Biotransformation of one monoterpene by sporulated surface cultures of Aspergillus niger and Penicillium sp.

Article abstract of DOI:10.1080/14786410802547341

In this study, biotransformation of menthol by sporulated surface culture of Aspergillus niger and Penicillium sp. was studied. The main bioconversion product obtained from menthol of A. niger was cis-p-menthan-7-ol and the main products obtained by surfa

Full text of DOI:10.1080/14786410802547341

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Biotransformation of one monoterpene
by sporulated surface cultures of
Aspergillus niger and Penicillium sp.
Akbar Esmaeili a , Shirin Sharafian a , Shila Safaiyan b , Shamsali
Rezazadeh c & Abdolhossein Rustaivan d
a Department of Chemistry, North Tehran Branch, Islamic Azad
University, Tehran, Iran
b Department of Marine Sciences and Technologies, Islamic Azad
University, North Tehran Branch, Tehran, Iran
c Iranian Acdemic Center for Education, Culture &
Research(ACECR)–Institute of Medicinal Plant Research, Tehran,
Iran
d Department of Chemistry, Science and Research Campus, I.A.
University, Tehran, Iran
Published online: 29 Oct 2009.
To cite this article: Akbar Esmaeili , Shirin Sharafian , Shila Safaiyan , Shamsali Rezazadeh &
Abdolhossein Rustaivan (2009) Biotransformation of one monoterpene by sporulated surface
cultures of Aspergillus niger and Penicillium sp., Natural Product Research: Formerly Natural
Product Letters, 23:11, 1058-1061, DOI: 10.1080/14786410802547341
To link to this article: http://dx.doi.org/10.1080/14786410802547341
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Natural Product Research
Vol. 23, No. 11, 20 July 2009, 1058–1061
Biotransformation of one monoterpene by sporulated surface cultures
of Aspergillus niger and Penicillium sp.
a
a
b
c
*
Akbar Esmaeili , Shirin Sharafian , Shila Safaiyan , Shamsali Rezazadeh and
Abdolhossein Rustaivand
aDepartment of Chemistry, North Tehran Branch, Islamic Azad University, Tehran, Iran;
bDepartment of Marine Sciences and Technologies, Islamic Azad University, North Tehran Branch,
Tehran, Iran; cIranian Acdemic Center for Education, Culture & Research(ACECR) – Institute of
Medicinal Plant Research, Tehran, Iran; dDepartment of Chemistry, Science and Research Campus,
I.A. University, Tehran, Iran
(Received 5 August 2008; final version received 10 October 2008)
In this study, biotransformation of menthol by sporulated surface culture of
Aspergillus niger and Penicillium sp. was studied. The main bioconversion product
obtained from menthol of A. niger was cis-p-menthan-7-ol and the main products
obtained by surface Penicillium sp. were limonene, p-cymene and ꢀ-terpinene
using sporulated surface culture. The pathways involved in the biotransformation
of menthol by A. niger and Penicillium sp. to main products are also discussed.
Keywords: Aspergillus niger; Penicillium sp.; biotransformation; bioconversion;
limonene; p-cymene; ꢀ-terpinene; cis-p-menthane-7-ol
1. Introduction
The work related to the bioconversion of monoterpene alcohols by Aspergillus niger and
Penicillium sp. was investigated. In recent years, the biotechnological production of
natural aromatic chemical (NACS) has been stimulated by consumer demand for natural
and healthy products (Sehilling & Hauslre, 1997).
This interest in natural flavours instead of synthetic flavours has led to increasing
research to focus on the microbial production of the so-called ‘bioflavours’ (Berger,
Drawert, & Tiefel, 1992; Drawert, 1988; Schreier, 1989, 1992). Nearly 80% of the flavours
and fragrances used worldwide are produced chemically (Demyttenaere & Kimpe, 2001).
The bioconversion of geranyl and neryl acetate by A. niger has been described
(Madyastha & Krishna Murthy, 1988a, 1988b). The main reaction found was hydrolysis of
terpene acetates to the corresponding alcohols, followed by further hydroxylation
experiments using liquid cultures of A. niger. Citral transformation to menthols by
liquid phase over Ni supported H–MCM-41 and H–Y, has also been examined by a group
in Finland (Arvela et al., 2005).
Biotransformation of some monoterpenes (geraniol, nerol and citral) by A. niger has
been studied. Linalool, ꢁ-terpineol and limonene were the main products obtained from
*Corresponding author. Email: akbaresmaeili@yahoo.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410802547341
http://www.informaworld.com

