Fungal Metabolism of Toluene: Monitoring of Fluorinated Analogs by ¹⁹ F Nuclear Magnetic Resonance Spectroscopy

Abstract

We used isomeric fluorotoluenes as model substrates to study the catabolism of toluene by five deuteromycete fungi and one ascomycete fungus capable of growth on toluene as the sole carbon and energy source, as well as by two fungi (Cunninghamella echinulata and Aspergillus niger) that cometabolize toluene. Whole cells were incubated with 2-, 3-, and 4-fluorotoluene, and metabolites were characterized by 19 F nuclear magnetic resonance. Oxidation of fluorotoluene by C. echinulata was initiated either at the aromatic ring, resulting in fluorinated o-cresol, or at the methyl group to form fluorobenzoate. The initial conversion of the fluorotoluenes by toluene-grown fungi occurred only at the side chain and resulted in fluorinated benzoates. The latter compounds were the substrate for the ring hydroxylation and, depending on the fluorine position, were further metabolized up to catecholic intermediates. From the 19 F nuclear magnetic resonance metabolic profiles, we propose that diverse fungi that grow on toluene assimilate toluene by an initial oxidation of the methyl group.In bacteria, five different metabolic pathways for the complete aerobic degradation of toluene and its assimilation are known (9, 16, 22, 23; B. Kaphammer, J. J. Kukor, and R. H. Olsen, Abstr. 90th Annu. Meet. Am. Soc. Microbiol., abstr. K-145, p. 243, 1990). Depending upon the strain, toluene is initially oxidized either at the methyl group or at the aromatic ring. Fungi also can oxidize toluene at both molecular sites. Cultures of Mortierella isabellina converted toluene into benzyl alcohol (12). Smith and Rosazza (17) identified two zygomycetes and three deuteromycetes that hydroxylated toluene at the aromatic ring to produce o-cresol and, in some cases, p-cresol. Mineralization of toluene has been reported for the white-rot fungus Phanerochaete chrysosporium, but the metabolic pathway was not determined (25). The fungal degradation of toluene in these cases occurred only by cometabolism and, consequently, it did not support growth. We previously identified and described a Cladosporium sphaerospermum strain that can grow on toluene as the sole carbon and energy source (20). Oxygen consumption experiments with whole cells and enzyme activities in cell extracts suggest that the initial oxidation of toluene takes place at the methyl group, rather than at the aromatic ring. Recently, we identified five additional fungi that also can assimilate toluene (14).19 F nuclear magnetic resonance ( 19 F NMR) has been used previously to characterize the degradation of fluorine-containing aromatic compounds by fungi (2, 13, 18). Fluorine, with its small size, can replace hydrogen in an organic substrate with few steric consequences. It also influences the conversion rate of many enzyme reactions (21). The 19 F isotope, with a natural abundance of 100% and a broad chemical shift range, is a very sensitive NMR-active nucleus that can be advantageously used in the identification and quantification of fluorinated intermediates by 19 F NMR spectroscopy. Our obj...

Substrate Interactions during the Biodegradation of Benzene, Toluene, Ethylbenzene, and Xylene (BTEX) Hydrocarbons by the Fungus Cladophialophora sp. Strain T1

“…strain T1, toluene is first hydroxylated to benzyl alcohol and subsequently converted to benzoic acid, prior to hydroxylation and cleavage of the aromatic ring (18). Here we have shown that o-and m-xylenes were also oxidized at the side chain to form dibenzoic acids, which were not metabolized further.…”

Correlation of Biological Activity and Reactor Performance in Biofiltration of Toluene with the Fungus Paecilomyces variotii CBS115145

“…The presence of p-cresol can be attributed to the ring hydroxylation. Prenafeta-Boldú et al (17) that this microorganism has more than one toluene metabolic pathway. The production of the intermediates was lower when the efficiency of the system increased, suggesting that the fungus was capable of overcoming the metabolic bottlenecks.…”

“…Weber et al (27) reported Cladosporium sphaerospermum growth on toluene with initial hydroxylation on the methyl group. Recently, Prenafeta-Boldú et al (17) used isomeric fluorotoluenes as model substrates for toluene catabolism by five fungi that grow on toluene and two others that cometabolize toluene with glucose (Cunningamella echinulata and Aspergillus niger). With the toluene-utilizing fungi and A. niger, initial hydroxylation occurred only at the methyl group and resulted in fluorinated benzoates.…”

“…In C. echinulata, hydroxylation was also initiated at the aromatic ring, and o-cresol and benzoate were detected. For toluene, it has been proposed that fungi preferentially hydroxylate the methyl group (13,17,27). Parallel pathways for the degradation of aromatic compounds have been shown elsewhere in fungi.…”

The biodegradation vs. biotransformation of fluorosubstituted aromatics

“…Besides the side chain oxidation products mentioned above, mono-and dihydroxylated benzoic acids were detected as well as 3-fluorocatechol and its ortho ringfission product, 2-fluoromuconic acid. In particular, free fluoride ion was produced only by the toluene utilizing fungi (Prenafeta-Boldú et al 2001).…”