1. IntroductionPhoto-Fenton processes which are based in the light induced reaction of ferrous and/or ferric ions with hydrogen peroxide to generate reactive oxygen species such as protonated superoxide (HO2•) and •OH radical, have recently risen as a promising method to inactivate waterborne bacteria [1-5].
Recent studies reported that solar photo-Fenton process at near neutral pH (5.5) achieved a complete inactivation of wild Salmonella sp., a pathogen bacterial strain resistant to solar disinfection [6], without re-growth after 72 h of dark storage by simple addition of H2O2 in natural waters containing iron at neutral pH [7]. Furthermore at pH 5.5 it was demonstrated that ferric ions undergo a strong interaction with E. coli cells avoiding its precipitation and inactivating efficiently the bacteria cells [4]. However all disinfection studies performed with photoFenton processes either at acid or near neutral pH have been carried out using plate count methods on solid agar to quantify the microorganisms [2,4,5,7,8]; this procedure could give a false positive since it takes only into account the cell culturability neglecting its viability. Several studies have suggested that when bacteria undergo oxidative stress by the presence of UV light or toxic substances such as radicals, they could enter in the transient viable but nonculturable state (VBNC), produced by oxidative stress; VBNC state is a condition where microorganisms lose its capacity to grow in nutritive media [9,10]. Nevertheless, when the oxidative stress ceases, microbial cells can undergo resuscitation where they recover their culturability under specific conditions [10]. This later fact is a key point since some authors have suggested Salmonella typhimurium cells could maintain their virulence during VBNC state, and this could imply a serious threat to human health in disinfection processes [11-13].
There are methodologies that can determinate the cell viability. Among them the coupling of Direct Viable count (DVC) with Fluorescence in situ Hybridation (FISH) seems to be effective to determinate viability of waterborne bacterial cells [14]. Moreover the coupling of DVC with FISH has successfully been used to determinate viability in Escherichia coli cells from natural waters and seems to be a powerful technique to evaluate the viability of waterborne enteric bacteria [15,16].
Herein, for the first time, culturability and viability were measured simultaneously by PC, MPN and DVCFISH of S. typhimurim being exposed to the photo-Fenton processes induced by simulated solar light irradiation at pH 5.5. It is suggested that this photochemical process could induce on the bacterial cells VBNC state avoiding their growing on solid agar and liquid media after 240 min of simulated solar irradiation. This fact is extremely important since it might allow evaluate precisely the ability and limitations of photo-Fenton process to inactive pathogen waterborne bacteria.
2. Materials and Methods2.1. Photochemical and Photo-Fenton Experiments at pH 5.5Pyrex glass bottles of 80 mL (3.5 cm in diameter and 10 cm high) were used as batch reactors. 2 mg L−1 of Fe3+ (FeSO4·7H2O (Merck-Germany)) and 20 mg L−1 of H2O2 (Merck-Germany 30% (v/v)) were added to 80 mL of an isotonic solution containing distilled water and NaCl (Sigma-Aldrich Germany) (8.0 g L−1), KCl (Merck Germany) (0.8 g L−1) in each reactor. The initial pH was adjusted by NaOH (Merck Germany) addition until reaching 5.5, not buffer was used. Solar irradiation was simulated by a Hanau Suntest solar simulator (Germany) having a wavelength spectral distribution with about 0.5% of emitted photons <300 nm (UV-C range) and about 7% between 300 and 400 nm (UV-B, A range). The emission spectrum between 400 and 800 nm follows the solar spectrum (Figure 1). Light intensity in all experiments was 500 W∙m−2 and it was monitored with a Kipp & Zonen (CM3) power meter (Omni instruments,
Dundee, UK). Pyrex bottles containing inoculated S. ty-phimurium were illuminated in the presence and absence of Fe3+ and H2O2 during 4 h and samples (1.0 mL) were taken at different time intervals and serial dilutions were performed in saline solution.
The irradiation experiments were performed at room temperature (25˚C) and the temperature of the solution increased up to approximately 30˚C during irradiation. All experiments were carried out in equilibrium agitating at 700 rpm in triplicate and the error bars were added on the graphs.
