Influence of whole microalgal cell immobilization and organic solvent on the bioconversion of androst-4-en-3,17-dione to testosterone by Nostoc muscorum

Article abstract of DOI:10.1016/j.molcatb.2009.10.006

The use of free, immobilized and reused immobilized cells of the microalga Nostoc muscorum was studied for bioconversion of androst-4-en-3,17-dione (AD) to testosterone in hexadecane. Among polymers such as agar, agarose, κ-carrageenan, polyacrylamide, polyvinyl alcohol, and sodium alginate that were examined for cell entrapment, sodium alginate with a concentration of 2% (w/v) proved to be the proper matrix for N. muscorum cells immobilization. The bioconversion characteristics of immobilized whole algal cells at ranges of temperatures, substrate concentrations, and shaking speeds were studied followed by a comparison with those of free cells. The conditions were 30°C, 0.5. g/L, and 100. rpm, respectively. The immobilized N. muscorum showed higher yield (72 ± 2.3%) than the free form (24 ± 1.3%) at the mentioned conditions. The bioconversion yield did not decrease during reuse of immobilized cells and remained high even after 5 batches of bioreactions while Na-alginate 3% was used; however, reuse of alginate 2% beads did not give a satisfactory result.

Full text of DOI:10.1016/j.molcatb.2009.10.006

Journal of Molecular Catalysis B: Enzymatic 62 (2010) 213–217
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Influence of whole microalgal cell immobilization and organic solvent on the
bioconversion of androst-4-en-3,17-dione to testosterone by Nostoc muscorum
a
a
a,b,∗
H. Arabi , M. Tabatabaei Yazdi , M.A. Faramarzi
a
Department of Pharmaceutical Biotechnology, Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences,
P.O. Box 14155-6451, Tehran 14174, Iran
Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran 14174, Iran
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 March 2009
Received in revised form 11 October 2009
Accepted 14 October 2009
Available online 10 November 2009
The use of free, immobilized and reused immobilized cells of the microalga Nostoc muscorum was stud-
ied for bioconversion of androst-4-en-3,17-dione (AD) to testosterone in hexadecane. Among polymers
such as agar, agarose, ␬-carrageenan, polyacrylamide, polyvinyl alcohol, and sodium alginate that were
examined for cell entrapment, sodium alginate with a concentration of 2% (w/v) proved to be the proper
matrix for N. muscorum cells immobilization. The bioconversion characteristics of immobilized whole
algal cells at ranges of temperatures, substrate concentrations, and shaking speeds were studied followed
Keywords:
Nostoc muscorum
Algae
◦
by a comparison with those of free cells. The conditions were 30 C, 0.5 g/L, and 100 rpm, respectively.
The immobilized N. muscorum showed higher yield (72 ± 2.3%) than the free form (24 ± 1.3%) at the
mentioned conditions. The bioconversion yield did not decrease during reuse of immobilized cells and
remained high even after 5 batches of bioreactions while Na-alginate 3% was used; however, reuse of
alginate 2% beads did not give a satisfactory result.
Steroid
Immobilization
Organic media
Hexadecane
© 2009 Elsevier B.V. All rights reserved.
1
. Introduction
Whole cell biocatalyst activity and stability during biocon-
is the toxicity of solvents, which may lead to the disruption of
the cell membrane, denaturation of membrane-bound enzymes,
and cytolysis [4,5]. Although much work has been reported on the
utilization of immobilized bacteria and fungi in organic media for
the conversion of organic compounds [1,7,8], no data are available
for the application of both techniques for the biotransformation of
steroids by microalgae [9].
The use of algal biotechnology has received considerable atten-
tion in recent decades. These photosynthetic organisms have been
used in food, cosmetics, aquaculture, and pharmaceutical indus-
tries [10]. However, the small size of microalgal cells implies a
problem in the application of biotechnology processes to those
organisms. Microalgal cell immobilization has been developed to
solve the mentioned difficulties.
In the this study, we applied immobilized microalga Nostoc mus-
corum for biotransformation of androst-4-en-3,17-dione (AD) to
testosterone in an organic medium. In our previous study, vari-
ous organic solvents were examined to find out the proper organic
media that caused no toxicity effect on N. muscorum cells [11].
Hexadecane and tetradecane proved to increase the solubility of
the lipophilic steroid substrate and enhanced enzyme activity. To
mask the inhibition effects of the solvent and substrate toxicity and
provide the possibility of reusing the algal cells, we tried to immo-
bilize N. muscorum by entrapment methods in different matrices.
