O R I G I N A L R E S E A R C H P A P E R

Biotransformation of menthol and geraniol by hairy root
cultures of Anethum graveolens: effect on growth
and volatile components

Jorge M. S. Faria

Æ Ineˆs S. Nunes Æ

A. Cristina Figueiredo

Æ Luis G. Pedro Æ

Helena Trindade

Æ Jose´ G. Barroso

Received: 13 January 2009 / Revised: 20 January 2009 / Accepted: 23 January 2009 / Published online: 11 February 2009
Ó Springer Science+Business Media B.V. 2009

Abstract

Two oxygen-containing monoterpene sub-

strates, menthol or geraniol (25 mg l

-1

), were added

to Anethum graveolens hairy root cultures to evaluate
the influence of the biotransformation capacity on
growth and production of volatile compounds. Growth
was assessed by the dissimilation method and by fresh
and dry weight measurement. The volatiles were
analyzed by GC and GC–MS. The total constitutive
volatile component was composed, in more than 50%,
by falcarinol (17–52%), apiole (11–24%), palmitic
acid (7–16%), linoleic acid (4–9%), myristicin (4-8%)
and n-octanal (2-5%). Substrate addition had no
negative influence on growth. The relative amount of
menthol quickly decreased 48 h after addition, and the
biotransformation product menthyl acetate was con-
comitantly formed. Likewise, the added geraniol
quickly decreased over 48 h alongside with the pro-
duction of the biotransformation products. The added
geraniol was biotransformed in 10 new products,
the alcohols linalool, a-terpineol and citronellol, the

aldehydes neral and geranial, the esters citronellyl,
neryl and geranyl acetates and linalool and nerol oxides.

Keywords

Anethum graveolens

Biotransformation

Geraniol Hairy roots

Menthol

Volatiles

Introduction

Chemical synthesis is still the major source of
important phytochemicals used in the pharmaceutical
and food industries. Nevertheless, in the past few
years, a renewed interest in the use of natural
products has motivated various industries in attempt-
ing alternative production procedures, namely by
plant biotechnology.

Hairy root cultures offer a flexible and versatile

system that is a promising technology for large scale
production of valuable phytochemicals (Srivastava
and Srivastava

2007

). Moreover, the production, in

these systems, can be qualitatively and/or quantita-
tively different from the whole plant (Santos et al.

2002

). Phytochemical production by hairy roots can be

improved by substrate feeding which stimulates the
plant cell enzymatic machinery—mainly enzymes that
can undergo reactions such as reductions, oxidations,
methylations and, particularly, glycosylation, that are
very common in plant cells (Li et al.

2003

).

Monoterpenes are of great importance in Apiaceae

and, in some cases, represent their dominant volatile

Electronic supplementary material

The online version of

this article (doi:

10.1007/s10529-009-9934-3

) contains

supplementary material, which is available to authorized users.

J. M. S. Faria

I. S. Nunes A. C. Figueiredo (

&)

L. G. Pedro

H. Trindade J. G. Barroso

Faculdade de Cieˆncias de Lisboa, Departamento de
Biologia Vegetal, Instituto de Biotecnologia e
Bioengenharia, Centro de Biotecnologia Vegetal,
Universidade de Lisboa, C2, Campo Grande,
1749-016 Lisbon, Portugal
e-mail: [e-mail]

123

Biotechnol Lett (2009) 31:897–903
DOI 10.1007/s10529-009-9934-3

components (Croteau

1980

). In modern society,

terpene alcohols play an essential role in the food
industry, as flavours, and in the perfume industry, as
fragrances. Considering the importance of monoter-
pene alcohols and given the lack of information on the
biotransformation of these compounds by hairy root
cultures, this study evaluates the biotransformation
capacity of Anethum graveolens hairy root cultures by
studying the influence of the addition of two oxygen-
containing monoterpenes, menthol and of geraniol, on
growth and volatiles composition.

