The objective of this study was to understand how the non-microbial factors of L-AD effluent affected the microbiome composition and successions in the SS-AD digesters using both Illumina sequencing and qPCR quantification of major genera of methanogens. The SS-AD digesters started with a feedstock/total effluent (F/Et) ratio 2.2 (half of the effluent was autoclaved) performed stably, while the SS-AD digesters started with a 4.4 F/Et ratio (no autoclaved effluent) suffered from digester acidification, accumulation of volatile fatty acids, and ceased biogas production two weeks after startup. Some bacteria and methanogens were affected by non-microbial factors of the L-AD fluent. Alkalinity, the main difference between the two F/Et ratios, may be the crucial factor when SS-AD digesters were started using L-AD effluent. (C) 2015 Elsevier Ltd. All rights reserved.