Metabolomics-based estimation of activated sludge microbial composition and prediction of filamentous bulking.

Understanding the structure and activity of activated sludge (AS) microbiome is key to ensuring optimal operation of wastewater treatment processes. While high-throughput metagenomics offers a comprehensive view of AS microbiome, its cost and time demands warrant alternative approaches. This study employed machine learning methods to integrate metabolomic and metagenomic data, enabling predictions of selected microbial abundances from metabolite profiling. Model training relied on rich microbial and metabolite abundance data collected in an intensively sampled AS system, including a period of filamentous bulking, as well as a few other AS systems. Multiple linear regression out-competed other three algorithms in achieving relatively high prediction accuracy (R2 = 0.70±0.02) for the abundances of 10 selected, either keystone or core metagenome-assembled genomes (MAGs). The model predicted the abundances of filamentous Microtrichaceae and Thiotrichaceae during bulking with an error range of 14-17.8 %. This predictive power extends beyond the specific system studied, showcasing potentials for broader applications across other AS systems. Aspartate, glycine, and folate were the most influential metabolite features contributing to model performance, which were also effective indicators for filamentous bulking, with up to one week of early warning potential. This study pioneers the application of metabolomics for fast, relatively accurate and cost-effective prediction of AS community composition, enabling proactive management of AS systems towards improved efficiency and stability.

Protective effects of antibiotic resistant bacteria on susceptibles in biofilm: Influential factors, mechanism, and modeling.

In environmental biofilms, antibiotic-resistant bacteria facilitate the persistence of susceptible counterparts under antibiotic stresses, contributing to increased community-level resistance. However, there is a lack of quantitative understanding of this protective effect and its influential factors, hindering accurate risk assessment of biofilm resistance in diverse environment. This study isolated an opportunistic Escherichia coli pathogen from soil, and engineered it with plasmids conferring antibiotic resistance. Protective effects of the ampicillin resistant strain (AmpR) on their susceptible counterparts (AmpS) were observed in ampicillin-stress colony biofilms. The concentration of ampicillin delineated protective effects into 3 zones: continuous protection (<1 MIC of AmpS), initial AmpS/R dependent (1-8 MIC of AmpS), and ineffective (>8 MIC of AmpS). Intriguingly, Zone 2 exhibited a surprising "less is more" phenomenon tuned by the initial AmpS/R ratio, where biofilm with an initially lower AmpR (1:50 vs 50:1) harbored 30-90 % more AmpR after 24 h growth under antibiotic stress. Compared to AmpS, AmpR displayed superiority in adhesion, antibiotic degradation, motility, and quorum sensing, allowing them to preferentially colonize biofilm edge and areas with higher ampicillin. An agent-based model incorporating protective effects successfully simulated tempo-spatial dynamics of AmpR and AmpS influenced by antibiotic stress and initial AmpS/R. This study provides a holistic view on the pervasive but poorly understood protective effects in biofilm, enabling development of better risk assessment and precisely targeted control strategies of biofilm resistance in diverse environment.

Adaptive strategies and ecological roles of phages in habitats under physicochemical stress.

Bacteriophages (phages) play a vital role in ecosystem functions by influencing the composition, genetic exchange, metabolism, and environmental adaptation of microbial communities. With recent advances in sequencing technologies and bioinformatics, our understanding of the ecology and evolution of phages in stressful environments has substantially expanded. Here, we review the impact of physicochemical environmental stress on the physiological state and community dynamics of phages, the adaptive strategies that phages employ to cope with environmental stress, and the ecological effects of phage-host interactions in stressful environments. Specifically, we highlight the contributions of phages to the adaptive evolution and functioning of microbiomes and suggest that phages and their hosts can maintain a mutualistic relationship in response to environmental stress. In addition, we discuss the ecological consequences caused by phages in stressful environments, encompassing biogeochemical cycling. Overall, this review advances an understanding of phage ecology in stressful environments, which could inform phage-based strategies to improve microbiome performance and ecosystem resilience and resistance in natural and engineering systems.

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.

Enhanced Bacterium-Phage Symbiosis in Attached Microbial Aggregates on a Membrane Surface Facing Elevated Hydraulic Stress.

