CE 10079 - Bioprocess Engineering Fundamentals Flashcards
What are intensive properties?
Properties independent of the size of a system.
E.g. concentration, temperature, velocity, pressure.
What are extensive properties?
Properties which depend on the size of a system.
E.g. volume, flow rate, mass, energy, force.
What’s bioengineering?
The application of the various branches of engineering, including mechanical, electrical, and chemical engineering to biological systems.
What are the typical elements of bioengineered reactor systems?
Influent (flowing in) Effluent (flowing out) Bioreactors Connections/links Recirculation Aeration processes
What’s a model?
It is a experimental nor mathematical tool to make predictions on the behavior of real systems subject to different disturbances.
It helps us to improve our understanding of the behavior of reality.
It’s analysis is more effective than direct observation of reality.
What’s a simulation?
A simulation implements a model of a system.
It allows us to perform virtual (non physical) experiments and to arrive at results, which
are transferable to reality.
What is advection?
The transfer of heat or matter by the flow of a fluid, especially horizontally in the atmosphere or the sea.
[If you have silt suspended in the water and heat it then you will get convection of the water and advection of the silt.]
What dispersion?
Advection averaged over space.
What’s sedimentation?
Directed movement relative to water.
The process of settling or being deposited as a sediment.
What’s turbulent diffusion?
Turbulent diffusion is the transport of mass, heat, or momentum within a system due to random and chaotic time dependent motions.
Turbulent diffusion or dispersion is the process by which a substance is moved from one place to another under the action of random turbulent fluctuations in the flow.
What are the 3 classifications of lipids?
Simple (fats and oils / triglycerides)
Compound (phospholipids and glycolipids)
Derived (steroids/cholesterols and carotenoids)
What are fatty acids?
Molecules which form fats and oils, consisting of a hydrophobic hydrocarbon tail and a hydrophilic carboxyl head.
They can be saturated and unsaturated.
What are triglycerides?
Molecules of glycerol and 3 fatty acids (bonded by ester bonds).
What are micelles?
Aggregates, composed of single tail lipids with hydrophilic heads and hydrophobic tails.
Their centres are usually hydrophobic also.
They form when critical micelle concentration (CMC) is reached.
How is micelle shape parameter calculated?
Ns = V / AL
Where: Ns is shape parameter V is volume of the tail A is area of the head L is max length of the tail
How is the Gibbs energy of micellization calculated?
ΔG micelle =RTln(CMC)
Where:
- ΔG is Gibbs energy of micellization
- R is universal gas constant
- T is the minimum temperature micelle forms
- CMC is critical micelle concentration (in M)
What are phospholipids?
Molecules which generally consist of two hydrophobic (nonpolar) fatty acid “tails” and a hydrophilic (polar) phosphate “head”, joined together by a glycerol molecule.
What are liposomes?
A spherical vesicle composed of phospholipids.
They often have hydrophilic centres.
What are the 2 means of liposome or micelle uptake by cells?
Phagocytosis
Receptor mediated endocytosis (RME)
How are liposomes and micelle taken up by the cell via phagocytosis?
They’re engulfed by the cell.
The cell surrounded the molecule then forms a vesicles around it.
How are liposomes and micelles taken up by cells via receptor mediated endocytosis (RME)?
The liposomes or micelles bind to receptors on the cell surface membrane, which are then engulfed and taken into vesicles.
What does K D represent? (D in subscript)
Dissociation constant (for binding between receptors and liposomes/ligands)
How is rate of association and rate of dissociation calculated (for when liposomes/micelles bind to receptors)?
Rf = kf [R] [L] Where: - Rf is rate of association - kf is association rate constant - [R] receptor conc' - [L] is ligand (liposome) conc'
Rr = kr [C] Where: - Rr is dissociation constant - kr is dissociation rate constant - [C] is conc' of complexes / occupied receptors
How is K D (dissociation constant) calculated?
K D = [R] [L] / [C] = kr / kf
Where:
- kf is association rate constant
- kr is dissociation rate constant
- [R] receptor conc’
- [L] is ligand (liposome) conc’
- [C] is conc’ of complexes / occupied receptors
How is the fraction of saturated / occupied receptors calculated?
r = [L] / [K D] + [L]
- [L] is ligand (liposome) conc’
- [K D] is dissociation constant
What’s catabolism and anabolism?
Catabolism - the breaking down of molecules, which releases energy.
Anabolism - the synthesis of complex molecules.
What is the typical notation for substrates [in bioengineering]?
Readily biodegradable, growth-limiting substrate
S - soluble materials
X - particulate materials
Subscripts - denote material type
What does the substrate notation Ss represent (an example for chemoheterotrophs)?
