Introduction Water resource recovery facilities face escalating challenges in solids handling as disposal costs rise, influent characteristics vary, and many plants operate close to or at the limits of their solids-processing capacity. The Maple Creek Wastewater Treatment Plant (MCWWTP) in Greer, South Carolina, a 5 MGD sequencing batch reactor facility treating mixed municipal and industrial wastewater, sought to improve thickening and dewatering performance without major capital investment. To meet this need, MCWWTP implemented a targeted optimization program built around an emerging application of nanobubble technology: introducing nanobubbles directly into the dilution water used for polymer hydration. The goal was to enhance polymer activation, strengthen floc formation, improve solids capture, increase hydraulic throughput, and reduce polymer consumption. Background MCWWTP had historically encountered high polymer usage, inconsistent thickening performance, limited throughput, and influent-driven variability. Nanobubble technology was selected for evaluation due to its potential to interact directly with polymer chemistry, modify hydration dynamics, and bolster the performance of existing thickening and dewatering equipment. Nanobubble-Enhanced Polymer Activation Nanobubbles are ultra-fine gas bubbles (< 200 nm) that remain suspended in liquid and exhibit unique physicochemical properties. Due to their neutral buoyancy, high internal gas pressure, and charged interfaces, nanobubbles persist in solution far longer than conventional bubbles. This stability allows them to interact with dissolved ions, water molecules, and polymer chains during flocculant preparation. Through R&D testing, and literature, when nanobubbles are introduced into polymer dilution water it is hypothesized, they participate directly in the hydration phase where polymer chains uncoil, absorb water, and develop their effective charge density. The presence of nanobubbles influences this process through multiple reinforcing mechanisms. Their small size enables them to adsorb onto polymer chains, encouraging a more extended coil conformation that increases reactive surface area. Their charged surfaces alter the local electrochemical environment, enhancing the apparent charge density of the polymer as it hydrates. Nanobubbles also promote more uniform polymer chain expansion, which increases bridging efficiency between particles. Collectively, these effects strengthen floc formation, enhance particle capture, increase floc resiliency, and reduce the amount of polymer required to achieve target performance as confirmed by full scale demonstrations. Methods Full-scale onsite trials were conducted using nanobubble-enriched dilution water to prepare polymer solutions for thickening and dewatering. Multiple high-charge, high-molecular-weight polymer formulations were evaluated to determine which chemistries most effectively leveraged nanobubble interactions. Performance monitoring included measurements of thickened sludge solids, cake dryness, filtrate clarity, hydraulic throughput, polymer consumption, and operator observations regarding equipment performance and process stability. Results and Discussion Nanobubble-enhanced polymer conditioning produced consistent and measurable improvements across all solids-separation indicators. Surface charge was measured with various polymers being utilized for thickening and dewatering and are shown in Table 1. Thickened sludge concentrations increased from approximately 4 percent to approximately 6 percent, reducing the volume of sludge requiring dewatering. Dewatered cake solids improved from about 13 percent to about 16 percent, generating drier material that reduces hauling and disposal costs. Dewatering throughput increased by approximately 50 to 100 gallons per minute, improving process resilience during periods of higher solids loading. Polymer consumption dropped by 20 to 35 percent, depending on polymer type and operating conditions. Operators also reported clearer filtrate, more stable floc formation, and reduced day-to-day performance variability, particularly during periods of influent fluctuations. Conclusions This full-scale evaluation demonstrates that nanobubble technology can significantly enhance polymer hydration, floc formation, and solids separation performance in municipal wastewater treatment. At MCWWTP, nanobubble-conditioned dilution water resulted in measurable improvements in thickening efficiency, dewatering throughput, cake solids, and polymer reduction. These gains translate directly into operational cost savings, improved process reliability, and greater resilience under variable influent conditions. For facilities seeking low-footprint strategies to improve solids handling without major capital investment, nanobubble-enhanced polymer activation represents a promising, scalable, and high-value solution.

Introduction Global populations have been estimated to rise to 9.8 billion by 2050, subsequently raising demand for natural resources (Nagarajan et al., 2019). With global populations rising, animal products will continue to rise to ensure food security. Pork is one of the most common proteins for consumption, averaging 45 lb of pork meat per person, per year (Zhou et al., 2021) and produced in centralized Confined Animal Feeding Operations (CAFOs) or local small-scale livestock farming. From these farms, large volumes of manure are produced, averaging about 5 L of wastewater per day per pig. In recent decades, concentrated swine farming has greatly increased in the United States with the number of hogs and pigs reaching 75.0 million head on U.S. farms (Zhang et al., 2018). This causes a significant amount of waste that needs to be properly processed instead of being released into the environment and therefore causing the release of excess nutrients like nitrogen and phosphorus into the environment as well as the larger potential of wasted carbon emitted as methane from manure storage and disposal. Fermentation of swine wastewater is a way to valorize the organic carbon available in swine manure, allowing capture in the form of short and medium chain volatile fatty acids (VFAs) (Deng et al., 2023; Li et al., 2020). Most VFAs, valuable short-chain fatty acids (SCFAs, C2-C6), are currently produced from petrochemicals, though biomanufacturing from waste streams shows strong potential (Agnihotri et al., 2022; Ramos-Suarez et al., 2021). VFAs-such as acetate, propionate, and butyrate-are intermediates of anaerobic digestion, which involves hydrolysis, acidogenesis, acetogenesis, and methanogenesis. Suppressing methanogens via pH control (acidic or alkaline) promotes VFA accumulation, with production efficiency ranging from 15-25% at pH 5 to 35-50% at pH 9. Separation methods include membranes, ion exchange, and distillation, though further development is needed (Zhu et al., 2023; Singh et al., 2023). Mixed-culture VFAs can serve as external carbon sources in nutrient removal and as feedstocks for bioplastics, chain elongation, and synthetic aviation fuels. This study explores VFA production from swine wastewater at pH 5 and 9 providing baseline data for ongoing AnMBR integration and co-digestion experiments with fats, oils and greases., and greases (FOG). Materials and Methods Activated sludge was collected from the anoxic selector basin at the wastewater treatment plant located in Manhattan, Kansas. The activated sludge was allowed to be settled for 24 hours and 3L of the thickened solids were allocated equally to two 2-L reactors to serve as the inoculum. Each fermenter received 1.5L ml of settled sludge in a 2-L bottle. After sludge analysis, casein, and cellulose, representative proteins, and carbohydrate substrates were dosed to the fermentation reactors at 3.93 g/L, each to aid with selective enrichment.. After the addition of casein and cellulose, TCOD and SCOD4 were monitored periodically during successive batch operations with casein and cellulose addition, along with VFAs. Once an increase followed by a stabilization of the SCOD and VFAs was noted, a second dose of casein and cellulose was added again at 3.93 g/L, 10 days after the first dose. Analysis of TCOD/SCOD and VFA concentrations were measured in same frequency until an additional increase in VFA and stabilization occurred. The initial TCOD and SCOD concentrations were 19.9 and 6.3 g/L, whereas total solids (TS) and volatile solids (VS) of the substrate were 12.8 and 10.1 g/L, respectively. Following acclimation of inoculum, swine wastewater was introduced as the substrate for VFA production. The reactors have been maintained at a 10-day hydraulic retention time (HRT), 30 degrees Celsius, and stirred at 350 RPM for more than 600 days as of writing. The pH of both fermenters were maintained at 5 and 9 respectively. Removed fermentation broth was centrifuged, and the supernatant was discarded after samplings for VFA analysis. The remaining solids are then resuspended in influent wastewater and added back into the fermenters to maintain a high solids retention time (SRT). Ideally, SRT would be infinite so as to maintain higher microbe populations and therefore higher VFA production, but some solids are lost during centrifugation. The actual SRT of the fermenters have been calculated by finding the total solids content of the bulk fermentation liquid and the supernatant after centrifugation. The average SRT of the pH 5 fermenter is 27 days and the average SRT of the pH 9 fermenter is 50 days. The characterization of total and volatile suspended solids was carried out weekly, according to Standard Methods (2540D and 2540E; Standard Methods Water and Wastewater). Weekly total chemical oxygen demand (TCOD) and SCOD sludge analysis was performed using HACH 822 TNT kits ranging from 20-1500 mg/L concentration using HACH DR 3900 spectrophotometer at 1:500 and 1:250 dilutions, respectively. The pH was measured and readjusted daily using Oakton pH 550 benchtop pH meter using HCl 4M and NaOH 5M. Centrifugation of the effluent was done at 15054 rcf with Eppendorf 5920 R (Eppendorf, Hamburg, Germany). Hydrogen and methane were analyzed using Gas-Chromatography (GC) with a thermal conductivity detector (TCD) using Argon as a carrier gas at a flow rate of 8.8 mL/min and pressure of 88.8 kPa. Temperatures of injection, column, and detector were 200, 80, and 150 °C, respectively. For VFA quantification, the HPLC (Shimadzu LC-20AT, USA) with an Aminex HPX-87H column (300 mm X 7.8 mm, Bio Rad Corp., USA) equipped with two detectors: photodiode array and refractive index detectors was used at 0.65 mL/min pump flow rate and 50 °C oven temperature with 2.5 mM sulfuric acid as eluent. Results and Discussion The KSU team has been operating bench-scale 2L swine waste fermenters at pH 5 and 9 for over two years and has preliminary data that will assist in the conversion of a pilot-scale AnMBR from anaerobic digestion to fermentation. Current experiments with the fermenters are on the effect of heat shocking the broth at 60 °C to evaluate its effect on fermentation efficiency and solids retention. The bench-scale fermenters have issues with solids retention due to the limitations of the centrifuge the team uses to separate out the solids when removing and replacing broth with fresh swine waste. Ideally, all solids are separated out using the centrifuge and can be added back to the fermenters, but the maximum speed of the centrifuge is not enough to fully separate the broth. This results in some loss of solids and subsequently lowers fermentation efficiency. Heat shocking the broth before centrifugation has improved solids separation and improved fermentation efficiency. The team does not expect this solids separation issue during operation of the pilot-scale AnMBR as the membrane will allow for effective dynamic separation of VFA-rich permeate from the solids in the system, resulting in minimal solids loss. The pH 5 fermenter has a variable profile of VFAs produced, with the primary products being acetate, propionate, and iso-caproate. The concentrations of each product can fluctuate greatly over time and is also influenced by the variable strength of the organics (COD) in the input swine wastewater. Figure 1 below shows the VFA profile of the pH 5 fermenter. Figure 2 shows methane concentration of gas in headspace and fermentation efficiency, which typically ranges from ~20-30%. The order of predominance of VFAs from swine wastewater selective fermentation at pH 5 is as follows: Acetate > Propionate > Iso-Caproate. As the pH is acidic, the VFAs are in solution rather than their conjugate bases, resulting in high surface tension and a greater tendency for foaming events. Foaming is the primary operational challenge for fermentation at pH 5 and this will be taken into account during testing of the pilot-scale AnMBR. The pH 9 bench-scale fermenter exhibited a very consistent VFA profile, as seen in Figure 1(c). The primary product is acetate, with it making up at least 70% of the total amount of VFAs produced. This lends towards fermentation at pH 9 being a better candidate for selective fermentation, allowing for optimized downstream processes. Fermentation efficiency is also significantly higher in the pH 9 fermenter, occasionally peaking above 70%, as seen in Figure 1(d).

