Exploring the roles of starch for microbial encapsulation through a systematic mapping review.

Microorganism encapsulation protects them from stressful conditions and assists in maintaining their viability, being especially beneficial when the carrier material is a renewable and biodegradable biopolymer, such as starch. Here, a systematic mapping was performed to provide a current overview on the use of starch-based systems for microbial encapsulation. Following well-established guidelines, a systematic mapping was conducted and the following could be drawn: 1) there was a significant increase in publications on microbial encapsulation using starch over the past decade, showing interest from the scientific community, 2) ionotropic gelation, emulsification and spray drying are the most commonly used techniques for starch-based microbial encapsulation, and 3) starch play important functions in the encapsulation matrix such as assisting in the survival of the microorganisms. The information gathered in this systematic mapping can be useful to guide researchers and industrial sectors on the development of innovative starch-based systems for microbial encapsulation.

Cellulose nanostructures obtained using enzymatic cocktails with different compositions.

Cellulose nanostructures obtained from lignocellulosic biomass by the enzymatic route can offer advantages in terms of material properties and processing sustainability. However, most of the enzymatic cocktails commonly used in the saccharification of biomass are designed to promote the complete depolymerization of the cellulose structure into soluble sugars. Here, investigation was made of the way that the action of different commercially available cellulase enzyme cocktails can affect the production of nanocellulose. For this, enzymatic cocktails designed for complete or partial saccharification were compared, using eucalyptus cellulose pulp as a model feedstock. The results showed that all the enzymatic cocktails were effective in the formation of nanocellulose structures, with the complete saccharification enzymes being more efficient in promoting the coproduction of glucose (36.5 g/L, 87% cellulose conversion). The presence of auxiliary enzymes, especially xylanases, acted cooperatively to favor the production of nanostructures with higher crystallinity (up to 79%), higher surface charge (zeta potential up to -30.9 mV), and more uniform dimensions within the size range of cellulose nanocrystals (80 to 350 nm). Interestingly, for the enzymatic cocktails designed for partial saccharification, the xylanase activity was more important than the endoglucanase activity in the production of nanocellulose with improved properties. The findings showed that the composition of the enzymatic cocktails already used for complete biomass saccharification can be suitable for obtaining nanocellulose, together with the release of a glucose stream, in a format compatible with the biorefinery concept.

Unveiling the Solubilization of Potassium Mineral Rocks in Organic Acids for Application as K-Fertilizer.

Organic acids produced by soil microorganisms can be useful to promote the release of potassium (K) from potassium mineral rocks (KR), but the complexity of low reactivity minerals limits K solubilization and their use as fertilizer. Here, we investigate the ways that different organic acids (gluconic, oxalic, and citric) can affect the solubilization of potassium minerals, in order to propose process strategies to improve their solubility. For this, evaluations were performed using the model minerals KRpolyhalite (sedimentary mineral), KRfeldspar (igneous mineral), and KCl (commercial fertilizer). For KCl and KRpolyhalite, complete solubilization was achieved using all the organic acids, while for KRfeldspar, the highest K+ solubilization (34.86 mg L-1) was achieved with oxalic acid. The solubility of KRfeldspar was further investigated under submerged cultivation with the filamentous fungus Aspergillus niger, as well as after a mechanochemical grinding treatment. The biotechnological route resulted in solubilized K up to 63.2 mg L-1. The mechanochemical route, on the other hand, increased the release of K by about 8.6 times (993 mg L-1) compared to the natural mineral, due to the greater fragmentation of the particles after the treatment (with a surface area about 2.5 times higher than for the in natura KRfeldspar). These findings demonstrated the potential of applying biotechnological and mechanochemical routes with organic acids to improve the solubilization of K present in low reactivity mineral rocks, indicating the possible use of these minerals in more sustainable agricultural practices.

Asbestos cement waste treatment through mechanochemical process with KH2PO4 for its utilization in soil pH correction and nutrient delivery.

The manufacture of asbestos materials has been banished worldwide due to their toxicity, but discarding the existing wastes remains a challenge. We investigated an alternative mechanochemical method to treat asbestos-cement materials by loading them with potassium and phosphorus from KH2PO4 during the milling process to obtain a product used as liming and soil conditioner. The results showed total asbestos fibrous elimination after 7 to 8 h of milling. The materials showed a slow-release fertilizer profile. The liming property is maintained when the asbestos-cement weight proportion used is equal to or higher than KH2PO4. A comparative soil experiment with limestone also indicates that lower doses of the K- and P-enriched detoxified asbestos cement were required to reach similar liming effects. Maize cultivation (greenhouse) was used to evaluate its performance showing higher biomass production for the sample loaded with potassium and phosphorous.

