1. Cell Lysis and Disruption

2. Laboratory techniques: Industrial technology:
Cell disruption
Laboratory techniques:
Sonication
Enzyme treatment
High mechanical shear
Industrial technology:
Ball mill grinding
Homogenization (orifice type)
Heat generation
*Chemical method + mechanical method combination

3. Product of Interest: Intracellular or Extracellular ?
Table 1. Cell Disruption Technique
Method
Technique
Principle
Stress
on Product
Cost
Examples
Chemical
Osmotic Shock
Osmotic rupture of
membrane
Gentle
Cheap
Rupture of red blood cells
Enzyme digestion
Cell wall digested,
providing disruption
Expensive
Micrococcus lysodeikticus treated with egg lysosyme
Solubilization
Detergents solubilize
cell membrane
Moderate-
expensive
Bile salts acting on E. coli
Lipid dissolution
Organic solvent dissolves
in cell wall, and so
destabilizes it
Moderate
Toluene disruption of yeast
Alkali treatment
Saponification of lipids
solubilizes membrane
Harsh
Mechanical
Homogenization
(blade type)
Cells chopped in
Waring blender
Animal tissue and cells
Grinding
Cell ruptured by grinding
with abrasives
Ultrasonication
Cells broken with
ultrasonic cavitation
Cell suspensions at least on small scale
(orifice type)
Cells forced through
small hole are broken
by shear
Large scale treatment of cell suspensions, except of bacteria
Crushing in
ball mill
Cells crushed between
glass or steel balls
Large scale treatment of cell suspensions and plant cells

4. Chemical Methods (not popular in industry)
Enzyme digestion
Enzyme cost
Alkali treatment
Harsh condition, degradation
(PHB separation)
Osmotic Shock
Cells are put into pure water
Solutes in the cells cause an osmotic flow of water into the cell
(Note: Plant cell are difficult to be burst.)
Solubilization : by detergent
Concentrated detergent solution is added to disrupts the cell membrane
Detergent - Ex: SDS (Sod Dodecylsulfate)
Solubilize cell wall lipid

5. Figure 1. Chemical structures of selected surfactants.
Lipid Dissolution
A volume of solvent( toluene) about 10% of the biomass is added to a cell suspension
The cell wall lipid is solubilized.

6. Mechanical Disruption
Homogenizer
(b)
(a)
Figure 2. Homogenizer Assembly. (a) A typical homogenizer and (b) a homogenization valve.
Cell passing through this valve areruptured by both shear and mechanical stress.

7. *How long for operation?
Time, min
Alcohol Dehydrogenase
Activity
Fumarase Activity
Apparent
Particle Size
Figure 3. Homogenization versus time. Mechanical disruption of cells reduces
particle size but some may also denature some of the products in the cell.
*How long for operation?

8. Ball Mill (Sand Grinder)
Feed in
Cooling Jacket
screen
Sand (bead)
Figure 4. Schematic diagram for ball mill equipment.
*batch, continuous type
*paint, dyestuff industry
*멧돌

9. Enzymatic Lysis dissolve the outer mannoprotein layer
production of protoplasts from yeast and bacteria
endo-β(1,3) glucanase
Lytic protease
Preparation of lytic enzymes
Lysis experiment for various enzymes -required

10. Cell Disruption Equipment: Bead Mill
Sept. 21st, 2011
Esther H. Kim

11. Bead mill Media volume 5.5L Media volume 0.4L
Laboratory bead mill (Dyno mill and DMQ-07)
Production machine (DMQ-10)

12. Scale-up & Application
Laboratory
Scale-up

13. Bead mill – Grinding media
Weaknesses of the sand mill
Transition to closed mills
ottawa sand  grinding beads
Grinding media (beads)
Steel, zirconium oxide, aluminum oxide, Si/Al/Zr mixed oxide (SAZ), steatite (modification of talc), glass and plastics
Diameter lies in the range from 0.1 to 3 mm.
The harder the beads, the greater the intensity of dispersion.
The number of beads is proportional to 1/d3  use smallest beads possible.
Translational and rotational movement: compressive stress and shear

14. Application in research
Horizontal bead mill used for cell rupture
(a) General view, (b) details of (i) stainless steel and (ii) polyurethane impellers

15. Application in research
Bead-mill homogenization
Lysis of microbial cells in soils and sediments for large scale
relatively inexpensive
widely applicable
Compared to chemical and enzymatic method

16. What is a French Press? 2011 - 9 - 21 Inae Kim
Bioseparation Engineering
What is a French Press?
Inae Kim

17. Contents What is a French Press? Principle Operation Characteristics
Manufacturer

18. What is a French Press? 기계적으로 세포를 파괴하는 방법 염록체, 혈구세포 및 단세포 생물의 분해와
동물조직 및 생물체 입자의 균등질(homogenate) 제조에 이용
세포핵은 손상시키지 않고 세포벽만을 분해
Sonication보다 균일하고 완전한 분해 가능
고압(최대 40,000 psi)과 빠른 감압을 이용
단단한 세포도 쉽게 분해 가능
Stainless steel  샘플의 오염 방지
Figure 1. French Press.

19. Principles of the French Press
Flow valve
Outlet tube
Cylinder body
Piston
Sample
Figure 2. French Press Cylinder.
Figure 3. Diagram of French Press Cylinder.

20. Operations of French Press① ② ③
Figure 5. French Press .
① Pump ON/OFF
② Ratio Selector valve (MED/HIGH)
- 유효 피스톤 면적 결정
③ Pressure valve (increase/decrease)
Figure 4. Installed French Press cylinder in the press .

21. Characteristics of the French Press장점
시료의 생화학적 성질 보존
시료의 손실 위험이 적음
시료 전처리 과정이 없음
크기가 작은 시료는 거의 완전히 분해 가능 단점
시료의 부피가 클 때는 부적절
부품의 무게가 무거워서 조작이나 세척이 다소 어려움
배출구가 막힐 수 있음

22. Manufacturers of the French Press