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Characterization of monoterpene
biotransformation in two pseudomonads
ARTICLE in JOURNAL OF APPLIED MICROBIOLOGY JANUARY 2009
Impact Factor: 2.39 DOI: 10.1111/j.1365-2672.2008.03923.x Source: PubMed

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Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Characterization of monoterpene biotransformation in two

pseudomonads
J.L. Bicas1,2, P. Fontanille2, G.M. Pastore1 and C. Larroche2
1 Laboratorio de Bioaromas, Departamento de Ciencia de Alimentos FEA, Universidade Estadual de Campinas, Rua Monteiro Lobato,
Campinas-SP, Brasil
2 Laboratoire de Genie Chimique et Biochimique PolytechClermont-Ferrand, Universite Blaise Pascal, Aubie`re Cedex, France

Keywords
a-pinene, b-pinene, biphasic system,
limonene, Pseudomonas.
Correspondence
Juliano Lemos Bicas, Laboratorio de
Bioaromas, Departamento de Ciencia de
Alimentos FEA-UNICAMP. Rua Monteiro
Lobato 80, 13083-862, Campinas-SP, Brazil.
E-mail: jlbicas@gmail.com

2008 0374: received: 3 March 2008, revised

5 June 2008 and accepted 6 June 2008
doi:10.1111/j.1365-2672.2008.03923.x

Abstract
Aims: To study the metabolic profile of Pseudomonas rhodesiae and Pseudomonas fluorescens in waterorganic solvent systems using terpene substrates for
both growth and biotransformation processes and to determine the aerobic or
anaerobic status of these degradation pathways.
Materials and Methods: Substrates from pinene (a-pinene, a-pinene oxide,
b-pinene, b-pinene oxide, turpentine) and limonene (limonene, limonene-1,2oxide, orange peel oil) families were tested. For the bioconversion, the terpenegrown biomass was concentrated and used either as whole cells or as a crude
enzymatic extract.
Conclusion: Pseudomonas rhodesiae was the most suitable biocatalyst for the
production of isonovalal from a-pinene oxide and did not metabolize limonene.
Pseudomonas fluorescens was a more versatile micro-organism and metabolized
limonene in two different ways. The first (anaerobic, cofactor-independent,
noninducible) allowed limonene elimination by synthesizing a-terpineol. The
second (aerobic, cofactor-dependent) involved limonene-1,2-oxide as an intermediate for energy production through a b-oxidation process.
Significance and Impact of the Study: Enzymatic isomerization of b- to
a-pinene was described for the first time for both strains. Alpha-terpineol
production by P. fluorescens was very efficient and appeared as a promising
alternative for the commercial production of this bioflavour.

Introduction
Terpenes are secondary metabolites of plants that are
produced, in part, as a defense against micro-organisms
and insects, in addition to their pollinator-attractive
properties (Gershenzon and Dudareva 2007). The simpler terpenes (mono- and sesquiterpenes) and terpenoids
are the major constituents of essential oils and are
widely used in the flavour industry. They may be incorporated in the formulation of foods, cosmetics, hygiene
and household products, acting not only as flavourings
(Bauer et al. 2001), but also as antibacterial agents (Griffin
et al. 1999; Bakkali et al. 2007). Moreover, some functionalized terpenes also present bioactivity against certain

types of tumour cells (Crowell 1999; Jun et al. 2006). In

this context, the interest in such compounds is constantly growing.
R-(+)-limonene and a-pinene are the most widespread
monoterpenes in nature. Owing to their abundance and
relatively low cost, these two monoterpenes have been
used as the main precursors for the synthesis of their
oxygenated counterparts. Oxygenation reactions may be
chemically catalysed, but the use of biotechnological tools
to perform them has been thoroughly investigated in the
last years, as they proceed under mild conditions, have
high regio- and enantioselectivities, do not generate toxic
waste and the products obtained can be labelled as
natural (Serra et al. 2005).

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1991

Degradation of monoterpenes

J.L. Bicas et al.

