Total Extracellular Polymeric Substances Research Articles

Sources for isolation of extracellular polymeric substances (EPSs) producing bacterial strains which are capable of using wastewater sludge as solo substrate

ABSTRACTIsolation of extracellular polymeric substances (EPSs) producing bacterial strains capable of using sludge as low-cost growth substrate was carried out in this study. A total of 110 EPS-producing strains were isolated from different sources, which include sludge of beer and winery wastewater treatment plant (WWTP); young, 2-month-old and 10-year-old leachate. Thirty-seven isolated strains showed good growth in sludge medium with cell count varying from 106 to 1010 most probable number (MPN)/mL and total EPS concentration from 2737 to 6639 mg/L. Twenty-one strains produced EPS with high flocculation activity (FAmax varied from 72.0% to 80.2%). The highest FAmax (80.2%) was observed with EPS produced by strain BES 19, which was isolated from sludge of beer WWTP. Sludge of beer WWTP, young leachate and 10-year-old leachate were good sources for isolation of EPS-producing bacteria.

The investigation of paper mill industry wastewater treatment and activated sludge properties in a submerged membrane bioreactor.

The paper mill industry produces high amounts of wastewater and, for this reason, stringent discharge limits are applied for sustainable reclamation and reuse of paper mill industry wastewater in many countries. Submerged membrane bioreactor (sMBR) systems can create new opportunities to eliminate dissolved substances present in paper mill wastewater including. In this study, a sMBR was operated for the treatment of paper mill industry wastewater at 35 h of hydraulic retention time (HRT) and 40 d of sludge retention time (SRT). The chemical oxygen demand (COD), NH3-N and total phosphorus (TP) removal efficiencies were found to be 98%, 92.99% and 96.36%. The results demonstrated that sMBR was a suitable treatment for the removal of organic matter and nutrients for treating paper mill wastewater except for the problem of calcium accumulation. During the experimental studies, it was noted that the inorganic fraction of the sludge increased as a result of calcium accumulation in the reactor and increased membrane fouling was observed on the membrane surface due to the calcification problem encountered. The properties of the sludge, such as extracellular polymeric substances (EPS) and soluble microbial products (SMP), relative hydrophobicity, zeta potential and floc size distribution were also monitored. According to the obtained results, the total EPS was found to be 43.93 mg/gMLSS and the average total SMP rejection by the membrane was determined as 66.2%.

Operation performance and membrane fouling of a spiral symmetry stream anaerobic membrane bioreactor supplemented with biogas aeration

The performances and membrane fouling of the spiral symmetry stream anaerobic membrane bioreactor (SSS-AnMBR) during stable operation period with and without biogas aeration were studied. The organic loading rate (OLR) and chemical oxygen demand (COD) removal rate of the SSS-AnMBR with aeration biogas condition were 2.28 kgCOD/(m3d) and 98.24%, respectively, higher than those without biogas aeration (2.13 kgCOD/(m3d) and 97.58%, respectively). Biogas aeration could reduce effectively membrane fouling, and the service time of the membrane was twice as much as without biogas aeration. Without biogas aeration, the main causes of membrane fouling were high total solids, volatile suspended solids and total extracellular polymeric substances (EPS) on the membrane surface; with biogas aeration, the main causes of membrane fouling were high slime-EPS, polysaccharide (PS) and small value of PN/PS on the membrane surface.

Effect of free ammonium and free nitrous acid on the activity, aggregate morphology and extracellular polymeric substance distribution of ammonium oxidizing bacteria in partial nitrification

Successful partial nitrification not only guarantees the inhibition of nitrite oxidation, but also does not excessively retard the ammonia oxidation rate. Therefore, the performance of ammonium oxidizing bacteria (AOB) during partial nitrification is fundamental to this process. In this study, two lab-scale partial nitrification bioreactors containing different inhibition conditions-one with free ammonium (FA) inhibition, the other with free nitrous acid (FNA) inhibition-were used to compare the differences between activity, quantity, aggregation morphology and extracellular polymeric substance (EPS) distribution of AOB. The results showed that although stable, long-term, partial nitrification was achieved in both reactors, there were differences in AOB activity, microbial spatial distribution and EPS characteristic. In the FA bioreactor, FA concentration was conducted at more than 40mg/L, which had a strong impact on the metabolism of AOB. The activity and quantity decreased by 50%. Higher EPS (42.44±2.31mgg-1 mixed liquor volatile suspended solids [MLVSS]) and protein were introduced into the EPS matrix. However, in the FNA bioreactor, the FNA concentration was about 0.23mg/L. It did not reach a level to affect AOB metabolism. The AOB activity and quantity were maintained at high levels and the total EPS content was 28.29±2.04mgg-1 MLVSS. Additionally, the microscopic results showed that in the FA bioreactor, AOB cells aggregated in microcolonies, while they appeared to be self-flocculating with no specific conformation in the other reactor. β-polysaccharides located inside sludge flocs in the FA bioreactor but only accumulated around the outer layer of activated sludge flocs in the FNA condition.

