Acidophilic heterotrophs: basic aspects and technological applications.

Acidophiles comprise a group of microorganisms adapted to live in acidic environments. Despite acidophiles are usually associated with an autotrophic metabolism, more than 80 microorganisms capable of utilizing organic matter have been isolated from natural and man-made environments. The ability to reduce soluble and insoluble iron compounds has been described for many of these species and may be harnessed to develop new or improved mining processes when oxidative bioleaching is ineffective. Similarly, as these microorganisms grow in highly acidic media and the chances of contamination are reduced by the low pH, they may be employed to implement robust fermentation processes. By conducting an extensive literature review, this work presents an updated view of basic aspects and technological applications in biomining, bioremediation, fermentation processes aimed at biopolymers production, microbial electrochemical systems, and the potential use of extremozymes.

K2CO3-Modified Smectites as Basic Catalysts for Glycerol Transcarbonation to Glycerol Carbonate.

A novel and cost-effective heterogeneous catalyst for glycerol carbonate production through transesterification was developed by impregnating smectite clay with K2CO3. Comprehensive structural and chemical analyses, including X-ray diffraction Analysis (XRD), Scanning Electron Microscopy (SEM)-Electron Dispersion Spectroscopy (EDS), Fourier Transform Infrared Spectroscopy (FTIR), and Brunauer-Emmett-Teller (BET) surface area analysis measurements, were employed to characterize the catalysts. Among the various catalysts prepared, the one impregnated with 40 wt% K2CO3 on smectite and calcined at 550 °C exhibited the highest catalytic activity, primarily due to its superior basicity. To enhance the efficiency of the transesterification process, several reaction parameters were optimized, including the molar ratio between propylene carbonate and glycerol reactor loading of the catalyst and reaction temperature. The highest glycerol carbonate conversion rate, approximately 77.13% ± 1.2%, was achieved using the best catalyst under the following optimal conditions: 2 wt% reactor loading, 110 °C reaction temperature, 2:1 propylene carbonate to glycerol molar ratio, and 6h reaction duration. Furthermore, both the raw clay and the best calcined K2CO3-impregnated catalysts demonstrated remarkable stability, maintaining their high activity for up to four consecutive reaction cycles. Finally, a kinetic analysis was performed using kinetic data from several runs employing raw clay and the most active K2CO3-modified clay at different temperatures, observing that a simple reversible second-order potential kinetic model of the quasi-homogeneous type fits perfectly to such data in diverse temperature ranges.

Hereditary Optic Neuropathies: A Systematic Review on the Interplay between Biomaterials and Induced Pluripotent Stem Cells.

Hereditary optic neuropathies (HONs) such as dominant optic atrophy (DOA) and Leber Hereditary Optic Neuropathy (LHON) are mitochondrial diseases characterized by a degenerative loss of retinal ganglion cells (RGCs) and are a cause of blindness worldwide. To date, there are only limited disease-modifying treatments for these disorders. The discovery of induced pluripotent stem cell (iPSC) technology has opened several promising opportunities in the field of HON research and the search for therapeutic approaches. This systematic review is focused on the two most frequent HONs (LHON and DOA) and on the recent studies related to the application of human iPSC technology in combination with biomaterials technology for their potential use in the development of RGC replacement therapies with the final aim of the improvement or even the restoration of the vision of HON patients. To this purpose, the combination of natural and synthetic biomaterials modified with peptides, neurotrophic factors, and other low- to medium-molecular weight compounds, mimicking the ocular extracellular matrices, with human iPSC or iPSC-derived cell retinal progenitors holds enormous potential to be exploited in the near future for the generation of transplantable RGC populations.

Overcoming Biochemical Limitations of Galactose Oxidase through the Design of a Solid-Supported Self-Sufficient Biocatalyst.

Galactose Oxidase (GalOx) has gained significant interest in biocatalysis due to its ability for selective oxidation beyond the natural oxidation of galactose, enabling the production of valuable derivatives. However, the practical application of GalOx has been hindered by the limited availability of active and stable biocatalysts, as well as the inherent biochemical limitations such as oxygen (O2 ) dependency and the need for activation. In this study, we addressed these challenges by immobilizing GalOx into agarose-based and Purolite supports to enhance its activity and stability. Additionally, we identified and quantified the oxygen supply limitation into solid catalysts by intraparticle oxygen sensing showing a trade-off between the amount of protein loaded onto the solid support and the catalytic effectiveness of the immobilized enzyme. Furthermore, we coimmobilized a heme-containing protein along with the enzyme to function as an activator. To evaluate the practical application of the immobilized GalOx, we conducted the oxidation of galactose in an instrumented aerated reactor. The results showcased the efficient performance of the immobilized enzyme in the 8 h reaction cycle. Notably, the GalOx immobilized into dextran sulfate-activated agarose exhibited improved stability, overcoming the need for a soluble activator supply, and demonstrated exceptional performance in galactose oxidation. These findings offer promising prospects for the utilization of GalOx in technical biocatalytic applications.

