Luciana Rocha Barros Gonçalves

Extraction of lipids from microalgae by ultrasound application: Prospection of the optimal extraction method

Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20-50% of dry weight) of lipids. A successful and economically viable algae based oil industry will depend on the selection of appropriate microalgal strains and the selection of the most suitable lipid extraction method. In this paper, five extraction methods were evaluated regarding the extraction of lipids from Chlorella vulgaris: Bligh and Dyer, Chen, Folch, Hara and Radin, and Soxhlet. Furthermore, the addition of silica powder was studied to evaluate the introduction of more shear stress to the system as to increase the disruption of cell walls. Among the studied methods, the Bligh and Dyer method assisted by ultrasound resulted in the highest extraction of oil from C. vulgaris (52.5% w/w). Addition of powder silica did not improve the extraction of oil.

Bioprospecting for oil producing microalgal strains: Evaluation of oil and biomass production for ten microalgal strains

Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20-50% of dry weight) of lipids. A successful and economically viable algae based oil industry depends on the selection of appropriate algal strains. In this study ten species of microalgae were prospected to determine their suitability for oil production: Chaetoceros gracilis, Chaetoceros mulleri, Chlorella vulgaris, Dunaliella sp., Isochrysis sp., Nannochloropsis oculata, Tetraselmis sp., Tetraselmis chui, Tetraselmis tetrathele and Thalassiosira weissflogii. The study was carried out in 3 L glass flasks subjected to constant aeration and controlled artificial illumination and temperature at two different salinities. After harvesting, the extraction of oil was carried out using the Bligh and Dyer method assisted by ultrasound. Results showed that C. gracilis presented the highest oil content and that C. vulgaris presented the highest oil production.

Alkaline hydrogen peroxide pretreatment of cashew apple bagasse for ethanol production: Study of parameters

The alkaline hydrogen peroxide (AHP) pretreatment of cashew apple bagasse (CAB) was evaluated based on the conversion of the resultant cellulose into glucose. The effects of the concentration of hydrogen peroxide at pH 11.5, the biomass loading and the pretreatment duration performed at 35°C and 250 rpm were evaluated after the subsequent enzymatic saccharification of the pretreated biomass using a commercial cellulase enzyme. The CAB used in this study contained 20.56 ± 2.19% cellulose, 10.17 ± 0.89% hemicellulose and 35.26 ± 0.90% lignin. The pretreatment resulted in a reduced lignin content in the residual solids. Increasing the H2O2 concentration (0-4.3% v/v) resulted in a higher rate of enzymatic hydrolysis. Lower biomass loadings gave higher glucose yields. In addition, no measurable furfural and hydroxymethyl furfural were produced in the liquid fraction during the pretreatment. The results show that alkaline hydrogen peroxide is effective for the pretreatment of CAB.

Kinetic study of biosurfactant production by Bacillus subtilis LAMI005 grown in clarified cashew apple juice

In this work a low cost medium for the production of a biosurfactant by Bacillus subtilis LAMI005 and the kinetics of surfactin production considering the effect of initial substrate concentration were investigated. First, cashew apple juice supplementation for optimal production of biosurfactant by B. subtilis LAMI005 was studied. The medium formulated with clarified cashew apple juice and distilled water, supplemented with 1.0 g/L of (NH(4))(2)SO(4), proved to be the best among the nutrients evaluated. The crude biosurfactant had the ability to decrease the surface tension of water to 30 dyne/cm, with a critical micelle concentration (CMC) of 63.0 mg/L. Emulsification experiments indicated that this biosurfactant effectively emulsified kerosene (IE(24)=67%) and soybean oil (IE(24)=64%). Furthermore, the emulsion stability was always very high. It was shown by biochemical analysis, IR spectra, that there is no qualitative differences in the composition of the crude biosurfactant from a standard sample of surfactin from B. subtilis.

Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan

Abstract - The objective of this work was to study enzyme immobilization on chitosan activated with glutaraldehyde, aiming to produce a cheap biocatalyst. Two different immobilization strategies were studied: one-point and multipoint covalent attachment to the solid matrix. The multipoint covalent attachment derivative had an 82% immobilization yield. It was4.9-fold more stable than the free enzyme at 50°C and 4.5-fold more stable than soluble enzyme at pH 10.0. The one-point derivativehad an 85% immobilization yield. It was 2.7-fold more stable than the free enzyme at 50°C and 3.8-fold more stable than soluble PGA at pH 10.0. Results indicated that chitosan can be loaded with PGA above 330 IU/g. Intraparticle diffusive effects, however,limited hydrolysis of penicillin G catalyzed by those derivatives at 37°C and 25°C. Operational stability assays were performed and the multipoint derivative exhibited a half-life of 40 hours. Keywords : Stabilization of enzymes; Penicillin G acylase; Chitosan and immobilization of enzymes.

Ultrasound-assisted production of biodiesel and ethanol from spent coffee grounds.

This study evaluates the production of biodiesel and ethanol from spent coffee grounds (SCG). The extraction of oil from SCG, biodiesel production and ethanol production processes were studied. The liquid-to-solid ratio and temperature were evaluated in the ultrasound-assisted extraction of the oil from SCG. The highest yield (12%) was obtained using 4 mL g(-1) liquid-to-solid ratio at 60°C for 45 min. The process to produce biodiesel showed a yield of 97% into fatty acid methyl esters (FAME). The highest glucose yield (192 mg g SCG(-1)) was obtained by hydrolysis with 0.4 mol L(-1) sulfuric acid at 121°C for 15 min. The hydrolysate was used as fermentation medium for ethanol production by Saccharomyces cerevisiae obtaining 19.0 g L(-1) at 10h of process of ethanol with a yield of ethanol and productivity of 0.50 g g(-1) and 1.90 g L(-1)h(-1), respectively. Spent coffee grounds were considered a potential feedstock for biodiesel and ethanol production.

