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In the context of biorefinery, researchers have been looking for lignocellulosic biomasses and ideal treatments to produce economically viable biofuels. In this scenario, the bamboo culm appears as a plant matrix of great potential, given the high cellulose content of low crystallinity. Thus, the objective and differential of this work was to determine the best conditions for enzymatic hydrolysis of cellulose extracted from bamboo culm and to evaluate its potential application in the production of bioethanol through Separate Hydrolysis and Fermentation (SHF) and Saccharification and Simultaneous Fermentation (SSF) by Saccharomyces cerevisiae modified via CRISPR/Cas9. The average cellulose extraction yield was 41.87 % with an extraction efficiency of 86.76 %. In general, as the hydrolysis time increased, an increase in glucose production was observed in almost all assays, with higher hydrolysis efficiency values at 72 h. The results ranged from 2.09 to 19.8 g/L of glucose obtained with efficiency values of 10.47 to 99 %. The best conditions were found in test 5 (temperature of 36 °C and pH 5.0, with only 10 FPU/g of substrate Cellic Ctec2 Novozymes ® cocktail). It is observed that for all hydrolysis times the independent variables pH and temperature were significant under the hydrolysis efficiency, showing a negative effect, indicating that higher values of the same promote lower values of the response variable. For bioethanol production, a maximum concentration of 7.84 g/L was observed for the SSH process after 4 h of fermentation, while for the SSF process it was 12.6 g/L after 24 h of fermentation, indicating the large potential of the simultaneous process together with the application of bamboo culm biomass for high production of biofuel.

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The conversion of bioethanol to ethylene in gas phase and atmospheric pressure was investigated over γ-Al2O3 supported copper and nickel catalysts. These catalysts were prepared by co-precipitation and pre-treated with hydrogen at 450 °C. Six catalysts were studied at 450 °C under a nitrogen atmosphere. It was found that the monometallic Cu/γ-Al2O3 catalyst exhibited the highest ethylene concentration, with a selectivity of around 90 %. The bioethanol conversion obtained was between 57 %-86 %. Another catalyst that exhibited high concentration values was the NiCu1 : 7 bimetallic catalyst. The catalysts were characterised using XRD, SEM, EDS, TEM, TGA, FTIR, Raman, and N2-physisoption techniques. Furthermore, the Cu/γ-Al2O3 catalyst was studied under different reduction temperatures and gas flow conditions. It was found that the catalysts reduced at 350 °C and 35 ml/min N2 flow presented ethylene concentrations between (0.18-0.21) g/L. Moreover, the catalyst deactivation was identified to be first order and the equation of the Cu/γ-Al2O3 catalyst deactivation model was determined. Carbonaceous deposits over the used sample were not detected by Raman and FTIR. It was determined that the Cu/γ-Al2O3 catalyst deactivation could be mainly attributed to the blocking of the catalytic sites by strongly adsorbed compounds and hydroxylation of the catalyst surface.

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d-Xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species. The performance of 9 selected xylose-metabolizing yeast strains was evaluated and compared across 3 oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption, and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction compared with other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens, and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. Spathaspora passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all 3 conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses. ONE-SENTENCE SUMMARY: This work aims to expand the knowledge of xylose utilization in different yeast species, with a focus on how oxygenation impacts xylose assimilation.

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The paper industry is a composite one constituting different types of mills, processes, and products. The paper industries consume large amounts of resources, like wood and water. These industries also create huge amounts of waste that have to be treated. In our study, 23 endophytic bacteria were isolated from Argemone mexicana, and 16 endophytic bacteria were isolated from Papaver rhoeas. Seventeen and 15 bacterial endophytes from A. mexicana and P. rhoeas, respectively, showed cellulose-degrading activity. The biochemical and molecular characterization were done for endophytic bacteria with cellulolytic activity. The consortium of cellulose-degrading endophytic bacteria from A. mexicana showed endoglucanase activity (0.462 IU/ml) and FPCase enzyme activity (0.269 IU/ml) and from P. rhoeas gave endoglucanase activity (0.439 IU/ml) and FPCase enzyme activity (0.253 IU/ml). Degraded carboxy methylcellulose and filter paper were further treated by Saccharomyces cerevisiae and bioethanol was produced. Cellulose-degrading endophytic bacteria were also tested for auxin, siderophore production, and phosphate solubilization activities. Individual cellulose-degrading endophytic bacteria with plant growth-promoting activities were used as biofertilizers, tested for plant growth-promoting activities using Basmati Pusa 1121 rice, and plant growth parameters were recorded. The degraded paper enhances the growth of rice plants. Selected bacterial endophytes and their consortia from A. mexicana and P. rhoeas were powerful cellulose degraders, which can be further employed for ethanol production and as significant biofertilizers in agriculture.