The microbial transformation of terpenes started 40

50 years ago, when soil pseudomonads were used for the
degradation of limonene (Dhavalikar and Bhattacharyya
1966; Dhavalikar et al. 1966), a- and b-pinenes (Shukla
and Bhattacharyya 1968; Shukla et al. 1968). Fungalmediated oxidation of terpenes was described in the same
period, after an Aspergillus niger capable of metabolizing
a-pinene to oxygenated products was selected among
different moulds (Bhattacharyya et al. 1960; Prema and
Bhattacharyya 1962a,b).
The mechanism of a-pinene degradation described in
the first studies passed through limonene and perillyl
alcohol to form isopropyl pimelic acid, besides four other
possible pathways (Shukla and Bhattacharyya 1968). Some
years later, other authors described another a-pinene
degradation pathway passing by limonene but with other
intermediate acids (Gibbon and Pirt 1971; Tudroszen
et al. 1977). A third metabolic route (pathway 1a in
Fig. 1), a variation of the last, was proposed for Pseudomonas fluorescens NCIMB 11671, which was capable of
completely degrading a-pinene (Best et al. 1987). A similar pathway was also evidenced for Nocardia sp. (Griffiths
et al. 1987a,b). Further reports suggest a different
dynamic for this pathway, explaining the formation of
novalal by isomerization of isonovalal (Zorn et al. 2004;
Laroche et al. 2006; pathway 1b in Fig. 1). Studying this
process more in depth, an optimized method for isonovalal production from a-pinene oxide by Pseudomonas
rhodesiae has been developed (Fontanille et al. 2002;
Fontanille and Larroche 2003). More recently, Yoo and
Day (2002) reported another a-pinene degradation pathway for Pseudomonas PIN. This novel route integrates
a-pinene, b-pinene, limonene and p-cymene and leads to
the formation of perillic acid, cumic acid and a-terpineol.
The degradation pathways for b-pinene have not yet
been fully elucidated. It is known that some A. niger
strains are capable of transforming it into a-terpineol as
the main product (Toniazzo et al. 2005; Rozenbaum et al.
2006). Different hydroxylated products have also been
identified for Botrytis cinerea (Farooq et al. 2002) and
Armillariella mellea (Draczynska et al. 1985; pathways 17
and 18 in Fig. 1). For bacteria, a common route for limonene, a- and b-pinene has already been described for
Bacillus pallidus BR425. In this case, myrtenol and pino-

carveol are the main products from b-pinene (Savithiry

et al. 1998). Conversely, the fermentation of b-pinene by
Pseudomonas PL resulted in different products (Shukla
and Bhattacharyya 1968; pathway 20 in Fig. 1). The main
metabolic pathways for a- and b-pinene are summarized
in Fig. 1.
It is observed that limonene is commonly found as an
intermediate in the metabolism of a- and b-pinene.
Therefore, it is possible to assume that, for some microorganisms, these two substrates may also follow a pathway similar to that observed for limonene. Marostica and
Pastore (2007a) have recently reviewed the main
metabolic routes for limonene and Fig. 2 illustrates its six
different degradation pathways, which are: (i) oxidation
of carbon 7 to perillyl compounds; (ii) ring double bond
epoxidation, followed by the corresponding diol formation and its oxidation; (iii) carbon 6 oxidation to form
carveol, carvone and dihydrocarvone; (iv) carbon 8
hydroxylation to directly form a-terpineol; (v) oxidation
of carbon 3 to form isopiperitenol and isopiperitenone
and (vi) 8,9 double bond epoxidation to form limonene8,9-epoxide. Other papers describe the catabolism of
acyclic monoterpenes (Forster-Fromme et al. 2006 and
references therein), the degradation of monoterpenes in
anaerobic conditions (Harder and Probian 1995) and
review the biotransformation of limonene (Duetz et al.
2003) and other terpenes (Van Der Werf et al. 1997; De
Carvalho and Da Fonseca 2006).
The two strains tested in this study, P. rhodesiae CIP
107491 and P. fluorescens NCIMB 11671, are recognized
for their ability to use a-pinene as sole carbon source and
to convert it into different oxygenated terpenoids. However, the information on the utilization of other terpenes
is limited. Therefore, different terpene sources were tested
as sole carbon sources and as substrates for biotransformation of these micro-organisms.
Materials and methods
Micro-organisms and chemicals
The two strains employed in this work were P. rhodesiae
CIP107491 and P. fluorescens NCIMB 11671. Alphapinene (Acros, 97% purity), ())-b-pinene (Fluka, >99%

