Anammox-based Processes Research Articles

Achieving advanced nitrogen removal with anammox and endogenous partial denitrification driven by efficient hydrolytic fermentation of slowly-biodegradable organic matter

Anammox-based processes are pivotal for elevating nitrogen removal efficiency in municipal wastewater treatment. This study established a novel HF-EPDA system combined in-situ hydrolytic fermentation (HF) with endogenous partial denitrification (EPD) and anammox. Slowly-biodegradable organic matter (SBOM) was degraded and transformed into endogenous polymers for driving production of sufficient nitrite by EPD, further promoted the nitrogen removal via anammox process. Processes above formed positive feedback, guaranteeing the robustness and recoverability of system. After a 92-day suspension during operation, advanced nitrogen removal was still achieved with excellent nitrogen removal efficiency of 95.84 ± 1.73 %, treating with actual domestic wastewater and synthetic nitrate wastewater. Candidatus Brocadia and Candidatus Competibacter were dominant bacteria on biofilms responsible for the anammox and EPD process respectively, while the main hydrolytic fermentation organisms norank_o SBR1031 was enriched in floc sludge. This study highlights the reliable potential for expanding anammox application with simultaneous improvement of SBOM utilization.

Metagenomic insights into microbial metabolic mechanisms of a combined solid-phase denitrification and anammox process for nitrogen removal in mainstream wastewater treatment

To overcome the significant challenges associated with nitrite supply and nitrate residues in mainstream anaerobic ammonium oxidation (anammox)-based processes, this study developed a combined solid-phase denitrification (SPD) and anammox process for low-strength nitrogen removal without the addition of nitrite. The SPD step was performed in a packed-bed reactor containing poly-3-hydroxybutyrate-co-3-hyroxyvelate (PHBV) prior to employing the anammox granular sludge reactor in the continuous-flow mode. The removal efficiency of total inorganic nitrogen reached 95.7 ± 1.2% under a nitrogen loading rate of 0.18 ± 0.01 kg N·m3·d−1, and it required 1.02 mol of nitrate to remove 1 mol of ammonium nitrogen. The PHBV particles not only served as biofilm carriers for the symbiosis of hydrolytic bacteria (HB) and denitrifying bacteria (DB), but also carbon sources that facilitated the coupling of partial denitrification and anammox in the granules. Metagenomic sequencing analysis indicated that Burkholderiales was the most abundant HB genus in SPD. The metabolic correlations between DB (Betaproteobacteria, Rhodocyclaceae, and Anaerolineae) and anammox bacteria (Candidatus Brocadiac and Kuenenia) in the granules were confirmed through microbial co-occurrence networks analysis and functional gene annotations. Additionally, the genes encoding nitrate reductase (Nap) and nitrite reductase (Nir) in DB primarily facilitated nitrate reduction, thereby supplying nitric oxide to anammox bacteria for subsequent nitrogen removal with hydrazine synthase (Hzs) and hydrazine dehydrogenase (Hdh). The findings provide insights into microbial metabolism within combined SPD and anammox processes, thus advancing the development of mainstream anammox-based processes in engineering applications.

Assigning strategic COD flow to enhance denitrification coupled with anaerobic digestion and anammox for systematic upgradation of advanced nitrogen removal from food waste digestate

Anammox is a low-carbon and efficient technology for treating high-strength ammonium in food waste digestate (FWD). However, achieving superior low-nitrogen effluent levels to meet strict discharge standards remains challenging. This study proposes an anammox-based process that integrates simultaneous denitrification and methanogenesis (SDM), partial nitrification (PN), and simultaneous anammox and denitrification (SAD) to realize advanced nitrogen removal from FWD by strategically utilizing influent organic matter. The SAD process achieved an inorganic nitrogen removal efficiency of 94.0 %, with the anammox reaction contributing 96.0 %. Residual nitrate in the SAD effluent was recirculated into SDM, maintaining a removal efficiency of 95 %–97 %. Profiling wastewater and material flows in the integrated process showed a 97.2 % nitrogen removal efficiency, with effluent nitrogen levels meeting discharge standards (<70 mg/L for municipal sewer systems). 3D-fluorescence analysis indicated that quasi-humic acid was effectively removed through SDM. Soluble microbial by-products of FWD served as carbon source for the denitrifying bacteria in SAD. Controlling influent bypass flow and effluent recirculation allowed for strategic allocation of chemical oxygen demand (COD) flow, achieving a total removal efficiency of 71.8 %. SDM removed 45.8 % COD, followed by PN at 13.4 % and SAD at 12.6 %. Compared with those of conventional nitrification and denitrification process, the economic cost, greenhouse gas emission, and total environmental impact of this integrated process were reduced by 85.8 %, 35.8 %, and 84.1 %, respectively. This study provides new insights into the application of anammox-based process for attaining advanced nitrogen removal while minimizing environmental impact.

