Annals of Microbiology, 59 (2) 349-351 (2009)
Microbial biotransformation of some monoterpene hydrocarbons Katayoun JAVIDNIA1,2*, Farzaneh ARAM1,3, Mahmood SOLOUKI3, Ahmad REZA MEHDIOPOUR1, Maryam GHOLAMI1, Ramin MIRI1,2 1Medicinal and Natural Product Chemistry Research Centre, 2Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran, P. O. Box: 71345-1149 3Department of Plant Breeding and BioCenter, Faculty of Agriculture, Zabol University, Zabol, Iran
Received 2 February 2009 / Accepted 7 May 2009
Abstract - High commercial value compounds can be obtained through the microbial biotransformation of monoterpenes. Some of these monoterpenic substances are not expensive and produced in a variety of plant species. Biotransformation of some monoterpene hydrocarbons such as A-pinene, β-pinene, myrcene and p-cymene by 7 strain bacteria and 2 strain fungi was investigated. It was observed that some of microorganisms transformed monoterpenes to oxygenated monoterpenes in a good yield which among them Staphylococcus epidermidis showed higher yields. Key words: biotransformation; monoterpene hydrocarbons; bacteria; fungi.
Aromas and fragrances are used everywhere in the modern world, they find a wide range of applications in the food, cosmetic, chemical and pharmaceutical sciences (Vandamme and Soetaert, 2002). Globally, the market demand for natural flavourings continues to increase. Most available flavour compounds are now produced via chemical synthesis or extraction. Drawbacks of such chemical processes are the formation of undesirable racemic mixtures and the growing aversion of the consumer towards chemicals added to his food, cosmetics and other household products (Janssens et al., 1992; Krings and Berger, 1998). The biotechnological approaches suggest advantages over such conventional methods (Krings and Berger, 1998). Monoterpene hydrocarbons such as A-pinene, β-pinene and p-cymene are inexpensively available in large quantities. Oxygenated monoterpenes are valuable compounds and used in flavour and fragrance industries. The biotransformation of monoterpene hydrocarbons to oxygenated ones is of interest because it allows the production of stereoselectively pure compounds under mild conditions and also the products may be considered as natural products (deCarvalho and daFonseca, 2006). Biotransformation of pinenes (A and β), myrcene and p-cymene have been reported previously. Farooq et al. (2002a, 2002b) used the plant pathogenic fungus, Botrytis cinerea to convert A-pinene and β-pinene into their oxidized forms. In another approach, Picea abies cells immobilized on alginate were used to transform A-pinene by Vanek et al. (2005). The main products were cis- and trans-verbenol, the later being further
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transformed to verbenone. All of these studies showed the possibility of production of oxygenated terpene by biotransformation of their corresponding monoterpenes. Erlenmeyer flasks of 100 ml volume were used for all experiments. Thirty ml of sterilized culture medium (Mueller Hinton broth pH 7.0 for bacteria and Sabouraud broth pH 5.0 for fungi), was added to each flask. Then flasks were autoclaved at 120 °C for 20 min. After cooling down, 300 μl of liquid stock of each microorganism were added to flasks. Bacteria strains were grown at 37 °C, Candida albicans was incubated at 26 °C and Aspergillus niger was incubated at 30 °C. Each monoterpene (1% v/v, 300 μl) was added into media directly in liquid phase after 16-24 h of incubation period. Then, After 7 days, 6 ml diethyl ether was added to flasks and they were shaken for 1 h then diethyl ether with solved products were extracted by syringe. The solvent was dried by anhydrous sodium sulphate and concentrated to 0.5 ml under a steam of nitrogen. Concentrated extracts were stored. Then, the extracts were analyzed by GC and GC/ Mass. GC analysis was carried out using HP-6890 Gas chromatograph (FID) with a HP-5 column (30 m x 0.25 mm, 0.25 μm film thickness). The oven temperature was programmed from 60 °C 2 min and 60 °C to 240 °C at the rate of 6 °C/min. The injector and detector temperatures were 240 and 250 °C, respectively. Helium (He) was used as the carrier gas with a flow rate of 0.9 ml/min. Quantitative data was obtained from electronic integration of peak areas without the use of correction factors. GC/MS analysis was carried out using a Hewlett-Packard 6890 machine operating at 70 eV ionization energy, equipped with a Hp-5 capillary column (phenyl methyl siloxane, 30 cm x 0.25 mm, 0.25 μm film thickness) with He as the carrier gas and a split ratio of 1:20. Retention indices were determined by using retention
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K. JAVIDNIA et al.
TABLE 1 - Biotransformation products of A-pinene by various bacteria and fungi Microorganism
A-pinene oxide
trans-Verbenol
Verbenone
Total
19.0 45.6 30.6 29.2 3.4 ND
40.2 23.8 34.0 31.9 3.2 0.15
40.8 21.3 24.0 19.1 2.0 0.05
100 90.8 88.6 80.8 8.6 0.2
Bacillus subtilis
ND
ND
ND
ND
Candida albicans
ND
ND
ND
ND
Aspergillus niger
ND
ND
ND
ND
Escherichia coli Staphylococcus epidermidis Pseudomonas aeruginosa Salmonella typhi Staphylococcus aureus Klebsiella peneumoniae
ND: Not detected.
