Detection and Quantification with 16S rRNA Probes of Planktonic Methylotrophic Bacteria in a Floodplain Lake

Abstract

Two approaches employing 16S rRNA oligonucleotide probes, in situ hybridization combined with ³³P-autoradiography and ³²P-labeled slot-blot hybridizations on nitrocellulose filters, were used to enumerate methylotrophic bacteria in the water column of Ryans 1 Billabong, a small floodplain lake in northeastern Victoria, Australia. Methylotrophic bacterioplankton numbered 0.6–1.2 × 10⁹ cells liter⁻¹ in the winter of 1994, and 0.8–5.5 × 10⁹ cells liter⁻¹ in the summer of 1994–95. This was equivalent to 10–46% of total bacterioplankton cell counts, determined via epifluorescence microscopy. Methylotrophic bacteria were not detected in the water column of the nearby Kiewa River, and a set of laboratory controls indicated that the high abundance of methylotrophs in the billabong samples was not a methodological artifact. There was no change, with water depth, in total bacterioplankton or methylotroph abundance in winter, a result consistent with the water column being well mixed at this time of year (dissolved O₂ concentrations 5–7 mg liter⁻¹; dissolved methane concentrations <60 μg liter⁻¹, or <5% methane saturation, at all depths). In summer the billabong became diurnally stratified (dissolved O₂ concentrations <2 mg liter⁻¹ at water depths of >45 cm; dissolved methane concentrations <100 μg liter⁻¹ at the surface, but >500 μg liter⁻¹ near the sediments) and there was a correspondingly marked increase in the abundance of total bacterioplankton and methylotrophs with depth. In situ hybridizations and slot-blot hybridizations both indicated that type II methylotrophs (detected with a probe specific for the 9-α subgroup of Proteobacteria) were markedly less abundant than were type I and X methylotrophs (detected with a probe specific for the 10-γ subgroup of Proteobacteria).