Micro-ecology of peat: minimally invasive analysis using confocal laser scanning microscopy, membrane inlet mass spectrometry and PCR amplification of methanogen-specific gene sequences

Abstract

A peat monolith (15 cm diameter, 35 cm length) from Ellergower Moss (New Galloway, Scotland), kept outdoors and maintained water-saturated, was investigated for the distributions of gases (O 2, CO 2, CH 4), micro-organisms, total archaeal DNA and methanogen DNA. From the water table (at the surface of the Sphagnum), a steep oxycline gave <0.25 μM O 2 at 2 cm depth (as shown by membrane inlet mass spectrometry and oxygen electrode methods) and <10 nM O 2 at 6 cm depth (photobacterium gas diffusion probe). Redox potential measurements indicated a steep decline between 6 cm and 13 cm to a value of −90 mV. At the oxic surface of the peat, CO 2 measured 0.5 mM and CH 4 <1 μM. Below 7 cm both gases increased to plateaux at 2 mM and 550 μM, respectively; CH 4 concentrations also indicated two distinct zones (7 μM to 2.5 cm depth, then to 28 μM at between 3 and 6 cm). Confocal laser scanning microscopy using the fluorophores 5-cyano-2,3-ditolyl tetrazolium chloride or 3,3-dihexyloxacarbocyanine iodide was used to image micro-organisms with redox active electron transport activities or transmembrane electrochemical potentials, respectively. Samples from 1–5 cm depth showed the presence of active aerobic organisms, whereas those from 10 and 20 cm depth were more active anaerobically, and especially so under H 2. Archaeal DNA was present throughout the core; strongest hybridisation was below 9 cm. Two methanogen-specific primers, ME1 and ME2 (which amplify a region of the α-subunit of methyl coenzyme M reductase), hybridised with DNA extracted from below 9 cm depth. Here we describe the concerted application of a number of techniques providing direct information on the precise location and activities of microbes involved in the flux of gases from peatlands.