Iron particle-integrated anammox granules in baffled reactor: Enhanced settling property and nitrogen removal performance

This study evaluates iron particle-integrated anammox granules (IP-IAGs) to enhance wastewater treatment efficiency. The IP-IAGs resulted in notable improvements in settleability and nitrogen removal. The settling velocity of IP-IAGs increased by 17.91 % to 2.92 ± 0.20 cm/s, and the total nitrogen removal efficiency in batch mode improved by 6.82 %. These changes indicate enhanced biological activity for effective treatment. In continuous operation, the IP-IAGs reactor showed no accumulation of nitrite until 40 d, reaching a peak nitrogen removal rate (NRR) of 1.54 kg-N/m3·d and a nitrogen removal efficiency of 82.61 %. Furthermore, a partial nitritation-anammox reactor that treated anaerobic digestion effluent achieved a NRR of 1.41 ± 0.09 kg-N/m3·d, proving the applicability of IP-IAGs in real wastewater conditions. These results underscore the potential of IP-IAGs to enhance the efficiency and stability of anammox-based processes, marking a significant advancement in environmental engineering for wastewater treatment.

Novel anammox granules formation from conventional activated sludge for municipal wastewater treatment through flocs management

The direct integration of anammox process into municipal wastewater treatment has caused widespread concern, but the lack of anammox seeds limited its real application. This study successfully cultivated anammox granules (322.0 μm) from conventional activated sludge treating municipal wastewater. Through ultra-low floc sludge retention times of 8d, nitrifiers on flocs were eliminated and partial nitrification was realized. Furthermore, highly bacteria-enriched granules were initially formed, with Nitrosomonas and Ca. Competibacter 4-fold higher than that of flocs. Specific staining results revealed the microbial interaction with Ca. Brocadia, considering that Ca. Competibacter and Nitrosomonas correspondingly identified in the inner and outer layers of granules. The percentage of Ca. Brocadia present on the granules increased substantially from 0.0 % to 3.0 %, accompanied by a nitrogen removal rate of 0.3 kg·m−3·d−1. Our findings revealed a valuable reference for the anammox bacteria in-situ enrichment under mainstream conditions, which provides theoretical guidance for anammox-based processes practical application.

Kinetic and elemental characterization of HAP-based high-rate partial nitritation/anammox system orienting stability and inorganic elemental requirements

Anammox-based processes are attractive for biological nitrogen removal, and the combination of anammox and hydroxyapatite (HAP) is promising for the simultaneous removal of nitrogen and phosphorus from wastewater. However, the kinetics of one-stage partial nitritation/anammox (PNA) in which ammonia-oxidizing bacteria (AOB) and anammox bacteria (AnAOB) exist in a reactor are poorly understood. Moreover, inorganic elements are required to promote microbial cell synthesis and growth; therefore, monitoring of elements to prevent the limitation and inhibition of the process is critical. The minimum amounts of inorganic elements required for a one-stage PNA process and the elemental flow remain unknown. Therefore, in this study, kinetics, stoichiometry, and element flow in the long-term, high-rate, continuous, one-stage HAP-PNA process with microaerobic granular sludge at 25 °C were determined using process modeling, parameter estimation, and mass balance. The biomass elemental composition was determined to be CH2.2O0.89N0.18S0.0091, and the biomass yield (Yobs) was calculated to be 0.0805 g/g NH4+-N. Therefore, a stoichiometric reaction equation for the one-stage HAP-PNA system was also proposed. The maximum specific growth rate (μm) of AnAOB and AOB were 0.0360 and 0.0982 d−1 with doubling times of 19 and 7.1 d, respectively. Finally, the elemental requirements for stable and high-rate performance were determined using element flow analysis. These findings are essential for developing the anammox-based process in a stable and resource-efficient manner and determining engineering applicability.

