Biological Reaction Engineering Flashcards
What’s a bioprocess?
A process that uses a biocatalyst.
Biocatalyst are: •Cells that can manufacture: •Microbial •Animal •Plant
Biocatalysts can be enzymes or microorganisms.
What are the features of process engineering?
Most reactions are aqueous phase
Solids are low density, high water content particles
Low diffusion rates; high viscosity
Cell growth is limited by nutrient mass transfer
Low concentrations (high water content)
Stability with regard to pH, temperature is low
Complex process streams
What’s the difference between stoichiometric and catalytic reactions?
Stoichiometric - conc’ of species scale with those of the substrates
Catalytic - conc’ of catalytic intermediates relative to substrate concentrations change as a function of substrate concentration.
Rate behaviour at high and low conc’ may not be the same.
What’s enzyme kinetics?
Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products
- substrate binds reversibly to the enzyme
- enzyme-substrate complex (Michaelis complex)
- catalytic step
- release product
What’s the quasi-equilibrium assumption?
If a reversible step in a reaction network remains practically in equilibrium through the reaction, the step is treated as equilibrium.
What’s the Bodenstein steady state assumption?
If the intermediate concentration is very small compared to the substrate concentration then the rate of change in concentration of the intermediate species will also be very small, and we may set this rate equal to zero
What are the 3 main ways (plots) that can be used to graphically determine Vmax and Km?
Lineweaver-Burk plot
Eadie-Hofstee plot
Hanes-Dixon plot
What is the zero order kinetic derivation?
So - S = kt
However, learn to derive, NOT memorise!
Remember rate = dP/dt = -dS/dt
What’s the first order kinetic derivation?
S / So = e^(-kt)
However, learn to derive, NOT memorise!
Remember rate = dP/dt = -dS/dt
Which kinetic equation is used when enzymes are used (as a biocatalyst)?
The Michaelis-Menten equation (using v max and Km)
Enzymes are dead.
Which kinetic equation is used when microorganisms are used (as a biocatalyst)?
The Monod equation (using mu max and Ks)
Microorganisms are alive.
From what foundations can the MM (Michaelis Menten) equation be derived?
k1 k2
E + S ES -> E + P
k2
And the fact that rate of product formation,
r = k2*[ES]
However, [ES] can’t be measured so an alternative must be found.
How is the deactivation of an enzyme equation derived?
[E]a = [E]o e^(-kdt)
Which is derived from:
r = -dEa/dt = kd[E]a
Learn to derive, not memorise
How is the half life of an enzyme found?
Half life reached when:
[E]a = [E]o/2
Substitute this into enzyme deactivation equation and solve.
Half life:
t (1/2) = ln 2 / kd
What are substrates used for for enzymatic and microorganism catalysed reactions?
Enzymatic:
Production
Microorganism:
Growth
Maintenance
Production
What are yield coefficients?
Biological variables used to relate ratio between consumption and production rates of mass and energy.
They are assumed to be time-independent.
How is the rate of biomass growth (batch growth) calculated?
rx = dX/dt = mu*X
Mu is NOT a constant, however mu max IS!
What happens in the lag phase (batch reactor)?
Occurs immediately after inoculation
- No increase in cell numbers (growth rate~0)
- Cells adapt to the new environment
- Length depends
- nutrient composition
- age of cells
•Reduce lag by
–Active inoculum,
–Same growth medium
–Large inoculum
What’s an inoculum?
A substance used for inoculation.
What’s doubling time?
Time taken for initial amount of substance to double.
X = 2*Xo
What’s the Monod equation?
mu = mu max * [S] / (Ks + [S])
What’s the Michaelis-Menten equation?
v = v max * [S] / (Km + [S])
What happens when products are directly linked to energy generation?
The rate of substrate consumption does not contain a separate term for product synthesis
Why do enzyme and microbe catalysed reactions differ in chemical reactions and kinetic expressions?
