Effect of NaClO and ClO2 on the bacterial properties in a reclaimed water distribution system: efficiency and mechanisms

Extensive application of reclaimed water alleviated water scarcity obviously. Bacterial proliferation in reclaimed water distribution systems (RWDSs) poses a threat to water safety. Disinfection is the most common method to control microbial growth. The present study investigated the efficiency and mechanisms of two widely used disinfectants: sodium hypochlorite (NaClO) and chlorine dioxide (ClO2) on the bacterial community and cell integrity in effluents of RWDSs through high-throughput sequencing (Hiseq) and flow cytometry, respectively. Results showed that a low disinfectant dose (1 mg/L) did not change the bacterial community basically, while an intermediate disinfectant dose (2 mg/L) reduced the biodiversity significantly. However, some tolerant species survived and multiplied in high disinfectant environments (4 mg/L). Additionally, the effect of disinfection on bacterial properties varied between effluents and biofilm, with changes in the abundance, bacterial community, and biodiversity. Results of flow cytometry showed that NaClO disturbed live bacterial cells rapidly, while ClO2 caused greater damage, stripping the bacterial membrane and exposing the cytoplasm. This research will provide valuable information for assessing the disinfection efficiency, biological stability control, and microbial risk management of reclaimed water supply systems.


Introduction
Reclaimed water was widely used in industrial recycling, agricultural irrigation, ecological rivers, and artificial wetlands, replenishing to alleviate the global water scarcity (Hu et al., 2020). However, the higher nutrient content of transported water could enhance bacterial proliferation in reclaimed water distribution systems (RWDSs) (Jjemba et al., 2010). These bacteria in RWDSs usually cause some detrimental consequences, such as the destruction of biostability, deterioration of taste or odor, discoloration, biocorrosion, and a series of undesirable water quality changes (Ma et al., 2020;Sun et al., 2014;Yang et al., 2014). Thus, it is crucial and urgent to control bacterial multiplication for RWDSs (Liu et al., 2017).
Among the methods to control microorganism growth, disinfection, such as applying chlorine, sodium hypochlorite (NaClO), chlorine dioxide (ClO 2 ), monochloramine, and ozone, is widely utilized (Luo et al., 2021a;Ramseier et al., 2011). However, once the disinfectant enters the RWDSs, it will also react with inorganic or organic substances in the mains water and pipe wall sediment, inducing the reduction of residual disinfectants (Ng et al., 2015;Zhu et al., 2020). Therefore, the regrowth of bacteria or opportunistic pathogens (OPs) often occurs, as well as the change in the bacterial activity, abundance, and community (Liu et al., 2018). Previous studies have also confirmed that a range of microorganisms was multiplicated under different disinfectant doses in RWDSs (Wang et al., 2019a;Wang et al., 2019b). Most previous studies investigated bacterial characteristics under disinfection based on cultivation-dependent techniques such as the heterotrophic plate count (HPC) and indicator organisms (Kwon et al., 2011). Recently, the newly-emerged culture-independent techniques such as highthroughput sequencing and flow cytometry (FCM) provided new possibilities for determining microbial properties systematically and accurately (Lu et al., 2013;Safford and Bischel, 2019).
High-throughput sequencing provided more information on the bacterial characteristics in water distribution system, such as the abundance, diversity, and taxonomy of microbial communities. Differences in the bacterial community caused by diverse disinfection dosing are more significant than those by time variables and pipe materials in water transportation (R 2 = 0.283, p < 0.01) (Zhang et al., 2019a). Some studies suggested that low-dose disinfectant (NaClO 0.04-0.56 mg/L, ClO 2 0.15-0.39 mg/L) promotes bacterial diversity, while high dose (NaClO 1.02-4.00 mg/L, ClO 2 0.60-2.00 mg/L) reduces the bacterial diversity in drinking water distribution systems (Ma et al., 2020;Mi et al., 2015;Song et al., 2020). However, when the disinfectant concentration (NaClO and ClO 2 ) increases to 2-4 mg/L, the original characteristics of the intrinsic community are reduced, while some disinfectant-resistant bacteria multiplied (Ersoy et al., 2019;Mi et al., 2015;Zhu et al., 2021). The relative abundance of Flavobacterium, Phreatobacter, and Porphyrobacter increased with elevated ClO 2 concentrations, and the relative abundance of Mycobacterium and Legionella increased with enhanced of NaClO concentrations in drinking water distribution system (Luo et al., 2021b;Ma et al., 2020;Mi et al., 2015). However, the effect of disinfection on bacterial property evolution in RWDSs is still unclear.
