Phthalate Esters Released from Plastics Promote Biofilm Formation and Chlorine Resistance.

Phthalate esters (PAEs) are commonly released from plastic pipes in some water distribution systems. Here, we show that exposure to a low concentration (1-10 µg/L) of three PAEs (dimethyl phthalate (DMP), di-n-hexyl phthalate (DnHP), and di-(2-ethylhexyl) phthalate (DEHP)) promotes Pseudomonas biofilm formation and resistance to free chlorine. At PAE concentrations ranging from 1 to 5 µg/L, genes coding for quorum sensing, extracellular polymeric substances excretion, and oxidative stress resistance were upregulated by 2.7- to 16.8-fold, 2.1- to 18.9-fold, and 1.6- to 9.9-fold, respectively. Accordingly, more biofilm matrix was produced and the polysaccharide and eDNA contents increased by 30.3-82.3 and 10.3-39.3%, respectively, relative to the unexposed controls. Confocal laser scanning microscopy showed that PAE exposure stimulated biofilm densification (volumetric fraction increased from 27.1 to 38.0-50.6%), which would hinder disinfectant diffusion. Biofilm densification was verified by atomic force microscopy, which measured an increase of elastic modulus by 2.0- to 3.2-fold. PAE exposure also stimulated the antioxidative system, with cell-normalized superoxide dismutase, catalase, and glutathione activities increasing by 1.8- to 3.0-fold, 1.0- to 2.0-fold, and 1.2- to 1.6-fold, respectively. This likely protected cells against oxidative damage by chlorine. Overall, we demonstrate that biofilm exposure to environmentally relevant levels of PAEs can upregulate molecular processes and physiologic changes that promote biofilm densification and antioxidative system expression, which enhance biofilm resistance to disinfectants.

Microplastics and chemical leachates from plastic pipes are associated with increased virulence and antimicrobial resistance potential of drinking water microbial communities.

There is increasing recognition of the potential impacts of microplastics (MPs) on human health. As drinking water is the most direct route of human exposure to MPs, there is an urgent need to elucidate MPs source and fate in drinking water distribution system (DWDS). Here, we showed polypropylene random plastic pipes exposed to different water quality (chlorination and heating) and environmental (freeze-thaw) conditions accelerated MPs generation and chemical leaching. MPs showed various morphology and aggregation states, and chemical leaches exhibited distinct profiles due to different physicochemical treatments. Based on the physiological toxicity of leachates, oxidative stress level was negatively correlated with disinfection by-products in the leachates. Microbial network analysis demonstrated exposure to leachates (under three treatments) undermined microbial community stability and increased the relative abundance and dominance of pathogenic bacteria. Leachate physical and chemical properties (i.e., MPs abundance, hydrodynamic diameter, zeta potential, total organic carbon, dissolved ECs) exerted significant (p < 0.05) effects on the functional genes related to virulence, antibiotic resistance and metabolic pathways. Notably, chlorination significantly increased correlations among pathogenic bacteria, virulence genes, and antibiotic resistance genes. Overall, this study advances the understanding of direct and indirect risks of these MPs released from plastic pipes in the DWDS.

Nano-sized polystyrene and magnetite collectively promote biofilm stability and resistance due to enhanced oxidative stress response.

Despite the growing prevalence of nanoplastics in drinking water distribution systems, the collective influence of nanoplastics and background nanoparticles on biofilm formation and microbial risks remains largely unexplored. Here, we demonstrate that nano-sized polystyrene modified with carboxyl groups (nPS) and background magnetite (nFe3O4) nanoparticles at environmentally relevant concentrations can collectively stimulate biofilm formation and prompt antibiotic resistance. Combined exposure of nPS and nFe3O4 by P. aeruginosa biofilm cells stimulated intracellular reactive oxidative species (ROS) production more significantly compared with individual exposure. The resultant upregulation of quorum sensing (QS) and c-di-GMP signaling pathways enhanced the biosynthesis of polysaccharides by 50 %- 66 % and increased biofilm biomass by 36 %- 40 % relative to unexposed control. Consistently, biofilm mechanical stability (measured as Young's modulus) increased by 7.2-9.1 folds, and chemical stress resistance (measured with chlorine disinfection) increased by 1.4-2.0 folds. For P. aeruginosa, the minimal inhibitory concentration of different antibiotics also increased by 1.1-2.5 folds after combined exposure. Moreover, at a microbial community-wide level, metagenomic analysis revealed that the combined exposure enhanced the multi-species biofilm's resistance to chlorine, enriched the opportunistic pathogenic bacteria, and promoted their virulence and antibiotic resistance. Overall, the enhanced formation of biofilms (that may harbor opportunistic pathogens) by nanoplastics and background nanoparticles is an overlooked phenomenon, which may jeopardize the microbial safety of drinking water distribution systems.

