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The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 10 9 colony-forming units (CFU)/g within 14 days. Subsequently, G Control , G Lecithin , G Break-Thru , G Bentonite, and G Organic compost+Break-Thru formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. G Control , G Break-Thru , G Bentonite , and G Organic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations G Control and G Bentonite remained consistent for > 3 months at ambient temperature, while in G Bentonite and G Organic compost+Break-Thru formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 10 4 to 5 × 10 6  CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life. KEY POINTS: • Innovative "bioreactor-in-a-granule" system for T. asperelloides is devised. • Dry granules of aerial conidia remain highly viable for 24 months at 4 °C. • Effective control of white-mold sclerotia via soil application of Trichoderma-based granules., (© 2024. The Author(s).)

This study explores the enhancement of phosphate rock (PR) solubilization through solid-state fermentation (SSF) by optimizing oxalic acid production using Aspergillus niger. Key process parameters, including the use of agro-industrial by-products (sugarcane bagasse (SCB), wheat bran (WB), soybean bran (SB)), pH levels, sucrose supplementation, and methanol addition, were systematically evaluated through sequential experimental designs. The results identified SCB and SB in a 1:1 ratio as the most effective substrate. Remarkably, the inclusion of methanol (7 %) and sucrose (0.5 %) resulted in a 3-fold increase in oxalic acid production. Under these optimized conditions, significant phosphorus solubilization of Bayóvar, Itafós, and Registro PRs was achieved, with Bayóvar rock releasing up to 12.1 g/kgds of soluble P (63.8 % efficiency). Additionally, the SSF process effectively released organic phosphorus from the agro-industrial substrates. These findings hold promise for advancing the bio-based economy and developing future industrial biofertilizers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Despite being composed of recyclable materials, the main technological challenge of multilayer carton packs involves the efficient decompatibilization of the cellulosic, polymeric, and metallic phases. Here, a simple two-step mechanochemical process is described that uses only aqueous media and mechanical force to promote phase separation in order to fully recycle multi-layer carton packaging. The first step produces value-added micro- and nanocellulose, while in the second step, aluminum is extracted, forming precipitated aluminum and aluminum oxyhydroxides. Solid polyethylene (PE) remains with a degree of purity defined by the process efficiency. The results show that cellulose is efficiently extracted and converted into micro- and nanocellulose after 15 min of milling. In the second stage, approximately 90% of the aluminum is extracted from the PE after 15 min of milling. Due to the separation and drying medium conditions, the finely divided particles of extracted aluminum also have oxyhydroxides in their composition. It is believed that a passivation layer forms on the metallic aluminum particle. The techno-economic analysis revealed a positive net present value (NPV) of $17.5 million, with a minimum selling price of 1.62 USD/kg of cellulose. The environmental analysis concluded that most of the environmental impact of the process is associated with the entry of carton packages into the system, incorporating a small environmental load related to the industrial process. The results indicate a promising option toward a circular economy and carbon neutrality., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The use of Bacillus as biofertilizer is a sustainable strategy to increase agricultural productivity, but it still requires the development of formulations to protect cells from stressful conditions. Ionotropic gelation using a pectin/starch matrix is a promising encapsulation strategy to achieve this goal. By incorporating additives such as montmorillonite (MMT), attapulgite (ATP), polyethylene glycol (PEG), and carboxymethyl cellulose (CMC), the properties of these encapsulated products could be further improved. In this study, we investigated the influence of these additives on the properties of pectin/starch-based beads for the encapsulation of Bacillus subtilis. FTIR analysis indicated pectin and Ca 2+ ions interactions, while the XRD showed good dispersion of clays in the materials. SEM and X-ray microtomography revealed differences in the morphology of the beads due to the use of the additives. The viabilities at the encapsulation were higher than 10 10  CFU g -1 for all formulations, with differences in the release profiles. In terms of cell protection, the pectin/starch, pectin/starch-MMT and pectin/starch-CMC formulations showed the highest cell viability after exposure to fungicide, while the pectin/starch-ATP beads showed the best performance after UV exposure. Moreover, all formulations maintained more than 10 9  CFU g -1 after six months of storage, which meets values required for microbial inoculants., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 KR polyhalite (sedimentary mineral), KR feldspar (igneous mineral), and KCl (commercial fertilizer). For KCl and KR polyhalite , complete solubilization was achieved using all the organic acids, while for KR feldspar , the highest K + solubilization (34.86 mg L -1 ) was achieved with oxalic acid. The solubility of KR feldspar 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 KR feldspar ). 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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The use of phosphate rocks as low-solubility phosphorus fertilizers has been promoted to reduce the environmental impacts of agriculture, but adequate nutrient uptake by plants depends on solubilization of the rock, driven by soil microorganisms. Here, investigation was made of the microbial solubilization of low-solubility phosphate rocks, together with simultaneous bioprotective action involving the biocontrol of microorganisms. The aim was to enhance function and value by delivering two effects using a single bio-based product, in accordance with the concept of a "bioreactor-in-a-granule" system. A composite structure was developed, based on a starch matrix, comprising a combination of Trichoderma asperelloides, as a biocontrol agent, and Aspergillus niger, as an acidulant. A significant increase of up to 150% in P solubilization was achieved, indicating the positive effect of the microorganism-composite interaction. In vitro assays showed that the ability of T. asperelloides to inhibit Fusarium oxysporum mycelial growth was maintained in the presence of A. niger. Moreover, the estimated cost of the composite granule (0.35 US$/kg of product on a dry basis) revealed competitive. The results indicated that the association of T. asperelloides and A. niger is an effective way to increase nutrient availability and to inhibit plant pathogens, opening up possibilities for the design of multifunctional bio-based fertilizer composites., (© 2022 American Institute of Chemical Engineers.)

