1
Q
What does is Red Biotechnology contain?
A
1) Health applications
2) Medical applications
e.g.
Gene therapy, Drug developement
2
Q
What does Green Biotechnology contain?
A
1) Agriculture applications
2) Farming apllications
e.g.
Pest resistant crops, Probiotics for farm animals
3
Q
What does white biotechnology contain?
A
1) Manufacturing
2) Industrial
e.g.
Biopolymer production, Biofuel-producing microalgae
4
Q
What does grey biotechnology contain?
A
1) Environmental
e.g.
Bioremeditation of chemical spills, Gene drives to control invasive species.
5
Q
What does Pink biotechnology contain?
A
1) Human Welfare
2) leisure
e.g.
Anti-hangover probiotics, Glowing bacterial lamp
6
Q
What does Yellow BioTechnology contain?
A
1)Food processing
2) Nutrition
e.g.
Brewing, lactose free dairy
7
Q
What does Brown Biotechnology contain?
A
1) Improving living conditions in arid and desertic areas
8
Q
What does Blue biotechnology contain?
A
1) Sustaining water resources
e.g. GM fishes for fish farms wastewater
9
Q
What are key properties of Cell Factories?
A
1) Saftety
- Non-pathogenic.
- Safe for environment.
2) Versatility
- Can utilize different substrates.
- Produce multiple products.
3) Defined metabolic and genetic background
- Genome sequence is available.
- Most important metabolic pathways are known.
10
Q
What types of microorganisms are there?
A
- Bacteria
- Fungi
- Yeast
11
Q
What cell cultures exists?
A
- Plant cells
- Insect cells
- Animal cells
12
Q
What facts do you know about bacteria?
- Eukaryotic or Prokaryotic (compartments or not)?
- size?
- Growth?
- Low, medium or high Metabolic activity?
- Amount of medium required?
- GRAS?
A
- Prokaryotic (with no compartments)
- size: 0.2-20um
- Growth: t_2 = 0.2-1 hour (or longer)
- High metabolic activity
- Ease of cultivation = minimum medium required.
- Many GRAS
13
Q
What facts do you know about Yeast?
- Eukaryotic or Prokaryotic?
- size?
- Growth?
- Ease of cultivation in what?
- low or high pH tolerance?
- GRAS?
A
- Eukaryotic
- size: 10 um
- Growth: t_2 = 2-10hour
- ease of cultivation in defined media.
- Tolerance of low pH (2-5)
- Mainly GRAS
14
Q
Saccharomyes cerevisial:
- What type of microorganism ?
- What does it produce?
A
- Yeast
- Beer and baking yeast: ethanol
- Protein and enzyme expression
15
Q
Komagataella phaffi (pichia pastoris):
- What type of microorganism ?
- What does it produce?
A
- Yeast
- enzyme and protein expression
- Methylotrophic (methanol utilization)
16
Q
Yarrow Lipolytica:
- What type of microorganism ?
- What does it produce?
A
- Yeast
- Oleaginous yeast = lipid producing.
- Organic acids and sugar alcohols
- Protein and enzyme expression
17
Q
What facts do you know about Fungi?
- Eukaryotic or Prokaryotic ( compartments or no)?
- size?
- Ease of cultivation in what?
- low or high pH tolerance?
- GRAS?
A
- Eukaryotic, with compartments
- up to 100um
- Ease of cultivation in defined media.
- Tolerance of low pH (2-3).
- Mainly GRAS
18
Q
What does GRAS mean?
A
Generally Recognized As Safe
19
Q
Aspergillus niger:
- What type of microorganism ?
- What does it produce?
A
- Fungi
- Produces: citric acid
20
Q
Penicillium chrysogenum:
- What type of microorganism ?
- What does it produce?
A
- Fungi
- Produces: Penicilin.
21
Q
Trichoderma reesei:
- What type of microorganism ?
- What does it produce?
A
- Fungi
- Produces: cellulases and hemicellulases.
22
Q
What do you know about ethanol production?
- produced by?
- produced from?
- type of process (and why)?
- Bioreactor scale?
A
- Produced by baker’s yeast: saccharomyces cerevisiae.
