Screening of filamentous fungi for production of enzymes of biotechnological interest

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Assessment of fungi in soils of sugarcane crops and their potential for production of biomass-degrading enzymes

Soil management practices are konwn to affect the biomass and enzyme activities of microbial soil communities. To assess whether burning of sugarcane prior to harvesting affects the community of soilborne fungi, we collected soil simples in two sites: burned sugarcane culture prior harvesting (BS) and non-burned sugarcane culture (NBS). A total of 75 filamentous fungal isolates were recovered from soils in both sites. Trichoderma was the most prevalent genus in both sites, followed by Fusarium, Cunninghamella and Aspergillus. The Sorensen's index (0.60) suggested a slight difference in fungi associated with both areas, with high number of fungal isolates found on BB soil. The abundance of Trichoderma isolates in NBS soil was higher than BS soil; however, the abundance of Fusarium, Aspergillus and Cunninghamella was higher in the latter type of soil. In addition, fungi isolated from BS soil showed the highest production of xylanase and laccase in comparision with fungi isolated form NBS soil. Our results indicate that the different types of sugarcane harvesting apparently did not interfere with the diversity of fungal communnities as revealed by culture-dependent methods. In addition, our data indicates the potencial of fungi from soils of sugarcane crops to produce relevant enzymes related to biomass conversion.

Thermophilic fungi as new sources for production of cellulases and xylanases with potential use in sugarcane bagasse saccharification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The capability of endophytic fungi for production of hemicellulases and related enzymes

Abstract Background There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G. Results The ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, β-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial β-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media. Conclusions The selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.

The capability of endophytic fungi for production of hemicellulases and related enzymes

Abstract BACKGROUND: There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G. RESULTS: The ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, β-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial β-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media. CONCLUSIONS: The selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.

In Vivo Determination Of The Volatile Metabolites Of Saprotroph Fungi By Comprehensive Two-dimensional Gas Chromatography.

In this work, we discuss the use of multi-way principal component analysis combined with comprehensive two-dimensional gas chromatography to study the volatile metabolites of the saprophytic fungus Memnoniella sp. isolated in vivo by headspace solid-phase microextraction. This fungus has been identified as having the ability to induce plant resistance against pathogens, possibly through its volatile metabolites. Adequate culture media was inoculated, and its headspace was then sampled with a solid-phase microextraction fiber and chromatographed every 24 h over seven days. The raw chromatogram processing using multi-way principal component analysis allowed the determination of the inoculation period, during which the concentration of volatile metabolites was maximized, as well as the discrimination of the appropriate peaks from the complex culture media background. Several volatile metabolites not previously described in the literature on biocontrol fungi were observed, as well as sesquiterpenes and aliphatic alcohols. These results stress that, due to the complexity of multidimensional chromatographic data, multivariate tools might be mandatory even for apparently trivial tasks, such as the determination of the temporal profile of metabolite production and extinction. However, when compared with conventional gas chromatography, the complex data processing yields a considerable improvement in the information obtained from the samples. This article is protected by copyright. All rights reserved.

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.

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe(3+)-reductants. Phenolates were the major compounds with Fe(3+)-reducing activity in both fungi and displayed Fe(3+)-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe(3+) and H(2)O(2) (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum-a model brown rot fungus-other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.

Role of entomopathogenic fungi in the control of Tetranychus evansi and Tetranychus urticae (Acari: Tetranychidae), pests of horticultural crops

The spider mites Tetranychus urticae Koch and Tetranychus evansi Baker and Pritchard are important pests of horticultural crops. They are infected by entomopathogenic fungi naturally or experimentally. Fungal pathogens known to cause high infection in spider mite populations belong to the order Entomophthorales and include Neozygites spp. Studies are being carried out to develop some of these fungi as mycoacaricides, as standalone control measures in an inundative strategy to replace the synthetic acaricides currently in use or as a component of integrated mite management. Although emphasis has been put on inundative releases, entomopathogenic fungi can also be used in classical, conservation and augmentative biological control. Permanent establishment of an exotic agent in a new area of introduction may be possible in the case of spider mites. Conservation biological control can be achieved by identifying strategies to promote any natural enemies already present within crop ecosystems, based on a thorough understanding of their biology, ecology and behaviour. Further research should focus on development of efficient mass production systems, formulation, and delivery systems of fungal pathogens.

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 A degrees C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 A degrees C and pH 6.5 for A. terricola, and 65 A degrees C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 A degrees C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t (50) of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4-3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and beta-xylosidase were detected which might act synergistically with xylanase.

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4-5 days in liquid medium at 40A degrees C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60-70A degrees C. The enzymes were stable for 30 min at 60A degrees C, maintaining 95-98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5-5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0-8.0, while the enzyme from A. niveus was more stable at pH 4.5-6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.

Correlation of fungi and endotoxin with PM2.5 and meteorological parameters in atmosphere of Sao Paulo, Brazil

Particulate matter, especially PM2.5, is associated with increased morbidity and mortality from respiratory diseases. Studies that focus on the chemical composition of the material are frequent in the literature, but those that characterize the biological fraction are rare. The objectives of this study were to characterize samples collected in Sao Paulo, Brazil on the quantity of fungi and endotoxins associated with PM2.5, correlating with the mass of particulate matter, chemical composition and meteorological parameters. We did that by Principal Component Analysis (PCA) and multiple linear regressions. The results have shown that fungi and endotoxins represent significant portion of PM2.5, reaching average concentrations of 772.23 spores mu g(-1) of PM2.5 (SD: 400.37) and 5.52 EU mg(-1) of PM2.5 (SD: 4.51 EU mg(-1)), respectively. Hyaline basidiospores, Cladosporium and total spore counts were correlated to factor Ba/Ca/Fe/Zn/K/Si of PM2.5 (p < 0.05). Genera Pen/Asp were correlated to the total mass of PM2.5 (p < 0.05) and colorless ascospores were correlated to humidity (p < 0.05). Endotoxin was positively correlated with the atmospheric temperature (p < 0.05). This study has shown that bioaerosol is present in considerable amounts in PM2.5 in the atmosphere of Sao Paulo, Brazil. Some fungi were correlated with soil particle resuspension and mass of particulate matter. Therefore, the relative contribution of bioaerosol in PM2.5 should be considered in future studies aimed at evaluating the clinical impact of exposure to air pollution. (C) 2010 Elsevier Ltd. All rights reserved.