Microbial community characterization in advanced water reclamation for potable reuse.

This study investigated the microbial community structure and composition across two treatment steps used in advanced water reclamation for potable reuse applications, namely Coagulation/Flocculation/Clarification/Granular Media Filtration (CFCGMF) and Ozone-Biological Activated Carbon filtration (O3/BAC). The study examined the richness, variations, and similarities of the microorganisms involved at each treatment step to better understand the role of ecology and the dynamics on unit process performance and the microbial community developed within it. The bacterial microbiomes at each treatment step were independently characterized using 16S metagenomic sequencing. Combining both treatment steps, a total of 3801 species were detected. From the total species detected, 38% and 98% were identified at CFCGMF and O3/BAC, respectively. The most abundant phyla were Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes in both treatment steps. The identified species were classified based on their preferences to free-living style (59%) vs attached-living style (22%) showing a relatively low richness in the BAC media, but higher diversities. At the taxonomic class level, Betaproteobacteria was the predominant in both system processes. Additionally, a list of eight genera were identified as potential bacterial pathogens present in both process effluents. They are Aeromonas, Clostridium, Enterobacter, Escherichia, Flavobacterium, Legionella, Mycobacterium, and Pseudomonas. CFCGMF effluent yielded less pathogenic bacteria than both the ozone and BAC filter effluent from the O3/BAC process unit; their relative abundance accounted for about 2% and 8% for CFCGMF and O3/BAC, respectively. Detailed studies to characterize the microbial communities are crucial in interpreting the mechanisms and synergies between processes performance and microorganisms by identifying the needs and best practices to ensure public health protection. Key points • Microbial communities of two treatment processes are characterized using 16S rRNA sequencing. • Organisms that can tolerate ozone and form biofilms define microbial community in subsequent biofilters. • In relatively low abundances, potential pathogenic bacteria are detected in the treated water.

Phosphorus Recovery by Methods Beyond Struvite Precipitation.

Unintentional phosphate precipitation at water resource recovery facilities (WRRFs) causes operation and maintenance challenges. With global phosphorus (P) scarcity looming and receiving water eutrophication caused by excess discharges of P, intentional P recovery at WRRFs has been gaining traction. To date, struvite recovery as slow release fertilizer has been the focus of P recovery. However, struvite recovery is not always the most cost-effective approach, especially when phosphate fertilizers cost considerably less than the cost to recover struvite as fertilizer. The aim of this state-of-an-art review paper is to discuss P recovery as calcium phosphate, which could be a fertilizer feedstock and incurs less chemical costs to produce. Calcium phosphate also offers broader applications for other industrial uses beyond fertilizers since the composition is close to mined phosphate rock. A strategic approach for a regional reclaimed phosphate reserve is proposed to secure the most economical future supply of P.

Sustainability Assessment for Indirect Potable Reuse: A Case Study from Reno, Nevada.

A triple bottom line (TBL) approach was used to examine the trade-offs between potential reclaimed water management strategies in a closed basin. The goals of the water management strategy included minimizing water source shortages, ensuring safe and resilient future water supplies, and protecting inland ecosystems through adequate surface flows. The TBL approach consisted of quantitative and qualitative impact assessments of social, environmental, and economic criteria. This research examined how potable reuse of reclaimed water addresses water needs in a closed basin such as maintaining water quality, managing reclaimed water disposal, meeting growing water demand, balancing groundwater extraction rates with inflows, preserving inland ecosystems, and ensuring a locally controlled safe drinking water source. The TBL assessment first evaluated water stress based on water demand and supply under status quo conditions. The results were compared with the potable reuse scenario, which provides more environmental and social benefits than the status quo scenario.

Directed evolution to produce sludge communities with improved oxygen uptake abilities.

Two activated sludge cultures, seeded with activated sludge from the same source, were cultivated for 370 days in synthetic wastewater. Both cultures were transferred weekly to fresh medium; one culture was operated at high dissolved oxygen (DO) (near saturation) and the other at low DO (0.25 mg O2/L). There were significant changes in the abundances of bacterial species and phyla present in each culture throughout the 370-day operational period. In the low DO culture, over time, there was a continuously increasing proportion of cells of species known to encode truncated hemoglobins (Hbs). These are the types of Hbs which may enhance delivery of oxygen to the respiratory chain, to enhance ATP production, especially under low aeration conditions. The levels of heme b, the heme found in Vitreoscilla hemoglobin, increased in parallel to the increase in Hb-encoding species, to much higher levels in the low DO culture than in the high DO culture. Specific oxygen uptake rates increased by 3 % for the high DO culture near the end of the 370-day period, while those for the low DO culture increased steadily to a level 28 % higher than that of the starting culture. Thus, imposition of low DO conditions may, due to selection for Hb-expressing species, be useful in developing bacterial communities with enhanced ability to function efficiently in aerobic wastewater treatment, especially under low aeration conditions.

Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation.

Since its first use in 1990 to enhance production of α-amylase in E. coli, engineering of heterologous hosts to express the hemoglobin from the bacterium Vitreoscilla (VHb) has become a widely used strategy to enhance production of a variety of bioproducts, stimulate bioremediation, and increase growth and survival of engineered organisms. The hosts have included a variety of bacteria, yeast, fungi, higher plants, and even animals. The beneficial effects of VHb expression are presumably the result of one or more of its activities. The available evidence indicates that these include oxygen binding and delivery to the respiratory chain and oxygenases, protection against reactive oxygen species, and control of gene expression. In the past 4 to 5 years, the use of this "VHb technology" has continued in a variety of biotechnological applications in a wide range of organisms. These include enhancement of production of an ever wider array of bioproducts, new applications in bioremediation, a possible role in enhancing aerobic waste water treatment, and the potential to enhance growth and survival of both plants and animals of economic importance.

Laboratory Evaluation of Sorptive Filtration Media Mixtures for Targeted Pollutant Removals from Simulated Stormwater.

Urban stormwater pollutants including nutrients, trace metals, polycyclic aromatic hydrocarbons (PAHs), total suspended solids (TSS) and indicator microorganisms (E. coli) were the target pollutants in this study. Their simultaneous removal from synthetic stormwater using different sorptive media was investigated through laboratory-scale column experiments. The media considered for testing were calcite (C), sand (S), zeolite (Z) and iron filings (Fe(0)). These media are easily/commercially available, environmentally benign, long lasting (non-biodegradable), highly permeable and effective in removing multiple target pollutants. Based on individual media testing, three different mixed media compositions were selected (1) C-Z-S, (2) C-S-Fe(0) and (3) C-Z-S-Fe(0), for simultaneous removal of target pollutants using column experiments. The mixed media composition, C-Z-S-Fe(0) showed significantly better simultaneous removal of all the target pollutants as compared to the other two compositions. These compositions can be tailored to meet site-specific stormwater pollutant removal in a passive in-ground sorptive media filter.

Toward Universal Half-Saturation Coefficients: Describing Extant K(s) as a Function of Diffusion.

Observed (extant) K(s) is not a constant and it is strongly influenced by diffusion. This paper argues that diffusion can be used to describe bacterial kinetic effects that are sometimes attributed to "K-strategists" and, in fact, the physics of the system is the dominant mechanism affecting the apparent (extant) Ks--not intrinsic biological characteristics--in real water resource recovery facility systems. Four different biological processes have been modeled using the "porter-diffusion" model that was originally developed by Pasciak and Gavis (1974) for aquatic systems. The results demonstrate that diffusion is the dominant mechanism affecting K(s) in all four biological processes. Therefore, the authors argue that for treatment processes in which substrate concentrations are low, it is important to consider shifting to variable extant K(s) values or explicitly modeling the effects of diffusion.

Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions.

We have recently reported that expression of an unidentified heme protein is enhanced in a nitrifying activated sludge community under low (0.1 mg O2/L) dissolved oxygen (DO) conditions. A preliminary assessment suggested it may be a type of hemoglobin (Hb) or a lesser-known component of the energy-transducing pathways of ammonia-oxidizing bacteria (AOB) (particularly an oxidase or peroxidase). Here, additional work was done to characterize this protein. Due to the unfeasibility of identifying the protein using gene-based methods, our approach was to carry out assays that target the activity and function of the protein, its location in the cell, and determination of the organisms that express it. Using CO-difference spectra, it was shown that the protein is expressed by AOB preferentially in the cytoplasm, while the pyridine hemochromogen method demonstrated that it has heme c as its prosthetic group. Peroxidase and oxidase assays were carried out on the soluble fraction of the low DO-grown cells; neither the peroxidase nor oxidase activities matched those of the CO-binding heme protein detected. Even though it is not possible to conclusively identify the protein detected as a Hb, all other known possibilities have been ruled out. Further work is needed to verify the identity of the heme protein as a Hb and to determine its type and biochemical role under low oxygen conditions.