Enzymes are dead - their concentration can only decrease
Mivoorganisms are alive and can grow - their concentration can increase and decrease
What is the specific growth rate constant, mu, equal to in batch reactors?
mu = mu max
This is for batch reactors only (in growth phase)
Otherwise, mu = mu max * [S] / (Ks + S)
How can fermentation products be classified?
According to the relationship between product synthesis and energy generation within the cell/
- Main product appears as a result of primary energy metabolism. I.e. product will be formed whenever there is growth (ethanol, butanol).
The simple growth stoichiometry still holds, just add another product term - The main product arises indirectly from energy metabolism. (Citric acid ).
The stoichiometry do not hold, need another equation. - The product is a secondary metabolite, no clear coupling (penicillin, vitamins).
The stoichiometry do not hold. Product formation is typically completely uncoupled with cell growth, need another equation
How is rate of product formation, considering product kinetics, calculated?
r(P) = dP/dt = q(P)*X
= (Y p/x * mu + m(p))*X
How is the rate of substrate uptake determined?
r(S) = dS/dt = q(S)*X
r(S) = r(X) / Y(x/s) + m(S)*X
= ((mu max * [S])/Yx/s (Ks + S) + ms) *X
What are the key considerations and choices in reactor design?
Reactor configuration – stirred tank, packed bed, airlift etc
Reactor size - what size of reactor is required to achieve desired productivity
Processing conditions in reactor
•temperature, pH, control etc
•mode of operation
•batch, continuous, series etc
Key output
- High productivity – how can this be optimised?
The bioreactor and operating strategy determines:
• product concentration, substrate utilisation, degree of impurities.
The choice of reactor must consider:
•catalyst/cells reactor
•separation and purification
•The two simple extremes: batch or a continuous stirred tank reactor
What are main reactors used in bioprocesses?
Stirred tank
Bubble column
Airlift
Packed bed
(Batch)
What are features of CSTR in bioprocess?
Complete mixing
70-80% of the volume is filled with liquid, the remaining headspace is for gas disengagement and to accommodate foam
Mixing and gas dispersion are achieved by mechanical agitation (high input of energy/ vol)
A wide variety of impeller designs are available (Rushton turbine, marine propeller)
Heat exchange is carried out with an external jacket and/or internal cooling coils
What’s are features of a bubble column for bioprocesses?
Rising bubbles cause the fluid to mix
Simple design and cheap
–No mechanical stirring, no moving parts
Hard to predict mass transfer
Homegeneous/ heterogeneous flow based on bubble sized
Good for low-viscosity culture
What are features of airlift reactors?
Air circulates up and down the column.
Patterns of liquid flow are more defined than in bubble columns – physical separation of up- and down-flowing streams
Low shear reactor, used for mammalian and plant cell culture
Generally better mixing than bubble columns, and offers greater stability with respect to high gas flowrates
Have a high height-to-diameter-ratio (~10)
Not suitable for high viscous culture
What are features of packed bed reactors?
Can be packed bed, fluidised bed or trickle reactor.
Used for immobilised or particulate catalysts
Medium can be fed from the bottom or at the top (see also - trickle bed reactor)
Minimal damage from particle attrition
Aeration is often carried out in a separate vessel to minimise gas channelling in the reactor
What main operating conditions should be considered?
Aseptic conditions:
–Minimal number of fitted internal structures
–No stagnant zones
–Filters to ensure sterile feed
–Sterile air for slight positive pressure
–Ensure sterile stirrer seals
–Inoculum is often blown in
Materials
–Withstand steam sterilisation and cleaning cycles
–Non-reactive and non-absorptive
What does innoculum mean?
The amount of microbes added at the beginning of a process to start a reaction.
If there’s no innoculum, there’s nothing to start the reaction.
How can foaming in reactors be reduced?
By using a foam breaker or a surfactant within the reactor.
List the assumptions required to estimate the time required for a reaction to reach stationary phase in a batch reactor:
1) no lag phase
2) mu = mu max
3) Kd «_space;mu max
4) becomes stationary when S = 0
5) all energy is used for growth (not ms or qp)
What does PCR stand for?
Polymerase chain reaction
What are primers/probes?
