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

EMP062Dominant denitrifiers in grassland and forest soils areAlpha- and Gammaproteobacteria as determined byisolation and next generation sequencingM. Selzer*, A. Goessner, R. Mertel, H.L. Drake, M.A. HornDepartment of Ecological Microbiology, University of Bayreuth, Bayreuth,GermanyDenitrifying prokaryotes are facultative aerobes that catalyse the reductionof nitrate and nitrite to nitrous oxide and molecular nitrogen. Soildenitrification is the main source but also a temporary sink of thegreenhouse gas nitrous oxide and depends on the denitrifying community.The diversity of denitrifiers in soils was assessed with isolation andmolecular approaches, which detect different prokaryotic groups. Twograssland and two forest soils under contrasting land use were studied.Maximum velocity of denitrification in these soils varied from 0.27 to 1.87μmol(N 2O) h -1 g -1 DW and most probable numbers of denitrifiers from 2*10 5to 1*10 7 g -1 DW . Neither maximum velocity of denitrification nor cellnumbers of denitrifiers were significantly different in grassland and forestsoils. Five different isolation approaches selective for denitrifiers yielded179 isolates. These isolates were affiliated to 22 different families fromProteobacteria, Bacilli, Actinobacteria, and Negativicutes. 7 of the isolatesrepresented putative novel species. Alpha- and gammaproteobacterialisolates were dominant in both grassland and forest soils, whileActinobacteria were also found in forest soils. 454 pyrosequencing of nitritereductase encoding genes (nirK/S) yielded 3,000 nirK sequences thatgrouped into 48 species-level OTUs that affiliated with 7 families withinAlphaproteobacteria and Gammaproteobacteria and Nitrospira. Grasslandsoils were less diverse than forest soils. 7,000 nirS sequences were groupedinto 30 species level OTUs belonging to 7 alpha-, beta andgammaproteobacterial families. Three and 24 OTUs were only found inforest and grassland soils, respectively. NirS diversity was higher ingrassland soils than in forest soils. Phylogenetic analyses indicated manynovel nirK and nirS OTUs. A higher family-level diversity was obtainedwith cultivation methods than with cultivation-independent methods, butmore novelty was detected with the latter approach. Both methods indicatedthat denitrifiers in grassland soils are different to those found in forest soils.The collective data indicates that Alphaproteobacteria andGammaproteobacteria are dominant denitrifiers in both grassland and forestsoils.EMP063Carbon stable isotope fractionation ofhexachlorocyclohexane isomers during aerobic andanaerobic dechlorinationS. Bashir*, K. Hitzfeld, C. Vogt, H.-H. Richnow, I. NijenhuisDepartment of Isotope Biogeochemistry, Helmholtz Center forEnvironmental Research (UFZ), 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 stable isotope fractionation of organic contaminants such as thepesticide Lindane (γ-hexachlorocyclohexane (γ-HCH)) may be used toassess their degradation in the environment. The extent of in situtransformation may therefore be inferred by using experimentallydetermined compound specific isotope fractionation factors duringbiotransformation by defined microbial cultures. In this study, carbonisotope fractionation factors were determined for the dechlorination of γ-hexachlorocyclohexane (γ-HCH) by the anaerobic strain Clostridiumpasteurianum DSM 525 and the aerobic strain Sphingobium indicum DSM16412. C. pasterianum dechlorinated γ-HCH in two weeks, and themetabolites γ-3,4,5,6-pentachlorocyclohexane (γ-PCCH) and chlorobenzene(CB) were formed. S. indicum, known to mineralize α, β and γ-HCH,degraded γ-HCH in four weeks with 1,2,4-TCB as metabolite. For bothstrains the carbon isotope fractionation of γ-HCH dechlorination wasquantified by using the Rayleigh equation. The bulk enrichment factor (εC)of - 4.8 ± 0.6 for C. pasteurianum was similar to the one previously reportedfor sulfate reducing