Investigation of the effect of ammonium sulfate on populations of ambient methane oxidising bacteria by 13 C-labelling and GC/C/IRMS analysis of phospholipid fatty acids Z.M. Crossman a , Z.-P. Wang b , P. Ineson c , R.P. Evershed a, * a Organic Geochemistry Unit, Bristol Biogeochemistry Research Centre, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK b Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, People’s Republic of China c Department of Biology, University of York, P.O. Box 373, York, YO10 5YW, UK Received 14 January 2005; received in revised form 13 July 2005; accepted 21 August 2005 Available online 23 September 2005 Abstract The oxidation of atmospheric methane by methanotrophic bacteria residing in soils constitutes an important terrestrial methane sink with previous studies having revealed the inhibition of microbially mediated methane oxidation in the presence of salt ions. The bacteria responsible for ambient methane oxidation are not amenable to currently available methods of culturing, resulting in the need for a method of in situ analysis. A combination of phospholipid fatty acid (PLFA) analysis and stable isotopic labelling has been employed in this investigation as a means of cultivation-independent bacterial analysis. Soil samples were treated with an ammonium sulfate solution at a concentration that was known to inhibit methane oxidation or with distilled water, serving as a control, and incubated with 13 C-labelled methane. PLFAs were analysed by GC/C/IRMS in order to determine their 13 C content and, hence, the PLFA distribution of the methane oxidising bacteria. Ammonium sulfate treatment reduced the amount of 13 C incorporated into the majority of PLFAs except the i17:0 PLFA in the presence of high concentrations of methane. These results implied a shift in the composition of the methane oxidising bacterial community in the soils treated with ammonium ions, with the treatment appearing to suppress one group of organisms more than another. q 2005 Published by Elsevier Ltd. Keywords: 13 C-labelling; GC/C/IRMS; Phospholipid fatty acids; Ambient methane oxidation; Ammonium; Inhibition; Forest soil 1. Introduction Methane is a greenhouse gas estimated at contributing 15% to global warming (Rohde, 1990). A major terrestrial sink of methane is its oxidation by methane oxidising bacteria inhabiting soils. The inhibitory effect of nitrogen fertilisation on bacterially mediated methane oxidation was first reported by Steudler et al. (1989), who found that after 4 months of fertilisation with NH 4 NO 3 methane consumption was reduced by up to 15% in a hardwood stand and up to 24% in a pine stand. Due to the important role played by methane oxidation in soils, further investigations into its responses to various salt treatments have been published (King and Schnell, 1994, 1998; Boeckx et al., 1997; Gulledge et al., 1997; Gulledge and Schimel, 1998). The mechanism of inhibition is still unclear and differs between study sites, appearing to be influenced by a variety of factors. Due to the physicochemical similarities between CH 4 and NH 3 it was initially postulated that these two compounds competed for the enzyme active sites so that NH 3 oxidation competitively inhibited CH 4 oxidation (Be ´dard and Knowles, 1989). However, in many cases when studying methane oxidation in soils the kinetics of NH C 4 inhibition could not be so easily explained by competitive inhibition. For example, King and Schnell (1994) noted that increasing atmospheric methane concentration increased the inhibitory effect that ammonium salts had on soil methane consumption. They proposed that nitrite formation from methanotrophic ammonium oxidation accounted for much of the observed inhibition. Gulledge and Schimel (1998), however, demon- strated the importance of nonammoniacal salts on methane oxidation in soils by treating soils with K 2 SO 4 , (NH 4 ) 2 SO 4 , Na 2 SO 4 , KCl or NH 4 Cl. They found that methane oxidation in temperate soils was inhibited to some extent by all the salts with NH 4 Cl and KCl being the most inhibitory followed by (NH 4 ) 2 SO 4 and finally K 2 SO 4 . As levels of natural NH C 4 in the soils were low it was thought that this effect was not a result of Soil Biology & Biochemistry 38 (2006) 983–990 www.elsevier.com/locate/soilbio 0038-0717/$ - see front matter q 2005 Published by Elsevier Ltd. doi:10.1016/j.soilbio.2005.08.011 * Corresponding author. Tel.: C44 117 928 7671; fax: C44 117 925 1295. E-mail address: r.p.evershed@bris.ac.uk (R.P. Evershed).