2.2. Preparation of Salmonella Typhimurium StrainA strain of S. typhimurium ATCC 15490 (Washington DC, USA) was used during these experiments. Strain was preserved in a 30% glycerol solution to avoid membrane rupture during freezing storage. The bacterial reactivation was achieved by inoculating 10 mL of nutrient broth (Oxoid) with 100 µL of bacterial suspension, and then incubating for 18 h at 35˚C. A second inoculum was transferred from this culture to a new tube with 10 mL of nutrient broth and again incubated for 18 h at 35˚C. This second culture was washed by centrifuging at 3000 rpm for 10 min; the supernatant was carefully extracted so the pellet can be suspended and diluted in 10 mL of 0.1% peptonized bi-distillated water (Milli-Q water), agitated and centrifuged again. This procedure was repeated three times, leaving the last cell pellet suspended in 10 mL of 0.1% peptonized bi-distillated water. The final concentration of the washed cell suspension was adjusted according to the McFarland scale #0.5 at 1.5 × 108 CFU mL−1. Consecutive dilutions (10−1 - 10−4) of the adjusted cell suspension (1.5 × 108 CFU mL−1) were made in tubes with 9 mL of 0.1% peptonized water (Oxoid), and seeded onto nutrient agar to be incubated for 18 h at 35˚C to establish the initial concentration. This cell suspension was used to inoculate the batch reactors containing the isotonic saline distillated water with Fe3+ (FeSO4·7H2O (Merck-Germany)), and H2O2 (Merck-Germany), to initiate irradiation experiments.
2.3. Culturability Measurement of Salmonella Typhimurium by MPN and PC2.3.1. PC Method100 μL from each dilution were seeded onto Petri dishes containing Nutrient Agar (Merck-Germany) and incubated at 37˚C ± 2˚C for 24 h to obtain the CFU measuring.
2.3.2. MPN Method1 mL from each dilution were inoculated onto tubes with 9 mL of lactose broth (Difco) and incubated at 35˚C ± 2˚C for 24 h in order to initiate the presumptive phase. The positives for bacterial growth were then recovered in Rappaport broth (Merck-Germany) before being seeded on Hektoen (Merck-Germany), Xylose, Lysine Desoxycholate agar (XLD agar) (Difco-France) and Nutrient agar (Merck-Germany) to finalize with the confirmatory phase.
2.4. Viability Measurement of Salmonella Typhimurium by DVC FISHThe FISH-DAPI coloration procedure suggested by Amman et al. [17] was used for the total cell enumeration, while viable cells were enumerated using the DVCFISH method proposed by Kogure et al. [18] and Regnault et al. [19] with the following modifications.
2.4.1. DVC-FISHThe remaining 8.8 mL sample in each dilution tube were treated with Nalidixic acid (40 μg mL−1) and 1 mL of lactose broth (Difco-France) and yeast extract (DifcoFrance), then incubated in the dark for 18 h at 35 ± 2˚C. Cells were harvested by centrifugation at 5000 g. Depending on the expected concentration, 100 or 300 μL were re-suspended in 10 x phosphate buffered saline (PBS) solution to reach 1 mL and fixated with a paraformaldehyde fixation buffer 30% (v/v), stored overnight at 4˚C, and harvested by centrifugation at 1000 g for 10 min. The pellets were washed with PBS solution. This washing process was repeated two times to ensure the complete removal of p-formaldehyde, and the final pellets were re-suspended in 500 μL of 1 × PBS and 500 μL of ethanol 100%. 10 μL of the resulting cell suspension was transferred to each well of a 8-well slide, previously covered with gelatine (0.1% w/v) and KCr(SO4)2 (MerckGermany) (0.01% w/v). The loaded slides were dehydrated with ethanol and incubated for 2 h in the dark with 10 μL of hybridization solution pre-warmed at 45˚C and containing 24 ng∙μL−1of S. typhimurium probe [16,20], marked with 5’ Cy3 (prepared by Microsynth GmbH Switzerland). Finally, cells were washed with a washing solution at 45˚C and then rinsed with distilled water.
