Continuous Production of Manganese Peroxidase by Phanerochaete chrysosporium Immobilized on Polyurethane Foam in a Pulsed Packed-Bed Bioreactor M. T. Moreira, G. Feijoo, C. Palma, J. M. Lema Department of Chemical Engineering, University of Santiago de Compostela, E-15706 Santiago de Compostela, Galiza, Spain; telephone: 34-81-563100; fax: 34-81-595012; e-mail: jmlema@usc.es Received 1 November 1996; accepted 7 February 1997 Abstract: The bottleneck of the application of manganese peroxidase (MnP) on an industrial scale in pulp bio- bleaching or in degradation of hazardous compounds is the lack of an efficient production system. Three main problems arise for the continuous production of MnP during secondary metabolism of Phanerochaete chryso- sporium: enzyme production occurs only under specific physiological conditions corresponding to Cor N limita- tion, high O 2 tension, and adequate Mn +2 concentration; the enzyme that is produced is destabilized by extracel- lular proteases; and excessive growth of the mycelium blocks effective oxygen transfer. To overcome these drawbacks, continuous production of MnP was opti- mized by selecting a suitable bioreactor configuration and the environmental and operating conditions affect- ing both enzyme production and stability. The combina- tion between a proper feed rate and the application of a pulsation in a packed-bed bioreactor permitted the main- tenance of continuous secretion of MnP while limiting mycelial growth and avoiding bed clogging. Environ- mental factors as an Mn +2 concentration of 5000 μM and high oxygen tension enhanced MnPproduction. The hy- draulics of the bioreactor corresponding to a plug flow model with partial mixing and an operating hydraulic rentention time of 24 h were optimal to achieve stable operating conditions. This policy allowed long operation periods, obtaining higher productivities than the best re- ported in the literature. © 1997 John Wiley & Sons, Inc. Bio- technol Bioeng 56: 130–137, 1997. Keywords: manganese peroxidase; Phanerochaete chrysosporium; pulsed packed-bed bioreactor INTRODUCTION Ligninolytic enzymes such as lignin peroxidase (LIP), man- ganese peroxidase (MnP), and laccase have been the focus of fundamental research in recent years because of their potential ability to degrade toxic pollutants (Feijoo et al., 1995; Field et al., 1992) and to carry out other environmen- tally clean processes such as biopulping and biobleaching of pulps (Kondo et al., 1994; Paice et al., 1995). Their appli- cations in industrial processes on a large scale requires the use of an efficient production system. Different authors have investigated the role of environ- mental factors, such as C and N regulation (Jeffries et al., 1981; Keyser et al., 1978), oxygen tension (Dosoretz et al., 1990a; Li et al., 1995), and Mn +2 concentration (Bonnarme and Jeffries, 1990) on promoting MnP synthesis. Neverthe- less, when continuous production of MnP during secondary metabolism is attempted, these factors should only be con- sidered as prerequisites. The main objective to be achieved in a continuous operation is the maintenance of a sustained secretion of MnP in steady-state conditions. A second im- portant objective is to avoid extracellular protease secretion that is mainly responsible for destabilization of the pro- duced enzymes (Dosoretz et al., 1990b). As reported for LIP, a residual glucose concentration minimizes extracellu- lar proteases synthesis, thus avoiding enzyme inactivation (Dosoretz et al., 1990b; Dosoretz et al., 1990c). Another key factor related to the proper operation in a bioreactor is to avoid the excessive growth of filamentous fungi that would imply operational difficulties such as fouling of the fer- menter probes and bed clogging. In brief, the aspects that must be taken into account for MnP production in a continuous fashion would comprise: a continuous supply of a suitable medium to keep secondary metabolism conditions necessary for enzymatic production and to avoid secretion of extracellular proteases; high oxy- gen tension; an adequate bioreactor hydraulics; and the con- trol of excessive hyphal growth. Thus, the bioreactor con- figuration should be selected as a compromise solution after analyzing all factors. In a previous work, we successfully achieved a continu- ous production of MnP by free pellets of Phanerochaete chrysosporium in a pulsed-flow expanded-bed bioreactor Correspondence to: J. M. Lema Contract grant sponsor: Spanish Commission of Science and Technol- ogy Contract grant number: BIO95-0377 Contract grant sponsor: European Commission Contract grant number: CI * CT940086 © 1997 John Wiley & Sons, Inc. CCC 0006-3592/97/020130-08