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

EMP049Identification and characterization of aerobicchloroethene degrading bacteriaS. Mungenast* 1 , T. Teutenberg 2 , T. Schwartz 2 , K.R. Schmidt 1 , A. Tiehm 11 Department of Environmental Biotechnology, Water Center Technology(TZW), Karlsruhe, Germany2 Department of Interface Microbiology, Karlsruhe Institute of Technology(KIT), Karlsruhe, GermanyThe extensive use of chloroethenes as solvents and synthetic feed stocksover decades made those compounds to a major source of groundwater andsoil contamination. The bioremediation of chlorinated ethenes such as vinylchloride (VC) in groundwater via oxidation by aerobic microorganisms is acost-effective alternative to physical and chemical approaches. Metabolicpathways that use the target pollutant as growth substrate are favourable forbioremediation processes, as compared to cometabolic degradation in thepresence of auxiliary substrates. Several mixed cultures and pure bacterialstrains that can use VC as sole carbon and energy source have beenpublished and examined in regard to application as bioremediation agent.Recently, metabolic cis-1,2-dichloroethene (cDCE) degradation has beenreported for a mixed culture enriched at TZW [1; 2]. Concentration andtemperature range as well as starvation capacity and effects of cocontaminatingchloroethenes were determined. In our current joint study,also molecular biological approaches are applied. Two aerobic metabolicallyVC-degrading isolates from two different sites in Germany were identifiedby sequence analysis at KIT. Hydrogenophaga taeniospiralis andMycobacterium tusciae were identified as VC-degrading bacteria. PCR-DGGE and 16S-DNA sequence analysis allowed the identification andcharacterization of the degrading organisms using the basic local alignmentsearch tool of the NCBI database. The design of PCR primers andfluorescence in-situ hybridisation (FISH) probes for bacteria involved in theprocess of aerobic degradation is part of this project.Funding by BMWi and AiF (grant no. 16224 N) is gratefully acknowledged.[1] Schmidt K.R. et al (2010): Aerobic biodegradation of cis-1,2-dichloroethene as sole carbon source:Stable carbon isotope fractionation and growth characteristics. Chemosphere 78:527-532.[2] Zhao H.-P. et al (2010): Inhibition of aerobic metabolic cis-1,2-di-chloroethene biodegradation byother chloroethenes. Water Research 44:2276-2282.EMP050Occurrence of acidophilic and halotolerant Fe(II)-oxidizing microorganisms in high saline mine tailingsfrom the Atacama desert, ChileH. Korehi* 1 , D. Kock 1 , B. Dold 2 , A. Schippers 11 Federal Institute for Geosciences and Natural Resources , Microbiology,Hannover, Germany2 Institute of Applied Economic Geology (GEA), Department ofMicrobiology, University of Concepcion, Concepcion, ChileA marine shore copper mine waste tailings deposit in arid climate atChañaral, Chile, was studied to understand the influence of high salinity onthe microbial community composition and biogeochemical processes in thisextreme environment. Samples were taken from the oxidized zones atseveral sites which had a paste pH in the range of 2-8. The microbialcommunity was quantitatively analyzed using different methods: 1) total cellnumbers by SYBR Green II direct counting, 2) quantitative real-time PCR,3) most probable number cultivation of acidophilic Fe(II)-oxidizers. Theresults showed that the composition of microbial communities and the cellnumbers of different microbial groups are highly variable at differentsampling sites. Depth profiles of cell numbers of the mine tailings depositshowed total cell numbers in the range of 10 4 - 10 8 cells g -1 tailings. Bacteriadominated over Archaea in the mine tailings. The acidophilic Fe(II)- and/orsulfur-oxidizing Acidithiobacillus spp. dominated over the acidophilicFe(II)-oxidizing Leptospirillum spp. among the Gram-negative Bacteria. Inparallel to the microbial community analyses, novel acidophilic halotolerantFe(II)-oxidizing microorganisms were enriched at salt concentrations of upto 1 M probable suitable for metal bioleaching using seawater. Growth of upto 10 7 cells/ml was observed in case of complete oxidation of ferrous iron inthe medium. The growth of these microorganisms and its ability to oxidizeferrous iron were depended on pH, temperature, initial concentration offerrous iron, and the inoculum. High ferric to ferrous ratios exhibited aninhibitory