Reaction Engineering

1
Reaction Engineering
2
Reaction Engineering

• -gt Fermentation Technology (reactors for
  microbial convertions)

• 1st lecture Introduction into Fermentation
  Technology
• 2nd lecture Main reactor types, Monod kinetics,
  mass balance and
• growth kinetic for Batch
  reactor
• 3rd 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

3
Fermentation Technology

• SOME SIGNIFICANT DATES IN FERMENTATION
  BlOTECHNOLOGY
• -gt ca. 3000 B.C. Ancient urban
  civilizations of Egypt and Mesopotamia are
  brewing beer.
• -gt 1683 A.D. Leeuwenhoek first
  describes observations of bacteria
• -gt 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.)
• -gt 1943 Industrial
  microbiological production of penicillin begins
• -gt 1978 Perlman's formal
  redefinition of fermentation as any commercially
  useful
• microbial
  product.

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Fermentation Technology
5
Fermentation Technology

• -gt Fermentation from latin -gt fervere -gt to
  boil (describing the anaerobic process of yeast
  producing CO2 on fruit extracts)
• -gt Nowadays more broad meaning!!!!
• The five major groups of commercially important
  fermentations
• -gt Process that produces microbial cells
  (Biomass) as a product
• -gt Process that produces microbial enzymes as a
  product
• -gt Process that produces microbial metabolites
  (primary or secondary) as a product
• -gt Process that produces recombinant products
  (enzymes or metabolite) as a product
• -gt Process that modifies a compound that is added
  to the fermentation transformation process

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Fermentation
Respiration
Oxidant terminal e--acceptor
No added terminal e--acceptor
ATP substrate level phosphorylation
ATP (e--transport) oxidative phosphoryl.
Glucose
Glucose

• 2 ATP
• 2 NADH

2 Pyruvate

• 2 ATP
• 2 NADH

2 Glyceraldehyde-3-P
CO2
2 Acetyl-CoA
2 Pyruvate

• CO2
• GTP
• NADH, FADH

Citric acid cycle
Regeneration of NAD
Acetaldehyde 2 CO2
2 Lactate 2 H
Acetate Formate
?ATP
H2O
O2
in
2 Ethanol
H2 CO2
Cytoplasmic membrane
H
H
H
H
H
H
out
1 Glucose ? 2 ATP
1 Glucose ? 38 ATP
Slow growth/low biomass yield
Fast growth/high biomass yield
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Fermentation Technology
Streptococcus
Hyaluronic acid lactic acid production
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Growth cycle of yeast during beer fermentation
From Papazian C (1991), The New Complete Joy of
Home Brewing.
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Alternate modes of energy generation
(H2S, H2, NH3)
(in autotrophs)
Fermentation
Fermentation
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Products of Anaerobic Metabolism
11
Growth basic concepts
Precursors
Anabolism biosynthesis
Catabolism reactions to recover energy (often
ATP)
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Fermentation Technology

• -gt Process that produces microbial cells
  (Biomass) as a product
• mainly for -gt baking industry (yeast)
• -gt human or animal food
  (microbial cells)

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Fermentation Technology
14
Fermentation Technology

• -gt Process that produces microbial enzymes as a
  product
• mainly for -gt food industry

15
Fermentation Technology

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

16
Fermentation Technology

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

17
Fermentation Technology

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

18
Fermentation Technology

• -gt 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
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Fermentation Technology

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

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Fermentation Technology
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Fermentation Technology
Bacterial growth
Growth increase in of cells (by binary
fission) generation time 10 min - days
Growth rate ?cell number/time or ?cell
mass/time
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Growth of bacterial population

• Exponential growth
• Geometric progression of the number 2.
• 21-22 1 and 2 number of generation that has
  taken place
• Arithmetic scale - slope
• Logaritmic scale - straight line

arithmetic scale
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Bacterial growth exponential growth
Semilogarythmic plot
Straight line indicates logarithmic growth
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Bacterial growth logarithmic growth
X cell mass at time t X0 cell mass at time t0
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Bacterial growth calculate the generation time
t time of exponential growth (in min, h) g
generation time (in min, h) n number of
generations
t
g
n
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Bacterial growth batch culture
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Turbidimetric measurements -gt Optical Density
Limits of sensitivity at high bacterial
density rescattering? more light reaches
detector consequence -gt no relyable values over
0.7
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Typical pattern of growth cycle during batch
fermentation

1. Lag phase
2. Acceleration phase
3. Exponential (logarithmic) phase
4. Deceleration phase
5. Stationary phase
6. Accelerated death phase
7. Exponential death phase
8. Survival phase

From EL-Mansi and Bryce (1999) Fermentation
Microbiology and Biotechnology.
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Batch culture Lag phase
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Batch culture exponential phase (balanced growth)
Max growth rate -gt smallest doubling time
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Batch culture Deceleration Phase
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Batch culture stationary phase
m 0
Growth rate -gt
33
Batch culture death phase
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Diauxie
When two carbon sources present, cells may use
the substrates sequentially. Glucose the
major fermentable sugar glucose repression.
Glucose depletedcells 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
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Factors affecting microbial growth

• Nutrients
• Temperature
• pH
• Oxygen
• Water availability

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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
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Temperature
3 cardinal temperatures
Temperature class of Organisms
Usually ca. 30C
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Maximum temperature
Thermal protein inactivation

• - Covalent/ionic interactions weaker at high
  temperatures.
• Thermal denaturation covalent or non-covalent
• reversible/ irreversible
• - heat-induced covalent mod. deamidation of Gln
  and Asn

Genetics
- Missense mutations reduced thermal stability
(Temp.-sens. mutants) - Heat shock response
proteases, chaperonins (i.e. DnaK Hsp70)
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Minimal Temperature

• Proteins
• Greater a-helix content
• more polar amino acids
• less hydrophobic amino acids

Membranes - temperature dependent phase
transition
Thermotropic Gel Hexagonal arranged
Fluid mosaic
Tm
??
Membrane proteins inactive (mobility/insertion)
Protein function normal
- homoviscous adaptation (adjustment of membrane
fluidity)
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Homoviscous adaptation
Homoviscous adaptation adjustment of membrane
fluidity
- high Tm - Few cis double bonds - optimal
hydrophobic interactions
- lowered Tm - More cis-double bonds - Reduced
hydrophobic interactions
- thermophiles
- mesophiles
Fatty acid composition of plasma membrane as
total fatty acids E. coli grown
at 10C 43C C16 saturated (palmitic) 18
48 C16 cis-9-unsat. (palmitoleic) 26 10
C18 cis-11-unsat. (cis-vaccinic) 38 12
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Growth at high temperatures
Molecular adaptations in thermophilic bacteria
Proteins

• Protein sequence very similar to mesophils
• 1/few aa substitutions sufficient
• more salt bridges
• densely packed hydrophobic cores

lipids

• more saturated fatty acids
• hyperthermophilic Archaea C40 lipid monolayer

DNA

• sometimes GC-rich
• potassium cyclic 2,3-diphosphoglycerate K
  protects from depurination
• reverse DNA gyrase (increases Tm by
  overwinding)
• archaeal histones (increase Tm)

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Bacterial growth pH
Most natural habitats
(extremes pH 4.6- 9.4)
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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
Growth at high 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

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Bacterial growth Oxygen
O2 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)
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Fermentation Process
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Fermenter
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Fermenter
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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)
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Fermenter Regulation versus Biological Processes
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Biotechnological processes of growing
microorganisms in a bioreactor

• 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 bakers 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.

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Biotechnological processes of growing
microorganisms in a bioreactor
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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)
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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.