The role of extracellular polymeric substances on carbon capture in a high rate activated sludge A-stage system

This paper quantifies the effect of varying solids retention time (SRT), hydraulic retention time (HRT) and dissolved oxygen (DO) concentrations on extracellular polymeric substances (EPS) production and subsequently effluent quality, carbon capture (bioflocculation) and carbon redirection (settling) in a high rate activated sludge A-stage system treating domestic wastewater. Two pilot-scale A-stage reactors were set-up with HRTs of 30 and 60min. Cascade DO control was used to maintain 3 DO set-points of 0.5, 1.0 and 1.5mg/L. A mixed liquor suspended solids (MLSS) concentration of 3000mg/L was maintained and the waste activated sludge (WAS) flow was varied to achieve SRTs of 0.28 and 0.56day. EPS fractions and the protein and polysaccharide concentrations of the mixed liquor were measured. Operation at the 0.56day SRT and 1.0mg/L DO resulted in the highest total suspended solids (TSS), total COD (tCOD), particulate COD (pCOD), and colloidal (cCOD) removal. The best overall performance in terms of bioflocculation (cCOD removal) and carbon capture (percent COD in the WAS) occurred at the 0.56day SRT and coincided with decreasing total EPS concentrations but the settling characteristics of the sludge were better at the 0.28day SRT. Overall, low correlations were found between EPS production and system performance. It is likely that at the high loading rate of the A-stage system, EPS production did not play a major role compared to the influence of operating parameters on effluent quality, carbon capture and redirection.

Rapid formation of biofilm grown on gas-permeable membrane induced by famine incubation

A new approach to rapidly initiate biofilm induced by famine incubation was developed in a membrane-aerated biofilm reactor (MABR). The correlation between extracellular polymeric substances (EPS) component, surface physiochemical properties of biofilm, and biofilm formation rate was evaluated. The results indicated that famine incubation appeared to stimulate bacteria to secrete more bound EPS and resulted in improvement in surface properties of cells in the culture favoring the accumulation of biofilm flocs onto the membrane surface. High contents of bound EPS and bound protein along with the reduced surface charges enhanced hydrophobicity of bacteria. Biofilm formation rate strongly correlated with EPS component and bindability rather than the concentration of total EPS. The study suggested that famine incubation may provide an alternative tool to initiate the rapid formation of biofilm by regulating the component and surface characteristics of biofilm-EPS matrix, which enhanced the efficacy of membrane-aerated biofilm (MAB) for high-rate organic carbonaceous pollutant biodegradation, volatile organic pollutant removal, and nitrification/denitrification processes.

Influence of operational conditions on the stability of aerobic granules from the perspective of quorum sensing

Integrated aerobic granules were first cultivated in two sequencing batch reactors (SBRs) (A1 and A2). Then, A1's influent organic loading rate (OLR) was changed from alternating to constant (cycling time was still 6h), while A2's cycling time varied from 6 to 4h (influent OLR strategy remained alternating). After 30-day operation since the manipulative alternations, granule breakage happened in two reactors at different operational stages, along with the decrease of granule intensity. Granule diameter in A1 declined from the original 0.84 to 0.32cm during the whole operation, while granules in A2 dwindled to 0.31cm on day 22 with similar size to A1. Both the amount of total extracellular polymeric substances (EPSs) and the protein were declining throughout the operation, and the large molecular weight of protein was considered closely related to the stability of aerobic granules. The relative AI-2 level decreased at the same time, and influent OLR strategy might had more evident impact on quorum sensing (QS) ability of sludge compared with starvation period. Combined with microbial results, the decline of total EPS amount in two reactors could be concluded as follows: During the reactor operation, some functional bacteria gradually lost their dominance and were eliminated from the reactors, which finally caused granule disintegration. In summary, the results further confirmed that alternating OLR and proper starvation period were two major factors in effective cultivation and stability of aerobic granules from the perspective of QS.

