Methodological Advances Filter centrifugation as a sampling method for miniaturization of extracellular fungal enzyme activity measurements in solid media J. HEINONSALO a , G. KABIERSCH a, *, R.M. NIEMI b , S. SIMPANEN b,c , H. ILVESNIEMI d , M. HOFRICHTER e , A. HATAKKA a , K.T. STEFFEN a a Viikki Biocenter, Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland b Finnish Environment Institute, Research Programme for Biodiversity, P.O. Box 140, FIN-00251 Helsinki, Finland c Department of Environmental Sciences, University of Helsinki, Niemenkatu 73, 15140 Lahti, Finland d Finnish Forest Research Institute, Vantaa Research Station, P.O. Box 18, FIN-01301 Vantaa, Finland e Department of Environmental Biotechnology, International Graduate School of Zittau, Markt 23, 02763 Zittau, Germany article info Article history: Received 16 December 2010 Revision received 12 May 2011 Accepted 20 July 2011 Available online 21 September 2011 Corresponding editor: Petr Baldrian Keywords: Enzyme extraction Fungi Peroxidase Sampling method Screening abstract A novel sampling method to evaluate extracellular fungal enzyme activities was developed and the validity tested for agar media. The method is based on centrifugation of small agar pieces taken from growing fungal solid-state cultures. Centrifuge tubes that allow spinning liquid out from small samples containing, for example, the hyphal front of a growing mycelium are essential for the protocol. Centrifugation recovers a liquid phase from the samples, which contains soluble material including many enzymes. The recovery of two added model enzymes, namely laccase and manganese peroxidase (MnP), from agar media was sufficient (ranging from 50 % to 75 %) but the addition of humic material into agar decreased the observed MnP activity significantly to approx. 25 % of the stock solution. Using growing cultures, the presence of humus as well as Scots pine sawdust on Hagem’s agar plates induced the production of laccase and peroxidase in certain fungi, which indicates that the method is suitable for screening enzyme activities on different growth media or with variable additives or growth conditions. The use of the presented sampling method for functional enzyme fingerprinting of different fungi may be a promising tool for investigating the behaviour and ecological role of forest soil fungi. This method also allows obtaining spatial data from very small and defined areas of solid fungal cultures, e.g. from microcosms. ª 2011 Elsevier Ltd and The British Mycological Society. All rights reserved. Introduction Analysis of enzymatic activities in samples obtained from solid media or solid environmental samples usually requires the addition of water and/or buffer. This may lead to the dilution of enzymes below the detection limit. As a conse- quence, additional laborious and costly working steps to concentrate the sample may be required. A common way to extract enzymes from solid media is to add buffer (e.g. 50 mM sodium acetate, pH 5.0) or water, soak and shake the samples * Corresponding author. Tel.: þ358 9 191 59 318; fax: þ358 9 191 59 322. E-mail address: grit.kabiersch@helsinki.fi (G. Kabiersch). available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/funeco 1754-5048/$ e see front matter ª 2011 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2011.07.008 fungal ecology 5 (2012) 261 e269