Inactivation of Candida albicans in Water Using Advanced Oxidation Processes ^†

Abstract

Pathogenic microorganisms such as bacteria, viruses, fungi and protozoa have played a central role in the safety of drinking water, since they spread easily in the water network, constituting a health risk for humans and animals. Currently in water treatments, advanced oxidative processes (AOPs) have been increasing in importance in the microbiological disinfection of water. The present study aimed to inactivate C. albicans, a commensal yeast species in Vertebrates that can cause disease, using AOPs. To achieve this objective, a powerful oxidant (hydrogen peroxide) was combined with UV radiation to promote the inactivation of C. albicans. Initially, the inactivation capacity of the H₂O₂ was assessed and it was verified that the application of 2.5 mM, 5 mM and 10 mM H₂O₂ reached a cell reduction of 3 log after 180, 360 and 300 min, respectively. Subsequently, the combination with UV-A radiation (λ = 365 nm) proved to be even more promising, as the H₂O₂ + UV-A system, using the same H₂O₂ concentrations, reached an inactivation of 3 log after 240, 180 and 60 min, respectively. These results support that UV-A radiation promotes the generation of hydroxyl radicals, which have a comparatively higher oxidation potential (2.8 eV) to the H₂O₂ (1.8 eV), responsible for the inactivation of C. albicans cells. Thus, the UV-A/H₂O₂ process can reduce this microorganism in an aqueous matrix, avoiding potential hazards to human and animal health.

1. Introduction

Approximately 1.7 million deaths per year worldwide, particularly in immunocompromised individuals, are caused by fungal infections [1]. Since the 1970s, infections caused by the genus Candida have steadily increased, due to the increased risk of opportunistic infections, the improvement of clinical procedures that identify fungi that cause nosocomial infections, as well as the development of antifungal resistance to prolonged exposure treatments [2,3].

In recent years, different countries and international organizations have legislated or published guidelines to regulate the reuse of treated wastewater [4,5,6]. These laws, or guidelines, consider the type of water reuse (urban, agricultural, industrial, recreational or environmental) and establish the maximum acceptable concentration of contaminants. The increasing demand for water and the scarcity of available water sources boosted the treatment and reuse of wastewater [7]. The advanced oxidation processes (AOPs) generated reactive free radicals, the most important of which is the hydroxyl radical (HO^•) with an oxidizing power of 2.80 V, which react with non-selective organic compounds [8,9]. The HO^• radical has numerous advantages, including not being toxic or corrosive, not generating waste, and having a very limited lifespan [10,11].

In this work, it was decided to test the effect of H₂O₂ and H₂O₂/UV-A, as hydrogen peroxide has been widely used in the removal of low levels of pollutants from wastewater (chlorine, nitrites, sulphites, hypochlorites, etc.) and as disinfectant. As mentioned by [12], the H₂O₂/UV process initially occurs in the photolytic degradation of hydrogen peroxide, through the scission of a H₂O₂ molecule that produces two hydroxyl radicals.

The main objective of this research is to evaluate the inhibition potential of C. albicans in water samples, through the addition of a powerful oxidant (hydrogen peroxide) and combining the effect of this oxidant with UV-A radiation.

2. Material and Methods

2.1. Microorganism and Reagents

For the inactivation study, cells of the strain C. albicans ATCC 90028 were grown on yeast malt extract agar (YMA). Hydrogen peroxide (H₂O₂) was purchased from Labkem. All reagents used were analytical grade. The inoculum was prepared from a culture of C. albicans with 48 h of growth, in which a loopful was suspended in 5.0 mL of sterilized saline solution (0.85% NaCl). The turbidity of the suspension was adjusted to 0.5 on the McFarland scale (1.5 × 10⁸ CFU/mL).

2.2. UV-A LEDS

All the experiments were carried out in a self-designed lab-scale reactor with a capacity of 110 cm³. The UV-A LED system was composed of 12 Indium Gallium Nitride (InGaN) LEDs lamps (Roithner APG2C1-365E LEDs, Vienna, Austria) with λ_max = 365 nm. Each UV-A LED has a nominal consumption of 1.4 W when the current is 350 mA with an optical power of 135 mW and an opening angle of 120°, eliminating shadow zones. The radiation was emitted in continuous mode for all the 12 UV-A LEDs being controlled by a power MOSFET in six different current settings, resulting in irradiance levels from 16 up to 85 W m⁻² measured at a 5 cm distance with a UVA Light Meter (Linshang model LS126A, Shenzhen, China). The UV-A LED system was located 5 cm above the solution surface in a parallel position.

2.3. Experimental Procedure

The cell inactivation process was carried out in a 500 mL reactor. Figure 1 shows the experimental procedure used for C. albicans inactivation. The microbial suspension was added to the reactor with 200 mL of saline solution (0.85% NaCl), a solution that preserves yeast cell homeostasis to obtain a microorganism concentration of 10⁵ colony-forming units (CFU)/mL.

Initially, experiments with the addition of H₂O₂ were carried out. Three different concentrations of H₂O₂ were evaluated under the same experimental conditions: 2.5 mM, 5 mM and 10 mM. In a second round of experiments, UV-A LEDs were added to the H₂O₂ concentrations previously tested. During all the oxidation processes, the reaction temperature was recorded and samples were taken over a period of 360 min. Microbiological analyses were performed by the spread plate method (Standard Method 9215C, [13]) after 10⁻¹ dilution in tubes with saline solution (0.85%). After incubation at 25 °C for 48 h, the colonies were quantified and the results expressed in log CFU/mL.

The entire procedure was performed under total aseptic conditions in a Biosafety BSL2 chamber in order to avoid any type of contamination.

