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Acremonium chrysogenum is cultured worldwide to produce approximately 2,500 tons of cephalosporin derivates annually (Schmitt et al., 2004) and production of cephalosporin C has doubled several times over the last few decades (Christensen et al., 2000). However, the cost of substrates used in antibiotic production, such as glucose or various plant oils, has increased. Recently, optimization of nutritional factors such as novel carbon sources was reported based on statistical analysis of the production of ß-lactam antibiotics by filamentous fungi (Saudagar and Singhal, 2007), and the development of efficient methods of producing cephalosporin C from low-priced materials using various carbon sources and optimization of parameters has also gained attention (Brakhage et al., 2004). Crude glycerol is created as a byproduct of biodiesel production (Galan et al., 2009); accordingly, the amount of glycerol produced as a result of increased biodiesel production worldwide has increased dramatically during the last 10 years, and the industrial value of glycerol has decreased from approximately 650-700 €ton in 1996 to 300-350 €ton in 2004 and to 150-200 €ton in 2005 (Peters, 2007). Due to this reduction in price, glycerol is now an attractive feedstock for the production of useful chemicals.In this study, the effects of glycerol on cephalosporin C production by Acremonium chrysogenum M35 were evaluated. The addition of glycerol increased cephalosporin production by up to 12-fold. Glycerol caused the upregulation of the transcription of the isopenicillin synthase (pcbC) and transporter (cefT) genes in early exponential phase, and affected the cell morphology since hyphal fragments differentiated into arthrospores. These results indicate that glycerol effectively enhances cephalosporin C production via stimulation of cell differentiation.