The spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by fluorescence in situ hybridization (FISH), beta imaging and microsensors. FISH results revealed a layered structure of microorganisms in the granule, where Chloroflexi was present in the outermost layer, Smithella spp. and Syntrophobacter spp. were found in a depth of ca. 100 μm, and Archaea was restricted to the inner layer (below ca. 300 μm from the surface). Substrate uptake patterns elucidated by beta imaging demonstrated that glucose uptake was highest at 50 μm depth, whereas propionate uptake had a peak at 200 μm depth. In addition, microsensor measurements revealed that acid was produced mainly at 100 μm depth and H2 production was detected at a depth from 100 to 200 μm. H2 consumption and corresponding CH4 production were found below 200 μm from the surface. Direct comparison of these results implied sequential degradation of complex organic compounds in anaerobic granules; Chloroflexi contributed to fermentation of organic compounds and acid production in the outermost layer, volatile fatty acids were oxidized and H2 was produced mainly by Smithella spp. and Syntrophobacter spp. at a depth from 100 to 200 μm, and Archaea produced CH4 below ca. 300 μm from the surface.

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