The anammox coupled partial-denitrification process in an integrated granular sludge and fixed-biofilm reactor developed for mainstream wastewater treatment: Performance and community structure

This study describes an integrated granular sludge and fixed-biofilm (iGB) reactor innovatively designed to carry out the anammox/partial-denitrification (A/PD) process for nitrogen removal with mainstream municipal wastewater. The iGB-A/PD reactor consists of anammox granules inoculated in the lower region of reactor and an acclimated fixed-biofilm positioned in the upper region. Compared to the other reported A/PD systems for mainstream wastewater treatment, this iGB-A/PD reactor is notable due to its higher quality effluent with a total inorganic nitrogen (TIN) of ∼3 mg•L⁻¹ and operation at a high nitrogen removal rate (NRR) of 0.8 ± 0.1 kg-N•m⁻³•d⁻¹. Reads-based metatranscriptomic analysis found that the expression values of hzsA and hdh, key genes associated with anammox, were much higher than other functional genes on nitrogen conversion, confirming the major roles of the anammox bacteria in nitrogen bio-removal. In both regions of the reactor, the nitrate reduction genes (napA/narG) had expression values of 56–99 RPM, which were similar to that of the nitrite reduction genes (nirS/nirK). The expression reads from genes for dissimilatory nitrate reduction to ammonium (DNRA), nrfA and nirB, were unexpectedly high, and were over the half of the levels of reads from genes required for nitrate reduction. Kinetic assays confirmed that the granules had an anammox activity of 16.2 g-NH₄⁺-N•kg⁻¹-VSS•d⁻¹ and a nitrate reduction activity of 4.1 g-N•kg⁻¹-VSS•d⁻¹. While these values were changed to be 4.9 g- NH₄⁺-N•kg⁻¹-VSS•d⁻¹and 4.3 g-N•kg⁻¹-VSS•d⁻¹ respectively in the fixed-biofilm. Mass flux determination found that PD and DNRA was responsible for ∼50% and ∼25% of nitrate reduction, respectively, in the whole reactor, consistent with high effluent quality and treatment efficiency via a nitrite loop. Metagenomic binning analysis revealed that new and unidentified anammox species, affiliated with Candidatus Brocadia, were the dominant anammox organisms. Myxococcota and Planctomycetota were the principal organisms associated with the PD and DNRA processes, respectively.