Identification of Benzo[ a ]pyrene-Metabolizing Bacteria in Forest Soils by Using DNA-Based Stable-Isotope Probing

Abstract

c DNA-based stable-isotope probing (DNA-SIP) was used in this study to investigate the uncultivated bacteria with benzo[a]pyrene (BaP) metabolism capacities in two Chinese forest soils (Mt. Maoer in Heilongjiang Province and Mt. Baicaowa in Hubei Province). We characterized three different phylotypes with responsibility for BaP degradation, none of which were previously reported as BaP-degrading microorganisms by SIP. In Mt. Maoer soil microcosms, the putative BaP degraders were classified as belonging to the genus Terrimonas (family Chitinophagaceae, order Sphingobacteriales), whereas Burkholderia spp. were the key BaP degraders in Mt. Baicaowa soils. The addition of metabolic salicylate significantly increased BaP degradation efficiency in Mt. Maoer soils, and the BaP-metabolizing bacteria shifted to the microorganisms in the family Oxalobacteraceae (genus unclassified). Meanwhile, salicylate addition did not change either BaP degradation or putative BaP degraders in Mt. Baicaowa. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) genes were amplified, sequenced, and quantified in the DNA-SIP 13 C heavy fraction to further confirm the BaP metabolism. By illuminating the microbial diversity and salicylate additive effects on BaP degradation across different soils, the results increased our understanding of BaP natural attenuation and provided a possible approach to enhance the bioremediation of BaP-contaminated soils. P olycyclic aromatic hydrocarbons (PAHs), a class of persistent organic pollutants (POPs), enter the environment through both natural and anthropogenic pathways. PAHs are released into the environment by means of natural processes, such as forest fires and direct biosynthesis under the action of microbes and plants (1). The primary artificial source of PAHs is the incomplete combustion of organic matter at high temperatures caused by human activities (2). Their presence in the environment poses a severe threat to public and ecosystem health because of their known acute toxicity and mutagenic, teratogenic, and carcinogenic features; they are therefore classified as priority pollutants by the U.S. Environmental Protection Agency (3). Furthermore, the persistence and genotoxicity of PAHs increase with molecular weight, and the presence of high-molecular-weight (HMW) PAHs in the environment is of greater concern. Benzo[a]pyrene (BaP), a representative HMW PAH with a five-ring structure, is a widespread pollutant with potent mutagenic and carcinogenic properties (4, 5). BaP is therefore identified as the first class of "human carcinogens" according to the report of the World Health Organization (WHO) International Agency for Research on Cancer (6). Generally, in soils with no industrial contamination, BaP concentrations vary from 3.5 to 3,700 g/kg, with a median concentration of 16 g/kg of soil; in contaminated soils and sediments, BaP concentrations range from 82 to 536 mg/kg (7).Bacteria possessing the ability of BaP utilization are readily isolated from contaminated soils or se...

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

“…There was no significant difference in PAHs removal between samples amended with 13 C-labeled and unlabeled PAHs [5,13,[26][27][28], and all the PAHs analysis in this study was then carried out for soil samples amended with unlabeled PAHs. More precisely, the soil samples (sterilize controls and unlabeled PAHs amended treatments) were sacrificed on day 3, 9 and 14 after the initiation of cultivation, according to previous instruction [26,27].…”

“…After sealing with rubber stoppers and aluminum seals, unlabeled PAHs (99%, Cambridge Isotope Laboratories, Inc.) or 13 C-labeled PAHs (ring-13 C 6 , 99%, Cambridge Isotope Laboratories, Inc.) were added to the bottles via gas-tight syringe to obtain a final concentration of 10 mg kg −1 for each PAHs. The cover of the bottles was opened each day for 1 h to keep oxygen in the media close to natural environments as described previously [26,27]. The four treatments included: sterile controls (nonbioactive, with PAHs additive), negative controls without PAHs addition, unlabeled PAHs amended treatments ( 12 C-ANT, 12 C-PHE and 12 C-FLT), and 13 C-labeled PAHs amended treatments ( 13 C-ANT, 13 C-PHE and 13 C-FLT).…”

“…More precisely, the soil samples (sterilize controls and unlabeled PAHs amended treatments) were sacrificed on day 3, 9 and 14 after the initiation of cultivation, according to previous instruction [26,27]. Briefly, soil samples were freeze-dried, homogenized, pulverized, spiked with 1000 ng of deuterated PAHs as surrogate standards, and extracted into dichloromethane (DCM) in a Soxhlet apparatus for 48 h, with the addition of activated copper to remove sulfur.…”

“…DNA concentrations were determined using an ND-2000 UV-vis spectrophotometer (NanoDrop Technologies, Wilmington, DE). Then DNA was prepared as previously for isopycnic ultracentrifugation and gradient fractionation [26,27]. Briefly, approximately 10,000-ng DNA was added to Quick-Seal polyallomer tubes (13 × 51 mm, 5.1 mL, Beckman Coulter), along with a Tris-EDTA (TE, pH 8.0)/CsCl solution with a final buoyant density (BD) of ∼1.77 g mL −1 .…”

“…After sealed with a cordless quick-seal tube topper, the tubes were transferred to a Beckman ultracentrifuge and centrifuged at 178,000 × g (20 • C) for 48 h. Following centrifugation, 150-L fractions were collected from each tube using a fraction recovery system (Beckman). The BD value of each fraction was then measured and CsCl was removed by glycogen-assisted ethanol precipitation [26,27].…”