Extracellular Polymeric Substances Layer Research Articles

Synchronous in-situ sludge reduction and enhanced denitrification through improving electron transfer during endogenous metabolisms with Fe(Ⅱ) addition

The creation of large amounts of excess sludge and residual nitrogen are critical issues in wastewater biotreatment. This study introduced Fe(II) into an oligotrophic anaerobic reactor (OARFe) that was implemented to modify an anoxic-oxic process to motivate in-situ sludge reduction and enhance denitrification under an effective electron shuttle among organic matter, nitrogen, and Fe. The addition of 15 mg L−1 Fe(II) resulted in a sludge reduction efficiency reached 32.0% with a decreased effluent nitrate concentration of 33.3%. This was mostly attributed to the electron transfer from Fe(II) to organic matters and nitrogen species in OARFe. The participation of Fe(II) led to the upregulation of Geothrix and Terrimonas, which caused active organic matter hydrolysis and cell lysis to stimulate the release of extracellular polymeric substances (EPS) and substance transfer between each layer of EPS. The higher utilization of released bioavailable dissolved organic matter improved endogenous denitrification, which can be combined with iron autotrophic denitrification to realize multiple electron donor-based nitrogen removal pathways, resulting in an increased nitrate removal rate of 58.2% in the absence of external carbon sources. These functional bacteria associated with the transformation of nitrogen and carbon and cycling between ferrous and ferric ions were enriched in OARFe, which contributed to efficient electron transport occurred both inside and outside the cell and increased 2,3,5-triphenyltetrazolium chloride electronic transport system activity by 46.9%. This contributed to the potential operational costs of chemical addition and sludge disposal of Fe-AO being 1.9 times lower than those of conventional A2O processes. These results imply that the addition of ferrous ions to an oligotrophic anaerobic zone for wastewater treatment has the potential for low-cost pollution control.

Pivotal role of intracellular oxidation by HOCl in simultaneously removing antibiotic resistance genes and enhancing dewaterability during conditioning of sewage sludge using Fe2+/Ca(ClO)2

Pre-acidification has been shown to be crucial in attenuating antibiotic resistance genes (ARGs) during the conditioning of sewage sludge. However, it is of great significance to develop alternative conditioning approaches that can effectively eliminate sludge-borne ARGs without relying on pre-acidification. This is due to the high investment costs and operational complexities associated with sludge pre-acidification. In this study, the effects of Fe2+/Ca(ClO)2 conditioning treatment on the enhancement of sludge dewaterability and the removal of ARGs were compared with other conditioning technologies. The dose effect and the associated mechanisms were also investigated. The findings revealed that Fe2+/Ca(ClO)2 conditioning treatment had the highest potential, even surpassing Fenton treatment with pre-acidification, in terms of eliminating the total ARGs. Moreover, the effectiveness of the treatment was found to be dose-dependent. This study also identified that the •OH radical reacted with extracellular polymeric substance (EPS) and extracellular ARGs, and the HOCl, the production of which was positively correlated with the dose of Fe2+/Ca(ClO)2, could infiltrate the EPS layer and diffuse into the cell of sludge flocs, inducing the oxidation of intracellular ARGs. Furthermore, this study observed a significant decrease in the predicted hosts of ARGs and MGEs in sludge conditioned with Fe2+/Ca(ClO)2, accompanied by a significant downregulation of metabolic pathways associated with ARG propagation, thereby contributing to the attenuation of sludge-borne ARGs. Based on these findings, it can be concluded that Fe2+/Ca(ClO)2 conditioning treatment holds great potential for the removal of sludge-borne ARGs while also enhancing sludge dewaterability, which mainly relies on the intracellular oxidation by HOCl.

Migration and morphological transformation patterns of heavy metals on sludge cells and extracellular polymeric substances (EPS) under the influence of different treatments.

