Biomst Ii - Biotechnological Tasks in Microsystem Technology II

1
BioMST II Biotechnological Tasks in

Microsystem Technology II
Felix von Stetten
3 Bioprocess engineering
Lecture 2
2
1.1 & 1.2 Overview
3. Bioprocess engineering
3.1 Introduction
3.2 Bioreactors
3.3 Upstream processes
3.4 Fermentation
3.5 Downstream processes
3.1 Introduction

3
1.1 & 1.2 Overview
3.2 Bioreactors
3.2.1 Structure and function
3.2.2 Bioreactor types
3.2.3 Miniature bioreactors
3.2.4 Balance equations
3.2.5 Ventilation and transportation of oxygen
3.2.6 Power input
3.2.7 Scaling up / Numbering up
3.2.8 Simulation

3.2 Bioreactors

4
Classification of different types of Bioreactors
Surface reactors
Submersed reactors
Solid culture medium (mushroom on horse manure)
Static surface culture
Bed reactors
Membrane reactors
Mechanical (mixers)
Pneumatic
Hydraulic
Batch (step by step)
Fed batch ( feed process)
Continuous
Chemostat (constant inflow of substrate)
Turbidostat (constant turbidity)
Application of energy (mixing)
Mode of operation
Surface
Control of cell growth
3.2 Bioreactors

source: Schwister,
Taschenbuch der
Verfahrenstechnik
5
Bioreactor Requirements
Creation of a large interface (gas/fluid)
High oxygen transfer rates
Homogeneous dispersion of all reactants in the reactor
Ability to stir viscous media (mycelium of fungi!)
Avoid foaming (foam dissolver e.g. silicone oil)
Use of inert materials (no cytotoxicity)
Possibility of sterile process control
Stirrer with low energy consumption and low shear forces
Option of temperature control
Flexibility regarding different process requirements
Ability to scale-up
Possibility of on-line process control
Possibilities of automation
gas bubble

O
2
cells

transmission of oxygen
mycelium of fungi
foaming
3.2 Bioreactors

source: Schwister, Taschenbuch der Verfahrenstechnik
6
Surface reactors
Static surface culture Bed reactors Membrane reactors
Shell Reactor
culture of fungi for citric acid
production 1923
applicable to bacteria and
mammalian cells
1. filling filling
2. draining
Fixed-bed reactor
vinegar production by
inoculation of wood chips
with bacteria
air
cells
medium
membrane
Hollow fiber membrane reactor
Only low shear forces
cultivation of animal cells
high cell density at low volume
3.2 Bioreactors

sources: Schwister, Taschenbuch der Verfahrenstechnik; Storhas Bioverfahrensentwicklung; Schmid, Taschenatlas der Biotechnologie und Gentechnik
exhaust air
collecting space
chip
space
air
mash
vinegar
mash circulation
7
Application of energy in Submersed reactors (I)
Pneumatic
Bubble column
highly distributed
gas bubbles
Airlift reactor
low shear forces
cultivation of animal
cells
low energy
consumption
supply
air
exhaust
air
Mammoth-loop reactor
wastewater treatment
supply air
exhaust
air
supply
air
inner
catalyst
tube
3.2 Bioreactors

8
Application of energy in Submersed reactors (II)
Hydraulic
Immersion jet reactor
motive jet 10 m/s
high turbulences
gassing of highly
stressed wastewater
yeast production
Tube loop reactor
reaction in tube
foam separation in the cyclone

Jet loop reactor
motive jet 20 m/s
wastewater treatment
supply air exhaust
air
exhaust
air
supply
air
exhaust air
supply air
inner
catalyst
tube
cyclone
damage of microorganisms by shear forces
problem of sterilization and sterile process control of external cycle

3.2 Bioreactors

9
Application of energy in Submersed reactors (III)
Mechanical (stirred tank reactors with different stirrers)
Propeller stirrer
Axial flow
For low viscosity media
Disc stirrer
Radial flow
For high viscosity media
Most commonly used bioreactor type
High energy consumption
supply air supply air
deflector
plate
deflector
plate
3.2 Bioreactors

10
Application of energy in Submersed reactors (III)
Mechanical (wave bioreactor, energy supply by luffing)
Careful cultivation of mammalian cells, disposable

http://pecf.epfl.ch/page62178.html
11
Mode of operation of Submersed reactors
Fed-batch (method of feed)
fed-batch reactor
3.2 Bioreactors

Continuous
start of continuous
culture
b
i
o
m
a
s
s

time t
1
Batch (method of operation)
batch reactor (batch tank)
b
i
o
m
a
s
s

time
medium
continuous flow
reactor
substrate
reservoir
production of penicillin with glucose feed
sensor,
e.g. balance
growth curve
feedback
transition region
extended transition
region
static state
product
growth curve
p
e
n
i
c
i
l
l
i
n

c
o
n
c
e
n
t
r
a
t
i
o
n

b
i
o
m
a
s
s

c
o
n
c
e
n
t
r
a
t
i
o
n

g
l
u
c
o
s
e

f
e
e
d

glucose
feed
time
12
Possibilities of control/ regulation of cell growth in continuous
reactors
Chemostat Turbidostat Plug-flow-reactor
Produkt product
medium medium
continuous feed of growth
limiting substrates goal:
extended siege to transition
phase
homogeneous mixing
Stabilized constant cell
concentration (turbidity) by
regulating medium feed
homogeneous mixing
growth phase space-
resolved
flow of plug without
backmixing
suitable for phototrophs

turbidity
sensor
f
e
e
d
b
a
c
k

medium
product
cell feedback
(inoculum)
product
medium
3.2 Bioreactors

13
1.1 & 1.2 Overview
3.2 Bioreactors
3.2.3 Miniature-bioreactors
3.2.6 Power input
3.2.8 Simulation

3.2 Bioreactors

3.2.3 miniature-bioreactors
14
Miniature-bioreactors types (examples)
Shaking flasks
50 500 ml
www.hitec-zang.de
Stirred tank reactors
5-20 ml
www.Fluorometrix.com
Microtiter plates
3 ml, 200 l, 50 l
(6-, 96-, 384-well)
Microtiter plates with
integrated fluidics
Choi et al., Lab Chip, 2007,
7, 550556 | 551

reactor array
5 l 1 ml
www.bioprocessors.com
segmented flow reactor
1 nl - 1 ml
www.raindancetechnologies.
com

commonly used
future
source: J. Betts et al.: Miniature bioreactors. Microbial Cell Factories 2006, 5: 21 www.microbialcellfactories.com/content/5/1/21
3.2 Bioreactors

15
Applications of Miniature-bioreactors
Advantages
Possibility of parallelization
Low substrate consumption
Low space requirement
High-Throughput-Screening (HTS)

Applications
Media development
Strain development
Process optimization

Integrated downstream processing
(currently hardly realizable)
sampling (also individual cells)
product harvest
product purification
product analysis

Miniature bubble column reactor
3.2 Bioreactors

16
Potential savings through micro bioreactors
3.2 Bioreactors

17
Conflict between Parallelization and Process control
p
r
o
c
e
s
s

-
c
o
n
t
r
o
l

a
n
d

-
i
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g
r
a
t
i
o
n
:

p
r
o
c
e
s
s

a
n
a
l
y
s
i
s
t
s

(
p
H
,

p
O
2
,

O
D
)

i
n
d
i
v
i
d
u
a
l

p
r
o
c
e
s
s

r
e
g
u
l
a
r
i
z
a
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i
o
n

d
o
w
n
s
t
r
e
a
m

p
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o
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e
s
s
i
n
g

(
p
r
o
d
u
c
t

r
e
c
o
v
e
r
y
,

p
u
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i
f
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c
a
t
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o
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,

a
n
a
l
y
s
i
s
)

c
o
n
t
r
o
l

&

i
n
t
e
g
r
a
t
i
o
n

parallelization
3.2 Bioreactors

18
Requirements of a Universal micro bioreactor
*
* e.g. microtiter plates compatibility
3.2 Bioreactors

19
Does an universal miniature bioreactor make sense?
Different applications have different requirements

Conclusion: Requirement for systems, adjusted to application.
example of application main requirement
Media screening High Throughput Screening
(parallelization)
Process optimization Process analysis and process
regulation/control
Screening of drug producing
microorganisms
Integrated downstream
processing, adjusted to
microorganisms and agent being
analyzed
3.2 Bioreactors

20
Segmented flow Miniature Bioreactors
Incubation module disc with 3 m
Teflon-capillary tube suitable for 550
compartments
Segmented flow of an aqueous
phase in Tetradecane
ratio 1:5
sample volumes 8 - 300 nl
Example of companies and institutes
source: Vortrge der Jahrestagung Mikrosysteme fr die
Biotechnologie, 21.-23. Juni 2006, Bremen,
AK Mikrosystemtechnik fr die Biotechnologie
3.2 Bioreactors

