Microbial Characteristics of the Combined Ozone and Tea Polyphenols Or Sodium Hypochlorite Disinfection in the Pipe Network

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Article Microbial Characteristics of the Combined Ozone and Tea Polyphenols or Sodium Hypochlorite Disinfection in the Pipe Network

Cuimin Feng 1,2,*, Na Zhu 1,2, Ying Li 1,2, Zhen Xu 1,2 and Ziyu Guo 3

1 Key Laboratory of Urban Stormwater System and Water Environment, Ministry of Education, Beijing University of Civil Engineering and Architecture, Beijing 100044, China; [email protected] (N.Z.); [email protected] (Y.L.); [email protected] (Z.X.) 2 National Demonstration Center for Experimental Water Environment Education, Beijing University of Civil Engineering and Architecture, Beijing 100044, China 3 Beijing General Municipal Engineering Design & Research Institute Co., Ltd., Beijing 100082, China; [email protected] * Correspondence: [email protected]

Abstract: Microbiological safety of water in the pipe network is an important guarantee for safe drinking water. Simulation tests of stainless steel pipe network were carried out using te4a polyphenols and sodium hypochlorite as auxiliary disinfectants for ozone disinfection to analyze the persistent disinfection effects of different combined disinfection methods by measuring the changes in total bacterial colonies in the water. High-throughput sequencing of microorganisms in the pipe network was

performed to analyze the differences in the community structure of microorganisms in the water and pipe wall under different disinfection methods. The results showed that the application of auxiliary

Citation: Feng, C.; Zhu, N.; Li, Y.; Xu, disinfectants had a relatively long-lasting inhibitory effect on the bacterial colonies in the water, and the Z.; Guo, Z. Microbial Characteristics diversity of microorganisms in the pipe network varied signiﬁcantly. As an auxiliary disinfectant for of the Combined Ozone and Tea ozone disinfection, tea polyphenols are more powerful than sodium hypochlorite in killing pathogens Polyphenols or Sodium Hypochlorite and chlorine-resistant bacteria, so they are more beneﬁcial to ensure the microbiological safety of Disinfection in the Pipe Network. water in stainless steel pipe networks. Water 2021, 13, 1835. https:// doi.org/10.3390/w13131835 Keywords: drinking water; ozone; tea polyphenols; disinfection; microorganisms; pipe network

Academic Editor: Helvi Heinonen-Tanski 1. Introduction Received: 3 June 2021 The safety of drinking water is vital to human health, and disinfection is an important Accepted: 29 June 2021 Published: 30 June 2021 guarantee for safety. Chlorine disinfection is widely used in water plant practice due to its better bactericidal effect and sustained action [1], but at the same time, a large num-

Publisher’s Note: MDPI stays neutral ber of disinfection by-products have emerged, and more than 700 chlorine disinfection with regard to jurisdictional claims in by-products have been detected, with different toxic effects posing a great threat to human published maps and institutional afﬁl- health [2]. Ozone is highly oxidizing and has obvious disinfection advantages compared to iations. chlorine disinfection, which can achieve effective killing of bacteria, viruses and protozoa at low concentrations within a short contact time [3,4]. Ozone can also reduce the organic content of water, provide odor and smell control, and oxidize heavy metals, etc. [5] How- ever, ozone is more active and lacks the persistent disinfection ability, so it is not used for disinfection alone [5]. In order to avoid the shortcomings of single disinfection technology, Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. two or more disinfection methods are usually used in combination [6,7]. Therefore, the This article is an open access article combined application of auxiliary disinfectants after ozone disinfection is considered to distributed under the terms and improve the safety of drinking water. conditions of the Creative Commons Compared with liquid chlorine, sodium hypochlorite has the advantages of high Attribution (CC BY) license (https:// safety, convenient use and easy storage [8]. Sodium hypochlorite forms hypochlorous creativecommons.org/licenses/by/ acid through hydrolysis, then further decomposes to form nascent oxygen [O], which has 4.0/). a strong oxidizing effect on microorganisms and achieves a killing effect [8]. Therefore,

Water 2021, 13, 1835. https://doi.org/10.3390/w13131835 https://www.mdpi.com/journal/water Water 2021, 13, 1835 2 of 15

the combined ozone and sodium hypochlorite disinfection can be applied to extend the sterilization time and reduce the dosage of sodium hypochlorite [9]. Tea polyphenols are a class of polyphenols extracted from tea leaves with high content and variety, which have broad-spectrum resistance to a variety of bacteria, fungi and viruses, and their antibacterial effects are manifested in both inhibition and killing [10–12]. Due to the superior antibacterial properties and sustained action of tea polyphenols, they can be used as disinfectants to provide security for drinking water, and the research related to this has progressed. Xie et al. [13] showed that tea polyphenols can be used for disinfection of different water sources, which can achieve good disinfection effect. However, tea polyphenols are used as the only disinfectant with high dosage and poor economy, so it is recommended to be used as an auxiliary disinfectant in conjunction with the main disinfection process [14,15]. Feng et al. [16] used the response surface method to design the combined disinfection effect test of ozone and tea polyphenols, and the ideal working conditions were obtained by simulation. The microbiological safety needs to be ensured in the drinking water distribution system, so there is a need for overall consideration of the water and pipe wall microorganisms as microorganisms on the pipe wall will enter the water under the effect of water ﬂow or by aging off and become a source of bacteria in the water [17]. Therefore, the effect of disinfection methods from the perspective of microorganisms themselves need to be studied in depth. In this study, stainless steel pipe was chosen as the research object, which has good wear resistance, corrosion resistance, ductility and toughness, and is gradually being used in water supply networks [18,19]. The effects of ozone disinfection (O3), the combined ozone/tea polyphenols disinfection (O3/TP) and the combined ozone/sodium hypochlorite disinfection (O3/NaClO) were analyzed by using a pipe network simulation system, to investigate the effects of different disinfection methods on the distribution characteristics of microorganisms in water and pipe walls. Then, the disinfection ability from the perspective of microorganisms was analyzed in order to clarify the feasibility of tea polyphenols as auxiliary disinfectants, providing a certain theoretical basis for the application of combined ozone/tea polyphenols disinfection technology.

2. Materials and Methods 2.1. Test Material The National I test piece was selected as the simulated pipe wall hanging piece, made of stainless steel, with the speciﬁcations of 50.0 mm × 25.0 mm × 2.0 mm (surface area of 28 cm2) and surface roughness of ∆7. The tea polyphenols were purchased from Beijing Zhongke Quality Control Biotechnol- ogy Co., Ltd., Beijing, China, and were in the form of yellow powder at room temperature with a purity of AR99.0%. The test results showed that the content of catechins accounted for 90%, and the content of Epigallocatechin gallate (EGCG) accounted for 56%. The test raw water was taken from the city water supply network and left for 2–3 days to remove the residual chlorine, which was used to simulate the ﬁltered water from the water plant. The water quality test results are as follows: pH 7.81, turbidity 0.96 NTU, chroma 1 degree (◦), total bacteria 540 CFU·mL−1 and residual chlorine 0.01 mg·L−1.

2.2. Test Device and Operating Conditions The dynamic simulation system of the pipe network is shown in Figure1. The test adopted a non-circular operation mode to simulate the stainless steel water supply pipe network system, and explored the characteristics of microorganisms in water and pipe walls where ozone, the combined ozone/tea polyphenols method and the combined ozone/sodium hypochlorite method were applied as disinfectants. The core of this device is biological annular reactor (BAR) with an effective volume of 6 L, and 24 test pieces were added to each reactor. During the test, the reactors were wrapped in tinfoil to avoid changes in water temperature caused by direct sunlight, and the water temperature was maintained at 22 ◦C by temperature sensors and temperature control rods. Water 2021, 13, x FOR PEER REVIEW 3 of 15

added to each reactor. During the test, the reactors were wrapped in tinfoil to avoid Water 2021, 13, 1835 changes in water temperature caused by direct sunlight, and the water temperature3 was of 15 maintained at 22 °C by temperature sensors and temperature control rods.