- Produced from sucrose
- Fed-batch ( to avoid
catabolite repression + continuous ethanol removal to avoid excess biomass formation. - Bioreactor scale = 500m^3
23
Q
What do you know about Citric Acid production
- Produced by?
- What substances are highly needed?
- What pH is needed?
- Citric acid used in?
A
- Produced by Aspergillus niger.
- High substrate (glucose) and oxygen needed
- Low pH
24
Q
What do you know about Glutamic Acid Production?
- Produced by?
- substrate = ?
- Derived from?
- Glutamic Acid used as?
A
- Produced by C. glutamicum
- Substrate = cheap sugar sources e.g. starch.
- Derived from TCA (tricarboxylic acid)
- used as flavour inhancer (“umami”)
25
What do you know about 1,3-propanediol production?
- produced by?
- How is microorganism engineered to enable this production?
- produced by Escherichia coli.
- Metabolically enginnered by deletion of sideways.
26
What forms of recombinant proteins exists?
- Soluble protein (e.g. cytoplasm)
- Inclusion bodies (IB) = aggregated expressed protein
- Fusion proteins = fusion partner can make protein purification easier
27
What recombinant protein products exist?
- Hormones
- Growth factors
- Cytokines
- Enzymes
- Blood clotting factors
- Vaccines
- Monoclonal antibodies
28
Four examples of technical substrates and their contents.
1) Mono-/disaccherides
- Fruit juices (10-30%)
- starch/cellulose hydrolysates (70%)
- Glucose hydrate
2) Sucrose
- Sugar beets, sugar cane (15%-22%)
- raw suger
- molasses
3) malt extract
- fermentable sugars
- nitrogen, vitamins (1%)
4) Lactose: milk/whey (3-8%)
- Natural form or dried
- pure lactose
29
What is the Crabtree effect?
Higher glucose concentration in fermentation broth (>100mg/L) leads to ethanol formation in the presence of oxygen (S.cerevisiae)
30
What is the law of conservation?
Mass in a closed system remains the same during a chemical reaction
Matter cannot be destroyed but rearranged:
mass of educts = mass of products
31
What process types exists?
- Batch Process
- Semi-batch Process
- Fed-Batch process
- Continuous process
32
What is characteristic about a Batch process?
- All materials are added to the system at the start of the process.
- Closed through reaction
- End products removed when the reaction is complete
33
What is characteristic about the semi-batch process?
- Allows either input or output of mass (not both).
34
What is characteristic about the fed-batch process?
- Allows input of material into the system but no output.
35
Whats is characteristic about the continuous process?
- Allows matter to flow in and out of the system
- If rates of mass input and output are equal, then continuous process can be operated indefinetly.
36
Formular: Rate of accumulation of mass within system
dM_i/dt = [Accumulation] = [in]-[out]-[consumption]
37
Formular: Convective mass flow rate.
[convective mass flow rate] = Volumetric flow rate * m/V
38
Fick's Law (Diffusion of components)
j_i=-D_i * d c_i/d Z_i
j_i = diffusion flux of component
c_i = concentration of component
D_i = diffusioncoefficient of component
Z = position (dZ = position difference)
39
Formular: total mass flow
total mass flow = F*ρ
F = force (A*v)
*A = area, v = velocity
ρ = density
40
Formular: Component mass flow (M_i)
M_i = F*c_i
F = force (A*v)
*A = area, v = velocity
c_i = concentration of component
41
Formular: substrate consumption rate (r_s)
r_s = r_x/ (Y_x/s) * V
r_s = substrate consumption rate
r_x = growth rate per volume
Y_x/s = yield = mass produced per substrate
42
How many available electrons does the following atoms have: C,H,O,P,S, N(ammonia, nitrate, molecular).
C = 4
H = 1
O = -2
P = 5
S = 6
N (amonia) = -3
N (nitrate) = 5
N (molecular) = 0
43
Formular: Degree of reduction?
DoR = (Available electrons_i*Number of that atom_i)/Number of carbon atoms
44
What are the molecular components of biomass?
- Protein: 32-55%
- Carbohydrates: 9-49%
- Lipids: 7-8%
- Nucleic Acids: 5-23%
- Ash: 5-10%
45
What are the growth phases in batch cultures?