Advancing the Comprehension of Iron Ions with Different Valences in Anammox-Based Processes: Insight into the Response and Mechanism Based on Prediction and Classification Machine Learning Models

Advancing the Comprehension of Iron Ions with Different Valences in Anammox-Based Processes: Insight into the Response and Mechanism Based on Prediction and Classification Machine Learning Models

Special Issue on “Anammox-Based Processes for Wastewater Treatment”

Anaerobic ammonium oxidation (anammox, A) has become an appealing bioprocess for the water sector as a method to remove nitrogen (N) from wastewater using low-energy and organic carbon inputs [...]

Succession of microbial communities reveals the inevitability of anammox core in the development of anammox processes

The lack of anammox seeds is regarded as the bottleneck of anammox-based processes. Although the interactions in anammox consortia have attracted increasing attention, little is known about the influence of inoculated sludge populations on the growth of anammox bacteria. In this study, four sludge of distinct communities mixed with anammox sludge (the relative abundance of Ca. Kuenenia was 1.96 %) were used as the seeds, respectively for the start-up of anammox processes. Notably, all these mixed microbial communities tend to form a similar microbial community, defined as the anammox core, containing anammox-bacteria (22.9 ± 5.9 %), ammonia-oxidizing-bacteria (0.8 ± 0.7 %), nitrite-oxidizing-bacteria (0.2 ± 0.2 %), Chloroflexi-bacteria (0.7 ± 0.4 %), and heterotrophic-denitrification-bacteria (0.3 ± 0.2 %). It also elucidated that the communities of Nitrosomonas-dominated sludge were the closest to the anammox core, and achieved the highest nitrogen-removal rate of 0.73 kg-N m−3 d−1. This study sheds light on the solution to the shortage of anammox seeds in the full-scale wastewater treatment application.

Role of quorum sensing-based regulation in development of anaerobic ammonium oxidation process

Shortage of anaerobic ammonium oxidation (anammox) sludge greatly limits the extensive full-scale application of anammox-based processes. Although numerous start-up strategies have been proposed, the interaction among microbial consortia and corresponding mechanism during the process development remain unknown. In this study, three reactors were established based on different seed sludges. After 27 days, the anammox process inoculated with anammox granules and activated sludge (1:5) was firstly achieved, and the highest nitrogen removal rate was 1.17 kg N m−3 d−1. Correspondingly, the anammox activity and abundances of related functional genes increased. Notably, the dominant anammox bacteria shifted from Candidatus Kuenenia to Candidatus Brocadia. Metagenomic analysis indicated that quorum sensing-based regulation mainly contributed to the proliferation and accumulation of anammox bacteria. This work provides an insight into the quorum sensing (QS)-regulated microbial interactions in the anammox and activated sludge consortia during the process development.

Microbial dynamics reveal the adaptation strategies of ecological niche in distinct anammox consortia under mainstream conditions

The feasibility of anammox-based processes for nitrogen-contained wastewater treatment has been verified with different anammox bacteria, however, the ecological niche of anammox bacteria under mainstream conditions is still elusive. In this study, six sludge samples collected from different habitats were utilized to culture anammox bacteria under mainstream conditions, and two distinct anammox genera (Ca. Kuenenia and Ca. Brocadia) with a relative abundance of 6.31% (C1) and 3.09% (C3), respectively, were identified. Notably, the microbial dynamics revealed that anammox bacteria (AMX), ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), Chloroflexi bacteria (CFX), and heterotrophic denitrification bacteria (HDB) were the core members in anammox consortia. However, Ca. Kuenenia and Ca. Brocadia occupied different ecological niches in anammox consortia. The dissolved oxygen and microbial structures of the anammox-continuous stirred tank reactor systems were the main factors to affect their niche differentiation. Meanwhile, comammox might exist in the systems and occupy the ecological niche of AOB in nitrogen cycling. The network analysis suggested that Ignavibacterium could be the associated bacteria in Ca. Kuenenia-dominated consortia, while Ca. Nitrotoga was that in the Ca. Brocadia-dominated consortia. Our findings reveal a valuable reference for the observation of distinct anammox genera under mainstream conditions, which provides theoretical guidance for the engineering application of mainstream anammox-based processes.

