Investigation of UV-Tio2 Photocatalysis and Its Mechanism

Home , Photocatalysis

effects were observed when UV light was applied as a primary disinfectant followed by free chlorine as a sec- ondary disinfectant.

References Bolton, J.R., Linden, K.G. “Standardization of Methods for fluence (UV Dose). Determination in Bench - Scale UV Experiments”. Journal of Environmental Engineering 129, 209-21 (2003). Cho, M., Gandhi, V., Hwang, T.M., Lee, S., Kim, J.H. “Investigation synergism during sequential inactivation of MS-2 phages and Bacillus subtilis spores with UV/H2O2 followed by free chlorine”. Water Research 45 (3), 1063-1070 (2011). Clevenger, T., DeGruson, E., Brazos, B., Banerji, S. “Comparision of the inactivation of bacillus subtilis spores and MS2 bacteriophage by MIOX, Clortec and hypochlorite”. Applied Microbiology 103 (2007). Driedger, A.M., Rennecker, J.L., Mariñas, B.J. “Sequential inactivation of Cryptosporidium parvum oocysts with ozone and free chlorine”. Water Research 34, 3591-3597 (2000). ISO 10705-1: Water quality - Detection and enumeration of bacteriophages - part 1: Enumeration of F-specific RNA bacteriophages (1995). USEPA Utraviolet Disinfection Guidance Manula for the final Long Term 2 Enhanced Surface Water Treatment Rule (Office of Water (4601) EPA 815-R-06-007, November 2006).

Investigation of UV-TiO2 Photocatalysis and its Mechanism on Bacillus Subtilis Spores Inactivation Yongji Zhang1, Yiqing Zhang1, Lingling Zhou2 1 Key Laboratory of Yangtze River Water Environment, Ministry of Education (Tongji University), Shanghai 200092, PR China 2 State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science & Engineering, Tongji University, Shanghai 200092, PR China

Abstract Keywords: UV; TiO2; Bacillus subtilis; spore; disinfection; lipid peroxidation The inactivation levels of Bacillus subtilis spores for various disinfection processes (UV, TiO2 and UV-TiO2) were compared. The results showed that compared with UV Introduction treatment alone, the inactivation effect increased sig- Ultraviolet (UV) technology has shown its high efficiency, nificantly with the addition of TiO2. Increases in the operational convenience and low yield of DBPs formation irradiance or TiO2 concentration both contributed to the in drinking water and wastewater disinfection [1]. UV increasing inactivation effect. Malondialdehyde (MDA) treatment is found to be effective with some chlorine- was used as an index to determine the extent of the lipid resistant microorganisms, such as Cryptosporidium and peroxidation. The MDA concentration surged with the Giardia [2]. However, due to the limitations of physical simultaneous process. At the same time the cell mem- disinfection processes such as UV, other chemicals are brane was totally damaged and cellular contents were required in order to maintain the capability of continuous completely lysed by UV-TiO2 treatment. disinfection and to improve the inactivation level [3, 4].

20 IUVA News | VOL. 15 | No. 2 Advanced oxidation processes (AOPs), especially UV- based processes, are able to produce highly reactive radicals by photolysis [5, 6]. UV-TiO2 is one of the most promising photochemical catalytic technologies. It was reported for the first time by Matsunaga et al. [7] that What If… by contacting microbial cells with TiO2/Pt particles under UV exposure for 60 to 120 min, they could be completely inactivated. Since then, UV-TiO2 has attracted an ever-increasing intensive research interest in the field of compound degradation. There was a way to monitor your UV-TiO2 is being studied to see if it is capable of inac- tivating microorganisms in water. Benabbou et al. [8] UV system confirmed that the combination of UV and TiO2 was a promising alternative for E. coli inactivation. Cho et al. anytime, [9] found that the addition of TiO2 contributed to the anywhere? efficiency of the UV treatment of MS-2 phage. Despite the many investigations on the inactivation behaviors of UV-TiO2, an understanding of its photochemical mechanism is far from complete.

AOPs exhibit a phenomenon called lipid peroxidation, There Is! namely the oxidative degradation of polyunsaturated fatty acids. The process is attributed to the oxidation of cell membrane lipids by free radicals, leading to membrane destruction [10]. This leads inevitable to lethality, since cell function depends heavily on an intact membrane structure [11]. As a result, lipid peroxidation has been proposed as the most hazardous effect on cells by free radicals [12].

