Mechanism insights into polyhydroxyalkanoate-regulated denitrification from the perspective of pericytoplasmic nitrate reductase expression

Abstract

For enhanced biological nutrient removal (BNR) process, the polyhydroxyalkanoate (PHA) can be used as an eco-friendly internal as well as external substrate for regulating the growth of heterotrophic denitrifiers and promoting the denitrification process for deep nitrogen removal from wastewater. However, the exact mechanisms by which PHA impacts bacterial metabolism and affects the electron transfer of denitrification remain unknown. In this study, the in-depth mechanism investigation for PHA-mediated denitrification based on the jointly applied transcriptomic, proteomic and Western Blotting techniques was performed on a model denitrifier, Pseudomonas stutzeri. Results showed that PHA dramatically fostered the growth of Pseudomonas stutzeri, resulting in improved nitrate removal efficiency from 32.8% to 45.8%. Comparison of protein expression profiles indicated that PHA promoted the expression of enzyme NapB and NapA by approximately 10.34 and 20.01 times, respectively, which were both in charge of reduction from nitrate to nitrite. Based on transcriptional sequencing and Tandem Mass Tags, the correlation results also showed that differential proteins and genes with the same expression trend were positively correlated (R² = 0.427, p-value<0.033). Western Blotting approach was further developed to confirm the up-regulated expression of target protein with the higher proportion of PHA in carbon source of the medium, which proved the reliability of proteomics results. All the findings presented here are believed to deepen the understanding of microbial mechanism about PHA-enhanced denitrification from the novel perspective of associated electron-transfer enzymatic proteins.

Keywords: Denitrification; Polyhydroxyalkanoate (PHA); Proteomics; Pseudomonas stutzeri; Solid biopolymer.