Lecture 4: Downstream Processing

Question 1

What is mass transfer?

Answer 1

“process of transport from one phase to another”

Phases can be: liquids, solids or gases

Phases can be miscible or separate e.g., gas & liquid, solid & liquid, oil & water

Concentration difference typically drives transferW

Question 2

What are the applications of mass transfer?

Answer 2

Distillation
Crystallisation
Gas adsorption
Nutrient supply

Question 3

What are the phases of mass transfer

Answer 3

1. Transfer within gas/oil to liquid interface
2. Transfer across gas/solid/oil interface into liquid phase
3. Transfer from liquid to cell boundary
4. Diffusion across cell boundary to cell surface
5. Diffusion into cell to reaction center

Question 4

What are the principles of mass transfer?

Answer 4

1. Mass transfer can limit growth by restricting nutrient supply
2. Defined by Ficks Law
   “J = -D deltaC/deltaX”
3. Growth is limited via:
   A. in phase transfer: slow
   diffusion or long
   distance
   B. Between phase transfer:
   exchange across
   interface can be rate
   limiting. Large clusters
   of cells can deprive
   middle cells of nutrients
   C. Across cell membrane:
   unless transporters,
   diffusion can be slow

Question 5

What are the effects of limited mass transfer?

Answer 5

1. limitation of output:

a. overall reaction rate = slower. Eg. formation of Glucoronic acid from glucose by Gluconobacteroxydans

b. Alternate metabolic pathways switched on. E.g., preparation of bakers yeast

2. Large scale bioreactors designed to optimise mass transfer

Question 6

How can mass transfer be optimised in bioreactors?

Answer 6

1. mixing: without shear force, heterogeneity in the chemostat
2. Particle size: small separate granules dissolve faster, smaller gas bubbles increase SA
3. Pressure: can help with gas/liquid driving force

Question 7

What are the different types of bioreactor?

Answer 7

1 Stirred tank reactors
2 bubble column bioreactors
3 airlift bioreactors
4 solid & fluid phase bed reactors

Question 8

What is the stirred tank bioreactor?

Answer 8

1. stirred tank vessels
2. aeration via gas exchange
3. baffles connected to stirrer
4. mixing through stirring, controls nutrient and gas distribution
5. Typically multiple baffles to increase mixing
6. impeller type, length, and liquid viscosity are related to “shear rate” (measure of mixing efficiency)
7. Shear rate is related to “shear stress” (which can damage cells) & also E req to turn impellers in viscose liquids
8. Shear stress can be minimised by larger fermenters, stirring at 120rpm for animal and plant cells, filamentous and mycelical cultures. Planktonic microbials >300rpm

Question 9

What is a bubble column bioreactor?

Answer 9

1. Gas stream forms tiny bubbles at base of reactor
2. density causes bubbles to rise
3. diffusion of gas through the reactor from bubbles
4. mixing of nutrients through bubbling

Question 10

What are the pros and cons of bubble column bioreactors?

Answer 10

+ high aeration with mixing
- Foaming a problem
+ suited for wastewater and less viscous fermentations
- poorly controlled and can be poor mixing

Question 11

What are airlift bioreactors?

Answer 11

1. Based on bubble column
2. gas used to create a flow, causing nutrient mixing
3. concentric is most common
4. multiple types of ALR:
   a. internal-loop split
   b. internal-loop concentric tube reactor
   c. External loop

Question 12

What are fluid and solid phase bioreactors?

Answer 12

1. Particles used to prod product from feed
2. Particles sink away from effluent which is harvested
3. solid phase can contain bacterial biofilms, enzymes, etc
4. no gas, so generally anaerobic

Question 13

What are hollow fiber bioreactos?

Answer 13

1. often used for mammalian cells
2. porous fibers stimulate blood vessels
3. nutrients and waste flow through hollow fibers
4. cells can grow on surface of fibers
5. cells and secreted proteins can accumulate in the interfiber space

Question 14

What is downstream processing?

Answer 14

1. Products of microbial activity include: biomass & or products trapped inside cells, soluble & secreted products
2. most of processing can determine viability of commercial process
3. yield is lost at each step of an isolation or purification
4. multiple products can sometimes be obtained from a single fermentation

Question 15

What is clarification

Answer 15

1. separation of cells from liquid
2. multiple methods depending on cells:
   A. waste water/brewing use settling/floccation
   B. centrifuging can be used on smaller cells
   C. Filtration often used to remove small/trace cells

Question 16

What is cell lysis

Answer 16

1. Mechanical: bread mills, homogenisers, ultrasonic disruption
2. Non-mechanical: osmotic shock, chemolysis (surfactants)

Question 17

Concentration of products in liquid?

Answer 17

1. Evaporation: can help for volatile products as part of distillation. Liquid can be frozen (lyophilisation)
2. Precipitation: addition of salts (NH4)2SO4 to precipitate proteins. Addition of organic solvents to precipitate organic compounds (will denature proteins)

Question 18

How are products purification

Answer 18

1. Extraction: Many organics are extracted using organic solvents (antibiotics, vitamins, alcohols, etc)

A. add non-miscible solvent (eg iso-butanol) with higher solubility for organic agitate. The organic will be extracted into solvent according to the “partition coefficient”

2. Crystallisation

A. Solvents can be evaporated and organics can be extracted as solid crystals

Question 19

Ultrapurification

Answer 19

1. chromatography is often used to obtain >99.99%
2. req for pharma grade products
3. Chromatography is expensive for yield, materials and time, so not used for low value products
4. proteins often pruified

Question 20

What is protein column chromatography?

Answer 20

1. Gel filtration (yield typically <90%)
2. adsorption
3. ion exchange
4. affinity chromatography

All bar gel filtration, yield typically <70%.

3 column purification can lose over 60% of product

difficult to seperate all the proteins within a cell

Question 21

What are sigma products?

Answer 21

Alkaline phosphatase from bovine intestinal mucosa.

Unit = 1mol 4-nitrophenyl phosphate hydrolysed min-1

P7460 10 units/mg. 11.6p/unit