Transcript Reaction Engineering - AAU -uddannelser, forskning og

Reaction Engineering
-> 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
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 CO2 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
Fermentation
Respiration
No added terminal e--acceptor
Oxidant = terminal e--acceptor
ATP: substrate level phosphorylation
ATP: (e--transport) oxidative phosphoryl.
Glucose
Glucose
2 Glyceraldehyde-3-P
2 ATP
2 NADH
2 Pyruvate
Regeneration of
2 Lactate
+ 2 H+
Acetaldehyde
+2 CO2
2 Pyruvate
CO2
2 Acetyl-CoA
Citric acid
cycle
NAD+
Acetate
+ Formate
H2O
in
2 Ethanol
H2 + CO2
CO2
GTP
NADH, FADH
O2
ATP
Cytoplasmic membrane
out
1 Glucose  2 ATP
Slow growth/low biomass yield
2 ATP
2 NADH
H+ H+ H+ H+ H+ H+
1 Glucose  38 ATP
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
(H2S, H2, NH
)
(in 3autotrophs)
Fermentation
Fermentation
Products of Anaerobic Metabolism
Growth: basic concepts
Precursors
Anabolism = biosynthesis
Catabolism = reactions to
recover energy (often ATP)
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
Bacterial growth
Growth rate = Δcell number/time
or Δcell mass/time
1 generation
Growth = increase in # of cells
(by binary fission)
generation time: 10 min - days
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
Bacterial growth: exponential growth
Semilogarythmic plot
Straight line
indicates
logarithmic
growth
Bacterial growth: logarithmic growth
X cell mass at time t
X0 cell mass at time t0
Bacterial growth: calculate the generation time
t
g= 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.
II.
III.
IV.
V.
VI.
VII.
VIII.
Lag phase
Acceleration phase
Exponential (logarithmic) phase
Deceleration phase
Stationary phase
Accelerated death phase
Exponential death phase
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
Maximum growth rates μmax
„midexponential“: bacteria often used for functional studies
Max growth rate -> smallest doubling time
Batch culture: Deceleration
Phase
Batch culture: stationary phase
Growth rate ->
m = 0
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
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
Complex
Defined
Purpose
Grow most heterotrophic organisms
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
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)
Minimal Temperature
Proteins:
- Greater a-helix content
- more polar amino acids
- less hydrophobic amino acids
Membranes:
- temperature dependent phase transition
Thermotropic Gel:
Hexagonal arranged
Membrane proteins
inactive (mobility/insertion)
Tm

„Fluid mosaic“
Protein function normal
- homoviscous adaptation (adjustment of membrane fluidity)
„Homoviscous adaptation“
Homoviscous adaptation = adjustment of membrane fluidity
- lowered Tm
- More cis-double bonds
- Reduced hydrophobic interactions
- high Tm
- Few cis double bonds
- optimal hydrophobic interactions
- mesophiles
- thermophiles
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 %
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)
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
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
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)
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)
2)
3)
4)
5)
6)
7)
Products may be required only in a small quantities at any given time.
Market needs may be intermittent.
Shelf-life of certain products is short.
High product concentration is required in broth for optimizing downstream
processes.
Some metabolic products are produced only during the stationary phase of the
growth cycle.
Instability of some production strains require their regular renewal.
Compared to continuous processes, the technical requirements for batch
culture is much easier.