Appropriate bioreaction conditions such as temperature, agitation
rate, and substrate concentration were also studied to maximize
the bioconversion yield.
version reactions may be influenced by parameters such as
temperature, shaking speed, and substrate concentration [1], which
can be reduced with cell immobilization techniques. The main
advantages claimed for applying immobilized whole cells include
the higher reaction rates due to increased cell densities, possibil-
ities for regenerating the biocatalytic activity, ability to conduct
continuous operations at a high dilution rate without washout, eas-
ier control of the fermentation process, long-term stabilization of
cell activity, reusability of the biocatalyst, and higher specific yields
[
2–5]. The combination of cell immobilization methods and organic
solvent systems in biotransformation processes decreases the dif-
ficulties of the low solubility of high concentrations of lipophilic
precursors and products, and provides environmental protection
for biocatalysts [6].
The use of immobilized cells for bioconversion of steroids in
organic media is a valuable method to increase the solubility of
poor water-soluble steroid compounds. However, the critical point
∗
Corresponding author at: Department of Pharmaceutical Biotechnology,
Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medi-
cal Sciences, P.O. Box 14155-6451, Tehran 14174, Iran. Tel.: +98 21 66954712;
fax: +98 21 66954712.
E-mail address: faramarz@tums.ac.ir (M.A. Faramarzi).
1
381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.10.006

2
14
H. Arabi et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 213–217
2
. Materials and methods
cell volume. The mixtures were placed in Petri dishes and allowed
to solidify by standing at 4 C. Hard gels were cut into small pieces
◦
2.1. Chemicals and instruments
and entrapped cells removed by washing with distilled water [15].
Androst-4-en-3,17-dione (AD) and ␬-carrageenan were pur-
2.3.2. Alga entrapment in ꢀ-carrageenan
chased from Sigma Chemical Co. (St. Louis, MO, USA). Agar, agarose,
polyvinyl alcohol, and polyacrylamide were obtained from Merck
A defined portion of N. muscorum cells was mixed with the same
volume of ␬-carrageenan solution (2%, w/v). The resulting mixture
was pumped through a needle (30-mL syringe, fitted with a wide-
bore needle approximately 2 mm diameter for droplet formation)
into a 2% (w/v) potassium chloride solution. The formed beads con-
taining algal cells were gently mixed with a magnetic stirring bar
for 1 h at room temperature. The beads were washed with distilled
water before use [16].
(
Darmstadt, Germany). Sodium alginate was supplied by B.D.H.
Chemical Ltd. (Poole, England). All other reagents and solvents were
of the highest purity grade available (Merck, Darmstadt, Germany)
unless otherwise mentioned.
The high-performance liquid chromatography (HPLC) apparatus
consisted of a Jasco model PU-986 pump, a UV-1570 UV variable-
wavelength detector, and an online degasser, all from Jasco (Tokyo,
Japan). Samples were injected in a Jasco AS-950 injector system
with an auto-injector. The data were acquired and processed with
Borwin chromatography software (version 1.5) from Jasco (Tokyo,
Japan). Chromatographic separation was achieved on a Finepack SIL
C18 reverse-phase column (C18, 15 cm × 0.46 cm i.d., 5-␮m particle
size) from Teknokroma (Barcelona, Spain).
2.3.3. Acrylamide entrapment
N. muscorum cells (200-mg wet weight equal to double the
packed cell volume) were added to 6 mL of Tris–HCl buffer, pH
7.8, containing 105 mg of acrylamide and 3 mg of bis-acrylamide.
The mixture was added drop by drop using the needle men-
tioned before into 100 mL of mineral oil containing 84 mg of
ammonium persulfate and 84 ␮L of tetramethylethylenediamine
(TEMED). Polymerization was performed at room temperature for
2.2. Algal strain and incubation condition
2
4 h. The formed beads were washed with sterile water to remove
An axenic culture of N. muscorum [12,13] was grown in BG-
1 medium [10] and maintained at 4 C on BG-11 agar slants.
undesired residue [17].
◦
1
The BG-11 medium contained (g/L) NaNO , 1.5; K HPO , 0.04;
2.3.4. Sodium alginate immobilization
3
2
4
MgSO ·7H O, 0.075; CaCl ·2H O, 0.036; ferric ammonium cit-
Algal cells were mixed homogenously with the same volume of
sodium alginate solution (2%, w/v). The mixture was added into
0.2 M of cold calcium chloride solution by a syringe and left to
harden for 1 h. The alginate beads were washed with distilled water
before use [3].