S - it’s a soluble molecule
s - it’s a growth substrate
Used as e– donors and carbon (C) source.
Limiting means it is essential for heterotrophic growth and cell maintenance; Examples: glucose, sucrose, fructose, maltose, or galactose, acetate.
What does the substrate notation So represent (an example for chemoheterotrophs)?
Terminal electron acceptors
Acronyms used: S - soluble o - oxygen Used as terminal e– acceptor They are growth limiting substrates.
Examples: oxygen, nitrate(SNO3), nitrite(SNO2), sulfate (SSO4).
What currency units are used in metabolic calculations?
- Electrons (e-) chemicals participating in redox reactions.
- Mass of N nitrogen containing chemicals (NH4+, NO3-, amino-acids) (N: 14 g/mol)
- Mass of P phosphate containing chemicals (e.g., PO43-, polyphosphate) (P: 31 g/mol)
What does the (change of) Gibbs free energy of a cell indicate?
The energy gain made by the cell.
It also indicates the direction of spontaneous reaction. (If negative, the reaction occurs spontaneously from left to right).
What does TOD stand for?
Theoretical oxygen demand
What is theoretical oxygen demand, TOD?
A concept to quantify e- in redox reactions.
Instead of directly referring to the number of electrons, the mass of oxygen required to accept the number of e- is measured.
It can be positive or negative.
How is TOD, theoretical oxygen demand, calculated?
Molar e- equivalents (eeq) are converted into oxygen mass equivalents.
The mass of oxygen accepting 1 electron can be determined:
1 mol O2 weighs 32g and can accept 4 electrons (2 e- per O)
Therefore 1 electron corresponds to 8g O2, thus
1 e- + 1 eeq = 8g TOD
What are the 2 fundamental types of nucleic acids?
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA)
What does the structure of a nucleotide consist of?
5 carbon pentose sugar
Nitrogenous base
Phosphate group
What are the purines and pyrimidines?
They’re nitrogenous bases.
Purines - guanine and adenine
Pyrimidines - thymine and cytosine (and uracil)
What is harder to separate, GC base pairs or AT base pairs?
Cytosine-guanine.
They form 3 hydrogen bonds between bases whereas adenine-thymine only form 2.
How do hydrophobic effects stabilise and fold the DNA helix into specific shapes?
DNA is in aqueous solution.
The hydrophilic regions are outwards and the hydrophobic regions are inwards (which keeps the DNA stable)
What are the formulae for exponential growth in cells?
dX/dt = mX
X = X0e^μt
Where: X is the cell number t is time μ is the growth constant X0 is the initial number of cells
What are the 2 types of cell death?
Necrosis (caused by trauma. Cells damaged irreversibly)
Apoptosis (programmes cell death. Natural form of death where a cell provokes it’s own death in response to a stimulus)
How does necrosis differ from apoptosis?
- Necrosis: Pathologic (due to disease) Due to cell trauma/injury Uncontrolled The cell swells and the plasma membrane collapses There is no DNA fragmentation Energy isn’t required
- Apoptosis: Physiological or pathologic Genetically programmed / suicide Controlled The cell shrinks and condenses and the plasma membrane integrity is maintained There is DNA fragmentation Energy is required
What are the formulae for the mathematical description of apoptosis?
dX/dt = mX
X = X0e^at
Where: X is the cell number t is time a is the apoptosis constant (-ve) X0 is the initial number of cells
What’s COD?
Chemical oxygen demand.
The total amount of oxygen required to chemically oxidise the bio degradable and non-biodegradable matter.
What are proteins?
Nucleic acids (polynucleotides) made from polynucleotides.
They have 7 functional classification groups:
- Structural
- Enzyme and catalytic
- Transport
- Hormonal
- Contractile
- Storage
- Defence
What are the properties of globular proteins?
Spherical or oval. Hydrophobic groups point into the centre of the globule
Usually soluble in water
Functional purpose (e.g. enzymes, transport, hormones etc.
Sensitive to pH and temp’
Irregular amino acid sequence
Examples include haemoglobin, histones and albumin
What are the properties of fibrous proteins?
Elongated shape with long chains parallel to each other. Chains are cross-linked for stability.
Usually insoluble in water
Have structural purposes e.g. bones, skin and hair.
They’re less sensitive to pH and temp’ than globular proteins
Have a repetitive amino acid structure
Examples include keratin, collagen, elastin
What do the different types of enzymes: transferases, hydrolases and lyases do?
