Towards the bio-production of trans-carveol and carvone from limonene: Induction after cell growth on limonene and toluene

Article abstract of DOI:10.1016/j.tetasy.2003.09.039

Rhodococcus opacus PW4 cells were found to produce trans- and cis-carveol and/or carvone as result of limonene metabolism, depending on the type and concentration of the carbon source used for cell growth. In aqueous systems, cells grown on ethanol and toluene only produced trans-carveol, whilst cells grown on limonene and on toluene with a larger head-space available produced both trans-carveol and carvone. In biphasic systems, limonene was converted to trans- and cis-carveol as well as to carvone, regardless of the carbon source used, although carveol and carvone production rates were higher in toluene and limonene grown cells, respectively. A good and stable emulsion was obtained in a magnetically stirred two-phase reactor but both trans-carveol and carvone were produced at low rates: 0.08 and 0.02 nmol/min mg prot, respectively. No cis-carveol was formed. When (-)-carveol was added, carvone production increased 4.7 fold to 0.12 nmol/min mg prot. Using an aerated two-phase reactor, carvone production was enhanced even with cells grown on toluene. The highest trans- and cis-carveol and carvone production rates were attained with cells grown on limonene by continuously supplying limonene to the reactor through the air stream, carvone production reaching 0.58 nmol/min mg prot. The best trans-/cis-carveol ratio (2.26) was observed with cells grown on toluene when limonene was supplied in the gas phase. When 50 mM limonene was added initially, carvone was produced 27.9 and 141.4 times faster than trans-carveol with cells grown on toluene and limonene, respectively.

Full text of DOI:10.1016/j.tetasy.2003.09.039

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 3925–3931
Towards the bio-production of trans-carveol and carvone from
limonene: induction after cell growth on limonene and toluene
Carla C. C. R. de Carvalho and M. Manuela R. da Fonseca*
Centro de Engenharia Biolo´gica e Qu´ımica, Instituto Superior Te´cnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
Received 2 September 2003; accepted 18 September 2003
Abstract—Rhodococcus opacus PW4 cells were found to produce trans- and cis-carveol and/or carvone as result of limonene
metabolism, depending on the type and concentration of the carbon source used for cell growth. In aqueous systems, cells grown
on ethanol and toluene only produced trans-carveol, whilst cells grown on limonene and on toluene with a larger head-space
available produced both trans-carveol and carvone. In biphasic systems, limonene was converted to trans- and cis-carveol as well
as to carvone, regardless of the carbon source used, although carveol and carvone production rates were higher in toluene and
limonene grown cells, respectively. A good and stable emulsion was obtained in a magnetically stirred two-phase reactor but both
trans-carveol and carvone were produced at low rates: 0.08 and 0.02 nmol/min mg prot, respectively. No cis-carveol was formed.
When (−)-carveol was added, carvone production increased 4.7 fold to 0.12 nmol/min mg prot. Using an aerated two-phase
reactor, carvone production was enhanced even with cells grown on toluene. The highest trans- and cis-carveol and carvone
production rates were attained with cells grown on limonene by continuously supplying limonene to the reactor through the air
stream, carvone production reaching 0.58 nmol/min mg prot. The best trans-/cis-carveol ratio (2.26) was observed with cells
grown on toluene when limonene was supplied in the gas phase. When 50 mM limonene was added initially, carvone was
produced 27.9 and 141.4 times faster than trans-carveol with cells grown on toluene and limonene, respectively.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
perillyl aldehyde by Bacillus stearothermophilus BR388;4
production of trans and cis-carveol and carvone by
enzymes from the plants Solanum aviculare and
Dioscorea deltoidea;5 formation of trans and cis-carveol
and carvone by the basidiomycete Pleurotus sapidus;6
production of carvone from limonene (produced from
geranyl diphosphate) in the fruit of caraway, Carum
carvi;7 conversion of (−)- and (+)-limonene into, respec-
tively, (−)- and (+)-carvone in the plant Mentha car-
diaca;8 production of perillyl alcohol and a-terpineol,
Terpenes are spread throughout nature and some of
them, such as limonene, are readily available at low
cost, which motivates their use as reaction substrates. A
high number of their oxygenated derivatives, known as
terpenoids, are used as flavours and fragrances in the
food and perfume industries.