- Lag
- Acceleration
- Growth
- Decline
- Stationary
- Death
46
What is the michalis-menten equation?
v = v_max*[S]/(K_m+[S])
47
What is the Lineweaver-Burke equation?
1/v=K_m/v_max*1/[S]+1/v_max
48
What is competitive inhibition?
And what is the formular?
- Substrate and inhibitor are similar and compete for binding to the enzyme.
v = v_max* [S]/(k_m*(1+[S]/[I])+[S])
49
What is uncompetitive inhibition?
And what is the formular?
Inhibitor does not bind the free enzyme but the enzyme-substrate complex.
v = v_max*[S]/(k_m+[S] *(1+([I]/K_I)))
K_I = inhibitions constant
50
What is non-competitive inhibition?
And what is the formular?
Inhibitior can bind to enzyme or enzyme-substrate complex.
v = v_max*[S]/((1*[S]/K_I)*(K_m+[S]))
51
Formular: Mass balance accumulation in bioreactor.
[Accumulation] = V_R* dc_i/dt + c_i*dV_R/dt
52
What is the T-R-Y metrics?
- Titer of product [g/L] "How much?"
- volumetric Rates [g/L*h]
"How fast?"
- Yield [g/g]
"How efficient?"
53
What is a volumentric rate (+ formular)?
r_i = change in concentration
r_i = dc_i/dt
54
Yield Coefficient
Y_i/j = dc_i/dc_j
e.g.
Y_i/j = dc_i/dc_j = (2.5-0)/(10-0) = 0.25 g/g
55
What is the respitatory coefficient? And how is it calculated?
- Gives information about metabolic activity. '
- Vales should be around 1
- if RQ is not = 1, then the product has low molecular weight.
RQ = CTR/OUR
CTR = carbondioxide production rate
OUR = oxygene uptake rate
56
Formulars: Batch-process mass balance (3 formulars)
- Biomass: r_x = dc_x/dt = v*[x]
- Substrate: r_s = d [S]/dt = r_x/Y_x/s
- Product: d[p]/dt = (k_1+k_2*v) * [x]
57
Formulars: fed-batch-process mass balance (3 formulars)
- Biomass: r_x = q_x * [x] = v*[x]
- Substrate: r_s = q_s * [x]
- Product: r_p = q_p * [x]
--> Needs to increase the substrate constantly to keep a constant growth rate (need for more substrate as more cells are produced).
58
Formular: substrate uptake.
q_s = a * v + b * q_p + m_s
q_s = substarte uptake rate
a = a coefficient
v = growth rate
b = b coefficient
q_p = product formation rate
m_s = substrate rate used for maintanance.
59
What are shifts in transient characterization?
- Needed during start-up of contineuos culture
- Can be used to establish "new" steady state conditions
- e.g. shift of D/v, pH, substrate in feed.
60
What is pulse?
- Addition of compound to a continuous culture in steady state.
-Removal of compound also represents a pulse but only possible for gaseous compounds, not liquid.
61
What types of Auxostats do you know?
- Chemostat (constant)
- Turbidostat/ permittistat (Optical density)
- pH-auxo (pH)
- nutristat (substrate concentration)
62
What is a chemostat?
- system in which the chemical composition is kept at a controlled and constant level.
- Fresh medium is continuously added at same rate as product is removed.
- v < vmax
63
What is a Turbidostat?
- A continuous microbiological device.
- Has no limiting nutrient
- Turbidity/ permittivity of the medium is constant
- Dilution rate varies
- Turbidity/Permittivity of the medium is constant
64
Formular: Oxygen Transer Rate (OTR).
OTR = N_a = K_L * a * (c*-c)
K_L = can be changed through the process
c* = max concentration of gas in reaction
c = concentration measured
(c*-c) = the driving force
65
What bioreactors exists?
- Stirred tank reactor (STR)
- vibromixer
- Bubble colums
- Tubular tower fermenter
- Airlift bioreactors
- Fluidized bed bioreactor (FBR)
- Trickle-bed bioreactor
- disposable bioreactor
66
What are the advantages of a Stirred Tank Reactor (STR) ?
- Homogeneous distribution
- Adequate mixing and aeration
- Temperature control
- Aseptic and reliable
- low energy demand
- pH demand
- easy to handle
- Material stability
67
What are the typical scales for production of different product types sterile and unsterile?
