Research in Microbiology 159 (2008) 103e109 www.elsevier.com/locate/resmic
Melanoidin-containing wastewaters induce selective laccase gene expression in the white-rot fungus Trametes sp. I-62 Tania Gonza´lez1, Marı´a Carmen Terro´n, Susana Yagu¨e, Howard Junca2, Jose´ Marı´a Carbajo3, Ernesto Javier Zapico4, Ricardo Silva5, Ainhoa Arana-Cuenca6, Alejandro Te´llez6, Aldo Enrique Gonza´lez* Department of Molecular Microbiology, Centro de Investigaciones Biolo´gicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Received 24 July 2007; accepted 23 October 2007 Available online 21 November 2007
Abstract Wastewaters generated from the production of ethanol from sugar cane molasses may have detrimental effects on the environment due to their high chemical oxygen demand and dark brown color. The color is mainly associated with the presence of melanoidins, which are highly recalcitrant to biodegradation. We report here the induction of laccases by molasses wastewaters and molasses melanoidins in the basidiomycetous fungus Trametes sp. I-62. The time course of effluent decolorization and laccase activity in the culture supernatant of the fungus were correlated. The expression of laccase genes lcc1 and lcc2 increased as a result of the addition of complete molasses wastewater and its high molecular weight fraction to fungal cultures. This is the first time differential laccase gene expression has been reported to occur upon exposure of fungal cultures to molasses wastewaters and their melanoidins. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Melanoidin; Molasses wastewaters; Laccase; Decolorization; Fungi; Ligninolytic enzymes; Basidiomycete
1. Introduction
* Corresponding author. Tel.: þ34 9 1837 3112x4413/4414; fax: þ34 9 1536 0432. E-mail address: aldo@cib.csic.es (A.E. Gonza´lez). 1 Present address: Departamento de Microbiologı´a, Instituto Cubano de los Derivados de la Ca~ na de Azu´car (ICIDCA), Vı´a Blanca 804 y Carretera Central, San Miguel del Padro´n, Havana, Cuba. 2 Present address: Biodegradation Research Group e GBF, Environmental Microbiology, Mascheroder Weg 1 D-38124, Braunschweig, Germany. 3 Present address: CIFOR-INIA, Crta. de La Coru~na Km. 7, 28040 Madrid, Spain. 4 Present address: Biotechnology Department, Technische Universita¨t Hamburg-Harburg, Biotechnology II, Denickestrasse, 15, 21073 Hamburg, Germany. 5 Present address: Fac. Ciencias Forestales, Dpto. de Ingenierı´a de la Madera, Universidad de Chile, Santa Rosa, 11315 Santiago, Chile. 6 Present address: Departamento Biotecnologı´a, Universidad Polite´cnic de Pachuca, Ex-Hacienda de Santa Ba´rbara Km 20, Carretera Pachuca-Ciudad Sahagun, Zempoala, CP. 43830, Estado de Hidalgo, Mexico. 0923-2508/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2007.10.005
Final effluents produced from alcoholic fermentation of molasses are among the most environmentally harmful wastewaters generated by the sugar and byproduct industries. These effluents, also known as vinasses, contain persistent toxic chemicals which have a harmful impact on aquatic ecosystems, not only by increasing the chemical oxygen demand (COD) but also as a result of their dark brown color. This leads to a reduction in penetration of sunlight in rivers, lakes and lagoons, which in turn decreases oxygenation by photosynthesis and causes multiple damaging effects to aquatic life. The organic matter can be degraded by conventional anaerobic-aerobic treatments, but the colored compounds of molasses effluents appear to be recalcitrant to biodegradation [31]. The characteristic very dark color is mainly due to the presence of melanoidins. These brown polymers, which are formed by amino-carbonyl reactions, are widely distributed in nature and are not readily susceptible to microbial degradation.
