Decolorization of salt-alkaline effluent with industrial reactive dyes by laccase-producing Basidiomycetes strains.

The discharge of highly coloured synthetic dye effluents into rivers and lakes is harmful to the water bodies, and therefore, intensive researches have been focussed on the decolorization of wastewater by biological, physical or chemical treatments. In the present study, 12 basidiomycetes strains from the genus Pleurotus, Trametes, Lentinus, Peniophora, Pycnoporus, Rigidoporus, Hygrocybe and Psilocybe were evaluated for decolorization of the reactive dyes Cibacron Brilliant Blue H-GR and Cibacron Red FN-2BL, both in solid and liquid media. Among the evaluated fungi, seven showed great ability to decolorize the synthetic textile effluent, both in vivo (74-77%) or in vitro (60-74%), and laccase was the main ligninolytic enzyme involved on dyes decolorization. Pleurotus ostreatus, Trametes villosa and Peniophora cinerea reduced near to 60% of the effluent colour after only 1 h of treatment. The decolorization results were still improved by establishing the nitrogen source and amount to be used during the fungal strains cultivation in synthetic medium previous their action on the textile effluent, with yeast extract being a better nitrogen source than ammonium tartarate. These results contribute for the development of an effective microbiological process for decolorization of dye effluents with reduced time of treatment.

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products.

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of α-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

Extraction of manganese peroxidase produced by Lentinula edodes.

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to LiP of Phanerochaete chrysosporium and a total inhibition of LiP was observed.

Response of Wolfiporia cocos to iron availability: alterations in growth, expression of cellular proteins, Fe3+-reducing activity and Fe3+-chelators production.

AIMS: The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability. METHODS AND RESULTS: W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe3+-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe3+-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible. CONCLUSIONS: W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.

Degradation of eucalypt waste components by Lentinula edodes strains detected by chemical and near-infrared spectroscopy methods.

There are many changes, both qualitative and quantitative, in eucalypt waste during growth and fructification of Lentinula edodes. Wet chemical analysis and near-infrared (NIR) spectroscopy were used in conjunction with multivariate regression and principal components analysis to monitor biodegradation of eucalyptus waste during growth of several L. edodes strains. Weight and component losses of eucalypt residue after biodegradation by L. edodes strains were compared for periods of 1 to 5 mo. Decrease in cellulose, hemicellulose, and lignin contents occurred, however it was not concomitant. Measurement of lignin degradation by NIR and wet chemical analysis indicated its attack in the early stages of biodegradation. Selective lignin degradation by L. edodes was observed up to 2 mo of biodegradation for strains DEBIQ and FEB-14. One group of degraded substrate was identified based on the principal component analysis (PCA) of the data on their biodegradation time. Samples treated for 5 months by L. edodes strains (DEBIQ, UFV or FEB-14) differed from other, but no discrimination was observed among them. By the end of 5 mo, NIR analyses showed decrease of about 18-47% cellulose, 35-47% polyose and 39-60% lignin. These data were used for comparison with those obtained by wet chemical method for the degradation of the substrate by other five L. edodes strains cultivated at the same conditions. NIR calibration developed in this study was proven to be perfectly suitable as an analytical method to predict the changes in lignocellulose composition during biodegradation.

Application of statistical experimental design to the treatment of bleaching kraft mill effluent using a mediated free radical system.

The aim of this work was to evaluate the effect of chelator mediated-Fenton reaction (CMFR) on the chemical oxygen demand (COD) removal from bleaching kraft mill effluent. Effluent treatments were carried out to study the effect of the chelator 3, 4-dihydroxyphenylacetic acid (DOPAC), Fe3+ and H2O2 concentrations on COD removal. For optimization of COD removal, the methodology of statistical experimental design was employed. The estimated second-order polynomial multiple regression model predicts a maximum COD removal of 75.5% (COD/COD0 = 0.245) at the confidence level of 95%. Analysis of variance (ANOVA) showed a good coefficient of determination (R2) value of 0.90, thus ensuring a satisfactory adjustment of the second-order regression model with the experimental data. Indeed, CMFR proved to be a potential process to treat industrial effluents characterized by its high COD.

Metal-chelating compounds produced by ectomycorrhizal fungi collected from pine plantations.