On-farm anaerobic digestion (AD) is an established technology for sustainable manure management that offers farmers the opportunity to produce renewable biogas and reduce the carbon footprint of their operations. Digesters are generally located on large dairy farms, with 250 to 1,000 cattle, that solely digest dairy manure as a liquid slurry or co-digest with commercial organic wastes. Dairy farms are optimal sites for AD because their daily operations meet the requirements of digestion applications: -Dairy manure is typically a low-solids liquid or slurry that is easily digested; -Dairy manure contains minimal bedding like straw or shavings; and -Manure collection occurs daily and can be continually fed to a digester. On-farm AD is less common for other livestock operations as the manure may be considered to be too high in solids, heterogeneous, contain too much cellulo-lignose bedding materials, or manure collection may only occur intermittently, thus inhibiting continuous feed to the digester. Feasibility of on-farm digesters is further dependent on final outlets for biogas utilization. Typically the biogas is used in on-farm operations (dairy farms have larger on-farm demand to power or heat milking operations and general farm buildings), to generate electricity for sale to the local utility, or is upgraded to renewable natural gas (RNG) and injected and sold to a local natural gas pipeline. Lystek's patented Thermal-Alkaline Hydrolysis Process (Lystek THP®) uses high-speed shearing, low-pressure steam heating, and alkali addition of potassium hydroxide within a Reactor to hydrolyze biological cells and particulate organic matter to release water trapped in the organic matrix. This hydrolysis process transforms dewatered or solid feedstocks into a low-viscosity material with liquid properties while maintaining a high total solids content, typically up to 16%. This technology enhances the digestibility of organics through multiple mechanisms: particle size reduction, feedstock homogenization, and solubilization of recalcitrant organic carbon. The hydrolyzed substrate has demonstrated greater digestion kinetics. The application of this technology to improve AD performance has been well demonstrated with biosolids, food and beverage processing residuals, as well as source-separated organics. This technology has been applied in a pre-feed (THP upstream of mesophilic AD), refeed (post-THP with a recirculation loop), and more recently in an innovative combined digestion and storage application. In this passive digestion application hydrolyzed biosolids are intermittently discharged to sealed anaerobic reservoirs, with no supplementary heating or mixing, and anaerobic digestion is efficiently occurring. The purpose of this study is to gain an understanding of how a range of livestock waste feedstocks are transformed using this technology and if/how the AD potential is affected. This research is funded by Natural Resources Canada under the Energy Innovation Program to investigate renewable resource opportunities in rural farming communities. The project has included over 30 samples from a range of livestock types, including dairy and beef cattle, swine, sheep, goats, broiler meat chickens, egg layer hen chickens, and turkeys. The variety of livestock types were selected to generate a large data set that can inform general trends of where this technology is most technically applicable and operationally feasible. All testing and laboratory analysis in the LysteMize Livestock Waste Characterization Study was completed in 2025. This paper will summarize the findings across all livestock manure samples provided and include depth discussion of select feedstocks that were further analyzed in Phase Two of this research. Bench-scale processing demonstrated the ability to transform multiple feedstocks, typically considered to not be operationally feasible for AD, into a liquified slurry with reduced viscosity sufficient for AD. Biochemical methane potential (BMP) testing and characterization was completed to quantify the rate, quantity, and quality of biogas from each sample, comparing the hydrolyzed sample to the unprocessed manure sample. Enhancement of dairy cattle manures is presented below in Figure 1 with average methane generation from materials that have been processed through laboratory-scale hydrolysis compared to unprocessed samples. The dairy manure, a common substrate for on-farm digestion, demonstrated a 70% increase in methane yield on average across the samples collected from two dairy farms with similar infrastructure. The homogeneity of the final manure product after hydrolysis is shown in Figures 2 and 3 to highlight the concentrated nature of the high-solids liquid. Solid dairy manure would typically be considered to have too many large particles, bedding or straw, that would require removal upstream of a conventional digester. The THP process effectively blends the material into a homogenous thickened liquid that is easily pumpable and, therefore, transportable. A critical drawback of conventional AD is that it produces a dilute liquid effluent. While the liquid is rich in nutrients, its dilute nature makes storage and application of the material expensive, operationally cumbersome, and inefficient. The final residual from this application is a concentrated liquid product that can be more efficiently stored and land applied using conventional equipment. Less typical on-farm digestion substrates include poultry manures due to their higher solids content, higher baseline ammonia levels, and infrequent collection methods that are incompatible with conventional AD. Samples from multiple turkey, broiler meat chicken, and egg layer hen chicken were analyzed, with more focus on layer hens as the study progressed to investigate the ammonia effects on digestion. The hydrolyzed, high-solids liquid manure sample in Figures 4 and 5 show a 40% methane yield increase and improved digestion kinetics without inhibitory effects observed from ammonia as high as 5,700 mg/L. Many poultry farms face challenges managing large volumes of solid, nutrient-rich manure. The excessive nitrogen or phosphorous concentrations in poultry manure can limit land application rates to ensure nitrogen or phosphorous limits defined by local agricultural regulations or best management practices are not exceeded. The thickened liquid nature of the hydrolyzed manure product offers opportunities for on-farm digestion to the poultry industry where it was not previously feasible with a solid material, as well as a more convenient manure management strategy that mitigates intensive nutrient concentrations in solid layer hen manure. Co-digestion of poultry manure feedstocks with low-solids liquid cattle and swine manure was also considered in the project to emulate a regional approach to on-farm digestion. This regional approach will further broaden the accessibility for on-farm digestion in rural communities. Solid and liquid manure feedstocks co-processed effectively to generate a homogenous, concentrated liquid feedstock for anaerobic digestion. Preliminary analyses of co-digested animal manures demonstrate comparatively positive increases in methane yield and digestion kinetics as the digestion of these feedstocks independently. A regional, co-digestion approach of various animal manure feedstocks not online increases the scale of the on-farm digestion operations but will optimize processing by increasing the solids concentrations of typically more dilute feedstocks (liquid dairy manure) and dilute high-solids manures with substantial cellulo-lignose concentrations (such as solid poultry manures up to 49%TS). This research demonstrates an increasing scope of size and type of livestock operations whereby AD is an accessible pathway to renewable energy generation via biogas production from manure wastes. Hydrolysis can be used on large cattle farms which have existing anaerobic digestion infrastructure to boost gas production. The THP system also offers other livestock farms with less conventionally digestible feedstocks, such as poultry manure, another approach to AD and biogas collection by transforming the material into a high-solids liquid that can be handled more efficiently. When considering the expanded operational feasibility of on-farm AD, this technology can be easily started and stopped to align with intermittent manure collection activities without losing process efficiency. Conventional digestion requires continuous operation and meticulous care to ensure the health and productivity of the bacterial mass. This technology remains effective with a more hands-off operational approach, which allows discontinuous manure collection methods or smaller scale farms access to digestion technology and energy production without impacts to their major farming activities.

