1. Reaction Engineering

2. Model to describe what is going on in a Bio-reactor
Mass balance : depentend on reactor type -> S, P, X
Growth Kinetics: -> Monod model (substrate depleting model)
-> Describes what happens in the reactor in steady state (constant conditions)
1. Mass Ballance: In – Out + Reaction = Accumulation
Biomass: FX0 - FX + ∫r dV = dn/dt dn/dt = d(XV)/dt
r = dX/dt = µ X
dn/dt=V (dX/dt) + X (dV/dt)
2. Monod Kinetics:
3. Steady state: dX/dt = 0 (NOT for Batch reactor!!!)

3. Continuous culture Fin = Fout ≠ 0 V = const.
Control:
Concentration of a limiting nutrient
Dilution rate
-> both influences X and P
steady state = cell number, nutrient status remain constant
-> Chemostat

5. Continuous culture: the chemostat
1. Concentration of a limiting nutrient
2. Dilution rate
Results from a batch culture
Monod Kinetics applies!!!
D is dilution rate
F is flow rate
V is volume
Substrate depletion kinetics !!

6. Chemostat: CSTR for Microbial GrowthCV
V = const.
Fin = Fout ≠ 0
Mass Balance: In – Out Reaction = Accumulation
Math: FX FX  r V = dX/dt V
Rearrange: F/V •(X0 –X) + r = dX/dt
Output
Growth

7. Chemostat: CFSTR for Microbial Growth
Take limits as X and t  0
F/V •(X0 –X) + r =
Substitute exponential growth equation for “r”
Set X0 = 0 (no influent cells)
Make steady state (SS) assumption
(no net accumulation or depletion):
 Let F/V = D = dilution rate
Rearrange:
D = m  

9. Determination of Monod Parameters
Cell Growth in Ideal Chemostat
In Chemostat: µg=D, varying D obtains D~S
Washed out: If D is set at a value greater than µm (D > µm),
the culture cannot reproduce quickly enough to maintain itself.
µm = 0.2 hr-1
Chemostat technique: reliable, constant environment, operation may be difficult.

18. Fed batch fermentation
-> In batch reactor, S and X are high. No transport of S or X and no control on µ.
-> In chemostat, S and X are low. Transport of S or X and control on µ.
-> In fed batch reactor. Substrate transport in, not out. No biomass transport.
Why fed batch?
Low S  no toxicity / osmotic problem
High X  high P  easier downstream processing
Control of µ?

19. Fed batch fermentation
Start feeding S0 S
Feeding phase under substrate limited conditions
S = 1 – 50 mg/l.
Batch phase
S0  5000 – mg/l
time
In substrate limited feeding phase, S is very low. Thus, one can use the pseudo steady state condition for substrate mass balance
-> Useful for Antibiotic fermentation
-> to overcome substrate inhibition!!

21. Mass Balance: In – Out + Reaction = Accumulation r = dX/dt = µ X
Biomass: FX0 - FX + r V = dn/dt dn/dt = d(XV)/dt

22. Fed batch Substrate balance – no outflow (Fcout = 0), sterile feed
St = SV and Xt = XV (mass of substrate or cells in reactor at a given time)
S0 = substrate in feed stream
substrate in
substrate
consumed
Substrate
balance
no substrate out (Flow out = 0)
Cell
balance

23. Fed-batch Cell balance – sterile feed
This can be a steady state reactor if substrate is consumed as fast as it enters (quasi-steady-state).
Then dX/dt = 0 and μ = D, like in a chemostat.
Recall, D = F / V

25. Fed batch What this means
the total amount of cells in the reactor increases with time
-> with increasing V
dilution rate and μ decrease with time in fed batch culture
Since μ = D, the growth rate is controlled by the dilution rate.

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

28. 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

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

30. 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

31. 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

32. 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)

33. Minibioreactors Test tubes: -> useful for developing inoculums
-> screening
-> volumne 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

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

35. 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

36. 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

37. 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

38. 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

39. 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 !!!)

40. 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

41. 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

42. Minibioreactors
NanoLiterBioReactor (NLBR):

43. Minibioreactors
NanoLiterBioReactor (NLBR):

45. 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 !!!

46. 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

47. 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

49. Class Exercise Problem 6.17
E. coli is cultivated in continuous culture under aerobic conditions with glucose limitation. When the system is operated at D= 0.2 hr-1, determine the effluent glucose and biomass concentrations assuming Monod kinetics (S0 = 5 g/l, mm= 0.25 hr-1 , KS = 100 mg/L, Y x/s = 0.4 g/g)

52. Class Exercise – 9.4
Penicillin is produced in a fed-batch culture with the intermittent addition of glucose solution to the culture medium. The initial culture volume at quasi-steady state is V0= 500 L, and the glucose containing nutrient solution is added with a flow rate of F = 50 L/h. X0 = 20 g/L, S0 = 300 g/L, mm = 0.2 h-1, Ks = 0.5 g/L and Y x/s= 0.3 g/g
Determine culture volume at t = 10 h
Determine concentration of glucose at t = 10 h
Determine the concentration and total amount of cells at t = 10 h
If qp = 0.05 g product.g cells h and P0 = 0.1 g/L, determine the product concentration at t = 10 h