+ Alpha-Pinene
+ Verbenol
Alpha-Pinene oxide
times of n-alkenes that had been injected after the oil under the same chromatographic conditions. The retention indices for all the components were determined according to the Van Den Dool method using n-alkanes as standard (Van Den Dool and Kratz, 1963). The compounds were identified by comparison of retention indices (RRI, HP-5) with those reported in the literature and by comparison of their mass spectra with the Wiley and Mass finder 3 libraries or with the published mass spectra (Adams, 2001). The studies on the microbial metabolism of A-pinene by seven different microorganisms showed the presence of three known oxidized metabolites. As seen in Table 1 Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa and Salmonella typhi could transform A-pinene to its oxidized metabolites: A-pinene oxide, trans-verbenol and verbenone in appropriate yields while Bacillus subtilis ad two fungi could not transform A-pinene even in very low yields. In this case, main product was different in each microorganism; while for E. coli,
Verbenone
the main products were trans-verbenol and verbenone (40.2 and 40.8%), in S. epidermidis, this was A-pinene oxide (45.6%) and for P. aeruginosa and S. typhi was trans-verbenol (34.0 and 31.9%). Staphylococcus epidermidis and Klabsiella pneumoniae could transform β-pinene to sabinol, pinocarvone, myrtenol and myrtenal while transformatiom of β-pinene by other microorganism was negligible. In both cases, the major product was sabinol (32.0% for S. epidermidis and 14.2% for K. pneumoniae). Sabinol is identified for the first time as product of a microbial biotransformation of β-pinene. Metabolites of β-pinene biotransformation and their percentage are shown in Table 2. Only, S. epidermidis was capable of production of p-cymene-8-ol in the presence of p-cymene in high yield (40.2%) (Table 3). On the other hand, none of microorganisms could transform myrcene. In summary, by simple and feasible methods and media we can produce chemical compounds that are often difficult or impossible to obtain by conventional chemical means. Therefore, high commercial value compounds can be obtained through the
TABLE 2 - Biotransformation product of β-pinene by various bacteria and fungi Microorganism Staphylococcus epidermidis Klebsiella peneumoniae Bacillus subtilis Pseudomonas aeruginosa Aspergillus niger Escherichia coli Staphylococcus aureus Salmonella typhi Candida albicans
Sabinol
Myrtenol
Myrtenal
Pinocarvone
Total
32.0 14.2 1.8 0.5 0.2 0.1 0.1 0.1 0.1
19.8 10.2 2.9 ND 0.2 0.1 0.1 t 0.1
25.3 4.9 ND ND ND 0.1 0.1 0.1 0.1
18.3 4.4 1.6 0.3 0.1 0.1 0.1 0.1 t
95.4 33.9 6.3 0.8 0.5 0.4 0.4 0.3 0.3
ND: Not detected, t: trace (< 0.05%).
OH
OH
+ Beta-Pinene
Sabinol
O
+ Myrtenol
+ Myrtenal
O
Pinocarvone
Ann. Microbiol., 59 (2), 349-351 (2009)
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TABLE 3 - Biotransformation products of p-cymene by various bacteria and fungi Microorganism
p-cymene-8-ol
Staphylococcus epidermidis
40.2
Klebsiella pneumoniae
1.6
Salmonella typhi
1.1
Escherichia coli
ND
Pseudomonas aeruginosa
ND
Staphylococcus aureus
ND
Bacillus subtilis
ND
Candida albicans
ND
Aspergillus niger
ND
Acknowledgment This work was supported by a grant from Shiraz University of Medical sciences.
REFERENCES Adams R.P. (2001). Identification of essential oil components by Gas Chromatography-Quadropole Mass Spectroscopy, Allured, Publ. Corp. Carol Stream, IL. DeCarvalho C.C., De Fonseca M.M. (2006). Biotransformation of terpenes. Biotechnol. Adv., 24: 134-142. Farooq A., Choudhary M.I., Tahara S., Rahman A.U., Baser K.H., Demirci F. (2002a). The microbial oxidation of (-)-betapinene by Botrytis cinerea. Z. Naturforsch. [C], 57: 686690.
ND: Not detected.
Farooq A., Tahara S., Choudhary M.I., Atta ur R., Ahmed Z., Husnu C.B., et al. (2002b). Biotransformation of (-)-a-pinene by Botrytis cinerea. Z. Naturforsch. [C], 57: 303-306.
OH p-cymene
p-cymene-8-ol
microbial biotransformation of monoterpenes. Since, monoterpenic hydrocarbons are not expensive and produced in a variety of plant species, their transformation to oxidized metabolites which had high value in industry would get great importance. In general, the biotransformation of A-pinene, β-pinene and p-cymene result in the formation of oxidized products through the insertion of oxygen in the substrate molecule. Some of microorganisms used in this study transformed monoterpenes to oxygenated monoterpenes in good yields. Among them, S. epidermidis seems to be a potential microorganism to transform most of the monoterpenes and further studies about this bacterium will be useful.
Janssens L., DePooter H.L., Schamp N.M., Vandamme E.J. (1992). Production of flavours by microorganisms. Process Biochem., 27: 195-215. Krings U., Berger R.G. (1998). Biotechnological production of flavours and fragrances. Appl. Microbiol. Biotechnol., 49: 1-8. Vandamme E.J., Soetaert W. (2002). Bioflavours and fragrances via fermentation and biocatalysis. J. Chem. Tech. Biotech., 77: 1323-1332. Van Den Dool H., Kratz P.D. (1963). A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr., 11: 463. Vanek T., Halík J., Vanková R., Valterová I. (2005). Formation of trans-verbenol and verbenone from alpha-pinene catalysed by immobilised Picea abies cells. Biosci. Biotechnol. Biochem., 69: 321-325.