Journal of Photochemistry and Photobiology B: Biology 134 (2014) 9–15

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Factors affecting UV/H2O2 inactivation of Bacillus atrophaeus spores in drinking water Yongji Zhang a, Yiqing Zhang a, Lingling Zhou b,⇑, Chaoqun Tan a a b

Key Laboratory of Yangtze River Water Environment, Ministry of Education, Tongji University, Shanghai 200092, PR China State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science & Engineering, Tongji University, Shanghai 200092, PR China

a r t i c l e

i n f o

Article history: Received 28 August 2013 Received in revised form 26 February 2014 Accepted 26 March 2014 Available online 13 April 2014 Keywords: UV H2O2 Bacillus atrophaeus spores Inactivation

a b s t r a c t This study aims at estimating the performance of the Bacillus atrophaeus spores inactivation by the UV treatment with addition of H2O2. The effect of factors affecting the inactivation was investigated, including initial H2O2 dose, UV irradiance, initial cell density, initial solution pH and various inorganic anions. Under the experimental conditions, the B. atrophaeus spores inactivation followed both the modified Hom Model and the Chick’s Model. The results revealed that the H2O2 played dual roles in the reactions, while the optimum reduction of 5.88 lg was received at 0.5 mM H2O2 for 10 min. The inactivation effect was affected by the UV irradiance, while better inactivation effect was achieved at higher irradiance. An increase in the initial cell density slowed down the inactivation process. A slight acid condition at pH 5 was considered as the optimal pH value. The inactivation effect within 10 min followed the order of pH 5 > pH 7 > pH 9 > pH 3 > pH 11. The effects of three added inorganic anions were investigated and 2  compared, including sulfate (SO2 4 ), nitrate (NO3 ) and carbonate (CO3 ). The sequence of inactivation 2  effect within 10 min followed the order of control group > SO4 > NO3 > CO2 3 . Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction The existence of microorganisms has threatened the safety of drinking water. A wealth of waterborne diseases caused by microorganisms has attracted broad attention. Among these microorganisms, Bacillus atrophaeus, once known as Bacillus subtilis [1], is a kind of non-pathogenic, Gram-positive bacteria, and extremely resistant to traditional disinfection methods [2,3]. It can survive under extreme environment with the help of a tough, protective endospore. Advanced oxidation processes (AOPs), especially UV-based technologies, have been wildly investigated, such as UV/TiO2 [4], UV/O3 [5], and UV/persulfate [6]. Among various AOP methods, the application of UV/H2O2 was proposed as an extremely promising technology in organic pollutants degradation [7,8]. However, it is still uncertain that whether this method is effective in microorganism inactivation. Mamane et al. [9] found that hardly did UV/ H2O2 exhibit any effect in B. subtilis spores inactivation, for the protection of spore coat layers. Gardner and Shama [10], on the contrary, confirmed that the inactivation of B. subtilis spores treated with UV/H2O2 process was 5.3 times higher than with UV

⇑ Corresponding author. Tel./fax: +86 21 65986313. E-mail address: [email protected] (L. Zhou). http://dx.doi.org/10.1016/j.jphotobiol.2014.03.022 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

treatment alone. The difference may be concerned with different kinds of UV sources, diverse UV or H2O2 doses and dissimilar microorganism initial concentrations. As a result, confirming the real performance of the B. atrophaeus spores inactivation by the UV treatment with addition of H2O2 proved to be significant. Furthermore, some experimental parameters could make significant contributions to affect B. atrophaeus spores inactivation. Larson and Mariñas [11] indicated that the fastest inactivation rate with ozone or monochloramine were observed at pH 10 and 6, respectively. Wang et al. [12] demonstrated that vacuum-UV treatment alone at 254 nm or 222 nm was much better than at 172 nm. Zhang et al. [13] investigated the effect of O3 dose with micro-bubble ozonation system. Some researches have also reported the effect of parameters on various organic pollutants degradation by UV/H2O2, such as ametryn [7], diethyl phthalate [14], and N-nitrosamines [15]. However, there has not been investigation into the effect of parameters affecting inactivation of microorganisms such as B. atrophaeus spores by UV/H2O2. This study aims at determining the kinetics of B. atrophaeus spores inactivated by UV/H2O2 under various factors. In addition, the effects of initial H2O2 dose, UV irradiance, initial cell density and initial solution pH on B. atrophaeus spores inactivation were investigated. In addition, since inorganic anions may react with  OH, which played a dominant role in UV/H2O2 process, it is essential to consider the existence of the anions [16]. The experiments

