From laboratory to pilot-scale: Assessing dissolved organic matter, biological stability and per- and polyfluoroalkyl substances removal on managed aquifer recharge performance.

Managed aquifer recharge (MAR) is a promising technique for enhancing groundwater resources and addressing water scarcity. Particularly, this research highlights the novelty and urgent need for MAR facilities in the Chungcheongnam-do region of South Korea as a solution to augment groundwater resources and combat water scarcity. This research encompasses a comprehensive assessment, ranging from laboratory-scale column experiments to pilot-scale tests, focusing on dissolved organic matter (DOM) characterization, natural organic matter (NOM) removal, and water quality improvement, including biological stability. In the laboratory, DOM characteristics of source water and recharged groundwater were analyzed using advanced dissolved organic characteristic tools, and their potential impacts on water quality, as well as per- and polyfluoroalkyl substances (PFASs) were assessed. DOM, total cell counts, and several PFASs with molecular weights >450 Da (particularly long-chain PFASs showing >99.9 % reduction) were effectively reduced in a laboratory-scale experiment. A laboratory-scale column study revealed that most selected PFASs were not effectively removed. Moving to the pilot-scale, a series of experiments were conducted to assess NOM removal during soil passage. Similar to the results of the laboratory-scale experiment, MAR demonstrated significant potential for reducing NOM concentrations, thus improving water quality. Regarding biological stability, assimilable organic carbon in production well (i.e., final produced water by MAR process) was lower than both two sources of surface water (e.g., SW1 and SW2). This suggests that water derived from PW (i.e., production well) exhibited biological stability, undergoing effective biodegradation by aerobic bacteria during soil passage. The findings from this study highlight the critical importance of implementing MAR techniques in regions facing water scarcity, emphasizing its potential to significantly enhance future water security initiatives.

Environmental consequences of transformation products from an antibiotic mixture and their mitigation in a wastewater microbiome using an HCl-modified adsorbent.

This study enhanced our understanding of antibiotic mixtures' occurrence, transformation, toxicity, and ecological risks. The role of acid-modified biochar (BC) in treating antibiotic residues was explored, shedding light on how BC influences the fate, mobility, and environmental impact of antibiotics and transformation products (TPs) in an activated sludge (AS) microbiome. A mixture of oxytetracycline and sulfamethoxazole was found to synergistically (or additively) inhibit cell growth of AS and disrupt the microbiome structure, species richness/diversity, and function. The formation of TPs with potentially higher toxicity and persistence than the original compounds was identified, explaining the microbiome disruption. Agricultural waste-derived BC was optimized for contaminant adsorption, leading to a reduction in toxicity when added to AS by sequestering TPs on its surface. This work highlighted adsorbents as a practical engineering strategy for mitigating liquid-phase contaminants' toxicological consequences, proactively controlling the fate and effects of antibiotics and TPs.

Integrating biological ion exchange with biological activated carbon treatment for drinking water: A novel approach for NOM removal, trihalomethane formation potential, and biological stability.

Ion exchange resins (IEX) are used in drinking water utilities to remove natural organic matter (NOM) from surface water; however, the disposal of used brine can be a major drawback. Recently, biological ion exchange (BIEX) has been proposed as an alternative to biological activated carbon (BAC) for removing natural organic matter (NOM). The present study is, to the best of our knowledge, the first attempt to use a hybrid BIEX and BAC (BIEX+BAC) system for drinking water treatment. The removal of NOM, assimilable organic carbon, and trihalomethane formation potential was investigated by operating four columns comprising IEX, BIEX, BAC, and BIEX+BAC with 18,000 bed volumes. The BIEX+BAC system was the most effective at removing dissolved organic carbon (59.9%). Based on fluorescence excitation-emission matrix spectroscopy, the BIEX+BAC column showed the maximum removal rates in all peak regions of T1, T2, and A. Using liquid chromatography-organic carbon detection, resin-containing columns were found to effectively remove humic substances, which are the principal precursors of trihalomethanes. The lowest potential for trihalomethane formation was observed in BIEX+BAC. BIEX+BAC also had the highest assimilable organic carbon removal efficiency (61.2%) followed by BIEX (52.3%), BAC (49.5%), and IEX (47.1%). The BIEX+BAC hybrid was found to be the most effective method for removing NOM fractions and reducing the formation of disinfection byproducts.

Predicting heterotrophic plate count exceedance in tap water: A binary classification model supervised by culture-independent data.