AIMS: To investigate the in vitro production of metal-chelating compounds by ectomycorrhizal fungi collected from pine plantations in southern Chile. METHODS AND RESULTS: Scleroderma verrucosum, Suillus luteus and two isolates of Rhizopogon luteolus were grown in solid and liquid modified Melin-Norkans (MMN) media with and without iron addition and the production of iron-chelating compounds was determined by Chrome Azurol S (CAS) assay. The presence of hydroxamate and catecholate-type compounds and organic acids was also investigated in liquid medium. All isolates produced iron-chelating compounds as detected by CAS assay, and catecholates, hydroxamates as well as oxalic, citric and succinic acids were also detected in all fungal cultures. Scleroderma verrucosum produced the greatest amounts of catecholates and hydroxamates whereas the highest amounts of organic acids were detected in S. luteus. Nevertheless, the highest catecholate, hydroxamate and organic acid concentrations did not correlate with the highest CAS reaction which was observed in R. luteolus (Yum isolate). CONCLUSIONS: Ectomycorrhizal fungi produced a variety of metal-chelating compounds when grown in liquid MMN medium. However, the addition of iron to all fungi cultures reduced the CAS reaction, hydroxamate and organic acid concentrations. Catecholate production was affected differently by iron, depending on the fungal isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: The ectomycorrhizal fungi described in this study have never been reported to produce metal-chelating compound production. Moreover, apart from some wood-rotting fungi, this is the first evidence of the presence of catecholates in R. luteolus, S. luteus and S. verrucosum cultures.

Purification and properties of a xylanase from Ceriporiopsis subvermispora cultivated on Pinus taeda.

The production of hemicellulose and cellulose degrading enzymes by the white-rot fungus Ceriporiopsis subvermispora was determined while growing in Pinus taeda wood chips. Enzymes produced by the fungus were extracted after 30 days of cultivation and at least two different xylanases were secreted. An endo-(1,4)-beta-xylanase was purified by means of ultrafiltration, anion exchange chromatography and gel filtration. Its molecular mass was 29 kDa and the pH and temperature optima were 5.0 and 60 degrees C, respectively. The endo-xylanase was able to hydrolyze xylan to principally xylotriose and xylotetraose and it has different activities against different xylans. With birchwood xylan as substrate, the enzyme showed a K(m) of 1.93 mg/ml and specific activity of 538 units/mg protein at 50 degrees C.

Effect of cereal brans on Lentinula edodes growth and enzyme activities during cultivation on forestry waste.

AIMS: To develop strategies for increasing the growth of Lentinula edodes in eucalyptus residues. To this end, we have examined the effects of cereal brans additions on production of mycelial biomass and enzymes. METHODS AND RESULTS: Three isolates of the mushroom shiitake, L. edodes (Berk. Pegler), were evaluated for enzyme and ergosterol production on eucalyptus residue supplemented with 5, 10, 15 and 20% (w/w) of soya, wheat or rice brans. Nitrogen imput on eucalyptus residues accelerated mycelial growth by supplying the L. edodes with this limiting nutrient. High levels of enzymes activities were produced in eucalyptus residues supplemented by soya bran. Comparison of cellulose and xylanase production with manganese peroxidase (MnP) at 20% soya bran indicated that hydrolytic enzymes, but oxidative enzymes were reduced. CONCLUSIONS: Mycelial growth measurements revealed that eucalyptus residues supplemented with cereal brans supported fast growth of L. edodes, indicating that mycelium extension is related to the bioavailability of nitrogen. The type and concentration of nutrient supplement has a considerable effect both on substrate colonization and on the type of hydrolytic and oxidative enzymes produced. These characteristics may be useful for mushroom growing. SIGNIFICANCE AND IMPACT OF THE STUDY: Lentinula edodes is commercially important for edible mushroom production and supplements which enhance growth and enzymes production might also be beneficial for mushroom yields.

Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus.

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.

Evaluating the basidiomycetes Poria medula-panis and Wolfiporia cocos for xylanase production.