Evaluating filterability of different types of sludge by statistical analysis: The role of key organic compounds in extracellular polymeric substances

An investigation was conducted for 20 different types of sludge in order to identify the key organic compounds in extracellular polymeric substances (EPS) that are important in assessing variations of sludge filterability. The different types of sludge varied in initial total solids (TS) content, organic composition and pre-treatment methods. For instance, some of the sludges were pre-treated by acid, ultrasonic, thermal, alkaline, or advanced oxidation technique. The Pearson's correlation results showed significant correlations between sludge filterability and zeta potential, pH, dissolved organic carbon, protein and polysaccharide in soluble EPS (SB EPS), loosely bound EPS (LB EPS) and tightly bound EPS (TB EPS). The principal component analysis (PCA) method was used to further explore correlations between variables and similarities among EPS fractions of different types of sludge. Two principal components were extracted: principal component 1 accounted for 59.24% of total EPS variations, while principal component 2 accounted for 25.46% of total EPS variations. Dissolved organic carbon, protein and polysaccharide in LB EPS showed higher eigenvector projection values than the corresponding compounds in SB EPS and TB EPS in principal component 1. Further characterization of fractionized key organic compounds in LB EPS was conducted with size-exclusion chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND). A numerical multiple linear regression model was established to describe relationship between organic compounds in LB EPS and sludge filterability.

Study of extracellular polymeric substances in the biofilms of a suspended biofilter for nitric oxide removal.

The extraction and quantitative analysis of extracellular polymeric substances (EPS) have been frequently reported in studies of activated sludge. However, little is currently known about the EPS in the biofilms of biofilter systems. This study investigates the EPS in biofilms of Chelatococcus daeguensis TAD1 established in a suspended biofilter for nitric oxide (NO) removal under thermophilic conditions. Polysaccharide was the main EPS component under all experimental operation conditions of the aerobic biofilter, although the EPS contents and components varied under different operating conditions. As the concentration of the inlet NO varied from 200 to 2000mg/m3, the EPS and protein contents generally increased. At the highest inlet concentration (2000mg/m3), the EPS and protein contents reached 0.118 and 0.055mg/g, respectively (representing increases of 7.3 and 35%, respectively, over the inlet concentration of 200mg/m3). In contrast, the polysaccharide content was quite stable against inlet NO concentration. Decreasing the empty bed residence time increased the EPS and polysaccharide contents, but exerted little effect on the protein content. Varying the pH of the circulating fluid from 4 to 8 changed the EPS and its components in complex ways. We also found a strong correlation between the total EPS content and the NO removal efficiency. Therefore, it is possible to take EPS into consideration for biofilter control.

Calcium carbonate formation on mica supported extracellular polymeric substance produced by Rhodococcus opacus

Extracellular polymeric substance (EPS) extracted from Rhodococcus opacus bacterial strain was used as a matrix for calcium carbonate precipitation using the vapour diffusion method. The total exopolymer and water-soluble exopolymer fraction of different concentrations were spread on the mica surface by the spin-coating method. The obtained layers were characterized using the atomic force microscopy measurement and XPS analysis. The effects of polymer concentration, initial pH of calcium chloride solution and precipitation time on the obtained crystals properties were investigated. Raman spectroscopy and scanning electron microscopy were used to characterize the precipitated minerals. It was found that the type of precipitated CaCO3 polymorph and the crystal size depend on the kind of EPS fraction. The obtained results indicates that the water soluble fraction favours vaterite dissolution and calcite growth, whereas the total EPS stabilizes vaterite and this effect is stronger at basic pH. It seems to be due to different contents of the functional group of EPS fractions.

Promoting the bio-cathode formation of a constructed wetland-microbial fuel cell by using powder activated carbon modified alum sludge in anode chamber.