A quantified nitrogen metabolic network by reaction kinetics and mathematical model in a single-stage microaerobic system treating low COD/TN wastewater

A single-stage intermittent aeration microaerobic reactor (IAMR) has been developed for the cost-effective nitrogen removal from piggery wastewater with a low ratio of chemical oxygen demand (COD) to total nitrogen (TN). In this study, a quantified nitrogen metabolic network was constructed based on the metagenomics, reaction kinetics and mathematical model to provide a revealing insight into the nitrogen removal mechanism in the IAMR. Metagenomics revealed that a complex nitrogen metabolic network, including aerobic ammonia and nitrite oxidation, anammox, denitrification via nitrate and nitrite, and nitrate respiration, existed in the IAMR. A novel method for solving kinetic parameters with high stability was developed based on a genetic algorithm. Use this method to calculate the kinetics of various reactions involved in nitrogen metabolism. Kinetics revealed that simultaneous partial nitritation-anammox (PN/A) and partial denitrification-anammox (PDN/A) were the dominant approaches to nitrogen removal in the IAMR. Finally, a kinetics-based model was proposed for quantitatively describing the nitrogen metabolic network under the limitation of COD. 58% ∼ 67% of nitrogen was removed via the anammox-based processes (PN/A and PDN/A), but only 7% ∼ 12% and 1% ∼ 2% of nitrogen were removed via heterotrophic denitrification of nitrite and nitrate, respectively. The half-inhibition constant of dissolved oxygen (DO) on anammox was simulated as 0.37 ∼ 0.60 mg L−1, filling the gap in quantifying DO inhibition on anammox. High-frequency intermittent aeration was identified as the crucial measure to suppress nitrite-oxidizing bacteria, although it has a high affinity for DO and NO2−-N. In continuous aeration mode, the simulated NO3−-N in the IAMR would rise by 39.6%. The research provides a novel insight into the nitrogen removal mechanism in single-stage microaerobic systems and provides a reliable approach to practicing PN/A and PDN/A for cost-effective nitrogen removal.

Effects of antimicrobials in anammox mediated systems: critical review

Anammox-mediated systems are thought to be cost-effective and efficient technologies for removing nitrogen from wastewater by converting nitrite and ammonium into dinitrogen gas. However, there are inhibitory substances that reduce the effectiveness and efficiency of these processes, preventing their widespread application. Antimicrobial agents are among these substances that have been observed to inhibit anammox-mediated processes. Therefore, this review provides a comprehensive overview of the effects of various antimicrobials on the anammox-based systems with emphasis on the effects in different reactor configurations, sludge types and microbial population of anammox-based systems. In addition, this review also discusses the mechanisms by which nitrifying bacteria are inhibited by the antimicrobials. Gaps in knowledge based on this review as well as future research needs have also been suggested. This review gives a better knowledge of antimicrobial effects on anammox-based systems and provides some guidance on the type of system to use to treat antimicrobial-containing wastewater using anammox-based processes.

Application of intermittent aeration in nitrogen removal process: development, advantages and mechanisms

• Applications of intermittent aeration in N removal are critically reviewed. • Strategies and mechanisms of enhanced N removal efficiency are presented. • Variations in N transformation and microbial interactions are discussed. • Advantages of intermittent aeration in the integrated anammox process are outlined. • Directions for future work in N removal with intermittent aeration are suggested. Intermittent aeration has been shown to be one of the most effective and cost-effective strategies for biological wastewater treatment. This review presents an overview of the application and advantages of intermittent aeration, with the mechanisms of improvement in process stability discussed. Intermittent aeration was applied to maximize the utilization of organic carbon for denitrification rather than oxidized by O 2 . Under that condition, advanced nitrogen removal and sludge settleability were achieved and the energy consumption and N 2 O emissions were reduced. With the discovery that nitrite oxidation bacteria are selectively inhibited while ammonium oxidation bacteria are less affected under intermittent aeration conditions, intermittent aeration has been applied to the partial nitrification process. Furthermore, the non-aerated phases of intermittent aeration have been found to facilitate the synergism of anaerobic ammonium oxidizing bacteria (AnAOB) and denitrifying bacteria, providing a suitable environment for AnAOB growth. Therefore, intermittent aeration is considered to be an effective operational strategy for anammox-based processes, such as single-stage partial nitrification-anammox. In addition, simultaneous nutrient removal and sludge reduction could also be achieved. In order to regulate the synergism of microbial species, dissolved oxygen (DO) concentrations and the durations of aerated and non-aerated periods can be adjusted based on the monitored parameters such as nitrogen concentrations, pH and oxidation reduction potential profiles. Finally, the current limitations, gaps in knowledge and the areas requiring further research are proposed.

