Droplet Digital PCR Facilitates the Revelation of Metabolic Interactions Among Key Microorganisms in the Integrated Simultaneous Desulfurization and Denitrification Process

The integrated simultaneous desulfurization and denitrification (ISDD) process offers a viable solution for coremoval of sulfate and nitrate while recovering elemental sulfur. However, the metabolic interactions among the functional microorganisms involved in the bioconversion of carbon, nitrogen, and sulfur in this process remain poorly understood. In this study, a quantitative framework was established to measure the absolute abundances of individual bacterial and archaeal taxa by integrating the precision of droplet digital PCR with the high-throughput 16S rRNA gene amplicon sequencing. Quantitative measurements of absolute abundances identified the dominant species as COD/sulfate ratio varied from 1 to 3 and then back to 1. As the COD/sulfate ratio increased from 1 to 3, sulfate removal efficiency improved from 57.67% to 94.69%, accompanied by increased hydrogen and methane production. Desulfobulbus exhibited a competitive advantage in facilitating metabolite transfer and enhancing methane production efficiency. Furthermore, higher influent COD upregulated apr, omcS, and nirK genes, while downregulating the sqr gene. Five pairs of electron-shuttling systems among Methanobacterium, Methanosaeta, Anaerolineaceae, Desulfobulbus and Sulfurovum were explained through structural equation modeling (SEM). These findings offer valuable insights into microbial interactions that could enhance ISDD system performance.