The recently developed FCM technology represents an efficient, accurate, and cultivation-independent approach to determine viable cells in cell suspensions (Flores-Barbosa et al., 2020;Garner et al., 2018). FCM is an online sensor technology that can detect the total cell count data (Farhat et al., 2020). Some studies have used FCM to distinguish dead and living bacteria (Wang et al., 2019a), while it is generally applied to evaluate the membrane integrity and permeability of disinfectant-inactivated bacteria in drinking water (Jia et al., 2020;Liu et al., 2016). In recent years, flow cytometry has been gradually introduced into the investigations of bacterial activity in rivers and landscaped lakes . However, a detailed analysis of bacterial viability in reclaimed water is still lacking.
The complexity of the survival mechanisms of microorganisms makes it difficult to determine the effects of disinfectants on shaping the microbial community, cell decay, and these survival properties (Kennedy et al., 2021;Pinel et al., 2020). Therefore, the main objective of this study was to investigate the impacts of different disinfectants (NaClO and ClO 2 ) at various doses (1, 2, and 4 mg/L) on bacterial properties in effluents of RWDSs. High-throughput sequencing technology was utilized to analyze the microbial diversity and community characteristics in the effluents. More detailed flow cytometry was used to detect the survival ability of bacterial cell integrity when exposed to continuous disinfection. Furthermore, the disinfection efficiency and mechanisms of NaClO and ClO 2 on bacteria were also discussed. These findings may help to refine microbial risk management from new perspectives, which may, in turn, allow operators to choose effective disinfection measures to guarantee the safety of reclaimed water more accurately.

Experiment setup
Annular reactors (ARs) with an effective volume of 1.5 L were used to simulate RWDSs (Wang et al., 2012). The ARs were sterilized by ultraviolet lamps on an ultra-clean platform for 30 min before use. Absolute ethanol was pumped into the ARs for further cleaning, and then, the reclaimed water was pumped in. Each AR comprised of a rotating inner cylinder driven by a brushless direct current motor, with a simulated pipe flow rate of approximately 0.5 m/s. A peristaltic pump was used to pump the reclaimed water into the ARs. The rotor speed of the peristaltic pump was 1 r/min, and the corresponding flow rate was 0.052 mL/s to maintain the hydraulic retention time (HRT) of ARs at 8 h.
The reclaimed water used in this experiment was obtained from a reclaimed water plant in North China; the pretreatment process of the plants is coagulation → sedimentation → decolorization process (ozone oxidation) → continuous microfiltration (CMF) technology. According to the Standard Test Methods for Drinking Water in China (GB/T 5750.1) and Analysis Method for Water and Waste Water (Wei et al., 2022), the used reclaimed water quality parameters and the detection methods can be seen in Table 1.

Experimental operation and design
Three groups of ARs (Supporting Information figure 1) were used to simulate three working status (two parallel per group), which were operated continuously for 90 days. The 1 3 first group of ARs was filled with reclaimed water without disinfectant as a reference (W1). For the second (W2) and third groups (W3), reclaimed water was disinfected with NaClO or ClO 2 , respectively. In the disinfection experiments, reclaimed water treated with NaClO or ClO 2 at 1, 2, and 4 mg/L were pumped in ARs successively for 30 days, respectively, corresponding to stages I (days 0-30), II (days 31-60), and III (days 61-90). About the disinfectant concentration selection, 1 and 2 mg/L are the most commonly used ones in water distribution systems, while 4 mg/L is the maximum ones stipulated by standard methods (standards for drinking water quality, GB5749-2022) (Wei et al., 2022). Highly concentrated sodium hypochlorite (NaClO) solution was diluted with deionized water to prepare the disinfectant solutions at the required concentrations (Jiangtian Chemical Co., Ltd., Tianjin, China). Each concentration of chlorine dioxide (ClO 2 ) solution was prepared using chlorine dioxide powder (Hualong Xingyu Technology Development Company, Beijing, China). The residual chlorine and chlorine dioxide concentrations were measured with a HACH pocket colorimeter (HACH DR1890 colorimeter, HACH Company, Loveland, CO, USA).