Sterile:
- Tissue culture = 100L
- DNA products = < 5M^3
- Vaccines < 5 m^3
- Insulin = 40 m^3
- Ethanol = 70 m^3
- Protease = 4-100 m^3
- yeast = 200 m^3
- Penecilin = 40 - 200 m^3
- Beer = 250 m^3
- Enzymes = 200 - 800 m^3
Unsterile:
- Wastewater treatment = 20000 m^3
68
What are advantages and disadvantages of a vibromixer reactor?
+
- Gentle mechanic mixing
- Little shear forces
- Very good oxygen transfer (OTR)
- high efficiency of mixing plate
- Used to generate emulsions
%
- complicated construction and difficult scale-up
69
What are advantages and disadvantages of a Bubble column reactor?
+
- pneumatic mixing
- simple design and little shear forces
%
- High air consumption
- long mixing times
- foam formation
70
What are advantages and disadvantages of a Turbular Tower Fermentor (reactor)?
+
- perforated plates installed better OTR (use of air)
%
- difficult mixing of cells
71
How does a Fluidized bed bioreactor (FBR) work?
- Fluid pumped through solid carriers
- Materials (enzyme, cells) at lower fluid velocity = carriers remain in place
- Fluid velocity increases = carriers swirl around = fluidized bed reactor
- often used in gasoline and polymer industry
72
How does a Trickle-bed bioreactor work?
- Liquid pumped through solid carriers
- fine film formed on particles
- continuous operation possible
%
- complex hydrodynamics and mass transfer events.
73
What are advantages and limitations about disposable bioreactors?
+
- No sterilization or cleaning required
- Less validation effort
- Increased flexibility especially in multipurpose facilities.
- Increased turnover time.
%
- Limited scale (< 3000L)
- High consumable cost
- extractables
74
How does a Solid State fermentor work (SSF)?
- microorganisms grown on a solid support
- Used for filamentous fungi (e.g. cellulose production)
- Rotating drum bioreactor
75
Formular: How is the Reynolds number (RE) calculated?
RE = (D * u * ρ)/ μ
D = diameter of pipe [m]
u = avaerage linear velocity [m/s]
ρ = fluid density [kg/m^3]
μ = fluid viscosity [Pa*s][kg/(m*s)]
76
Formular: For specific Reynolds number, how is the Re_i calculated?
Re_i = (N_i*D_i*ρ)/μ
D = diameter of pipe [m]
N_i = stirrer speed [1/s]
ρ = fluid density [kg/m^3]
μ = fluid viscosity [Pa*s][kg/(m*s)]
77
What are shear forces in a bioreactor?
- Interaction between cells and fluid turbulance.
- collision with other cells
- Bubbles traveling through liquid
- As turbulence increases eddy size will decrease and shear forces will increase
78
How can you reduce shear stress?
-Medium addtives e.g. pluronic68
- reduce gas flow
- adapt stirring configuration
- smaller systems: bubble free aeration
79
What does the required power for bioreactors depend on?
- stirrer speed
- impeller shape and size
- tank geometry
- fluid density
- viscosity
80
Formular: What is the power number (Np)?
Np = P/(ρ*N_i^3*D_i^5)
Np = power number [dimensionless]
ρ = density [kg/m^3]
N_i = stirring speed [s^-1]
D_i = diamenter of impeller
81
What types of aeration in bioreactor are there?
- Head space aeration
- self-priming stirrer
- pressure aeration
- membrane aeration
82
What is the definition of defusion?
- Molecule movement in mixture due to concentration difference
- Diffusion occurs in direction to distroy the concentration gradient.
- Molecular diffusion is described by Fick's 1st law of diffusion
83
Formular: (diffusion coefficient) Stokes-Einstein Equation
D_AB = k*T/6*pi*η*r_H
k = Boltzmann constant
T = temp. [K]
η = dynamic viscosity of the liquid
r_H = hydrodynamic radius of particle [m]
84
Which formular is Mass trasfer rates formular identical to?
Oxgygen tranfer rate (OTR)
= k_L*a*(c*-c)
85
Formular: What is Henry's law?
