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 !!!!