Acidophilic Methanotrophic Communities from Sphagnum Peat Bogs

Abstract

Highly enriched methanotrophic communities (>25 serial transfers) were obtained from acidic ombrotrophic peat bogs from four boreal forest sites. The enrichment strategy involved using media conditions that were associated with the highest rates of methane uptake by the original peat samples, namely, the use of diluted mineral medium of low buffering capacity, moderate incubation temperature (20°C), and pH values of 3 to 6. Enriched communities contained a mixture of rod-shaped bacteria arranged in aggregates with a minor contribution ofHyphomicrobium-like cells. The growth stoichiometry of isolates was characteristic of methanotrophic bacteria (CH₄/O₂/CO₂=1:1.1:0.59), with an average apparent yield of 0.41 ± 0.03 g of biomass C/g of CH₄-C. DNA from each enrichment yielded a PCR product of the expected size with primers for both mmoX andmmoY genes of soluble methane monooxygenase. Two types of sequences were obtained for PCR-amplified fragments ofmmoX. One of them exhibited high identity to themmoX protein of the Methylocystis-Methylosinusgroup, whereas the other showed an equal level of divergence from both the Methylosinus-Methylocystis group andMethylococcus capsulatus (Bath) and formed a distinct branch. The pH optimum for growth and for CH₄ uptake was 4.5 to 5.5, which is very similar to that for the optimum CH₄ uptake observed in the original peat samples. These methanotrophs are moderate acidophiles rather than acidotolerant organisms, since their growth rate and methane uptake were much lower at neutral pH. The growth of the methanotrophic community was enhanced by using media with a very low salt content (20 to 200 mg/liter), more typical of their natural environment. All four enriched communities grew on N-free medium.