4
2
2
2
rate, 0.006; citric acid, 0.006; Na EDTA, 0.001; Na CO , 0.02;
2
2
3
trace element solution, 1 mL; distilled water up to 1000 mL. The
trace element solution contained (g/L) H BO , 2.86; MnCl ·4H O,
3
3
2
2
1
.81; ZnSO ·7H O, 0.222; Na MoO ·2H O, 0.3; CuSO ·5H O, 0.079;
4
2
2
4
2
4
2
Co(NO ) ·6H O, 0.494; distilled water up to 1000 mL.
3
2
2
The alga was transferred to fresh medium every two months. To
2.3.5. Polyvinyl alcohol (PVA) immobilization
prepare adequate cell mass, N. muscorum was inoculated into two
The reaction mixture was prepared by dissolving PVA (15%, w/v)
◦
◦
5
00-mL Erlenmeyer flasks, each containing 100 mL of BG-11 liquid
in distilled water at 100 C. After cooling down to 30–40 C, one
portion of PVA aqueous solution was mixed with an equal volume
of packed algal cells. The resulting mixture was put drop wise into
the saturated boric acid solution through a needle and gently stirred
for 1 h to form spherical beads. The PVA beads were screened with
a mesh, and then rinsed with tap water to remove the residual
boric acid. The formed gel beads were transferred to a 1-M sodium
phosphate solution pH 7 for 30 min to harden. The phosphorylated
beads were separated by filtration and rinsed with distilled water
[18].
◦
medium (pH 7.0), and incubated at 25 C under continuous illumi-
nation from all sides at an irradiance of 60 ␮mol photons/m s
2
1
with cool-white fluorescent lamps. Cells were harvested in the
3
late exponential phase and centrifuged at 2.0 × 10 g for 5 min.
Each 5-mL packed wet cell volume [14], which is equal to 100 mg
dry cell weight, was used to inoculate a 100-mL Erlenmeyer flask
containing 20 mL of organic solvent. The control aqueous biotrans-
formation medium was prepared using the same procedure except
that the organic system was replaced by 20 mL of BG-11 medium.
The viability of algal cells in the presence of hexadecane was
proved by the cells appearance and re-culturing in fresh BG-11
medium. The optimum reaction temperature and substrate con-
2.4. Bioconversion
◦
centration were 30 C and 0.5 mg/L, respectively.
The prepared beads of the immobilized algal cells were sep-
arately suspended in 100-mL Erlenmeyer flasks; each containing
20 mL of BG-11 medium for the aqueous system and 20 mL of hex-
adecane for the organic mono-phase system. A defined amount of
AD, 10 mg, dissolved in 100 ␮L of chloroform was added to each
flask. Free whole cells of N. muscorum were used as control. The
2.3. Immobilization technique
According to a recent study [11], hexadecane with log poctanol
8
.2 is the proper organic medium for biotransformation of AD to
◦
its single reductive derivative by the alga N. muscorum. Therefore,
the following immobilization experiments in the selected matrices
were carried out in hexadecane under the conditions mentioned
above (see Section 2.2). In all experiments, free whole cells of N.
muscorum were used as control. Before each immobilization exper-
iment, algal cells were harvested in the late exponential phase,
flasks were incubated on a rotary shaker at 30 C with a shaking
speed of 100 rpm. After 5 days, the samples were taken followed
by AD and testosterone analysis by HPLC. To validate the repro-
ducibility of the data, each bioconversion run was carried out in
triplicate using different batches.
3
centrifuged at 2.0 × 10 g for 5 min and provided a 5-mL packed
2.5. Quantitative analyses
wet cell volume.
HPLC was applied for quantitative studies to obtain bioconver-
sion yield in aqueous and organic systems as well as time course
study, influence of temperature, substrate concentration, and shak-
ing speed. In this order, each 100 ␮L of samples was diluted with
400 ␮L of acetonitrile (ACN). A total of 10 ␮L was injected into
2
.3.1. Cell entrapment in agar and agarose
The reaction mixtures were separately prepared by dissolving
◦
agar or agarose (2%, w/v) in distilled water at 100 C, and then
cooled at 40 C and mixed with an equal volume of packed algal
◦

H. Arabi et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 213–217
215
Fig. 1. Algal transformation of AD (I) to testosterone (II) by N. moscurom.
a C18 reverse-phase column. The mobile phase was water/ACN
with a gradient of 0–100% ACN over 33 min and a flow rate of
ces showed lower yields. Since agar, agarose, ␬-carrageenan, and
polyvinyl alcohol gels (with bioconversion yields of 25 ± 1.8%,
19.8 ± 1.2%, 31 ± 3.1%, and 3.6 ± 0.6%, respectively) are formed by
lowering the temperature, the strength of the resulting gels is
somewhat lower. The additional disadvantage is due to the diffi-
culty in the preparation of the beads, and many of the cells were
lost or damaged due to the rather high temperature necessary to
keep the gels molten. The toxicity of the acrylamide matrix affects
algal cell viability, and most of them are lost (Table 1). In contrast,
Na-alginate gel presents a higher suitable rigidity, and the prepa-
ration of the beads is quite easy. Thus, Na-alginate was selected for
the entrapment of N. muscorum.