Culture-independent data can be utilized to identify heterotrophic plate count (HPC) exceedances in drinking water. Although HPC represents less than 1% of the bacterial community and exhibits time lags of several days, HPC data are widely used to assess the microbiological quality of drinking water and are incorporated into drinking water standards. The present study confirmed the nonlinear relationships between HPC, intact cell count (ICC), and adenosine triphosphate (ATP) in tap water samples (stagnant and flushed). By using a combination of ICC, ATP, and free chlorine data as inputs, we show that HPC exceedance can be classified using a 2-layer feed-forward artificial neural network (ANN). Despite the nonlinearity of HPC, the best binary classification model showed accuracies of 95%, sensitivity of 91%, and specificity of 96%. ICC and chlorine concentrations were the most important features for classifiers. The main limitations, such as sample size and class imbalance, were also discussed. The present model provides the ability to convert data from emerging measurement techniques into established and well-understood measures, overcoming culture dependence and offering near real-time data to help ensure the biostability and safety of drinking water.

Biomass formation and organic carbon migration potential of microplastics from a PET recycling plant: Implication of biostability.

The growth of the polyethylene terephthalate (PET) mechanical recycling industry has resulted in the challenge of generating microplastics (MPs). However, little attention has been given to investigating the release of organic carbon from these MPs and their roles in promoting bacterial growth in aquatic environments. In this study, a comprehensive method is proposed to access the potential of organic carbon migration and biomass formation of MPs generated from a PET recycling plant, and to understand its impact on the biological systems of freshwater habitats. Various MPs sizes from a PET recycling plant were selected to conduct a series of tests, including the organic carbon migration test, biomass formation potential test, and microbial community analysis. The MPs smaller than 100 µm, which are difficult to remove from the wastewater, exhibited greater biomass in the observed samples (1.05 × 1011 bacteria per gram MPs). Moreover, PET MPs altered the microbial diversity, with Burkholderiaceae becoming the most abundant, while Rhodobacteraceae was eliminated after being incubated with MPs. This study partly revealed that organic matter adsorbed on the surface of MPs was a significant nutrient source that increased biomass formation. PET MPs acted not only as carriers for microorganisms but also for organic matter. As a result, it is crucial to develop and refine recycling methods in order to decrease the production of PET MPs and minimize their adverse effects on the environment.

Effects of biochar addition on the fate of ciprofloxacin and its associated antibiotic tolerance in an activated sludge microbiome.

This study investigated the effects of adding biochar (BC) on the fate of ciprofloxacin (CIP) and its related antibiotic tolerance (AT) in activated sludge. Three activated sludge reactors were established with different types of BC, derived from apple, pear, and mulberry tree, respectively, and one reactor with no BC. All reactors were exposed to an environmentally relevant level of CIP that acted as a definitive selective pressure significantly promoting AT to four representative antibiotics (CIP, ampicillin, tetracycline, and polymyxin B) by up to two orders of magnitude. While CIP removal was negligible in the reactor without BC, the BC-dosed reactors effectively removed CIP (70-95% removals) through primarily adsorption by BC and biodegradation/biosorption by biomass. The AT in the BC-added reactors was suppressed by 10-99%, compared to that without BC. The BC addition played a key role in sequestering CIP, thereby decreasing the selective pressure that enabled the proactive prevention of AT increase. 16S rRNA gene sequencing analysis showed that the BC addition alleviated the CIP-mediated toxicity to community diversity and organisms related to phosphorous removal. Machine learning modeling with random forest and support vector models using AS microbiome data collectively pinpointed Achromobacter selected by CIP and strongly associated with the AT increase in activated sludge. The identification of Achromobacter as an important AT bacteria revealed by the machine learning modeling with multiple models was also validated with a linear Pearson's correlation analysis. Overall, our study highlighted Achromobacter as a potential useful sentinel for monitoring AT occurring in the environment and suggested BC as a promising additive in wastewater treatment to improve micropollutant removal, mitigate potential AT propagation, and maintain community diversity against toxic antibiotic loadings.

A shift from chemical oxygen demand to total organic carbon for stringent industrial wastewater regulations: Utilization of organic matter characteristics.

