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Article

Effect of Pipe Materials and Interspecific Interactions on Biofilm Formation and Chlorine Resistance: Turn Enemies into Friends

by
Lili Shan
1,2,
Yunyan Pei
2,
Siyang Xu
2,3,
Yuhong Cui
1,*,
Zhengqian Liu
1,
Zebing Zhu
2,* and
Yixing Yuan
4
1
School of Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, China
2
School of Civil Engineering and Architecture, East China Jiao Tong University, Nanchang 330013, China
3
Comprehensive Transportation Development Research Center of Jiangxi Provincial, Department of Transportation of Jiangxi Province, Nanchang 330036, China
4
State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China
*
Authors to whom correspondence should be addressed.
Water 2024, 16(20), 2930; https://doi.org/10.3390/w16202930
Submission received: 13 September 2024 / Revised: 3 October 2024 / Accepted: 9 October 2024 / Published: 15 October 2024
(This article belongs to the Topic Towards Energy-Positive and Carbon-Neutral Technology for Wastewater Treatment and Reclamation)
Browse Figures
Versions Notes

Abstract

:
Drinking water distribution systems (DWDSs) may be contaminated to various degrees when different microorganisms attach to the pipe walls. Understanding the characteristics of biofilms on pipe walls can help prevent and control microbial contamination in DWDSs. The biofilm formation, interspecific interactions, and chlorine resistance of 10 dual-species biofilms in polyethylene (PE) and cast iron (CI) pipes were investigated in this paper. The biofilm biomass (heterotrophic bacterial plate count and crystal violet) of dual species in CI pipes is significantly higher than that in PE pipes, but the biofilm activity in CI pipes is significantly lower than that in PE pipes. The interspecific interaction of Sphingomonas-containing group presented synergistic or neutral relationship in PE pipes, whereas the interspecific interaction of the Acidovorax-containing group showed a competitive relationship in CI pipes. Although interspecific relationships may help bacteria resist chlorine, the chlorine resistance was more reliant on dual-species groups and pipe materials. In CI pipes, the Microbacterium containing biofilm groups showed better chlorine resistance, whereas in PE pipes, most biofilm groups with Bacillus exhibited better chlorine resistance. The biofilm groups with more extracellular polymeric substance (EPS) secretion showed stronger chlorine resistance. The biofilm in the PE pipe is mainly protected by EPS, while both EPS and corrosion products shield the biofilms within CI pipe. These results supported that dual-species biofilms are affected by pipe materials and interspecific interactions and provided some ideas for microbial control in two typical pipe materials.

1. Introduction

Biofilms on pipe walls and planktonic microorganisms in bulk water can affect the water safety of drinking water distribution systems (DWDSs) [1]. In DWDSs, about 95% of total microbial population proliferated in biofilms, as they can be well-protected [2,3]. With high-throughput deep sequencing, many microbial communities have been found in DWDSs. Some chlorine-resistant bacteria (CRB), including Bacillus, Pseudomonas, Mycobacterium, and Acinetobacter can still grow at high chlorine levels (>0.6 mg/L) [4]. Many CRB are pathogens or conditionally pathogens [3,5]. The CRB, such as Sphingomonas, Acidovorax, Bacillus, and Acinetobacter, have been reported by many studies since they may pose a threat to drinking water quality [6,7,8]. Ahmed et al. [9] have reviewed many studies and found that Microbacterium, Acinetobacter, Bacillus, and Sphingomonas may enhance the biofilm biomass or the resistance against disinfectants. The CRB can cause microbial growth as biofilms, which would lead to multiple issues, including turbidity, chroma, odors, pipe corrosion, and outbreaks of diseases [2,10,11,12,13,14,15]. Therefore, it is necessary initially to focus on the biofilm formation of CRB in DWDSs.
Microbial interspecific interactions may lead to different outcomes, providing benefits to one or all the interacting groups, deleterious or not, and influencing either group respectively [16]. The synergistic, neutral, and competitive interspecific interactions may have an influence on biofilm biomass and resistance to disinfectants [17]. The interspecific interaction between the same bacterial groups may be altered by nutrients [18] and space. Polyethylene (PE) and cast iron (CI) are two kinds of pipe materials commonly used in DWDSs. The significant strength of CI makes it an optimal pipe material [10], while PE is lightweight and not easily corroded and has been widely used for water distribution pipes with diameters of less than 300 mm [19]. It has been reported that nutrients can be dissolved from plastic pipes that are beneficial to the growth of microorganisms [20], while the surface of CI is easily corroded, which facilitates the attachment and growth of microorganisms [21]. Although these factors may affect microbial interspecific interactions, research on the effects of pipe materials on microbial interspecific interactions is scarce.
The microbial resistance to disinfectants can be altered by interspecific interactions [17]. Simoes et al. [22] have found that the dual-species biofilm of Pseudomonas and Bacillus exhibited more resistance to chemicals than mono-species biofilm, whereas Lindsay et al. [23] noted that Bacillus increased Pseudomonas biofilm sensitivity to disinfectants. In addition, it has also been indicated that the reaction between pipeline materials and disinfectants can reduce the disinfection effect [24,25]. Pipe materials and interspecific interactions also have various effects on biofilms. However, the interactive impacts of pipe materials, bacterial species, and chlorination on biofilms in DWDSs are unknown. Hence, it is necessary to study the biofilm formation of various representative bacteria and pipe materials in DWDSs under consistent cultivation conditions to obtain more useful results.
The aim of this study was to identify the effects of pipe material on biofilm formation, interspecific interactions, and chlorination. Actinobacteria were found to be highly abundant in PE and CI, with Bacilli only having a higher abundance in CI [26]. Bacillus of the Bacilli and Acidovorax of the β-Proteobacteria may lead to iron corrosion [27], and the corroded CI may have an influence on biofilm in DWDSs. The CRB of Sphingomonas, Acidovorax, Bacillus, Microbacterium, and Acinetobacter can reduce potable water quality [6,7,28,29,30]. The five CRB isolates were combined into ten dual-species groups for the representation of dual-species biofilms in DWDSs. The biofilm formation, interspecific interactions, and chlorine resistance of these ten dual-species groups in PE and CI pipes were investigated, and the effects of pipe materials on interspecific interactions and chlorine resistance were analyzed in depth. This could greatly promote the application of pipe materials to microbial control in DWDSs.

2. Materials and Methods

2.1. Bacterial Strains and Pipe Materials

Sphingomonas sp., Acidovorax defluvii, Acinetobacter sp., Bacillus cereus, and Microbacterium laevaniformans were selected as the representative CRBs, whose characteristics were described in a previous study [17]. Those CRB strains were chosen for being representative of drinking water bacteria due to their ability to form complex biofilms. The strains of Sphingomonas sp., Acidovorax defluvii, and Acinetobacter sp. affiliated with Proteobacteria were Gram-negative (G−), while Bacillus cereus affiliated with Firmicutes and Microbacterium laevaniformans affiliated with Actinobacteridae were Gram-positive (G+). All sequences of tested bacterial strains were deposited in the Genbank of the National Center for Biotechnology Information (NCBI) under accession numbers MT279967 to MT279971. The five-CRB strain suspension was cultured on an R2A medium, and ten dual-species groups were formed with equal density (OD600) of the two-strain suspension.
Two kinds of pipe coupons (PE and CI) were used to form biofilm in simulated DWDSs, and the features of the coupons are listed in Table S1. Before the experiment, the CI pipe coupons were polished step by step to keep the same roughness. Large coupons were cut for the biofilm EPS analysis, while smaller ones were cut for the determination of biofilm biomass, activity, and morphology. Iron concentration and the corrosion level of CI were measured according to Zhu‘s method [8].

2.2. Biofilm Formation and Chlorination Experiments

Biofilm formation and chlorination experiments were carried out by our previous modified methods [11]. In brief, equal volumes of bacterial culture with an OD600 value of 0.05 (approximately 106 cells/mL in R2A broth) were mixed and placed into a sterile centrifuge tube with a piece of pipe. The biofilm formation experiments were conducted under standardized conditions of 100 rpm and 25 °C with fresh R2A broth (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) in a shaking incubator. The biofilms that formed after 5 days of incubation were disinfected with 4 mg/L free chlorine, which is the maximum limit in DWDSs in China and the United States [17]. The experiment was carried out in a shaking incubator at 100 rpm and 25 °C for 1 h to make the biofilm fully contact the NaClO (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). The tube was rinsed twice with 0.5% (w/v) Na2S2O3 (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) to stop the chlorination reaction and rinsed once with sterile water to remove the loosely attached planktonic bacteria. Then, the biomass, extracellular polymeric substances (EPS), and morphology of the biofilm before and after chlorine disinfection were measured.