DNA extraction and sequencing
W0 represented raw reclaimed water (during the experiment, this water was renewed every 2 days to ensure that the influents were stable). The microbial samples in effluents of ARs were collected on days 3, 10, 20, 30, 60, and 90. The sampled bacteria were labeled as "Wndx," where "n" represented the AR groups 1, 2, and 3, and "x" represented the sampled days.
The Illumina HiSeq 2500 platform was used to analyze the microbial diversity and community in the effluents (Novo Gene Biological Information Technology Co., Ltd, Beijing, China). First, 0.45 μm filter membranes were used to filter the microorganisms in the water. The Water DNA Kit (Omega, D5525-02) was used to extract DNA from the filter membrane. The obtained DNA was then examined by 1% agarose gel electrophoresis (AGE) and observed by an ultraviolet analyzer, and the strip was detected for eligibility. DNA samples were stored in the refrigerator at −20 °C, and PCR amplicon libraries were constructed with the bacterial primers 515F (5′-GTG CCA GCMGCC GCG G-3′) and 806R (5′-GGA CTA CHVGGG TWT CTAAT-3′) targeting V3 and V4 hypervariable regions. The original data sequence was divided into the bacterial types with Mothur v.1.32.0 under 97% similarity. The Chao index was used to analyze the alpha diversity of sample community groups. A principal component analysis (PCA) was used to analyze the beta diversity of grouped sample communities. The raw reads were deposited into NCBI (http:// www. ncbi. nlm. nih. gov/) and reported in the sequence read archive (SRA), as SRP333244 and SRP333125.
The bacterial communities between influents and biofilm were also compared. The biofilm was removed by scraping with sterile spatulas and sterile cotton, and the subsequent extraction process was as previously described (Zhang et al., 2019b). The numbers are as follows: W1: mixed samples in reference effluents on days 30, 60, and 90; W2 and W3: corresponding samples in NaClO and ClO 2 dosing experiments; G1, G2, and G3: mixed samples in the biofilm of reference experiments; and NaClO and ClO 2 dosing experiments, respectively.

Apoptosis status analysis of microbial cells
Advanced BD FACSCalibur flow cytometry was used to determine the apoptosis status of microbial cells in effluents after disinfection (FACSCalibur; BD annexin V: FITC Apoptosis Detection Kit). Before the test, cells were pretreated with the following steps: first, cells were washed once with pre-cooled phosphate buffer solution (PBS), then re-suspended with binding buffer, and finally, the cell concentration was adjusted to 1-5 × 10 6 cells/mL. Propidium iodide (PI) was used to determine membrane integrity and cellular activity in effluents. Cell suspension (100 μL) was sampled; then, 5 μL annexin V and 5 μL PI were added and mixed gently and incubated for 20 min at room temperature in the dark. Binding buffer (400 μL) was added and then placed into the flow cytometer for further analysis. The effects of impurities on bacterial detection were excluded through the differences between the side and forward light scatter of FCM reactions (Jia et al., 2020). The changes in cellular activity were observed by the shifts in fluorescence positions of bacterial clusters (Berney et al., 2008;Liu et al., 2016).

Residual disinfectant concentration variations
In the process of reclaimed water transportation, the attenuation of disinfectant is inevitable. The residual disinfectant concentration actually worked on the microbes. Residual disinfectant concentrations in ARs treated with NaClO and ClO 2 at the different operation times are shown in Fig. 1.
In the first 30 days, the residual disinfectant concentration in effluents of ARs treated with both NaClO or ClO 2 was less than 0.5 mg/L (initial disinfectant dosing: 1 mg/L), showing an apparent attenuation. Subsequently, when the initial disinfectant dosing concentration increased to 2 mg/L, the residual disinfectant concentration increased slowly and achieved a relatively stable value in both ARs. The residual chlorine concentration was 1.0 mg/L in the NaClO dosing experiments, and the residual ClO 2 concentration was 0.8 mg/L in the ClO 2 dosing experiments, representing a clear difference (p < 0.05). In summary, the attenuation of ClO 2 was relatively higher than that of NaClO in the first 60 days. This could be related to the higher oxidation capacity of ClO 2 towards iron corrosion in the pipe surface (Zhu et al., 2020). Our previous study has confirmed that disinfectant attenuation was consistent with the iron corrosion rate tendency, and higher corrosion rate was found in ClO 2 dosing environments (Zhang et al., 2019b).
