Reaction Engineering

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22 Φεβ 2013 (πριν από 4 χρόνια και 5 μήνες)

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Reaction Engineering

-
> Fermentation Technology (reactors for
microbial convertions)


1
st

lecture:
Introduction into Fermentation Technology


2
nd

lecture:
Main reactor types, Monod kinetics, mass balance and


growth kinetic for Batch reactor


3
rd

lecture:
Main reactor types, mass balance and growth kinetic


for Continuous culture and Fed
-
batch reactor and


applications in the range of micro
-

and nano
-

reactors


Fermentation Technology

SOME SIGNIFICANT DATES IN FERMENTATION
BlOTECHNOLOGY


-
>
ca. 3000 B.C. Ancient urban civilizations of Egypt and Mesopotamia
are brewing beer.


-
>
1683 A.D. Leeuwenhoek first describes observations of bacteria


-
>
1856 Pasteur demonstrates that microorganisms produce
fermentations and that



different organisms produce
different fermentation products. (His



commercial
applications include the "pasteurization" of wine as
well as milk.)


-
> 1943
Industrial microbiological production of penicillin
begins


-
> 1978
Perlman's
formal redefinition of fermentation
as any
commercially
useful



microbial product.





Fermentation Technology

Fermentation Technology

-
> Fermentation: from latin
-
> ”
fervere

-
> to boil (describing the
anaerobic process of yeast
producing CO
2

on fruit extracts)


-
> Nowadays: more broad meaning!!!!


The five major groups of commercially important fermentations:


-
> Process that produces
microbial cells (Biomass)
as a product

-
> Process that produces
microbial enzymes
as a product

-
> Process that produces
microbial metabolites (primary or secondary)
as a product

-
> Process that produces
recombinant products (enzymes or metabolite)
as a product

-
> Process that
modifies a compound that is added to the fermentation


transformation process

Regeneration of NAD
+

Fermentation

Respiration

No added terminal e
-
-
acceptor

Oxidant = terminal e
-
-
acceptor

ATP: substrate level phosphorylation

ATP: (e
-
-
transport) oxidative phosphoryl.

Glucose

2 Glyceraldehyde
-
3
-
P


2 ATP


2 NADH

2 Pyruvate

2
Lactate

+ 2
H
+

Acetaldehyde

+2
CO
2

2
Ethanol

Acetate

+ Formate

H
2

+
CO
2

Glucose


2 ATP


2 NADH

2 Pyruvate

2 Acetyl
-
CoA

CO
2

Citric acid

cycle


CO
2


GTP


NADH, FADH

Cytoplasmic membrane

out

in


ATP

H
+

H
+

H
+

H
+

H
+

H
+

O
2

H
2
O

1 Glucose


2 ATP

1 Glucose


38 ATP

Slow growth/low biomass yield

Fast growth/high biomass yield

Fermentation Technology

Streptococcus

Hyaluronic acid + lactic acid production

Growth cycle of yeast
during beer
fermentation

From: Papazian C (1991), The New
Complete Joy of Home Brewing.

Alternate modes of energy generation

(H
2
S, H
2
, NH
3
)

(in autotrophs)

Fermentation

Fermentation

Products of Anaerobic Metabolism

Growth: basic concepts

Anabolism

= biosynthesis

Catabolism

= reactions to
recover energy (often ATP)

Precursors

Fermentation Technology

-
> Process that produces
microbial cells (Biomass)
as a product




mainly for
-
> baking industry (yeast)


-
> human or animal food (microbial cells)


Fermentation Technology

Fermentation Technology


-
> Process that produces
microbial enzymes
as a product



mainly for
-
> food industry


Fermentation Technology

-
> Process that produces
microbial metabolites (primary or secondary)
as a
product





Fermentation Technology

-
> Process that produces
microbial metabolites (primary or secondary)
as a
product





Fermentation Technology

-
> Process that produces
microbial metabolites (primary or secondary)
as a
product





Fermentation Technology

-
> Process that produces
microbial metabolites (primary or secondary)
as a
product





Typical fermentation profile for a filamentous
microorganism producing a
secondary metabolite

Time course of a typical
Streptomyces

fermentation for an
antibiotic

Fermentation Technology

-
> Process that produces
microbial metabolites (primary or secondary)
as a
product





Fermentation Technology

Fermentation Technology

Growth

= increase in # of cells



(by binary fission)



generation time: 10 min
-

days

Bacterial growth

Growth rate

=
Δ
cell number/time



or
Δ
cell mass/time


1 generation

Growth of bacterial population


Exponential growth


Geometric progression of the number 2.