2.3. Biofilm Analysis Method

The biofilms formation was evaluated by detecting the biomass from the 1st to 5th days of incubation in PE and CI pipe. The classification method for biofilm formation was based on the determination of crystal violet (CV) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) staining, as described by Stepanovic [31] and Zhu [30]. On the 5th day, the biofilm was disinfected, and the biofilm biomass, activity, and morphology before and after disinfection were measured to evaluate the effect of chlorination on the biofilm. The extraction of biofilm was carried out using the method of Zhu [17], and then the heterotrophic plate count (HPC) and CV were measured. The specific respiratory activity was analyzed using the XTT (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) method [32] and an ATP assay kit [33]. The morphology of the biofilm in the pipe was observed using field emission scanning electron microscopy (FE-SEM) (Apreos, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA) characterization, as described by Siddam [34]. The extraction and determination of EPS in biofilms, as described by Zhu [30], includes the contents of protein (PN), polysaccharide (PS), and extracellular DNA. Each experiment was performed three times.

2.4. Analysis of Variance and Correlation

The analysis of variance (ANOVA) and Spearman correlation for investigating biofilm biomass, activity, and EPS was carried out using SPSS 26.0 for Windows software, as described by Vaz-Moreira [35] and Zhu [8].

3. Results and Discussion

3.1. Biofilm Formation Ability

The biofilm formation ability of ten dual-species in PE and CI pipe are exhibited in Figure 1a,b, respectively. Biofilm formation abilities were significantly (p < 0.05) influenced by both pipe materials and the dual-species group. During the first day, Sphingomonas sp. + Microbacterium laevaniforman and Bacillus cereus + Microbacterium laevaniforman in the PE pipe presented weak biofilm formation ability, whereas only Acinetobacter sp. + Microbacterium laevaniforman exhibited weak biofilm formation ability in the CI pipe. This indicated that dual-species bacterial adhesion on the CI pipe surface was stronger than on the PE pipe, possibly due to its high absolute roughness (Table S1).
The CV values and biofilm formation ability of ten dual-species groups in the two pipe materials increased with growth time. As compared to the PE pipe, according to the CV values in the CI pipe, almost all the dual-species groups showed a faster increase. On the fifth day, all 10 groups in the CI pipe exhibited strong biofilm formation ability, perhaps because the CI pipe was more susceptible to corrosion, making it more suitable for microorganisms to grow on the corroded surface [21]. While, in PE pipe, strong biofilm formation ability was observed in eight dual-species groups, except Bacillus cereus + Microbacterium laevaniforman and Acidovorax defluvii + Microbacterium laevaniforman. The CV values in the PE pipe exhibited slight differences from the results of our previous biofilm formation on a 96-well microplate [29], but the biofilm formation in the first three days was similar (Figure 1a), which may be attributed to different cultivation days and pipe materials.
HPC evaluation results revealed that the biofilm biomass in the PE pipe was significantly lower (p < 0.001) than those in the CI pipe on day 5 (Figure 1c), which is consistent with Hemdan’s studies [24]. It should be mentioned that Acidovorax [12], Bacillus [13], Sphingomonas, and Acinetobacter [14,15] would corrode CI and cause a rougher surface, which could concentrate organic nutrients [21]. In general, the dual-species relationship would be deemed a synergistic relationship if the dual-species biofilm biomass was more than the sum of mono-species biomasses in the same environment. The dual-species relationship is classified as a competitive or neutral relationship when the biomass is lower than the sum of the biomass or shows no significant difference in the sum of the biomass [17]. In the PE pipe, Acidovorax defluvii + Microbacterium laevaniforman, Bacillus cereus + Microbacterium laevaniforman, and Bacillus cereus + Acinetobacter sp. exhibited competitive relationships, Sphingomonas sp. + Microbacterium laevaniforman and Acinetobacter sp. + Microbacterium laevaniforman showed synergism, and other dual-species groups presented neutralism. Furthermore, dual-species groups with Sphingomonas sp. did not exhibit competition in the PE pipe, implying that the presence of Sphingomonas sp. may allow for a more harmonious interspecific relationship in the biofilm. On the contrary, in the CI pipe, Sphingomonas sp. + Microbacterium laevaniforman and Sphingomonas sp. + Bacillus cereus showed synergism, Bacillus cereus + Microbacterium laevaniforman and Acinetobacter sp. + Microbacterium laevaniforman presented neutralism, and other groups exhibited competition. Moreover, groups with Acidovorax defluvii presented competition, suggesting that the addition of Acidovorax defluvii may be helpful in controlling dual-species biofilm in CI pipes. These differences in the relationships between the same bacterial groups in different pipes could be due to the combined effects between pipe materials and dual-species groups. The PE pipe environment did not cause stress to the dual-species groups under nutritional sufficiency. In contrast, the high iron concentration in CI pipes can impair microbial growth (Figure S1) [36]. Although dual-species groups in the CI pipe always showed competition, the biofilm biomass in the CI pipe is still higher than that in the PE pipe. This indicated that the influence of the pipe materials on biofilm biomass is significantly more than that of interspecific interaction.

