- The document summarizes the MSc thesis of Mauro José Castanho Claudino which aimed to screen and characterize different immobilization methods for whole-cell steroid bioconversion using Mycobacterium sp. NRRL B-3805. - Silicone was found to be the most suitable immobilization support, providing efficient cell adsorption, good thermal stability, and ability to catalyze the biotransformation of β-sitosterol to 4-androstene-3,17-dione over multiple batches. Kinetic studies showed Michaelis-Menten behavior and storage stability could be modeled using bi-exponential equations. - The work demonstrated the feasibility of using small-scale bioreactors to

1. Characterization of 4-androstene-3,17-dione
Production through the use of Immobilized
Cells in Minireactors
Lisbon, 08th
January 2008
IST – Instituto Superior Técnico
Centre for Biological and Chemical Engineering (CEBQ)
BioEngineering Research Group – BERG
Supervisor: Dr. Pedro Fernandes
Co-Supervisor: Prof. Dr. Joaquim Sampaio Cabral
Institute for Biotechnology and
Bioengineering
M.Sc. Thesis
Mauro José Castanho Claudino
1

2. Aim of this Thesis:
Screening of different immobilization procedures (adsorption, encapsulation
and entrapment) for sterol bioconversion and systematically characterize the
most suitable immobilization method, using a small scale approach.
Objective
Parameters evaluated:
 Cell loading capability;
 Substrate and product partition effects;
 Temperature, pH and hydrodynamic conditions;
 Stability (thermal and storage);
 Reaction kinetics;
 Biocatalyst reusability. 2

3. Small-scale bioreactors Microtitre plates, test tubes and shaken reactors
Advantages:
 Parallel and automated experimental set-ups;
 Cost reduction for media components due to low working volumes used;
 Less space requirements as compared to the use of conventional
systems (e.g. Erlenmeyer flasks);
 Wide array of data output with significant time and cost savings;
 Provide the basis for rational set-up of the evaluating systems.
Disadvantages:
 Suitable online monitoring of operational parameters;
 Reduced volumes for conventional evaluation;
Introduction
3

4. Introduction
Advantages and purposes of immobilization in whole-cell
biocatalysis:
 Cell retention within the support/bioreactor
 High cell concentrations Enhanced volumetric productivities;
 Control of biocatalyst microenvironment
 Eased separation of biocatalyst from product
Possible biocatalyst reuse;
Contamination avoided;
High dilution rates;
Protection against shear forces;
Lower costs in recovery,
recycling and downstream
processing;
Increased cell stability;
4

5. Introduction
Potential limitations of cell-immobilized systems:
 Increased costs of biocatalyst production
 Loss of biocatalytic activity
 Empiricism
Immobilization
procedure
Matrix nature
Reaction step
pH, temperature extremes;
Toxic reagents;
High shear/mechanical
conditions;
Exclusion of molecules;
Local pH shifts;
Mass transfer limitations;
Cell leakage;
Inhibitors build-up;
Need for case specific, multi-parameter optimization;
Difficult process modelling and control.
5

6. Introduction
Whole-cell immobilization methods:
Adapted from Kourkoutas et al., 2004;
Food Microb. (21) 377-397
Silicone
Celite 560
PU-foam
Scotch-brite®
PVA
PVA-LentiKats®
PVA/Alginate
Alginate
Alginate/polyurea
Ca-alginate
6

7. Bioconversion System: case study
Selective side-chain cleavage of β-sitosterol to 4-androstene-3,17-dione (AD)
performed by Mycobacterium sp. NRRL B-3805 resting cells
Main features of biotransformation:
 Multi-enzymatic oxidative biotransformation requiring cofactors (involves the
use of nine catabolic enzymes in a 14-step metabolic pathway);
 Mycobacterium sp. is a relatively slow growth microorganism;
 Oxygen required for reaction;
 Low solubility of substrate and products in aqueous media (<1.0 mM); 7
HO
O
O
O
O
Pharmaceutical
steroids
4-androstene-3,17-dione (AD)
1,4-androstadiene-3,17-dione (ADD)
β-Sitosterol
HO
O
O
O
O
Pharmaceutical
steroids
4-androstene-3,17-dione (AD)
1,4-androstadiene-3,17-dione (ADD)
β-Sitosterol

