Laccase production by Pycnoporus cinnabarinus grown on sugar-cane bagasse: Influence of ethanol vapours as inducer
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 gethanol 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 gethanol 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.
Section snippets
Fungal strain
The P. cinnabarinus strain used in this study is the monokaryotic strain ss3 obtained from the Banque de Ressources Fongiques de Marseille (BRFM, France). It was isolated from the fruit-like structure of the dikaryotic strain I-937 (Collection Nationale de Culture de Micro-organismes, Institut Pasteur, Paris, France), according to Herpoel et al. [6]. The strain was maintained on malt agar slants at 4 °C.
Media and culture conditions
P. cinnabarinus was obtained from 10-day-old non-agitated precultures grown in Roux flasks
Ethanol adsorption on sugar-cane bagasse
Adsorption of ethanol vapours on sugar-cane bagasse was studied in columns, packed with previously sterilised bagasse. The ethanol outlet concentration reached the inlet value after 5 days showing that adsorption by the wet support material was slow. The quantity adsorbed was obtained by integrating numerically the difference between the measured inlet and outlet concentrations. Wet bagasse presented a high capacity to adsorb ethanol (0.0269 gethanol/gwet bagasse), but dissolution into the
Conclusions
The present study showed that P. cinnabarinus ss3 was able to grow under SSF culture conditions on sugar-cane bagasse for the production of laccase. It was demonstrated that ethanol vapours served as an inducer of laccase. Maximum laccase production was obtained for an ethanol concentration about 7 g m−3. Under these conditions, laccase activity rose over the first 14 days and then remained steady over a long period (15 days). In order to obtain the highest laccase production, ethanol has to be
Acknowledgements
Juan Carlos Meza is grateful to CONACyT (Consejo Nacional de Ciencia y Technologia, Mexico) for providing his doctoral financial support. Authors wish to thank Roland Ponge, Lionel Merino (Ecole Supérieure des Ingénieurs de Luminy, Marseille) and David Navarro (UMR 1163, INRA Marseille) for their technical support.
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