K.K Lecture 3&4

Question 1

Describe the process of Cell Immobilisation

Answer 1

1. Containment of cells in membrane
2. Attach Cells to solid substrate
3. Entrapment of cells in gel
4. Flocculation

Question 2

Advantages of cell immobolisation

Answer 2

1. HIGH CELL DENSITY/BIOMASS
2. NO WASH OUT OF CELLS.
3. REDUCE CONTAMINATION
4. REDUCE GROWTH INHIBITION
5. SEPARATE CELLS AND MEDIUM
6. INCREASE PRODUCT YIELD
7. INCREASE SPEED OF FERMENTATION

Question 3

Disadvantages of Cell Immobilisation

Answer 3

1. Maintain Yeast Viability
2. Stability over time

Question 4

Containment of cell in membrane bioreactors

Answer 4

To trap inside a membrane:
– POROUS MATERIAL
– MICROFILTERS
– DIALYSIS MEMBRANE

Question 5

What is a problem associated with containment of cell in membrane bioreactors?

Answer 5

Membrane clogging

Question 6

How is the Attachment of cells to solid substrate performed?

Answer 6

Can be done electrostatically or covalently

Use gluteraldehyde to cross link

Question 7

Entrapement of cells in alginate beads

Answer 7

2-4%(w/v) SODIUM ALGINATE + CaCl2 SOLUTION
– BIOCHEMICALLY INERT GEL.
– POROUS
– STABLE
– DIFFUSABLE.
USE: BATCH, CRBF, CONTINUOUS
FREE, PACKED COLUMNS

(CRBF - cell recyle batch fermentation)

Question 8

Advantages of Entrapment of cells in alginate beads

Answer 8

*Increase ethanol Production
*Efficient Fermentations

Question 9

Disadvantages of Entrapement of cells in alginate beads

Answer 9

*Calcium Chelating Agent
(Phosphate) - can cause disruption

*Gas diffusion problems

Question 10

Flocculation

Answer 10

Flocculation is natural process in yeast and fungi.
At the end of their growth their cell walls become adhesive and they begin to adhere together into a clump at high cell density.

No need to centrifuge

Question 11

Downstream Processing

Answer 11

Is a method of pulling out and concentrating the product.

1. Maximum Recovery of product.
2. Process must be Reliable & Continuous.
3. Large reductions in Volume.
4. Final broth must be usable.
5. Processing commence as Broth leaves fermenter.
6. Cost considerations.

Question 12

Process of Cell Disruption

Answer 12

*Sonification
*Agitation with Glass Beads
*French Press
*Cell Wall Degredation
*Controlled Autolysis

Question 13

Mechanical Methods of Cell Breakage

Answer 13

Grinding, Pressure, Ultrasound

Question 14

Non-mechanical methods of cell breakage

Answer 14

*Dessication
*Lysis - osmotic, detergents and enzymatic

Question 15

Means of Seperating

Answer 15

*SIZE:ULTRACENTRIFUGATION
GEL CHROMATOGRAPHY

*DIFFUSIVITY: DIALYSIS

*IONIC CHARGE: ELECTROLYSIS

*SOLUBILITY: SOLVENT EXTRACTION

*DENSITY: CENTRIFUGATION

Question 16

Isolation of Intracellular Enzymes

Answer 16

1. Remove nucleic acids from cell debris nuclease
2. Precipitation of enzymes -Ammonium Sulphate ppt - salting out of proteins
   broad fractions
3. Microsomal Enzymes
   -ppt with PEG
   -Microsomes (Microsomal fraction)
   -Membrane Bound

Question 17

What are the typical products at the end of fermentation?

Answer 17

*Vitamin B12
*Antibiotics
*Lipids
*SCP
*Ethanol

Question 18

Engineering Barely

Answer 18

Source of: a-amylase, b-amylase, b-glucanase
ALSO: contaminants (Fusarium spp)
Integrate enzymes coding for enzymes into genome

Transformed Barley: thermostable b-glucanase
(Stable after 2hr at 65oC)
Reduced wort viscosity.