Materials and methods

Hairy root cultures

Anethum graveolens hairy roots, established as pre-
viously described by Santos et al. (

2002

), were

maintained in SH medium (Schenk and Hildebrandt

1972

) with 30 g sucrose l

-1

, in darkness at 24

°C on

orbital shakers (80 rpm). Routinely the cultures were
subcultured every three weeks.

Biotransformation

Erlenmeyer flasks with 100 ml SH medium were
aseptically inoculated with 1 g (fresh wt) of dill hairy
roots and maintained as above. Fifteen days following
subculture, 2% (v/v) or 2% (w/v) for geraniol or
menthol in methanol, respectively was added to each
culture flask, at 25 mg l

-1

. Growth and volatiles

production were evaluated after 1, 4, 8, 24, 32 and 48 h
and weekly during the following 4 weeks. Two
independent experiments were separately run, for each
substrate, and two replicates of each flask were used in
each experiment.

Control cultures (=without substrates) were grown

simultaneously. Substrate evaporation and decompo-
sition control experiments were performed by adding
the same amount of substrate to flasks containing only
basal culture medium, and keeping them in the
same conditions as the culture flasks throughout the
experiment.

Determination of hairy root growth

Hairy roots growth was measured, both in control and
substrate added cultures, by the dissimilation method,

and by fresh and dry weight determination, as
previously described (Santos et al.

2002

).

Isolation of the volatile components

Volatiles were isolated from dill hairy roots by
distillation–extraction, for 3 h, using a Likens-Nick-
erson type apparatus (Likens and Nickerson

1964

)

using distilled n-pentane as organic solvent. The
volatile oils were stored at -20

°C in the dark until

analysis.

GC and GC–MS

The isolated volatiles were analyzed by GC and GC–
MS as previously described (Costa et al.

2008

).

Results and discussion

Hairy root growth

Independently of the growth evaluation procedure
(Fig.

1

), dill hairy roots showed an initial latent phase

of about seven days, followed by a short exponential
phase, subsequently a linear growth phase that
reached approximately the 30th day and then the
stationary phase. This growth profile is similar to that
obtained previously, with dill hairy root cultures
(Santos et al.

2002

), indicating a stable growth

pattern for this in vitro system.

Menthol or geraniol addition, in the linear phase

(15th day), did not greatly affect A. graveolens hairy
roots growth, as shown by the growth curves similar
to those of the control cultures (Fig.

1

a, b). Similar

results were obtained with Achillea millefolium cell
suspension cultures (Figueiredo et al.

1996

) and with

Levisticum officinale hairy root cultures (Nunes et al.

2009

).

Constitutive volatile components

Forty-two components were identified in the constitu-
tive volatile fraction of dill hairy roots, for 6 weeks,
in a total relative amount [82% (Table

1

). Falcarinol

(17–52%), apiole (11–24%) and palmitic acid (7–16%)
were the dominant compounds. Other major compo-
nents were linoleic acid (4–9%), myristicin (4–8%) and
n-octanal (2–5%). Polyacetylenes constituted the major

898

Biotechnol Lett (2009) 31:897–903

123

group of volatile components, represented only by
falcarinol, also named panaxynol, which is a powerful
anti-fungal substance (Seigler

1998

) and a potential

anti-cancer drug (Zheng et al.

1999

). Phenylpropa-

noids (16-31%) and fatty acids (13-25%) were also
present in considerable amounts. The terpene fraction
was constituted only by monoterpenes and didn’t
exceed 8%. These results are in agreement with those
obtained previously by Santos et al. (

2002

) with the

same in vitro system, suggesting that A. graveolens
hairy roots have a relatively stable production of
volatile compounds, as the cultures have been main-
tained for over twelve years with a routine subculture
every three weeks. The higher relative amount of fatty
acids, found in the present study, when compared to
that obtained by Santos et al. (

2002

), may be due to the

different culture medium used, which may alter the
volatile composition as was found in L. officinale hairy
roots (Costa et al.

2008

).

