Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw

The potential of enzymes recycling after hydrolysis and fermentation of wheat straw under a variety of conditions was investigated, monitoring the activity of the enzymes in the solid and liquid fractions, using low molecular weight substrates. A significant amount of active enzymes could be recovered by recycling the liquid phase. In the early stage of the process, enzyme adsorb to the substrate, then gradually returning to the solution as the saccharification proceeds. At 50 °C, normally regarded as an acceptable operational temperature for saccharification, the enzymes (Celluclast) significantly undergo thermal deactivation. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Indeed, the amount of enzymes in the liquid phase increased with its thermostability and hydrolytic efficiency. This study contributes towards developing effective enzymes recycling strategies and helping to reduce the enzyme costs on bioethanol production.The authors acknowledge funding through FP7 KACELLE (Kalundborg Cellulosic Ethanol) project for supporting this work. We also thank Dra. Lucilia Domingues for supplying the yeast Saccharomyces cerevisiae CEN PK 113 wild type

Production and characterization of a new dextrin based hydrogel

Dextrin is a polymer composed of α-(1→4) D-glucose units produced by partial hydrolysis of starch. In this work, the transesterification of the soluble polysaccharide with vinyl acrylate (VA) was carried out in anhydrous dimethylsulfoxide (DMSO). The effect of the water activity and of the enzyme Proleather, on the reaction rates, was analysed. Different degrees of substitution (DS) ranging from ca. 10% to 70% were obtained by controlling the molar ratio of VA to dextrin. Gels were obtained by free radical polymerization of dextrin-VA, with different degrees of substitution and monomer concentration, in water. A comprehensive solid state-NMR analysis of the hydrogels was performed. These hydrogels are being developed as scaffold materials for bioactive molecule and cell delivery, tissue engineering and a variety of other biomedical applications

Cradle-to-gate assessment of bacterial cellulose production

In an increasing environmental conscious world, Life Cycle Assessment (LCA) is an important tool for estimate environmental impact of processes and products. This work aims to determine the environmental impacts of the production of bacterial cellulose (BC) intended to be used as a food additive. BC is a biopolymer produced mainly by Gram-negative acetic acid bacteria such as Glunoacetobacter xylinum. BC structure consist in glucose monomers arranged in a unique nanostructure, exhibiting numerous great properties, leading to wide range of applications in different areas including as a food product or as food addictive, and in high-value-added niche markets such as medical applications and cosmetic industry[1]. The LCA (cradle-to-gate) was simulated in GaBi Software (ThinkStep), using ReCiPe 2016 method to estimate the Life Cycle Impact Assessment (LCIA). LCIA translates emissions and resource extractions into a limited number of environmental impact scores (environmental impact categories) by means of the so-called characterization factors. The study includes not only the production of BC, the energy and the equipment used, but also the extraction of natural resources and their transformation and the disposal of the produced waste (Cradle-to-gate). The functional unit of this LCA was defined as 1 kg of BC packed in a plastic container and a carton box. Results presented in 18 midpoints impact categories show that the sodium hydroxide, carton package, and plastic package productions are responsible for considerable impacts. The production of sodium hydroxide and carton package are also responsible for considerable amount of water resources and emissions to the environment.info:eu-repo/semantics/publishedVersio

Characterization of dextrin hydrogels by FTIR spectroscopy and solid state NMR spectroscopy

Fourier transform infrared (FTIR) and 13C solid state nuclear magnetic resonance (NMR) spectroscopy were used to study dextrin structural changes occurring upon hydrogel formation by vinyl acrylate (VA) grafting and subsequent free radical polymerization. The degrees of VA substitution (DS) and polymerization (DP) were quantified up to 40%VA by FTIR intensity measurements and partial least squares (PLS)/FTIR, the latter being a faster and less error-prone method. Above 40%VA, both parameters are underestimated by FTIR. A spin counting NMR experiment showed high carbon observabilities for hydrogels and improved PLS/NMR models were achieved for DS and DP determination. Alternative NMR integration methods are hindered by the broad VA peaks and need for area correction, due to their CP dynamics. NMR changes in C1 profile showed that a single helical conformation predominates at lower %VA, being replaced by disordered conformations as %VA increases. Furthermore, a correlation FTIR/NMR study indicated that ring conformations are significantly affected in hydrogels, compared to unpolymerized dextrin.Funding is acknowledged from the Foundation for Science and Technology, Portugal, for project POCI/QUI/ 56229/2004 and grants for H.G. and C.G. (SFRH/22242/ 2005). The authors also wish to acknowledge CICECO for partial funding of this work.Fundação para a Ciência e a Tecnologia (FCT