Synthesis of Glycerol Carbonate from Ethylene Carbonate Using Zinc Stearate as a Catalyst: Operating Conditions and Kinetic Modeling.

With the advent of biodiesel as a substitute/additive for diesel, the production of glycerol has experienced an increase, as it is an unavoidable byproduct of the biodiesel process; therefore, novel products and processes based on this triol are being very actively researched. Glycerol carbonate emerges as an advanced humectant from glycerol and a monomer for diverse polycarbonates. Its production in high yields and amounts can be achieved through the solventless transcarbonation of glycerol with other organic carbonates driven by alkaline catalysts, standing out amongst the cyclic carbonates due to its reactivity. Here, we have studied the main operational variables that affect the transcarbonation reaction of glycerol and ethylene carbonate catalyzed by zinc stearate: catalyst concentration, reagent molar ratio, and temperature. Subsequently, an appropriate kinetic model was fitted to all data obtained at 80 °C and several catalyst concentrations as well as reagent molar ratios. Finally, the selected kinetic model was extended and validated by fitting it to data obtained at several temperatures, finding that the activation energy of this reaction with this catalyst is around 69.2 kJ·mol-1. The kinetic model suggests that the reaction is bimolecular and elemental and that the process is interfacial in essence, with the catalyst dispersed in a narrow space between polar (glycerol) and nonpolar (ethylene carbonate) phases.

Chemical Reaction Engineering to Understand Applied Kinetics in Free Enzyme Homogeneous Reactors.

Chemical reaction engineering is interested in elucidating the reaction kinetics through the determination of the fundamental influencing variables. The understanding of enzyme kinetics is needed to implement the potential of enzymes to satisfy determined production targets and for the design of the reactor. The quantification of the enzyme kinetics is implemented by the elucidation and building of the kinetic model (it includes one or more kinetic equations). In the context of process development, the kinetic model is not only useful to identify feasibility and for optimizing reaction conditions but also, at an early stage of development it is very useful to anticipate implementation bottlenecks, and so guide reactor setup. In this chapter we describe theoretical and practical considerations to illustrate the methodological framework of kinetic analysis. We take as study cases four archetypal kinetic cases by using as example the hydrolysis of cellobiose catalyzed by a beta-glucosidase. We show the different experimental data that can be obtained by the monitoring of enzymatic reactions in different configuration of free enzyme homogeneous ideal reactors; we show step-by-step the visualization, treatment, and analysis of data to elucidate kinetic models and the procedure for the quantification of kinetic constants. Finally, the performance of different reactors is compared in the interplay with the enzyme kinetics. This book chapter aims at being useful for a broad multidisciplinary audience and different levels of academic development.

A simple procedure to obtain a medium-size oligogalacturonic acids fraction from orange peel and apple pomace wastes.

Pectin oligosaccharides, which can be obtained from fruit wastes, have proven their potential as plant immune-system elicitors. Although the precise size of active species is still under investigation, medium size oligosaccharides have been reported as the most active. Three defined oligogalacturonic acid (OGAs) mixtures were produced from commercial pectin, orange peel and apple pomace residues. The methodology developed involves two sequential acid treatments followed by stepwise ethanol precipitation. Without the need of chromatographic separations, three different fractions were obtained. The fractions were analyzed by high performance anion exchange chromatography (HPAEC) and were completely characterized by mass spectrometry, showing that the small size, medium size and large size fractions contained OGAs of degree of polymerization 3 to 9, 6 to 18, and 16 to 55, respectively.

Production of MCM-41 Nanoparticles with Control of Particle Size and Structural Properties: Optimizing Operational Conditions during Scale-Up.