21
Fluidic Unit Operations
Segmenting/Shaping/formulating Combining
source:
www.raindancetechnologies.com
3.2 Bioreactors

22
Mixing Separating
Fluidic unit operations
source:
3.2 Bioreactors

23
Preserving Storing
source:
3.2 Bioreactors

24
Detecting Sorting
source:
3.2 Bioreactors

25
Analysis of mixing effects in a compartment
Friction between interfaces
results in induced internal-
phase flows
fluid / channel wall
fluid 1 / fluid 2
Particle Imaging Velocimetry for internal-phase flow analysis.
Results: Best mixing effects in curved channels.
source: Dr. Thomas Henkel, IPHT
Jena
3.2 Bioreactors

water water
direction of transportation
tetradecane
26
Example: Optimization of microbial ethanol retrieval
using screening procedures
Use of segmented flow nano bioreactors for process development
Professional Laboratory System
Chip layout
Microorganisms library for secretion of target enzymes.
Incubation library testing different conditions (pH, C/N ratio,
temperature)
NanoReactor Chip
source:
3.2 Bioreactors

27
Micro bioreactor array An example from research
3.2 Bioreactors

28
Best available technology
3.2 Bioreactors

29
Functional principle of the micro bioreactor array
Figure of 4 reactors
cross section I:
Peristaltic oxygenating
mixture
fluid reservoir with
pressure chamber
material: PDMS
Top view:
Optical sensors for pH,
OD, DO (Dissolved
Oxygen)
Peristaltic mixer
Fluid injector
cross section II:
Interface for
compressed air and
system fluids
Peristaltic valves
3.2 Bioreactors

30
Fabrication of the micro bioreactor array out of PDMS
3.2 Bioreactors

31
Interfaces, sensors for OD and fluorescence measurement
Pneumatic feed lines
copper plate
fiber optics for OD excitation
fluorescence sensor (pH, DO) OD (optical density)
LED
optode
Two micro bioreactor modules on thermostat
detection fiber
aperture
aperture
cavity
excitation
fiber
fiber
optics
3.2 Bioreactors

32
Characterization of the micro bioreactor
Which parameters are relevant?
Mixing time
Input power
Oxygen Transfer Rate (OTR), k
L
a-value
Gas content, useable volume (not required for membrane
micro bioreactors)
Bubble flow (not required for membrane micro bioreactors)
Growth curves with relevant organisms
turbidity and biomass respectively
pH-value
DO (Dissolved Oxygen)
Comparison with larger scale (reference reactor)
3.2 Bioreactors

33
Result
Characterization mixing time
Method

Discoloring of a bromthymol blue
solution after injection of acid and
base

3.2 Bioreactors

34
Characterization OTR, k
L
a
Model for stirred-tank reactor Model for membrane micro-bioreactor
OUR C C a k
dt
dC
L
= ) (
*
: OTR (oxygen rate of feed)

k
L
a: mass transfer coefficient
C: dissolved oxygen (DO)
C*: saturation concentration
OUR: oxygen uptake rate (O
2
-Verbrauchsrate)
dt
dC
simplified illustration, for details see publication
( ) OUR
z
C
z D
z t
C
|
.
|
\
|
=
o
o
o
o
o
o
w p
ss L
D
L
K D
Ld
a k
2
1
2
+
=
z: Thickness of PDMS (-d<z<0) and cavity (0<z<L)
respectively
D: Diffusion coefficient of PDMS (p) and water (w)
respectively
K: Ratio of oxygen saturation concentration in water /
PDMS
q: Diffusion enhancement factor by mixing
(dont learn equations, but be able to explain them)
3.2 Bioreactors

35
Characterization OTR, k
L
a
Method
Dynamic gassing
Parameter
Membrane thickness 70
mm
Mixing 40 Hz, 8 psi ( input
power)
Result; k
L
a-value for micro-
membrane bioreactor
k
L
a, measured with mixing
k
L
a, measured without mixing
theoretical (model 1)
theoretical (model 2)
3.2 Bioreactors

q=13
q=0
36
Characterization growth curve
Method
OD measurement (E. coli)