strains. In the case of the aerobic strain a similar trendfor the isotopic fractionation was observed.EMP064Alphaproteobacteria are prevalent methylotrophs inaerated soils as determined by cultivation andpyrosequencing of structural genesA. Stacheter*, S. Hetz, L. Ebertsch, B. Apelt-Glowik, H.L. Drake, S. KolbDepartment of Ecological Microbiology, University of Bayreuth, Bayreuth,GermanyMethanol augments the formation of ozone in the troposphere. Aerobicmethylotrophic microorganisms reduce the emission of methanol fromplant-derived carbon and soil organic carbon. Most methylotrophs utilizemethanol. The biogeography and diversity of methanol-utilizingmethylotrophs in soils is not well resolved. Methylotrophic communities ofmineral soils from two forests and two grasslands (Nationalpark Hainich,Germany) were analyzed by cultivation and gene marker based methods. 77strains were isolated on methanol-containing mineral medium and belongedto seven classes of Bacteria. The most abundant class wasAlphaproteobacteria (39%). Viable cell numbers of methylotrophs variedbut averaged 5 x 10 7 g -1 DW . 19,000 sequences of mxaF (gene of thealphasubunit of methanol dehydrogenase), mch (methenyltetramethanopterinedehydrogenase), and fae (formaldehyde-activatingenzyme) were retrieved by tagged amplicon sequencing. The majority ofthese genes were affiliated with Alphaproteobacteria. Only one-third of thedetected genotypes occurred in all four soils, indicating that uniformlycommon genotypes were the minority genotypes. Molecular finger printingof fae revealed seasonal differences. The detectable class-level diversityobtained by cultivation was higher than the class-level diversity detectedwith gene markers. Nonetheless, both approaches suggested thatAlphaproteobacteria (e.g. Hyphomicrobium) were prevalent methanolutilizingmethylotrophs in aerated soils.EMP065Novel hydrogenase gene transcripts indicative of activefacultative aerobes and obligate anaerobes in theearthworm gutO. Schmidt*, P.K. Wüst, S. Hellmuth, K. Borst, M.A. Horn, H.L. DrakeDepartment of Ecological Microbiology, University of Bayreuth, Bayreuth,GermanyMucus-derived saccharides provide ideal in situ conditions for ingestedfermentative bacteria in the anoxic earthworm gut, and high concentrationsof H 2 as well as organic acids are indicative of ongoing fermentation. H 2production during fermentation is catalyzed by [FeFe]- and [NiFe]-hydrogenases that are present in obligate anaerobes and facultative aerobes,respectively. The main objective of this study was to resolve transcriptdiversities of [FeFe]- and [NiFe]-hydrogenases of potentially active H 2producers in anoxic gut content microcosms of Lumbricus terrestrissupplemented with glucose, a representative sugar found in gut contents.177 of 178 [FeFe]-hydrogenase gene transcripts affiliated to theClostridiales (65-81% amino acid sequence identity) whereas the remainingtranscript had an 84% identity (based on translated amino acid sequence) toa hydrogenase of Pelobacter carbinolicus (Deltaproteobacteria). The[FeFe]-hydrogenase gene transcripts yielded 13 distinct OTUs (based on anamino acid sequence similarity cut-off of 80%). 21% and 79% of 86 [NiFe]-hydrogenase gene transcripts were affiliated to Aeromonadaceae andEnterobacteraceae, respectively. Verrucomicrobia- and Firmicutesaffiliated[NiFe]-hydrogenases gene sequences were also detected, mainly ofwhich were highly novel. The collective findings (a) indicate that themicrobial community of the earthworm gut hosts microbes containinghitherto undetected [FeFe]- and [NiFe]-hydrogenases, (b) suggest thatobligate anaerobes of the Clostridiales and facultative aerobes of theEnterobacteriaceae were the main H 2 producers in glucose-supplementedgut enrichments, and (c) reinforce previous RNA-based stable isotopeprobing studies that identified Clostridiaceae and Enterobacteriaceae asimportant glucose-fermenting taxa in earthworm gut content.spektrum | Tagungsband 2011