Figure 1 Main metabolic pathways of a- and b-pinene described in the literature. Pathways: 1a reported by Best et al. (1987) and Griffiths et al.
(1987a,b); 1b by Laroche et al. (2006); Zorn et al. (2004); 24 and 1315 by Schrader (2007); 5 by Agrawal et al. (1999); Agrawal and Joseph
(2000a,b); Draczynska et al. (1985); Prema and Bhattacharyya (1962a); Rozenbaum et al. (2006); Van Dyk et al. (1998); and Wright et al. (1986);
6 by Gibbon and Pirt (1971); Savithiry et al. (1998); Tudroszen et al. (1977) and Yoo and Day (2002); 7 by Chatterjee et al. (1999); Draczynska
et al. (1985); Rozenbaum et al. (2006) and Wright et al. (1986); 810, 21 and 22 by Savithiry et al. (1998); 11 and 20 by Shukla and Bhattacharyya (1968) and Shukla et al. (1968); 12 by Draczynska et al. (1985); Prema and Bhattacharyya (1962a) and Wright et al. (1986); 16 by Rozenbaum
et al. (2006) and Toniazzo et al. (2005); 17 by Farooq et al. (2002); 18 by Draczynska et al. (1985); 19 by Van Dyk et al. (1998) and 23 by
Savithiry et al. (1998) and Yoo and Day (2002). The wide arrows represent the metabolism of pinenes by Pseudomonas rhodesiae and Pseudomonas fluorescens and the dash arrows correspond to the isomerization described in this study.

1992

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J.L. Bicas et al.

Degradation of monoterpenes

Isonovalal

HO

Isonovalic acid

CHO

COOH

1a

COOH

1a
Carveol

Carvone

1a,b

1b
CHO

4
O

Dimethyl
pentanoic
acid

COOH

1a

1a,b
1a,b

-pinene
oxide

Thujone

Novalic acid

Novalal

2
OH

Borneol
Verbenol

OH

HO
6 -hydroxy- -pinene

O
Verbenone

+
OH
HO

Fig. 2
5

9-hydroxy- pinene

Limonene
15

16

OH

23

OH

14
O
OH
4 -hydroxy- pinene-6-one
13

OH
OH
4,5-dihydroxy -pinene
+

OH
-terpineol

17

-pinene

2 ,3 -dihydroxy- -pinene
+

-pinene

22
10
CH2OH
OH

O
12

Pinocarveol

Pinocamphone

21

Myrtenol

11

CH2O

OH

20
O

Pinocarvone

OH
trans-sobrerol

O
OH
4 -hydroxy- -pinene-6-one

19

18

CH2OH

Pinocamphone
Myrtenal
OH
7-hydroxy- -terpineol

COOH

COOH

COOH

COOH

OH
+

Borneol

OH
OH
Oleuropeic acid

or

4-hydroxy
phellandric acid
(from -pinene)

COOH

OH
4-hydroxydihydro
phellandric acid
(from -pinene)

-isopropyl
pimelic acid

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1993

Degradation of monoterpenes

J.L. Bicas et al.

CH2OH

COH

COOH

COSCoA

-oxidation

Perillyl alcohol

Perillyl aldehyde

Perillic acid

OH
O

OH

OH

perillyl-CoA
O

O
O

SCoA

Limonene1,2-oxide

Limonene1,2-diol

HO

1-hydroxy-2oxolimonene

3-isopropenyl-6oxoheptanoate

3-isopropenyl-6oxoheptanyl-CoA

Carvone

Carveol

Dihydrocarvone

OH
-teripenol

OH

Isopiperitenone

Isopiperitenol

O
Limonene-8,9-oxide

purity), R-(+)-limonene (Fluka, 98% purity), ())a-pinene oxide (Aldrich, 97% purity), b-pinene oxide
(Acros, mixture of cis trans isomers, 75% purity), (+)limonene-1,2-oxide (Aldrich, mixture of cis trans isomers,
97% purity) and (+)-carvone (Acros, 98% purity) were
used as substrates in n-hexadecane (SDS, 99% purity) as
organic phase. The complex monoterpene mixtures, turpentine and orange peel oil, obtained in the Brazilian
market, were also tested.
Preculture preparation
Three full loops of a 24-h-old culture on a petri dish were
transferred to a 500 ml conical flask that contained 10 g
1994

