO 2 concentrations and increasingpressure from 1 to 5000 kPa were performed in self constructed pressurevessels to simulate conditions typically occurring in deep aquifers. Thecombination of pressure and high CO 2 concentrations reduced significantlythe viability of all tested strains. These results give first information for aconcrete risk evaluation of the CCS technology and potentially leakagerelatedmicrobiological changes in shallow aquifers.BIOspektrum | Tagungsband 2012