The oxidation of limonene at the 6-position has been
reported: production of cis- and trans-carveol, carvone,
cis and trans-p-menth-2,8-dien-1-ol, p-menth-1,8-dien-
4-ol, perillyl alcohol and p-menth-8-ene-1,2-diol by
Penicillium digitatum and P. italicum;1 production of
a-terpineol and 6-hydroxycarveol by the fungus Armil-
lareira mellae;2 formation of isopiperitenone, limonene-
1,2-diol, cis-carveol, perillyl alcohol, isopiperitenol and
a-terpineol by the fungus Aspergillus cellulosae;3 pro-
duction of carveol, a-terpineol, perillyl alcohol, and
after cloning the genes encoding
a thermostable
limonene hydratase, in Escherichia coli.9
Recently, Duetz et al.10 presented a toluene-degrading
strain, Rhodococcus opacus PWD4, which is able to
produce enantiomerically pure trans-carveol with traces
of carvone by hydroxylation of
D-limonene exclusively
at the 6-position. These authors used toluene as the
carbon and energy source for cell growth and achieved
a maximum specific activity of 14.7 U (g
of
dry weight
cells). R. globerulus PWD8, also able to degrade tolu-
ene, oxidised the trans-carveol formed after limonene
oxidation in carvone, although at low rates.
* Corresponding author. Tel.: +351-21-841 76 81; fax: +351-21-841 90
62; e-mail: manuela.fonseca@ist.utl.pt
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.09.039

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C. C. C. R. de Car6alho, M. M. R. da Fonseca / Tetrahedron: Asymmetry 14 (2003) 3925–3931
Previous work carried out in our laboratory, concern-
ing the biotransformation of limonene-1,2-epoxide and
(−)-carveol carried out with whole cells of R. erythropo-
lis DCL14, showed that biotransformation rates could
be enhanced by growing the cells in limonene as carbon
source and by performing the reaction in an
organic:aqueous system.11–14 The conversion rates
depended widely on the carbon source used for cell
growth and on the solvent chosen as organic phase. The
present work is aimed at finding favourable growth and
bioconversion conditions to enhance limonene hydroxyl-
ation by R. opacus PWD4 cells.
carveol from limonene, although cells grown on 0.25%
(v/v) toluene produced also a small amount of cis-
carveol. Cells grown on toluene in flasks with a larger
head-space and thus more air (B-Tol) and on limonene
produced both trans-carveol and carvone. When a con-
centration of limonene and toluene of 0.125% was used,
carvone production rate was around 80% that of trans-
carveol, whilst for an initial concentration of 0.25%,
carvone production rate decreased to 60 and 40% the
value of trans-carveol production rate for cells that
metabolised limonene and toluene, respectively. In
biphasic systems, R. opacus PWD4 cells converted
limonene into trans and cis-carveol and carvone, inde-
pendent of the carbon source used for cell growth. Cells
grown on ethanol, toluene and 0.125% B-Tol, produced
three products at similar rates. The best trans-carveol
and carvone production rates were attained with cells
grown on 0.125% limonene. Carveol selectivity was also
improved as the production of cis-carveol was very low.
When metabolising 0.25% of limonene, the carvone
production rate was also higher than that obtained with
the other carbon sources, but cells produced more
cis-carveol as well. Cells grown on 0.25% B-Tol pro-
duced cis- and trans-carveol at similar rates and almost
no carvone.
2. Results and discussion
2.1. Carbon source
Duetz et al.10 found that R. opacus PWD4 cells grown
on glucose were unable to carry out the formation of
trans-carveol or any other product resulting from the
oxidation of limonene, whilst cells grown on toluene as
the sole carbon source produced enantiomerically pure
(+)-trans-carveol with traces of (+)-carvone.
Herein, it has been found that the production of both
trans- and cis-carveol and carvone depends on the type
and concentration of carbon source used for cell
growth (Fig. 1). In single aqueous phase systems, cells
grown on ethanol and toluene produced only trans-
The results indicate that if the cells grow on toluene
with a higher amount of air available, the conversion of
limonene to carvone will be induced in single aqueous
systems, probably due to an activation of oxidative
steps in the metabolic pathway. On the other hand, in
organic:aqueous systems, these cells produced a low
amount of carvone whilst cis-carveol was produced at a
higher rate than when the cells grew with less oxygen
available. The ratio between the production rates of
carvone and trans-carveol obtained with cells grown on
limonene was also lower in biphasic systems than in
single aqueous systems (data not shown). It could be
considered that, since carveol partitions preferentially
to the organic phase, in biphasic systems cells could
have a more difficult access to this compound, and
thus, a lower carvone production. However, since the
reaction is carried out inside the cell, carveol should be
readily available for carvone production. The fact that
cis-carveol production is induced in biphasic systems
could be the result of a higher initial limonene concen-
tration allowed in these systems, in which the organic
phase acts as substrate reservoir.
