Deciphering the Roles of Extracellular Polymeric Substances (EPS) in Shaping Disinfection Kinetics through Permanent Removal via Genetic Disruption.

Extracellular polymeric substances (EPS) ubiquitously encapsulate microbes and play crucial roles in various environmental processes. However, understanding their complex interactions with dynamic bacterial behaviors, especially during the disinfection process, remains very limited. In this work, we investigated the impact of EPS on bacterial disinfection kinetics by developing a permanent EPS removal strategy. We genetically disrupted the synthesis of exopolysaccharides, the structural components of EPS, in Pseudomonas aeruginosa, a well-known EPS-producing opportunistic pathogen found in diverse environments, creating an EPS-deficient strain. This method ensured a lasting absence of EPS while maintaining bacterial integrity and viability, allowing for real-time in situ investigations of the roles of EPS in disinfection. Our findings indicate that removing EPS from bacteria substantially lowered their susceptibility threshold to disinfectants such as ozone, chloramine B, and free chlorine. This removal also substantially accelerated disinfection kinetics, shortened the resistance time, and increased disinfection efficiency, thereby enhancing the overall bactericidal effect. The absence of EPS was found to enhance bacterial motility and increase bacterial cell vulnerability to disinfectants, resulting in greater membrane damage and intensified reactive oxygen species (ROS) production upon exposure to disinfectants. These insights highlight the central role of EPS in bacterial defenses and offer promising implications for developing more effective disinfection strategies.

Antimicrobial and antibiofilm activity of specialized metabolites isolated from Centaurea hyalolepis.

The discovery of plant-derived compounds that are able to combat antibiotic-resistant pathogens is an urgent demand. Over years, Centaurea hyalolepis attracted considerable attention because of its beneficial medical properties. Phytochemical analyses revealed that Centaurea plant species contain several metabolites, such as sesquiterpene lactones (STLs), essential oils, flavonoids, alkaloids, and lignans.The organic extract of C. hyalolepis plant, collected in Palestine, showed significant antimicrobial properties towards a panel of Gram-negative and Gram-positive bacterial strains when the Minimal Inhibitory Concentration (MIC) values were evaluated by broth microdilution assays. A bio-guided fractionation of the active extract via multiple steps of column and thin layer chromatography allowed us to obtain three main compounds. The isolated metabolites were identified as the STLs cnicin, 11ß,13-dihydrosalonitenolide and salonitenolide by spectroscopic and spectrometric analyses. Cnicin conferred the strongest antimicrobial activity among the identified compounds. Moreover, the evaluation of its antibiofilm activity by biomass assays through crystal violet staining revealed almost 30% inhibition of biofilm formation in the case of A. baumannii ATCC 17878 strain. Furthermore, the quantification of carbohydrates and proteins present in the extracellular polymeric substance (EPS) revealed the ability of cnicin to significantly perturb biofilm structure. Based on these promising results, further investigations might open interesting perspectives to its applicability in biomedical field to counteract multidrug resistant infections.

Bacteria contribute exopolysaccharides to an algal-bacterial joint extracellular matrix.

Marine ecosystems are influenced by phytoplankton aggregation, which affects processes like marine snow formation and harmful events such as marine mucilage outbreaks. Phytoplankton secrete exopolymers, creating an extracellular matrix (ECM) that promotes particle aggregation. This ECM attracts heterotrophic bacteria, providing a nutrient-rich and protective environment. In terrestrial environments, bacterial colonization near primary producers relies on attachment and the formation of multidimensional structures like biofilms. Bacteria were observed attaching and aggregating within algal-derived exopolymers, but it is unclear if bacteria produce an ECM that contributes to this colonization. This study, using Emiliania huxleyi algae and Phaeobacter inhibens bacteria in an environmentally relevant model system, reveals a shared algal-bacterial ECM scaffold that promotes algal-bacterial aggregation. Algal exudates play a pivotal role in promoting bacterial colonization, stimulating bacterial exopolysaccharide (EPS) production, and facilitating a joint ECM formation. A bacterial biosynthetic pathway responsible for producing a specific EPS contributing to bacterial ECM formation is identified. Genes from this pathway show increased expression in algal-rich environments. These findings highlight the underestimated role of bacteria in aggregate-mediated processes in marine environments, offering insights into algal-bacterial interactions and ECM formation, with implications for understanding and managing natural and perturbed aggregation events.

