Evaluation of Saccharomyces cerevisiae modified via CRISPR/Cas9 as a cellulosic platform microorganism in simultaneously saccharification and fermentation processes.

The nonrenewable character and deleterious effects of fossil fuels foster the need for cleaner and more inexhaustible energy sources, such as bioethanol. Especially from lignocellulosic biomasses. However, the economic viability of this product in the market depends on process optimization and cost reduction. This research applied a sequential experimental project to investigate the process of enzymatic saccharification and simultaneous fermentation to produce ethanol with sugarcane bagasse. The differential of the work was the application of the strain of Saccharomyces cerevisiae AGY001 which was improved by evolutionary engineering to become thermotolerant and by a heterologous expression based on genomic integration by CRISPR/Cas9 to produce endoglucanase and ß-glucosidase (AsENDO-AsBGL). The maximum ethanol yield found was 89% of the maximum theoretical yield (released sugars), obtained at temperature concentrations, sugarcane bagasse and inoculum at 40 °C, 16.5%, and 4.0 g/L, respectively (12.5 FPU/g bagasse). The mathematical model obtained can predict approximately 83% of the data set with 95% confidence. Therefore, these findings demonstrated the potential of sugarcane bagasse and S. cerevisiae AGY001 strain (CRISPR/Cas9 modified) in bioethanol production without the need for impractical selection media on an industrial scale, in addition to providing useful insights for the development of SSF processes.

Deconstruction of banana peel for carbohydrate fractionation.

The deconstruction of banana peel for carbohydrate recovery was performed by sequential treatment (acid, alkaline, and enzymatic). The pretreatment with citric acid promoted the extraction of pectin, resulting in a yield of 8%. In addition, xylose and XOS, 348.5 and 17.3 mg/g xylan, respectively, were also quantified in acidic liquor as a result of partial depolymerization of hemicellulose. The spent solid was pretreated with alkaline solution (NaOH or KOH) for delignification and release of residual carbohydrates from the hemicellulose. The yields of xylose and arabinose (225.2 and 174.0 mg/g hemicellulose) were approximately 40% higher in the pretreatment with KOH, while pretreatment with NaOH promoted higher delignification (67%), XOS yield (32.6 mg/g xylan), and preservation of cellulosic fraction. Finally, the spent alkaline solid, rich in cellulose (76%), was treated enzymatically to release glucose, reaching the final concentration of 28.2 g/L. The mass balance showed that through sequential treatment, 9.9 g of xylose, 0.5 g of XOS, and 8.2 g of glucose were obtained from 100 g of raw banana peels, representing 65.8% and 46.5% conversion of hemicellulose and cellulose, respectively. The study of the fractionation of carbohydrates in banana peel proved to be a useful tool for valorization, mainly of the hemicellulose fraction for the production of XOS and xylose with high value applications in the food industry.

Cello-oligosaccharides production from lignocellulosic biomass and their emerging prebiotic applications.

Cello-oligosaccharides (COS) are linear oligosaccharides composed of ß-1,4-linked glucopyranose units. They comprise a group of important new oligosaccharides of significant interest and potential applications in the pharmaceutical, food, chemical, and feed industries, currently emerging as potential prebiotic compounds. COS from lignocellulosic biomass, specifically the agro-industrial residues and by-products of the forestry industry, constitute a new attractive process that imposes the sustainable use of biomass resources. Two main strategies have been used for the production of COS: acid-based and enzyme-based cellulose hydrolysis. The latter has been considered more attractive due to the use of milder reaction conditions and less production of monomers. This review summarizes that although COS is emerging as a potential prebiotic with also other potential applications, there is a lack of information regarding the large-scale production, which could be associated with the recalcitrant nature of cellulose compared to other polysaccharides, which hinders the hydrolysis of its dense network.

Cellulase and oxidative enzymes: new approaches, challenges and perspectives on cellulose degradation for bioethanol production.

Second-generation bioethanol is a sustainable energy source that can be produced from different renewable materials. However, there is a challenge we must overcome to significantly enhance bioethanol production: the hydrolysis of lignocellulosic biomass to fermentable sugars. Synergistic enzymes, such as endoglucanases, ß-glucosidases, cellobiohydrolases, and, more recently, lytic polysaccharide monooxygenases and cellobiose dehydrogenases have been used with great success to hydrolyze pretreated biomass. Further advances in the field of second-generation bioethanol production will likely depend on an increased understanding of the interactions between enzymes and lignocellulosic substrates, the development of enzyme engineering, and the optimization of enzyme mixtures to enhance cellulose hydrolysis.

