Identification Of Three Strains Of TMPD Degrading Bacteria

Oxytropis.glacialis is one kind of Locoweeds that only distributed in the area of Ali, Tibet. 2,2,6,6-Tetramethyl-4-piperidone (TMPD) is a main kind of toxical alkaloids which is separated and extracted from aerial parts of Oxytropis.glacialis. Though Oxytropis.glacialis is toxic, its nutritive value is high. There will be great economic returns if it is detoxicated and used properly. TMPD toxin degradation with microorganism have been reseached. In this paper, identificating these three strains with morphological, biochemical and physiological characteristics, 16S rDNA sequence homology and phylogentic analysis. The results are as follows.1 The cells of strain LF-1 were straight rods that were 0.5～0.9μm×1.0～2.0μm,Gram negative, with capsules, without gemmas, without mobility and existed as single, double and acervate. Its coenobiums were round, slick, moist, caelate, fringe-concinnous, gray and lucent. This strain that was facultative anaerobic could use glucose as carbon source and produce gases. Indole, M.R, nitrate reduction, use of citrate and malonate, catalaes, urase test were positive. H2S production, gelatin liquefaction, oxidase test were negative. V-P test was instable. The slant and bottom of TSI medium changed yellow and produced gases. The coenobiums on MCA medium were pink. Through 16S rDNA sequence homology and phylogentic analysis, the phylogentic tree shows: the genetic evolutionary distance between strain LF-1 and Klebsiella were the nearest, and 16S rDNA sequence of strain LF-1 shared 99. 52% homologies with Klebsiella oxytoca isolate DR.Y14(DQ226215), which was a published sequence of relative bacteria from GenBank, and both sequences constituted a branch in the phylogenetic tree. Therefore, strain LF-1 was considered as one strain of Klebsiella oxytoca.2 The cells of strain LF-2 were spherical that were 0.8～1.0μm, Gram positive, without capsules, without gemmas, without mobility and existed as single, double and acervate. Its coenobiums were round, slick, moist, caelate, fringe-concinnous, gray and lucent. This strain that was facultative anaerobic could use glucose as carbon source but produce no gases. M.R, slarch hydrolysis, nitrate reduction, catalaes, urase, 5% and 7% NaCl gtowth test were positive. Indole, V-P, oxidase, coagulase test were negative. Through 16S rDNA sequence homology and phylogentic analysis, the phylogentic tree shows: the genetic evolutionary distance between strain LF-2 and Staphylococcus were the nearest, and 16S rDNA sequence of strain LF-3 shared 99. 80% homologies with Staphylococcus pasteuri (AJ717376), which was a published sequence of relative bacteria from GenBank, and both sequences constituted a branch in phylogenetic tree. Therefore, strain LF-2 was considered as one strain of Staphylococcus pasteuri.3 The cells of strain LF-3were straight rods that were 1.0～1.2μm×1.5～5.0μm, Gram negative, without capsules, with gemmas that were elliptic or columnar, and mesial, with mobility and existed as single, double and acervate. Its coenobiums were hyphostroma shape, not slick, dry, flat, fringe-unconcinnous, gray and lucent. This strain that was strictly aerobiotic could use slight glucose as carbon source but produce no gases. Slarch hydrolysis, gelatin liquefaction, catalaes, 5% and 7% NaCl gtowth test were positive. Indole, M.R, V-P, nitrate reduction, use of citrate and malonate, H2S production, oxidase, urase, 50℃growth test were negative. Through 16S rDNA sequence homology and phylogentic analysis, the phylogentic tree shows: the genetic evolutionary distance between strain LF-3 and Bacillus were the nearest, and 16S rDNA sequence of strain LF-3 shared 99. 80% homologies with Bacillus flexus (DQ837542), which was a published sequence of relative bacteria from GenBank, and both sequences constituted a branch in phylogenetic tree. Therefore, strain LF-3 was considered as one strain of Bacillus flexus.`...