Methanogenic archaea and CO2‐dependent methanogenesis on washed rice roots

Abstract

Washed excised roots of rice (Oryza sativa) immediately started to produce CH₄ when they were incubated in phosphate buffer under anoxic conditions (N₂ atmosphere), with initial rates varying between 2 and 70 nmol h⁻¹ g⁻¹ dry weight of root material (mean ± SE: 20.3 ± 5.9 nmol h⁻¹ g⁻¹ dry weight; n = 18). Production of CH₄ continued for at least 500 h, with rates usually decreasing slowly. CH₄ production was not significantly affected by methyl fluoride, an inhibitor of acetoclastic methanogenesis. Less than 0.5% of added [2-¹⁴C]-acetate was converted to ¹⁴CH₄, and conversion of ¹⁴CO₂ to ¹⁴CH₄ indicated that CH₄ was almost exclusively produced from CO₂. Occasionally, however, especially when the roots were incubated without additional buffer, CH₄ production started to accelerate after about 200 h reaching rates of > 100 nmol h⁻¹ g⁻¹ dry weight. Methyl fluoride inhibited methanogenesis by more than 20% only in these cases, and the conversion of ¹⁴CO₂ to ¹⁴CH₄ decreased. These results indicate that CO₂-dependent rather than acetoclastic methanogenesis was primarily responsible for CH₄ production in anoxically incubated rice roots. Determination of most probable numbers of methanogens on washed roots showed highest numbers (10⁶g⁻¹ dry roots) on H₂ and ethanol, i.e. substrates that support CH₄ production from CO₂. Numbers on acetate (10⁵ g⁻¹ dry roots) and methanol (10⁴ g⁻¹ dry roots) were lower. Methanogenic consortia enriched on H₂ and ethanol were characterized phylogenetically by comparative sequence analysis of archaeal small-subunit (SSU) ribosomal RNA-encoding genes (rDNA). These sequences showed a high similarity to SSU rDNA clones that had been obtained previously by direct extraction of total DNA from washed rice roots. The SSU rDNA sequences recovered from the H₂/CO₂-using consortium either belonged to a novel lineage of methanogens that grouped within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales or were affiliated with Methanobacterium bryantii. SSU rDNA sequences retrieved from the ethanol-using consortium either grouped within the genus Methanosarcina or belonged to another novel lineage within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales. Cultured organisms belonging to either of the two novel lineages have not been reported yet.