Factors influencing the inactivation of Bacillus subtilis by epigallocatechin gallate (EGCG)

The disinfection process of drinking water is essential to ensure safe drinking water quality and human health. Pollution of drinking water resources is a prominent problem due to agricultural runoff, wastewater, illegal dumping of chemicals, atmospheric pollution, and algal blooms (Prasse et al. 2022). A common method of preventing bacterial regeneration is to disinfect drinking water before it enters the network using chlorine, chlorine dioxide, or chloramine and to maintain a certain concentration of disinfectant residue. However, some bacteria in the network can still survive and even reproduce in a high chlorine residual environment, and these bacteria are more resistant to chlorine than ordinary bacteria, so they are called ‘chlorine-resistant bacteria’ (Wang et al. 2019). The regrowth of chlorine-resistant bacteria in drinking water networks may cause biological contamination, leading to deterioration of water quality and threatening human health (Wu et al. 2022). Chlorine-resistant bacteria themselves pose a significant risk to drinking water safety, and some chlorine-resistant bacteria are pathogenic or conditionally pathogenic, and their colonization of pipe networks can greatly increase the risk of waterborne diseases (Choi & Kim 2022). For example, there have been cases of food poisoning in humans caused by drinking water contaminated with Bacillus cereus, which causes symptoms such as vomiting and diarrhea (Stelder et al. 2018). In addition to this, the colonization of some non-pathogenic chlorine-resistant bacteria can lead to a decrease in the biostability of the water supply network by affecting the color, turbidity and odor of the water, or by causing corrosion of the pipes and overgrowth of biofilms (Vreeburg & Boxall 2007; Wang et al. 2019).

Therefore, increasing the chlorine dose to ensure microbial safety in the pipe network is not a proven method. Inadequate chlorine dosage not only leads to an increased risk of chemical contamination in drinking water due to the production of disinfection byproducts (DBPs), but it also fails to enhance the efficiency of inactivating chlorine-resistant bacteria (Richardson et al. 2007; Liang et al. 2022). Chloramine showed a low disinfection effect on chlorine-resistant bacteria at both high and low doses (Xu et al. 2023). In 2014, an abnormal bacterial bloom was observed in a drinking water plant in southern China, which could not be controlled by sodium hypochlorite, and the increasing dosage of chlorine in the disinfection process did not improve the situation (Ding et al. 2019). Since chlorine disinfection is not effective in controlling chlorine-resistant bacteria, ozone and ultraviolet light are used to reduce DBPs and improve the removal rate of chlorine-resistant bacteria. Ozone is a commonly used means of disinfection and has also been gradually used by researchers to inactivate chlorine-resistant bacteria due to its greater oxidizing capacity (He et al. 2023). Ding et al. (2019) found that ozone can destroy the cell structure and gene fragment of Bacillus, which is an effective disinfectant for chlorine-resistant bacteria and their spores in the water. Sun et al. (2013) found that 4 mg/l chlorine disinfection for 240 min could only reduce the viability of Sphingomonas sphaericus by 5%, while 40 mJ/cm² of UV light could inactivate Sphingomonas sp. by more than 3-log (99.9%). In addition, researchers have developed UV-based combination disinfection processes such as UV/chlorine, UV/ozone, and ozone/chlorine (Rattanakul & Oguma 2017; Zhang et al. 2019). However, these ozone- and UV-based disinfection methods alone or in combination lack sustained disinfection capacity and need to be supplemented with chlorine agents to safeguard the biostability of the water in the network (Zyara et al. 2016). At least 95% of microorganisms in water supply networks grow on pipe wall biofilms (Ma & Bibby 2017), and chlorine supplementation promotes the growth of chlorine-resistant bacteria in pipe wall biofilms (Rizzo et al. 2012) and increases the risk of DBPs in the network. In summary, the existing disinfection methods cannot satisfy the chemical and biological safety of pipe water at the same time, and new drinking water disinfection methods are required urgently to improve the safety of drinking water.

