Effect of selected monoterpenes on methane oxidation, denitrification, and aerobic metabolism by bacteria in pure culture

Abstract

Selected monoterpenes inhibited methane oxidation by methanotrophs (Methylosinus trichosporium OB3b, Methylobacter luteus), denitrification by environmental isolates, and aerobic metabolism by several heterotrophic pure cultures. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation by Methylosinus trichosporium OB3b with 1.1 mM (-)-alpha-pinene lasted for more than 2 days with a culture of optical density of 0.05 before activity resumed. Inhibition was greater under conditions under which particulate methane monooxygenase was expressed. No apparent consumption or conversion of monoterpenes by methanotrophs was detected by gas chromatography, and the reason that transient inhibition occurs is not clear. Aerobic metabolism by several heterotrophs was much less sensitive than methanotrophy was; Escherichia coli (optical density, 0.01), for example, was not affected by up to 7.3 mM (-)-alpha-pinene. The degree of inhibition was monoterpene and species dependent. Denitrification by isolates from a polluted sediment was not inhibited by 3.7 mM (-)-alpha-pinene, gamma-terpinene, or beta-myrcene, whereas 50 to 100% inhibition was observed for isolates from a temperate swamp soil. The inhibitory effect of monoterpenes on methane oxidation was greatest with unsaturated, cyclic hydrocarbon forms [e.g., (-)-alpha-pinene, (S)-(-)-limonene, (R)-(+)-limonene, and gamma-terpinene]. Lower levels of inhibition occurred with oxide and alcohol derivatives [(R)-(+)-limonene oxide, alpha-pinene oxide, linalool, alpha-terpineol] and a noncyclic hydrocarbon (beta-myrcene). Isomers of pinene inhibited activity to different extents. Given their natural sources, monoterpenes may be significant factors affecting bacterial activities in nature.

FIG. 1

Representative structural formulae of the monoterpenoids used. 1, (R)-(+)-limonene; 2, (+)-limonene oxide; 3, (+)-α-pinene oxide; 4, α-pinene; 5, (−)-β-pinene; 6, γ-terpinene; 7, α-terpinene; 8, α-terpineol; 9, β-myrcene; 10, linalool.

FIG. 2

Effects of different levels of (−)-α-pinene on CH₄ oxidation by Methylosinus trichosporium OB3b. α-Pinene at final concentrations of 0 mM (○), 0.37 mM (▪), 0.73 mM (▴), and 1.10 mM (▾) was added to cultures having an initial OD₆₀₀ of 0.05. The values are means ± standard errors of the means determined from triplicate experiments. d, day.

FIG. 3

Changes in the levels of total CH₄ (A) and headspace (−)-α-pinene (B) during CH₄ oxidation by Methylosinus trichosporium OB3b. Control, inoculated flasks were incubated without (−)-α-pinene (○). (−)-α-Pinene (1.10 mM) was added to both inoculated (▪) and uninoculated (▴) flasks. An initial OD₆₀₀ of 0.045 was used. The values are means ± standard errors of the means determined from duplicate experiments. d, day.

FIG. 4

Changes in the levels of total CH₄ (A) and headspace (−)-α-pinene (B) in preincubated (▪) and nonpreincubated (▴) flasks inoculated with Methylosinus trichosporium OB3b. Preincubated flasks were supplemented with (−)-α-pinene and shaken for 3 days before inoculation. Nonpreincubated flasks were inoculated immediately after (−)-α-pinene was added. Control flasks were incubated without (−)-α-pinene (○). An initial cell OD₆₀₀ of 0.075 and 1.80 mM (−)-α-pinene were used. The values are means ± standard errors of the means determined from triplicate experiments. d, day.

FIG. 5

Effects of selected monoterpenes on denitrification (N₂O accumulation) by environmental isolates from Hamilton Harbour (HH1)(A) and Mt. St. Hilaire (D1)(B). Flasks were incubated with acetylene (○), monoterpene (•), and acetylene plus monoterpene (▪). The initial culture densities (OD₆₀₀) were 0.12 (for pinene and terpinene additions) and 0.03 (for myrcene additions). The monoterpene concentrations were 3.70 mM. The results for isolate HH1 (A) are representative of the results obtained with isolates HH3, HH4, HH6, and the results for isolate D1 (B) are similar to the results obtained with isolate D3 (see text). The values are means ± standard errors of the means determined from duplicate experiments. d, day.

FIG. 6

Effects of different levels of (−)-α-pinene on aerobic metabolism (CO₂ production) of E. coli (•) and B. subtilis (▪) over a 38-h period. The initial cell OD₆₀₀ was 0.01. The values are means ± standard errors of the means determined from duplicate experiments.