2
1
-
2
2
1 and 2 number of generation that has taken place


Arithmetic scale
-

slope


Logaritmic scale
-

straight line


arithmetic

scale

Bacterial growth: exponential growth

Semilogarythmic plot

Straight line
indicates
logarithmic
growth

Bacterial growth: logarithmic growth

X cell mass at time t


X
0

cell mass at time t
0

Bacterial growth: calculate the generation time

g =

t

n

t = time of exponential growth (in min, h)

g = generation time (in min, h)

n = number of generations

1 generation

Bacterial growth: batch culture

Turbidimetric measurements
-
> Optical Density

Limits of sensitivity at high bacterial density

„rescattering“


more light reaches detector



consequence
-
> no relyable values over 0.7

Typical pattern of growth cycle during batch
fermentation

I.
Lag phase

II.
Acceleration phase

III.
Exponential (logarithmic) phase

IV.
Deceleration phase

V.
Stationary phase

VI.
Accelerated death phase

VII.
Exponential death phase

VIII.
Survival phase


From: EL
-
Mansi and Bryce (1999)

Fermentation Microbiology

and Biotechnology.

Batch culture: Lag phase

no Lag phase:

Inoculum from exponential phase grown in the same media

Lag phase:

Inoculum from stationary culture (depletion of essential constituents)

After transfer into poorer culture media (enzymes for biosynthesis)

Cells of inoculum damaged (time for repair)

Batch culture: exponential
phase (balanced growth)

Exponential phase = log
-
phase

„midexponential“: bacteria often used for functional studies

Maximum growth rates
μ
max

Max growth rate
-
> smallest doubling time

Batch culture: Deceleration
Phase

Batch culture: stationary phase

Bacterial growth is limited:

-

essential nutrient used up

-

build up of toxic metabolic products in media

Stationary phase:

-

no net increase in cell number

-

„cryptic growth“ (cell growth rate =cell death rate)

-

energy metabolism, some biosynthesis continues

-

specific expression of „survival“ genes

-

secondary metabolites produced

m


=
0

Growth rate
-
>

Batch culture: death phase

Bacterial cell death:

-

sometimes associated with cell lysis

-

2 Theories:

-


programmed
“: induction of viable but non
-
culturable

-

gradual deterioration
:

-

oxidative stress: oxidation of essential molecules

-

accumulation of damage

-

finaly less cells viable

Diauxie

When two carbon sources present,
cells may use the substrates
sequentially.


Glucose


the major fermentable
sugar


glucose repression.


Glucose depleted

cells derepressed


induction of respiratory enzyme
synthesis




oxidative consumption of the
second carbon source (lactose)





a second phase of exponential
growth called diauxie.

E.coli

ML30 on equal molar concentrations (0.55
mM) of glucose and lactose

Factors affecting microbial growth


Nutrients


Temperature


pH


Oxygen


Water availability

Microbial growth media

Media


Purpose

Complex


Grow most heterotrophic organisms

Defined


Grow specific heterotrophs and are often mandatory for




chemoautotrophs, photoautotrophs and for microbiological



assays


Selective

Suppress unwanted microbes, or encourage desired microbes

Differential

Distinguish colonies of specific microbes from others

Enrichment

Similar to selective media but designed to increase the numbers of


desired microorganisms to a detectable level without stimulating



the rest of the bacterial population

Reducing


Growth of obligate anaerobes

MacConkey Agar:

Temperature

3 cardinal temperatures:

Usually ca. 30
°
C

Temperature class of Organisms

Maximum temperature

-

Covalent/ionic interactions weaker at high temperatures.

-

Thermal denaturation:


covalent or non
-
covalent


reversible/ irreversible

-

heat
-
induced covalent mod.: deamidation of Gln and Asn

Thermal protein inactivation:

-

Missense mutations: reduced thermal stability (Temp.
-
sens. mutants)

-

Heat shock response: proteases, chaperonins (i.e. DnaK ~ Hsp70)

Genetics:


Proteins:

-

Greater
a
-
helix content

-

more polar amino acids

-

less hydrophobic amino acids

Membranes:


-

temperature dependent phase transition

Thermotropic Gel:
Hexagonal arranged


-

homoviscous adaptation (
adjustment of membrane fluidity)



„Fluid mosaic“

Membrane proteins

inactive (mobility/insertion)