However, after 60 days, the residual disinfectant concentration of effluents in ARs treated with ClO 2 sharply increased and became higher than that in ARs treated with NaClO (p < 0.05), suggesting the relatively higher disinfectant attenuation in the latter. As the corrosion scales accumulate, the variation in disinfectant after 60 days might be related to the corrosion scale characteristics. It is likely that ClO − with a smaller ion radius than ClO 2 could penetrate the thick corrosion scales more easily and interact with iron accumulations more effectively, thus causing the higher NaClO attenuation after 60 days (Guo, 1984;Zhang et al., 2019b;Zheng et al., 2007). Previous studies also confirmed that high concentrations of chlorine disinfectant could impact the morphology and characteristics of oxide films, making them became loose and porous and inducing the ferrous iron and microorganisms penetrating further. Then, in the inner of corrosion scales, the chlorine will attenuate continuously (Sharafimasooleh et al., 2016).

Effects of disinfectant type and dosage on biodiversity
A total of 53349 high-quality microbial V3-V4 sequences were obtained from effluents of ARs with or without disinfection. These sequences were classified into 284 operational taxonomic units (OTUs) at a 97% similarity level (Supporting Information 2). Abundance-rank curves indicated that the sequence depths of all the samples were adequate (Supporting Information 3). The alpha diversity variations of influents and effluents in ARs can be seen in Table 2.
In the reference experiment (W1 group), Chao indices in effluents increased over time, demonstrating the proliferation of microorganisms with AR operation. In the first 30 days, there were no significant differences in the Chao index for disinfected effluents and the reference ones, suggesting that the relatively low disinfectant concentration (1 mg/L) did change the microbial abundance significantly (p > 0.05). The Chao indices of samples W2d60 and W3d60 were lower than that of W1d60, showing that 2 mg/L NaClO and ClO 2 could reduce the bacterial abundance effectively. However, when the disinfectant concentration gradually increased to 4 mg/L, the Chao indices in disinfected samples (W2d90) became higher than that in reference effluents (W1d90), demonstrating that a higher disinfectant dose did not reduce the biodiversity further. This might be related with the emergence of disinfectant-resistant bacteria (DRB) (El-Chakhtoura et al., 2018;Ma et al., 2020;Moraga-McHaley et al., 2013;Wang et al., 2019a). Further details of these bacteria will be discussed in "Relative abundance of species at the phylum level and class level" section and "Analysis of bacterial community structure transformation" section.
The beta diversity represented by PCoA plots can be seen in Fig. 2.
Distinct differences were observed between influents (W0 group), reference effluents (W1 group), and disinfectant by NaClO (W2 group) or ClO 2 (W3 group). The W1 group was far away from W0, showing a marked shift of the microbial community in the undisinfected effluents, confirming the bacterial growth in water supply system. The W2 group was farthest from the W0 group, suggesting that NaClO dosing reshaped the bacterial community in effluents significantly. However, the W3 group was relatively closer to the W0 group, indicating that ClO 2 disinfection changed the microbial community in effluents a little. The specific cluster analysis of the bacteria between the samples is discussed below.

Relative abundance of species at the phylum level and class level
The bacterial properties (community and dominant species) in different disinfection environments also changed significantly. The unweighted pin-group method with arithmetic mean (UPGMA) is a common cluster analysis method used to analyze the similarity between samples. Figure 3a shows the relative abundance of microorganisms and UPGMA  194 cluster tree analysis at the phylum level in effluents. Figure 3b illustrates the relative bacterial abundance in effluents of each AR at the class level. In sample W0, Proteobacteria was the dominant phylum, accounting for approximately 96% of the total bacterial community, followed by Firmicutes and Bacteroidetes, both of which accounted for more than 4% of the total community. At the class level, Gammaproteobacteria, Betaproteobacteria, Alphaproteobacteria, Bacillus, Clostridia, and Flavobacteria predominated in raw influents (W0). With the increase of operational time, the abundance of Proteobacteria decreased, while Firmicutes increased in effluents without disinfection (W1d60, W1d90) at the phylum level, and proportion of Bacilli and Clostridia increased, while the abundance of Gammaproteobacteria decreased at class level, demonstrating the transformations of bacteria in reclaimed water distribution process (Hu et al., 2021).