The advantages of epigallocatechin gallate (EGCG), such as broad spectrum of bacterial inhibition, good bacterial inhibition, persistence of disinfection, and a variety of beneficial bioactivities, as well as the reported absence of toxic byproducts, have led researchers to study it extensively as a potential disinfectant for drinking water. Feng et al. (2017) used high performance liquid chromatography to investigate the effects of initial EGCG concentration, pH, and light on the attenuation of EGCG and proposed a kinetic model for the attenuation of EGCG disinfection. In order to improve the sustainability of the disinfection technology, the researchers explored the possibility of using tea polyphenols as an adjunct disinfectant to ozone (Feng et al. 2020) and UV (Liu et al. 2020). The results show that tea polyphenols can make up for the lack of sustained disinfection ability of these processes and exhibit good disinfection effects. Besides, nanotechnology and electrochemical processes may interact with EGCG, providing new ideas to improve disinfection efficiency (Huo et al. 2023). The sustained disinfection effect of EGCG as a drinking water disinfectant has been widely confirmed, and the advantage of EGCG in reducing the DBPs is obvious. However, the killing effect and mechanism of EGCG on B. subtilis and other chlorine-resistant bacteria in drinking water under different conditions have not been fully studied, and further experiments are needed to demonstrate this. The study of the factors affecting the inactivation of B. subtilis by EGCG and its mechanism will be helpful to further explore the effectiveness and potential use of EGCG against chlorine-resistant B. subtilis in water. Furthermore, it will contribute to the control of chlorine-resistant bacteria and the biostability of pipeline networks, providing a theoretical basis for the application of EGCG in drinking water treatment.

In the tests, EGCG, which has the highest content and the strongest antibacterial activity among tea polyphenols, was used as a disinfectant to inactivate B. subtilis. EGCG was purchased from Nanjing Guangrun Biological Products Co. Ltd. The purity of EGCG is more than 98% (98.76%), and the molecular weight is 458.4. It is white or off-white powder at room temperature and should be stored under dry and light-proof conditions.

B. subtilis spores (ATCC 6633) were prepared from the corresponding strain in a liquid broth medium. To restore the activity of B. subtilis, the first generation culture was obtained by dissolving the lyophilized powder of B. subtilis in nutrient broth and incubating it at 37 °C for 24 h, followed by incubation in a solid nutrient agar medium. The first-generation culture was inoculated on solid nutrient agar slants and incubated at 37 °C for 24 h to obtain the second-generation culture. Typical colonies from the second-generation cultures were picked and inoculated on nutrient agar slants and incubated at 37 °C for 24 h to obtain the third-generation cultures. A 3.0 ml of tryptone saline solution was aspirated in a nutrient agar slant test tube and blown repeatedly. The wash solution was added to the penicillin bottle and shaken well, and then the B. subtilis suspension was diluted to 10³–10⁴CFU/ml with saline for bacterial propagule inactivation test.

To investigate the effect of EGCG concentration on the inactivation of B. subtilis, the suspension of B. subtilis was diluted to 10⁴ CFU/ml and then 100 ml of each suspension was added to nine conical flasks. Different concentrations of EGCG were added to the conical flasks so that the concentrations of EGCG in the suspensions reached 100, 200, 400, 600, 800, 1,000, 1,200, and 1,400 mg/l, respectively, which were filtered through a 0.22 μm membrane to remove the bacteria and then used. After sterilization for 30 min, the culture was counted and the inactivation rate was calculated by the pouring method.

To investigate the effect of sterilization time on the inactivation effect of B. subtilis, the suspensions of 10³–10⁴ CFU/ml were placed on a magnetic stirrer and injected with 100, 400, and 800 mg/l of EGCG, respectively. Samples were taken at 0, 10, 15, 30, and 60 min of stirring and culturable B. subtilis cells and spores were counted using standard plate counting methods.

In order to investigate the effect of pH on the inactivation of B. subtilis, the suspension of B. subtilis was diluted to 10⁴ CFU/ml, and then 100 ml of suspension was added into five conical flasks. The pH of suspension was adjusted to 6.5, 7.0, 7.5, 8.0, and 8.5 with hydrochloric acid and sodium hydroxide, respectively, and the concentration of EGCG reached 800 mg/l. Samples were taken from the conical flasks at 5, 15, 30, and 60 min of sterilization, and the inactivation rate of B. subtilis was calculated using the pouring method.

To investigate the effect of water temperature on the inactivation of B. subtilis, the suspension was diluted to 10⁴ CFU/ml. In total, 100 ml of the suspension was then added into five conical flasks, and the pH of the suspension was adjusted to 7.0. EGCG was added to each conical flask to reach a concentration of 800 mg/l. The conical flasks were placed in a refrigerator (at 5 °C), in a room (at 15 °C), and in a water bath (adjust the temperature to 25, 35, and 45 °C). Samples were taken from the conical flasks at 5, 15, 30, and 60 min of sterilization, and the inactivation rate of B. subtilis was calculated by culture counting using the pouring method.

