Fraunhofer-Institut für Atmosphärische Umweltforschung IFU

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  • Publication
    Improved method for detection of methanotrophic bacteria in forest soils by PCR
    ( 2001)
    Steinkamp, R.
    ;
    Zimmer, W.
    ;
    Papen, H.
    A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the alpha -subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils.
  • Publication
    Identification and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7. Sequencing and functional analysis of the gene locus
    ( 1998)
    Zimmer, W.
    ;
    Wesche, M.
    ;
    Timmermans, L.
    The root-associated bacterium Azospirillum brasilense Sp7 produces the growth-stimulating phytohormone indole-3-acetic acid (=IAA) via the indole-3-pyruvate pathway. The DNA region containing ipdC, the structural gene for indole-3-pyruvate decarboxylase, was identified in a cosmid gene library of strain Sp7 by hybridization and has been sequenced. Upstream of the gene, two other ORF homologous to gltX and cysS were sequenced that are transcribed in the opposite direction. A functional analysis of the cloned ipdC region has been performed. To test the expression of the gene, a lacZ-Km cartridge was introduced into the gene. By this construct, tryptophan-dependent stimulation of gene expression in A. brasilense Sp7 was observed. Evidences for the existence of another copy of the ipdC gene in the Azospirillum genome are also reported.
  • Publication
    Physiological and molecular biological characterization of ammonia oxidation of the heterotrophic nitrifier Pseudomonas putida
    ( 1998)
    Daum, M.
    ;
    Zimmer, W.
    ;
    Papen, H.
    ;
    Kloos, K.
    ;
    Nawrath, K.
    ;
    Bothe, H.
    The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N2O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized gamma-aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C2H2, indicated that P putida expressed an ammonia monooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers.
  • Publication
    Production of nitric oxide in nitrosomonas europea by reduction of nitride.
    ( 1990)
    Conrad, R.
    ;
    Remde, A.
    Nitrosomonas europea and Nitrosovibrio sp. produced NO and N sub 2 O during nitrification of ammonium. Less than 15 % of the produced NO was due to chemical decomposition of nitrite. Production of NO and especially of N sub 2 O increased when the bacteria were incubated under anaerobic conditions at decreasing flow rates of air, or at increasing cell densities. Low concentrations of chlorite (10 myM) inhibited the production of NO and N sub 2 O, but not of nitrite indicating that NO and N sub 2 O were not produced during the oxidative conversion of ammonium to nitrite. NO and N sub 2 O were produced during reduction of nitrite with hydrazine as electron donor in almost stoichiometric quantities indicating that reduction of nitrite was the main source of NO and N sub 2 O.
  • Publication
    Heterotrophic nitrification by Alcaligenes faecalis - NO2-, NO3-, N2O, and NO production in exponentially growing cultures.
    ( 1989)
    Hinkel, I.
    ;
    Papen, H.
    ;
    Rennenberg, H.
    ;
    Thoene, B.
    ;
    Berg, R. von
    Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations: Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2-, and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.
  • Publication
    Hydrogen turnover by psychotrophic nomoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment.
    ( 1989)
    Bak, F.
    ;
    Seitz, H.J.
    ;
    Thebrath, B.
    ;
    Mayer, H.P.
    ;
    Conrad, R.
    ;
    Schütz, H.
