Microbial biotransformation is a pertinent strategy to overcome difficulties and problems arising from the chemical synthesis, in order to have access to regiospecific and stereospecific compounds and also explain the inactivity of essential oils or chemicals against some microorganisms. We evaluate in this research work, the bioconversion of (R)-limonene dominating the chemical composition of peels essential oil of the Tunisian Citrus aurantium by Trichoderma harzianum and Saccharomyces cerevisiae. In order to understand the exact and specific effects of these two yeasts on (R)-limonene, a negative control in the presence of air oxygen was carried out. Six oxygenated monoterpenes were produced and identified by GC–MS. Following the same protocol in the presence of these two yeasts, eight and seven secondary metabolites were formed, respectively. The results showed specific bioconversions and the fungi used involved to complete the full conversion of limonene and catalyze certain successive reactions in a specific way.
Limonene (4-isopropenyl-1-methylcyclohexene) is a natural monocyclic monoterpene very attractive, renewable resource  and the most abundant structure and it is widely available as a hydrocarbon monoterpene in the world belonging to the family of terpenoids which represent the largest class of secondary plant metabolites. Its derivatives are numerous and widely studied [1,2,3].
In recent years, much research has been carried out on the metabolism of terpenoids; mainly limonene which is known by its various biological activities such as cytotoxicity , hypoglycemic , anti-inflammatory [5, 6], antioxidant [6, 7] antitumoral , antifungal , antibacterial  and an effect protective against oxidative stress . Chemically, it was used as a precursor in many reactions affording new molecules used in several application fields [12, 13], as flavors and fragrances for perfume and cosmetic products , as drugs in the pharmaceutical industry  and in the petrochemical industry . For biotransformation, it was studied with various microorganisms (bacteria, yeasts and fungi) in order to search ways of producing different oxygenated terpenes [16,17,18,19] that have been reported as biosynthetic products, compounds of higher value. This bioconversion reaction led chemists to overcome difficulties and problems of regio-stereospecificity and enantioselectivity in chemistry in order to access the target compounds.
The inactivity of peel essential oil of Citrus aurantium towards Trichoderma harzianum, which we noted in one of our previous work , lets us to suppose that this fungus very probably has bioconverted the major component, limonene (73.60% of the toal oil) into no phytopathogenics derivatives. This hypothesis motivated us to treat the pure limonene by T. harzianum  and check its eventual bioconvertion into other derivatives.
For that, we started with a negative assay without fungi but in the presence of oxygen leading to six oxygenated monoterpenes. Then, we bioconverted limonene by T. harzianum we obtained eight oxygenated monoterpenes. In order to expand the bioconversion mechanism and understand the behavior of this monoterpene against other fungi, we chose the baker’s yeast, (Saccharomyces cerevisiae) because of its availability and its use in several biotransformations [22, 23]. Our aim was to identify the eventual formed metabolites. This enzymatic reaction resulted in the identification of seven oxygenated derivatives.
The (R)-limonene was purchased from Aldrich Chemical (Co., 90% purity, 98% ee).
Trichoderma harzianum was collected from the Laboratory of Phytopathology, Higher School of Agronomy, Chott Meriam, Sousse, Tunisia. A conidial suspension of the tested fungi was prepared (104–105 CFU/mL) and added to PDA medium (for phytopathogenic strains) cooled at 45 °C and supplemented with streptomycin sulfate (300 mg/mL).
The baker’s yeast (S. cerevisiae) was collected from a bakery used for the preparation of bread.
A mixture of 750 μL of (R)-limonene and 75 mL of the culture medium PDB (Potato Dextrose Broth) was stirred at room temperature for 7 days.
The mixture was poured into water and extracted twice with diethyl ether. The organic layer was dried over Na2SO4. Then the solvent was removed in vacuo and the residue was analyzed by GC-FID and GC/MS.
In Presence of Trichoderma harzianum
A mixture of 750 μL of (R)-limonene, 75 mL of the culture medium PDB (Potato Dextrose Broth) and the fungal T. harzianum (a disc of 6 mm) was stirred at room temperature for seven days. The mixture was poured into water and extracted twice with diethyl ether. The organic layer was dried over Na2SO4. Then the solvent was removed under reduced pressure and the residue was analyzed by GC-FID and GC/MS.
In Presence of Saccharomyces cerevisiae
A solution of 300 mL of distilled water, 2 g of baking yeast and 3 g of sucrose was stirred for one hour at 30 °C. 250 μL (R)-limonene is added to this solution. The mixture was stirred at room temperature for seven days. The mixture was poured into water and extracted twice with diethyl ether. The organic layer was dried over Na2SO4. Then, the solvent was removed in vacuo and the residue was analyzed by GC-FID and GC–MS.
GC-FID and and GC–MS Analyses
The identification of the formed metabolites was achieved by using a GC-FID composed of a gas chromatograph (HP 5890 II set) equipped with Flame ionization detectors (FID). Two types of columns have been used: an apolar capillary column HP-5, (30 m x 0.25 mm ID, film thickness 0.25 µm). Its stationary phase was 5% of biphenyl and 95% of dimethyl polysiloxane and a HP polar column INNOWAX (30 m × 0.25 mm ID, film thickness 0.25 µm). Injector and detector temperature: 250 °C and 280 °C, respectively. Helium was used as a vector gas (flow rate: l.2 mL/min), under a pressure of 9 Psi. The temperature of the two columns used was programmed as follows: 50 °C (1 min), 50–280 °C at 5 °C/min, 280 °C (20 min) and 50 °C (1 min), 50 - 250 °C at 5 °C/min, 250 °C (20 min), respectively. The sample volume injected was 1 µL (1% diluted in hexane). The percentages of the compounds were calculated by electronic integration of FID peak areas using HP-5 capillary column without the use of response factor correction. The identification of compounds was done by the comparison of their retention times with those of authentic samples and by means of their linear retention index (LRI), relative to the series of n-hydrocarbons [24, 25].
GC–MS analyses were achieved with the help of a Hewlett-Packard 5972 MSD System. The same two columns previously used in GC-FID analysis have been also employed: a HP-5MS, (30 m x 0.25 mm ID, film thickness 0.25 µm) and a HP polar column INNOWAX (30 m x 0.25 mm ID, film thickness 0.25 µm). The conditions of programming were the following: the temperatures of the injector, the source and the interface were 250 °C, 175 °C and 280 °C, respectively. Helium was as vector gas (flow rate: l.2 mL/min), under a pressure of 9 Psi. The same oven temperature programming previously used in the GC-FID analysis was also applied. The sample volume injected was 1 µL (1% diluted in hexane). The acquirement of the spectral data is achieved in fashion of sweep (2 SCAN/ses), it is from 50 to 550 u.m.a. The energy of broad cast is 70 eV. The spectroscopic analysis of the compounds is achieved by comparison with their counterparts with the help of the NBS75K.L spectral bank and WILLEY275.L.
Results and Discussion
We start this study of bioconversion of (R)-limonene by his oxidation with air oxygen.
Chemical Conversion of (R)-Limonene by Oxygen
Without of any fungi, 88.09% of (R)-limonene was transformed into six oxygenated monoterpenes (alcohols (56.32%) and ketones (31.77%). This fraction is dominated by carvone (29.30%), trans-carveol (20.80%) and limonene glycol (18.90%). The remaining compounds are present in smaller amounts such as p-mentha-trans-2,8-dien-1-ol (9.30%), cis-carveol (7.32%) and cis-dihydrocarvone (2.47%) (Fig. 1).
These findings showed that limonene is converted into oxygenated monoterpenes, in particular alcohols and ketones by a simple contact with oxygen. After seven days only 11.91% of (R)-limonene were not oxidized, hence the sensitivity of limonene to this kind of contact.
Biotransformation of (R)-Limonene by Trichoderma harzianum
When (R)-limonene was treated by T. harzianum, the entire amount was bioconverted into eleven compounds of which eight have been identified which represent 97.98% of the total. The results obtained (Fig. 2) showed that limonene was bioconverted into alcohols (53.16%) and ketones (32.45%) and an epoxide (12.37%) in the presence of T. harzianum. However, in addition to p-mentha-trans-2,8-dienol, carvone, trans-carveol, cis-carveol and limonene glycol, already formed in the absence of T. harzianum, three additional derivatives were biosynthesized, trans-limonene oxide (12.37%), perillic alcohol (4.21%) and the piperitenone (2.62%).
Looking at the structure of the compounds formed by the bioconversion of limonene by T. harzianum we fancy that this yeast catalyzes the formation of some products such as trans-limonene oxide, piperitenone and perillic alcohol. It also continues the bioconversion of some derivatives already obtained from the chemical oxidation (Fig. 1) to access to their structural analogues such as the dehydrogenation of cis-dihydrocarvone and the oxidation of cis- and trans-carveol in order to afford carvone.
Biotransformation of (R)-Limonene by Baker’s Yeast (Saccharomyces cerevisiae)
The biotransformation of (R)-limonene by baker’s yeast resulted in the formation of seven terpenoids representing 98.03% of all compounds identified, distributed as follows: alcohols (89.70%), ketones (6.63%) and an epoxide (1.70%). The results obtained showed that (R)-limonene was completely bioconverted. In addition to carvone, trans-carveol, cis-carveol and p-mentha-trans-2,8-dienol already formed in the absence of S. cerevisiae, three additional derivatives were biosynthesized, α-terpineol (2.75%), trans-limonene oxide (1.70%) and linalool (1.17%) (Fig. 3).
Table 1 lists all the compounds identified with the LRI values using apolar and polar columns, the MS data and the identification methods.
The difference in proportions of most compounds formed by comparison to the negative control and the biotransformation with T. harzianum shows that S. cerevisiae interacts with (R)-limonene in a specific manner (Fig. 4). As in the case of the biotransformation with T. harzinum, hydroxylation, oxidation and isomerization reactions were recorded.
The disappearance of cis-dihydrocarvone, the decrease in the proportion of carvone and the increase in proportion of trans-carveol and cis-carveol by comparison to the negative control (Fig. 4) could be explained by the dehydrogenation of cis-dihydrocarvone to carvone which is reduced in a stereoselective manner in cis- and trans-carveol. The current use of this yeast in similar stereoselective and sterespecific reduction reactions of ketones in alcohols was in favor of the obtained results [26,27,28]. This reaction does not seem obvious when observing the relatively high proportion of carvone (29.83%) compared to those of cis- and trans-carveol (8.89% and 13.26%, respectively) when using T. harzianum.
By examining the obtained results, we are allowed to note that the main factor in the conversion of limonene is, without doubt, oxygen that mostly favors hydroxylation and oxidation reactions then come the fungi, either to accelerate or also inhibit these reactions or to catalyze specific biconversions such as the stereoselective reduction of carvone in cis- and trans-carveol by baker’s yeast.
Oxidation of (R)-limonene by air oxygen and via its bioconversion by T. harzianum mainly led to the formation of the carvone (29.30–29.83%, respectively), which is known by its various properties, such as the protection against paclitaxel-induced retinal and optic nerve cytotoxicity , the prevention and relief against hepatic steatosis in rat , the anticancer  and the immunomodulatory  effects.
Saccharomyces cerivisiae leads the bioconversion of (R)-limonene into two enantiomers, trans-carveol (38.82%) and cis-carveol (38.77%), both have specific odor and flavor that resemble those of caraway and spearmint. They are employed as a fragrance in cosmetics and as a flavor in the food industry . They have been also found to display chemoprevention against breast cancer .
These fairly useful and encouraging data can motivate manufacturers to think of taking advantage of the increased availability of limonene, either extracted or synthetic, to use it as starting material to access the two geometric isomers trans- and cis-carveol which will be separated by fine fractional distillation and exploited in various fields as mentioned above.
Most previous studies which have documented the bioconversion of limonene in aerobic conditions by fungi or bacteria did not mention the role of oxygen in this kind of reaction. Indeed, we show through our results that the greatest amount of limonene is converted by oxygen and fungi are in this case the catalysts of specific reactions that depend on enzymes that constitute them.
Hypothetical biosynthetic pathways, some of which inspired from literature [35,36,37] are proposed to explain the formation of α-terpineol, trans-limonene oxide, carvone, piperitenone, perillic alcohol and linalool (Schemes 1, 2, 3).
In this study, the biotransformation of (R)-limonene by T. harzianum and S. cerivisiae was evaluated in aerobic conditions. The idea was to verify what these two fungi can do on limonene in the presence of oxygen. The results of the GC–MS analysis showed that 88.09% of limonene was oxidized by oxygen, leading to the formation of six oxygenated derivatives mainly alcohols and ketones. In the presence of T. harzianum and S. cerivisiae, (R)-limonene was further completely bioconverted, affording eight and seven oxygenated monoterpenes (alcohols, ketones and epoxide), respectively through oxidation, hydroxylation and isomerization reactions, and specific modifications have been noted with each microorganism. Hypothetical biosynthetic pathways of some formed oxygenated derivatives have been proposed. The negative control has shown that air oxygen is the main factor of transformation of (R)-limonene, and fungi can be a catalyst of specific reactions which depend on the nature of the enzymes present therein.
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The authors acknowledge the Ministry of Higher Education, Scientific Research and Technology of Tunisia for its financial support (LR11ES39).
Conflict of interest
The authors declare no conflict of interest.
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Ben Bnina, E., Daami-Remadi, M. & Ben Jannet, H. Access to Oxygenated Monoterpenes via the Biotransformation of (R)-Limonene by Trichoderma harzianum and Saccharamyces cerevisiae. Chemistry Africa (2020). https://doi.org/10.1007/s42250-020-00154-3
- Oxygenated monoterpenes
- Saccharomyces cerevisiae
- Trichoderma harzianum