Characterization and DNA Stable-Isotope Probing of Methanotrophic Bioaerosols

Abstract

The growth and activity of bacteria have been extensively studied in nearly every environment on Earth, but there have been limited studies focusing on the air. Suspended bacteria (outside of water droplets) may stay in the atmosphere for time frames that could allow for growth on volatile compounds, including the potent greenhouse gas methane. We investigated the ability of aerosolized methanotrophic bacteria to grow on methane in the airborne state in rotating gas-phase bioreactors. The physical half-life of the aerial bacterium-sized particles was 3 days. To assess the potential for airborne growth, gas-phase bioreactors containing the aerosolized cultures were amended with 1,500 ppmv ¹³CH₄ or ¹²CH₄. Three of seven experiments demonstrated ¹³C incorporation into DNA, indicating growth in air. Bacteria associated with the genera Methylocystis and Methylocaldum were detected in ¹³C-DNA fractions, thus indicating that they were synthesizing new DNA, suggesting growth in air. We conclude that methanotrophs outside of water droplets in the air can potentially grow under certain conditions. Based on our data, humidity seems to be a major limitation to bacterial growth in air. Furthermore, low biomass levels can pose problems for detecting ¹³C-DNA synthesis in our experimental system. IMPORTANCE Currently, the cellular activities of bacteria in the airborne state outside of water droplets have not been heavily studied. Evidence suggests that these airborne bacteria produce ribosomes and metabolize gaseous compounds. Despite having a potentially important impact on atmospheric chemistry, the ability of bacteria in the air to metabolize substrates such as methane is not well understood. Demonstrating that bacteria in the air can metabolize and grow on substrates will expand knowledge about the potential activities and functions of the atmospheric microbiome. This study provides evidence for DNA synthesis and, ultimately, growth of airborne methanotrophs.

Keywords: aerobiology; bacterial growth; humidity; methane oxidation; water availability.

FIG 1

Bacterial compositions of the methanotrophic air enrichment and methanotrophic maple leaf enrichment cultures. (A) Relative abundances of the different ASVs at the family level based upon partial 16S rRNA gene sequences. (B) Compositions of the cultures at the genus level for methanotrophs present in the cultures based upon partial pmoA gene sequences.

FIG 2

Bioaerosol characteristics. Two gas-phase bioreactors rotating at 1 rpm were filled with bioaerosols of a mixture of a methanotrophic air enrichment and S. aerolata NW12 for 30 min in 5-min increments. (A) Bioaerosols are characterized by the filling profile showing particle size distribution in the bioreactors; (B) physical retention (half-life) of particles in different particle size bins over a 5-day period; (C) average particle size distribution at days 0 and 5.

FIG 3

Liquid-phase growth of methanotrophs. (A and B) Duplicate sets of the methanotrophic air enrichment culture in liquid medium incubated with 4% [¹²C]methane or [¹³C]methane (vol/vol, gas headspace) over 6 days. The ¹²C- and ¹³C-labeled DNA was separated by ultracentrifugation and gradient fractionation. The presence of the DNA of methanotrophs across the density gradient was detected by qPCR of the pmoA gene (normalized to the highest copy number of the pmoA gene in a single sample). The pmoA amplicons of fractions at select densities were sequenced, and the relative abundances of different genera were identified as Methylocystis, Methylocaldum, or unknown based upon pmoA sequencing.

FIG 4

Isotopic incorporation of ¹³C into DNA from [¹³C]methane by aerosolized methanotrophic enrichment cultures. Each graph shows the average amounts of pmoA copies determined from technical replicates of a fraction with a specific buoyant density. The pmoA copy number was normalized to the highest copy number of the collected fractions for a sample (left y axis). Amplicons from qPCR of experiments 2 and 4 were sequenced (right y axis), and methanotrophs were identified. (A) Experiment 5; (B) experiment 2; (C) experiment 7; (D) experiment 4.