Figure 1. Schematic diagram of test equipment. In theFigure test 1. operation, Schematic diagram the raw of water test equipment. entered the ozone contact column through the lifting pump and after disinfection by ozone, the effluent entered BARs through the multi-channelIn the test peristalticoperation,pump, the raw then water no enter auxiliaryed the disinfectant ozone contact was column addedto through BAR1, the tea liftingpolyphenols pump and after sodium disinfection hypochlorite by ozone, were the added effluent to BAR2 enter anded BAR BAR3,s through respectively. the multi The- channeldisinfection peristaltic effect pump was affected, then no by auxiliary the amount disinfectant of ozone was dosage, added ozone to BAR contact1, tea timepolyphe- and nolsauxiliary and sodium disinfectant hypochlorite dosage. Inlet were flow added of BARs to BAR2 were and controlled BAR3, respectively as 0.6 L·h−1 and. The simulated disinfec- tionhydraulic effect retentionwas affected time by was the 10 amount h during of ozone the test. dosage, ozone contact time and auxiliary disinfectant dosage. Inlet flow of BARs were controlled as 0.6 L·h−1 and simulated hydrau- lic2.3. retention Testing Indexes time was and 10 Methods h during the test. Ferrous tartrate-standard curve method was used to determine the concentration of 2.3.tea polyphenols.Testing Indexes First and theMethods regression equation of the standard curve was determined, and thenF theerrous regression tartrate equation-standard was curve used method to calculate was used the concentration to determine ofthe tea concentration polyphenols of in teathe polyphenols. water during First the test. the regression Iodometry equation method wasof the used standard to determine curve was the det concentrationermined, and of thenozone the in regression the water, equation and starch was solution used to was calculate used as the an concentration indicator. of tea polyphenols in the waterThe totalduring number the test. of bacteriaIodometr iny themethod water was was used determined to determine according the concentration to the Standard of ozoneExamination in the Methodswater, and for Drinkingstarch solution Water (GB/T was used 5750.12-2006) as an indicator. by the plate count method [20]. The testThe runtotal time number was of for bacteria a total in of the 35 days,water everywas determined 5 days for according each reactor to the in theStandard same Examinationlocation of the Methods simulated for Drinking pipe wall Water hanging (GB/T piece 5750.12 of colony-2006) count by the test. plate count method [20]. TheFor microorganismstest run time was in for the a water,total of 6 35 L ofdays, water every samples 5 days were for each taken reactor fromeach in th reactore same locationon the 35th of the day simulated of the dynamic pipe wall test. hanging Then, filterpiece membranes of colony count with test. a pore size of 0.22 µm wereFor selected microorganisms and the water in the samples water, 6 were L of water filtered samples with a were vacuum taken extraction from each bottle reactor to onenrich the the35th amount day of ofthe bacteria dynamic and test extract. Then DNA., filter After membrane passings thewith test, a pore the V3–V4size of region0.22 μm of w16Sere rRNA selected was and amplified the water by samples PCR, and were high-throughput filtered with a sequencing vacuum extraction was performed bottle to after en- richsuccessful the amount amplification. of bacteria Microbial and extract community DNA. After analysis passing was the performed test, the V3 according–V4 region to the of 16Ssequencing rRNA was results. amplified by PCR, and high-throughput sequencing was performed after successfulFor microorganisms amplification. M onicrobial the pipe community wall, 10 mock analysis pipe was wall performed hangings wereaccording taken to from the sequencingeach reactor results. on the 35th day of the dynamic test. First, they were placed in a beaker containing 300 mL of sterile water, and then placed in ultrasonic cleaning shaking. The For microorganisms on the pipe wall, 10 mock pipe wall hangings were taken from same filter membranes with a pore size of 0.22 µm were selected, and then the above each reactor on the 35th day of the dynamic test. First, they were placed in a beaker con- procedure was repeated. taining 300 mL of sterile water, and then placed in ultrasonic cleaning shaking. The same filter3. Results membrane and Discussions with a pore size of 0.22 μm were selected, and then the above procedure was3.1. Analysisrepeated. of Disinfection Effect

3. ResultsA stainless and Discussion steel network simulation system was set up to analyze the persistent disinfection effect of different disinfection methods by detecting the total amount of bacteria 3.1.in the Analysis efﬂuent of Disinfection of the three Effect BARs. Ozone and tea polyphenols were dosed with reference to theA resultsstainless obtained steel network in the preliminarysimulation system work ofwas the set research up to analyze group: the ozone persistent dosage dis- of infection2.5 mg·L −effect1, ozone of different contact timedisinfection of 25 min, methods and tea by polyphenols detecting the dosage total amount of 20 mg of· Lbacteria−1 [16]. inSodium the effluent hypochlorite of the three was dosedBARs. atOzone 2 mg and·L−1 teawith polyphenol references to were the operatingdosed with experience reference of the water plant during the test [21,22]. The total bacterial colonies in the water under different disinfection methods were measured at intervals of 5 days, as shown in Figure2.

Water 2021, 13, x FOR PEER REVIEW 4 of 15

to the results obtained in the preliminary work of the research group: ozone dosage of 2.5 mg·L−1, ozone contact time of 25 min, and tea polyphenols dosage of 20 mg·L−1 [16]. So- dium hypochlorite was dosed at 2 mg·L−1 with reference to the operating experience of the Water 2021, 13, 1835 water plant during the test [21,22]. The total bacterial colonies in the water under different4 of 15 disinfection methods were measured at intervals of 5 days, as shown in Figure 2.

