Variable carbon isotope fractionation expressed by aerobic CH4-oxidizing bacteria

Carbon isotope fractionation factors reported for aerobic bacterial oxidation of CH₄(α_CH4-CO2) range from 1.003 to 1.039. In a series of experiments designed to monitor changes in the carbon isotopic fractionation of CH₄ by Type I and Type II methanotrophic bacteria, we found that the magnitude of fractionation was largely due to the first oxidation step catalyzed by methane monooxygenase (MMO). The most important factor that modulates the (α_CH4-CH3OH) is the fraction of the total CH₄ oxidized per unit time, which strongly correlates to the cell density of the growth cultures under constant flow conditions. At cell densities of less than 0.1 g/L, fractionation factors greater than 1.03 were observed, whereas at cell densities greater than 0.5 g/L the fractionation factors decreased to as low as 1.002. At low cell densities, low concentrations of MMO limit the amount of CH₄ oxidized, while at higher cell densities, the overall rates of CH₄ oxidation increase sufficiently that diffusion of CH₄ from the gaseous to dissolved state and into the cells is likely the rate-determining step. Thus, the residual CH₄ is more fractionated at low cell densities, when only a small fraction of the total CH₄ has been oxidized, than at high cell densities, when up to 40% of the influent CH₄ has been utilized. Therefore, since Rayleigh distillation behavior is not observed, δ¹³C values of the residual CH₄ cannot be used to infer the amount oxidized in either laboratory or field-studies. The measured (α_CH4-CH3OH) was the same for both Type I and Type II methanotrophs expressing particulate or soluble MMO. However, large differences in the δ¹³C values of biomass produced by the two types of methanotrophs were observed. Methylosinus trichosporium OB3b (Type II) produced biomass with δ¹³C values about 15‰ higher than the dissimilated CO₂, whereas Methylomonas methanica (Type I) produced biomass with δ¹³C values only about 6‰ higher than the CO₂. These effects were independent of the magnitude of the initial carbon isotope fractionation caused by MMO and were relatively constant despite changing ratios of assimilatory to dissimilatory carbon transformation by the organisms. This suggests that the difference in biomass carbon isotopes is primarily due to differences in the fractionation effect at the formaldehyde branch point in the metabolic pathway, rather than assimilation of CO₂ by Type II methanotrophs.