Response of methanogenic archaeal community to nitrate addition in rice field soil

Addition of nitrate strongly inhibits CH₄ production from anoxic soil. The main mechanisms were considered to be the substrate competition and the toxic effect of denitrification intermediates on the methanogenesis. However, it is unclear whether these inhibitory effects are reflected in the structure and dynamics of methanogenic community in the soil. In the present study, the response of methanogenic archaeal community to nitrate addition was determined using terminal restriction fragment length polymorphisms in combination with cloning and sequencing of archaeal 16S rRNA genes. When nitrate was added at the beginning of an anoxic incubation of rice field soil, denitrification occurred rapidly and the denitrification intermediates were detected only for a short time. Total production of CH₄ was reduced, but no obvious effect on the structure of methanogenic community was observed. In contrast, when nitrate was added 20 days after the anoxic incubation, the denitrification intermediates obviously accumulated. CH₄ production was completely suppressed for 7 and 16 days from treatments of 5 and 10 mM nitrate respectively. The dynamics of methanogenic community also diverged greatly from the control. While the hydrogenotrophic methanogens increased and the acetoclastic methanogens decreased with the incubation in the control soil, the structure and abundance of the methanogenic community remained unchanged after the addition of nitrate. Methanogenesis resumed when the denitrification intermediates were depleted in soil. The analysis of carbon isotopic signals revealed that hydrogenotrophic methanogenesis recovered faster than acetoclastic methanogenesis. Our study suggests that the accumulation of denitrification intermediates has a strong inhibitory effect on the activity but not the structure of methanogenic community.