Earthworm-assisted constructed wetlands: A multidisciplinary investigation of organic matter and nitrogen removal using hydrodynamic, metagenomic, and water quality approaches

Earthworm (EW) soil-based filtration technologies offer a promising solution for decentralized wastewater (WW) treatment in urban areas. This study examined the effects of inoculating three Lumbricidae species (Eisenia fetida, Eisenia andrei, and Dendrobaena hortensis) on hydrodynamics, microbial communities, and purification performance in EW-assisted constructed wetlands (CWs). Conducted on mesocosm-scale systems (0.2 m² units) treating raw campus effluent, the experiment showed that EW inoculation slightly delayed the reduction of hydraulic conductivity over time without inducing short-circuiting, thereby preserving and slightly increasing mean hydraulic residence time. Organic matter and nitrogen removal remained comparable to controls. A 65 % increase in bacterial gene abundance was observed in EW-assisted CW substrates, without contributing to further bioclogging. Metagenomic analysis of interstitial biofilms revealed increases in the relative and absolute abundances of traits associated with the degradation of complex, recalcitrant organic molecules, but no significant changes in nitrogen-related processes. Traits related to recalcitrant organic molecules degradation represented only 3–6 % of all organic matter removal functions considered, consistent with the absence of significant differences in bulk organic matter removal. Overall, hydraulic, microbial, and water quality observations were consistent with each other, however the absence of a measurable effect of EW inoculation on overall treatment performance under the tested conditions limits mechanistic interpretation. The same integrative approach should be applied under different, potentially more favorable operational conditions to better reveal EW-driven effects (e.g. longer-term monitoring, higher organic loading to induce more severe clogging, alternative substrate types) and disentangle the respective contributions of hydraulic and microbial processes.