INNOVATION AND IMPACT Over the past two years, NEW Water, the brand of the Green Bay Metropolitan Sewerage District, completed full-scale demonstration testing of low dissolved oxygen (DO) setpoints to investigate the dynamics of simultaneous nitrification and denitrification (SND). The testing completed at NEW Water gave insight into: -SND dynamics quantified both in the field and via bench scale testing related to COD availability, DO setpoint impact, and COD type -Confirmation that SND does occur in both the field and bench scale testing -Identification of controlling parameters for SND, including COD:TN, floc size and DO setpoint -Identification of emissions risks, and the importance of SRT and aeration control to minimize emissions With the results from NEW Water, we can start to have a better prediction of the level of SND that will occur in our biological nutrient removal (BNR) designs. INTRODUCTION/BACKGROUND NEW Water is collaborating with Black & Veatch and EnviroMix (Xylem) to pilot a transition from their existing anaerobic-oxic process to an anaerobic-anoxic-oxic (A2O) process at low DO concentrations. The full-scale A2O test basin has an average primary effluent (PE) flow of 7 mgd and includes the addition of EnviroMix mixing technology and aeration and process control strategy (Figure 1). The demonstration at NEW Water has been operational for two years. Three different low DO aeration control strategies have been compared, as shown in Table 1, including static low DO setpoints of 0.3 mg/L and 0.5 mg/L, and FlexZone operation, an aeration and mixing control strategy to dynamically transition DO setpoints. METHODOLOGY Sensors: Two YSI VARiON ion-selective electrode (ISE) sensors measuring ammonia (NH4) and nitrate (NO3) were installed within the demonstration basin to support continuous, in-situ nitrification and denitrification rate calculations. One sensor was located at the end of the anoxic zone, while the second was installed in the middle of subzone 2 (SZ2), approximately one-third of the way through the low DO aeration zone. DO was monitored in each aerated zone using optical sensors, and biomass concentration was estimated using a total suspended solids (TSS) sensor. In-situ Rate Calculations: Probe data was used to calculate in-situ nitrification and denitrification rates occurring in SZ1 and SZ2 under low DO conditions. The in-situ rates were calculated based on NH4 and NO3 values from both sensors on a one-minute frequency and utilizing a TSS probe and assumed MLVSS/MLSS ratio. Bench-Top Rate Testing: The impact of DO setpoint on nitrification rate was measured using benchtop nitrification rate testing. A DO controller was used to maintain a constant DO setpoint during three-hour rate tests ranging between 0.1 mg/L and 4 mg/L. Similarly, the denitrification rate was measured on the bench top with a range of DO setpoints, carbon-to-nitrogen ratios (C/N), and carbon sources, including endogenous decay products (no external carbon), acetate, and a synthetic fermentate mixture. RESULTS Full Scale SND Simultaneous nitrification-denitrification has been observed under conditions where bulk oxygen is limiting, reduced oxygen diffusion into flocs creates a thin aerobic layer over an anoxic core, and organisms with greater oxygen affinity are favored. This has resulted in reported reductions in total effluent nitrogen in the literature (Rieger et al. 2012; Regmi et al. 2022). Here, SND was observed across low DO control strategies. Mass balance calculations indicated SND increased with decreasing DO setpoints (Figure 2). The calculated nitrification and denitrification rates grouped by test phase are shown in Figure 3, with the correlation of nitrification rate and denitrification rate show for different DO bins. Figure 4 shows how the PE COD:TKN related to denitrification rate. Dissolved nitrous oxide (N2O) was also measured throughout testing to understand emission dynamics (Figure 5). Key observations from full-scale SND measurements include: -SND was consistently quantified using overall mass balance calculations in the aerobic zones during low DO testing. -At low DO conditions (<0.5 mg/L), the denitrification rate was consistently between 2 and 2.5 mgN/gVSS-hr despite the nitrification rate ranging from 2 to 6 mgN/gVSS-hr, indicating that denitrification was likely limited by carbon availability. -At higher DO setpoints (>0.5 mg/L), the denitrification rate was strongly correlated to the nitrification rate, with the denitrification rate typically 30% of the nitrification rate. The maximum denitrification rate occurred at high nitrification rates (>6 mgN/gVSS-hr). At this high nitrification rate, the denitrification rate matched those observed at low DO setpoints (approximately 2 mgN/gVSS-hr). This indicates that nitrate availability is a critical aspect of SND at slightly higher DO setpoints. This may be associated with diffusion into flocs or possibly denitrification kinetics. -Although the lower COD:TKN data set was limited, a significant decrease in denitrification rate in the low DO zones was observed when COD:TKN was less than 6 in the PE. Denitrification rates averaged a more similar number at higher COD:TKN. -Emissions were dependent on an ecology that is stabilized for low DO operation. Benchtop Scale Testing Nitrification and denitrification kinetics were further explored with benchtop testing. Nitrification followed a similar pattern to previously reported conditions (Sabba et al. 2024, Jimenez et al. 2024) (Figure 6). Benchtop denitrification rates were also measured at low DO conditions. With no external carbon added, no denitrification was observed in the lab. The greatest denitrification rate was observed with a complex carbon source at the highest C/N ratio. Additionally, when the DO setpoint was above 0.1 mg/L, denitrification was reduced significantly (Figure 7). Key observations from bench-scale SND measurements include: -Nitrification would likely not be limiting until the DO reached 0.1 mg/L. At this DO, the nitrification rate (1.2 mgN/gVSS-hr) is less than the average denitrification rate under low DO conditions -The denitrification rate at 0.1 mg/L is significantly higher than at a 0.25 mg/L DO setpoint and is highly dependent on the availability of carbon. -Interestingly, the denitrification rate in the full-scale (2 to 2.5 mgN/gNSS-hr) at <0.5 mg/L DO is similar to the denitrification rate in benchtop experiments at 0.1 mg/L DO. This may be an indicator that the DO profiles within the aerobic zone are critical, as small pockets of lower DO (~0.1 mg/L) may be a large driver for SND rates in full-scale aeration basins. The benchtop results also highlight the criticality of floc size, as a larger floc may experience more volume at less than 0.1 mg/L and drive higher SND rates. CONCLUSIONS AND SIGNIFICANCE SND was confirmed to be occurring with full-scale data based on lab measurements and online sensors. In addition, bench scale experimentation examined dynamics related to DO concentration, COD availability, and COD type on nitrification and denitrification rates at low DO concentrations (<0.5 mg/L). The results provide insights into why SND has been variable at different sites, and why the occurrence of SND was a hotly debated topic at WEF RBITT 2025. This project provides the following key insights: -Carbon matters. Higher PE COD:TKN ratios resulted in higher rates of denitrification in the low DO zones. When a facility has a COD:TKN ratio of less than 6, it may result in decreased rates of SND. -Denitrification is highly sensitive to DO concentration. In lab tests, the denitrification rate almost disappeared at a DO of 0.25 mg/L, but was similar to the observed rate at a DO of 0.1 mg/L. This relatively small change in DO likely lead to large variations in observed SND rates, and site-specific mixing and airflow distribution likely contribute to variations in observed SND in aeration basins. Maintaining a DO of less than 0.5 mg/L also appears to be a critical control factor. -Nitrification rate influences denitrification rate under low DO conditions. When the DO is greater than 0.5 mg/L, the denitrification rate is correlated to the nitrification rate. This means that SRT and HRT will have a large impact on the SND rates in the system. -Floc size will be a critical factor. This relates to the DO and nitrification rate influences on SND rates. The smaller the floc, the more sensitive SND will be to DO concentration as the micro-gradients in the floc will play a critical role in SND. -SRT and controls matter for SND and emissions. More stable SRTs and allowing for biological adaptation were critical for both SND rates as well as N2O production rates. SND designs should consider the design SRT, control equipment, and control loops carefully to maximize the level of SND occurring in the system.