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Y. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 134 (2014) 9–15

were carried out in compliance with the rules of changing single variable at each time and keeping other parameters constant. 2. Materials and methods 2.1. Experimental apparatus The facility used in the experiments was a Collimated Beam Apparatus containing a low-pressure mercury lamp (Philips TUV 36 T5 SP 40W, Netherland), as illustrated in Fig. 1. The monochromatic UV radiation emitting by this lamp was directed to the surface of the test samples. The average irradiance at 254 nm measured by a UV-M radiometer (Beijing Normal University Experiment Company, China) was 113.0, 56.5, and 28.3 lW/cm2, respectively, based on the Bolton and Linden protocol [17]. 2.2. B. atrophaeus spores culture and enumeration Pure cultured B. atrophaeus spores (ATCC 9372), provided by China General Microbiological Culture Collection Center, were rehydrated aseptically with Nutrient Broth (Peptone 10 g/L, NaCl 5 g/L, Beef extract 3 g/L). The bacterial suspension was incubated for 24 h and sporulation medium (Yeast extract 0.7 g/L, Glucose 1 g/L, Peptone 1 g/L, MgSO47H2O 0.2 g/L, (NH4)2SO4 0.2 g/L), respectively, for 48 h at 37 °C in a shaker. After that, the spores were centrifuged (6000 rpm, 10 min) and redissolved 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 106–108 colony forming units per milliliter (CFU/mL). The viable spore suspension was serially diluted depending on the order of magnitudes. Then 0.1 mL of the suspension was injected onto nutrient agar medium. Each dilution was plated in triplicate, and incubated with nutrient agar medium (37 °C, 24 h) to enumerate the B. atrophaeus spores [18]. 2.3. Materials The solutions were adjusted to the desired pH by addition of HCl or NaOH. Since the spores were redissolved in 10% NaCl solution and H2O contained large amount of OH, the added inorganic anions Cl and OH had no inhibition influence on inactivation  effect. Solutions with tested inorganic anions, such as SO2 4 , NO3 2 and CO3 were obtained via adding Na2SO4, NaNO3 and Na2CO3

into certain amount of deionized water, respectively. All the reagents, offered by Sinopharm Chemical Reagent Company Limited (China), were analytical reagent grade. Distilled water for analytical use was from Direct-Q3 (MilliPore, USA). Reagents and materials used in this experiment were sterilized by autoclaving for 20 min at 120 °C. 2.4. Experimental methods Petri dishes (90 mm diameter) with 40 mL samples were exposed to the UV in the Collimated Beam Apparatus and stirred gently by a magnetic stirring apparatus. H2O2 (30%) was diluted and added to the B. atrophaeus spores samples to achieve various final concentrations. As soon as the designed exposure time was finished, 1 mL Na2SO3 was added to cease further oxidation process. Then 1 mL sample was taken out, serially diluted and incubated on culture medium in the dark. Samplings were performed at various intervals from 0 to 10 min. An adjustable volume pipette was used to transfer the liquid. 2.5. Data presentation In order to evaluate the effect of disinfection, the inactivation effect of B. atrophaeus spores is usually analyzed via various models [19–22]. This study selected two different models to fit the experimental results. 2.5.1. The Hom Model The Hom Model is given as

lgðN0 =NÞ ¼ k0 t h

ð1Þ

where N0 and N are microbial concentrations (CFU/mL) before and after disinfection, t refers to the exposure time (min), k0 refers to the constant rate and h refers to a second parameter. This equation exists an initial shoulder delay when h > 1, or a final trailing curve when h < 1. Since it is incapable to illustrate the simultaneous presence of both phenomena, the modified Hom Model was tested instead, shown as follows: k3

lgðN0 =NÞ ¼ k1 ½1  expðk2 tÞ

ð2Þ

This three-parameter model is suitable to fit the disinfection processes with a shoulder delay at the beginning, a log-linear region in the middle and a tail behavior in the end [24]. 2.5.2. The Chick’s Model The Chick’s Model conveyed the primary principles of conventional disinfection processes [23]. It is described as linear relationship between the inactivation effect and the exposure time [25]:

lgðN0 =NÞ ¼ kt

ð3Þ

where k, the slope of the line, is the pseudo-first order rate constant (min1). The inactivation of B. atrophaeus spores shows a pseudofirst-order kinetic behavior, while the goodness of fit is presented by correlation coefficient (R2) and the slope of the line is stated as pseudo-first order rate constant (k). The Chick’s Model has been widely utilized in the literatures to compare the inactivation effect through the values of k. In addition, other parameters, such as standard deviation and root mean square error, were also calculated. 3. Results and discussion 3.1. Effect of initial H2O2 dose

Fig. 1. Schematic diagram of Collimated Beam Apparatus.

The experiments were carried out to investigate the effect of initial H2O2 dose on B. atrophaeus spores inactivation at irradiance

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Y. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 134 (2014) 9–15

of 113.0 lW/cm2, initial cell density of 107 CFU/mL and initial pH 7. As shown in Fig. 2, at a contact time of 10 min, the inactivation effect increased from 3.85 to 5.88 lg with the H2O2 dosage increased from 0 to 0.5 mM, indicating that UV/H2O2 was more efficient than UV treatment alone. However, the inactivation effect reduced to 5.28 lg when H2O2 concentration increased to 0.9 mM, referring that the exorbitant H2O2 dose exhibited a negative effect. The fitting results of the modified Hom Model were illustrated in Fig. 2(a), while the fitting equations were presented in Table 1. It is no doubt that the modified Hom Model successfully fit the experimental data, since the correlation coefficients were close to 1. However, the three-parameters-model made it complicated to compare the inactivation effect. In order to give a striking contrast, the Chick’s Model was proposed in Fig. 2(b) and the pseudo-firstorder rate constants (k) of various initial H2O2 dose were exhibited in the inset of Fig. 2(b). It was apparent that k increased from 0.365 to 0.597 min1 with the increasing H2O2 concentration from 0 to 0.5 mM, while dropped to 0.544 min1 at 0.9 mM H2O2. As a result, an optimum value at about 0.5 mM for the H2O2 concentration was observed in this study. Although this conclusion is valid only under the reported experimental conditions, it can be used to guide the H2O2 dose selection in inactivating B. atrophaeus spores by UV/ H2O2 to some extent. The root mean squares for the inactivation effects shown in Fig. 2 were presented in Table SI 1. This phenomenon could be explained by dual roles H2O2 played in the reactions, both promoter and scavenger of OH [27–29]. At low H2O2 concentration, the quantity of OH is the critical factor influencing photolysis rate. Through trapping photogenerated electrons and photolysis, increasing dosage of H2O2 was able to improve quantities of OH (Eq. (4)). However, terminal reactions may also be involved to reduce inactivation efficiency at high

(a)

6

lg (N0 / N)

5 4

2

0 mM, R2=0.994 0.1mM, R2=0.992 0.3 mM, R2=0.994 0.5 mM, R2=0.999 0.7 mM, R2=0.998 0.9 mM, R2=0.997

1 0 0

2

4

6

8

10

time (min) 6

k (min-1)

0.7

5

lg (N0 / N)

H2O2 dose (mM)

Fitting equation

0

lgðN 0 =NÞ ¼ 12:08½1  expð0:059tÞ1:43

0.1

lgðN 0 =NÞ ¼ 6:209½1  expð0:216tÞ2:22

0.3

lgðN 0 =NÞ ¼ 13:26½1  expð0:056tÞ1:151

0.5

lgðN 0 =NÞ ¼ 10:96½1  expð0:107tÞ1:458

0.7

lgðN 0 =NÞ ¼ 12:39½1  expð0:081tÞ1:306

0.9

lgðN 0 =NÞ ¼ 8:724½1  expð0:122tÞ1:430

H2O2 concentration. H2O2 in excess acts as a scavenger of highly reactive OH to form HO2 and O2 with less oxidizing capacity (Eqs. (5) and (6)). Moreover, high concentration of OH will readily self-recombine to produce H2O2 (Eq. (7)), which is undesirable.

