Search

Cocoa pod husks (CPH) and cocoa bean shells (CBS) are the main by-products of the cocoa industry and a source of bioactive compounds. These residues are not completely used and thrown in the fields without any treatment, causing environmental problems. Looking for a holistic valorization, the aim of this work was first to deeply characterize CPH and CBS in their chemical composition, amino acid, and fatty acid profiles, as well as their application as antioxidants. CBS had a high level of protein (17.98% DM) and lipids (16.24% DM) compared with CPH (4.79 and 0.35% DM respectively). Glutamic acid and aspartic acid were the predominant amino acids. The total phenolic compounds (TPC) detected in the ethanolic extracts of CPH and CBS were similar to pyrogallol as the main detected polyphenol (72.57 mg/L). CBS ethanolic extract showed a higher antioxidant activity than CPH. Both extracts increased the oxidation stability of soybean oil by 48% (CPH) and 32% (CBS). In addition, alkaline pretreatment of CPH was found suitable for the release of 15.52 ± 0.78 g glucose/L after subsequent saccharification with the commercial enzyme Cellic®. CTec2. Alkaline hydrolyzed and saccharified CPH (Ahs-CPH) was assessed for the first time to obtain polyhydroxy alkanoate (PHAs) and bioethanol. Ahs-CPH allowed the growth of both Cupriavidus necator DSM 545 and Saccharomyces cerevisiae Fm17, well-known as PHA- and bioethanol-producing microbes, respectively. The obtained results suggest that such agricultural wastes have interesting characteristics with new potential industrial uses that could be a better alternative for the utilization of biomass generated as million tons of waste annually. [ABSTRACT FROM AUTHOR]

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route. [ABSTRACT FROM AUTHOR]

The production of lignocellulosic ethanol calls for a robust fermentative yeast able to tolerate a wide range of toxic molecules that occur in the pre-treated lignocellulose. The concentration of inhibitors varies according to the composition of the lignocellulosic material and the harshness of the pre-treatment used. It follows that the versatility of the yeast should be considered when selecting a robust strain. This work aimed at the validation of seven natural Saccharomyces cerevisiae strains, previously selected for their industrial fitness, for their application in the production of lignocellulosic bioethanol. Their inhibitor resistance and fermentative performances were compared to those of the benchmark industrial yeast S. cerevisiae Ethanol Red, currently utilized in the second-generation ethanol plants. The yeast strains were characterized for their tolerance using a synthetic inhibitor mixture formulated with increasing concentrations of weak acids and furans, as well as steam-exploded lignocellulosic pre-hydrolysates, generally containing the same inhibitors. The eight non-diluted liquors have been adopted to assess yeast ability to withstand bioethanol industrial conditions. The most tolerant S. cerevisiae Fm17 strain, together with the reference Ethanol Red, was evaluated for fermentative performances in two pre-hydrolysates obtained from cardoon and common reed, chosen for their large inhibitor concentrations. S. cerevisiae Fm17 outperformed the industrial strain Ethanol Red, producing up to 18 and 39 g/L ethanol from cardoon and common reed, respectively, with ethanol yields always higher than those of the benchmark strain. This natural strain exhibits great potential to be used as superior yeast in the lignocellulosic ethanol plants. [ABSTRACT FROM AUTHOR]

Wheat bran, generated from the milling of wheat, represents a promising feedstock for the production of bioethanol. This substrate consists of three main components: starch, hemicellulose and cellulose. The optimal conditions for wheat bran hydrolysis have been determined using a recombinant cellulase cocktail (RCC), which contains two cellobiohydrolases, an endoglucanase and a β-glucosidase. The 10% (w/v, expressed in terms of dry matter) substrate loading yielded the most glucose, while the 2% loading gave the best hydrolysis efficiency (degree of saccharification) using unmilled wheat bran. The ethanol production of two industrial amylolytic Saccharomyces cerevisiae strains, MEL2[TLG1-SFA1] and M2n[TLG1-SFA1], were compared in a simultaneous saccharification and fermentation (SSF) for 10% wheat bran loading with or without the supplementation of optimised RCC. The recombinant yeast S . cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] completely hydrolysed wheat bran’s starch producing similar amounts of ethanol (5.3 ± 0.14 g/L and 5.0 ± 0.09 g/L, respectively). Supplementing SSF with RCC resulted in additional ethanol production of about 2.0 g/L. Scanning electron microscopy confirmed the effectiveness of both RCC and engineered amylolytic strains in terms of cellulose and starch depolymerisation. This study demonstrated that untreated wheat bran could be a promising ready-to-use substrate for ethanol production. The addition of crude recombinant cellulases improved ethanol yields in the SSF process and S . cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] strains can efficiently convert wheat bran’s starch to ethanol. [ABSTRACT FROM AUTHOR]

