Manganese peroxidase production by Bjerkandera sp. BOS55 1. Regulation of enzymatic production M. T. Moreira, G. Feijoo, J. M. Lema Abstract The white-rot fungus Bjerkandera sp. BOS55 has been suggested as a good alternative for the production of ligninolytic enzymes, specially Manganese peroxidase MnP), by its potential ability to degrade complex com- pounds. However, the application of this fungus requires the complete knowledge of the fermentation pattern in submerged cultures, conditions similar to those existing in industrial size reactors. For this purpose, the nutritional and environmental factors enabling high ligninolytic ac- tivity were studied. According to the results, under limi- tation and suf®ciency of nitrogen, there is a threshold concentration for nitrogen from which MnP is produced. However, under nitrogen excess, the ligninolytic stage of the fungus was coincident with growth, with no apparent substrate limitation according to existing levels of carbon and nitrogen. Concerning carbon concentration, MnP synthesis took place independently of glucose concentra- tion, this indicating that carbon limitation does not seem to be the triggering factor for MnP secretion. Other two environmental factors were studied: oxygenation and agi- tation, but no signi®cant effect on MnP production was observed, a quite different aspect from the behaviour of other known fungi like Phanerochaete chrysosporium. 1 Introduction The importance of lignin-degrading fungi and their lig- ninolytic enzymes has been related to their possible ap- plication in biotechnological processes of environmental concern, acting as natural oxidants of recalcitrant com- pounds. The most ubiquitous peroxidase in most white-rot fungi corresponds to manganese peroxidase MnP), ®rst described by Gold and Glenn 1988). Production of MnP has been considered as an event associated to secondary metabolism conditions for many white-rot fungi. This fact implies that its potential application has been hindered by the low production achieved in batch cultures. This drawback can be partially overcome by selecting white-rot fungi with ability to overproduce enzymes during primary metabolism or in nutrient rich medium. Bjerkandera sp. BOS55 was reported to express high ligninolytic activity under non-nitrogen limiting condi- tions in static cultures [2]. It has also shown high ability to the degradation of some toxic PAH compounds [3], bio- bleaching of kraft pulp [4] and detoxi®cation of highly- coloured ef¯uents [5]. These features suggest this strain for further applications at a larger scale and make it in- teresting to scale-up ligninolytic production. To achieve this objective, however, it is necessary to know about its growth and substrate utilisation in submerged agitated cultures, as an initial step to larger scale fermentations. Therefore, the regulation of MnP activity under different nutritional conditions and environmental conditions was evaluated in batch experiments. These conditions com- prised different nitrogen and carbon concentrations, ox- ygenation supply as well as agitation rate. The analysis of the obtained data in shaken ¯ask cultures will give insight into the main variables in the fermenter operation, such as nutrient concentrations and degree of aeration and agita- tion, which would directly affect enzymatic regulation, fungal growth and morphology and rheological properties of the broth. 2 Materials and methods 2.1 Microorganism Bjerkandera sp. strain BOS55 was isolated and determined as described before [6] and it was deposited as ATCC 90940 in the American Type Culture Collections Rock- ville, MD). It was cultured in malt extract plates per litre, 15.0 g of agar, 3.5 g of malt extract, 10.0 g of glucose) at 30 °C for 5 days, from which ®ve 6 mm agar plugs were transferred to Fernsbach ¯asks. 2.2 Culture conditions The inoculum was prepared by homogenising 5 day cul- tures grown in a 1-l Fernsbach with 100 ml of the prein- oculum medium. Pellets were formed in 250 ml Erlenmeyer ¯asks containing 90 ml of the culture medium and 10% vol/vol) homogenised mycelium. The cultures were incubated in an orbital shaker New Brunswick Sci- enti®c, Innova 4000) at 30 °C and 150 rpm. In the exper- iment with oxygen, the headspace was aseptically ¯ushed with O 2 gas 0.8 bar manometric pressure) for 3 min at the Bioprocess Engineering 23 2000) 657±661 Ó Springer-Verlag 2000 657 Received: 20 December 1999 M. T. Moreira, G. Feijoo, J. M. Lema &) Department of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, E-15706 Santiago de Compostela, Spain This work was funded by the Spanish Commission of Science and Technology CICYT), Project BIO98-610 and 1FD97-0854).