The impediment of sludge resource utilization stems from the presence of heavy metals within the sludge matrix. To optimize heavy metal removal techniques from undried sludge, it is essential to study the distribution of heavy metals in the sludge flocs structure and the changes in morphology in the sludge cells after different treatments. In this study, the sludge was subjected to chemical treatments using citric acid (CA), EDTA, and saponin, as well as electrokinetic treatment at 2 V/cm. The distribution and migration of Cu, Ni, and Zn in sludge flocs after various treatment methods were analyzed. The heavy metals were found to migrate from intracellular to extracellular polymeric substances (EPS) without causing extensive sludge cell lysis. They gradually diffused outward with the dispersion of the EPS layer. The migration efficiency of the three heavy metals in the sludge flocs was Zn, Ni, and Cu. This was mainly related to the initial distribution and morphology of the heavy metals. Under the influence of chemicals and an electric field, the acid-soluble and reducible heavy metals in the cells partially migrated to the EPS, while the stable heavy metals transformed into an unstable state. Furthermore, the order of chemical reagents in terms of their effect on the migration efficiency of heavy metals was CA > EDTA > Saponin, owing to the varying binding strengths of heavy metals and their impact on the degree of loosening of the EPS. Especially after CA treatment a greater proportion of Cu, Ni, and Zn were transferred from the cells to the EPS. The acidification effect near the anode during electrokinetic treatment intensifies the migration of heavy metals. This study provides basic research for subsequent engineering optimization aimed at removing heavy metals from sludge.

Enhancement of sewer sediment control and disruption of adhesive gelatinous sediment structure using low-dose calcium peroxide

Large quantities of sediments in urban sewer systems pose significant risk of pipe clogging and corrosion. Owing to their gel-like structure, sewer sediments have strong resistance to hydraulic shear stress. This study proposed a novel approach to weaken the erosion resistance of sewer sediments by destroying viscous gel-like biopolymers in sediments with low doses of calcium peroxide (CaO2). After treatment with 10–50 mg g−1 TS of CaO2, the critical erosion shear stress was significantly reduced by 25.7%–59.9%. The sediment aggregates gradually disintegrated into small diameter particles with increasing CaO2 dosage. Further analysis showed that the strong oxidizing and alkaline environment induced by CaO2 treatment led to cell lysis and changes in the composition and property of extracellular polymeric substances (EPS). After CaO2 treatment, aromatic proteins and humic acid-like substances associated with adhesion translocated from the inner EPS layers to outer layers while being disintegrated into small organic molecules. Concomitantly, CaO2 treatment disrupted the main functional groups (–OH, COO−, C–N, CO, and CN) in inner EPS layers, thus weakening EPS adhesion. Analysis of protein secondary structure and zeta potential reflected the reduced aggregation capacity of sediment microorganisms and loosening of sediment structure after CaO2 treatment. Thus, CaO2 treatment facilitated fragmentation and disaggregation of the gelatinous structure of sewer sediments. Such green strategy decreased the cost of sewer sediment disposal by 42.10–68.95% when compared to water flushing, and it would improve the self-cleaning capacity of sewer system and efficiency of dredging equipment.

Nitrogenous organics in sewage sludge: New insights into bottlenecks of anaerobic transformation

As the most crucial carrier of carbon and nitrogen in sludge, nitrogenous organics, are essential in governing the efficiency of sludge anaerobic digestion (AD). An in-depth understanding of the anti-degradation mechanism of nitrogenous organics in sludge at various occurrence states is needed. This study classified nitrogenous organics in sludge into extracellular nitrogenous organics (e-NO) and intracellular nitrogenous organics (i-NO). Further, it identified the hindrances of anaerobic degradation of e-NO and i-NO in sludge by comparing the structural and AD performance differences between sludge and other nitrogen source substrates (food waste, straw, chicken manure, soybean, and beef). Experimentally, soybean and beef (with excellent e-NO degradation rates) and chicken manure (with excellent i-ON degradation efficiency) were further compared. We found that the relative abundance of peptidase genes in sludge was generally lower than in soybean and beef. Also, the pyruvate (a volatile fatty acid precursor) metabolism of some amino acids (Gly, Ala, Phe) with high content was inactive. Refractory hydrolysis and incomplete fermentation of nitrogenous organics in sludge are the main reasons for the low e-ON degradation rate. Further experiments showed that the anaerobic degradation rate of i-NO in the sludge was lower than that of e-NO. However, the former was significantly enhanced by extracellular polymeric substances (EPS) removal. Thus, the distribution and structure of EPS in the sludge were compared with that found in the chicken manure. We observed that the proportion of tightly bound EPS (TB-EPS) in sludge was higher, and the refractory glycoconjugates were mainly distributed in TB-EPS. Meanwhile, organic-bound Fe was higher in the sludge EPS. These results indicate that the difficulties of anaerobic degradation of i-NO arise from a tight EPS layer protecting the cell body. Future studies may consider combining enhanced hydrolysis methods (i.e., additional enzymes) and cell-breaking pretreatment techniques to facilitate the degradation of nitrogenous organics in sludge. In addition, refractory amino acids can also be studied to produce functionalized organic matter (such as humic substances) for high-value applications.