P_AG = H * C_AL* = P_T * Y_AG
P_AG = gas partial pressure of A in gas [bar]
H = Henry constant [mol/m^3*Pa]
C*_AL = max solubility of A in the liquid [kg/m^3]
p_T = total gas pressure [bar]
Y_AG = conc. A in the gas (mole fraction)
86
What is foam in reactor tank?
- Foam = trapped gas bubbles in a liquid
87
What can you use against foam and how does it work?
- ANti foam = organic and silicone based types
- They destabilizes foam lamellas.
88
Formular: What is the formular for heat transfer?
Q = U * A * dT
Q = rate of heat transfer [w]
U = heat transfer coefficient [w/(m^2*k)]
A = area of heat transfer [m^-2]
dT = temp.diff. (reactor cooling) [K].
89
What is the goal of a sccale up?
To increase the working volume, but to keep physiology and productivity constant
90
Formular: What is the scale-up criterion:
P/V = Np*P*N^3*D^5/V
91
What it the scale up criterion for mixing time?
t_m = V/N*d^3
V = working volume
N = impeller rotational speed [rpm]
d = impeller diameter
t_m = mixing time
92
In the topic sensors what are CCPs?
Control and monitor Critical Process parameter (CCPs)
93
In the topic sensors what are CQAs?
ensure Critical Quality Attributes (CQAs)
94
Sensors - what to consider?
- types of sensing - what is being monitored
- How is parameter measured
- precision and accuracy
- Resolution (range)
- Calibration and repeatability
- stability
- linearity
- response time
- power consumption
- costs
95
What classifications of sensors are there?
- Acoustic
- Magnetic
- Mechanical
- Electrical
- Temperature
- Optical
96
How does Gas Chromatography work (GC)?
- Used for analysing and seperating compounds that can be vaporized (without decomposition)
- seperates compounds through a stationary phase and a mobile phase (carrier gas)
97
Formular: Capacitance (Di-electric spectroscopy)
C = Q/U
C = capacity [farad]
Q = charge [coulomb]
U = voltage [Volt]
98
What is flow cytometry (FCM)
- is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles
--> number of cells
--> morphological characteristics
- Lasers used to produce both scattered and flouresent light signals
Flourosent reagtens:
- DNA binding dyes
- ion indicator dyes
- fluorescently binding dyes.
99
What are advantages and disadvantages of flow cytometry?
ADVANTAGES:
- Characterization at single cell level
- Several attributes measured at once
- Good differentiation between OM and IM
DISADVANTAGES:
- Expensive and toxic
- high sample dilution strains
- complex automation
100
What types of spectroscopy are there?
- IR- spectroscopy
- RAMAN spectroscopy
- Fluoresent spectroscopy
- NMR spectroscopy
101
How does spectroscopy work?
- Spectroscopy is electromagnetic radiation as a function of its wavelength or frequency in order to obtain information concerning the structure and properties of matter
102
What is high Performance liquid chromatography? What can there be seperated based on?
- Based on interaction of analyte with stationary phase and mobile phase.
Seperation principles:
- Normal phase (stationary phase polar)
- Reversed phase (stationary phase polar)
- Ion exchange
- Size exclusion
- Affinity
103
What is IM integretity and OM? integretity?
The cell envelope of gram-negative bacteria, a structure comprising an outer (OM) and an inner (IM) membrane, is essential for life.
IM Integretity= Im integretity marker = substances occuring in the cytoplasm
OM = OM Integretity marker = substances occouring in the periplasm
104
What tools can be used to measure density?
- hydrometer
- pycnometer
- hydrostatic balance
105
What tools can be used to measure viscosity?
- ubbelohole viscosimeter
- Rotational viscosimeter
- vibrational viscosimeter
106
What is PAT?
PAT = technology to design, analyse and control pharmaceutical manufacturing processes.
1) multivariate data aquistion
2) Process analytic tools
3) knowlegde mangement tools
107
What is downstream processing (DSP)?
Downstream processing (DSP)
= Recovery and purification of biosynthetic products.
GOALS:
- concentration (removal of water)
- Purification (removal of contaminants)
- Polishing (removal of adventitious agents).
108
What are the differences between upstream and downstream processing?
