reaction engineering -> fermentation technology (reactors for microbial convertions) 1 st...
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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 TechnologySOME 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: 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
Regeneration of NAD+
Fermentation RespirationNo added terminal e--acceptor Oxidant = terminal e--acceptor
ATP: substrate level phosphorylationATP: (e--transport) oxidative phosphoryl.
Glucose
2 Glyceraldehyde-3-P 2 ATP2 NADH
2 Pyruvate
2 Lactate+ 2 H+
Acetaldehyde+2 CO2
2 Ethanol
Acetate+ Formate
H2 + CO2
Glucose2 ATP2 NADH
2 Pyruvate
2 Acetyl-CoACO2
Citric acidcycle
CO2
GTPNADH, FADH
Cytoplasmic membrane
out
inATP
H+H+H+H+H+H+
O2H2O
1 Glucose 2 ATP 1 Glucose 38 ATPSlow growth/low biomass yield Fast growth/high biomass yield
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
-> 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
Growth = increase in # of cells (by binary fission) generation time: 10 min - days
Bacterial growth
Growth rate = Δcell number/time or Δcell mass/time
1 g
en
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tion
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: calculate the generation time
g =tn
t = time of exponential growth (in min, h)g = generation time (in min, h)n = number of generations
1 g
en
era
tion
Turbidimetric measurements -> Optical Density
Limits of sensitivity at high bacterial density„rescattering“ more light reaches detector
consequence -> no relyable values over 0.7
I. Lag phaseII. Acceleration phaseIII. Exponential (logarithmic) phase IV. Deceleration phaseV. Stationary phaseVI. Accelerated death phaseVII. Exponential death phaseVIII. 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 μmax
Max growth rate -> smallest doubling time
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
= 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
DiauxieDiauxie
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
Microbial growth media
Media PurposeComplex Grow most heterotrophic organismsDefined Grow specific heterotrophs and are often mandatory for
chemoautotrophs, photoautotrophs and for microbiological assays
Selective Suppress unwanted microbes, or encourage desired microbesDifferential Distinguish colonies of specific microbes from othersEnrichment 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:
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 -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 proteinsinactive (mobility/insertion)
Protein function normal
Tm
Minimal Temperature
„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
Fatty acid composition of plasma membrane as % total fatty acidsE. coli grown at: 10°C 43°CC16 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: C40 lipid monolayer
lipids
- sometimes GC-rich- potassium cyclic 2,3-diphosphoglycerate: K+ protects from depurination- reverse DNA gyrase (increases Tm by „overwinding“)- archaeal histones (increase Tm)
DNA
Growth at low pH
Fungi: - often more acid tolerantthan bacteria (opt. pH5)
Obligate acidophilic bacteria:Thiobacillus ferrooxidans
Obligate acidophilic Archaea:SulfolobusThermoplasma
Most critical: cytoplasmic membraneDissolves 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
O2 as electron sink for catabolism toxicity of Oxygen species
Aerobes: growth at 21% oxygenMicroaerophiles: growth at low oxygen concentrationFacultative aerobes: can grow in presence and absence of oxygenAnaerobes: lack respiratory systemAerotolerant anaerobesObligate anaerobes: cannot tolerate oxygen (lack of detoxification)
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)
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.
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)
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.
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