Metatranscriptomic Analysis Reveals Synergistic Activities of Comammox and Anammox Bacteria in Full-Scale Attached Growth Nitrogen Removal System.

Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is a poor understanding of how operational parameters, in particular, dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial community's transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility where nitrogen removal is predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated, relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supplies anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations.

Efficacy and Mechanism of Antibiotic Resistance Gene Degradation and Cell Membrane Damage during Ultraviolet Advanced Oxidation Processes.

Combinations of UV with oxidants can initiate advanced oxidation processes (AOPs) and enhance bacterial inactivation. However, the effectiveness and mechanisms of UV-AOPs in damaging nucleic acids (e.g., antibiotic resistance genes (ARGs)) and cell integrity represent a knowledge gap. This study comprehensively compared ARG degradation and cell membrane damage under three different UV-AOPs. The extracellular ARG (eARG) removal efficiency followed the order of UV/chlorine > UV/H2O2 > UV/peracetic acid (PAA). Hydroxyl radical (•OH) and reactive chlorine species (RCS) largely contributed to eARG removal, while organic radicals made a minor contribution. For intracellular ARGs (iARGs), UV/H2O2 did not remove better than UV alone due to the scavenging of •OH by cell components, whereas UV/PAA provided a modest synergism, likely due to diffusion of PAA into cells and intracellular •OH generation. Comparatively, UV/chlorine achieved significant synergistic iARG removal, suggesting the critical role of the RCS in resisting cellular scavenging and inactivating ARGs. Additionally, flow cytometry analysis demonstrated that membrane damage was mainly attributed to chlorine oxidation, while the impacts of radicals, H2O2, and PAA were negligible. These results provide mechanistic insights into bacterial inactivation and fate of ARGs during UV-AOPs, and shed light on the suitability of quantitative polymerase chain reaction (qPCR) and flow cytometry in assessing disinfection performance.

Gradual Recovery of Building Plumbing-Associated Microbial Communities after Extended Periods of Altered Water Demand during the COVID-19 Pandemic.

COVID-19 pandemic-related building restrictions heightened drinking water microbiological safety concerns post-reopening due to the unprecedented nature of commercial building closures. Starting with phased reopening (i.e., June 2020), we sampled drinking water for 6 months from three commercial buildings with reduced water usage and four occupied residential households. Samples were analyzed using flow cytometry and full-length 16S rRNA gene sequencing along with comprehensive water chemistry characterization. Prolonged building closures resulted in 10-fold higher microbial cell counts in the commercial buildings [(2.95 ± 3.67) × 105 cells mL-1] than in residential households [(1.11 ± 0.58) × 104 cells mL-1] with majority intact cells. While flushing reduced cell counts and increased disinfection residuals, microbial communities in commercial buildings remained distinct from those in residential households on the basis of flow cytometric fingerprinting [Bray-Curtis dissimilarity (dBC) = 0.33 ± 0.07] and 16S rRNA gene sequencing (dBC = 0.72 ± 0.20). An increase in water demand post-reopening resulted in gradual convergence in microbial communities in water samples collected from commercial buildings and residential households. Overall, we find that the gradual recovery of water demand played a key role in the recovery of building plumbing-associated microbial communities as compared to short-term flushing after extended periods of reduced water demand.

Spatial dynamics of bacterial community in chlorinated drinking water distribution systems supplied with two treatment plants: An integral study of free-living and particle-associated bacteria.

With the expansion of cities, the deterioration of drinking water quality undergoing complex and long-distance distribution is gaining increasing attention. However, spatial variations between free-living bacteria (FLB) and particle-associated bacteria (PAB) in chlorinated drinking water distribution systems (DWDSs) have not been fully explored, especially in complex water supply areas with multiple interconnected DWDSs. To fill this gap, this study utilized 16S rRNA approaches to characterize the spatial patterns of FLB and PAB in DWDSs with intersection regions. Based on distance-decay analysis, transportation distance is a potential driver of bacterial variation for both FLB (Pearson's r = -0.476, p < 0.01) and PAB. (Pearson's r = -0.352, p < 0.01). Moreover, the influence of transportation distance was further confirmed by a 1.20-99.45% decline in microbial contribution to the source of FLB and PAB communities in pipe water along the transportation pipelines. Meanwhile, significant difference (PERMANOVA, R2 = 0.14, p < 0.01) was found between FLB and PAB in DWDSs. Average proportions of Pseudomonas spp. were 59.84% and 45.59% for the PAB and intersection regions based on the 16S rRNA results, respectively, suggesting that PAB are potential reservoirs for high-risk bacteria, and a greater microbial risk may exist in intersection regions. In summary, transportation distance and pipeline intersection exerted significant impacts on the FLB and PAB in DWDSs. Therefore, precautionary strategies for controlling microbial risks that consider different microbial components and intersection regions in long-distance and multi-plant DWDSs should be implemented.