The synthesis of Mobil Composition of Matter 41 (MCM-41) mesoporous silica nanoparticles (MSNs) of controlled sizes and porous structure has been performed at laboratory and pilot plant scales. Firstly, the effects of the main operating conditions (TEOS -Tetraethyl ortosilicate- addition rate, nanoparticle maturation time, temperature, and CTAB -Cetrimonium bromide- concentration) on the synthesis at laboratory scale (1 L round-bottom flask) were studied via a Taguchi experimental design. Subsequently, a profound one-by-one study of operating conditions was permitted to upscale the process without significant particle enlargement and pore deformation. To achieve this, the temperature was set to 60 °C and the CTAB to TEOS molar ratio to 8. The final runs were performed at pilot plant scale (5 L cylindrical reactor with temperature and stirring speed control) to analyze stirring speed, type of impeller, TEOS addition rate, and nanoparticle maturation time effects, confirming results at laboratory scale. Despite slight variations on the morphology of the nanoparticles, this methodology provided MSNs with adequate sizes and porosities for biomedical applications, regardless of the reactor/scale. The process was shown to be robust and reproducible using mild synthesis conditions (2 mL⋅min-1 TEOS addition rate, 400 rpm stirred by a Rushton turbine, 60 min maturation time, 60 °C, 2 g⋅L-1 CTAB, molar ratio TEOS/CTAB = 8), providing ca. 13 g of prismatic short mesoporous 100-200 nm nanorods with non-connected 3 nm parallel mesopores.

Enzymatic hydrolysis of several pretreated lignocellulosic biomasses: Fractal kinetic modelling.

Enzymatic hydrolysis of three pre-treated lignocellulosic biomasses -LCB- (wheat straw-WS-, corn stover-CSV- and cardoon stems -CS-) is studied. These biomasses were pre-treated by two methods: diluted sulfuric acid and acid ethanol-water extraction at six severity levels (H values). Pretreated solid fractions were hydrolyzed with commercial enzyme cocktails at standard conditions. A first-order kinetic fractal model was fitted to the experimental results. This model accurately describes the hydrolysis of all biomasses at all pre-treatment conditions studied. The results show that the formal first-order kinetic constant k depends on the biomass nature. The hydrolysis rate increases as the pre-treatment severity does, while the fractal exponent value h decreases. With these pre-treatments, and in terms of k and h, WS is highly reactive and, at medium H with EW pretreatment, highly accessible; CSV has a low reactivity and high accessibility and CS has the lowest reactivity and an increasing accessibility as severity rises.

High 2,3-butanediol production from glycerol by Raoultella terrigena CECT 4519.

Bioconversion of biodiesel-derived glycerol into 2,3-butanediol has received recently much attention due to its increasing surplus and its multiple uses in industry as bulk chemical. The influence of initial glycerol concentration on 2,3-butanediol production in batch runs has been studied. A concentration higher than 140 g/L produces an inhibitory effect on the final 2,3-butanediol concentration and its production rate. In batch mode, the highest yield respect to the theoretical maximum yield (71%) was reached employing 140 g/L as initial concentration 140 g/L. Based on these results, a high 2,3-butanediol production has been achieved through a fed-batch strategy. The reached 2,3-butanediol concentration was 90.5 g/L from pure glycerol and 80.5 g/L from raw glycerol. The 2,3-butanediol yield respect to the theoretical maximum yield was also improved through the fed-batch operation (90%). To date, this concentration is the highest produced amount employing as biocatalyst a non-pathogenic bacterium (level 1).

Liquor re-use strategy in lignocellulosic biomass fractionation with ethanol-water mixtures.

Liquor recycle in lignocellulosic biomass fractionation with ethanol-water has been studied. Runs have been carried out in a 6 L tank reactor with liquor recirculation. The liquors obtained in six successive fractioning operations have been analyzed together with the solid phase remnant. Experimental results revealed that the number of re-uses reduces solids recovery (from 52.2 to 42.6%) and cellulose recovery (from 28.1 to 23.3%) with minor or no effect on the hemicelluloses and lignin removal. The more remarkable effect is an increase of the glucose yield (from 76.7 to 95.3% after enzymatic hydrolysis during 72 h). The accumulation of acetic acid in the spent liquors (until 1.3 g/L) seems to be responsible of the higher enzymatic hydrolysis yield, from 76.3 (first use) to 87.7% (fifth re-use). Liquor re-use is effective to improve the sustainability of the pre-treatment obtaining a cellulose-rich solid easy to hydrolysate to sugars reducing energy consumption.