Characterization of supports activated with divinyl sulfone as a tool to immobilize and stabilize enzymes via multipoint covalent attachment. Application to chymotrypsin

Divinyl sulfone (DVS) has been used to activate agarose beads. The DVS activated agarose resulted quite stable in the pH range 5–10 at 25 °C under wet conditions, and can react rapidly with α-amides of Cys and His, at pH 5–10, with Lys mainly at pH 10 and with Tyr in a much slower fashion. After blocking with different nucleophiles, the support lost all reactivity, confirming that this protocol could be useful as an enzyme–support reaction end point. Then, chymotrypsin was immobilized on this support at pH 5, 7 and 10. Even though the enzyme was immobilized at all pH values, the immobilization rate decreased with the pH value. The effect of the immobilization on the activity depended on the immobilization pH, at pH 7 the activity decreased (to 50%) more than at pH 10 (by a 25%), while at pH 5 the immobilization has no effect. Then, the effect of blocking with different reagents was analyzed. It was found that blocking with ethylenediamine improved the enzyme activity by 70% and gave the best stability. The stability of all enzyme preparations improved when 24 h incubation was performed at pH 10, but the qualitative stabilization depended on the inactivation conditions. The analysis of the amino acids of the preparation immobilized at pH 10 showed that Lys, Tyr and Cys residues were involved in the immobilization, involving a minimum of 10 residues (glyoxyl agarose gave 4 Lys involved in the immobilization). The new preparation was 4–5 fold more stable than glyoxyl agarose preparation, considered a very stable one, and in some instances was more active than the free enzyme (170% for the enzyme immobilized at pH 10). Thus, DVS activated supports are very promising to permit the multipoint covalent attachment of enzymes, and that way to improve their stability.

Improving the catalytic properties of immobilized Lecitase via physical coating with ionic polymers

Lecitase Ultra has been immobilized on cyanogen bromide agarose (via covalent attachment) and on octyl agarose (via physical adsorption on the hydrophobic support by interfacial activation). Both immobilized preparations have been incubated in dextran sulfate (DS) or polyethylenimine (PEI) solutions to coat the enzyme surface. Then, the activity versus different substrates and under different experimental conditions was evaluated. The PEI coating generally produced a significant increase in enzyme activity, in some cases even by more than a 30-fold factor (using the octyl-Lecitase at pH 5 in the hydrolysis of methyl phenyl acetate). In opposition, the DS coating usually produced some negative effects on the enzyme activity. The rate of irreversible inhibition of the covalent preparation using diethyl p-nitrophenylphosphate did not increase after PEI coating suggesting that the increase in Lecitase activity is not a consequence of the stabilization of the open form of Lecitase. Moreover, the coating greatly increased the stability of the immobilized Lecitase, for example using DS and the covalent preparation, the half-life was increased by a 30-fold factor in 30% acetonitrile. The stabilizing effect was not found in all cases, in certain cases even a certain destabilization is found (e.g., octyl-Lecitase-DS at pH 7). Thus, the effects of the ionic polymer coating strongly depend on the substrate, experimental conditions and immobilization technique employed.

Enzymatic synthesis of prebiotic oligosaccharides.

Prebiotic oligosaccharides are nondigestible carbohydrates that can be obtained by enzymatic synthesis. Glucosyltransferases can be used to produce these carbohydrate through an acceptor reaction synthesis. When maltose is the acceptor a trisaccharide composed of one maltose unit and one glucose unit linked by an α-1,6-glycosidic bond (panose) is obtained as the primer product of the dextransucrase acceptor reaction. In this work, panose enzymatic synthesis was evaluated by a central composite experimental design in which maltose and sucrose concentration were varied in a wide range of maltose/sucrose ratios in a batch reactor system. A partially purified enzyme was used in order to reduce the process costs, because enzyme purification is one of the most expensive steps in enzymatic synthesis. Even using high maltose/sucrose ratios, dextran and higher-oligosaccharide formation were not avoided. The results showed that intermediate concentrations of sucrose and high maltose concentration resulted in high panose productivity with low dextran and higher-oligosaccharide productivity.

Immobilization of Candida antarctica Lipase B by Covalent Attachment to Green Coconut Fiber

The objective of this study was to covalently immobilize Candida antarctica type B lipase (CALB) onto silanized green coconut fibers. Variables known to control the number of bonds between enzyme and support were evaluated including contact time, pH, and final reduction with sodium borohydride. Optimal conditions for lipase immobilization were found to be 2 h incubation at both pH 7.0 and 10.0. Thermal stability studies at 60°C showed that the immobilized lipase prepared at pH 10.0 (CALB-10) was 363-fold more stable than the soluble enzyme and 5.4-fold more stable than the biocatalyst prepared at pH 7.0 (CALB-7). CALB-7 was found to have higher specific activity and better stability when stored at 5°C. When sodium borohydride was used as reducing agent on CALB-10 there were no improvement in storage stability and at 60°C stability was reduced for both CALB-7 and CALB-10.