Biochemical Engineering CEN 551

1
Biochemical EngineeringCEN 551

• Instructor Dr. Christine Kelly
• Chapter 11 Recovery and Purification of Products

2
Schedule

• Thursday 3/4
• chapter 11HW due
• take home exam
• homework solutions
• Tuesday 3/16 Haowen, Ashutosh, Nilay
• Thursday 3/18 take home exam due

3
(No Transcript)
4
(No Transcript)
5
(No Transcript)
6
General Approach

1. Separation of insoluble products or components.
2. Primary isolation or concentration and removal of
   water.
3. Purification and removal of contaminated
   chemicals.
4. Product preparation.

7
(No Transcript)
8
(No Transcript)
9
Factors that impact difficulty and cost of
recovery

• Product can be biomass, intracellular or
  extracellular component.
• Fragile or heat sensitive.
• Concentration or titer in the broth.
• Typically recovery and purification is more than
  50 of total manufacturing costs

10
(No Transcript)
11
(No Transcript)
12
(No Transcript)
13
Insoluble Products or Components

• Filtration
• Centrifugation
• Coagulation and Flocculation

14
Filtration

• Most cost-effective, most common in industrial
  biotechnology.
• Rotary vacuum precoat filters traditional.
  Penicillin mold.
• Cross flow ultrafiltration 0.02-0.2 um
  bacterial separations
• Cross flow microporous filtration0.2-2 um for
  yeast

15
Rotary vacuum precoat filters
16

• V volume of filtrate
• A surface area of filter
• ?p pressure drop through the cake and filter
  medium
• u viscosity of filtrate
• rm resistance of filter medium
• rc resistance of cake

17

• Substitute, integrate, linearize
• ? specific resistance of cake, C cake
  weight/volume filtrate
• Plot t/V vs. V, slope 1/K, intercept 2Vo
• Can find rm and ?

18

• Assumes incompressible cake.
• Fermentation cakes are compressible.
• Filter aid is added to decrease the cake
  resistance.
• pH and fermentation time can affect resistance.
• Heat treatment can reduce cake resistance.

19
Centrifugation

• Used to separate solids of size 0.1 um to 100 um
  using centrifugal forces.
• Being replaced by microfiltration.
• Fc2Uo?
• Fc flow, Uo free settling velocity
• ?centrifugation coefficient re?2Vc/gLe
• Reradius of rotation, ? angular velocity,
  Lesettling distance,

20
(No Transcript)
21
Coagulation and Flocculation

• Pretreatment to centrifugation, gravity settling
  or filtration to improve separation.
• Coagulation formation of small flocs of cells
  using coagulating agents, electrolytes.
• Flocculation formation of agglomeration of
  flocs into settleable particles using
  flocculating agents, polyelectrolytes or CaCl2.
• Used wastewater treatment processes to improve
  clarification.

22
Cell Disruption Intracellular Products

• Mechanical Methods
• Sonication
• Bead beating
• Pressing
• Non-Mechanical methods
• Osmotic shock
• Freeze-thaw
• Enzymatic

23

• Ultrasound disrupts cell membrane. Mostly used
  at the laboratory scale.
• Pressing extrude cell paste at high pressure.
• Bead beating grind cells with glass, metal
  beads.
• Heat dissipation is a problem with all of these
  methods.

24

• Osmotic shock Salt differences to cause the
  membrane to rupture. Common.
• Freeze-thaw Causes cell membrane to rupture.
  Common.
• Enzymatic Lysozyme attacks the cell wall.
• Can use a combination of these methods.

25
Separation of Soluble Products

• Liquid-liquid extraction
• Aqueous two phase extraction
• Precipitation
• Adsorption
• Dialysis
• Reverse osmosis
• Ultrafiltration and microfiltration
• Cross-flow filtration and microfiltration
• Chromatography
• Electrophoresis
• Electrodialysis

26
Liquid-Liquid Extraction

• Separate inhibitory fermentation products from
  broth.
• Based on solubility difference for the compound
  between the phases.
• Distribution coefficient KD YL/XH
• YLconcentration in the light phase
• XNconcentration in the heavy phase

27

• Mass balance assuming immiscibility yields
• X1/X0 1/(1E) where E extraction factor
  LKD/H
• Percent extraction f(E and the number of
  stages)
• Antibiotics are extracted using liquid-liquid
  extraction.

28
http//www.facstaff.bucknell.edu/mvigeant/field_gu
ide/kandle01/
29
http//www.liquid-extraction.com/
30
(No Transcript)
31
Precipitation

1. Salting out inorganic salts (NH4)2SO4 at high
   ionic strength
2. Solubility reduction at low temperatures (less
   than 5oC) by adding organic solvents

32
(No Transcript)
33
Adsorption

• Removal of solutes from aqueous phase onto a
  solid phase.
• Chromatography is based on adsorption.

34
(No Transcript)
35
Dialysis

• Membrane separation used to remove low molecular
  weight solutes.
• For example, removal of urea from urine medical
  treatment dialysis for diabetic patients.
• Used to remove salts from protein solutions.
• Transport occurs due to a concentration gradient
  driving force.

36
(No Transcript)
37
Reverse Osmosis (RO)

• Osmosis Transport of water molecules from a
  high to a low concentration pure water to salt
  water.
• In RO, pressure is applied to salt phase causing
  water to move against a concentration gradient.
• Salt phase becomes more concentrated.

38
(No Transcript)
39
Ultrafiltration and Microfiltration

• Pressure driven molecular sieve to separate
  molecules of different size.
• Dead end filtration retained components
  accumulate on the filter. Gel layer formed on
  the filter.
• Cross flow filtration retained components flow
  tangentially across the filter

40
(No Transcript)
41
Cross-flow filtration
42
(No Transcript)
43
(No Transcript)
44
Types of filtration equipment
45
(No Transcript)
46
(No Transcript)
47
http//www.lcsupport.com/home.htm
http//www.gewater.com/equipment/membranehousing/1
193_Membrane_elements.jsp
48
http//www.gewater.com/equipment/membranehousing/1
193_Membrane_elements.jsp
49
Configurations of filtration equipment
50
(No Transcript)
51
(No Transcript)
52
(No Transcript)
53
(No Transcript)
54
Effect of pressure and protein concentration on
flux
55
(No Transcript)
56
(No Transcript)
57
(No Transcript)
58
Costs of filtration equipment
59
(No Transcript)
60
(No Transcript)
61
Chromatography

• Separates mixtures into components by passing the
  mixture through a bed of adsorbent particles.
• Solutes travel at different speeds through the
  column resulting in the separation of the solutes.

62
(No Transcript)
63
(No Transcript)
64
(No Transcript)
65
(No Transcript)
66
http//sepragen.com/products/columns/process_colum
ns.html
67
Affinity Chromatography

• Highly specific interaction between a ligand on
  the particle and a component in the mixture.
  Often based on antibodies.

68
(No Transcript)
69
(No Transcript)
70
Electrophoresis

• Separation of molecules based on size and charge
  in an electric field.

71
Electrodialysis

• Membrane separation to separate charged molecules
  from a solution.

72
Finishing Steps

• Crystallization
• Drying