Pulsed Addition of Limiting-Carbon During Aspergillus oryzae Fermentation Leads to Improved Productivity of a Recombinant Enzyme Swapnil Bhargava, 1 Kevin S. Wenger, 2 Mark R. Marten 1 1 Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, Maryland 21250; telephone: 410-455-3400; fax: 410-455-1049; e-mail: marten@umbc.edu 2 Novozymes, North America, Inc., Franklinton, North Carolina Received 7 March 2002; accepted 11 September 2002 DOI: 10.1002/bit.10548 Abstract: Fungal morphology in many filamentous fun- gal fermentations leads to high broth viscosity which limits oxygen mass transfer, and often results in reduced productivity. The objective in this study was to determine if a simple, fed-batch, process strategy—pulsed addition of limiting-carbon source—could be used to reduce fun- gal broth viscosity, and increase productivity of an indus- trially relevant recombinant enzyme (glucoamylase). As a control, three Aspergillus oryzae fed-batch fermenta- tions were carried out with continuous addition of limit- ing-carbon. To determine the effect of pulse-feeding, three additional fermentations were carried out with lim- iting-carbon added in 90-second pulses, during repeated five-minute cycles. In both cases, overall carbon feed- rate was used to control dissolved oxygen concentration, such that increased oxygen availability led to increased addition of limiting-carbon. Pulse-fed fermentations were found to have smaller fungal mycelia, lower broth viscosity, and improved oxygen mass transfer. As a re- sult, more carbon was added to pulse-fed fermentations that led to increased enzyme productivity by as much as 75%. This finding has significant implications for the bio- processing industry, as a simple process modification which is likely to cost very little to implement in most production facilities, has the potential to substantially in- crease productivity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 111–117, 2003. Keywords: fed-batch; morphology; filamentous fungi; fermentation INTRODUCTION The world market for industrial enzymes has been estimated to be worth over $1.6 billion, with almost half of these enzymes produced in filamentous fungal fermentations (Mu ¨ller 2001). This is because many strains of filamentous fungi have been classified GRAS (generally regarded as safe), can grow on relatively inexpensive substrates, and can both produce and secrete tremendous amounts of recombi- nant protein (van den Hondel et al., 1992; Ward et al., 1992). Unfortunately, the fungal morphology in many of these fermentations leads to high viscosity that can impede mixing and mass transfer, and ultimately leads to reduced productivity. Despite ongoing study, there has been rela- tively little success in reducing broth viscosity in industrial- scale systems (Olsvik and Kristiansen, 1994). Early ap- proaches involved addition of water or media to dilute the broth (Buckland et al., 1988b; Olsvik and Kristiansen, 1992a; Taguchi and Miyamoto, 1966) or increased agitation to fragment the mycelia (Olsvik and Kristiansen 1992b; Smith et al. 1990; van Suijdam and Metz, 1981); they have not proven to be consistently effective (Olsvik and Kris- tiansen, 1994). More recent attempts to use metabolic en- gineering to modify morphology (McIntyre et al. 2001; Mu ¨ller, 2001) illustrate a clever approach and appear quite promising , but will apparently involve significant genetic manipulation and thus may be difficult to implement. The filamentous fungus Aspergillus oryzae has been used for more than 2000 years for the production of food prod- ucts in Asia (Cook and Campbell-Platt, 1994), and today is widely used in the bioprocessing industry for the production of a variety of both native and recombinant enzymes (Chris- tensen, 1994). In a previous study using A. oryzae, we showed a relatively simple process modification during fed- batch fermentation could be used to reduce fungal broth viscosity (Bhargava et al., 2002). An A. oryzae wild-type strain was grown in pilot-scale fermentors, with either con- tinuous or pulsed addition of glucose. In both pulse-fed and continuously fed batches, the same total amount of glucose was added. We found pulsing had no effect on growth or extracellular (native) protein production, but made a sub- stantial difference in fungal morphology. Fungal elements grown in pulse-fed fermentations were significantly Correspondence to: Mark Marten Contract grant sponsors: National Science Foundation; Novozymes North America, Inc. Contract grant numbers: BES-9876012; BES-9906586 © 2003 Wiley Periodicals, Inc.