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Process parameters and reusability of the free cell mass of Torulaspora delbrueckii
for the production of L-phenylacetylcarbinol (L-PAC)
V.B. Shukla and P.R. Kulkarni*
Food and Fermentation Technology Division, University Department of Chemical Technology,
University of Mumbai, (UDCT), Nathalal Parekh Marg, Matunga, Mumbai 400 019, India
*Author for correspondence: Tel.: +91-22-4145 616, Fax: +91-22-4145 5614, E-mail: rekha@foodbio.udct.ernet.in
Received 26 October 2000; accepted 15 March 2001
Keywords: Benzaldehyde, biotransformation, L-phenylacetylcarbinol, process parameters, reusability, Torulaspora
delbrueckii
Summary
The e€ect of process parameters on the biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC)
using a yeast isolate identi®ed as Torulaspora delbrueckii was studied. The maximum yield of L-PAC obtained was
(331 mg) per 100 ml biotransformation medium (glucose 3%, peptone 0.6% and at pH 4.5) from 600 mg of
benzaldehyde with 8 h of reaction at 30 ‹ 2 °C. Growing the organism in presence of 3% glucose reduced the
biotransformation time to 120 min. Addition of 0.6% acetaldehyde (30±35%) lead to an increase in L-PAC yield to
450 mg%. Semi-continuous feeding of benzaldehyde (200 mg) and acetaldehyde (200 ll) four times at 30 min
intervals could produce 683 mg of L-PAC/100 ml biotransformation medium. Chiral HPLC analysis of puri®ed L-
PAC and PAC-diol showed 99% enantiomeric purity. The cell mass was found to be reusable for biotransformation
up to nine times when benzaldehyde and acetaldehyde levels were maintained at (350 mg and 350 ll)±(400 mg and
400 ll). At concentrations from 450 mg and 450 ll to 600 mg and 600 ll, however the cell mass could give ecient
biotransformation only during one use.
Introduction
Biotransformation of benzaldehyde to L-phenylacetyl-
carbinol (L-PAC) by yeast has been studied extensively
(Gupta et al. 1979; Agrawal et al. 1987; Agrawal & Basu
1989). Alternating current (Elliah & Krishna 1988),
cofactors (Smith & Hendlin 1953), chemical modi®ca-
tion of substrate (Long & Ward 1989b), size of inoculum
(Gupta et al. 1979), aeration and reduced temperature
(Zeeman et al. 1992) are known to in¯uence L-PAC
production. The rate of L-PAC production is reported
to be dependent on cell mass, sucrose, and benzaldehyde
level (Long & Ward 1989a). All the L-PAC-producing
strains like Saccharomyces cerevisiae, S. carlsbergensis
and Candida utilis that have been examined produce by-
products like benzyl alcohol and PAC-diol (Gupta et al.
1979; Agrawal et al. 1987; Shin & Roger 1996). The
toxicity and inhibitory e€ects of substrate as well as
products (Long & Ward 1989a) in this transformation
make it impossible to reuse the cell mass for many times
and reports on the need for reuse of cell mass in L-PAC
production to economize the process are available
(Coughlin et al. 1991). In the production of L-PAC by
biotransformation, toxicity and inhibitory e€ects of
substrate as well as products are also reported (Long &
Ward 1989a). Use of cyclodextrin to decrease the
toxicity of benzaldehyde for bioconversion using immo-
bilized cells has been reported (Mahmoud et al. 1990;
Coughlin et al. 1991). The work reported in this respect
is mostly on the yeast S. cerevisiae.
A yeast strain identi®ed as Torulaspora delbrueckii
capable of converting benzaldehyde to L-PAC has been
recently isolated in our laboratory (Shukla 1999; Shukla
& Kulkarni 2000). The present communication reports
an examination of the process parameters on production
of L-PAC by this new isolate. The reusability of the cell
mass at various concentrations of benzaldehyde and
acetaldehyde was also studied.