ABSTRACT Global warming remains a critical issue, with nitrous oxide (N2O) emissions from wastewater treatment (WWT) systems contributing significantly. N2O measurements are limited, making data-driven predictions essential for mitigation. In this study, we quantify N2O emissions from BNR systems using a USEPA-endorsed protocol and developed an eXtreme Gradient Boosting (XG Boost) machine learning model for prediction and mitigation of N2O. The SHapley Additive exPlanations (SHAP) and partial dependence plot (PDP) highlighted ammonia (NH3), dissolved oxygen (DO), nitrite (NO2), and solids retention time (SRT) as key drivers. These insights enable emission reduction and process optimization, supporting resilient and sustainable wastewater treatment systems. INTRODUCTION Nitrous oxide (N2O) is a potent greenhouse gas (GHG) and ozone-depleting substance, with wastewater treatment plants (WWTPs) increasingly recognized as substantial anthropogenic sources(Ahn et al., 2010; Ravishankara et al., 2009). N2O is produced during the nitrification through nitrifier nitrification and nitrifier denitrification pathway, and in denitrification, through nitrifier denitrification and inhibition of N2O reductase(Rassamee et al., 2011). Machine learning (ML) models offer valuable insights into process performance and drivers of N2O emissions, with supervised ML approaches showing strong potential for predicting emissions in biological nutrient removal (BNR) systems(Khalil et al., 2023). However, existing models are limited by minimal datasets and lack interpretability(Khalil et al., 2023). N2O emissions are directly linked to nitrogen dynamics and serve as an indicator of BNR performance. This study integrates both historical and newly quantified data with supervised ML to capture interactions among operational conditions and N2O emissions, providing actionable insights for optimizing WWTP performance. MATERIALS AND METHODS Gaseous N2O concentrations from BNR processes were quantified using an USEPA-endorsed protocol. Measurements employed the Surface Emission Isolated Flux Chamber (SEIFC), the only EPA-approved floating body designed for measuring gaseous nitrogen fluxes from activated sludge tanks(Ahn et al., 2010). N2O concentrations were recorded at 1/min using an infrared gas filter correlation analyzer (Teledyne API, San Diego, California), while off-gas flow was simultaneously measured with a hot-wire thermo anemometer to determine fluxes in aerated and non-aerated zones. Each location was monitored continuously for 24 hours to capture diurnal variability. Data were separated into aerobic and anoxic zones, pre-processed to address missing/non-numeric values, log-transformed and min-max normalized [0,1]. The dataset was split into 75% training and 25% testing, with k-fold cross-validation for hyperparameter tuning. Extreme Gradient Boosting (XG Boost) models were developed and interpreted using Shapley Additive exPlanations (SHAP) and Partial Dependence plot (PDP) in R Studio. RESULTS AND DISCUSSION Interpretable SHAP analysis was used to uncover both the magnitude and directionality of variables influencing predicted N2O flux. Among all plant operational variables trained, the key variables driving N2O emissions are shown in Figure 1A-G. In the aerobic zone, SHAP results indicate that localized, zone-specific variables such as ammonia (NH3), dissolved oxygen (DO), and nitrite (NO2) positively influence increased N2O flux at high concentrations (Figure 1A-C). This underscores the importance of optimizing DO levels in conjunction with appropriate NH3 concentrations to sustain effective nitrification(Brotto et al., 2015). The global variable, solids retention time (SRT), shows that SRT values below 10 days negatively influence reduced N2O flux (Figure 1D). For the anoxic zone, SHAP results demonstrate that local variables like DO and NO2 positively influence N2O flux at high concentrations (Figure 1E,F), potentially linking to DO-mediated inhibition of N2O reduction to N2. A bi-phasic response of N2O flux to SRT was observed (Figure 1G), suggesting possible interactions with DO and NO2 concentrations (Ahn et al., 2010). The process optimization approach identified operating ranges for SRT as the principal process parameter, coupled with process performance response that concurrently minimizes off-gas N2O fluxes, using PDP analysis. In the aerobic zone, Figure 2A shows that NH3 concentrations < 4 mg L-1 and DO concentrations < 3 mg L-1 at SRT < 15 days resulted in less N2O flux. However, higher NH3 concentrations under both low and high DO conditions produced and emitted N2O (Figure 2A). This indicates that N2O production occurs through both nitrifier nitrification and nitrifier denitrification pathways in the aerobic zone. Thus, optimizing DO and SRT in conjunction with NH3 concentration is critical to improving process performance and reducing N2O fluxes. In the anoxic zone, the conditions that minimize N2O flux required maintaining DO concentrations < 1 mg L-1 coupled with a total process SRT < 20 days (Figure 2B). However, N2O flux increased when operating DO concentrations exceeded 1 mg L-1 under the same SRT conditions (Figure 2B). At DO concentrations > 1 mg L-1, NO2 accumulation in the anoxic zone was promoted, which in turn elevated N2O fluxes (Figure 2B)(Ahn et al., 2010; Rassamee et al., 2011). The results highlight the importance of strict DO control in anoxic zones to prevent NO2 buildup and mitigate emissions. CONCLUSION This study established ML as a powerful framework for disentangling the complex interactions governing N2O emissions from BNR systems. By integrating real-time and historical data, coupling supervised models with interpretable tools such as SHAP and PDP analyses, we quantified the relative contributions of operational and water quality parameters and identified conditions that exacerbate or suppress emissions. Results demonstrate that aerobic zone N2O fluxes are primarily driven by NH3 dynamics, with DO exerting a nonlinear influence that highlights the sensitivity of nitrification pathways to aeration control. Conversely, anoxic zone N2O fluxes were strongly modulated by DO and NO2, reflecting incomplete denitrification and inhibition of N2O reductase under suboptimal conditions. The observed biphasic response of SRT across both zones underscores its central role in shaping microbial activity and nitrogen transformation. Optimization analyses further delineated operational windows that minimize emissions while sustaining treatment performance. Aerobic zones operated at DO < 3 mg L-1 and NH3 concentration < 4 mg L-1 with SRT below 15 days, and anoxic zones maintained under strict oxygen limitation of DO < 1 mg L-1 with SRT under 20 days. These findings directly link predictive modeling outcomes to process-level strategies, providing actionable pathways for emission mitigation. Beyond prediction accuracy, the integration of ML with bioprocess fundamentals advances a mechanistic understanding of N2O dynamics, enabling data-driven optimization of wastewater treatment. The framework presented here illustrates how interpretable ML can support regulatory compliance, guide plant-wide control strategies, and ultimately reduce the climate and environmental footprint of wastewater treatment infrastructure.

Background Sludge dewatering is a critical and cost-intensive operation for municipal wastewater utilities, directly affecting biosolids handling, hauling, disposal, and overall lifecycle costs. Across a wide range of solids separation technologies including centrifuges, belt filter presses, and dissolved air flotation systems, polymer is widely used to improve floc formation, solids capture, and cake dryness. Historically, improvements in dewatering performance have been pursued through increased polymer dosing, an approach that can yield diminishing returns while escalating chemical costs, disposal costs, and operational complexity. As utilities near capacity or pursue process intensification, they increasingly operate sludge dewatering systems closer to mechanical and hydraulic limits. Field experience has shown that under these conditions, polymer solvation and activation can become a limiting factor influencing the dewatering performance of solids separation technologies. Nanobubbles, defined as ultra-fine gas bubbles less than 200 nanometers in diameter, are neutrally buoyant, highly stable, and electrochemically active. These properties enable nanobubbles to interact directly with polymer chains during solvation. Conditioning polymer makedown water with nanobubbles enhances polymer activation prior to contact with sludge, offering a mechanism to improve dewatering efficiency of solids separation technologies. Nanobubble-conditioned makedown water was first identified unexpectedly during a field trial when nanobubble-treated secondary effluent was repurposed as polymer makedown water. This observation prompted controlled studies within Moleaer's research laboratories and testing facilities, where mechanistic evaluations confirmed that nanobubbles interact with polymer chains during hydration, altering coil conformation, effective charge density, and bridging efficiency. These findings established the scientific basis for subsequent shop-scale testing and full-scale utility pilots, positioning the technology along a continuous path from research discovery to applied engineering and operational impact. This project evaluates nanobubble-conditioned makedown water as a novel and scalable approach to improving polymer efficiency, with a primary focus on full-scale centrifuge dewatering at the Rock Creek Water Resource Recovery Facility (WRRF). Supporting observations from additional municipal WRRF field studies indicate that polymer efficiency improvements have been observed across multiple dewatering technologies and sludge types. Methods Clean Water Services conducted full-scale onsite trials at the Rock Creek WRRF to evaluate polymer performance using nanobubble-conditioned makedown water for centrifuge dewatering. The pilot followed a structured multi-phase protocol that included baseline operation, functional testing, trial operation with nanobubbles, polymer dose optimization, and repeated baseline validation periods. Performance was evaluated using time-series operational data and direct comparisons of polymer dose expressed as pounds of active polymer per dry ton of feed, biosolids cake total solids, centrate clarity, makedown water quality, hydraulic throughput, and operator observations of equipment performance and process stability. These field evaluations were informed by bench-scale mechanistic testing and shop-scale engineering evaluations that established both the scientific basis and the operational feasibility of nanobubble-enhanced polymer activation. Results Full-scale centrifuge trials demonstrated that nanobubble-conditioned makedown water enabled a reduction in polymer dose while maintaining dewatering performance within mechanical limits. At Rock Creek, polymer dose was reduced from approximately 28 to 23 pounds of active polymer per dry ton of feed, representing a 17 percent reduction in polymer usage with no observed degradation in dewatering performance (Figures 1 and 2). Centrifuge cake solids were observed to plateau at approximately 23 percent total solids due to mechanical limitations of the centrifuges; therefore, performance improvements were realized primarily through reduced polymer demand rather than increased cake solids. Time-series results showed stable operation across multiple baseline and trial periods, indicating robust and repeatable performance of nanobubble-conditioned makedown water. Findings Dose-response analysis demonstrated a shift in the polymer efficiency curve when nanobubble-conditioned makedown water was applied, with comparable cake solids achieved at lower polymer doses relative to baseline operation (Figure 2). These results indicate improved polymer utilization rather than simple dose reduction. Field observations from multiple municipal wastewater treatment plants further support the transferability of the approach. Polymer reductions on the order of approximately 20 to 35 percent have been observed across different sludge types and dewatering configurations, accompanied by improvements in dewaterability, cake dryness, and filtered water quality. The consistency of these outcomes suggests that nanobubble-enhanced polymer activation represents a dewatering-technology-agnostic improvement to polymer performance. Conclusion Full-scale field data demonstrate that meaningful reductions in polymer usage can be achieved without sacrificing dewatering performance, resulting in significant potential reductions in chemical costs and biosolids hauling and disposal expenses. By targeting polymer activation upstream of the dewatering device rather than increasing polymer dose or modifying equipment, this approach offers utilities a scalable, easy-to-implement solution with broad applicability across dewatering technologies without requiring equipment modification. When considered alongside the bench-scale mechanistic findings and shop-scale validations, the results indicate a mature, transferable technology capable of delivering sustained operations and maintenance benefits across the wastewater industry.

