1. Production of lipopeptides by Bacillus subtilis in a metal packing biofilm reactor and analysis by X-ray tomography Quentin Zune (Thesis funded by FRIA) Promotor : Frank Delvigne 1st Meeting BIBR 20th December 2013

2. Context of the study Environmental Food Pharmaceutical Energy Microorganisms White Biotechnologies Product and services Eco friendly Competitive Efficient

3. Scale-up stress Inhibitor by- products Cell sensitivity Substrate feed zone Need  bioreactor best suited for microbial physiology! Lower productivities

4. Qureshi, 2005. Microbial Cell Factories Environmental applications  biofilm reactor Organic or inert support persistence of microbial system bioconversion Other applications… microbial catalysis  metabolites (low to medium added value) fine chemicals But… small-scale system low specific area

5. Objectives Rosche, 2009. Trends in Biotechnology Air supply Liquid phase Mono-species biofilm reactor  HAV biomolecules 750 m²/m³ Tank of 20L

6. VS Biofilm reactor Stirred tank bioreactor Bacillus subtilis Lipopeptides  (surface active and antibiotic properties) Performance of excretion Free biomass Attached biomass (Biofilm)

7. Macroscale (Mass balance) Mesoscale (X-ray tomography) Microscale (Biofilm composition)

8. Results Characterisation of the biofilm reactor  Macroscale  Mesoscale  Microscale Comparison of lipopeptides profiles  Qualitative  Quantitative

9. Macroscale : Mass balance 94 % After 72 hours of fermentation Adhesion and biofilm growth

10. Mesoscale approach : X-ray tomography Data and image processing X-ray source X-ray detector Rotational motion Packing (white pixels) Void (black pixels)

11. Mesoscale approach : X-ray tomography

12. 17 cm Biofilm (white pixels) Packing (gray pixels) Void (black pixels) Mesoscale approach : X-ray tomography

13. Top (5%) Bottom (25%)

14. Microscale : Biofilm composition High water content  EPS hydration, cell osmosis and transport of nutrients Large part of C & N required for matrix synthesis

15. Lipopeptides profiles : Qualitative Biosurfactant  economic interest Several functions in the biofilm : - surface colonization, - diffusion of nutriments, - inducer of matrix synthesis, etc. 3 families of lipopeptides - surfactin - fengycin - iturin

16. Lipopeptides profiles : Qualitative VS BfR (biofilm reactor) STR (stirred tank reactor) Lipopeptides profile Surfactin Fengycin Iturin Surfactin 1,25 times greater in the biofilm reactor Fengycin and iturin have lower contents in the biofilm reactor

17. Lipopeptides profiles : Quantitative 350 mg / L 280 mg / L  20 % of the total amount of surfactin remains trapped in the biofilm matrix ! STRBfR mg surfactin / g of dry biomass 22,8±2,837,9±2,2

18. Cellular stateSessilePlanktonic ≠ secretion profile Technological view point No foam formation Anti-foaming agent is essential ! Downstream process operations Lipopeptides Simple Complex, expensive However…. Inhomogeneous biofilm distribution and clogging areas bad mass transfer of metabolites/nutrients inside the biofilm matrix Biofilm reactor Stirred tank bioreactor

19. Conclusion improve liquid distribution to control thickness of the biofilm stacking of several packings for process intensification Perspectives Metal structured packing  biofilm formation X-ray tomography  relevant tool for biofilm monitoring Conical distribution and clogging of the biofilm Better surfactin production VS submerged culture in STR Technological progress  no foam formation !!!!