Acclimation of denitrifying activated sludge to a single vs. complex external carbon source during a start-up of sequencing batch reactors treating ammonium-rich anaerobic sludge digester liquors.

In this study, denitrification of ammonium-reach anaerobic sludge digester liquor was investigated during start-up periods of two laboratory-scale "fill-and-draw" reactors. One reactor was fed with a single carbon source (ethanol), whereas the other reactor was fed with a complex carbon source (fusel oil). During two acclimation experiments, the structure of microbial community involved in denitrification was analyzed using 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis fingerprints and fluorescent in situ hybridization. The characteristics of the mixed liquor were additionally supported by regular measurements of nitrate uptake rates. The addition of fusel oil and ethanol resulted in a significant enhancement of the denitrification rate and efficiency combined with the increasing volumetric addition of sludge digester liquor up to 15 % of the reactor volume. The microbiological analyses revealed that the addition of sludge digester liquor as well as both external carbon sources (fusel oil and ethanol) did not affect the structure of microbial communities in a severe way. In both reactors, Curvibacter sp. and Azoarcus sp. were found as the most abundant representatives of denitrifiers.

Enhanced stabilization of digested sludge during long-term storage in anaerobic lagoons.

The goal of this work was to study changes in anaerobically stored digested sludge under different lengths of storage time to evaluate the quality of final product biosolids. The analyses of collected data suggest the organic matter degradation occurrence in the anaerobic environment of the lagoon approximately within the first year. After that, the degradation becomes very slow, which is likely caused by unfavorable environmental conditions. The performance of lagoon aging of digested sludge was also compared to the performance of lagoon aging of anaerobically digested and dewatered sludge. It was concluded that both of these processes result in biosolids of comparative quality and that the former provides more economical solution to biosolids handling by eliminating the need for mechanical dewatering.

Seasonal and influent characteristic effects on hydrogen sulfide generation at a water reclamation plant.

Correlations between sulfide generation and seasonal influent wastewater characteristics were identified based on a long-term monitoring program in summer and winter at a water reclamation plant. During summer, the emission rates of hydrogen sulfide (H2S) from the liquid treatment processes increased substantially compared to those during winter due to the increased wastewater temperature. The open tanks/clarifiers were the least significant H2S emission contributors throughout the year. For solids-handling processes, the H2S emission rates did not change during the year due to similar sludge characteristics in the different seasons. The fate of sulfide in liquid treatment processes was investigated as an alternative to estimation of H2S emissions. H2S emission from the wet well and screens was proven to be robustly associated with the wastewater temperature, flow rate, 5-day biochemical oxygen demand and total Kjeldahl nitrogen levels. However, the correlation between influent parameters and H2S emission from aerated grit chambers was not statistically significant.

Indicator and pathogenic virus removal in bench scale soil aquifer treatment.

The demonstration of enteric virus removal for indirect potable reuse of advanced purified water is necessary to ensure safe water reclamation practices. This study evaluated the efficacy of soil treatment in reducing concentrations of Pepper Mild Mottle Virus (PMMoV), Hepatitis A (HAV), and Norovirus (NoV) gene markers through bench scale unsaturated soil columns. Three different infiltration rates were evaluated to determine their impact on viral gene marker removal. The concentrations of viral markers in the column influent and effluent samples were measured through RNA extraction and then RT-qPCR, and the log reduction values (LRVs) were calculated to quantify the effectiveness of removal across the columns. The LRVs achieved for PMMoV were 2.80 ± 0.36, 2.91 ± 0.48, and 2.72 ± 0.32 for infiltration rates of 4.9 mm/h, 9.4 mm/h, and 14.0 mm/h, respectively. A one-way ANOVA indicated no statistically significant differences in LRVs among the various infiltration rates (p-value = 0.329). All samples measured for HAV were below the detection limit both in the influent and effluent of the soil columns. While NoV GI and GII markers were measurable in the soil column influent, they were removed to below the detection limit in the effluent. The use of half the Limit-of-Detection (LoD) for effluent values enabled the estimation of log removals, which were calculated as 1.42 ± 0.07, 1.64 ± 0.29, and 1.74 ± 0.18 for NoV GI and 1.14 ± 0.19, 1.58 ± 0.21, and 1.87 ± 0.41 for NoV GII at infiltration rates of 4.9 mm/h, 9.4 mm/h, and 14.0 mm/h. This highlights the efficacy of soil treatment in reducing virus gene marker concentrations at various infiltration rates, and that spreading basins employed for reclaimed water recharge to ground water aquifers are an effective method for reducing the presence of viral contaminants in indirect potable reuse systems.