Figure 2 The six main metabolic pathways for limonene (Van Der
Werf et al. 1999; Marostica and Pastore 2007a). The wide arrows
represent the metabolism of limonene by Pseudomonas fluorescens
described in this study.

of carbon source (sodium lactate for P. rhodesiae and

glucose for P. fluorescens), 025 g of (NH)4SO4, 5 ml of
Hutner solution (Fontanille 2002), 10 ml of solution A
(65 g of K2HPO4, 828 g of KH2PO4 in 250 ml distilled
water) and 235 ml of distilled water. The flasks were
incubated at 30C and 200 rev min)1 for 24 h, to reach
an optical density close to 40 at 600 nm (OD600).
Cell cultures
Twenty millilitres of the preculture were aseptically transferred to a 500 ml conical flask with the same culture
medium as described before. However, the sole carbon
source consisted of 05 g of one of the terpene substrates

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J.L. Bicas et al.

Degradation of monoterpenes

diluted in 125 ml of n-hexadecane. The flasks were left at

30C and 200 rev min)1 for 24 h.

concentrations measured at a given time always gave differences less that 10%.

Recovery of the biomass

Analytical conditions

After centrifuging the culture at 2600 g for 10 min, the

supernatant was eliminated and the resulting biomass was
resuspended in 25 ml of 20 mmol l)1 of phosphate buffer
pH 75. This biomass was either used directly (biotransformation with fresh cells) or was frozen ()18C) for the
trials using crude enzymatic extracts. The biomass concentration was determined indirectly by the OD600 of the
medium, through the following experimental correlations:
P. rhodesiae (Fontanille et al. 2002): biomass (gram per
litre of the medium) = 041 OD600; P. flourescens: biomass (gram per litre of the medium) = 035 OD600.

The products obtained were analysed in a HP 5890 gas

chromatograph with a flame ionization detector (GCFID). A SBP-5 (Supelco) capillary column of 30 m
032 mm 025 lm id was employed. Nitrogen was the
carrier gas, with a constant pressure in the head of the
column of 08 bar and split ratio of 1 : 5. The temperature programme used was as follows: initial temperature
of 80C for 5 min, rising at 20C min)1 until 200C, then
held for 5 min. The temperatures in the injector and
detector were both 250C. The quantification was performed using 10 ml of heptadecane (Fluka, 98% purity)
per litre of n-hexadecane as internal standard and the
yield was calculated as the ratio of the amount of product
recovered (g) to the mass of substrate consumed (g). The
products obtained were identified by a HP 6890 gas chromatograph coupled with a HP 5973 mass selective detector (GC-MS). Helium was the carrier gas and the split
ratio was 1 : 5. The capillary column and the temperature
programme was the same as before. The MS system operated with an electron impact of 70 eV, an acceleration
voltage of 11 kV and an emission current of 35 lA. The
temperatures of the quadrupole, the ionic source and the
GC-MS interface were 150C, 230C and 280C, respectively. The isomers obtained, i.e. novalal and isonovalal,
were identified on the basis of their retention times and
mass spectra. An example of a GC analysis is shown in
Fig. 3.

Production of the crude enzymatic extracts

The concentrated biomass of P. rhodesiae (see preceding
paragraph) was thawed and treated for 1 h at 30C and
200 rev min)1 with 6% (v v) diethyl ether (Fontanille
and Larroche 2003). The P. fluorescens strain employed
in this study has shown a relatively high resistance to
cell permeabilization, in comparison with P. rhodesiae.
Hence, the standard protocol used for this last microorganism allowed 7090% of the initial biomass to
remain viable. The cells were then disrupted by a threecycle passage in a two-stage high-pressure homogenizer
(model APV 2000; APV Systems, Alberstlund, Denmark),
using 1000 bars in the first stage and 100 bars in the
second. This methodology was able to reduce the final
number of viable cell counts to 5% of the initial value.
The suspension resulting from this operation was membrane filtered (045 lm) to remove the remaining viable
cells.
Biotransformation procedure
Twenty-five millilitres of the concentrated biomass and
the same volume of n-hexadecane were transferred to a
250 ml conical flask. The substrate was added to reach a
final concentration of 40 g l)1 of n-hexadecane. The flasks
were incubated at 30C and 200 rev min)1. Samples were
periodically taken from the organic and aqueous phases
to follow substrate consumption and product formation.
The organic phase was directly injected (1 ll) in the gas
chromatograph while the aqueous phase had to be acidified (20 ll ml)1 concentrated sulfuric acid) and extracted
with the same volume of etherhexane (1 : 1, v v) before
the organic layer was injected (1 ll) in the gas chromatograph. The procedure for the crude enzymatic extract was
similar. Experiments were carried out in triplicate and