Wearable sensors made with solution-blow spinning poly(lactic acid) for non-enzymatic pesticide detection in agriculture and food safety.

On-site monitoring the presence of pesticides on crops and food samples is essential for precision and post-harvest agriculture, which demands nondestructive analytical methods for rapid, low-cost detection that is not achievable with gold standard methods. The synergy between eco-friendly substrates and printed devices may lead to wearable sensors for decentralized analysis of pesticides in precision agriculture. In this paper we report on a wearable non-enzymatic electrochemical sensor capable of detecting carbamate and bipyridinium pesticides on the surface of agricultural and food samples. The low-cost devices (

Multifunctional Ginger Nanofiber Hydrogels with Tunable Absorption: The Potential for Advanced Wound Dressing Applications.

In this study, ginger residue from juice production was evaluated as a raw material resource for preparation of nanofiber hydrogels with multifunctional properties for advanced wound dressing applications. Alkali treatment was applied to adjust the chemical composition of ginger fibers followed by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation prior to nanofiber isolation. The effect of alkali treatment on hydrogel properties assembled through vacuum filtration without addition of any chemical cross-linker was evaluated. An outstanding absorption ability of 6200% combined with excellent mechanical properties, tensile strength of 2.1 ± 0.2 MPa, elastic modulus of 15.3 ± 0.3 MPa, and elongation at break of 25.1%, was achieved without alkali treatment. Furthermore, the absorption capacity was tunable by applying alkali treatment at different concentrations and by adjusting the hydrogel grammage. Cytocompatibility evaluation of the hydrogels showed no significant effect on human fibroblast proliferation in vitro. Ginger essential oil was used to functionalize the hydrogels by providing antimicrobial activity, furthering their potential as a multifunctional wound dressing.

Cellulolytic enzymes production guided by morphology engineering.

Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. ß-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or ß-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.

Synergy of Aspergillus niger and Components in Biofertilizer Composites Increases the Availability of Nutrients to Plants.

Intensive fertilization has been required to provide nutrients for plant growth under the current agricultural practices being applied to meet the global food demands. Micronutrients such as zinc, manganese, and copper are required in small quantities when compared to macronutrients (such as nitrogen, phosphorus and potassium), but they are essential for the plant growth cycle and consequently for increasing productivity. Mineral oxides such as ZnO, MnO, and CuO are used in agriculture as micronutrient sources, but their low solubility limits practical applications in plant nutrition. Similarly, elemental sulfur (S0) can provide a high-concentration source of sulfate, but its availability is limited by the ability of the soil to promote S0 oxidation. We propose here the integration of these nutrients in a composite based on a biodegradable starch matrix containing mineral oxides and S0 in a dispersion that allowed encapsulation of the acidifying agent Aspergillus niger, a native soil fungus. This strategy effectively improved the final nutrient solubility, with the composite starch/S0/oxidemixture multi-nutrient fertilizer showing remarkable results for solubilization of the oxides, hence confirming a synergic effect of S0 oxidation and microbial solubilization. This composite exhibited an extended shelf life and soil-plant experiments with Italian ryegrass (Lolium multiflorum Lam.) confirmed high efficiencies for dry matter production, nutrient uptake, and recovery. These findings can contribute to the development of environmentally friendly fertilizers towards a more sustainable agriculture and could open up new applications for formulations containing poorly soluble oxide sources.

Relation between pellet fragmentation kinetics and cellulolytic enzymes production by Aspergillus niger in conventional bioreactor with different impellers.

