Denitrification associated with Groups I and II methanotrophs in a gradient enrichment system

The occurrence and distribution of Groups I and II methanotrophs and their potential impact on denitrification were studied in a diffusion column model system simulating CH₄ and O₂ sources and delivery in the environment. We used NO₃⁻- or NH₄⁺-containing mineral salts media and three different inoculum sources: a swamp soil, a lake sediment and a cultivated humisol. The methylotrophic community structure which developed in the diffusion columns was characterized using oligodeoxynucleotide probes specific for ribulose monophosphate pathway (Group I; 10γ probe) and serine pathway (Group II; 9α probe) methylotrophs. Methanotrophs that grew near the top of the columns in zones of low CH₄ and high O₂ concentration, were generally from Group I; those growing at the bottom of the columns in zones of high CH₄ and low O₂ concentration were from Group II. Only in the humisol were both Group I and II detected at the top of the column. Concomitant production of N₂O with CH₄ consumption, observed in the diffusion columns, was confirmed in enrichment cultures. At least three denitrifiers associated with methanotrophic growth and activity were isolated. Methanotrophs that grew under high CH₄ and low O₂ conditions were associated with a Hyphomicrobium-like bacterium capable of denitrifying with methanol. Methanotrophic activity supported denitrification by (i) reducing the O₂ tension, and (ii) supplying organic compounds to the denitrifiers. Because this model system mimics many of the natural environments of methanotrophs, it is likely that the observed segregation of physiological types of methanotrophs and their interaction with denitrifiers also occur in nature.