Wastewater surveillance for bacterial targets: current challenges and future goals.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.

Sulfide alters microbial functional potential in a methane and nitrogen cycling biofilm reactor.

Cross-feeding of metabolites between coexisting cells leads to complex and interconnected elemental cycling and microbial interactions. These relationships influence overall community function and can be altered by changes in substrate availability. Here, we used isotopic rate measurements and metagenomic sequencing to study how cross-feeding relationships changed in response to stepwise increases of sulfide concentrations in a membrane-aerated biofilm reactor that was fed with methane and ammonium. Results showed that sulfide: (i) decreased nitrite oxidation rates but increased ammonia oxidation rates; (ii) changed the denitrifying community and increased nitrous oxide production; and (iii) induced dissimilatory nitrite reduction to ammonium (DNRA). We infer that inhibition of nitrite oxidation resulted in higher nitrite availability for DNRA, anammox, and nitrite-dependent anaerobic methane oxidation. In other words, sulfide likely disrupted microbial cross-feeding between AOB and NOB and induced cross-feeding between AOB and nitrite reducing organisms. Furthermore, these cross-feeding relationships were spatially distributed between biofilm and planktonic phases of the reactor. These results indicate that using sulfide as an electron donor will promote N2 O and ammonium production, which is generally not desirable in engineered systems.

Phage phylogeny, molecular signaling, and auxiliary antimicrobial resistance in aerobic and anaerobic membrane bioreactors.

Phage emit communication signals that inform their lytic and lysogenic life cycles. However, little is known regarding the abundance and diversity of the genes associated with phage communication systems in wastewater treatment microbial communities. This study focused on phage communities within two distinct biochemical wastewater environments, specifically aerobic membrane bioreactors (AeMBRs) and anaerobic membrane bioreactors (AnMBRs) exposed to varying antibiotic concentrations. Metagenomic data from the bench-scale systems were analyzed to explore phage phylogeny, life cycles, and genetic capacity for antimicrobial resistance and quorum sensing. Two dominant phage families, Schitoviridae and Peduoviridae, exhibited redox-dependent dynamics. Schitoviridae prevailed in anaerobic conditions, while Peduoviridae dominated in aerobic conditions. Notably, the abundance of lytic and lysogenic proteins varied across conditions, suggesting the coexistence of both life cycles. Furthermore, the presence of antibiotic resistance genes (ARGs) within viral contigs highlighted the potential for phage to transfer ARGs in AeMBRs. Finally, quorum sensing genes in the virome of AeMBRs indicated possible molecular signaling between phage and bacteria. Overall, this study provides insights into the dynamics of viral communities across varied redox conditions in MBRs. These findings shed light on phage life cycles, and auxiliary genetic capacity such as antibiotic resistance and bacterial quorum sensing within wastewater treatment microbial communities.

Mainstream partial denitrification-anammox in sand and expanded clay deep-bed polishing filters under practical loading rates and backwashing conditions.

This study focused on evaluating the feasibility of expanded clay and sand as media types for mainstream partial denitrification-anammox (PdNA) in deep-bed single-media polishing filters under nitrogen and solids loading rates as well as backwash conditions similar to conventional denitrification filters. The surface roughness and iron content of the expanded clay were hypothesized to allow for enhanced anammox retention, nitrogen removal rates, and runtimes. However, under the tested loading rates and backwash conditions, no clear benefit of expanded clay was observed compared with conventional sand. This study showed the feasibility of PdNA in filters with both sand and expanded clay with PdN efficiencies of 76% and 77%, PdNA rates of 840 and 843 g N/m3 /d and TIN removal rates of 960 and 964 g N/m3 /d, respectively. Glycerol demands were 1.5-1.6 g COD added per g TIN removed , thus indicating potential carbon savings up to 75% compared with conventional denitrification. Overall, this study showed for the first time PdNA filters performing at nitrogen removal rates double that of previous PdNA studies under realistic conditions while providing insights into the media choice and backwashing conditions. Future research on expanded clay backwash conditions is needed to provide its full potential in PdNA filters. PRACTITIONER POINTS: Hydraulic and TSS loading rates similar to conventional denitrification can be applied in PdNA filters. Conventional sand can be used when retrofitting conventional denitrification filters into PdNA filters. Carbon savings up to 75% can be achieved with glycerol when retrofitting conventional filters into PdNA filters.

Media selection for anammox-based polishing filters: Balancing anammox enrichment and retention with filtration function.

