ARTICLE An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall-Degrading Enzyme Activity of Fungal Culture Extracts Brian C. King, 1 Marie K. Donnelly, 2 Gary C. Bergstrom, 1 Larry P. Walker, 2 Donna M. Gibson 1,3 1 Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York 14853; telephone: 607-255-2359; fax: 607-255-2739; e-mail: dmg6@cornell.edu 2 Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853 3 USDA Agricultural Research Service, Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 Received 30 June 2008; revision received 25 August 2008; accepted 22 September 2008 Published online 29 September 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22151 ABSTRACT: Developing enzyme cocktails for cellulosic bio- mass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of poly- saccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethyl- cellulose, and arabinoxylan was quantified using a minia- turized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bio- prospecting for new sources of novel enzymes for biofuel production. Biotechnol. Bioeng. 2009;102: 1033–1044. ß 2008 Wiley Periodicals, Inc. KEYWORDS: bioethanol; biomass conversion; cellulase; cell wall hydrolysis; high throughput screening; plant pathogens Introduction Hydrolysis of the plant cell wall polysaccharides cellulose and hemicellulose to fermentable sugar monomers is a critical step in the conversion of lignocellulosic material to ethanol (Jørgensen et al., 2007; Merino and Cherry, 2007). The most effective means of lignocellulosic hydrolysis uses a cocktail of different cellulolytic enzymes, but conversion is still not optimal (Berlin et al., 2007; Cherry and Fidantsef, 2003; Irwin et al., 1993; Jeoh et al., 2002; Kabel et al., 2006). There is a need to develop more effective cocktails with a range of properties complementary to current cellulase systems, at a significantly reduced cost. Multiple reports have underscored this necessity in order to reduce ethanol production costs from 10–25 cents to 3–5 cents per gallon (Gray et al., 2006; Jørgensen et al., 2007; Lin and Tanaka, 2006; USDOE, 2004, 2005, 2006; Zhang et al., 2006). New cocktails must be adaptable to a wide range of cellulosic feedstocks, from dedicated energy crops to agricultural and forestry residues, depending on regional feedstock avail- ability. A diverse library of cellulolytic enzymes will allow development of cocktails specific to any available feedstock. Trichoderma reesei (teleomorph ¼ Hypocrea jecorina) is the major industrial source for cellulases and hemicellulases. However, genome sequencing and analysis have revealed Brian C. King and Marie K. Donnelly contributed equally to this work. Correspondence to: D.M. Gibson Contract grant sponsor: Cornell University Contract grant sponsor: Department of Plant Pathology and Plant-Microbe Biology Contract grant sponsor: USDA-ARS Contract grant sponsor: New York State Foundation for Science, Technology and Innovation (NYSTAR) ß 2008 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 102, No. 4, March 1, 2009 1033