3.2. Dual-Species Biofilm Activity

The ATP level represents a vital index of cell metabolic viability and activity [37], and scholars have employed XTT as a measure of cell-specific respiratory activity [29]. Whether PE pipe (r = 0.839, p < 0.001) or CI pipe (r = 0.839, p < 0.001), the ATP concentration showed a significant correlation with the specific respiratory activity (Figure 2) in the present study. Interestingly, the CI dual-species groups presented greater biofilm biomass but lower bacterial activity compared with those in the PE pipe (p < 0.001). Different culture conditions can alter bacterial activity [38]. In the research, the CI was highly corroded (Figure S2), with high concentrations of iron ions released to the environment, and ATP was consumed for iron ion expulsion from the cells [39]. As shown in Figure S2, bacterial cells are enveloped by corrosion products in the CI pipe. Previous studies have shown that bacterial cells require substantial energy for survival in deteriorating environments [40], which may be the reason for the negative correlation between iron concentration and bacterial activity observed in the present study (Figure S4). Moreover, it has been reported that bacteria may consume energy to secrete some substances to gain a competitive advantage [41], which may result in lower bacterial activity in the CI pipes. It also can be found that the synergistic groups exhibited higher bacterial activity.

3.3. Dual-Species Biofilm Biomass Reduction after Chlorination

The reduction in dual-species biofilm biomass in the two pipe materials after chlorination is shown in Figure 3. It can be seen that the HPC reduction was lower than the CV reduction. Despite the high chlorine concentration, the chlorination effect was clearly weakened by the short disinfection time and the thicker biofilm [42]. Thus, many bacterial cells were only damaged without rupture, and then they could revive under appropriate conditions [43], which led to lower HPC reduction. Dual-species groups showed different chlorine resistance levels in the two pipe materials. Only Sphingomonas sp. + Microbacterium laevaniforman exhibited synergism and exhibited higher chlorine resistance in the two pipes. Notably, the chlorine resistance of Acinetobacter sp. + Microbacterium laevaniforman in the PE pipe and Sphingomonas sp. + Bacillus cereus in the CI pipe was weak, despite their synergistic interactions. In addition, Acidovorax defluvii + Microbacterium laevaniforman, which showed competition in both pipes, showed better chlorine resistance. Therefore, the chlorine resistance of dual-species groups depended more on pipe materials and bacterial species than interspecific interactions.
In addition, the combined effects of bacterial species, pipe materials, and chlorination were notable (p < 0.001) on the dual-species biofilms. The dual-species groups containing Microbacterium laevaniforman showed better chlorine resistance in the CI pipe, which means that Microbacterium laevaniforman in the CI pipe may enhance the chlorine resistance of some dual-species groups. However, the result was not observed in the PE pipe, suggesting that the role of bacteria in chlorination may be affected by pipe materials [25]. The dual-species groups with Bacillus cereus demonstrated better chlorine resistance in the PE pipe, except for Acidovorax defluvii + Bacillus cereus. Notably, Bacillus exhibited strong chlorine resistance, as they can form spores [35] and secrete EPS. Nevertheless, Acidovorax defluvii + Bacillus cereus exhibited lower chlorine resistance because of their moderate biofilm biomass and low EPS content. The biofilm resistance to disinfectants can be improved under certain conditions when certain bacteria exist in a biofilm [3], and the resistance of bacterial biofilms may change with pipe materials [24,44]. Furthermore, different bacteria can cause pipe corrosion to different extents (Figure S2), and the corrosion products will react with the chlorine to reduce the chlorination effect [25]. All these factors can lead to diverse chlorination effects on the same dual-species biofilm in different pipe materials [17,23].

