Untargeted metabolomics elucidated biosynthesis of polyhydroxyalkanoate by mixed microbial cultures from waste activated sludge under different pH values.

Waste activated sludge has been frequently used as mixed substrate to produce polyhydroxyalkanoate (PHA). However, insufficient research on microbial metabolism has led to difficulties in regulating PHA accumulation in mixed microbial cultures (MMCs). To explore the variation of functional genes during domestication and the effect of different pH conditions on metabolic pathways during PHA accumulation, MMCs were domesticated by adding acetate and propionate with aerobic dynamic feeding strategy for 60 days. As the domestication progressed, the microbial community diversity declined and PHA-producing bacteria, Brevundimonas, Dechloromonas and Hyphomonas, were enriched. Through bacterial function prediction by PICRUSt the gene rpoE involved in starvation resistance of bacteria was enriched after the domestication. The pH value of 8.5 was the best condition for PHA accumulation in MMCs, under which a maximum PHA content reached 23.50% and hydroxybutyric (HB)/hydroxyvaleric (HV) reached 2.22. Untargeted metabolomics analysis exhibited that pH conditions of 7 and 8.5 could promote the up-regulation of significant differential metabolites, while higher alkaline conditions caused the inhibition of metabolic activity. Functional annotation showed that pH condition of 8.5 significantly affected Pyrimidine metabolism, resulting in an increase in PHA production. Regarding the pathways of PHA biosynthesis, acetoacetate was found to be significant in the metabolism of hydroxybutyric, and the alkaline condition could restrain the conversion from hydroxybutyric (HB) to the acetoacetate to protect PHB accumulation in MMCs compared with neutral condition. Taken together, the present results can advance the fundamental understanding of metabolic function in PHA accumulation under different pH conditions.