Fate of antibiotic resistance genes and bacteria under sequential redox conditions within biofilm reactors

Antibiotic resistance (AR) is a major health threat to global populations. However, mortal infections are most profound in Low-Middle-Income Countries (LMICs) where wastewater treatment is not universal and rarely precedes urbanisation. Therefore, reducing waste- and water-borne AR exposures through improved wastewater treatment is a high priority; however, few small-scale and economical technologies are available for application in LMICs. This thesis studied low-energy, sponge-core bioreactors, called Denitrifying Downflow Hanging Sponge (DDHS) systems, as a technology reducing AR genes and bacteria from domestic wastewater. The technology uses sequential redox conditions (i.e., aerobic-anoxic), an option previously shown to enhance AR reduction in wastewater ecosystems. Here, DDHS systems were co-optimised for total nitrogen (TN) and AR genes removal, using a 20% influent wastewater bypass (by volume of total influent) to enhance denitrification in the second-stage anoxic unit. Under such conditions, removals of 2.0 to 3.0 log AR genes, >79% carbon and 71% TN were achieved. Subsequent 16S rRNA amplicon sequencing and microbiome characterisation indicated the wastewater bypass positively influenced resident microbial communities, especially increasing reactor biodiversity (Shannon diversity index for 0% bypass = 5.92 ± 0.05 and 20% bypass = 6.15 ± 0.03), which in turn, translated to improved overall treatment performance. To better explain AR fate in the DDHS reactors, independent experiments assessed the impact of different redox conditions on relative transmission of AR gene-bearing plasmids. Biofilm and liquid phase samples from aerobic, anoxic and anaerobic bioreactors were collected and assessed for in situ horizontal gene transfer, tracked using a fluorescent-labelled AR plasmid assay (developed here) from a recombinant E. coli host added to the systems at seeding concentrations of 106 cells/mL. Overall, plasmid hosts disappeared more rapidly in the aerobic bioreactors (2.0 log net reduction; final concentrations = 4.4 ~ 4.7 x104 cells/mL after 72 hours) and survived much longer in oxygen-free systems, especially in anaerobic biofilms (1.0 log net reduction; final concentrations = 1.6 ~ 2.7 x 105 cells/mL after 72 hours). However, evident conjugal transfer of the AR plasmid was limited in native biofilm communities. vi Final work tested DDHS systems at pilot-scale in Southern Malaysia to operationalise and validate the technology for field application. A semi-optimised configuration was developed, effectively removing C and TN (respective percentage load removal at 55% and 53%; satisfying local discharge standards), micropollutants, and reducing AR genes by 1.0 to 2.0 log from the wastewater community. Promising field results warrant further development of future prototypes to fuel the uptake of the DDHS technology, especially for LMIC applications.