3.4. Dual-Species Biofilm Activity Reduction after Chlorination

Dual-species biofilm activity reduction in two pipe materials after chlorination is shown in Figure 4. The dual-species biofilm activity reduction in the PE pipe was greater than that in the CI pipe. In the CI pipe, due to the low initial biofilm activity before chlorination, it was not easy to further reduce biofilm activity. In the PE pipe, the biofilm activity reduction of Acidovorax defluvii + Microbacterium laevaniforman was the lowest, whereas those of Acinetobacter sp. + Microbacterium laevaniforman, Acidovorax defluvii + Acinetobacter sp., and Acidovorax defluvii + Bacillus cereus were higher. Biofilm activity was reduced to a lesser extent in the dual-species groups with Bacillus cereus, except for Acidovorax defluvii + Bacillus cereus. In the CI pipe, Sphingomonas sp. + Bacillus cereus exhibited the highest reduction in biofilm activity, whereas the dual-species groups with Microbacterium laevaniforman demonstrated a low reduction in biofilm activity.

3.5. Dual-Species Biofilm EPS Content before and after Chlorination

EPS can promote biofilm formation [36]. As shown in Figure 5a, the content of EPS, especially polysaccharides and proteins, was significantly lower in the CI pipe than in the PE pipe (p < 0.001). The biofilm bacteria in the CI pipe can be protected by the reaction between the corroded surface and disinfectants [25], leading to the replacement of the EPS function and a decrease in EPS content. These results revealed that dual-species groups with high biofilm biomass may not have high EPS contents, which is consistent with the findings reported in many studies [17,45,46]. In addition, synergism can also increase the EPS content [47]. In the two outdoor pipes, Acidovorax defluvii + Acinetobacter sp. showed the highest EPS content, followed by groups presenting synergism, which were among the top four groups with high EPS content. Most of the dual-species groups in the PE pipe exhibited synergism and neutralism, whereas the majority of the groups indicated competitive interaction in the CI pipe. These findings may also explain the significant correlation between the EPS content and biofilm biomass in the PE pipe but not the CI pipe (Figure S4). According to the results of this study, bacteria species and pipe materials had a more significant influence on EPS content than interspecific interaction.
The EPS content of the biofilms in the two pipes decreased after chlorination. As illustrated in Figure 5b, the EPS content was higher in the PE pipe than in the CI pipe. Biofilm bacteria would be protected by EPS when subjected to environmental stress [48], and corrosion substances on CI pipes can react with some disinfectants, surrounding bacterial cells and preventing them from chlorination in CI pipes, thereby protecting the bacteria [25,49]. Therefore, although the dual-species biofilm in PE pipes showed higher EPS content, there was no significant difference in the biofilm biomass reduction between PE pipes and CI pipes. Although the Sphingomonas sp. + Bacillus cereus in the PE pipe displayed better chlorine resistance, their chlorine resistance is poor in CI pipes, which may be due to the different protective effects of EPS. The EPS content of Sphingomonas sp. + Bacillus cereus in CI pipes was the second highest before chlorination but reached a low level after chlorination. Dual-species groups with Microbacterium laevaniforman in the CI pipe exhibited low EPS reduction (Figure S3) and better chlorine resistance. The EPS and biomass reduction of the biofilms were significantly correlated (Figure S4) in CI (r = 0.826, p < 0.001) and PE pipes (r = 0.646, p < 0.001), demonstrating a positive correlation between higher EPS reduction and more biomass reduction. This result indicated that a more robust EPS can protect bacteria better during the chlorination process, mainly by inhibiting disinfectant diffusion [50,51].

3.6. Biofilm Morphological Changes between Pre- and Post-Chlorinations

The microbial cells of dual-species biofilms in different pipe materials were more exhaustively examined by using SEM. Three groups of representative dual-species biofilm with low, moderate, and high biomasses were selected for SEM observation to highlight the difference between the two pipe materials. As illustrated in Figure 6, CI and PE provided distinct growth environments for the dual-species biofilms. The CI pipes were severely corroded by bacteria, while the PE pipes remained unchanged. The corrosion products on the CI pipe, which were identified as lepidocrocite (γ-FeOOH) [52] and akageneite (α-Fe2O3) [53], were enveloped with different sizes of bacterial cells. Moreover, the EPS was also detected around bacterial cells. However, after chlorination, the bacterial cells in the biofilm were greatly dispersed, the integrity and strength of the biofilm structure were fully reduced, and the number of cells significantly decreased. In addition, bacterial cell morphologies have been altered after chlorination, and some cells were damaged or deformed. In addition, the EPS content was reduced in PE pipes, while there were still some corrosive substances encircling the bacterial cells in CI pipes.