Result

4 L stirred-tank reactor (reference)
pH and DO regulated (DO 50 %)
pH not regulated
3.2 Bioreactors

37
Fermentation under controlled conditions is possible
Cell density similar to stirred-tank reactor (up to 14 g L
-1
)
Array micro-bioreactor eliminates compromise between
throughput and process control
Conclusion
3.2 Bioreactors

38
1.1 & 1.2 Overview
3.2 Bioreactors
3.2.6 Power input
3.2.8 Simulation

3.2 Bioreactors

3.2.4 balance equations
39
Types of balancing
Balance environment
Conservative Laws apply to
Energy (heat)
Impulse
Mass (material)

balances
Heat balance
Impulse balance
Mass balance (substrate,
biomass)

Actions in balancing environment
Transport
Storage
Conversion (reaction)

source: Storhas,
Bioverfahrensentwicklung, S. 209
Balance environment
in out
Drain / Source
Storage
Conversion

Transport
3.2 Bioreactors

40
Mass balance and heat balance
Discontinuous system Continuous system
m
1
m
2
m
Ges
Mass balance
Heat balance
1
m
2
m
A
3
m
4
m
Ges
m m m = +
2 1 4 3 2 1
m m m m m

+ + A = +
1 1
h m
( )
misch
h m 0 , A

( ) H h m h m h m
Ges
= = +
2 2 1 1
2 2
h m
( )
misch Ges
h m 0 ,
1 1
h m
2 2
h m
3 3
h m
4 4
h m
( )
4 4 3 3 2 2 1 1
h m h m h m h m h m + + A = +

source: Kessler, Lebensmittel- und
Bioprocess engineering, S. 31
h: specific enthalpy; H: enthalpy; m=cV; c: concentration
3.2 Bioreactors

41
Enthalpy and Heat capacity
Enthalpy
Heat content at constant
pressure, i.e. at isobaric state
change
symbol H (Heat content)
unit [Joule, J]

Specific Enthalpy

h = H / m [kJ/kg]

h = 0 J/kg for water at 0 C.

Heat capacity
Capability of a body to store
thermal energy of a body

symbol C
unit [J]
Specific heat capacity
Amount of heat quantity, which
raises the temperature of one kg
of a material by 1 K
(at same physical state and
constant pressure)

material c
p
[kJ / kgK]
water 4,2
ice 2,0
water vapor 1,9
polystyrene 1,3
air 1,0
Bioprocess engineering, S. 28
3.2 Bioreactors

42
Calculation of Enthalpy using Temperature and Heat capacity
For liquids, solids and gases at
constant pressure

At the phase change from liquid to
solid (freezing)

At phase change from liquid to gas
(water steam mixture)

0
s
boiling temperature
0 final temperature
0
sch
melting temperature
c
fl
specific heat of liquid

c
f
specific heat of solidified liquid
c
pD
specific heat of steam
r
sch
enthalpy of fusion at 0
sch

(333 kJ/kg for pure ice)
r enthalpy of evaporation at 0
s

(2200 kJ/kg for water at 100 C)
0 = c h
( )
sch fest sch sch fl
c r c h 0 0 0 + =
( )
s pD s fl
c r c h 0 0 0 + =
Bioverfahrenstechnik, S. 29
3.2 Bioreactors

43
Application: Calculation of a mixing temperature
What is the temperature in a continuous mixing system?

Approach:

Explanation:
The mass flow can be calculated using the concentration c and the volume flow
( ), the mass is obtained using mass balance.
Transition may be neglected, in large reactors
Conversions have a dominant impact on the temperature of liquid, in miniature
reactors
Relevance of this approach
Addition of sewage sludge into biogas plant

( )
misch
h m 0 , A

1 1
h m
2 2
h m
3 3
h m
4 4
h m
( )
capacity heat specific :
.
follows, with
;
4 4 3 3 2 2 1 1
4 4 3 3 2 2 1 1
c
c m c m h m h m
c h
h m h m h m h m
misch
0
0
+ = +
=
+ = +

V c m

=
3.2 Bioreactors

44
Balancing for complete mixture
Balance environment: Uniform change of the elements i in the
entire space

Balance

Volumetric rate of product formation r

( )
V r c V c V
dt
dV
c
dt
dc
V
dt
c V d
i
Aus
i
Aus Ein
i
Ein
i
i i
= + =

n
i i
c k r =
reaction term
+ forming
- degrading
Ein
i
Ein
c V
i
Aus
i
Aus
c c V =
V
c
i
k reaction rate constant
n order of reaction
source: Storhas,
3.2 Bioreactors

