Minibioreactors

-> Volumes below 100 ml

Characterized by:

-> area of application-> mass transfer-> mixing characteristics

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

Minibioreactors

- shake-flasks- microtiter plates- test tubes

- stirred bioreactors

- special reactors

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

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

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)

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

Minibioreactors

Stirred Systems:

-> homogeneous environment

-> sampling, online monitoring,

control possible without disturbance of culture

-> increased mixing (stirring) + mass transfer (gassing rate)

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

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

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

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

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 !!!)

MinibioreactorsNanoLiterBioReactor (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

MinibioreactorsNanoLiterBioReactor (NLBR):

Circular with central post (CP-NBR)Chamber: 825 μm in diameterVolumne: 20 μl

Perfusion Grid (PG-NBR)Similar VolumneIncorporated sieveWith openings 3-8 μm-> small traps for cells

Multi trap (MT-NBR)larger VolumneIncorporated sieveOpening 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

MinibioreactorsNanoLiterBioReactor (NLBR):

MinibioreactorsNanoLiterBioReactor (NLBR):

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 !!!

MinibioreactorsMicro/Nanofluidic Device for Single cell based assay:

-> used a microfluidic chip to capture passively a single cell and have nanoliter injection of a drug

MinibioreactorsMicro/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