Insight into the roles of soluble, loosely bound and tightly bound extracellular polymeric substances produced by Enterobacter sp. in the Cd2+ and Pb2+ biosorption process: Characterization and mechanism

Extracellular polymeric substances (EPSs) are macromolecular polymers formed by metabolic secretion, and they have great potential for removing heavy metal (HM) ions from the aquatic phase. In this study, the contributions of soluble EPSs (S-EPSs), loosely bound EPSs (LB-EPSs) and tightly bound EPSs (TB-EPSs) secreted by Enterobacter sp. to Cd2+ and Pb2+ adsorption were analyzed. The results indicated that in a solution containing both Cd2+ and Pb2+, pH= 6.0 was best suited for the adsorption process, and adsorption equilibrium was reached in approximately 120 min. Moreover, the mechanism for adsorption of Cd2+ and Pb2+ by the different layers of EPSs involved spontaneous chemical processes. However, Cd2+ adsorption by the three layers of the EPSs was an exothermic process (∆H0 <0), but Pb2+ adsorption by the three layers of the EPSs was an endothermic process (∆H0 >0). The variations in zeta potentials indicated that ion exchange occurred during Cd2+ and Pb2+ adsorption. FT-IR, XPS and 3D-EEM analyses indicated that the functional groups of the EPSs involved in adsorption were mainly the CO, C-O and C-O-C groups of the polysaccharides; furthermore, fulvic acid-like substances, humic-like substances and tyrosine-like proteins played important roles in the adsorption of Cd2+ and Pb2+ by the different EPS layers.

Multispecies biofilms on reverse osmosis membrane dictate the function and characteristics of the bacterial communities rather than their structure

The main reason for the deterioration of membrane operation during water purification processes is biofouling, which has therefore been extensively studied. Biofouling was shown to reduce membrane performance reflected by permeate flux decline, reduced selectivity, membrane biodegradation, and consequently, an increase in energy consumption. Studies of biofouling focused on the identification of the assembled microbial communities, the excretion of extracellular polymeric substances (EPS), and their combined role in reduced membrane performance and lifetime. However, the link between the structure and function of biofouling communities has not been elucidated to date. Here, we provide a novel insight, suggesting that bacterial functions rather than composition control biofouling traits on reverse osmosis (RO) membranes. We studied the potential activity of RO biofilms at metatranscriptome resolution, accompanied by the morphology and function of the biofouling layer over time, including microscopy and EPS composition, adhesion, and viscoelastic properties. To that end, we cultivated natural multispecies biofilms in RO membranes under treated wastewater flow and extracted RNA to study their taxonomies and gene expression profiles. Concomitantly, the biofilm structure was visualized using both scanning electron microscopy and laser scanning confocal microscopy. We also used quartz crystal microbalance with dissipation to characterize the affinity of EPS to membrane-mimetic sensors and evaluated the viscoelasticity of the Ex-Situ EPS layer formed on the sensor. Our results showed that different active bacterial taxa across five taxonomic classes were assembled on the RO membrane, while the composition shifted between 48 and 96 h. However, regardless of the composition, the maturation of the biofilm resulted in the expression of similar gene families tightly associated with the temporal kinetics of the EPS composition, adhesion, and viscoelasticity. Our findings highlight the temporal selection of specific microbial functions rather than composition, featuring the adhesion kinetics and viscoelastic properties of the RO biofilm.

