Green process for microbial decontamination based on light and humidity control – GreenDeconta

The GreenDeconta project aimed at developing a system of microbial inactivation based on high power visible light. Treating with a high irradiance, the point was to define if the damages generated by the treatment were similar to those reported when long treatments at lower intensity are applied. Several prototypes of high-power LEDs reactors were conceived during the project. Microscopic tools and flow cytometry are used to characterize the state and the damages of the treated microbial cells. The survival of microorganisms was also determined with subsequent growth on nutritive medium in order to create inactivation mathematical modeling. The efficacy of the light treatments was studied on microorganisms poured on nutritive medium, on pieces of surface commonly found in food industry or on food products. The effect of air humidity, due to its potential effect on light transmission in the air or on microorganisms themselves, was also investigated.

The results of the GreenDeconta project identified the wavelengths around 400 nm were the most lethal for all the microorganisms tested (S. cerevisiae, P. digitatum, B. cereus). Based on this knowledge, a high power LED-based reactor was developed and used to perform rapid treatments. This reactor made it possible to process samples at 385 and / or 405 nm at powers of up to 5000 W / m² for each wavelength. This prototype showed very convincing results in vegetative species with reductions of several log order, but also a reduction in the spore population of B. cereus after treatments of few minutes. The study of the simultaneous application of the two wavelengths did not reveal any synergistic effects. However, this made it possible to specify that the 385 nm wavelength was more effective than that of 405 nm. The cellular mechanisms leading to the death of microorganisms following treatments at 385 and 405 nm were then studied on the yeast S. cerevisiae. Using a cytometric approach, it has been shown that these light treatments induce a significant production of reactive oxygen species. This production is greater at 385 nm than at 405 nm. This explains the greater efficiency of the lower wavelength. The effect of wavelengths was also observed on the loss of membrane integrity with greater permeabilization at 385 nm. The use of mutant yeast strains deleted for enzymes involved in oxidative defense systems has confirmed the role of photo-oxidation in causing cell death. However, the correlation between loss of cultivability and loss of plasma membrane integrity was not complete. Experiments have also shown that the withdrawal of oxygen during treatment leads to cell death, but to a lesser extent. This suggests that other mechanisms unrelated to oxidation are involved in light inactivation. Many microbial contaminations are linked to interfacial microorganisms present on the surface of food or technological surfaces. The resistance of interfacial microorganisms was finally studied after depositing yeasts on coupons representative of technological surfaces. Very short treatments (less than 5 min) allowed complete elimination of S. cerevisiae from all treated surfaces. However, the inactivation rates have been shown to depend on the nature of the surface for shorter treatments. For example, a treatment of 2 min on stainless steel leads to an inactivation of about 4 log while 1.5 log is destroyed on the glass under the same conditions. These differences are explained by surface characteristics such as absorption and reflection of light radiation.

From a fundamental point of view, the knowledge acquired during the GreenDeconta project has allowed a better understanding of the cellular effects of high intensity visible light on microorganisms. The advances made have allowed us to highlight lethal effects that are not exclusively linked to photo-oxidation. These mechanisms will have to be researched to finally describe more precisely the scenario of events leading to cell death during these treatments. The results of the project have also highlighted the applicative potential of high power LED light treatments for surface decontamination. Food technological surfaces could be treated but these treatments could also be applied to surfaces in other environments (public places, hospitals...) Finally, the results of the GreenDeconta project have led to the funding of 3 new projects around this theme. The first is the FEDER Virolux project (2020-2021) in which the effect of the treatments developed is studied on the Norovirus at the origin of viral gastroenteritis. The second is the ANR project ClostAbat (2022-2026) in which one of the tasks is dedicated to the decontamination of surfaces contaminated by pathogenic bacteria such as Clostridium in slaughterhouses. The last one is an Inter-Carnot Qualiment/Plant2Pro project (Effinox, 2022-2025) in which the effects of light treatments on food matrices in the wine and cereal industries will be studied. Two CIFRE thesis projects are also under discussion with the companies NLX (LED lighting solutions for professionals) and STERIXENE (Expert in decontamination by pulsed light and LED).