Butanol production by Saccharomyces cerevisiae: perspectives, strategies and challenges.

The search for gasoline substitutes has grown in recent decades, leading to the increased production of ethanol as viable alternative. However, research in recent years has shown that butanol exhibits various advantages over ethanol as a biofuel. Furthermore, butanol can also be used as a chemical platform, serving as an intermediate product and as a solvent in industrial reactions. This alcohol is naturally produced by some Clostridium species; however, Clostridial fermentation processes still have inherent problems, which focuses the interest on Saccharomyces cerevisiae for butanol production, as an alternative organism for the production of this alcohol. S. cerevisiae exhibits great adaptability to industrial conditions and can be modified with a wide range of genetic tools. Although S. cerevisiae is known to naturally produce isobutanol, the n-butanol synthesis pathway has not been well established in wild S. cerevisiae strains. Two strategies are most commonly used for of S. cerevisiae butanol production: the heterologous expression of the Clostridium pathway or the amino acid uptake pathways. However, butanol yields produced from S. cerevisiae are lower than ethanol yield. Thus, there are still many challenges needed to be overcome, which can be minimized through genetic and evolutive engineering, for butanol production by yeast to become a reality.

Enzymatic removal of inhibitory compounds from lignocellulosic hydrolysates for biomass to bioproducts applications.

The physicochemical pretreatment is an important step to reduce biomass recalcitrance and facilitate further processing of plant lignocellulose into bioproducts. This process results in soluble and insoluble biomass fractions, and both may contain by-products that inhibit enzymatic biocatalysts and microbial fermentation. These fermentation inhibitory compounds (ICs) are produced during the degradation of lignin and sugars, resulting in phenolic and furanic compounds, and carboxylic acids. Therefore, detoxification steps may be required to improve lignocellulose conversion by microoganisms. Several physical and chemical methods, such as neutralization, use of activated charcoal and organic solvents, have been developed and recommended for removal of ICs. However, biological processes, especially enzyme-based, have been shown to efficiently remove ICs with the advantage of minimizing environmental issues since they are biogenic catalysts and used in low quantities. This review focuses on describing several enzymatic approaches to promote detoxification of lignocellulosic hydrolysates and improve the performance of microbial fermentation for the generation of bioproducts. Novel strategies using classical carbohydrate active enzymes (CAZymes), such as laccases (AA1) and peroxidases (AA2), as well as more advanced strategies using prooxidant, antioxidant and detoxification enzymes (dubbed as PADs), i.e. superoxide dismutases, are discussed as perspectives in the field.

Increased biomass saccharification by supplementation of a commercial enzyme cocktail with endo-arabinanase from Bacillus licheniformis.

OBJECTIVES: The use of endo-arabinanase from Bacillus licheniformis (ABNase) for sugarcane saccharification has been evaluated by enzyme immobilization and commercial cocktail supplement with the immobilized heterologous protein. RESULTS: Biochemical characterization of the purified ABNase showed that the catalytic activity was strongly inhibited by 5 mM Cu(2+), Zn(2+) or Fe(3+). The optimum pH and temperature for activity were 5.5-6.5 and 35-40 °C, respectively. The enzyme stability increased 128-fold when immobilized with glyoxyl agarose, and the hydrolysis of pretreated sugar cane biomass increased by 15 % when a commercial enzyme cocktail was supplemented with immobilized ABNase. CONCLUSION: Pectin hydrolysis by recombinant ABNase plays a role in the effective application of enzymatic cocktails for biomass saccharification.

Development of hemicellulolytic enzyme mixtures for plant biomass deconstruction on target biotechnological applications.

