Effect of Medium Composition on the Production of Tetanus Toxin by Clostridium tetani Fernando Fratelli,* ,† Tatiana Joly Siquini, † Sally Mu 1 ller Affonso Prado, † Hisako Gondo Higashi, † Attilio Converti, § and Joa ˜ o Carlos Monteiro de Carvalho ‡ Division of Technology and Production Development of Institute Butantan, Av. Vital Brazil, 1500, 05504-900 Sa ˜ o Paulo-SP, Brazil, Department of Chemical and Process Engineering “G.B. Bonino”, University of Genoa, Italy, and Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of Sa ˜ o Paulo, Brazil The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G 0 ) and N-Z Case TT (NZ 0 ) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 L f /mL), achieved at G 0 ) 9.7 g/L and NZ 0 ) 43.5 g/L, was 80% higher than that obtained with standard cultivations (G 0 ) 8.0 g/L and NZ 0 ) 25.0 g/L). Introduction Tetanus is a devasting disease of muscle spam and autonomic instability with a high mortality (1), whose first medical description can be encountered even in the works of Hippocrates (2). Although its incidence is still uncertain, it can be estimated to be in the range of 400,000-1,000,000 cases per year, mainly of neonatal tetanus (3, 4). Almost no evidence exists to support any therapeutic intervention in tetanus (4); nevertheless, it is one of the few human diseases that can be nearly completely controlled by vaccination, the absence of which constitutes a strong risk factor (1). In Brazil, vaccination against tetanus is part of the National Program of Immunization. Great attention has been paid to toxological and immunologic studies on applications of different toxins. However, there are only a few recent references in the scientific literature on Clostridium tetani cultivation for the production of tetanus toxin as precursor of tetanus toxoid. This means that although such a microorganism has been largely utilized by private enterprises mainly to produce antitetanic vaccine for animal use, the results of these studies, devoted to increase in the production capacity, were only scarcely published to preserve the industrial secret. Nevertheless, the production of tetanus toxin is under investigation mainly in academic labora- tories and governmental institutions. For example, De Luca et al. (5) have developed a system for stripping the flue fermentation gases by nitrogen supply, which led to significant increase in tetanus toxin activity. Recently, attempts have also been made toward purification of the tetanus toxin (6) and tetanus toxoid (7). There are several parameters impacting on the micro- bial cultivation performance, among which is the com- position of the medium, which is as a crucial factor for the success of any fermentation process (8). In particular, the carbon source plays a fundamental role in the formulation of any cultivation medium, because it is usually the factor limiting microbial growth and provides either the energy for the anabolic activities and cell maintenance or the carbon for the synthesis of bio- molecules, including peptides. A further element of great significance in the formulation of cultivation media is nitrogen, which provides the amino group for compounds such as nucleic acids and amino acids; therefore, it actually takes part in cell growth and peptide synthesis. When using complex organic nitrogen sources, these are commonly able to provide the cultivation medium not only with nitrogen but also with vitamins, amino acids, and salts. Although C. tetani was shown to grow well on thio- glycolate-containing medium, which is usually employed for anaerobics (2), this medium proved to be inadequate for the step of toxin production, therefore it has only been used for strain propagation (9), including recovery of the lyophilized cells (5). The media employed up to 1962 in the step of toxin production contained hearth infusion as an essential factor, which however contained several contaminating proteins. This bottleneck was overcome by the use of a pancreatic digest of casein; the inclusion of different nutrients in the basic medium formulation, including amino acids, uracil, vitamins, and inorganic salts (10); and adjustment of the level of reduced iron. Mueller and Miller (11) had previously demonstrated the significance of other treatments in the preparation of the * To whom correspondence should be addressed. Fax: ++55-11-37267222, r. 2068. E-mail: fratelli@butantan.gov.br. † Division of Technology and Production Development of Insti- tute Butantan. § University of Genoa. ‡ University of Sa ˜ o Paulo. 756 Biotechnol. Prog. 2005, 21, 756-761 10.1021/bp049571b CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/25/2005