Stable-Isotope Probing of Bacteria Capable of Degrading Salicylate, Naphthalene, or Phenanthrene in a Bioreactor Treating Contaminated Soil

Abstract

[ 13 C 6 ]salicylate, [U-13 C]naphthalene, and [U-13 C]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 [ 13 C]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 13 C during the incubation did not appear to be an issue in this experiment.

Response of bacterial pdo1, nah, and C12O genes to aged soil PAH pollution in a coke factory area

“…However, it is well known that culture-independent approaches allow for a broader recognition of microbial populations responsible for PAH degradation in environments (Baldwin et al 2003;Jurelevicius et al 2012). Recently, the abundance and composition of PAHdegrading microbial populations present in PAH-polluted environments have been investigated to some extent through direct extraction of DNA from the environment and analysis of PAH-degrading genes (Singleton et al 2005;Lillis et al 2010;Paissé et al 2012). pdo1, nah, and C12O are three important PAH-degrading genes which have been detected widely from environments polluted by aromatic compounds or bacteria isolated from those environments (Baldwin et al 2003;Johnsen et al 2006;Khan et al 2009;Tuan et al 2011).…”

Who eats what, where and when? Isotope-labelling experiments are coming of age

“…As a result, several research groups have pioneered in-house chemical and biological synthesis for the preparation of their desired substrates. Singleton et al (2005Singleton et al ( , 2006 employed chemical synthesis of commercially unavailable 13 C-labelled salicylate, naphthalene, phenanthrene and pyrene from the 'building blocks' of purchased 13 C-phenol, succinic acid and phenol. In a recent study of soil cellulolytic bacteria, the 13 Clabelled cellulose was purified from the growth medium of Acetobacter xylinus, which was grown on 13 C-glucose as a sole source of carbon (el Zahar Haichar et al, 2007).…”

Bacteria capable of degrading anthracene, phenanthrene, and fluoranthene as revealed by DNA based stable-isotope probing in a forest soil

“…SIP is suitable for identifying microbial populations within complex communities that are responsible for the degradation of targeted contaminants [5,9]. To date, SIP has been successfully applied to identify soil bacteria capable of metabolizing BTEX (benzene, toluene, ethylbenzene, and xylenes) [5], biphenyl [10], benzoate [10], and PAHs (naphthalene [4], anthracene [11,12], phenanthrene [4], pyrene [6], fluoranthene [13], and benz[a]anthracene [13]). Particularly for PAHs which are always found as complex mixtures in soils, some identified PAHs degraders can utilize a wide range of PAHs as sole carbon sources [14], or in some cases are only capable of degrading specific PAHs molecules.…”

“…However, most SIP studies address PAHs degraders in heavily contaminated soils [4,15] and little attention addressed their metabolism in forest soils. From global distillation theory [16], persistent organic pollutants (POPs) undergo long-range transport at global scale and eventually accumulated in media with organic carbon-rich reservoirs.…”

“…PAHs contamination results primarily from the The primary process of PAHs attenuation in soil is microbial degradation, and bioremediation provides an environmentally friendly and cost-effective option for removing these compounds from the soil environment [3]. To date, both culture dependent and independent techniques have been used to study the organisms responsible for PAHs degradation [4,5]. Bacterial species possessing the ability to metabolize PAHs are readily isolated from contaminated soils or sediments.…”