The BIOSURFING project: New-to-nature biosurfactants by metabolic engineering: production and application October 2011 – September 2015 KBBE.2011.3.3-03: Cellular, metabolic and genetic engineering for novel compounds Biosurfactants ecological alternative to traditional chemically produced detergents good skin compatibility low toxicity good biodegradability 100% plant based produced via natural production process by yeast

Ecological alternative for petro-chemical surfactants BIOsurfactants Surfactants or detergent-like molecules are applied in a wide range of industries and products: cleaning, personal care, cosmetics, agriculture, oil recovery, paper industry, etc. Hence, they are produced in big amounts: about 18 million tons annually and Europe is estimated to be the largest consumer. As about 50% is used in normal households for laundry and cleaning, a large volume of surfactants ends up in the environment or wastewater after use, resulting in a huge ecological load. In addition, most currently used surfactants are still produced from petroleum, a limited resource. Biosurfactants on the other hand offer a worthy alternative to their chemical derived counterparts with equal performance. They are produced from plant renewable resources by a biological process and offer clear ecological advantages such as low ecotoxicity and good biodegradability. Why does the EU invest? The European Union has a lead position in terms of volume and revenue of biosurfactants and is therefore investing in research on these biobased molecules. Indeed, biosurfactants offer great opportunities, but the current lack of diversity limits their penetration in a broad range of applications; biosurfactants are applied, but only in specific niche products. Therefore a consortium of European academic and industrial partners are working together to develop new types of biosurfactants. By developing a wider range of biosurfactants and evaluating their use in various applications (cleaning, cosmetics, medical and nanoscience), the ultimate goal of the project is to get biosurfactants to the supermarket shelf in day-to-day products available for every EU-citizen. New biosurfactants Broader applicability Greener society

Introduction to the BIOSURFING project A yeast living in bumblebee nests was found to be a very efficient biosurfactant producer. We could identify the key steps in this biological process and thanks to this knowledge we created new types of biosurfactants by using metabolically engineered yeasts. For each target molecule, a specific production process was developed and scale-up from a small laboratory synthesis to a bigger production was conducted. In a third phase, the molecules were evaluated for various applications: cleaning, cosmetics, medics and nanoscience. Our expertise The project thus covers the whole innovation chain from basic research to production and application development. To achieve this goal, a complementary consortium of European academic and industrial partners (including a large participation of small and medium enterprises) has been formed that covers the whole range of required expertises. The individual partners are present at the back of this paper.

Our project results 1 At Ghent University (Belgium) several new yeast strains were created starting from the bumblebee yeast, each producing a novel kind of biosurfactant. Some of these strains were not as performant as the original one or required different treatments. These yeast were investigated in a fundamental way to understand why. The strains were transferred to the Bio Base Europe Pilot Plant where a specific green production process was developed for each product : locally grown renewable ingredients are used water and green solvents are applied low carbon footprint Long term and stable storage of these biological products requires processing to a dry product, whereas concentrated solutions can be the future end-product once a supply chain is operational. Next, the novel biosurfactants were tested by the application partners: Ecover tested the new biosurfactants on their properties to be incorporated in cleaning and laundry products. Aspects like water solubility, foam, surface tension and aquatic toxicity were evaluated during the project. work will be conducted for finding the correct formulation and conditions for optimal performance. Furthermore, Werner & Mertz evaluated the biosurfactants in several cleaners and compared them with commercially available surfactants. The molecules did perform, but could not outcompete conventional plant-based surfactants in the tested conditions. Further work

Our project results 2 The company Cosmetic did some extended testing regarding applications in cosmetics. The new lactonic biosurfuctant was found to be a good active ingredient for mild deodorant and acne treatment products. More specifically it kills Corynebacterium Xerosis (malodour causing   The work performed at Ulster University identified that at low concentrations, biosurfactants constrain the growth of several clinically relevant pathogenic bacteria.  Furthermore, we found that minor amounts of biosurfactants can lower the concentration of certain antibiotics required to kill bacteria.  These characteristics are extremely useful in terms of formulating new treatments for chronic wounds or surgical site infections, particularly in regard to the challenge of emerging antibiotic resistance. Our findings have also demonstrated the pharmaco-therapeutic capabilities of a specific novel biosurfactant against bowel cancer in pre-clinical testing. The molecules have shown ability to specifically target and inflict toxic effects against the harmful cancer cells leaving the normal healthy cells alive and thriving. causing bacterium of the arm pit) and Propionbacterium acnes (the acne causing agent). One biosurfactant displayed good solubilising properties. Solubilisers are commonly used in various applications such as personal care cosmetics to dissolve the perfume or other oily ingredients and to give transparent solutions.

Our project results 3 Future The UPMC University wants to understand the behavior in water of the new biosurfactants. Detergents form nanoscale objects in water (diameter of about 0.000000005 meters) lying at the basis of the cleaning process. Compared to classical detergents, the tailored biosurfactants have a unique composition, which has a high impact on the shape and dimension of the structures they form in water. On this basis, UPMC has: demonstrated that the different novel biosurfactants can form a broad variety of nanoscale objects: globules, fibers, infinite layers, hollow beads, etc. used the most powerful tools (Transmission Electron Microscopes and X-ray radiation using Europe’s most powerful Synchrotrons) to precisely describe the nanoscale objects developed new materials using the nanoscale assemblies, such as porous silica supports and highly stable magnetic nanoparticles. Future Thanks to the Biosurfing project, several novel biosurfactants were generated and their potential in various applications was evaluated. We feel that there is much more to be discovered and tested, and that a huge potential lies within these innovative biological derived compounds. Hence, our research will continue also after the project end date and the project partners will keep in contact and continue to exchange information. Image credit: S. Abel, E. Karakas (CEA, France)

Project partnership More information can be found on www.biosurfing.ugent.be Ghent University Belgium (coordinator) Cellectis SA France University of Ulster United Kingdom Bio Base Europe Pilot Plant VZW Belgium Werner & Mertz GmbH Germany Cosmetic sp Greece Ecover Belgium Université Pierre et Marie Curie France

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