Climate change, unpredictable weather patterns, and droughts are depleting water resources in some parts of the globe, where recycling and reusing wastewater is a strategy for different purposes. To counteract this, the EU regulation for water reuse sets minimum requirements for the use of reclaimed water for agricultural irrigation, including a reduction in human enteric viruses. In the present study, the occurrence of several human enteric viruses, including the human norovirus genogroup I (HuNoV GI), HuNoV GII, and rotavirus (RV), along with viral fecal contamination indicator crAssphage was monitored by using (RT)-qPCR methods on influent wastewater and reclaimed water samples. Moreover, the level of somatic coliphages was also determined as a culturable viral indicator. To assess the potential viral infectivity, an optimization of a capsid integrity PMAxx-RT-qPCR method was performed on sewage samples. Somatic coliphages were present in 60% of the reclaimed water samples, indicating inefficient virus inactivation. Following PMAxx-RT-qPCR optimization, 66% of the samples tested positive for at least one of the analyzed enteric viruses, with concentrations ranging from 2.79 to 7.30 Log10 genome copies (gc)/L. Overall, most of the analyzed reclaimed water samples did not comply with current EU legislation and contained potential infectious viral particles.

Sewage contamination of environmental waters is increasingly assessed by measuring DNA from sewage-associated microorganisms in microbial source tracking (MST) approaches. However, DNA can persist through wastewater treatment and reach surface waters when treated sewage/recycled water is discharged, which may falsely indicate pollution from untreated sewage. Recycled water discharged from an advanced wastewater treatment (AWT) facility into a Florida stream elevated the sewage-associated HF183 marker 1,000-fold, with a minimal increase in cultured Escherichia coli. The persistence of sewage-associated microorganisms was compared by qPCR in untreated sewage and recycled water from conventional wastewater treatment (CWT) and AWT facilities. E. coli (EC23S857) and sewage-associated markers HF183, H8, and viral crAssphage CPQ_056 were always detected in untreated sewage (6.5-8.7 log10 GC/100 mL). Multivariate analysis found a significantly greater reduction of microbial variables via AWT vs CWT. Bacterial markers decayed ~4-5 log10 through CWT, but CPQ_056 was ~100-fold more persistent. In AWT facilities, the log10 reduction of all variables was ~5. In recycled water, bacterial marker concentrations were significantly correlated (P ≤ 0.0136; tau ≥ 0.44); however, CPQ_056 was not correlated with any marker, suggesting varying drivers of decay. Concentrations of cultured E. coli carrying the H8 marker (EcH8) in untreated sewage were 5.24-6.02 log10 CFU/100 mL, while no E. coli was isolated from recycled water. HF183 and culturable EcH8 were also correlated in contaminated surface waters (odds ratio β1 = 1.701). Culturable EcH8 has a strong potential to differentiate positive MST marker signals arising from treated (e.g., recycled water) and untreated sewage discharged into environmental waters.
OBJECTIVE: Genes in sewage-associated microorganisms are widely accepted indicators of sewage pollution in environmental waters. However, DNA persists through wastewater treatment and can reach surface waters when recycled water is discharged, potentially causing false-positive indications of sewage contamination. Previous studies have found that bacterial and viral sewage-associated genes persist through wastewater treatment; however, these studies did not compare different facilities or identify a solution to distinguish sewage from recycled water. In this study, we demonstrated the persistence of bacterial marker genes and the greater persistence of a viral marker gene (CPQ_056 of crAssphage) through varying wastewater treatment facilities. We also aim to provide a tool to confirm sewage contamination in surface waters with recycled water inputs. This work showed that the level of wastewater treatment affects the removal of microorganisms, particularly viruses, and expands our ability to identify sewage in surface waters.

