Biology & Bioprocesses

CE10079
Dr Christopher Chuck C.chuck@bath.ac.uk
What makes a good fermentation process?

Department of Chemical Engineering

What we did last time

Environmental, structural, molecular and genetic mechanisms of regulation.

Co-factors and co-enzymes (link with MDL).

Feedback regulation: inhibition and repression.
Explain the mechanisms of concerted, co-operative, cumulative, sequential,

and iso-enzyme regulation of metabolic pathways.

Protein synthesis: transcription and translation, and where it happens in cells
in terms of structure (link to MDL).
Induction and repression of protein synthesis.
Example of Metabolic Engineering on an industrial scale

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What we are covering this time around

Disciplines involved in developing bioprocesses.

Ideal features of micro-organisms, raw materials and processes.

Bioreactors and scale-up from lab to process.
A typical batch fermentation
the sequence of stages in growth and substrate concentration,
effect on cell growth,
the Monod equation and a mass balance / kinetic analysis.

Stages in a typical fermentation process.

NREL lignocellulosic bioethanol process flow diagram

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Basic disciplines in Bioprocessing:

Microbiology Microbial Physiology
Biochemistry Enzymology

Genetics Molecular biology

Engineering ..
.

Process Design & Control

Economic Evaluation

Marketing & Sales

.
.

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Selecting the appropriate micro-organism :

A micro-organism that forms a desirable end product.
The organism must have the correct metabolic pathways present

Metabolic / genetic engineering can be used to enhance this products

production

A micro-organism which can be cultivated artificially

..

An organism that is biologically uniform

Genetically stable (has very low mutation rates)
Stable metabolic processes (predictable yields).

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Selecting the appropriate raw materials:

Are the raw materials (substrates) ..?
Are the raw materials ?
Are the raw materials ?
Are they from a first generation feedstock
Starch from corn or potatoes, sugars from sugarcane

What other inputs are necessary beyond the main feedstocks?

. (N & P)
Minerals (metals)
Vitamins

Protein

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Developing the ideal process

Are the genetics and metabolic pathways understood?

Do we get good conversion of raw materials into product?

The S. cerevisiae ethanol process has been finessed over many years

..?
This can be the main factor in producing an economical process

Are the products readily recoverable and can they be purified

and packaged?

..?

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Going up in scale
Production of -1-antitrypsin
From bench or lab scale to pilot scale. for treatment of cystic fibrosis
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Department of Chemical Engineering

Production of glutamic acid on an industrial scale

Production bioreactors ............

..
..

the precursor for mono-sodium

glutamate (MSG).

Each bioreactor has a working volume

of 250,000 litres. They are about 30
metres tall.

Bioreactors for industrial bioethanol

production are of similar scale.
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A typical batch fermentation:

2

2.5

1.6

1.4
1.2

1.5

1
0.8

0.6
0.4

0.5

0.2
0

0
0

Time

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10

12

14

ln (substrate concentration)

ln (cell concentration)

1.8

Cell reproduction binary fission:

1, then 2, then 4, then 8, then 16, then 32 then

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A typical batch fermentation:

1.8

ln (cell concentration)

Cell need time to adapt to their

environment lag phase

2
1.6
1.4

In the acceleration phase growth starts

1.2
1

0.8
0.6

In the growth phase, the growth rate is

0.4
0.2

at its highest

0
0

Time

10

In the decline phase the growth rate starts to fall due to lack of resources or
inhibitory compounds in the medium
In the stationary phase the growth rate ceases
In the death phase the growth rate becomes negative more cells are
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of created
Chemical Engineering
lysing
than being

15

Bio-reactor engineering (kinetics):

Important to note that cells will be dying and lysing constantly, it is just the
overall growth rate that matters
Simplest (but surprisingly accurate) kinetic description of an organism in a
batch fermentation is the MONOD equation

= specific growth rate h-1

max = maximum specific growth rate h-1
KS = substrate affinity constant g l-1
kd = biomass death rate h-1
S = substrate concentration in the bioreactor g l-1

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The MONOD equation

ADVANTAGES

Simple to calculate and derive

Originally based on experimental data
.

..
LIMITATIONS
Does not take into account

................................................................................................................
Assumes one nutrient only is limiting factor
Constant environmental conditions

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That metabolic pathways always function to maximum capacity

Bio-reactor engineering (mass balance):

Mass balances of both the cell biomass and substrate volume vital part of
bioreactor engineering

Mass balance on biomass (X g l-1):

which can be integrated to give:

dX
X
dt

X t X 0 e t

And now by taking natural logs:

Mass balance on substrate (S g

l-1):

dS
X

dt
Y( X / S )

Y(X/S) = biomass yield on substrate g g-1

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Flow sheet for a

fermentation process
Stock
culture

Shake
flasks

Media
formulation

Seed
fermenter

Product
fermenter
Stabilised Liquid

Cell
separation

Spray dried
powder
Product
formulation

Product
concentration

Final
purification

Filtration

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Insulin Production Flowsheet