Phages are increasingly recognized for their importance in microbial aggregates, including their influence on microbial ecosystem services and biotechnology applications. However, the adaptive strategies and ecological functions of phages in different aggregates remain largely unexplored. Herein, we used membrane bioreactors to investigate bacterium-phage interactions and related microbial functions within suspended and attached microbial aggregates (SMA vs AMA). SMA and AMA represent distinct microbial habitats where bacterial communities display distinct patterns in terms of dominant species, keystone species, and bacterial networks. However, bacteria and phages in both aggregates exhibited high lysogenicity, with 60% lysogenic phages in the virome and 70% lysogenic metagenome-assembled genomes of bacteria. Moreover, substantial phages exhibited broad host ranges (34% in SMA and 42% in AMA) and closely interacted with habitat generalist species (43% in SMA and 49% in AMA) as adaptive strategies in stressful operation environments. Following a mutualistic pattern, phage-carried auxiliary metabolic genes (pAMGs; 238 types in total) presumably contributed to the bacterial survival and aggregate stability. The SMA-pAMGs were mainly associated with energy metabolism, while the AMA-pAMGs were mainly associated with antioxidant biosynthesis and the synthesis of extracellular polymeric substances, representing habitat-dependent patterns. Overall, this study advanced our understanding of phage adaptive strategies in microbial aggregate habitats and emphasized the importance of bacterium-phage symbiosis in the stability of microbial aggregates.

The association of prokaryotic antiviral systems and symbiotic phage communities in drinking water microbiomes.

Prokaryotic antiviral systems are important mediators for prokaryote-phage interactions, which have significant implications for the survival of prokaryotic community. However, the prokaryotic antiviral systems under environmental stress are poorly understood, limiting the understanding of microbial adaptability. Here, we systematically investigated the profile of the prokaryotic antiviral systems at the community level and prokaryote-phage interactions in the drinking water microbiome. Chlorine disinfectant was revealed as the main ecological driver for the difference in prokaryotic antiviral systems and prokaryote-phage interactions. Specifically, the prokaryotic antiviral systems in the microbiome exhibited a higher abundance, broader antiviral spectrum, and lower metabolic burden under disinfectant stress. Moreover, significant positive correlations were observed between phage lysogenicity and enrichment of antiviral systems (e.g., Type IIG and IV restriction-modification (RM) systems, and Type II CRISPR-Cas system) in the presence of disinfection, indicating these antiviral systems might be more compatible with lysogenic phages and prophages. Accordingly, there was a stronger prokaryote-phage symbiosis in disinfected microbiome, and the symbiotic phages carried more auxiliary metabolic genes (AMGs) related to prokaryotic adaptability as well as antiviral systems, which might further enhance prokaryote survival in drinking water distribution systems. Overall, this study demonstrates that the prokaryotic antiviral systems had a close association with their symbiotic phages, which provides novel insights into prokaryote-phage interactions and microbial environmental adaptation.

Benzo[a]pyrene stress impacts adaptive strategies and ecological functions of earthworm intestinal viromes.

The earthworm gut virome influences the structure and function of the gut microbiome, which in turn influences worm health and ecological functions. However, despite its ecological and soil quality implications, it remains elusive how earthworm intestinal phages respond to different environmental stress, such as soil pollution. Here we used metagenomics and metatranscriptomics to investigate interactions between the worm intestinal phages and their bacteria under different benzo[a]pyrene (BaP) concentrations. Low-level BaP (0.1 mg kg-1) stress stimulated microbial metabolism (1.74-fold to control), and enhanced the antiphage defense system (n = 75) against infection (8 phage-host pairs). Low-level BaP exposure resulted in the highest proportion of lysogenic phages (88%), and prophages expressed auxiliary metabolic genes (AMGs) associated with nutrient transformation (e.g., amino acid metabolism). In contrast, high-level BaP exposure (200 mg kg-1) disrupted microbial metabolism and suppressed the antiphage systems (n = 29), leading to the increase in phage-bacterium association (37 phage-host pairs) and conversion of lysogenic to lytic phages (lysogenic ratio declined to 43%). Despite fluctuating phage-bacterium interactions, phage-encoded AMGs related to microbial antioxidant and pollutant degradation were enriched, apparently to alleviate pollution stress. Overall, these findings expand our knowledge of complex phage-bacterium interactions in pollution-stressed worm guts, and deepen our understanding of the ecological and evolutionary roles of phages.

