*2.7. E*ff*ect of [Fe2*+*] on Responses*

Statistical analyses identified [Fe2+] as an important factor for the three types of responses studied (D% MB, kapp MB and IuLog EC). For both the D% MB analysis and for kapp MB the increasing of [Fe2+] value (studied at 6.0 <sup>×</sup> <sup>10</sup>−<sup>4</sup> , 8.0 <sup>×</sup> <sup>10</sup>−<sup>4</sup> and 1.0 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mol/L) exhibited a significant positive effect on these responses, while for the IuLog EC analysis this parameter showed a significant negative effect. In Equation (1) it is observed that the formation of ·OH due to the oxidation of Fe2<sup>+</sup> to Fe3<sup>+</sup> in the presence of H2O<sup>2</sup> will increase with a higher concentration of Fe2+. A single ·OH attack on the MB structure leads to its decolorization (D% MB and kapp MB), which seems to agree with the importance of [Fe2+] found in the present research. However, if the ·OH also had a preponderant role in the inactivation of *E. coli* K12, then the [Fe2+] values should not have a significant negative effect on the IuLog EC response. Some authors [44–47] suggest that although ·OH oxidize most simple organic compounds (such as recalcitrant compounds), the inactivation of a bacterium (such as *E. coli* K12) is not directly affected by the production of these radicals. These researchers indicate that the formation of singlet oxygen (1O2) is responsible for the inactivation of bacteria. Consequently, the IuLog EC decreases with increasing values of [Fe2+], since excess Fe2<sup>+</sup> could react with the ·OH formed (Equation (5)), decreasing the possibility that the ·OH react to form <sup>1</sup>O<sup>2</sup> (Equations (3) and (4)).

### *2.8. E*ff*ect of pH on Responses*

The pH value (studied at pH 3.0, 4.0 and 5.0) showed a significant effect on the D% MB and IuLog EC responses but did not show a significant effect on the kapp MB response. The Fenton reaction (Fe2<sup>+</sup> and <sup>H</sup>2O2), with a rate constant 76 L·mol−<sup>1</sup> s −1 [48], form ·OH quickly, consume Fe2<sup>+</sup> and produce Fe3+. The Fenton-like reaction (Fe3<sup>+</sup> and H2O2) has a much slower rate constant (0.01 L·mol−<sup>1</sup> s −1 ) than the Fenton reaction [48] and it only produces O2· <sup>−</sup>, a much less reactive radical than ·OH. Additionally it has been established that the species of Fe(II) that prevails in the working pH range (3.0–5.0) is Fe2<sup>+</sup> [49], while in the same pH range the speciation of Fe(III) demonstrates the formation of Fe(OH)2<sup>+</sup> and Fe(OH)<sup>2</sup> <sup>+</sup> species, species that are less reactive than Fe3<sup>+</sup> [22]. Based on this information, it is expected that the rate of ·OH formation from the Fenton reaction, at least in the first minutes of reaction that directly influence the determination of kapp MB, will not be greatly altered when changing system pH between 3.0 and 5.0. However, D% MB and IuLog EC, which are obtained in a final time of 15 min, will be influenced by both the Fenton reaction and the subsequent Fenton-like reaction. Considering this, the participation of the Fenton-like reaction implies that the pH and its effect on Fe(III) speciation have a greater influence on the D% MB and IuLog EC responses, as observed in this investigation.

#### **3. Materials and Methods**

#### *3.1. Chemicals and Materials*

Iron sulfate heptahydrate (FeSO4·7H2O), hydrogen peroxide (H2O2, 30%), sodium hydroxide (NaOH), hydrochloric acid (HCl), Luria Bertani (LB) medium and methylene blue (C16H18N3SCl·3H2O) were purchased from Merck S.A. (Santiago, Chile).

