J Appl Phycol (2011) 23:975–981 DOI 10.1007/s10811-010-9625-4

Biotransformation of monoterpenes by immobilized microalgae Sara Rasoul-Amini & Elham Fotooh-Abadi & Younes Ghasemi

Received: 5 June 2010 / Revised and accepted: 1 November 2010 / Published online: 16 November 2010 # Springer Science+Business Media B.V. 2010

Abstract This paper reports the biotransformation of carvone, limonene, β-pinene, thymol, and linalool using whole-cell-immobilized microalgal strains isolated from paddy fields of Iran. The strains was recognized by morphological characterization and assigned according to amplified 16S/18S rRNA genes by PCR. Ten unialgal strains including Chlorella, Oocystis, Chlamydomonas, and Synechococcus were immobilized in calcium alginate beads. After a 24-h incubation with substrates, characterization and identification of biotransformation products were done by GC/MS. None of the isolated immobilized microalgae converted β-pinene. In contrast, most of these strains biotransformed carvone and limonene to the related compounds. Some strains only reduced the C=C double bond to yield the dihydrocarvone isomers while others reduced the ketone to give the dihydrocarveol. The transformation ratio showed that Oocystis sp. MCCS 033 and Synechococcus sp. MCCS 035 produced dihydrocarvone isomers with the highest efficiency. Furthermore, limonene was converted into a mixture of five corresponding products and the maximum yield was 52.1% S. Rasoul-Amini : E. Fotooh-Abadi : Y. Ghasemi (*) Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran e-mail: [email protected] S. Rasoul-Amini : Y. Ghasemi Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-158, Shiraz, Iran S. Rasoul-Amini Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran

for carvone, the bioconverted product. Only one strain, Synechococcus sp. MCCS 034, oxidized thymol, and the product obtained from thymol was thymoquinone. Also, linalooloxide isomers and dihydrolinalool were obtained from linalool, and finally dihydrolinalool was the main product. These results showed a novel conversion pathway of linalool-forming dihydrolinalool. Keywords Microalgae . Biotransformation . Monoterpenes . Cell immobilization . GC/MS

Introduction Monoterpene molecules (synthesized from isoprene units) are the largest family of secondary plant metabolites (Van Der Werf et al. 1999; 2000; Van Der Werf and Boot 2000) found in nature as the main constituents of many essential oils of aroma plants (Misra et al. 1996; Chatterjee and Bhattacharyya 2001). Traditionally, monoterpenes have been widely used in flavor and fragrance industry (Misra et al. 1996; Velankar and Heble 2003), but now monoterpenes have drawn increasing commercial attention because of better understanding of their roles in acting in the prevention and therapy of several diseases, due to their ecological activity as natural insecticides and antimicrobial agents and also because they are used as solvents and can be building blocks in the synthesis of many highly valued compounds (de Carvalho and da Fonseca 2006). Over the last years, the biotechnological production of these compounds has gained a growing interest (de Carvalho and da Fonseca 2006). Biotransformation is a valuable tool for the production of stereo- and regiospecific monoterpenes (Tecelão et al. 2001; Hamada et al.

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1997; Simmonds and Robinson 1997; Demyttenaere and De Kimpe 2001) since it allows the produced monoterpenes to be labeled as “natural” compounds (Onken and Berger 1999). These processes are performed by many prokaryotic and eukaryotic microorganisms, which convert monoterpenes to several products (Menéndez et al. 2002; Speelmans et al. 1998; de Carvalho and da Fonseca 2006) and microalgae and their enzymes have been proven to be versatile biocatalysts for biotransformation of various monoterpenes (Hook et al. 2003; Noma et al. 1991; 1992). Although information on the biotransformation of monoterpenes by microalgae is far less than for bacteria, fungi, and yeast, some studies indicate that they were capable of biotransforming the monoterpenes (Farooq and Hanson 1995; Van Rensburg et al. 1997; de Carvalho and da Fonseca 2006; Semple et al. 1999). Interestingly, microalgae are photoautotrophic and can use sunlight as their sole energy source. Moreover, the largescale culture of microalgae is simpler and cheaper than that of other microorganisms (Hook et al. 2003). These are the important advantages of microalgae as biocatalysts in biotransformation of readily available chemicals such as terpenes into valuable compounds. The aim of this study is to investigate the biotransformation of these compounds by immobilized microalgae. Immobilization of cells provided a method of regulating metabolism and hence product formation. Furthermore, systems using immobilized cells may allow the recovery and reuse of cells, resulting in usually cheaper and simpler processes than those requiring extraction and purification of enzymes (de Carvalho and da Fonseca 2006; Velankar and Heble 2003; Hamada et al. 2003). In addition, the metabolic production and activity remains constant on long period (Moreno-Garrido 2008) and few algal immobilizations have been reported for conversion of monoterpene compounds.

