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
Context of the study
• Environmental
• Food
• Pharmaceutical
• Energy
Microorganisms
White Biotechnologi
es
Product and
services
• Eco friendly
• Competitive
• Efficient
Scale-up stres
s
Inhibitor by-products
Cell sensitivity
Substrate feed zone
Need bioreactor best suited for microbial physiology!
Lower productiviti
es
Qureshi, 2005. Microbial Cell Factories
Environmental applications
biofilm reactor
• Organic or inert support
• persistence of microbial system
• bioconversionOther applications…
• microbial catalysis metabolites (low to medium added value)
• fine chemicals
But…
• small-scale system
• low specific area
Objectives
Rosche, 2009. Trends in Biotechnology
Air supply
Liquid phase
Mono-species biofilm reactor HAV biomolecules
750 m²/m³
Tank of 20L
VS
Biofilm reactor
Stirred tank
bioreactor
Bacillus subtilis
Lipopeptides (surface active and antibiotic properties)
Performance of excretion
Free biomass
Attached biomass (Biofilm)
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
94 %
After 72 hours of fermentation
Adhesion and biofilm growth
Mesoscale approach : X-ray tomography
Data and image
processing
X-ray source
X-ray detector
Rotational motion
Packing (white pixels)
Void (black pixels)
Mesoscale approach : X-ray tomography
17 cm
Biofilm (white pixels)
Packing (gray
pixels)
Void (black pixels)
Mesoscale approach : X-ray tomography
Top (5%)
Bottom (25%)
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
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
Lipopeptides profiles : Quantitative
350 mg / L
280 mg / L
20 % of the total amount of surfactin remains trapped in the biofilm matrix !
STR BfRmg surfactin / g of
dry biomass 22,8±2,8 37,9±2,2
Cellular state
Sessile Planktonic
≠ secretion profile
Technological view point
No foam formatio
n
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
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 !!!!