<i>Propionibacterium freudenreichii</i>-Assisted Approach Reduces N₂O Emission and Improves Denitrification via Promoting Substrate Uptake and Metabolism

N2O emission is often encountered during biodenitrification. In this paper, a new approach of using microorganisms to promote substrate uptake and metabolism to reduce denitrification intermediate accumulation was reported. With the introduction of Propionibacterium freudenreichii to a biodenitrification system, N2O and nitrite accumulation was, respectively, decreased by 74 and 60% and the denitrification efficiency was increased by 150% at the time of 24 h with P. freudenreichii/groundwater denitrifier of 1/5 (OD600). Propionate, produced by P. freudenreichii, only accelerated nitrate removal and was not the main reason for the decreased intermediate accumulation. The proteomic and enzyme analyses revealed that P. freudenreichii stimulated biofilm formation by upregulating proteins involved in porin forming, putrescine biosynthesis, spermidine/putrescine transport, and quorum sensing and upregulated transport proteins, which facilitated the uptake of the carbon source, nitrate, and Fe and Mo (the required catalytic sites of denitrification enzymes). Further investigation revealed that P. freudenreichii activated the methylmalonyl-CoA pathway in the denitrifier and promoted it to synthesize heme/heme d1, the groups of denitrification enzymes and electron transfer proteins, which upregulated the expression of denitrifying enzyme proteins and enhanced the ratio of NosZ to NorB, resulting in the increase of generation, transfer, and consumption of electrons in biodenitrification. Therefore, a significant reduction in the denitrification intermediate accumulation and an improvement in the denitrification efficiency were observed.