Effect of Incubation Conditions on the Enrichment of Pyrene-degrading Bacteria Identified by Stable-isotope Probing in an Aged, PAH-contaminated Soil

Abstract

To determine whether the diversity of pyrene-degrading bacteria in an aged polycyclic aromatic hydrocarbon-contaminated soil is affected by the addition of inorganic nutrients or by slurrying the soil, various incubation conditions (all including phosphate buffer) were examined by mineralization studies and stable-isotope probing (SIP). The addition of nitrogen to either continuously mixed slurry or static field-wet soil incubations increased the rate and extent of mineralization of [(14)C]pyrene, with the most rapid mineralization observed in slurried, nitrogen-amended soil. Microcosms of slurry and static field-wet soil amended with nitrogen were also examined by SIP with [U-(13)C]pyrene. Recovered (13)C-enriched deoxyribonucleic acid (DNA) was analyzed by denaturing-gradient gel electrophoresis (DGGE) and 16S ribosomal ribonucleic acid (rRNA) gene clone libraries. DGGE profiles of (13)C-enriched DNA fractions from both incubation conditions were similar, suggesting that pyrene-degrading bacterial community diversity may be independent of treatment method. The vast majority (67 of 71) of the partial sequences recovered from clone libraries were greater than or equal to 97% similar to one another, 98% similar to sequences of pyrene-degrading bacteria previously detected by SIP with pyrene in different soil, and only 89% similar to the closest cultivated genus. All of the sequences recovered from the field-wet incubation and most of the sequences recovered from the slurry incubation were in this clade. Of the four sequences from slurry incubations not within this clade, three possessed greater than 99% similarity to the 16S rRNA gene sequences of phylogenetically dissimilar Caulobacter spp.

Biostimulation Reveals Functional Redundancy of Anthracene-Degrading Bacteria in Polycyclic Aromatic Hydrocarbon-Contaminated Soil

“…SIP has been used to identify phylogenetically diverse bacteria capable of growth on PAHs ranging from 2 to 4 rings, including naphthalene ( Jeon et al, 2003;Singleton et al, 2005;Yu and Chu, 2005;Gutierrez et al, 2011;Jones et al, 2011a;Uhlik et al, 2012), anthracene ( Jones et al, 2011b;Zhang et al, 2011bZhang et al, , 2012, phenanthrene (Singleton et al, 2005(Singleton et al, , 2007Cébron et al, 2011;Jones et al, 2011a;Martin et al, 2012), pyrene (Singleton et al, 2006(Singleton et al, , 2007Jones et al, 2008;Jones et al, 2011a), fluoranthene ( Jones et al, 2011a), and benz [a]anthracene ( Jones et al, 2011a). A previous SIP study was conducted in this lab to identify aerobic anthracenedegrading bacteria in PAH-contaminated soil sampled from the site of a former manufactured gas plant ( Jones et al, 2011b).…”

Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil

“…Stable-isotope probing (SIP) is one cultivation-independent molecular technique that can link the identity of a microorganism with its metabolic function without isolating that organism from its natural environment (38). DNA-based SIP has been used to identify bacteria capable of degrading aromatic hydrocarbons in PAH-contaminated environments, and in some cases, it has revealed novel bacterial groups (15,16,33,35,43). Earlier SIP studies of pyrenedegrading bacteria conducted in our lab revealed members of previously uncultivated beta-and gammaproteobacterial groups, neither of which is related to any cultivated genus (16,35).…”

“…DNA-based SIP has been used to identify bacteria capable of degrading aromatic hydrocarbons in PAH-contaminated environments, and in some cases, it has revealed novel bacterial groups (15,16,33,35,43). Earlier SIP studies of pyrenedegrading bacteria conducted in our lab revealed members of previously uncultivated beta-and gammaproteobacterial groups, neither of which is related to any cultivated genus (16,35). SIP investigations have also facilitated the isolation of ecologically relevant organisms (15,19,36) and have been used to reduce the complexity of community DNA slated for metagenomic analysis (4).…”

Description of Immundisolibacter cernigliae gen. nov., sp. nov., a high-molecular-weight polycyclic aromatic hydrocarbon-degrading bacterium within the class Gammaproteobacteria, and proposal of Immundisolibacterales ord. nov. and Immundisolibacteraceae fam. nov.

“…One group, designated 'Pyrene Group 2' or 'PG2', had no cultivated relatives and could only be classified to the class Gammaproteobacteria. Subsequent SIP experiments using pyrene, benz[a]anthracene or fluoranthene [2,3] further implicated bacteria belonging to PG2 in the degradation of high-molecularweight PAHs as well as the low-molecular-weight PAH phenanthrene [4] and potentially the low-molecular-weight PAH anthracene [5]. The 16S rRNA gene sequences comprising PG2 from these SIP projects possessed a relatively high (!96 %) intragroup 16S rRNA gene identity.…”

“…Strain TR3.2 T displayed growth on pyruvate as a carbon source with both nitrogen sources tested. Nitrate reduction was evaluated using the culture grown in sRB2 medium substituted with 5 mM KNO 3 . No nitrate reduction to nitrite beyond what was required for assimilation and growth was observed.…”