1
mL/min. Detection was done by UV at 254 nm. The starting mate-
rial and product were separated by HPLC with retention times of 21
and 23 min, respectively. No other metabolite was detected in the
transformation medium. The validation method has been described
previously [11]. The product’s relative peak area was calculated and
the amount of residual substrate was determined quantitatively by
subtracting from the quantity of starting material.
2.6. Reuse of immobilized cell beads
The reusability of immobilized biocatalysts is often expected.
Therefore, immobilized N. muscorum cells were examined for
this purpose. After the maximum production of testosterone was
attained in 5 days, the beads were harvested, washed with dis-
tilled water, and added to fresh medium that included 20 mL of
hexadecane for the next use. The process was repeated for 7 batches
until the product formation decreased. Each bioreaction was con-
ducted in 20 mL of hexadecane supplemented with 10 mg of steroid
3
.2. Optimum concentration of alginate beads
The concentration of utilized sodium alginate varied from 1% to
%. As shown in Table 2, a yield of 72 ± 2.3% of testosterone was
4
achieved when beads containing 2% alginate were applied. Higher
concentrations resulted in yield reduction due to the difficulty of
substrate accessibility to the immobilized cells. Beads containing
◦
substrate at 30 C and an agitation rate of 100 rpm for 5 days.
1
% (w/v) Na-alginate also resulted in yield reduction due to the
formation of weak beads, with the escape of the cells.
3
. Result and discussion
Bioconversion of the starting material (I) by immobilized N. mus-
corum cells led to accumulation of the sole bio-product (II) in the
3.3. Culture condition for biotransformation with alginate
immobilized cells
algal medium (Fig. 1).
Bioconversion of AD to testosterone was studied under temper-
◦
◦
3.1. Selection of immobilization method
atures between 25 and 55 C with an interval of 5 C employing
alginate immobilized N. muscorum cells. When the temperature
◦
Immobilized algal cells entrapped in the applied polymers of
was increased gradually from 25 to 55 C, the highest yields of trans-
◦
agar, agarose, ␬-carrageenan, polyacrylamide, polyvinyl alcohol,
and sodium alginate were compared for their bioconversion ratios.
Table 1 shows the effect of each immobilizing carrier on testos-
terone production. Na-alginate entrapped cells could effectively
convert AD with a yield of 72 ± 2.3%, whereas the other matri-
formation were obtained at 30 C for both immobilized and free
cells. However, the immobilized cells were more stable at higher
temperatures than the free cells (Fig. 2). This is probably due to
significant water evaporation in the free wet cells compared to
that of the immobilized cells. The decrease in the water layer sur-
rounding bio-molecules, which is essential for their activity, may
be deleterious to the cells.
Table 1
Influence of type of matrices used for cell immobilization on the bioconversion yield.
Bioconversion was carried out in a 100-mL Erlenmeyer flasks containing 20 mL hex-
◦
adecane at 30 C and 100 rpm for 5 days. The cell volume of immobilized alga was
Table 2
5
mL packed cell volume and substrate concentration was 10 mg/20 mL. The con-
Influences of Na-alginate concentrations on the bioconversion of AD to testosterone.
Bioconversion was carried out in a 100-mL Erlenmeyer flasks containing 20 mL hex-
adecane at 30 C and 100 rpm for 5 days. The cell volume of immobilized alga was
version percentage is expressed as the value as testosterone produced per initial
substrate.
◦
5
mL packed cell volume and substrate concentration was 10 mg/20 mL. The con-
Type of matrix
Testosterone
concentration
Residual AD
(g/L)
Conversion (%)
version percentage is expressed as the value as testosterone produced per initial
substrate.
