Multi-feedstock biorefinery concept: Valorization of winery wastes by engineered yeast.

The wine industry produces significant amounts of by-products and residues that are not properly managed, posing an environmental problem. Grape must surplus, vine shoots, and wine lees have the potential to be used as renewable resources for the production of energy and chemicals. Metabolic engineering efforts have established Saccharomyces cerevisiae as an efficient microbial cell factory for biorefineries. Current biorefineries designed for producing multiple products often rely on just one feedstock, but the bioeconomy would clearly benefit if these biorefineries could efficiently convert multiple feedstocks. Moreover, to reduce the environmental impact of fossil fuel consumption and maximize production economics, a biorefinery should be capable to supplement the manufacture of biofuel with the production of high-value products. This study proposes an integrated approach for the valorization of diverse wastes resulting from winemaking processes through the biosynthesis of xylitol and ethanol. Using genetically modified S. cerevisiae strains, the xylose-rich hemicellulosic fraction of hydrothermally pretreated vine shoots was converted into xylitol, and the cellulosic fraction was used to produce bioethanol. In addition, grape must, enriched in sugars, was efficiently used as a low-cost source for yeast propagation. The production of xylitol was optimized, in a Simultaneous Saccharification and Fermentation process configuration, by adjusting the inoculum size and enzyme loading. Furthermore, a yeast strain displaying cellulases in the cell surface was applied for the production of bioethanol from the glucan-rich cellulosic. With the addition of grape must and/or wine lees, high ethanol concentrations were reached, which are crucial for the economic feasibility of distillation. This integrated multi-feedstock valorization provides a synergistic alternative for converting a range of winery wastes and by-products into biofuel and an added-value chemical while decreasing waste released to the environment.

Nanocellulose Production: Exploring the Enzymatic Route and Residues of Pulp and Paper Industry.

Increasing environmental and sustainability concerns, caused by current population growth, has promoted a raising utilization of renewable bio-resources for the production of materials and energy. Recently, nanocellulose (NC) has been receiving great attention due to its many attractive features such as non-toxic nature, biocompatibility, and biodegradability, associated with its mechanical properties and those related to its nanoscale, emerging as a promising material in many sectors, namely packaging, regenerative medicine, and electronics, among others. Nanofibers and nanocrystals, derived from cellulose sources, have been mainly produced by mechanical and chemical treatments; however, the use of cellulases to obtain NC attracted much attention due to their environmentally friendly character. This review presents an overview of general concepts in NC production. Especial emphasis is given to enzymatic hydrolysis processes using cellulases and the utilization of pulp and paper industry residues. Integrated process for the production of NC and other high-value products through enzymatic hydrolysis is also approached. Major challenges found in this context are discussed along with its properties, potential application, and future perspectives of the use of enzymatic hydrolysis as a pretreatment in the scale-up of NC production.

Molecular and physiological basis of Saccharomyces cerevisiae tolerance to adverse lignocellulose-based process conditions.

Lignocellulose-based biorefineries have been gaining increasing attention to substitute current petroleum-based refineries. Biomass processing requires a pretreatment step to break lignocellulosic biomass recalcitrant structure, which results in the release of a broad range of microbial inhibitors, mainly weak acids, furans, and phenolic compounds. Saccharomyces cerevisiae is the most commonly used organism for ethanol production; however, it can be severely distressed by these lignocellulose-derived inhibitors, in addition to other challenging conditions, such as pentose sugar utilization and the high temperatures required for an efficient simultaneous saccharification and fermentation step. Therefore, a better understanding of the yeast response and adaptation towards the presence of these multiple stresses is of crucial importance to design strategies to improve yeast robustness and bioconversion capacity from lignocellulosic biomass. This review includes an overview of the main inhibitors derived from diverse raw material resultants from different biomass pretreatments, and describes the main mechanisms of yeast response to their presence, as well as to the presence of stresses imposed by xylose utilization and high-temperature conditions, with a special emphasis on the synergistic effect of multiple inhibitors/stressors. Furthermore, successful cases of tolerance improvement of S. cerevisiae are highlighted, in particular those associated with other process-related physiologically relevant conditions. Decoding the overall yeast response mechanisms will pave the way for the integrated development of sustainable yeast cell-based biorefineries.

HAA1 and PRS3 overexpression boosts yeast tolerance towards acetic acid improving xylose or glucose consumption: unravelling the underlying mechanisms.