45
Balance environment: Axial inhomogeneity, Radial homogeneity

balance
Balance equations for spatial inhomogeneity
Ein
i
Ein
c V
Aus
i
Aus
c V
0 = x L x =
dx
x
c u
dx
dc
D
ax

infinitesimal small volume
with axial homogeneity
n
i
i
i
i
c k
dx
dc
D c u
dx
d
dt
dc

|
.
|
\
|
=
u flow velocity in x direction
D diffusion coefficient
diffusive part
convective part
(dont learn equations,
but be able to explain them)
source: Storhas,
3.2 Bioreactors

46
1.1 & 1.2 Overview
3.2 Bioreactors
3.2.6 Power input
3.2.8 Simulation

3.2 Bioreactors

3.2.5 ventilation and transportation of oxygen
47
Importance of Ventilation
Supply the cells with oxygen
Removal of gaseous products (CO
2
,N
2
)
Low solubility of oxygen in water
continuous additional supply required
Solubility of oxygen depends on temperature and pressure

source: Schwister, Verfahrenstechnik

Ventilation of bioreactor with air or oxygen

3.2 Bioreactors

temperature
solubility solubility
48
Gas exchange between Gas bubbles and cell
GB gas bubble
GF gas film
FF liquid film
C* O
2
-concentration in gas bubble
C O
2
-concentration in fluid

source: Schwister, Verfahrenstechnik
O
2
-
c
o
n
c
e
n
t
r
a
t
i
o
n

Gas Bubble
* C
C
3.2 Bioreactors

cell
interface
gas/liquid
interface
gas/cell
liquid
cell
49
Two film model
Gas phase and liquid phase
oxygen transport via convection
Stable boundary on both sites of the gas/liquid interface
Oxygen transport via diffusion
Liquid film
Gas film (10
4
times faster diffusion than in water negligible)

Oxygen diffusion rate through the interface
R
C C
dt
dC
=
*
) (
*
C C a k OTR
dt
dC
L
= =
a k A K
R
L L
1 1
=
=
with
OTR, Oxygen Transfer Rate,
accumalation term

k
L
a volume related oxygen transfer
coefficient
C concentration of dissolved O
2
(DO)

C* O
2
saturation concentration
OUR O
2
uptake rate (O
2
consumption rate)
dt
dC
source: Schwister, Verfahrenstechnik, S. 475
results
R Boundary layer resistance
A gas-fluid interface
K
L
liquid film oxygen transition coefficient
dt
dC
3.2 Bioreactors

OUR C C a k OTR
dt
dC
L
= = ) (
*
in case oxygen is consumed following applies:
50
Method to determine k
L
a value
Dynamic method (without reaction: OUR = 0)
Gradual change in saturation concentration C*

C* calculated with Henrys law
C measure with oxygen sensor

Steady state method ( Accumulation term negligible: )
Sulfite method (oxidation of sulfite to sulfate) oxygen drain

) (
1
*
C C dt
dC
a k
L
=
0 ~
dt
dC
) (
*
C C
OUR
a k
L
=
H
O
k
p
C
2
* =
Co
2+
/ Cu
2+
Na
2
SO
3
+0,5 O
2
Na
2
SO
4
C* O
2
saturation concentration
C concentration of dissolved oxygen
p
O2
oxygen partial pressure in the fluid
k
H
Henry's law constant
OUR O
2
-absorption rate

dt
dC
Accumulation term
sources: http://www.uni-saarland.de/fak8/heinzle/de/teaching/Technische_Chemie_Prakt/kla_shakeflask.pdf
Robert Puskeiler, Miniaturisierte Parallelreaktoren..., Dissertation 2004, TU-Mnchen

3.2 Bioreactors

51
How can the oxygen transport be improved?
Increase solubility of O
2

Pressure increase from 100 to 200 kPa
Increase O
2
-percentage in air
Enrichment of supply air with O
2

Use of pure O
2

Change in the phase boundary (gas/fluid)
Size and dispersion of gas bubbles
Contact time of gas phase and liquid phase
Viscosity of medium
decrease of viscosity increase in relative velocity of gas bubbles more
thin liquid film increase in k
L
a-value
Use of surface active substances (discussed controversial)

3.2 Bioreactors


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BioMST II Biotechnological Tasks in

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