Protein function normal

T
m

Minimal Temperature

„Homoviscous adaptation“

Homoviscous adaptation = adjustment of membrane fluidity

-

lowered T
m

-

More cis
-
double bonds

-

Reduced hydrophobic interactions

-

high T
m

-

Few cis double bonds

-

optimal hydrophobic interactions

Fatty acid composition of plasma membrane as % total fatty acids

E. coli

grown at:



10
°
C


43
°
C

C16 saturated (palmitic)


18 %


48 %

C16 cis
-
9
-
unsat. (palmitoleic)

26 %


10 %

C18 cis
-
11
-
unsat. (cis
-
vaccinic)

38 %


12 %

-

thermophiles

-

mesophiles

Growth at high temperatures

Molecular adaptations in thermophilic bacteria

-

Protein sequence very similar to mesophils

-

1/few aa substitutions sufficient

-

more salt bridges

-

densely packed hydrophobic cores

Proteins

-

more saturated fatty acids

-

hyperthermophilic Archaea: C
40

lipid monolayer

lipids

-

sometimes GC
-
rich

-

potassium cyclic 2,3
-
diphosphoglycerate: K
+

protects from depurination

-

reverse DNA gyrase (increases T
m

by „overwinding“)

-

archaeal histones (increase T
m
)

DNA

Bacterial growth: pH

(extremes: pH 4.6
-

9.4)

Most
natural
habitats

Growth at low pH



Fungi:
-

often more acid tolerant


than bacteria (opt. pH5)


Obligate acidophilic bacteria:


Thiobacillus ferrooxidans


Obligate acidophilic Archaea:


Sulfolobus


Thermoplasma


Most critical: cytoplasmic membrane

Dissolves at more neutral pH

-

Few alkaliphiles (pH10
-
11)

-

Bacteria:
Bacillus

spp.

-

Archaea

-

often also halophilic

-

Sometimes: H
+

gradient replaced by
Na
+

gradient (motility, energy)

-

industrial applications (especially
„exoenzymes“):

-
Proteases/lipases for detergents
(
Bacillus licheniformis
)

-
pH optima of these enzymes: 9
-
10

Growth at high pH

Bacterial growth: Oxygen

O
2

as electron sink for catabolism


toxicity of Oxygen species

Aerobes
: growth at 21% oxygen

Microaerophiles
: growth at low oxygen concentration

Facultative aerobes
: can grow in presence
and

absence of oxygen

Anaerobes
: lack respiratory system

Aerotolerant anaerobes

Obligate anaerobes
: cannot tolerate oxygen (lack of detoxification)

Fermentation Process

Fermenter

Fermenter

Major functions of a fermentor

1) Provide operation free from contamination;

2) Maintain a specific temperature;

3) Provide adequate mixing and aeration;

4) Control the pH of the culture;

5) Allow monitoring and/or control of dissolved oxygen;

6) Allow feeding of nutrient solutions and reagents;

7) Provide access points for inoculation and sampling;

8) Minimize liquid loss from the vessel;

9) Facilitate the growth of a wide range of organisms.

(Allman A.R., 1999: Fermentation Microbiology and Biotechnology)

Fermenter Regulation versus Biological Processes

Biotechnological processes of growing
microorganisms in a bioreactor

1)
Batch culture:

microorganisms are inoculated into a fixed volume of medium
and as growth takes place nutrients are consumed and products of growth
(biomass, metabolites) accumulate.


2)
Semi
-
continuous:

fed batch
-
gradual addition of concentrated nutrients so that
the culture volume and product amount are increased (e.g. industrial production
of baker’s yeast);

Perfusion
-
addition of medium to the culture and withdrawal of an equal volume of
used cell
-
free medium (e.g. animal cell cultivations).


3)
Continuous:

fresh medium is added to the bioreactor at the exponential phase
of growth with a corresponding withdrawal of medium and cells. Cells will grow at
a constant rate under a constant condition.

Biotechnological processes of growing
microorganisms in a bioreactor

Batch culture versus continuous culture

Continuous systems: limited to single cell protein, ethanol productions, and
some forms of waste
-
water treatment processes.


Batch cultivation: the dominant form of industrial usage due to its many
advantages.


(Smith J.E, 1998: Biotechnology)

Advantages of batch culture versus continuous
culture

1)
Products may be required only in a small quantities at any given time.

2)
Market needs may be intermittent.

3)
Shelf
-
life of certain products is short.

4)
High product concentration is required in broth for optimizing downstream
processes.

5)
Some metabolic products are produced only during the stationary phase of the
growth cycle.

6)
Instability of some production strains require their regular renewal.

7)
Compared to continuous processes, the technical requirements for batch
culture is much easier.