Use of treated and disinfected wastewater for irrigation can be a good solution for conserving drinking water in areas with limited natural water resources. Existing methods of bacterial eradication in wastewater involve the use of aggressive chemicals, such as chlorine-based compounds and ozone, or power-consuming physical methods, such as UV radiation. Photodynamic treatment with the help of photosensitizers may comprise an alternative approach to wastewater disinfection [1
Photosensitizers (PSs) are colored compounds which are excited under illumination by visible light and can either transfer their excitation energy or exchange an electron with other substances [3
]. Upon excitation, PSs can respectively follow two pathways, named Type I and Type II reactions. In the Type I mechanism, PS molecules react with bio-organic molecules, producing active free radicals and radical ions of the PS or another organic substrate which further react with oxygen producing peroxides, superoxide ions and hydroxyl radicals [4
]. The Type II reaction is accompanied by energy transfer to molecular oxygen dissolved in an aqueous phase [5
]. The photosensitizers typically interact with triplet oxygen species to produce reactive oxygen species (ROS), such as singlet oxygen, superoxide anion, hydroperoxyl radical, hydrogen peroxide, and hydroxyl radical [6
]. In nature, singlet oxygen is generated in neutrophils and macrophages for killing microorganisms. Microorganism cells produce superoxide dismutases, catalases and peroxidases in order to defend themselves against radical- and reduced-oxygen species. However, these enzymes are not effective against singlet oxygen [6
]. Contrary to antimicrobials, acquired resistance to singlet oxygen by a bacterium, fungus, or virus has never been reported. Gram-positive bacteria are more sensitive to singlet oxygen than Gram-negative bacteria [8
]. In the case of viruses, enveloped species are inactivated by singlet oxygen more easily than non-enveloped viruses [10
PSs can be applied for eradication of microorganisms in a free form, encapsulated into liposomes or immobilized onto solid supports. Application of free PS for wastewater disinfection was examined by Jemli et al. [1
] who showed that free MB, RB, and meso-substituted cationic porphyrin were effective for inactivating faecal bacteria under illumination and proposed treating wastewater by PSs in order to reuse the treated wastes. Introducing water-soluble PSs into the aqueous phase necessitates further extraction of the former, whereas using PSs immobilized onto a solid phase makes this additional stage redundant. Application of immobilized PSs for water disinfection affords several additional advantages over the use of free disinfectants: 1. Easy separation between the disinfecting agent and the treated water; 2. The possibility of designing a continuous process; 3. The possibility of reusing the immobilized PSs in batch schemes; and 4. Increased resistance of immobilized PSs to bleaching by light and oxygen over free PSs.
Immobilization of PSs can be performed by covalent bonding, by formation of ionic bonds between ion-exchange resins and PSs, by adsorption onto a solid support or by incorporation into polymers [13
Covalent attachment of a large number of PSs, such as Rose Bengal (RB), eosin, fluorescein, chlorophyllin, hematoporphyrin, and Zn(II) phthalocyanine tetrasulfonic acid to various supports, including silica gel, poly(styrene-co-vinylbenzyl chloride), poly[(N
-isopropylacrylamide)-co-(vinylbenzyl chloride)], poly[(sodium p
-styrenesulfonate)-co-(4-vinylbenzyl chloride)], and chitosan, has been reported [13
]. PSs covalently attached to polymers demonstrated high (up to 0.91) quantum yields of singlet oxygen [23
Using immobilized PSs for water disinfection was tested by Bonnet et al. [13
], where PSs were immobilized on chitosan. The group of Orellana [26
] proposed immobilizing PSs from a polyazaheterocyclic Ru(II) group onto porous silicone in order to apply them for water disinfection. The main topic in Garcia-Fresnadillo’s [7
] review on photoinactivation of microorganisms in water is dedicated to water treatment in photoreactors with the help of PSs based on fullerenes and Ru(II) complexes immobilized in polymeric supports, where sunlight is used as a light source. In all of the above cases, the immobilized PSs demonstrated high efficacy in bacterial eradication.
We have previously reported inclusion of PSs into a polymeric film by mixing solutions of PSs in chloroform with solutions of polystyrene, polycarbonate or poly(methyl methacrylate) in the same solvent, with subsequent air evaporation of the latter. The polymeric films obtained via this procedure were effective in eradication of Gram-positive and Gram-negative bacteria under moderate white light illumination [16
Most PS immobilization methods include complicated chemical schemes using toxic and expensive reagents and/or organic solvents. Although the resulting immobilized PSs exhibit high cytotoxic activity, the high ecological “price” of these processes is not compensated by the environmental and economic profits gained from using disinfected wastewater instead of drinking water for irrigation.
The aim of the current work was to develop a green reagent-less PS-immobilization method for disinfection of contaminated water.