600

) O /TP -1 500 3

mL O3/NaClO ·

400

300

200

Total number of colonies/(CFUTotal 100

0 5 10 15 20 25 30 35 Operating days/d

Figure 2. The total bacterial colonies in the water under different disinfection methods. Figure 2. The total bacterial colonies in the water under different disinfection methods. It can be seen that the total bacterial colonies in the water was less than 100 CFU·mL−1 −1 withinIt can 5 be days seen after thatthe the combinedtotal bacterial ozone colonies and teain the polyphenols water was less or sodium than 100 hypochlorite CFU·mL withindisinfection 5 days after (Figure the2 ),combined which meets ozone the and requirements tea polyphenols of the or Standards sodium hypochlorite for Drinking dis- Wa- infectionter Quality (Figure (GB5749-2006) 2), which meets [20]. the After requirements that, until 35of days,the Standards the total for bacterial Drinking colonies Water in Qualitythe water (GB5749 with- tea2006) polyphenols [20]. After that, as auxiliary until 35 disinfectantdays, the total were bacterial always colonies maintained in the below wa- ter100 with CFU tea·mL polyphenols−1 and continued as auxiliary to show disinfectant a decreasing were trend. always However, maintained there was below a rebound 100 CFUphenomenon·mL−1 and c withontinued sodium to show hypochlorite a decreasing as an auxiliarytrend. However, disinfectant there within was a 10–35rebound days, phe- and nomenonthe water with quality sodium did hypochlorite not meet the as standard an auxiliary requirements. disinfectant It showswithin that10–35 the days, combined and theozone/tea water quality polyphenols did not disinfectionmeet the standard has a relatively requirements. long-lasting It shows inhibitory that the effect combined and the ozone/teawater quality polyphe ofnols the stainlessdisinfection steel has pipe a relatively network long is safer-lasting among inhibitory the three effect disinfection and the watermethods. quality The of reason the stainless may be that steel during pipe network the disinfection is safer process, among the the action three of disinfection tea polyphe- methods.nols is stable The reason and lasts may for be a that long during time, while the disinfection sodium hypochlorite process, the has action a rapid of tea effect poly- and phenolsdecays is quickly stable atand the lasts same for time. a long Hence, time, the while synergistic sodium effect hypochlorite of sodium has hypochlorite a rapid effect and andozone decays has quickly a better at inhibitory the same time. effect Hence, on microorganisms the synergistic in effect the early of sodium stage. hypochlorite However, the andattenuation ozone has of a teabetter polyphenols inhibitory is effect slower, on somicroorganisms its continuous actionin the makesearly stage. the control Howe effectver, theon attenuation bacteria stronger. of tea polyphenols In addition, theis slower, oxidizing so its free continuous radicals generated action makes in the the water control after effectozone on dosingbacteria will stronger. cause teaIn addition, polyphenols the oxidizing to exhibit free pro-oxidation radicals generated at low concentrations, in the water afterincreasing ozone dosing the amount will cause of ·OH tea in polyphenols the water, and to enhancingexhibit pro the-oxidatio sterilizationn at low effect concentra- of O3/TP tions,combined increasing disinfection. the amount of ·OH in the water, and enhancing the sterilization effect of O3/TP combined disinfection. 3.2. Microbial Diversity Analysis 3.2. MicrobialHigh-throughput Diversity Analysis sequencing of the bacteria in the water and the pipe wall samples at theHigh end-throughput of the dynamic sequencing operation of the of bacteria the three in BARs,the water then and the the OTU pipe at wall 97% samples similarity atlevel the end were of subjectedthe dynamic to bioinformatic operation of the statistical three BARs, analysis then after the de-redundancyOTU at 97% similarity to reﬂect levelthe were abundance subjected and to diversity bioinformatic of the statistical microbial analysis communities after de by-redundancy the Alpha diversity to reflect index the abundance(Table1). and diversity of the microbial communities by the Alpha diversity index (Ta- ble 1). Table 1. The Alpha diversity index (water/pipe wall).

Disinfection Coverage Shannon ACE Chao1 Simpson Method

O3 1.00/1.00 0.78/1.57 256.18/410.12 229.30/368.33 0.69/0.41 O3/TP 1.00/1.00 1.89/2.50 258.46/353.86 243.64/319.12 0.34/0.14 O3/NaClO 1.00/0.95 5.72/3.22 1407.10/474.57 1403.53/413.18 0.01/0.22

ACE index and Chao1 index are often used in ecology to estimate the total number of species, with larger indices indicating higher microbial richness. Shannon index and Simpson index are often used in ecology to describe biodiversity, with larger Shannon and smaller Simpson indices indicating higher community diversity [23]. It can be concluded Water 2021, 13, 1835 5 of 15

that ACE, Chao1 and Shannon increased and Simpson decreased in the water when the auxiliary disinfectants were applied in combination with ozone compared to ozone disinfection from Table1. This indicates that the microbial species diversity and richness in the water increased with combined disinfection compared to ozone alone. Among them, ACE and Chao1 increased slightly and Simpson decreased from 0.69 to 0.34 when tea polyphenols was used as auxiliary disinfectant, while ACE and Chao1 increased significantly and Simpson decreased from 0.69 to 0.01 when sodium hypochlorite was used as auxiliary disinfectant. Combined with Figure2, it can be seen that the combined O 3/TP disinfection has better bacterial inhibition effect and lower diversity of microorganisms in the water compared with the combined O3/NaClO disinfection. Microbial diversity analysis of the pipe wall found ACE and Chao1 were 353.86 and 319.12 respectively when the combined disinfection of O3/TP was used, both of which decreased compared to ozone disinfection. This indicates that the microbial richness of the pipe wall was reduced, which may be due to the effect of metal ions in stainless steel on the disinfection of tea polyphenols. Studies have shown that the phenolic hydroxyl groups in the structure of tea polyphenols can complex with most metal ions (such as Cd2+, Ca2+, Cu2+, Fe3+, etc.), forming tea polyphenols-metal complexes with some differences in bacterial inhibitory activity [24]. In contrast, the Alpha diversity indices for the combined O3/NaClO disinfection reflected an increase in the microbial diversity and richness on the pipe wall compared to the ozone disinfection. This indicates that different auxiliary disinfectants have different effects on the biofilm of the pipe wall. Therefore, it is necessary to analyze the microbial characteristics of the water and the pipe wall to investigate the differences in the effectiveness of different combined disinfection from the bacteria themselves.

3.3. Analysis of Microbial Characteristics in the Water

The combined O3/TP disinfection has the bacterial inhibition advantage over the combined O3/NaClO disinfection, and the microorganisms in the water have a lower diversity. However, the speciﬁc characteristics of bacterial community structure need to be further analyzed.

3.3.1. Microbial Diversity Analysis Based on Phylum Level

The raw water was separately disinfected by O3,O3/TP and O3/NaClO, then stayed in the stainless steel pipe network dynamically for 10 h. After that, high-throughput sequencing was performed on microorganisms in the water, and the colony structure at phylum level is shown in Figure3. The bacteria were mainly Proteobacteria in the water after ozone disinfection, whose content accounted for 97.8%, followed by Euryarchaeota accounted for 2.1%. Proteobacteria are all Gram-negative bacteria, containing a variety of metabolic species, both aerobic and anaerobes, both autotrophic and heterotrophic, and they are common bacteria in drinking water pipe networks [25]. Proteobacteria are currently the largest phylum within the bacteria domain, including several pathogens such as Escherichia coli, Salmonella, Vibrio, Pseudomonas aeruginosa, etc. [26] As a large branch of archaea, Euryarchaeota contain most species such as Methanogenus and Halobacterium. The microbial community structure in the water became simpler when the combined disinfection of O3/TP was used, with Proteobacteria reaching 99.95% and Euryarchaeota not exceeding 0.01%. In contrast, the diversity of bacteria increased signiﬁcantly after the combined O3/NaClO disinfection. In addition to 48.62% of Proteobacteria, there were Firmicutes (19.69%), Bacteroidetes (9.38%), Actinobacteria (4.14%), Nitrospirae (3.26%) and Euryarchaeota (0.61%), etc. The unicity of the colony structure in the water after the combined O3/TP disinfection shows that this method has a more obvious inhibitory effect on Euryarchaeota, Firmicutes, Bacteroides, etc., than the combined O3/NaClO disinfection. Water 2021, 13, x FOR PEER REVIEW 6 of 15

archaea, Euryarchaeota contain most species such as Methanogenus and Halobacterium. The microbial community structure in the water became simpler when the combined disinfection of O3/TP was used, with Proteobacteria reaching 99.95% and Euryarchaeota not exceeding 0.01%. In contrast, the diversity of bacteria increased significantly after the combined O3/NaClO disinfection. In addition to 48.62% of Proteobacteria, there were Firmicutes (19.69%), Bacteroidetes (9.38%), Actinobacteria (4.14%), Nitrospirae (3.26%) and Euryarchaeota (0.61%), etc. The unicity of the colony structure in the water after the combined O3/TP Water 2021, 13, 1835 disinfection shows that this method has a more obvious inhibitory effect on Euryarchaeota6 of 15, Firmicutes, Bacteroides, etc., than the combined O3/NaClO disinfection.