The objective of this study was to demonstrate and con- trast the potential inactivation effects of UV, TiO2 and the simultaneous process of UV-TiO2. The spores of B. subtilis were selected as the indicator microorganism. Since bacterial inactivation was not the only issue to be concerned about, cell death caused by disruption of the cell membrane was also taken into account. To produce Introducing a better understanding of the mechanisms, the impact of photocatalytic disinfection on lipid peroxidation was studied. MDA, the degradation product of lipid peroxida- UVConnect tion, was used as an index to determine the extent of the reaction. In order to observe changes in cell ultrastructure caused by lipid peroxidation reaction, transmission electron microscope (TEM) images were taken to provide a deep insight into the cell morphology. P: 859.341.0710 www.aquionics.com

Aquionics UV Delivered™ is a trademark of Aquionics.

Summer 2013 21 Experimental lection Center and were rehydrated aseptically with Nu- Reagents trient Broth (Peptone 10 g/L, NaCl 5 g/L, Beef extract The photocatalyst used in this study was Degussa P-25 3 g/L). The bacterial suspension was incubated for 24 h and then added to sporulation medium (Yeast extract TiO2 (Sigma, German). Other reagents were supplied by Sinopharm Chemical Reagent Company Limited, and 0.7 g/L, Glucose 1 g/L, Peptone 1 g/L, MgSO4•7H2O they were analytical reagent grade. Distilled water for 0.2 g/L, (NH4)2SO4 0.2 g/L) and incubated for 48 h at analytical use was from Direct-Q3 (MilliPore, USA). The 37 °C in a shaker. Almost 90% of the vegetative cells malondialdehyde (MDA) test kit used in this research with spores. The spores were recovered by centrifugation was provided by Nanjing Jiancheng Biology Corporation at 6000 rpm for 10 min and resuspended in 10% NaCl (China). Reagents and materials used were sterilized by solution. Then the bacterial suspension was placed in a autoclaving (120 °C, 20 min). water bath to kill the remaining vegetative cells (80 °C, 10 min). The ultimate cell density was approximately Experimental apparatus 107 colony forming units per milliliter (CFU/mL). The A Collimated Beam apparatus with a 75 W low-pressure viable spore suspension was serially diluted depending on (LP) mercury lamp (Philips, Netherland) was utilized the order of magnitude. Then 0.1 mL of the suspension (Figure 1.). The monochromatic UV radiation emitted by was injected onto nutrient agar medium. Each dilution this lamp was directed to the surface of the test samples. was plated in triplicate, and incubated on nutrient agar The average irradiance in the solution and the UV dose medium (37 °C, 24 h) to enumerate the B. subtilis spores. were determined based on the Bolton and Linden protocol [1], using a UV-M radiometer (Beijing Normal University Experimental methods Experiment Company, China). Several other parameters, A petri dish (90 mm diameter) containing a 40 mL sample including solution volume, solution absorbance, distance was exposed to UV while it was being stirred gently by a from the lamp to the water surface, were taken into ac- magnetic stir bar in the collimated beam apparatus. The count following the separate Excel spreadsheets available irradiances detected at the solution surface were 0.18, 2 at www.iuva.org. 0.10, 0.008 mW/cm , respectively. TiO2 was spiked into the samples to produce concentrations of 5 and 10 mg/L. 2.3 B. subtilis spores culturing and enumeration The UV-TiO2 process was based on previous UV expo- Pure cultures of B. subtilis spores (ATCC 9372) were sures with the addition of TiO2. The MDA content was provided by China General Microbiological Culture Col- determined by the thiobarbituric acid (TBA) method with a DR 5000 spectrophotometer (HACH, USA) [13], and it was expressed as nmol/mg of cell dry weight [11],. The TEM samples with and without treatment were prepared following the methods described by Ou et al. [14] and examined using a JEM-1230 TEM (JEOL, Japan).

Data presentation To evaluate the effect of disinfection, the inactivation level of B. subtilis spores is usually analyzed by the microbial inactivation credit (MIC = log-credits) [15]. The relationship between the MIC (log) and the microbial concentration (CFU/mL) is described as follows: MIC = log (N0/N) (1) where, N0 and N (CFU/mL) are the microbial concentrations before and after the disinfection process.