From 2022, industrial wastewater discharge regulations in South Korea will replace chemical oxygen demand (CODMn) with total organic carbon (TOC). A shift from CODMn to TOC is a pioneering change in protecting water bodies from organic contaminants. However, several industries are struggling to meet these TOC requirements even though their effluents met the CODMn limits. Effluent CODMn/TOC ratios (1.28 ± 0.64) found in our study were lower than the CODMn/TOC coefficients (1.33-1.80) suggested by the Ministry of Environment in South Korea. Aliphatic and particulate organic matter contents in effluents likely influenced the CODMn/TOC ratio. Regardless of the industrial category, dissolved organic carbon often consists of low molecular weight neutrals, hydrophobic organic carbon, and protein-like substances in raw and treated industrial wastewaters. The present study also revealed that TOC and CODMn represented different organic matter fractions in the paper mill and oil refinery wastewater, whereas the industrial park wastewater showed similar dissolved organic matter characteristics. Specifically, CODMn was effective in the determination of humic content in paper mill wastewater but was underestimated in oil refinery wastewater. Additionally, only paper mill effluents exceeded the TOC requirements (4 of 6 samples) and required an additional post-treatment process owing to higher organic loads.

Lab experiments on hybridization of managed aquifer recharge with river water via sand column, pre-oxidation, and nanofiltration.

A hybridization of managed aquifer recharge (MAR) with pre-oxidation processes was conducted in this study to investigate changes in dissolved organic matter characteristics and the attenuation of selected trace organic contaminants (TrOCs). Potassium permanganate, chlorine, and ozone treatments were used for pre-oxidation, which effectively attenuated some TrOCs, particularly the combination of MAR with ozone achieved 84-99% attenuation. The pre-oxidation step using potassium permanganate showed high removal of carbamazepine (96%). Moreover, MAR was also combined with nanofiltration (NF) as a multi-barrier concept for the removal of persistent TrOCs after MAR. A short-chain polyfluoroalkyl substance (PFAS) was effectively removed after combining MAR columns with NF membranes. Thus, pre-oxidation coupled with MAR followed by NF could potentially enhance the removal of selected TrOCs.

Intermittent Water Supply Impacts on Distribution System Biofilms and Water Quality.

Intermittent water supplies (IWS) are routinely experienced by drinking water distribution systems around the world, either due to ongoing operational practices or due to one off interruptions. During IWS events changing conditions may impact the endemic biofilms leading to hydraulic mobilisation of organic and inorganic materials attached to pipes walls with a resulting degradation in water quality. To study the impact of IWS on the microbiological and physico-chemical characteristics of drinking water, an experimental full-scale chlorinated pipe facility was operated over 60 days under realistic hydraulic conditions to allow for biofilm growth and to investigate flow resumption behaviour post-IWS events of 6, 48 and 144 hours. Turbidity and metal concentrations showed significant responses to flow restarting, indicating biofilm changes, with events greater than 6 hours generating more turbidity responses and hence discolouration risk. The increase in pressure when the system was restarted showed a substantial increase in total cell counts, while the subsequent increases in flow led to elevated turbidity and metals concentrations. SUVA254 monitoring indicated that shorter times of non-water supply increased the risk of aromatic organic compounds and hence risk of disinfection-by-products formation. DNA sequencing indicated that increasing IWS times resulted in increased relative abundance of potential pathogenic microorganisms, such as Mycobacterium, Sphingomonas, and the fungi Penicillium and Cladosporium. Overall findings indicate that shorter IWS result in a higher proportion of aromatic organic compounds, which can potentially react with chlorine and increase risk of disinfection-by-products formation. However, by minimising IWS times, biofilm-associated impacts can be reduced, yet these are complex ecosystems and much remains to be understood about how microbial interactions can be managed to best ensure continued water safe supply.

Assessment of organic carbon migration and biofilm formation potential on polymeric tubes in contact with water.

Biofilm formation has been frequently identified as a pathway of nosocomial infection in polymeric tubes used for patients of all ages. Biofilm formation on tube surfaces can lead to hygienic failure and cause diarrhea, stomach pain, inflammation, and digestive system disease. This study investigated the influence of polymeric tube materials in contact with water on the biomass formation potential and migration potential of microbially available carbon from plasticizers using a BioMig test. The thermoplastic elastomer tube, which is reusable, leached a relatively low amount of assimilable organic carbon to water. In contrast, the assimilable organic carbon migration potential of polyurethane was the most significant, 6-fold greater than that of the thermoplastic elastomer. Moreover, the same materials (e.g., silicone) produced via different manufacturing processes showed significant differences in migration behaviors. The potential biomass formation observed in polyurethane was approximately 7 × 109 cells cm-2 for both Aeromonas hydrophila and Escherichia coli strains. This study highlights the importance of choosing the correct material characteristics of polymeric tubes in contact with water to protect them from bacterial contamination. Therefore, manufacturers can use the BioMig test to evaluate and produce more hygienic and biostable tubes.