Natural Product Research
1059
H+
(b)
+
-H2O
+
OH
OH2
Menthol
-H2O
Shift carbocation
+
OH
cis-p-menthan-7-ol
Scheme 1. Biotransformation of menthol by Aspergillus niger (a); biosynthesis of menthol to
cis-p-menthan-ol (b).
nerol and citral by sporulated surface culture (Demyttenare, Herrea, & Kimpe, 2000).
Using a surface culture of the organism and adding a methanolic solution of the terpene
after a good mat had been developed, the evidence was found to suggest that geraniol was
converted to linalool and partially oxidized to citral (Wood, 1969).
In this article, the biotransformation of a monoterpene (menthol) by sporulated surface
cultures of A. niger strain and Penicillium sp. strain is compared.
2. Experimental
2.1. Microorganisms
A strain of Penicillium sp. and A. niger was isolated from the soil of our laboratories in
Tehran prefecture, and was identified according to its physiological and morphological
characteristics. Aspergillus niger (P.T.C.C.5011) and Penicillium sp. (P.T.C.C.5074) were
identified according to its Persian-Type Culture-e Collection, Iranian Research
Organization for Science & Technology, Tehran, Iran. A spore suspension of A. niger and
Penicillium sp. was prepared in nutrient broth solution for inoculating fungi in Petri dishes.
2.2. Growth medium and conditions
For the isolation, growth and conservation of the fungi in Petri dishes one and the same
solid medium was used: Sabouraud Dextrose Agar (SDA) medium contained mycological
peptone 1.0%, glucose 4.0% and agar 1.5%.
The solid agar medium was inoculated with spores of A. niger and Penicillium sp. First,
germination of the spores and mycelial growth took place, then the growth medium was
stored at room temperature. After 1 week, the surfaces of Petri dishes were covered with
spores and the biotransformation reaction started.

1060
A. Esmaeili et al.
OH
+
P. Sp.
+
OH
OH
Limonene
Menthol
γ
-Terpinene
OH
-2H2
P. Sp.
OH
OH
Menthol
P-Cymene
α
-Terpinene
Scheme 2. Biotransformation of menthol by Penicillium sp.
2.3. Experiments with spore suspension
In both spores were recovered from 1-week-old surface cultures of A. niger and Penicillium
sp. grown in Petri dishes on SDA. This was done by adding 10 mL of a sterile Tween 80
solution and 0.2% Tween 80 in distilled water to each culture, bringing the spores into
suspension. A total spore suspension of 50 mL was obtained and shaken in a 250 mL
conical flask.
To this spore suspension of 1 mL, a solution of 5% menthol in ethanol was added, and
the suspension was placed on a shaker at 180 rpm. In each process, it was taken out and
extracted with Et2O three consecutive times after 7 days, and the products were directly
analysed by GC ad GC/MS.
2.4. Analysis of the sample with GC/MS
Analysis was performed using a Hewlett–Packard 6890 with a DB-5 capillary column
(30 m  0.25 mm; film thickness 0.25 mm) and programmed as follows: 60ꢀC for 5 min and
programmed to 220ꢀC at a rate of 4ꢀC/min. The flow rate to helium as carrier gas with
(2 mL minÀ1) MS were taken at 70 eV. The retention indices of C9–C28 n-alkanes were
computer matched with the Wiley 275 Library, as well as by comparison of their mass
spectrums with those of authentic samples or with data already available in the literature
(Adams, 2001; Davies, 1990).
3. Results and discussion
In this experiment, the biotransformation of menthol by sporulated surface cultures of
A. niger (P.T.C.C.5011) and Penicillium sp. (P.T.C.C.5074) grown on some medium
culture flasks was monitored similarly for only 1 week. Cultures were grown in Petri dishes
on solid medium Sabouraud Dextrose Agar (SDA) containing menthol. After incubation,

Natural Product Research
1061
SDA culture was extracted – see Section 2. It was noticed that after 3 days the cultures
with 0.05 menthol were fully grown and sporulation had occurred. The cultures with 0.1%
menthol covered only a part of the surface. The suspension was extracted with Et2O three
consecutive times and directly analysed by GC and GC/MS.
In these analyses various chemicals were obtained. The main products obtained in the
bioconversion of A. niger of menthol and bioconversion of Penicillium sp. of menthol were
cis-p-menthan-7-ol and, limonene, p-cymene and ꢀ-terpinene, respectively. From the data
in Scheme 1 it can be concluded that menthol has been converted much more than
limonene. Scheme 2 shows that menthol can be bioconverted by A. niger to cis-p-menthan-
7-ol. Pot synthesis of p-cymene directly from menthol can, however, be a difficult task
because it includes selective dehydration of menthol (Scheme 1). Synthesis of limonene and
ꢀ-terpinene from menthol showed converted dehydration by tertiary hydrogens.
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