3.7. Mechanism of Dual-Species Biofilm Formation and Chlorine Resistance

The mechanisms of dual-species biofilm formation and chlorine resistance in the two pipe materials were summarized in Figure 7. Although PE and CI pipes are both common outdoor pipes, these two types of pipe materials create different suitable habitats for microbial growth. The PE pipe promoted microbial growth by dissolving nutrients [20], while the CI pipe was easily corroded, resulting in an increase in surface roughness and release of iron ions, which would be beneficial for the adhesion of bacteria in the biofilm. The biofilm in the CI pipe had more biomass and more dispersed bacterial cells, which were surrounded by corrosion products. Interspecific interaction also affected the dual-species groups and varied with different pipe materials, with more than half of the population exhibiting competitive relationships. In the competitive interaction of dual-species biofilm, bacterial cells may consume energy to impair other bacterial cells. In addition, higher concentrations of iron ions may inhibit microbial growth [36], and the unfavorable environment of the CI pipe decreased the bacterial activity, which led to a low EPS content. In contrast, most of the dual-species groups in PE pipes showed synergism or neutralism, and the bacterial cells were more aggregated and grew well, resulting in a higher EPS content.
The biomass of both types of outdoor pipe biofilms significantly decreased after disinfection and was affected by various factors. The chlorine resistance of dual-species groups depended more on pipe materials and bacterial species than interspecific interactions. In both PE and cast iron pipes, the EPS on biofilms exerted a certain protective effect on microorganisms, but the effect depended on the type of microorganisms [8]. The corrosion products in CI pipes could also react with chlorine to act as a “shelter” to protect microorganisms from chlorination [54,55]. There is also an increase in iron ions released into the environment from CI pipes, which also affects the growth of microorganisms [8]. In the CI pipe, the presence of Microbacterium laevaniforman in the dual-species groups improved their chlorine resistance. Most of the dual-species biofilms with Microbacterium laevaniforman in the CI pipe performed synergism or neutralism, and their EPS content was stable. In PE pipes, the presence of Bacillus cereus (which can produce spores) in dual-species groups can enhance their chlorine resistance, and most of the groups with Bacillus cereus showed better chlorine resistance, but the biofilm biomass of Acidovorax defluvii + Bacillus cereus decreased significantly, and its chlorine tolerance was lower. In summary, dual-species biofilms displayed different levels of chlorine resistance due to variations in pipe materials, biofilm biomass, EPS, and interspecific interaction. Chlorination can cause some bacterial cells to deform or rupture, ultimately reducing the biomass of biofilms. More stable EPS can improve chlorine resistance, and the bacteria are more protected by corrosion products. In addition, the groups of bacteria forming spores also have stronger chlorine resistance. Due to the lower biofilm biomass only being protected by EPS, PE pipe material is more suitable for outdoor DWDSs with a diameter of less than 300 mm [19].

4. Conclusions

Outdoor pipe materials and dual-species groups displayed significant interaction effects on biofilm biomass and chlorine resistance. The biofilm biomass in the CI pipe was more than in the PE pipe. In the PE pipe, only three dual-species groups exhibited competition, whereas in the CI pipe, over half the groups presented competition. The dual-species groups exhibited different interspecific relationships between the two pipe materials. After the corrosion of the CI pipe, a large amount of iron ions were leached, the corrosion products surrounded bacterial cells, and the excess iron ions decreased ATP and EPS levels in the dual-species groups.
The effects of chlorination on dual-species biofilms in both pipes were significantly affected by many factors. In both pipe materials, the biofilm biomass significantly decreased after chlorination and was lower in the PE pipe than in the CI pipe. Although interspecific relationships may contribute to bacterial cell resistance to chlorination, the resistance to chlorination was significantly influenced by pipe material, bacterial species, biofilm biomass, EPS, and chlorine resistance mechanisms. In addition, the bacterial cells in PE pipes were predominantly protected from chlorination by EPS, while those in CI pipes were protected by both corrosion substances and EPS.
These results reveal the significant role of pipe materials and interspecific interactions on biofilm formation and indicate the factors that affect chlorination and threaten the safety and quality of water. Moreover, if conditions permit, choosing PE pipes instead of cast iron pipes can better ensure the safety of drinking water. In the future, additional pipe materials, multi-species biofilms, or different disinfectants should be explored and studied to build on the current findings and address remaining knowledge gaps.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/w16202930/s1, Table S1: the properties of the tested pipe material segments; Figure S1: iron ion concentration and the corrosion rate of cast iron pipe; Figure S2: SEM-EDS of the represented bacteria in two pipe materials; Figure S3: dual-species biofilm EPS reduction in two pipe materials after chlorination; Figure S4: correlation heat map of all data of two pipe materials.