100

80 Planctomycetes Chloroflexi Acidobacteria 60 Nitrospirae Other Euryarchaeota Bacteroidetes 40 Actinobacteria Firmicutes Proteobacteria

Proportion of bacterial species/% 20

0 O O /TP O /NaClO 3 3 3 FigureFigure 3. 3. ColonyColony structure structure at at phylum phylum level. level. 3.3.2. Microbial Diversity Analysis Based on Class Level 3.3.2. Microbial Diversity Analysis Based on Class Level There is no obvious difference in the characteristics of the bacterial community be- There is no obvious difference in the characteristics of the bacterial community be- tween ozone and the combined O3/TP disinfection at phylum level, further analysis is tween ozone and the combined O3/TP disinfection at phylum level, further analysis is needed at class level (Figure4). It can be seen that the dominant flora of Proteobacteria after needed at class level (Figure 4). It can be seen that the dominant flora of Proteobacteria after the three disinfection methods was different at class level. The dominant flora is Gammapro- the three disinfection methods was different at class level. The dominant flora is Gammap- teobacteria with 82.92%, followed by Alphaproteobacteria with 14.67% after ozone disinfection. roteobacteriaWhen teapolyphenols with 82.92%, were followed used by as anAlphaproteobacteria auxiliary disinfectant, with 14.67%Alphaproteobacteria after ozone disin-is pre- fection.dominant When with tea 74.86%, polyphenols followed were by usedBetaproteobacteria as an auxiliary(18.51%) disinfectant, and Gammaproteobacteria Alphaproteobacteria is(6.58%). predominant This indicates with 74.86%, that teafollowed polyphenols by Betaproteobacteria are more effective (18.51%) in killing and Gammaproteobac-Gammaproteobacte- teriaria. (6.58%). The species This and indicates genetic that diversity tea polyphenols of Proteobacteria are moreis extremely effective in rich killing [26], Gammaprote- among them, obacteriaAlphaproteobacteria. The speciesinclude and genetic plant diversity symbiotic of bacteria,Proteobacte animalria is extremely symbiotic rich bacteria, [26], among and are them,widely Alphaproteobacteria present in the water include environment. plant symbioticBetaproteobacteria bacteria, animalinclude symbiotic many aerobic bacteria, bacteria and areor widely facultative present bacteria, in the which water can environment. be detected inBetaproteobacteria environmental samplesinclude many such as aerobic wastewater bac- teriaor soil. or facultativeGammaproteobacteria bacteria, include which somecan be taxa detected that are in important environmental in medicine samples and such scientific as wastewaterresearch, such or soil. as Enterobacteraceae,Gammaproteobacteria Vibrionaceae include some and taxa Pseudomonadaceae, that are important in etc. medicine [26]. Mi- andcroorganisms scientific research, in the water such were as Enterobacteraceae, more complex after Vibrionaceae the combined and OPseudomonadaceae3/NaClO disinfection,, etc. [26]with. MicroorganismsBetaproteobacteria in, Gammaproteobacteriathe water were moreand complexAlphaproteobacteria after the combinedaccounting O3/NaClO for 21.94%, disinfection,15.99% and with 9.22%, Betaproteobacteria, respectively. Gammaproteobacteria Besides, Bacilli, Clostridia and Alphaproteobacteria, Actinobacteria, Bacteroidia accountingand forSphingobacteriia 21.94%, 15.99%also and had 9.22%, high percentagesrespectively. of Besides, 9.68%, 7.86%,Bacilli, 4.14%, Clostridia 3.88%, Actinobacteria and 3.75%, respec-, Bac- teroidiatively. andBacilli Sphingobacteriiaand Clostridia also belong had to high Firmicutes, percentages and Gramof 9.68%, staining 7.86%, is positive4.14%, 3.88% [27]. Manyand 3.75%,Firmicutes respectively. can produce Bacilli budding and Clostridia spores belong to resist to dehydration Firmicutes, and and Gram extreme staining environments. is posi- tiveActinobacteria [27]. Many areFirmicutes also a groupcan produce of Gram-positive budding spores bacteria to resist [27], dehydration about 80% of and widely extreme used environments.antibiotics are Actinobacteria produced by are various also aActinobacteria group of Gram, such-positive as streptomycin, bacteria [27] oxytetracycline,, about 80% of widelytetracycline, used antibiotics gentamicin, are etc.produced [28]. Bacteroidia by variousand ActinobacteriaSphingobacteriia, suchbelong as streptomycin, to Bacteroidetes ox- , ytetracycline,and many bacteria tetracycline, of Bacteroidia gentamicin,live inetc. the [28] intestinal. Bacteroidia tract and of humansSphingobacteriia or animals, belong and to in Bacteroidetessome cases,, and become many pathogens. bacteria of The Bacteroidia test results live show in the that intestinal these groups tract of of humans bacteria or are ani- tol- mals,erant and to chlorine in some but cases, sensitive become to tea pathogens. polyphenols, The whichtest results are related show tothat the these characteristics groups of of cell membranes of different classes of bacteria [29]. This indicates that the growth of many common microorganisms are better controlled during the combined O3/TP disinfection compared to the combined O3/NaClO disinfection, and has better inhibitory effects on chlorine-resistant groups of Bacilli, Actinobacteria, and Sphingobacteriia, etc. Water 2021, 13, x FOR PEER REVIEW 7 of 15

bacteria are tolerant to chlorine but sensitive to tea polyphenols, which are related to the characteristics of cell membranes of different classes of bacteria [29]. This indicates that the growth of many common microorganisms are better controlled during the combined O3/TP disinfection compared to the combined O3/NaClO disinfection, and has better in- Water 2021, 13, 1835 hibitory effects on chlorine-resistant groups of Bacilli, Actinobacteria, and Sphingobacteriia7 of 15, etc.

100

Negativicutes Planctomycetia 80 Acidobacteria_Gp4 Anaerolineae Deltaproteobacteria Nitrospira 60 Bacteroidia Clostridia Actinobacteria Thermoplasmata 40 Bacilli Sphingobacteriia Gammaproteobacteria Proportion of bacterial species/% 20 Betaproteobacteria Alphaproteobacteria Other