Degradation of several monoterpenes, namely (+)-(R)-
limonene, (−)-carveol (mixture of enantiomers), (−)-
(R)-carvone, (+)-(S)-carvone and limonene-1,2-epoxide,
in dodecane:aqueous phase systems, indicated the steps
used by R. opacus cells in the metabolism of these
compounds (data not shown). Degradation of limonene
with limonene grown cells resulted in the production of
carvone and almost no production of either trans- or
cis-carveol, although the production rate of the three
compounds increased with increasing initial limonene
concentrations. However, the degradation of limonene
was strongly inhibited by its initial concentration:
almost nothing was produced for an initial limonene
concentration of 30 mM, referred to the aqueous phase.
Figure 1. Trans- and cis-Carveol and carvone production
rates in single aqueous and iso-octane:aqueous systems with
cells grown on 0.125 or 0.25% (v/v) of ethanol (EtOH),
limonene (Lim), toluene (Tol) and toluene in flasks with more
air available (B-Tol); 30°C and 200 rpm with an initial
biomass correspondent to 0.39 g/L of protein; limonene initial
concentration referred to the aqueous phase.

C. C. C. R. de Car6alho, M. M. R. da Fonseca / Tetrahedron: Asymmetry 14 (2003) 3925–3931
3927
Toluene grown cells produced mainly cis- and trans-
carveol from limonene and almost no carvone.
Bioconversion of (−)-carveol by limonene grown cells
resulted in the production of (−)-carvone by degrada-
tion of mainly trans-carveol. For a carveol concentra-
tion range of 3–50 mM, referred to the aqueous phase,
little variation in the carvone production rate was
observed, the average being 0.15 nmol/min mg prot.
Cells grown on toluene produced (−)-carvone, almost
independently of the initial carveol concentration, by
degradation of trans-carveol at a low rate, the average
being 0.08 nmol/min mg prot.
When (−)-carvone was added to biphasic systems with
limonene grown cells, for concentrations referred to the
aqueous phase lower than 16 mM, trans-carveol was
produced, while for concentrations higher than 30 mM,
R. opacus cells produced cis-carveol. Cells grown on
toluene produced both trans- and cis-carveol (the cis
isomer at higher rates) for initial carvone concentra-
tions higher than 16 mM. In all cases, the production
rates were very low (around 0.007 nmol/min mg prot).
Figure 2. trans-Carveol and carvone production rates
obtained with cells grown on agar-based mineral medium
plates with different initial amounts of limonene.
than 80 mL of limonene (concentrations higher than
0.32%, v/v), trans-carveol and carvone production rates
were almost constant in the tested concentrations, car-
vone being produced 7 fold faster than carveol.
The (+)-(S)-carvone tested had a purity of 96%, the 4%
being (+)-trans and (+)-carveol. When this carvone was
used as substrate, it was not metabolised by R. opacus
cells, but the (+)-carveol present was rapidly consumed,
yielding (+)-carvone. Cells grown on limonene showed
a 0.87 nmol/min mg prot production rate, whilst tolu-
ene grown cells attained 4.35 nmol/min mg prot. The
result indicates that R. opacus cells are much better
degraders of (+)-carveol than of (−)-carveol. Limonene-
1,2-diol was formed by conversion of 50 mM (referred
to the aqueous phase) limonene-1,2-epoxide at 3.83 and
8.5 nmol/min mg prot by cells grown on limonene and
toluene, respectively. In both cases, the diol production
rate increased with increasing epoxide concentrations.
Extraction 1:1 (v/v) of the aqueous phase with ethyl
acetate showed that limonene-1,2-diol was also pro-
duced during the experiments (data not shown). The
diol was only detected 24 h after the beginning of the
experiment, which could be the reason why Duetz et al.