An essential step in the conversion of lignocellulosic biomass to ethanol and other biorefinery products is conversion of cell wall polysaccharides into fermentable sugars by enzymatic hydrolysis. The objective of the present study was to understand the mode of action of hemicellulolytic enzyme mixtures for pretreated sugarcane bagasse (PSB) deconstruction and wheat arabinoxylan (WA) hydrolysis on target biotechnological applications. In this study, five hemicellulolytic enzymes-two endo-1,4-xylanases (GH10 and GH11), two α-L-arabinofuranosidases (GH51 and GH54), and one ß-xylosidase (GH43)-were submitted to combinatorial assays using the experimental design strategy, in order to analyze synergistic and antagonistic effects of enzyme interactions on biomass degradation. The xylooligosaccharides (XOSs) released from hydrolysis were analyzed by capillary electrophoresis and quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Based on this analysis, it was possible to define which enzymatic combinations favor xylose (X1) or XOS production and thus enable the development of target biotechnological applications. Our results demonstrate that if the objective is X1 production from WA, the best enzymatic combination is GH11 + GH54 + GH43, and for xylobiose (X2) production from WA, it is best to combine GH11 + GH51. However, if the goal is to produce XOS, the five enzymes used in WA hydrolysis are important, but for PSB hydrolysis, only GH11 is sufficient. If the final objective is bioethanol production, GH11 is responsible for hydrolyzing 64.3 % of hemicellulose from PSB. This work provides a basis for further studies on enzymatic mechanisms for XOS production, and the development of more efficient and less expensive enzymatic mixtures, targeting commercially viable lignocellulosic ethanol production and other biorefinery products.

Non-animal protein hydrolysates from agro-industrial wastes: A prospect of alternative inputs for cultured meat.

In light of the growing demand for alternative protein sources, laboratory-grown meat has been proposed as a potential solution to the challenges posed by conventional meat production. Cultured meat does not require animal slaughter and uses sustainable production methods, contributing to animal welfare, human health, and environmental sustainability. However, some challenges still need to be addressed in cultured meat production, such as the use of fetal bovine serum for medium supplementation. This ingredient has limited availability, increases production costs, and raises ethical concerns. This review explores the potential of non-animal protein hydrolysates derived from agro-industrial wastes as substitutes for critical components of fetal bovine serum in cultured meat production. Despite the lack of standardization of hydrolysate composition, the potential benefits of this alternative protein source may outweigh its disadvantages. Future research holds promise for increasing the accessibility of cultured meat.

Performance of Saccharomyces cerevisiae strains against the application of adaptive laboratory evolution strategies for butanol tolerance.

Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds.

Thermostability Enhancement of GH 62 α-l-Arabinofuranosidase by Directed Evolution and Rational Design.

GH 62 arabinofuranosidases are known for their excellent specificity for arabinoxylan of agroindustrial residues and their synergism with endoxylanases and other hemicellulases. However, the low thermostability of some GH enzymes hampers potential industrial applications. Protein engineering research highly desires mutations that can enhance thermostability. Therefore, we employed directed evolution using one round of error-prone PCR and site-saturation mutagenesis for thermostability enhancement of GH 62 arabinofuranosidase from Aspergillus fumigatus. Single mutants with enhanced thermostability showed significant ΔΔG changes (<-2.5 kcal/mol) and improvements in perplexity scores from evolutionary scale modeling inverse folding. The best mutant, G205K, increased the melting temperature by 5 °C and the energy of denaturation by 41.3%. We discussed the functional mechanisms for improved stability. Analyzing the adjustments in α-helices, ß-sheets, and loops resulting from point mutations, we have obtained significant knowledge regarding the potential impacts on protein stability, folding, and overall structural integrity.

Technological and prebiotic aspects of young bamboo culm flour (Dendrocalamus latiflorus) combined with rice flour to produce healthy extruded products.

Young bamboo culm flour (YBCF) has proved to be a healthy and sustainable ingredient, due to its high fiber content and high yield of bamboo crops. The present study evaluated the effects of YBCF from Dendrocalamus latiflorus on the physicochemical, technological properties and prebiotic activity of rice-based extrudates aiming to expand its application. The extrudates were produced in a twin-screw extruder with different RF:YBCF concentrations (100:0; 95:5, 90:10, and 85:15 %). During the process, the specific mechanical energy increased as YBCF content increased because of the high shear favored by YBCF particles. With increasing RF replacement by YBCF, the extruded products presented a significant (p < 0.05, by the Scott-Knott test) increase in hardness (57.37 to 82.01 N) and water solubility index (12.80 to 34.10 %), as well as a decrease in color luminosity (L*=85.49 to 82.83), expansion index (2.68 to 1.99), and pasting properties. In addition, all extrudate samples presented bifidogenic activity. Therefore, YBCF exhibited attractive technological properties and can be used as an ingredient in the production of healthy and sustainable extruded products.

Influence of cocoa varieties on carbohydrate composition and enzymatic activity of cocoa pulp.