#### *3.2. Fenton Experiments*

Methylene blue, a dye that does not generate toxic byproducts when reacting with ·OH [50–52], was used as a model of recalcitrant compound, while *E. coli* K12, a non-pathogenic *E. coli* [53], was used as a model of bacteria. Experiments were performed in 20 mL glass reactors containing the MB solution (5.0 <sup>×</sup> <sup>10</sup>−<sup>5</sup> mol/L) or *E. coli* K12 (10<sup>6</sup> CFU), kept under magnetic stirring at room temperature (25 ◦C) [43,44]. First, FeSO4·7H2O solution was added to each sample according to the experimental design. The pH of each sample was adjusted by using NaOH (0.25 mol/L) or HCl (0.10 mol/L) solutions. Reactions were started by adding an aliquot of H2O<sup>2</sup> solution. After the experimental time elapsed (15 min), for *E. coli* K12 analysis, 0.2 mL of each sample was collected for its enumeration. The decolorization of MB was studied by determining its kinetic constants of color decay and the degree of decolorization. After 2 min of maintaining the reaction under constant agitation, samples (3.0 mL) were withdrawn, and immediately injected into a cuvette for analysis at time intervals of 3, 6, 9, 12 and 15 min. The analyses in samples were performed spectrophotometrically by UV–Vis spectrophotometry (Shimadzu UV-1800, Shimadzu Inc., Kyoto, Japan) at 668 nm using quartz cells with path lengths of 1 cm. A calibration curve was constructed (5.30 <sup>×</sup> <sup>10</sup>−7–1.30 <sup>×</sup> <sup>10</sup>−<sup>5</sup> mol/L; R<sup>2</sup> <sup>=</sup> 0.999). Fitting decolorization kinetics and the rate constant was obtained by Sigma Plot 11.0 software (Systat Software, Inc., San Jose, CA, USA).

#### *3.3. Detection and Enumeration of E. coli K12*

Strain samples were stored in cryo-vials containing 20% glycerol at −20 ◦C. To prepare the bacterial pellet for the experiments, one colony was picked from the precultures and loop-inoculated into a 50 mL sterile PE eppendorf flask containing the Luria Bertani (LB) medium. The flask was then incubated aerobically at 37 ◦C and 150 rpm in a shaker incubator (Gerhardt THO500, Gerhardt GmbH & Co., Königswinter, Germany) until the stationary physiological phase was reached. After 24 h, cells were centrifuged (SIGMA 2-16P, Sigma Laborzentrifugen GmbH, Steinheim, Germany) and diluted until optical density 0.5 a.u. (i.e., 10<sup>6</sup> CFU/mL) at 600 nm [43,44]. Component of LB medium included sodium chloride (10 g), tryptone (10 g) and yeast extract (5 g) in 1 L of deionized water; this solution was then sterilized by autoclaving for 20 min at 121 ◦C. The bacterial pellet was resuspended and washed three times with a saline solution (NaCl/KCl). The final pellet was resuspended in saline solution. This procedure resulted in a cell density of approximately 10<sup>9</sup> colony forming units (CFU) per milliliter. The pH of the solution was adjusted to 7.0 and the solution was then sterilized by autoclaving for 30 min at 121 ◦C. The bacterial solution was diluted in reactors to the required cell density corresponding to 10<sup>6</sup> CFU/mL [43,44].

CFU were performed by plating on plates (PCA method). Of the samples 0.2 mL was withdrawn. Samples were diluted (10% *v*/*v*) and 0.1 mL poured on plates. Plates were aerobic incubated for 24 h at 37 ◦C (Heraeus B6, Kendro, Langenselbold, Germany) and the CFU were counted manually. All experiments were performed in triplicates. The enumeration of colonies was expressed as CFU (colony forming units) per 100 mL of sample. These concentrations were transformed to log<sup>10</sup> and the removal of bacteria, uLog = log(N<sup>t</sup> /N0), was calculated from the initial bacteria concentration (N0) and the remaining bacteria population at "t" time (Nt).

#### *3.4. Experimental Design*

To determine the optimal experimental conditions for the decolorization of MB and the inactivation of *E. coli* K12 by Fenton technology, a Box–Behnken design was performed. pH, Fe2<sup>+</sup> concentration ([Fe2+], mol/L) and molar concentration ratio of Fe2<sup>+</sup> and H2O<sup>2</sup> ([H2O2]/[Fe2+]) were selected as independent variables in the experimental design (Table 6).

**Table 6.** Independent variables and levels used in the Box–Behnken design for Fenton technology.


Three replicates were performed at the central point, with 15 runs performed for each study. The chosen levels of the independent variables were based on literature reports [54]. The experimental

responses were the degree of MB decolorization (D% MB), the apparent kinetic constant of MB decolorization (kapp MB), and removal of bacteria in uLog units (IuLog EC) for variables showed in Table 6.

A second-order linear polynomial regression model (Equation (7)) was obtained to analyze the data. Data were statistically evaluated and an analysis of variance (ANOVA) was applied at with a confidence level of 95% using software Design Expert version 10 (Stat-Ease Inc., Minneapolis, MN, USA). Responses of the experimental tests were compared to the estimated values, and the fit of model was assessed. Experimental tests, performed under optimal conditions, were performed to achieve maximal D% MB, kapp MB and IuLog EC.