2. Materials and Methods
Low-density polyethylene (PE) beads (5 mm diameter) and polypropylene (PP) beads (3 mm diameter) were purchased from Carmel Olefins Ltd., Haifa, Israel.
Rose Bengal disodium salt (RB), purity 95%, Rose Bengal lactone (RBL), purity 95%, methylene blue chloride·3H2O (MB), purity 97%, and hematoporphyrin (HP), purity 97%, were purchased from Sigma-Aldrich, St. Louis, MO, USA.
2.2. Thermogravimetric Analysis
PS were tested for thermostability by TGA using a TGA-DSC instrument (Mettler Toledo International Inc., Grefensee, Switzerland). PS samples of 5–10 mg were places into standard aluminum crucibles and heated from 25 to 300 °C at the rate of 10 °C min−1 in the flow of nitrogen supplied at a flow of 50 mL min−1.
2.3. Immobilization of PSs
Immobilization of the PSs in polymers was performed by co-extrusion using an extruder (Allspeeds Ltd., Accrington, England) under an inlet temperature of 80–90 °C and an outlet temperature of 150–200 °C. For this purpose, a mixture of polymer beads and PS powder were placed in a feed and the extruder was activated to melt the mixture at 43 rpm. The resulting fluid composition was then pushed through a die with a 5 mm round section or a flat 1 × 19.6 mm section. This procedure yielded polymeric rods with the incorporated PS. The rods were chopped into 3 mm beads or used as is.
2.4. Evaluation of PS Inclusion into Polymers
The inclusion yield of PSs into polymers was determined after the immobilization of RB, RBL, and MB, taking the amounts of PS applied for immobilization and the non-incorporated amounts into account. The latter were evaluated by washing the inner chamber of the extruder with water after the extrusion and measuring the absorbance of the washings at an appropriate wavelength (544 nm for RB, 557 nm for RBL and 665 nm for MB) using a Cary 100-Bio UV–VIS spectrophotometer (Varian, Sydney, Australia). The amount of PS not incorporated into the polymeric matrix in the measured volume of washings was calculated using calibration curves. Since HP has poor solubility in water, its inclusion was determined gravimetrically by weighting the HP and the polymer before the extrusion and the immobilized HP after the extrusion.
2.5. Testing PS Leakage from the Polymers
Leakage of PSs from the polymeric matrices was evaluated by soaking a known amount of co-extruded polymer-PS pellets in a bath with a known volume of tap water at ambient temperature for washing from non-entrapped PS. The pellets did not undergo preliminary washing before the experiment. Water was changed twice a day for five days, where all washings were monitored using a spectrophotometer at the appropriate wavelength. The amount of leaked PS was calculated after measuring the absorbance in the washings using calibration curves at the wavelengths mentioned above for RB, RBL, and MB, and at 615 nm for HP. In the case of RB immobilized in PE, RB leakage was also tested for 260 g polymer placed in a bath with 1.5 L of tap water using continuous washing by tap water for three weeks under illumination at a flow rate of 2 mL/min provided by a multi-channel peristaltic pump (Ecoline, Ismatec, Glattbrugg, Switzerland). The washings at the outlet were tested by HPLC analysis with a Jasco LC model (JASCO International Co., Tokyo, Japan) on a RP-18 column YMC-Triart C18, 75 × 3.0 mm, bead size 1.9 μm, in the isocratic regime using an eluent composed of 10:40:50 v/v of 20 mM ammonium acetate: acetonitrile:methanol. The RB concentration in the washings was calculated using calibration curves.
2.6. Bacterial Growth
Cultures of Staphylococcus aureus (ATCC 25923), S. epidermidis (ATCC 12228) and Escherichia coli (ATCC 10798) were grown on brain-heart agar (BHA, Acumedia, Lansing, MI, USA) for 24 h, after which they were transferred into brain-heart broth (BH, Acumedia, Lansing, MI, USA) and grown at 37 °C and shaking at 170 rpm to OD600 = 0.3. Cells were harvested by centrifugation, washed twice with sterile 0.05 M phosphate-buffered saline (PBS), pH 6.5, diluted with PBS to OD600 = 0.1 which corresponded to a final concentration of 108 cells mL−1 and then serially diluted in two to four 10-fold dilutions.
2.7. Antibacterial Activity Assay
The antibacterial activity of the polymer-PS compositions was studied in batch experiments as follows: 25 mL portions of a S. aureus, S. epidermidis, or E. coli suspension at a concentration of 105 cells mL−1 in sterile PBS were dispensed into Petri dishes with 0.1–10 g of immobilized PS beads. In all the experiments the beads were thoroughly pre-washed before a use. The plates were illuminated from the top for periods of 30 min to 24 h with a white luminescent lamp emitting in the range of 400–700 nm with a fluence rate of 1.25 mW cm−2 (light doses of 4–194 J cm−2). The light intensity was measured with a LX-102 Light-meter (Lutron, Taiwan). The distance between the lamp and Petri dishes was 40 cm. Control experiments were carried out in the dark in the presence of the immobilized PS and under illumination with bacterial suspensions in the presence of polymeric pellets not containing PS as well as in the absence of any beads.