(2001) never detected it. However, the production rate
of the diol was nearly 7.9 nmol/min mg prot, by cells
grown on the two highest limonene concentrations
tested. This shows that R. opacus cells also contain a
limonene epoxidase which enables them to convert
limonene in limonene-1,2-epoxide. Furthermore, when
both R. opacus and R. erythropolis DCL14 (which
shows a high carveol dehydrogenase activity and thus
should improve carvone production12) were used to
carry out the conversion of limonene, no carvone or
carveol were produced (data not shown). Only accumu-
lation of limonene-1,2-diol was observed, independently
of the carbon source used for growing R. opacus cells.
Apparently, and considering the results obtained, R.
opacus PWD4 cells contain at least a limonene hydrox-
ylase (conversion of limonene to carveol), a carveol
dehydrogenase (carveol to carvone) and a limonene
epoxide hydrolase (limonene epoxide to limonene diol).
The conversion of carvone to carveol may be achieved
by the same carveol dehydrogenase or by a different
carvone hydrogenase. Further studies concerning these
enzymes need to be made to elucidate the whole
limonene metabolic pathway in this strain.
In all assays described, no differences were observed
when they were carried out with 100 mM glucose as
compared to the those assays performed in the absence
of glucose. Nevertheless, trans-carveol production rates
as high as those presented by Duetz et al.10 were never
attained.
Assays carried out with cells grown on agar-based
mineral medium plates with different amounts of
limonene showed that the production of trans-carveol
and carvone depended on the concentration of
limonene used as carbon source (Fig. 2). Carvone pro-
duction rate decreased with increasing concentrations
of limonene in the petri dish, up to a concentration of
0.16% (v/v) limonene, whilst trans-carveol production
increased. The minimum carvone and maximum
carveol production rates were attained when cells had
grown in the presence of 40 mL of limonene (0.16%).
The converse was observed for higher limonene concen-
trations. When the cells grew in the presence of more
2.2. Magnetically stirred two-phase reactor
In order to increase trans-carveol and carvone produc-
tion rates and to evaluate the production of limonene-
1,2-diol during the experiments, a magnetically stirred
two phase reactor was tested, at 30°C. Iso-octane was
used as organic phase to follow diol accumulation, as
some interference with the diol peak would occur dur-
ing GC analysis if n-dodecane were used.
This reactor allowed the formation of a good and stable
emulsion, which permitted a high area of contact
between the cells and the organic phase containing

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C. C. C. R. de Car6alho, M. M. R. da Fonseca / Tetrahedron: Asymmetry 14 (2003) 3925–3931
limonene. Nevertheless, trans-carveol and carvone were
produced at low rates: 0.08 and 0.02 nmol/min mg prot,
respectively (Fig. 3). No cis-carveol formation was
detected. Diol production was only significant after 25
h of bioconversion, but a production rate of 0.53
nmol/min mg prot was attained onwards.
When commercial carveol was added to the system at
126.7 h, carvone production was enhanced 4.7 fold to
0.12 nmol/min mg prot (data not shown), suggesting that
the step from limonene to carveol is the limiting step in
carvone production.
2.3. Aerated two-phase reactor
The aim of developing an aerated two-phase reactor was
both to increase the amount of air available to the cells,
so as to increase carvone production, and to feed
continuously limonene to the system, while preventing
high concentrations that might be toxic to the cells. The
highest production rates were attained with cells grown
on limonene and when limonene was fed through the air
stream (Fig. 4). In this case, cells produced trans-carveol
nearly 40 times faster than cells grown on toluene while
carvone production rate was, on average, 7 fold higher.
This also means that carvone production was enhanced
even in toluene grown cells, as compared to trans-carveol
production.
Figure 4. trans- and cis-Carveol and carvone production rates
during the biotransformation of limonene on an aerated
two-phase reactor with cells grown on toluene (Tol) or
limonene (Lim). Limonene was supplied either through the air
stream (lim-air stream) or was added initially (lim-added).
The best trans/cis selectivity was obtained with cells
grown on toluene when limonene was supplied through
the air stream, trans-carveol being produced at a rate 2.26
times faster than the cis-isomer (Fig. 5). Cells grown on
toluene and carrying out the conversion in the situation
in which limonene was added at the beginning of the
assay presented a similar trans/cis ratio to those grown
on limonene but performing the bioconversion of
limonene supplied by the gas phase. In these cases, the
cis-isomer was produced faster than trans-carveol.