0 O3 O3/TP O3/NaClO

Figure 4. Colony structure at class level. Figure 4. Colony structure at class level. 3.3.3. Microbial Diversity Analysis Based on Genus Level 3.3.3. TheMicrobial differences Diversity in the Analysis flora in theBased pipe on network Genus Level water are very obvious at genus level, as shownThe differences in Figure5 .in The the microbial flora in the species pipe network were relatively water are simple very when obvious ozone at genus disinfection level, aswas shown used, in with FigurePseudomonas 5. The microbialas the dominant species were genus relatively accounting simple for about when 80%, ozone followed disinfec- by tBlastomonasion was usedat, aboutwith Pseudomonas 10%. Whilethe as percentagesthe dominant of genusPseudomonas accountingand Blastomonas for about 80%,detected followedwere significantlyby Blastomonas reduced at about after 10%. the While combined the percentage disinfection,s of Pseudomonas indicating that and the Blastomonas auxiliary detecteddisinfectants were can significantly effectively reducedcontrol the after growth the combined of these two disinfection, types of bacteria. indicatingBlastomonas that the auxiliaryis Gram-negative disinfectants bacteria can thateffective reproducely control by germination,the growth ofPseudomonas these two typesis widely of bacteria. present Blastomonasin water, soil is andGram atmosphere-negative bacter by flagellaria that movement, reproduce byand germination Gram stain, isPseudomonas also negative, is widelyamong present which P. in aeruginosa water, soil, P. and pseudomallei atmosphereand P.by fluorescens flagellar movement,are all opportunistic and Gram pathogens stain is alsopresent negative, in the among environment which [ 30P. ].aeruginosa As a representative, P. pseudomallei of this and genus, P. fluorescensP. aeruginosa are hasall oppor- several tunisticpathogenic pathogen factors,s present and the in presencethe environment of it was [30]. detected As a representative by Anversa etof al.this [31 genus,] during P. aeruginosaan investigation has several of the pathogenic quality of factors, public and water the suppliespresence inof municipalitiesit was detected in by S ãAnversao Paulo etState, al. [31] Brazil, during indicating an investigation the ability of of the this quality bacterium of public to resist water conventional supplies in water municipalities treatment. inHuang São Paulo et al. State [32] found, Brazil, that indicating the sources the ability of contamination of this bacterium were diversified to resist conventional through the wateranalysis treatment. of the contamination Huang et al. of[32]P. aeruginosafound thatin the water sources sources, of contamination drinking water, were and diversi- pipeline fiedwater through supply the systems. analysis Since of thePseudomonas contaminationhas certain of P. aeruginosa resistance in to water UV, ozone sources, and drinking chlorine water,disinfection, and pipeline its proportion water supply was relatively systems. high Since during Pseudomonas ozone disinfection has certain inresistance the experiment, to UV, ozoneand the and percentage chlorine disi decreasednfection, significantlyits proportion after was the relatively addition high of during auxiliary ozone disinfectants. disinfec- tionYi et in al. the [33 experiment,] showed that and tea the polyphenols percentage have decreased a significant significantly effect on theafter membrane the addition protein of auxiliaryof P. aeruginosa disinfectants., promoting Yi et the al. disorder[33] showed of bacterial that tea metabolismpolyphenols and have gradually a significant destroying effect onthe the cell membrane structure, protein which of play P. aeruginosa a role in inhibitory., promoting After the disorder sodium of hypochlorite bacterial metabolism is added, andit is gradually hydrolyzed destroying to produce the hypochlorouscell structure, acid,which which play a acts role on in microorganismsinhibitory. After throughsodium hypochloritevarious mechanisms is added, to it achieveis hydrolyzed disinfection to produce [8]. Alcaligenes hypochlorousand Rhizobacteracid, which, whichacts on have mi- croorganismsslightly different through flagella various morphological mechanisms characteristics to achieve disinfection from Pseudomonas [8]. Alcaligenes, were and detected Rhi- zobacterwith sodium, which hypochlorite have slightly anddifferent tea polyphenols flagella morphological as auxiliary characteristics disinfectants, from respectively, Pseudo- monasaccounting, were fordetected a relatively with smallsodium percentage. hypochlorite and tea polyphenols as auxiliary disinfectants,It can respectively, be seen that accounting the microbial for a community relatively small structure percentage. within the water samples was more complex during combined disinfection from Figure5. Variovorax, Porphyrobacter, Methylophilus, and Sphingobium accounted for 10.39%, 5.24%, 5.02% and 4.33%, respectively, when the combined O3/TP disinfection was used. While Bacillus, Achromobacter, Stenotrophomonas, Citrobacter, Escherichia and Klebsiella accounted for a higher percentage with 6.09%, 5.34%, 2.87%, 2.65%, 2.05% and 1.65%, respectively, when the combined

O3/NaClO disinfection was used. Among them, Citrobacter, Escherichia and Klebsiella belong to Enterobacteriaceae and are used as a microbiological indicator of water quality and fecal contamination. Additionally, they have varying degrees of drug resistance, as opportunistic pathogens have a greater impact on human health [34]. In contrast, these Enterobacteriaceae microorganisms accounted for a smaller proportion after the combined O3/TP disinfection, Water 2021, 13, 1835 8 of 15

indicating that tea polyphenols are more effective than sodium hypochlorite as an auxiliary disinfectant in controlling the above-mentioned opportunistic pathogens, and the biosafety of the water was higher. Liu et al. [35] found that UV/tea polyphenols combined disinfection was signiﬁcantly more effective than UV/sodium hypochlorite combined disinfection in killing most of the common environmental microbial populations, and also had a strong Water 2021, 13, x FOR PEER REVIEW ability to kill pathogens and chlorine-resistant bacteria, which is similar to the results8 of 15 of this study. That is, the use of tea polyphenols as an auxiliary disinfectant in combination with the main disinfection process is more secure for controlling water microbial safety.

100 Blastomonas Clostridium Klebsiella Escherichia 80 Enterococcus Azospira Citrobacter Stenotrophomonas Nitrospira 60 Alcaligenes Achromobacter Bacillus Herminiimonas 40 Rhizobacter Novosphingobium Sphingobium Variovorax Proportion of bacterial species/% 20 Pseudomonas Acinetobacter Methylobacterium Methylophilus 0 Porphyrobacter Other O3 O3/TP O3/NaClO

Figure 5. Colony structure at genus level. Figure 5. Colony structure at genus level. 3.4. Analysis of Microbial Characteristics of the Pipe Wall ItMicroorganisms can be seen that willthe microbial use the water community body organic structure matter within for metabolism,the water samples growth was and morereproduction, complex during and they combined attach to disinfection the pipe wall from to form Figure biofilm 5. Variovorax during, thePorphyrobacter operation of, Methylophilusthe pipe network., and Sphingobium Many bacteria accounted will grow for and 10.39%, reproduce 5.24%, in 5.02% the microbial and 4.33%, community respectively,on the when pipe the wall combined and enter O the3/TP water disinfection under was the used action. While of water Bacillus flushing, Achromobacter or aging off, Sten- [36]. otrophomonasFrom the overall, Citrobacter safety, Escherichia of the pipe and network, Klebsiella the accounted community for structurea higher percentage of the pipe with wall 6.09%,microorganisms 5.34%, 2.87%, is analyzed. 2.65%, 2.05% and 1.65%, respectively, when the combined O3/NaClO disinfection was used. Among them, Citrobacter, Escherichia and Klebsiella belong to Entero- bacteriaceae3.4.1. Microbial and are Diversity used as Analysisa microbiological Based on indicator Phylum Levelof water quality and fecal contamination.The Additionally, colony structure they have of the varying pipe wall degrees at phylum of drug level resistance, derived as from opportunistic high-throughput path- ogenssequencing have a is greater shown impact in Figure on6, human which is health similar [34]. to the In distributioncontrast, these of microorganismsEnterobacteriaceae in microorganismsthe water. The microorganisms accounted for a weresmaller all dominatedproportion byafterProteobacteria the combined, accounting O3/TP disinfection for 97.91%,, indicating98.82%, and that 87.17%, tea polyphenols respectively, are after more the effective disinfection than of sodium O3,O3/TP hypochlorite and O3/NaClO. as an auxil- While iarythe disinfectant percentage in of controllingFirmicutes, theBacteroidetes above-mentionedand Actinobacteria opportunisticwere pathogens, also higher and after the bi- the osafetycombined of the O 3water/NaClO was disinfection, higher. Liu et with al. [35] 4.62%, found 3.72%, that andUV/tea 0.94%, polyphenols respectively. combined This is disinfectionsimilar to the was finding significantly of Sun etmore al. [ 25effective] that the than larger UV/sodium population hypochlorite on biofilm combined is Proteobacteria dis- , infectionfollowed in by killingFirmicutes most ,ofActinobacteria the common, etc.,environmental in the drinking microbial water population distributions, and system. also hadThat a strong is, these ability bacteria to kill are pathogens widely present and chlorine in the-resistant water supply bacteria, system, which adaptable is similarand to thewidespread. results of this Studies study. have That also is, the shown use of that tea the polyphenols main factor as affecting an auxiliary the structuredisinfectant of thein combinationbiofilm community with the on main the inner disinfection wall of the process pipeline is more at phylum secure level for iscontrolling the pipe material water mi- [36], crobialfollow-up safety. on this point of view needs to be analyzed through comparative experiments.

3.4. Analysis of Microbial Characteristics of the Pipe Wall Microorganisms will use the water body organic matter for metabolism, growth and reproduction, and they attach to the pipe wall to form biofilm during the operation of the pipe network. Many bacteria will grow and reproduce in the microbial community on the pipe wall and enter the water under the action of water flushing or aging off [36]. From the overall safety of the pipe network, the community structure of the pipe wall microorganisms is analyzed.

3.4.1. Microbial Diversity Analysis Based on Phylum Level The colony structure of the pipe wall at phylum level derived from high-throughput sequencing is shown in Figure 6, which is similar to the distribution of microorganisms in the water. The microorganisms were all dominated by Proteobacteria, accounting for 97.91%, 98.82%, and 87.17%, respectively, after the disinfection of O3, O3/TP and O3/NaClO. While the percentage of Firmicutes, Bacteroidetes and Actinobacteria were also higher after the combined O3/NaClO disinfection, with 4.62%, 3.72%, and 0.94%, respectively. This is

Water 2021, 13, x FOR PEER REVIEW 9 of 15

similar to the finding of Sun et al. [25] that the larger population on biofilm is Proteobacteria, followed by Firmicutes, Actinobacteria, etc., in the drinking water distribution system. That is, these bacteria are widely present in the water supply system, adaptable and wide- spread. Studies have also shown that the main factor affecting the structure of the biofilm Water 2021, 13, 1835 9 of 15 community on the inner wall of the pipeline at phylum level is the pipe material [36], follow-up on this point of view needs to be analyzed through comparative experiments.

100

80 Planctomycetes Chloroflexi Acidobacteria 60 Nitrospirae Other Euryarchaeota Bacteroidetes 40 Actinobacteria Firmicutes Proteobacteria

Proportion of bacterial species/% 20

0 O O3/TP O3/NaClO 3 Figure 6. Figure 6. ColonyColony structure structure at at phylum phylum level. level. 3.4.2. Microbial Diversity Analysis Based on Class Level 3.4.2. Microbial Diversity Analysis Based on Class Level The colony structure at class level is shown in Figure7. Gammaproteobacteria was the dominantThe colony bacteria structure after at class ozone level disinfection, is shown in accounting Figure 7. Gammaproteobacteria for 61.27%, followed was by theAl- dominant bacteria after ozone disinfection, accounting for 61.27%, followed by Alphapro- phaproteobacteria (29.94%) and Betaproteobacteria (6.68%). When the combined O3/TP dis- teobacteriainfection was(29.94%) used, andAlphaproteobacteria Betaproteobacteriawas (6.68%). the dominant When the bacteria combined with O 61.07%,3/TP disinfection followed wasby Gammaproteobacteriaused, Alphaproteobacteria(21.44%) was the and dominantBetaproteobacteria bacteria (16.29%).with 61.07%, When followed the combined by Gam- 3 maproteobacteriaO3/NaClO disinfection (21.44%) was and used, Betaproteobacteria the percentage (16.29%). of Proteobacteria When thewas combined 87.11% onO / theNaClO pipe disinfectionwall, much greaterwas used, than the 47.15% percentage in the of water, Proteobacteri with thea specificwas 87.11% distribution on the pipe of Betaproteobac- wall, much greaterteria (68.92%), than 47.15%Gammaproteobacteria in the water, (11.51%) with the and specificAlphaproteobacteria distribution (6.68%). of Betaproteobacteria In addition, (68.92%),Clostridia ,GammaproteobacteriaSphingobacteriia, Bacilli (11.51%)and Actinobacteria and Alphaproteobacteriaalso had high (6.68%). percentages In addition, with 2.57%, Clos- tridia2.19%,, Sphingobacteriia 1.74%, and 0.94%,, Bacilli respectively. and Actino Thebacteria above also data had indicate high percentages that tea polyphenols with 2.57%, as 2.19%,an auxiliary 1.74%, disinfectant and 0.94%, hasrespectively. a stronger The inhibitory above data effect indicate on Betaproteobacteria that tea polyphenolscompared as an to auxiliarysodium hypochlorite.disinfectant ha Lis eta stronger al. [37] showed inhibitory that effectBetaproteobacteria on Betaproteobacteriaand Gammaproteobacteria compared to so- diumwere morehypochlorite. resistant Li to et chlorine al. [37] thanshowedAlphaproteobacteria that Betaproteobacteriain biofilms, and Gammaproteobacteria which is similar to werethe results more resistant of O3/NaClO to chlorine disinfection than Alphaproteobacteria in this experiment. in biofilms,Betaproteobacteria which is similarare the to most the resultswidely of distributed O3/NaClO classdisinfection in nature in this and experiment. are widely Betaproteobacteria found in lakes, riversare the and most drinking widely distributedwater biofilms class [38 in,39 nature]. It has and been are shownwidely thatfoundBetaproteobacteria in lakes, rivershave and drinking the ability water to secrete bio- filmsExtracellular [38,39]. It Polymeric has been Substancesshown that (EPS), Betaproteobacteria which make have microorganisms the ability to adhere secrete to Extracel- the inner lularsurface Polymeric of pipes, Substances enhance the (EPS), hydrophobicity which make of microorga cells, andnisms aggregate adhere into to clusters the inner with surface other ofmicroorganisms pipes, enhance [40 the]. Underhydrophobicity the protection of cells, of EPS, and microorganisms aggregate into clusters on biofilm with are other protected mi- croorganismsfrom the direct [40]. action Under of disinfection the protection and of have EPS, access microorganisms to more nutrients. on biofilmLiu et are al. protected [41] stud- fromied the the effects direct of action tea polyphenols of disinfection on the and quorum have sensingaccess to and more virulence nutrients. factors Liu of etKlebsiella al. [41] sPneumoniaetudied the effectsand found of tea that polyphenols tea polyphenols on the inhibitedquorum sensing population and sensing virulence activity factors and of Klebsiellareduced theirPneumoniae motility, and protease found andthat extracellulartea polyphenols polysaccharide inhibited population production, sensing then inhibited activity andbiofilm reduced formation. their motility, Huber etproteas al. [42e] and also extracellular reported that polysaccharide plant polyphenols production could, interferethen in- hibitedwith bacterial biofilm populationformation. sensingHuber et and al. inhibit[42] also bacterial reported biofilm that plant formation. polyphenols Liu et al.could [35] interferefound that with tea bacterial polyphenols population probably sensing destroyed and inhibit tyrosine-like bacterial proteins biofilm andformation. degraded Liu hu- et al.mic [35] acids, found thereby that teaffectinga polyphenols the growth probably of pipe destroyed wall microorganisms tyrosine-like proteins by analyzing and de- the gradedfluorescence humic properties acids, thereby of organic affecting matter the growth in pipe of wall pipe biofilm wall microorganisms when UV/tea polyphenols by analyz- ingcombined the fluorescence disinfection. properties Moreover, of catechins organic and matter epicatechins in pipe wallwere biofilmdetected when in the UV/tea biofilm of the pipe network, which indicated that tea polyphenols would interact with the pipe wall microorganisms [35]. Therefore, the characteristics and environmental factors of the tea polyphenols disinfection pipe wall microorganisms in pipe networks still need to be

studied in depth with a focus on the dual role and conditions of tea polyphenols on the inhibition and assimilation of pipe wall microorganisms to clarify the microbial safety of tea polyphenol disinfection of pipe wall networks. Water 2021, 13, x FOR PEER REVIEW 10 of 15

polyphenols combined disinfection. Moreover, catechins and epicatechins were detected in the biofilm of the pipe network, which indicated that tea polyphenols would interact with the pipe wall microorganisms [35]. Therefore, the characteristics and environmental factors of the tea polyphenols disinfection pipe wall microorganisms in pipe networks still need to be studied in depth with a focus on the dual role and conditions of tea polyphenols Water 2021, 13, 1835 10 of 15 on the inhibition and assimilation of pipe wall microorganisms to clarify the microbial safety of tea polyphenol disinfection of pipe wall networks.