Materials and Methods
Materials
Microbial media components (Hi-Media Ltd., Mum-
bai), solvents and chemicals (AR grade), (S.D. Fine
Chemicals Ltd., Mumbai and Merck India Ltd., Mum-
bai), Co-enzymes (Sigma Chemicals Ltd., St. Louis,
USA), Molasses (Vasant Co-operative Sugar Factory,
Erandol, Dist. Jalgaon, India) and Corn-steep liquor
donated by Anil Starch Pvt. Ltd., Ahmedabad, India,
were used. The yeast isolate from molasses identi®ed as
World Journal of Microbiology & Biotechnology 17: 301±306, 2001. 301
Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.

T. delbrueckii was used. The standards for GC analysis
viz. benzaldehyde and benzyl alcohol were obtained
from Sigma Chemicals Co., St. Louis (USA), while L-
PAC and PAC-diol were obtained by puri®cation of the
biotransformation broth (Shukla & Kulkarni 1999). The
composition of the maintenance medium (Long & Ward
1989a) used was: glucose 2%, peptone 1%, yeast extract
1%, agar 1% and had pH 5.5. The growth medium
(Long & Ward 1989a) had composition glucose 2%,
peptone 2%, yeast extract 1% and had pH 5.5. The
biotransformation medium (Nikolova & Ward 1991)
having composition glucose 5%, peptone 0.6% and
pH 4.5 was used.
Growing the culture
1 ml of 24 h old suspension of T. delbrueckii containing
106
cells was inoculated into 9 ml of growth medium
and incubated on a rotary shaker at 30 ‹ 2 °C at
240 rev/min for 24 h. The culture so obtained was
inoculated into 100 ml of the same medium and allowed
to grow for 24 h under the same conditions. The growth
was centrifuged at 17,000 Â g for 15 min at 15 °C. The
biomass so obtained was used for biotransformation
studies.
Study of process parameters for biotransformation
of benzaldehyde to L-PAC using T. delbrueckii
E€ect of time of biotransformation
The cell mass obtained as above was inoculated into
100 ml biotransformation medium and incubated for
1 h at 30 ‹ 2 °C on a rotary shaker for adaptation.
Benzaldehyde (0.6%) was then added and biotransfor-
mation continued under same conditions for di€erent
time intervals.
E€ect of glucose as carbon source
on the biotransformation
To 100 ml biotransformation medium containing cell
mass 2.6% (wet wt.) 600 mg benzaldehyde was added.
Glucose was added at di€erent levels (0±5%) and the
reaction time maintained at 8 h, which was found to be
optimum from the above experiment.
E€ect of peptone as a nitrogen source on
biotransformation
E€ect of varying peptone concentration (0±1%) was
studied at glucose concentration of 3% optimized in the
previous experiment and keeping other conditions same
as described earlier.
E€ect of initial pH of the biotransformation medium
The initial pH of the medium was varied from 3.0 to 7.5
with glucose at 3%, peptone 0.6% (optimized as above)
keeping the other parameters the same.
E€ect of level of ingredients of growth medium
The organism was grown in growth medium by varying
glucose level at 2, 3 and 4%, yeast extract at 1, 2 and 3%
and peptone at 2, 3, 4% by changing one factor at a time
keeping the other factors constant. The biotransforma-
tion pro®les of the yeast as grown in this manner were
then studied under conditions of process parameters
optimized as above.
Study of bioconversion in presence of di€erent levels
of acetaldehyde
Acetaldehyde (30±35% aq. solution) was added at levels
varying from 0 to 3000 ll at pH 4.5 keeping other
parameters same as above.
E€ect of semicontinuous feeding of benzaldehyde
and acetaldehyde
Benzaldehyde and acetaldehyde were fed at 150±250 mg
and 150±250 ll respectively in the ®rst set at intervals of
30 min and in the second set at interval of 60 min
keeping all other conditions same as above.
Analysis of biotransformation products
This was carried out as described earlier (Shukla &
Kulkarni 1999). Each experiment was repeated three
times. The results of the three parallel results were found
to be reproducible within ‹5%.
