Investigation of UV-TiO Photocatalysis and its Mechanism on ......Water Research 34, 3591-3597 (2000). ISO 10705-1: Water quality - Detection and enumeration of bacterio-phages - part

20 IUVA News | VOL. 15 | No. 2

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

referencesBolton, 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 bacterio-phages - 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 InactivationYongji Zhang1, Yiqing Zhang1, Lingling Zhou21 Key Laboratory of Yangtze River Water Environment, Ministry of Education (Tongji University), Shanghai 200092, PR China2 State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science & Engineering, Tongji University, Shanghai 200092, PR China

abstractthe inactivation levels of Bacillus subtilis spores for vari-ous disinfection processes (UV, tiO2 and UV-tiO2) were compared. the results showed that compared with UV treatment alone, the inactivation effect increased sig-nificantly with the addition of tiO2. Increases in the irradiance or tiO2 concentration both contributed to the increasing inactivation effect. Malondialdehyde (MdA) was used as an index to determine the extent of the lipid peroxidation. the MdA concentration surged with the simultaneous process. At the same time the cell mem-brane was totally damaged and cellular contents were completely lysed by UV-tiO2 treatment.

Keywords: UV; tiO2; Bacillus subtilis; spore; disinfection; lipid per-oxidation

introductionUltraviolet (UV) technology has shown its high efficiency, operational convenience and low yield of dBPs formation in drinking water and wastewater disinfection [1]. UV treatment is found to be effective with some chlorine-resistant microorganisms, such as Cryptosporidium and Giardia [2]. however, due to the limitations of physical disinfection processes such as UV, other chemicals are required in order to maintain the capability of continuous disinfection and to improve the inactivation level [3, 4].

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Advanced oxidation processes (AOPs), especially UV-based processes, are able to produce highly reactive radi-cals 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 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.

UV-tiO2 is being studied to see if it is capable of inac-tivating microorganisms in water. Benabbou et al. [8] confirmed that the combination of UV and tiO2 was a promising alternative for E. coli inactivation. Cho et al. [9] found that the addition of tiO2 contributed to the 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, 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 a better understanding of the mechanisms, the impact of photocatalytic disinfection on lipid peroxidation was studied. MdA, the degradation product of lipid peroxida-tion, was used as an index to determine the extent of the reaction. In order to observe changes in cell ultrastruc-ture caused by lipid peroxidation reaction, transmission electron microscope (teM) images were taken to provide a deep insight into the cell morphology.

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Experimentalreagentsthe photocatalyst used in this study was degussa P-25 tiO2 (sigma, German). Other reagents were supplied by sinopharm Chemical Reagent Company Limited, and they were analytical reagent grade. distilled water for analytical use was from direct-Q3 (MilliPore, UsA). the malondialdehyde (MdA) test kit used in this research was provided by Nanjing Jiancheng Biology Corporation (China). Reagents and materials used were sterilized by autoclaving (120 °C, 20 min).

Experimental apparatusA Collimated Beam apparatus with a 75 w low-pressure (LP) mercury lamp (Philips, Netherland) was utilized (Figure 1.). the monochromatic UV radiation emitted by this lamp was directed to the surface of the test samples. the average irradiance in the solution and the UV dose were determined based on the Bolton and Linden protocol [1], using a UV-M radiometer (Beijing Normal University experiment Company, China). several other parameters, including solution volume, solution absorbance, distance from the lamp to the water surface, were taken into ac-count following the separate excel spreadsheets available at www.iuva.org.

2.3 B. subtilis spores culturing and enumerationPure cultures of B. subtilis spores (AtCC 9372) were provided by China General Microbiological Culture Col-

lection Center and were rehydrated aseptically with Nu-trient Broth (Peptone 10 g/L, NaCl 5 g/L, Beef extract 3 g/L). the bacterial suspension was incubated for 24 h and then added to sporulation medium (Yeast extract 0.7 g/L, Glucose 1 g/L, Peptone 1 g/L, MgsO4•7H2O 0.2 g/L, (Nh4)2sO4 0.2 g/L) and incubated for 48 h at 37 °C in a shaker. Almost 90% of the vegetative cells with spores. the spores were recovered by centrifugation at 6000 rpm for 10 min and resuspended in 10% NaCl solution. then the bacterial suspension was placed in a water bath to kill the remaining vegetative cells (80 °C, 10 min). the ultimate cell density was approximately 107 colony forming units per milliliter (CFU/mL). the viable spore suspension was serially diluted depending on the order of magnitude. then 0.1 mL of the suspension was injected onto nutrient agar medium. each dilution was plated in triplicate, and incubated on nutrient agar medium (37 °C, 24 h) to enumerate the B. subtilis spores.

Experimental methodsA petri dish (90 mm diameter) containing a 40 mL sample was exposed to UV while it was being stirred gently by a magnetic stir bar in the collimated beam apparatus. the irradiances detected at the solution surface were 0.18, 0.10, 0.008 mw/cm2, respectively. tiO2 was spiked into the samples to produce concentrations of 5 and 10 mg/L. the UV-tiO2 process was based on previous UV expo-sures with the addition of tiO2. the MdA content was 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 pre-pared following the methods described by Ou et al. [14] and examined using a JeM-1230 teM (JeOL, Japan).

data presentationto evaluate the effect of disinfection, the inactivation level of B. subtilis spores is usually analyzed by the mi-crobial 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 con-centrations before and after the disinfection process.