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

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

Cell density and extracellular matrix composition mitigate bacterial biofilm sensitivity to UV-C LED irradiation.

Ultraviolet-C light-emitting diodes (UV-C LEDs) are an emerging technology for decontamination applications in different sectors. In this study, the inactivation of bacterial biofilms was investigated by applying an UV-C LED emitting at 280 nm and by measuring both the influence of the initial cell density (load) and presence of an extracellular matrix (biofilm). Two bacterial strains exposing diverging matrix structures and biochemical compositions were used: Pseudomonas aeruginosa and Leuconostoc citreum. UV-C LED irradiation was applied at three UV doses (171 to 684 mJ/cm2) on both surface-spread cells and on 24-h biofilms and under controlled cell loads, and bacterial survival was determined. All surface-spread bacteria, between 105 and 109 CFU/cm2, and biofilms at 108 CFU/cm2 showed that bacterial response to irradiation was dose-dependent. The treatment efficacy decreased significantly for L. citreum surface-spread cells when the initial cell load was high, while no load effect was observed for P. aeruginosa. Inactivation was also reduced when bacteria were grown under a biofilm form, especially for P. aeruginosa: a protective effect could be attributed to abundant extracellular DNA and proteins in the matrix of P. aeruginosa biofilms, as revealed by Confocal Laser Scanning Microscopy observations. This study showed that initial cell load and exopolymeric substances are major factors influencing UV-C LED antibiofilm treatment efficacy. KEY POINTS: • Bacterial cell load (CFU/cm2) could impact UV-C LED irradiation efficiency • Characteristics of the biofilm matrix have a paramount importance on inactivation • The dose to be applied can be predicted based on biofilm properties.

Bioprospecting of Chlamydomonas reinhardtii for boosting biofuel-related products production based on novel aggregation-induced emission active extracellular polymeric substances nanoprobes.

Biofuel production from microalgae has been greatly restricted by low biomass productivity and long-term photosynthetic efficacy. Here, a novel strategy for selecting high-growing, stress-resistant algal strains with high photosynthetic capacity was proposed based on biocompatible extracellular polymeric substances (EPS) probes with aggregation-induced emission (AIE) properties. Specifically, AIE active EPS probes were synthesized for in-situ long-term monitoring of the EPS productivity at different algal growth stages. By coupling the AIE-based fluorescent techniques, algal cells were classified into four diverse populations based on their chlorophyll and EPS signals. Mechanistic studies on the sorted algal cells revealed their remarkable stress resistance and high expression of cell division, biopolymer production and photosynthesis-related genes. The sorted and subcultured algal cells consistently exhibited relatively higher growth rates and photosynthetic capacities, resulting in an increased (1.2 to 1.8-fold) algal biomass production, chlorophyll, and lipids. This study can potentially open new strategies to boost microalgal-based biofuel production.

Non-filamentous bulking of activated sludge induced by graphene oxide: Insights from extracellular polymeric substances.

Widespread use of nanomaterials raises concerns. The underlying mechanism by which graphene oxide (GO) nanoparticles causes poor settleability of activated sludge remains unclear. To explore this mechanism, three reactors with different GO concentrations were established. Extended Derjaguin-Landau-Verwey-Overbeek theory indicated that GO destroyed the property of extracellular polymeric substances (EPS), increasing the energy barrier between bacteria. Low levels of uronic acid and hydrogen bonding in exopolysaccharide weakened the EPS gelation increasing aggregation repulsion. Lager amounts of hydrophilic amino acid and looser structure of extracellular proteins for exposing inner hydrophilic groups significantly contributed to the hydrophilicity of EPS. Both changes implied deterioration in EPS structure under GO stress. Metagenome demonstrated a decrease in genes responsible for capsular polysaccharide colonization and genes regulated the translocation of loose proteins were increased, which increased repulsion between bacteria. This study elucidated that changes in EPS secretion under GO exposure are the underlying causes of poor settleability.

Bacterial biofilm growth and perturbation by serine protease from Bacillus sp.