1. Background and Motivation Rotating biological contactors (RBCs) are energy-efficient, attached-growth systems that tolerate hydraulic and organic variability but can experience ecological shifts from off-design conditions. A common example is persistent oligochaete (red worm) proliferation, signaling low F/M ratios, extended biomass age, and detrital buildup. This case study of a New York RBC facility shows chronic infestation results from long-term low F/M operation, low shear, and sustained nitrification, and is an expected biological outcome rather than an anomaly. 2. Facility Description and Study Approach The facility evaluated consists of two parallel RBC trains, each configured as two stages in series designed for combined BOD removal and nitrification. The evaluation integrated: -Water quality data (BOD, sBOD, NH₄-N, TSS, alkalinity) -Organic, hydraulic, and nitrogen loading rates normalized to RBC surface area -Seasonal temperature trends -Microscopic analysis of biofilm -Visual documentation of worm populations Key operating parameters were compared against widely cited RBC design ranges for various design treatment levels. Table 1 summarizes the typical RBC organic loading design ranges used as the benchmark for evaluation. 3. Organic Loading, F/M Ratio, and Biomass Age Historical organic and nitrogen loading data, expressed as g tBOD/m²·d and g NH4-N/ m²·d, show the RBC system has operated consistently below the recommended range for combined BOD removal and nitrification. Observed loadings regularly approach or fall below values associated with dedicated nitrification stages. Figures 1 and 2 illustrate the long-term organic and nitrogen loading relative to recommended design ranges. From a ecology perspective, sustained low organic loading results in: -Reduced net bacterial growth -High endogenous respiration -Long effective solids retention time on the media -Accumulation of decaying biomass and extracellular polymeric substances (EPS) These conditions define a late-successional attached-growth ecosystem, which is highly favorable to detritivorous metazoans such as oligochaetes. 4. Hydraulic Loading and Physical Disturbance Hydraulic loading rates normalized to RBC surface area were evaluated against design guidance. Figure 3 shows the system frequently operates at the low end or below the recommended hydraulic loading range. Low hydraulic loading reduces: -Biofilm shear and natural sloughing -Transport of detached solids out of RBC basins -Physical disturbance of benthic organisms In attached-growth systems, hydraulic shear is a primary ecological control mechanism. Its absence allows thick biofilms and settled solids to persist, creating stable habitats for oligochaete colonization. 5. Nitrogen Loading and Nitrification Performance Multi-year effluent data demonstrate robust nitrification across seasons, with NH4-N concentrations consistently reduced from influent levels >20 mg/L to near-zero in the effluent. Figure 4 presents effluent NH₄-N, BOD, TSS, and temperature trends. While desirable for compliance, robust nitrification further confirms that the RBCs operate with long biomass age and low competition for surface area, reinforcing conditions favorable to oligochaete. 6. Organism Identification and Ecological Role Microscopic examinations and photographic evidence confirm that the organisms present are oligochaete worms rather than chironomid larvae. However, the seasonal presence of chironomid larvae during warmer months of the year has been observed by operations staff. Key distinguishing features for the oligochaete worms include thin, threadlike morphology, formation of tangled mats, absence of a head capsule, and reproduction by fragmentation. Microbial analysis results are presented in Figures 5 and 6. Oligochaetes communities observed are shown in Figure 7. Oligochaetes assimilate nitrogen for biomass but do not nitrify. Instead, they promote ammonification by mineralizing organic nitrogen. They can disrupt process stability by grazing on nitrifiers and damaging biofilms, reducing nitrification efficiency. High densities often indicate sludge bulking or aging, common under low F/M ratios and long sludge retention times. 7. Synthesis: Why the Infestation Occurred Taken together, the data indicates that oligochaete proliferation is the expected biological response to: 1.Chronic underloading relative to RBC design criteria 2.Long effective biomass age on media 3.Low hydraulic shear and limited physical disturbance 4.Persistent accumulation of detrital solids 5.Temperature conditions supportive of metazoan survival Therefore, the worms are best understood not as a failure of operation, but as a biological indicator that the system has drifted into a low-F/M, late-successional operating regime. 8. Implications for RBC Design and Operation This study shows design margins in attached-growth systems can unintentionally favor higher trophic organisms as influent loads decline. Viewing oligochaete growth as an ecological signal, not a nuisance, enables proactive adjustments instead of reactive removal. 9. Conclusions Oligochaete infestation in RBC systems is a predictable consequence of sustained low F/M operation, low hydraulic loading, and long biomass age. These findings underscore the importance of periodically reassessing attached-growth systems against their original design assumptions and adapting operational strategies as influent conditions evolve.

As wastewater treatment facilities face increasing constraints on footprint, energy consumption, and capital expansion, there is growing interest in treatment technologies that intensify performance within existing process boundaries. Conventional primary clarification is largely limited to gravity-driven particulate solids removal and provides minimal attenuation of soluble organic matter, thereby placing the full burden of biological oxidation on downstream secondary treatment processes. This conventional separation of physical and biological treatment functions results in large reactor volumes, high aeration demand, and limited operational flexibility. This study evaluates an integrated primary biofiltration approach that introduces fixed-film biological activity into a primary treatment configuration, effectively redefining primary treatment as an intensified, multifunctional unit process capable of combining physical filtration and biological oxidation. An up-flow floating media biofiltration pilot system was designed and operated over a seven-month period (August 2024-March 2025) at the Linda County Water District municipal wastewater treatment plant in Northern California. The reactor consisted of two vertically stacked floating media zones: a lower, unaerated filtration zone providing physical capture of suspended solids, and an upper, aerated zone supporting attached biofilm growth and biological oxidation. This configuration enabled concurrent solids removal and biological treatment within a single vessel, while maintaining short hydraulic retention times compatible with primary treatment applications. Pilot testing was conducted at empty bed contact times (EBCTs) of 20, 30, 60, and 120 minutes to evaluate system performance across a range of hydraulic and organic loading conditions representative of both high-rate and extended-contact operation. Treatment performance was assessed using total suspended solids (TSS), five-day biochemical oxygen demand (BOD), and soluble chemical oxygen demand (sCOD) as key indicators of physical and biological treatment efficacy. Results demonstrate that integrating fixed-film biological activity into a primary treatment configuration can substantially intensify organic removal at hydraulic retention times far shorter than those required for conventional secondary treatment. At EBCTs of 20-30 minutes, the system consistently achieved TSS removals on the order of 80%, exceeding values typically reported for conventional primary clarification, while simultaneously achieving measurable reductions in soluble organics (50-59% sCOD removal). These results indicate that the system provided meaningful attenuation of both particulate and soluble organic loading under short-contact conditions relevant to high-rate treatment. At an EBCT of 60 minutes, average BOD removal exceeded 80% and sCOD removal exceeded 70%, demonstrating that a significant fraction of biological oxidation typically assigned to secondary treatment could be achieved upstream within a compact primary-scale reactor. Analysis of effluent sCOD:BOD ratios revealed a progressive shift toward more stabilized organic fractions with increasing EBCT, confirming that soluble organic removal was driven by biological oxidation within the attached biofilm rather than physical filtration alone. Importantly, solids removal performance remained stable across all tested EBCTs, indicating that biological activity did not compromise filtration performance and that the integrated system was resilient to hydraulic variability. These findings suggest that the combined physical-biological configuration can decouple solids capture from biological kinetics while maintaining consistent treatment performance. High oxygen transfer performance was observed within the aerated biofiltration zone, sustaining biofilm growth and enabling biological oxidation at elevated areal and volumetric rates. Measured oxygen transfer efficiencies per unit depth ranged from 1.10 to 1.69% OTE·ft⁻¹ exceeding values commonly reported for conventional activated sludge systems, including those equipped with fine-pore diffusers. These elevated oxygen transfer efficiencies are notable given the relatively shallow reactor depth and primary-scale configuration, and they suggest favorable gas-liquid mass transfer conditions associated with the floating media architecture and internal hydraulics. At extended EBCTs, partial nitrification was observed, further confirming the biological functionality of the integrated system and indicating that the reactor environment supported both heterotrophic and autotrophic microbial activity. Similar performance trends were independently documented at a separate facility in Ohio in 2025, supporting the broader applicability of the observed mechanisms. Collectively, these results demonstrate that biologically active primary biofiltration can shift a meaningful portion of organic oxidation upstream of secondary treatment, thereby reducing the biological load imposed on downstream processes. From a treatment-train perspective, this redistribution of treatment functions has implications for secondary reactor sizing, aeration demand, and solids production dynamics. By consolidating physical filtration and biological oxidation within a single intensified unit process, integrated primary biofiltration offers a pathway toward more compact, energy-efficient, and adaptable wastewater treatment configurations. This work reframes primary treatment not as a passive, gravity-driven step, but as an active and biologically relevant process with the potential to simplify treatment trains and enhance overall system performance.

