Fungal Morphology and Fragmentation Behavior in a Fed-Batch Aspergillus oryzae Fermentation at the Production Scale Zheng Jian Li, 1 Vivek Shukla, 1 Andrew P. Fordyce, 2 Annemarie Gade Pedersen, 2 Kevin S. Wenger, 3 Mark R. Marten 1 1 University of Maryland, Baltimore County (UMBC), Department of Chemical and Biochemical Engineering, 1000 Hilltop Circle, Baltimore, Maryland 21250; telephone (410) 455-3439; fax (410) 455-1049; E-mail: marten@umbc.edu 2 Novo Nordisk A/S, Enzyme Research, Bagsvaerd, Denmark 3 Novo Nordisk BioChem North America, Inc., Franklinton, North Carolina Received 22 November 1999; accepted 14 May 2000 Abstract: It is well known that high-viscosity fermenta- tion broth can lead to mixing and oxygen mass transfer limitations. The seemingly obvious solution for this problem is to increase agitation intensity. In some pro- cesses, this has been shown to damage mycelia, affect morphology, and decrease product expression. How- ever, in other processes increased agitation shows no effect on productivity. While a number of studies discuss morphology and fragmentation at the laboratory and pi- lot scale, there are relatively few publications available for production-scale fungal fermentations. The goal of this study was to assess morphology and fragmentation behavior in large-scale, fed-batch, fungal fermentations used for the production of protein. To accomplish this, a recombinant strain of Aspergillus oryzae was grown in 80 m 3 fermentors at two different gassed, impeller power-levels (one 50% greater than the other). Impeller power is reported as energy dissipation/circulation func- tion (EDCF) and was found to have average values of 29.3 ± 1.0 and 22.0 ± 0.3 kW m –3 s -1 at high and low power levels, respectively. In all batches, biomass concentra- tion profiles were similar and specific growth rate was < 0.03 h -1 . Morphological data show hyphal fragmenta- tion occurred by both shaving-off of external clump hy- phae and breakage of free hyphae. The fragmentation rate constant (k frag ), determined using a first-order model, was 5.90 and 5.80 h -1 for high and low power batches, respectively. At the end of each batch, clumps accounted for only 25% of fungal biomass, most of which existed as small, sparsely branched, free hyphal ele- ments. In all batches, fragmentation was found to domi- nate fungal growth and branching. We speculate that this behavior was due to slow growth of the culture during this fed-batch process. © 2000 John Wiley & Sons, Inc. Bio- technol Bioeng 70: 300–312, 2000. Keywords: filamentous fungi; fermentation, morphol- ogy; fragmentation; agitation INTRODUCTION Filamentous fungi offer many benefits over other types of cells when used for the production of chemicals and phar- maceuticals and, as a result, are used to produce products comprising a large fraction of the world’s pharmaceutical and biotechnology market (Arora et al., 1992). The wide- spread use of fungi in biotechnology stems from the unique ability of fungi to economically produce many different types of products, including some of the most important antibiotics (e.g., penicillins and cephalosporins; Masurekar, 1991; Rambosek, 1991), commodity chemicals (e.g., citric acid; Zidwick, 1991), and commercial enzymes (e.g., pro- teases and amylases; Bigelis, 1991; Arora et al., 1992). For the production of enzymes and therapeutic proteins, fungi offer several advantages over other types of cells. First among these is the large amount of protein (greater than 50 g/L in some cases) fungi are able to both produce and se- crete to the growth medium. In addition, fungi generally exhibit correct glycosylation/posttransitional modification of proteins, are often classified as GRAS (generally re- garded as safe) by the FDA (Food and Drug Administra- tion), and can be grown on a wide variety of inexpensive substrates. In addition, recent improvements in host cells and expression vectors enable filamentous fungi to be con- sidered as a practical alternative to bacteria, yeast, insect, or mammalian expression systems for production of heterolo- gous gene products (Fowler and Berka, 1991). While filamentous fungi have been used for many years to produce a vast array of products, many fungal fermenta- tions suffer from the same problem—high broth viscosity that often leads to oxygen mass-transfer limitations (Morris et al., 1973; Metz et al., 1979; Atkinson and Mavituna, 1991; McNeil and Harvey, 1993; Olsvik and Kristiansen, 1994). In a mechanically agitated tank, one frequently used method to overcome oxygen limitations is simply to in- crease impeller power, thus increasing oxygen mass- Correspondence to: M.R. Marten Contract grant sponsor: the National Science Foundation Contract grant number: BES-9876012 © 2000 John Wiley & Sons, Inc.