#### **4. Conclusions**

The present study provided a comprehensive description regarding the application of the Fenton technology as a process for MB decolorization and *E. coli* K12 inactivation in aqueous solutions at different [H2O2]/[Fe2+] values (1.0, 2.0 and 3.0), [Fe2+] values (6.0 <sup>×</sup> <sup>10</sup>−<sup>4</sup> , 8.0 <sup>×</sup> <sup>10</sup>−<sup>4</sup> and 1.0 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mol/L) and pH values (3.0, 4.0 and 5.0) up to 15 min of reaction. It was found that the Box–Behnken model could effectively predict and optimize the performance of Fenton technology for MB decolorization and *E. coli* K12 inactivation.

The maximum D% MB of 94.57% was predicted at [H2O2]/[Fe2+] <sup>=</sup> 2.9, [Fe2+] <sup>=</sup> 1.0 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mol/<sup>L</sup> and pH = 3.2; for kapp MB the maximum of 2.08 min−<sup>1</sup> was predicted at [H2O2]/[Fe2+] = 1.7, [Fe2+] <sup>=</sup> 1.0 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mol/L and pH <sup>=</sup> 3.7 and the maximum IuLog EC of 0.89 uLog was predicted at [H2O2]/[Fe2+] <sup>=</sup> 2.9, [Fe2+] <sup>=</sup> 7.6 <sup>×</sup> <sup>10</sup>−<sup>4</sup> mol/L and pH <sup>=</sup> 3.2. This analysis revealed good agreement between experimental results and the RSM predictions, further illustrating that RSM is a suitable approach to optimize the MB decolorization and *E. coli* K12 inactivation.

The Pareto and perturbation analysis of the model terms showed that all parameters analyzed have different effects on the responses. The [H2O2]/[Fe2+] values show a significant positive effect only on D% MB. The pH values show a significant negative effect on D% MB and IuLog EC, which could involve the main role of speciation of Fe(II) and Fe(III) species in the total process of MB decolorization and *E. coli* K12 inactivation by Fenton technology. The positive and negative significant effect of the [Fe2+] values on the MB decolorization (D% MB and kapp MB) and *E. coli* K12 inactivation (IuLog EC) respectively, suggest that different oxidizing species are involved in these processes.

Thus, considering that bacteria are larger than dye molecules, the complex self-repair mechanisms of bacteria and the different external structures of bacteria compared to the dyes structure, the *E. coli* inactivation proved to be less effective than MB decolorization by Fenton processes.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4344/10/12/1483/s1, Figure S1: Three-dimensional response surface plots (a, c and e) and their corresponding contour plots (b, d and f) representing de modeled D% MB as a function of: [H2O<sup>2</sup> ]/[Fe2+] and [Fe2+] (a, b), pH and [H2O<sup>2</sup> ]/[Fe2+] (c, d), [Fe2+] and pH (e, f) at central point values of other parameters, Figure S2: Three-dimensional response surface plots (a, c and e) and their corresponding contour plots (b, d and f) representing de modeled kapp MB as a function of: [H2O<sup>2</sup> ]/[Fe2+] and [Fe2+] (a, b), pH and [H2O<sup>2</sup> ]/[Fe2+] (c, d), [Fe2+] and pH (e, f) at central point values of other parameters, Figure S3: Three-dimensional response surface plots (a, c and e) and their corresponding contour plots (b, d and f) representing de modeled IulogEC as a function of: [H2O<sup>2</sup> ]/[Fe2+] and [Fe2+] (a, b), pH and [H2O<sup>2</sup> ]/[Fe2+] (c, d), [Fe2+] and pH (e, f) at central point values of other parameters.

**Author Contributions:** Conceptualization, P.S. and G.V.; methodology, P.S. and G.V.; software, P.S. and J.L.F.; validation, P.S. and J.L.F.; formal analysis, P.S. and G.V.; investigation, J.L.F.; writing—original draft preparation, P.S. and G.V.; writing—review and editing, P.S. and G.V.; supervision, G.V.; project administration, G.V.; funding acquisition, P.S. and G.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by ANID/FONDAP/15130015 and ANID FONDECYT/Postdoctorado 3180566.

**Acknowledgments:** José Luis Frontela thanks ERASMUS mobility program.

**Conflicts of Interest:** The authors declare no conflict of interest.