(
g/L)
Sodium alginate
concentration (%,
w/v)
Testosterone
concentration
(g/L)
Residual AD
(g/L)
Conversion (%)
Whole free cell
Agar
Agarose
0.12
0.38
24 ± 1.3
25 ± 1.8
19.8 ± 1.2
31 ± 3.1
0.0
0.125
0.099
0.155
0.0
0.375
0.401
0.345
0.50
0.482
0.14
␬
-Carrageenan
1
2
3
4
0.09
0.36
0.16
0.105
0.41
0.14
0.34
0.395
18 ± 1.6
72 ± 2.3
32 ± 1.5
21 ± 0.6
Polyacrylamide
Polyvinyl alcohol
Sodium alginate 2% (w/v) 0.36
0.018
3.6 ± 0.6
72 ± 2.3

2
16
H. Arabi et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 213–217
Fig. 4. Effect of incubation time on the production of testosterone by alginate (2%,
w/v) immobilized N. muscorum cells. Bioconversion was carried out in 20 mL hex-
◦
adecane supplemented with 10 mg steroid substrate at 30 C and an agitation rate
of 100 rpm. (ꢀ) Immobilized cells and (ꢁ) free cells.
Fig. 2. Effect of temperature on production of testosterone by alginate (2%, w/v)
immobilized N. muscorum cells. Bioconversion was carried out in 20 mL hexadecane
supplemented with 10 mg steroid substrate at an agitation rate of 100 rpm for 5
days. (ꢀ) Immobilized cells and (ꢁ) free cells.
A similar observation was found in the literature [19–23].
Entrapment is the physical confinement of enzymes within micro-
spaces formed in matrix structures. In this method, enzymes do
not chemically bond to polymeric matrices. Therefore, the three-
dimensional structure of the enzymes may not be affected by the
immobilization procedure, and thus, the optimum parameters of
immobilized and free-form cells may be identical [21,23,24].
Similar to the free cells, the optimum substrate concentration
that could be maximally biotransformed to product was found
to be 10 mg/20 mL. Further increase in substrate concentration
decreased the product formation; however, the immobilized cells
appeared to be less affected than the free cells. It is probably due to
immobilization protection against substrate toxicity (Fig. 3).
The optimum incubation time for the production of testosterone
for immobilized cells was the same as for the free cells, and the
highest yield of testosterone was achieved after 5 days in both the
free and immobilized cells (Fig. 4).
Fig. 5. Effect of shaking speed on the production of testosterone by alginate (2%, w/v)
immobilized N. muscorum cells. Bioconversion was performed in 20 mL hexadecane
supplemented with 10 mg steroid substrate at 30 C for 5 days. (ꢀ) immobilized cells
and (ꢁ) free cells.
◦
External mass transfer limitation can be prevented by a suit-
able hydrodynamic condition. This experiment reflects, to a certain
extent, the stability of the biocatalyst against the mechanical stress
caused by shaking. An increase in the shaking speed from 50 to
1
00 rpm increased the yield from 44.3 ± 0.78% to 72 ± 1.8% (Fig. 5).
Fig. 6. Reusability of immobilized N. muscorum cells for 7 times. Each bioreaction
Fig. 3. Effect of AD concentrations on the production of testosterone by alginate
was conducted in 20 mL of hexadecane supplemented with 10 mg steroid substrate
(
2%, w/v) immobilized N. muscorum cells. Bioconversion was performed in 20 mL
◦
at 30 C and an agitation rate of 100 rpm for 5 days (ꢀ) Na-alginate, 2% and (ꢁ)
◦
hexadecane supplemented with steroid substrate at 30 C and an agitation rate of
Na-alginate, 3%.
1
00 rpm for 5 days. (ꢀ) Immobilized cells and (ꢁ) free cells.

H. Arabi et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 213–217
217
Further increase in the shaking speed caused a decrease in the
amount of formed testosterone. Increasing the shaking speed up
to 200 rpm probably caused cracks in the membranes. This phe-
nomenon may be explained by the variation in the susceptibility
of different microalgae to hydrodynamic forces caused by shaking
and the spontaneous agglomeration of hydrogel particles in non-
polar organic solvents [25]. The latter was previously reported for
hexane, and considerable abrasion of the gel matrix was observed
following the stirring or shaking used for the particles dissociations
free cells in hexadecane. This probably results from the protec-
tive effect of immobilization against organic solvent toxicity. Reuse
of immobilized beads (5 times) gave satisfactory results without
decreasing the enzyme activity and reduced the cost of cell recovery
and recycling.
Acknowledgments
This work was supported by a grant from Biotechnology
Research Center, Tehran University of Medical Sciences, Tehran
[
26–28].
1
4174, Iran. The useful advice of Dr. Sina Adrangi (Tehran University
3.4. The reusability of immobilized algal cell beads
of Medical Sciences) is also acknowledged.
Immobilized cells on alginate beads (3%) were reused in five suc-
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are used. Freely suspended cells were difficult to handle in repeated
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