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.

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.

Rapid Simulation of Decade-Scale Charcoal Aging in Soil: Changes in Physicochemical Properties and Their Environmental Implications.

In situ aging can change biochar properties, influencing their ecosystem benefits or risks over time. However, there is a lack of field verification of laboratory methods that attempt simulation of long-term natural aging of biochar. We exploited a decade-scale natural charcoal (a proxy for biochar) aging event to determine which lab-aging methods best mimicked field aging. We oxidized charcoal by ultraviolet A radiation (UVA), H2O2, or monochloramine (NH2Cl), and compared it to 10-year field-aged charcoal. We considered seven selected charcoal properties related to surface chemistry and organic matter release, and found that oxidation with 30% H2O2 most representatively simulated 10-year field aging for six out of seven properties. UVA aging failed to approximate oxidation levels while showing a distinctive dissolved organic carbon (DOC) release pattern. NH2Cl-aged charcoal was the most different, showing an increased persistent free radical (PFR) concentration and lower hydrophilicity. All lab oxidation techniques overpredicted polycyclic aromatic hydrocarbon release. The O/C ratio was well-correlated with DOC release, PFR concentration, surface charge, and charcoal pH, indicating the possibility to accurately predict biochar aging with a reduced suite of physicochemical properties. Overall, our rapid and verified lab-aging methods facilitate research toward derisking and enhancing long-term benefits of biochar application.

Mechanism of ß-blocker biodegradation by wastewater microorganisms.

The recalcitrant ß-blockers have been widely detected in aquatic environments up to several hundred µg/L, which are major contributors to ß1 antagonistic activities in wastewater. Their biodegradation mechanisms remain obscure, hindering the development of efficient removal techniques. This study constructed the biodegradation pathways for three typical ß-blockers, namely atenolol, metoprolol, and propranolol, assessed the toxicity of their major biotransformation products, and identified the key enzyme catalyzing the O-dealkylation reaction leading to pollutant mineralization. Atenolol and metoprolol degradation was more efficient than that of propranolol by activated sludge, producing metoprolol acid (MTPA) as a major intermediate. Hydrogenophaga sp. YM1 isolated from activated sludge possess the α-ketoglutarate dependent dioxygenase (TfdA) responsible for O-dealkylation of MTPA and propranolol, producing 4-hydroxyphenylacetic acid (4-HPA) that can be further degraded and ultimately enters the TCA cycle. The role of TfdA was verified by proteomics, enzyme stimulation/inhibition tests, and gene knockout experiments. Molecular docking suggests its different interactions with MTPA and propranolol. Acetate facilitated the degradation of ß-blockers efficiently. The results may shed light on enhanced biological removals of broader ß-blockers and their transformation products in the environment.

Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity.

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control.

Chlorination (but Not UV Disinfection) Generates Cell Debris that Increases Extracellular Antibiotic Resistance Gene Transfer via Proximal Adsorption to Recipients and Upregulated Transformation Genes.

To advance the understanding of antibiotic resistance propagation from wastewater treatment plants, it is important to elucidate how different effluent disinfection processes affect the dissemination of predominantly extracellular antibiotic resistance genes (eARGs). Here, we show that, by facilitating proximal adsorption to recipient cells, bacterial debris generated by chlorination (but not by UV irradiation) increases the natural transformation frequency of their adsorbed eARG by 2.9 to 7.2-fold relative to free eARGs. This is because chlorination increases the bacterial surface roughness by 1.1 to 6.7-fold and the affinity toward eARGs by 1.6 to 5.8-fold, and 98% of the total eARGs released after chlorination were adsorbed to cell debris. In contrast, UV irradiation released predominantly free eARGs with 18% to 56% lower transformation frequency. The collision theory indicates that the ARG donor-recipient collision frequency increased by 35.1-fold for eARGs adsorbed onto chlorination-generated bacterial debris, and the xDLVO model infers a 29% lower donor-recipient contact energy barrier for these ARGs. Exposure to chlorination-generated bacterial debris also upregulated genes associated with natural transformation in Vibrio vulnificus (e.g., tfoX encoding the major activator of natural transformation) by 2.6 to 5.2-fold, likely due to the generation of chlorinated molecules (5.1-fold higher Cl content after chlorination) and persistent reactive species (e.g., carbon-centered radicals) on bacterial debris. Increased proximal eARG adsorption to bacterial debris was also observed in the secondary effluent after chlorination; this decreased eARG decay by 64% and increased the relative abundance of ARGs by 7.2-fold. Overall, this study highlights that different disinfection approaches can result in different physical states of eARGs that affect their resulting dissemination potential via transformation.