H2 O2 þ hm ƒƒƒ! 2 OH

ð4Þ

H2 O2 þ  OH ƒƒƒ! H2 O þ  HO2

ð5Þ



OH þ  HO2 ƒƒƒ! H2 O þ O2

ð6Þ



OH þ  OH ƒƒƒ! H2 O2

ð7Þ

Some results have been reported to prove the existence of H2O2 optimum concentration using UV/H2O2. Chang et al. [30] discovered that highest azo dye decomposition rate was obtained at H2O2 concentration of 8.42 mM with azo dye concentration of 20 mg/L. Zhang et al. [27] investigated the rapid photocatalytic decolorization of methylene blue, indicating that 2.94 mM was found to be the optimum H2O2 concentration. He et al. [31] proved the threshold concentration of H2O2 to be 0.882 mM under 0.27 mW/cm2 irradiance and 1 lM microcystin-LR in microcystin-LR degradation. The variation could be ascribed to different material characteristics. Commonly, microorganisms required less H2O2 than organic compounds, since they possessed comparatively larger sizes and more feasible to be attacked [26]. 3.2. Effect of UV irradiance

3

(b)

Table 1 Fitting equation at different initial H2O2 dosages (irradiance = 113.0 lW/cm2; initial cell density = 107 CFU/mL and initial solution pH = 7).

0.5

4

0.3 0.0

0.3 0.6 0.9 H2O2 dose (mM)

3 2

0 mM, R2=0.984 0.1mM, R2=0.979 0.3 mM, R2=0.993 0.5 mM, R2=0.995 0.7 mM, R2=0.996 0.9 mM, R2=0.993

1 0 0

2

4

6

8

10

time (min) Fig. 2. Effect of initial H2O2 dosages on B. atrophaeus spores inactivation: (a) the modified Hom Model, (b) the Chick’s Model (irradiance = 113.0 lW/cm2; initial cell density = 107 CFU/mL and initial solution pH = 7). Inset of (b) shows the variation of pseudo-first-order rate constant with respect to H2O2 dose. The plot represents the mean value, and the error bar represents the standard deviation value. The number of data for each plot was 3.

In order to investigate the effect of irradiance on B. atrophaeus spores inactivation, three reaction solutions were prepared and exposed to irradiance of 113.0, 56.5, 28.3 lW/cm2, respectively, while initial cell density was 107 CFU/mL and initial pH was 7. The chosen H2O2 concentration was 0.5 mM, which was the optimum H2O2 concentration under reported experimental condition. Fig. 3 illustrates that better inactivation effect was received at higher irradiance. Taking exposure time of 10 min as an example, the inactivation effect of B. atrophaeus spores decreased from 5.88 to 3.08 lg with the irradiance decreased from 113.0 to 28.3 lW/cm2, namely a substantial 47.6% decrease. The fitting curves of the modified Hom Model were demonstrated in Fig. 3(a), while the fitting equations were shown in Table 2. Obviously, the modified Hom Model matched the experimental results very well. However, it is unable to make a comparison of the inactivation effect. As a result, the Chick’s Model was utilized (Fig. 3(b)) and the pseudo-first-order rate constants (k) were presented in the inset of Fig. 3(b), which showed that k decreased from 0.597 to 0.308 min1 with the irradiance decreased from 113.0 to 28.3 lW/cm2. The root mean squares for the inactivation effects shown in Fig. 3 were presented in Table SI 2. This could be attributed to the increasing amount of radiation photon by the improvement of irradiance. According to Eq. (2), the quantity of OH was closely related to the number of radiation photon. Increasing UV irradiance improved the OH produced in

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Y. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 134 (2014) 9–15

(a)

6

6

5

5

4

lg (N0 / N)

lg (N0 / N)

7

3 2

1

0 0

2

4

6

8

3 2

113.0 µW/cm2, R2=0.999 56.5 µW/cm2, R2=0.988 28.3 µW/cm2, R2=0.989

1

4

0

10

0

time (min) 0.9

6 5

(b)

0.6

3

4

6

8

10

0.7 0.5 0.3

5

lg (N0 / N)

lg (N0 / N)

0.0 0

7 6

0.3

4

2

time (min)

k (min-1)

(b)

106 CFU/mL, R2=0.963 107 CFU/mL, R2=0.999 108 CFU/mL, R2=0.993

k (min-1)