Thermal and operational deactivation of Aspergillus fumigatus ß-glucosidase in ethanol/water pretreated wheat straw enzymatic hydrolysis.

The stabilization effects on a novel commercial ß-glucosidase preparation from Aspergillus fumigatus during saccharification of ethanol-water pretreated wheat straw were analysed in comparison to this enzyme stability during cellobiose hydrolysis. For this purpose, the kinetics of ß-glucosidase residual activity during cellobiose hydrolysis from 40 till 70 °C were studied, resulting in the fitting of a first-order partial deactivation model. Furthermore, a subsequent fitting of a kinetic model including this first-order deactivation equation and a Michaelis-Menten equation with double competitive inhibition by glucose and uncompetitive inhibition by cellobiose to released glucose was successful. Finally, global enzyme deactivation and prospective deactivation of enzyme remaining in the liquid phase were evaluated during wheat straw hydrolysis at 50 °C as a relevant saccharification process. Results suggest that the presence of a solid substrate dramatically reduces the global deactivation rate of the enzyme and, in addition, there is no loss the stability of the enzyme in the liquid phase along the saccharification process, even for 72 h.

Pre-treatment of corn stover, Cynara cardunculus L. stems and wheat straw by ethanol-water and diluted sulfuric acid: Comparison under different energy input conditions.

Ethanol-water (EW) and diluted sulfuric acid (DSA) pre-treatment have been studied for lignocellulosic biomass (corn stover, Cynara cardunculus L. stems and wheat straw). Both pre-treatments have been compared taken into account: solids recovery, glucans recovery, xylans removed, delignification and glucose yield. In all cases, the amount of energy involved has been taken as a criterion for sustainability. In general terms, EW is more efficient to remove lignin and DSA more appropriate to hydrolysate xylans. The combined effect of delignification and xylans removal is responsible for the improvement in the enzymatic cellulose hydrolysis. Under conditions of moderate-low energy inputs, EW pre-treatment yields better results than DSA with glucose yields in the range of 50-60% for EW pre-treated corn stover and cardoon stems; while wheat straw pulps reach up to 80%. So, multiple raw materials biorefinery needs a previous study to fit the type and conditions of the pre-treatment to each feedstock.

Physico-chemical kinetic modelling of hydrolysis of a steam-explosion pre-treated corn stover: A two-step approach.

A physico-chemical kinetic model for the hydrolysis of pre-treated corn stover is proposed. This model takes into account two reactions in series, the hydrolysis of cellulose to cellobiose and the production of glucose from cellobiose. Experiments have been carried out with an industrial enzymatic cocktail from Trichoderma reesei containing endo and exoglucanases and a very low activity of ß-glucosidase. Kinetic parameters were calculated by fitting the proposed model to experimental data of cellulose and glucose concentrations with time. The kinetic parameters fulfilled all relevant statistical and physical criteria. The kinetic model has been validated with published saccharification data regarding differently pre-treated corn stover and enzymatic cocktail, in this case with a very high ß-glucosidase activity (as it is common in modern industrial cellulase cocktails). In both cases, the kinetic model proposed could be fitted very appropriately to cellulose hydrolysis data.

Wheat straw fractionation by ethanol-water mixture: Optimization of operating conditions and comparison with diluted sulfuric acid pre-treatment.

The fractionation of wheat straw by ethanol-water (EW) pre-treatment was studied regarding its main operating conditions: time, temperature, L/S ratio and ethanol percentage were optimized by using an orthogonal experimental design (Taguchi). Afterwards, diluted sulfuric acid (DSA) hydrolysis and EW treatments have been compared in terms of energy consumption and yield of a cellulosic solid residue able to be enzymatically hydrolyzed to glucose. Experimental results show that temperature is the only variable of EW with a significant effect on the quality of the pretreated solids. EW pre-treatment of wheat straw is more effective than DSA hydrolysis due to its higher capacity of delignification. Moreover, a high glucose yield (80%) can be obtained by enzymatic hydrolysis of a solid pretreated with a moderate energy input EW (160 °C, 45 min) while wheat straw needs of a higher energy input during DSA to produce a similar yield of glucose after saccharification.

Modification of Bacterial Cellulose Biofilms with Xylan Polyelectrolytes.