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results and discussionEffect 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, 200, 400 and 600 s resulted in MICs of 0.81, 1.60, 2.97 and 3.95 log, respectively. Moreover, the MIC increases 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 mw/cm2, respectively, with an exposure time of 200 s. the MIC of B. subtilis spores by UV exposure was lower than bacteria without endospores. Only 0.81 log of MIC was received at an irradiance of 0.18 mw/cm2 for 100 s. According to Liu and Zhang [16], E. coli, Staphylococ-cus aureus and Candida albicans achieved 4.98, 5.31 and 4.93 log at the same irradiance and exposure time. In addition, wang et al. [17] believed that B. subtilis spores presented higher resistance than E. coli when treated 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-dospore can survive in hostile environment, such as high temperature, dryness or UV treatment [18].

Effect of tio2 alone in the dark two concentrations of tiO2 (5 and 10 mg/L) and four 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].

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Effects of irradiance and tio2 concentration by UV-tio2 Fig. 3 illustrates the effects of irradiance and tiO2 con-centration by UV-tiO2 on B. subtilis spores inactivation. As demonstrated in Fig. 3A, UV-tiO2 showed a significant inactivation effect compared with UV treatment alone, under an irradiance of 0.10mw/cm2. Furthermore, with the tiO2 concentration increasing from 5 to 10 mg/L, 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 was concluded that the inactivation effect of UV-tiO2 was superior to that of UV treatment alone, because the tiO2 contributed to photochemical catalysis by generat-ing Oh radicals with high activity.

Fig. 3B shows the effect of UV irradiance on B. subtilis spores inactivation by UV-tiO2 with 5mg/L tiO2. Com-pared with tiO2 alone in the dark, the inactivation effect increased significantly with the presence of UV treatment. As the UV dose increased, the MIC rose as well. similar phenomenon can be noticed in other published research [20, 21]. this may be explained by higher UV doses produced increased numbers of radiation photons, facili-tating tiO2 to generate more Oh radicals and resulting in less possibility of the repair of injured enzymes [22].

Effect of various parameters on lipid peroxidation productionIt is obvious that UV-tiO2 gave rise to an improvement in bacterial inactivation efficiency, since it reduced viable cell counts by the inhibition of the ability to replicate. however, bacterial inactivation was not the only issue explored in this research. Cell death caused by disrup-tion of the cell membrane was also taken into account.

In order to discover the relationship between membrane damage and the disinfection process, the level of lipid peroxidation was determined by measuring the MdA concentration with various conditions (Fig. 4). As shown 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 in the dark (10 mg/L) for 600 s, respectively. however, when B. subtilis spores were exposed to UV treatment together with tiO2 addition under the same condition, the MdA concentration increased to 3.24 nmol MdA/mg dry cell. this indicated that both UV treatment and tiO2 addition were essential for lipid peroxidation. spores without disinfection, as a blank control, resulted in 0.13 nmol MdA/mg dry cell, which demonstrated that the MdA preexisted but there was a negligible concentra-tion in the cells.

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Fig. 4B shows the lipid peroxidation production at vari-ous irradiances and tiO2 concentrations, at an exposure time of 600 s. It is apparent that the MdA concentration increased significantly with the increase in the tiO2 con-centration. Moreover, the increases in the UV dose led to a small increase in the MdA concentration. It was in line with the phenomenon that the MIC increased with the increasing tiO2 concentrations or UV dose, as described in section 3.3.

tEm imagesIn order to observe changes in cell ultrastructure caused by the lipid peroxidation reaction, cell morphology pic-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 be-ing 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, sug-gesting 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.

Conclusionthe inactivation effect of B. subtilis spores by UV treat-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-ing UV doses or tiO2 concentration. Lipid peroxidation was found to be the underlying mechanism of inactivation


with UV combined with tiO2. the MdA concentration surged significantly with the combination disinfection, which indicated that both UV and tiO2 are essential for lipid peroxidation. Moreover, UV-tiO2 was able to totally damage the cell membrane as well as completely lyse the cellular contents, suggesting that lipid peroxidation was responsible for the death of cells.

acknowledgethis work was supported by the National Natural science Foundation of China (Grant No. 51178323, 51108329), China Postdoctoral science Foundation funded project (No. 2012t50413), and the Fundamental Research Funds for the Central Universities (No. 0400219205).

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6. Chen, F., Zou, W., Qu, W. and Zhang, J. “Photocatalytic performance of a visible light TiO2 photocatalyst prepared by a surface chemical modification process”, Catal. Commun. 10(11):1510-1513 (2009).

7. Matsunaga, T., Tomoda, R., Nakajima, T. and Wake, H. “Photoelec-trochemical sterilization of microbial cells by semiconductor powders”, Fems Microbiol. Lett. 29(1–2):211-214 (1985).

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9. Cho, M., Chung, H., Choi, W. and Yoon, J. “Different inactivation behaviors of MS-2 phage and Escherichia coli in TiO2 photocatalytic disinfection”, Appl. Environ. Microb. 71(1):270-275 (2005).

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14. Ou, H., Gao, N., Deng, Y., Qiao, J., Zhang, K., Li, T. and Dong, L. “Mechanistic studies of Microcystic aeruginosa inactivation and deg-radation by UV-C irradiation and chlorination with poly-synchronous analyses”, Desalination 272(1–3):107-119 (2011).

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Investigation of UV-TiO Photocatalysis and its Mechanism on ......Water Research 34, 3591-3597 (2000). ISO 10705-1: Water quality - Detection and enumeration of bacterio-phages - part