1. Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 78:949–951 (online: 2003)
DOI: 10.1002/jctb.863
Comparative studies on bioconversion
of benzaldehyde to L-phenylacetylcarbinol
(L-PAC) using calcium alginate- and barium
alginate-immobilized cells of Torulaspora
delbrueckii
Vilas B Shukla and Pushpa R Kulkarni∗
Food & Fermentation Technology Division, University Institute of Chemical Technology, University of Mumbai (UICT), Nathalal Parekh Marg,
Matunga, Mumbai–400 019, India
Abstract: Comparison of the production of L-phenylacetylcarbinol (L-PAC) from benzaldehyde by cells
of Torulaspora delbrueckii immobilized in calcium alginate and barium alginate showed that cells
immobilized in calcium alginate beads were usable for up to six cycles compared with nine cycles for
barium alginate-immobilized cells. Barium alginate-immobilized cells yielded a total of 2.17 g of L-PAC
in nine cycles whereas in the case of calcium alginate-immobilized cells, a total of 1.17 g of L-PAC was
formed when 400 mg benzaldehyde and 400 mm3
of acetaldehyde were used in each cycle as substrate and
co-substrate respectively.
 2003 Society of Chemical Industry
Keywords: benzaldehyde; bioconversion; immobilization; calcium alginate; barium alginate; L-phenylacetyl-
carbinol; Torulaspora delbrueckii
INTRODUCTION
Bioconversion of benzaldehyde to L-phenylacetyl-
carbinol (L-PAC), the key intermediate for the synthe-
sis of L-ephedrine1
by yeast cells, is well established.2,3
In this bioconversion calcium alginate-immobilized
yeast cells were reported to withstand a high concen-
tration of benzaldehyde (up to 6 g dm−3
) and produce
correspondingly more L-PAC. In semi-continuous fer-
mentation by calcium alginate-immobilized cells, the
total amount of L-PAC produced in seven cycles was
found to be five-fold higher than that obtained in
a single batch fermentation.4
No reports on yeast
cells immobilized in any other alginate or comparative
accounts of calcium alginate-immobilized cell mass
with any other alginate matrix for this bioconversion
have been found. Therefore comparative studies on the
isolate, Torulaspora delbrueckii, able to produce L-PAC
from benzaldehyde, were undertaken using calcium
and barium alginates for immobilization.
EXPERIMENTAL
Materials
All the solvents and chemicals used were of AR
grade and were purchased from SD Fine Chemicals
Ltd, Mumbai, India. Media ingredients (Hi Media
Laboratories Pvt Ltd Mumbai) and sodium alginate
(Loba Chemie Pvt Ltd, Mumbai) were used.
An isolate of Torulaspora delbrueckii isolated from
molasses in our laboratory2
was used.
Methods
Growth of the isolate Torulaspora delbrueckii
One cm3
of a suspension of Torulaspora delbrueckii
containing 106
cells was inoculated into 9 cm3
of
growth medium (composition (g dm−3
): glucose 30,
peptone 20, yeast extract 10; pH 5.5) and incubated
on a rotary shaker at 240 rpm and 30 ± 2 ◦
C for 24 h.
The culture obtained was inoculated into 100 cm3
of
the same medium, allowed to grow for 24 h under
the same conditions and centrifuged at 17 000 × g for
15 min at 15 ◦
C.
Immobilization of cells
Three g (wet wt) of the cell mass obtained was
subjected to immobilization by suspending it in 10 cm3
of sterile distilled water at 10 ◦
C and mixing with
sterile sodium alginate (35 cm3
; 40 g dm−3
) solution
at 10 ◦
C. This mixture was dropped by a 10 cm3
pipette having a narrow mouth, into 100 cm3
sterile
∗ Correspondence to: Pushpa R Kulkarni, Food & Fermentation Technology Division, University Institute of Chemical Technology, University
of Mumbai (UICT), Nathalal Parekh Marg, Matunga, Mumbai–400 019, India
E-mail: rekha@foodbio.udct.ernet.in
(Received 2 September 2002; revised version received 6 January 2003; accepted 5 April 2003)
 2003 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2003/$30.00 949

2. VB Shukla, PR Kulkarni
solution of CaCl2 (20 g dm−3
) at 10 ◦
C under gentle
stirring using a magnetic stirrer; the volume of each
drop of the mixture was approximately 0.2 cm3
. The
beads so obtained, having diameters of 2–3 mm, were
kept for curing for 1 h at 15 ◦
C in the same salt solution.
