Innovative Minibioreactors for Optimized Bioprocesses and Research

1. Reaction Engineering

2. Minibioreactors -> Volumes below 100 ml Characterized by: -> area of application -> mass transfer -> mixing characteristics

3. Minibioreactors • Why do we want to scale down ? • - Parallelization (optimization, screening) • automatization • cost reduction • What can you optimize? • Biocatalyst (organism) design • medium (growth conditions) design • process design

4. Minibioreactors • shake-flasks • microtiter plates • test tubes • stirred bioreactors • special reactors

5. Minibioreactors Shaking flasks: -> easy to handle -> low price -> volumne 25 ml – 5 L (filled with medium 20% of volumne) -> available with integrated sensors (O2, pH) -> limitation: O2 limitation (aeration) -> during growth (improved by 1. baffled flasks 2. membranes instead of cotton -> during sampling

6. Minibioreactors Microtiter plates: -> large number of parallel + miniature reactors -> automation using robots -> 6, 12, 24, 48, 96, 384, 1536 well plates -> volumne from 25 μl – 5 ml -> integrated O2 sensor available Increased throughput rates allow applications: - screening for metabolites, drugs, new biocatalysts (enzymes) - cultivation of clone libraries - expression studies of recombinant clones - media optimization and strain development

7. Minibioreactors Microtiter plates: -> Problems: - O2 limitation (aeration) -> faster shaking -> contamination - cross-contamination - evaporation -> close with membranes - sampling (small volumne -> only micro analytical methods + stop shaking disturbs the respiration)

8. Minibioreactors Test tubes: -> useful for developing inoculums -> screening -> volumne 2 -25 ml (20% filled with medium) -> simple and low costs -> O2 transfer rate low -> usually no online monitoring (pH and O2) -> interruption of shaking during sampling

9. Minibioreactors Stirred Systems: -> homogeneous environment -> sampling, online monitoring, control possible without disturbance of culture -> increased mixing (stirring) + mass transfer (gassing rate)

10. Minibioreactors Stirred Systems – Stirred Minibioreactor -> T, pH, dissolved O2 can be controlled -> Volumne from 50 ml – 300 ml -> small medium requirenments -> low costs (isotope labeling) -> good for research -> good for continous cultivation -> Limitation: - system expensive due to minimization (control elements) - not good for high-throughput applications

11. Minibioreactors Stirred Systems – Spinner flask -> designed to grow animal cells -> high price instrument -> shaft containing a magnet for stirring -> shearing forces can be too big -> side arms for inoculation, sampling, medium inlet, outlet, ph probe, air (O2) inlet, air outlet -> continous reading of pH and O2 possible

12. Minibioreactors Special Devices – Cuvette based microreactor -> optical sensors (measuring online: pH, OD, O2) -> disposable -> volumne 4 ml -> air inlet/outlet -> magnet bead -> stirring -> similar performance as a 1 L batch reactor

13. Minibioreactors Special Devices – Miniature bioreactor with integrated membrane for MS measurement: -> custom made -> expensive -> a few ml -> online analysis of H2, CH4, O2, N2, CO2, and many other products, substrate,... -> used to follow respiratory dynamics of culture (isotope labeling) -> stirred vessel with control of T, O2, pH -> MS measurements within a few seconds to minutes -> continous detection -> fast kinetic measurements, metabolic studies

14. Minibioreactors Special Devices – Microbioreactor: -> Vessel 5 mm diameter round chamber -> Really small working volumne -> 5 μl -> integrated optical sensors for OD, O2, pH -> made out of polydimethylsiloxane (PDMS) -> transparent (optical measurements), permeable for gases (aeration) -> E. coli sucessfully grown -> batch and continous cultures possible -> similar profile as 500 ml batch reactors -> limitation: sampling (small volumne -> analytical methods !!!)

15. Minibioreactors NanoLiterBioReactor (NLBR): -> used for growing up to several 100 mammalien cells -> culture volumne around 20 μl -> online control of O2, pH, T -> culture chamber with inlet/outlet ports (microfluidic systems) -> manufactured by soft-lithography techniques -> made out of polydimethylsiloxane (PDMS) -> transparent (optical measurements), permeable for gases (aeration) -> direct monitoring of culture condition -> PDMS is transparent -> flourescence microscope -> limitation: batch culture very difficult-> too small volumne -> suffers from nutrient limitation -> But in principle system allows -> batch, fed-batch, continous

16. Minibioreactors NanoLiterBioReactor (NLBR): Circular with central post (CP-NBR) Chamber: 825 μm in diameter Volumne: 20 μl Perfusion Grid (PG-NBR) Similar Volumne Incorporated sieve With openings 3-8 μm -> small traps for cells Multi trap (MT-NBR) larger Volumne Incorporated sieve Opening similar -> multi trap system -> Seeding was necessary (Introduction of cells into chamber) -> 30 μm filtration necessary -> to prevent clogging in the chamber (aggregated cells) -> Flow rate of medium: 5-50 nl/min

17. Minibioreactors NanoLiterBioReactor (NLBR):

18. Minibioreactors NanoLiterBioReactor (NLBR):

20. Minibioreactors • Why do we want micro-and nano reactors? • Applications in: • Molecular biology • Biochemistry • Cell biology • Medical devices • Biosensors • > with the aim to look at single cells !!!

21. Minibioreactors Micro/Nanofluidic Device for Single cell based assay: -> used a microfluidic chip to capture passively a single cell and have nanoliter injection of a drug

22. Minibioreactors Micro/Nanofluidic Device for Single cell based assay: -> used a microfluidic chip to capture passively a single cell and have nanoliter injection of a drug Microchannel height: 20 μm (animal cells are smaller than 15 μm in diameter) -> If channel larger than 5 μm in diameter -> hydrophilic -> if channel smalles than 5 μm in diameter -> hydrophobic Gray area is hydrophobic -> air exchange possible -> no liquide (medium) can leak out