Chloride-Mediated Enhancement in Heat-Induced Activation of Peroxymonosulfate: New Reaction Pathways for Oxidizing Radical Production.

This study is the first to demonstrate the capability of Cl- to markedly accelerate organic oxidation using thermally activated peroxymonosulfate (PMS) under acidic conditions. The treatment efficiency gain allowed heat-activated PMS to surpass heat-activated peroxydisulfate (PDS). During thermal PMS activation at excess Cl-, accelerated oxidation of 4-chlorophenol (susceptible to oxidation by hypochlorous acid (HOCl)) was observed along with significant degradation of benzoic acid and ClO3- occurrence, which involved oxidants with low substrate specificity. This indicated that heat facilitated HOCl formation via nucleophilic Cl- addition to PMS and enabled free chlorine conversion into less selective oxidizing radicals. HOCl acted as a key intermediate in the major oxidant transition based on temperature-dependent variation in HOCl concentration profiles, kinetically retarded organic oxidation upon NH4+ addition, and enabled rapid organic oxidation in heated PMS/HOCl mixtures. Chlorine atom that formed via the one-electron oxidation of Cl- by the sulfate radical served as the primary oxidant and was involved in hydroxyl radical production. This was corroborated by the quenching effects of alcohols and bicarbonates, reactivity toward multiple organics, and electron paramagnetic resonance spectral features. PMS outperformed PDS in degrading benzoic acid during thermal activation operated in reverse osmosis concentrate, which was in conflict with the well-established superiority of heat-activated PDS.

Effects of powdered activated carbon and calcium on trihalomethane toxicity of zebrafish embryos and larvae in hybrid membrane bioreactors.

This study investigated the effect of powdered activated carbon and calcium on trihalomethane toxicity in zebrafish embryos and larvae in hybrid membrane bioreactors. Two hybrid membrane bioreactors were configured with the addition of powdered activated carbon or calcium to reduce the trihalomethane formation potential. Trihalomethane formation decreased by approximately 37.2% and 30.3% in membrane bioreactor-powdered activated carbon and membrane bioreactor-calcium, respectively. Additionally, the toxic effect of trihalomethane formation was examined on zebrafish embryos and larvae. About 35% of the embryos exposed to trihalomethanes (800 ppb) showed signs of deformation, with the majority displaying coagulation within 24 h after exposure. Color preference tests, which were conducted to identify any abnormal activities of the embryos, showed an increase in preference from short to longer wavelengths upon exposure to high levels of trihalomethanes. This may indicate damage to the optical organs in zebrafish when exposed to trihalomethanes. Behavioral analysis showed reduced mobility of zebrafish larvae under different trihalomethane concentrations, indicating a decrease in the average activity time with an increasing trihalomethane concentration. The membrane bioreactor effluents were toxic to zebrafish embryos and larvae in the presence of high trihalomethane concentrations. To understand the mechanism behind trihalomethane toxicity, further studies are needed.

Feasibility of membrane distillation process for potable water reuse: A barrier for dissolved organic matters and pharmaceuticals.

In this study, the feasibility of the membrane distillation (MD) process as a wastewater reclamation system for portable reuse was investigated. The flux was stably maintained at about 20 L/m2h (LMH) at ΔT 30 °C, compared to higher flux at ΔT 50 °C, which showed a rapid decrease in the flux due to severe fouling. MD produced excellent quality of potable water satisfied the drinking water standards of Korea from effluent of sewage treatment plant (ESTP). The fractions of the hydrophobic OC (HOC) and chromatographic DOC (CDOC) from LC-OCD analysis was firstly suggested to understand different organic transport during the MD process. The transport of organic matters across the MD membrane mitigated at low operation temperature and the transported organics in all the tested waters were mostly volatile low molecular weight organics, aromatic amino acids. All of thirteen selected pharmaceuticals were completely removed by MD, regardless of their properties. In order to retard the membrane fouling of the MD process, coagulation and filtration pre-treatments were applied. The pre-treatment process coupled MD process could successfully remove impurities including NH4-N without severe membrane fouling. Moreover, coagulation pretreatment reduced transport of ammonia due to decrease in pH.

