Nitrate removal performance and diversity of active denitrifying bacteria in denitrification reactors using poly(L-lactic acid) with enhanced chemical hydrolyzability

Poly(L-lactic acid) (PLLA) can be used as an external electron donor in denitrification reactors to treat drinking water, aquaculture water, and industrial wastewater with an imbalanced carbon/nitrogen ratio. However, for PLLA to function in these applications, its chemical hydrolyzability requires improvement. Although the adjustment of the crystallinity (Xc) is effective in improving the hydrolyzability of PLLA, the condition for the Xc of PLLA, in which a sufficient amount of lactic acid is released for denitrification, must be clarified. Therefore, this study investigated the effective Xc range and optimal PLLA content as an electron donor for continuous nitrate removal in denitrification reactors. This study also explored the abundance, succession, and diversity of active denitrifying bacteria in denitrification reactors. The nitrate removal activity of activated sludge using the highly crystalline PLLA (Xc = 39.4%) was 1.8 mg NO3− -N g MLSS−1 h−1, which is 2.4 times higher than that using the nearly amorphous PLLA (Xc = 0.9%). During the 57 days of operation, the denitrification reactor with 3% (w/v) highly crystalline PLLA continued to completely remove nitrate, with a maximum nitrate removal activity of 22.8 mg NO3− -N g MLSS−1 h−1. The 16S rRNA amplicon sequencing and clone library analyses are using transcripts of two nitrite reductase genes, encoding cytochrome cd1 nitrite reductase, and copper-containing nitrite reductase revealed that bacteria belonging to the families Comamonadaceae, Rhodocyclaceae, and Alcaligenaceae were active denitrifying bacteria in the denitrification reactor using PLLA.