Results
Characterization of the complex substrates
Turpentine and orange peel oil are cheap and widespread
terpene sources and could be involved in bioconversion
processes, either as inducing agents for biocatalyst production or as precursors for the reaction itself.
As for any other agroindustrial product, their composition may vary depending on seasonal conditions. The
composition of turpentine and orange peel oil, shown in
Table 1, was determined by GC-FID and GC-MS. The
concentration of the main compound in each mixture
was consistent with already reported values (Table 1).
Utilization of the terpene substrates for bacterial growth
The two micro-organisms were first tested for their ability
to grow on terpene substrates as sole carbon and energy
sources. The growth was stopped after 24 h because the

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Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 19912001

1995

Solvent

-pinene oxide

Isonovalal

-pinene

-pinene
FID signal

Internal standard

J.L. Bicas et al.

Novalal

Degradation of monoterpenes

10

12

Time (min)

Table 1 Main constituents of turpentine and orange peel oil used in

this study

Component

Proportion
(%, peak area in
gas chromatography)

Turpentine
a-pinene

671

Camphene
R-(+)-limonene
Others
Orange peel oil
R-(+)-limonene

Cis- and translimonene-1,2-oxide

Carvone
Others

Reported values (%)

6070 (Bauer et al.

2001);
4389 (Coppen et al.
1998)

206
46
77
877

8397 (Shaw 1979);

943 (Marostica and
Pastore 2007b)

36
16
71

biomass obtained at this time was a good indicator of the

ability of a given substrate to support bacterial growth.
The utilization of a given terpene indicated that a
metabolic pathway involving it was actually active in the
corresponding bacterial strain.
It can be observed in Table 2 that a-pinene, b-pinene
and a-pinene oxide were substrates for both micro-organisms. As expected, turpentine was also a substrate for the
two bacteria, probably operating as an a-pinene source.
The other constituents of turpentine were not toxic to the
bacteria, at least at the concentration assayed. Limonene
and b-pinene oxide were substrates only for P. fluorescens,
while limonene-1,2-oxide and orange peel oil were
substrates for none of the two strains. All the degradable
1996

14

Figure 3 Example of a gas-chromatographic

analysis: bioconversion of b-pinene by fresh
cells of Pseudomonas rhodesiae grown on
b-pinene. Experimental conditions are
described in Table 3.

Table 2 Utilization of the terpene substrates by Pseudomonas rhodesiae and Pseudomonas fluorescens for growth. The inoculum
consisted of a culture grown on a medium with 025 g of (NH)4SO4,
5 ml of Hutner solution (Fontanille 2002), 10 ml of solution A (65 g
of K2HPO4, 828 g of KH2PO4 in 250 ml of distilled water), 1 g of
Na-lactate (P. rhodeisae) or 1 g of glucose (P. fluorescens) and 235 ml
of distilled water. Cell growth was developed in the same medium
using 05 g of the terpene substrate in 125 ml of n-hexadecane as
sole carbon source
Terpene
substrate
a-pin
b-pin
Lim
a-pin ox
b-pin ox
Lim ox
Turp
OPO

Growth after 24 h
P. rhodesiae

P. fluorescens

+ (09)
+ (18)
)
+ (03)
)
)
+ (11)
)

+ (17)
+ (16)
+ (14)
+ (08)
+ (09)
)
+ (18)
)

a-pin, a-pinene; b-pin, ())-b-pinene; lim, R-(+)-limonene; a-pin ox, ())a-pinene oxide; b-pin ox, b-pinene oxide; lim ox, (+)-limonene-1,2oxide; Turp, turpentine; OPO, orange peel oil; +, growth; ), no
growth.
Numbers in parentheses are the approximate final biomass concentration (g l)1).

terpene substrates produced biomass with concentrations

ranging from 03 to 18 g l)1 and these were usually lower
for the epoxides (Table 2).
Biocatalytic activity of Pseudomonas rhodesiae and
Pseudomonas fluorescens
To define the metabolic pathways through which the terpene substrates were degraded and to find activities that
could be exploited at a preparative scale, bioconversion

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J.L. Bicas et al.