In nature, bacteria are ubiquitous and can be categorized as beneficial or harmless to humans, but most bacteria have one thing in common which is their ability to produce biofilm. Biofilm is encased within an extracellular polymeric substance (EPS) which provides resistance against antimicrobial agents. Protease enzymes have the potential to degrade or promote the growth of bacterial biofilms. In this study, the effects of a recombinant intracellular serine protease from Bacillus sp. (SPB) on biofilms from Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were analyzed. SPB was purified using HisTrap HP column and concentrated using Amicon 30 ultra-centrifugal filter. SPB was added with varying enzyme activity and assay incubation period after biofilms were formed in 96-well plates. SPB was observed to have contrasting effects on different bacterial biofilms, where biofilm degradations were observed for both 7-day-old A. baumannii (37.26%) and S. aureus (71.51%) biofilms. Meanwhile, SPB promoted growth of P. aeruginosa biofilm up to 176.32%. Compatibility between protein components in S. aureus biofilm with SPB as well as a simpler membrane structure morphology led to higher biofilm degradation for S. aureus compared to A. baumannii. However, SPB promoted growth of P. aeruginosa biofilm due likely to its degrading protein factors that are responsible for biofilm detachment and dispersion, thus resulting in more multi-layered biofilm formation. Commercial protease Savinase which was used as a comparison showed degradation for all three bacterial biofilms. The results obtained are unique and will expand our understanding on the effects that bacterial proteases have toward biofilms.

Effect of salinity on denitrification, membrane fouling and bacterial community in a fixed-bed biofilm membrane reactor.

In this study, a fixed-bed biofilm membrane bioreactor was used to assess denitrification and carbon removal performance, membrane fouling, composition, and the dynamics of microbial communities across 10 salinity levels. As salinity levels increased (from 0 to 30 g/L), the removal efficiency of total nitrogen and chemical oxygen demand decreased from 98 and 86% in Phase I to 25 and 45% in Phase X, respectively. Beyond a salinity level of 10 g/L, membrane fouling accelerated considerably. The analysis of fouling resistance distribution suggested that soluble microbial products (SMPs) were the primary cause of this phenomenon. The irregularity in microbial community succession reflected the varying adaptability of different bacteria to different salinity levels. The relative abundance of Sulfuritalea, Lentimircobium, Thauera, and Pseudomonas increased from 20.2 to 47.7% as the experiments progressed. Extracellular polymeric substances-related analysis suggested that Azospirillum plays a positive role in preserving the structural integrity of the biofilm carrier. The SMP-related analysis showed a positive correlation between Lentimircobium, Thauera, Pseudomonas, and the SMP content. These results suggested that these three bacterial genera significantly promoted the release of SMP under salt stress, which in turn led to severe membrane fouling.

Biochar inoculated with Rhodococcus biphenylivorans altered microecological regulation by promoting quorum sensing and electron transfer: Up-regulation of related genes and enhancement of phenol and ammonia degradation.

Bioaugmentation is an efficient method for improving the efficiency of coking wastewater removal. Nevertheless, how different immobilization approaches affect the efficiency of bioaugmentation remains unclear, as does the corresponding mechanism. With the assistance of immobilized bioaugmentation strain Rhodococcus biphenylivorans B403, the removal of synthetic coking wastewater was investigated (drying agent, alginate agent, and absorption agent). The reactor containing the absorption agent exhibited the highest average removal efficiency of phenol (99.74 %), chemical oxygen demand (93.09 %), and NH4+-N (98.18 %). Compared to other agents, the covered extracellular polymeric substance on the absorption agent surface enhanced electron transfer and quorum sensing, and the promoted quorum sensing benefited the activated sludge stability and microbial regulation. The phytotoxicity test revealed that the wastewater's toxicity was greatly decreased in the reactor with the absorption agent, especially under high phenol concentrations. These findings showed that the absorption agent was the most suitable for wastewater treatment bioaugmentation.

Deciphering the Interrelationship of arnT Involved in Lipid-A Alteration with the Virulence of Salmonella Typhimurium.

The lipopolysaccharide (LPS) that resides on the outermost surface and protects Gram-negative bacteria from host defenses is one of the key components leading to Salmonella infection, particularly the endotoxic lipid A domain of LPS. Lipid A modifications have been associated with several genes such as the arnT that encodes 4-amino-4-deoxy-L-arabinose transferase, which can be critical for bacteria to resist cationic antimicrobial peptides and interfere with host immune recognition. However, the association of arnT with virulence is not completely understood. Thus, this study aimed to elucidate the interrelationship of the major lipid A modification gene arnT with Salmonella Typhimurium virulence. We observed that the arnT-deficient S. Typhimurium (JOL2943), compared to the wild type (JOL401), displayed a significant decrease in several virulence phenotypes such as polymyxin B resistance, intracellular survival, swarming, and biofilm and extracellular polymeric substance (EPS) production. Interestingly, the cell-surface hydrophobicity, adhesion, and invasion characteristics remained unaffected. Additionally, LPS isolated from the mutant induced notably lower levels of endotoxicity-related cytokines in RAW and Hela cells and mice, particularly IL-1ß with a nine-fold decrease, than WT. In terms of in vivo colonization, JOL2943 showed diminished presence in internal organs such as the spleen and liver by more than 60%, while ileal infectivity remained similar to JOL401. Overall, the arnT deletion rendered the strain less virulent, with low endotoxicity, maintained gut infectivity, and reduced colonization in internal organs. With these ideal characteristics, it can be further explored as a potential attenuated Salmonella strain for therapeutics or vaccine delivery systems.

