Environ. Sci. Technol. 2006, 40, 2421-2425
Photosensitized Destruction of Chlorella vulgaris by Methylene Blue or Nuclear Fast Red Combined with Hydrogen Peroxide under Visible Light Irradiation CATHY MCCULLAGH AND PETER K. J. ROBERTSON* Centre for Research in Energy and the Environment, The Robert Gordon University, Schoolhill, Aberdeen, AB10 1FR, U.K.
A considerable number of investigations have started to elucidate the essential roles biological agents play in the biodeterioration of stone. Chemical biocides are becoming increasingly banned because of the environmental and health hazards associated with these toxic substances. The present study reports the photodynamic effect of Methylene Blue (MB) and Nuclear Fast Red (NFR) in the presence of hydrogen peroxide (H2O2) on the destruction of the algae Chlorella vulgaris (C. vulgaris) under irradiation with visible light. Illumination of C. vulgaris in the presence of MB or NFR combined with H2O2 results in the decomposition of both the algal species and the photosensitizer. The photodynamic effect was investigated under aerobic and anaerobic conditions. Differences in mechanism type are reported and are dependent on both the presence and the absence of oxygen. The behavior of each photosensitizer leads to a Type II mechanism and a Type I/Type II combination for MB and NFR, respectively, being concluded. This novel combination could be effective for the remediation of biofilm-colonized stone surfaces.
Directive 67/548/EEC (Directive 92/32/EEC) (9) have accelerated the search for more environmentally and toxicologically safe biocides. Photodynamic therapy (PDT) is a method that utilizes chemicals that require the application of light for their activity (10). Photodynamic therapy has most commonly found application within the field of medicine where PDT agents are site nonspecific drugs; that is, they do not target a specific enzyme or receptor (11). Photodynamic therapy has also been investigated within cancer research (11-15) and as a potential method for the treatment of antibiotic resistant microbial species (16, 17). In this paper, we examine the photodynamic effect as an alternative method for the destruction of algal growth on stone. Nuclear Fast Red (NFR) and Methylene Blue (MB) have been investigated as photosensitizers for the destruction of Chlorella vulgaris using the photodynamic effect. Methylene Blue (MB), from the phenothiazine family, is a photosensitizer (18, 19) that has been used for a variety of applications, including solar energy conversion and PDT (16, 20). Nuclear Fast Red is an anthraquinone dye, which is commonly used as a histological stain. Many anthraquinones possess the ability to mediate one electron transfer to molecular oxygen to form superoxide anion radicals and to generate ROS upon visible light illumination (21). Upon illumination of the photosensitizers, MB or NFR, the production of singlet oxygen and other radical species are produced, which affect the viability of the algal cells. Cell viability was monitored via the fluorescence of chlorophyll within the algal cells. The effect of H2O2 on the activity of NFR and MB toward the photosensitized destruction of Chlorella vulgaris was also investigated. It was anticipated that the radical species produced from the photosensitizers would react with H2O2 producing hydroxyl radicals, thereby increasing the amount of radical species in solution available to penetrate the cell wall and cause disruption to the cell function and ultimately death. The effect of aerobic and anaerobic conditions was examined to determine whether the photosensitizers proceeded via a Type 1 or Type II mechanism.
Experimental Section Introduction Colonization of building surfaces by microorganisms causes aesthetic and physical damage to the structure and can lead to human health problems (1). The most common microorganisms on the exterior surfaces of buildings are fungi, algae, and cyanobacteria, which can resist repeated cycles of drying and rehydration (2). Deterioration of stone, including man-made construction materials, exposed to the natural environment occurs through physical, chemical, and biological processes in isolation or in combination. It has been estimated that approximately 20-30% of stone deterioration is a result of biological activity (3). In the United Kingdom alone, the building cleaning and restoration industry has an estimated annual market of over £100 m (4). Stone surfaces have been traditionally treated using physical or chemical methods such as sand blasting or the application of chemical biocides (5). Current market-based biocides are hazardous to the environment and to public health (6, 7). External pressures including the approval of the European Directive 98/8/EC (8) concerning placing biocidal products on the market and the 7th Amendment to * Corresponding author phone: +44 1224 262301; fax: +44 1224 262222; e-mail:
[email protected]. 10.1021/es052542s CCC: $33.50 Published on Web 03/08/2006
2006 American Chemical Society
Reagents. Methylene Blue ∼85% (remaining 15% primarily salt) and Nuclear Fast Red ∼97% were purchased from Aldrich and used in aqueous solution (Milli Q water). Hydrogen peroxide (>30% w/v) was purchased from Fisher. All chemicals were used as received. Chlorella vulgaris was purchased from Sciento. It was subcultured in a common algal culturing media, BG11, and incubated with continuous illumination at 21 °C. The algae were sampled in the stationary phase of growth. Photochemical Reactions. The experiments were performed with 4.51 × 106 cells/mL of Chlorella vulgaris, sampled from 2.25 × 107 cells/mL stock of Chlorella vulgaris. MB (6 µM) and NFR (10 µM) were added separately to a volume of Chlorella vulgaris. In separate experiments, the effect of H2O2 (1 M) with MB and NFR on Chlorella vulgaris was investigated. The solutions (30 mL) were exposed to illumination from a 500 W tungsten halogen lamp in open Pyrex flasks for a period of 240 min. Samples were taken at 15/30 min intervals. Each experiment was repeated three times, and dark controls were carried out simultaneously. For the inert experiments, the solutions were placed in a sealed vessel and were bubbled with argon for 15 min prior to commencing the experiment. The solutions were purged with argon for the duration of the experiment. The irradiated samples were analyzed using a luminescence spectrometer (Perkin-Elmer LS 50B). The VOL. 40, NO. 7, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Dark control experiments of MB alone and with H2O2 and NFR alone and with H2O2, and dark and light control of Chlorella vulgaris ([ MB (dark); 9 MB/H2O2 (dark); 2 NFR (dark); × NFR/H2O2 (dark); ] no biocide (dark); 4 no biocide (light)).
FIGURE 2. The effect of illumination, MB, and MB with H2O2 on the fluorescence of C. vulgaris under aerobic conditions (b no biocide; 0 MB; 2 H2O2; × MB + H2O2).
excitation and emission wavelengths for fluorescence monitoring of algae, MB, and NFR were 420 nm:685 nm; 667 nm: 691 nm; and 545 nm:595 nm, respectively.
Results and Discussion PDT of C. vulgaris with MB or NFR and H2O2 under Aerobic Conditions. The viability of C. vulgaris was monitored via the fluorescence of its pigment, chlorophyll. The decrease in fluorescence indicated a decrease in the amount of intact chlorophyll. As this is the pigment responsible for photosynthesis in algae, it is reasonable to suggest that a decrease in the fluorescence of the pigment would signify cell death. The concentration of MB (6 µM) and NFR (10 µM) investigated in this study was that which had the maximum absorption of visible light. At lower concentrations, slower kinetics for the process was observed, while at higher concentrations MB formed dimeric compounds (22, 23). Higher concentrations of NFR lead to quenching of the fluorescence signal by collisions between excited and ground-state molecules. A series of dark controls were carried out to investigate the dark toxicity of the photosensitizers alone and in combination with H2O2 toward C. vulgaris (Figure 1). Under dark conditions in the presence of H2O2 and MB, an increase in the fluorescence of C. vulgaris was observed (
