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A novel two-phase anaerobic treatment technology was developed to treat high-concentration organic cassava bioethanol wastewater. The start-up process and contribution of organics (COD, total nitrogen, and NH4+-N) removal in spatial succession of the whole process and spatial microbial diversity changing when sampling were analyzed. The results of the start-up phase showed that the organic loading rate could reach up to 10 kg COD/m3d, with the COD removal rate remaining over 90% after 25 days. The sample results indicated that the contribution of COD removal in the pre-anaerobic and anaerobic phases was 40% and 60%, respectively, with the highest efficiency of 98.5%; TN and NH4+-N had decreased to 0.05 g/l and 0.90 g/l, respectively, and the mineralization rate of total nitrogen was 94.8%, 76.56% of which was attributed to the anaerobic part. The microbial diversity changed remarkably among different sample points depending on the physiological characteristics of identified strains. Moraxellaceae,Planococcaceae, and Prevotellaceae were dominant in the pre-anaerobic phase and Bacteroidetes, Campylobacterales,Acinetobacter, Lactobacillus, Clostridium, and Bacillus for the anaerobic phase. Methanosarcinaceae and Methanosaeta were the two main phylotypes in the anaerobic reactor.

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