Promoting vivianite recovery: Crucial role of tightly-bound extracellular polymeric substances

Vivianite is an important bio-induced product of dissimilated iron reduction (DIR), which exists widely in activated sludge and digested sludge of wastewater treatment plants (WWTPs). Extracellular polymeric substances (EPS) surrounding dissimilatory iron-reducing bacteria (DIRB) were reported to be transient media for microbial extracellular electron transfer (EET) and DIR. However, what happens when a layer of EPS envelops microbial cells during vivianite recovery? This study tried to assess the relationship between iron reduction and electron transfer by stripping EPS layer-by-layer, then clarified the delamination of different EPS layers on the performance of vivianite recovery. After stripping total EPS (PC-EPS batches) of DIRB, the Fe (III) reduction efficiencies (RFe), Fe (III) reduction rate (VFe), and vivianite formation efficiency (RV) decreased by 33%, 37%, and 30%, respectively. Tightly bound-EPS (TB-EPS) was confirmed to play a crucial role in vivianite formation, which contributed 28–51% to vivianite recovery and 34–55% to iron reduction. Electronic shuttle substances were contained in TB-EPS, which enhanced the EET rate and efficiency between cells and Fe (III). The polysaccharide content of TB-EPS was positively related to adhesiveness between cells and mineral, as well as the iron reduction efficiency. Furthermore, redox proteins (PpcA, OmcB) were embedded in EPS, which shortened the distance of electron hopping to across the EPS layer and further accelerated vivianite recovery from wastewater. Long-term operation of anaerobic reactors confirmed that TB-EPS played critical roles in bio-induced vivianite formation.

Spectroscopic fingerprints profiling the polysaccharide/protein/humic architecture of stratified extracellular polymeric substances (EPS) in activated sludge

Extracellular polymeric substances (EPS), with a stratified structure including tightly-bound EPS (TB-EPS), loosely-bound EPS (LB-EPS), and soluble EPS (S-EPS) surrounding the microbial cells, are known to vitally affect the physicochemical and biological functions of activated sludge in wastewater treatment. Polysaccharides (PS), proteins (PN), and humic acids (HA) are key components of EPS but their roles in constructing the multi-layer architecture are still unclear. This study explored the EPS characteristics in relation to the components using spectroscopic fingerprinting techniques. Ultraviolet-visible (UV-vis) spectra demonstrated stark difference between TB-EPS and other EPS. Fluorescence excitation-emission matrix (FEEM) and apparent quantum yield revealed further detailed differences. Fluorescence quotient analysis highlighted the dominance of TB-EPS, LB-EPS, and S-EPS in the excitation/emission wavelength (Ex/Em) region of Em = 350–400 nm, Em > 400 nm, and low-Stokes shift band (Em − Ex < 25 nm), respectively. Wavelength-wise prediction of the FEEM intensity was achieved through multiple linear regression against the chemical composition and variance partitioning analysis witnessed binary interactions of PS×HA and PS×PN in S-EPS, PN×HA and PS×PN in LB-EPS, and ternary interaction of PS×PN×HA in TB-EPS as well as the wavelength-specific fluorescence responses of these interactions. Further, X-ray photoelectron spectroscopy, infrared spectra, and circular dichroism spectra corroborated the differences in primary, secondary, and tertiary structures across the EPS layers. Ultrahigh-performance liquid chromatography-mass spectrometry detected molecular fragments confirming the multi-component hybridization among PS, PN, and HA. This study demonstrates a spectroscopic approach to sensitively fingerprint the fine structure of EPS, which has the potential for rapid monitoring of EPS and related sludge properties in wastewater treatment systems.

Malodorous volatile organic compounds (MVOCs) formation after dewatering of wastewater sludge: Correlation with the extracellular polymeric substances (EPS) and microbial communities