A total of 300 ml of B. subtilis suspension was prepared and dispensed into three sterile conical flasks. The group without EGCG was used as a blank control, and the remaining two conical flasks were added with 100 mg/l (representative of low concentration) and 800 mg/l (representative of high concentration) of EGCG solution, respectively. After sterilization for 30 min, the suspensions were transferred to 50 ml centrifuge tubes and centrifuged at 1,000 rpm for 10 min. The precipitates were rinsed three times by adding sterile phosphate buffer to the centrifuge tubes after discarding the supernatant. The B. subtilis precipitate was transferred to a 1.5 ml spike tube, and 2.5% glutaraldehyde fixative was slowly added along the wall of the centrifuge tube until full. The centrifuge tubes were stored in the refrigerator at 4 °C for 12 h. Subsequently, the samples were subjected to gradient dehydration, permeabilization, embedding, sectioning, and staining. Ultrathin sections of each sample were observed under a transmission electron microscope and photographed at magnifications of 4,000, 10,000, and 20,000 times, respectively.

In order to investigate the extent of damage to the cell wall of B. subtilis by different concentrations of EGCG, the alkaline phosphatase (AKP) content in the bacterial suspension after EGCG sterilization was detected by using the AKP kit. AKP exists between the bacterial cell wall and the cell membrane, and when the cell wall is damaged, AKP leaks out of the cell so that the content of the AKP in the bacterial suspension will be elevated (Guo et al. 2018). The supernatant was centrifuged from the suspensions without EGCG 100 and 800 mg/l.

For investigating the destructive effect of different concentrations of EGCG on the cell membrane of B. subtilis, the soluble protein content in the bacterial suspension was determined by the method of Caulobacter Brilliant Blue. The destructive effect of EGCG on the cell membrane will lead to an increase in the permeability of the cell membrane, which will lead to the leakage of soluble proteins in the bacterial cells. The degree of disruption of B. subtilis cell membrane by different concentrations of EGCG was demonstrated by measuring the amount of soluble protein leakage (Luo et al. 2021; Zhang et al. 2023). Standard concentrations of protein and koammas brilliant blue mixed solutions were prepared, by measuring the absorbance value of each solution at 595 nm and the protein-absorbance standard curve was plotted using a UV spectrophotometer. Using sterile water as a blank control, different concentrations of EGCG were added to the suspension of B. subtilis so that the concentrations of EGCG were 100 and 800 mg/l, respectively. The samples were taken at 15 min, 30 min, 1, 2, 4, 6, and 12 h, respectively, and added into centrifuge tubes and centrifuged at 4,500 r/min for 10 min. The absorbance of the supernatant after centrifugation was measured at 595 nm by UV spectrophotometer and the soluble protein content was calculated by referring to the protein-absorbance standard curve.

To investigate the effects of different concentrations of EGCG on the respiration and energy metabolism of B. subtilis, the glucose content of glucose-containing suspensions of B. subtilis was examined using a kit. B. subtilis can oxidize and decompose organic matter such as glucose through aerobic respiration to provide energy for life. The change of glucose content in the suspension can reflect the strength of respiration and energy metabolism of B. subtilis. A 0.3% glucose solution was prepared with purified water and the suspension was diluted to 10⁴ CFU/ml. The suspension with sterile water was used as a blank control group, and different concentrations of EGCG were added to the suspension to make the concentrations of EGCG 100 and 800 mg/l, respectively. The samples were taken at 1, 2, 4, 6, and 12 h, respectively, and were added into centrifuge tubes for 10 min at 12,000 r/min. The precipitate was removed, and the supernatant was discarded, washed with phosphate buffer and resuspended. The cells were broken by rapid tissue cell disrupter and centrifuged again. Finally, the ATP content in the supernatant was determined using an ATP kit.

In this experiment, an enzyme kit was used to measure the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in the samples, and the upregulation of SOD indicated that excessive superoxide anion was produced by the damaged bacteria; the upregulation of CAT indicated that hydrogen peroxide was produced by the bacteria. The upregulation of GSH could accelerate the decomposition of intracellular free radicals produced by the bacteria stimulated by external factors, and protect the sulfhydryl-containing proteins from oxidative damage. A decrease in GSH content indicates a decrease in the antioxidant capacity of the bacteria, which is a potential signal of apoptosis.

As can be seen from Figure 5(b), the content of CAT in B. subtilis under the action of 100 mg/l EGCG increased rapidly within 1 h, and then decreased gradually, and all of them were higher than that of the control group. This may be attributed to the excessive production of hydrogen peroxide in B. subtilis under the stimulation of low concentration of EGCG, and at the same time, the SOD also generates a part of hydrogen peroxide during the conversion of superoxide anion radicals. The excessive generation of hydrogen peroxide promoted the expression of CAT synthesis-related genes, leading to an increase in CAT content. The content of CAT in EGCG-administered B. subtilis was always higher than that in the control group, whereas the content of SOD was always lower than that in the control group, which may be due to the fact that the consumption rate of CAT is lower than that of SOD. The level of CAT under the action of 800 mg/l EGCG is lower than that of 100 mg/l EGCG. This suggests that the higher the concentration of EGCG, the greater the inhibition of the CAT synthesis pathway in B. subtilis.

