VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

acids, indicating that pyruvate is a substantial basis for primary metabolites.Our results provided detailed information on the carbon metabolism of thestrain GD1, indicating a metabolic versatile lifestyle in situ.EMP074Detection of tar oil degrading bacteria with PCR and anovel most probable number (MPN) testC. Zawadsky*, C. Stoll, C. Kraffert, S. Krassnitzer, A. TiehmDepartment of Environmental Biotechnology, Water Technology Center(WTZ), Karlsruhe, GermanyAbandoned manufactured gas plant sites and landfills containing gasworksresidues are important emittants of tar oil pollutants. Additionally to BTEXand PAH, the NSO-heterocyclic aromatic compounds are increasinglyconsidered in monitoring programmes. The presence of bacteria with thecapability to degrade tar oil pollutants is a pre-requisite for bioremediation.In our study, molecular biological methods (PCR) and culture techniques(MPN) for the detection of pollutant degrading bacteria are examined.PCR-analysis of oxygenases, key enzymes of the aromatic metabolism ofaerobic bacteria, represents a promising approach to detect hydrocarbondegrading bacteria. A qPCR method for the detection of mono- anddioxygenases was established. Specific aromatic dioxygenase (nahAc, PAH-RHD) and monooxygenase (tmoA) genes were demonstrated to occur in taroil contaminated groundwater.Bacterial numbers of BTEX, PAH and NSO-HET degraders in groundwatersamples are additionally determined by the MPN (Most Probable Number)microplate technique. For the detection of 2- and 3-ring NSO-HETdegraders a novel MPN method was established. Mixtures of 2- and 3-ringheterocyclic compounds were provided over the gas atmosphere. MTT, atetrazolium salt, is absorbed by living cells only and reduced by the bacteriametabolically active. Formation of blue formazan crystals enabled theenumeration of NSO-HET degrading bacteria.Funding by the BMWi (project no KF2265706SA9) is gratefullyacknowledged.EMP075Establishment of a standard operational procedure forcharacterization of work related microbial exposure levelM. Elena, U. Jäckel, C. Klinger*Federal Institute for Occupational Safety and Health (BAuA), BiologicalAgents, Berlin, GermanyToday’s large-scale poultry production is often accompanied by highconcentrations of airborne microorganisms at working places. Tocharacterise the work related microbial exposure level (WoRMEL)standardisied operational procedures are essential to compare the exposurelevel on the manifold agricultural working places.Current quantitative detection methods of airborne bacteria are based oncultivation approaches using non selective culture medium like TSA.However, a qualitative statement can not be made because of the timeconsuming but necessary isolation procedure. Furthermore, nonviable ordead bacterial cells which can also cause negative health effects remainundetected. Here, molecular approaches can be a useful alternative.Therefore, the aim of this study was the establishment of a cultivationindependent assay to characterise the bacterial diversity in bioaerosolsobtained from poultry processing plants. A fundament within this assay is anoptimal DNA extraction from collected bioaerosols. To establish a validatedDNA isolation protocol four commercial DNA extraction kits (GenElute TMPlant Genomic DNA Miniprep Kit, Sigma-Aldrich; innuPREP BacteriaDNA Kit, Analytik Jena Biometra; peqGOLD Bacterial DNA Kit, Peqlab;FastDNA® Spin Kit for Soil, MP Biomedicals) each in combination withmechanical treatment were used in parallel. To compare the results onedefined bioaerosol sample from an exhaust air flue of a broiler shed wereinvestigated. The amount of extracted DNA was determined fluorometically.The qualitative comparison was done by RFLP analysis of 16S rRNA genePCR products using agarose gel electrophoresis and automated chip basedelectrophoresis (Biorad Experion TM ). Preliminary results showed that two ofthe four used DNA extraction kits were unusable because no positive PCRproducts could be obtained. RFLP analyses of 16S rRNA PCR productsobtained from DNA which was extracted by the remaining kits resulted inquite similar restriction pattern indicating an equivalent quality. For moredetailed qualitative analyses PCR products will be analyzed by generating16S rRNA gene