Biochar-mediated DNRA pathway of anammox bacteria under varying COD/N ratios

Coupling dissimilatory nitrate reduction to ammonium (DNRA) pathway with anammox process has a prominent advantage in enhancement of nitrogen removal. However, the anammox bacteria driven-DNRA is difficult to proceed at normal autotrophic circumstance. Herein, for the first time, biochar (prepared by bamboo) was used as a mediator to stimulate the DNRA pathway of anammox bacteria under varying chemical oxygen demand (COD) to nitrogen (COD/N) ratios (0.1–0.7), and the underlying stimulation mechanism was elucidated by metagenomics sequencing analysis. Results showed that biochar addition (10 g/L) stimulated DNRA pathway of anammox bacteria at low COD/N ratios (0.1–0.5), thus enhancing the nitrogen removal efficiency (NRE) of the anammox system by 7.2%–16.4% and 0.9%–3.0%, respectively, compared to that of tests without sodium acetate and biochar (p<0.05). This enhancement was attributed to the improved extracellular electron accepting capacity of anammox biomass by biochar. The easily obtained electrons (from sodium acetate) further increased the relative abundances of anammox-related (hzs) and complete DNRA-related (napAB and nrfAH) genes (p<0.05), which catalyze electron-consuming reactions. The stimulated anammox pathway and DNRA pathway further increased the specific anammox activity and the relative abundance of anammox bacteria (especially Ca. Jettenia) by 15.5%–23.0% and 11.3%–82.6% compared with that without biochar, respectively. Metagenomics sequencing also revealed that anammox bacteria, Ca. Jettenia caeni, was the main bacteria for DNRA metabolism in this system. Our findings reveal that biochar could selectively stimulate DNRA pathway of anammox bacteria affiliated by a low amount of carbon, which provides a novel strategy to improve the nitrogen removal of anammox-based processes.

An Innovative Carbon and Nitrogen Control Strategy Based on Post-Anoxic with Partial Feeding (Pa-Pf) for Advanced Treatment of Mainstream Wastewater Using Multiple Anammox-Based Processes

An Innovative Carbon and Nitrogen Control Strategy Based on Post-Anoxic with Partial Feeding (Pa-Pf) for Advanced Treatment of Mainstream Wastewater Using Multiple Anammox-Based Processes

Recent advancements in the biological treatment of high strength ammonia wastewater

The estimated global population growth of 81 million people per year, combined with increased rates of urbanization and associated industrial processes, result in volumes of high strength ammonia wastewater that cannot be treated in a cost-effective or sustainable manner using the floc-based conventional activated sludge approach of nitrification and denitrification. Biofilm and aerobic granular sludge technologies have shown promise to significantly improve the performance of biological nitrogen removal systems treating high strength wastewater. This is partly due to enhanced biomass retention and their ability to sustain diverse microbial populations with juxtaposing growth requirements. Recent research has also demonstrated the value of hybrid systems with heterogeneous bioaggregates to mitigate biofilm and granule instability during long-term operation. In the context of high strength ammonia wastewater treatment, conventional nitrification-denitrification is hampered by high energy costs and greenhouse gas emissions. Anammox-based processes such as partial nitritation-anammox and partial denitrification-anammox represent more cost-effective and sustainable methods of removing reactive nitrogen from wastewater. There is also growing interest in the use of photosynthetic bacteria for ammonia recovery from high strength waste streams, such that nitrogen can be captured and concentrated in its reactive form and recycled into high value products. The purpose of this review is to explore recent advancements and emerging approaches related to high strength ammonia wastewater treatment.

