Utilization of vegetable oil in the production of clavulanic acid by Streptomyces clavuligerus ATCC 27064 G.L. Maranesi, A. Baptista-Neto, C.O. Hokka and A.C. Badino* Department of Chemical Engineering, Universidade Federal de Sa ˜o Carlos, Cx. Postal 676, CEP 13565-905 Sa ˜o Carlos SP, Brazil *Author for Correspondence: Tel.: +55-16-3351-8001, Fax: +55-16-3351-8266, E-mail:badinojr@power.ufscar.br Keywords: Beta-lactamase inhibitor, clavulanic acid, lipid as substrate, soybean oil, Streptomyces clavuligerus, vegetable oil Summary Production of clavulanic acid (CA) by Streptomyces clavuligerus ATCC 27064 in shake-flask culture (28 °C, 250 rev min )1 ) was evaluated, with media containing different types and concentrations of edible vegetable oil. Firstly, four media based on those reported in the literature were examined. The medium containing soybean oil and starch as carbon and energy source gave the best production results. This medium, with the starch replaced by glycerol, and with various soybean oil concentrations (16, 23 and 30 g l )1 ) was utilized to further investigate CA production. Medium containing 23 g l )1 led to the highest CA productivity (722 mg l )1 in 120 h) and that one containing 30 g l )1 gave the highest CA titre (753 mg l )1 in 130 h). Also, substitution of corn and sunflower edible oils furnished similarly good results in terms of CA titre and productivity. It can be concluded that easily available vegetable oil is a very promising substrate for CA production, since it is converted slowly to glycerol and fatty acids, which are the main carbon and energy source for the microorganism. Introduction The use of antibiotics to control infectious diseases is greatly hindered by bacterial resistance. One of the most important resistance mechanisms exhibited by a variety of Gram-positive and Gram-negative bacteria is their ability to produce beta-lactamases, which inactivate penicillins and cephalosporins by hydrolysing their beta-lactam ring. Clavulanic acid, (CA), is a beta- lactam antibiotic with a low antibacterial activity; it is, however, a potent inhibitor of the beta-lactamases produced by many pathogenic microorganisms resistant to beta-lactam antibiotics (Butterworth 1984). The combination of CA with amoxicillin is the most successful example of the use of a beta-lactam antibiotic sensitive to beta-lactamase together with an inhibitor of these enzymes (Mayer & Deckwer 1996). CA is pro- duced industrially by strains of Streptomyces clavulige- rus (Butterworth 1984) in medium containing soybean flour as nitrogen source and soluble starch together with glycerol as carbon and energy sources. S. clavuligerus was first named and described as a new species by Higgens & Kastner (1971) from a Streptomycetes strain isolated from South American soil. These authors performed physiological tests and investigated carbon- utilization patterns and found that, among the 13 carbon sources tested, including glucose, sucrose and fructose, only one, maltose, showed positive utilization. Brown et al. (1976) reported the isolation of CA, and Reading & Cole (1977) described the conditions for the cultivation of the organism and detection and isolation of this novel beta-lactamase inhibitor. Since then much research has been published dealing with the biosyn- thetic pathway, molecular genetics, regulation and physiology of CA production by S. clavuligerus. Ahar- onowitz & Demain (1978) found glycerol to be a substrate for growth and antibiotic production. They studied the effect of increasing glycerol concentration and observed that glycerol was the factor limiting growth in a medium containing L -asparagine as nitro- gen source. Based in this work, Garcia-Dominguez et al. (1989) suggested that S. clavuligerus cells are unable to carry out active transport of glucose. Glycerol has been extensively utilized as the carbon source in this process with CA titres up to 3.25 g l )1 being reported (Mayer & Deckwer 1996; Gouveia et al. 1999; Kim et al. 2001; Chen et al. 2002; Roubos et al. 2002). The role of glycerol as a CA precursor and an inhibitor of CA production rate has been speculated upon (Romero et al. 1984; Chen et al. 2002). According to the review work of Liras & Rodriguez-Garcia (2000), the pathway of CA biosynthesis is now partially understood, with the World Journal of Microbiology & Biotechnology (2005) 21:509–514 Ó Springer 2005 DOI 10.1007/s11274-004-2393-z