Reaction Engineering

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Reaction Engineering
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Model to describe what is going on in a
Bio-reactor

• Mass balance depentend on reactor type -gt S, P,
  X
• Growth Kinetics -gt Monod model (substrate
  depleting model)
• -gt 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/dtV (dX/dt) X
(dV/dt) 2. Monod Kinetics 3. Steady state
dX/dt 0 (NOT for Batch reactor!!!)
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Continuous culture
Fin Fout ? 0 V const.

• Control
• Concentration of a limiting nutrient
• Dilution rate
• -gt both influences X and P

steady state cell number, nutrient status
remain constant -gt Chemostat
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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 !!
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Chemostat CSTR for Microbial Growth
V const. Fin Fout ? 0
Growth
Output
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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
?
?
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Determination of Monod Parameters
Cell Growth in Ideal Chemostat
In Chemostat µgD, varying D obtains DS
Washed out If D is set at a value greater than
µm (D gt µm), the culture cannot reproduce quickly
enough to maintain itself.
µm 0.2 hr-1
Chemostat technique reliable, constant
environment, operation may be difficult.
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Fed batch fermentation

• -gt In batch reactor, S and X are high. No
  transport of S or X and no control on µ.
• -gt In chemostat, S and X are low. Transport of S
  or X and control on µ.
• -gt 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?

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Fed batch fermentation
Start feeding
S0 S
Feeding phase under substrate limited
conditions S 1 50 mg/l.
Batch phase
S0 ? 5000 20000 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 -gt Useful
for Antibiotic fermentation -gt to overcome
substrate inhibition!!
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Mass Balance In Out Reaction
Accumulation r dX/dt µ X Biomass
FX0 - FX r V dn/dt dn/dt
d(XV)/dt
0
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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

no substrate out (Flow out 0)
Substrate balance
Cell balance
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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

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Fed batch

• What this means
• the total amount of cells in the reactor
  increases with time
• -gt 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.

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Minibioreactors
-gt Volumes below 100 ml Characterized by -gt
area of application -gt mass transfer -gt mixing
characteristics
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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

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Minibioreactors

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

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Minibioreactors
Shaking flasks -gt easy to handle -gt low
price -gt volumne 25 ml 5 L (filled with medium
20 of
volumne) -gt available with integrated sensors
(O2, pH) -gt limitation O2 limitation
(aeration) -gt during growth
improved by 1. baffled flasks

2. membranes instead of cotton
-gt during sampling

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Minibioreactors
Microtiter plates -gt large number of parallel
miniature reactors -gt automation using robots -gt
6, 12, 24, 48, 96, 384, 1536 well plates -gt
volumne from 25 µl 5 ml -gt 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
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Minibioreactors
Microtiter plates -gt Problems - O2 limitation
(aeration) -gt faster shaking -gt contamination
- cross-contamination
- evaporation -gt close with membranes
- sampling (small volumne -gt
only micro analytical methods
stop shaking disturbs the
respiration)

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Minibioreactors
Test tubes -gt useful for developing
inoculums -gt screening -gt volumne 2 -25 ml (20
filled with medium) -gt simple and low costs -gt O2
transfer rate low -gt usually no online monitoring
(pH and O2) -gt interruption of shaking during
sampling
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Minibioreactors
Stirred Systems -gt homogeneous environment -gt
sampling, online monitoring, control possible
without disturbance of culture -gt increased
mixing (stirring) mass transfer (gassing rate)
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Minibioreactors
Stirred Systems Stirred Minibioreactor -gt T,
pH, dissolved O2 can be controlled -gt Volumne
from 50 ml 300 ml -gt small medium
requirenments -gt low costs (isotope labeling)

-gt good for research
-gt good for continous
cultivation -gt Limitation - system expensive
due to minimization (control elements)
- not good for high-throughput
applications
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Minibioreactors
Stirred Systems Spinner flask -gt designed to
grow animal cells -gt high price instrument -gt
shaft containing a magnet for stirring -gt
shearing forces can be too big -gt side arms for
inoculation, sampling, medium inlet, outlet, ph
probe, air (O2) inlet, air outlet -gt
continous reading of pH and O2 possible
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Minibioreactors
Special Devices Cuvette based microreactor
-gt optical sensors (measuring online pH, OD,
O2) -gt disposable -gt volumne 4 ml -gt air
inlet/outlet -gt magnet bead -gt stirring -gt
similar performance as a 1 L batch reactor
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Minibioreactors
Special Devices Miniature bioreactor with
integrated membrane for MS measurement -gt
custom made -gt expensive -gt a few ml -gt online
analysis of H2, CH4, O2, N2, CO2, and many
other products, substrate,... -gt used to follow
respiratory dynamics of culture (isotope
labeling) -gt stirred vessel with control of T,
O2, pH -gt MS measurements within a few seconds to
minutes -gt continous detection -gt fast kinetic
measurements, metabolic studies
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Minibioreactors
Special Devices Microbioreactor -gt Vessel 5
mm diameter round chamber -gt Really small working
volumne -gt 5 µl -gt integrated optical sensors
for OD, O2, pH -gt made out of polydimethylsiloxane
(PDMS) -gt transparent (optical
measurements), permeable for gases (aeration) -gt
E. coli sucessfully grown -gt batch and continous
cultures possible -gt similar profile as 500 ml
batch reactors -gt limitation sampling (small
volumne -gt analytical methods !!!)
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Minibioreactors
NanoLiterBioReactor (NLBR) -gt used for growing
up to several 100 mammalien cells -gt culture
volumne around 20 µl -gt online control of O2, pH,
T -gt culture chamber with inlet/outlet ports
(microfluidic systems) -gt manufactured by
soft-lithography techniques -gt made out of
polydimethylsiloxane (PDMS) -gt
transparent (optical measurements), permeable for
gases (aeration) -gt direct monitoring of culture
condition -gt PDMS is transparent -gt
flourescence microscope -gt limitation batch
culture very difficult-gt too small volumne
-gt
suffers from nutrient limitation -gt But in
principle system allows -gt batch, fed-batch,
continous
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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 -gt small traps for cells
Multi trap (MT-NBR) larger Volumne Incorporated
sieve Opening similar -gt multi trap system
-gt Seeding was necessary (Introduction of cells
into chamber) -gt 30 µm filtration necessary
-gt to prevent clogging in the chamber (aggregated
cells) -gt Flow rate of medium 5-50 nl/min
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Minibioreactors
NanoLiterBioReactor (NLBR)
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Minibioreactors
NanoLiterBioReactor (NLBR)
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Minibioreactors

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

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Minibioreactors
Micro/Nanofluidic Device for Single cell based
assay
-gt used a microfluidic chip to capture passively
a single cell and have nanoliter injection of a
drug
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Minibioreactors
Micro/Nanofluidic Device for Single cell based
assay
-gt 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) -gt If channel
larger than 5 µm in diameter -gt hydrophilic -gt if
channel smalles than 5 µm in diameter -gt
hydrophobic
Gray area is hydrophobic -gt air exchange
possible -gt no liquide (medium) can leak out
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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)

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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