The hydrodynamic environment in bioreactors affects the oxygen transfer rate and the shear conditions during microbial cultivations. Therefore, assessment of the effect of the hydrodynamic environment on cellular morphology can contribute to favoring the production of metabolites of interest. The aim of this work was to use image analysis in order to quantify the fragmentation of Aspergillus niger pellets in a conventional bioreactor operated using different impeller speeds, air flow rates, and impeller configurations including Rushton turbines and Elephant Ear impellers, with evaluation of the influence of the hydrodynamic environment on the production of cellulolytic enzymes. An empirical kinetic model was proposed to describe the dynamics of pellet fragmentation and quantify the shear conditions. The results showed that the agitation speed affected the dynamics of pellet fragmentation in two ways, by accelerating the damage process and by increasing the magnitude of the fragmentation. Both endoglucanase and ß-glucosidase production exhibited a linear relationship with the pellet fragmentation percentage, which was directly related to the shear conditions. Interestingly, ß-glucosidase production was favored under high shear conditions, while the highest endoglucanase production occurred under low shear conditions. These findings may be useful for defining suitable systems and operating conditions for the production of metabolites including enzymes in bioreactors, as well as defining conditions that favour a specific pre-determined enzyme cocktail.

Performance targets defined by retro-techno-economic analysis for the use of soybean protein as saccharification additive in an integrated biorefinery.

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.

ß-Mannanase Production Using Coffee Industry Waste for Application in Soluble Coffee Processing.

Soluble coffee offers the combined benefits of high added value and practicality for its consumers. The hydrolysis of coffee polysaccharides by the biochemical route, using enzymes, is an eco-friendly and sustainable way to improve the quality of this product, while contributing to the implementation of industrial processes that have lower energy requirements and can reduce environmental impacts. This work describes the production of hydrolytic enzymes by solid-state fermentation (SSF), cultivating filamentous fungi on waste from the coffee industry, followed by their application in the hydrolysis of waste coffee polysaccharides from soluble coffee processing. Different substrate compositions were studied, an ideal microorganism was selected, and the fermentation conditions were optimized. Cultivations for enzymes production were carried out in flasks and in a packed-bed bioreactor. Higher enzyme yield was achieved in the bioreactor, due to better aeration of the substrate. The best ß-mannanase production results were found for a substrate composed of a mixture of coffee waste and wheat bran (1:1 w/w), using Aspergillusniger F12. The enzymatic extract proved to be very stable for 24 h, at 50 °C, and was able to hydrolyze a considerable amount of the carbohydrates in the coffee. The addition of a commercial cellulase cocktail to the crude extract increased the hydrolysis yield by 56%. The production of ß-mannanase by SSF and its application in the hydrolysis of coffee polysaccharides showed promise for improving soluble coffee processing, offering an attractive way to assist in closing the loops in the coffee industry and creating a circular economy.

Hydroxyapatite nanoparticles modified with metal ions for xylanase immobilization.

Hydroxyapatite (HA) nanoparticles are promising materials for enzyme immobilization, since they provide a high specific surface area for enzyme loading and can also be modified with metal ions (HA-Me2+) to enable interaction with proteins. The adsorption of proteins on HA-Me2+ has been explored for purification purposes, while the use of this material as a support for the immobilization of enzymes remains to be further investigated. Xylanase is an enzyme of considerable industrial interest, being used in the biofuel, pharmaceutical, pulp, and food & beverage sectors, among others. The immobilization of xylanase can enable recovery of the enzyme after biocatalysis, so that it can be reused several times, hence reducing the costs of industrial processes. Here, a systematic study was performed of the immobilization of xylanase on HA nanoparticles modified with metal ions (Cu2+ and Ni2+). A simple, fast, and efficient immobilization protocol was established using statistical experimental design as a tool, generating derivatives by interactions involving complexation of metals of the support with electron donor groups of the enzyme. The affinity towards xylanase was higher for the HA-Cu2+ support, compared to HA and HA-Ni2+. The pH and temperature profiles for the immobilized enzyme activity remained the same as for the soluble enzyme, indicating that the xylanase did not undergo major changes in its conformational state after immobilization. The HA-Cu2+ support was the most effective in reuse assays, retaining up to 80% activity in the second cycle. The results showed that xylanase could be immobilized on HA nanoparticles modified with Cu2+ and Ni2+ metal ions, using a simple and effective method, indicating the promising potential of the system for applications in different industrial processes.

Phytase Immobilization on Hydroxyapatite Nanoparticles Improves Its Properties for Use in Animal Feed.