2.4. Statistical Analysis

The data were analyzed using the EXCEL 2011 and OriginLab 2019 (Northampton, MA, USA) software. The analysis involved descriptive statistics (means and standard-deviation), and the one-way ANOVA with the post-hoc Tukey test.

3. Results and Discussion

Table 1. C. albicans inactivation time dynamics (log CFU/mL) by H₂O₂ alone and combined with UV-A ($\stackrel{-}{\mathrm{x}}$ ± sd). n.d.—not detectable.

	H2O2				H2O2 + UV-A
	Log (CFU/mL)				Log (CFU/mL)
Time (min)	0 mM	2.5 mM	5 mM	10 mM	0 mM	2.5 mM	5 mM	10 mM
1	3.1 ± 2.0	3.0 ± 0.0	2.6 ± 2.5	2.6 ± 0.0	3.1 ± 0.5	2.8 ± 1.0	2.4 ± 1.5	2.8 ± 3.5
30	3.1 ± 0.5	2.7 ±1.5	2.4 ± 0.5	2.6 ± 0.0	2.9 ± 5.5	2.6 ± 2.0	2.0 ± 0.0	2.7 ± 0.5
60	2.9 ± 1.0	2.7 ± 1.5	2.6 ± 2.0	2.4 ± 1.5	3.0 ± 1.5	2.6 ± 0.0	2.0 ± 0.0	n.d.
120	3.0 ± 0.5	2.2 ± 0.5	1.7 ± 0.5	2.6 ± 0.0	3.1 ± 0.5	2.7 ± 3.0	1.7 ± 0.5	n.d.
180	3.2 ± 0.0	n.d.	n.d.	n.d.	3.0 ± 0.0	1.7 ± 0.5	n.d.	n.d.
240	3.2 ± 1.0	n.d.	n.d.	1.7 ± 0.5	3.0 ± 3.5	n.d.	n.d.	n.d.
300	3.1 ± 2.5	n.d.	1.7 ± 0.5	n.d.	2.7 ± 0.5	n.d.	n.d.	n.d.
360	3.0 ± 2.0	n.d.	n.d.	n.d.	2.9 ± 2.0	n.d.	n.d.	n.d.

shows the results of the inactivation of C. albicans after the application of H₂O₂ and the combination of H₂O₂ with UV-A LED radiation.

As shown in Table 1, the efficacy of H₂O₂ against C. albicans is time- and, to a less extent for the tested concentration range, dose-dependent. The ANOVA one way analysis did not find any differences (p > 0.05) between either of the controls (without H₂O₂ versus without H₂O₂ + UV-A). As the concentration of H₂O₂ rises, oxidative stress increases, triggering responses from C. albicans cells. In fact, this species is well adapted to oxidative stress induced by macrophages that includes an enzymatic arsenal and morphological changes [14,15]. Also, in the presence of H₂O₂, C. albicans presents a rough and wrinkled surface, according to images obtained by SEM, indicating that H₂O₂ can damage the cell wall and cell permeability [16]. In another study, peroxymonosulphate (PMS) combined with UV-A LED was used to inactivate C. albicans, and the authors reported that due to its greater resilience to oxidative stress, higher doses (5 mM) were required [17]. C. albicans cells appear to be more resistant to H₂O₂, cationic stress and disinfectant agents than C. auris [18].

The application of H₂O₂ at a concentration of 2.5 mM or above achieved 3-log inactivation of C. albicans after 180 min of treatment (Table 1). Punctually, and at concentrations 5 mM and 10 mM, and after 180 min of treatment, growth was noted (corresponding to 1 UFC/plate) that which may indicate differences among C. albicans cells to oxidative stress caused by H₂O₂.

In general, the combination of H₂O₂ with UV-A LED radiation was more efficacious against C. albicans at higher H₂O₂ concentrations (5 and 10 mM; p < 0.05). The highest microbial inactivation rate was achieved in 60 min using 10 mM of H₂O₂ combined with UV-A LED radiation, with a 3-log reduction and no detectable (re)growth afterwards. Contrary, the total inactivation with H₂O₂ alone was achieved much later (3-log at 300 min). Also, for lower H₂O₂ concentrations (2.5 and 5.0 mM) combined with UV-A, no regrowth or recovery was detected after 240 and 180 min, respectively. Therefore, the combination of H₂O₂ and UV-A radiation induced higher and faster C. albicans inactivation rates. Some authors claim that, when H₂O₂ is introduced into the process, the degree of inactivation of C. albicans with the effect of UV radiation tends to increase [19], a statement that is in line with the results obtained. The rate of photodecomposition of H₂O₂ determines the efficiency of the process depending on the intensity of UV radiation, as well as the nature of the impurities and concentrations [20,21,22]. These responses may explain the effect of H₂O₂ and the combined effect of H₂O₂ and UV-A on the inactivation rate of C. albicans.

Comparing the results obtained in both treatments with those of the respective controls, without the addition of H₂O₂, the results were satisfactory, as it appears that the inactivation of C. albicans cells occurred throughout the process.

4. Conclusions

The data obtained in this study draw attention to the importance of finding an effective procedure for disinfecting water and inactivating pathogenic microorganisms such as C. albicans. Based on these results, it can be concluded that (1) UV-A radiation enhances the conversion of H₂O₂, leading to a higher production of hydroxyl radicals that are responsible for the inactivation of C. albicans cells; (2) the H₂O₂/UV-A process can reduce this species in an aqueous matrix, avoiding potential hazards to human and animal health; (3) UV-A LED radiation is an attractive alternative to the use of conventional UV lamps in microbial inactivation processes, since LEDs are environmental friendly, have a low operating cost and high energy efficiency.