A mass balance approach for quantifying the role of natural decay and fate mechanisms on SARS-CoV-2 genetic marker removal during water reclamation.

The recent SARS-CoV-2 outbreak yielded substantial data regarding virus fate and prevalence at water reclamation facilities (WRFs), identifying influential factors as natural decay, adsorption, light, pH, salinity, and antagonistic microorganisms. However, no studies have quantified the impact of these factors in full scale WRFs. Utilizing a mass balance approach, we assessed the impact of natural decay and other fate mechanisms on genetic marker removal during water reclamation, through the use of sludge and wastewater genetic marker loading estimates. Results indicated negligible removal of genetic markers during P/PT (primary effluent (PE) p value: 0.267; preliminary and primary treatment (P/PT) accumulation p value: 0.904; and thickened primary sludge (TPS) p value: 0.076) indicating no contribution of natural decay and other fate mechanisms toward removal in P/PT. Comparably, adsorption and decomposition was found to be the dominant pathway for genetic marker removal (thickened waste activated sludge (TWAS) log loading 9.75 log10 GC/day); however, no estimation of log genetic marker accumulation could be carried out due to high detections in TWAS. PRACTITIONER POINTS: The mass balance approach suggested that the contribution of natural decay and other fate mechanisms to virus removal during wastewater treatment are negligible compared with adsorption and decomposition in P/PT (p value: 0.904). During (P/PT), a higher viral load remained in the (PE) (14.16 log10 GC/day) compared with TPS (13.83 log10 GC/day); however, no statistical difference was observed (p value: 0.280) indicting that adsorption/decomposition most probably did not occur. In secondary treatment (ST), viral genetic markers in TWAS were consistently detected (13.41 log10 GC/day) compared with secondary effluent (SE), indicating that longer HRT and the potential presence of extracellular polymeric substance-containing enriched biomass enabled adsorption/decomposition. Estimations of total solids and volatile solids for TPS and TWAS indicated that adsorption affinity was different between solids sampling locations (p value: <0.0001).

Temporal assessment of SARS-CoV-2 detection in wastewater and its epidemiological implications in COVID-19 case dynamics.

This research evaluated the relationship between daily new Coronavirus Disease 2019 (COVID-19) cases and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) concentrations in wastewater, followed by effects of differential SARS-CoV-2 shedding loads across various COVID-19 outbreaks. Linear regression analyses were utilized to examine the lead time of the SARS-CoV-2 signal in wastewater relative to new COVID-19 clinical cases. During the Delta wave, no lead time was evident, highlighting limited predictive capability of wastewater monitoring during this phase. However, significant lead times were observed during the Omicron wave, potentially attributed to testing capacity overload and subsequent case reporting delays or changes in shedding patterns. During the Post-Omicron wave (Febuary 23 to May 19, 2022), no lead time was discernible, whereas following the lifting of the COVID-19 state of emergency (May 30, 2022 to May 30, 2023), the correlation coefficient increased and demonstrated the potential of wastewater surveillance as an early warning system. Subsequently, we explored the virus shedding in wastewater through feces, operationalized as the ratio of SARS-CoV-2 concentrations to daily new COVID-19 cases. This ratio varied significantly across the Delta, Omicron, other variants and post-state-emergency phases, with the Kruskal-Wallis H test confirming a significant difference in medians across these stages (P < 0.0001). Despite its promise, wastewater surveillance of COVID-19 disease prevalence presents several challenges, including virus shedding variability, data interpretation complexity, the impact of environmental factors on viral degradation, and the lack of standardized testing procedures. Overall, our findings offer insights into the correlation between COVID-19 cases and wastewater viral concentrations, potential variation in SARS-CoV-2 shedding in wastewater across different pandemic phases, and underscore the promise and limitations of wastewater surveillance as an early warning system for disease prevalence trends.

Contaminants of emerging concern reduction and microbial community characterization across a three-barrier advanced water treatment system.

This research investigated the removal of contaminants of emerging concern (CECs) and characterized the microbial community across an advanced water treatment (AWT) train consisting of Coagulation/Flocculation/Clarification/Granular Media Filtration (CFCGMF), Ozone-Biological Activated Carbon Filtration (O3/BAC), Granular Activated Carbon filtration, Ultraviolet Disinfection, and Cartridge Filtration (GAC/UV/CF). The AWT train successfully met the goals of CECs and bulk organics removal. The microbial community at each treatment step of the AWT train was characterized using 16S rRNA sequencing on the Illumina MiSeq platform generated from DNA extracted from liquid and solid (treatment media) samples taken along the treatment train. Differences in the microbial community structure were observed. The dominant operational taxonomic units (OTU) decreased along the treatment train, but the treatment steps did impact the microbial community composition downstream of each unit process. These results provide insights into microbial ecology in advanced water treatment systems, which are influenced and shaped by each treatment step, the microbial community interactions, and their potential metabolic contribution to CECs degradation.