Effects of phosphate and hydrogen peroxide on the performance of a biological activated carbon filter for enhanced biofiltration.

Biofilm formation on biofilters can influence their hydraulic performance, thereby leading to head loss and an increase in energy use and costs for water utilities. The effects of a range of factors, including hydrogen peroxide and phosphate, on the performance of biological activated carbon (BAC) and biofilm formation were investigated using laboratory-scale columns. Head loss, total carbohydrates, and proteins were reduced in the nutrient-enhanced, oxidant-enhanced, and nutrient + oxidant-enhanced BAC filters. However, there were no changes in the removal of dissolved organic matter, trihalomethane formation potential, or selected trace organic contaminants. The biofilm formation on polyvinyl chloride and stainless steel coupons using the laboratory biofilm reactor system was lower when the effluent from a nutrient-enhanced column was used, which indicated that there was less biofilm formation in the distribution systems. This may have been because the effluent from the nutrient-enhanced column was more biologically stable. Therefore, enhanced biofiltration could be used not only to reduce head loss in biofilters, but also to delay biofilm formation in distribution systems.

Evaluation of organic migration and biomass formation on polymeric components in a point-of-use water dispenser.

To minimize the aesthetic and hygienic concerns regarding tap water (e.g., odor, taste, suspended solids, and microorganisms), point-of-use (POU) water dispensers and filters are used in households worldwide. However, the POU water dispenser itself can adversely impact water quality. This study investigated the bacterial growth through a POU water dispenser fed with chlorinated tap water; specifically, the heterotrophic plate count increased from 0.01 to 20.01 × 103 of colony-forming units per ml. The BioMig test, which evaluates the biostability of polymeric materials based on the migration potential and the biofilm formation potential, was firstly applied for the water dispenser system. Organic migration and biofilm formation varied by the polymer type used in the water dispenser components (e.g., tubing, fittings, and reservoir). Assimilable organic carbon migration in cold water (23 ±â€¯2 °C) was better correlated with the biofilm formation potential (R = 0.93) than that of warm water (60 ±â€¯2 °C) migration (R = 0.62). The most problematic test material was silicone based on assimilable organic carbon migration and biofilm formation, whereas approved materials such as polyethylene and polyvinyl chloride were relatively stable. Polymeric component examination of an actual POU water dispenser revealed highly accumulated biofilms on the silicone tube used in the device (118 × 103 CFU cm-2). The use of polymers with high biofilm formation should be minimized in water dispensers, whereas approved polymeric components contribute to biological stability in the dispensed drinking water.

Comparison of pre-oxidation between O3 and O3/H2O2 for subsequent managed aquifer recharge using laboratory-scale columns.

A hybrid process of managed aquifer recharge with pre-oxidation was investigated as part of a multiple-barrier approach for safe water production. This study evaluated O3 and O3/H2O2 for the pre-oxidation of urban surface water prior to managed aquifer recharge (MAR) and compared their effectiveness with respect to trace organic contaminants (TrOCs), biostability, and trihalomethane formation potential. The combination of pre-oxidation and MAR was performed using long-term column studies, and the results confirmed the removal of 64 and 56% dissolved organic carbon by using O3 and O3/H2O2, respectively. MAR combined with O3 and O3/H2O2 achieved >50% removal of dissolved organic carbon with the first 5 days of residence time. O3 alone showed better performance in alleviating trihalomethane formation potential during chlorination compared to using O3/H2O2. The pre-oxidation of urban surface water was effective in attenuating selected TrOCs (35 - >99% removal), and subsequent MAR achieved >99% removal of selected TrOCs within the first 5 days, regardless of pretreatment methods examined in this study. The results of this study provide an understanding of the effects of O3 and O3/H2O2 as pre-oxidation processes on urban surface water prior to MAR, as well as the resulting impact on MAR.

Reducing bacterial aerosol emissions from membrane bioreactors: The impact of SRT and the addition of PAC and calcium.