Biotransformation products

The detailed relative amounts of the substrates and
their biotransformation products during the time-
course study are given in Supplementary Table

1

and

a biosynthetic scheme showing the probable relation-
ship between these various compounds is given in
Supplementary Fig.

1

.

Menthol addition, 15 days after subculture, dras-

tically altered the constitutive volatiles composition
of A. graveolens hairy root cultures. Menthol was
quickly biotransformed into menthyl acetate, decreas-
ing from a maximum of 51% in the first hour, to 11%,
48 h after addition (Fig.

2

a). Concomitantly, menthyl

acetate increased from 4% in the 1st hour to a
maximum of 55%, 48 h after menthol addition. From
this point on both compounds slowly decreased
throughout the next 4 weeks.

Menthol has been most thoroughly studied in

species of the genus Mentha, particularly in Mentha
9 piperita. In this species, Martinkus and Croteau
(

1981

) have identified an acetyl-CoA:monoterpenol

acetyltransferase that is responsible for the transfor-
mation of l-menthol into menthyl acetate. According
to Lange et al. (

2000

), studying the same species, the

enzyme menthol acetyltransferase was responsible for
menthol acetylation. Probably an enzyme of this
acetyltransferase family is active in A. graveolens
hairy root cultures that acetylates menthol, with no
detectable effects in growth. Nevertheless this capac-
ity seems to be species specific, as in Levisticum
officinale hairy root system this capacity was not
detected (Nunes et al.

2009

) and in A. millefolium

cell suspension cultures (Figueiredo et al.

1996

),

menthyl acetate was produced, though in trace
amounts.

The addition of geraniol to A. graveolens hairy

root cultures resulted in the formation of 10 new

0

2

4

6

8

10

12

0

14

21

28

35

42

Time (days)

D

is

s

imila

tio

n

(m

g

.ml

-1

)

(a)

0

5

10

15

20

25

30

35

0

14

21

28

35

42

Time (days)

Fresh weight (g)

0

1

2

3

Dry weight (g)

(b)

7

7

Fig. 1

Growth curves of dill hairy roots evaluated by the

dissimilation method (a) and by fresh and dry weight methods
(b). Dissimilation growth curves: control cultures [=without
substrates, r (standard deviation 0–2 mg l

-1

)], menthol [j

(standard deviation 0–3 mg l

-1

)] and geraniol [m (standard

deviation 0–1 mg l

-1

)] added cultures. Fresh and dry weight

growth curves: control [r (standard deviation 0–5 g), e
(standard deviation 0–1 g), respectively], menthol [j (standard
deviation 1–5 g), h (standard deviation \0.5 g)] and geraniol
[m (standard deviation 0–4 g), D (standard deviation \0.5 g)]
added cultures, respectively

Biotechnol Lett (2009) 31:897–903

899

123

Table 1

Percentage composition of the constitutive volatiles isolated from dill hairy roots maintained for 6 weeks in SH medium, in

darkness at 24

°C and 80 rpm

Components

RI

Anethum graveolens hairy root cultures

Time (days)