Degradation of monoterpenes

Table 3 Main products accumulated in the bioconversion of the a-pinene, b-pinene and limonene families by Pseudomonas rhodesiae and Pseudomonas fluorescens. The culture grown on a terpene substrate as sole carbon source (24 h at 30C and 200 rev min)1) was concentrated 10
times and the resulting biomass was either used fresh or as a crude enzymatic extract (1 h at 30C and 200 rev min)1, in the presence of 6%
ether, for P. rhodesiae, or three cycle passage in a two-stage APV Systems homogenizer, using 1000 bar in the first stage and 100 bar in the second, for P. fluorescens, before being used). The bioconversion trials consisted of a 250 ml conical flask filled with 25 ml of concentrated biomass,
25 ml of n-hexadecane and 1 g of terpene substrate incubated for 24 h at 30C and 200 rev min)1
Terpene substrates

Products accumulated after 24 h

P. rhodesiae

P. fluorescens

Cell
growth

Bioconversion

Fresh

Crude enzymatic
extract

Fresh

a-pin
a-pin
a-pin ox
Turp
Turp
b-pin
b-pin
b-pin ox
Lim
Lim

a-pin
a-pin ox
a-pin ox
Turp*
a-pin ox
b-pin
b-pin ox
b-pin ox
Lim
OPO

DMPA (10)
Isonov (6), nov (11), nov ac (44)
Isonov (47), nov (7), nov ac (2)
DMPA (8)
Isonov (13), nov (24), nov ac (35)
a-pin (14), DMPA (7)
Autodegrad

0
Isonov (80)

0
Isonov (75)
a-pin (04)
Autodegrad

DMPA (2)
Nov (48), nov ac (18)
Nov (10), nov ac (17)
a-pin ox (3), nov (6)

a-pin (20), DMPA (1)

Autodegrad
Autodegrad
a-ter (50)
**

Lim

Lim ox

Lim diol (27), HOL (77)

Crude enzymatic
extract
0
Isonov (66)

a-pin (03)
Autodegrad
Autodegrad
a-ter (91)
a-ter (05),
Lim diol (02)
Lim diol (05)

0, no products; , not done; a-pin, a-pinene; a-pin ox, a-pinene oxide; a-ter, a-terpineol; b-pin, ())-b-pinene; b-pin ox, b-pinene oxide; autodegrad, the same products obtained in the control experiments (inactive biomass); DMPA, 3,4-dimethyl pentanoic acid; HOL, 1-hydroxy-2-oxolimonene; isonov, isonovalal; lim, R-(+)-limonene; lim diol, limonene-1,2-diol; lim ox, limonene-1,2-oxide; nov, novalal; nov ac, novalic acid; OPO,
orange peel oil; turp, turpentine. Numbers in parentheses are the approximate yield (%).
*only a-pinene is metabolized in turpentine, while the other constituents are not consumed.
After 6 h bioconversion.
Product concentration (g l)1) after 24 h (too low substrate consumption and product formation to calculate a reliable yield).
No growth on the carbon source.
Limonene and part of limonene-1,2-oxide are metabolized in orange peel oil.
**Orange peel oil permeabilizes the biomass.

experiments were carried out with either concentrated

fresh cells or crude enzymatic extracts. The latter could
be considered as a form of the biocatalyst unable to perform cofactor-dependent reactions. Results obtained are
presented in Table 3, with the main products accumulated after 24 h of bioconversion and their respective
approximate yields. When product accumulation and
substrate consumption were very low, the parameter
amount produced was chosen as a more reliable option.
Alpha and b-pinene
The results shown in Table 3 indicate that both precursors were consumed and all the products accumulated by
both strains were basically from the same metabolic pathway (pathway 1b in Fig. 1). When a fresh biomass of
P. rhodesiae grown on a-pinene was used to convert
a-pinene, 3,4-dimethyl pentanoic acid (DMPA) was the
major product accumulated after 24 h. The same feature
was observed for fresh P. fluorescens cells, but with lower
yields. Alpha-pinene oxide, the first intermediate in

