Determination of mass transfer limitations in E. coli encapsulated beads

1. Determination of mass transfer limitations in E. coli encapsulated beads From Team M-4 Leader: Waifong Chan George Hammer Yimin Tang

2. 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.

3. 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.

4. 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

5. 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

6. Thiele Modulus • Observable Thiele Modulus 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

7. Weisz criteria 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

8. Apparatus • Ring stand • Centrifuge tube • Syringe • Air jet • Air rotameter • Petri-dish

9. Apparatus (Cont’d) • Oxygen Probe

10. 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

11. Results (Bead size) Fig.1: Alginate bead at air flow rate of 60 SCFH.

12. Results (oxygen uptake)

13. Results (oxygen uptake) Ф < 0.3 ηi= 1 negligible mass transfer limitation

14. Results from other literatures • From XiaomingXu 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.

15. 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 Weiszcriteria, alginate beads was demonstrated to have no mass transfer limitation. • Other literatures shows that smaller size of alginate beads can have higher mass transfer.

16. 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.

17. References • Team M-5, Phase II Memo, 04/05/2010 • G. W. Vandenberg, C. Drolet, S. L. Scott1 and J. de la Noüe, “Factors affecting protein release from alginate–chitosancoacervate microcapsules during production and gastric/intestinal simulation Journal of Controlled Release”, Volume 77, Issue 3, 13 December 2001, pages 297-307. • aUlfPr¨usse*, bLucaBilancetti, cMarekBučko, dBrankoBugarski, eJozefBukowski, cPeterGemeiner, eDorotaLewi´nska, dVericaManojlovic, fBenjaminMassart, b Claudio Nastruzzi, gViktorNedovic, hDenisPoncelet, iSwenSiebenhaar, hLucienTobler, bAzzurraTosi, cAlicaVikartovska, aKlaus-Dieter Vorlop., “Comparison of different technologies for alginate beads production”, Chemical Papers 62 (4) 364–374 (2008) • XiaomingXu, 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 • Rigini M Papi, Sotiria A Chaitidou, Fotini A Trikkaand Dimitrios A Kyriakidis, Encapsulated Escherichia coli in alginate beads capable of secreting a heterologous pectin lyase, Microbial Cell Factories 2005, 4:35. • Mehmetoglu, U. "Oxygen Diffusivity in Calcium Alginate Gel Beads Containing GluconobacterSuboxydans." Artificial Cells, Blood Substitutes, and Biotechnology 24.2 (196): 91‐106. Web. 28 Mar 2010. http://www.informaworld.com/smpp/content~content=a789260358