**3. Solar-driven Fenton and Fenton-like inactivation of pathogenic microorganisms**

Homogeneous processes rely on the generation of hydroxyl radicals. Nevertheless, it has been proposed that other highly oxidant species could also be involved in pollutant degradation (Anipsitakis and Dionysiou, 2004). Fenton and Fenton-like processes are among the most widely studied methodologies. When a Fenton process uses ultraviolet (UV) radiation, visible light or a combination of both, the resulting process (known as photo-Fenton) has several advantages, including the increase of degradation rate and the flexibility of using alternative energy sources (i.e., solar radiation) for driving the process (Bandala et al., 2007; Bandala and Estrada, 2007; Fernandez et al., 2005). From the economic point of view, the possibility of using solar energy to promote both homogeneous and heterogeneous photocatalytic processes is an interesting alternative technology for use in developing countries (Bolton, 2001).

Several reports on the use of photo-Fenton process for deactivating pathogenic microorganisms have been published in the near past. In these studies, a wide variety of analysis have been conducted and reported, such as: the effects of the iron salt and pH on deactivating *E. coli* (Spuhler et al., 2010), the capability of solar driven photo-Fenton process to achieve simultaneous degradation of natural organic matter (NOM) and water disinfection (Moncayo-Lasso et al., 2009) as well as the effect of many other specific parameters as reported in a recent review by Malato et al. (2009).

(Dunlop *et al*., 2008). Cells are used to regulate iron adsorption as a defense mechanism against hydroxyl radical; however once hydroxyl radicals are generated in the intracellular media, as a result of the Fenton-like process by direct attack of the ROS, they are free for

radical and hydrogen peroxide (H2O2), as a result of cell respiration. However, these oxidizing species are in equilibrium with the immune system defense mechanism through anti-oxidizing enzyme production related to superoxide dismutase (SODs), catalase (CAT) and glutathione peroxidase (GPX) families (Castillo-Ledezma et al., 2011). When microorganisms are exposed to a major oxidative stress, for example ROS produced during a photocatalytic process, enzyme production is no longer capable of eliminating excess radical allowing deep cell damage. In the same way, ROS may produce additional oxidative stress in the cells through Fenton and Heber-Weiss reaction (Dunlop *et al*., 2008) generating damage in all the cell components including proteins, lipids and DNA. In the case of DNA damage, produced by pyrimidine dimmers formation by the generation of covalent bonds among the bases in the same DNA chain (Sichel *et al*., 2009), it generates mutations that may lead to loss of functional capability and death of cell (Malato *et al*., 2009). At the same time, when microorganisms are exposed to ultraviolet radiation (UV, λ ≤ 400 nm) during the photocatalytic reaction, DNA damage occurred directly through the radiation absorption by cell chromophores, which absorb radiation and produce heat. This interaction leads to an increase in ATP and RNA synthesis, jointly with the increase of ROS production. Microorganisms receiving a sub-lethal dose of UV radiation may become resistant to induced oxidative stress, partially recover their defense mechanisms and adapt to oxidative stress generated by exposure to UV radiation alone (tanning effect) (Bandala et al., 2011b).

•), hydroxyl

During cellular metabolism some ROS are produced, such as superoxide (O2

**3. Solar-driven Fenton and Fenton-like inactivation of pathogenic** 

Homogeneous processes rely on the generation of hydroxyl radicals. Nevertheless, it has been proposed that other highly oxidant species could also be involved in pollutant degradation (Anipsitakis and Dionysiou, 2004). Fenton and Fenton-like processes are among the most widely studied methodologies. When a Fenton process uses ultraviolet (UV) radiation, visible light or a combination of both, the resulting process (known as photo-Fenton) has several advantages, including the increase of degradation rate and the flexibility of using alternative energy sources (i.e., solar radiation) for driving the process (Bandala et al., 2007; Bandala and Estrada, 2007; Fernandez et al., 2005). From the economic point of view, the possibility of using solar energy to promote both homogeneous and heterogeneous photocatalytic processes is an interesting alternative technology for use in

Several reports on the use of photo-Fenton process for deactivating pathogenic microorganisms have been published in the near past. In these studies, a wide variety of analysis have been conducted and reported, such as: the effects of the iron salt and pH on deactivating *E. coli* (Spuhler et al., 2010), the capability of solar driven photo-Fenton process to achieve simultaneous degradation of natural organic matter (NOM) and water disinfection (Moncayo-Lasso et al., 2009) as well as the effect of many other specific

parameters as reported in a recent review by Malato et al. (2009).

reacting with biomolecules (Darlymple *et al*., 2009).