Determination of the enantiomeric excess
of biotransformation products
For this purpose, cell mass (3 g wet wt.) obtained from
the growth medium (optimized as above) was added to
100 ml biotransformation medium. After 1 h of adap-
tation 600 mg benzaldehyde was added and reaction
was carried out for 2 h in biotransformation medium
containing 3% glucose, 0.6% peptone and having an
initial pH 4.5 (optimized as above). After extraction by
diethyl ether the sample was subjected to puri®cation by
column chromatography (Shukla & Kulkarni 1999).
The optical rotations of puri®ed L-PAC and PAC-diol
were recorded using a Jasco-360 polarimeter (Jasco
Corporation, Japan) in chloroform. Both compounds
were then subjected to chiral separation using chiral
HPLC. For this purpose a Waters HPLC connected to
u.v. detector at 215 nm and chiralyzer detector with
OD-R column (Dicel Chemical Industries Ltd., Tokyo,
Japan) was used. Other conditions for HPLC were as
follows: mobile phase 40% acetonitrile in water, column
dimensions 250 mm Â 4.6 mm, ¯ow rate of solvent
0.5 ml/min. Along with the chiral column, a diode array
detector (chiralyzer) connected to a polarimetric detec-
tor in series with a u.v. detector was used to obtain
additional information.
Study of the reusability of cell mass
The biomass (3 g wet wt.) obtained by growing in the
growth medium standardized as above was used for
biotransformation studies. The biotransformation me-
dium (standardized as above) had composition (g%) ±
302 V.B. Shukla and P.R. Kulkarni

glucose 3%, peptone 2% and pH 4.5. The biotransfor-
mation medium was kept for adaptation for 1 h at
30 ‹ 2 °C and 240 rev/min. Combinations of benzal-
dehyde and acetaldehyde (350 mg and 350 ll)±(600 mg
and 600 ll) were then added. The reaction was allowed
to take place for 2 h, cell mass centrifuged out and
added into the fresh 100 ml biotransformation medium.
After adaptation for 1 h the same combinations of
benzaldehyde and acetaldehyde were added and the
biotransformation was allowed to take place in a similar
way.
Results and Discussion
For studying the e€ect of important process parameters
on the biotransformation of benzaldehyde to L-PAC
using T. delbrueckii, initially the time of biotransforma-
tion was varied. The maximum yield of L-PAC (331 mg/
100 ml) was observed at 8 h of the reaction, (Figure 1).
L-PAC production is reported to take place in
S. cerevisiae in three phases with the middle phase
producing most L-PAC (Voet et al. 1973) which is also
observed in the present case. The lower L-PAC produc-
tion in the early phase was due to the lag to adapt to
benzaldehyde, which seems to have been overcome in
the middle phase, resulting in more production of L-
PAC. In the last phase due to the combined toxic e€ect
of product, byproduct and substrate on the cells, a
decrease in the rate of L-PAC production was observed.
The e€ect of concentration of glucose as carbon
source on the product pro®le was then studied for a
period of 8 h. It was observed from Figure 2 that L-
PAC as well as benzyl alcohol formation increased with
increasing level of glucose upto 3%, remaining un-
changed thereafter. PAC-diol formation was also lowest
at 3% glucose level, whereas at 1 and 2% of glucose it
increased (Figure 2). Glucose has also been shown to be
a better carbon source than other sources like maltose,
mannitol, citric acid and lactic acid in S. cerevisiae
(Gupta et al. 1979).
In medium without peptone, formation of both L-
PAC as well as benzyl alcohol was much less, with the
addition of 0.2% of peptone, benzyl alcohol production
was drastically increased, while the increase in L-PAC
was comparatively less. From 0.2 to 0.6% level of
peptone, L-PAC increased at a higher rate than benzyl
alcohol (Figure 2). Probably this could be due to
peptone accelerating the level of pyruvate decarboxy-
lase. Gupta et al. (1979) suggested that there is a
relation between source of nitrogen used, its level and
PAC production. In the present case it is established
that when the L-PAC level is lower the reaction appears
to shift to more production of benzyl alcohol and
PAC-diol.