Almost no trans-carveol was observed by degradation of
limonene, with limonene grown cells, when 50 mM
limonene was added at the beginning of the experiment.
Cells grown on toluene produced trans-carveol at similar
rates when limonene was added through the air stream
and when 50 mM were added initially (maintaining the
aeration). However, in this latter case, both cis-carveol
and carvone were produced at higher rates than when
limonene was supplied in the gas phase. Cells grown on
limonene, produced the three compounds at much higher
rates when limonene was supplied through the air stream.
In all cases, carvone was always the main product of
limonene conversion (Fig. 5). Even when limonene was
added through the air stream (which also enhanced
trans-carveol production), carvone production rate was
18.7 and 4.7 fold higher than that of trans-carveol for
cells grown on toluene and limonene, respectively. When
50 mM limonene was added initially, carvone was
produced 27.9 times faster than trans-carveol with cells
grown on toluene, the ratio achieving 141.4 for cells
grown on limonene.
The blank assay revealed that the time of residence of
limonene inside the reactor was very short when
limonene was supplied through the air stream, specially
when cells were not present (data not shown). This
suggested that a larger amount of solvent should be
present to maintain higher quantities of limonene inside
the reactor. When the organic:aqueous phase ratio was
increased to 2:5 the amount of trans- and cis-carveol and
carvone was much lower than in systems with a
Figrue 3. Time course of the biotransformation in a magneti-
cally stirred two-phase reactor. Concentrations referred to the
organic phase.

C. C. C. R. de Car6alho, M. M. R. da Fonseca / Tetrahedron: Asymmetry 14 (2003) 3925–3931
3929
Figure 5. trans-/cis-Carveol and carvone/trans-carveol production rate ratios observed during limonene conversion in an aerated
two-phase reactor. Abbreviations as in Figure 4.
ratio of 1:5 (data not shown). The decrease was almost
10 fold when limonene was supplied through the gas
phase, independent of the carbon source used for cell
growth. When 50 mM limonene was added initially, the
decrease was even more significant reaching almost 30
fold. The results thus show that this phase ratio did not
improve limonene oxidation. Although higher amounts
of limonene were present, at this phase ratio, only
about a quarter of the organic phase was emulsified
since the extent of coalescence was also high, and thus
the interfacial area was much smaller than in 1:5
systems.
different carvone hydrogenase. The results also suggest
that R. opacus cells degrade (+)-carveol at higher rates
than (−)-carveol.
The production rates of trans-carveol and carvone
depended also on the concentration of limonene used as
carbon source. Carvone production rate decreased with
increasing initial concentrations of limonene, up to a
concentration of 0.16% (v/v), whilst trans-carveol pro-
duction increased. For higher limonene concentrations
the converse was observed. At limonene concentrations
higher than 0.32% (v/v), trans-carveol and carvone
production rates were almost constant in the tested
concentrations, carvone being produced 7 fold faster
than carveol.
3. Conclusions
A good and stable emulsion was obtained in a magnet-
ically stirred two-phase reactor but both trans-carveol
and carvone were produced at low rates: 0.08 and 0.02
nmol/min mg prot, respectively. No cis-carveol was
formed but limonene-1,2-diol was produced from 25 h
onwards at 0.53 nmol/min mg prot. When (−)-carveol
was added, carvone production increased 4.7 fold to
0.12 nmol/min mg prot.
Production of trans- and cis-carveol and/or carvone as
result of limonene metabolism by R. opacus PW4 cells
was found to depend on both the type and concentra-
tion of the carbon source used for cell growth. In
aqueous systems, cells grown on ethanol and toluene
only produced trans-carveol. However, cells grown on
limonene and on toluene with a larger head-space pro-
duced both trans-carveol and carvone. In biphasic sys-
tems, limonene was converted to trans- and cis-carveol
as well as to carvone, regardless of the carbon source
used, although carveol and carvone production rates
were higher in toluene and limonene grown cells,
respectively.
Using an aerated two-phase reactor, carvone produc-
tion was enhanced even with cells grown on toluene.
The highest production rates were attained with cells
grown on limonene and when limonene was fed to the
reactor through the air stream. The best trans-/cis-
carveol ratio (2.26) was observed with cells grown on
toluene when limonene was supplied in the gas phase.