Structure-dependent antimicrobial mechanism of quaternary ammonium resins and a novel synthesis of highly efficient antimicrobial resin.

The demand for powerful and multifunctional water-treatment materials and reagents is increasing, because we are facing worse raw water quality, various tolerant bacteria, and risky disinfection by-products (DBPs) in drinking water. Quaternary ammonium resins (QARs) are promising candidates for water disinfection and purification, but their limited bactericidal capacities are difficult to improve because of the lack of guidelines for enhancing antibacterial efficiency. Therefore, we first systematically studied the structure-dependent antimicrobial mechanism of QARs and found that the best resin skeleton is acrylic-type, the optimal bactericidal alkyl is hexyl or octyl, the most applicable sizes are 80-100 meshes, the best counter anion is iodide ion, and the optimum quaternization reagent is iodoalkane. Moreover, the antibacterial capacity was demonstrated to depend on surficial N+ groups, correlating with surficial N+ charge density (R2 of 0.98) but not with exchange capacity (R2 of 0.26), physical adsorption of resin skeleton, or electrostatic adsorption of N+ groups. Based on these principles, we synthesized a new resin, Ac-81, with a surficial antibacterial design, which simultaneously exhibited better antimicrobial efficiency (two orders of magnitude) as well as higher contaminant removal potential (61.92%) compared to the traditional Ac-8C antibacterial resin. Furthermore, the new resin showed remarkable broad-spectrum antibacterial effects against Gram-negative E. coli and P. aeruginosa and Gram-positive B. subtilis and S. aureus in simulated water and actual water. Simultaneously, water quality was significantly improved, with HCO3-, SO42-, TN, TP, and TOC reduced by 79-90%, >99%, 66-85%, >99%, and 22-26%, respectively. Ac-81 is characterized by facile reusability, high treatment capacity of 1500 bed volume, and good adaptability for treating actual water, providing a promising alternative for drinking-water disinfection and purification.

Surficial N+ charge density indicating antibacterial capacity of quaternary ammonium resins in water environment.

The antibacterial effects of quaternary ammonium resins (QARs) have been reported for decades, but there are few practical applications because of limited improvements in bactericidal capacity and the absence of an efficient antibacterial-indicating parameter. An in-situ determination method of surficial N+ groups for QARs, defined as surficial N+ charge density, was first established to merely quantify the exposed surficial quaternary ammonium groups (QAs). The mechanism of the new method depends on the tetraphenylboron sodium standard solution (TS), which is a colloidal solution with high steric hindrance, making it difficult to permeate into QARs and further react with the inner QAs. The results showed that the antibacterial efficacy of QARs correlates with the surficial N+ charge density with R2 > 0.95 (R2 of 0.97 for Escherichia coli, R2 of 0.96 for Staphylococcus aureus) but not with the strong-base group exchange capacity or zeta potential. Furthermore, the surficial N+ charge density was demonstrated efficient to indicate the antibacterial capacities against both gram-negative and gram-positive bacteria for commercial QARs, including acrylic, styrene and pyridine resin skeletons, especially for the QARs with similar skeletons and similar QAs. Based on the finding that the bactericidal groups merely involve the surficial QAs of QARs, this study proposes a new direction for improving the antibacterial capacity by enriching the surficial QAs and enhancing the bactericidal property of these surficial QAs, and provides a practicable synthesis with two-step quaternization.

Metagenomic profiling of antibiotic resistance genes and their associations with bacterial community during multiple disinfection regimes in a full-scale drinking water treatment plant.

For comprehensive insights into the effects of multiple disinfection regimes on antibiotic resistome in drinking water, this study utilized metagenomic approaches to reveal the changing patterns of antibiotic resistance genes (ARGs) and bacterial community as well as their associations. A total of 297 ARGs within 17 types were detected in the drinking water, and their total relative abundance ranged from 195.49 ± 24.85 to 626.31 ± 38.61 copies of ARGs per cell. The total ARG abundance was significantly increased after the antimicrobial resin and ultraviolet (AR/UV) disinfection while significantly decreased after the ozone and chlorine (O3/Cl2) disinfection and remained stable after AR/Cl2 disinfection. Overall, 18 ARGs including bacA, mexT, and blaOXA-12, mainly affiliated to bacitracin, multidrug, and beta-lactam, were persistent and discriminative during all the disinfection strategies in drinking water, and they were considered as key ARGs that represent the antibiotic resistome during drinking water disinfection. Additionally, possible hosts of 50% key ARGs were revealed based on co-occurrence network. During multiple disinfection processes, the change of Fusobacteriales and Aeromonadaceae in abundance mainly contributed to the abundance shift of bacA, and Pseudomonas mainly increased the abundance of mexT. These findings indicated that bacterial community shift may be the key factor driving the change of antibiotic resistome during disinfection. The strong association between antibiotic resistome alteration and bacterial community shift proposed in this study may enhance our understanding of the underlying mechanism of the disinfection effects on antibiotic resistance and benefit effective measures to improve safety of drinking water.