2.4.2. DAPIAfter hybridization, all samples were washed in distilled water and stained with 9 μL of DAPI (Organic Research-Ireland) (4’,6’-diamidino-2-phenylindole) 0.0001% (w/v) for 10 minutes, washed again in distilled water and dried (to aid cell localization, slides were cover with antifade citi-fluor solution). Microscopic cell count (DAPI) and oligonucleotide probe-positive count (Cy3) were performed using a Nikon Eclipse E800 microscope (Japan) equipped with a specific DAPI filter and G-2A filter sets. A minimum of 20 view squares were enumerated for each well; two wells were examined in total.
2.5. Assays of Growing in Mineral Enriched MediaFour tubes containing 20 mL of mineral media containing distilled water and NaCl (Sigma-Aldrich Germany) (8.0 g∙L−1), KCl (Merck Germany) (0.8 g∙L−1) and 20% of Glucose (Merck Germany) or Peptone (DIFCO UK), respectively, were inoculated with 1 mL of irradiated bacteria. The turbidity at 650 nm was measured after 18, 24, and 96 h of incubation and the increase in 0.1 or more turbidity units was reported as positive growth.
3. Results and Discussion3.1. Effect of Simulated Solar Light Arradiation on Culturability and Viability of S. Typhimurium Cells in Absence of Ferric Ions and H<sub>2</sub>O<sub>2</sub>The initial S. typhimurium concentration before irradiation events, evaluated with DVC FISH and MPN methods, achieved concentrations two logs higher than the PC method (Figure 2). Although there is no difference between the nutrient composition of a liquid and a solid culture media, in practice the culture broths have a higher
rate of cell recovery. In solid media (PC method) the bacteria growing is limited by the availability of water and nutrients close to the colony’s growing area while the liquid media (MPN and Nalidixic media) ensures constant and direct contact of cell with nutrients. This could explain why DVC-FISH and MPN gave initial concentrations higher than PC.
On the other hand, results obtained by MPN and PC (Figure 2) revealed that after 240 min of solar simulated irradiation, the former method showed that Salmonella population underwent a decreasing of almost 2 logs while the later did not show any decreasing of bacterial concentration. Furthermore, DVC FISH viability count underwent a slight decreasing of its initial concentration (~1 log) after of 240 min of solar simulated irradiation. The experiments were followed by 48 h in dark conditions and the results showed that DVC FISH and MPN did not undergo neither decreasing nor re-growth on the bacterial population. However PC method showed a slight decreasing of almost 1 log probably due to the fact that under these conditions weakened microorganisms (by UV irradiation) could undergo an additional stress by the lack of nutrients. This decreasing of E. coli population after a photocatalytic treatment (in presence of TiO2 and solar simulated light) under dark conditions was already observed by Rincon and Pulgarin [21] and referred as post-irradiation events.
In summary, the three methods used in this study (DVC FISH, MPN and PC) revealed that direct solar simulated irradiation had slight negative effect on the S. typhimurium population. It is well known that UV-C component (220 - 280 nm) of UV light is the most lethal to the microorganisms since it might produce chemical changes on the DNA components [22]. Solar simulator emits few UV-C light irradiation (Figure 1) and this fact could explain why light irradiation did not affect in a larger extent of the initial concentration of S. typhimurium. However, the UV-A (320 - 400 nm) is emitted by the solar simulator and some studies have argued that this UV component could affect the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and could also produce partial degradation of iron-containing proteins, releasing Fe (II) ions into the cell [23, 24]. Thus, only the presence of UV-A light could generate a bacteria weakening by diminishing its antioxidant capacity without producing a lethal damage.
3.2. Effect of Photo-Fenton Process at near Neutral pH on Culturability and Viability of S. typhimurium CellsFigure 3 shows that culturability measured by MPN and PC was strongly affected by photo-Fenton process at pH 5.5. Results obtained by MPN revealed a diminution of 4 logs after 240 min of simulated solar irradiation and PC