The enzyme phytase has important applications in animal feed, because it favors the bioavailability of phosphorus present in phytate, an antinutritional compound widely found associated with plant proteins. However, for feed applications, the phytase must withstand high temperatures during the feed pelleting process, as well as the gastrointestinal conditions of the animal. This work evaluates the feasibility of immobilizing phytase on hydroxyapatite (HA) nanoparticles, in order to improve its properties. HA is a material with excellent physicochemical characteristics for enzyme immobilization, and it can also act as an inorganic source of phosphorus and calcium in animal feed. The strong affinity of the phytase for the support resulted in rapid adsorption, with total immobilization yield and recovered activity greater than 100%. After immobilization, the phytase showed a broader activity profile in terms of pH and temperature, together with considerably higher thermoresistance at 80 and 90 °C. As a proof of concept, it was shown that the phytase immobilized on HA presented good resistance to acidic conditions and resistance to proteolysis when passing through simulated gastrointestinal conditions of fish. The findings showed that phytase immobilized onto HA presents suitable properties and has great potential for use in animal feed.

Correction to: Phytase Immobilization on Hydroxyapatite Nanoparticles Improves Its Properties for Use in Animal Feed.

In the original version of this article, under Calculation of Immobilization Parameters heading, the presentation of the equations are incorrect. The correct presentation of the equations are given below.

High cellulolytic activities in filamentous fungi isolated from an extreme oligotrophic subterranean environment (Catão cave) in Brazil.

Isolation and screening of new fungal strains from extreme and understudied environments, such as caves, is a promising approach to find higher yields enzyme producers. Cellulolytic fungal strains isolated from a Brazilian cave were evaluated for their enzymatic production after submerged (SmF) and solid-state fermentation (SSF). After SmF, three strains were selected for their high enzymatic activities: Aspergillus ustus for endoglucanase (4.76 U/mg), Talaromyces bruneus for ß-glucosidase (11.71 U/mg) and Aspergillus sp. (CBMAI 1926) for total cellulase (1.70 U/mg). After SSF, these strains, showed better yields compared to the reference strain Aspergillus niger 3T5B8. Aspergillus sp. (CBMAI 1926) stood out as a new species that expressed activity of total cellulases (0.10 U/mg) and low protein concentration (0.44 mg/mL). In conclusion, these isolated strains have a more efficient and promising cellulolytic enzyme complex that can be used in fermentation and saccharification processes with a lower protein concentration and a higher enzymatic activity than the reference strain. Therefore, beside the new genetic material characterized, our study highlights the benefits of cave extreme environments exploitation to find new potentially valuable strains.

Adaptive laboratory evolution of nanocellulose-producing bacterium.

Adaptive laboratory evolution through 12 rounds of culturing experiments of the nanocellulose-producing bacterium Komagataeibacter hansenii ATCC 23769 in a liquid fraction from hydrothermal pretreatment of corn stover resulted in a strain that resists inhibition by phenolics. The original strain generated nanocellulose from glucose in standard Hestrin and Schramm (HS) medium, but not from the glucose in pretreatment liquid. K. hansenii cultured in pretreatment liquid treated with activated charcoal to remove inhibitors also converted glucose to bacterial nanocellulose and used xylose as carbon source for growth. The properties of this cellulose were the same as nanocellulose generated from media specifically formulated for bacterial cellulose formation. However, attempts to directly utilize glucose proved unsuccessful due to the toxic character of the lignin-derived phenolics, and in particular, vanillan and ferulic acid. Adaptive laboratory evolution at increasing concentrations of pretreatment liquid from corn stover in HS medium resulted in a strain of K. hansenii that generated bacterial nanocellulose directly from pretreatment liquids of corn stover. The development of this adapted strain positions pretreatment liquid as a valuable resource since K. hansenii is able to convert and thereby concentrate a dilute form of glucose into an insoluble, readily recovered and value-added product-bacterial nanocellulose.

Alternative Low-Cost Additives to Improve the Saccharification of Lignocellulosic Biomass.

A potential strategy to mitigate problems related to unproductive adsorption of enzymes onto lignin during the saccharification of lignocellulosic biomass is the addition of lignin-blocking agents to the hydrolysis reaction medium. However, there is a clear need to find more cost-effective additives for use in large-scale processes. Here, selected alternative low-cost additives were evaluated in the saccharification of steam-exploded sugarcane bagasse using a commercial enzymatic cocktail. The addition of soybean protein, tryptone, peptone, and maize zein had positive effects on glucose release during the hydrolysis, with gains of up to 36% when 8% (w/w) soybean protein was used. These improvements were superior to those obtained using bovine serum albumin (BSA), a much more expensive protein that has been widely reported for such an application. Moreover, addition of soybean protein led to a saving of 48 h in the hydrolysis, corresponding to a 66% decrease in the reactor operation time required. In order to achieve the same hydrolysis yield without the soybean additive, the enzyme loading would need to be increased by 50%. FTIR spectroscopy and nitrogen elemental analysis revealed that the additives probably acted to reduce unproductive binding of cellulolytic enzymes onto the lignin portion of the sugarcane bagasse.