Per- and poly-fluoroalkyl substances removal in multi-barrier advanced water purification system for indirect potable reuse.

The study evaluated the removal efficacy of per- and poly-fluoroalkyl substances (PFAS) across various advanced water treatment (AWT) processes in a field-scale AWT train using secondary effluent samples from a full-scale water reclamation facility (WRF). Samples collected from April to October 2020 revealed PFCAs as the dominant PFAS compounds in the WRF secondary effluent, with PFPeA having the highest average concentration and PFSAs in notably lower amounts. Temporal fluctuations in total PFAS concentrations peaked in September 2020, which may reflect the seasonality in PFAS discharges related to applications like AFFFs and pesticides. In assessing AWT processes, coagulation-flocculation-clarification-filtration system showed no notable PFAS reduction, while ozonation resulted in elevated PFBS and PFBA concentrations. Biological activated carbon (BAC) filtration effectively removed long-chain PFAS like PFOS and PFHxS but saw increased concentrations of short-chain PFAS post-treatment. Granular activated carbon (GAC) filtration was the most effective treatment, reducing all PFSAs below the detection limits and significantly decreasing most PFCAs, though short-chain PFCAs persisted. UV treatment did not remove short-chain PFCAs such as PFBA, PFPeA, and PFHxA. The findings highlight the efficacy of AWT processes like GAC in PFAS reduction for potable reuse, but also underscore the challenge presented by short-chain PFAS, emphasizing the need for tailored treatment strategies. PRACTITIONER POINTS: Secondary effluents showed higher concentrations of PFCAs compared to PFSAs. Advanced water treatment effectively removes long-chain PFAS but not short-chain. Ozonation may contribute to formation of short-chain PFAS. BAC is less effective on short-chain PFAS, requiring further GAC treatment.

Enhanced heme protein expression by ammonia-oxidizing communities acclimated to low dissolved oxygen conditions.

This study has investigated the acclimation of ammonia-oxidizing communities (AOC) to low dissolved oxygen (DO) concentrations. Under controlled laboratory conditions, two sequencing batch reactors seeded with activated sludge from the same source were operated at high DO (near saturation) and low DO (0.1 mg O2/L) concentrations for a period of 220 days. The results demonstrated stable and complete nitrification at low DO conditions after an acclimation period of approximately 140 days. Acclimation brought about increased specific oxygen uptake rates and enhanced expression of a particular heme protein in the soluble fraction of the cells in the low DO reactor as compared to the high DO reactor. The induced protein was determined not to be any of the enzymes or electron carriers present in the conventional account of ammonia oxidation in ammonia-oxidizing bacteria (AOB). Further research is required to determine the specific nature of the heme protein detected; a preliminary assessment suggests either a type of hemoglobin protein or a lesser-known component of the energy-transducing pathways of AOB. The effect of DO on AOC dynamics was evaluated using the 16S rRNA gene as the basis for phylogenetic comparisons and organism quantification. Ammonium consumption by ammonia-oxidizing archaea and anaerobic ammonia-oxidizing bacteria was ruled out by fluorescent in situ hybridization in both reactors. Even though Nitrosomonas europaea was the dominant AOB lineage in both high and low DO sequencing batch reactors at the end of operation, this enrichment could not be linked in the low DO reactor to acclimation to oxygen-limited conditions.

Dissolved organic nitrogen removal and its mechanisms during simulated soil aquifer treatment.