Upstream:
- Live organisms
- Large volumens
- many components
- Heterogeneous mixture
- capital intensive
- Automated
- Few operations
- lower cost materials
Downstream:
- No organisms
- Progressively smaller volumes
- Progressively fewer components
- homogeneous solution
- labor intensive
- manual
- many operations
- higher cost materials
109
How can cells be lysed?
- Osmotic shock
- Thermal (freezing)
- Chemical Treatments
110
Formular: What is the Bernoulli principle?
e = ( u^2/2 )+ ( p/ρ ) + g*z
e = energy
u = velocity
p = pressure
ρ = density
g = acceleration
z = height/distance
111
Formular: What is Stoke's law?
Describes the movement of a sphere/particle in a gravitational field:
v = d^2*g*(ρ_p-ρ_s)/18*η
v = velocity
d = particle diameter
g = gravity
ρ = density (solvent or particle)
112
What is the sigma factor and its formular?
Factor to compare centrifuges and assist in scale-up:
Sigma = Q/u_T
Sigma [m^2]
Q = [m^3/s]
u_T = thermal velocity of particles
113
What types of centrifugations are there?
- Tubular Bowl centrifuge
+ high centrifugal accelaration
% low sigma factor
- Multichamber separator
- Disc stack centrifuge
+ continuous operation possible
% high price
114
What is Normal Flow Filtration (NFF) and its characteristics?
- dead end filtration (DEF)
- single pass through filter
+ easy toperate
+ low residence time of product
% large membrane area required
% expensive scale-up
115
What is Cross Flow Filtration (CFF) and its characteristics?
- Tangential Flow Filtration (TFF)
- continuous feed stream
+More efficient use of mambrane
+ linear scale up
-product recirculated in retenrate flow (long recidense time).
116
What are performance factors for protein chromatograpy?
- column efficiency
- chromatographic resolution R_s
- Dynamic Binding Capacity
117
Formular: What is Lambert-Beers law?
A = ε * b * c
A = absorbance
ε = molar extrinction coef.
b = path length (1cm)
c = concentration
118
What are different methods for continuous chromatography?
- Periodic countercurrent chromatography (PCC)
- Continuous countercurrent Tangential chromatography (CCTC)
- Simulated Moving Bed Chromatography (SMB)
- Continuous Annular chromatography (CAC)
119
What is a medium called that contains only glucose, ammonia, salts and trace elements in defined but not optimized concentrations?
Synthetic medium
120
What examples do you have of products that Aspergillus niger is used to produce industrially?
- citric acid production
- Amylase (and other enzymes)
- Pectinase
- Other organic acids
121
Which metabolically engineered microorganisms is used for commercial production of 1,3-propanediol?
Escherichia coli
- deletion of side pathways
122
What is an inclusion body?
Aggregated misfolded proteins
- Formed due to overexpression of recombinant protein.
123
What is mass/matter conservation?
Mass of educts = mass of products
- Mass in a closed system remains the same during a chemical reaction. Matter cannot be destroyed but rearranged.
124
Which inputs are used for differential mass balance?
Differential balance:
- mass balance based on rates
Integral balance:
- mass balance based on mass quantities
125
What is the number of available electrons for glucose (C6H12O6)?
Available electrons = 4*6 + 1*12 - 2*6 = 24
126
What is the degree of reduction of ethanol C2H6O?
Available electrons = 4*2 + 1*6 -1*2 = 12
Degree of reduction = 12/2 = 6
127
What does the ks value of a microorganism tell about its relationship to a substrate?
It defines at what substrate concentration the enzyme will have reached it's 1/2 vmax.
- It tells how efficient the enzyme works. the smaller ks is the more efficient the enzyme works.
128
What bioreactor volume is needed to make 4.8 kg citric acid per day when A.niger shows a productivity of 10g/(L*h)?
10g/(L*h) * 24h * V[L] = 4800g
240 (g/L) * V[L] = 4800g
V[L] = 4800g/240g
V[L] = 20
129
What is the specific growth rate μ of cells in retentostat operated at a retention rate of 0.9 and dilution rate D of 0.2 h^-1?
Cell retention systems (retentostat) = rate of cell retention can be controlled.
μ = (1-R) * D
μ = (1-0.9) * 0.2 = 0.02
130
What can be used to simulate scale-up?
CFC = Computer Fluid Dynamics
Biochemical Engineering
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