Biocatalyst engineering of Thermomyces Lanuginosus lipase adsorbed on hydrophobic supports: Modulation of enzyme properties for ethanolysis of oil in solvent-free systems.

Different immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL) exhibited different properties for the ethanolysis of high oleic sunflower oil in solvent-free systems. TLL immobilized by interfacial adsorption on octadecyl (C-18) supports lost its 1,3-regioselectivity and produced more than 99% of ethyl esters. This reaction was influenced by mass-transfer limitations. TLL adsorbed on macroporous C-18 supports (616 Å of pore diameter) was 10-fold more active than TLL adsorbed on mesoporous supports (100-200 Å of pore diameter) in solvent-free systems. Both derivatives exhibited similar activity when working in hexane in the absence of diffusional limitations. In addition, TLL adsorbed on macroporous Purolite C-18 was 5-fold more stable than TLL adsorbed on mesoporous Sepabeads C-18. The stability of the best biocatalyst was 20-fold lower in anhydrous oil than in anhydrous hexane. Mild PEGylation of immobilized TLL greatly increased its stability in anhydrous hexane at 40 °C, fully preserving the activity after 20 days. In anhydrous oil at 40 °C, PEGylated TLL-Purolite C-18 retained 65% of its initial activity after six days compared to 10% of the activity retained by the unmodified biocatalyst. Macroporous and highly hydrophobic supports (e.g., Purolite C-18) seem to be very useful to prepare optimal immobilized biocatalysts for ethanolysis of oils by TLL in solvent-free systems.

Nanoimmobilization of ß-glucosidase onto hydroxyapatite.

ß-Glucosidase is an enzyme of great industrial interest that is used in biorefineries and in the pharmaceutical, food, and beverage sectors, among others. These industrial processes would benefit from the use of immobilized enzyme systems that allow several reuses of the enzyme. A promising inorganic and nontoxic material for such application is hydroxyapatite (HA), which can be synthesized at nanometric scale, hence providing good accessibility of the substrate to the catalyst. Here, we carried out a systematic study to evaluate the feasibility of immobilizing ß-glucosidase on HA nanoparticles. The immobilization process was highly effective over wide ranges of pH and ionic strength, resulting in immobilization yields and recovered activities up to 90%. Investigation of the type of interaction between ß-glucosidase and HA (using FT-IR, zeta potential measurements, and desorption tests with different salts) indicated the formation of coordination bonds between Ca2+ sites of HA and COO- of amino acids. Even after 10 cycles of reuse, the immobilized ß-glucosidase retained about 70% of its initial activity, demonstrating the operational stability of the immobilized enzyme. The results showed that ß-glucosidase could be efficiently immobilized on HA nanoparticles by means of a very simple adsorption protocol, offering a promising strategy for performing repeated enzymatic hydrolysis reactions.

On-Site Production of Cellulolytic Enzymes by the Sequential Cultivation Method.

The conversion of renewable lignocellulosic biomass into fuels, chemicals, and high-value materials using the biochemical platform has been considered the most sustainable alternative for the implementation of future biorefineries. However, the high cost of the cellulolytic enzymatic cocktails used in the saccharification step significantly affects the economics of industrial large-scale conversion processes. The on-site production of enzymes, integrated to the biorefinery plant, is being considered as a potential strategy that could be used to reduce costs. In such approach, the microbial production of enzymes can be carried out using the same lignocellulosic biomass as feedstock for fungal development and biofuels production. Most of the microbial cultivation processes for the production of industrial enzymes have been developed using the conventional submerged fermentation. Recently, a sequential solid-state followed by submerged fermentation has been described as a potential alternative cultivation method for cellulolytic enzymes production. This chapter presents the detailed procedure of the sequential cultivation method, which could be employed for the on-site production of the cellulolytic enzymes required to convert lignocellulosic biomass into simple sugars.