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

143[2], van Ginkel, C. G., 1996. Biodegradation 7:151-164.[3], Schleheck, D.et al.,2004. Appl Environ Microbiol 70:4053-4063.[4], Schleheck, D. et al., 2010. Appl Environ Microbiol 76:196-202.OTP023Use of transcription factors to visualize small-molecules at thesingle cell level, and application for metabolic engineeringG. SchendzielorzForschungszentrum Jülich Gmbh, IBG1: Biotechnologie, Jülich, GermanySuccessful mutant development in microbial biotechnology relies onrandom mutations and combinatorial approaches. A current limitation isthe subsequent screening of bacterial populations for cells with increasedproduction properties. We developed sensors to quantify metaboliteswithin a single cell. Together with FACS this enables the isolation ofsingle producer cells from large mutant libraries. The system is based on atranscriptional regulator and its target gene fused to eyfp. Sincetranscriptional regulators exist which naturally sense numerous smallmolecules,our technology enables a various new applications.As one example we use the transcriptional regulator LysG of C.glutamicum sensing basic amino acids. Introducing the sensor pSenLys ina C. glutamicum mutant producing L-lysine or L-arginine resulted instrong fluorescent cells, which was not the case with controls. The keyenzyme of L-arginine synthesis is the argB encoded acetylglutamatkinasewhich is inhibited in its activity by L-arginine. A plasmid-encoded argBmutant library was generated via epPCR and introduced intoC.glutamicum carrying pSenLys. Applying FACS selection, sequencingand acetylglutamatkinase activity determination 16 argB alleles wereisolated carrying 22 different mutations. Whereas wild type argB isinactive at 0.5 mM L-arginine, mutant alleles were selected which retainedfull activity at 4 mM L-arginine.As another example we treated the wild type of C.glutamicum carryingpSenLys with N-methyl-N-nitro-N-nitrosoguanidine. Out of 6.5 x 10 6cells 270 cells were selected, of which 225 accumulated 3-38 mM L-lysine. Targeted sequencing identified 13 new chromosomal mutations inthe known targets lysC and hom. From 10 mutants with no mutation inknown targets the entire genome was sequenced using Illumina HiSeq2000 technology. A murE mutation was identified which when introducedinto existing L-lysine producers improved the L-lysine titers significantlyOTP024Correlations between process parameters and the microcosmof biogas fermentersN. Krakat*, P. SchererHAW, LifeSciences, Hamburg, GermanyThe influence of the process parameters hydraulic retention time (HRT),organic loading rate (OLR), substrate and temperature upon bacterialdiversity was analyzed in automated fermenters. Therefore, a mesophilic(41°C) and thermophilic (55 and 60°C) anaerobic fermentation of beetsilage as model substrate for renewable biomass was monitored by theamplified ‘‘ribosomal DNA’’ restriction analysis (ARDRA).Surprisingly, a predominant population of hydrogen utilizingEuryarchaeota (represented by Methanobacteriales, Methanomicrobiales)was observed under all operating modes. The acetotrophic Methanosaetassp. and Methanosarcina spp. played apparently only a minor role amongthe operational taxonomic units (OTUs) found. Under thermophilicconditions Methanosaeta spp. could even not be detected.This contradicts to common models for anaerobic digestion processes. Animportant finding was that under thermophilic conditions a change intemperature from 60 °C to 55 °C and back to 60 °C again was an importantparameter to impact reversibly the morphological diversity ofmethanogenic Euryarchaeota. They changed from a mixture ofmethanogenic cocci and rods to an exclusive appearance of rods and vice versa.Under mesophilic conditions the temperature was held constant andvariations of the hydraulic retention time (HRT) influenced remarkably thediversity of methanogens. Long HRTs (e.g. 37 days) kept the level ofmethanogenic species richness low, while quickly decreased HRTs (e.g. 8days) induced a higher diversity and similar diversity patterns, respectively.This study also revealed that the population dynamics, the species richnessand diversity of hydrolytic and fermentative Bacteria was higher comparedto the diversity of methanogenic Archaea.Under mesophilic andthermophilic fermentation temperatures, most of the detected OTUs couldbe assigned to the Phyla Firmicute, Bacteroidetes andProteobacteria,while Chloroflexi appear to play an important but yetunknown role during a mesophilic biogas process with high nutrient levelsof renewable biomass like beets. Astonishingly, only single bacterial phylacould be impacted. One explanation of this phenomenon could be thefunctional redundancy of carbohydrate degraders. The presence of the taxaPlanctomycetes, Actinobacteria and Alcaligenaceae was related to