100

Negativicutes Planctomycetia 80 Acidobacteria_Gp4 Anaerolineae Deltaproteobacteria Nitrospira 60 Bacteroidia Clostridia Actinobacteria Bacilli 40 Sphingobacteriia Gammaproteobacteria Betaproteobacteria Proportion of bacterial species/% 20 Alphaproteobacteria Other

0 O3 O3/TP O3/NaClO

Figure 7. Colony structure at class level. Figure 7. Colony structure at class level. 3.4.3. Microbial Diversity Analysis Based on Genus Level 3.4.3. Microbial Diversity Analysis Based on Genus Level The colony structure at genus level is shown in Figure8. It can be seen that the distri- butionThe of colony microorganisms structure at was genus relatively level is simple shown after in Figure ozone disinfection,8. It can be seen with thatPseudomonas the dis- tributiondominating of microorganisms about 60.67%, followed was relatively by Blastomonas simple after(13.69%), ozone Methylobacteriumdisinfection, with(13.11%), Pseudo- monasand Curvibacter dominating(3.86%). aboutPseudomonas 60.67%, followedwere found by Blastomonas to exhibit excellent (13.69%), biofilm Methylobacterium formation in (13.11%),a variety and of environments, Curvibacter (3.86%). and they Pseudomonas provide protection were found against to exhibit chemical excellent and biofilm mechanical for- mationattack [in43 a]. variety In contrast, of environments, the percentage and of theyPseudomonas provide protectionwere significantly against chemical reduced and and mechanicalthe dominant attack genus [43]. changed In contrast, when the ozone percentage and auxiliary of Pseudomonas disinfectants were were significantly combined re- for duceddisinfection. and the The dominant microorganisms genus changed with a higher when percentage ozone and were auxiliaryMethylobacterium disinfectants(23.62%), were combinedRhizobacter for(15.13%), disinfectionMethylophilus. The microorganisms(6.17%), Pseudomonas with a higher(6%) and percentageCurvibacter were(3.91%) Methylo- after bacteriumthe combined (23.62%), O3/TP Rhizobacter disinfection. (15.13%), The dominant Methylophilus bacterium (6.17%),Methylobacterium Pseudomonas belongs(6%) and to CurvibacterMethylobacteriaceae (3.91%) andafterAlphaproteobacteria the combined . O Tsagkar3/TP disinfection et al. [44] found. The that dominant it is a key bacterium strain in Metthehylobacterium formation of belongs bacterial to aggregates Methylobacteriaceae in water and subsequentAlphaproteobacteria surface. Tsagkar biofilm formation,et al. [44] foundwhich that may it account is a key for strain its high in the percentage formation in theof bacterial pipe wall aggregates flora. The percentage in water and of Curvibac- subse- quentter increased surface significantlybiofilm formation, to about which 46.87% may after account the combined for its high O3/NaClO percentage disinfection, in the pipe and wallthis flora bacterium. The percentage belongs to ofComamonas Curvibacter, Betaproteobacteria increased significantly, a new to genus about identified 46.87% after by Ding the combinedand Yokota O3 of/NaClO the University disinfection, of Tokyo and this in 2004bacterium [45]. In belongs addition, to thereComamonas were Pseudomonas, Betaproteo- bacteria(6.71%),, a Duganellanew genus(4.87%), identifiedAchromobacter by Ding and(2.19%), YokotaNovosphingobium of the University(1.84%), of TokyoAlcaligenes in 2004 [45].(1.46%) In addition, and Bacillus there(1.39%) were Pseudomonas under this (6.71%), method. Duganella That is, (4.87%), the pipe Achromobacter wall microorganisms (2.19%), Novosphingobiumare more complex (1.84%), when Alcaligenes sodium hypochlorite (1.46%) and isBacillus used as(1.39%) an auxiliary under this disinfectant. method. That The is,distribution the pipe wall of microorganisms are in the more water complex and the when pipe sodium wall has hypochlorite some correlation is used and as andifference, auxiliary whichdisinfectant. needs toThe be distribution compared and of analyzed.microorganisms in the water and the pipe wall has some correlation and difference, which needs to be compared and analyzed. 3.5. Comparative Analysis of Microorganisms in the Water and the Pipe Wall From the analysis in 2.3 and 2.4, it is clear that the bacterial community composition is significantly different under the action of different disinfection methods, which has a greater correlation with the nature of the bacteria themselves. In this study, the analysis of biodiversity can clarify the differences in bacterial tolerance to the environment under the three disinfection methods. The differences in bacterial community structure at class level are presented as an example (Table2).

Water 2021,, 13,, x 1835 FOR PEER REVIEW 1111 of of 15 15

100 Clostridium Duganella Hyphomicrobium 80 Curvibacter Blastomonas Azospira Alcaligenes 60 Achromobacter Bacillus Rhizobacter 40 Novosphingobiu Sphingobium Variovorax Pseudomonas Proportion of bacterial species/% 20 Methylobacteriu Methylophilus Porphyrobacter Other 0 O3 O3/TP O3/NaClO