M e d ia

P -6 / A F - 1 0 2

F e rme nta tio n S e c tio n

P rima ry R e cov e ry S e c tio n

S -1 0 8

A ir F ilt r a t io n

S -1 0 7

E D T A

W a te r

S o lu t io n
T rit o n - X - 1 0 0
L iq W a s t e 2

P -1 / V -1 0 1

P -2 / S T -1 0 1

M ix in g

S -1 0 6

L iq w a s t e 1

H e a t S t e r iliz a t io n

S -1 4 2
P -1 1 / H G -1 0 1
S -1 0 1

H o m o g e n iz a t io n

P -3 8 / V - 1 0 9
B le n d in g

P -9 / D S - 1 0 1
A m m o n ia

P -5 / A F - 1 0 1
A ir F ilt r a t io n

S -1 1 8

S -1 0 4
/

P -1 3 / D S - 1 0 1

S to ra g e

P -1 0 / V - 1 1 0

C e n t r if u g a t io n

P -7 / V -1 0 2
P -1 9 / V - 1 0 6

F e rm e n t a t io n

B le n d in g

C e n t r if u g a t io n

S to ra g e

S to ra g e
S -1 4 3
A ir

S -1 1 0
M rE T O H / U re a
P -4 / G - 1 0 1

W F I -1

C N B r/ H C O O H

C e n t r if u g a l C o m p r e s s io n

L iq W a s t e 3

S -1 2 7
G ua n

H C l

S -1 3 2

W F I -2
S -1 2 1

S -1 1 5
S -1 1 4
P -1 4 / D S - 1 0 1

P -1 6 / D F -1 0 1
P -1 7 / D E - 1 0 1
P -1 8 / C S P - 1 0 1
R o t a ry

S -1 2 9

D e a d -E n d

E v a p o ra t o r

F ilt r a t io n

S o lu b iliz a t io n

L iq W a s t e 4

P -2 0 / V - 1 0 5

D ia f ilt r a t io n

IB

P -1 5 / V - 1 0 3
C N B r C le a v a g e

P -2 1 / D F -1 0 1

C e n t r if u g a t io n

P -8 / V -1 0 3

D ia f ilt r a t io n

L iq W a s t e 5
S -1 5 8
S -1 5 9

S u lf it o ly s is

L iq W a s t e 6

S -1 6 0

W F I -4

E n zym e s
S -1 4 6
S -1 4 7
S -1 4 8

S -1 3 4
S -1 3 5

R e a ctions S e ctio n

W F I -3

S -1 5 4

S -1 4 9
S -1 0 2

S -1 3 6

S -1 5 6

S -1 2 3
S -1 5 2

S -1 5 3
P -2 5 / C - 1 0 3

S -1 1 9

P -2 7 / D F -1 0 2

L iq W a s t e 9

P -2 6 / V - 1 0 8

P -2 2 / C - 1 0 2

S -1 4 4
S -1 4 1

L iq W a s t e 7

H IC

E n zym e

C o lu m n

S -S e p h a ro s e

L iq W a s t e 1 1

D ia f ilt r a t io n

C o n v e rs io n
L iq W a s t e 1 0

P -3 6 / C - 1 0 1

S -1 6 1

P -2 4 / D F -1 0 2

S -S e p h a ro s e

P -2 3 / V - 1 0 7

D ia f ilt r a t io n

L iq W a s t e 8

F ina l P urific a tion S e ctio n

M rE t O H R e f o l d i n g

S -1 7 3

S -1 6 5

S -1 7 4

S -1 6 6

S -1 6 2

S -1 7 5

S -1 2 6
S -1 8 6

W F I -5

S -1 6 7
W F I -6

S -1 3 7
S -1 2 4
S -1 7 2

P ro d u c t
P -3 5 / F D R - 1 0 1
F re e z e

P -3 3 / D F -1 0 4
S -1 5 7

D ry in g

P -3 1 / C - 1 0 5

D ia f ilt r a t io n

G e l F ilt r a t io n
P -1 2 / V - 1 1 1
P -3 4 / B C F -1 0 1

P -2 8 / C - 1 0 4

D ia f ilt r a t io n

D ia f ilt r a t io n
R P -H P L C

C r y s t a lliz a t io n

B a s k e t C e n t rif u g a t io n
L iq W a s t e 1 7

P -3 0 / D F -1 0 3

P -2 9 / D F -1 0 3

L iq W a s t e 1 5
L iq W a s t e 1 6

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L iq W a s t e 1 4

L iq W a s t e 1 3

L iq W a s t e 1 2

Bioprocess Products:
Concentration in the bioreator (kg m-3)

water

fuels

1000

amino acids

100
10

citric acid, MSG

Penicillin

antibiotics

Threonine

ethanol
Gold

Glucose oxidase

bulk enzymes
0.1

Amylase

0.01

Hepatitis B vaccine

Rennin

0.001

Research and diagnostic

enzymes

0.0001
0.00001
1.E-02

Insulin

1.E+00

1.E+02

1.E+04

Therapeutic
enzymes
Luciferase
1.E+06

Selling Price($ kg-1)

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Urokinase
1.E+08

1.E+10

Bioethanol from lignocellulose

Process flow sheet for bioethanol production from lignocellulose
Lignocellulose is made up of lignin, cellulose and hemicellulose
Subject of the final lecture
An example of bioprocessing on a huge scale
Taken from National Renewable Energy Laboratory (NREL) data 2011

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USA Bioethanol from lignocellulose

Bioethanol from sugars is generally
unsustainable and competes with food
production
New ethanol technologies need to
expand to encompass second
generation feedstocks

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Raw
feedstock

Feed handling

Nutrients
Chemicals

feedstock

Pretreatment
and
conditioning

Enzyme
production

Hydrolysate

enzymes

Enzymatic
hydrolysis
sugars

Wastewater
treatment

Yeast
Stillage
Fermentation

Anaerobic
biogas

Distillation,
dehydration,
and solids
separation

Burner / boiler
turbogenerator
Biomass

Beer

Ethanol
Electricity

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