Malodorous volatile organic compounds (MVOCs) are often the key odorants in determining sludge odor character and odor impact. However, the emission characterization and generation mechanisms of MVOCs from various dewatered sludge have not been sufficiently understood partly due to the diverse and complex composition and low concentration of odorants. In this study, waste activated sludge (WAS) was collected to examine the variation of MVOC emission from sludge after different dewatering treatment in lab-scale trials. The MVOCs were measured using the electronic nose (eNose), headspace gas chromatography-coupled ion mobility spectrometry (HS-GC-IMS), and headspace solid-phase microextraction gas chromatography–mass spectrometry (HS-SPME-GC–MS). The results showed that centrifugation treatment promoted the generation of various odorous volatiles. The identified key odorants included dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), geosmin, and p-cresol according to their odor activity values (OAVs). The effects of the enhanced dewatering on volatile production were greater than thickening, however, the key odorants of dewatered sludge using gravity thickening varied more greatly than sludges from centrifuge thickening. The distribution of extracellular polymeric substances (EPS) and variation of microbial community showed correlations with the production of key odorants. Tryptophan-like substances in the inner layer of EPS (LB-EPS and TB-EPS) were better correlated with the key odorants. The bound EPS released by centrifugation may play the role of precursor for odorous microbial volatiles. According to the predicted functions of differential microbial genera, Desulfobulbus (Desulfobacterota), Gordonia (Actinobacteriota), and Hyphomicrobium (Proteobacteria) were associated with the production of DMS, DMDS, and DMTS, while Gordonia and Hyphomicrobium were related to p-cresol production.

Insights into the resistance of different extracellular polymeric substance (EPS) layers to the fermentation environment in sludge anaerobic digestion

• EPS layer delamination had great promotion for the methane production. • The semi-rigid structure of sludge flocs was damaged after EPS layer delamination. • EPS layer delamination reduced metal contents and the apparent activation energy. Extracellular polymeric substance (EPS) is key in maintaining microbial cell stability and agglomeration in sludge, which may play as a protective layer for cells. This study tried to assess the digestive performances of sludge after delamination of different EPS layers, aiming to understand the resistance of different EPS layers to the external environment. Results indicated that, after EPS layer delamination, the cumulative methane yield in anaerobic digestion (AD) was improved from 148.8 to 199.9 mL/g COD. The total methane yield for the EPS layers and EPS extracted sludge increased by 32.2% compared to the raw sludge. Moreover, with the delamination of EPS layers, the apparent activation energy (AAE) of the sludge samples decreased, and the interactions between metals and organic substances were destroyed, resulting in lower energy barrier in organic solubilization. Additionally, the treatment changed the distribution of organic substances in sludge, and the residual organic substances presented higher theoretical methane production (TMP). Therefore, both the hydrolytic efficiency and the conversion efficiency of organic substances to methane in AD were improved. By understanding the resistance effects of different EPS layers to the fermentation environment, this study will help us develop more targeted methods in sludge pretreatment for improving its digestive performance.

Unraveling pH Effects on Ultrafiltration Membrane Fouling by Extracellular Polymeric Substances: Adsorption and Conformation Analyzed with Localized Surface Plasmon Resonance.

Extracellular polymeric substances (EPSs) can conform and orient on the surface according to the applied aquatic conditions. While pH elevation usually removes EPSs from membranes, small changes in pH can change the adsorbed EPS conformation and orientation, resulting in a decrease in membrane permeability. Accordingly, EPS layers were tested with localized surface plasmon resonance (LSPR) sensing and quartz crystal microbalance with dissipation monitoring (QCM-D) using a hybrid sensor. A novel membrane-mimetic hybrid QCM-D-LSPR sensor was designed to indicate both "dry" mass and mechanical load ("wet" mass) of the adsorbed EPS. The effect of pH on the EPS layer's viscoelastic properties and hydrated thickness analyzed by QCM-D corroborates with the shift in EPS areal concentration, ΓS, and the associated EPS conformation, analyzed by LSPR. As pH elevates, the processes of (i) elevation in EPS layer's thickness (QCM-D) and (ii) decrease in the EPS areal density, ΓS (LSPR), provide a clear indication for changes in EPS conformation, which decrease the effective ultrafiltration (UF) membrane pore diameter. This decrease in the pore diameter together with the increase in surface hydrophobicity elevates UF membrane hydraulic resistance.

