Inhibitory effects of nitrate, nitrite, NO and N2O on methanogenesis by Methanosarcina barkeri and Methanobacterium bryantii

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In order to elucidate the mechanism of the inhibitory effect of nitrate and its denitrification intermediates nitrite, NO and N2O on methanogenesis in anoxic environments, we tested possible toxic effects of these N-compounds on the methanogenic bacteria Methanosarcina barkeri and Methanobacterium bryantii which are ubiquitous in methanogenic rice field soils. The different N-compounds inhibited H2-dependent methanogenesis by these bacteria to different extents. Nitrate showed the weakest inhibition of methanogenesis in both bacteria, followed by N2O and nitrite for Ms. barkeri, and nitrite and N2O for Mb. bryantii, respectively. In both bacteria, the strongest inhibition was caused by NO. Concentrations of 30 mM nitrate still enabled a CH4 production rate of 25–40% of that before the addition of the N-compound, whereas NO completely inhibited methanogenesis at concentrations ≥0.8–1.7 μM (equivalent to 50–100 Pa NO partial pressure). Removal of NO by replacing the atmosphere with H2/CO2 (8:2) resulted in resumption of methanogenesis only if the bacteria had been treated with NO concentrations ≤0.8 μM (50 Pa). Removal of N2O from the cultures resulted in resumption of methanogenesis if Mb. bryantii had been treated with ≤95 μM N2O (500 Pa) or Ms. barkeri with ≤950 μM N2O (5 kPa). These results show that the denitrification products of nitrate can inhibit CH4 production both reversibly and irreversibly depending on the type of methanogenic bacterium and the applied concentration of the N-compound. In a separate experiment with methanogenic rice field slurries addition of nitrate resulted in immediate inhibition of CH4 production. Nitrate was consumed resulting in the sequential accumulation of nitrite, NO and N2O which were subsequently utilized. Nitrite and N2O reached maximum concentrations that would have been inhibitory in the methanogenic bacterial cultures examined.

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