Economic & environmental assessment of bacterial cellulose production

MultiBiorefinery (SAICTPAC/0040/2015 - POCI-01-0145-FEDER-016403)NORTE-01-0145-FEDER-00000

Life cycle assessment (cradle-to-gate) of bacterial cellulose production

In a world, increasingly conscious of environmental issues, Life Cycle Assessment (LCA) is an important tool for estimating the environmental impact of processes and products. This work aims to determine the possible environmental impacts of the production of bacterial cellulose intended to be used as a food additive. For this, the LCA (cradle-to-gate) was simulated with GaBi Software, using ReCiPe 2016 method. Results show that the production of materials was responsible for the major part of the input and output flows (in kg). Wastewater treatment, materials production and cooling and heating agents production are responsible for major impacts in several impact categories.info:eu-repo/semantics/publishedVersio

Recycling of cellulases in lignocellulosic hydrolysates using alkaline elution

The recovery of cellulases from lignin, lignocellulosic hydrolysates and cellulose by alkaline washes at pH 9 and 10 was examined. The effect of the pH on the structural stability of purified Cel7A was analyzed by circular dichroism. Purified Cel7A showed conformational changes at pH 9 and 10 that were reversible at pH 4.8. Temperature influenced the enzymatic hydrolysis of wheat straw and may be critical for the efficiency of cellulase recycling from wheat straw hydrolysates. Operation at moderate temperatures (37 °C) resulted in a rate of saccharification 19% higher than that obtained at 50° C, improving cellulase recycling by 49%. Over 60% of the enzyme activity on the synthetic substrate 4-methylumbelliferyl-β-d-cellobioside (MUC) may be recovered by using a simple alkaline wash. This is thus a promising strategy for enzyme recycling that is simple to implement at industrial scale, economical and effective.The authors acknowledge funding through FP7 KACELLE (Kalundborg Cellulosic Ethanol) project for supporting his work. We also thank Dr. Mai Ostergaard Haven for critical reviewing of the manuscript and Dr. Jane Lindedam for supplying the lignin

Hyaluronic acid-amphotericin B nanocomplexesa: a promising anti-leishmanial targeted drug delivery system

Leishmaniasis has been classified as one of the most neglected tropical diseases, causing 50 thousand deaths and 1.5 to 2 million new cases every year, according to the World Health Organization. This disease, promoted by protozoan parasites of the genus Leishmania, has a high incidence affecting 89 countries worldwide. Nowadays, current treatment strategies still rely on the antifungal agent amphotericin B (AmB) but are rather inadequate due to the high prevalence of the disease within low-income population of sub-developed regions, the intracellular location of the parasite and the emergence of parasite resistance. Thus, other strategies have been pursued to improve the therapeutic efficacy and to reduce the toxicity of AmB such as the use of biocompatible polysaccharides as carriers. In this work, a simple and inexpensive production process using hyaluronic acid (HA, 50 kDa) was used in order to develop water-soluble hyaluronic acid-amphotericin B nanocomplex (HA-AmB). HA is the main ligand of CD44 receptor, thus being favorably internalized by macrophages that overexpress this receptor upon infection. Therefore, HA arises as a suitable polysaccharide to target the AmB delivery to the leishmania-infected macrophages. The nanocomplex, obtained by simply processing the mixture of the polysaccharide with the drug in a nanospray dryer (HA-AmB SD), was characterized in terms of size/zeta potential (DLS) and morphology (SEM and Cryo-SEM). Furthermore, an HPLC-MS detection method was optimized and used to determine the AmB content in the nanocomplex. Also, to ascertain the interaction between AmB and the HA, FTIR, DSC and PXRD analysis were performed. Cytotoxic and hemolytic effects were assessed on different cell lines through the resazurin test and in dogs blood, respectively. Anti-leishmanial activity was assessed in vitro in axenic cultures of Leishmania by resazurin and in infected bone marrow-derived macrophages (BMM) stained with different fluorescent probes using high-content microscopy. Our results shown that the produced material has a spherical morphology in aqueous solution with a mean hydrodynamic diameter of 318.4 ± 34.7 nm and low polydispersity (0.239 ± 0.02). Moreover, this material that presents an AmB content of 13.56 ± 3.49 %, has a good colloidal stability due to the highly negative surface charge (-39.45 ± 1.12 mV). DSC and PXRD analysis strongly suggested the formation of an amorphous inclusion complex between AmB and the complex polysaccharide chain networks, explaining the high solubility of the drug in water. The in vitro assays showed that compared to free-AmB, the nanocomplex had significantly less cytotoxicity against BMM and HEK293T cell lines, significant less hemolytic effect and inhibited the infection in the Leishmania-infected BMM. Exploratory in vivo assays are being conducted in mice. In conclusion, this work has shown that the hyaluronic acid-AmB nanocomplex is a promising system for the treatment of Leishmaniasis, possessing similar effects to the free-AmB against Leishmania-infected macrophages and Leishmania axenic cultures, with reduced cytotoxicity. Given the affordability, simplicity, low-toxicity and facile scale up of the developed formulation, the hyaluronic acid-AmB nanocomplex may represent an alternative to the expensive nanoformulations available.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for supporting this study under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01- 0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Ricardo Silva-Carvalho also acknowledges FCT for the PhD scholarship SFRH/BD/118880/2016.info:eu-repo/semantics/publishedVersio