Acetic acid tolerance and xylose consumption are desirable traits for yeast strains used in industrial biotechnological processes. In this work, overexpression of a weak acid stress transcriptional activator encoded by the gene HAA1 and a phosphoribosyl pyrophosphate synthetase encoded by PRS3 in a recombinant industrial Saccharomyces cerevisiae strain containing a xylose metabolic pathway was evaluated in the presence of acetic acid in xylose- or glucose-containing media. HAA1 or PRS3 overexpression resulted in superior yeast growth and higher sugar consumption capacities in the presence of 4 g/L acetic acid, and a positive synergistic effect resulted from the simultaneous overexpression of both genes. Overexpressing these genes also improved yeast adaptation to a non-detoxified hardwood hydrolysate with a high acetic acid content. Furthermore, the overexpression of HAA1 and/or PRS3 was found to increase the robustness of yeast cell wall when challenged with acetic acid stress, suggesting the involvement of the modulation of the cell wall integrity pathway. This study clearly shows HAA1 and/or, for the first time, PRS3 overexpression to play an important role in the improvement of industrial yeast tolerance towards acetic acid. The results expand the molecular toolbox and add to the current understanding of the mechanisms involved in higher acetic acid tolerance, paving the way for the further development of more efficient industrial processes.

Recent advances in biorefineries based on lignin extraction using deep eutectic solvents: A review.

Considering the urgent need for alternative biorefinery schemes based on sustainable development, this review aims to summarize the state-of-the-art in the use of deep eutectic solvent pretreatment to fractionate lignocellulose, with a focus on lignin recovery. For that, the key parameters influencing the process are discussed, as well as various strategies to enhance this pretreatment efficiency are explored. Moreover, this review describes the challenges and opportunities associated with the valorization of extraction-derived streams and highlights recent advancements in solvent recovery techniques. Furthermore, the utilization of computational models for process design and optimization is introduced, as the initial attempts at the economic and environmental assessment of this lignocellulosic bioprocess based on deep eutectic solvents. Overall, this review offers a comprehensive perspective on the recent advances in this emerging field and serves as a foundation for further research on the potential integration of deep eutectic pretreatment in sustainable multi-product biorefinery schemes.

Current Options in the Valorisation of Vine Pruning Residue for the Production of Biofuels, Biopolymers, Antioxidants, and Bio-Composites following the Concept of Biorefinery: A Review.

Europe is considered the largest producer of wine worldwide, showing a high market potential. Several wastes are generated at the different stages of the wine production process, namely, vine pruning, stalks, and grape marc. Typically, these residues are not used and are commonly discarded. Portugal generates annually approximately 178 thousand metric tons of wine production waste. In this context, the interest in redirecting the use of these residues has increased due to overproduction, great availability, and low costs. The utilization of these lignocellulosic biomasses derived from the wine industry would economically benefit the producers, while mitigating impacts on the environment. These by-products can be submitted to pre-treatments (physical, chemical, and biological) for the separation of different compounds with high industrial interest, reducing the waste of agro-industrial activities and increasing industrial profitability. Particularly, vine-pruning residue, besides being a source of sugar, has high nutritional value and may serve as a source of phenolic compounds. These compounds can be obtained by bioconversion, following a concept of biorefinery. In this framework, the current routes of the valorisation of the pruning residues will be addressed and put into a circular economy context.

Resveratrol production for the valorisation of lactose-rich wastes by engineered industrial Saccharomyces cerevisiae.

Resveratrol is an antioxidant with applications in the food and cosmetic industries. Its biosynthesis can side the hindrances of its extraction from plants. The dairy industry generates tonnes of lactose-rich wastes, which can be a carbon source. Saccharomyces cerevisiae is an industrial workhorse for biotechnological processes, being unable to naturally metabolise lactose. Here, an S. cerevisiae strain was engineered for de novo production of resveratrol from lactose. A resveratrol titre of 210 mg/L from 100 g/L of lactose in synthetic media was achieved. Process optimization increased by 35% the production by a two-stage process, one favouring ethanol production and a subsequent one with stronger agitation favouring ethanol and lactose consumption with conversion into resveratrol. Resveratrol production from cheese whey was further attained. To the best knowledge of the authors, this is the first report on resveratrol production from lactose, relevant in dairy wastes, establishing grounds for future resveratrol-producing lactose-based processes.

Current breakthroughs in the hardwood biorefineries: Hydrothermal processing for the co-production of xylooligosaccharides and bioethanol.