The glucose content of the bacterial suspensions under 100 and 800 mg/l EGCG conditions increased from 0 to 30 min. Among them, the glucose content increased from 0.3 to 0.32% at 15 min with 100 mg/l EGCG and from 0.3 to 0.34% at 30 min with 800 mg/l EGCG. This may be due to the leakage of glucose as EGCG disrupts the bacterial membrane. After 30 min, the glucose content in the bacterial suspensions under both 100 and 800 mg/l EGCG conditions decreased gradually with time. Among them, the glucose in the bacterial suspensions under 100 mg/l EGCG condition decreased faster than that under 800 mg/l EGCG condition from 30 min to 12 h. The glucose content in the bacterial suspensions under 100 mg/l EGCG condition decreased faster than that under 800 mg/l EGCG condition. This may be because the higher the concentration of EGCG, the stronger its ability to inhibit bacterial respiration, which led to a higher degree of blockage of glucose metabolism and a slower rate of glucose decline. In addition, the glucose content in the bacterial suspensions decreased to different degrees after 12 h regardless of the addition of EGCG.

When the pH of the reaction system was acidic, EGCG mainly carried out the reaction of Equation (2), and the acidic conditions promoted the reaction equation to proceed in the positive direction, so that EGCG produced more H₂O₂ in water, resulting in the enhanced inactivation ability of EGCG against B. subtilis. Meanwhile, the acidic condition inhibited the ionization reaction of the phenolic hydroxyl group of EGCG in water, so that the phenolic hydroxyl group, which is the main inhibitory functional group of EGCG, could act more on B. subtilis. When the pH of the reaction system was alkaline, a large amount of OH^– in water made Equation (3) proceed positively, leading to an increasing number of EGCG radicals, which were then oxidized to quinone, as shown in Equations (4) and (5). EGCG was consumed in large quantities in alkaline conditions, and its rapid decay led to a slower sterilizing rate and poorer sterilizing effect against B. subtilis. Therefore, EGCG is suitable for dosing under acidic conditions, which can improve its inactivation effect on chlorine-resistant B. subtilis.

This study aimed to investigate the inactivation effect and mechanism of EGCG on B. subtilis under different conditions, with the goal of providing a theoretical basis for its application in water treatment. The findings of the study are as follows:

• (1) This study thoroughly investigated the inactivation effect of EGCG on B. subtilis under various conditions and elucidated the underlying mechanisms. The inactivation rate of B. subtilis by EGCG is positively correlated with the duration of exposure, with the most rapid inactivation occurring within the initial minutes. Over time, the rate slows as EGCG interacts with and degrades within bacterial cells. Additionally, the study highlighted that water temperature significantly affects EGCG's bactericidal efficiency. While rapid inactivation is observed at both low (5 °C) and high (45 °C) temperatures, the stability of EGCG at lower temperatures makes it more effective over time. The optimal temperature range for EGCG's antimicrobial activity is between 25 and 45 °C, balancing EGCG stability with bacterial metabolic inhibition.

• (2) The optimal concentration for inactivating B. subtilis using EGCG was found to be 800 mg/l. This concentration can be used as a reference dosage for EGCG in water treatment.

• (3) Acidic conditions, specifically a pH of 6.5, significantly enhanced the inactivation effect of EGCG. Therefore, adjusting the pH is recommended to optimize the disinfection effect of EGCG.

• (4) EGCG's inactivation mechanism targets the structural integrity, energy metabolism, and antioxidant system of B. subtilis. TEM observations showed concentration-dependent cell damage, including blurred cell boundaries and intracellular leakage. Furthermore, EGCG reduces the levels of SOD, CAT, and GSH, compromising the bacterium's antioxidant defenses. This multifaceted action impairs B. subtilis's respiration and ATP synthesis, effectively inactivating the bacteria and presenting EGCG as a viable eco-friendly disinfectant for drinking water.

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by T.W., Z.X.C., and X.Y. The first draft of the manuscript was written by W.Q.Y. and J.L. Review and editing was written by C.M.F. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

This research was funded by the National Natural Science Foundation of China (51678026), Beijing University of Civil Engineering Postgraduate Innovation Project (PG2023060), and Open Project of Key Laboratory of Urban Stormwater System and Water Environment, Ministry of Education, Beijing University of Civil Engineering and Architecture (2020).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.