Abstract
The aim of this study was to extend the results of our previous stable isotope probing (SIP) investigation: we identified a soil fungus involved in phenol biodegradation at an agricultural field site. DNA extracts from our previous study were examined using fungi-specific PCR amplification of the 18S-28S internal transcribed spacer (ITS) region. We prepared an 80-member clone library using PCR-amplified, (13)C-labeled DNA derived from field soil that received 12 daily doses of (13)C-phenol. Restriction-fragment-length-polymorphism screening and DNA sequencing revealed a dominant clone (41% of the clone library), the ITS sequence of which corresponded to that of the fungal genus Trichosporon. We successfully grew and isolated a white, filamentous fungus from site soil samples after plating soil dilutions on mineral salts agar containing 250 p.p.m. phenol. Restreaking on both yeast extract-peptone-galactose and Sabouraud dextrose agar plates led to further purification of the fungus, the morphological characteristics of which matched those of the genus Trichosporon. The ITS sequence of our isolated fungus was identical to that of a clone from our SIP-based library, confirming it to be Trichosporon multisporum. High-performance liquid chromatography and turbidometeric analyses showed that the culture was able to metabolize and grow on 200 p.p.m. phenol in an aqueous mineral salts medium within 24 h at room temperature. Gas chromatography-mass spectrometry analysis of (13)CO(2) respiration from laboratory soil incubations demonstrated accelerated phenol mineralization in treatments inoculated with T. multisporum. These findings show that T. multisporum actively degraded phenol in our field-based, soil experiments.
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