    The effect of temperature on CH4 production, turnover of dissolved H2, and enrichment of H2-utilizing anaerobic bacteria was studied in anoxic paddy soil and sediment of Lake Constance. When anoxic paddy soil was incubated under an atmosphere of H2/CO2, rates of CH4 production increased with time at temperatures higher than 25 degree C, but decreased at temperatures lower than 20 degree C. Chloroform completely inhibited methanogenesis in anoxic paddy soil and lake sediment, but did not or only partially inhibit the turnover of dissolved H2, especially at low incubation temperatures. Cultures with H2 as energy source resulted in the enrichment of chemolithotrophic homoacetogenic bacterial whenever incubation temperatures were lower than 20 degree C. Hydrogenotrophic methanogens could only be enriched at 30 degree C from anoxic paddy soil. A homoacetogen (strain HP4) and a methanogen (strain Bab1) were isolated from enrichtment cultures with lake sediment at 4 degree C and with anoxic p addy soil at 30 degree C, respectively. The two strains greatly differed in cardinal temperatures of growth. Whereas the methanogen was mesophilic, the homoacetogen was psychrotrophic. This adaptation possibly allows homoacetogenic bacteria to contribute to the turnover of dissolved H2 at low in situ temperatures of anoxic soils and lake sediments.
  • Publication
    Microbial production and uptake of nitric oxide in soil.
    ( 1989)
    Slemr, F.
    ;
    Conrad, R.
    ;
    Remde, A.
    Fluxes of NO from three different soils have been studied by a flow-through system in the laboratory as a function of gas flow rate, of NO mixing ratio, and of incubation conditions. The dependence of net NO fluxes on gas flow rates and on NO mixing ratios could be described by a simple model of simultaneous NO production and NO uptake. By using this model, rates of gross NO production, rate constants of NO uptake, and NO compensation mixing ratios could be determined as function of the soil type and the incubation condition. Gross NO production rates were one or two orders of magnitude larger under anaerobic than under aerobic conditions. NO uptake rate constants, on the other hand, were only 5-8 times larger so that the compensation mixing ratios of NO were in a range of about 1600-2200 ppbv under anaerobic and of about 50-600 ppbv under aerobic conditions. The different soils exhibited similar NO uptake rate constants, but the gross NO production rate and compensation mixing ratio w as significantly higher in an acidic (pH 4.7) sandy clay loam than in the other less acidic soils. Experiments with autoclaved soil samples showed that both NO production and NO uptake was mainly due to microbial metabolism.
  • Publication
    Selenium increased hydrogenase expression in autotrophically cultures Bradyrhizobium japonicum and is a constituent of the purified enzyme.
    ( 1988)
    Boursier, P.
    ;
    Hanus, F.J.
    ;
    Papen, H.
    ;
    Russell, S.A.
    ;
    Becker, M.
    ;
    Evans, H.J.
    We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122 DES cultured in a medium purified to remove selenium compounds. In addition hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 myM significantly increased total cell protein and hydrogenase derepression by 133 %, whereas VO3, AsO2 high 2 minus, SO2 high 2 minus, and TeO3 high 2 minus failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se readioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 plus minus 0.13 molof Se per mol of enzyme. These results in dicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.
  • Publication
    Temperature limitation of hydrogen turnover and methanogenesis in anoxic paddy soil
    ( 1987)
    Babbel, M.
    ;
    Conrad, R.
    ;
    Schütz, H.
    The shift of incubation temperature in anoxic paddy soil from 30 to 15 degrees C resulted in a reversible decrease of the methane production rate and of the H2 steady state partial pressure. Only at 30 degrees C but not at 17 degrees C, total CH4 production rates were enhanced by the addition of H2, acetate, or cellulose compared to the control. Apparent activation energies which were calculated from the temperature dependence of CH4 production were higher in presence than in absence of excess H2. Decrease of temperature caused a decrease of the H2 turnover rate constant and of the Gibbs free energy of H2-dependent methanogenesis, and also resulted in a smaller contribution of H2 to total methanogenesis. However, H2-dependent methanogenesis was significantly stimulated by excess H2 and slightly inhibited by acetate at low as well as high temperature. The results show that H2-producing bacteria were limited by temperature to a greater extent than the methanogens so that the methanogenic microbial community in paddy soil was limited by the supply of H2. At lows as well as high temperatures, excess H2 apparently enabled part of the methanogenic community to shift from acetate-dependent to H2-dependent CH4 had only this effect, but with increasing temperature, excess H2 additionally stimulated total methanogenic activity and eventually even growth. (IFU)