Sponge iron strengthens the activity of anammox biofilm under low nitrogen conditions in a two-stage fixed-bed biofilm reactor.

Strengthening the activity competitiveness of anaerobic ammonium oxidation (anammox) bacteria (AnAOB) under low nitrogen conditions is indispensable for mainstream anammox application. This study demonstrates that sponge iron addition (42.8 g/L) effectively increased apparent AnAOB activity and extracellular polymeric substance (EPS) production of low load anammox biofilms cultivated under low (influent of 60 mg N/L) and even ultra-low (influent of 10 mg N/L) nitrogen conditions. In-situ batch tests showed that after sponge iron addition the specific AnAOB activity in the low and ultra-low nitrogen systems further increased to 1.18 and 0.47 mmol/g VSS/h, respectively, with an apparent growth rate for AnAOB of 0.011 ± 0.001 d-1 and 0.004 ± 0.001 d-1, respectively. The averaged EPS concentration of anammox biofilm in both low (from 35.84 to 71.05 mg/g VSS) and ultra-low (from 44.14 to 57.59 mg/g VSS) nitrogen systems increased significantly, while a higher EPS protein/polysaccharide ratio, which was positively correlated with AnAOB activity, was observed in the low nitrogen system (3.54 ± 0.34) than that in the ultra-low nitrogen system (1.82 ± 0.10). In addition, Candidatus Brocadia was detected as dominant AnAOB in the anammox biofilm under the low (12.2 %) and ultra-low (24.7 %) nitrogen condition. Notably, the genus Streptomyces (26.3 %), capable for funge-like codenitrification, increased unexpectedly in the low nitrogen system, but not affecting the nitrogen removal performance. Therefore, using sponge iron to strengthen AnAOB activity under low nitrogen conditions is feasible, providing support for mainstream anammox applications.

Tertiary treatment of municipal wastewater in an IBFR dominated by PD/A with unique niche.

To explore the feasibility of biofilter reactor to treat municipal secondary effluent deeply without extra carbon source, this paper proposed an integrated biofilter reactor (IBFR) coupling partial denitrification (PD) with anammox (A) to treat the secondary effluent and raw sewage with the flow ratio of 3:1 together. The results show that the effluent concentration of TN and COD in IBFR could be reduced to 10 mg/L and 15 mg/L, respectively, under hydraulic retention time of 1.5 h and nitrogen loading rate of 0.55 kg/(m3·d). The highest specific anammox activity (19.2 mg N/(g TVS·d)) and the maximum extracellular polymeric substance (EPS) content (107.21 mg/g TVS) occurred at the 25-50 cm section of IBFR, where Thauera, Candidatus Anammoximicrobium and Candidatus Brocadia were the dominant denitrifiers and anammox bacteria. Furthermore, the cyclic self-stratification occurred along the reactor height, where the utilization, decomposition, transformation and cross-feeding of EPS enhanced the performance stability of nitrogen and carbon removal, strengthened the niche structure and promoted the synergistic symbiosis. In conclusion, IBFR coupling PD and A demonstrated the possibility to treat secondary effluent without additional carbon sources, which is expected as an alternative approach for tertiary treatment of municipal wastewater.

Reactive oxygen species mediated extracellular polymeric substances production assisting the recovery of Thalassiosira pseudonana from polystyrene micro and nanoplastics exposure.