Bacterial aerosols resulting from membrane bioreactor (MBR) processes, which require excessive aeration in a confined space, are important to investigate because of their possible adverse effects on human health. This study investigated the influence of solid retention time (SRT) on bacterial aerosols from MBRs. Moreover, powdered activated carbon (PAC) and calcium were used to attenuate bacterial aerosol emissions from MBRs. The particulate matter (PM) emitted from the MBRs was reduced by 30.5 and 25.2% at SRTs of 20 and 80 d, respectively, compared to the level emitted at an SRT of 10 d. Total cell counts were similarly reduced at SRTs of 20 and 80 d. Longer SRTs also led to greater reductions in the particle size distribution of the sludge within 10 µm. Several factors in the MBR influenced the behavior of the bacterial aerosol emissions from the MBRs. This study showed that changes in viscosity and particle size induced by the SRT influenced the bacterial aerosol emissions in MBRs. Therefore, SRT was identified as an important design parameter affecting bacterial aerosol emissions in MBR processes. The amounts of particulate matter and bacterial aerosols were reduced in MBRs using PAC and calcium, both of which exerted an immediate effect on the bacterial aerosol emissions in MBRs by increasing the aerosol-particle size.

A proof of concept study for wastewater reuse using bioelectrochemical processes combined with complementary post-treatment technologies.

This article describes a proof-of-concept study designed for the reuse of wastewater using microbial electrochemical cells (MECs) combined with complementary post-treatment technologies. This study mainly focused on how the integrated approach works effectively for wastewater reuse. In this study, microalgae and ultraviolet C (UVC) light were used for advanced wastewater treatment to achieve site-specific treatment goals such as agricultural reuse and aquifer recharge. The bio-electrosynthesis of H2O2 in MECs was carried out based on a novel concept to integrate with UVC, especially for roust removal of trace organic compounds (TOrCs) resistant to biodegradation, and the algal treatment was configured for nutrient removal from MEC effluent. UVC irradiation has also proven to be an effective disinfectant for bacteria, protozoa, and viruses in water. The average energy consumption rate for MECs fed acetate-based synthetic wastewater was 0.28±0.01 kWh per kg of H2O2, which was significantly more efficient than are conventional electrochemical processes. MECs achieved 89±2% removal of carbonaceous organic matter (measured as chemical oxygen demand) in the wastewater (anolyte) and concurrent production of H2O2 up to 222±11 mg L-1 in the tapwater (catholyte). The nutrients (N and P) remaining after MECs were successfully removed by subsequent phycoremediation with microalgae when aerated (5% CO2, v/v) in the light. This complied with discharge permits that limit N to 20 mg L-1 and P to 0.5 mg L-1 in the effluent. H2O2 produced on site was used to mediate photolytic oxidation with UVC light for degradation of recalcitrant TOrCs in the algal-treated wastewater. Carbamazepine was used as a model compound and was almost completely removed with an added 10 mg L-1 of H2O2 at a UVC dose of 1000 mJ cm-2. These results should not be generalized, but critically discussed, because of the limitations of using synthetic wastewater.

Effects of hydraulic loading rate and organic load on the performance of a pilot-scale hybrid VF-HF constructed wetland in treating secondary effluent.

This study evaluated the performance of a pilot-scale hybrid constructed wetland system for secondary effluent and investigated bulk organic matter characteristics. The hybrid constructed wetland consisted of a vertical-flow (VF) bed followed by a horizontal-flow (HF) bed. We also investigated the effects of hydraulic loading rates and influent organic load on the performance of the pilot-scale VF-HF hybrid constructed wetland. The results showed a high removal efficiency for suspended solids (>95%) and organic matter as determined by total organic carbon (>98.5%) and dissolved organic carbon (>70%), but no significant change in nitrogen removal was observed. The wetland treatment efficiency for suspended solids and organic matter showed a good buffer capacity even when hydraulic loading rates increased from 750 to 1500 L m-2 d-1 and 500-1000 L m-2 d-1 during the VF and HF stages, respectively. Moreover, there was no significant change in the performance when influent organic load increased eight-fold. Fluorescence excitation-emission matrix and liquid chromatography-organic carbon detection (LC-OCD) were used to investigate the dissolved organic matter characteristics in the hybrid VF-HF constructed wetland. Fluorescence excitation-emission matrix spectroscopy showed that both protein- and humic-like substances did not significantly change in the effluent when hydraulic loading rates and organic load increased by two- and eight-fold, respectively. Biopolymers determined using LC-OCD were effectively removed via the VF and HF stage wetlands, indicating the occurrence of biodegradation. Fluorescence excitation-emission matrix spectroscopy and LC-OCD provided the fate of dissolved organic matter characteristics in the hybrid VF-HF constructed wetland.