8. Materials and Methods
Free-suspended cell growth medium
 Di-sodium/potassium Phosphate Buffer 0.1 M pH 7.0
 Yeast Extract (10 g.l-1
)
 Glycerol (10 g.l-1
)
 NH4Cl (4.0 g.l-1
)
 Tween®
20 (0.8 g.l-1
)
 MgSO4⋅7H2O (0.14 g.l-1
)
 Substrate: β-sitosterol (activity inducer, 1.0 g.l-1
)
Conditions: 30 ºC at 200 rpm for 36-hrs in 2.0 L Erlenmeyer flasks
Cell recovery: Vacuum filtration (wet cell-paste, 60-70% humidity), washed with
phosphate buffer and stored at -20ºC
8

9. Biocatalyst Preparation
Surface adsorption (Bio)encapsulation
Silicone PU-foam Scotch-brite®
fabric Celite 560
Orbital shaking
(30ºC, 200 rpm, 48-hrs)
Harvest
Supports in growth
medium + cell inoculum
Storage -20ºC
50 ml Erlenmeyer
Concentrated
cell suspension
50 g/L
CaCl2 solution with
xanthan gum and
Tween 20
Na-alginate
solution
Ca-alginate
capsules
Cell suspension
with thickener and
surfactant
D = 5-6 mm
Storage -20ºC
180 mg of solid
carriers
Interfacial
polymerization
reaction
9

10. Biocatalyst Preparation
Entrapment
Spherical beads PVA-LentiKats®
disks
Plastic Petri-dish
Stabilizing bath with
activation medium
(Sloughing and re-swelling)
Lenticular shaped
particles
(optimum geometry)
gelation
Cell suspension in
melted LentiKats®
Liquid
(40 g/L)
Cell suspension in
Na-alginate or in
polyvinyl alcohol
(PVA)
CaCl2 solution or
saturated boric-acid
solution
or Ca-alginate
beads
PVA beads
Polyurea coating
D = 2.5 mm
Alginate coated with
polyurea
(beads more hydrophobic)
D = 3 mm
http://www.geniaLab.de/download/tt-english.pdf
6-hrs evaporation
10

11. Bioconversion Trials
Reaction in aqueous medium
Biocatalyst + 1 ml 0.1M Tris-HCl buffer (pH 7.5) +
120 µl of β-sitosterol (24 mM) in EtOH 96% (v/v)
Organic-aqueous two-liquid phase reaction
Biocatalyst + 0.5 ml 0.1M Tris-HCl buffer (pH 7.5) +
0.5 ml of β-sitosterol in BEHP (12 mM)
Reaction in predominantly organic medium
Biocatalyst + 1 ml of β-sitosterol in BEHP (12 mM)
Analytical methods: HPLC Lichrospher Si-60 column (5 µm particle size)
Isocratic elution (1 ml.min-1
);
Mobile phase: n-heptane/EtOH (92:8, v/v);
UV detection (β-sitosterol, 220 nm; AD, 254 nm).
Protein determination by Lowry Method
Protein estimation: [Total protein] (mg.l-1
) = 304 × [Dry biomass] (mg.ml-1
) + 0.8
11
Incubation conditions
Reaction flasks: 15 ml screw-capped
vessels (minireactors, 80% headspace);
35ºC, 250 rpm, 24-hrs.

12. Results
I – Screening of several immobilization methods
No biocatalytic activity detected
OverproductionUnderproductionBiocatalyst form
Relative specific AD production (%)
(based on biomass weight)
No biocatalytic activity detectedl
100 % (± 7.6)
149 % (± 7.7)
119 % (± 8.6)
108 % (± 9.5)
124 % (± 8.2)
27 % (± 10.3)
132 % (± 5.4)
81 % (± 3.0)
24 % (± 8.0)
12
Aqueous media
Silicone 1 mm

13. Results
II – Adsorption capacity
Celite 560 Silicone 1 mm
Dry biomass content
Protein content
Carrier
Partition studies (incubation: 40-hrs, 35ºC, 250 rpm)
Biocatalyst
(mg of dry biomass)
Global AD
Accumulation
(mM)
Global AD specific
accumulation
(mmol⋅g-1
dry biomass)
AD in
aqueous
phase (mM)
Sitosterol in
aqueous
phase (mM)
AD
adsorbed
onto
support
(mmol⋅g-1
support)
Sitosterol
adsorbed
onto
support
(mmol⋅g-1
support)
Free cells (0.8) 0.262 0.328 -------- -------- -------- --------
Immobilized cells:
Silicone 1mm (1.4) 0.436 0.312 0.321 (76%) 2.7×10-3
2.0×10-3
6.0×10-3
Celite 560 (1.2) 0.220 0.099 0.190 (86%) 0.165 4.0×10-4
1.3×10-3
13