As emerging pollutants in the aquatic environments, micro- and nano-plastics (MNPs) aroused widespread environmental concerns for their potential threats to the ecological health. Previous research has proved that microalgae growth could recover from the MNPs toxicities, in which the extracellular polymeric substances (EPS) might play the key role. In order to comprehensively investigate the recovery process of microalgae from MNPs stress and the effecting mechanisms of EPS therein, this study conducted a series of experiments by employing two sizes (0.1 and 1 µm) of polystyrene (PS) MNPs and the marine model diatom Thalassiosira pseudonana during 14 days. The results indicated: the pigments accumulations and photosynthetic recovery of T. pseudonana under MPs exposure showed in the early stage (4-5 days), while the elevation of reactive oxygen species (ROS) and EPS contents lasted longer time period (7-8 days). EPS was aggregated with MNPs particles and microalgal cells, corresponding to the increased settlement rates. More increase of soluble (SL)-EPS contents was found than bound (B)-EPS under MNPs exposure, in which the increase of the protein proportion and humic acid-like substances in SL-EPS was found, thus facilitating aggregates formation. ROS was the signaling molecule mediating the overproduction of EPS. The transcriptional results further proved the enhanced EPS biosynthesis on the molecular level. Therefore, this study elucidated the recovery pattern of microalgae from MNPs stress and linked "ROS-EPS production changes-aggregation formation" together during the growth recovery process, with important scientific and environmental significance.

Biological soil crust elicits microbial community and extracellular polymeric substances restructuring to reduce the soil erosion on tropical island, South China Sea.

Soil erosion stands as the preeminent environmental concern globally, attaining heightened significance, particularly within islands where land resources prove notably scarce. Biological soil crusts, referred to as biocrusts, assume a pivotal ecological role in soil conservation. Notably, they augment the horizontal stability of the substrate through the exudation of microbial extracellular polymeric substances (EPS), thereby shielding the soil against shear stress, exemplified in the form of water erosion. While extant research has delved into the anti-erosion mechanisms of biocrusts in arid landscapes, a conspicuous lacuna persists in the exploration of coral island environments. In this study, we collected and assessed 30 samples encompassing dark biocrusts, light biocrusts, and bare soil to scrutinize the potential anti-erosion efficacy of tropical coral island biocrusts within the South China Sea. Employing a cohesive strength meter, we quantified soil shear stress across various stages of biocrust development, revealing a discernible enhancement in soil erosion resistance during the formation of biocrusts. Relative to the exposed bare soil, the soil shear stress exhibited an escalation from 0.33 N m-2 to 0.61 N m-2 and 1.31 N m-2 in the light biocrusts and dark biocrusts, respectively. Mechanistically, we assayed microbial EPS contents, exposing a positive correlation between EPS and soil anti-erodibility, encompassing extracellular protein and polysaccharide. Concurrently, bacterial abundance displayed a significant augmentation commensurate with biocrust formation and development. In pursuit of elucidating the origin of EPS, high-throughput amplicon sequencing was executed to identify microorganisms contributing to biocrust development. Correlation analysis discerned Cyanobacteria, Chloroflexi, Deinococcota, and Patescibacteria as potential microbials fostering EPS production and fortifying erosion resistance. Collectively, our study presents the first evidence that biocrust from tropical coral reef island in the South China Sea promotes resistance to soil erosion, pinpointing key EPS-producing microbials against soil erosion. The findings would provide insights for island environment restoration.

Coupling of gene regulation and carrier modification manipulates bacterial biofilms as robust living catalysts.

The biofilm of an engineered strain is limited by slow growth and low yield, resulting in an unsatisfactory ability to resist external stress and promote catalytic efficiency. Here, biofilms used as robust living catalysts were manipulated through dual functionalized gene regulation and carrier modification strategies. The results showed that gene overexpression regulates the autoinducer-2 activity, extracellular polymeric substance content and colony behavior of Escherichia coli, and the biofilm yield of csgD overexpressed strains increased by 79.35 % compared to that of the wild type strains (p < 0.05). In addition, the hydrophilicity of polyurethane fibres modified with potassium dichromate increased significantly, and biofilm adhesion increased by 105.80 %. Finally, the isoquercitrin yield in the catalytic reaction of the biofilm reinforced by the csgD overexpression strain and the modified carrier was 247.85 % higher than that of the untreated group. Overall, this study has developed engineered strains biofilm with special functions, providing possibilities for catalytic applications.

Understanding microplastic aging driven by photosensitization of algal extracellular polymeric substances.

