1.

Mass balance : depentend on reactor type -> S, P, X

2. 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 = X
dn/dt=V (dX/dt) + X (dV/dt)
max S
2. Monod Kinetics:
Ks S
3. Steady state: dX/dt = 0 (NOT for Batch reactor!!!)
 Fin = Fout 0
V = const.

Control:
1. Concentration of a
limiting nutrient
2. Dilution rate

-> both influences X dS

0
and P dt
dX
steady state = cell 0
number, nutrient dt
status remain
constant

-> Chemostat
 1. Concentration of a limiting nutrient 2. Dilution rate

Results from a batch culture

Monod Kinetics applies!!!

F D is dilution rate
D F is flow rate
Substrate depletion kinetics !! V V is volume
 V = const.
CV Fin = Fout 0

Mass Balance: In Out + Reaction = Accumulation

Math: FX0 - FX + rV = dX/dt V

Rearrange: F/V (X0 X) + r = dX/dt

Output Growth
 Take limits as X and t 0

F/V (X0 X) + r =
dX
dt dX
Substitute exponential growth equation for r
X r
dt
Set X0 = 0 (no influent cells)
Make steady state (SS) assumption F
(no net accumulation or depletion): dX D
Let F/V = D = dilution rate
0 V
dt
Rearrange:

F
V
X X F
V
D=
 Cell Growth in Ideal Chemostat

In Chemostat: g=D, varying D obtains D~S m S

g D
KS S
1 K 1 1
S ( Lineweaver - Burk)
D m S m

4 0.3
DX
3.5
0.25
3 Washed out: If D is set at
0.2

DX (g/L-hr)
2.5 a value greater than m (D
S, X (g/L)

m = 0.2 hr-1 X S
2 0.15 > m),
1.5 the culture cannot
0.1
1 reproduce quickly enough
0.5
0.05 to maintain itself.
0 0
0 0.05 0.1 0.15 0.2 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 .
-> In chemostat, S and X are low. Transport of S or X
and control on .
-> 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 ?
 Start feeding

S0 Feeding phase under substrate

limited conditions
S Batch phase
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!!
Mass Balance: In Out + Reaction = Accumulation r = dX/dt = X
0
Biomass: FX0 - FX + r V = dn/dt dn/dt = d(XV)/dt
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 substrate
in consumed

Substrate dS t
X t
no substrate out
balance FS0 (Flow out = 0)

dt YX / S
Cell t
dX
balance X t

dt
Cell balance sterile feed

rfi X
KS D
dX S
( D) X m D
dt

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 = D, like in a chemostat.
dX
Recall, D = F / V 0
dt
 X X FYX / SS0 t
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 decrease with time in fed batch culture

Since = 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 l 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 l
-> 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 l
-> 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 m in diameter
Volumne: 20 l
Perfusion Grid (PG-NBR) Multi trap (MT-NBR)
Similar Volumne larger Volumne
Incorporated sieve Incorporated sieve
With openings 3-8 m Opening similar
-> small traps for cells -> multi trap system

-> Seeding was necessary (Introduction of cells into chamber)

-> 30 m 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 Microchannel height: 20 m (animal cells are smaller than
-> air exchange possible 15 m in diameter)
-> no liquide (medium) can leak out -> If channel larger than 5 m in diameter -> hydrophilic
-> if channel smalles than 5 m 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 hr-
1 , 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 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, 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