a-pinene degradation, allowed the accumulation of isonovalal, novalal, novalic acid and traces of DMPA with
P. rhodesiae, while isonovalal could not be detected with
P. fluorescens. The behaviour of the crude enzymatic
extracts (Table 3) demonstrated that, for both strains,
epoxidation of a-pinene was a cofactor-dependent process, while aldehyde synthesis from the epoxide was cofactor-independent.
The bioconversion of b-pinene by fresh cells of P. rhodesiae and P. fluorescens preliminarily grown on the same
carbon source resulted in the formation of a-pinene,
DMPA and traces of intermediates shown in pathway 1b
in Fig. 1 (Fig. 3). The same reaction with the crude enzymatic extracts of both strains gave only a low a-pinene
accumulation, not observed in the control experiments
(Table 3).
When b-pinene oxide was used as substrate, the products obtained were always the same as those found in
the control experiments (without biomass). This behaviour was evidenced for all kinds of biomass and strains

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Degradation of monoterpenes

J.L. Bicas et al.

used in this work (Table 3). It was thus concluded that

the main phenomenon was a precursor auto-oxidation in
the aqueous phase, which took place without any significant biocatalytic activity.
Limonene
Pseudomonas rhodesiae did not grow on limonene as the
sole carbon source, it was thus not considered for the
limonene bioconversion trials shown in Table 3. For
the fresh cells and crude enzymatic extract of P. fluorescens biomass, the sole metabolite accumulated was a-terpineol, the yield being higher with the extract. The
production obtained in this process, around 1011 g l)1
of n-hexadecane after 3040 h at a maximum rate of c.
1 g l)1 h)1 (data not shown), was in fact, to the best of
our knowledge, the highest already reported for the bioproduction of a-terpineol.
Interestingly, it was noticed that whole cells of this
micro-organism were also able to accumulate limonene1,2-diol and 1-hydroxy-2-oxolimonene from limonene1,2-oxide, a process practically inoperative with the crude
extract (Table 3).
Turpentine and orange peel oil
The bioconversion of turpentine (source of a-pinene) by
fresh P. rhodesiae cells grown on the same substrate gave
almost the same profile as that observed for the conversion of pure a-pinene. This result demonstrated that this
essential oil was also able to act as a cell inducer for
a-pinene degradation. The other major constituents of
turpentine (camphene, limonene) were never metabolized
(Table 3). The substitution of a-pinene by turpentine for
the cell growth in the process of isonovalal production
(Fontanille and Larroche 2003) resulted in similar results
to that obtained in the standard process, i.e. maximal
concentration of c. 80 g l)1 obtained after 4 h, with an
initial maximum rate of c. 40 g l)1 h)1 and a yield of 75
80% (data not shown).
It was not possible to perform the bioconversion of
orange peel oil with fresh P. fluorescens biomass because,
after a short period (<6 h), the cells were dead, as evidenced by the absence of growth on the agar medium. The
biocatalyst could thus be considered as permeabilized and
thus behaving like a crude enzymatic extract after this period, which might explain why the micro-organism grew on
limonene but not on orange peel oil (c. 88% limonene) as
the sole carbon source (Table 2). Moreover, the limonene
biotransformation activity using orange peel oil as substrate appeared dramatically lower than that achieved with
pure limonene (Table 3). It was thus assumed that orange
peel oil, in addition to its inactivating action, also acted as
an enzyme inhibitor. Trials with mixtures of R-(+)limonene, limonene-1,2-oxide and carvone in the same
1998

