This paper reports biotransformation of (L)-citronellal to (L)-citronellol using free and immobilized cells of Rhodotorula minuta. Optimum culture conditions for obtaining maximum cell growth of R. minuta were studied. The suitable culture conditions for (L)-citronellal biotransformation for free and immobilized cells of R. minuta. have been compared. The maximum product concentrations on use of free and immobilized cells were 3.5 gl^-1 and 3.3 gl^-1 respectively at (L)-citronellal concentration of 4.47 gl^-1. Immobilized cells could be reused twice after the first run.

Monoterpenes are widely distributed in nature and find extensive applications in the flavor and fragrance industry. Citronellal (3,7-Dimethyl-6-octanal), a monoterpene that occurs in the L or D form, bears distinct odor characteristics and also occurs as a constituent of essential oils in Eucalyptus citriodora (Betts, 2000). Citronellal can be further hydrogenated via biotransformation to produce citronellol (3,7-Dimethyl-6- octanol), which is a commercially important product due to its peculiar rose-like odor characteristics (Guenther, 1950). The optimization of process parameters remains critical due to several limitations posed by monoterpenes such as toxicity and volatility, by-product formation, immiscibility and low yields of the product (Krasnobajew, 1984).

The use of immobilized viable cells for biotransformation offers several advantages. In the present work, the optimal conditions for R. minuta growth and its application in biotransformation of (L)-citronellal to (L)-citronellol by free and immobilized cells is reported.The reuse of immobilized cells is presented in this work. The biotransformation reaction using R. minuta cells is schematically presented.

Nutrient media components and other reagents were obtained from standard sources. (L)-citronellal and (L)-citronellol were purchased from Sigma Chemicals Co., USA.

Microorganism and Cultural conditions

Strain of Rhodotorula minuta (NCIM 3359) was obtained from NCL, India and maintained on potato/dextrose/agar slants or broth.

Growth

R. minuta cell growth in different media (Atlas, 1997) such as PDB, YEPD, MEB (malt extract broth), MDP (malt extract/dextrose/peptone broth) and GYPB (glucose/yeast extract/peptone broth) was estimated and the growth curves [Dry wt. (gl^-1) vs. Time (hrs)] were plotted.

Immobilization of R. minuta cells

Equivalent cell concentration (13.1 gl^-1) of R. minuta cells was immobilized by entrapment in agarose (D'Souza and Nadkarni, 1980), hen egg white (HEW) (D'Souza et al. 1985), acrylamide (D'Souza and Nadkarni, 1980) and alginate (Rotmann and Rehm, 1990). Polyethyleneimine (PEI) treated glasswool (D'Souza et al. 1986) was used for adsorption of R. minuta cells. Immobilized cells were activated in nutrient medium (PDB) at 27ºC, 100 rpm for 12 hrs.

Biotransformation

Either the free cells after 44 hrs of growth (13.1 gl^‑1) in PDB or the immobilized cells after activation in PDB were added to 100 ml YEPD containing 4.47 gl^-1 (L)-citronellal. 0.1 gl^-1 methanol was used as the solubilizing agent. Optimum culture conditions for biotrans-formation was studied.

Extraction and analysis

On completion, the contents of the flasks were extracted twice with petroleum ether (40ºC - 60ºC), de-moisturized by addition of 1 gl^-1 sodium sulphate and distilled on water bath at 60ºC. The products were further identified by GC and GCMS analysis.

For optimum R. minuta cell growth, PDB was found to be the suitable media composition where maximum cell concentration of 13.1 gl^-1 and optimum growth rate of 0.086 h^-1 was obtained. The pH and temperature values of pH 5.5 and 27ºC were found to be suitable for growth.

Optimization studies to determine suitable culture conditions for biotransformation of (L)-citronellal to (L)-citronellol by free cells of R. minuta were conducted. Maximum (L)-citronellol formation (3.3 gl^-1) was obtained in PDB at pH 5.5,27ºC, 150 rpm and in 8 hrs. The maximum (L)-citronellal concentration that could be employed was 4.47 gl^-1.

Different solubilizers/emulsifiers namely dimethyl-sulfoxide, methanol, isopropanol and octane were used for solubilizing/emulsifying immiscible the substrate. The addition of 0.1 gl^-1 methanol was found to be suitable for increasing the product yields to 3.5 gl^-1.

Amongst the different matrices used for R. minuta immobilization, alginate entrapped cells could effectively biotransform 4.47 gl^-1 (L)-citronellal to 3.2 gl^-1 (L)-citronellol. The product concentrations were lower when R. minuta cells were immobilized by other entrapment and adsorption methods. In our studies, incubation of immobillized cells in nutrient rich media (YEPD) was found to be useful in prolonging cell activity. Single regeneration treatment in nutrient medium (YEPD) before the first run proved useful and product concentrations of 2.6 gl^-1 and 1.6 gl^-1 were obtained during the first and second reuse respectively.

The biotransformation of (L)-citronellal to (L)-citronellol was studied under different pH and temperature conditions employing alginate immobilized R. minuta cells. The pH and temperature values in the range of pH 5.5 - 6 and 27ºC were found to be suitable for obtaining 3.3 gl^-1 product concentration after incubation in YEPD for 8 hrs. Similar to free cells, the optimum substrate concentration that could be maximally biotransformed to the product (3.2 gl^-1) was found to be 4.47 gl^-1. The addition of solvents/emulsifiers for increasing (L)-citronellol formation was more effective for alginate-immobilized cells. The overall effectivity of solvents/emulsifiers for immobilized cell catalyzed (L)-citronellal biotransformation could be attributed to increased availability of the substrate through the immobilization matrix. The highest product concentration of 3.3 gl^-1 was obtained in case of 0.1 gl^-1 methanol, followed by 0.1 gl^-1 octane and 0.1 gl^-1 DMSO where product concentration of 3.2 gl^-1 was obtained.

Immobilized cells that were activated in nutrient media prior to the first run could be re-employed twice after the first use. The product concentrations during the first and second reuse were 2.63 gl^-1 and 1.56 gl^-1 respectively.

In this paper, we report the optimum culture conditions for R. minuta growth and (L)-citronellal biotransformation. The important advantage of immobilized cell reuse twice after the first run has been investigated. The data is important considering the scale-up potential of (L)-citronellal biotransformation.

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