10th International Aerosol Conference September 2 - September 7, 2018 America's Center Convention Complex St. Louis, Missouri, USA
Abstract View
Carbon Stable Isotope Probing Demonstrates Growth of Airborne Methanotrophs
VALDIS KRUMINS, Kevin Dillon, Donna Fennell, Rutgers, The State University of New Jersey
Abstract Number: 829 Working Group: Bioaerosols
Abstract Outdoor air holds 104 – 105 cells m-3, which until recently have been assumed to be inactive while aloft. We have previously shown that airborne bacteria – outside of water droplets or other aggregates – express proteins and produce ribosomes in response to volatile organic substrates. Here we examine the activity of airborne methanotrophic bacteria using DNA stable isotope probing, and demonstrate that bacteria also are capable of growth (production of DNA) in bulk air.
Methanotrophic cultures were established from rinsate of a maple leaf (dominated by type II methanotrophs) and from an outdoor air sample (containing mostly type I methanotrophs). The cultures were enriched and maintained in minimal media using methane as the sole carbon source. For each reactor run, up to 2 L of liquid culture (fed 12C methane) were concentrated by centrifugation and aerosolized into rotating aerosol bioreactors using a single pass aerosol generator (SPAG, CH Technologies, Westwood, NJ, USA). The 0.32 m3 paired reactors hold cells aloft for days, and are equipped with on-board sensors for temperature, pressure, relative humidity, CO2 and methane. After filling with bioaerosols, the reactors were amended with 1500 ppmv methane. One of the paired reactors received 13C-labeled methane; the other, unlabeled methane. The reactors were rotated at 3 rpm and held at at 22 oC for five days. At the end of incubation, ~107 cell-sized particles remained airborne. These airborne cells were collected onto 0.8 μm SUPOR membrane filters and immediately frozen (-80 oC). DNA extracted from the filters was ultracentrifuged (48,000 x g) for 48 hours on a CsCl gradient. The gradients were then separated into 24 fractions. The particulate methane monooxygenase gene (pmoA) and methanotroph-specific 16S rRNA genes (type I or type II methanotrophs, depending on the culture) were quantified in each fraction using real-time polymerase chain reaction (qPCR). In the reactors which received 13C labeled methane, separate DNA peaks representing 13C DNA and 12C DNA were detected in the gradient; while in reactors receiving unlabeled methane, a single 12C DNA peak was detected.
Detection of 13C DNA in the reactors amended with 13CH4 is evidence of production of new DNA and thus growth by airborne methanotrophs. The results suggest that microorganisms can metabolize methane, a potent greenhouse gas, while airborne.