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 KH 2 PO 4 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 KH 2 PO 4 . 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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 (

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. β-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 β-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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

The manner in which added non-catalytic proteins during enzymatic hydrolysis of lignocellulosic substrates enhances hydrolysis mechanisms is not completely understood. Prior research has indicated that a reduction in the non-specific adsorption of enzymes on lignin, and deactivation of enzymes exposed to air-liquid interface provide rationale. This work investigated root causes including effects of the air-liquid interface on non-catalytic proteins, and effects of lignin on endoglucanase. Three different experimental designs and three variables (air-liquid interfacial area, the types of lignin (acid or enzymatic lignin), and the presence of non-enzymatic protein (bovine serum albumin [BSA] or soy proteins ) were used. The results showed that acid isolated lignin adsorbed almost all endoglucanase activity initially present in supernatant, independent of air interface conditions (25 or 250 ml flasks) with the presence of BSA preventing this effect. Endoglucanase lost 30%-50% of its activity due to an air-liquid interface in the presence of lignin while addition of non-enzymatic protein helped to preserve this enzyme's activity. Langmuir and Freundlich models applied to experimental data indicated that the adsorption increases with increasing temperature for both endoglucanase and BSA. Adsorption of the enzyme and protein were endothermic with an increase in entropy. These results, combined, show that hydrophobicity plays a strong role in the adsorption of both endoglucanase and BSA on lignin., (© 2021 American Institute of Chemical Engineers.)

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 (S 0 ) can provide a high-concentration source of sulfate, but its availability is limited by the ability of the soil to promote S 0 oxidation. We propose here the integration of these nutrients in a composite based on a biodegradable starch matrix containing mineral oxides and S 0 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/S 0 /oxide mixture multi-nutrient fertilizer showing remarkable results for solubilization of the oxides, hence confirming a synergic effect of S 0 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.

Trichoderma harzianum has attracting attention for its potential alternative use in biofuel production, due to a recognized competence for high diversity glycoside hydrolases (GH) enzyme complex, including higher β-glucosidases and auxiliary proteins, using low-cost carbon sources. This strain constitutively overexpressed the global regulator putative methyltransferase - LAE1, in order to improve the GHs production. The recombinant strain achieved 79-fold increase in lae1 expression and high GHs productivity. The evaluation of the LAE1 impact to induce the GHs used soluble and lignocellulose inexpensive carbon sources in a stirred-tank bioreactor. Using sugarcane bagasse with sucrose, the overexpression of lae1 resulted in significantly increment of gh61b (31x), cel7a (25x), bgl1(20x) and xyn3 (20x) genes expression. Reducing sugar released from pretreated sugarcane bagasse, which hydrolyzed by recombinant crude enzyme cocktail, achieved 41% improvement. Therefore, lae1 overexpression effectively is a promising improving GHs target for biomass degradation by T. harzianum., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 β-glucosidase production exhibited a linear relationship with the pellet fragmentation percentage, which was directly related to the shear conditions. Interestingly, β-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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