Materials and methods The strains used in this study were isolated during a screening program from soil samples of paddy fields of Iran (July to November 2008). Soil samples were suspended in specific volume of distilled water and were transferred onto solid BG-11 medium. Petri dishes were stored in culture room under constant illumination (∼25 μmol photons m−2 s−1) with white fluorescent lamps at 25±2°C. After colonization, the isolation and purification were performed using plate agar method to obtain unialgal cultures (Ghasemi et al. 2003). All isolated microalgae were grown at room temperature in liquid BG-11 medium with shaking at 70 rpm. The taxonomic identification was done following the keys of Desikachary (1959) and John et al. (2003). In order to

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confirm and determine the species, the sequence of small subunit of rRNAs was studied using molecular markers. Genomic DNA of microalgal strains was prepared according to Rasoul-Amini et al. (2009). DNA fragments of ∼800 and ∼700 bp were amplified from genomic DNA of microalgal strains with polymerase chain reaction (PCR) by using universal primers against the 16S/18S rRNA genes, respectively. The forward 16S universal primer was 5′-CAGCCGCGGTAATAC-3′ and 5′-ACGGGCGGTGTG TAC-3′ using as the reverse primer (Billi et al. 2001). The universal eukaryotic primers 5′-GTCAGAGGTGAA ATTCTTGGATTTA-3′ as forward primer and 5′-AGGG CAGGGACGTAATCAACG-3′ as reverse primer, amplify a ∼700-bp region of the 18S rRNA gene (Ghasemi et al. 2008). PCR amplifications were determined by 1% (w/v) agarose gel electrophoresis in TBE buffer. PCR products were purified form agarose gel with the CoreBio PCR purification kit (Cat No. GE-100) and used as templates in sequencing reactions by CinnaGene Company. 16S/18S rRNA sequences were analyzed by using the BLAST program and annotations of all microalgal strains were deposited in GeneBank under specific accession numbers. Then the environmentally isolated microalgae were kept in the liquid nitrogen and lyophilized in order to add into Microalgal Culture Collection of Shiraz University of Medical Science (MCCS). Immobilization of microalgae cells in calcium alginate Current advantages of immobilized living cells in comparison to the free cells include advantages such as culture collection handling, increasing the retention time in the reactor and removing the risk of washout (Moreno-Garrido 2008). As suggested by Mallick (2004), the alginate beads were prepared using the microalgal culture in log phase. Microalgal cells were separated and harvested by centrifugation at 2,000×g at 4°C for 3 min. The cells were resuspended in 500 mL of saline (0.85% NaCl solution) and mixed with 60 mL of alginate (4%) using magnetic stirring at room temperature (Hamada et al. 2003). The mixture was titrated into cold CaCl2 solution (2%) and slowly stirred with a syringe. Pump pressure and the needle gauge were used to control the bead size. The produced Ca2+-alginate beads were washed with 0.85% (w/v) saline and cultured in fresh BG-11 medium. Biotransformation experiments Of the monoterpene substrates (from Merck Company), 20 μl were added to 40 beads of the immobilized microalgal cells. The cells were cultured in 250-mL conical flasks, containing 20 mL of fresh BG-11 medium, and the reaction mixture incubated at a temperature of 25±2°C for

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Fig. 1 PCR-amplified segments of 16S/18S rRNA genes from ten unialgal strains: M, 100 bp DNA marker; 1, Chlo. sp. No.1; 2, Chlo. sp. No.2; 3, Chlo. sp. No.3; 4, hlo. sp. No.4; 5, Oo. sp. No.1; 6, Oo. sp. No.2; 7, Chla. sp. No.1; 8, Syn. sp. No.1; 9, Chla. sp. No. 2; and 10, Syn sp. No.2

24 h with shaking at 70 rpm. The occurrence of compounds was also examined to determine whether the reactions were formed chemically or biologically. Thus, in an initial biotransformation experiments, chemical blank (culture medium with monoterpene substrates and without microalgae) and biological blank (culture medium with immobilized microalgae and without substrates) were performed. After incubation, the cell-free medium was separated. Then the culture broth containing the reaction products (5 mL) was mixed and extracted with 5-mL chloroform. The solvent phase was collected and dried over sodium sulphate. Concentration was performed under N2 gas (Shams-Ardakani et al. 2005), and the concentrated extract was used for gas chromatography-mass spectroscopy (GC/ MS) analysis.

