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To investigate the effect of ammonium addition on the species composition and activity of freshwater methane oxidizing bacteria, intact sediment cores were labeled with 13CH4 and incubated under ambient and elevated ammonium concentrations. After 7 days, methanotroph activity was assessed by quantifying the isotopic composition of the carbon in membrane lipids. The 16-carbon rather than the 18-carbon methanotroph-specific biomarkers showed a clear enrichment in 13C, suggesting the importance of group I methanotrophs in these sediments. Ammonium addition resulted in a depleted isotopic signal compared to ambient controls, suggesting that high ammonium concentrations inhibit methane incorporation into cellular components. These results compare favorably with studies that showed ammonium inhibition of methane oxidation, and extend these findings by demonstrating the effect of nitrogen fertilization on methanotroph lipid synthesis.