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-Me 2+ ) to enable interaction with proteins. The adsorption of proteins on HA-Me 2+ 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 (Cu 2+ and Ni 2+ ). 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-Cu 2+ support, compared to HA and HA-Ni 2+ . 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-Cu 2+ 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 Cu 2+ and Ni 2+ metal ions, using a simple and effective method, indicating the promising potential of the system for applications in different industrial processes., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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.

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 β-mannanase production results were found for a substrate composed of a mixture of coffee waste and wheat bran (1:1 w / w ), using Aspergillus niger 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 β-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., Competing Interests: The authors declare no conflicts of interest.

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.

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., (© 2019 Wiley Periodicals, Inc.)

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 β-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.

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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

β-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 β-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 β-glucosidase and HA (using FT-IR, zeta potential measurements, and desorption tests with different salts) indicated the formation of coordination bonds between Ca 2+ sites of HA and COO - of amino acids. Even after 10 cycles of reuse, the immobilized β-glucosidase retained about 70% of its initial activity, demonstrating the operational stability of the immobilized enzyme. The results showed that β-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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The efficient use of renewable lignocellulosic feedstocks to obtain biofuels and other bioproducts is a key requirement for a sustainable biobased economy. This requires novel and effective strategies to reduce the cost contribution of the cellulolytic enzymatic cocktails needed to convert the carbohydrates into simple sugars, in order to make large-scale commercial processes economically competitive. Here, we propose the use of the whole solid-state fermentation (SSF) medium of mixed filamentous fungi as an integrated one-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production. Ten different individual and mixed cultivations of commonly used industrial filamentous fungi (Aspergillus niger, Aspergillus oryzae, Trichoderma harzianum, and Trichoderma reesei) were performed under SSF and the whole media (without the extraction step) were used in the hydrolysis of pretreated sugarcane bagasse. The cocultivation of T. reesei with A. oryzae increased the amount of glucose released by around 50%, compared with individual cultivations. The release of glucose and reducing sugars achieved using the whole SSF medium was around 3-fold higher than obtained with the enzyme extract. The addition of soybean protein (0.5% w/w) during the hydrolysis reaction further significantly improved the saccharification performance by blocking the lignin and avoiding unproductive adsorption of enzymes. The results of the alcoholic fermentation validated the overall integrated process, with a volumetric ethanol productivity of 4.77 g/L.h, representing 83.5% of the theoretical yield. These findings demonstrate the feasibility of the proposed one-pot integrated strategy using the whole SSF medium of mixed filamentous fungi for on-site enzymes production, biomass hydrolysis, and ethanol production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:671-680, 2018., (© 2018 American Institute of Chemical Engineers.)

The endophytic fungi represent a potential source of microorganisms for enzyme production. However, there have been only few studies exploiting their potential for the production of enzymes of industrial interest, such as the (hemi)cellulolytic enzymatic cocktail required in the hydrolysis of lignocellulosic biomass. Here, a collection of endophytic fungi isolated from mangrove tropical forests was evaluated for the production of carbohydrolases and performance on the hydrolysis of cellulose. For that, 41 endophytic strains were initially screened using a plate assay containing crystalline cellulose as the sole carbon source and the selected strains were cultivated under solid-state fermentation for endoglucanase, β-glucosidase, and xylanase enzyme quantification. The hydrolysis of a cellulosic material with the enzymes from endophytic strains of the Aspergillus genus resulted in glucose and conversion values more than twofold higher than the reference strains (Aspergillus niger F12 and Trichoderma reesei Rut-C30). Particularly, the enzymes from strains A. niger 56 (3) and A. awamori 82 (4) showed a distinguished saccharification performance, reaching cellulose conversion values of about 35% after 24 h. Linking hydrolysis performance to the screening steps played an important role towards finding potential fungal strains for producing enzymatic cocktails with high saccharification efficiency. These results indicate the potential of mangrove-associated endophytic fungi for production of carbohydrolases with efficient performance in the hydrolysis of biomass, thus contributing to the implementation of future biorefineries.