Author Contributions

Conceptualization, Y.C., Z.L., Z.Z. and Y.Y.; methodology, S.X. and Z.Z.; software, L.S.; validation, Y.P.; formal analysis, Y.P. and S.X.; investigation, Y.P. and S.X.; resources, Z.Z.; data curation, Y.P. and S.X.; writing—original draft preparation, S.X.; writing—review and editing, L.S.; visualization, Y.P. and S.X.; project administration, L.S.; funding acquisition, L.S. and Z.Z. 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 (52460009), the Science and Technology Research Project of Jiangxi Provincial Department of Education (GJJ2200652, GJJ2200645), and the Natural Science Foundation of Jiangxi Province (20242BAB25314, 20232BAB204096).

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 16 02930 g001
Figure 1. Dual-species biofilm formation ability and biofilm biomass in two pipe materials. (a) Crystal violet of PE pipe during the biofilm formation, (b) crystal violet of cast iron pipe during the biofilm formation, (c) HPC of the 5-day biofilm. a, b, and c in the figure represent the results of a cluster analysis. a1, b1, and c1 are the results of the PE pipe, while a2, b2, and c2 are the results of cast iron pipe. I: Sphingomonas sp. + Acidovorax defluvii, II: Sphingomonas sp. + Bacillus cereus, III: Sphingomonas sp. + Acinetobacter sp., IV: Sphingomonas sp. + Microbacterium laevaniformans, V: Acidovorax defluvii + Bacillus cereus, VI: Acidovorax defluvii + Acinetobacter sp., VII: Acidovorax defluvii + Microbacterium laevaniformans, VIII: Bacillus cereus + Acinetobacter sp., IX: Bacillus cereus + Microbacterium laevaniformans, X: Acinetobacter sp. + Microbacterium laevaniformans. The groups corresponding to I~X in the following figure are the same as those in Figure 1.
Figure 1. Dual-species biofilm formation ability and biofilm biomass in two pipe materials. (a) Crystal violet of PE pipe during the biofilm formation, (b) crystal violet of cast iron pipe during the biofilm formation, (c) HPC of the 5-day biofilm. a, b, and c in the figure represent the results of a cluster analysis. a1, b1, and c1 are the results of the PE pipe, while a2, b2, and c2 are the results of cast iron pipe. I: Sphingomonas sp. + Acidovorax defluvii, II: Sphingomonas sp. + Bacillus cereus, III: Sphingomonas sp. + Acinetobacter sp., IV: Sphingomonas sp. + Microbacterium laevaniformans, V: Acidovorax defluvii + Bacillus cereus, VI: Acidovorax defluvii + Acinetobacter sp., VII: Acidovorax defluvii + Microbacterium laevaniformans, VIII: Bacillus cereus + Acinetobacter sp., IX: Bacillus cereus + Microbacterium laevaniformans, X: Acinetobacter sp. + Microbacterium laevaniformans. The groups corresponding to I~X in the following figure are the same as those in Figure 1.
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Figure 2. Dual-species biofilm bacteria activity in two pipes. (a) ATP concentration, (b) specific respiratory activity. a, b, and c in the figure represent the results of a cluster analysis. a1, b1, and c1 are the results of the PE pipe, while a2, b2, c2 are the results of the cast iron pipe.
Figure 2. Dual-species biofilm bacteria activity in two pipes. (a) ATP concentration, (b) specific respiratory activity. a, b, and c in the figure represent the results of a cluster analysis. a1, b1, and c1 are the results of the PE pipe, while a2, b2, c2 are the results of the cast iron pipe.
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Figure 3. Dual-species biofilm biomass reduction in the two pipe materials after chlorination. (a) CV reduction rate, (b) HPC reduction rate. a, b, c, and d in the figure represented the results of a cluster analysis. a1, b1, c1, and d1 are the results of the PE pipe, while a2, b2, c2, and d2 are the results of the cast iron pipe.
Figure 3. Dual-species biofilm biomass reduction in the two pipe materials after chlorination. (a) CV reduction rate, (b) HPC reduction rate. a, b, c, and d in the figure represented the results of a cluster analysis. a1, b1, c1, and d1 are the results of the PE pipe, while a2, b2, c2, and d2 are the results of the cast iron pipe.
