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BI311 > K.K Lecture 3&4 > Flashcards

K.K Lecture 3&4 Flashcards

1
Q

Describe the process of Cell Immobilisation

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

Advantages of cell immobolisation

A
  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
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3
Q

Disadvantages of Cell Immobilisation

A
  1. Maintain Yeast Viability
  2. Stability over time
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4
Q

Containment of cell in membrane bioreactors

A

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

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5
Q

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

A

Membrane clogging

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6
Q

How is the Attachment of cells to solid substrate performed?

A

Can be done electrostatically or covalently

Use gluteraldehyde to cross link

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7
Q

Entrapement of cells in alginate beads

A

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)

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8
Q

Advantages of Entrapment of cells in alginate beads

A

*Increase ethanol Production
*Efficient Fermentations

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9
Q

Disadvantages of Entrapement of cells in alginate beads

A

*Calcium Chelating Agent
(Phosphate) - can cause disruption

*Gas diffusion problems

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10
Q

Flocculation

A

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

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11
Q

Downstream Processing

A

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.
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12
Q

Process of Cell Disruption

A

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

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13
Q

Mechanical Methods of Cell Breakage

A

Grinding, Pressure, Ultrasound

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14
Q

Non-mechanical methods of cell breakage

A

*Dessication
*Lysis - osmotic, detergents and enzymatic

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15
Q

Means of Seperating

A

*SIZE:ULTRACENTRIFUGATION
GEL CHROMATOGRAPHY

*DIFFUSIVITY: DIALYSIS

*IONIC CHARGE: ELECTROLYSIS

*SOLUBILITY: SOLVENT EXTRACTION

*DENSITY: CENTRIFUGATION

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16
Q

Isolation of Intracellular Enzymes

A
  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
17
Q

What are the typical products at the end of fermentation?

A

*Vitamin B12
*Antibiotics
*Lipids
*SCP
*Ethanol

18
Q

Engineering Barely

A

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.

19
Q

Lactic Acid Starter Cultures in Malting

A
  1. Use bacteria to out compete Fusarium species and so reduce amount of myotoxins in wort.
  2. Out compete aerobic bacteria during malting
20
Q

Myotoxins

A

Toxic, produce gushing.

21
Q

Wort

A

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

22
Q

Brewing Technology

A

Possibility of continuously operated system

Problems: New equipment
New technology

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

23
Q

Yeast

A

Ethanol and Flavour Compounds

24
Q

Flavour Compounds

A

Primary Fermentation but some in Secondary Fermentation

25
Q

Diacetyl:

A

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.

26
Q

Altering Flocculation to Increase Yield

A

Autoaggregation of microbes

-Brewing
-Wine making
-Wastewater treatment
-Bioconversion

In brewing: At the end of fermentation flocculation causes yeast to clump and precipitate

27
Q

How Does flocculation occur?

A

Colloid Theory
Calcium Bridging
Lectin Theory

28
Q

Colloid Theory

A
  1. COLLOID THEORY:
    Inorganic salts –> Flocculation.
    Charge neutralisation
29
Q

Calcium Bridging

A

Ca2+ links cells BUT mannose blocks adherence

30
Q

Lectin-like theory

A
  1. LECTIN-LIKE THEORY:
    Lectin adheres to a-mannan carbohydrates on yeast cell wall.
31
Q

Molecular Basis of Yeast Flocculation

A

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

32
Q

Describe Cell Surface Hydrophobicity and Flocculation? (CSH)

A

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

33
Q

What leads to the creation of super-flocculent yeast?

A

BREWING CONDITIONS
Constituents in Wort –> increase Flocculation.

Creation of super-flocculent yeast

34
Q

Immobilised Yeast in Brewing

A

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

35
Q

Continous Fermentation

A

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

36
Q

Fermentation of xylose by yeasts

A

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

37
Q

Discovery of Xylose fermenting yeast.

A

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

38
Q

Pachysolen tannophilus

A
  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)

BI311 (14 decks)

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