effect on bacterial growth.EMP051Metagenome approach of two microbial biofilms in abiogas systemA. Rademacher* 1 , M. Zakzewski 2 , A. Schlüter 2 , M. Klocke 11 Department of Biotechnology, Leibniz Institute for Argicultural TechnologyPotsdam-Bornim e.V., Potsdam, Germany2 Centrum für Biotechnologie (CeBiTec), University of Bielefeld, Bielefeld,GermanyBiogas production forms a substantial component amongst the renewableenergy technologies converting biomass to methane. In order to improvebiogas production, a profound knowledge about the involvedmicroorganisms is essential.We investigated two biofilms of a thermophilic (55°C) two-phase leach-bedsystem in laboratory scale using a metagenome approach. This biogassystem with two internal circulations of leachate consisted of a gastighthydrolysis reactor, an effluent storage reactor and a downstream anaerobicfilter reactor. The retention time of the rye silage and the winter barley straw(w/w 10/1) was 21 days. Afterwards, samples of the digestate of thehydrolysis reactor (cellulolytic biofilm) and from a tower packing of theanaerobic filter reactor (methanogenic biofilm) were taken. The extractedDNA was sequenced by means of 454-pyrosequencing technology in aGenome Sequencer FLX Titanium System resulting in altogether 552,268reads with a total of 218 mb sequence information. The average read lengthwas 395 bases. Efficient characterization based on reads was applied usingseveral software pipelines as RDP classifier or CARMA software fortaxonomical analyses and Pfam or COG classification for functionalanalyses.Up to 30% of the obtained reads could be assigned to taxonomic ranksindicating that many up to now unknown microorganisms are participatingin the formation of methane. However, Clostridia, Thermotogae and Bacilliare the most prevalent classes among the bacteria in the cellulolytic biofilmsample and therefore may play a key role in carbohydrate degradation. Pfamcharacterization of enzymes also revealed Clostridia and Bacilli as prevalentfor carbohydrate degradation supporting the previous findings. In contrast,pfam analysis of the methanogenic biofilm sample showed a high abundanceof methanogenic enzymes for Methanobacteriales, whereas taxonomicalanalyses revealed that Methanosarcinales is highly abundant. These resultssuggest that the biofilm-based methanogenesis is not only driven by thehydrogenothrophic, but also by the acetoclastic pathway.In conclusion, the two biofilms, sampled from one biogas reactor, revealedstrong differences in taxonomical and functional analysis caused presumablyby reactor compartmentation.EMP052Genetic and biochemical characterization of ahydrothermal vent enrichment with autotrophichydrogen oxidizersM. Hansen*, M. PernerDepartment of Microbiology and Biotechnology, University of Hamburg,Hamburg, GermanyAn enrichment culture for autotrophic hydrogen oxidizing microbes,inoculated with diffuse fluids from the hydrothermal vent system SistersPeak (5°S on the Mid-Atlantic Ridge) was investigated with respect to themicrobial community composition, as well as the genetic and biochemicalfeatures with regard to hydrogen oxidation and CO 2 fixation.According to phylogenetic analyses (16S rRNA genes) Alpha- andGammaproteobacteria were detected, whereby the majority of sequenceswas related to Thalassospira. In contrast, 16S rRNA sequences generatedfrom isolated RNA by RT-PCR were mostly assigned to Thiomicrospiracrunogena. Interestingly, the relative abundances of Alpha- andGammaproteobacteria were 10 % and 11 %, respectively, whereas nearly 80% of all DAPI-stained cells could not be assigned to any bacterial group,although common probes targeting different Proteobacteria were used.None of the identified species are known for their ability to oxidize H 2.The only NiFe uptake hydrogenase (responsible for energy yielding H 2oxidation) identified by a PCR based screening of a metagenomic libraryfrom the culture using different primer sets was most similar to therespective gene from T. crunogena, which up to now has not been cultivatedwith H 2 as electron donor. But the transcription of this gene could not beconfirmed by RT-PCR yet. Recent investigations hint at the presence of ahydrogenase from Alteromonas macleodii, which was not amplified duringthe screening. But this needs further investigations to be confirmed. Theuptake hydrogenase activity of membrane associated proteins wasspektrum | Tagungsband 2011