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Enhanced aerobic/anoxic Downflow Hanging Sponge (DHS) bioreactors were assessed for carbon (C) and total nitrogen (TN) removal for decentralised domestic wastewater treatment applications. The initial design included upper aerobic and lower anoxic sponge layers, and effluent recirculation, and achieved >80% COD and >90% NH-N removal. However, effluent TN was higher. It was concluded the anoxic layer was C-limited for denitrification, therefore an influent bypass was added to the anoxic layer to provide supplemental C. Differed bypass ratios were compared, including 0%, 10%, 20% and 30% (% of total influent), and effluent TN declined with increasing bypass; i.e., 50.1±23.3mg-N/L, 49.9±27.8mg-N/L, 31.9±18.4mg-N/L and 10.7±5.8mg-N/L, respectively, and all reactors removed >80% COD. This design has potential because it uses limited energy, tolerates variable flows, and simultaneously removes C and TN; all key for effective decentralised treatment applications.

Wastewater is a common pathway for the spread of antibiotic resistance (AR) genes and bacteria into the environment. Biological treatment can mitigate this path, but horizontal gene transfer (HGT) between bacteria also occurs in such processes, although the influence of bioreactor habitat and ecology on HGT frequency is not well understood. Here, we quantified how oxidation-reduction (redox) conditions impact the fate of a Green fluorescent protein (Gfp)-tagged AR plasmid (pRP4-gfp) within an E. coli host (EcoFJ1) in the liquid and biofilms of multi-phase bioreactors. Replicate bioreactors treating domestic wastewater were operated under stable aerobic (+195 ± 25 mV), anoxic (-15 ± 50 mV), and anaerobic (-195 ± 15 mV) conditions, and flow cytometry and selective plating were used to quantify donor strain, EcoFJ1(pRP4-gfp), and putative transconjugants over time. Plasmid pRP4-gfp-bearing cells disappeared rapidly in aerobic ecosystems (~2.0 log reduction after 72 h), especially in the liquid phase. Conversely, EcoFJ1(pRP4-gfp) and putative transconjugants persisted longest in anaerobic biofilms (~1.0 log reduction, after 72 h). Apparent plasmid transfer frequencies also were higher under anaerobic conditions. Further, protozoan abundances were over 20 times higher in aerobic reactors relative to anaerobic reactors, and protozoa numbers significantly inversely correlated with pRP4-gfp signals across all reactors (p < 0.05). Overall, HGT frequency was consequentially impacted by bioreactor habitat conditions and trophic effects, especially relative oxygen level and evident predation. These observations provide guidance for new and alternate bioreactor designs aimed at minimizing AR HGT during biological wastewater treatment.

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Inadequate sanitation can lead to the spread of infectious diseases and antimicrobial resistance (AMR) via contaminated water. Unfortunately, wastewater treatment is not universal in many developing and emerging countries, especially in rural and peri-urban locations that are remote from central sewers. As such, small-scale, more sustainable treatment options are needed, such as aerobic-Denitrifying Downflow Hanging Sponge (DDHS) bioreactors. In this study, DDHS reactors were assessed for such applications, and achieved over 79% and 84% removal of Chemical Oxygen Demand and Ammonium, respectively, and up to 71% removal of Total Nitrogen (TN) from domestic wastes. Elevated TN removals were achieved via bypassing a fraction of raw wastewater around the top layer of the DDHS system to promote denitrification. However, it was not known how this bypass impacts AMR gene (ARG) and mobile genetic element (MGE) levels in treated effluents. High-throughput qPCR was used to quantify ARG and MGE levels in DDHS bioreactors as a function of percent bypass (0, 10, 20 and 30% by volume). All systems obtained over 90% ARG reduction, although effluent ARG and TN levels differed among bypass regimes, with co-optimal reductions occurring at ~20% bypass. ARG removal paralleled bacterial removal rate, although effluent bacteria tended to have greater genetic plasticity based on higher apparent MGE levels per cell. Overall, TN removal increased and ARG removal decreased with increasing bypass, therefore co-optimization is needed in each DDHS application to achieve locally targeted TN and AMR effluent levels.

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