Aggregation and construction mechanisms of microbial extracellular polymeric substances with the presence of different multivalent cations: Molecular dynamic simulation and experimental verification.

Interactions between cations and extracellular polymeric substances (EPS) play an important role in the formation of microbial aggregates and have key effects on the physical properties of activated sludge across wastewater and sludge treatment process. Here, a molecular model of EPS cluster in activated sludge was constructed and simulated by molecular dynamics (MD) to probe the structural properties of EPS and the interaction between EPS and prevalent multivalent cations (Ca2+, Mg2+, Al3+). Then the predicted changes in physical properties were validated against the dynamic light scattering, XAD resin fractionation and rheology test. The binding dynamics and interactions mechanisms between multivalent cations and EPS functional groups were further investigated using MD in combination with spectroscopic analysis. Results suggest that biopolymers are originally aggregated by electrostatic and intermolecular interactions forming dynamic clusters with negatively charged surface functional groups, which induced electrostatic repulsion preventing further agglomeration of biopolymer clusters. In the presence of multivalent cations, surface polar functional groups in biopolymers are connected, causing the rearrangement of EPS molecular conformation that forms larger and denser agglomerates. Reduced solvent accessible surface area, enhanced hydrophobicity, and increased binding free energy lead to a strong gel-like network of EPS. Ca2+ and Al3+ predominantly interact with functional groups in polysaccharides, promoting agglomeration of macromolecules. In contrast, Mg2+ and Al3+ disrupted the secondary structure of proteins, exposing hydrophobic interaction sites. Al3+ can better agglomerate biopolymers with its higher positive charge and shorter coordination distance as compared to Ca2+ and Mg2+, but compromised by the effect of hydration. This work offers a novel approach to explore the construction and molecular aggregation of EPS, enriching the theoretical basis for optimization of wastewater and sludge treatment.

Biofilm-responsive encapsulated-phage coating for autonomous biofouling mitigation in water storage systems.

Biofilms in water storage systems may harbor pathogens that threaten public health. Chemical disinfectants are marginally effective in eradicating biofilms due to limited penetration, and often generate harmful disinfection byproducts. To enhance biofouling mitigation in household water storage tanks, we encapsulated bacteriophages (phages) in chitosan crosslinked with tri-polyphosphate and 3-glycidoxypropyltrimethoxysilane. Phages served as self-propagating green biocides that exclusively infect bacteria. This pH-responsive encapsulation (244 ± 11 nm) enabled autonomous release of phages in response to acidic pH associated with biofilms (corroborated by confocal microscopy with pH-indicator dye SNARF-4F), but otherwise remained stable in pH-neutral tap water for one month. Encapsulated phages instantly bind to plasma-treated plastic and fiberglass surfaces, providing a facile coating method that protects surfaces highly vulnerable to biofouling. Biofilm formation assays were conducted in tap water amended with 200 mg/L glucose to accelerate growth and attachment of Pseudomonas aeruginosa, an opportunistic pathogen commonly associated with biofilms in drinking water distribution and storage systems. Biofilms formation on plastic surfaces coated with encapsulated phages decreased to only 6.7 ± 0.2% (on a biomass basis) relative to the uncoated controls. Likewise, biofilm surface area coverage (4.8 ± 0.2 log CFU/mm2) and live/dead fluorescence ratio (1.80) were also lower than the controls (6.6 ± 0.2 log CFU/mm2 and live/dead ratio of 11.05). Overall, this study offers proof-of-concept of a chemical-free, easily implementable approach to control problematic biofilm-dwelling bacteria and highlights benefits of this bottom-up biofouling control approach that obviates the challenge of poor biofilm penetration by biocides.

UV-aging of microplastics increases proximal ARG donor-recipient adsorption and leaching of chemicals that synergistically enhance antibiotic resistance propagation.