Compared with samples of the W1 group (W1d3, W1d10, W1d30) in the reference experiments, the microbial composition changed slightly in the NaClO (W2d3, W2d10, W2d30) and ClO 2 experiments (W3d3, W3d10, W3d30) with a low concentration of disinfectant (1 mg/L), showing that low concentration of disinfectant dosing (1 mg/L NaClO or ClO 2 ) could not change the bacterial community structure at phylum and class level obviously.
As the disinfectant concentration increased, the residual disinfectant concentration increased correspondingly. When the disinfectant concentration was maintained at 2 mg/L in influent (W2d60), the relative abundance of Proteobacteria and Firmicutes of effluents at phylum level did not change significantly, while the relative abundance of Actinobacteria increased compared with that in reference (W1d60), suggesting that Actinobacteria was resistant to 2 mg/L NaClO. Actinobacteria, as a typically chlorineresistant bacteria, have also been reported in drinking water (Jin et al., 2015;Luo et al., 2021b). Additionally, this intermediate disinfectant dosing (2 mg/L NaClO or ClO 2 ) reduced the bacterial abundance of Gammaproteobacteria, Betaproteobacteria, and Clostridia at class level effectively. However, different phenomena occurred in the ClO 2 experiment (2 mg/L). Proteobacteria, Bacteroidetes, and Actinobacteria were far less abundant in sample W3d60 than that in W1d60, showing that 2 mg/L ClO 2 inactivated these bacteria groups effectively.
Subsequently, when the disinfectant concentration increased to 4 mg/L in influents, the relative abundance of Proteobacteria and Actinobacteria at phylum level decreased (W2d60) compared with that in reference (W1d60), showing that 4 mg/L NaClO inactivated Proteobacteria and Actinobacteria effectively. When enhancing the ClO 2 dosage to 4 mg/L, the relative abundance of Proteobacteria increased, while the proportion of Firmicutes decreased, showing that the high ClO 2 dosage favors the growth of Proteobacteria. Proteobacteria is the largest bacterial phylum, which can shift into various species to adapt to extreme environments (Madigan and Martinko, 2007). The same phenomenon of Proteobacteria regrowth has also been reported in drinking water distribution systems using chloramine disinfection and UV/Cl 2 disinfection (Zhu et al., 2020). Moreover, at the class level, the high concentration of disinfectant (4 mg/L NaClO or ClO 2 ) could not efficiency inactivate the bacteria, while some disinfectant-tolerant species survived and multiplied, such as Alphaproteobacteria and Bacilli in NaClO dosing environment and Betaproteobacteria and Alphaproteobacteria in ClO 2 dosing environment. Generally, Bacilli can produce spores with special resistance to adverse conditions, enabling them to colonize and survive under disinfection, consistent with results reported by Roy and Ghosh (Rozalski et al., 1997). The Alphaproteobacteria and Betaproteobacteria showed significant variability, and the resistance of Alphaproteobacteria to ClO 2 has been demonstrated previously (Shams et al., 2011). Moreover, the high NH 2 Cl concentration could also induce the increment of Betaproteobacteria (Mi et al., 2015).

Analysis of bacterial community structure transformation
The microbial community transformation tree in effluents caused by disinfection (kingdom → phylum → class → order → family → genus → species) was drawn based on the Mothur software (http:// www. Mothur. org/), as shown in Fig. 4. Groups are distinguished by different colors, and the bacterial abundance at different levels is represented in the pie charts. The bacteria were taken as the starting point of transformation and were normalized (100%). The percentage of each transformed branch represents the proportion of upperlevel communities that shifted to next-level communities.