14. Results
III – Temperature, pH and shaking speed
50
Relativespecificactivity(%)
Temperature (ºC) pH
Shaking speed (rpm)
Relativespecificactivity(%)
Optimum temperature: 35ºC
6.5 < pH < 8.0
Shaking speed: 150 – 300 rpm
250 rpm, pH 7.5 250 rpm, 35ºC
35ºC, pH
7.5
14
 Silicone 1 mm
 Celite 560
 Free cells

15. IV – Thermal and storage stabilities
Results
Incubation time (days) Storage time (days)
Relativespecificactivity
0
( )
( )
( ) ( ) ( )1
0
α β= × − × + − × − ×
E t
B exp t B exp t
E
Adapted from Aymard and Belarbi (2000)
Enz. Microb. Technol. (27) 612-618
15
 Silicone 1 mm
 Celite 560
 Free cells
Modelling deactivation profiles:

16. Results
V – Kinetic studies and reusability of the silicone immobilized
biocatalyst
Aqueous β-sitosterol concentration (mM)
Specificactivity
(mmolAD⋅g-1
drybiomass⋅h-1
)
Relativeproductyield,(%)
Relativeamountofbiomass
retainedinsupport,(%)
Batch number #
Michaelis-Menten equation*
*Apparent kinetic parameters obtained using
Leonora®
software (Cornish -Bowden, 1995):
Vmáx, imm = 0.145 mmol AD.g-1
dry biomass.h-1
Km, imm = 0.14 mM
Silicone 1 mm
Celite 560
16
[ ]
[ ]
×
=
+
máx ,imm
m ,imm
v S
v
K S
 Silicone 1 mm
 Celite 560
 Free cells

17. Conclusions
 Sitosterol side-chain cleavage pathway is susceptible to prolonged drying
at room temperatures (LentiKats®
) and to relatively harsh chemical
manipulations (alginate coated with polyurea). Hydrogels provided efficient
cell retention but are limited to their hydrophilic nature;
 Apparently silicone slabs provide an efficient carrier for cell-surface
adsorption displaying catalytic activity for sitosterol side-chain cleavage;
 A cell-loading capacity of 6 mg dry biomass per gram of support was
achieved;
 Hydrophobic nature of silicone favours both cell-adhesion and the
substrate partition to the surface, while retaining low quantities of AD
formed;
 Immobilization provides good stability of biocatalyst preparation under
operating conditions up to 300 rpm and 45ºC with an Topt of 35ºC;
 The pH/activity profile was not considerably altered as a result of
immobilization;
17

18.  Michaelis-Menten type kinetics adequately described the bioconversion
system in the substrate range evaluated. Low apparent Km, imm value suggests
high affinity to β-sitosterol;
 All biocatalytic systems displayed thermal and storage deactivation.
Deactivation profiles can be accurately modelled using a 3 parameter bi-
exponential equation;
 Repeated batch biotransformations were feasible and simpler to perform
when silicone immobilized cells were used. Marked decay of product
formed occurred mainly due to loss of cell oxidative potential;
 Except for cell-loading capacity, silicone based biocatalysts performed
better than Celite immobilized cells, and usually outperformed free cells;
 Experiments proved the feasibility of using 15-ml screw-capped shaken
bioreactors for screening purposes and system characterization in aqueous
medium;
Conclusions
18

19.  Biocatalytic activity using bioencapsulation could be improved using more
biocompatible hydrophobic matrix and reducing particle size;
 Biocapsules could provide a good approach but are limited to high particle
size. Reducing membrane thickness along with particle diameter could be
the solution while providing a suitable internal microenvironment for
bioconversion to occur;
 The use of PPG, Ionic Liquids (IL’s) and more hydrophobic materials may
help facilitate substrate and oxygen availabilities and partition effects;
 For adsorption experiments it is suggested the use of smaller carrier
particles (crushed silicone slabs, micronized liquid silicone and/or small
latex particles);
 Evaluation of silicone hydrophobicity  Mycobacteria cell wall, sitosterol
and AD;
 Assess cell-to-support adsorption profile along fermentation time and
correlate to viable biomass and displayed catalytic activity;
 Compare results with those to obtain by using 24-well microplates;
Future Work
19

20. Acknowledgements
 Professor Doctor Joaquim Sampaio Cabral
 Doctor Pedro Fernandes
 Marco Marques
 Fellow colleagues of IBB and M.Sc. Course
 My outstanding family and friends
20

21. The End
Thanks for your attention
Questions?
maurojcclaudino@gmail.com
08th
January 2008
21