Impact of magnesium ions on lysozyme-triggered disintegration and solubilization of waste activated sludge

Lysozyme can efficiently accelerate solubilization and hydrolysis of waste activated sludge (WAS) for anerobic digestion. However, the effect of lysozyme was easily to be inhibited by metal ions in WAS. The impact of magnesium ions (Mg2+) on lysozyme catalyze WAS disintegration was investigated in this study. The effect of lysozyme on WAS hydrolysis could be hindered by Mg2+. Relatively high concentrations (>50 mg/L) of Mg2+ in sludge significantly reduced the release of soluble polysaccharides and proteins from WAS, while sulfate ions or chloride ions caused no such effect. Proteins were difficult to be extracted from extracellular polymeric substances (EPS) of WAS in the presence of Mg2+ (>10 mg/L) due to the divalent cation bridging (DCB) behavior, while the extraction of polysaccharides was not significantly affected. The polysaccharides and proteins in the inner EPS layer were transferred to the outer layer during the lysozyme treatment, and total quantities of both components maintained constantly. At least 23.1% lysozymes were trapped in the liquid phase of 100 mg Mg2+/L in the first hour. Mg2+ could significantly affect the transfer of lysozyme from liquid phase to the inner layer of sludge. Mg2+ neutralized the negative surface charge of the sludge particles, which hindered the absorption of positively charged lysozyme molecules by sludge flocs from the liquid phase. The proteins of TB-EPS had higher ratios of α-helixes and tighter structures than those in LB-EPS, which could impede the lysozyme transfer to the cell wall.

Ebselen’s Potential to Inhibit Planktonic and Biofilm Growth of Neisseria mucosa

Background: Antibiotic resistance of various bacterial communities remains a global burden in the healthcare industry. Biofilm formation is one of the resistance mechanisms acquired by bacterial communities in order to reverse the action of antibiotics. There is an urgent need for the discovery of novel antimicrobials and novel approaches to tackle this problem. However, it is very expensive and challenging to develop new antibiotics. Drug repurposing is an efficient strategy which reduces time and cost associated with drug discovery. Objective: In the current study, anti-microbial and antibiofilm potential of an organoselenium clinical molecule Ebselen against Neisseria mucosa has been elucidated. Methods: Ebselen Antibacterial studies include Minimum Inhibitory Concentration (MIC), growthkill, Colony Forming Unit (CFU) assays and intracellular Reactive Oxygen Species (ROS) accumulation studies. Antibiofilm studies included inhibition, eradication and cell surface hydrophobicity assays, quantification of Extracellular Polymeric Substance (EPS) and eDNA and for anti-quorum sensing activity, protease and urease enzyme activities were elucidated. Results: Ebselen showed efficient bactericidal activity as indicated by its low MIC values, bacterial growth inhibition over time and its ability to prevent clonal propagation in this bacterium. Increased accumulation of ROS in Ebselen treated cells indicates radical mediated induction of bacterial death. Interestingly, Ebselen inhibited and distorted matured biofilms by degrading the eDNA component of the EPS layer. Ebselen also attenuated quorum-sensing pathway as indicated by decreased urease and protease enzyme activities. Conclusion: Taken together, these results paved the way to repurpose Ebselen as a potential drug target to curb Neisseria mucosa infections.

Adsorptive removal of gallium from aqueous solution onto biogenic elemental tellurium nanoparticles

This study investigated the adsorption of gallium onto chemical and biogenic elemental tellurium nanoparticles. Biogenic elemental tellurium nanoparticles (BioTeNPs) were found to be a good adsorbent for the removal and recovery of gallium from aqueous solution with an adsorption capacity of 74 mg of Ga3+ per g BioTeNPs. The adsorption mechanism followed pseudo second order kinetics (R2 = 0.999) and the experimental data fitted the monolayer adsorption theory-based Langmuir model. BioTeNPs had a size of 5.30–86.1 nm, a negative zeta potential (–32 mV) and were coated with a layer of extracellular polymeric substances (EPS). Further, a high desorption efficiency of 99% from Ga3+ adsorbed onto the BioTeNPs was achieved using oxalic acid (0.1 M) as eluent. This study proposes a novel removal and recovery method for Ga3+ by adsorption on BioTeNPs produced in bioreactors treating tellurium oxyanion rich wastewaters.