The dominant species varied depending on the disinfection condition, based on their resistance to the disinfectants. In raw water (W0), Proteobacteria was the dominant bacteria, which primarily transformed into two branches: the Proteobacteria → Gammaproteobacteria → Pseudomonadales → Moraxellaceae → Acinetobacter and Proteobacteria → Betaproteobacteria → Burkholderiales → Acidovorax, respectively. Pseudomonadaceae is a potential pathogen (Kwon et al., 2011). Moraxellaceae is a family of Gammaproteobacteria, including several pathogenic species, primarily found in water or soil (Douterelo et al., 2014). Huang et al. (Huang et al., 2014) reported that Alphaproteobacteria was the dominant taxonomic class in drinking water, while Kwon et al. (Kwon et al., 2011) demonstrated that Betaproteobacteria (40%) was more abundant than Alphaproteobacteria (21%). However, Gammaproteobacteria was found to be the main evolutionary bacterial genus in this study, which might be related to the water quality (e.g., pH, TOC, salinity) of the transported water (Holmfeldt et al., 2009). Both Pseudomonadaceae and Moraxellaceae were also found in effluents with a low disinfectant concentration (1 mg/L NaClO or ClO 2 ), indicating that a low concentration could not effectively inactivate these two pathogenic species. However, higher NaClO and ClO 2 dosing (2-4 mg/L) could inhibit their growth effectively. Meanwhile, the bacteria mainly shifted to Firmicutes in effluents with higher disinfectant dosage (> 4mg/L NaClO or > 2 mg/L ClO 2 ), suggesting that this branch of Firmicutes showed significant chlorine resistance. Additionally, the chlorine-resistant Firmicutes branch shifted to Bacilli → Bacillales → Bacillaceae → Bacillus. A similar phenomenon of Firmicutes surviving NaClO or ClO 2 disinfection has also been reported in drinking water treatment systems (Ma et al., 2020). Bacillaceae is a class of aerobic or facultatively anaerobic, Gram-positive rod-shaped bacilli (Ziganshina et al., 2018). Previous studies have shown that Bacillus is resistant to low chlorine concentrations (0.5-1.0 mg/L) (Bhojani et al., 2018). This study confirmed that Firmicutes could survive in high disinfectant environments (2, 4 mg/L), which might be due to its biofilm encapsulation. Therefore, disinfectant dosing also changed the bacterial community structure evolution.

Differences of microorganisms between biofilm and effluents
Previously, our group has investigated the biofilm microbial community using the same disinfection strategies, sampling time, and experimental apparatus (Zhang et al., 2019b). The presented study also conducted a comparative analysis of microorganisms between biofilm and effluents, as shown in Fig. 5. Tukey's test was applied to make a comparative analysis of microorganisms between biofilm and effluents. LDA effect size (LEfSe) is an algorithm for discovering and characterizing biomarkers with statistical differences between groups (Segata et al., 2011).
The group species differences between G2-W2 and G3-W3 were lower than those in G1-W1, demonstrating that disinfection reduced the difference of microorganisms between biofilm and effluents. Distinct species appeared on biofilm, and effluents presented different conditions in reference and disinfected environments. More distinct species were found on biofilm (G1) in reference environments, and among them, Alphaproteobacteria was accounted for the highest abundance, with multiple classes including o_Caulobacterales-f_Caulobacteraceae, o_Rhizobiales-(f_Bradyrhizobiaceae and f_Rhizobiaceae), o_Sphingomonadales-f_Sphingomonadaceae, and f_Alcaligenaceae. Less distinct species were found in effluents, only including c_Betaproteobacteria-o_Burkholderiales-f_Comamonadaceae.
However, in NaClO-disinfected samples, more differential species were present in effluents (W2) than that in biofilm (G2). p_Bacteroidetes-Flavobacteriia-Flavobacteriales-Flavobacteriaceae were significantly enriched in effluents, while g_Rhizobium and g_Sphingomonas were enriched in biofilm (G2). In the ClO 2 disinfection process, species in biofilm (G3) and effluents (W3) showed some similarity, and the species differences were minimal. The species that showed significant differences were o_Caulobacterales-f_Caulobacteraceae, o_Rhizobiales-f_Bradyrhizobiaceae, f_Oxalobacteraceae, and o_Xanthomonadales-f_Xanthomonadaceae, which were the main evolutionary branches of Proteobacteria. Additionally, in this study, the LDA score of Sphingomonas in G2 and G3 was higher than that in G1, suggesting that Sphingomonas showed significant resistance to ClO 2 and NaClO disinfection ( Fig. 5(b 2 ), (c 2 )). However, Pseudomonas was not observed in G2 or G3, indicating that Pseudomonas on biofilm was sensitive to NaClO and ClO 2 .