22 IUVA News | VOL. 15 | No. 2 Results and Discussion Effect of UV treatment alone As shown in Fig. 2, higher UV doses produced higher MIC with the same irradiance. Take UV irradiance of 0.18 mW/cm2 as an example. Exposure times of 100, What If… 200, 400 and 600 s resulted in MICs of 0.81, 1.60, 2.97 and 3.95 log, respectively. Moreover, the MIC increases There was a wth UV dose as well. For example, the MIC was 0.71, 1.20 and 1.60 log at the UV irradiance of 0.008, 0.10 and 0.18 closed vessel unit mW/cm2, respectively, with an exposure time of 200 s. that was

The MIC of B. subtilis spores by UV exposure was lower single ended, than bacteria without endospores. Only 0.81 log of MIC was received at an irradiance of 0.18 mW/cm2 for 100 resulting in a s. According to Liu and Zhang [16], E. coli, Staphylococ- smaller footprint, cus aureus and Candida albicans achieved 4.98, 5.31 and 4.93 log at the same irradiance and exposure time. In and full access for addition, Wang et al. [17] believed that B. subtilis spores presented higher resistance than E. coli when treated maintenance? with OH radicals. This phenomenon is attributed to the existence of endospores, a kind of global or oval dormant body produced by some specific microorganism. The en- There Is! dospore can survive in hostile environment, such as high temperature, dryness or UV treatment [18].

Introducing

Effect of TiO2 alone in the dark Two concentrations of TiO2 (5 and 10 mg/L) and four InLine+SE intervals of contact time (100, 200, 400 and 600 s) were selected to investigate the inactivation effect of TiO2 alone in the dark. It was found that TiO2 alone in the dark hardly inactivated any B. subtilis spores, as was reported previously [19]. P: 859.341.0710 www.aquionics.com

Aquionics UV Delivered™ is a trademark of Aquionics.

Summer 2013 23 Effects of irradiance and TiO2 concentration by Effect of various parameters on lipid peroxidation UV-TiO2 production Fig. 3 illustrates the effects of irradiance and TiO2 con- It is obvious that UV-TiO2 gave rise to an improvement centration by UV-TiO2 on B. subtilis spores inactivation. in bacterial inactivation efficiency, since it reduced viable As demonstrated in Fig. 3A, UV-TiO2 showed a significant cell counts by the inhibition of the ability to replicate. inactivation effect compared with UV treatment alone, However, bacterial inactivation was not the only issue under an irradiance of 0.10mW/cm2. Furthermore, with explored in this research. Cell death caused by disrup- the TiO2 concentration increasing from 5 to 10 mg/L, tion of the cell membrane was also taken into account. the MIC was raised from 4.58 to 5.45 log, which was 1.13 and 2.0 log higher than UV treatment alone for 600 s. It In order to discover the relationship between membrane was concluded that the inactivation effect of UV-TiO2 damage and the disinfection process, the level of lipid was superior to that of UV treatment alone, because the peroxidation was determined by measuring the MDA TiO2 contributed to photochemical catalysis by generat- concentration with various conditions (Fig. 4). As shown ing OH radicals with high activity. in Fig. 4A, 0.38 and 0.25 nmol MDA/mg dry cell resulted from only UV treatment (0.10 mW/cm2) and TiO2 alone Fig. 3B shows the effect of UV irradiance on B. subtilis in the dark (10 mg/L) for 600 s, respectively. However, spores inactivation by UV-TiO2 with 5mg/L TiO2. Com- when B. subtilis spores were exposed to UV treatment pared with TiO2 alone in the dark, the inactivation effect together with TiO2 addition under the same condition, increased significantly with the presence of UV treatment. the MDA concentration increased to 3.24 nmol MDA/ As the UV dose increased, the MIC rose as well. Similar mg dry cell. This indicated that both UV treatment and phenomenon can be noticed in other published research TiO2 addition were essential for lipid peroxidation. Spores [20, 21]. This may be explained by higher UV doses without disinfection, as a blank control, resulted in 0.13 produced increased numbers of radiation photons, facili- nmol MDA/mg dry cell, which demonstrated that the tating TiO2 to generate more OH radicals and resulting MDA preexisted but there was a negligible concentra- in less possibility of the repair of injured enzymes [22]. tion in the cells.