concentrations as found in orange peel oil led to consider

that the growth inhibition agent was not one of the main
constituents of the oil and that limonene-1,2-oxide was
the main compound responsible for the enzymatic inhibitory effect (data not shown). More detailed studies are
however still needed to get a better understanding of the
inhibition phenomenon owing to orange peel oil.
Discussion
In accordance with previous results (Best et al. 1987; Fontanille et al. 2002), the metabolism of a-pinene by P. rhodesiae and P. fluorescens was found to be basically the
same (pathway 1b in Fig. 1), although some discrepancies
in the level of enzymatic activities were noticed between
them. For example, in contrast to the well-known feature
of P. rhodesiae, the fresh cells of P. fluorescens did not
accumulate isonovalal during the bioconversion of
a-pinene oxide. Also, a-pinene and its metabolites seemed
to be almost completely metabolized by this strain after
24 h, while the fresh biomass of P. rhodesiae accumulated
DMPA with a significant yield.
It was also evidenced that the carbon source used for
biomass production acted as an inducing agent and had a
major effect on the yields and rates achieved during the
bioconversion phase. For example, the use of a-pinene
oxide for cell growth considerably increased isonovalal
accumulation in fresh P. rhodesiae and lowered novalal
accumulation in whole cells of P. fluorescens, in comparison with data observed with a-pinene used as carbon
source. It was demonstrated that turpentine (source of
a-pinene) could be used as carbon source for biocatalyst
production for further bioconversion of a-pinene oxide.
This feature opened a possibility to lower the cost of biocatalyst production.
Epoxidation of a-pinene did not occur in the presence
of the crude enzymatic extracts of either P. rhodesiae or
P. fluorescens, which confirmed that this conversion was
carried out by a NADH-dependent monooxygenase (Best
et al. 1987). As no b-pinene oxide was formed in the
bioconversion of b-pinene, it was assumed that this
enzyme had narrow substrate specificity, just like
the following enzyme in the pathway, a-pinene oxide
lyase.
The results obtained for b-pinene revealed that this
compound could be metabolized by both strains used in
this study. This result was, at our knowledge, evidenced
for the first time. It was supposed that this compound
was metabolized by a cofactor-independent isomerase via
a-pinene as described in pathway 1b in Fig. 1, as the
pathway reported by Yoo and Day (2002) involved limonene as intermediate, which was never detected in our
experiments.

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J.L. Bicas et al.

The biotransformation of limonene to a-terpineol

(pathway 4 in Fig. 2) has already been reported and studied for many other micro-organisms (Kraidman et al.
1969; Tan and Day 1998a,b; Tan et al. 1998; Adams et al.
2003; Marostica and Pastore 2007b), including a Pseudomonas species (Cadwallader et al. 1989), although not for
P. fluorescens NCIMB 11671. The results obtained with
the crude enzymatic extracts in this work have shown that
this reaction was catalysed by a cofactor-independent
enzyme, similar to a-terpineol dehydratase from Pseudomonas gladioli (Cadwallader et al. 1992). Also, as for
Fusarium oxisporum (Marostica and Pastore 2007b), the
enzyme responsible for this conversion seemed non
inducible, because a-terpineol was still produced by a biocatalyst grown on glucose (data not shown). The lack of
a-terpineol production during the bioconversion of turpentine by P. fluorescens might be explained by a catabolic
repression of a-pinene in limonene biotransformation
(Yuste et al. 1998).
The accumulation of limonene-1,2-diol and 1-hydroxy-2-oxolimonene during the limonene-1,2-oxide bioconversion led to suggest that P. fluorescens might have
another pathway for limonene, similar to that described
by Van Der Werf et al. (1999; pathway 2 in Fig. 2). The
first enzyme of this metabolic route, limonene-1,2-epoxide hydrolase, was cofactor-independent, but the following steps necessarily needed cofactors and, thus, could
be performed only by aerobically operated whole cells. It
was thus proposed that the two limonene metabolic
routes took place simultaneously in whole P. fluorescens
cells (pathways 2 and 4 in Fig. 2). The first, passing
through limonene-1,2-oxide, was an aerobic process
leading to energy production, while the second, leading
to a-terpineol, was oxygen-independent and could be
viewed as a process for detoxification of the medium
(Griffin et al. 1999; Bakkali et al. 2007; Bicas and
Pastore 2007).
Conclusion
Results achieved in this study demonstrated that P. rhodesiae could be considered as a specialist for the degradation of the a- and b-pinene family through the aldehyde
(pathway 1b in Fig. 1).
Pseudomonas fluorescens, on the other hand, appeared
to be a more versatile micro-organism. Besides degrading
a- and b-pinene in the same way as P. rhodesiae, this
strain also metabolized limonene through two pathways,
the most efficient being the synthesis of a-terpineol.
Results obtained with complex terpene mixtures confirmed that the toxicity of these compounds is highly
variable. Turpentine did not contain inhibitors for the
two strains used in this study and could thus be used

Degradation of monoterpenes

as an a-pinene source. It was not the case for orange

peel oil, which could not be used as a limonene
supplier.
Acknowledgements
J.L. Bicas acknowledges CAPES for financial support
(fellowship number 0725 07-2).
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