Considering that more differentiated species were present at the genus level between biofilm and effluents, the Wilcox rank sum tests were applied to the T-test analysis to analyze the significant differences in species diversity between biofilm and effluents (significance at p < 0.05). Figure 6 shows the species difference at the genus level between biofilm and effluents.
As shown in Fig. 5)(a, more differentiated species (12 species) were observed in group W1-G1 (reference experiments). The abundance of Acidovorax (p = 0.025) in effluents was significantly higher than that in biofilm, while the abundance of other species was higher in biofilm. Sphingomonas and Pseudomonas were the main bacterial genera detected in biofilm in reference experiments (G1) (Fig. 5(a)). The species difference between group W3-G3 (ClO 2 disinfection) exhibited the second largest. The abundance of Stenotrophomonas, Sphingomonas, Shinella, and Devosia were relatively higher in biofilm samples (G3) than that in effluents (W3) (Fig. 5c). Thus, it can be concluded that ClO 2 disinfection can significantly reduce the planktonic concentration in effluents, while having little effect on biofilm bacteria (Gagnon et al., 2005). As a potential pathogen, Stenotrophomonas was often detected in reclaimed water distribution system (Garner, et al., 2018), which was also reported that could not eliminate when maintaining the free chlorine at 1.20 ± 0.23 mg/L (Zhu et al., 2014). Especially, Sphingomonas is a typical disinfectant-resistant bacteria due to its ability to form a protective layer and wrap itself (Hu et al., 2021;Yang et al., 2017). As reported previously, the inactivation rate of Sphingomonas is less than 1-log reduction under 0.2 mg/L free chlorine (Wang et al., 2019b). Here, results also confirmed that Stenotrophomonas, Sphingomonas, Shinella, and Devosia on biofilm were also resistant to even 4 mg/L ClO 2 dosing. , and (c 1 ) represent the branching diagram of G1-W1, G2-W2, and G3-W3 groups, respectively; (a 2 ), (b 2 ), and (c 2 ) represent the LDA distribution histogram of G1-W1, G2-W2, and G3-W3 groups, respectively. Notes: in (a 1 ), (b 1 ), and (c 1 ), the circles radiating from the inside to the outside represent the classi-fication level from phylum to species, and the diameter of the circle is proportionate to the relative abundance. Species with no significant difference are uniformly colored yellow, while the red and green nodes indicate the main microorganisms in biofilm and effluents, respectively However, the species difference between group W2-G2 (NaClO disinfection) was minimum, and only Paenibacillus was predominant in biofilm (Fig. 5b). It was speculated that the disinfectant type and the scale characteristics were critical factors in determining this difference. There was greater species abundance in effluents of the reference experiments, and this was reduced after disinfection (NaClO or ClO 2 ). Notably, the lowest abundance was found in biofilm and effluents disinfected with NaClO. It was speculated that the ClO − in transported water with a smaller ionic radius could more easily penetrate the corrosion scale and affect the bacteria on biofilm (Supporting Information 4), making the bacterial variation basically the same as in effluents (Williams and Braun-Howland, 2003). On the contrary, the higher abundance of different species in effluents and biofilm following ClO 2 disinfection suggests that ClO 2 was hard to pass through the scale, which might be because ClO 2 has a larger molecular radius and a V-shaped structure ( Figure S7) (Guo, 1984;Zheng et al., 2007).

Detection of bacterial apoptosis by flow cytometry
Flow cytometry with double staining of cells by PI and fluorescein isothiocyanate (FITC) was utilized to evaluate the inactivation mechanism of disinfectant on cell integrity in reclaimed water (Berney, 2006;Liu et al., 2016). The Fig. 6 Species differences at the genus level between biofilm and effluents from T-test: a without disinfectant; b NaClO disinfection process; c ClO 2 disinfection process. The left hand panel shows the abundance of the differentiated species, and the right hand panel shows the confidence specific states can be seen in Fig. 7, and the exact numerical information can be seen in Supporting Information 5.
Overall, the cells of raw water (W0) were concentrated in the Q4 quadrant. The proportion of live cells accounted for 81.80% of total cells in raw influents (W0), while those of early apoptotic cells (Q3), membrane compromised cells (Q1), and dead cells (Q2) accounted for 4.88%, 9.93%, and 3.39% respectively, showing the high bioactivity of bacteria in raw reclaimed water. In ARs without disinfection, the percentage of viable live cells in effluents changed slightly (W1d30, W1d60, and W1d90), demonstrating that live bacterial cells could not die naturally; thus, disinfection is a necessary step.