MFC centered hybrid technologies have attracted attention during the last few years due to their compatibility and dual advantages of energy recovery and wastewater treatment. In this study, a MFC was integrated into a dewatered alum sludge (DAS)- based vertical upflow constructed wetland (CW). Powder activate carbon (PAC) was used in the anode area in varied percentage with DAS to explore its influences on the performance of the CW-MFC system. The trial has demonstrated that the inclusion of PAC improved the removal efficiencies of COD, TN and RP. More significantly, increasing the proportion of PAC from 2% to 10% can significantly enhance the maximum power densities from 36.58 mW/m2 to 87.79 mW/m2. The induced favorable environment for bio-cathode formation might be the main reason for this improvement since the content of total extracellular polymeric substances (TEPS) of the substrate in the cathode area almost doubled (from 44.59 μg/g wet sludge to 87.70 μg/g wet sludge) as the percentage of PAC increased to 10%. This work provides another potential usage of PAC in CW-MFCs with a higher wastewater treatment efficiency and energy recovery.

Characteristics of extracellular polymeric substances from sludge and biofilm in a simultaneous nitrification and denitrification system under high salinity stress.

The composition and distribution of extracellular polymeric substance (EPS) both from suspended sludge and attached biofilm were investigated in a simultaneous nitrification and denitrification (SND) system with the increase of the salinity from 1.0 to 3.0%. Fourier-transform infrared (FTIR) spectroscopy and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy were used to examine proteins (PN), polysaccharides (PS) and humic substances (HS) present in EPS. High total nitrogen removal (above 83.9%) via SND was obtained in the salinity range of 1.0-2.5%. Total EPS in the sludge increased from 150.2 to 200.6mg/gVSS with the increase of salinity from 1.0 to 3.0%, whereas the corresponding values in the biofilm achieved the maximum of 288.6mg/g VSS at 2.0% salinity. Dominant composition of EPS was detected as HS in both sludge and biofilm, having the percentages of 50.6-68.6 and 41.1-69.9% in total EPS, respectively. Both PN and PS contents in soluble EPS (S-EPS), loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) of sludge and biofilm increased with the increased salinity. The FTIR spectrum and 3D-EEM fluorescence spectroscopy of S-EPS, LB-EPS and TB-EPS in the sludge and biofilm showed the changes of functional groups and conformations of the compositions in EPS with the increase of salinity. The results demonstrated that the characteristics of EPS varied from sludge to biofilm. The obtained results could provide a better understanding of the salinity effect on the EPS characteristics in a SND system.

Temporal changes in extracellular polymeric substances on hydrophobic and hydrophilic membrane surfaces in a submerged membrane bioreactor

Membrane surface hydrophilic modification has always been considered to mitigating biofouling in membrane bioreactors (MBRs). Four hollow-fiber ultrafiltration membranes (pore sizes ∼0.1 μm) differing only in hydrophobic or hydrophilic surface characteristics were operated at a permeate flux of 10 L/m2 h in the same lab-scale MBR fed with synthetic wastewater. In addition, identical membrane modules without permeate production (0 L/m2 h) were operated in the same lab-scale MBR. Membrane modules were autopsied after 1, 10, 20 and 30 days of MBR operation, and total extracellular polymeric substances (EPS) accumulated on the membranes were extracted and characterized in detail using several analytical tools, including conventional colorimetric tests (Lowry and Dubois), liquid chromatography with organic carbon detection (LC-OCD), fluorescence excitation - emission matrices (FEEM), fourier transform infrared (FTIR) and confocal laser scanning microscope (CLSM). The transmembrane pressure (TMP) quickly stabilized with higher values for the hydrophobic membranes than hydrophilic ones. The sulfonated polysulfone (SPSU) membrane had the highest negatively charged membrane surface, accumulated the least amount of foulants and displayed the lowest TMP. The same type of organic foulants developed with time on the four membranes and the composition of biopolymers shifted from protein dominance at early stages of filtration (day 1) towards polysaccharides dominance during later stages of MBR filtration. Nonmetric multidimensional scaling of LC-OCD data showed that biofilm samples clustered according to the sampling event (time) regardless of the membrane surface chemistry (hydrophobic or hydrophilic) or operating mode (with or without permeate flux). These results suggest that EPS composition may not be the dominant parameter for evaluating membrane performance and possibly other parameters such as biofilm thickness, porosity, compactness and structure should be considered in future studies for evaluating the development and impact of biofouling on membrane performance.

Enzyme disintegration with spatial resolution reveals different distributions of sludge extracellular polymer substances.