7

14

21

28

35

42

Benzaldehyde

927

0.1

0.3

0.9

0.3

0.3

0.6

a-Pinene

930

0.5

t

0.8

0.1

t

0.3

n-Heptanol

952

0.1

0.2

0.5

0.2

0.1

0.2

Sabinene

958

t

t

t

t

t

t

1-Octen-3-ol

961

t

t

t

t

t

t

b-Pinene

963

0.4

0.9

1.4

0.8

0.8

0.7

2-Octanone

967

0.2

0.1

0.2

0.1

0.2

0.1

2-Pentyl furan

973

t

0.1

0.2

0.7

0.1

t

n-Octanal

973

2.6

3.8

3.2

1.9

3.5

4.5

Benzene acetaldehyde

1002

0.3

0.2

0.7

0.2

0.3

0.3

p-Cymene

1003

0.3

0.2

0.8

t

t

t

Limonene

1009

0.3

0.8

1.1

0.4

0.3

1.1

n-Octanol

1045

0.1

t

0.5

t

0.1

t

2-Nonanone

1058

0.3

0.8

0.6

0.3

0.4

0.5

Terpinolene

1064

0.3

0.1

0.2

t

t

t

2-Hexyl furan

1064

t

t

t

t

t

t

Phenyl ethyl alcohol

1064

t

t

t

t

t

t

Nonanal

1073

0.4

1.3

1.0

0.5

0.9

0.9

Vertocitral C*

1077

0.3

0.1

0.2

0.2

0.2

0.3

trans-Tagetone

1116

0.3

0.5

1.1

0.8

0.9

1.5

cis-Tagetone

1123

t

t

0.2

t

t

0.4

2- trans -Nonen-1-al

1114

0.9

0.9

1.2

1.1

1.2

2.1

Decanal

1180

0.4

0.3

0.2

0.3

0.2

0.4

cis-Ocimenone

1200

0.7

0.1

0.6

0.3

0.3

0.5

trans-Ocimenone

1207

t

t

0.1

t

t

0.1

2-trans-Decenal

1224

0.5

1.0

0.7

0.5

0.5

0.8

2 trans-4-cis-Decadienal

1242

0.1

0.1

0.1

0.2

0.1

0.2

2-Undecanone

1271

0.4

0.1

0.1

0.3

0.3

0.5

Carvacrol

1286

0.7

1.1

1.0

2.2

2.5

2.3

2 trans-4 trans-Decadienal

1286

0.5

0.2

t

t

0.2

0.5

trans-2-Undecenal

1323

t

0.8

0.2

0.6

0.2

0.4

trans-4-Undecenal*

1402

0.6

0.6

0.3

0.5

1.1

1.1

Myristicin

1493

4.2

7.5

7.9

3.5

4.8

3.7

c-Undecalactone*

1535

t

0.6

t

t

t

t

2,4-Dimethoxyacetophenone

1544

t

0.1

t

t

0.2

t

Dill apiole

1587

2.6

1.8

1.3

1.2

1.7

1.7

Apiole

1640

23.7

20.5

15.2

11.2

13.0

17.6

Myristic acid

1723

0.5

0.2

t

0.2

0.5

0.1

Pentadecanoic acid*

1778

t

1.0

0.6

0.6

0.8

1.4

Palmitic acid

1908

16.4

14.6

14.7

6.7

8.1

12.3

Falcarinol

2002

17.2

23.8

27.4

51.6

42.6

25.1

900

Biotechnol Lett (2009) 31:897–903

123

biotransformation products. These were the alcohols
linalool, a-terpineol and citronellol, the aldehydes
neral and geranial, the esters citronellyl, neryl and

geranyl acetates and, in traces, linalool and nerol
oxides. Geranial and neral occurred in similar relative
amounts throughout the time-course study. L. officin-
alle (Apiaceae) hairy root cultures, grown under
similar conditions, were able to biotransform geraniol
into nerol and neral but not into geranial (Nunes et al.

2009

). According to several authors (in Iijima et al.

2004

), it is not yet established if citral (mixture of

geranial and citral) is formed from geraniol by the
action of an alcohol dehydrogenase or by an oxidase,
not even if geraniol is the only substrate in the
formation of citral, since nerol can also be a precursor.

All the new biotransformation products, with the

exception of linalool and nerol oxides, were detected
1 h after

geraniol

addition

(Fig.

2

b).

Geraniol

decreased quickly from 20%, in the first hour, to
\3%, 48 h after addition. The new alcohols formed,
didn’t exceed 10%, throughout the time-course study
(Fig.

2

b). Citral and citronellol reached a maximum

8 h after substrate addition and linalool attained a
maximum only 32 h after geraniol addition.

