Effects of alkaline pretreatment on the distribution of amino acid enantiomers and their biofunctions during the anaerobic digestion of waste activated sludge

The types and concentrations of L- and D-type amino acids (AAs) produced by sludge hydrolysis increased with increasing pH of alkali treatment, with the highest content of cysteine and a higher degree of racemization of threonine, cysteine, and alanine. There was a strong correlation between methane production and the following AAs (D-leucine, D-aspartic acid, L-threonine, and L-cysteine). The addition of representative AAs (L-cysteine and D-cysteine) increased cumulative methane production, and L-type promoted better than D-type, with the highest cumulative methane production 906.3 mL/g-VS with the addition of 50 mg/L of L-cysteine, which was 1.20 times that of the CK group (digestion time 21 d); while the methane production with the addition of 200 mg/L of L-cysteine would be inhibited. The L-type AAs promoted better than the D-type in the solubilization, hydrolysis, acidification, and hydrogen-consumption methanogenesis stages, whereas the homoacetogenesis process and acetate-consumption methanogenesis stages did not play a significant role. The dominant genera of bacteria at the genus level belonged to unclassified_c_Acitinobacteria, unclassified_p_Chloroflexi, unclassified_c_Acidimicrobiia and Mycobacterium, while Methanothrix and Methanobacterium were the dominant archaea in the digesters. The top 4 metabolic functions were biosynthesis of secondary metabolites, microbial metabolism in diverse environments, biosynthesis of AAs and carbon metabolism. Metagenome results showed that, with L-cysteine addition, enriching Methanothrix and Methanobacterium showed enhanced contribution to the main pathways, especially the biosynthesis of secondary metabolites and carbon metabolism.