Background To understand the intrinsic role of hydrolytic enzymes in sludge treatment, particularly their effect on the digestibility and dewaterability of sludge, activated sludge flocs were disintegrated using various techniques that included different enzymes (amylase, cellulase, proteinase, DNase, and polygalacturonase), pH adjustment, and temperature adjustment. The effectiveness of each enzyme treatment was pinpointed by quantifying the spatial distribution of each type of organic matters (protein, polysaccharide, DNA, fluorescent organics) in outer layer extracellular polymeric substances (EPS), inner layer EPS, and cells. ResultsMost hydrolytic enzymes functioned only owing to a temperature or pH effect. The release of organic matter from the interior fraction of EPS to the exterior fraction was prompted under high pH and temperature even without enzyme addition. The effectiveness of enzyme addition was only significant for cellulase and polygalacturonase treatments. Polygalacturonase unexpectedly increased the total EPS polysaccharides up to seven times, accompanied with improved dewaterability, while the amount of EPS proteins was almost unchanged. Combining chemical and morphological evidence, a new conceptual model considering the spatial distribution of polygalacturonic acid-like matter, proteins, cellulose, and other organics in EPS was proposed.ConclusionsPolygalacturonic acid-like matter hydrolysis caused significant release of polysaccharides. Polygalacturonase released polysaccharides while keeping proteins unreleased. Temperature and pH adjustment were as effective as enzyme at sludge disintegration. Cellulose hydrolysis led to massive release of all kinds of organic matters. A new conceptual sludge structure model regarding organic components is proposed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0444-y) contains supplementary material, which is available to authorized users.

Influence of COD:N ratio on sludge properties and their role in membrane fouling of a submerged membrane bioreactor

The effect of COD:N ratio on sludge properties and their role in membrane fouling were examined using a well-controlled aerobic membrane bioreactor receiving a synthetic high strength wastewater containing glucose. Membrane performance was improved with an increase in the COD/N ratio (100:5–100:1.8) (i.e. reduced N dosage). Surface analysis of sludge by X-ray photoelectron spectroscopy (XPS) indicates significant differences in surface concentrations of elements C, O and N that were observed under different COD/N ratios, implying changes in the composition of extracellular polymeric substances (EPS). Fourier transform-infrared spectroscopy (FTIR) revealed a unique characteristic peak (CO bonds) at 1735 cm−1 under nitrogen limitation conditions. Total EPS decreased with an increase in COD/N ratio, corresponding to a decrease in the proteins (PN) to carbohydrates (CH) ratio in EPS. There were no significant differences in the total soluble microbial products (SMPs) but the ratio of PN/CH in SMPs decreased with an increase in COD/N ratios. The results suggest that EPS and SMP composition and the presence of a small quantity of filamentous microorganisms played an important role in controlling membrane fouling.

The role of autoinducer-2 in aerobic granulation using alternating feed loadings strategy

Quorum sensing (QS) plays an important role in aerobic granulation while how QS system regulates the formation of aerobic granules needs further discussion. This study cultivated activated sludge in two identical sequencing batch reactors (R1 and R2) at different influent organic loading rate (OLR) strategies: R1 was operated using constant OLR (around 8.0kg/m3d), while R2 was operated at alternating OLR (4.0–17.0kg/m3d). Microbial aggregates appeared in R2 on day 19, while the morphology of sludge in R1 changed little compared with the initial sludge. The concentration of autoinducer-2 (AI-2) in R2 showed an ascending trend, along with the increase of cell adhesiveness. The total extracellular polymeric substances (EPS) amount and large molecular weight EPS of R2 rose steadily, which was different from R1. Some bacteria able to self-aggregate and promote EPS secretion were exclusive in R2. A mechanism about aerobic granulation at alternating OLR was proposed.

Variations in distribution and composition of extracellular polymeric substances (EPS) of biological sludge under potassium ferrate conditioning: Effects of pH and ferrate dosage

The effect of pH on chemical conditioning with potassium ferrate (K2FeO4) for improving sludge filtration dewatering performance has been studied. The variation of extracellular polymeric substances (EPS) in conditioning process was investigated in detail to unravel the reaction mechanism. The results indicated that sludge dewaterability was improved by decreasing solution pH in terms of filtration rate and cake solids content. At acid conditions, protonation of EPS resulted in reduction of negative charge and densification of sludge floc. Sludge conditioning efficiency was improved with decrease in pH. Ferrate can solubilize EPS through oxidation process and also remove a portion of soluble EPS (SEPS) by charge neutralization and interfacial adsorption of hydrolyzed ferric ions, consequently compressing EPS and decreasing total extractable EPS content. In addition, when pH and potassium ferrate were 3 and 0.1g/gTSS, the sludge filtration dewatering rate and extent reached the maximum. Deterioration of sludge dewaterability was observed as the ferrate was overdosed (>0.2g/gTSS). This could be attributed to the release of a large amount of bound EPS (BEPS) as a result of ferrate oxidation, consequently increasing the sludge filtration resistance.

