Reaction Engineering -> Fermentation Technology (reactors for microbial convertions) 1 st lecture: Introduction into Fermentation Technology 2 nd lecture:

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 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


Streptococcus

Hyaluronic acid + lactic acid production

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

Alternate modes of energy generation

(H2S, H2, NH3)(in autotrophs)

FermentationFermentation

Products of Anaerobic Metabolism

Growth: basic concepts

Anabolism = biosynthesis

Catabolism = reactions to recover energy (often ATP)

Precursors


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



-> Process that produces microbial enzymes as a product

mainly for -> food industry


-> 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





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

era

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: logarithmic growth

X cell mass at time t

X0 cell mass at time t0

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

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

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: 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

= 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

Factors affecting microbial growth

• Nutrients• Temperature• pH• Oxygen• Water availability

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:

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 -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

Bacterial growth: pH

(extremes: pH 4.6- 9.4)

Most natural habitats

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)

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

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

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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Reaction Engineering -> Fermentation Technology (reactors for microbial convertions) 1 st lecture: Introduction into Fermentation Technology 2 nd lecture: