Transcript Reaction Engineering

Fin = Fout ≠ 0
V = const.
Control:
1. Concentration of a
limiting nutrient
2. Dilution rate
-> both influences X
and P
steady state = cell
number, nutrient
status remain
constant
-> Chemostat
dS
0
dt
dX
0
dt
1. Concentration of a limiting nutrient
2. Dilution rate
Results from a batch culture
Monod Kinetics applies!!!
Substrate depletion kinetics !!
F
D
V
D is dilution rate
F is flow rate
V is volume
V = const.
Fin = Fout ≠ 0
CV
Mass Balance:
Math:
Rearrange:
In –
Out
FX0 t -
FX t +
+
F/V •(X0 –X) + r
Output
Growth
Reaction = Accumulation
r V t
= X/t
=
X V
Take limits as X and t  0
F/V •(X0 –X) + r =
dX
dt
Substitute exponential growth equation for “r”
Set X0 = 0 (no influent cells)
Make steady state (SS) assumption
dX
(no net accumulation or depletion):

 Let F/V = D = dilution rate
dt
Rearrange:
F
X  X
V

F

V

dX
 μ X  r
dt
0
F
D
V
D=
Cell Growth in Ideal Chemostat
In Chemostat: µg=D, varying D obtains D~S
g  D 
K 1
1
1
 S

( Lineweaver- Burk)
D m S m
DX
3.5
0.25
0.2
2.5
S
X
2
0.15
1.5
0.1
1
0.05
0.5
0
0
0.05
0.1
0.15
0.2
DX (g/L-hr)
S, X (g/L)
3
µm = 0.2
KS  S
0.3
4
hr-1
m S
Washed out: If D is set at
a value greater than µm (D
> µm),
the culture cannot
reproduce quickly enough
to maintain itself.
0
0.25
D (1/hr)
Chemostat technique: reliable, constant environment, operation may be difficult.
-> 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?
1. Low S  no toxicity / osmotic problem
2. High X  high P  easier downstream processing
3. Control of µ?
Start feeding
S0
S
Batch phase
Feeding phase under substrate
limited conditions
S = 1 – 50 mg/l.
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
-> Useful for Antibiotic fermentation
-> to overcome substrate inhibition!!
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
balance
Cell
balance
substrate
consumed
dS
X
 FS0 
dt
YX / S
t
t
t
dX
t
 X
dt
no substrate out
(Flow out = 0)
Cell balance – sterile feed
rfi  X
dX
 (   D) X
dt
KS D
S
m  D
This can be a steady state reactor if substrate is consumed as
fast as it enters (quasi-steady-state).
dS
0
dt
Then dX/dt = 0 and μ = D, like in a chemostat.
dX
0
Recall, D = F / V
dt
X  X  FYX / SS0t
t
t
0
•What this means
•the total amount of cells in the reactor increases with time
-> dX  0
with increasing V
dt
•dilution rate and μ decrease with time in fed batch culture
•Since μ = D, the growth rate is controlled by the dilution
rate.
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 !!!)
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
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
Minibioreactors
NanoLiterBioReactor (NLBR):
Minibioreactors
NanoLiterBioReactor (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 !!!
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
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
Gray area is hydrophobic
-> air exchange possible
-> no liquide (medium) can leak out
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
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, m= 0.25 hr1 , K = 100 mg/L, Y
S
x/s = 0.4 g/g)
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 quasisteady 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, m = 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