Introduction Thermal hydrolysis (THP) is widely deployed to improve sludge treatment performance by enhancing anaerobic digestion (AD), increasing biogas production, boosting dewaterability, and achieving hygienization. However, THP also generates biologically recalcitrant compounds-particularly refractory chemical oxygen demand (rCOD) and refractory dissolved organic nitrogen (rDON)-that can resist downstream biological treatment and contribute to effluent residuals. This challenge is increasingly important as utilities face tightening discharge standards for COD and nitrogen and rising interest in high load solids strategies. This full scale study at the Dijon EauVitale WRRF (France) investigates how THP operating temperature (140-165  °C) influences the quantity and typology of refractory organics, their fate through digestion, and microbial community resilience. We combine high resolution chemometric fingerprints (UPLC SEC with UV/fluorescence detection) and artificial neural network (ANN) feature selection with parsimonious multiple linear regressions (MLRs) to build predictive models for rCOD and rDON using interpretable descriptors. We further assess PCDD/Fs concentrations and Toxic Equivalents (TEQ) around THP and characterize bacterial/archaeal diversity in digestate to evaluate operational robustness. A set of mass balances at full scale complements bench scale degradability tests to quantify how much refractory material is formed by THP and retained/released through AD and dewatering. Materials & Methods The study was conducted at the Dijon EauVitale WRRF equipped with thermal hydrolysis (140-165  °C) and mesophilic anaerobic digestion. Samples were collected at key process stages (pre-THP, post-THP, digestate, effluent) across multiple campaigns. Refractory COD and DON were quantified using standard colorimetric assays, while molecular fingerprints were characterized by UPLC-SEC with UV/fluorescence detection. Predictive models were developed using artificial neural networks and multiple linear regressions based on selected spectral descriptors. Microbial community structure was assessed via 16S rRNA sequencing and diversity metrics. Results & Discussion 1) Temperature vs Quantity and Typology of Refractory Organics Linear scaling of refractory formation. THP increased rCOD proportion linearly from 140 to 165  °C: ≈2.9 fold at 140  °C and ≈4 fold at 165  °C relative to PWAS. Despite quantity scaling, the rDON/rCOD ratio remained stable at ~4.5% (mean; 95% CI ≈3.8-5.1%), indicating a constant nitrogen share in refractory pools across the studied range. This stability suggests that temperature mainly scales the refractory quantity, not the nitrogenization of the refractory fraction. Chemometric fingerprints: typology conserved. PCA/HCA of the 144 spectral ratios revealed three robust clusters: (i) raw PWAS, (ii) THP/digestate streams, and (iii) final effluent. Analytical descriptors most discriminant (ANOVA, p < 0.05) were dominated by 20-40 kDa signals combining tryptophan like and humic /melanoidin like features (e.g., TRY1/MEL, TRY1/HA, TRY2/AF2). Across temperatures, these fingerprints remained consistent, supporting that THP temperature does not materially alter molecular typology, even though it amplifies the amount of refractory material. ANN MLR predictive modeling. ANNs prioritized a subset of descriptors, from which compact MLRs reproduced measured rCOD and rDON with R² = 0.972 and 0.964 using 4-5 interpretable ratios per model. Coefficient signs provided mechanistic insight: low MW protein-melanoidin and humic normalized protein/fulvic contrasts acted as negative predictors of recalcitrance, while 20-40 kDa protein/humic enrichments were positive predictors, consistent with the cluster discrimination. Practically, these descriptors can be embedded into monitoring to track refractory risk and guide temperature setpoints. Operational implication. For design/operations, increasing THP temperature predictably raises refractory load without altering its spectral signature, enabling utilities to use ANN MLR monitors as early indicators and control levers (e.g., temperature windows, blend strategies) rather than expecting typology shifts to ease downstream treatment. 2) Fate Through Anaerobic Digestion and Return Pathways AD mitigation (~31%). Full scale mass balances combined with degradability assays indicated that AD reduces refractory loads by ~30.6 ± 2.3% (95% CI ≈20-40%). Evidence points to physical mechanisms-adsorption/copolymerization/association with biomass-more than biodegradation, consistent with literature on hydrophobic recalcitrants binding to flocs. This mitigation means not all THP derived refractory material returns to headworks; a significant share remains associated with digested sludge and exits with dewatered cake. Centrate contribution and effluent context. At Dijon, THP derived refractory accounted for ~23-34% of effluent refractory COD during campaigns, with the remainder dominated by external industrial leachates. Even so, the AD mediated reduction increases the liquids line's headroom, limiting residual build up during high load operation. For centralized solids hubs, this synergy suggests greater acceptance capacity for external sludges than expected if considering THP alone. Operational implication. Coupling THP with robust AD provides a two stage strategy: push hydrolysis for digestion gains while buffering refractory recirculation through physical capture in digesters and solids handling. Monitoring centrates and adjusting cake handling strategies (e.g., capture, conditioning) becomes essential when effluent residuals are rate limiting for compliance. 3) PCDD/Fs Risk Profile at THP Temperatures Measured concentrations and TEQ. Around THP, PCDD/Fs totals showed negligible change at 140  °C (PWAS→HWAS; within method uncertainty) but increased at 165  °C, with TEQ rising to ~9.2 ng TEQ/kg TS. Although higher than at 140  °C (~2.9 ng TEQ/kg TS), values remained below applicable agronomic thresholds currently discussed in the EU context (e.g., France's Socle Decree limit: 20 ng TEQ/kg TS for land application). The total mass increase at 165  °C was accompanied by toxicity weighted increase, suggesting formation/enrichment of more dioxin like congeners at the upper end of the THP window. Operational implication. Within 140-165  °C, TEQ remains manageable, but facilities should adopt periodic screening when operating ≥160-165  °C, especially if feedstocks or legacy contamination could bias congener profiles. Pressure effects and catalyst/ash interactions warrant further study under THP conditions. 4) Microbial Diversity and Process Robustness Stable alpha diversity. Across THP temperatures, both bacterial and archaeal communities exhibited high and stable diversity (Shannon/Simpson), indicating that THP setpoints do not compromise the functional breadth of digestion. Archaeal communities remained dominated by hydrogenotrophic methanogens (e.g., Methanoculleus) typical of high ammonia sludge digestion, while bacteria showed moderate compositional shifts (e.g., Fastidiosipila vs. Proteiniphilum/Brevefilum) consistent with changes in substrate complexity rather than loss of core hydrolytic/acidogenic functions. Beta diversity insight. Bray-Curtis ordination highlighted greater sensitivity in bacterial assemblages to temperature than archaea, aligning with their broader roles in hydrolysis/acidogenesis. Functionally, the core trophic network remained intact, supporting operational resilience across the THP window. Operational implication. Utilities can tune THP temperature for digestion/performance gains without destabilizing microbial function, provided ammonia management and organic loading remain within design envelopes. Conclusions and Practical Guidance - Temperature effect: Increasing THP temperature (140→165  °C) linearly amplifies refractory organics without changing their molecular fingerprint; rDON shares remain proportionally constant. - Predictive control: A small, interpretable set of chemometric descriptors predicts rCOD/rDON with high fidelity, enabling ANN MLR monitoring to proactively manage refractory risk and temperature setpoints. - AD synergy: Anaerobic digestion removes ~31% of THP derived refractory material-primarily via physical association-meaning not all recalcitrants recirculate to the liquids line. - PCDD/Fs vigilance: TEQ remains below agronomic thresholds at 165  °C but rises versus 140  °C; adopt periodic screening when operating at the upper THP range. - Microbial resilience: High and stable diversity and conserved core functions confirm process robustness across the studied THP temperatures.