24 IUVA News | VOL. 15 | No. 2 What If…

There was a drinking water Fig. 4B shows the lipid peroxidation production at various irradiances and TiO2 concentrations, at an exposure system that time of 600 s. It is apparent that the MDA concentration did not require an increased significantly with the increase in the TiO2 concentration. Moreover, the increases in the UV dose led to action spectra a small increase in the MDA concentration. It was in line with the phenomenon that the MIC increased with the correction factor? increasing TiO2 concentrations or UV dose, as described in section 3.3.

TEM images In order to observe changes in cell ultrastructure caused by the lipid peroxidation reaction, cell morphology pic- There Is! tures with and without disinfection were taken by TEM (Fig. 5). Fig. 5A shows a typically healthy B. subtilis cell, possessing an inflated and intact cell structure. The cell is rod-shaped, containing cell wall, cell membrane and other cytoplasmic inclusion bodies. However, after being treated with UV at 0.10 mW/cm2 for 600 s, the cell membrane shrunk and the intracellular structure became indistinct (Fig. 5B). Furthermore, as shown in Fig. 5C, cell disruption was observed with the addition of 10 mg/L TiO2. The cell membrane was totally damaged and the cytoplasmic inclusions were completely lysed, suggesting that the cell was beyond repair. Above all, it can be inferred that lipid peroxidation, caused by UV-TiO2 photocatalysis, was responsible for the death of cells. Introducing Conclusion The inactivation effect of B. subtilis spores by UV treat- InLine+D ment was below the bacteria without endospores. TiO2 alone in the dark inactivates only a very small number of B. subtilis spores. Compared with respective disinfection process of UV or TiO2 treatment, TiO2 has a significant synergetic effect when combined with UV treatment. The inactivation effect increased significantly with the increas- P: 859.341.0710 ing UV doses or TiO2 concentration. Lipid peroxidation www.aquionics.com was found to be the underlying mechanism of inactivation Aquionics UV Delivered™ is a trademark of Aquionics.