(a)

50 100 150 UV irradiance (µW/cm2)

0.1 105 106 107 108 109 cell density (CFU/mL)

4 3

2 2

113.0 µW/cm2, R2=0.995 56.5 µW/cm2, R2=0.971 28.3 µW/cm2, R2=0.985

1

1

0 0

2

4

6

8

106 CFU/mL, R2=0.980 107 CFU/mL, R2=0.995 108 CFU/mL, R2=0.962

0

10

0

time (min)

2

4

6

8

10

time (min)

Fig. 3. Effect of UV irradiances on B. atrophaeus spores inactivation: (a) the modified Hom Model, (b) the Chick’s Model (initial H2O2 dose = 0.5 mM; initial cell density = 107 CFU/mL and initial solution pH = 7). Inset of (b) shows the variation of pseudo-first-order rate constant with UV irradiance. The plot represents the mean value, and the error bar represents the standard deviation value. The number of data for each plot was 3.

Fig. 4. Effect of initial cell densities on B. atrophaeus spores inactivation: (a) the modified Hom Model, (b) the Chick’s Model (initial H2O2 dose = 0.5 mM, irradiance = 113.0 lW/cm2 and initial solution pH = 7). Inset of (b) shows the variation of pseudo-first-order rate constant with initial cell density. The plot represents the mean value, and the error bar represents the standard deviation value. The number of data for each plot was 3.

Table 2 Fitting equation at different UV irradiances (initial H2O2 dose = 0.5 mM; initial cell density = 107 CFU/mL and initial solution pH = 7).

in 5.88 lg and 2.78 lg, respectively, with the initial cell density increased from 107 to 108 CFU/mL, namely a 53% reduction. However, reducing initial cell density from 107 to 106 CFU/mL did not enhance the inactivation efficiency of B. atrophaeus spores. The observed inactivation effect within 10 min was 5.99 lg for 106 CFU/mL, indicating that the increase was only 2%. As demonstrated in Fig. 4(a), the modified Hom Model perfectly matched the experimental results, with the fitting equations shown in Table 3. While in the light of the Chick’s Model (Fig. 4(b)), at initial cell densities from 106 to 108 CFU/mL, all the reactions exhibited pseudo-first-order kinetic behaviors. These pseudo-first-order rate constants decreased from 0.641 to 0.253 min1 with the initial density increased from 106 to 108 CFU/mL, as shown in the inset of Fig. 4(b). As expected, an increase in the initial cell density slowed down the inactivation efficiency. The root mean squares for the inactivation effects shown in Fig. 4 were presented in Table SI 3. The phenomenon could be ascribed to two reasons [30,36]:

UV irradiance (lW/cm2)

Fitting equation

113

lgðN 0 =NÞ ¼ 10:96½1  expð0:107tÞ1:458

56.5

lgðN 0 =NÞ ¼ 5:326½1  expð0:254tÞ2:146

28.3

lgðN 0 =NÞ ¼ 6:001½1  expð0:1tÞ1:466

the UV/H2O2 system, resulting in enhanced inactivation efficiency [32]. As a result, higher irradiance gave rise to better inactivation effect [14,26]. It has been reported that irradiance plays a significant role in microcystin-LR removal [31], DNA photorepair of Escherichia coli [33] and dimethyl phthalate degradation [34] by UV/ H2O2. Benabbou et al. [26] found that the required time for totally inactivating E. coli by UV treatment increased from 90 to 180 min, when the irradiance decreased from 3.85 to 0.48 mW/cm2. Meanwhile, the irradiance trend remained the same on E. coli inactivation with addition of 1.5 g/L TiO2. Wu et al. [35] reported that UV irradiation plays a significant character removal in UV/Ag–TiO2/ O3 system. The inactivation effect of Dunaliella salina improved from 0.98 to 1.17 lg with the irradiance increased from 1.2 to 6.5 mW/cm2, within an exposure time of 15 s. Although other biochemical responses may occur, since D. salina is a photosynthetic eukaryote, it still demonstrates the importance of UV irradiation to some extent.

(1) The limited OH generated in the UV/H2O2 process dominates the reaction rate. As high initial cell density was able to arouse a high internal optical density of solution, it Table 3 Fitting equation at different initial cell densities (initial H2O2 dose = 0.5 mM, irradiance = 113.0 lW/cm2 and initial solution pH = 7).