longHRTs and short OLRs, while the Phylum Acidobacteria was governed byshort HRTs and high OLRs, respectively.OTP025Identification of Klebsiella pneumoniae’s strains isolated from« urine » as a human pathological product and evaluation of theirantibiotic resistanceK. Bensalem*, H. ChettibiBadji Mokhtar University, Laboratory of Microbiology. Department ofBiochemistry, ANNABA, AlgeriaOur study was about the biochemical identification of Klebsiellapneumoniae’s strains which were isolated from “urine” as a humanpathological product, in addition to the evaluation of their sensibility toantibiotics. The results synthesized from this research have shown that:K.pneumoniae has the ability to produce “acetoin” from “pyruvic acid”,hence it is characterized by a positive Voges-Proskauer reaction.The results of “the antibiogram” have confirmed the efficiency of“colistin” as an antibiotic on our strains. We have also shown theproduction of BLSE enzymes (Beta Lactamases with Extended Spectrum)by some strains. In addition to this, we have tested the effect of“inoculum” on the resistance to “cefotaxim” and to the association“amoxicillin + clavulanic acid” and the results have shown a widening ofthe circle’s diameter surrounding the antibiotic’s disc after dilution, whichexplains a higher sensibility of strains to antibiotics. This experience of“inoculum’s effect” has shown us that from a lower inoculum (afterdilution) results a higher sensibility.OTP026Isomer and enantioselective carbon stable isotope fractionation ofhexachlorocyclohexane during aerobic biodegradation bySpingobium sppS. Bashir*, H.-H. Richnow, I. NijenhuisHelmholtz Centre for Environmental Research GmbH - UFZ, Isotopebiogeochemistry, Leipzig, GermanyIn biochemical processes the preferential reactivity of the lighter stableisotope over the heavier stable isotope results in enrichment of the heavierisotopes in the residual substrate and relative enrichment of the lighterisotope in the products. The isomer and enantioselective carbon stableisotope fractionation of organic contaminants such ashexachlorocyclohexane and its chiral isomers (-HCH) may be used toassess their fate in the environment. The extent ofin situtransformationmay therefore be inferred by using experimentally determined compoundspecific isotope fractionation factors during biotransformation by definedmicrobial cultures. In this study, carbon isotope fractionation factors weredetermined for the biotransformation of and -HCH using two aerobicbacterial strains: Sphingobium indicum B90A and Sphingobium japonicumUT26. Batch culture biodegradation experiments were performed and thecarbon isotope fractionation of -HCH degradation was quantified by theRayleigh equation. The bulk enrichment factor for -HCH was highlysimilar (C= -1.8) for both S. japonicum UT26 and S. indicum B90A, butless compared previously reported values for anaerobic HCHdechlorination (-3.9±0.6) [1]. Additionally, the carbon isotopefractionation for -HCH and its enantiomers was quantified. Interestingly,carbon isotope fractionation of -HCH by S. japonicum was in a similarrange to -HCH; for S. indicum fractionation was about 3 fold higher.Similarly, preliminary investigation showed that fractionation of -HCHenantiomers was corresponding to the bulk isotope fractionation of -HCH. The differences in fractionation may be due to the presence andactivity of the different dehalogenases (Lin) in these organisms. Therefore,although a qualitative assessment of biodegradation of HCHin situmay bepossible, a quantitative assessment requires further investigations.[1]Badeaet al.(2009) Environmental Science & Technology 43(9), 3155-3161.OTP027The ability of Iranian traditional dairy bacterial strains todetoxification of Aflatoxin B1P. Jafari* 1,2 , M. Tajabadi Ebrahimi 2,3 , S.D. Hosseini 2,3,41 Islamic Azad University, Arak barnch, Microbiology, Science faculty, Tehran, Iran2 Islamic Azad University (IAU), Arak Branch, Microbiology, Science faculty,Arak, Iran3 Islamic Azad University, Central Tehran Branch, cellular and molecularbiology, Tehran, Islamic Republic of Iran4 Razi Vaccination and Serum Research, Cellular and Molecular Biology, Arak,Islamic Republic of IranIntroduction: Aflatoxins such as Aflatoxin B1 (AFB1) are highly toxic,mutagenic, teratogenic and carcinogenic compounds produced by somespecies ofAspergillus.They are found in many foods and feeds andconsidered as a major public health problem especially in developing countries.This study was conducted investigate the AFB1 detoxification ability of 60probiotic bacteria isolated from Iranian traditional dairy products.Method: A working solution of 5 g/ml of AFB1 was prepared inphosphate-buffered saline (PBS, pH 7.3). Bacterial suspension wasprepared by culturing the strains in MRS broth at 37°C for 20h. TheseBIOspektrum | Tagungsband 2012