The effect of the addition of two [4-butyltrimethylammonium]-xylan chloride polyelectrolytes (BTMAXs) on bacterial cellulose (BC) was evaluated. The first strategy was to add the polyelectrolytes to the culture medium together with a cell suspension of the bacterium. After one week of cultivation, the films were collected and purified. The second approach consisted of obtaining a purified and homogenized BC, to which the polyelectrolytes were added subsequently. The films were characterized in terms of tear and burst indexes, optical properties, surface free energy, static contact angle, Gurley porosity, SEM, X-ray diffraction and AFM. Although there are small differences in mechanical and optical properties between the nanocomposites and control films, the films obtained by BC synthesis in the presence of BTMAXs were remarkably less opaque, rougher, and had a much lower specular gloss. The surface free energy depends on the BTMAXs addition method. The crystallinity of the composites is lower than that of the control material, with a higher reduction of this parameter in the composites obtained by adding the BTMAXs to the culture medium. In view of these results, it can be concluded that BC-BTMAX composites are a promising new material, for example, for paper restoration.

Study on the effects of several operational variables on the enzymatic batch saccharification of orange solid waste.

In this work, batch enzyme-aided extraction and enzymatic saccharification of blade-milled orange waste was studied. The operation variables for this process were thoroughly analysed. It was determined that batch runs with initial pH values of 5.0 and 5.2 controlled during the first hour, 50°C and 300-500r.p.m. agitation resulted in the best yields, with a limited total and partial first-order enzyme deactivation (for cellulases and polygalacturonidase, respectively). Orange peel waste (OPW) at 6.7% w/w dry solid, 0.22 filter paper units (FPU)/g DS and proportional activities of other enzymes led to over 40g/L free monosaccharides and global yields to glucose over 80%. When using 10.1% w/w DS in these conditions, glucose yield was 63%, with total monosaccharide concentration on top of 50g/L. Similar concentrations were obtained by additional partial drying of OPW to 60% humidity at DS/L ratios near 7.5% (glucose yield >80%).

Enzymatic saccharification of acid pretreated corn stover: Empirical and fractal kinetic modelling.

Enzymatic hydrolysis of corn stover was studied at agitation speeds from 50 to 500rpm in a stirred tank bioreactor, at high solid concentrations (20% w/w dry solid/suspension), 50°C and 15.5mgprotein·gglucane(-1). Two empirical kinetic models have been fitted to empirical data, namely: a potential model and a fractal one. For the former case, the global order dramatically decreases from 13 to 2 as agitation speed increases, suggesting an increment in the access of enzymes to cellulose in terms of chemisorption followed by hydrolysis. For its part, the fractal kinetic model fits better to data, showing its kinetic constant a constant augmentation with increasing agitation speed up to a constant value at 250rpm and above, when mass transfer limitations are overcome. In contrast, the fractal exponent decreases with rising agitation speed till circa 0.19, suggesting higher accessibility of enzymes to the substrate.

Influence of fluid dynamic conditions on enzymatic hydrolysis of lignocellulosic biomass: Effect of mass transfer rate.

The effect of fluid dynamic conditions on enzymatic hydrolysis of acid pretreated corn stover (PCS) has been assessed. Runs were performed in stirred tanks at several stirrer speed values, under typical conditions of temperature (50°C), pH (4.8) and solid charge (20% w/w). A complex mixture of cellulases, xylanases and mannanases was employed for PCS saccharification. At low stirring speeds (<150rpm), estimated mass transfer coefficients and rates, when compared to chemical hydrolysis rates, lead to results that clearly show low mass transfer rates, being this phenomenon the controlling step of the overall process rate. However, for stirrer speed from 300rpm upwards, the overall process rate is controlled by hydrolysis reactions. The ratio between mass transfer and overall chemical reaction rates changes with time depending on the conditions of each run.

Characterization of purified bacterial cellulose focused on its use on paper restoration.

Bacterial cellulose (BC) synthesized by Gluconacetobacter sucrofermentans CECT 7291 seems to be a good option for the restoration of degraded paper. In this work BC layers are cultivated and purified by two different methods: an alkaline treatment when the culture media contains ethanol and a thermal treatment if the media is free from ethanol. The main goal of these tests was the characterization of BC layers measured in terms of tear and burst indexes, optical properties, SEM, X-ray diffraction, FTIR, degree of polymerization, static and dynamic contact angles, and mercury intrusion porosimetry. The BC layers were also evaluated in the same terms after an aging treatment. Results showed that BC has got high crystallinity index, low internal porosity, good mechanical properties and high stability over time, especially when purified by the alkaline treatment. These features make BC an adequate candidate for degraded paper reinforcement.