The salt solution was then drained off carefully under
aseptic conditions and the beads were washed twice
with sterile distilled water and used for bioconversion.
The cell mass was also immobilized in a similar manner
using a 20 g dm−3
solution of BaCl2.
Bioconversion using immobilized cells
The beads so obtained were added to 100 cm3
bio-
conversion medium having the following composition
(g dm−3
): glucose 30, peptone 6; pH 4.5. The immo-
bilized cell mass was allowed to adapt to the bio-
conversion medium for 1 h under shaking conditions
of 240 rpm at 30 ± 2 ◦
C. Either 400 mg benzaldehyde
and 400 mm3
acetaldehyde or 600 mg benzaldehyde
and 600 mm3
acetaldehyde were added and bio-
conversion allowed to continue for 200 min under
conditions of shaking (240 rpm), at 30 ± 2 ◦
C.
After bioconversion, the supernatant was separated
from the beads, then extracted thrice with 100 cm3
of diethyl ether. The combined extracts were dried
over anhydrous sodium sulfate, concentrated under
reduced pressure and subjected to GC analysis.6
The
beads were added to 100 cm3
fresh bioconversion
medium to which the same combinations of benzalde-
hyde and acetaldehyde were added and bioconversion
continued similarly. Each experiment was repeated
three times. The results of these parallel experiments
were found to be reproducible within ±5%.
RESULTS AND DISCUSSION
In view of reports on the resistance of barium
alginate beads to swelling, pH changes and chemical
agents,7–9
the reusability of calcium alginate- and
barium alginate-immobilized cells was studied at two
different levels of benzaldehyde and acetaldehyde. In
the case of calcium alginate-immobilized cells the
beads were found to weaken gradually as the number
of cycles increased at both levels of benzaldehyde and
acetaldehyde tried. The effect was more prominent
at 600 mg and 600 mm3
. At 400 mg of benzaldehyde
and 400 mm3
of acetaldehyde gradual increases in
L-PAC and residual benzaldehyde were observed as
the number of cycles increased (Fig 1), with relatively
minor fluctuations of benzyl alcohol in cycles 2 to 6.
In the case of 400 mg of benzaldehyde and 400 mm3
of acetaldehyde a total quantity of 1.17 g L-PAC was
formed over six cycles of use.
At 600 mg of benzaldehyde and 600 mm3
of
acetaldehyde a marked decrease in L-PAC and benzyl
alcohol but an increase in residual benzaldehyde
was observed up to the sixth cycle (Fig 2); a
total of 0.7 of L-PAC was formed in the first
three cycles, after which the L-PAC production was
reduced drastically (Fig 2). A corresponding increase
0
Productprofile(mg)
50
100
150
200
250
300
350
1 2 3 4
Cycle number
5 6
Residual benzaldehyde Benzyl alcohol L-PAC
Figure 1. Reusability of calcium alginate-entrapped cells at 400 mg
of benzaldehyde and 400 mm3 of acetaldehyde.
1 2 3 4 5 6
Cycle number
0
Productprofile(mg)
50
100
150
200
250
300
350
Residual benzaldehyde Benzyl alcohol L-PAC
Figure 2. Reusability of calcium alginate-entrapped cells at 600 mg
of benzaldehyde and 600 mm3 of acetaldehyde.
1 2
300
250
200
150
100
50
0
3 4 5
Cycle number
Productprofile(mg)
6 7 8 9
Residual benzaldehyde Benzyl alcohol L-PAC
Figure 3. Reusability of barium alginate-entrapped cells at 400 mg of
benzaldehyde and 400 mm3
of acetaldehyde.
in residual benzaldehyde was also observed as the
number of cycles increased. Probably the higher levels
of benzaldehyde and acetaldehyde used were toxic to
the cells so that the bioconversion was slowed down
and residual benzaldehyde accumulated. In the case of
cells immobilized in barium alginate, with 400 mg of
benzaldehyde and 400 mm3
of acetaldehyde the beads
were found to be intact up to the ninth cycle. As shown
in Fig 3 there was an increase of L-PAC production
in every cycle up to the fifth cycle, the peak level
950 J Chem Technol Biotechnol 78:949–951 (online: 2003)

3. Calcium and barium-alginate immobilized cells for L-phenylacetylcarbinol production
being maintained through cycles 6 to 8. The benzyl
alcohol level increased up to the third cycle, after
which it started decreasing. The residual benzaldehyde
increased slowly up to the eighth cycle, but increased
sharply in the ninth cycle. A total quantity of 2.17 g of
L-PAC was formed over the nine cycles.
