Production of lipopeptides by Bacillus subtilis in a
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 1 st Meeting BIBR 20 th December 2013
Context of the study White Biotechnologies • Environmental • Food • Pharmaceutical • Energy Microorganisms Product and services • Eco friendly • Competitive • Efficient
stress Sc al e- up Substrate feed zone Cell sensitivity Lower productivities Inhibitor byproducts Need bioreactor best suited for microbial physiology!
Environmental applications biofilm reactor • Organic or inert support • persistence of microbial system Qureshi, 2005. Microbial Cell Factories • bioconversion Other applications… • microbial catalysis metabolites (low to medium added value) • fine chemicals But… • small-scale system • low specific area
Objectives 750 m²/m³ Air supply Rosche, 2009. Trends in Biotechnology Liquid phase Tank of 20 L Mono-species biofilm reactor HAV biomolecules
Bacillus subtilis Lipopeptides (surface active and antibiotic properties) Performance of excretion Free biomass Attached biomass (Biofilm) VS Stirred tank bioreactor Biofilm reactor
Macroscale (Mass balance) Mesoscale (X-ray tomography) Microscale (Biofilm composition)
Results • Characterisation of the biofilm reactor Macroscale Mesoscale Microscale • Comparison of lipopeptides profiles Qualitative Quantitative
Macroscale : Mass balance After 72 hours of fermentation 94 % Adhesion and biofilm growth
Mesoscale approach : X-ray tomography X-ray detector Rotational motion X-ray source Data and image processing Packing (white pixels) Void (black pixels)
Mesoscale approach : X-ray tomography
Mesoscale approach : X-ray tomography Biofilm (white pixels) Packing (gray pixels) Void (black pixels) 17 cm
Bottom (25%) Top (5%)
Microscale : Biofilm composition • High water content EPS hydration, cell osmosis and transport of nutrients • Large part of C & N required for matrix synthesis
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
Lipopeptides profiles : Qualitative VS Lipopeptides profile • Surfactin STR (stirred tank reactor) • Fengycin • Iturin Bf. R (biofilm reactor) Surfactin 1, 25 times greater in the biofilm reactor Fengycin and iturin have lower contents in the biofilm reactor
Lipopeptides profiles : Quantitative 350 mg / L 280 mg / L 20 % of the total amount of surfactin remains trapped in the biofilm matrix ! mg surfactin / g of dry biomass STR Bf. R 22, 8± 2, 8 37, 9± 2, 2
Biofilm reactor Cellular state Sessile Downstream process operations Planktonic ≠ secretion profile Lipopeptides Technological view point Stirred tank bioreactor No foam formation Simple Anti-foaming agent is essential ! Complex, expensive However…. Inhomogeneous biofilm distribution and clogging areas bad mass transfer of metabolites/nutrients inside the biofilm matrix
Conclusion • 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 !!!! Perspectives • improve liquid distribution to control thickness of the biofilm • stacking of several packings for process intensification
- Slides: 19