Enrichment of a dioxin-dehalogenating Dehalococcoides species in two-liquid phase cultures.
Enrichment cultures capable of reductively dechlorinating 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) were shown to dechlorinate 1,2,3-trichlorobenzene (1,2,3-TrCB) to 1,3-dichlorobenzene. To test if this activity can be used to enrich for dioxin-dechlorinating bacteria, a two-liquid phase cultivation with 200 mM 1,2,3-TrCB dissolved in hexadecane was established. During the dechlorination of 1,2,3-TrCB, the number of 1,2,4-TrCDD-dechlorinating bacteria increased by four orders of magnitude, eventually accounting for 11% of the total cell number. Characterization of the bacterial communities of the initial dioxin-dechlorinating culture and of the trichlorobenzene enrichments by restriction fragment length polymorphism (RFLP) analysis of cloned 16S rRNA genes revealed a proportional increase of nine different sequence types, one representing a Dehalococcoides strain. Inhibition of methanogens further enhanced the rate of chlorobenzene dehalogenation and also resulted in a rapid dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin that was applied via a hexadecane phase. The further enrichment was monitored by terminal RFLP, quantitative real-time PCR and microscopy, and aimed at the reduction of the accompanying non-dehalogenating populations by using different combinations of electron donors and the application of antibiotics. Hydrogen as the sole electron donor proved to be less efficient due to the co-enrichment of acetogens. The novel Dehalococcoides strain DCMB5 was enriched up to 50% by the cultivation with organic acids, hydrogen and vancomycin, and was finally purified by conventional isolation techniques.
Bunge
Michael
M
Institute of Biology/Microbiology, Martin-Luther-Universität Halle-Wittenberg, Germany; and Institute of Microbiology, Universität Innsbruck, Austria.
Wagner
Anke
A
Fischer
Marco
M
Andreesen
Jan R
JR
Lechner
Ute
U
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002264:Carboxylic Acids / Q000378:metabolism; D002722:Chlorobenzenes / Q000378:metabolism; D041862:Chloroflexi / Q000254:growth & development* / Q000302:isolation & purification / Q000378:metabolism*; D015169:Colony Count, Microbial; D003470:Culture Media / Q000737:chemistry; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D006859:Hydrogen / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D008853:Microscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D000072317:Polychlorinated Dibenzodioxins / Q000031:analogs & derivatives* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D014640:Vancomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D002264:Carboxylic Acids; D002722:Chlorobenzenes; D003470:Culture Media; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D000072317:Polychlorinated Dibenzodioxins; D012336:RNA, Ribosomal, 16S; C036720:trichlorobenzene; C090523:1,2,4-trichlorodibenzo-1,4-dioxin; D014640:Vancomycin; C042146:1,3-dichlorobenzene; D006859:Hydrogen