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Figure 4. Dual-species biofilm bacteria activity reduction in two pipe materials after chlorination. (a) ATP concentration reduction, (b) specific respiratory activity reduction. a, b, and c in the figure represent the results of a cluster analysis. a1, b1, and c1 are the results of the PE pipe, while a2, b2, and c2 are the results of the cast iron pipe.
Figure 4. Dual-species biofilm bacteria activity reduction in two pipe materials after chlorination. (a) ATP concentration reduction, (b) specific respiratory activity reduction. a, b, and c in the figure represent the results of a cluster analysis. a1, b1, and c1 are the results of the PE pipe, while a2, b2, and c2 are the results of the cast iron pipe.
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Figure 5. Dual-species biofilm EPS contents before (a) and after (b) chlorination in two pipe materials. PS represents polysaccharides, and PN represents protein. a, b, c, and d in the figure represent the results of a cluster analysis. a1, b1, and c1 are the result of the PE pipe, while a2, b2, c2, and d2 are the results of the cast iron pipe.
Figure 5. Dual-species biofilm EPS contents before (a) and after (b) chlorination in two pipe materials. PS represents polysaccharides, and PN represents protein. a, b, c, and d in the figure represent the results of a cluster analysis. a1, b1, and c1 are the result of the PE pipe, while a2, b2, c2, and d2 are the results of the cast iron pipe.
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Figure 6. Biofilm morphology before and after chlorination in two pipe materials. (a) PE pipe before chlorination, (b) PE pipe after chlorination, (c) cast iron pipe before chlorination, (d) cast iron pipe after chlorination. IV: Sphingomonas sp. + Microbacterium laevaniforman, V: Acidovorax defluvii + Bacillus cereus, VI: Acidovorax defluvii + Acinetobacter sp., VII: Acidovorax defluvii + Microbacterium laevaniforman, VIII: Bacillus cereus + Acinetobacter sp., X: Acinetobacter sp. + Microbacterium laevaniforman. Triangular tips: amorphous extracellular matrix; arrows swollen, damaged, and deformed cells; tips: spores.
Figure 6. Biofilm morphology before and after chlorination in two pipe materials. (a) PE pipe before chlorination, (b) PE pipe after chlorination, (c) cast iron pipe before chlorination, (d) cast iron pipe after chlorination. IV: Sphingomonas sp. + Microbacterium laevaniforman, V: Acidovorax defluvii + Bacillus cereus, VI: Acidovorax defluvii + Acinetobacter sp., VII: Acidovorax defluvii + Microbacterium laevaniforman, VIII: Bacillus cereus + Acinetobacter sp., X: Acinetobacter sp. + Microbacterium laevaniforman. Triangular tips: amorphous extracellular matrix; arrows swollen, damaged, and deformed cells; tips: spores.
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Figure 7. Schematic of dual-species biofilm formation and chlorine resistance in two pipe materials.
Figure 7. Schematic of dual-species biofilm formation and chlorine resistance in two pipe materials.
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Shan, L.; Pei, Y.; Xu, S.; Cui, Y.; Liu, Z.; Zhu, Z.; Yuan, Y. Effect of Pipe Materials and Interspecific Interactions on Biofilm Formation and Chlorine Resistance: Turn Enemies into Friends. Water 2024, 16, 2930. https://doi.org/10.3390/w16202930

AMA Style

Shan L, Pei Y, Xu S, Cui Y, Liu Z, Zhu Z, Yuan Y. Effect of Pipe Materials and Interspecific Interactions on Biofilm Formation and Chlorine Resistance: Turn Enemies into Friends. Water. 2024; 16(20):2930. https://doi.org/10.3390/w16202930

Chicago/Turabian Style

Shan, Lili, Yunyan Pei, Siyang Xu, Yuhong Cui, Zhengqian Liu, Zebing Zhu, and Yixing Yuan. 2024. "Effect of Pipe Materials and Interspecific Interactions on Biofilm Formation and Chlorine Resistance: Turn Enemies into Friends" Water 16, no. 20: 2930. https://doi.org/10.3390/w16202930

APA Style

Shan, L., Pei, Y., Xu, S., Cui, Y., Liu, Z., Zhu, Z., & Yuan, Y. (2024). Effect of Pipe Materials and Interspecific Interactions on Biofilm Formation and Chlorine Resistance: Turn Enemies into Friends. Water, 16(20), 2930. https://doi.org/10.3390/w16202930

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