In this study, evidence for two novel metabolic processes catalyzed by a filamentous fungus, Graphium sp. strain ATCC 58400, is presented. First, our results indicate that this Graphium sp. can utilize the widely used solvent diethyl ether (DEE) as the sole source of carbon and energy for growth. The kinetics of biomass accumulation and DEE consumption closely followed each other, and the molar growth yield on DEE was indistinguishable from that with n-butane. n-Butane-grown mycelia also immediately oxidized DEE without the extracellular accumulation of organic oxidation products. This suggests a common pathway for the oxidation of both compounds. Acetylene, ethylene, and other unsaturated gaseous hydrocarbons completely inhibited the growth of this Graphium sp. on DEE and DEE oxidation by n-butane-grown mycelia. Second, our results indicate that gaseous n-alkane-grown Graphium mycelia can cometabolically degrade the gasoline oxygenate methyl tert-butyl ether (MTBE). The degradation of MTBE was also completely inhibited by acetylene, ethylene, and other unsaturated hydrocarbons and was strongly influenced by n-butane. Two products of MTBE degradation, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), were detected. The kinetics of product formation suggest that TBF production temporally precedes TBA accumulation and that TBF is hydrolyzed both biotically and abiotically to yield TBA. Extracellular accumulation of TBA accounted for only a maximum of 25% of the total MTBE consumed. Our results suggest that both DEE oxidation and MTBE oxidation are initiated by cytochrome P-450-catalyzed reactions which lead to scission of the ether bonds in these compounds. Our findings also suggest a potential role for gaseous n-alkane-oxidizing fungi in the remediation of MTBE contamination.
The growth of a filamentous fungus, a Graphium sp., on n-alkanes (C 2 to C 4) was inhibited by low concentrations of acetylene, propyne, 1-butyne, ethylene, and propylene. Acetylene and other unsaturated hydrocarbons had no effect on the growth of the Graphium sp. on potato dextrose broth, ethanol, or acetate. Our results suggest that n-alkynes and n-alkenes are selective inhibitors of a nonspecific monooxygenase enzyme responsible for the initial oxidation of n-alkanes. Graphium sp. strain ATCC 58400 is an example of the limited number of fungal species that grow on gaseous n-alkanes (1, 2, 10, 11, 15, 18). This organism was first isolated from an enrichment culture which used natural gas as a carbon and energy source (18). Originally it was suspected that this organism grew on methane (18), although subsequent studies demonstrated that its growth was being supported by the low levels of ethane found in domestic natural gas supplies (15, 16). It has also been demonstrated that this Graphium sp. and other fungal species also grow on other gaseous saturated hydrocarbons, including propane and n-butane. In contrast to these earlier studies, the aim of the present study was to examine the interaction between the Graphium sp. and gaseous unsaturated hydrocarbons. Our results demonstrate that gaseous unsaturated hydrocarbons do not support the growth of the Graphium sp. but that they are effective inhibitors of the growth of this organism on gaseous n-alkanes. In all experiments the Graphium sp. was grown for 5 to 7 days under axenic conditions in shake flasks (500 ml). The growth medium (100 ml) was either potato dextrose broth (PDB) (24 g/liter) or a mineral salts medium containing (per liter) (NH 4) 2 SO 4 (1 g), CaCl 2 ⅐ 2H 2 O (0.1 g), MgSO 4 ⅐ 7H 2 O (0.5 g), NaCl (0.1 g), and FeCl 3 ⅐ 6H 2 O (0.58 mg) added as a solution in 50 mM Na EDTA, pH 7, and 1 ml of a trace elements solution (4). The medium was adjusted to an initial pH of 7 by the addition of 1 ml of an aqueous sterile solution of K 2 HPO 4 (10% [wt/vol]). The flasks were inoculated with conidia (between 10 5 and 10 6 /100 ml) obtained from mycelia grown for 6 to 8 days at 25ЊC on potato dextrose agar plates under constant illumination. Following inoculation, the flasks were stoppered with butyl rubber stoppers, and sterile gaseous hydrocarbon substrates (10% gas phase [vol/vol]) were added as an overpressure by using plastic syringes fitted with sterile needles and Acrodisc filters (Gelman Sciences, Ann Arbor, Mich.). The flasks were incubated at 24ЊC in an orbital shaker (125 rpm). Yields of mycelia were determined from dry weight measurements which were made following vacuum filtration of the culture medium through paper filters (VWR Scientific, Los Angeles, Calif.) which had been previously dried and weighed. After filtration, the filter papers and mycelia were dried (65ЊC for 24 h) and reweighed. The data presented are representative results of experiments which were typically repeated three
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.