Photodynamic Inhibition of Biofilm Forming Microorganisms on Surfaces
Authors: Roeder Beate , Preuß Annegret , Pohl Jakob , Bornhuetter Tobias 
Published: February 2020

Microorganisms play an important role in our everyday life and in an ecological context. While a great number of species provide a benefit to humans, some of them can be terribly harmful due to the diseases they trigger. To remove or hinder those unwanted microorganisms has been a goal since their discovery. There are classical approaches to accomplish that, but the adaptability of microorganisms – especially the alarming development of antibiotic resistant strains - always calls for new methods. Once microorganisms grow in biofilms they are hard to come by and inadvertently spread infections are a fatal risk for immunodeficient people. Photodynamic Inactivation (PDI) has good potential as antimicrobial agent, which avoids most of aforementioned problems. Singlet oxygen is a highly reactive molecule against which there has been no development of countermeasures in biological systems besides provision of quenching molecules inside the cells. Therefore there is also little to no risk of resistance development when using PDI against microorganisms. Easy availability of light and oxygen in many of the potential target sites are just some advantages of PDI.
Even though much less often associated with maladies, fungi can be similarly harmful. Due to their asexual life cycle and sporulation they are more robust against changes in environmental conditions. Their high robustness requires highly toxic biocides which in turn come with the risk of damaging the environment. Even seemingly, harmless microorganisms like algae and cyanobacteria show increasing tendencies to harm humans directly or indirectly and in symbiosis with fungi, they form even more resistant lichen, which pose new challenges concerning their removal.
Biofilms on outdoor walls cause aesthetic problems and economic damage. Therefore, wall colors are often mixed with conventional biocides. However, biocides are increasingly designed towards higher efficacy while remaining environmentally persistent. This combination implies serious toxicity problems for all living organisms and whole ecosystems. Thus, an eco-friendly and non-harmful to human health alternative to conventional biocides in wall color is strongly recommended. The well-known photosensitizer TMPyP was used as an additive in commercially available facade paint. The generation of singlet molecular oxygen was shown using time resolved 2D measurements of the singlet oxygen luminescence. The photodynamic activity of the photosensitizer in the facade paint was demonstrated by phototoxicity tests with defined mold fungi and a mixture of microorganisms harvested from native outdoor biofilms as model organisms.
Phototrophic biofilms are an important factor in biofouling and biodeterioration of building materials. The aim of this study was to examine the potential of PDI through generation of singlet oxygen as a possible alternative to biocides preventing the growth and formation of biofilms of these phototrophic organisms. Four photosensitizers were investigated according to their photoinhibitive effect on two strains of green algae using visible light for photoexcitation. The two anionic photosensitizers showed no significant phototoxicity whereas the two cationic photosensitizers caused a drastic reduction of biomass on a short time scale and also displayed long term inhibition of algae growth. Furthermore, to investigate the intrinsic mechanisms of PDI we studied the singlet oxygen luminescence kinetics - a method that proved to be a very effective approach towards understanding mechanisms on a cellular level. Here we present the first two-dimensional measurement of singlet oxygen kinetics in phototrophic microorganisms on surfaces during PDI. We established a system of reproducible algae samples on surfaces, incubated with two different cationic, antimicrobial potent photosensitizers. Fluorescence microscopy images indicate that one photosensitizer localizes inside the green algae while the other accumulates along the outer algae cell wall. A newly developed setup allows for the measurement of singlet oxygen luminescence on the green algae sample surfaces over several days. The kinetics of the singlet oxygen luminescence of both photosensitizers show different developments and a distinct change over time, corresponding with the differences in their localization as well as their photosensitization potential. While the complexity of the signal reveals a challenge for the future, this study incontrovertibly marks a crucial, inevitable step in the investigation of photodynamic inactivation of biofilms: it shows the feasibility of using the singlet oxygen luminescence kinetics to investigate photodynamic effects on surfaces and thus opens a field for numerous investigations.

Figure 1