From Team M-4
Leader: Waifong Chan
George Hammer
Yimin Tang
Introduction of Cell Encapsulation
 Commonly employed in bioprocesses to
encapsulate bioactive species.
 Immobilize the cells.
 Protect the cells from shear forces.
 Increase the surface area and allow higher
permeability.
 Promote the level of cell viability.
Project Overview
“I would like to know what parameters may control the average diameter of the beads
produced and whether encapsulation imposes mass-transfer limitations for the bacteria.” -Dr.
Ima Manager.
 To examine one method for encapsulating cells in
alginate beads.
 To demonstrate the parameters which control the bead
size.
 To determine the reaction is limited either by the
reaction rate or the mass transfer limitation.
Previous Work
 Phase-1:
 Safety
 Standard operating procedure
 Beads diameter regarding the changing flow rate.
 Phase-2: determine the change of oxygen uptake
by changing…
 Bead size
 E. coli concentration
Theory of mass transfer limitation
Assumption:
 Steady State
 Particle is isothermal
 Mass transfer by diffusion only
 DAE, effective diffusivity is constant
 Particle is homogeneous
 Zero-kinetic
 Reaction rate is independent on the substrate
concentration.
 Reaction rate = rate constant * particle volume
Thiele Modulus
 Observable Thiele Modulus
2
 V p  rA,obs
   
 S x  DAeC As
2
 R  rA,obs
 
 3  DAeC As
Where:
 Vp= catalyst volume
 Sx = External surface area
 R= radius of bead
 rA,obs= unit oxygen uptake rate =
 DAe= Effective Diffusivity of oxygen in the beads
 CAs= Concentration of oxygen at the surface
Weisz criteria
  .3 
i  1
 3
 i  1
ηi: the internal effectiveness factor
ηi = (observed rate)/(rate that would occur if CA = CAS)
 If ηi ≈ 1, negligible mass transfer limitation
 If ηi < 1, mass transfer deficiencies throughout the bead
Apparatus
 Ring stand
 Centrifuge tube
 Syringe
 Air jet
 Air rotameter
 Petri-dish
Apparatus (Cont’d)
 Oxygen Probe
Methods
 Prepare solution---- 0.5ml 3% Sodium Alginate & 0.5ml E.Coli
 Transfer solution----To a syringe plunger with a 22 gauge needle
 Secure syringe----use rubber band that needle protrudes 1 mm
 Prepare Petri dish----filled with CaCl2
 Turn on air jet----to 60 SCFH and place it coaxially with syringe
 Collect beads----record volume used and drain CaCl2
 Calibrate oxygen probe
 O2 calibration----in 30ml LB & beads mixture with parafilm
 Record O2 concentration----on every 5 min after stabilized
Results (Bead size)
Air Flow
Diameter Sample
1
Diameter Sample
2
Diameter Sample
3
Diameter Sample
4
60 SCFH
50 SCFH
225µm
275µm
280µm
275µm
328µm
300µm
300µm
330µm
Average Diameter
282µm
295µm
Fig.1: Alginate bead at air flow rate
of 60 SCFH.
Results (oxygen uptake)
Dissolved oxygen content (mg/L)
Dissolved oxygen content (mg/l)
6
5.5
y = -0.0081x + 5.2862
R² = 0.8745
5
4.5
4
3.5
3
0
50
100
150
Time elapsed (s)
200
250
300
350
Results (oxygen uptake)
rA,obs
Average Bead
Diameter (m)
Average Unit Bead
Volume (m3)
Number of
Beads used
Overall Oxygen Uptake
Rate (mg/l)
(mg/l)
2.82E-04
1.17E-11
8.52E+04
8.10E-03
9.51E-08
R (m)
1.41E-04
rA,obs (mg/l)
9.51E-08
Dae (m2/s)
2.56E-09
Cas (mg/l)
5.76
Φ
1.42E-08
Ф < 0.3
ηi = 1
negligible mass transfer limitation
Results from other literatures
 From Xiaoming Xu et al[4]
 Mass transfer is inversely related to beads’ diameter.
 Some suggest that[2] [3]
 Needle inside diameter and the viscosity of the alginate
also contribute to the variability of bead’s diameter.
Conclusion
 The main parameter to determine the bead size is the
co-axle air flow rate.
 Higher flow rate corresponds to smaller beads but in
better spherical shape.
 Using Thiele modules and Weisz criteria, alginate
beads was demonstrated to have no mass transfer
limitation.
 Other literatures shows that smaller size of alginate
beads can have higher mass transfer.
Recommendations
 Using at least 1 ml of alginate beads in
order to observe significant change in
oxygen consumption.
 Install a heater to incubate alginate beads
at 37 °C.
 Using magnetic stirring bar with suitable
glassware to minimize the vibration created
by the oxygen probe.
References
1.
Team M-5, Phase II Memo, 04/05/2010
2.
G. W. Vandenberg, C. Drolet, S. L. Scott1 and J. de la Noüe, “Factors affecting protein release from alginate –
chitosan coacervate microcapsules during production and gastric/intestinal simulation Journal of Controlled
Release”, Volume 77, Issue 3, 13 December 2001, pages 297-307.
3.
aUlf Pr¨usse*, bLuca Bilancetti, cMarek Bučko, dBranko Bugarski, eJozef Bukowski, cPeter Gemeiner, eDorota
Lewi´nska, dVerica Manojlovic, fBenjamin Massart, b Claudio Nastruzzi, gViktor Nedovic, hDenis Poncelet, iSwen
Siebenhaar, hLucien Tobler, bAzzurra Tosi, cAlica Vikartovska, aKlaus-Dieter Vorlop., “Comparison of different
technologies for alginate beads production”, Chemical Papers 62 (4) 364–374 (2008)
4.
Xiaoming Xu, Philip S. Stewart *, Xiao Chen Transport limitation of chlorine disinfection of Pseudomonas aeruginosa
entrapped in alginate beads, Biotechnology and bioengineering 1996, vol. 49, no1, pp. 93-100 (25 ref.) 1996 John Wiley
& Sons, Inc
5.
Rigini M Papi, Sotiria A Chaitidou, Fotini A Trikka and Dimitrios A Kyriakidis, Encapsulated Escherichia coli in
alginate beads capable of secreting a heterologous pectin lyase, Microbial Cell Factories 2005, 4:35.
6.
Mehmetoglu, U. "Oxygen Diffusivity in Calcium Alginate Gel Beads Containing Gluconobacter Suboxydans."
Artificial Cells, Blood Substitutes, and Biotechnology 24.2 (196): 91‐106. Web. 28 Mar 2010.
http://www.informaworld.com/smpp/content~content=a789260358