Transferases - Transfer various groups from one molecule (donor) to another (acceptor)
Hydrolases - Use water to cleave a bond in a molecule
Lyases - create a double bond or ring
What are the 2 parts of an enzyme?
Apoenzyme (protein part)
Coenzyme / co-factor (non-protein part) which supports the role of enzymes
What are the 2 models used to explain enzyme-substrate binding?
Lock and key (complementary and precise shapes)
Induced fit (active site adjusts its shape)
How is the reaction rate of spontaneous chemical reactions defined?
For the reaction: nS -> P
Reaction rate is defined:
r = k[S]^n
r = d[P]/dt
Where: r = rate of reaction k = rate constant for the reaction n = stoichiometric coefficient / order of reaction t = time [S] = Concentration of substrate [P] = Concentration of product
What is Km?
The Michaelis-Menten constant.
It shows the concentration of the substrate when the reaction velocity is equal to one half of the maximal velocity for the reaction.
It measures affinity of the enzyme for the substrate.
What is the Michaelis-Menten equation and its inverse, the Lineweaver-Burke equation?
MM:
v = (v max * [S]) / ([S] + Km)
LB:
1/v = 1/v max + (Km/v max)(1/[S])
What do the x and y intercepts and gradient of the Lineweaver-Burke plot represent?
[1/v against 1/s]
Gradient - Km/v max
x intercept - -1/Km
y intercept - 1/ v max
What do kf, kr and kb represent in enzyme-substrate reactions?
kf - rate constant of ES formation
kr - rate constant of ES dissociation
kb - rate constant for P release from ES complex
What does k cat represent?
The catalytic constant.
It is also known as the turnover number of the enzyme - defining the maximum number of substrate molecules converted to product per unit time
How is k cat, catalytic constant, calculated?
k cat = kb = v max / [E]
What does the symbol, 𝜂, represent?
The catalytic efficiency of an enzyme.
The greater the 𝜂 value, the more efficient the enzyme.
𝜂 max is reached when kb»kr
How is the catalytic efficiency of an enzyme, 𝜂, calculated?
𝜂 = kcat / Km
= kf*kb/(kr + kb)
In the reactions:
EI E + I
and
ESI ES + I
how are constants K EI and K ESI determined?
K EI = [E][I] / [EI]
K ESI = [ES][I] / [ESI]
How can the rate of a reaction (velocity) in the presence of an inhibitor be determined?
By modification of the general equation:
v = v max / (a’ + aKm/[S])
Where:
a’ = 1 + [I] / K ESI
a = 1 + [I] / K EI
The formula can also be inversed to form an LB plot:
1/v = a’/v max + (aKm / v max)(1/[S])
What are the 3 modes of inhibition:
Competitive
Uncompetitive
Non-competitive
How does the LB plot graph of 1/v against 1/s (1/velocity vs 1/substrate) for a competitive inhibitor compare the graph for no inhibitor?
The enzyme’s affinity for the substrate is lowered
Max rate for reaction isn’t changed.
- Gradient of the graph increases (Km / v max)
- y intercept (1/vmax) is the same
- x intercept (-1/Km) increases/becomes less negative
What’s uncompetitive inhibition?
When an inhibitor binds to a site of the enzyme that is not the active site, but only if the substrate is already present.
(Non-competitive binds to allosteric site and changes active site shape)
How does the LB plot equation for a competitive inhibitor differ to that of an uncompetitive inhibitor?
Competitive:
1/v = 1/v max + (aKm/v max)(1/[S])
Uncompetitive:
1/v = a’/v max + (Km/v max)(1/[S])
How does the LB plot graph of 1/v against 1/s (1/velocity vs 1/substrate) for an uncompetitive inhibitor compare the graph for no inhibitor?
Maximum rate of reaction decreases - more time required for product to leave the active site
Km gets smaller (greater affinity for inhibitor)
- Same gradient (Km / v max)
- y intercept (1/v max) is different (larger)
- x intercept (-1/Km)is different (more negative)
How does the LB plot graph of 1/v against 1/s (1/velocity vs 1/substrate) for a non-competitive inhibitor compare the graph for no inhibitor.
- Gradient is steeper (larger Km/v max)
- y intercept (1/v max) is larger
- x intercept (-1/Km) is the same
What are the special rules that apply for the calculation of TOD of chemicals containing N and S that undergo redox reactions?
Multiply the change in oxidation state of N and S by:
- -8 gTOD/mol for e- acceptors (chemicals reduced)
- 8gTOD/mol for e- donors (chemicals oxidised)
Consider O and H atoms, and charges in the molecules with TOD = 0.
E.g. NO3-, NO2-, NH4+, H2S, SO4-
What does the symbol, Y x/s, represent?
True growth yield
This is the fraction of electrons which go to anabolism.
What is true growth yield, Y x/s?
How is it determined?
This is the fraction of electrons which go to anabolism.
Y x/s = anabolic e- uptake / total e- donated by organic substrates
What does the symbol, f𝒸, represent?
Catabolic electron fraction
This is the fraction of electrons going to catabolism.
What is (respiratory) catabolic electron fraction? How is it determined?
The fraction of electrons going to catabolism
f𝒸 = 1 - Y x/s
What does the symbol, rho, represent?
The reaction process rate
How can reaction process rate, rho, be determined?
rho = r / v
Where:
rho is the process rate
r is the reaction rate of a species
v is the associated species’ stoichiometric coefficient
(Value of stoichiometric coefficient, v, is +/- 1 as it is used to normalise other species
What are typical units of mass-based and molar-based stoichiometric coefficients?
v j,i = r j,i/rho j
stoichiometric coefficient = species rate / process rate
Thus,
Molar-based:
mol(i)/mol(j,p)
Mass-based:
M i/M j,p
How is the growth process rate, rho growth, calculated?
rho growth = dX/dt = mu*X
Where:
- rho growth is growth process rate
- Mu [1/T] is specific growth rate (parameter varying as a function of substrate conc’)
- X [M/L^3] is microbial biomass conc’
How is the decay process rate, rho decay, calculated?
rho decay = dX/dt = b*X
Where:
- b [1/T] is specific decay rate (constant for a given culture)
- X [M/L^3] is microbial biomass conc’
How is specific growth rate, mu, calculated by the Monod kinetic equation with a single substrate?
mu = mu max *(Ss/(Ks + Ss))
Where:
- mu max [1/T] is max specific growth rate
- Ks is the half-saturation coefficient
- Ss is the growth substrate (e- donor) conc’
How is specific growth rate, mu, calculated by the Monod kinetic equation with 2 substrates?
mu = mu max *(Ss/(Ks + Ss))(S O2/(K O2 + S O2))
Where:
- mu is the specific growth rate
- Ss is growth substrate conc’
- S O2 is dissolved oxygen (terminal e- acceptor) conc’
When using 2+ switching terms (2+ substrates) the mu value will correspond to the lowest S (Ss or S O2) value.
How does the Ks value indicate microbe affinity to substrate Ss?
The lower the Ks value (and steeper the mu/Ss graph gradient), the greater the microbe affinity to substrate.
How is the process rate for X B,H defined (using Ss and S O2), by using substitution of the catalytic electron fraction equation?
Catalytic electron fraction:
f𝒸 = 1 - Y x/s
Therefore,
Ss + x S O2 -> x X B
can be written:
(1/Y x/s)Ss + ((1-Y x/s)/Y x/s)S O2 -> X B
What effect does increasing temperature have on enzymes and the rate of reaction?
It increases reaction rate by increasing kb.
Stability of the enzyme decreases.
What effect does temperature have on enzyme activity?
Increased temperature leads to more energetic collisions between molecules, more collisions per unit time and the heat of molecules in the system will increase.
What is the Arrhenius equation?
It describes how reaction rate kr varies with temperature.
It’s a measure of the rate of successful collisions between reactants.
kr = Ae^(-Ea/RT)
How is the Arrhenius equation plotted?
What are features of the plot?
ln kr = lnA - Ea/RT
ln k against 1/T plotted
Gradient = -Ea/R
Y intercept = ln A
How can the Arrhenius equation be used for enzymes, using kb, to calculate V max for enzymes?
kb = Ae^(-Ea/RT)
and kb = V max/[E]
Therefore:
Vmax = A[E]e^(-Ea/RT)
How do enzymes become destabilized due to temperature?
At high temperatures, high kinetic energy leads to inter and intra molecular bonds breaking. This causes the protein to denature.
[E native] -> [E unfolded]
Which can be written:
d[E native]/dt = -kD [E native]
How can the rate of native enzyme destabilization be determined?
d[E native]/dt = -kD [E native]
[E native] = [E native 0]e^-kDt
Where:
[E native] is conc’ of native enzymes
kD is the dissociation constant
What are carbohydrates?
A biological molecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms.
Roles include: storing energy, structural components and nutrition.
Can be found as monosaccharides, disaccharides and polysaccharides.
What are Daltons?
Unit to express molecular weight of proteins, equivalent to atomic mass.
What are homo and heteropolysaccharides?
Homo - one type of monosaccharides
Hetero - more than one type of monosaccharide
How is elastic (young) modulus calculated?
