Determination of mass transfer limitations in E. coli encapsulated

beadsFrom Team M-4

Leader: Waifong ChanGeorge Hammer

Yimin Tang

Introduction of Cell EncapsulationCommonly 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

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.

“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.

Previous WorkPhase-1:

Safety Standard operating procedureBeads diameter regarding the changing flow

rate.

Phase-2: determine the change of oxygen uptake by changing…Bead sizeE. coli concentration

Theory of mass transfer limitationAssumption:Steady StateParticle is isothermalMass transfer by diffusion onlyDAE, effective diffusivity is constantParticle is homogeneousZero-kinetic

Reaction rate is independent on the substrate concentration.

Reaction rate = rate constant * particle volume

Thiele ModulusObservable 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

AsAe

obsA

CD

rR ,2

3

AsAe

obsA

x

p

CD

r

S

V ,

2

Weisz criteria

ηi: the internal effectiveness factor

ηi = (observed rate)/(rate that would occur if CA = CAS)If ηi ≈ 1, negligible mass transfer limitationIf ηi < 1, mass transfer deficiencies throughout

the bead

13. i13 i

ApparatusRing standCentrifuge tubeSyringeAir jetAir rotameterPetri-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 60 SCFH 50 SCFH

Diameter Sample 1 225µm 275µm

Diameter Sample 2 280µm 275µm

Diameter Sample 3 328µm 300µm

Diameter Sample 4 300µm 330µm

Average Diameter 282µm 295µm

Fig.1: Alginate bead at air flow rate of 60 SCFH.

Results (oxygen uptake)

0 50 100 150 200 250 300 3503

3.5

4

4.5

5

5.5

6

f(x) = − 0.00805238095238095 x + 5.28619047619048R² = 0.874486209815902

Dissolved oxygen content (mg/L)

Time elapsed (s)

Dis

solv

ed o

xygen

con

ten

t (m

g/l

)

Results (oxygen uptake)Average Bead Diameter (m)

Average Unit Bead Volume (m3)

Number of Beads used

Overall Oxygen Uptake Rate (mg/l)

rA,obs

(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 = 1negligible mass transfer limitation

Results from other literaturesFrom 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.

ConclusionThe 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.

RecommendationsUsing 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.

References1. 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