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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Produced waste-activated sludge (WAS) can be recovered as a promising carbon source in wastewater treatment plants. However, the cell integrity is always ignored, which results in the unwanted release of N and P. In this work, an alginate-degrading microbial consortium (ADC) with a higher percentage of genus Bacteroides (>90%) was enriched. The role of enriched ADC on the productions of methane and volatile fatty acids (VFAs) and cell integrity (via lactate dehydrogenase (LDH) activity and DNA concentrations) was then investigated in WAS fermentation. The results showed that dosing with ADC increased methane production by 53% without P release. Methane production of 440 mL from extracted extracellular polymeric substances was comparable to that from WAS (445 mL). After inhibiting methanogenesis, VFA concentrations via dosing with ADC were comparable to those of alkali fermentation, while expectantly showing lower N (114 vs 367 mg/L) and P (0 vs 122 mg/L) release. Since N and P are mainly stored within cells, such better selectivity was attributed to the ADC not affecting cell integrity, which was also verified by LDH activity and DNA concentrations. Consequently, these results provide a highly selective microbial method to produce biochemicals from WAS while maintaining cell integrity.

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