Use of the same lignocellulosic biomass as feedstock for enzymes and ethanol production has been suggested as a lower cost option in future biorefineries. Here, we propose a closed-loop strategy to produce the cellulolytic enzymes required for biomass hydrolysis using sugarcane bagasse liquefied by a home-made enzymatic cocktail as carbon source and inducer. The fed-batch liquefaction conditions were firstly evaluated using commercial enzymes. Subsequently, the effects of different liquefied materials and solids loadings on endoglucanase production by Aspergillus niger cultivated in submerged fermentation were investigated. The liquefied bagasse produced using the home-made cocktail was more favorable for endoglucanase production, resulting in improvement up to 17%, compared to bagasse liquefied by commercial enzymes. The results indicated that liquefied bagasse produced by home-made enzymatic cocktail could provide a cost-effective carbon source and inducer for cellulolytic enzyme production, and could contribute to closing loops within the biorefinery, thus reducing costs and minimizing waste., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

One of the main challenges of second generation (2G) ethanol production is the high quantities of phenolic compounds and furan derivatives generated in the pretreatment of the lignocellulosic biomass, which inhibit the enzymatic hydrolysis and fermentation steps. Fast monitoring of these inhibitory compounds could provide better control of the pretreatment, hydrolysis, and fermentation processes by enabling the implementation of strategic process control actions. We investigated the feasibility of monitoring these inhibitory compounds by ultraviolet-visible (UV-Vis) spectroscopy associated with partial least squares (PLS) regression. Hydroxymethylfurfural, furfural, vanillin, and ferulic and p-coumaric acids generated during different severities of liquid hot water pretreatment of sugarcane bagasse were quantified with highly accuracy. In cross-validation (leave-one-out), the PLS-UV-Vis method presented root mean square error of prediction (RMSECV) of around only 5.0%. The results demonstrated that the monitoring performance achieved with PLS-UV-Vis could support future studies of optimization and control protocols for application in industrial processes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

The feasibility of integration of cellulosic ethanol production with the manufacture of cellulose nanofibers (CNF) and cellulose nanocrystals (CNC) was evaluated using eucalyptus cellulose pulp as feedstock and employing the biochemical route alone. For the enzymatic hydrolysis step, experimental central composite design (CCD) methodology was used as a tool to evaluate the effects of solids loading (SL) and enzymatic loading (EL) on glucose release and cellulose conversion. Glucose concentrations from 45 to 125 g/L were obtained after 24 h, with cellulose conversions from 35 to 96%. Validation of the statistical model was performed at SL of 20% and EL of 10 mg protein/g, which was defined by the desirability function as the optimum condition. The sugars released were used for the production of ethanol by Saccharomyces cerevisiae, resulting in 62.1 g/L ethanol after 8 h (yield of 95.5%). For all the CCD experimental conditions, the residual solids presented CNF characteristics. Moreover, the use of a new strategy with temperature reduction from 50 to 35°C after 24 h of enzymatic hydrolysis enabled CNC to be obtained after 144 h. The CNC showed a crystallinity index of 83%, length of 260 nm, diameter of 15 nm, and aspect ratio (L/D) of 15. These characteristics are suitable for many applications, such as reinforcement in polymeric materials and other lower volume higher value bio-based products. The findings indicate the viability of obtaining ethanol and CNC using the biochemical route exclusively, potentially contributing to the future implementation of forest biorefineries. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1085-1095, 2017., (© 2017 American Institute of Chemical Engineers.)