The development of lignocellulosic biorefineries requires a first stage of pretreatment which enables the efficient valorization of all fractions present in this renewable material. In this sense, this review aims to show the main advantages of hydrothermal treatment as a first step of a biorefinery infrastructure using hardwood as raw material, as well as, main drawback to overcome. Hydrothermal treatment of hardwood highlights for its high selectivity for hemicelluloses solubilization as xylooligosaccharides (XOS). Nevertheless, the suitable conditions for XOS production are inadequate to achieve an elevate cellulose to glucose conversion. Hence, several strategies namely the combination of hydrothermal treatment with delignification process, in situ modification of lignin and the mixture with another renewable resources (concretely, seaweeds, and by-products generated in the food industry with high sugar content) were pinpointed as promising alternative to increase the final ethanol concentration coupled with XOS recovery in the hydrolysate.

Potential and prospects for utilization of avocado by-products in integrated biorefineries.

The industrial processing of avocado to extract oil, and produce guacamole or sauces generates enormous quantities of peels and seeds (around 2 million tons worldwide in 2019) without commercially valuable applications. However, various studies have suggested the presence of a wide range of interesting compounds in the composition of these by-products. This review depicts a thorough outline of the capacity of avocado residues to be converted into a portfolio of commodities that can be employed in sectors such as the food, cosmetics, pharmaceuticals, environment, and energy industries. Therefore, a novel biorefinery strategy to valorize avocado-processing residues to obtain a polyphenolic extract, pectooligosaccharides, and succinic acid was presented. Additionally, the prospects and challenges facing a biorefinery based on the valorization of avocado residues are presented, particularly its techno-economic feasibility on an industrial scale, aiming for a resource-efficient circular bio-economy.

Fast-growing Paulownia wood fractionation by microwave-assisted hydrothermal treatment: A kinetic assessment.

Microwave hydrothermal treatment (MHT), a novel advanced technology, was proposed for the fractionation of Paulownia wood (PW) at temperatures ranging 200-230 °C and residence times of 0-50 min, corresponding to severities of 2.93-4.70. This procedure allowed 80% of xylan recovery as xylooligosaccharides and an average of 95% cellulose recovery in the pretreated PW biomass, showing the selectivity of the treatment, that was also compared to conduction-convection heating autohydrolysis. Finally, a kinetic model was proposed for the prediction of PW fractionation using MHT, with the ultimate goal of being applied to a wide range of feedstocks and minimizing the number of parameters used. For that, two strategies were approached, allowing the reduction of 80 to 34 parameters, without significant influence in the kinetic fitting. To the best of our knowledge, this is the first kinetic modelization of MHT of PW, taking into account all the lignocellulosic fractions.

Microwave hydrothermal processing of the invasive macroalgae Sargassum muticum within a green biorefinery scheme.

This study deals with the multiproduct valorization of the invasive macroalgae Sargassum muticum within a green biorefinery concept using microwave hydrothermal treatment. Temperatures of 160 and 180 °C for 0-60 min (severities 1.62-3.54) were evaluated, allowing a recovery of a liquid phase rich in fucoidan-derived compounds (up to 4.81 g/L), oligomers and phenolics with antioxidant capacity (up to 2.85 g TE/L by ABTS assay), and a high-enzymatically susceptible solid (glucan to glucose conversion 76-100% in 9 h) suitable for bioethanol production (20.5 g/L in 18 h, corresponding to 96% ethanol yield). Moreover, energy consumption of the pretreatments' temperature-time binomial was evaluated showing significant differences, demonstrating the advantages of microwave as alternative heating pretreatment.

Resveratrol Production from Hydrothermally Pretreated Eucalyptus Wood Using Recombinant Industrial Saccharomyces cerevisiae Strains.

Resveratrol is a phenolic compound with strong antioxidant activity, being promising for several applications in health, food, and cosmetics. It is generally extracted from plants or chemically synthesized, in both complex and not sustainable processes, but microbial biosynthesis of resveratrol can counter these drawbacks. In this work, resveratrol production by microbial biosynthesis from lignocellulosic materials was assessed. Three robust industrial Saccharomyces cerevisiae strains known for their thermotolerance and/or resistance to inhibitory compounds were identified as suitable hosts for de novo resveratrol production from glucose and ethanol. Through the CRISPR/Cas9 system, all industrial strains, and a laboratory one, were successfully engineered with the resveratrol biosynthetic pathway via the phenylalanine intermediate. All strains were further screened at 30 °C and 39 °C to evaluate thermotolerance, which is a key feature for Simultaneous Saccharification and Fermentation processes. Ethanol Red RBP showed the best performance at 39 °C, with more than 2.6-fold of resveratrol production in comparison with the other strains. This strain was then used to assess resveratrol production from glucose and ethanol. A maximum resveratrol titer of 187.07 ± 19.88 mg/L was attained from a medium with 2% glucose and 5% ethanol (w/v). Lastly, Ethanol Red RBP produced 151.65 ± 3.84 mg/L resveratrol from 2.95% of cellulose from hydrothermally pretreated Eucalyptus globulus wood, at 39 °C, in a Simultaneous Saccharification and Fermentation process. To the best of our knowledge, this is the first report of lignocellulosic resveratrol production, establishing grounds for the implementation of an integrated lignocellulose-to-resveratrol process in an industrial context.

Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial Saccharomyces cerevisiae as efficient whole cell biocatalysts.

BACKGROUND: Consolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which contains xylose as a major component in concentrations that can reach up to 40% of the total biomass in hardwoods and agricultural residues. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiae-the most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose. RESULTS: These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 g/g of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. CONCLUSIONS: These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.

Comparative study of biorefinery processes for the valorization of fast-growing Paulownia wood.

In this work, valorization of Paulownia wood (PW) was proposed following several process configurations for biofuels and value-added compounds production. Firstly, autohydrolysis and ethanol-organosolv strategies were assessed separately for the fractionation of PW to enhance the enzymatic digestibility of cellulose. A third strategy focused on a sequential process (autohydrolysis and organosolv) was explored. Two temperatures were selected for the first stage of the combined process. High concentration of oligosaccharides (26.29 g/L) and high concentration of degradation products (17.21 g/L) were obtained at 210 and 230 °C, respectively. The solids obtained from both pretreatments were subjected to organosolv delignification (200 °C, 3 h and 50% ethanol) achieving delignification of 58 and 30% for the autohydrolyzed biomass at 210 °C and 230 °C, respectively. The combined process resulted in susceptible biomass able to produce 64 g/L of ethanol. Therefore, the strategies explored in this work open the possibility to build a refinery around Paulownia wood.

Aqueous solutions of deep eutectic systems as reaction media for the saccharification and fermentation of hardwood xylan into xylitol.

The aim of this study was to evaluate the effect of aqueous solutions of deep eutectic solvent, Cholinium Chloride:Urea ([Ch]Cl:U) at 50 wt% and 20 wt%, using different molar ratios (1:1, 2:1 and 1:2) on the enzymatic hydrolysis of xylan for xylose production and its subsequent bioconversion into xylitol using a recombinant yeast strain. The lowest xylan conversion into xylose (45%) was obtained using 1:2 [Ch]Cl:U molar ratio. On the other hand, the 1:1 [Ch]Cl:U molar ratio, at 20 wt% in water, improved this conversion, achieving the highest xylose yield (81.4%). The xylitol production was then optimized with [Ch]Cl:U (1:1) at 20 wt% by simultaneous saccharification and fermentation process, attaining 23.67 g/L, corresponding to 66.04% of xylitol yield. This study reveals the possibility of using xylan solubilized in DES aqueous solutions directly for xylitol production, thus assembling a one-step process.

Recent trends on seaweed fractionation for liquid biofuels production.

Concerns about fossil fuels depletion has led to seek for new sources of energy. The use of marine biomass (seaweed) to produce biofuels presents widely recognized advantages over terrestrial biomasses such as higher production ratio, higher photosynthetic efficiency or carbon-neutral emissions. In here, interesting seaweed sources as a whole or as a residue from seaweed processing industries for biofuel production were identified and their diverse composition and availability compiled. In addition, the pretreatments used for seaweed fractionation were thoroughly revised as this step is pivotal in a seaweed biorefinery for integral biomass valorization and for enabling biomass-to-biofuel economic feasibility processes. Traditional and emerging technologies were revised, with particular emphasis on green technologies, relating pretreatment not only with the type of biomass but also with the final target product(s) and yields. Current hurdles of marine biomass-to-biofuel processes were pinpointed and discussed and future perspectives on the development of these processes given.

Engineering aspects of hydrothermal pretreatment: From batch to continuous operation, scale-up and pilot reactor under biorefinery concept.