As global effects of water scarcity raise concerns and environmental regulations evolve, contemporary wastewater treatment plants (WWTPs) face the challenge of effectively removing a diverse range of contaminants of emerging concern (CECs) from municipal effluents. This study focuses on the assessment of advanced oxidation processes (AOPs), specifically UV-C/H2O2 and UV-C/Chlorine, for the removal of 14 target CECs in municipal secondary effluent (MSE, spiked with 10 μg L-1 of each CEC) or in the subsequent MSE nanofiltration retentate (NFR, no spiking). Phototreatments were carried out in continuous mode operation, with a hydraulic retention time of 3.4 min, using a tube-in-tube membrane photoreactor. For both wastewater matrices, UV-C photolysis (3.3 kJ L-1) exhibited high efficacy in removing CECs susceptible to photolysis, although lower treatment performance was observed for NFR. In MSE, adding 10 mg L-1 of H2O2 or Cl2 enhanced treatment efficiency, with UV-C/H2O2 outperforming UV-C/Chlorine. Both UV-C/AOPs eliminated the chronic toxicity of MSE toward Chlorella vulgaris. In the NFR, not only was the degradation of target CECs diminished, but chronic toxicity to C. vulgaris persisted after both UV-C/AOPs, with UV-C/Chlorine increasing toxicity due to potential toxic by-products. Nanofiltration permeate (NFP) exhibited low CECs and microbial content. A single chlorine addition effectively controlled Escherichia coli regrowth for 3 days, proving NFP potential for safe reuse in crop irrigation (<1 CFU/100 mL for E. coli; <1 mg L-1 for free chlorine). These findings provide valuable insights into the applications and limitations of UV-C/H2O2 and UV-C/Chlorine for distinct wastewater treatment scenarios.

The water crisis may be solved by utilizing reclaimed water. Three reclaimed water sources have restored the lower sections of the Licun River, forming a landscaped river. In this paper, the river\'s water quality was monitored for a year, and the ecological concerns were analyzed using luminescent bacteria, chlorella, and zebrafish. The results indicated that although basic water quality indicators like COD and ammonia fluctuated along the river, the classification of water quality was primarily affected by factors such as flow rate and water depth. Under experimental conditions, the toxic inhibitory effect of river water on luminescent bacteria, chlorella, and zebrafish was related to the treatment process of reclaimed water. It was found that the reclaimed water produced by the MBR, along with the UV disinfection process, showed no detectable toxicity. In contrast, the MBBR process, when combined with coagulation, sedimentation, filtration, ozonation, and chlorination, seemed to be the source of this toxicity. Along the river, the results of water quality assessments and ecological risk assessments were different, indicating that both should be conducted to evaluate rivers replenished with reclaimed water.

Enteric virus concentration in large-volume water samples is crucial for detection and essential for assessing water safety. Certain dissolution and suspension components can affect the enrichment process. In this study, tangential flow ultrafiltration (TFUF) was used as an enrichment method for recovering enteric virus in water samples. Interestingly, the bacteriophage MS2 recovery in reclaimed water and the reclaimed water without particles were higher than that in ultrapure water. The simulated reclaimed water experiments showed that humic acid (HA) (92.16% ± 4.32%) and tryptophan (Try) (81.50 ± 7.71%) enhanced MS2 recovery, while the presence of kaolin (Kaolin) inhibited MS2 recovery with an efficiency of 63.13% ± 11.17%. Furthermore, Atomic force microscopy (AFM) revealed that the MS2-HA cluster and the MS2-Try cluster had larger roughness values on the membrane surface, making it difficult to be eluted, whereas MS2-Kaolin cluster had compact surfaces making it difficult to be eluted. Additionally, the MS2-HA cluster is bound to the membrane by single hydrogen bond with SO, whereas both the MS2-Try cluster and the MS2-Kaolin cluster are bound to the membrane by two hydrogen bonds, making eluting MS2 challenging. These findings have potential implications for validating standardized methods for virus enrichment in water samples.

Biofilms attached to submerged macrophytes play an important role in improving the water quality of the water environment supplemented with reclaimed water. In order to explore the effects of reclaimed water quality and submerged macrophyte species on the characteristics of an epiphytic bacterial community, different types of submerged macrophytes were selected as research objects in this study. 16S rRNA high-throughput sequencing technology was used on the epiphytic bacteria and the surrounding environmental samples to analyze the bacterial community structure and functional genes. The results showed that approximately 20%-35% of the nitrogen and phosphorus nutrients were absorbed and utilized in the water environment supplemented with reclaimed water. However, the COD, turbidity, and chroma of the downstream water were significantly increased. The bacterial community of the biofilms attached to submerged macrophytes was significantly different from that in the surrounding environment (soil, sediment, and water body) and in the activated sludge that was treated by reclaimed water. In terms of bacterial community diversity, the richness and diversity were significantly lower than those of soil and sediment but higher than those of plankton bacteria in water. In terms of bacterial community composition, dominant genera and corresponding abundances were also different from those of other samples. The main dominant bacterial genera were Sphingomonas, Aeromonas, Pseudomonas, and Acinetobacter, accounting for 7%-40%, respectively. Both macrophyte species and the quality of reclaimed water (BOD5, TN, NH4+-N, and TP) could affect the bacterial community. However, the effect of water quality of the bacterial community was greater than that of macrophytes species. Additionally, the quality of reclaimed water also affected the abundance of functional genes in the bacterial community, and the relative abundance of nitrogen and phosphorus cycling functional genes was higher in areas with higher nitrogen and phosphorus concentrations.