Studying Vitis vinifera L., grape berry mesocarp,

Luan et al. (

2005

) were able to identify a stereose-

lective geraniol reductase responsible for citronellol
production after labelled geraniol addition. Never-
theless they were unable to clarify its origin since
geraniol also yielded its isomer neral, which can also
be converted to citronellol. These authors have also
reported the production, although in small amounts,
of geranial and neral which requires the presence of
an oxidase and/or a dehydrogenase.

Table 1

continued

Components

RI

Anethum graveolens hairy root cultures

Time (days)

7

14

21

28

35

42

Linoleic Acid

2125

5.8

8.8

5.8

5.0

4.3

4.2

% Identification

81.7

93.5

91.0

92.5

90.7

86.4

Grouped components

Monoterpene hydrocarbons

1.8

2.0

4.3

1.3

1.1

2.1

Oxygen-containing monoterpenes

2.0

1.8

3.2

3.5

3.9

5.1

Polyacetylenes

17.2

23.8

27.4

51.6

42.6

25.1

Phenylpropanoids

30.5

29.8

24.4

15.9

19.5

23.0

Fatty acids

22.7

24.6

21.1

12.5

13.7

18.0

Others

7.5

11.5

10.6

7.7

9.9

13.1

RI Retention index relative to C

9

–C

22

n-alkanes on the DB-1 column, t traces (\0.1%)

* Identification based on mass spectra only

1

4

8

24

32

48

168 336

504 672

Menthol

Menthyl acetate

0

10

20

30

40

50

60

Relative amount (%)

Time after substrate addition (h)

(a)

1

4

8

24

32

48

168 336

504 672

Geraniol

Aldehydes

Alcohols

Esters

0

10

20

30

40

50

60

Relative amount (%)

Time after substrate addition (h)

(b)

Fig. 2

Time-course study of the relative amounts of substrates

and of their biotransformation products. a Menthol and the
biotransformation product, menthyl acetate. b Geraniol and the
biotransformation products, esters, alcohols and aldehydes

Biotechnol Lett (2009) 31:897–903

901

123

In comparison with the other A. graveolens hairy

root geraniol biotransformation products, the ester
acetates attained higher relative amounts (47%) but
were slower in reaching their peaks (Fig.

2

b). While

geranyl and neryl acetates attained their maximum
after 48 h, citronellyl acetate reached the highest
relative amount only one week after substrate addi-
tion. This delay may indicate that this compound can
be formed directly from citronellol, through acetyla-
tion, or from the reduction of geranyl acetate. Such
secondary transformations are common and were
reported by King and Dickinson (

2000

) in a study of

monoterpene alcohols biotransformation using yeast
cells. In that study, geraniol addition resulted in the
formation of, among others, linalool and a-terpineol,
and the addition of linalool resulted in the formation
of a-terpineol. This suggests that within the new
biotransformation products obtained, with A. graveo-
lens hairy roots, some may derive from secondary
reactions of geraniol primary biotransformation prod-
ucts. Geranyl acetate was also obtained from geraniol
biotransformation in lovage hairy root cultures under
the same conditions used in the present work (Nunes
et al.

2009

).

The relative amounts of all biotransformation

products slowly decreased with culture time, which
may reflect their loss due to high volatility (King
and Dickinson

2000

), or their incorporation into

non-volatile hairy roots components, by glycosyla-
tion, as it was found in other in vitro plant cul-
ture systems (Figueiredo et al.

1996

; Nunes et al.

2009

).

Everitt and Lockwood (

1995

) reported the bio-

transformation

capacity

of

A.

graveolens

cell

suspension cultures. In that study, geraniol, added in
10, 20, 30, 50 and 100 mg l

-1

, was readily converted

into nerol. No other product was detected and both
geraniol and nerol were reduced to trace amounts after
48 h. Our results suggest that, for the same species,
biotransformation products greatly depend on the in
vitro system used.

In conclusion

, Anethum graveolens hairy root

cultures show biotransformation ability related to a
group of biocatalysts, namely redutases, isomerases
and transacetylases, among others, that readily trans-
form geraniol and menthol into different biotransfor-
mation products and with no visible negative impact
on growth.

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