Biotransformation of menthol by Chlorella vulgaris and study of the pathways involved

Full text of DOI:10.1007/s10600-014-0849-4

Chemistry of Natural Compounds, Vol. 49, No. 6, January, 2014 [Russian original No. 6, November–December, 2013]
BIOTRANSFORMATION OF MENTHOL BY Chlorella vulgaris
AND STUDY OF THE PATHWAYS INVOLVED
Akbar Esmaeili*, Hamid Reza Mirmhadi, and Farnas Rafiee
UDC 547.913
The literature provides several examples of biotransformations of microalgae. Microalgae have been used to improve
the yield of already known substances or to produce novel substances [1]. Microalgae have been less considered in research on
biotransformation and bioconversion of terpenes, with most interest in this area placed on conversion of monoterpene compounds.
Earlier studies have already demonstrated the potential of microalgae for modification of steroids. In our own previous research,
the biotransformation of onopordopicrin by Aspergillus niger was investigated; four novel products were obtained and their
structures identified on the basis of chemical and spectroscopic data [2]. In other studies, biotransformation of citral, geraniol,
menthol, myrcene, and nerol by various fungi, including A. niger and Penicillium spp., produced such compounds as
1,8-cineole, 2,6-dimethyloctane, ꢀ-pinene, ꢀ-terpineol, cis-p-menthan-7-ol, dihydrolinalool, ꢁ-terpinene linalool, limonene,
p-cymene, p-menth-1-ene, sabinene, and trans-p-menthan-1-ol [3–7]. In the present study, microbial transformation of
the pure terpene alcohol menthol (1) was carried out using C. vulgaris. Biotransformation and bioconversion of menthol over
varying periods of 72, 92, and 120 h produced primarily isomenthol. The product of the biotransformation was extracted with
diethyl ether and analyzed with the use of Fourier transform infrared spectroscopy (FT-IR), gas chromatography (GC), and gas
chromatography-mass spectroscopy (GC-MS).
Biotransformation of menthol by C. vulgaris produced cis-p-menth-1-en-3-ol (2), dihydroterpineol, and isomenthol
in high percentages. Similar products were obtained in previous research. The main bioconversion products of (–)-menthol by
sporulated surface culture of Mucor ramannianus were trans-p-menthan-8-ol, trans-menth-2-en-1-ol, p-menthane-3, 8-diol
isomenthol, sabinane and 1,8-cineole. Products obtained from menthol by surface grown Penicillium spp. included menthene
(5.8%), terpineol (10.6%), ꢀ-pinene (18.0%), sabinene (3.9%), 1,8-cineole (6.4%), and limonene (3.2%) [8]. Recent experimental
work suggests that microbial transformation of monoterpenes with Penicillium spp. and A. niger involves oxidation, resulting
in a more stable product. Using C. vulgaris for bioconversion makes possible to selectively obtain products selectively in high
proportion. Using periods of 72, 92, and 120 h, we identified one or two major components for each time period, representing
69.8%, 92.3%, and 95.2% respectively. Menthol converted with C. vulgaris for 72 h produced dihydroterpineol (3, 48.8%)
and isomenthol (4, 20.2%). When continued to 92 h, the main product was 4 (92.3%). At 120 h, the main compounds produced
were cis-p-menth-1-en-3-ol (2, 46.0%) and 3 (49.2%) (Scheme 1).
Scheme 1 shows the pathways involved in bioconversion of menthol to dihydroterpineol.
Microalgae have been less considered in biotransformation and bioconversion of monoterpene compounds. In a
previous study of biotransformation of menthol by sporulated surface cultures of A. niger, the main bioconversion product
obtained was cis-p-menthan-7-ol. In a similar study using Penicillium spp. the primary products were limonene, p-cymene,
and ꢁ-terpinene [9]. Success has documented maximizing product yields by solubilizing or emulsifying immiscible substrates.
In such a case, care should be paid to choice and concentration of the solvent as at lower concentrations many miscible
solvents are cytotoxic [9]. Biotransformation of steroid estrogens by C. vulgaris has been investigated [5, 10]. Biotransformation
of volatile monoterpenoids by fungi has also been investigated [3].
At each of these points volatile and monoterpene components mostly exit the first column. The best results were
obtained at 92 h. For the other time periods, the percentage of the components was small and hence could not be identified. All
were checked against the Eight Peak Index of Mass Spectra [11].
Department of Chemical Engineering, North Tehran Branch, Islamic Azad University, P.O. Box 19585-936, Tehran,
Iran. Published in Khimiya Prirodnykh Soedinenii, No. 6, November–December, 2013, pp. 995–996. Original article submitted
August 1, 2012.
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0009-3130/14/4906-1160 02014 Springer Science+Business Media New York

72 h
OH
Cation shift
120 h
3
+
OH
OH
OH
OH
2
1
2
4
3
120 h
92 h
Scheme 1
Menthol was converted primarily to dihydroterpineol, isomenthol, and cis-p-menth-1-en-3-ol. Tests of different pH
showed pH 5.5 to be optimum. Earlier research suggested that biotransformation of monoterpenes with Penicillium and
Aspergillus involves an oxidation reaction and results in a more stable product. But bioconversion using the sporulated surface
cultures and liquid methods with Penicillium showed that it was possible to obtain a similar product with high yield and
selectivity.
Biotransformation Experiments. Each batch experiment was repeated three times. Individual samples (500 ng)
were spiked separately into dark 250 mL Teflon bottles and dark or light 500 mL conical flasks. A gentle nitrogen stream was
utilized to blow each sample to dryness. An algal culture (200 mL) was transferred into the receptacles so to achieve an initial
–1
concentration of 2.5 g·L . This was then placed on a rotary shaker in the dark or aerated in the light with samples taken at 72,
96, and 120 h.
Identification of the constituents of the oil was made by comparison of their mass spectra and retention indices (RRI)
with those given in the literature and with authentic samples [5]. The mass spectra of a mixture of cis-p-menth-1-en-3-ol,
dihydroterpineol, and isomenthol had three main peaks.
+
–1
cis-p-Menth-1-en-3-ol (2). 154 [M ]: 84 (100), 41 (60), 54 (40), 91 (30), 105 (20), 128 (5); FT-IR (KBr, ꢂ , cm ):
max
3200, 1620, 1344.
+
–1
Dihydroterpineol (3). 156 [M ]: 43 (100), 71 (90), 57 (20), 67 (19), 87 (15); FT-IR (KBr, ꢂ , cm ): 3200, 1620,
max
1340, 1372.
Isomenthol (4). 156 [M ]: 41 (100), 95 (70), 71 (80), 55 (60), 81 (78), 67 (50); FT-IR (KBr, ꢂ , cm ): 3200, 1638.
+
–1
max
ACKNOWLEDGMENT
The authors wish to thank Ms. Vakili, Department of Marine Sciences and Technologies, Islamic Azad University,
North Tehran Branch, Tehran, Iran for assistance with the use of the laboratory.
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