Laccase production by Pycnoporus cinnabarinus grown on sugar-cane bagasse: Influence of ethanol vapours as inducer

Abstract

The influence of gaseous ethanol addition as inducer of laccase produced by Pycnoporus cinnabarinus ss3 in a biofilter packed with sugar-cane bagasse was investigated. Gaseous-ethanol concentration up to 13.3 g m⁻³ allowed an important increment of laccase activity. Maximum of laccase production of 90 U g⁻¹ dry support, which was 45 times higher than that without ethanol, was obtained for a concentration of about 7 g_ethanol m⁻³ (ethanol concentration in the liquid phase of 31 g L⁻¹). At this concentration, the maximum laccase activity was reached after 14 days, and then remained steady for 15 days. For other gaseous-ethanol concentrations, enzyme activity reached a maximum after 6–9 days, and then decreased. In spite of the inhibitory effect of ethanol on P. cinnabarinus ss3 growth, it provides an opportunity to improve the production of laccases. It is noteworthy that to obtain highest laccase production, ethanol vapours have to be added at the beginning of the experiment at a low air flow-rate of 40 mL min⁻¹. A gradient of laccase activity down along the column was observed and correlated with gaseous-ethanol concentrations.

Introduction

Laccases (EC 1.10.3.2) are copper-containing oxidases that catalyse the oxidation of phenolic compounds by molecular oxygen. They are able to delignify wood pulp [1], decolorize and detoxify effluents generated by the pulp and paper industry and degrade toxic environmental pollutants such as benzoprenes, dioxins and various xenobiotics [2]. Laccases are excreted by most of the basidiomycetes that cause white rot of wood. Among basidiomycetes, Pycnoporus cinnabarinus was reported to produce laccase as the predominant lignolytic enzyme [3]. This white-rot fungus has been used in the decolorization of dyes [4], in soil bioremediation [5] and for its lignolytic potential in the sequential treatment of wheat-straw chemical pulp [6]. Addition of inducers such as phenolic substrates (ferulic acid, 2,5-xylidine and veratryl alcohol), alcohols (ethanol and methanol) and sulphones (dimethylsulphoxide) with Trametes versicolor [2] and P. cinnabarinus submerged cultures, was shown to stimulate laccase production [3], [6], [7], [8]. As with inducers such as 2,5-xylidine and veratryl alcohol, the addition of 40 g_ethanol L⁻¹ resulted in a 20-fold increase of laccase production in the case of Trametes versicolor submerged cultures [2]. Laccase production by P. cinnabarinus increased four times after addition of 3% of dimethylsulphoxide (DMSO) up to 76,300 U L⁻¹, but only twice with methanol [8].

In the presence of 35 g_ethanol L⁻¹, an even higher level of laccase activity (266,600 U L⁻¹, equivalent productivity of 19,000 U L⁻¹day⁻¹), was found in P. cinnabarinus submerged cultures [7]. This amount was 155-fold higher than with non-ethanol induced cultures. In comparison with other inducers, the use of ethanol is attractive at a pilot scale to produce large quantity of laccases since it is much cheaper and environmentally safer than every other compound.

Most laccase is produced by liquid-substrate fermentation (LSF), which is the main process employed in pharmaceutical and food industries. Solid-substrate fermentation (SSF) is an alternative for enzyme production. It is defined as a cultivation allowing micro-organisms to growth on and in a moist water-insoluble solid material with a lack of free water. In some cases, SSF is most suitable for ligno-cellulosic enzyme production, including laccases [9]. A complete review of enzyme production by SSF can be found in literature [9]. The production of laccase by Pleurotus pulmonarius was studied in SSF using wheat-bran as a substrate [10]. This allowed high level of laccase production because of the presence of phenolic compounds (ferulic, vanillic and p-coumaric acids) in wheat-bran. Bagasse, an agricultural residue of the sugar industry, has also been used for cultivating several fungal strains to produce enzymes [9]. Maximum laccase production of 3.1 U g⁻¹ dry support by Pleurotus ostreatus in solid-state cultivation on sugar-cane-bagasse wheat-bran medium was obtained in the presence of 20 μmol g⁻¹ of veratryl alcohol [11].

Enzymic inducer or carbon source is generally added to the medium (liquid or solid) in liquid form. Very few studies have been focused on the use of volatile organic compounds as enzymic inducer or carbon source. In a biofilter packed with porous ceramic Rashig rings and inoculated with a methylotrophic yeast Pichia pastoris BN21, a strain previously transformed to produce a fungal laccase, methanol was used as carbon source to produce laccase [12]. The objective was to mineralise gaseous methanol and produce laccase simultaneously. For the duration of the experiment, laccase production was generally well correlated with the elimination capacity of the biofilter. A packed-bed reactor, filled with sugar-cane bagasse and inoculated with Candida utilis, was used to eliminate ethanol vapours [13]. In this case, ethanol served also as carbon source to investigate the potential of the yeast-enriched bagasse as food.

It is hypothesised that the aerial mycelia of fungi, which are in direct contact with gas, give a large superficial area and allow a direct mass transfer of the volatile compound. To our knowledge, only one work concerning the addition of a volatile compound to produce enzymes by yeast in SSF has been published [12]. The influence of gaseous-ethanol addition (as enzymic inducer) on the activity of laccase produced by P. cinnabarinus ss3 was studied in a biofilter packed with sugar-cane bagasse. The activity of laccase was studied regarding the optimum ethanol concentration.