(monoterpene substrates) was injected into a HP-5M capillary column (phenyl methyl siloxan, 25 m×0.25 mm i.d., Hewlett–Packard, USA). Helium with the flow rate of 1.2 mL min−1 was used as carrier gas. Column oven temperature was programmed from 85°C (5 min) to 265°C at the rate of 7°C min−1 where it was held at 265°C for 10 min. The mass spectrometer (Hewlett–Packard 5973, USA) was operated in EI (Electron ionization) mode at 70 eV. The interface temperature was 265°C and the mass range was 15–650 m/z. The split ratio was 1:20. Quantitative data was obtained from electronic integration of peak areas without the use of correction factors. 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 identification of products was performed by comparison of the fragmentation patterns of the mass spectra and the retention indices with those reported in the literature and also by comparing the obtained mass spectra with Wiley (275) libraries (Adams 2004). Relative percentage amounts of the separated compounds were calculated from total ion chromatograms through the computerized integrator.

Results and discussion The ten microalgal strains belonged to four families: Chlorellaceae, Oocystaceae, Chlamydomonadaceae, and Synechococcaceae. The PCR amplification of chromosomal DNA of the algae with forward and reverse primers revealed efficient amplification (Fig. 1). Based on chemotaxonomic and 16S/18S rRNA data, the microalgal strains were identified as shown in Table 1.

Analytical methods GC/MS analyses were carried out on a Hewlett–Packard 6890 USA, as described previously (Ghasemi et al. 2007). One microliter of each extract and standard chemicals Table 1 The isolated microalgal strains that were added into MCCS

Table 2 Biotransformation by immobilized microalgae and the products after a 42-h incubation period Substrates

Metabolites

Carvone (1)

Cis-dihydro carvone (2) Trans-dihydro carvone (3) Dihydro carveol (4) Cis-carveol (6) Trans-carveol (7) Carvone (8) Cis-limonene oxide (9) Trans-limonene oxide (10) – Thymoquinone (13) Cis-linalool oxide (15) Trans-linalool oxide (16) Dihydro linalool (17)

Strains Chlorella sp. MCCS028 Chlorella sp. MCCS029 Chlorella sp. MCCS030 Chlorella sp. MCCS031 Oocystis sp. MCCS032 Oocystis sp. MCCS033 Synechococcus sp. MCCS034 Synechococcus sp. MCCS035 Chlamydomonas sp. MCCS036 Chlamydomonas sp. MCCS037

Limonene (5)

β-Pinene (11) Thymol (12) Linalool (14)

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Comparative studies of nucleotide sequences of 16S/18S rRNA were chosen because of their appropriate length and sufficient sequence information. Ribosomal RNA sequences have been used successfully to predict genetic relatedness at the Fig. 2 Chemical structures of the substrates and the products biotransformed by immobilized microalgae

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genus and species level in different classes of algae (Neilan et al. 1997; Gunderson et al. 1987). These molecular markers are used as tools for estimating the phylogenetic relationships of different kinds of organisms (Olsen and Woese 1993).

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Biotransformation of monoterpenes

Table 4 Biotransformation (%) of substrate 5 by immobilized microalgae after a 24-h incubation period

In chemical and biological blank experiments, no products were detected in any of the culture media. This confirmed that the observed effects were not caused by a carryover of substrates or products from cultures. Our studies on the biotransformation of (R)-(+)-carvone (1), (D)-(+)-limonene (5), (+)-β-pinene (11), thymol (12), and (+)-linalool (14) with immobilized microalgae for 24 h, are summarized in Table 2 and Fig. 2. The nine immobilized microalgae reduced 1 to different degrees (Table 3). After 24-h incubation, Chlorella sp. MCCS 028, Chlorella sp. MCCS 029, Chlorella sp. MCCS 031, Oocystis sp. MCCS 032, Oocystis sp. MCCS 033, and Synechococcus sp. MCCS 035, only reduced the C=C double bond in 1 to yield cis- and trans-dihydrocarvone (2, 3), while Chlorella sp. MCCS 030, Synechococcus sp. MCCS 034 and Chlamydomonas sp. MCCS 037, also reduced the ketone to give dihydrocarveol (4). In this regard, the first chemical or enzyme attack may occur at the C=C double bond of carvone. These results also differ from those observed for Synechococcus sp. MCCS 035 where it is the most efficient strain in the reduction of the C=C bond and the regio-specific organism of the production of 3. The nonselective biotransformation of limonene to cis- and trans-carveol (6, 7) carvone (8) and cis- and trans-limonene oxide (9, 10) occurred by three immobilized microalgal strains (Table 4). Chlorella sp. MCCS 030 was the most effective microalgae for oxidized 5 to 8 in quite high yields of 52.1% within the 24-h incubation period. In this experiment, Chlorella sp. MCCS 030 was the stereoselective strain for the production of trans-carveol from limonene. Based on our results these microalgal strains are also able to oxidize the sixth position of 5 and contain the current enzyme pathway. However, allylic oxidation of 5 at the sixth position (reached 6, 7, and 8 compounds) were

Organisms

Table 3 Biotransformation (%) of substrate 1 by immobilized microalgae after a 24-h incubation period Organisms

Chlorella sp. MCCS 028 Chlorella sp. MCCS 029 Chlorella sp. MCCS 030 Chlorella sp. MCCS 031 Oocystis sp. MCCS 032 Oocystis sp. MCCS 033 Synechococcus sp. MCCS 034 Synechococcus sp. MCCS 035 Chlamydomonas sp. MCCS 037

Products 2 Yield (%)

3

4

2.3 6.1 5.1 2.5 4.2 41.1 3.4 0 1

3.6 23.4 16.2 14.1 11.4 27.1 12.7 53.7 7.5

0 0 2.8 0 0 0 6.7 0 2.1

Chlorella sp. MCCS 030 Chlorella sp. MCCS 031 Synechococcus sp. MCCS 034

Products 6 7 Yields (%)

8

9

10

0 25.2 38.1

52.1 4.1 2.3

6.6 5.1 4.3

1.2 3.2 2.3

2.3 4.1 1.3

reported via a few studies (Duetz et al. 2001 and Onken and Berger 1999). In addition, when limonene was used as a substrate by Chlorella sp. MCCS 030, we found that no 3 was detected throughout the incubation period. Whereas, the main transformation product of one with immobilized cells of Chlorella sp. MCCS 030 was 3. From these observations it may be evident that the incubation time was insignificant in subsequent biotransformation of 5 to yield 3. However, it might be attributed to the presence of different byproducts in degradation of 5, which had inhibitory effect. Also, the substrate utilization pattern for degradation of 1 and intermediate carvone might be different. The biotransformation of β-pinene was also investigated in this study, but no products derived from the biotransformation of 11 by any of the immobilized algae. Although previous researchers have reported further conversion of 11 (Ghasemi et al. 2009; Farooq et al. 2002), we feel that this may be explained by the fact that 11 was more toxic to all immobilized microalgal strains than the other substrates. Maximum biotransformation of Thymol was carried out by Synechococcus sp. MCCS 034 with 1.5% efficiency in producing Thymoquinone (13). This low level of transformation may be due to the toxicity of 12. On the other hand, 12 were hardly converted into its corresponding products. From the identification of transformation products of linalool, it became clear that three immobilized microalgal strains had the ability to reduce and oxidized 14. Compound 14 was transformed to 17 as the major product and 15 and 16 as the minor products (Table 5). All three microalgal strains produced dihydro linalool (17) as the main product. Also in the conversion of linalool, Table 5 Biotransformation (%) of substrate 14 by immobilized microalgae after a 24-h incubation period Organisms

Chlorella sp. MCCS 028 Chlorella sp. MCCS 029 Chlamydomonas sp. MCCS 036

Products 15 Yield (%)

16

17

1.4 0 0

1.4 0 0

3.2 4.1 3.3

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Chlorella sp. MCCS 028 oxidized 14 to give cis- and translinalool oxide (15, 16). As far as we know, this work reports for the first time the production of dihydro linalool (17) from 14. In conclusion, Chlorella sp. MCCS 028 contains a novel bioconversion pathway for linalool. In addition, the immobilized microalgal strains showed different efficiency for the biotransformation of terpenes. The current results confirm that the different metabolic activities are dependent on the structure of the added substrates which result in reduction, oxidation and isomerization reactions. Although microalgae are little studied in regard to monoterpene conversions, they may be considered as useful biocatalysts for biological conversions. As a part of an ongoing study into microalgal biotransformation, selecting the right microalgal strains/species, increasing the efficiency of the processes, and identifying new potentially useful bioconversions is continuing. Acknowledgments This work was supported by a grant from the Research Council of Shiraz University of Medical Science, Shiraz, Iran.

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