**microorganisms** 

developing countries (Bolton, 2001).

In addition to the study of *E. coli* cells, many other microorganisms have been tested and used as indicators for evaluating the performance of photo-Fenton processes such as *Salmonella spp.* (Sciacca et al., 2011), *Fusarium solani* spores (Polo-Lopez et al., 2011), helminth eggs (Bandala et al., 2011a,b) and *Bacillus subtilis* spores (Bandala et al., 2011c). In a recent work, several different Fenton reagent concentrations were tested in combination with UV-A radiation (λmax= 365 nm) to pursue deactivation of *B. subtilis* spores. The best spore deactivation conditions were found using [Fe(II)] = 2.5 mM and [H2O2] = 100 mM and UV-A radiation. As depicted in Figure 1, under these experimental conditions, over a 9-log reduction in spore viability was reached after 20 minutes of reaction. Interesting results were also observed from experiments conducted with low Fe(II) concentrations or even when no Fe(II) was added and only H2O2 and UV-A radiation were used. Under these experimental conditions, a lag phase –where no deactivation occurred- was observed during earlier stages of the disinfection process and much lower spore viability was determined after long time of irradiation. These results might suggest that microorganisms have the capability to generate defense mechanisms as a response to threatening environmental stresses. It is also suggested that the observed initial delays in the inactivation process may be due to the effect of defense mechanisms by the microorganisms against low ROS concentrations generated under these reaction conditions.

Fig. 1. Deactivation of *Bacillus subtilis* spores using photo-assisted Fenton reaction.

Furthermore, researchers have hypothesized that, when the iron salt is added to the reaction mixture, ROS generation increases dramatically and overwhelms the defense capability of the microorganisms leading to their immediate death without undergoing the lag phase observed previously.

The effects of ionic strength and natural organic matter (NOM) in spore deactivating kinetics have also tested and representative experimental results are illustrated in Figures 2 and 3, respectively. In both cases, an important decrease on the deactivating rate was observed when NOM concentration and ionic strength were increased. Experimental data has been modeled using a modification of the delayed Chick-Watson model, including the accumulated energy (*Qn*), rather than the traditional C×t factor (disinfectant concentration times contact time) used for chemical disinfection (Bandala et al., 2009). Model fitting parameters, including deactivating rate constants for the different experimental conditions, are reported in Table 3.

$$\frac{N}{N\_{\phi}} = \begin{cases} \frac{N}{N\_{\phi}} \text{ if } \underline{Q}\_{n} \le \underline{Q}\_{n \text{log}} = \frac{1}{k} \ln \left\{ \left( \frac{N\_{1}}{N\_{0}} \right) \left( \frac{N\_{0}}{N} \right)\_{\varepsilon} \right\} \\\frac{N\_{1}}{N\_{\phi}} e^{-k\underline{Q}\_{n}} \text{ if } \underline{Q}\_{n \text{log}} \le \underline{Q}\_{n} \le \underline{Q}\_{n \text{2}} \\\frac{N\_{2}}{N\_{\phi}} e^{-k\underline{Q}\_{n}} \text{ if } \underline{Q}\_{n} \ge \underline{Q}\_{n \text{2}} = \frac{1}{k\_{2} - k\_{1}} \ln \left( \frac{N\_{2}}{N\_{1}} \right) \end{cases} \tag{22}$$

It was demonstrated that the lag-phase described initially for spore deactivation was avoided by the use of photo-assisted Fenton reaction whereas very different results were obtained when natural organic matter was present in raw water.