At the end of the project, one scientific article has been accepted for publication in Applied Mircrobiology and Biotechnology and two other original articles are in the process of being finalized. The first paper deals with the development of an Ultra-High Intensity (UHI) reactor and the proof of efficacy of such treatment on yeasts. The second one will endeavor to describe the activity on more resistant forms. Finally, the last article aimed to describe the knowledge of the inactivation mechanisms.

1. Lang E., Thery T., Peltier C., Colliau F., Adamuz J., Grangeteau C., Dupont S., Beney L. Ultra-High Irradiance (UHI) blue light: Highlighting the potential of a novel LED-based device for short antifungal treatments of food contact surfaces, Applied Microbiology and Biotechnology (In press, doi.org/10.1007/s00253-021-11718-9)
2. Thery T., Grangeteau C., Beney L., Dupont S. Ultra-High Irradiance (UHI) LED inactivation of bacterial and fungal spores (submission scheduled for the first half of 2022 in Food Control)
3. Thery T., Grangeteau C., Beney L., Dupont S. Ultra-high irradiance blue light treatment : kinetics and mechanisms of inactivation (submission scheduled for the first half of 2022 in Frontiers in Microbiology)

On top of having pathogenic effects, the presence of microorganisms on the surface of food raw materials is at the origin of a major loss in agricultural production for end consumer consumption. The main current solution is the massive use of fungicides and bactericides which are directly applied on fruits and vegetables, but also on industrial food process contact surfaces or used to treat wash waters of food raw materials. However, this solution induces major environmental and toxicological issues. Thus, the search for sustainable alternative solutions or technologies has become a major concern.
The GreenDeconta project proposes to develop knowledge on the response of microorganisms (bacteria and fungi) to their exposure to certain wavelengths of the visible light (photo-oxidation) and on the cellular damages induced by such treatments. This knowledge could then be used to develop a reasoned and sustainable process for food raw material and surface decontamination from pathogenic and alteration microbiological flora. The microorganisms studied in the project will be bacteria (Escherichia coli and Bacillus cereus) and fungi (Saccharomyces cerevisiae and Penicillium digitatum). For spore-forming microorganisms, the resistance to light treatments of the vegetative form will be compared with that of spores. The GreenDeconta project is divided into 3 work packages (WP) to allow the identification of the possible application fields of such a technology. WP1 first deals with the identification of the wavelength of the visible light impacting the viability of the microorganisms but also the characterization of the cellular consequences for every wavelength. At the end of this WP, a light reactor using LED technology will be developed in order to treat samples with several wavelengths simultaneously. WP2 will then integrate the air relative humidity as a control parameter during the light treatment. This parameter could increase de decontaminating effect of the light treatments. On the one hand, a decrease in air humidity could affect microorganism physiology, and especially, their resistance to perturbations; on the other hand, a decrease in vapor content in the air, which decreases the light diffusion between the source and the samples, could improve the efficacy of the treatment. WP3 will then model the microbial inactivation for every microorganism of the project. For that, inactivation curves obtained in different conditions (liquid media and on inert or fruit surfaces) will be treated thanks to linear and non-linear models. The ideal model, i.e. presenting an optimal adjustment versus the experimental data, will be selected. These data will be of particular interest for the identification of the possible application fields of light treatments based on the combination of several wavelengths of the visible light. Finally, preliminary trials will be performed on fruits (apples and lemons) which would have beforehand been inoculated with different microorganisms.
This project will develop knowledge on the response and the resistance of microorganisms to photo-oxidation induced by visible light. In an original way, the affected cellular structures will be identified according to the wavelength used. This knowledge will allow the development of a LED reactor combining several wavelengths of the visible light. The impact of the control of air relative humidity on the efficacy of the treatment will also be characterized. The achieved results will allow the Young Researcher, who will coordinate the project, to develop a new research theme within its laboratory. These results will also be at the basis of a future development of a sustainable process for microbial decontamination.