3.3. Effect of initial cell density The influence of the initial cell density was shown in Fig. 4, with H2O2 dose of 0.5 mM, irradiance of 113.0 lW/cm2 and pH 7. The results showed that the inactivation effect within 10 min resulted

Cell density (CFU/mL)

Fitting equation

106

lgðN 0 =NÞ ¼ 7:94½1  expð0:171tÞ1:427

107

lgðN 0 =NÞ ¼ 10:96½1  expð0:107tÞ1:458 –a

108 a

Ambiguous.

Y. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 134 (2014) 9–15

prevented UV treatment from penetrating towards the solution immediately and slowed down the generation of OH. As a result, the improvement of initial cell density would result in a negative impact on inactivation effect, and vice versa. (2) As initial cell density increased, the ratio of H2O2 to cell decreased. Accordingly, the amount of cell inactivated by the OH was relatively lower against a higher initial density. This could give rise to the deceleration of the inactivation effect as well. The various effects of the initial density of treated material were also reported previously. Haji et al. [36] observed a high degradation rate of methyl orange dye at high dye concentration using UV–H2O2, since the possibility of collisions between OH and the stuff increased at higher density. Li et al. [37] believed that the microcystin-LR degradation rate decreased with its concentration increased using UV–H2O2. Sontakke et al. [38] showed the initial cell concentration exhibited a significant effect on the E. coli and Pichia pastoris inactivation using UV–Ag/TiO2, while decreasing the initial cell concentration of both microorganisms led to an increase in the inactivation rate. The variations could also be attributed to diverse nature of materials.

3.4. Effect of the initial solution pH Experiments were performed at five initial solution pH value varied from 3 to 11 at 0.5 mM H2O2, irradiance of 113.0 lW/cm2 and initial cell density of 107 CFU/mL to investigate the effect of initial pH. As shown in Fig. 5, at an exposure time of 10 min, the inactivation effect increased from 4.40 to 5.88 lg with pH increasing from 3 to 5, then decreased to 3.62 lg with pH increasing to 11.

(a)

7 6

lg (N0 / N)

5 4 3 2

pH=3, R2=0.990 pH=5, R2=0.999 pH=7, R2=0.999 pH=9, R2=0.992 pH=11, R2=0.986

1 0 0

2

4

6

8

10

time (min) 7 6

0.7 k (min-1)

(b)

0.5

lg (N0 / N)

5

0.3 3

4

5

7 9 pH

11

3 2

pH=3, R2=0.983 pH=5, R2=0.985 pH=7, R2=0.995 pH=9, R2=0.990 pH=11, R2=0.972

1 0 0

2

4

6

8

10

time (min) Fig. 5. Effect of initial solution pH on B. atrophaeus spores inactivation: (a) the modified Hom Model, (b) the Chick’s Model (initial H2O2 concentration = 0.5 mM, irradiance = 113.0 lW/cm2 and initial B. atrophaeus spores density = 107 CFU/mL). Inset of (b) shows the variation of pseudo-first-order rate constant with initial solution pH. The plot represents the mean value, and the error bar represents the standard deviation value. The number of data for each plot was 3.

13

The modified Hom Model method was applied to the experimental data as demonstrated in Fig. 5(a), resulting in the equations (Table 4) closely fit the data. While according to the fitting curves of the Chick’s Model (Fig. 5(b)) and the pseudo-first-order rate constants (k) (inset of Fig. 5(b)), it was obviously that the rate constants increased from 0.461 to 0.657 min1 with the pH increased from 3 to 5. Above the threshold pH value, rate constants varied significantly within the range of 0.657–0.389 min1 at pH 5–11. Pseudo-first-order rate constants followed the sequence: pH 5 > pH 7 > pH 9 > pH 3 > pH 11. It may give an instruction to the pH selection in B. atrophaeus spores inactivation by UV/H2O2. The root mean squares for the inactivation effects shown in Fig. 5 were presented in Table SI 4. The opposite effect of pH may be ascribed to the following reasons [14,34,39,40]. According to Eqs. (8) and (9), more OH were generated under alkaline conditions, while OH is more reactive than H2O2. However, according to Eqs. (10) and (11), the intermediate product hydroperoxide anion (HO 2 ) acted as a scavenger of  OH. Moreover, high pH may also strongly impel the self-decomposition of H2O2 through Eq. (12). In conclusion, a slight acid condition at pH 5 was considered as the optimal pH value.