On to the impact of low cost substrates for BNC production

Bacterial nanocellulose (BNC) is an exopolysaccharide produced by certain acetic acid bacteria. It has high crystallinity, high mechanical strength, high purity and high water-holding capacity. These properties make it useful in making artificial skin (1), electronic paper, composite reinforcement, development of food and cosmetic applications (2). The cost of fermentation media is believed to contribute significantly to the operational costs, especially if synthetic commercial media are used. Hence, much research on BNC production using low-cost substrates has been done focusing on lowering the production costs (3). Also, to meet the requirement for industrial applications, effective large-scale BNC production systems need to be developed, which involves improving the fermentation conditions and identifying high yield BNC-producing strains (4). However, as with many fermentation systems, while promoting the recycling of low value-added products, the use of complex substrates may in fact represent a bottleneck in the BNC fermentation processes. Some of these substrates present, comparatively to synthetic nutrients, high chemical oxygen demand (COD), total and volatile solids (TS and VS), total nitrogen (TN), antimicrobial components (such as phenols) Consequently, these alternative substrates may place an economic problem either downhill, due to the need for wastewaters treatments and/or, uphill, due to the need of substrates pre-treatment. In this work, the optimization of alternative BNC culture medium (Molasses-Corn Steep Liquor, MOL-CSL), using Response surface methodology central composite design was used to evaluate the effect of inexpensive and widely available nutrients sources, namely MOL, ethanol (EtOH), CSL and ammonium sulphate on BNC production yield under static culture by komagataeibacter xylinus BPR 2001. The optimized parameters for maximum BNC production were: % (m/v): MOL 5.38, CSL 1.91, ammonium sulphate 0.63, disodium phosphate 0.270, citric acid 0.115 and ethanol 1.38 % (v/v). The maximum BNC production yield were 7.5 ± 0.54 g/L versus 1.79 ± 0.04 g/L for MOL-CSL and synthetic medium (HS-EtOH) culture medium, respectively. The resulting wastewater from each culture medium was characterized regarding COD, TN, TS and VS, leading to the conclusion that the wastewaters generated using MOL-CSL are more heavily charged with organic matter, increasing the final costs of BNC production due to the higher costs associated to wastewater treatment. Anaerobic digestion (AD) was studied for wastewater treatment and biogas production from the wastewaters of the BNC fermentation and purification process. Finally, a preliminary Life Cycle Assessment of BNC production was performed and will be presented.info:eu-repo/semantics/publishedVersio

Numerical study of the unsteady flow in simplified and realistic iliac bifurcation models

Cardiovascular diseases are a major cause of death and disability worldwide and they are commonly associated with the occurrence of atherosclerotic plaque deposition in the vessel walls, a process denoted as atherosclerosis. This is a chronic and progressive inflammatory disease of large-/medium-sized blood vessels that affects blood flow profiles, with the abdominal aorta and its branches being one of the locations prone to the development of this pathology, due to their curvatures and bifurcations. In this regard, the effect of flow patterns was studied and compared for both a simplified three-dimensional model of aorta bifurcation on the iliac arteries and a realistic model of iliac bifurcation, which was constructed from a computational tomography medical image. The flow patterns were analyzed in terms of velocity and wall shear stress distribution, but a special focus was given to the size and location of the recirculation zone. The simulations were performed using the Computational Fluid Dynamics software, FLUENT, taking into account the cardiac cycle profile at the infrarenal aorta. The shear stress and the velocity distribution observed for both models indicated that higher shear stress occurred along the flow divider wall (inner wall) and low shear stress occurred along the outer walls. In addition, the results demonstrated that the wall shear stress profiles were deeply affected by the transient profile of the cardiac cycle, with the deceleration phase being the most critical phase to the occurrence of backflow.This work was supported by FCT—Fundação para a Ciência e Tecnologia through the R&D Units Project Scope: UIDB/00319/2020, UIDB/04077/2020, and NORTE-01-0145-FEDER-030171, funded by COMPETE2020, NORTE 2020, PORTUGAL 2020, and FEDER