On the acid side at pH 3.0 and 3.5 more residual
benzaldehyde was seen, which decreased drastically
above pH 4.0 and showed no change beyond this pH.
At pH 3.0 benzyl alcohol was much low, and increased
after pH 3.5. Maximum L-PAC formation was observed
in between pH 4.5 and 5.0 (Figure 2). This is in
agreement with reports by Nikolova & Ward (1991,
1992) and Gupta et al. (1979). The present observation
suggests that in the pH range of 4.0±4.5 probably,
maximum activity of PDC may be present, diverting
more benzaldehyde molecules towards L-PAC forma-
tion than towards benzyl alcohol.
From the studies on the product pro®le of the
biotransformation reaction by cell mass grown at
di€erent levels of glucose, peptone and yeast extract
3%, glucose 2%, peptone and 1% yeast extract were
found to give 320 mg% L-PAC production (Figure 3).
When the biotransformation reaction of cell mass
grown in 3% glucose medium was studied at di€erent
time intervals, a time interval of only 2 h was sucient
for the reaction (Figure 4) to reach maximum L-PAC
yield. Increased PDC and ADH with an increase in
glucose level in the medium are known (Entian et al.
1984) which could be responsible for this observation.
Figure 1. E€ect of time of biotransformation on product pro®le.
residual benzaldehyde; ( benzyl alcohol; L-PAC; PAC-diol.
Figure 2. E€ect of glucose level, peptone level and initial pH of
biotransformation medium on product pro®le. Product pro®le at
di€erent levels of glucose: (. . .j. . .) residual benzaldehyde; (. . .r. . .)
benzyl alcohol; (. . .m. . .) L-PAC; (. . .d. . .) PAC-diol. Product pro®le
at di€erent levels of peptone: (ÐjÐ) residual benzaldehyde; (ÐrÐ)
benzyl alcohol; (ÐmÐ) L-PAC; (ÐdÐ) PAC-diol. Product pro®le at
di€erent pH: ( j ) residual benzaldehyde; ( r ) benzyl alcohol;
( m ) L-PAC; ( d ) PAC-diol.
Production of L-phenylacetylcarbinol by Torulaspora delbrueckii 303

Controversial results on the e€ect of the addition of
acetaldehyde as hydrogen acceptor on the production of
L-PAC have been reported (Netraval & Vojtisek 1982;
Zeeman et al. 1992). In the present work (as shown in
Figure 5) at 300 and 600 ll acetaldehyde (30±35%), the
yield of benzyl alcohol decreased, followed by an
increased production of L-PAC (458 mg%). Acetalde-
hyde probably acts as substrate for alcohol dehydro-
genase using less benzaldehyde to decrease yield of
benzyl alcohol and increasing yield of L-PAC. Further
increase in the concentration of acetaldehyde resulted in
lower yields of benzyl alcohol, L-PAC was also com-
paratively lower.
As benzaldehyde and acetaldehyde are toxic and
inhibitory to the transforming organisms, semi-contin-
uous feeding of these was attempted at an interval of
30 min and the reaction was allowed to take place for
4 h. As shown in Figure 6 when the feeding level of
benzaldehyde was 175 mg and of acetaldehyde was
175 ll the production of L-PAC increased appreciably
to 483 mg% as compared to feeding at 150 mg of
benzaldehyde and 150 ll of acetaldehyde (361 mg%).
Also semi-continuous feeding of (200 mg and 200 ll)
and (225 mg and 225 ll) of benzaldehyde and acetalde-
hyde increased production of both L-PAC 683 and
603 mg% and benzyl alcohol 246 and 269 mg%, fol-
lowed by drastic decrease to 499 and 157 mg% at
250 mg and 250 ll levels of benzaldehyde and acetalde-
hyde. Long & Ward (1989a) reported that pulse feeding
to maintain lower benzaldehyde concentration resulted
in prolonged yeast viability and biotransformation
which may have resulted in higher overall yield of
product.