H2 O2 ƒƒƒ! HO2 þ Hþ

ð8Þ

HO2 þ Hþ þ hm ƒƒƒ! 2 OH

ð9Þ

HO2 þ  OH ƒƒƒ!  HO2 þ OH

ð10Þ

HO2 þ H2 O2 ƒƒƒ! H2 O þ O2 þ OH

ð11Þ

2H2 O2 ƒƒƒ! 2H2 O þ O2

ð12Þ

In previous researches, Aleboyeh et al. [39] reported that the highest decolorization rates of Acid Blue 74 were found at pH range between 3.5 and 5.5 using UV/H2O2. Xu et al. [34] observed that the optimum degradation effect for dimethyl phthalate removal by UV/H2O2 was at pH 4.0. Pelaez et al. [41] determined the highest initial reaction rate of microcystin-LR under visible light-activated TiO2 was achieved at pH 3.0. In conclusion, both organic contaminant and microcystin being treated with UV/H2O2 showed great similarity with this work. 3.5. Effect of different inorganic anions By adding 1 mM anions into the system respectively, the effects of three inorganic anions were investigated and compared, includ 2 ing sulfate (SO2 4 ), nitrate (NO3 ) and carbonate (CO3 ). As illustrated in Fig. 6, the sequence of inactivation effect within 10 min  followed the order of control group (5.88 lg) > SO2 4 (5.30 lg) > NO3 (3.52 lg) > CO2 (2.97 lg). 3 Compared with control group, only a slight reduction of 0.58 lg 2 was observed with the addition of SO2 4 , since SO4 was unable to compete with B. atrophaeus spores for OH [7]. In contrast, NO 3 resulted in a negligible decrease of 2.36 lg within 10 min, namely a 40.1% reduction. The inhibition effect of  NO 3 is attributed to its ability to react with OH during the photoly sis. In fact, NO3 showed a dual character in the process [42]. On one hand, it contributes to the production of additional OH under UV treatment (Eqs. (13)–(17)). On the other hand, it also acts as an inner filter and cuts down the absorbed radiation photon account. Whether character dominates relies heavily on the reaction conditions. Apparently the negative effect triumphs over the positive effect in this research.

NO3 þ hm ƒƒƒ! NO2 þ O

ð13Þ

NO3 þ hm ƒƒƒ!  O þ  NO2

ð14Þ

14

Y. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 134 (2014) 9–15

Table 4 Fitting equation at different initial solution pH (initial H2O2 concentration = 0.5 mM, irradiance = 113.0 lW/cm2 and initial B. atrophaeus spores density = 107 CFU/mL). pH

Fitting equation

3

lgðN 0 =NÞ ¼ 6:37½1  expð0:162tÞ1:627

5

lgðN 0 =NÞ ¼ 7:594½1  expð0:229tÞ1:921

7

lgðN 0 =NÞ ¼ 10:96½1  expð0:107tÞ1:458

9

lgðN 0 =NÞ ¼ 13:16½1  expð0:053tÞ1:094

11

lgðN 0 =NÞ ¼ 4:526½1  expð0:226tÞ

(a)

lg (N0 / N)

5 4

2 Control, R2=0.999 SO42-, R2=0.988 NO3-, R2=0.997 CO32-, R2=0.984

0

2

4

6

8

10

time (min)

OH þ HCO3 ƒƒƒ!  HCO3 þ OH

ð19Þ



HCO3 ƒƒƒ!  CO3 þ Hþ

ð20Þ



   OH þ CO2 3 ƒƒƒ! CO3 þ OH

ð21Þ



CO3 þ H2 O2 ƒƒƒ!  HO2 þ HCO3

ð22Þ

4. Conclusions

0.4

3 2-

O C

4 2-

N

3

O

0.2

3 -

4

2 Control, R2=0.995 SO42-, R2=0.981 NO3-, R2=0.972 CO32-, R2=0.980

1 0

0

2

4

6

8

10

time (min) Fig. 6. Effect of inorganic anions on B. atrophaeus spores inactivation: (a) the modified Hom Model, (b) the Chick’s Model (initial H2O2 dose = 0.5 mM; irradiance = 113.0 lW/cm2; initial cell density = 107 CFU/mL; initial solution pH = 7 and inorganic anions concentrations of 2 mM). Inset of (b) shows the variation of pseudo-first-order rate constant with respect to inorganic anions. The plot represents the mean value, and the error bar represents the standard deviation value. The number of data for each plot was 3.