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.

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.

Phage Predation Promotes Filamentous Bacterium Piscinibacter Colonization and Improves Structural and Hydraulic Stability of Microbial Aggregates.

Although bacteria-phage interactions have broad environmental applications and ecological implications, the influence of phage predation on bacterial aggregation and structural stability remains largely unexplored. Herein, we demonstrate that inefficient lytic phage predation can promote host filamentous bacterium Piscinibacter colonization onto non-host Thauera aggregates, improving the structural and hydraulic stability of the dual-species aggregates. Specifically, phage predation at 103-104 PFU/mL (i.e., multiplication of infection at 0.01-0.1) promoted initial Piscinibacter colonization by 10-15 folds and resulted in 29-31% higher abundance of Piscinibacter in the stabilized aggregates than that in the control aggregates without phage predation. Transcriptomic analysis revealed upregulated genes related to quorum sensing (by 15-92 folds) and polysaccharide secretion (by 10-90 folds) within the treated aggregates, which was consistent with 120-172% higher content of polysaccharides for the treated dual-species aggregates. Confocal laser scanning microscopic images further confirmed the increase of filamentous bacteria and polysaccharides (both with wider distribution) within the dual-species aggregates. Accordlingly, the aggregates' structural strength (via atomic force microscopes) and shear resistance (via hydraulic stress tests) increased by 77 and 42%, respectively, relative to the control group. In the long-term experiments, the enhanced hydraulic stability of the treated aggregates could facilitate dwelling bacteria propagation in flow-through conditions. Overall, our study demonstrates that phage predation can promote bacterial aggregation and enhance aggregate structural stability, revealing the beneficial role of lytic phage predation on bacterial symbiosis and environmental adaptivity.

Bacterial Inactivation and Biofilm Disruption through Indigenous Prophage Activation Using Low-Intensity Cold Atmospheric Plasma.

Biofilms can be pervasive and problematic in water treatment and distribution systems but are difficult to eradicate due to hindered penetration of antimicrobial chemicals. Here, we demonstrate that indigenous prophages activated by low-intensity plasma have the potential for efficient bacterial inactivation and biofilm disruption. Specifically, low-intensity plasma treatment (i.e., 35.20 W) elevated the intracellular oxidative reactive species (ROS) levels by 184%, resulting in the activation of prophage lambda (λ) within antibiotic-resistant Escherichia coli K-12 (lambda+) [E. coli (λ+)]. The phage activation efficiency was 6.50-fold higher than the conventional mitomycin C induction. Following a cascading effect, the activated phages were released upon the lysis of E. coli (λ+), which propagated further and lysed phage-susceptible E. coli K-12 (lambda-) [E. coli (λ-)] within the biofilm. Bacterial intracellular ROS analysis and ROS scavenger tests revealed the importance of plasma-generated ROS (e.g., •OH, 1O2, and •O2-) and associated intracellular oxidative stress on prophage activation. In a mixed-species biofilm on a permeable membrane surface, our "inside-out" strategy could inactivate total bacteria by 49% and increase the membrane flux by 4.33-fold. Furthermore, the metagenomic analysis revealed that the decrease in bacterial abundance was closely associated with the increase in phage levels. As a proof-of-concept, this is the first demonstration of indigenous prophage activations by low-intensity plasma for antibiotic-resistant bacterial inactivation and biofilm eradication, which opens up a new avenue for managing associated microbial problems.

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.