This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and β-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Addition of surfactants, polymers, and non-catalytic proteins can improve the enzymatic hydrolysis of lignocellulosic materials by blocking the exposed lignin surfaces, but involves extra expense. Here, soybean protein, one of the cheapest proteins available, was evaluated as an alternative additive for the enzymatic hydrolysis of pretreated sugarcane bagasse. The effect of the enzyme source was investigated using enzymatic cocktails from A. niger and T. reesei cultivated under solid-state, submerged, and sequential fermentation. The use of soybean protein led to approximately 2-fold increases in hydrolysis, relative to the control, for both A. niger and T. reesei enzymatic cocktails from solid-state fermentation. The effect was comparable to that of BSA. Moreover, the use of soybean protein and a 1:1 combination of A. niger and T. reesei enzymatic cocktails resulted in 54% higher glucose release, compared to the control. Soybean protein is a potential cost-effective additive for use in the biomass conversion process., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 °C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 °C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn +2 (45 %), while Cu +2 and Ag + ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K M 1.2 mg mL -1 ) and V max of 17.4 μmol min -1  mg -1 of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cultivation procedure and the fungal strain applied for enzyme production may influence levels and profile of the proteins produced. The proteomic analysis data presented here provide critical information to compare proteins secreted by Trichoderma reesei and Aspergillus niger when cultivated through submerged and sequential fermentation processes, using steam-explosion sugarcane bagasse as inducer for enzyme production. The proteins were organized according to the families described in CAZy database as cellulases, hemicellulases, proteases/peptidases, cell-wall-protein, lipases, others (catalase, esterase, etc.), glycoside hydrolases families, predicted and hypothetical proteins. Further detailed analysis of this data is provided in "Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation process: enzyme production for sugarcane bagasse hydrolysis" C. Florencio, F.M. Cunha, A.C Badino, C.S. Farinas, E. Ximenes, M.R. Ladisch (2016) [1].

The use of residues from the industrial processing of palm oil as carbon source and inducer for microbial lipase production can be a way to add value to such residues and to contribute to reduced enzyme costs. The aim of this work was to investigate the feasibility of using palm oil industrial waste as feedstock for lipase production in different cultivation systems. Evaluation was made of lipase production by a selected strain of Aspergillus niger cultivated under solid-state (SSF) and submerged fermentation (SmF). Lipase activity levels up to 15.41 IU/mL were achieved under SSF. The effects of pH and temperature on the lipase activity of the SSF extract were evaluated using statistical design methodology, and maximum activities were obtained between pH 4.0 and 6.5 and at temperatures between 37 and 55 °C. This lipase presented good thermal stability up to 60 °C and higher specificity towards long carbon chain substrates. The results demonstrate the potential application of palm oil industrial residues for lipase production and contribute to the technological advances needed to develop processes for industrial enzymes production.

High activity and stability are essential for (hemi)cellulolytic enzymes used in biomass conversion, while non-productive binding of cellulases to lignin reduces saccharification efficiency and needs to be avoided. One potential strategy is the addition of inexpensive metal ions. This paper describes the influence of divalent metal ions on the activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes produced in-house by Aspergillus niger under solid-state fermentation (SSF). The use of Mn(2+) provided the best (hemi)cellulolytic activity and stability, with an increase in endoglucanase activity of up to 57%. The use of Mn(2+) was then investigated in the saccharification of sugarcane bagasse submitted to acid, steam-explosion, and hydrothermal pretreatments. The addition of Mn(2+) ions at 10mM in the saccharification of acid-pretreated bagasse resulted in a 34% increase in glucose release. These positive effects appeared to be due to a reduction in non-productive enzyme adsorption. The findings suggest that the addition of inexpensive metal ions can help to improve activity, thermostability, and saccharification efficiency of (hemi)cellulolytic enzymes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.

