Bacterial bioaugmentation of woodchip bioreactors to increase nitrate removal in cold agricultural drainage water

Woodchip bioreactors (WBRs) are biological systems designed to prevent excess nitrate (NO₃^-) leaching from agricultural fields to aquatic ecosystems. Nitrate is removed by microbial denitrification, but the enzyme-mediated process slows down at cold temperatures (<10°C), where NO₃^- removal in WBRs can be less than 20%. We studied the use of bacterial bioaugmentation in replicated test-scale WBRs (∼0.1 m³) as an environmental technology to increase NO₃^- removal at cold temperatures. Nitrate removal rates increased following injection of a nitrate-reducing inoculum (<i>Pseudomonas proteolytica</i> and <i>Klebsiella</i> sp.), but the effect disappeared within a week and was reproduced in control WBRs by injection of sterile medium (phosphate buffer saline). Metagenome analyses showed a shift in the bacterial community composition after bioaugmentation in the planktonic phase of the woodchip reactors, but not in the solid phase (woodchip matrix). Only in the planktonic phase, <i>Pseudomonas</i> and <i>Klebsiella</i> increased their relative abundance as monitored by 16S rRNA gene sequences. In addition, an increased abundance of genes related to NO₃^- transformation after bacterial inoculation was observed in the metagenomic sequences. After one week, bacterial community composition became similar to its initial state, indicating resilience of the WBR microbial communities. We conclude that improved inoculation methods are needed to unlock the potential of bioaugmentation to increase NO₃^- removal at cold temperatures and make it a relevant technology for practical use at field-scale.