Background & Drivers: Columbus City Utilities (CCU), Indiana experienced an increase in biosolids production driven by higher-than-anticipated sludge yields from a legacy solids reduction system and then a rapid increase due to increased industrial loadings. Average influent TBOD rose from 10,500 lb/day (2022) to 17,200 lb/day (2023) when a new industrial customer began discharging to the plant. Even after pretreatment, CCU's post-pretreatment influent TBOD baseline remained 14,600 - 16,000 lb/day with planning projections to ~22,900 lb/day by 2042. Dewatered cake at 18-20% TS had to be frequently land applied (constrained by weather and availability) or hauled to a distant landfill, with high disposal costs, creating an operationally fragile and labor-intensive program. Goals for Utilities: CCU's program demonstrates how an indirect thermal drying solution, paired with flexible dewatering and silo storage, can convert a haul-limited, weather-dependent biosolids operation into a resilient Class A program. Key lessons include: 1) vet dryer technology constraints and options early to right-size equipment needs, facility sizes, building envelopes and electrical classifications; 2) design sludge feed to enable continuous drying and minimize start/stop energy and processing inefficiencies; 3) align conveyance and storage decisions (drag/pneumatic conveyance, hopper sizing, hauling) with O&M needs; 4) build a realistic model that reflects peak-month operation; and 5) develop a comprehensive permitting roadmap and site access plan to avoid late-stage obstacles. Solution Selection: CCU undertook an alternatives analysis for biosolids handling and selected thermal drying to achieve ~90% TS and Class A biosolids, explicitly to reduce haul volume and cost, decouple operations from seasonal constraints with land applying, and expand disposal and beneficial reuse options. Multiple technologies were considered, but an indirect screw/paddle dryer operating under nitrogen purge with heat provided by a thermal fluid system was chosen for its process stability, footprint fit, NFPA envelope compatibility and safeguards, and integration with existing dewatering upgrades. A condenser and chemisorption-based odor control train were included to minimize odor and return condensate to the forward flow treatment train. A retrofit inside the existing Dewatering Building proved infeasible due to clearances, ventilation, and NFPA Class II Div 1/2 electrical classification envelopes, so CCU planned for a new Dryer Building adjacent to the dewatering facilities. The new Dryer Building includes two 50-CY live-bottom hoppers (wet cake feed control), progressive cavity feed pumps, indirect dryer, thermal fluid heater, condenser, nitrogen generation, cooling conveyor, and SCADA/electrical rooms. The dried product will be conveyed to a 178 CY silo with drive-through loading and nitrogen purge. The silo will have 6 to 8 days of storage depending on operating and loading factors. The project includes site improvements for tractor trailer access, stormwater detention upgrades, and demolition of an existing tank to meet several site constraints. Operations Strategy: The design supports steady, continuous drying while dewatering operates primarily on staffed shifts. The operation and timing was discussed to allow for flexibility in desired operation needs, staff availability and hauling options. During average conditions, centrifuges run 40 hours/week while the dryer runs 100-130 hours/week. For projected 2042 maximum month, the dryer can operate up to 160 hours/week while centrifuges intermittently run 94 hours/week to maintain feed, stabilizing thermal performance and minimizing start/stop energy inefficiencies. The use of two hoppers provides several hours of buffer and the silo provides 6-8 days of dried product storage, improving resilience, weekend staffing flexibility, and outage management. Anticipated Performance and Benefits: Transitioning from 18-20% TS cake to 90% TS reduces hauled mass by 75-80%, reduces truck trips, reduces excessive maintenance and costs on utility hauling trucks, and mitigates weather-driven land-application limits. The Class A product broadens end uses and provides a more robust platform for addressing contaminants of emerging concern (e.g., PFAS) through reduced solids mass. CCU visited several full-scale dryer operations to inform the utility planning decisions such as reviewing facility dried product consistency meeting >90% solids, daily operator time requirements and staffing, and attention to dust/odor control. Energy & Utilities: The energy profile reflects real-world thermal drying. Estimated natural gas ranges from 43,000 to 73,000 mmBTU/year across 2024 daily average to 2042 maximum month, while electricity is 420,000 to 674,000 kWh/year over the same span. The annual energy costs were estimated at $74,000-$287,000 across conditions (unit costs based on 2025 rates). The dryer requires continuous and intermittent water streams with return of condensate to the headworks; the project includes gas, power distribution, and water service modifications to support the new process.

Evaluating Biochar Blending During Curing of THP Class A Biosolids: Impacts on Product Quality, Odor, Handleability, and PFAS Concentrations Background The Hampton Roads Sanitation District (HRSD) Atlantic Treatment Plant produces Class A/EQ biosolids using thermal hydrolysis followed by mesophilic anaerobic digestion. The resulting thermal hydrolysis process (THP) cake is nutrient-rich but can present challenges related to odor generation, moisture content, and product handleability during curing, storage, and distribution. HRSD has implemented a windrow 'curing' process to dry the product, reduce ammonia compounds, and improve odor and stabilization of the cured product. However, site odors associated with the curing process have been observed. To address both site and product odor challenges and improve the physical and chemical properties of Class A biosolids - including odor, moisture content, and availability of metals and organic compounds - HRSD, in collaboration with Material Matters (MM), conducted a pilot-scale blending trial incorporating wood-based biochar into the THP cake prior to curing. Biochar, a carbon-rich product made from the pyrolysis of biomass, is known for its adsorptive capacity, moisture retention characteristics, and potential to mitigate odors while stabilizing nutrients and pollutants. This pilot-scale blending trial aimed to determine whether varying biochar addition rates could improve overall product quality and reduce potential challenges associated with product storage and beneficial use. It was hypothesized that increasing the proportion of biochar added to THP cake, combined with the established curing process, would result in lower moisture and ammonia concentrations, and improve site and product odor, nutrient and pollutant concentrations, and handling characteristics. Objectives The research objectives are described below: 1. Determine the ideal range of biochar addition ratios relative to fresh cake biosolids on a dry weight basis to achieve odor and pollutant reduction; 2. Assess physical, chemical, and biological characteristics present in cured biosolids end-products; and 3. Provide recommendations for optimal biochar amendment ratio based on trial findings. Methodology A pilot-scale blending and curing trial was conducted using five biosolids piles (approximately 6 wet tons each): one unamended control (0% biochar) and four amended piles with biochar addition rates of 2%, 5%, 8%, and 10% on a dry weight basis (Figure 1). Initial blending ensured even distribution of biochar. All piles were managed under HRSD's standard six-week curing protocol (windrows turned three times per week, located on a paved, covered storage pad). Environmental exposure and management conditions were consistent across all piles to minimize variability unrelated to amendment rate. Composite solids sampling was performed immediately after blending (week 0), mid-curing (week 3), and at the end of curing (week 6). Analytical parameters included nutrients, metals, moisture content, soluble salts, maturity indicators, product odor characterization, handleability metrics, and per- and polyfluoroalkyl substances (PFAS). Following the six-week curing period, each product was moved to individual storage piles for up to ten months. Solids Sampling and General Analytical Parameters - At each sampling event, composite solids samples were collected from each pile using stainless steel scoops. Samples were obtained from seven evenly distributed locations within each pile, with four scoops collected per location (i.e., 28 scoops total per pile). The scoops from each pile were thoroughly homogenized prior to sub-sampling for laboratory analysis. Subsamples were submitted to accredited laboratories for analysis of nutrients, metals, moisture content, soluble salts, and compost maturity indicators, consistent with standard biosolids and compost characterization protocols. PFAS Sampling - PFAS analysis was conducted on solids collected from the 0% (control) and both 5% and 10% biochar-amended piles to evaluate potential effects of biochar addition on PFAS concentrations during curing. Odor Sampling - Odor sampling was conducted during three curing events to evaluate the effect of biochar amendment rate on biosolids odor potential. Odor samples were collected using a composite headspace sampling approach designed to minimize spatial variability within each pile. For each sampling event, composite solids from each pile were prepared as described above and placed into individual 25-L glass wide-mouth bubblers. Each bubbler contained homogenized material representing all pile locations. Following removal of solids subsamples for chemical and physical analysis, the remaining material in each bubbler was sealed with a Teflon transfer lid equipped with dedicated flux and sample tubing. Bagged gas samples were collected by fluxing the bubbler headspace with nitrogen gas at a rate of 5 L/min (e.g., fluxing rate) while simultaneously drawing sample air at 2.5 L/min (e.g., sampling flow rate) into 10-L sampling bags using SKC pumps. Samples were collected via a vacuum chamber system to prevent contamination and maintain consistent flow rates. Each bag was pre-conditioned through multiple fill-and-purge cycles prior to final sample collection to minimize background odors. Handleability - Handleability of each cured product was evaluated qualitatively and quantitatively at week 0 (immediately after blending), week 3 (mid-curing), and at week 6 (end of curing). Observations were recorded by operations staff using standardized evaluation criteria (rating on a scale of 1 to 4; with 4 indicating the most favorable score for that factor) to allow comparison across amendment rates. Findings and Current Status: Product Quality - Biochar-amended piles exhibited higher ammonium-N fractions and elevated solute salt concentrations relative to the control, consistent with biochar's adsorptive properties and its potential to retain ammonium during the curing process. These changes may influence nitrogen availability following land application, depending on crop demand, soil conditions, and application rate. Increased ammonium-N may be advantageous for beneficial use scenarios requiring readily plant-available nitrogen, while potentially limiting suitability for end uses the prioritize controlled nitrogen release or have low tolerance for elevated soluble salts and ammonium-related phytotoxicity. Other measured constituents, including nutrients and regulated metals, generally reflected dilution effects associated with biochar addition (Figure 2). Product Odor - Product odor improved substantially across all piles as a result of aerobic curing, indicating that curing was the primary driver of odor reduction, supported by both odor detection thresholds (Figure 3) and selected-ion flow-tube mass spectrometry (SIFT-MS) results (Figure 4). While biochar-amended piles exhibited odor profiles comparable to or slightly improved relative to the control at certain time points, overall results provided limited evidence that biochar addition at rates up to 10% produced measurable odor reductions beyond those achieved through curing alone. Product Handleability - Handleability improved moderately with curing across all piles. Improvements included reduced smear and increased friability as moisture content declined. PFAS Concentrations - PFAS results showed substantial variability over time, with evidence suggesting precursor transformation during aerobic curing. While biochar-amended piles generally exhibited lower PFAS concentrations than the control, reductions were likely reflective of dilution or sampling variability. Additional Research - HRSD staff are currently conducting additional research to understand the impact of medium to long-term product storage on the cured piles with respect to physical and chemical changes in the end-product with potential to impact market outlet acceptance. Conclusion Trial findings provide practical insights for utilities considering biochar as a biosolids amendment within existing curing operations. The preliminary findings of the trial suggest biochar addition (up to 10% by dry weight) to fresh THP cake biosolids does not drastically improve odor or physical product characteristics beyond the benefits of curing alone. Findings highlight the need for further research evaluating long-term storage after curing, higher amendment rates, potential impacts on PFAS mobility and leaching behavior, and assessing the replicability of these initial observations.