As observed in the experimental results, the photo-assisted Fenton reaction might represent an interesting alternative to deactivate recalcitrant microorganisms in water. In this particular study, the photo-assisted process was used to kill *B. subtilis* spores, which are currently considered among the most resistant bacteria. It has been demonstrated that, if the photo-Fenton reaction is capable to deactivate *B. subtilis* spores, it could be able to eliminate other less resistant pathogenic microorganisms present in water under the same reaction conditions (Bandala et al., 2011a).


Table 3. Kinetic data obtained using Chick-Watson model for homogeneous photocatalytic disinfection.

The effects of ionic strength and natural organic matter (NOM) in spore deactivating kinetics have also tested and representative experimental results are illustrated in Figures 2 and 3, respectively. In both cases, an important decrease on the deactivating rate was observed when NOM concentration and ionic strength were increased. Experimental data has been modeled using a modification of the delayed Chick-Watson model, including the accumulated energy (*Qn*), rather than the traditional C×t factor (disinfectant concentration times contact time) used for chemical disinfection (Bandala et al., 2009). Model fitting parameters, including deactivating rate constants for the different experimental conditions,

It was demonstrated that the lag-phase described initially for spore deactivation was avoided by the use of photo-assisted Fenton reaction whereas very different results were

As observed in the experimental results, the photo-assisted Fenton reaction might represent an interesting alternative to deactivate recalcitrant microorganisms in water. In this particular study, the photo-assisted process was used to kill *B. subtilis* spores, which are currently considered among the most resistant bacteria. It has been demonstrated that, if the photo-Fenton reaction is capable to deactivate *B. subtilis* spores, it could be able to eliminate other less resistant pathogenic microorganisms present in water under the same reaction

**Experimental conditions k2(min-1)** ܖܔ ൬ۼ

UV, pH = 3 - - - [H2O2] = 50 mM - 2.45 - [H2O2] = 100 mM 0.39 0 2.99 [Fe(II)] = 1mM; [H2O2] = 100 mM 0.83 2.53 15.23 [Fe(II)] = 2.5 mM; [H2O2] = 100mM 0.87 2.78 15.67 [Fe(II)] = 2.5 mM; [H2O2] = 100 mM; [Cl-] = 25 mgL-1 0.85 2.72 15.82 [Fe(II)]=2.5 mM; [H2O2] = 100 mM; [Cl-] = 50 mgL-1 0.02 2.77 16.61 [Fe(II)] = 2.5 mM; [H2O2] = 100 mM; [Cl-] = 100 mgL-1 0.02 2.69 16.11 [Fe(II)] = 2.5 mM; [H2O2] = 100 mM; [SR-NOM] = 2.5 mgL-1 0.74 2.53 17.05 [Fe(II)] = 2.5mM; [H2O2] = 100 mM; [SR-NOM] = 5.0 mgL-1 0.76 2.3 16.12 Table 3. Kinetic data obtained using Chick-Watson model for homogeneous photocatalytic

obtained when natural organic matter was present in raw water.

(22)

ۼ

ۼ൬ ܖܔ <sup>൰</sup> ۼ ൰

are reported in Table 3.

conditions (Bandala et al., 2011a).

disinfection.

Fig. 2. Effect of natural organic matter (NOM) on the efficiency of photo-assisted deactivation of *B. subtilis* spores.

Fig. 3. Effect of ionic strength on the efficiency of photo-assisted deactivation of *B. subtilis* spores.

Spore deactivation using photo-Fenton reaction is considerably affected by the ionic strength and natural organic matter, mainly by delaying the beginning of actual deactivation process. As shown in Figure 2, an increase in the concentration of natural organic matter has an important slowing effect on spore deactivation since it increases the duration of the lag phase.

The experimental results presented in Figures 2 and 3 suggest that the efficiency of photo-Fenton processes used for disinfection depend largely on the quality of background water. Therefore, if these processes are to be cost-effective, they should be coupled with pretreatment and/or other conventional drinking water processes.
