The use of Macronet resins to recover γ-decalactone produced by Rhodotorula aurantiaca from the culture broth
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- Alchihab, M., Aldric, J., Aguedo, M. et al. J Ind Microbiol Biotechnol (2010) 37: 167. doi:10.1007/s10295-009-0659-z
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During the biotransformation of castor oil into γ-decalactone, R. aurantiaca produced both the lactone form and its precursor (4-hydroxydecanoic acid). After six days of culture, a maximum yield of γ-decalactone of 6.5 g/l was obtained. The parameters of γ-decalactone adsorption on three Macronet resins (MN-202, MN-102 and MN-100) were investigated in water. Adsorption isotherms of γ-decalactone for the three Macronet resins were linear. The trapping of γ-decalactone produced by R. aurantiaca on these resins was then carried out. γ-Decalactone was effectively retained by all the studied Macronet resins. The resin MN-202 trapped γ-decalactone more efficiently than MN-102 and MN-100. The percentages of γ-decalactone adsorbed on the resins MN-202, MN-102 and MN-100 were, respectively, 85, 75 and 81%, whereas around 70% of the adsorbed γ-decalactone was then desorbed. We propose an industrial process that uses Macronet resins to extract γ-decalactone from culture broth of R. aurantiaca.
Keywordsγ-DecalactoneExtractionMacronet resinRhodotorula aurantiaca
Lactones are aroma compounds that are widely used in the flavouring industry . Among these lactones, γ-decalactone is a molecule with a fruity peach-like odour which, if produced by yeasts, can obtain a “natural” label. This aroma compound is produced by biotechnological methods using microorganisms, mainly yeast strains [2, 3]. The γ-decalactone concentrations reported in such processes vary from a few mg/l up to 11 g/l .
Product recovery is a difficult step in many bioprocesses, especially for flavour compounds because of their volatility and low solubility. The methods most commonly used to extract organic compounds from aqueous media involve solvent extraction, separation on specific membranes and adsorption on activated carbon [5–7]. In the last years, porous organic resins have replaced activated carbon as methods for extracting aroma compounds due to their hydrophobic nature and their high specific surface areas. Previous studies reported the adsorption of γ-decalactone onto activated carbon and hydrophobic resins (Porapak Q, Chromosorb 105 and SM4) by online extraction. However, while they limited the toxicity of the lactone towards the yeast, their presence in the bioconversion medium also decreased the production of γ-decalactone [8, 9].
Davankov and Tsyurupa  disclosed their new series of hypercrosslinked networks in 1969. The polymers were originally described as being macroreticulated, offering characteristics different from those of other polymeric resins. An optimal series of Hypersol-Macronet™ sorbent resins for industrial application was developed by Purolite International Ltd. However, crosslinked polystyrene resins have turned out to be the most efficient adsorbents for recovering flavour compounds from aqueous solution [12, 13]. Valderrama et al.  described the sorption of six polycyclic hydrocarbons from an aqueous solution on the Macronet polymeric sorbent MN-200. In the present work, the use of Macronet resins in culture broth to extract the lactone produced by R. aurantiaca was evaluated. To our knowledge, this is the first report discussing the utilization of Macronet resins for extracting lactones from production media.
Materials and methods
Yeast cultures and media
Rhodotorula aurantiaca is a psychrophilic strain previously isolated near the Antarctic Station Dumont d’Urville and deposited at the Mycotheque of the University of Louvain-la-neuve (MUCL), Belgium, under registration No. 40267. The yeast was grown in glucose medium as previously described . This yeast was used in the present study for γ-decalactone production. The biotransformation medium was composed of 6 g/l of peptone casein, 3 g/l of yeast extract and 20 g/l of castor oil.
Biotransformation of castor oil into γ-decalactone
A 20-l fermentor was used, with 12 l of biotransformation medium. All fermentor trials were conducted with aeration (1.5 vvm) and at constant temperature (14°C), pH (6.8) and agitation (350 rpm), by online control using Freelance 2000 software. The pH was controlled by the automatic addition of 6 M NaOH and 6 M H3PO4 solutions. The fermentation was stopped when the γ-decalactone concentration reached its maximum and cells entered the stationary phase. To increase the lactonization of 4-hydroxydecanoic acid into γ-decalactone, the fermentor content was acidified at pH 2.0 using concentrated sulfuric acid and heated at 95°C for 30 min . After cooling and centrifugation, γ-decalactone was then extracted from the culture broth by adsorption on hydrophobic Macronet resins (MN-202, MN-102 and MN-100).
Characteristics of the synthetic Macronet resins
Specific gravity (g/ml)
Specific area (m2/g)
Adsorption of γ-decalactone on Macronet resins
The adsorption of γ-decalactone on the different Macronet resins was achieved at 25°C. To investigate the adsorption kinetics of γ-decalactone on each resin, an aqueous solution of γ-decalactone at 0.6 g/l was prepared. The resins were added to the solution at a concentration of 30 g/l and stirred at 400 rpm. The influence of the ratio resin/γ-decalactone and of the regeneration of resins on the extraction of γ-decalactone was studied.
Recovery and quantification of the adsorbed γ-decalactone from the resins
Adopting the procedure of Matsukura et al. , the resin was removed from the culture broth, washed several times with distilled water, then placed over a paper filter and air-dried at room temperature for 1 h. The organic products were extracted with three volumes of ethanol at a ratio of 4 ml/g resin. For quantification, a sample of the ethanolic fraction was collected and dried overnight over anhydrous sodium sulfate, and γ-valerolactone was added as an internal standard. Direct injection of the ethanolic extract was then performed by GC.
In order to determine the purity of γ-decalactone, 50 mg of concentrated γ-decalactone produced by R. aurantiaca was dissolved in 100 ml of diethyl ether and compared by GC analysis to the commercial γ-decalactone (Sigma–Aldrich).
GC analyses were performed using a 5890 series II gas chromatograph from Hewlett-Packard (Palo Alto, CA, USA) equipped with a flame ionisation detector and an Alltech AT AQUAWAX column (30 m × 0.25 mm ID, film thickness 0.25 μm). The oven temperature was held at 40°C for 2 min, raised to 250°C at a rate of 10°C/min, then fixed at 250°C for 20 min. The injector and detector temperatures were 200 and 250°C, respectively. The carrier gas, helium, was adjusted to a linear velocity of 1 ml/min and 0.5 bars. One-microlitre samples were injected into the GC apparatus.
Results and discussion
Production of γ-decalactone and 4-hydroxydecanoic acid by R. aurantiaca in a 20-l fermentor
During the biotransformation of castor oil (a source of ricinoleic acid), R. aurantiaca produced four lactones: γ-octalactone, γ-nonalactone, γ-decalactone and γ-undecalactone. Among these lactones, γ-decalactone was the most abundant . In biotransformation medium, R. aurantiaca produced both γ-decalactone and its precursor 4-hydroxydecanoic acid. Maximum γ-decalactone and 4-hydroxydecanoic acid concentrations (2 and 4.5 g/l, respectively) were obtained at a medium pH of 6.8 (data not shown).
Influence of broth pH and heating on the recovery of γ-decalactone produced by R. aurantiaca after six days of culture
pH of medium
pH 2.0 and heating at 95°C for 30 min
The lactonization of 4-hydroxydecanoic acid into γ-decalactone was enhanced by heating the culture broth at 95°C for 30 min. After six days of culture, the concentration of γ-decalactone obtained by R. aurantiaca was around 6.5 g/l (Table 2). The results obtained using the yeast R. aurantiaca in the fermentor was on the order of the concentrations usually described in patents. Consequently, the bioproduction of γ-decalactone with R. aurantiaca includes a two-step procedure, i.e. the biotransformation of castor oil into 4-hydroxydecanoic acid and the lactonization of this acid (after acidification and heating).
γ-Decalactone was then extracted from the culture broth by adsorption on hydrophobic Macronet resins (MN-202, MN-102 and MN-100). Some aspects of the adsorption of γ-decalactone onto these three resins from aqueous media will be discussed below, before we describe their application in the production medium.
The adsorption kinetics of γ-decalactone on the three Macronet resins were studied in water with 0.6 g/l of γ-decalactone and 30 g/l of each resin. The decrease in the level of γ-decalactone in the aqueous solution was followed by analysis every 10 min until the remaining quantity of γ-decalactone reached equilibrium. The quantity adsorbed corresponded to the difference between the initial concentration and the final concentration (after the adsorption).
Initial adsorption rate of γ-decalactone on the Macronet resins (MN-202, MN-100 and MN-102) after 10 min
Resin (30 g/l)
Initial concentration of γ-decalactone (g/l)
Adsorbed quantity of γ-decalactone (g/l) after 10 min
Initial adsorption rate
0.83 × 10−3
0.33 × 10−3
1 × 10−3
Adsorption isotherms and the ratio resin/γ-decalactone
Desorption of γ-decalactone
Regeneration of the resin
The regeneration of the Macronet resins (MN-202, MN-102 and MN-100) was investigated by a batchwise method where ethanol was used to remove the adsorbed γ-decalactone (five cycles of extraction). We carried out a regeneration study of these resins by analyzing six cycles of adsorption at 25°C. The results obtained demonstrated that all of the resins can be reused at least six times. In all cases, and even after six cycles, the percentage adsorption was higher than 80%.
Adsorption of γ-decalactone produced by R. aurantiaca on Macronet resins
Studying the adsorption of γ-decalactone onto Macronet resins in water permitted us to determine the experimental conditions (adsorption time, amount of resin required to extract γ-decalactone, and volume of ethanol needed to desorb it) required for the recovery of γ-decalactone produced with R. aurantiaca from culture broth.
The adsorption of γ-decalactone produced by R. aurantiaca onto the resins was investigated. γ-Decalactone was strongly adsorbed onto all Macronet resins (from 75% for MN-102 to 85% for MN-202). Among these resins, MN-202 was the most efficient adsorbent for γ-decalactone. The percentage desorption was around 70% of the adsorbed γ-decalactone, with a purity of 80%. The level of adsorption of γ-decalactone from the culture broth of R. aurantiaca onto Macronet resins was lower than that observed in water. This difference can be attributed to the presence of castor oil and biomass in the medium, which adsorbed some of the compound. The castor oil used as substrate acts not only as a biotransformation precursor but also as a lactone extractant. However, this oily phase is progressively reduced during the process.
The adsorption of γ-decalactone onto Macronet resins is a suitable method for extracting γ-decalactone from culture broth. The usefulness of Macronet resins for industrial applications results from a number of advantages: the studied resins are not chemically reactive with the aroma compounds; they are easily regenerated; they are not expensive; and they can easily be adapted to an industrial scale. Moreover, their elution requires only small volumes of ethanol, which can also be recycled.
We thank the government of Syria for their financial support of this study.