E mod = stress / strain
What is the Monod equation?
Kinetic model to describe the growth rate of cells as a function of the substrate concentration.
= Smax/(Ks + S) = 1/X dX/dt
= mu = 1/t ln(X/X0)
X: cell number
t: time
μ: specific growth rate (1/time)
μmax: maximum specific rate growth (1/time)
S: substrate concentration
KS:‘half saturation’ constant => the value of S at
μmax/2
X0: initial number of cells in the reactor
What’s the general mass balance equation?
(Mass in) -(Mass out) + (Mass gen) - (Mass con) = (Mass accum)
What’s a differential mass balance?
A mass balance based on rates.
For continuous processes, the information is collected for a particular instant in time.
What’s an integral mass balance?
Mass balances for batch and semi batch processes, where information is collected over a period of time rather than a particular instant.
What is bioprocess/product yield, Y ps?
The ratio of amount of bioprocess produced to the amount of substrate consumed.
How is bioprocess/product yield, Y ps, calculated?
Y ps = mass product / mass substrate consumed
= nMr (p) / nMr (s)
What are the methods of entry of molecules into cells?
Diffusion
Facilitated diffusion (carrier proteins)
Active transport
What are the basic disciplines of bioprocessing?
- Microbiology – Microbial Physiology
- Biochemistry – Enzymology
- Genetics – Molecular biology
- Engineering – Mass & Energy Balances, Reaction Engineering, Fluid Mechanics
- Process Design & Control
- Economic Evaluation
- Marketing & Sales
- Public Relations
How is the appropriate microorganism selected?
A microorganism is needed that:
- forms a desirable end product
- can be artificially cultivated
- is biologically uniform
What’s considered when selecting the appropriate raw materials?
- Are the raw materials (substrates) economically viable?
- Are the raw materials readily available?
- Are the raw materials renewable or sustainable?
• What other inputs are necessary beyond the main
feedstocks?
What are the stages in a typical batch fermentation process?
Lag phase - cell needs time to adapt to environment. μ = 0
Acceleration phase - growth starts. 0< μ < μ max
Growth phase - growth rate is at its highest. μ ≈ μ max
Decline phase - growth rate starts to fall due to lack of resources or inhibitory compounds. μ < μ max
Stationary phase - growth rate stops. μ≈0
Death phase - negative growth rate, more cells are dying than being created. μ <0
(Of cell concentration. Meanwhile, substrate concentration is relatively constant and high, until the acceleration phase of the cell concentration. Substrate concentration then linearly decreases)
What is the Monod equation accounting also for cell death?
μ = [μ max S / (Ks + S) ] - kd
Where kd is the biomass death rate (typically per hr)
What are the (6) processes determined in the STT matrix?
Stoichiometric transfer-transport
Growth
Decay
Influent mass transport
Effluent mass transport
So mass transfer
Excess biomass wastage mass transport
What are the (6) processes determined in the STT matrix and what are their process rate equations?
(Stoichiometric transfer-transport)
Growth
μ·Xb
Decay
b·Xb
Influent mass transport
Q,inf/v · s,inf
Effluent mass transport
Q,eff/v · s,eff
So mass transfer
KLa ·(S*o - So)
Excess biomass wastage mass transport
Q,was/v · Xb [or Q,was/v · S]
What are the 6 process rate equations?
Growth
μ·Xb
Decay
b·Xb
Influent mass transport
Q,inf/v · s,inf
Effluent mass transport
Q,eff/v · s,eff
So mass transfer
KLa ·(S*o - So)
Excess biomass wastage mass transport
Q,was/v · Xb [or Q,was/v · S]
What’s kLa?
The volumetric mass-transfer coefficient that describes the efficiency with which oxygen can be delivered to a bioreactor (for a given set of operating conditions)
What is considered to produce the ideal (bio)process?
Are the genetic and metabolic pathways understood?
Is there good conversion of raw materials to product?
Is rate of fermentation reasonable?
Are the products readily recoverable and can be purified and packaged?
What are first and second generation feedstocks?
First - is feedstock/fuel produced directly from food crops (starch, sugar, animal fats and vegetable oil)
Second - is not produced from food crops
What are the main stages of the fermentation process?
Feedstock is fed into a reactor for pre-treatment.
This forms polysaccharides which undergo enzymatic hydrolysis to form sugars.
Yeast is also added to the sugars. They then undergo fermentation to form biomass (in the form of alcohol, cells etc.)
Filtration and separation are then carried, forming the products: bioethanol, biproducts and electricity.
What can lignocellulose be used for?
Formation of bioethanol.