In Brazil, after the witch's broom disease incidence, diverse cocoa hybrids were developed, and variations were reported on their composition and characteristics. Based on this, the present study aimed to evaluate the pulp composition of several cocoa hybrids in order to better understand these variations. Results show that cocoa pulp is composed, on average, of 76 % sugar, and a wide variation (20 %) was observed in sugar content between hybrids. Regarding the sugar profile, a prevalence of reducing sugars was observed. Pod origin also plays an important role in pulp composition, with variations between hybrids from Espírito Santo and Bahia states. In relation to the degree of ripeness, ripe pods showed higher fructose and glucose content, while unripe pods presented mainly sucrose. Similar to sugars, the cello-oligossacharides profile was influenced by the degree of pod ripeness and origin and most ripe samples presented mainly cellobiose, cellotriose and cellotetrose. In addition, the prebiotic potential of cocoa pulp was highlighted by cello-oligossacharides digestion assay which exhibited low rates of degradation. Varying enzymatic activity was observed amongst different pulp hybrids, with polyphenol oxidase showing a higher variation when compared to invertase and polygalacturonase ranging. This study shows that the pod hybrid, origin and ripening degree may change the cocoa pulp composition. Therefore, it is very important to understand and evaluate these variations, in order to obtain better results in pulp utilization either in cocoa fermentation or as a coproduct.

Fractionation of functional oligosaccharides produced from sugarcane straw using serial nanofiltration membranes and their influence on prebiotic potential.

Functional oligosaccharides are non-digestible by human gut enzymes and provide health benefits as fibers and prebiotics. The cello-oligosaccharides (COS) and xylooligosaccharides (XOS) are functional oligosaccharides obtained from xylan and cellulose, respectively, and are present in lignocellulosic material. The serial NF membranes process was performed to investigate the impact of the fractionation process on the prebiotic activity of oligosaccharides from xylan and cellulose. The NP030 (weight cut-off of 500-600 Da) and DK (weight cut-off of 150-300 Da) NF polymeric membranes were employed using defined operational conditions. The diafiltration (DF) was also investigated and it was determined that only a 1-time DF for NP030 was a more suitable strategy and improved the performance indices for short DP oligosaccharides. The short DP fractions obtained favored cell density for probiotic strains, which presented an increase on the optical density of up to 25 % after the fractionating process; enabling the use of short purified fractions in the food and pharmaceutical industry as a prebiotic ingredient.

Impact of adding xylooligosaccharides encapsulated in butter: Microstructural, optical, rheological and sensory aspects.

This study investigated the microstructure, rheological properties, and sensory characteristics of butters produced with free and encapsulated xylooligosaccharides (XOS). Four formulations of butter were processed: BCONT: 0 % w/w XOS (control); BXOS: 20% w/w free XOS; BXOS-ALG: 20% w/w XOS microencapsulated with alginate (XOS-alginate ratio of 3:1 w/w); and BXOS-GEL: 20% w/w XOS microencapsulated with alginate-gelatin (XOS-alginate-gelatin ratio of 3:1:1.5 w/w). The microparticles showed a bimodal distribution, low size and low span values, demonstrating physical stability to be included in emulsions. The XOS-ALG presented surface weighted mean diameter (D3.2) of 90.24 µm, volume-weighted mean diameter (D4.3) of 131.8 µm, and Span of 2.14. In contrast, the XOS-GEL presented D3.2 of 82.80 µm, D4.3 of 141.0 µm, and a Span of 2.46. Products with XOS were characterized by higher creaminess, sweet taste, and lower salty taste than the control. However, the addition form significantly impacted the other evaluated parameters. The utilization of XOS in a free form (BXOS) resulted in smaller droplet sizes (1.26 µm) than encapsulated XOS and control (XOS-ALG = 1.32 µm / XOS-GEL = 1.58 µm, / BCONT = 1.59 µm), and changes in the rheological parameters (higher values of shear stress, viscosity, consistency index, rigidity (J0), and Newtonian viscosity (ηN) and lower elasticity (τ)). Furthermore, it changed the color parameters (more yellow and dark color, lower L* and higher b* values). On the other hand, the utilization of micropaticles of XOS (BXOS-ALG and BXOS-GEL) kept shear stress, viscosity, consistency index, rigidity (J0), and elasticity (τ) more similar to control. The products had a less intense yellow color (lower b* values) and was perceived with more consistency and butter taste. However, the presence of particles was perceived by consumers. The results suggest that consumers were more attentive to reporting flavor-related attributes than texture. In conclusion, adding microparticles of XOS could improve butter's rheological and sensory properties. In conclusion, adding microparticles of XOS could improve butter's rheological and sensory properties.