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BACKGROUND: The selection of Saccharomyces cerevisiae strains with higher alcohol tolerance can potentially increase the industrial production of ethanol fuel. However, the design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. Although these undesirable side effects are often associated with acute selection regimes, the utility of using harsh ethanol treatments to obtain robust ethanologenic yeasts still has not been fully investigated. RESULTS: We conducted an adaptive laboratory evolution by challenging four populations (P1-P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68-82 cycles of 2-h ethanol shocks (19-30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c-P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c-P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production. CONCLUSIONS: Our study combined genomic, mutational, and fitness analyses to understand the genetic underpinnings of yeast evolution to ethanol shocks. Although fitness analyses revealed that most evolved mutations impose a cost for cell propagation, combination of key mutations cyr1A1474T/usv1Δ endowed yeasts with higher tolerance for growth in the presence of ethanol. Moreover, alleles selected for acute stress survival comprising the P1c genotype conferred stress tolerance and optimal performance under conditions simulating the Brazilian industrial ethanol production.

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Colombia's continuous contamination of water resources and the low alternatives to produce biofuels have affected the fulfillment of the objectives of sustainable development, deteriorating the environment and affecting the economic productivity of this country. Due to this reality, projects on environmental and economic sustainability, phytoremediation, and the production of biofuels such as ethanol and hydrogen were combined. The objective of this article was to design and develop a sustainable system for wastewater treatment and the generation of biofuels based on the biomass of the aquatic plant Eichhornia crassipes. A system that simulates an artificial wetland with live E. crassipes plants was designed and developed, removing organic matter contaminants; subsequently, and continuing the sustainability project, bioreactors were designed, adapted, and started up to produce bioethanol and biohydrogen with the hydrolyzed biomass used in the phytoremediation process, generating around 12 g/L of bioethanol and around 81 ml H2/g. The proposed research strategy suggests combining two sustainable methods, bioremediation and biofuel production, to preserve the natural beauty of water systems and their surroundings.

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Maize (Zea mays L.) is an important crop in Argentina. Aspergillus section Flavi can infect this crop at the pre-harvest stage, and the harvested grains can be contaminated with aflatoxins (AFs). During the production of bioethanol from maize, AF levels can increase up to three times in the final co-products, known as, dry and wet distiller's grain with solubles (DDGS and WDGS), intended for animal feed. Fungal enzymes like laccases can be a useful tool for reducing AF contamination in the co-products obtained from this process. The aim of the present study was to evaluate the ability of laccase enzymes included in enzymatic extracts (EE) produced by different species in the Basidiomycota phylum to reduce AF (AFB1 and AFB2) accumulation under the conditions of in vitro assays. Four laccase activities (5, 10, 15, and 20 U/mL) exerted by nine isolates were evaluated in the absence and presence of vanillic acid (VA), serving as a laccase redox mediator for the degradation of total AFs. The enzymatic stability in maize steep liquor (MSL) was confirmed after a 60 h incubation period. The most effective EE in terms of reducing AF content in the buffer was selected for an additional assay carried out under the same conditions using maize steep liquor obtained after the saccharification stage during the bioethanol production process. The highest degradation percentages were observed at 20 U/mL of laccase enzymatic activity and 1 mM of VA, corresponding to 26% for AFB1 and 26.6% for AFB2. The present study provides valuable data for the development of an efficient tool based on fungal laccases for preventing AF accumulation in the co-products of bioethanol produced from maize used for animal feed.

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Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.

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Cyanobacteria produce exopolysaccharides (EPSs) as an adaptative mechanism against ultraviolet radiation and desiccation. Cellulose is present in the extracellular polymeric substance in some cyanobacteria genera and it has been proposed as a raw material for biofuel production. The goal of this work was to evaluate the cellulose presence in EPS of Atacama cyanobacteria strains and its use as an alternative and innovative biological source to produce bioethanol. The presence of cellulose was evaluated using techniques of molecular biology, bioinformatics, and electronic microscopy. The conserved motif D,D,D,35QXXRW, characteristic of processive ß-glycosyltransferase in all cellulose-producing organisms, was identified in the genome of the LLA-10 strain. This is evidence that cellulose synthase in the LLA-10 strain is a functional enzyme. EPS from Atacama cyanobacteria was hydrolyzed by ß-glucosidases (cellobiase and cellulase) and the released glucose was yeast-fermented to ethanol. Ethanol production reached 172.69 ± 0.02 mg ethanol/g EPS after 48 h of incubation. These results are the first step in the evaluation of EPS produced by native cyanobacteria isolated from northern Chile for future biotechnological applications such as the production of bioethanol.

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Glucoamylases (GAs) are one of the principal groups of enzymes involved in starch hydrolysis and belong to the glycosylhydrolase family. They are classified as exo-amylases due to their ability to hydrolyze α-1,4 glycosidic bonds from the non-reducing end of starch, maltooligosaccharides, and related substrates, releasing ß-D-glucose. Structurally, GAs possess a characteristic catalytic domain (CD) with an (α/α)6 fold and exhibit five conserved regions within this domain. The CD may or may not be linked to a non-catalytic domain with variable functions depending on its origin. GAs are versatile enzymes with diverse applications in food, biofuel, bioplastic and other chemical industries. Although fungal GAs are commonly employed for these purposes, they have limitations such as their low thermostability and an acidic pH requirement. Alternatively, GAs derived from prokaryotic organisms are a good option to save costs as they exhibit greater thermostability compared to fungal GAs. Moreover, a group of cold-adapted GAs from psychrophilic organisms demonstrates intriguing properties that make them suitable for application in various industries. This review provides a comprehensive overview of the structural and sequential properties as well as biotechnological applications of GAs in different industrial processes.

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