Saccharolytic activity and its role as a limiting step in methane formation during the anaerobic degradation of rice straw in rice paddy soil

Rice straw polysaccharides are one of the major C sources for CH4 formation in anoxic rice paddy soils. We investigated the initial step of straw degradation by measuring the substrate-saturated activities of the polysaccharolytic enzymes beta-glucosidase, exo-beta-1,4-glucanase and xylosidase using substrates labelled with methylumbelliferone (MUF). The actual activity of the enzymes was measured by the release of reducing sugars after the inhibition of microbial carbohydrate uptake by toluene. The substrate-saturated enzyme activities increased during the first 11 days of incubation, while the actual activities decreased, presumably due to the decreasing access of straw polysaccharides to hydrolytic enzymes. The temporal progress of polysaccharide hydrolysis, transient accumulation of fermentation products and CH4 production indicated five distinct phases. In phase I (<8 h), the fermentation of sugar monomers released by hydrolysis of polysaccharides was limiting. In phase II (<day 3), sugars were fermented but the methanogcnic precursors H-2 and acetate accumulated since the activity of methanogens was low, though increasing exponentially. In phase III (days 3-10). H-2 was also consumed by respiratory processes (e.g. SO42- reduction) so that H-2-dependent methanogenesis sometimes became substrate limited. However, acetate still accumulated, probably due to the limiting activity of acetoclastic methano-ens. In phase IV (days 10-18), methanogenic activity was no longer limited and acetate was depleted to low concentrations. In phase V (>day 18), the methanogenic degradation of straw reached a quasi-steady state, when polysaccharide hydrolysis became the rate-limiting step for CH4 formation.