Soil aquifer treatment systems are known to further remove contaminants in wastewater effluent when applied through infiltration into the ground. Dissolved organic nitrogen (DON) in the effluent, a precursor for nitrogenous disinfection by-products (DBPs) such as N-nitrosodimethylamine (NDMA), is of great concern upon subsequent use of the groundwater infiltered into the aquifer. In this study, the vadose zone of the soil aquifer treatment system was simulated using 1 m laboratory soil columns under unsaturated conditions representing the vadose zone. The final effluent of a water reclamation facility (WRF) was applied to these columns to investigate the removal of N species with a focus on DON, as well as NDMA precursors. DON removal achieved was up to 99 % with an average of 68 % and was accompanied by a 52 % nitrate increase suggesting the occurrence of ammonification and nitrification through the soil columns. Around 62 % of total DON removal was seen at <10 cm travel distance, which was in accordance with higher adenosine triphosphate (ATP) concentrations at the top of the column due to more oxygen and organic matter availability. Total Dissolved N removal was drastically lowered to 4.5 % in the same column without microbial growth, which highlights the importance of biodegradation. The columns were capable of removing 56 % of the fluorescent dissolved organic matter (FDOM). Soil columns could remove NDMA precursors up to 92 % through the column with the initial concentration of 89.5 ng/L, possibly due to the removal of DON fractions. The results demonstrate the capability of the vadose zone in further treatment of DON and other organic matter before reaching the groundwater through infiltration or indirect discharge to surface water. Differences in applied water quality and the site-specific oxic conditions in SAT systems could lead to variable removal efficiencies.

Wildfire impact on disinfection byproduct precursor loading in mountain streams and rivers.

We investigated short (first post-fire precipitation)- and long-term (11-month) impacts of the Caldor and Mosquito Fires (2021 and 2022) on water quality, dissolved organic matter, and disinfection byproduct (DBP) precursors in burned and adjacent unburned watersheds. Both burned watersheds experienced water quality degradation compared to their paired unburned watersheds, including increases in dissolved organic carbon (DOC), dissolved organic nitrogen (DON), and DBP precursors from precipitation events. DBP precursor concentrations during storm events were greater in the Caldor Fire's burned watershed than in the unburned watershed; precursors of trihalomethanes (THMs), haloacetic acids (HAAs), haloacetonitriles (HANs), and haloacetamides (HAMs) were 533 µg/L, 1,231 µg/L, 64 and 58 µg/L greater. The burned watershed of the Mosquito Fire also had greater median concentrations of THM (44 µg/L), HAA (37 µg/L), HAN (7 µg/L), and HAM (13 µg/L) precursors compared to the unburned watershed during a storm immediately following the fire. Initial flushes from both burned watersheds formed greater concentrations of more toxic DBPs, such as HANs and HAMs. The Caldor Fire burn area experienced a rain-on-snow event shortly after the fire which produced the greatest degradation of water quality of all seasons/precipitation events/watersheds studied. Over the long term, statistical analysis revealed that DOC and DON values in the burned watershed of the Caldor Fire remained higher than the unburned control (0.98 mg C/L and 0.028 mg N/L, respectively). These short and long-term findings indicate that wildfires present potential treatment challenges for public water systems outside of the two studied here.

Understanding the influence of dissolved organic nitrogen characteristics on enhanced coagulation performance for water reuse.

With the recent focus on using advanced water treatment processes for water reuse, interest is growing for utilizing enhanced coagulation to remove dissolved chemical species. Up to 85% of the nitrogen in wastewater effluent is made up of dissolved organic nitrogen (DON), but there is a knowledge gap regarding its removal during coagulation, which can be influenced by DON characteristics. To address this issue, tertiary-treated wastewater samples were analyzed before and after coagulation with polyaluminum chloride and ferric chloride. Samples were size-fractionated into four molecular weight fractions (0.45 µm, 0.1 µm, 10 kDa, and 3 kDa) using vacuum filtration and ultrafiltration. Each fraction was further evaluated by coagulating it separately to assess DON removal during enhanced coagulation. The size fractionated samples were also separated into hydrophilic and hydrophobic fractions using C18 solid phase extraction disks. Fluorescence excitation-emission matrices were used to investigate the characteristics of dissolved organic matter contributing to DON during the coagulation process. The results showed that DON compounds of size <3 kDa constituted a majority of the total DON. Coagulation removed more than 80% DON from size fractions 0.45 µm-0.1 µm and 0.1 µm-10 kDa, but less than 20% was removed from 10 kDa to 3 kDa and <3 kDa fractions. Coagulation on pre-filtered samples removed 19% and 25% of the <3 kDa DON fraction using polyaluminum chloride and ferric chloride, respectively. In all molecular weight fractions, hydrophilic DON compounds were found to be dominant (>90%), and enhanced coagulation was not effective in removing hydrophilic DON compounds. LMW fractions respond poorly to enhanced coagulation due to their hydrophilic nature. Enhanced coagulation effectively removes humic acid-like substances, but poorly removes proteinaceous compounds such as tyrosine and tryptophan. This study's findings provide insights into DON behavior during coagulation and factors affecting its removal, potentially improving wastewater treatment strategies.