Stable-isotope probing of bacteria capable of degrading salicylate, naphthalene, or phenanthrene in a bioreactor treating contaminated soil.
[13C6]salicylate, [U-13C]naphthalene, and [U-13C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the "heavy" and "light" DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from Escherichia coli K-12 was added to each sample as an internal indicator of separation efficiency. While E. coli was not detected in most analyses of heavy DNA, a low number of E. coli sequences was recovered in the clone libraries associated with the heavy DNA fraction of [13C]phenanthrene incubations. The number of E. coli clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the Pseudomonas and Ralstonia genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the Acidovorax genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the 13C during the incubation did not appear to be an issue in this experiment.
Singleton
David R
DR
Department of Environmental Sciences and Engineering, School of Public Health, CB 7431, University of North Carolina, Chapel Hill, NC 27599-7431, USA.
Powell
Sabrina N
SN
Sangaiah
Ramiah
R
Gold
Avram
A
Ball
Louise M
LM
Aitken
Michael D
MD
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001673:Biodegradation, Environmental; D019149:Bioreactors; D002247:Carbon Isotopes; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism*; D010802:Phylogeny; D011549:Pseudomonas / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D043367:Ralstonia / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020156:Salicylic Acid / Q000378:metabolism*; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000378:metabolism*
D002247:Carbon Isotopes; D004269:DNA, Bacterial; D009281:Naphthalenes; D010616:Phenanthrenes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012989:Soil Pollutants; C031721:naphthalene; C031181:phenanthrene; D020156:Salicylic Acid