Mild hydrogen peroxide interceded bacterial disintegration of waste activated sludge for efficient biomethane production

Regardless of the issue of sludge management all over the world, the role of phase separated pretreatment prior to anaerobic digestion are more promising in terms of energy efficient biomethane production. However, the effect of phase separated pretreatment (dissociation of extracellular polymeric substances (EPS) followed by biological pretreatment in a two-step process) must be sensibly evaluated from various perceptions to consolidate its effectiveness in sludge management and bioenergy recovery. In this study, mild hydrogen peroxide induced bacterial pretreatment (H2O2-BP) was employed as phase separated pretreatment to investigate the effectiveness of EPS dissociation prior to biological pretreatment on sludge solubilization and biomethanation. The novelty of this study is the application of mild dosage of hydrogen peroxide at sludge pH for the removal of EPS layer with lesser formation of recalcitrant substances which thereby enhances the disintegration by enzyme secreting bacterial and methane generation. The outcome confirmed that the higher EPS dissociation was achieved at H2O2 dosage of 8 μL per 100 mL of sludge with negligible cell lysis. An extractable EPS of 172.8 mg/L was obtained after H2O2 treatment. The higher sCOD solubilization of 22% and the suspended solid reduction of 17.14% were achieved in hydrogen peroxide followed by bacterial pretreatment (H2O2-BP) as compared to of bacterial pretreatment alone (BP) (solubilization-11% and suspended solids reduction-9.3%) and control (C) sludges (solubilization-5% and suspended solids reduction-4.3%). The methane generation for H2O2-BP sludge is 0.174 L/gCOD which is higher than BP (0.078 L/gCOD,) and C sludge (0.02175 L/gCOD). A higher biomass solubilization and increased biomethanation in H2O2-BP revealed that dissociation of EPS prior to bacterial pretreatment increases the surface area for bacterial pretreatment facilitating easier accessibility of substrate and enhanced biomethanation.

Periodic chemical cleaning with urea: disintegration of biofilms and reduction of key biofilm-forming bacteria from reverse osmosis membranes

Biofouling is one of the major factors causing decline in membrane performance in reverse osmosis (RO) plants, and perhaps the biggest hurdle of membrane technology. Chemical cleaning is periodically carried out at RO membrane installations aiming to restore membrane performance. Typical cleaning agents used in the water treatment industry include sodium hydroxide (NaOH) and hydrochloric acid (HCl) in sequence. Rapid biofilm regrowth and related membrane performance decline after conventional chemical cleaning is a routinely observed phenomenon due to the inefficient removal of biomass from membrane modules. Since extracellular polymeric substances (EPS) make up the strongest and predominant structural framework of biofilms, disintegration of the EPS matrix should be the main target for enhanced biomass removal. Previously, we demonstrated at lab-scale the use of concentrated urea as a chemical cleaning agent for RO membrane systems. The protein denaturation property of urea was exploited to solubilize the proteinaceous foulants, weakening the EPS layer, resulting in enhanced biomass solubilization and removal from RO membrane systems. In this work, we investigated the impact of repeated chemical cleaning cycles with urea/HCl as well as NaOH/HCl on biomass removal and the potential adaptation of the biofilm microbial community. Chemical cleaning with urea/HCl was consistently more effective than NaOH/HCl cleaning over 6 cleaning and regrowth cycles. At the end of the 6 cleaning cycles, the percent reduction was 35% and 41% in feed channel pressure drop, 50% and 70% in total organic carbon, 30% and 40% in EPS proteins, and 40% and 66% in the peak intensities of protein-like matter, after NaOH/HCl cleaning and Urea/HCl cleaning, respectively. 16S ribosomal RNA (rRNA) gene sequencing of the biofilm microbial community revealed that urea cleaning does not select for key biofouling families such as Sphingomonadaceae and Xanthomonadaceae that are known to survive conventional chemical cleaning and produce adhesive EPS. This study reaffirmed that urea possesses all the desirable properties of a chemical cleaning agent, i.e., it dissolves the existing fouling layer, delays fresh fouling accumulation by inhibiting the production of a more viscous EPS, does not cause damage to the membranes, is chemically stable, and environmentally friendly as it can be recycled for cleaning.

