Groundwater nitrate contamination poses a threat to both the ecological environment and human health. This study investigated the potential of using saturated Ca(OH)2 to pretreat wheat straw and woodchips, aiming to enhance their efficacy as carbon sources for denitrification. The optimization of pretreatment conditions, and the elucidation of underlying mechanisms were explored. The pretreatment process involved the dissolution of lignin and hemicellulose, exposure of the cellulose structure, reduction of hydrogen bonds within cellulose, hydrolysis of polymerized cellulose, and the formation of cracks and hierarchical structures on the surface of the carbon source. These alterations improved the attachment and utilization of microorganisms. The maximum enzymatic reducing sugar yields for wheat straw and woodchips were achieved at solid-liquid ratios of 1:40 and soaking times of 5 and 2 days, respectively. The response surface predicted the optimal pretreatment conditions for wheat straw to be a solid-liquid ratio of 1:88.1 and a soaking time of 8.2 h. Alkaline treatment increased the denitrification rate of woodchips by fivefold and prevented the initial organic matter leaching rate of wheat straw, thereby reducing the risk of secondary pollution. The predominant microbial communities in all samples exhibited functions related to lignocellulose degradation and denitrification. The community composition of solid-phase carbon sources was found to be richer than that of liquid-phase carbon sources, and the pretreatment increased the abundance of lignocellulose degradation and denitrification functional microorganisms. The pretreatment liquid of wheat straw achieved the highest denitrification rate constant (0.43 h−1). Our result validated the feasibility of using the pretreatment liquid as a denitrification carbon source and presenting a novel approach for waste resource utilization.
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