The L-PAC obtained in the present case was then
analysed. The speci®c optical rotation at 30 °C for L-
PAC (0.107 g/l in CHCl3) observed was )387 which
matches the literature values of )380 for >95%
enantiomeric excess (Cadogen 1996a). In the case of
PAC-diol (0.101 g/l in CHCl3) out of possible enantio-
mers the value of speci®c optical rotation )43.56
matches the (1R, 2S) enantiomer which has the value
of )38.6 (3.2 g/l in CHCl3). Other two reported values
are )61.5 (4.3 g/l in CHCl3) for (1R, 2R), and for (1S,
2R) )18.3 (0.24 g/l in EtOH) (Cadogen 1996b).
To con®rm the optical purity of the compounds, they
were subjected to chiral HPLC analysis. The chroma-
tograms after chiral separation showed that both L-
PAC and PAC-diol produced and puri®ed had more
than 99% enantiomeric excess giving a single peak with
same optical rotation as analysed by chiralyzer diode
array detector.
Figure 3. E€ect of glucose, peptone and yeast extract level in the
growth medium on product pro®le. residual benzaldehyde; ( benzyl
alcohol; L-PAC; PAC-diol.
Figure 4. E€ect of time of biotransformation on product pro®le of the
cell mass grown in medium containing 3% glucose. residual
benzaldehyde; ( benzyl alcohol; L-PAC; PAC-diol.
Figure 5. E€ect of acetaldehyde (30±35%) levels on biotransformation
product pro®le. residual benzaldehyde; ( benzyl alcohol; L-PAC;
PAC-diol.
Figure 6. E€ect of feeding levels of benzaldehyde and acetaldehyde on
product pro®le. residual benzaldehyde; ( benzyl alcohol; L-PAC;
PAC-diol.

According to a patent (Seely et al. 1990a, b) yeast
cells cannot be used for multiple batches, as the L-
PAC content drops with increased exposure to the
substrate and product. Reusability of cell mass would
therefore be welcome. In the present work, the
maximum level of benzaldehyde and acetaldehyde
which the organism can tolerate and transform are
600 mg and 600 ll respectively. The reusability of cell
mass of T. delbrueckii was studied at various combi-
nations of benzaldehyde and acetaldehyde from sub-
optimal levels to maximum tolerable levels. It was
observed that at benzaldehyde and acetaldehyde con-
centrations over the range 350 mg and 350 ll±450 mg
and 450 ll, the L-PAC concentration decreased at
second cycle of use and thereafter increased continu-
ously till ninth cycle (Figure 7) and eighth cycle
(Figure 7) respectively. In case of 350 mg or ll of
benzaldehyde and acetaldehyde respectively residual
benzaldehyde disappeared completely by ®fth cycle,
also benzyl alcohol yield was more or less constant
and cell mass could be usable up to nine cycles. In
case of 400 mg or ll benzaldehyde and acetaldehyde
level, benzyl alcohol steadily increased up to the
eighth cycle, whereas in case of 450 mg and 450 ll of
benzaldehyde and acetaldehyde, residual benzaldehyde
increased after the ®fth cycle and decreased later on
benzyl alcohol started decreasing. The PAC yield after
third cycle remained more or less constant up to the
seventh cycle. Beyond 450 mg/ll concentration of
benzaldehyde and acetaldehyde reusability of cell mass
beyond the ®rst cycle was not possible. At the
concentration range of 500 mg and 500 ll±600 mg
and 600 ll maximum L-PAC was formed at the end
of the ®rst cycle itself and from the third cycle
onwards L-PAC yield was zero (Figure 8). The
present results suggest that at beyond 450 mg/ll
concentrations of benzaldehyde and acetaldehyde,
toxicity of these two ingredients hindered the reus-
ability of the cell mass as has been reported in case of
S. cerevisiae by others (Gupta et al. 1979; Long &
Ward 1989a).
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Production of L-phenylacetylcarbinol by Torulaspora delbrueckii 305

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