The aging of microplastics (MPs) is extremely influenced by photochemically-produced reactive intermediates (PPRIs), which are mediated by natural photosensitive substances. Algal extracellular polymeric substances (EPS) can produce PPRIs when exposed to sunlight. Nonetheless, the specific role of EPS in the aging process of MPs remains unclear. This work systematically explored the aging process of polystyrene (PS) MPs in the EPS secreted by Chlorella vulgaris under simulated sunlight irradiation. The results revealed that the existence of EPS accelerated the degradation of PS MPs into particles with sizes less than 1 µm, while also facilitating the formation of hydroxy groups on the surface. The release rate of dissolved organic matter (DOM) from PS MPs was elevated from 0.120 mg·L-1·day-1 to 0.577 mg·L-1·day-1. The primary factor contributing to the elevated levels of DOM was humic acid-like compounds generated through the breakdown of PS. EPS accelerated the aging process of PS MPs by primarily mediating the formation of triplet excited states (3EPS*), singlet oxygen (1O2), and superoxide radicals (O2∙-), resulting in indirect degradation. 3EPS* was found to have the most substantial impact. This study makes a significant contribution to advance understanding of the environmental fate of MPs in aquatic environments impacted by algal blooms.

From "resistance genes expression" to "horizontal migration" as well as over secretion of Extracellular Polymeric Substances: Sludge microorganism's response to the increasing of long-term disinfectant stress.

Glutaraldehyde-Didecyldimethylammonium bromides (GDs) has been frequently and widely employed in livestock and poultry breeding farms to avoid epidemics such as African swine fever, but its long-term effect on the active sludge microorganisms of the receiving wastewater treatment plant was keep unclear. Four simulation systems were built here to explore the performance of aerobic activated sludge with the long-term exposure of GDs and its mechanism by analyzing water qualities, resistance genes, extracellular polymeric substances and microbial community structure. The results showed that the removal rates of CODCr and ammonia nitrogen decreased with the exposure concentration of GDs increasing. It is worth noting that long-term exposure to GDs can induce the horizontal transfer and coordinated expression of a large number of resistance genes, such as qacE, sul1, tetx, and int1, in drug-resistant microorganisms. Additionally, it promotes the secretion of more extracellular proteins, including arginine, forming a "barrier-like" protection. Therefore, long-term exposure to disinfectants can alter the treatment capacity of activated sludge receiving systems, and the abundance of resistance genes generated through horizontal transfer and coordinated expression by drug-resistant microorganisms does pose a significant threat to ecosystems and health. It is recommended to develop effective pretreatment methods to eliminate disinfectants.

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.

Mechanistic insight into the aggregation ability of anammox microorganisms: Roles of polarity, composition and molecular structure of extracellular polymeric substances.

The chemical characteristics of extracellular polymeric substances (EPS) of anammox bacteria (AnAOB) play a crucial role in the rapid enrichment of AnAOB and the stable operation of wastewater anammox processes. To clarify the influential mechanisms of sludge EPS on AnAOB aggregation, multiple parameters, including the polarity distribution, composition, and molecular structure of EPS, were selected, and their quantitative relationship with AnAOB aggregation was analyzed. Compared to typical anaerobic sludge (anaerobic floc and granular sludge), the anammox sludge EPS exhibited higher levels of tryptophan-like substances (44.82-56.52 % vs. 2.57-39.81 %), polysaccharides (40.02-53.49 mg/g VSS vs. 30.22-41.69 mg/g VSS), and protein structural units including α-helices (20.70-23.98 % vs. 16.48-19.32 %), ß-sheets (37.43-42.98 % vs. 25.78-36.72 %), and protonated nitrogen (Npr) (0.065-0.122 vs. 0.017-0.061). In contrast, it had lower contents of ß-turns (20.95-27.39 % vs. 28.17-39.04 %). These biopolymers were found to originate from different genera of AnAOB. Specifically, the α-helix-rich proteins were mainly derived from Candidatus Kuenenia, whereas the extracellular proteins related to tryptophan and Npr were closely associated with Candidatus Brocadia. Critically, these EPS components could drive anammox aggregation through interactions. Substantial amounts of tryptophan-like substances facilitated the formation of ß-sheet structures and the exposure of internal hydrophobic clusters, which benefited the anammox aggregation. Meanwhile, extracellular proteins with high Npr content played a pivotal role in the formation of mixed protein-polysaccharide gel networks with the electronegative regions of polysaccharides, which could be regarded as the key component in the maintenance of anammox sludge stability. These findings provide a comprehensive understanding of the multifaceted roles of EPS in driving anammox aggregation and offer valuable insights into the development of EPS regulation strategies aimed at optimizing the anammox process.