Nutrient removal, microalgal biomass growth, harvesting and lipid yield in response to centrate wastewater loadings

The effects of wastewater, with four different nutrient loadings, from synthetic centrate on biomass production, nutrient removal, microalgal settling, and lipid production were investigated in photobioreactors under both batch and, subsequently, semi-continuous operations. At higher centrate concentration factors (17.2% and 36.2%), hydraulic retention time and pH adjustments could be employed to sustain acceptable microalgal growth rates and wastewater treatment. Similar nutrient removals efficiencies (>95%) and biomass production (0.42–0.51 g/L) were observed for the four centrate concentrations. Both the lipid productivity and lipid content decreased with increasing nutrient loading in the wastewater. The results also demonstrated that the mass ratio of carbohydrate to protein could provide a good indication of microalgal settling performance, rather than sole component composition or total extracellular polymeric substances. The highest settling efficiency (42.3 ± 0.04% after 24 h) and lowest lipid content (10.2 ± 1.6%) were observed for the lowest mass ratio of carbohydrate to protein (0.74 ± 0.15) noted in the microalgae cultivated in the wastewater with the highest centrate concentration factor (36.2%).

The influences of shear stress on Extracellular Polymeric Substances of activated sludge

Shear stress is one of the key factors in aggregation and detachment of microbial flocs, which will influence the production of Extracellular Polymeric Substances (EPS) as well. In this study, the components and composition of activated sludge EPS, including loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS), were analyzed under different shear stresses. To perform this, sequencing batch reactors were set up separately with various shear stresses (represented by superficial upflow air velocity). The results show that shear stress significantly affects EPS production in the SBRs. When the superficial upflow air velocity increased from 2.3 to 9.1 m/h, the contents of total EPS, TB-EPS, and LB-EPS first increased and then decreased. The highest total EPS (70.94 mg COD/gVSS) and LB-EPS (27.96 mg COD/gVSS) contents were obtained under superficial upflow air velocity of 5.7 m/h, while the maximum TB-EPS (45.03 mg COD/gVSS) content was attained when superficial upflow air velocity rose up to 6.8 m/h. The contents of polysaccharides, proteins and humic substances in EPS showed similar trends with an increase in the shear stress. Compared with the variation of protein and humic substance, the variation of polysaccharide was extremely dramatic in this study, especially in LB-EPS. It was also found the contents of EPS (TB-EPS, LB-EPS and total EPS) have a significant linear correlation with the sludge volume index (SVI). The highest correlation coefficient (R2 = 0.9522) showed the closest correlation between LB-EPS contents and SVI. These findings indicate that LB-EPS and polysaccharides rather than TB-EPS and proteins play important roles for the settling properties of activated sludge under various shear stresses.

Effects of CeO2 nanoparticles on production and physicochemical characteristics of extracellular polymeric substances in biofilms in sequencing batch biofilm reactor

Extracellular polymeric substances (EPS) are a major component of biofilms that act as a gel-like matrix, binding the cells together to form their three-dimensional structure. The effects of ceria nanoparticles (CeO2 NPs) on the production and physicochemical characteristics of EPS in biofilms in a sequencing batch biofilm reactor were investigated. Total EPS production, including loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS), increased by 35.41% compared to in control tests without CeO2 NPs. Protein production increased by 47.02% (LB-EPS) and 58.83% (TB-EPS) after 50mg/L CeO2 NP exposure. Three-dimensional excitation–emission fluorescence spectra revealed that tyrosine (LB-EPS) and aromatic (TB-EPS) protein-like substances formed after CeO2 NP exposure. Fourier transform infrared spectroscopy results indicated the susceptibility of –OH and –NH2 in EPS hydroxyl and amine groups to CeO2 NPs. Exposure to 50mg/L CeO2 NPs reduced the flocculating capacity of LB-EPS (51.78%) and TB-EPS (17.14%), consistent with the decreased zeta potential.