Enrichment and Isolation of Aerobic Methanotrophs

Enrichment cultures with both microbial mats and lake water (20 ml) collected from Movile Cave (April 2010) were set up in 120 ml serum vials. In order to reproduce the atmosphere found in the air bells, the headspace of the serum vials was flushed with O₂-free nitrogen and amended with 7% O₂ and 2.5% CO₂. Methane (10%) was introduced to the serum vial headspace as carbon source for enrichment of aerobic MOB. Following 2 weeks of enrichment, 50 μl aliquots, including a dilution series of 1:10 and 1:100 of the culture were spread onto Dilute Basal Salts (DBS) agar plates [12 ]. The plates were incubated in airtight plastic boxes with 10% CH₄ in the atmosphere and monitored for colony formation. Selected colonies were streaked onto fresh DBS agar plates and sub-culturing was performed until the cultures were deemed to be pure. Purity was determined by phase contrast microscopy (×1000) and lack of growth on R2A agar (Oxoid) plates were used to confirm the purity of the culture. Initial identification of the MOB isolate was determined by sequencing the 16S rRNA gene as described in [9 (link)].