Summer 2013 25 with UV combined with TiO2. The MDA concentration 9. Cho, M., Chung, H., Choi, W. and Yoon, J. “Different inactivation surged significantly with the combination disinfection, behaviors of MS-2 phage and Escherichia coli in TiO2 photocatalytic which indicated that both UV and TiO are essential for disinfection”, Appl. Environ. Microb. 71(1):270-275 (2005). 2 10. Marnett, L. J. “Lipid peroxidation—DNA damage by malondial- lipid peroxidation. Moreover, UV-TiO2 was able to totally dehyde”, Mutat. Res-Fund. Mol. M 424(1):83-95 (1999). damage the cell membrane as well as completely lyse the 11. Maness, P.-C., Smolinski, S., Blake, D. M., Huang, Z., Wolfrum, E. cellular contents, suggesting that lipid peroxidation was J. and Jacoby, W. A. “Bactericidal activity of photocatalytic TiO2 responsible for the death of cells. reaction: toward an understanding of its killing mechanism”, Appl. Environ. Microb. 65(9):4094-4098 (1999). 12. Miura, T., Muraoka, S. and Fujimoto, Y. “Lipid peroxidation induced Acknowledge by phenylbutazone radicals”, Life Sci. 70(22):2611-2621 (2002). This work was supported by the National Natural Science 13. Ganhao, R., Estevez, M. and Morcuende, D. “Suitability of the TBA method for assessing lipid oxidation in a meat system with Foundation of China (Grant No. 51178323, 51108329), added phenolic-rich materials”, Food Chem. 126(2):772-778 (2011). China Postdoctoral Science Foundation funded project 14. Ou, H., Gao, N., Deng, Y., Qiao, J., Zhang, K., Li, T. and Dong, L. (No. 2012T50413), and the Fundamental Research Funds “Mechanistic studies of Microcystic aeruginosa inactivation and deg- for the Central Universities (No. 0400219205). radation by UV-C irradiation and chlorination with poly-synchronous analyses”, Desalination 272(1–3):107-119 (2011). 15. Hijnen, W. A. M., Beerendonk, E. F. and Medema, G. J. “Inactivation References credit of UV radiation for viruses, bacteria and protozoan (oo)cysts 1. Bolton, J. R. and Linden, K. G. “Standardization of methods for in water: A review”, Water Res. 40(1):3-22 (2006). fluence (UV dose) determination in bench-scale UV experiments”, J. 16. Liu, W. and Zhang, Y. “Effects of UV intensity and water turbidity Environ. Eng. 129(3):209-215 (2003). on microbial indicator inactivation”, J. Environ. Sci. 18(4):650-653 2. Linden, K. G., Gwy-Am Shin, Faubert, G., Cairns, W. and Sobsey, (2006). M. D. “UV disinfection of Giardia lamblia cysts in water”, Environ. 17. Wang, D., Oppenlander, T., El-Din, M. G. and Bolton, J. R. “Com- Sci. Technol. 36(11):2519-2522 (2002). parison of the Disinfection Effects of Vacuum-UV (VUV) and UV 3. Yang, H., Chen, Y. and Guo, L. “Homogenous photocatalytic decom- Light on Bacillus subtilis Spores in Aqueous Suspensions at 172, 222 position of acetic acid using UV-Fe2+/Fe3+ system in the absence and 254 nm”, Photochem. Photobiol. 86(1):176-181 (2010). of oxygen”, Catal. Commun. 11(14):1099-1103 (2010). 18. Serrano, M., Zilhão, R., Ricca, E., Ozin, A. J., Moran, C. P. and 4. Qui, N. V., Scholz, P., Krech, T., Keller, T. F., Pollok, K. and On- Henriques, A. O. “A Bacillus subtilis secreted protein with a role in druschka, B. “Multiwalled carbon nanotubes oxidized by UV/H2O2 endospore coat assembly and function”, J. Bacteriol. 181(12):3632- as catalyst for oxidative dehydrogenation of ethylbenzene”, Catal. 3643 (1999). Commun. 12(6):464-469 (2011). 19. Ibáñez, J. A., Litter, M. I. and Pizarro, R. A. “Photocatalytic bac- 5. Malato, S., Fernández-Ibáñez, P., Maldonado, M., Blanco, J. and tericidal effect of TiO2 on Enterobacter cloacae: Comparative study Gernjak, W. “Decontamination and disinfection of water by solar with other Gram (-) bacteria”, J. Photochem. Photobiol. A 157(1):81- photocatalysis: Recent overview and trends”, Catal. Today 147(1):1- 85 (2003). 59 (2009). 20. Xu, B., Gao, N.-Y., Sun, X.-F., Xia, S.-J., Rui, M., Simonnot, M.- 6. Chen, F., Zou, W., Qu, W. and Zhang, J. “Photocatalytic performance O., Causserand, C. and Zhao, J.-F. “Photochemical degradation of of a visible light TiO2 photocatalyst prepared by a surface chemical diethyl phthalate with UV/H2O2”, J. Hazard. Mater. 139(1):132- modification process”, Catal. Commun. 10(11):1510-1513 (2009). 139 (2007). 7. Matsunaga, T., Tomoda, R., Nakajima, T. and Wake, H. “Photoelec- 21. Zhou, C., Gao, N., Deng, Y., Chu, W., Rong, W. and Zhou, S. “Fac- trochemical sterilization of microbial cells by semiconductor powders”, tors affecting ultraviolet irradiation/hydrogen peroxide (UV/H2O2) Fems Microbiol. Lett. 29(1–2):211-214 (1985). degradation of mixed N-nitrosamines in water”, J. Hazard. Mater. 8. Benabbou, A., Derriche, Z., Felix, C., Lejeune, P. and Guillard, C. 231–232(0):43-48 (2012). “Photocatalytic inactivation of Escherischia coli: Effect of concen- 22. Bohrerova, Z. and Linden, K. G. “Standardizing photoreactivation: tration of TiO2 and microorganism, nature, and intensity of UV Comparison of DNA photorepair rate in Escherichia coli using four irradiation”, Appl. Catal. B 76(3):257-263 (2007). different fluorescent lamps”, Water Res. 41(12):2832-2838 (2007).

26 IUVA News | VOL. 15 | No. 2