Retrofitting conventional denitrification filters into partial denitrification-anammox (PdNA)- or anammox (AnAOB)-based filters will reduce the needs for external carbon addition. The success of AnAOB-based filters depends on anammox growth and retention within such filters. Studies have overlooked the importance of media selection and its impact on AnAOB capacity, head loss progression dynamics, and shear conditions applied onto the AnAOB biofilm. The objective of this study was to evaluate viable media types (10 types) that can enhance AnAOB rates for efficient nitrogen removal in filters. Given the higher backwash requirement and lower AnAOB capacity of the conventionally used sand, expanded clay (3-5 mm) was recommended for AnAOB-based filters in this study. Owing to its surface characteristics, expanded clay had higher AnAOB activity (304- vs. 104-g NH4 + -N/m2 /day) and higher AnAOB retention (43% more) than sand. Increasing the iron content of expanded clay to 37% resulted in an increase in zeta potential, which led to 56% more anammox capacity compared to expanded clay with 7% iron content. This work provides insight into the importance of media types in the growth and retention of AnAOB in filters, and this knowledge could be used as basis in the development of PdNA filters. PRACTITIONER POINTS: Expanded clay showed the lowest head loss buildup and most likely will result in longer runtime for full-scale PdNA applications The highest AnAOB rates were achieved in expanded clay types and sand compared with smaller media typically used in biofiltration Expanded clay resulted in better AnAOB retention under shear, whereas sand could not withstand shear and required more frequent backwashing Expanded clay iron coating enhanced AnAOB enrichment and retention, most likely due to increased surface roughness and/or positive charge.

Sensor-mediated granular sludge reactor for nitrogen removal and reduced aeration demand using a dilute wastewater.

A sensor-mediated strategy was applied to a laboratory-scale granular sludge reactor (GSR) to demonstrate that energy-efficient inorganic nitrogen removal is possible with a dilute mainstream wastewater. The GSR was fed a dilute wastewater designed to simulate an A-stage mainstream anaerobic treatment process. DO, pH, and ammonia/nitrate sensors measured water quality as part of a real-time control strategy that resulted in low-energy nitrogen removal. At a low COD (0.2 kg m-3  day-1 ) and ammonia (0.1 kg-N m-3  day-1 ) load, the average degree of ammonia oxidation was 86.2 ± 3.2% and total inorganic nitrogen removal was 56.7 ± 2.9% over the entire reactor operation. Aeration was controlled using a DO setpoint, with and without residual ammonia control. Under both strategies, maintaining a low bulk oxygen level (0.5 mg/L) and alternating aerobic/anoxic cycles resulted in a higher level of nitrite accumulation and supported shortcut inorganic nitrogen removal by suppressing nitrite oxidizing bacteria. Furthermore, coupling a DO setpoint aeration strategy with residual ammonia control resulted in more stable nitritation and improved aeration efficiency. The results show that sensor-mediated controls, especially coupled with a DO setpoint and residual ammonia controls, are beneficial for maintaining stable aerobic granular sludge. PRACTITIONER POINTS: Tight sensor-mediated aeration control is need for better PN/A. Low DO intermittent aeration with minimum ammonium residual results in a stable N removal. Low DO aeration results in a stable NOB suppression. Using sensor-mediated aeration control in a granular sludge reactor reduces aeration cost.

Sulfide inhibition of nitrite oxidation in activated sludge depends on microbial community composition.

Increasingly, technologies that use sulfide as an electron donor are being considered for nitrogen removal; however, our understanding of how sulfide affects microbial communities in nitrifying treatment processes is limited. In this study, we used batch experiments to quantify sulfide inhibition of both ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) using activated sludge from two full-scale treatment plants with distinct treatment processes. The batch experiments showed that NOB were more vulnerable to sulfide inhibition than AOB, and that inhibition constants (KI) for NOB were distinct between the two treatment plants, which also had distinct nitrite oxidizing microbial communities. A Nitrospira-rich, less diverse NOB community was inhibited more by sulfide than a more diverse community rich in Nitrotoga and Nitrobacter. Therefore, sulfide-induced nitritation may be more successful in less diverse, Nitrospira-rich communities. Additionally, sulfide significantly influenced the activity of non-nitrifying microbial community members, as measured by 16S rRNA cDNA sequencing. Overall, these results indicate that sulfide has a strong impact on both nitrification and the activity of the underlying microbial communities, and that the response is community-specific.

Elucidating the impact of microbial community biodiversity on pharmaceutical biotransformation during wastewater treatment.

In addition to removing organics and other nutrients, the microorganisms in wastewater treatment plants (WWTPs) biotransform many pharmaceuticals present in wastewater. The objective of this study was to examine the relationship between pharmaceutical biotransformation and biodiversity in WWTP bioreactor microbial communities and identify taxa and functional genes that were strongly associated with biotransformation. Dilution-to-extinction of an activated sludge microbial community was performed to establish cultures with a gradient of microbial biodiversity. Batch experiments were performed using the dilution cultures to determine biotransformation extents of several environmentally relevant pharmaceuticals. With this approach, because the communities were all established from the same original community, and using sequencing of the 16S rRNA and metatranscriptome, we identified candidate taxa and genes whose activity and transcript abundances associated with the extent of individual pharmaceutical biotransformation and were lost across the biodiversity gradient. Metabolic genes such as dehydrogenases, amidases and monooxygenases were significantly associated with pharmaceutical biotransformation, and five genera were identified whose activity significantly associated with pharmaceutical biotransformation. Understanding how biotransformation relates to biodiversity will inform the design of biological WWTPs for enhanced removal of chemicals that negatively impact environmental health.