Hydrothermal pretreatment for the production of prebiotic oligosaccharides from tobacco stem.

Tobacco stem is an abundant and inexpensive renewable source to produce prebiotics by circular economy. In this study, hydrothermal pretreatments were evaluated on the release of xylooligosaccharides (XOS) and cello-oligosaccharides (COS) from the tobacco stem by a central composite rotational design associated with response surface methodology to evaluate the effects of temperature (161.72 to 218.3 °C) and solid load (SL) (2.93 to 17.07%). XOS were the main compounds released to the liquor. Desirability function was performed to maximize the production of XOS and minimize the effects of release of monosaccharides and degradation compounds. The result indicated yield of 96% w[XOS]/w[xylan] for 190 °C-2.93% SL. The highest value for COS and total oligomers content (COS + XOS) was 6.42 g/L and 17.7 g/L, respectively, for 190 °C-17.07% SL. The mass balance for the best yield XOS condition predicted 132 kg of XOS (X2-X6) from 1000 kg of tobacco stem.

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate.

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two ß-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both ß-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

Enzymatic generation of short chain cello-oligosaccharides from Miscanthus using different pretreatments.

Enzyme combinations producing short-chain cello-oligosaccharides (COS) as major bio-products from cellulose of Miscanthus Mx2779 accessed through different pretreatment methods were compared. Over short hydrolysis times, processive endoglucanase TfCel9a produced a high percentage of cellotetraose and cellopentaose and is synergistic with endoglucanase CcCel9m for producing short oligomers from amorphous cellulose but had low activity on untreated Miscanthus. Hydrolysis of the latter improved when these were combined with a mutant cellobio/triohydrolase OsCelC7(-105) and a lytic polysaccharide monooxygenase TrCel61a, a combination which also produced the highest COS yields from phosphoric acid swollen cellulose. Steam explosion pretreatment of Miscanthus increased COS yields, with/without phosphoric acid swelling, while increased swelling time (from 20 to 45 min) also increased yields but decreased the need for TrCel61a. The highest COS yields (933 mg/g glucan) and most stable product profile were obtained using ionic liquid [C2mim][OAc] pretreatment and the three enzyme mixture TfCel9a, Cel9m and OsCel7a(-105).

Xylo-Oligosaccharide Utilization by Engineered Saccharomyces cerevisiae to Produce Ethanol.

The engineering of xylo-oligosaccharide-consuming Saccharomyces cerevisiae strains is a promising approach for more effective utilization of lignocellulosic biomass and the development of economic industrial fermentation processes. Extending the sugar consumption range without catabolite repression by including the metabolism of oligomers instead of only monomers would significantly improve second-generation ethanol production This review focuses on different aspects of the action mechanisms of xylan-degrading enzymes from bacteria and fungi, and their insertion in S. cerevisiae strains to obtain microbial cell factories able of consume these complex sugars and convert them to ethanol. Emphasis is given to different strategies for ethanol production from both extracellular and intracellular xylo-oligosaccharide utilization by S. cerevisiae strains. The suitability of S. cerevisiae for ethanol production combined with its genetic tractability indicates that it can play an important role in xylan bioconversion through the heterologous expression of xylanases from other microorganisms.

Microalgae-based carbohydrates: A green innovative source of bioenergy.

Microalgae contribute significantly to the global carbon cycle through photosynthesis. Given their ability to efficiently convert solar energy and atmospheric carbon dioxide into chemical compounds, such as carbohydrates, and generate oxygen during the process, microalgae represent an excellent and feasible carbohydrate bioresource. Microalgae-based biofuels are technically viable and, delineate a green and innovative field of opportunity for bioenergy exploitation. Microalgal polysaccharides are one of the most versatile groups for biotechnological applications and its content can be increased by manipulating cultivation conditions. Microalgal carbohydrates can be used to produce a variety of biofuels, including bioethanol, biobutanol, biomethane, and biohydrogen. This review provides an overview of microalgal carbohydrates, focusing on their use as feedstock for biofuel production, highlighting the carbohydrate metabolism and approaches for their enhancement. Moreover, biofuels produced from microalgal carbohydrate are showed, in addition to a new bibliometric study of current literature on microalgal carbohydrates and their use.