Microbial Degradation of Soil-Aged 14C-Labeled Pyrene by an Environmental Mycobacterium sp. Strain S65 in Different Soil Environments

Abstract

Mycobacterium (designated Mycobacterium sp. strain S65 in Genbank), previously isolated and characterized from a jet-fuel contaminated site at the Sept-Iles, Quebec, Canada airport, was found to be competent in mineralizing selected recalcitrant polycyclic aromatic hydrocarbon (PAH) compounds of minimal water solubility and low bioavailability in soils, when grown in yeast extract-tryptone-soluble starch medium. In aerobic liquid microcosms, strain S65 was capable of 61% cumulative mineralization of pyrene over 14 days. Based on 14CO2 evolution, the mineralization capability and capacity of this organism for soil-aged 14C-labeled pyrene was assessed. Gamma-ray sterilized farm soil microcosms were spiked with 1.0 105 dpm 14C-labeled pyrene and allowed to age for up to 360 days. The experimental design consisted of a factorial combination of two types of soil (clay and sandy), two levels of soil organic matter (high and low), and two concentrations of pyrene (1000 and 500 mg L-1) with six replicates per treatment (48 microcosms per inoculation date). One set of 48 aged microcosms was inoculated after each aging period (0, 30, 180, or 360 days) with three replicates per treatment (i.e., 24 microcosms) each receiving 1.0 107 cfu of live Mycobacterium sp. strain S65 (i.e., treatment) and a further three replicates per treatment receiving heat-killed Mycobacterium (i.e., negative control) for an overall total of 192 microcosms. The 14CO2 that evolved over 1, 3, 6, or 9 days and was trapped in a KOH solution was measured by scintillation counter, and cumulative mineralization was calculated as a percent of initial pyrene. As a further basis of comparison, mineralization of un-aged pyrene in culture broth was also measured (i.e., positive control) over the same incubation period. Serial dilution and spread-plate techniques served to assess the possible presence of a viable culturable microorganism population (CFU g-1) in different sterile soils for a total of 4 4 3 = 48 (i.e., soils aging time replicate) microcosm bottles (i.e., sterile control). No microbial colonies were observed on solid media, indicating no culturable competitive microbial strain that might contribute to the mineralization of 14C-labeled pyrene, at any time during the aging process. Un-aged pyrene in soil (day 0) was utilized by Mycobacterium sp. strain S65 without any lag phase, and pyrene mineralization proceeded thereafter. Cumulative mineralization of pyrene added at concentrations of 1000 and 500 mg L-1 reached 19% and 18.5%, respectively, after 24 h and 21% and 20%, respectively, after 96 h in sand-loam, low organic matter soil. At 96 h, cumulative pyrene mineralization in clay-loam, high organic matter soil was only 15.5% and 14.5%, respectively, distinctly less than in the sandy, low organic matter soil. Sampling continued until no further mineralization occurred (i.e., cumulative mineralization reached a plateau). Soil-aged pyrene was metabolized at a moderate rate by Mycobacterium sp. strain S65, whereas un-aged pyrene was mineralized at a greater rate (61% cumulative mineralization) in an aerobic liquid microcosm. For initial additions of 1000 and 500 mg L-1 pyrene, followed by 30 days of aging, Mycobacterium sp. strain S65 showed a maximum cumulative mineralization of 14% and 13.25%, respectively, in sand-loam, low organic matter soil but only 12.5% and 11.5%, respectively, in clay-loam, high organic matter soil. For 180-day and 360-day aged pyrene, cumulative pyrene mineralization was less than 10%, a significant drop from less-aged pyrene-soil microcosms. This suggests that as the aging proceeds in the soil medium, the pyrene becomes less accessible for degradation. Nonetheless, this study supports the idea that Mycobacterium sp. strain S65 has the potential to be useful in bioremediation and biodetoxification of high molecular weight PAH-contaminated sites.