Antibacterial activity in a continuous regime was studied by flowing a suspension of bacteria in saline solution from the top down through a vertical column (1 × 50 cm) packed with 14 g of pre-washed RB/PE beads at flows of 0.2–0.9 mL/min using the multi-channel peristaltic pump. A control column, packed by PE beads not containing RB, was connected in a parallel mode to the same bacterial source. The columns were permanently illuminated from the side by two fluorescent lamps installed in a vertical mode parallelly to the columns at the fluence rate of 1.25 mW cm−2. Sampling was performed at the inlet and the outlet of the column and the bacterial concentration was estimated as the number of colony forming units (CFU) per mL determined by the live count method. The suspension of the source bacteria was replaced daily by a fresh one.
Photostability experiments were performed by placing 14 g of preliminary washed 1% RB immobilized in PP in a 3-liter bath with tap water into which suspensions of S. epidermidis bacteria were added daily. The control bath contained no polymeric material or PS. Both baths were permanently illuminated by a luminescent lamp. Samples from both baths were taken daily before addition of fresh portions of bacteria and the bacterial concentration was tested using the live count method.
2.9. Statistical Data Processing
The results obtained from at least three independent experiments carried out in duplicates were statistically analysed by single-factor or two-factor ANOVA analyses. The difference between results was considered significant when the p-value was less than 0.05.
All immobilized PSs examined in the present work were active against waterborne Gram-positive bacteria under moderate ilumination, and their relative efficiency was as follows: RBL/PE > HP/PE > RB/PE > MB/PE (Figure 3
and Figure 5
). Only three of the tested PSs were efficient against Gram-negative bacteria. In this case their relative antibacterial activity was: MB/PE > RBL/PE > RB/PE >> HP/PE (Figure 4
). Since Gram-negative bacteria in general, and coliform bacteria in particular, are a very essential part of the microbial population in wastewater, only those PSs which are active against them can be recommended for wastewater disinfection.
Varying the loadings of immobilized PSs enables presetting conditions for the necessary extent of bacterial eradication (Figure 5
). This finding, and the fact that the bacterial eradication rate depends on the initial bacterial concentration, indicate that direct contact between the bacteria and the surfaces of the immobilized PSs is necessary. In order to provide such contact, mass transfer can be improved by stirring floating beads of immobilized PSs in bacterial suspension in the case of batch processes, or by transferring polluted water via a column packed with PS/polymer beads, i.e., performing disinfection in a continuous mode. Reasonable time periods of water disinfection enable using immobilized PSs not only in batch schemes but also in continuous processes. Application of the latter mode is limited only by the photostability period of the immobilized PSs, which is estimated at approximately 10 days (Figure 9
), i.e., cartridges with PS/polymer beads can be used for water disinfection over the course of several days without replacement and with no need for introducing any additional external cytotoxic agents into the treated water. In any case, water disinfection by PACT requires good light penetration into the treated system. This method is, therefore, applicable only for wastewater after secondary treatment of wastes at the water treatment plants, which decreases the water turbidity.
The results of continuous eradication of both Gram-positive and Gram-negative bacteria (Figure 8
) show good applicability of the method for water disinfection. These experiments confirm, once again, that prolonged killing of bacteria is due to immobilized and not to leaked PSs, since only thoroughly pre-washed PS/polymer beads were used. The method for PS immobilization by the co-extrusion of PSs and polymers, suggested in the present work, does not require any additional chemicals, and can thus be considered as a reagent-less and totally “green” method. PSs are well-retained in the polymeric matrix, and minor PS leakage to an aqueous phase cannot interfere with applying immobilized PSs for water disinfection, since the leaked PS can also contribute to the overall antimicrobial activity. However, it does not lead to a significant increase in the total organic compounds load in the wastewater, since the concentration of leaked PSs does not exceed the ppm level even in the absence of preliminary washing of PS/polymer beads. The proposed PS immobilization method can compete with immobilization of PSs by adsorption [20
], which is also a reagent-less method of immobilization, except for the possibility of PS leakage and adsorption of other components from the treated wastewater.
Further development of the method may include using sunlight as the illumination source, as proposed previously by García-Fresnadillo [7
]. Since bacterial eradication by immobilized PSs occurs effectively even at a very moderate light fluence rate (1.25 mW/cm2
), exposure to much more intense sunlight is expected to significantly increase the efficiency of water disinfection. The problem of a dark period can be solved by accumulation of solar energy during the day and conversion of the accumulated energy into light energy at night.