At 600 mg of benzaldehyde and 600 mm3
acetalde-
hyde, the yield of L-PAC and benzyl alcohol increased
between the first and second cycles, after which the
yield of L-PAC decreased drastically (Fig 4). The level
of residual benzaldehyde increased gradually over the
cycles of reuse, up to the sixth cycle. This increase
was very drastic beyond the sixth cycle. Here too,
the higher levels of substrate and co-substrate used,
ie 600 mg and 600 mm3
respectively, were probably
toxic to the cells, resulting in the reduction of biocon-
version to L-PAC and accumulation of the residual
benzaldehyde. A total quantity of around 0.9 g L-PAC
was formed over the first three cycles in this case.
Thus, at the lower levels of substrate and co-
substrate the barium alginate-immobilized cells were
found to be usable satisfactorily for nine cycles (Fig 3),
while with calcium alginate it was only six times
(Fig 1). Mahmoud et al10
have reported reusability
of re-dissolved calcium alginate re-immobilized yeast
cell mass for up to seven cycles. In the present
Cycle number
1 2 3 4 5 6 7 8 9
Residual benzaldehyde Benzyl alcohol L-PAC
Productprofile(mg)
0
50
100
150
200
250
300
350
400
450
500
Figure 4. Reusability of barium alginate-entrapped cells at 600 mg of
benzaldehyde and 600 mm3
of acetaldehyde.
case, however, even without such treatment direct
reuse was possible with barium alginate for up to
nine cycles. When barium alginate beads without cell
mass were placed in the bioconversion medium with
benzaldehyde and acetaldehyde and shaken as before,
no damage was observed to the beads at the levels of
benzaldehyde and acetaldehyde used as above. The
above results suggest barium alginate to be a better
immobilizing matrix than calcium alginate, allowing
the use of immobilized cells for increased numbers of
cycles and subsequently a higher yield of L-PAC than
that obtained with calcium alginate-immobilized cells.
REFERENCES
1 Shukla VB, Madyar VR, Khadilkar BM and Kulkarni PR,
Bioconversion of benzaldehyde to L-phenylacetylcarbinol
(L-PAC) by Torulaspora delbrueckii and conversion to
ephedrine by microwave radiation. J Chem Technol Biotechnol
77:137–140 (2002).
2 Shukla VB and Kulkarni PR, Review: L-phenylacetylcarbinol
(L-PAC): biosynthesis and industrial applications. World J
Microbiol Biotechnol 16:499–506 (2000).
3 Tripathi CKM, Basu SK, Vora VC, Mason JR and Pirt SJ,
Biotransformation of benzaldehyde to L-acetylphenylcarbinol
(L-PAC) by immobilized yeast cells. Res and Ind 36:159–160
(1991).
4 Mahmoud WM, El Sayed AHMM and Coughlin RW, Produc-
tion of L-phenylacetylcarbinol by immobilized yeast cells: II
Semi continuous fermentation. Biotechnol Bioeng 36:55–63
(1990).
5 Nikolova P and Ward OP, Effect of support matrix on ratio
of product to byproduct formation in L-phenylacetylcarbinol
synthesis. Biotechnol Lett 16:7–10 (1994).
6 Shukla VB and Kulkarni PR, Downstream processing of
biotransformation broth for recovery and purification of L-
phenylacetylcarbinol (L-PAC). J Sci and Ind Res 58:591–593
(1999).
7 Tanaka H, Preparation of stable alginate gel beads in electrolyte
solutions using barium and strontium. Biotechnol Tech
2:115–120 (1988).
8 Tanaka H and Irie S, Immobilization of micro-organisms and
enzymes in alginate gel. JP Appl 87(5):887 (1987).
9 Tanaka H and Irie S, Immobilization of micro-organisms and
enzymes in alginate gel. Chem Abs 109:10 702 (1988).
10 Mahmoud WM, El Sayed AHMM and Coughlin RW, Produc-
tion of L-phenylacetylcarbinol by immobilized yeast cells:
Batch fermentation. Biotechnol Bioeng 36:47–54 (1990).
J Chem Technol Biotechnol 78:949–951 (online: 2003) 951