Xylanase, oxidative enzymes and iron-binding compounds were detected in the filtrates of Wolfiporia cocos and Poria medula-panis grown in wheat bran liquid medium. Xylanase and iron-binding compounds were produced at high levels by the brown-rot fungus (BR) W. cocos and at low levels by the white-rot fungus (WR) P. medula-panis. Phenoloxidase was produced only by P. medula-panis. Polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) showed a wide variety of bands for extracellular proteins produced by W.cocos, with low molecular weight (<30 kDa) and minor bands with molecular weight above 45 kDa. Two bands with xylanase activity derived from W. cocos extracts were detected in the gels, whereas many different bands with xylanase activity were found in the extracts from P. medula-panis. P. medula-panis is a selective lignin degrader, whereas W. cocos preferentially removes cellulose from wood.

A new xylanase from a Trichoderma harzianum strain.

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45 degrees C and was stable at 50 degrees C and pH 5.0 up to 4 h xylanase. XYL2 had a low Km with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains.

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28 degree C in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ss-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20. 45 cp. The efficiency of delignification was 33%.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28oC in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ß-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20.45 cp. The efficiency of delignification was 33

Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay.

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.

Screening of variables in xylanase recovery using BDBAC reversed micelles.

Xylanase recovery by reversed micelles using cationic surfactant (N-benzyl- N-dodecyl-N-bis(2-hydroxyethhyl)ammonium chloride) BDBAC was evaluated under different experimental conditions. A full factorial design with center point was employed to verify the influence of different factors on the recovery. A mathematical model was found to represent the xylanase yield (Y) as a function of BDBAC and hexanol: Y = 4.32 + 5.1B + 2.64D + 0.83B2 + 1.46D2, where B = BDBAC and D = hexanol. The highest xylanase recovery (27%), indicated by the model was attained at pH = 8.1, BDBAC = 0.38 M and hexanol = 8.6%. Under these conditions, and to test the model, a new xylanase extraction was performed in laboratory, giving 29.4% recovery yield, this value was similar to that predicted by the model.

Stability and chemical modification of xylanase from Aspergillus sp. (2M1 strain).

The 2M1 strain of Aspergillus sp., which showed high extracellular xylanolytic activities in a pre-screening, was studied. Oat-spelt, birch, eucalyptus and pine xylans were used as xylanolytic inductors. The following activities were found at 50 degrees C in the presence of 1% xylan: 120 units/ml (oat-spelt xylan), 132 units/ml (birch xylan), 107 units/ml (eucalyptus xylan), 67 units/ml (pine xylan) and 137 units/ml (larch-wood xylan). Xylanase induced by pine xylan exhibited a higher stability than those induced by the other xylans. The stability was improved by addition of glycerol. In the crude extract, reagents which were found to affect xylanase activity were 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide for amidation of carboxylic groups and N-bromosuccinimide at a concentration of 0.5 mM for indole oxidation. Methylene Blue, butane-2,3-dione, N-acetylimidazole, chloramine-T and iodoacetate had little effect on the enzyme activity (more than 97% of the original activity remained).

Production of extracellular xylanases by Penicillium janthinellum. Effect of selected growth conditions.

Xylanase production by Penicillium janthinellum using 10-100 mM of 2,2-dimethylsuccinate (DMS) buffer, in a range of pH 4.5-6.0 was studied. The enzyme activity was enhanced using oat xylan as the carbon source. Under these conditions a culture produced 1.14 mumol/min (11.4 U/mL or 84.4 U/mg) of beta-xylanase after 5 d of growth in a 10-mM buffer solution at pH 4.5. Protease was absent in the DMS buffer except when 100 mM phosphate buffer at pH 6.0 was used (4 U/mL). beta-Xylosidase was only found at a pH of 4.5 in all the buffer concentrations. At a 50 mM DMS buffer concentration at pH 4.5 beta-xylanases were induced by both oat and birch xylans, having a greater effect with oat spelt xylans. Electrophoretic analyses showed that the birchwood xylan induction exhibited different proteins profiles. No beta-xylosidase or beta-glucosidase was induced until d 5. The beta-xylanases were rapidly inactivated at 50 degrees C, however, birch xylanase appeared to be more stable than oat xylanase. Using oat xylan as an inductor, the beta-xylosidase and beta-glucosidase were 85 and 91 U/L, respectively, on d 7. The xylanase produced by induction from sugar cane bagasse hydrolyzate was used for pulp biobleaching. A 20% decrease on the Kappa value in Kraft pulp using the culture extract was obtained. These selective growth conditions led us to modulate the xylanase production for pulp delignification.