clone libraries. Results of phylogenetically assignment andcomparison of 16S rRNA gene sequences will be discussed at the posterpresentation.EMP076Microbial activity in schwertmanniteJ. Kipry*, C. Wiacek, M. SchlömannDepartment of Environmental Microbiology, University of Mining andTechnology, Freiberg, GermanyMining activities in Lusatia result in mine water that is loaded with highconcentrations of iron and sulfate. A biological technology has highpotential for mine water treatment, since the immobilization of iron canoccur by microbial iron oxidation. Such an approach is realized in a pilotplant at the open pit Nochten where indigenous bacteria oxidize iron whichsubsequently precipitates as the iron-oxyhydroxysulfate schwertmannite.As shown previously [1], bacteria can be found in the water as well as in theschwertmannite that is deposited on carrier material. For stabilizing theprocess of iron oxidation and increasing the iron oxidation rate, therecirculation of schwertmannite was considered. Therefore, the activity ofmicroorganisms in schwertmannite on carrier material and in agingschwertmannite was investigated.The investigations of the activity of bacteria in schwertmannite on carriermaterial showed that the total cell number decreases with increasing depthof the mineral layer. The determination of the percentages of living and deadcells with the LIVE/DEAD® BacLight TM Bacterial Viability Kit(Invitrogen) revealed a decrease of living cells and a corresponding increaseof dead cells with increasing depth of the schwertmannite layer. Furthermorethe composition of the microbial community was analyzed by fluorescencein situ hybridization (FISH) and T-RFLP. Heinzel et al. [2] reported that‘Ferrovum myxofaciens' and Gallionella-relatives dominated in the water. Inthe schwertmannite layer these two groups could be also found as thedominant species. However, a significant change in the structure of themicrobial community in the different depths of the schwertmannite layerwas not observed.In the aging schwertmannite the activity of the microorganisms, determinedwith LIVE/DEAD® BacLight TM Bacterial Viability Kit, was almost constantover a several weeks.The results suggest that a recirculation of schwertmannite could increase theoxidation rate, since relatively high bacterial activity exists in the mineralsludge.[1] Heinzel, E. et al (2009): Applied and Environmental Microbiology 75(3):858-861.[2] Heinzel, E. et al (2009): Environmental Science & Technology 43(16): 6138-6144.EMP077The Earthworm Aporrectodea caliginosa Augments theMicrobial Degradation of 2,4-Dichlorophenol inAgricultural SoilA. Ramm*, H.L. Drake, M.A. HornDepartment of Ecological Microbiology, University of Bayreuth, Bayreuth,GermanyEarthworms play an important role in processing soil organic matter andcontribute to the removal of organic pollutants from soil. 2,4-dichlorophenol(2,4-DCP) represents the initial degradation product of the widely usedherbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Aerobic microbialprocesses are important to mineralization in soil. ‘Hotspots’ of microbialactivity in soils include the drilosphere, i.e., earthworm gut content, cast, andburrows. Earthworms (Aporrectodea caliginosa) accelerated thedisappearance of 2,4-DCP in soil columns. Most probable numbers (MPNs)of 2,4-DCP degraders (a) in bulk soil of columns with and withoutearthworms approximated 6∙10 5 and 6∙10 3 g -1 DW , respectively, and (b) inburrow walls approximated 9∙10 6 g -1 DW . Mineralization of [U 14 C]-2,4-DCPwas enhanced in oxic microcosms of soil that was pre-incubated withearthworms. Over 300 2,4-DCP putative degraders were isolated under oxicand anoxic conditions, and belonged to 19 genera. The majority of theisolates belonged to the Gammaproteobacteria (i.e., Pseudomonadaceae,Enterobacteraceae). Approximately 30% of the aerobic isolates were notpreviously known to degrade 2,4-DCP. Analyses of tfdB (encodes a 2,4-DCP-hydroxylase) and pheA (encodes a phenol hydroxylase) as structuralmarker genes for 2,4-DCP degraders indicated the presence of novel anddiverse 2,4-DCP degraders in the drilosphere and soil. tfdB of burrow wallswere most diverse. The collective data indicates that (a) earthwormsstimulate the degradation of 2,4-DCP in soil by enhancing the growth of 2,4-spektrum | Tagungsband 2011