Biochemical Engineering Journal 67 (2012) 140–147 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journal journa l h o me pa ge: www.elsevier.com/locate/bej Regular article Enhancement of Dichomitus squalens tolerance to copper and copper-associated laccase activity by carbon and nitrogen sources Ranjani Kannaiyan a , Nader Mahinpey a,∗ , Thilakavathi Mani a , Robert J. Martinuzzi b , Victoria Kostenko c a Chemical and Petroleum Engineering Department, University of Calgary, 2500 University Drive NW, Calgary, T2N 1N4 Canada b Mechanical and Manufacturing Engineering Department, University of Calgary, 2500 University Drive NW, Calgary, T2N 1N4 Canada c Calgary Center for Innovative Technology, University of Calgary, 2500 University Drive NW, Calgary, T2N 1N4 Canada a r t i c l e i n f o Article history: Received 24 March 2012 Received in revised form 30 May 2012 Accepted 12 June 2012 Available online 21 June 2012 Keywords: Copper Tolerance Laccase activity Carbon/nitrogen ratio a b s t r a c t This study revealed a dual effect of copper on D. squalens laccase activity, inducing and suppressing. The addition of copper sulfate led to enhancement of the enzyme activity until it reached a maximum value at peak copper sulfate concentration (PC) followed by suppression and even complete termination of the enzyme activity. Maximum laccase yield is a function of fungi tolerance and media composition. Nutrients had a significant impact on copper-independent and copper-induced laccase activity. Some nutrients also improved tolerance of cells and laccase-producing system to copper load. The effect of specific nutrient depended on the ratio of carbon and nitrogen sources. This research can be used to predict the effective copper concentration and nutrient composition to stimulate the laccase production without compromis- ing the fungi growth. The methylated substrates (3-O-methylglucose, methylcellulose) in combination with casein and copper demonstrated high potential to support fungi tolerance and enhanced laccase activity. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The production of biofuels, e.g. bioalcohols, via the conversion of biomass is a rapidly growing industry. However, current biofuel generation faces supply-limitation pressures since most bioconver- sion technologies rely on the fermentation of ‘food crop’-derived sugars to bioalcohols and, hence, interfere with the food supply chain. An attractive approach is to replace food crops used for bio- fuel generation with lignocellulosic raw materials. Lignocellulosic materials constitute a large portion of the wastes produced by different industries including forestry, pulp and paper, and agri- culture. The basic element of this technology is the hydrolysis of biomass cellulose to sugars with subsequent sugar fermentation to bioalcohol. The utilization of waste cellulose increases the amount of biofuel that can be produced sustainably without competing with the food supply. The principal challenge to the ‘cellulosic’ biofuel generation is the complex structure of lignocellulose which locks the fermentable sugars within the lignin–hemicellulose matrix [1]. Lignin–hemicellulose networks normally protect the embed- ded cellulose from interaction with water, solvents and enzymes, and, hence, prevent cellulose hydrolysis to simple sugars and further fermentation to bioalcohols. Thus, efficient conversion of ∗ Corresponding author at: EEEL 417 B, 2500 University Drive NW, University of Calgary, Calgary AB, T2N 1N4 Canada. Tel.: +1 403 210 6503; fax: +1 403 284 4852. E-mail address: nader.mahinpey@ucalgary.ca (N. Mahinpey). lignocellulosic waste to biofuel requires the pre-removal of lignin and hemicellulose. Chemical delignification, widely used in biofuel generation, is expensive, energy consuming, and results in the release of inhibitors such as weak acids, furan and phenolic compounds which reduce hydrolysis and fermentation rates and overall bio- fuel yield [2]. Alternatively, lignin can be degraded by the action of lignin-degrading enzymes produced by white-rot fungi [3]. This approach is based on naturally occurring delignification and has the advantage of: low-energy demand; minimal waste and toxic com- pounds production; and negligible environmental impact. Some white-rot basidiomycetes such as Dichomitus squalens selectively degrade lignin [4] in a wide range of substrates such as hard or soft wood, and grass [5,6] without removing the cellulose subsequently needed for sugar release and fermentation [7]. The main D. squalens enzyme participating in delignification is laccase (EC.1.10.3.2). The activity of laccase is determined by cop- per, which is the active center and transcriptional regulator of the enzyme [8–10]. However, copper is toxic for fungi at relatively low concentrations and limits fungi growth, and consequently laccase production [11]. On the other hand, some carbon and nitrogen sources have been reported to enhance laccase activity in copper-containing media [12,13]. The mechanism for laccase activity enhancement by media modification is not elucidated. We hypothesize that some carbon and nitrogen sources and their combinations could impact fungi tolerance to copper and, hence, facilitate copper-associated laccase activity. To this end, D. squalens 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.06.007