Inhibition of Growth of a Graphium sp. on Gaseous n-Alkanes by Gaseous n-Alkynes and n-Alkenes

Abstract

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

The anaerobic degradation of gaseous, nonmethane alkanes — From in situ processes to microorganisms

“…A copper-containing monooxygenase, similar with the particulate methane monooxygenase and ammonia monooxygenase, was proposed for a strain of Nocardioides [21] . Activation of gaseous alkanes in fungi was proposed to be carried out by cytochrome P-450 monooxygenases [22] . The primary alcohols are further oxidized to aldehydes and fatty acids, which are channeled into central metabolic pathways, and typically oxidized completely to CO 2 [11] .…”

Utilisation of C2–C4 gaseous hydrocarbons and isoprene by microorganisms

“…Only two strains, Mycobacterium E2039 and Rhodococcus rhodochrous B‐27656 (formerly Nocardia corallina ), which grow well on both classes of hydrocarbon gases have been described. Where comparative testing has been carried out, ethylene, propylene and butylene could not be used for growth by other gaseous n ‐alkane‐utilising strains 36, 42, 44, 58, 60, 67, 79, 90…”

Fungal Communities in Hydrocarbon Degradation

“…The resulting fatty acids are then incorporated to the central catabolic pathways via p-oxidation, involving the initial activation of the fatty acid to form an acyl-CoA ester. Gaseous nalkenes and n-alkynes were reported as selective inhibitors of the CYP monooxygenase responsible for the initial n-alkane oxidation: growth of Scedosporium boydii ATCC 58400 on ethane was blocked when the fungus was exposed to low concentrations of propene and propyne (Curry et al 1996). The activity of an alkanespecific CYP monooxygenase in this fungus has recently been demonstrated via RNAi silencing (Trippe et al 2014).…”