FigureFigure 8. 8. ColonyColony structure structure at at genus genus level. level. Table 2. Percentage of microorganisms at class level (water/pipe wall). 3.5. Comparative Analysis of Microorganisms in the Water and the Pipe Wall Disinfection Method From the analysis in 2.3 and 2.4, it is clearO that(%) the bacterial O /TP community (%) O composition/NaClO (%) Bacterial Composition 3 3 3 is significantly different under the action of different disinfection methods, which has a Alphaproteobacteria 14.67/29.94 74.86/61.07 9.22/6.68 greater correlation with the nature of the bacteria themselves. In this study, the analysis Betaproteobacteria 0.21/6.68 18.51/16.29 21.94/68.92 of biodiversityGammaproteobacteria can clarify the differences in 82.92/61.27bacterial tolerance 6.58/21.44 to the environment 15.99/11.51 under the three disinfectionSphingobacteriia methods. The differences-/0.47 in bacterial community 0.01/0.57 structure 3.75/2.19 at class level are presentedBacilli as an example (Table 2). 0.02/0.42 0.02/0.09 9.68/1.74 Clostridia -/0.03 -/0.02 7.86/2.57 Table 2. PercentageBacteroidia of microorganisms at class level (water/pipe-/- wall). -/- 3.88/0.87 Nitrospira -/- -/- 3.26/0.52 DisinfectionPlanctomycetia method -/0.33 -/0.30 1.66/0.35 O3 (%) O3/TP (%) O3/NaClO (%) Bacterial compositionOther 1.36/0.04 0.02/0.04 12.98/2.49 Note: “-”Alphaproteobacteria means the bacterium is not detected14.67/29.94 or the percentage of detection74.86/61.07 is relatively small, which9.22/6.68 is classified as other. Betaproteobacteria 0.21/6.68 18.51/16.29 21.94/68.92 Gammaproteobacteria 82.92/61.27 6.58/21.44 15.99/11.51 It canSphingobacteriia be seen from Table2 that the-/0.47 type of disinfectant0.01/0.57 affects the composition3.75/2.19 and structure of theBacilli bacterial community0.02/0.42 and the dominant bacteria0.02/0.09 detect vary,9.68/1.74 however, the dominant bacteriaClostridia are consistent in the-/0.03 water and the pipe-/0.02 wall for the same7.86/2.57 disinfection method. InBacteroidia general, ozone disinfection can-/- effectively remove-/-Betaproteobacteria 3.88/0.87, and O 3/TP disinfectionNitrospira has a strong inhibitory effect-/- on Gammaproteobacteria-/- . The two3.26/0.52 also have a better removalPlanctomycetia effect on Bacilli, Clostridia-/0.33, Bacteroidia , etc.,-/0.30 which were poorly1.66/0.35 inhibited when O3/NaClOOther disinfection was used.1.36/0.04 However, O30.02/0.04/NaClO disinfection12.98/2.49 can better Note:remove “-” Alphaproteobacteriameans the bacterium thanis not thatdetected of O or3/TP the percentage disinfection. of detection Studies is have relatively shown small, that tea whichpolyphenols is classified have as antibacterialother. activity against both Gram-negative and Gram-positive bacteria, but have a stronger effect on the latter [46]. Most of Alphaproteobacteria are Gram- negativeIt can bacteria, be seen so from it accounts Table 2 for that a higherthe type proportion of disinfectant when Oaffects3/TP disinfectionthe composition was used.and structureDouterelo of et the al. bacterial [47] measured community significantly and the different dominant bacterial bacteria community detect vary, composition however, the in dominantbiofilms and bacteria water are samples consistent in an in experimental the water and drinking the pipe waterwall for distribution the same disinfection system and method.found that In Betaproteobacteria general, ozone disinfectionand Gammaproteobacteria can effectivelywere remove the dominant Betaproteobacteria bacteria in, and the Obiofilm3/TP disinfection in the pipeline, has a strong while inhibitoryAlphaproteobacteria effect on Gammaproteobacteriadominated in the water. The samples.two also have It is asimilar better toremoval these testeffect results, on Bacilli but Douterelo, Clostridia, et Bacteroidia al. chose, only etc., sodiumwhich were hypochlorite poorly inhibited as disin- whenfectant O during3/NaClO their disinfection study. Geng was et used. al. [48 However,] found that O3/NaClOBacillus subtilisdisinfectionspores can are better resistant re- moveto chlorine Alphaproteobacteria disinfection alone,than that but of the O3 inactivation/TP disinfection. effects Studies of them have are shown greatly that enhanced tea pol- yphenolswhen ozone/chlorine have antibacterial combined activity disinfection against both has aGram synergistic-negative effect. and TachikawaGram-positive et al. bac- [49] teria,found but that have the a sequential stronger effect disinfection on the oflatter ozone [46]. and Most hydrogen of Alphaproteobacteria peroxide showed are aGram good- negativesynergistic bacteria, effect, so and it accounts the removal for effecta higher of biofilmproportion was when better. O Combined3/TP disinfection disinfection was used. has Douterelocertain application et al. [47] prospects,measured significantly and its mechanism different still bacterial needs to community be explored composition in depth. For in

Water 2021, 13, 1835 12 of 15

the development of biological safety control measures for drinking water, the criteria measured include total bacteria, opportunistic pathogens or pathogens, changes in biodiversity, bioﬁlm and bioerosion, etc. [50]. Therefore, the analysis of microbial diversity in the pipe network is relevant for the selection of auxiliary disinfectants. It also provides scientiﬁc support for the application of ozone/tea polyphenols combined disinfection systems.

4. Conclusions (1) The total bacterial colonies in the water were well controlled when the auxiliary disinfectant was combined with ozone for disinfection in stainless steel pipe network simulation system. When using tea polyphenols and sodium hypochlorite as auxiliary disinfectants, the total bacterial colonies in the water remained below 100 CFU·mL−1 for 35 days and 5 days, respectively. This indicates that the combined ozone/tea polyphenols disinfection is more sustainable and the water quality in the pipe network is safer. (2) Microbial species diversity and richness in the water increased when combined with auxiliary disinfectants compared to ozone disinfection. The combined ozone/tea polyphenols disinfection has better bacterial inhibition effect and lower microbial diversity in the water compared with the combined ozone/sodium hypochlorite disinfection. Mi- crobial diversity increased and richness decreased on the pipe wall when the combined ozone/tea polyphenols disinfection was used, this may be due to the effect of metal ions in the stainless steel pipe on the disinfection of tea polyphenols. However, microbial diversity and richness on the pipe wall both increased when the combined ozone/sodium hypochlorite disinfection was used. This indicates that different auxiliary disinfectants combined with ozone have different effects on the biofilm organisms on the pipe wall. (3) For microorganisms in the water, the dominant bacteria were approximately the same at phylum and class levels before and after the application of auxiliary disinfectants. However, the proportions were different and the other bacterial groups varied considerably. Compared with sodium hypochlorite as an auxiliary disinfectant, the ozone/tea polyphenols combined disinfection was better at removing common microorganisms and chlorine- resistant bacteria such as Bacilli, Actinobacteria and Sphingobacteriia, etc. At genus level, the microbial species were simple after ozone disinfection, Pseudomonas and Blastomonas were the dominant genera with a total share of about 90%, while the killing effect on both was significant after combined disinfection. The combined ozone/tea polyphenol disinfection was better in killing Citrobacter, Escherichia, and other opportunistic pathogens, which improved the biosafety compared to sodium hypochlorite as an auxiliary disinfectant. (4) For microorganisms of the pipe wall, Proteobacteria were the main phylum under the three disinfection methods. The dominant genera at class level were Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, respectively, when ozone, the combined ozone/tea polyphenols disinfection and the combined ozone/sodium hypochlorite were applied as disinfectants, which were related to the difference in the effects of different disinfectants on bacteria. At genus level, the microbial distribution was highly variable, with Pseudomonas, Methylobacterium and Curvibacter, etc. all showing some biofilm formation ability. The distribution of microorganisms in water and pipe wall had some correlation and difference, and the correlation showed that the dominant genera in water and pipe wall were unified by different disinfection methods. The difference was manifested in the different growth ability of the same microorganism in water and pipe wall, for example, Betaproteobacteria tend to form biofilms on the inner wall of the pipe, so their proportion in the wall microorganisms is significantly larger than that in water. Microorganisms on the pipe wall will enter the water under the effect of water flow or aging off in actual operation and become the source of bacteria in the water. Therefore, it is necessary to take measures to suppress the microorganisms in the pipe network to ensure the safety of drinking water. (5) Combined disinfection has more advantages in microbiological safety control than ozone alone disinfection. In the stainless steel pipe network, the composition of bacteria in the water is complex and the microbial risk is high after the combined ozone/sodium hypochlorite disinfection. It has a persistent disinfection effect and small microbial diver- Water 2021, 13, 1835 13 of 15

sity in the water when tea polyphenols are used as auxiliary disinfectants, besides, the inhibitory effect on chlorine-resistant bacteria and opportunistic pathogens in the water is stronger than that of sodium hypochlorite. Therefore, the combined ozone/tea polyphenol disinfection method can better ensure the safety of drinking water in the pipe network.

Author Contributions: Conceptualization, C.F., N.Z. and Z.G.; methodology, Z.G. and N.Z.; software, Z.X. and N.Z.; validation, C.F., N.Z. and Y.L.; formal analysis, N.Z. and Z.X.; resources, C.F.; data curation, Z.G. and N.Z.; writing—original draft preparation, N.Z.; writing—review and editing, C.F., Y.L. and N.Z.; visualization, Z.X.; supervision, C.F.; project administration, C.F.; funding acquisition, C.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (51678026), Beijing University of Civil Engineering Postgraduate Innovation Project (PG2021047). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest: The authors declare no conflict of interest.

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