Environmental Microbiology (2005) doi:10.1111/j.1462-2920.2005.00786.x © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 20057Original Article Mono Lake methane oxidation and community compositionS. Carini, N. Bano, G. LeCleir and S. B. Joye Received 26 July, 2004; revised 22 November, 2004; accepted 22 November, 2004. *For correspondence. E-mail mjoye@uga.edu; Tel. (+1) 706 542 5893; Fax (+1) 706 542 5888. Aerobic methane oxidation and methanotroph community composition during seasonal stratification in Mono Lake, California (USA) Stephen Carini, Nasreen Bano, Gary LeCleir and Samantha B. Joye* Department of Marine Sciences, University of Georgia, Athens, GA 30602-3636, USA. Summary Patterns of aerobic methane (CH 4 ) oxidation and asso- ciated methanotroph community composition were investigated during the development of seasonal stratification in Mono Lake, California (USA). CH 4 oxi- dation rates were measured using a tritiated CH 4 radiotracer technique. Fluorescence in situ hybridiza- tion (FISH), denaturing gradient gel electrophoresis (DGGE) and sequence analysis were used to charac- terize methanotroph community composition. A tem- porally shifting zone of elevated CH 4 oxidation (59– 123 nM day -1 ) was consistently associated with a sub- oxycline, microaerophilic zone that migrated upwards in the water column as stratification progressed. FISH analysis revealed stable numbers of type I (4.1– 9.3 ¥ 10 5 cells ml -1 ) and type II (1.4–3.4 ¥ 10 5 cells ml -1 ) methanotrophs over depth and over time. Dena- turing gradient gel electrophoresis and sequence analysis indicated slight shifts in methanotroph com- munity composition despite stable absolute cell num- bers. Variable CH 4 oxidation rates in the presence of a relatively stable methanotroph population sug- gested that zones of high CH 4 oxidation resulted from an increase in activity of a subset of the existing methanotroph population. These results challenge existing paradigms suggesting that zones of elevated CH 4 oxidation activity result from the accumulation of methanotrophic biomass and illustrate that type II methanotrophs may be an important component of the methanotroph population in saline and/or alkaline pelagic environments. Introduction Aerobic methane-oxidizing bacteria (methanotrophs) are distinguished by their ability to use methane (CH 4 ) as their sole source of metabolic energy and structural carbon. Methanotrophs inhabit a variety of terrestrial and aquatic habitats and play an important role in global carbon, oxy- gen and nitrogen cycling (Cicerone and Oremland, 1988). Biological CH 4 oxidation is the predominant sink mitigating the flux of CH 4 , an important radiative trace gas, to the atmosphere (Topp and Hanson, 1991). Methanotrophic bacteria consume up to 80% of the CH 4 produced in freshwater and marine environments (Reeburgh et al., 1993) and may be a significant CH 4 sink in saline and alkaline lakes (Iverson et al., 1987; Joye et al., 1999; Khmelenina et al., 2000). Methanotrophs are grouped into two families based pri- marily on internal membrane arrangement and on the carbon assimilation pathway employed. Type I methan- otrophs have disc-shaped membrane bundles distributed throughout the cytoplasm, assimilate carbon as formalde- hyde via the RuMP pathway and comprise a distinct clus- ter within the gamma subclass of the Proteobacteria (g- Proteobacteria). Type II methanotrophs possess paired internal membrane structures aligned with the periphery of the cell, assimilate formaldehyde via the serine pathway and form a distinct cluster in the a-Proteobacteria (Han- son and Hanson, 1996). Type I methanotrophs have been described as the dominant or exclusive family of methan- otrophs observed in estuarine, marine and hypersaline environments (Holmes et al., 1996; Bourne et al., 2000; Khmelenina et al., 2000). Although type II methanotrophs are a significant component of the methanotroph popula- tions in freshwater sediments (Costello and Lidstrom, 1999), peat bogs (Deydysh et al., 2000) and rice paddy soils (Henckel et al., 1999; Eller and Frenzel, 2001), these organisms have not been documented previously in saline and/or alkaline environments such as Mono Lake. Saline lakes are numerous, geographically widespread, and comprise a significant part of the world’s inland aquatic ecosystems (Williams, 2002). Mono Lake is an alkaline salt lake located just east of the Sierra Nevada range in northern California (38∞N, 119∞W). During this study, the lake was meromictic and the monimolimnion (bottom water) had been isolated below the pycnocline since 1995. This isolation resulted in persistent anoxia and accumulation of high concentrations of dissolved CH 4 (50–100 mM) in the bottom waters. During winter, the lake was isothermal to the pycnocline. Increased solar heating