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The hydrolysis of extracellular polymeric substances (EPS) in waste-activated sludge (WAS) is considered as the rate-limiting step in anaerobic digestion. Uronic acids such as alginate are one of main polysaccharide components in EPS; however, their roles on WAS conversion are overlooked until now. Previously, we described alginatedegrading consortia (ADC) that have high activity for alginate conversion. In this work, ADC was studied for polysaccharide hydrolysis and methane production from WAS for the first time, which increased the methane production by 115%−185%. Dosing ADC also increased the values of biological methane potential from 131 to 172 mL/gVSS. An alginate-like exopolysaccharide was extracted from WAS, and the content was 65 mg/ g-VSS. Then, the molecular weight profiles at UV 254nm showed that disaccharides were the final hydrolysates of alginate by ADC enzyme. Extracted EPS could be utilized by ADC for methane production with acetate as the main intermediate. The mechanism was proposed that ADC played a key role in WAS conversion. These results indicated that alginate in EPS shall not be overlooked, which offers a new microbial method to enhance methane recovery from WAS. The microbial changes in ADC for the stability of WAS digestion should be investigated in the future.

Uronic acid in extracellular polymeric substances is a primary but often ignored factor related to the difficult hydrolysis of waste-activated sludge (WAS), with alginate as a typical polymer. Previously, we enriched alginate-degrading consortia (ADC) in batch reactors that can enhance methane production from WAS, but the enzymes and metabolic pathway are not well documented. In this work, two chemostats in series were operated to enrich ADC, in which 10 g/L alginate was wholly consumed. Based on it, the extracellular alginate lyase (∼130 kD, EC 4.2.2.3) in the cultures was identified by metaproteomic analysis. This enzyme offers a high specificity to convert alginate to disaccharides over other mentioned hydrolases. Genus Bacteroides (>60%) was revealed as the key bacterium for alginate conversion. A new Entner−Doudoroff pathway of alginate via 5-dehydro-4-deoxy-D-glucuronate (DDG) and 3deoxy-D-glycerol-2,5-hexdiulosonate (DGH) as the intermediates to 2-keto-3-deoxy-gluconate (KDG) was constructed based on the metagenomic and metaproteomic analysis. In summary, this work documented the core enzymes and metabolic pathway for alginate degradation, which provides a good paradigm when analyzing the degrading mechanism of unacquainted substrates. The outcome will further contribute to the application of Bacteroides-dominated ADC on WAS methanogenesis in the future.

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