Aimo Saano

Analysis of a 2,4,6-trichlorophenol-dehalogenating enrichment culture and isolation of the dehalogenating member Desulfitobacterium frappieri strain TCP-A

Abstract. An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescensT, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.

Nested PCR detection of Archaea in defined compartments of pine mycorrhizospheres developed in boreal forest humus microcosms

Archaea colonising defined compartments of Scots pine Suillus bovinus or Paxillus involutus mycorrhizospheres developed in forest humus-containing microcosms were investigated by nested polymerase chain reaction (PCR), cloning, restriction fragment length polymorphism (RFLP) and sequencing. Archaea representing six RFLP groups were detected in the system. Sequence analysis of clones representing the different RFLP types confirmed the presence of novel Finnish forest soil Crenarchaeota. Archaeal sequences were identified from mycorrhizas of both P. involutus and S. bovinus, at the margins of the external mycelium and in uncolonised humus but not from non-mycorrhizal short roots. Fungal and compartment-specific crenarchaeal occupation of mycorrhizospheres is discussed in relation to bacterial community distribution in similar systems.

Small scale extraction of DNA from soil with spun column cleanup

A general outline of procedures for isolation of DNA from soil by direct lysis is: 1) lysis of the cells; 2) separation of DNA from other cell components such as polysaccharides and proteins; 3) release of DNA from soil particles and purification of the DNA extract from soil compounds; 4) precipitation of DNA; and 5) further purification of DNA from soil compounds. Steps 2–4 do not proceed in succession in all of the published protocols, and 2 and 3 may have to be repeated after 4. However, in the protocol described below, the procedure follows the outline given above.

Detection of Archaeal Diether Lipid by Gas Chromatography from Humus and Peat

Abstract A suitable method based on gas chromatography to detect the diphytanylglycerol diether (archaeol), the domain membrane lipid of Archaea, was used to trace the presence of Archaea in humus and peat. The elution of the standard used (1,2-di-O-hexadecyl-rac-glycerol) was reproducible above a concentration of 1 mg 1 −1 (2 ng peak −1), which was the detection limit of the method. No archaeol was detected from the humus sample. This was verified using polymerase chain reaction (PCR) with primers specific to archaeal 16S rDNA gene region. Spiking the humus with an archaeon, Halobacterium salinarum, gave a positive response for both methods. This indicated that there were no Archaea in the specific humus sample. The peat samples used for extraction of diether lipids were first characterized for their CH4 production rate, which indicated the presence of methanogens (Archaea). With unlimited access to CO2/H2, the methane production rate peaked between 15 and 25 cm. The archaeol could be identified from all...

Detection of Rhizobium galegae from one-gram soil samples by non-radioactive DNA-DNA-hybridization

Abstract Non-radioactive DNA-DNA-hybridization was applied to identify Rhizobium galegae from l g soil samples. Total bacterial DNA from the samples was extracted using lysozyme and a freeze-thaw procedure and the sample DNA was attached to nitrocellulose or nylon membranes. During a 3 h hybridization 10 pg of sample DNA could be detected on a nylon filter with digoxigenin-dUTP-labelled R. galegae total DNA. The sensitivity was the same for either the colour-based or the chemiluminescence detection techniques.