Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains

The viability of converting biomass into biofuels and chemicals still requires further development towards the reduction of the enzyme production costs. Thus, there is a growing demand for the development of efficient procedures for selection of cellulaseproducing microorganisms. This work correlates qualitative screening using agar plate assays with quantitative measurements of cellulase production during cultivation under solid-state fermentation (SSF). The initial screening step consisted of observation of the growth of 78 preselected strains of the genus Trichoderma on plates, using microcrystalline cellulose as carbon source. The 49 strains that were able to grow on this substrate were then subjected to a second screening step using the Congo red test. From this test it was possible to select 10 strains that presented the highest enzymatic indices (EI), with values ranging from 1.51 to 1.90. SSF cultivations using sugarcane bagasse and wheat bran as substrates were performed using selected strains. The CG 104NH strain presented the highest EGase activity (25.93 UI·g −1 ). The EI results obtained in the screening procedure using plates were compared with cellulase production under SSF. A correlation coefficient (R 2 ) of 0.977 was obtained between the Congo red test and SSF, demonstrating that the two methodologies were in good agreement

Solubilização biológica de rocha fosfática por cultivo em estado sólido para produção de fertilizantes ecológicos

The objective of this work was to evaluate the solubilization of phosphorus from a phosphate rock by Aspergillus niger, under solid-state cultivation (SSC) in sugarcane (Saccharum officinarum) bagasse, by maximizing the efficiency of citric acid production. The phosphate rock (IPR) chosen for the study is a type of igneous rock with a very low phosphorus solubility, obtained from the Itafós company, in Arraias, in the state of Tocantins, Brazil. The rotatable central composite design (RCCD) was used as a statistical tool to evaluate the effect of the concentrations of the carbon source (sucrose) and of the IPR on the SSC medium as a strategy to improve P solubilization. In the process without the IPR, there was a citric acid production of up to 300 g per kilogram of substrate. The experiments in the RCCD showed that the addition of the IPR affected citric acid production, with values of solubilized P ranging from 1.44 to 2.72 g per kilogram of substrate and of solubilized P yield from 12.96 to 48.94%. The analysis of the solubilized P/sucrose ratio showed favorable conditions for P solubilization and citric acid production. The obtained solubilized P values are promising considering that the IPR has a very low solubilization, with only 8.6% P2O5. Overall, these findings could contribute to the development of biotechnological processes for producing eco-friendly phosphate fertilizers, as an alternative for a more sustainable agriculture.O objetivo deste trabalho foi avaliar a solubilização de fósforo de rocha fosfática por Aspergillus niger, em cultivo em estado sólido (CES) em bagaço de cana-de-açúcar (Saccharum officinarum), por meio da maximização da eficiência de produção de ácido cítrico. A rocha fosfática (IPR) escolhida para o estudo é um tipo de rocha ígnea com solubilização de fósforo muito baixa, obtida da empresa Itafós, em Arraias, no estado do Tocantins. Utilizou-se o delineamento composto central rotacional (DCCR) como ferramenta estatística para avaliar o efeito das concentrações da fonte de carbono (sacarose) e da IPR no meio para CES como estratégia para melhorar a solubilização de P. Para o processo sem a IPR, houve produção de ácido cítrico de até 300 g por quilograma de substrato. Os experimentos em DCCR mostraram que a adição da IPR afetou a produção de ácido cítrico, com valores de P solubilizado de 1,44 a 2,72 g por quilograma de substrato e rendimento de P solubilizado de 12,96 a 48,94%. Além disso, a análise da razão Psolubilizado/sacarose mostrou condições favoráveis para solubilização de P e produção de ácido cítrico. Os valores de P solubilizado são promissores, ao se considerar que a IPR apresenta baixa solubilização, com apenas 8,6% de P2O5. No geral, esses resultados podem contribuir para o desenvolvimento de processos biotecnológicos, para a produção de fertilizantes fosfatados ambientalmente favoráveis, como alternativa para uma agricultura mais sustentável

DESAFIOS RELACIONADOS À PRODUÇÃO E APLICAÇÃO DAS ENZIMAS CELULOLÍTICAS NA HIDRÓLISE DA BIOMASSA LIGNOCELULÓSICA

The second generation (2G) ethanol obtained from lignocellulosic biomass has been considered as a potential alternative source of energy for the transportation sector. However, the technologies to convert lignocellulose into fermentable sugars still face several challenges. One of the major challenges of this process is related to the cost of the cellulolytic enzymes. Thus, the development of bioprocesses for enzyme production and strategies to increase the final yield of the enzymatic hydrolysis reactions are required to ensure the economic feasibility of the biomass conversion process. In this review, it is discussed the progress related to fungal enzyme technology over the past few years with major emphasis on the biocatalysts needed for cellulose degradation as well as on cultivation strategies for the production of enzymes on-site. The potential of applying secretome analysis as a tool for the characterization of the enzymatic complexes and some limitations on the use of enzymatic cocktails in the saccharification step, such as the unproductive adsorption of enzymes onto lignin are also discussed in light of the recent literature

Caracterização de fungos isolados da região Amazônica quanto ao potencial para produção das enzimas envolvidas na conversão da biomassa vegetal

<p>A conversão da biomassa vegetal proveniente de resíduos agroindustriais e florestais em biocombustíveis e bioprodutos, dentro do conceito de biorrefinarias, é de grande interesse, principalmente para o Brasil, onde a agroenergia possui um enorme potencial de desenvolvimento. Entretanto, para garantir a viabilidade do processo de conversão, é fundamental reduzir o custo das enzimas utilizadas na etapa de hidrólise. Para isso, deve-se dispor da peça chave deste processo, que é o microrganismo. Nesse contexto, o objetivo deste trabalho foi avaliar fungos isolados da região Amazônica em relação ao potencial de produção das enzimas celulases e xilanases. De um total de 40 isolados cultivados por fermentação em estado sólido (FES), durante 10 dias, os fungos que se destacaram quanto à produção de endoglucanase (351,79Ug^-1 em 120h) e β-glicosidase (62,31Ug^-1em 72h) foi o P47C3 (A. niger), e na produção de xilanase (1076,94Ug^-1 em 72h) e FPase (2,46Ug^-1 em 120h) foram o P6B2 (A. oryzae) e o P40B3, respectivamente. Os resultados obtidos demonstram o enorme potencial de aplicação das enzimas produzidas pelos fungos isolados da Amazônia, contribuindo, assim, para gerar os avanços tecnológicos necessários para o aumento da eficiência do uso da biomassa vegetal como fonte de energia renovável</p

Stabilization of glycosylated β-glucosidase by intramolecular crosslinking between oxidized glycosidic chains and lysine residues

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.The authors thank the support from Research Institute of Food Science (CIAL), Embrapa Instrumentation of São Carlos, Foundation for Research Support of Espírito Santo (FAPES), and Federal University of Espírito Santo (UFES).Peer reviewe

Mechanochemically Synthesized Nitrogen-Efficient Mg- and Zn-Ammonium Carbonate Fertilizers

New scalable methods are needed to provide sustainable solutions for nutrient recovery in the form of solid fertilizer materials and their environmental stabilization from anaerobic digestion liquid byproducts. In this work, two nutrient metal containing double salts, Mg(NH4)2(CO3)2·4H2O and Zn(NH3)CO3, were synthesized using naturally abundant Mg- and Zn-carbonate minerals and ammonium (bi)carbonate salts. This constituted a conceptually new synthetic approach, different from the previous work in which the aqueous solution was utilized. We also showed that the materials could be easily scaled to 20 g quantities sufficient for soil testing. The crystalline structure of the resulting materials was confirmed using powder XRD and thermal analysis showed properties distinctly different from those of parent ammonium (bi)carbonates. Accordingly, a reduction in NH3 volatilization in soil was measured with up to 20% more NH4+ recovered after the soil experiments at 80% water holding capacity. Further, inhibition of the agriculture-beneficial bacteria Bacillus subtillis in a nutrient medium was dramatically reduced when compared to the ammonium bicarbonate alone, suggesting decreased negative effects on soil biota. Finally, Mg(NH4)2(CO3)2·4H2O and Zn(NH3)CO3 matched the kinetic nitrogen need of lettuce plants better than the ammonium carbonate control while also keeping it in a form that will be available in the future. The utility of magnesium and zinc double salts in agriculture is paramount if environmentally benign and nutrient-efficient fertilizers from liquid digestate waste are to be enabled