Rapid, efficient, and low-cost technologies for monitoring the fermentation process during second generation (2G) or cellulosic ethanol production are essential for the successful implementation of this process at the commercial scale. Here, the use of near-infrared (NIR) spectroscopy associated with partial least squares (PLS) regression was investigated as a tool for monitoring the production of 2G ethanol from lignocellulosic sugarcane residues including bagasse, straw, and tops. The spectral data was based on a set of 103 alcoholic fermentation samples. Models based on different pre-processing techniques were evaluated. The best root mean square error of prediction (RMSEP) values obtained in the external validation were around 3.02 g/L for ethanol and 6.60 g/L for glucose. The findings showed that the PLS-NIR methodology was efficient in accurately predicting the glucose and ethanol concentrations during the production of 2G ethanol, demonstrating potential for use in monitoring and control of large-scale industrial processes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: In the sugarcane industry, large amounts of lignocellulosic residues are generated, which includes bagasse, straw, and tops. The use of the whole sugarcane lignocellulosic biomass for the production of second-generation (2G) ethanol can be a potential alternative to contribute to the economic viability of this process. Here, we conducted a systematic comparative study of the use of the lignocellulosic residues from the whole sugarcane lignocellulosic biomass (bagasse, straw, and tops) from commercial sugarcane varieties for the production of 2G ethanol. In addition, the feasibility of using a mixture of these residues from a selected variety was also investigated., Results: The materials were pretreated with dilute acid and hydrolyzed with a commercial enzymatic preparation, after which the hydrolysates were fermented using an industrial strain of Saccharomyces cerevisiae. The susceptibility to enzymatic saccharification was higher for the tops, followed by straw and bagasse. Interestingly, the fermentability of the hydrolysates showed a different profile, with straw achieving the highest ethanol yields, followed by tops and bagasse. Using a mixture of the different sugarcane parts (bagasse-straw-tops, 1:1:1, in a dry-weight basis), it was possible to achieve a 55% higher enzymatic conversion and a 25% higher ethanol yield, compared to use of the bagasse alone. For the four commercial sugarcane varieties evaluated using the same experimental set of conditions, it was found that the variety of sugarcane was not a significant factor in the 2G ethanol production process., Conclusions: Assessment of use of the whole lignocellulosic sugarcane biomass clearly showed that 2G ethanol production could be significantly improved by the combined use of bagasse, straw, and tops, when compared to the use of bagasse alone. The lower susceptibility to saccharification of sugarcane bagasse, as well as the lower fermentability of its hydrolysates, can be compensated by using it in combination with straw and tops (sugarcane trash). Furthermore, given that the variety was not a significant factor for the 2G ethanol production process within the four commercial sugarcane varieties evaluated here, agronomic features such as higher productivity and tolerance of soil and climate variations can be used as the criteria for variety selection.

The development of new cost-effective bioprocesses for the production of cellulolytic enzymes is needed in order to ensure that the conversion of biomass becomes economically viable. The aim of this study was to determine whether a novel sequential solid-state and submerged fermentation method (SF) could be validated for different strains of the Trichoderma genus. Cultivation of the Trichoderma reesei Rut-C30 reference strain under SF using sugarcane bagasse as substrate was shown to be favorable for endoglucanase (EGase) production, resulting in up to 4.2-fold improvement compared with conventional submerged fermentation. Characterization of the enzymes in terms of the optimum pH and temperature for EGase activity and comparison of the hydrolysis profiles obtained using a synthetic substrate did not reveal any qualitative differences among the different cultivation conditions investigated. However, the thermostability of the EGase was influenced by the type of carbon source and cultivation system. All three strains of Trichoderma tested (T. reesei Rut-C30, Trichoderma harzianum, and Trichoderma sp INPA 666) achieved higher enzymatic productivity when cultivated under SF, hence validating the proposed SF method for use with different Trichoderma strains. The results suggest that this bioprocess configuration is a very promising development for the cellulosic biofuels industry.

