JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 88, No. 5, 488-494. 1999 Sophorolipid Production by Candida bombicola: Medium Composition and Culture Methods JO& A. CASAS AND FGLIX GARCiA-OCHOA* Departamento Ingenieria Quimica, Facultad CC. Q&micas, Universidad Complutense, 28040~Madrid, Spain Received 27 June 1997/Accepted 6 July 1999 Candida bombicola is able to produce sophorollpid molecules with surfactant properties when grown in a medium composed of two different carbon sources (usually sugar and oil) and a nitrogen source (frequently yeast extract). In this work, the composition of the medium and the culture method employed have been stu- died. The influences of glucose concentration, properties of oil and yeast extract concentration have been taken into account. Accordingly, a production medium composition is proposed (100 g/Z glucose, 100 g/l sunflower oil and 1 g/Z yeast extract). The most frequent culture methods reported in literature, i.e. batch, medium pulse mode and resting-cell methods, have been tested. The resting-cell method was found to produce the highest final concentration of sophorolipid, obtaining a good yield of carbon sources in a relatively short time. Under the best operational conditions and using the resting-cell method, 120 g/l sophorolipid was obtained in 8 d, with a carbon source yield of 0.60. Product distribution has also been investigated and the sophorolipid molecular structure of opened or cycled molecules has been determined under different operational conditions. Low yeast extract concentration and long fermentation time enhance the production of cycled structures by all the production methods studied. [Key words: sophorolipid production, Candida bombicola, resting cell, culture methods, product structure distkbution] - Sophorolipids are surfactant molecules produced and secreted into the medium by the yeast Torulopsis bombi- cola which is now classified under the genus Candida (13). These compounds contain the dimeric sugar so- phorose (p-(1 -2)glucose) linked to one long chain car- boxylic acid, with a hydroxyl function on the penulti- mate or terminal carbon (4). Sophorolipids can have two different structures, opened or cycled (l), as can be seen in Fig. 1. In most of these structures, the acid is at- tached to the carbohydrate by the hydroxyl group, leav- ing a free carboxyl function (acid or opened structure); when this carboxyl function is also linked to the so- phorose molecule, a cycled or la&one structure is form- ed. Both structures show similar tensioactive proper- ties (2), but only closed structures can inhibit the growth of different microorganisms (10). Sophorose is always present in sophorolipids but the length and/or nature of fatty acids can vary depending on the medium composition (8, 9, 17) with hydroxyl fatty acids 16 to 18 carbons long being the usual com- ponents (17). The production of sophorolipids by yeast has been known since Gorin et al. (6) reported the production of a mixture of molecules (sophorolipids) synthesised by T. bombicola from glucose, yeast extract (YE) and urea. They observed that the production of this biosurfactant can be increased using a second carbon source of lipophilic nature, for example oil or fatty acids (17). In later studies, different authors tested various production medium compositions using different hydrophilic and lipophilic carbon sources. Thus, the composition of production medium, usually composed of glucose, oil and complex extracts, has been studied, taking into ac- count the main variables of glucose concentration (1, 4, 7, 20), lipid source or properties of oil (1, 4, 7, 11, 19, * Corresponding author. 20) and nitrogen source (usually urea or complex ex- tracts, such as cornsteep liquor, yeast extract or peptone (4, 20)). Although these works demonstrate clear influences of the different variables, there is no agree- ment about the optimum concentration values for some nutrients, particularly for YE. The production media used in previous works were usually composed of either 100 g/l glucose, 100 g/l vegetable oil or complex extracts and salts (4, 10-12, 20). Although glucose was always used as the hydrophilic carbon source, the lipophilic source employed varied, with those more often employ- ed being safflower oil (4, 11, 20), soybean oil (1, 4, 14) and sunflower oil (4, 12). All these vegetable oils pro- duce sophorolipids in similar concentrations due to their similar compositions of triglycerides and fatty acids (4). Higher sophorolipid concentrations or yields have been reported with rapeseed oil ethyl esters (5), oleic acid (16) or canola oil (21), but these lipophilic carbon sources are more expensive than vegetable oils (15). In the production medium, a source of vitamins, nitro- gen and trace elements is needed. Different compounds such as cornsteep liquor (5), peptone (4), YE (4, 10) and mixtures of YE with urea (1, 7, 18, 20) or different salts (12, 14) have been employed for this role. YE is the most frequently used nitrogen source, but optimal con- centration of this nutrient is not clear. While Cooper and Paddock (4) found 5 g/l to be the optimal concentra- tion, Zhou et al. (20) obtained higher sophorolipid con- centrations using a lower YE concentration of between 2 and 3 g/l. The yield of sophorolipids could also be substantially modified by employing different culture methods, with batch (4, 6, 14, 19), fed-batch (1, 5, 14) and resting-cell (7) methods having been considered in previous works. When batch culture is used, the yield of sophorolipid de- pends very much on medium composition and operation- al conditions (temperature, aerating flow, stirrer speed, 488