When 50 mM limonene was added initially, carvone
was produced 27.9 and 141.4 times faster than trans-
carveol with cells grown on toluene and limonene,
respectively.
Degradation of (+)-(R)-limonene, (−)-carveol (mixture
of enantiomers), (−)-(R)-carvone, (+)-(S)-carvone and
limonene-1,2-epoxide, indicated that R. opacus PWD4
cells contain a limonene hydroxylase (conversion of
limonene to carveol), a carveol dehydrogenase (carveol
to carvone), a limonene epoxidase (conversion of
limonene to limonene-1,2-epoxide) and a limonene
epoxide hydrolase (limonene epoxide to limonene diol).
The conversion of carvone to carveol may be achieved
by the same mentioned carveol dehydrogenase or by a
Although our results show that biphasic systems may
largely increase trans-carveol and carvone productions
as compared to single aqueous systems, specially if air

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C. C. C. R. de Car6alho, M. M. R. da Fonseca / Tetrahedron: Asymmetry 14 (2003) 3925–3931
is supplied, the cells used were never able to produce
trans-carveol at the rate indicated by Duetz et al.,
assays, 100 mM glucose were also added initially. The
reaction started by adding 1, 5 or 17 mL of limonene.
The bioconversions were carried out in test tubes closed
with tin foil and tape, at 30°C and 200 rpm. The
limonene conversion was followed by monitoring
limonene consumption and product accumulation in
the organic phase by GC analysis.
which was 14.7 U/g
of cells, in a single aqueous
dry weight
system.10
4. Materials and methods
4.1. Strain
4.4.3. Biphasic systems magnetically stirred. 4 mL of
iso-octane containing 50 mM limonene were added to
20 mL of phosphate buffer containing R. opacus cells
(corresponding to 9.44 mg of protein) and 100 mM
glucose in a 100 mL flask closed with rubber bungs
wrapped in tin foil. The bioconversion was performed
at 30°C and 200 rpm and monitored by GC analysis of
the organic phase.
R. opacus PWD4 (DSMZ 44313) was supplied by Duetz
and co-workers.
4.2. Growth on solid media
4.2.1. On toluene. Cells were grown on agar-based
mineral medium plates, without any added carbon
source, and incubated in a desiccator with a solution of
10% (v/v) toluene in n-hexadecane. After 4 days at
room temperature, the cells were scrapped from the
agar surface and suspended in mineral medium with the
same composition as described by Wiegant and de
Bont.15
4.4.4. Biphasic systems with aeration. The reaction sys-
tem consisted of 20 mL of phosphate buffer containing
the cells (corresponding to 9.69 mg of protein) and 4
mL of n-dodecane. Limonene was either continuously
fed by the air stream (by bubbling the inlet air at 158.8
mL/min, through the sintered glass sparger of a glass
bottle containing limonene; the air stream entered each
reaction flask at 79.4 mL/min) or added initially (170
mL; air was supply directly to the reaction flask at 79.4
mL/min).
4.2.2. On limonene. Cells were grown at 30°C on agar-
based mineral medium plates to which 5, 10, 20, 40, 80
or 160 mL of limonene were added before solidification
of the agar (25 mL per Petri dish). The petri dishes were
wrapped in celophane paper to prevent limonene evap-
oration. Four days after inoculation colonies were
already visible on the plates. The cells were harvest and
suspended in mineral medium as described previously.
4.5. Chemicals
The terpenes used were (+)-(R)-limonene (97%), (−)-
carveol (97%), (−)-(R)-carvone (98%) and (+)-(S)-car-
vone (96%) from Aldrich Chemicals. The ethyl acetate
(99.5%) used for extracting the terpenes from the
aqueous phase was from Riedel-de Hae¨n. The organic
solvents used in biphasic systems were n-dodecane
(>99%) purchased from Aldrich and iso-octane
(>99.5%) from Riedel-de Hae¨n.
4.3. Growth on liquid media
Cells were grown on 1 L flasks, closed with rubber
stoppers, containing 150 mL of mineral medium. 4 mL
of cell suspension, previously grown on 1 g/L ethanol,
were used as inocculum. The carbon sources used were
0.125% (v/v) ethanol, limonene and toluene. Growth
was followed by measuring the optical density (O.D.) at
600 nm. When the value was higher than 1.5, cells were
harvested by centrifugation (7000 rpm, 10 min) and
used forthwith in the bioconversion studies.
