Exploring recycled agricultural wastes for high-rate removal of nitrogen in wastewater: Emphasizing on the investigation of the inner driving force and comparison with conventional liquid carbon sources.

In this study, four typical recycled agricultural wastes (AWs), corn cob, wheat straw, sawdust and walnut shells (named AW1, AW2, AW3 and AW4, respectively), were selected as external solid carbon sources to enhance the removal of nitrogen in wastewater, and specifically, the driving mechanism was thoroughly investigated. The leaching experiments showed that the dissolved organic carbon (DOC) release capacity followed the order of AW1>AW2>AW3>AW4, ranging from 6.21 to 31.92 mg/g. DOC released from AWs mainly consisted of protein-like substances, fulvic acid-like substances and humic-like substances. AW1 and AW2 achieved comparable NOx--N removal performance with a liquid carbon source of sodium acetate (SA) during the long-term denitrification experiments (>94.2%) but not for the other two AWs (only 16.8%-38.1%). Denitrification performance relied on DOC released from AWs at the beginning, while the enrichment of the functional CAZymes (including glycoside hydrolase and carbohydrate esterase) involved in cellulose and hemicellulose decomposition of AWs and functional genes (GAPDH, gap 2, PK, etc.) related to glycolysis were the inner driving force, which guaranteed the continuous supply of electron donors for denitrification. The relatively high abundances of napAB, narGHI, nirKS, norBC and nosZ, which encode nitrate reductase, nitrite reductase, NO reductase and N2O reductase, assured the better denitrification performance in the SA, AW1 and AW2 groups. In addition to denitrification-related functional genes, the relative abundances of nirBD and nrfAH associated with dissimilatory nitrate reduction were much higher in AW1 and AW2 groups than in SA group, implying that the nitrogen removal mechanism should be different in liquid carbon source and AW-based solid carbon source systems. In addition, GLU, gltBD and glnA, which participate in ammonia assimilation were the highest in the AW2 group, resulting in a large amount of organic nitrogen accumulation (peak concentration of approximately 24.5 mg/L), and this finally ruled it out as an alternative external carbon source. The abovementioned microbial mechanism was verified based on the correlation analysis of nutrient removal and functional genes combined with host bacterial analysis. Our study can provide valuable information for understanding the mechanism of using AWs as alternative external carbon sources to promote the removal of nitrogen in wastewater.