Influential mechanism of water occurrence states of waste-activated sludge: specifically focusing on the roles of EPS micro-spatial distribution and cation-dominated interfacial properties

The highly hydrated colloidal structure of waste-activated sludge (WAS) is the main obstacle of enhanced dewatering for sludge volume minimization. Extracellular polymeric substances (EPS) maintain the colloidal stability of bio-flocs in a three-dimensional matrix due to bindings with bivalent cations (i.e., Ca2+ and Mg2+) and hydrophobic interactions. However, few studies specifically focused on the quantitative relationships among spatial distribution of EPS, microstructure of bio-flocs and fractions of bound water (e.g. vicinal water and interstitial water). Thus, there may be still some debates on whether and what extent of the lysis or flocculation of sludge flocs is optimal for the dewaterability improvement. This study applied the gradient addition of cation exchange resin (CER) to remove EPS-complexed cations and loosen the spatial distribution of EPS. Consequently, how the spatial extension of EPS layers with relief of complex cations influenced the particle size distribution, fractal dimension, interfacial free energy and water occurrence states of WAS was systematically investigated. The quartz crystal microbalance with dissipation (QCM-D) was also applied to analyze the water-EPS interactions with and without the presence of Ca2+ and Mg2+. All the results confirmed that the dispersed EPS adhering layers led to the higher fractal dimension (Df) but the lower space filling degree of bio-flocs. Also, the 4-fold reduction in the polar/acid-based interfacial free energy could be induced by the removal of cations from EPS matrix, which indicated the significant increase in hydrophobicity. Predictably, the fractions of vicinal water and interstitial water were dominated by the polar/acid-based interfacial free energy and pore structure of microbial aggregates, respectively, which were confirmed by the strong Pearson correlation (Rp>0.80, p-value<0.04). These findings are expected to provide the improved mechanistic insights into the relationship between water occurrence states and colloidal structure of WAS, and can serve as the basis for the optimal combination of various sludge conditioning approaches towards regulating aggregation states of bio-flocs.

Modeling molecular structure and behavior of microbial extracellular polymeric substances through interacting-particle reaction dynamics

Extracellular polymeric substances (EPS) are essential for bacteria to interact with external environments and play a key role in the formation of microbial aggregates. Unveiling the black box of EPS has become an urgent topic in the field of microbiology, medical science and environmental science. Here, we develop an explicit approach to describe the molecular structure and behaviors of EPS using interacting-particle reaction dynamics (iPRD). Three representative states of EPS (i.e., normal EPS layer, metal bridging EPS layer and extracted EPS layer) are qualitatively simulated at molecular scale and validated with previous research results on EPS. Furthermore, an averaged concentration representation method is proposed to quantitatively model the EPS-oriented bioprocesses. Through this method, the contents of protein and polysaccharide in EPS extracted by cation exchange resin are accurately predicted by our model (R2>0.982). This work gives new insights into EPS at the molecular scale and opens up new avenues for further exploring and modeling complex molecular structure and behaviors of EPS.

Acceleration of the bio-reduction of methyl orange by a magnetic and extracellular polymeric substance nanocomposite

Extracellular electron transfer (EET) plays an important role in bio-reduction of environmental pollutants. Extracellular polymeric substances (EPS), a kind of biogenic macromolecule, contain functional groups responsible for acceleration of EET. In this study, azo dye-methyl orange (MO) was chosen as a model pollutant, and a Fe3O4 and EPS nanocomposite (Fe3O4@EPS) was prepared to evaluate its promotion on the bio-reduction of MO. The flower-like core-shell configuration of Fe3O4@EPS with a 12 nm of light layer of EPS was confirmed by TEM. The redox ability of EPS was well reserved on Fe3O4@EPS by FTIR and electrochemical test. The application of Fe3O4@EPS on sustained acceleration of MO decolorization were confirmed by batch experiments and anaerobic sequenced batch reactors. Due to biocompatibility of the biogenic shell, the as-prepared Fe3O4@EPS exhibited low toxic to microorganisms by the Live/dead cell test. Moreover, negligible leaching of EPS under high concentration of various anions and less than 10% of EPS was released under extreme acidic and basic pH condition. The results of study provided a new preparation method of biological intimate and environmentally friendly redox mediators and suggested a feasible way for its use on bio-reduction of pollutants.