This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting ...biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HAapp) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g−1 of gel for GLU, 7.76 mg g−1 of gel for GLY, and 7.65 mg g−1 of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g−1 of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.
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CEKLJ, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The use of additives in the enzymatic saccharification of lignocellulosic biomass can have positive effects, decreasing the unproductive adsorption of cellulases on lignin and reducing the loss of ...enzyme activity. Soybean protein stands out as a potential lignin-blocking additive, but the economic impact of its use has not previously been investigated. Here, a systematic evaluation was performed of the process conditions, together with a techno-economic analysis, for the use of soybean protein in the saccharification of hydrothermally pretreated sugarcane bagasse in the context of an integrated 1G-2G ethanol biorefinery. Statistical experimental design methodology was firstly applied as a tool to select the process variable solids loading at 15% (w/w) and soybean protein concentration at 12% (w/w), followed by determination of enzyme dosage at 10 FPU/g and hydrolysis time of 24 h. The saccharification of sugarcane bagasse under these conditions enabled an increase of 26% in the amount of glucose released, compared to the control without additive. The retro-techno-economic analysis (RTEA) technique showed that to make the biorefinery economically feasible, some performance targets should be reached experimentally such as increasing biomass conversion to ideally 80% and reducing enzyme loading to 5.6 FPU/g in the presence of low-cost soybean protein.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The yeast
Saccharomyces cerevisiae
is the most used microorganism for ethanol production, however, on its wild form it cannot assimilate xylose. The previous
ex-vivo
isomerization of xylose to ...xylulose catalyzed by the enzyme xylose isomerase (XI) is an alternative to overcome this limitation. The present work evaluated continuous 2G ethanol production through xylose simultaneous isomerization and fermentation (SIF) using xylose isomerase co-immobilized with yeast. From the initial studies carried out with five different industrial
S. cerevisiae
strains, Itaiquara
®
baker´s yeast was selected due to its good performance in terms of ethanol yield (0.34 g/g) and productivity (2.1 g/L/h) having xylose as a carbon source. Continuous xylose SIF in a fixed-bed reactor was run for 7 days with high values of ethanol yield (0.37 g/g) and productivity (1.9 g/L/h). The operation for longer periods could be possible upon implementing strategies for pH and contamination control, showing great progress to achieve a feasible industrial 2G ethanol production process.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Streptococcus pneumoniae
is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the ...vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects
S. pneumoniae
from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at −20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.
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CEKLJ, DOBA, EMUNI, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Various bio‐based processes depend on controlled micro‐aerobic conditions to achieve a satisfactory product yield. However, the limiting oxygen concentration varies according to the micro‐organism ...employed, while for industrial applications, there is no cost‐effective way of measuring it at low levels. This study proposes a machine learning procedure within a metabolic flux‐based control strategy (SUPERSYS_MCU) to address this issue. The control strategy used simulations of a genome‐scale metabolic model to generate a surrogate model in the form of an artificial neural network, to be used in a micro‐aerobic fermentation strategy (MF‐ANN). The meta‐model provided setpoints to the controller, allowing adjustment of the inlet air flow to control the oxygen uptake rate. The strategy was evaluated in micro‐aerobic batch cultures employing industrial Saccharomyces cerevisiae yeast, with defined medium and glucose as the carbon source, as a case study. The performance of the proposed control scheme was compared with a conventional fermentation and with three previously reported micro‐aeration strategies, including respiratory quotient‐based control and constant air flow rate. Due to maintenance of the oxidative balance at the anaerobiosis threshold, the MF‐ANN provided volumetric ethanol productivity of 4.16 g·L−1·h−1 and a yield of 0.48 gethanol.gsubstrate−1, which were higher than the values achieved for the other conditions studied (maximum of 3.4 g·L−1·h−1 and 0.35–0.40 gethanol·gsubstrate−1, respectively). Due to its modular character, the MF‐ANN strategy could be adapted to other micro‐aerated bioprocesses.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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•A protocol to in-house-production of xylan is proposed.•Eucalyptus bleached pulp (holocellulose) was used as raw material.•Xylan was extracted in alkaline solution, acid precipitated ...and freeze dried.•Xylanase activities were equivalent to the ones obtained with birchwood substrate.•The protocol was validated, allowing comparison between past and future xylanase works.
