title: Preferential cultivation of type II methanotrophic bacteria from littoral sediments (Lake Constance).
abstract: Most widely used medium for cultivation of methanotrophic bacteria from various environments is that proposed in 1970 by Whittenbury. In order to adapt and optimize medium for culturing of methanotrophs from freshwater sediment, media with varying concentrations of substrates, phosphate, nitrate, and other mineral salts were used to enumerate methanotrophs by the most probable number method. High concentrations (>1 mM) of magnesium and sulfate, and high concentrations of nitrate (>500 microM) significantly reduced the number of cultured methanotrophs, whereas phosphate in the range of 15-1500 microM had no influence. Also oxygen and carbon dioxide influenced the culturing efficiency, with an optimal mixing ratio of 17% O(2) and 3% CO(2); the mixing ratio of methane (6-32%) had no effect. A clone library of pmoA genes amplified by PCR from DNA extracted from sediment revealed the presence of both type I and type II methanotrophs. Nonetheless, the cultivation of methanotrophs, also with the improved medium, clearly favored growth of type II methanotrophs of the Methylosinus/Methylocystis group. Although significantly more methanotrophs could be cultured with the modified medium, their diversity did not mirror the diversity of methanotrophs in the sediment sample detected by molecular biology method.
authors.affiliation: LS Mikrobielle Okologie, Fachbereich Biologie, Universität Konstanz, Germany. ingeborg.bussmann@uni-konstanz.de
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Bussmann
authors.forename: Michael
authors.initials: M
authors.lastname: Pester
authors.forename: Andreas
authors.initials: A
authors.lastname: Brune
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schink
mesh_terms: D001431:Bacteriological Techniques; D002245:Carbon Dioxide / Q000378:metabolism; D015169:Colony Count, Microbial; D003470:Culture Media / Q000737:chemistry; D005618:Fresh Water / Q000382:microbiology*; D019015:Geologic Sediments / Q000382:microbiology*; D005858:Germany; D008697:Methane / Q000378:metabolism*; D042083:Methylocystaceae / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D020581:Methylosinus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics; D010802:Phylogeny; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
chemical_list: D003470:Culture Media; D002245:Carbon Dioxide; D010105:Oxygenases; C021555:methane monooxygenase; D008697:Methane; D010100:Oxygen