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Thus, there is an urgent need to develop alternative biotechnological processes to effectively remove light-absorptive compounds from molasses effluents. White-rot basidiomycetous fungi can degrade lignin and a broad range of environmentally persistent xenobiotics, organopollutants and industrial wastewaters [18,37]. Some of these fungi have also been shown to be effective in the decolorization of natural and synthesized melanoidins and molasses wastewaters (MWWs) [16]. A complex non-specific enzyme system secreted by these organisms has been shown to be associated with their degradative capacities. The ligninolytic system consists of two main groups of enzymes: peroxidases (lignin peroxidases and manganese peroxidases) and laccases [1,2,11,18]. Although the enzymatic system associated with decolorization of melanoidins appears to be related to the presence and activity of fungal ligninolytic mechanisms, this relation is as yet not completely understood [12]. Trametes sp. I-62 (CECT 20197) is a white-rot fungus strain with a high detoxification capacity towards molasses effluents. It also represents a model strain for studying the diversity [1,13,14,20], transcriptional [21,34] and postranslational regulation of laccases in a single organism. Four laccase genes have been reported thus far in this basidiomycete [14,20]. In the present work, we analyzed the relationship between the production of ligninolytic enzymes and decolorization of MWW by Trametes sp. I-62. The effect of molasses effluents and molasses melanoidins on laccase gene transcription was also evaluated.
2.4. Culture conditions Submerged cultures were prepared from 7-day-old cultures of the fungus grown on agar plates with Kirk medium [17]. Eight plugs (1 cm2) were cut and inoculated under sterile conditions in 500 ml culture flasks containing 300 ml of the same growth medium and four 1.5 cm diameter glass beads. They were incubated for 24 h at 28 C in an orbital shaker (100 rpm). A 1:10 (v/v) inoculum was transferred into 250 ml flasks containing 75 ml (total volume) of Kirk medium supplemented with 20% (v/v final concentration) of MWW. Controls were prepared in the same way, except that Kirk medium was not supplemented with the wastewater. Abiotic controls contained Kirk medium and the effluent, but were not inoculated with fungus. Fungal cultures were incubated at 28 C in an orbital shaker (100 rpm) for 16 days. COD, decolorization, lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activity measurements were monitored on a daily basis, in triplicate. To study the effect of both MWW and molasses melanoidins on lcc gene transcription, they were added to 8-day-old fungal cultures in Kirk medium (28 C, 100 rpm) at a final concentration of 0.37 mM total phenols in the culture medium. Laccase activity was monitored over a 43 h period and fresh 10 mg mycelium samples were harvested at different time points (7, 19, 31 and 43 h) following the addition of either effluent or melanoidins to the cultures. 2.5. Analytical methods
2. Materials and methods 2.1. Wastewater The final effluent from the distillation of ethanol produced from sugar-cane molasses was provided by a distillery in Havana, Cuba. MWW are complex organic mixtures including melanoidins, which results in acid dark-brown solutions. The effluent used in the present study has the following physical characteristics: pH 4.0, color units 60,923 100, and a COD of 55.5 1.2 g/l. 2.2. Separation of molasses melanoidins The MWW was centrifuged at 12,000 rpm for 15 min to eliminate suspended solids. The resultant supernatant was dialyzed against running tap water through a 10 kD membrane (Pierce) at room temperature for 2 days, and then against deionized water for another two days. The resulting solution of non-dialyzable compounds was used as a solution of molasses melanoidins. 2.3. Organism Basidiomycete Trametes sp. I-62 (CECT 20197) was isolated from decayed wood in Pinar del Rı´o, Cuba [21]. The fungal culture was maintained on agar plates with Medium-7 [20]. Plates were grown for 7 days at 28 C and stored at 4 C.
Color units and COD were determined according to CPPA [8] and ‘‘Standard methods for the examination of water and wastewater’’ [33], respectively. The concentration of total phenols was determined by the Folin-Ciocalteu method [30] with minor modifications [4] using gallic acid (Sigma Chemicals) as a reference standard. 2.6. Enzyme assays Laccase activity in the culture supernatant was determined by the method of Mansur and coworkers [20] using ABTS (2,20 -azinobis-3-ethylbenzthiazoline-6-sulfonate) as the substrate. Lignin and manganese peroxidases were determined as previously described by Tien and Kirk [35], and Pick and Keisare [27], using veratryl alcohol and phenol red, respectively, as substrates. Enzyme activities were expressed in units defined as 1 mmol product formed per min. 2.7. Total RNA preparation and cDNA synthesis RNA extraction was performed using the Fast RNA kit-Red, following the manufacturer’s instructions (BIO 101). In order to remove contaminating DNA, 1 unit per mg of RNA of RQ1 DNase enzyme (Promega) was added to each RNA sample and subsequently incubated for 30 min at 37 C. First-strand cDNA synthesis was carried out using 2 mg of total RNA as template and the cDNA synthesis kit from Roche.
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2.8. Multiplex PCR reaction Design and optimization of this method have been previously described [13]. Briefly, a PCR mix was prepared by adding together the three pairs of primers used to amplify lcc1, lcc2 and lcc3 (GenBank accession numbers AF548032, AF548034, AF548035, respectively) gene fragments. A total of 100 ml PCR mixtures also contained 5 ml of reverse transcription product, 2.5 U Taq polymerase (Perkin Elmer) and all the rest of the standard components of a PCR DNA amplification reaction [29]. PCR reactions were performed in a Rapidcycler (Idaho Technology) thermocycler. The basic program comprised an initial denaturizing step at 95 C for 1 min followed by 30 cycles of 95 C for 45 s, 30 s at the annealing temperature (59 C) and 72 C for 2 min, one final extension step at 72 C for 7 min and incubation at 4 C until further storage of reactions at 20 C. The same procedure was performed to amplify a fragment of the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase gene ( gpd1, GenBank acc. No. AF297874) from the RT reaction, which was used as a control to normalize differences in the total RNA input or in the reverse transcription reaction efficiencies. The only changes were that amplification was performed for 25 cycles and the annealing temperature was set at 55 C. 2.9. Quantitative and statistical analysis Three independent amplification reactions were performed for each condition assayed. PCR products (10 ml of each reaction) were separated by 1.5% agarose gel electrophoresis and visualized after staining for 10 min in a 1 mg/ml ethidium bromide solution. Densitometric analysis of Polaroid film gel images was performed using ‘‘Image Quant 3.3’’ software (Molecular Dynamics). Levels of lcc mRNAs were expressed in arbitrary units, as the rate between lcc transcript levels (previously normalized according to size differences) and those of gpd1 calculated by the following equation: laccase/ ( gpd1sample/gpd1average). For all experiments and determinations, variability coefficients between triplicate samples were calculated. Statistical differences were determined by the ‘‘t test’’ for mean comparison (with P < 0.001). 3. Results 3.1. Ligninolytic enzyme profile and MWW decolorization In a previous study we reported the optimization of different parameters in submerged cultures of Trametes sp. I-62 in order to attain the maximum reduction in color and COD of the MWW [12]. These optimal parameters were applied here to analyze the relationship between the production of ligninolytic enzymes and effluent decolorization. We monitored culture supernatant for the presence of both lignin peroxidase (LiP) and manganese peroxidase (MnP) activity as well as for laccase activity. Neither LiP nor MnP activities could be detected in the
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cultures with effluent nor in the controls during the 16 days of the experiment, under the assayed conditions. Laccase was the only detectable ligninolytic activity produced under these conditions. Levels of this enzyme in the medium supplemented with effluent were always significantly higher than those of the controls (Fig. 1A). Maximal differences of 13and 19-fold were achieved in the 8- and 16-day cultures, respectively. In contrast, decolorization of 76.9% and a COD reduction of 71% with respect to the initial values were achieved at the end of the experiment (Fig. 1 and data not shown). The time course of effluent decolorization and laccase activity detected in the culture supernatant of Trametes sp. I62 showed a similar trend. Statistical analysis revealed a significant correlation of 96% between the two variables. 3.2. Effect of MWW and of molasses melanoidins on laccase gene transcription The effect of complete MWW and of the high molecular weight fraction (corresponding to melanoidins) upon induction of laccase gene expression was compared. The time course analysis of laccase activity (Fig. 1B) indicated that 30 min after their addition, both the complete effluent and the isolated molasses melanoidins caused an increase in extracellular laccase activity. Although the time course was very similar, the maximal level of activity was slightly higher and more rapidly achieved (after 11 h) in the presence of the melanoidin fraction with respect to those media amended with complete MWW, in which maximal levels were detected after 24 h. Laccase activity was minimal in controls, and no significant changes were produced in these samples during the assay. Both complete molasses effluents and melanoidins selectively induced lcc1 and lcc2 laccase gene transcription (Figs. 1C and 2A,B). However, a higher increase in lcc transcript levels was observed in the presence of the complete molasses effluent, with induction being detected from the first sampling time (30 min) after the addition of the effluent. In contrast, at that time no lcc gene expression could be detected in the media supplemented only with the molasses melanoidins. Maximal induction of lcc1 transcripts was detected 7 h after supplementation with melanoidins and at 19 h following addition of the complete molasses effluent. In terms of lcc2 transcript levels, maximal expression of lcc2 was observed 7 h after the addition of both molasses effluent and melanoidins. However, no lcc3 gene expression could be detected in any of the samples. When the time course of total transcripts was analyzed (Fig. 2C), it could be noted that the highest lcc levels were observed 7 h after the addition of both MWW and melanoidins. However, overall induction of lcc gene expression was initially produced in the presence of the complete MWW and was slightly higher than induction observed with the isolated melanoidins. Nevertheless, the later decrease, after 19 h, was more pronounced in the presence of the complete MWW; indeed, after 31 h, laccase levels were lower than those corresponding to addition of the melanoidins. No lcc gene expression were detected in any of the control samples throughout the course of the experiment.
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Fig. 1. Effect of MWW and molasses melanoidins on Trametes sp. I-62 laccase activity and lcc transcript levels. (A) Biotreatment with the fungus grown in Kirk medium with 20% vinasses for 16 days. Laccase activity in cultures with effluent and corresponding decolorization rate values are represented. Controls were grown on Kirk medium without effluent. (B) Changes in laccase activity after the addition of the complete MWW or the melanoidin fraction to 8-day cultures of Trametes sp. I-62 in Kirk medium. (C) Effect on lcc transcript levels. Amplification of a fragment from the gpd1 gene was used as an internal control for each sample.
4. Discussion The application of basidiomycetous fungi to melanoidin decolorization has been studied for more than 30 years. Various strains can degrade these polymers and decolorize MWW
[12]. Those studies were reviewed by Coulibaly and coworkers [7] and by Pant and coworkers [26]. The results in our work compare with some of the best results reported in relation to the extent of decolorization and COD removal. Paradoxically, the first studies on the enzymatic system involved in these processes did not focus on ligninolytic enzymes. In fact, intracellular sugar oxidase enzymes were considered as having the most important role in decolorization [36]. Miyata and coworkers [23] subsequently proposed the participation of ligninolytic enzymes, particularly peroxidases, in the degradation of melanoidins. In the present work, MnP and LiP do not appear to be involved in decolorization of MWW by Trametes sp. I-62, since neither of these enzymatic activities could be detected under conditions that resulted in maximal color reduction. Laccase was the only enzyme which could be detected at high levels in culture supernatants. Previous studies to detect ligninolytic enzymes in this fungus showed that the major ligninolytic activity in culture supernatants of Trametes I-62 was laccase, in conjunction with small amounts of manganese peroxidase [20]. No lignin peroxidase has been detected thus far in this strain, even in culture conditions that permit the expression of this enzyme in a Trametes versicolor strain used as a control (data not published). A study on decolorization of colored effluents from textile, paper and pulp mill and distillery waste with a marine basidiomicetous fungus has been recently published by D’Souza and coworkers [10]. The authors also report laccase as being the dominant lignin-degrading enzyme, with very low activities of manganese-dependent peroxidase and no lignin peroxidase activity. Although induction of laccase activity in various basidiomycete grown on MWW has previously been reported [12,15] no clear correlation with effluent decolorization was observed. More recently, Rodrı´guez and coworkers [28] suggested an important role for laccases from Pleurotus ostreatus in MWW decolorization, as well as the involvement of other enzymes or mechanisms when nutrient levels become restrictive. On the other hand, D’Souza and co-workers [10] described induction of laccase activity by MWW and decolorization of the effluent with a partially purified laccase preparation. Results presented here strengthen the role of laccases in MWW color reduction, since decolorization correlated directly with laccase activity throughout the experiment. Recent works have focused on the use of different natural or synthetic compounds to induce laccases and to improve their secretion by white-rot fungi [3,15,22], but fewer have determined the inductive effect of these compounds on the expression of laccase genes [5,6,32]. Previous work by our group has shown that lcc gene expression in Trametes sp. I62 is induced by veratryl alcohol, by two of its isomers and by different aromatic monomers [13,21,34]. We now report for the first time that lcc gene expression in this fungus can also be induced by MWW and by molasses melanoidins. Maximal levels of lcc transcripts were detected in the presence of the complete and the high molecular weight fraction of the effluent, and they were similar to those observed following induction of the best aromatic monomers tested
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thus far ( p-coumaric acid, guaiacol and p-methoxyphenol) under identical culture conditions in Trametes sp. I-62 [34]. Nevertheless, both the complete MWW and the dialyzed fraction resulted in a more rapid increase in laccase activity than those obtained with aromatic monomers in the previous study. On the other hand, it would seem contradictory that maximal extracellular laccase levels occurred first in the presence of the isolated melanoidins, even when lcc genes were previously induced by the complete MWW. Various factors would explain these phenomena, considering that the effect of the effluent on laccase activity can occur at a number of different levels to produce more active protein. For instance, it has been reported that humic acids possess surfactant properties, which have been proposed to favor the liberation of enzymes in Trametes versicolor and Phanerochaete chrysosporium [9]. Thus, due to the structural similarity between humic acids and melanoidins, it seems reasonable to suggest that melanoidins play a similar role, resulting in increased release of enzymes from fungus grown on cultures amended by these compounds. In addition, melanoidins, as a result of the presence of phenolic groups in their structure, may also act as laccase stabilizers. Indeed, Mai and coworkers [19] reported enhancement of laccase stability in the presence of phenolics compounds. Prior to the addition of the effluent to Trametes sp. I-62 cultures, low levels of extracellular laccase were measured. If both the afore mentioned factors are considered, namely stimulation of laccase secretion from the fungus coupled with potential stabilization of the enzyme in culture media, then the more marked increment in laccase activity observed in media with isolated melanoidins, which occurs even before detection of lcc gene induction, can be explained. The rapid induction of lcc genes in media with complete molasses effluent may be associated with the presence of low molecular weight compounds that can be easily transported through fungal membranes. The inductive effect of melanoidins at the genetic level may, in fact, be mediated by the action of lower molecular weight compounds derived from their degradation. Another factor to consider is that melanoidins are potent copper chelators. It has been shown that their chromophore groups are related to this property, since liberation of chelated copper was detected when melanoidin degradation occurs as part of the decolorization process [24]. If we consider that laccase genes can be induced by copper [25,32], then degradation of melanoidins could result in induction of lcc gene expression as a consequence of the release of copper into the culture media. These are tentative explanations which require additional studies for confirmation.
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Fig. 2. Changes in the relative transcript levels of lcc1, lcc2 and lcc3 at different times following addition of the complete MWW (A), and of the melanoidin fraction (B) to 8-day old cultures in Kirk medium analyzed by multiplex RTPCR. Each data point represents the mean PCR product yield from two independent amplifications. Arbitrary units express the ratio between lcc transcript levels (normalized according to PCR product size) and those of glyceraldehyde 3-phosphate dehydrogenase ( gpd1). This means: laccase/( gpd1sample/ gpd1average). (C) Total lcc transcript levels calculated from the addition of the relative levels of lcc1, lcc2 and lcc3 mRNA in each sample, at different times, following addition of the complete MWW or the melanoidin fraction.
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In conclusion, results presented in this work indicate a close relationship between decolorization of MWW and selective induction of laccase activity in Trametes sp. I-62. Two laccase genes, lcc1 and lcc2, are overexpressed during the decolorization process. Differential laccase gene expression occurs upon exposure of fungal cultures to both molasses wastewaters and their melanoidins, the high molecular weight fraction of these effluents. These findings could have important implications for a better understanding of molecular processes involved in depollution of distillery industrial effluents. Acknowledgments We are grateful to G. del Solar, M. Espinosa and A. Dobson for their critical reading of the manuscript. We also acknowledge the valuable help of L. Rodo´n and F.J. Carbajo with some of the figures. This work was supported by projects BIO95-2065-E and BIO97-0655 from the Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT, Madrid, Spain). T. Gonza´lez acknowledges support from a Mutis Program doctoral grant from AECI (Spain) as well as from The International Foundation for Science (grant F/3899-1). M.C. Terro´n acknowledges a post-doctoral grant from the Consejerı´a de Educacio´n y Cultura de la Comunidad Auto´noma de Madrid (Spain).
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