Over the past decades, most studies with xylanases have used Birchwood xylan as the standard substrate for activity assays. However, recently, Birchwood xylan production was discontinued by major suppliers, creating an important demand for a substitute. Ongoing and future studies require a substrate with characteristics equivalent to the discontinued xylan, in order to enable the comparison of results. In this context, a protocol for the production of a substrate similar to the discontinued commercial Birchwood xylan is reported. Obtained from bleached Eucalyptus cellulose pulp, xylan was extracted using 4% w/v NaOH solution at 25 °C, precipitated with glacial acetic acid (HOAc), and freeze-dried. A thermal pretreatment in an autoclave for 15 min increased its solubility. The resulting xylan was characterized by infrared spectroscopy, thermogravimetry, and NMR. When assessing the activity of xylanases, the results were the same as those for commercial Birchwood xylan.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Anaerobic digestion (AD) of effluents in sugarcane biorefineries may improve the energy balance in the bioethanol production process and the environmental suitability of wastes disposal. This study ...analyses the inclusion of AD of vinasse (stillage) into different configurations of sugarcane biorefineries, using technical, economic and environmental metrics. The three base-case scenarios are a first generation (1G) ethanol plant, an integrated first and second generation (1G2G) ethanol plant with alcoholic fermentation of the hemicellulose fraction (C5), and an 1G2G ethanol plant with biodigestion of the C5 fraction. These three base scenarios are compared with the similar ones including the biodigestion of vinasse. The implementation of vinasse biodigestion had positive impact on the productivity and sustainability of sugarcane biorefineries. The use of biogas from vinasse for a 1G ethanol plant increased in 9.20% the surplus of electric energy yielded to the grid. This scenario had a positive net present value, NPV: + 11.5 × 10
6
USD, assuming minimum acceptable rate of return, MARR = 11%. For the 1G2G + C5 fermentation scenario the NPV was + 4.63 × 10
6
USD. Assuming NPV = 0, the IRR for 1G becomes 19.7% and for 1G2G + C5, 13.6%, an IRR higher than the MARR. The inclusion of AD of vinasse when the C5 fraction is biodigested was not an economically feasible option. In all scenarios, vinasse biodigestion reduced environmental impacts, and the 1G2G processes exhibit better results than the consolidated 1G for almost all environmental impact categories. The AD of vinasse can be considered an environmental-friendly and economical-feasible technology to improve the process in sugarcane biorefineries.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
•An enhanced surrogate assisted framework for constrained global optimization is proposed.•Maximizing probability of improvement approach is used for selecting infill points.•Kriging meta-models of ...objective and constraints functions are updated in every iteration.•Meta-model of objective function is local optimized when it’s sufficient mature.•Numerical results indicate that the framework is suitable for use in solving computationally expensive and constrained black-box optimization.
An enhanced surrogate assisted framework, based on Probability of Improvement (PI) method, is proposed in this paper. We made some modifications to the original PI approach to enhance the performance of the modeling and optimization framework, leading to fewer rigorous simulations to find the optimal solution without loss of accuracy. We also extended the algorithm for handling general constraints using a fully probabilistic approach. The behavior of the proposed framework was investigated through a set of 9 Unconstrained Test Functions (UTF), 7 Constrained Optimization Problems (COP) and 3 Chemical Engineering Problems (CEP). The numerical results indicate that a lower number of rigorous model simulations were needed for optimizing UTF compared to the classic PI method and that the proposed framework was capable of achieving sustained near optimal solutions for COP and CEP. These results indicate that the proposed framework is suitable for solving computationally expensive constrained black-box optimization problems.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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•Castor bean lipase solid extract catalyzed hydrolysis of soy bean oil in acidic pH.•Gum Arabic presence showed a negative effect in the reaction conversion under mechanical ...stirring.•A complex heterogeneous system was well represented by a simple kinetic model.•This model can be used in industrial engineering applications.
Soybean oil hydrolysis catalyzed by castor bean (Ricinus communis L.) seeds lipase was investigated. The optimum operational temperature (in the range 25–50 °C) was 37 °C, and Na+ increased reaction velocities. When stirring at 300 rpm, gum arabic enhanced the rate of hydrolysis, but at 1000 rpm the emulsifier decreased long-term conversions. Experiments using different particle sizes indicated the presence of diffusion delays above 300 μm. A parsimonious model for this complex system was proposed. Initial rates (V0) experiments, spanning 0.01–0.5 w/w of oil, showed a decrease in V0 for high substrate concentrations, depending on the stirring conditions, indicating coalescence of substrate droplets. This hypothesis was confirmed by experiments with more impeller paddles. A pseudo-homogeneous Michaelis–Menten model with substrate inhibition (to represent coalescence effects), fitted well to the initial rates. Product inhibition for high loads of substrate and enzyme deactivation were also considered. The kinetic model was validated by independent experiments, and thus may be useful for design and optimization of industrial enzymatic reactors.
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GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Xylose is a sugar that is plentiful in lignocellulosic biomass, but is currently underused. Despite it being a potential carbon source for 2G ethanol production, the native yeast Sacharomyces ...cerevisiae cannot assimilate xylose. One possible way to overcome this restriction would be the previous isomerization of xylose to xylulose, catalyzed by the enzyme xylose isomerase (XI). Due to the unfavorable chemical equilibrium of conversion of 5-xylose to 1-xylulose, this route requires simultaneous isomerization and fermentation (SIF), in order to shift the equilibrium. The present work describes a study of ethanol production from xylose in a SIF process, using a biocatalyst consisting of XI immobilized on chitosan and subsequently co-immobilized with baker's yeast in Ca-alginate gel. The effects of biocatalyst composition (enzyme and yeast loads) and temperature were evaluated. The biocatalyst composition was varied using enzyme loads from 5 to 20% (w/v) and cell concentrations from 5 to 17% (w/v). Productivity and yield increased together with the yeast concentration, while selectivity increased with the enzyme concentration. For a biocatalyst consisting of 10% (w/v) of yeast (50 g. L−1) and 20% of enzyme (120 × 103 IU.L−1), 98% conversion was achieved within 11 h, providing a yield of 0.35 ± 0.02 g. g−1, productivity of 2.07 ± 0.17 g. L−1.h−1, and ethanol/xylitol selectivity of 2.42 ± 0.01. The temperatures tested were 32, 35, and 37 °C, and ethanol yield and productivity were around 0.35 g. g−1 and 2.03 g .L−1.h−1, respectively, in all the experiments. Although higher temperatures favor XI activity, 35 °C was selected because it favored ethanol formation.
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•More robust and efficient biocatalyst developed for the SIF of xylose.•Incorporation of CaCO3 into biocatalyst beads allowed pH control.•Milder temperatures favored ethanol/xylitol selectivity.•Ratio of enzyme/yeast loads is a key factor in SIF performance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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