In the effluents of ARs disinfected with NaClO, the PI red fluorescent signal shifted to the Q2 area in sample W2d30, and the proportion of live cells decreased sharply to 34.2%, correspondingly. The proportion of membrane-compromised cells increased to 30.3%, showing that 1 mg/L NaClO rapidly inactivated the living cells effectively and induced the membrane compromised. This might be related that the NaClO could cause damage of the cellular integrity by changing the permeability of cell membranes (Cho et al., 2010). However, as the increasing of NaClO dosage and disinfection time, the percentage of live cells in 2 and 4 mg/L NaClO dosing experiments (30.60% and 30.80%, respectively) decreased slightly, showing that enhancing NaClO dosing (> 2mg/L) could not inactivate living cells further. The red fluorescent signal in Q2 gradually changed to a green fluorescent signal, which was largely concentrated in the Q3 quadrant, showing that membrane-compromised cells mainly transformed into early apoptotic cells.
In ClO 2 -disinfected environment, the PI red fluorescent signal was mainly concentrated in the Q4 quadrant (W3d30), and there were no observable differences in the proportion of live cells, showing the poor inactivation effect of low ClO 2 dosing (1 mg/L) on living cells. The PI red fluorescent signal mainly changed to a green fluorescent signal in samples W3d60 and W3d90 and spreading into the Q1 and Q2 quadrants, respectively, showing that the percentage of dead cells and membrane-compromised cells increased sharply. Previous studies have reported similar findings, with numerous dead cells observed in bulk water treated with ClO 2 (Song et al., 2020). Thus, it can be assumed that ClO 2 mainly degrades biological activity by destroying cellular integrity and exposing the nucleus. Therefore, ClO 2 is superior to NaClO in terms of destroying bacterial cells.
To summary, NaClO disturbed live bacterial cells rapidly, while ClO 2 caused more damage, stripping the bacterial membrane and exposing the nuclear material. This difference may be related to distinct inactivate mechanisms of NaClO and ClO 2 on cell integrity. NaClO, with a smaller ionic radius, could penetrate through the cell membrane, chlorinate proteins, and cause early apoptotic cell formation (Jia et al., 2020;Wang et al., 2019a). However, ClO 2 is difficult to pass through the cell membrane, which could oxidize the cell membrane as a whole and induce an electrochemical gradient deviation that damaged the permeability of the cell membrane further (Vives-Rego et al., 2000).

Conclusions and recommendations
This study provides a comprehensive investigation on bacterial properties in effluents of simulated RWDSs (ARs) disinfected with different concentrations of NaClO or ClO 2 . The bacterial community and cell integrity changed with continuous disinfection, determined by the disinfectant type and dosage, which provides a reference for further research on disinfection efficiency and microbiological risk management.
The specific conclusions are as follows: (1) The dominant species that regrew under each disinfection condition were distinct, based on their capacity to withstand the disinfectants. (2) The low disinfectant dosing (1 mg/L NaClO or ClO 2 ) did not affect the bacterial community in effluents significantly.
(3) Intermediate disinfectant dosing (2 mg/L NaClO or ClO 2 ) inactivated Gammaproteobacteria, Betaproteobacteria, and Clostridia. (4) The high concentration of disinfectant (4 mg/L NaClO or ClO 2 ) could not inactivate bacteria further, while some disinfectant-tolerant species survived and multiplied, such as Alphaproteobacteria and Bacilli in NaClO dosing environment and Betaproteobacteria and Alphaproteobacteria in ClO 2 dosing environment. (5) The bacterial community in effluents showed clear differences with those in biofilms, with the lowest differences found in ClO 2 -disinfected environments. (6) Flow cytometry demonstrated that NaClO disturbed live bacterial cells rapidly, while ClO 2 caused greater damage, stripping the bacterial membrane and exposing the cytoplasm.
In summary, 2 mg/L of NaClO or ClO 2 is the suggested disinfectant concentration to control microbial growth in RWDSs, and ClO 2 is the most recommended disinfectant based on cell integrity damage alone in the presented study. However, different raw water, treatment processes, and pipeline environments will cause the different bacterial communities colonized. Therefore, reasonable disinfection measures should be adopted comprehensively.