Question 19

Lactic Acid Starter Cultures in Malting

Answer 19

1. Use bacteria to out compete Fusarium species and so reduce amount of myotoxins in wort.
2. Out compete aerobic bacteria during malting

Question 20

Myotoxins

Answer 20

Toxic, produce gushing.

Question 21

Wort

Answer 21

Wort is the liquid extracted from the mashing process during the brewing of beer or whisky.

Question 22

Brewing Technology

Answer 22

Possibility of continuously operated system

Problems: New equipment
New technology

High gravity beers: add syrup and then dilute final product at
end of fermentation.

Question 23

Yeast

Answer 23

Ethanol and Flavour Compounds

Question 24

Flavour Compounds

Answer 24

Primary Fermentation but some in Secondary Fermentation

Question 25

Diacetyl:

Answer 25

Diacetyl: gives some flavour but unpleasant Reduce levels below 0.05mg/l a-Acetolactate --- Diacetyl --- Acetoin Engineer yeast to convert a-Acet to Acetoin Introduce genes for a-acetolactate decarboxylase from bacteria into brewing yeast.

Question 26

Altering Flocculation to Increase Yield

Answer 26

Autoaggregation of microbes -Brewing -Wine making -Wastewater treatment -Bioconversion In brewing: At the end of fermentation flocculation causes yeast to clump and precipitate

Question 27

How Does flocculation occur?

Answer 27

Colloid Theory Calcium Bridging Lectin Theory

Question 28

Colloid Theory

Answer 28

1. COLLOID THEORY: Inorganic salts --> Flocculation. Charge neutralisation

Question 29

Calcium Bridging

Answer 29

Ca2+ links cells BUT mannose blocks adherence

Question 30

Lectin-like theory

Answer 30

3. LECTIN-LIKE THEORY: Lectin adheres to a-mannan carbohydrates on yeast cell wall.

Question 31

Molecular Basis of Yeast Flocculation

Answer 31

Calcium ions Inhibit with mannose Agitate cells ---> Ca2+- dependent Lectin-like sugar interaction

Question 32

Describe Cell Surface Hydrophobicity and Flocculation? (CSH)

Answer 32

When cell stop dividing --> Increase flocculation. CSH: 1. Increase CSH before Flocculation. 2. Treat with protease -- decrease Flocculation. 3. Polycations -- increase CSH.

Question 33

What leads to the creation of super-flocculent yeast?

Answer 33

BREWING CONDITIONS Constituents in Wort --> increase Flocculation. Creation of super-flocculent yeast

Question 34

Immobilised Yeast in Brewing

Answer 34

*Higher yeast cell densities *Retard production of diacetyl *Faster fermentation *Retain flavour *Easier down stream processing

Question 35

Continous Fermentation

Answer 35

*Aspectic conditions *Use immobilised yeast in a Cns fermenter *Optimise fermentation conditions and ensure no effect on flavour/quality

Question 36

Fermentation of xylose by yeasts

Answer 36

Why? Economic potential - Biofuels Pollution Reduction Xylose: Aldopentose Second most abundant carbohydrate Located in Xylan Release: Enzymatically Dilute Acid Hydrolysis - 60% of sugar It was traditionally thought yeast incapable of fermenting xylose

Question 37

Discovery of Xylose fermenting yeast.

Answer 37

Schnedier et al., 1981: Pachysolen tannophilus Jeffries et al., 1981: Candida tropicalis Gong et al., 1981: Candida sp. Du Preez and Van der Walt, 1983: Candida shehatae Du Preez & Prior, 1985: Pichia stipitis

Question 38

Pachysolen tannophilus

Answer 38

1. Most studies Xylose fermenting yeast 2. Relatively poor fermenter 3. Possesses a genetic system 4. Easy to control fermentation (look at slides 14&15)