They are a short base-pair sequence of either DNA or RNA
- Designed to attach to a single stranded base pair sequence
- The longer the sequence, the more specific
- Vital in a wide range of microbiological tools (GE, FISH, PCR, Cloning, Sequencing etc..)
What’s a primer?
A strand of nucleic acids that serves as a starting point for DNA replication
- It’s necessary as DNA polymerase enzyme cannot start by itself – it can only add to an existing strand on the DNA
- Starts on the 3’-end and copies the opposite strand (ie copies 5’-3’)
Used to
• Sequence DNA
• Amplify DNA
What are probes?
Similar to primer = short base-pair sequence
- Designed to attach to either RNA/DNA
- Main difference from primer:
- Applied to whole cells (i.e. no need to extract the nucleic acids from the cells first)
- Designed to only bind to DNA/RNA, (i.e. it is not used as a starting point for DNA replication)
• Have a fluorochrome attached to allow detection
What purines and pyrimidines bind today?
A - T
A - U
C - G
(Purines: A G)
(Pyrimidines: C T U)
What does PCR (polymerase chain reaction) allow?
Allows amplification of DNA (1 to millions in 2 hours)
• Rapidly clone DNA in a test tube instead of in a cell
PCR applications:
• Basic molecular biology research
• Generate DNA for cloning/blotting probes
• Clinical testing
• Forensic science (paternity test, one little drop of blood from a crime scene, identify a mummy as a russian tsar …)
• Bioremediation, wastewater, food engineering etc..
What’s Taq polymerase?
A heat resistant enzyme that allows sequencing of DNA.
What are the requirements for PCR?
Thermocycler (allows rapid heating and cooling)
Template DNA to be amplified
Two primers (complimentary to the 5’ and 3’ region respectively)
Taq polymerase enzyme – Heat resistant enzyme that allows to sequence DNA
Deoxynucleoside triphosphates (dNTPs) (building blocks for new DNA strands)
Buffer (reaction media)
What are the (3) stages for PCR?
Not examined on each stage - just understand that there is heating, decomposition and cooling
Denaturation step:
Heat 94-98°C for 20-30 s. Melts DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step:
T= 50-65°C for 20-40 s. Annealing of the primers to the single-stranded DNA template.
Typically the annealing temperature is about 3-5°C below the Tm of the primers used.
Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence.
The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension:
T=T optimum polymerase enzyme used (Taq polymerase = 75-80°C). Synthesizes a new DNA strand complementary to the DNA template strand.
What does GE stand for?
Gel electrophoresis
What’s GE (gel electrophoresis)?
What are it’s features?
Technique that allows for separation of DNA, RNA or proteins
Extract the DNA/RNA/Proteins out of the cell first
Consist of a box that contain a gel (often a cross linked polymer)
Electrophoresis comes from the electromotive force that is applied of this gel to move the molecules through this matrix.
Separation is based on how fast sequences migrate through the gel
Often mobility is related to mass
Each separate sequence can be removed and analysed afterwards
What does FISH stand for?
Fluorescence in situ hybridisation
What is FISH (fluorescent in situ hybridisation) used for?
To localise the presence/absence of target DNA/RNA sequences in whole cells
Main advantages:
• Do not require extraction of DNA
• No need to cultivate (immediate result)
• Spatial location of cells or genes
Applied to:
• Identify bacteria/yeasts/cells of interest
• Detect where on the chromosome a specific DNA sequence is located
How does FISH work?
The first step is to prepare short sequences of single-stranded DNA that match a portion of the gene the researcher is looking for. These are called probes.
The next step is to label these probes by attaching one of a number of colors of fluorescent dye.
DNA is composed of two strands of complementary molecules that bind to each other like chemical magnets.
Since the researchers’ probes are single-stranded, they are able to bind to the complementary strand of DNA, wherever it may reside on a person’s chromosomes.
When a probe binds to a chromosome, its fluorescent tag provides a way for researchers to see its location.
How is specific rate of product formation, qp, calculated?
qp = Y xp * mu + mp
Therefore:
r p = qpX = (Y xp * mu + mp)*X
