USING STABLE ISOTOPE PROBING TO IDENTIFY SOIL MICROBIAL POPULATIONS INVOLVED IN PHENOL METABOLISM AT AN AGRICULTURAL FIELD SITE

Abstract

The global distribution and universal toxicity of phenolic compounds make their degradation of great interest. The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13CO2 respired from 13C-labeled phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using GC/MS, it was shown that 13C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of 13C-phenol, (ii) 11 daily doses of unlabeled-phenol followed by 1 dose of 13C-phenol, and (iii) 12 daily doses of 13C-phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13CO2 evolution by a factor of 10. Furthermore, imaging of 13C-treated soil using Secondary Ion Mass Spectrometry (SIMS) verified that individual bacteria inc orporated 13C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of ?-, ?-, and ?-proteobacteria, and high G+C Gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically-related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. Furthermore, fungi-specific PCR amplification and sequencing of the 18S-28S internal transcribed spacer region genes from soil-derived, 13C-DNA revealed a number of fungi involved in phenol degradation at this site. 13C-labeled fungal DNA was only detected in one of our treatments (that representing trophically-related carbon cross-feeders) which suggests that these organisms are potentially secondary consumers in phenol-degradation at this site. These data show that SIP has the potential to document population shifts caused by substrate pre-exposure and to follow the flow of carbon through terrestrial microbial food chains.