Different pretreatments strategies have been developed over the years mainly to enhance enzymatic cellulose degradation. In the new biorefinery era, a more holistic view on pretreatment is required to secure optimal use of the whole biomass. Hydrothermal pretreatment technology is regarded as very promising for lignocellulose biomass fractionation biorefinery and to be implemented at the industrial scale for biorefineries of second generation and circular bioeconomy, since it does not require no chemical inputs other than liquid water or steam and heat. This review focuses on the fundamentals of hydrothermal pretreatment, structure changes of biomass during this pretreatment, multiproduct strategies in terms of biorefinery, reactor technology and engineering aspects from batch to continuous operation. The treatise includes a case study of hydrothermal biomass pretreatment at pilot plant scale and integrated process design.

Intensifying ethanol production from brewer's spent grain waste: Use of whole slurry at high solid loadings.

This work targets the valorization of brewer's spent grain (BSG) waste by ethanol production, providing strategies for increasing titers in the multiple process steps involved. High solid loadings and use of whole slurry from the pretreatment were evaluated, aiming to achieve high ethanol concentration and yield. As variability in BSG chemical composition presents a challenge for their valorization, six different BSGs were chemically characterized, which allowed the selection of two with high polysaccharide content. High solid loadings (up to 25%) were employed for the pretreatment of selected BSGs by autohydrolysis, an environmentally friendly process, to improve enzymatic saccharification and extract fermentable sugars as oligosaccharides and monosaccharides. As a result, high glucose concentrations (43.7 and 57.7 g L-1) and glucose yield (85.9 and 70.6%) were obtained from the saccharification of the pretreated BSG whole slurry at 20 and 25% solid loading, respectively. Whole slurries from autohydrolysis were used as substrate for ethanol production by hybrid saccharification and fermentation. Two different Saccharomyces cerevisiae strains were evaluated and high ethanol concentration (42.27 g L-1) at a high yield (94.0%) was achieved. The results attained by the combined intensification approaches qualify BSG waste as a valuable renewable resource for cost-effective ethanol production.

Xylose fermentation efficiency of industrial Saccharomyces cerevisiae yeast with separate or combined xylose reductase/xylitol dehydrogenase and xylose isomerase pathways.

BACKGROUND: Xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways have been extensively used to confer xylose assimilation capacity to Saccharomyces cerevisiae and tackle one of the major bottlenecks in the attainment of economically viable lignocellulosic ethanol production. Nevertheless, there is a lack of studies comparing the efficiency of those pathways both separately and combined. In this work, the XI and/or XR/XDH pathways were introduced into two robust industrial S. cerevisiae strains, evaluated in synthetic media and corn cob hemicellulosic hydrolysate and the results were correlated with the differential enzyme activities found in the xylose-pathway engineered strains. RESULTS: The sole expression of XI was found to increase the fermentative capacity of both strains in synthetic media at 30 °C and 40 °C: decreasing xylitol accumulation and improving xylose consumption and ethanol production. Similar results were observed in fermentations of detoxified hydrolysate. However, in the presence of lignocellulosic-derived inhibitors, a positive synergistic effect resulted from the expression of both XI and XR/XDH, possibly caused by a cofactor equilibrium between the XDH and furan detoxifying enzymes, increasing the ethanol yield by more than 38%. CONCLUSIONS: This study clearly shows an advantage of using the XI from Clostridium phytofermentans to attain high ethanol productivities and yields from xylose. Furthermore, and for the first time, the simultaneous utilization of XR/XDH and XI pathways was compared to the single expression of XR/XDH or XI and was found to improve ethanol production from non-detoxified hemicellulosic hydrolysates. These results extend the knowledge regarding S. cerevisiae xylose assimilation metabolism and pave the way for the construction of more efficient strains for use in lignocellulosic industrial processes.

Microbial lipids from industrial wastes using xylose-utilizing Ashbya gossypii strains.

This work presents the exploitation of waste industrial by-products as raw materials for the production of microbial lipids in engineered strains of the filamentous fungus Ashbya gossypii. A lipogenic xylose-utilizing strain was used to apply a metabolic engineering approach aiming at relieving regulatory mechanisms to further increase the biosynthesis of lipids. Three genomic manipulations were applied: the overexpression of a feedback resistant form of the acetyl-CoA carboxylase enzyme; the expression of a truncated form of Mga2, a regulator of the main Δ9 desaturase gene; and the overexpression of an additional copy of DGA1 that codes for diacylglycerol acyltransferase. The performance of the engineered strain was evaluated in culture media containing mixed formulations of corn-cob hydrolysates, sugarcane molasses or crude glycerol. Our results demonstrate the efficiency of the engineered strains, which were able to accumulate about 40% of cell dry weight (CDW) in lipid content using organic industrial wastes as feedstocks.