The objective of this paper is to report liquefaction of pretreated and sterilized sugarcane bagasse for enhancing endoglucanase production through submerged fermentation by Aspergillus niger. After initial solid state fermentation of steam pretreated bagasse solids by A. niger, fed-batch addition of the substrate to cellulase in buffer over a 12h period, followed by 36h reaction, resulted in a liquid slurry with a viscosity of 0.30±0.07Pas at 30% (w/v) solids. Addition of A. niger for submerged fermentation of sterile liquefied bagasse at 23% w/v solids resulted in an enzyme titer of 2.5IUmL(-1) or about 15× higher productivity than solid-state fermentation of non-liquefied bagasse (final activity of 0.17IUmL(-1)). Bagasse not treated by initial solid-state fermentation but liquefied with enzyme gave 2IUmL(-1). These results show the utility of liquefied bagasse as a culture medium for enzyme production in submerged fermentations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Accessory enzymes that assist biomass degradation could be used to improve the recovery of fermentable sugar for use in biorefineries. In this study, different fungal strains isolated from the Amazon rainforest were evaluated in terms of their ability to produce feruloyl esterase (FAE) and xylanase enzymes, and an assessment was made of the contributions of the enzymes in the hydrolysis of pretreated sugarcane bagasse. In the selection step, screening using plate assays was followed by shake flask submerged cultivations. After carbon source selection and cultivation in a stirred-tank bioreactor, Aspergillusoryzae P21C3 proved to be a promising strain for production of the enzymes. Supplementation of a commercial enzyme preparation with 30% (v/v) crude enzymatic complex from A. oryzae P21C3 increased the conversion of cellulose derived from pretreated sugarcane bagasse by 36%. Supplementation with FAE and xylanase enzymes produced on-site can therefore be used to improve the hydrolysis of sugarcane bagasse., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The β-glucosidase (BG) enzyme plays a vital role in the hydrolysis of lignocellulosic biomass. Supplementation of the hydrolysis reaction medium with BG can reduce inhibitory effects, leading to greater conversion. In addition, the inclusion of immobilized BG can be a useful way of increasing enzyme stability and recyclability. BG was adsorbed on polyacrylic resin activated by carboxyl groups (BG-PC) and covalently attached to glyoxyl-agarose (BG-GA). BG-PC exhibited similar behavior to soluble BG in the hydrolysis of cellobiose, while BG-GA hydrolyzed the same substrate at a lower rate. However, the thermal stability of BG-GA was higher than that of free BG. Hydrolysis of pretreated sugarcane bagasse catalyzed by soluble cellulase supplemented with immobilized BG improved the conversion by up to 40% after 96 h of reaction. Both derivatives remained stable up to the third cycle and losses of activity were less than 50% after five cycles., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Sugarcane bagasse is a low-cost and abundant by-product generated by the bioethanol industry, and is a potential substrate for cellulolytic enzyme production. The aim of this work was to evaluate the effects of air flow rate (QAIR), solids loading (%S), sugarcane bagasse type, and particle size on the gas hold-up (εG) and volumetric oxygen transfer coefficient (kLa) in three different pneumatic bioreactors, using response surface methodology. Concentric tube airlift (CTA), split-cylinder airlift (SCA), and bubble column (BC) bioreactor types were tested. QAIR and %S affected oxygen mass transfer positively and negatively, respectively, while sugarcane bagasse type and particle size (within the range studied) did not influence kLa. Using large particles of untreated sugarcane bagasse, the loop-type bioreactors (CTA and SCA) exhibited higher mass transfer, compared to the BC reactor. At higher %S, SCA presented a higher kLa value (0.0448 s−1) than CTA, and the best operational conditions in terms of oxygen mass transfer were achieved for %S < 10.0 g L−1 and QAIR > 27.0 L min−1. These results demonstrated that pneumatic bioreactors can provide elevated oxygen transfer in the presence of vegetal biomass, making them an excellent option for use in three-phase systems for cellulolytic enzyme production by filamentous fungi.

The enzymatic cocktail of cellulases is one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels and other chemicals. Here, the influence of liquid hot water, dilute acid, alkali, and combined acid/alkali pretreatments on sugarcane bagasse (SCB) used for cellulase production was investigated by means of spectroscopic and imaging techniques. Chemical composition and structural characteristics, such as crystallinity (determined by X-ray diffraction), functional groups (Fourier transform infrared spectroscopy), and microstructure (scanning electron microscopy), were used to correlate SCB pretreatments with enzymatic biosynthesis by a strain of the filamentous fungus Aspergillus niger under solid-state fermentation. The combined acid/alkali pretreatment resulted in a SCB with higher cellulose content (86.7%). However, the high crystallinity (74%) of the resulting biomass was detrimental to microbial uptake and enzyme production. SCB pretreated with liquid hot water yielded the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and xylanase activities (0.4, 14.9, and 26.1 U g(-1), respectively). The results showed that a suitable pretreatment for SCB to be used as a substrate for cellulase production should avoid severe conditions in order to preserve amorphous cellulose and to enhance the physical properties that assist microbial access.

The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan.