The use of phosphate rocks as low‐solubility phosphorus fertilizers has been promoted to reduce the environmental impacts of agriculture, but adequate nutrient uptake by plants depends on ...solubilization of the rock, driven by soil microorganisms. Here, investigation was made of the microbial solubilization of low‐solubility phosphate rocks, together with simultaneous bioprotective action involving the biocontrol of microorganisms. The aim was to enhance function and value by delivering two effects using a single bio‐based product, in accordance with the concept of a “bioreactor‐in‐a‐granule” system. A composite structure was developed, based on a starch matrix, comprising a combination of Trichoderma asperelloides, as a biocontrol agent, and Aspergillus niger, as an acidulant. A significant increase of up to 150% in P solubilization was achieved, indicating the positive effect of the microorganism‐composite interaction. In vitro assays showed that the ability of T. asperelloides to inhibit Fusarium oxysporum mycelial growth was maintained in the presence of A. niger. Moreover, the estimated cost of the composite granule (0.35 US$/kg of product on a dry basis) revealed competitive. The results indicated that the association of T. asperelloides and A. niger is an effective way to increase nutrient availability and to inhibit plant pathogens, opening up possibilities for the design of multifunctional bio‐based fertilizer composites.
Enzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized ...lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions.
Immobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts. On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 μmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone.
Activity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.
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 cellulase-producing 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 (R2) of 0.977 was obtained between the Congo red test and SSF, demonstrating that the two methodologies were in good agreement.
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.
Resumo: 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 P solubilizado/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.
The manner in which added non‐catalytic proteins during enzymatic hydrolysis of lignocellulosic substrates enhances hydrolysis mechanisms is not completely understood. Prior research has indicated ...that a reduction in the non‐specific adsorption of enzymes on lignin, and deactivation of enzymes exposed to air–liquid interface provide rationale. This work investigated root causes including effects of the air–liquid interface on non‐catalytic proteins, and effects of lignin on endoglucanase. Three different experimental designs and three variables (air–liquid interfacial area, the types of lignin (acid or enzymatic lignin), and the presence of non‐enzymatic protein (bovine serum albumin BSA or soy proteins ) were used. The results showed that acid isolated lignin adsorbed almost all endoglucanase activity initially present in supernatant, independent of air interface conditions (25 or 250 ml flasks) with the presence of BSA preventing this effect. Endoglucanase lost 30%–50% of its activity due to an air–liquid interface in the presence of lignin while addition of non‐enzymatic protein helped to preserve this enzyme's activity. Langmuir and Freundlich models applied to experimental data indicated that the adsorption increases with increasing temperature for both endoglucanase and BSA. Adsorption of the enzyme and protein were endothermic with an increase in entropy. These results, combined, show that hydrophobicity plays a strong role in the adsorption of both endoglucanase and BSA on lignin.
The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and ...other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of
Trichoderma harzianum
followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.
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 role of xyr1 (xylanase regulator 1) in the cellulases production by Trichoderma harzianum.•Relevant improvement of genes expression encoding enzymes involved in biomass degradation by xyr1 clone ...overexpression.•New enzymatic complex by T. harzianuḿs xyr1 overexpression used in sugarcane bagasse saccharification.
This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and β-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum.
•Accessory enzymes produced on-site can improve biomass degradation.•Feruloyl esterase and xylanase improved sugarcane bagasse hydrolysis.•Sugarcane bagasse hydrolysis increased 36% with ...supplementation.
Accessory enzymes that assist biomass degradation could be used to improve the recovery of fermentable sugar for use in biorefineries. In this study, different fungal strains isolated from the Amazon rainforest were evaluated in terms of their ability to produce feruloyl esterase (FAE) and xylanase enzymes, and an assessment was made of the contributions of the enzymes in the hydrolysis of pretreated sugarcane bagasse. In the selection step, screening using plate assays was followed by shake flask submerged cultivations. After carbon source selection and cultivation in a stirred-tank bioreactor, Aspergillusoryzae P21C3 proved to be a promising strain for production of the enzymes. Supplementation of a commercial enzyme preparation with 30% (v/v) crude enzymatic complex from A. oryzae P21C3 increased the conversion of cellulose derived from pretreated sugarcane bagasse by 36%. Supplementation with FAE and xylanase enzymes produced on-site can therefore be used to improve the hydrolysis of sugarcane bagasse.
PROCESSING AND APPLICATION OF POLYMERIC BIOMATERIALS: RECENT ADVANCES AND PERSPECTIVES. Biomaterials have been intensively investigated due to the increase in the elderly population and high ...prevalence of several disorders, such as cardiovascular and orthopedic diseases. Polymeric and composite polymeric materials in combination with different processing techniques, such as electrospinning, solution blow spinning, ultrathin film preparation, and 3D printing are promising for obtaining biomaterials with patient-specific applications. Here, we provide a review on recent advances and perspectives for synthetic and natural polymers as well as composites in the design of biomaterials. After introducing basic information about biomaterials, we describe the fundamentals of manufacturing techniques and highlight possible biomedical applications.
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