Despite growing attention to environmental pollution by microplastics (MP), the effects of MP aging on bacterial horizontal gene transfer (HGT) have not been systematically investigated. Here, we used UV-aged polystyrene microplastics (PS-MPs) to investigate how aging affects antibiotic resistance genes (ARGs) transfer efficiency from various ARG vectors to recipient bacteria. The adsorption capacity of MP20 (20-day UV-aged PS-MPs) towards E. coli (harboring plasmid-borne blaTEM-1), plasmid pET29 (harboring blaNDM-1) and phage lambda (carrying the aphA1 ARG) increased by 6.6-, 5.2- and 8.3-fold, respectively, relative to pristine PS-MPs (MP0), due to increased specific surface area and affinity for these ARG vectors. Moreover, MP20 released more organic compounds (TOC 1.6 mg/g-MP20, versus 0.2 mg/g-MP0 in 4 h) -possibly depolymerization byproducts (verified by GC-MS), which induced intracellular ROS generation, increased cell permeability and upregulated HGT associated genes. Accordingly, MP20 enhanced ARG transfer frequency from E. coli, plasmid pET29 and phage lambda (relative to MP0) by 1.3-, 4.7- and 3.5-fold, respectively. The Bliss independence model infers that higher bacterial adsorption and exposure to chemicals released during MP aging synergistically enhanced ARG transfer. This underscores the need to assess the significance of this overlooked phenomenon to the environmental dissemination of antibiotic resistance and other HGT processes.

Inhibition of biofouling on reverse osmosis membrane surfaces by germicidal ultraviolet light side-emitting optical fibers.

Biofouling of membrane surfaces poses significant operational challenges and costs for desalination and wastewater reuse applications. Ultraviolet (UV) light can control biofilms while reducing chemical usage and disinfection by-products, but light deliveries to membrane surfaces in spiral wound geometries has been a daunting challenge. Thin and flexible nano-enabled side-emitting optical fibers (SEOFs) are novel light delivery devices that enable disinfection or photocatalytic oxidation by radiating UV light from light-emitting diodes (LEDs). We envision SEOFs as an active membrane spacer to mitigate biofilm formation on reverse osmosis (RO) membranes. A lab-scale RO membrane apparatus equipped with SEOFs allowed comparison of UV-A (photocatalysis-enabled) versus UV-C (direct photolysis disinfection). Compared against systems without any light exposure, systems with UV-C light formed thinner-but denser-biofilms, prevented permeate flux declines due to biofouling, and maintained the highest salt rejection. Results were corroborated by in-situ optical coherence tomography and ex-situ measurements of biofilm growth on the membranes. Transcriptomic analysis showed that UV-C SEOFs down-regulated quorum sensing and surface attachment genes. In contrast, UV-A SEOFs upregulated quorum sensing, surface attachment, and oxidative stress genes, resulting in higher extracellular polymeric substances (EPS) accumulation on membrane surfaces. Overall, SEOFs that deliver a low fluence of UV-C light onto membrane surfaces are a promising non-chemical approach for mitigating biofouling formation on RO membranes.

Changes in tetracycline partitioning and bacteria/phage-comediated ARGs in microplastic-contaminated greenhouse soil facilitated by sophorolipid.

The emerging mixed contamination of antibiotics and microplastics in greenhouse soil has made the control of antibiotic resistant gene (ARG) transmission a novel challenge. In this work, surfactant sophorolipid was applied to enhance the dissipation of tetracycline (TC) and tet genes in the presence of microplastics in greenhouse soil. During 49days of incubation, soil bacteria and phages were both found to be the crucial reservoirs of ARGs. Meanwhile, microplastic's presence significantly inhibited the dissipation of TC and ARGs in the soil. However, sophorolipid application was proved to outweigh the negative impact caused by microplastic existence, and lead to the highest dissipation of soil TC and ARGs. Significant positive correlation was detected between the dissipation rate of water-soluble and exchangeable TC content and bacteria/phage co-mediated ARG levels. This also held true between the two fractions of soil TC and the ratio of ARG level in the bacteria to that in the phages (BARGs/PARGs). The opposite impacts of microplastic presence and sophorolipid amendment on the TC/ARG dissipation found in this work provides new information for understanding ARG transmission between bacteria and phages in the mixed contaminated greenhouse soil.