The use of reclaimed water for urban river replenishment has raised concerns regarding its impact on water quality and aquatic ecosystems. This study aims to reveal the improvements seen in an urban river undergoing a practical water eco-remediation after being replenished with reclaimed water. A one-year monitoring of water quality, phytoplankton, and zooplankton was carried out in Dongsha River undergoing eco-remediation in Beijing, China. The results showed that compared to the unrestored river, the concentrations of COD, NH4+-N, TP, and TN decreased by 28.22 ± 7.88 %, 40.24 ± 11.77 %, 44.17 ± 17.29 %, and 28.66 ± 10.39 % in the restoration project area, respectively. The concentration of Chlorophyll-a in the restoration area was maintained below 40 μg/L. During summer, when algal growth is vigorous, the density of Cyanophyta in the unrestored river decreased from 46.84 × 104cells/L to 16.32 × 104cells/L in the restored area, while that of Chlorophyta decreased from 41.61 × 104cells/L to 11.87 × 104cells/L, a reduction of 65.16 % and 71.47 %, respectively. The dominant phytoplankton species were replaced with Bacillariophyta, such as Synedra sp. and Nitzschia sp., indicating that the restoration of aquatic plants reduces the risk of Cyanophyta blooms. Zooplankton species also changed in the restoration area, especially during summer. The density of pollution-tolerant Rotifer and Protozoa decreased by 31.06 % and 27.22 %, while the density of clean water indicating Cladocera increased by 101.19 %. We further calculated the diversity and evenness index of phytoplankton and zooplankton within and outside the restoration area. The results showed that the Shannon-Weaver index for phytoplankton and zooplankton in the restoration area was 2.1 and 1.91, which was higher than those in the river (1.84 and 1.82). This further confirmed that aquatic plant restoration has positive effects. This study can provide a practical reference and theoretical basis for the implementation of water ecological restoration projects in other reclaimed water rivers in China.

At the time when water resources are in short supply,wastewater recycling is both an important environmental protection strategy and also a resource strategy. Disinfection is essential to ensure the biological safety of reclaimed wastewater by killing pathogens and preventing the spread of waterborne diseases. However,the disinfection process could inevitably produce toxic disinfection byproducts(DBPs)due to the reaction between the disinfectants and wastewater organic matters. Regarding wastewater DBPs,this study reviewed their identification methods,formation conditions(including precursors,the effect of water quality,disinfectants,and operational parameters on DBPs),and control methods(including source control,process control,and end control). In addition,future research trends of wastewater DBPs were discussed.

Reclaimed water with nitrogen, phosphorus, and other contaminants may trigger algal blooms during its ecological utilization in replenishing rivers or lakes. However, the effect of reclaimed water on algal growth rates is not well understood. In this study, the growth potentials of algae in terms of Cyanophyta, Chlorophyta, and Bacillariophyta, as well as mixed algae in both regular culture medium and reclaimed water produced from treatment plants in Beijing with similar N and P concentrations, were compared to evaluate whether reclaimed water could facilitate algal growth. In addition, reclaimed water was also sterilized to verify the impact of bacteria\'s presence on algal growth. The results indicated that most algae grew faster in reclaimed water, among which the growth rate of Microcystis aeruginosa even increased by 5.5 fold. The growth of mixed algae in reclaimed water was not enhanced due to the strong adaptive ability of the community structure. Residual bacteria in the reclaimed water were found to be important contributors to algal growth. This work provided theoretical support for the safe and efficient utilization of reclaimed water.