Table 5 Fitting equation with inorganic anions (initial H2O2 dose = 0.5 mM; irradiance = 113.0 lW/cm2; initial cell density = 107 CFU/mL; initial solution pH = 7 and inorganic anions concentrations of 2 mM). Inorganic anions

Fitting equation

Control

lgðN 0 =NÞ ¼ 10:96½1  expð0:107tÞ1:458

SO2 4

lgðN 0 =NÞ ¼ 8:613½1  expð0:115tÞ1:27

NO 3

lgðN 0 =NÞ ¼ 4:066½1  expð0:275tÞ2:011

CO2 3

lgðN 0 =NÞ ¼ 6:157½1  expð0:099tÞ1:492

2 NO2 þ H2 O ƒƒƒ! NO2 þ NO3 þ 2Hþ

ð15Þ

O þ H2 O ƒƒƒ! 2 OH

ð16Þ

O þ H2 O ƒƒƒ!  OH þ OH

ð17Þ





0.6

on tr ol SO

lg (N0 / N)

5

k (min-1)

6

C

(b)

ð18Þ

As in the above case, the modified Hom Model well fitted the experimental data as illustrated in Fig. 6(a) and the results were shown in Table 5. While on the basis of the Chick’s Model (Fig. 6(b)), the constant rates gave a striking comparison. It indi 2 cated that the rates of control group, SO2 were 4 , NO3 and CO3 1 0.597, 0.558, 0.389 and 0.306 min , respectively. The rates of  CO2 3 and NO3 were far below that of control group, referring that the reaction between the two anions and OH slowed down the inactivation effect. The results are in agreement with other published investigations [7,15,37]. The root mean squares for the inactivation effects shown in Fig. 6 were presented in Table SI 5.

3

0

  CO2 3 þ H2 O ƒƒƒ! HCO3 þ OH

1:911

6

1

2.91 lg of decrease was observed within 10 min, namely a 49.5% reduction. Since the addition of CO2 would quickly form HCO 3 3 and reach the equilibration of carbonate system (Eq. (18)), both of the anions act as scavengers of OH (Eqs. (19)–(22)) Accordingly, it makes an inhibition effect by producing radicals with less oxidizing capacity [15,37].

In addition, CO2 exhibited a more significant inhibiting effect 3 and slowed down the inactivation efficiency evidently. About

The inactivation of B. atrophaeus spores followed both the modified Hom Model and the Chick’s Model. Inactivation effect of B. atrophaeus spores was affected by various parameters, such as initial H2O2 dose, UV irradiance, initial cell density, initial solution pH and various inorganic anions. H2O2 played both promoter and scavenger of OH in the reactions. The optimum H2O2 concentration was found to be 0.5 mM, which resulted in 5.88 lg reduction within 10 min. The inactivation effect exhibited a substantial 47.6% decrease when irradiance decreased with 113.0–28.3 lW/ cm2. Reducing initial cell density made a contribution to the deceleration of inactivation effect. The highest inactivation effect was observed at pH 5, following the order: pH 5 > pH 7 > pH 9 > pH 3 > pH 11. Inorganic anions exhibited a negligible inhibiting effect and slowed down the inactivation efficiency to different degrees. The inhibiting effect on B. atrophaeus spores inactivation followed  2 the order: SO2 4 < NO3 < CO3 . Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 51178323 and 51108329), China Postdoctoral Science Foundation funded project (No. 2012T50413), and the Fundamental Research Funds for the Central Universities (No. 0400219205). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2014. 03.022. References [1] D. Fritze, R. Pukall, Reclassification of bioindicator strains Bacillus subtilis DSM 675 and Bacillus subtilis DSM 2277 as Bacillus atrophaeus, Int. J. Syst. Evol. Microbiol. 51 (2001) 35–37.

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