Anammox-Based Processes for Mature Leachate Treatment in SBR: A Modelling Study

Mature landfill leachates are characterized by high levels of ammoniacal nitrogen which must be reduced for discharge in the sewer system and further treatment in municipal wastewater treatment plants. The use of anammox-based processes can allow for an efficient treatment of ammonium-rich leachates. In this work, two real scale sequencing batch reactors (SBRs), designed to initially perform partial nitritation/anammox (PN/A) and simultaneous partial nitrification and denitrification (SPND) for the treatment of ammonium-rich urban landfill leachate, were modelled using BioWin 6.0 in order to enable plant-wide modelling and optimizing. The constructed models were calibrated and validated using data from long- and short-term (one cycle) SBR operation and fit well to the main physical-chemical parameters (i.e., ammonium, nitrite and nitrate concentrations) measured during short-term (one cycle) operations. Despite the different strategies in terms of dissolved oxygen (DO) concentrations and aeration and mixing patterns applied for SBR operation, the models allowed for understanding that in both reactors the PN/A process was shown as the main contributor to nitrogen removal when the availability of organic carbon was low. Indeed, in both SBRs, the activity of nitrite oxidizing bacteria was inhibited due to high levels of free ammonia, whereas anammox bacteria were active due to the simultaneous presence of ammonium and nitrite and their ability to recover from DO inhibition. Increasing the external carbon addition, a prompt decrease of the anammox biomass was observed, with SPND becoming the main nitrogen removal mechanism. Models were also applied to estimate the production rates of nitrous oxide by aerobic ammonia oxidizing bacteria and heterotrophic denitrifiers. The models were found to be a robust tool for understanding the effects of different operating conditions (i.e, temperature, cycle phases, DO concentration, external carbon addition) on the nitrogen removal performances of the two reactors, assessing the contribution of the different bacterial groups involved.

Exploring the role of heterotrophs in partial nitritation-anammox process treating thermal hydrolysis process - anaerobic digestion reject water

Heterotrophic bacteria (HB) are generally prevalent in anammox-based processes, but their functional and ecological roles in partial nitritation-anammox (PN/A) process treating high-organics wastewater remained unclear. This study aimed to elucidate HB activities and microbial interactions in a one-stage PN/A treating thermal hydrolysis process (THP) - anaerobic digestion (AD) reject water. The PN/A reactor achieved a satisfactory nitrogen removal rate of 0.58 ± 0.06 g N/(L·d), and around 12% of COD in the THP-AD reject water was removed. N2O emission factors of the PN/A reactor were 1.15% ± 0.18% treating synthetic wastewater, and 0.95% ± 0.06% treating reject water. A balanced symbiotic relationship was maintained between HB and functional groups (i.e., anammox bacteria and aerobic-ammonia-oxidizing bacteria) over the reactor operation. The relative abundances of Anaerolineae spp. clearly increased, while Denitratisoma, capable of denitrification, slightly decreased when treating THP-AD reject water. The preference for electron donors of heterotrophs explained discrepant growth trends.

Removal of nitrogen components, bulk organics, and fluorophores during one-stage partial nitrification-Anammox treatment of landfill leachate

Anammox-based processes have been intensively studied for the nitrogen removal from leachate, but less attention was paid to monitoring the evolution of leachate organics. In this study, fluorescence Excitation-Emission Matrix (EEM) measurement coupling with Parallel Factor Analysis (PARAFAC) analysis was used to supplement the routine monitoring in order to reveal more insights into pollutants removal during the leachate treatment by a one-stage partial nitrification-Anammox process (PNA). During this PNA process, up to 96% of total nitrogen and 43% of COD were removed, indicating the potential of PNA process to simultaneously remove nitrogen and organics. Fluorescence intensities and ratios clearly showed the dynamics of influent fluorophores during seasonal variations, where a high amount of protein-like compound was observed during summer months. Protein-like compound was preferentially removed (43–63%) in the PNA process, whereas humic/fulvic-like compounds exhibited recalcitrance to biodegradation. The increase of oxygen supply promoted protein-like compound degradation, which could be associated with the aerobic oxidation pathway. Furthermore, the protein-like compound removal was linearly correlated with the reduction of NH4+-N at limited oxygen conditions (i.e., air flow rate ≤1.6 Lgas/h/Lreactor) (r = 0.97, n = 65). The fluorescence directly extracted at Ex/Em:230 nm/345 nm was strongly correlated with the biodegradable chemical oxidation demand (BCOD) (r = 0.92, n = 204), showing its high potential as an indicator for biodegradable organics. Given the relatively weak correlation between fluorescence parameters and COD, EEM coupled with multivariate partial least squares (PLS) modeling was proposed and exhibited good predictive power for COD, as exemplified by a mean error of 4.5%.