4.6. Analysis
Limonene consumption and carveol and carvone accu-
mulation was followed by gas chromatography on a
Hewlett Packard 5890 gas chromatograph with a FID
detector, connected to a HP3394 integrator. The capil-
lary column was a SGE HT5, 25 m in length and with
internal and external diameters of 0.22 and 0.33 mm,
respectively. The oven temperature was 120°C and that
of the injector 200°C. The detector was at 250°C.
4.4. Bioconversion assays
All assays were carried out at least in duplicate.
4.4.1. Single aqueous systems. To 2 mL of fresh phos-
phate buffer (50 mM, pH 7.0) containing the cells, 1 or
5 mL of limonene were added on a 10 mL test tube,
closed with tin foil and tape to prevent limonene losses.
The reactions were carried out at 30°C and 200 rpm. In
some of the assays, 100 mM of glucose were added
initially.10 The reaction was followed by gas chro-
matography (GC) after extraction of substrate and
products from the aqueous phase with 0.5 mL of ethyl
acetate.
4.7. Error analysis
The error associated with the GC quantification of
samples, injected without previous treatment, was 6%
and that of aqueous samples, extracted with ethyl ace-
tate prior to injections, was 8%. The errors were
calculated based on the standard deviation and sample
mean of seven repeated injections and are quoted for a
confidence interval of 95%. Biomass concentration
measurements (O.D.) had an associated error of 8%
based on the standard deviation and sample mean of
eight repeated samples, quoted for a confidence interval
of 95%.
4.4.2. Biphasic systems. To 2 mL of fresh phosphate
buffer (50 mM, pH 7.0) containing the cells, 0.4 mL of
either iso-octane or n-dodecane were added. In some

C. C. C. R. de Car6alho, M. M. R. da Fonseca / Tetrahedron: Asymmetry 14 (2003) 3925–3931
3931
Acknowledgements
6. Onken, J.; Berger, R. G. J. Bio-technol. 1999, 69, 163–
168.
7. Bouwmeester, H. J.; Gershenzon, J.; Konings, M. C. J.
M.; Croteau, R. Plant Physiol. 1998, 117, 901–912.
8. Rani, K.; Akhila, A. Fitoterapia 1998, 69, 337–348.
9. Savithiry, N.; Cheong, T. K.; Oriel, P. Appl. Biochem.
Biotech. 1997, 63–65, 213–220.
10. Duetz, W. A.; Fja¨llman, A. H. M.; Ren, S.; Jourdat, C.;
Witholt, B. Appl. Env. Microbiol. 2001, 67, 2829–2832.
11. de Carvalho, C. C. C. R.; van Keulen, F.; da Fonseca,
M. M. R. Biocatal. Biotrans. 2000, 18, 223–235.
12. de Carvalho, C. C. C. R.; da Fonseca, M. M. R. J. Mol.
Catal. B-Enzym. 2002, 19–20, 389–398.
This study was supported by a Ph.D. grant (PRAXIS
XXI/BD/21574/99) awarded to Carla da C.C.R. de
Carvalho by Fundac¸a˜o para a Cieˆncia e a Tecnologia,
Portugal. The authors would like to thank Dr. Wouter
A. Duetz and co-workers for supplying the strain R.
opacus PWD4 used in this study.
References
13. de Carvalho, C. C. C. R.; da Fonseca, M. M. R. J. Mol.
1. Bowen, E. R. Proc. Fla. State Horti. Soc. 1975, 304–308.
2. Draczynska, L. B. J. Basic Microbiol. 1987, 27, 191–196.
3. Noma, Y.; Yamasaki, S.; Asakawa, Y. Phytochemistry
1992, 31, 2725–2727.
4. Chang, H. C.; Oriel, P. J. J. Food Sci. 1994, 59, 660–662.
5. Vanek, T.; Valterova, I.; Vankova, R.; Vaisar, T. Bio-
technol. Lett. 1999, 21, 625–628.
Catal. B-Enzym. 2002, 19–20, 377–387.
14. de Carvalho, C. C. C. R.; van Keulen, F.; da Fonseca,
M. M. R. Tetrahedron: Asymmetry 2002, 13, 1637–
1643.
15. Wiegant, W. M.; de Bont, J. A. M. J. Gen. Microbiol.
1980, 120, 325–331.