Introduction It is a complicated proposition when Water Resource Recovery Facilities (WRRF) elect to sell biogas to a commercial 3rd party to make Renewable Natural Gas (RNG) or to make and sell RNG for themselves. These endeavors involve markets, contracts, commitments, uncertain outcomes, and partnerships that lie far outside the typical world of those working at or designing WRRFs. Engaging in this type of gas sales process can be daunting and risky if projects are not pursued in a manner that retains proper benefits and sets manageable responsibilities for the WRRF. The authors of this paper include biosolids and biogas engineers working at or on behalf of municipal WRRFs that have undertaken biogas and RNG sales contracting successfully. The case studies presented will highlight specific keys to successful outcomes in each. However, the authors feel there is one major aspect that generally drives success that the WRRF and engineers working on their behalf must take the lead on devising the gas sales agreements and partnerships with commercial 3rd parties. WRRFs can benefit significantly from gas sales agreements but only when commercial entities are forced to compete around the items that matter and when a true partner can be identified that is willing to share benefits and risks with the WRRF. Roles and Responsibilities in Biogas to RNG Understanding the main entities, roles, and their responsibilities is crucial for navigating biogas-to-RNG sales contracting. Figure 1 provides a schematic representation of the participating entities and their roles in a typical biogas-to-RNG contract. -Biogas Producer: The entity that owns and operates the digester infrastructure, which converts organic feedstock into raw biogas (typical for WRRFs). -Biogas Upgrader: The entity that owns and operates the biogas upgrading infrastructure, which converts raw biogas to RNG product gas (optional role for WRRFs). -Gas Broker: The entity that purchases the RNG Product Gas itself as a commodity, as well as the rights to the Environmental Attributes (EAs) that are generated. The Gas Broker is also typically responsible for registering the RNG project with appropriate authorities to generate EAs and conducting EA sales in response to tracking of market pricing. -Natural Gas Utility Pipeline: If RNG is not dispensed directly to end users, a NG Utility Pipeline will physically accept the RNG-produced gas into their pipeline infrastructure. -Final End User: The entity that ultimately purchases RNG from the Gas Broker and utilizes it in a variety of ways, including fueling compressed natural gas (CNG) vehicles, for campus heating, or for the generation of other energy products. Case 1 - City of Dayton Dayton identified selling of biogas to generate RNG as a priority project in their 2019 Masterplan. This was in the wake of the significant RIN price boom in 2017. Interestingly, Dayton had already constructed and installed a PSA upgrading system back in 2012 to clean gas ahead of their boilers and cogeneration engines. Dayton elected to solicit commercial 3rd parties to purchase their biogas for beneficial use. Under this type of arrangement, the solicitation and selection process is the essential tool that the WRRF has at its disposal to ensure it is identifying a true partner and entering into an agreement that adequately shares risks and rewards. Dayton's selection process is summarized below. RFQ Phase - An open solicitation was issued for interested parties. Ahead of the RFQ, Dayton conducted a pipeline interconnection feasibility study with the local NG utility and included detailed results in the RFQ (see Fig 2). Along with a technical approach, the solicitation called for respondents to provide evidence/experience that they could execute all necessary aspects of an RNG project (see Fig 3). This was key because there are many varied elements required for successful RNG projects and finding a partner that can do all and not some of them is the main goal of an RFQ. The Dayton solicitation received 12 responses of which 3 were shortlisted for invite only RFPs. RFP Phase - The RFP phase required significant detail from invitees that matched the approach promised in the RFQ. Dayton created 20-year pro-forma spreadsheets that matched the RFQ approaches and required respondents to fill in key values that elucidated both their projected risks and returns, and their offered royalty rate back to Dayton. Dayton also authored and provided a draft contract for respondents to review and provide exceptions. Exceptions were allowed but negatively affected the RFP response score. Thus, respondents were forced to compete not just on financial benefit, but on the contracting responsibilities they would accept. For example, Dayton provided historical biogas production information (see Fig 4). Risk was shared by Dayton's agreement to a minimum gas production floor and the biogas upgrader's agreement to accept gas up to a ceiling value exceeding current production. Outcome - Dayton selected to partner with DTE Vantage (DTE) for biogas sales. DTE offered extremely lucrative benefits due to the fact they already owned and operated an RNG system at a nearby landfill(see Fig 5), already had a functioning natural gas pipeline interconnection, and already had a vehicle offtaker and D3 RIN sales in place. None of this was known to Dayton prior to the solicitation so the thorough and comprehensive selection process resulted in major dividends. The Dayton biogas started flowing to DTE's pipeline in November 2024 and Dayton's net financial returns are currently estimated at approximately $750k annually. Case 2 - Capital Region Water Through biosolids and energy planning, Capital Region Water (CRW) in Harrisburg, PA identified upgrading biogas and selling it as RNG as a priority project. Electing to own and operate the RNG upgrading system themselves in order to maximize revenue and fund other fundamental infrastructure upgrades to their WRRF. Another aspect of their decision was the existence of a NG pipeline injection company (UGIES) who already owned and operated a NG pipeline interconnection into the low-pressure distribution system less than 500 feet from CRW's digesters. UGIES was also able to serve as the gas broker in addition to its role as the pipeline interconnection which further incentivized cooperation. With a known partner in hand, the key to CRW's success was crafting a series of agreements with UGIES that allowed successful execution of the gas sales. CRW and engineers working on their behalf crafted the principal versions of the following agreements to ensure benefits and reasonable responsibilities were maintained. Interconnection Agreement - This set the interconnection construction responsibilities of both parties, established methods of payment to UGIES for construction, and reserved unfettered access to the NG pipeline interconnection for CRW if gas quality met the required standards. Some benefits to highlight include delegating the construction and upkeep of jurisdictional pipeline to UGIES who already had certified labor and expertise for this work seeing as they are a NG pipeline company. It also involved eliminating the typical redundant Gas Chromatograph monitoring stations on both sides of the fence. Due to the extreme proximity and cooperative nature of the parties, a single monitoring station was owned and operated by UGIES and readings could be shared back to SCADA at CRW. Biomethane Transaction Agreement - This set the ground rules and the pricing structure under which UGIES would purchase both the commodity NG and the RNG related environmental attributes (EAs) from CRW. One notable benefit was the absence of an RNG supply floor minimum guarantee from CRW. The price paid to CRW for EAs was based on a percentage of a public price index (RIN pricing data as published by the EPA) to allow full price transparency. There was also an Alternatives Markets clause (which was exercised) if both parties agreed that pursuing RINs was not the most beneficial route for monetization. All direct costs which refer to the gas broker's logistical costs for monetizing EAs were kept solely as the responsibility of UGIES and could not be passed back to CRW. Outcome - The RNG upgrading and interconnection was completed and RNG was flowing to the pipeline in late 2025. CRW established an RNG sales pathway while retaining the vast majority of sales revenue to fund other critical infrastructure upgrades. Partnering with a single entity serving as both the gas broker and NG interconnection point significantly simplified the contracting structure CRW needed to pursue. It also provided mutual incentive to get the interconnection constructed in a timely manner and kept open allowing both parties to benefit from the production and sales of RNG.