Corn starch is currently the primary feedstock for bioethanol production, but concerns about food security have prompted the evaluation of alternative cereals as feedstock. Two potential candidates, triticale and sorghum, can be grown on marginal farming land and mainly serve as animal feed. This study assessed flour and bran-like fractions of triticale and sorghum grains as feedstocks for bioethanol production using a consolidated bioprocessing approach. A Saccharomyces cerevisiae Ethanol Red™-derived amylolytic strain (ERT12) achieved ethanol conversion efficiencies of 100 and 96% from sorghum (after 96 h) and triticale flour (after 168 h), respectively. Supplementation with 10% of the recommended STARGEN™ 002 enzyme loading halved the fermentation time for sorghum and triticale flour. The raw starch in sorghum flour was more digestible than in triticale flour under consolidated bioprocessing conditions, whereas simultaneous saccharification and fermentation proved more effective for triticale flour. The two substrates also showed different hydrolysis patterns, which may be linked to their amylopectin content. This study demonstrated that both substrates could be converted to ethanol using only an efficient amylolytic strain without nitrogen supplementation. However, process development for biofuel production may require different fermentation configurations to be optimised for each substrate. [Display omitted] [ABSTRACT FROM AUTHOR]

Bioethanol obtained from agro-food wastes could contribute to decrease the dependency on fossil resources, reduce the impact of fossil fuels on the environment, and mitigate the food versus fuel debate. This study is aimed to investigate the availability of residual inexpensive agro-food biomasses that could feed a second-generation bioethanol plant located in a specific area of North Eastern Italy. After the identification of all crops in the area, more than 40 agro-food residues were analyzed for their availability and compositions in terms of water, polysaccharides, and sugars potentially convertible into bioethanol. 574,166 Mg of residual wet lignocellulosic biomass corresponding to 297,325 Mg of dry material were found available for bioethanol conversion. The most promising substrates were wheat straw and vine shoots. Based on the chemical composition of residues, the potential attainable ethanol was determined. Theoretical potential ethanol production was estimated at nearly 72,000 Mg per year. This quantity extensively exceeds the minimum yearly capacity of a sustainable bioethanol plant previously identified as around 50,000 Mg of ethanol. Taken together, these results demonstrate that, in the analyzed area, agro-food residues are available in an amount that could sustain bioethanol production in a specific and restricted district. Techno-economical evaluations are in progress to assess the actual feasibility of installing a second generation bioethanol production plant in the area of interest. [ABSTRACT FROM AUTHOR]

Recent advances in amylolytic strain engineering for starch-to-ethanol conversion have provided a platform for the development of raw starch consolidated bioprocessing (CBP) technologies. Several proof-of-concept studies identified improved enzyme combinations, alternative feedstocks and novel host strains for evaluation and application under fermentation conditions. However, further research efforts are required before this technology can be scaled up to an industrial level. In this review, different CBP approaches are defined and discussed, also highlighting the role of auxiliary enzymes for a supplemented CBP process. Various achievements in the development of amylolytic Saccharomyces cerevisiae strains for CBP of raw starch and the remaining challenges that need to be tackled/pursued to bring yeast raw starch CBP to industrial realization, are described. Looking towards the future, it provides potential solutions to develop more cost-effective processes that include cheaper substrates, integration of the 1G and 2G economies and implementing a biorefinery concept where high-value products are also derived from starchy substrates. [ABSTRACT FROM AUTHOR]

In yeast engineering, metabolic burden is often linked to the reprogramming of resources from regular cellular activities to guarantee recombinant protein(s) production. Therefore, growth parameters can be significantly influenced. Two recombinant strains, previously developed by the multiple δ-integration of a glucoamylase in the industrial Saccharomyces cerevisiae 27P, did not display any detectable metabolic burden. In this study, a Fourier Transform InfraRed Spectroscopy (FTIR)-based assay was employed to investigate the effect of δ-integration on yeast strains' tolerance to the increasing ethanol levels typical of the starch-to-ethanol industry. FTIR fingerprint, indeed, offers a holistic view of the metabolome and is a well-established method to assess the stress response of microorganisms. Cell viability and metabolomic fingerprints have been considered as parameters to detecting any physiological and/or metabolomic perturbations. Quite surprisingly, the three strains did not show any difference in cell viability but metabolomic profiles were significantly altered and different when the strains were incubated both with and without ethanol. A LC/MS untargeted workflow was applied to assess the metabolites and pathways mostly involved in these strain-specific ethanol responses, further confirming the FTIR fingerprinting of the parental and recombinant strains. These results indicated that the multiple δ-integration prompted huge metabolomic changes in response to short-term ethanol exposure, calling for deeper metabolomic and genomic insights to understand how and, to what extent, genetic engineering could affect the yeast metabolome. [ABSTRACT FROM AUTHOR]

An engineered yeast producing all the cellulases needed for cellulose saccharification could produce ethanol from lignocellulose at a lower cost. This study aimed to express fungal β-glucosidases in Saccharomyces cerevisiae to convert cellobiose into ethanol. Furthermore, two engineering platforms (laboratory vs industrial strain) have been considered towards the successful deployment of the engineered yeast under simulated industrial conditions. The industrial S. cerevisiae M2n strain was engineered through the δ-integration of the β-glucosidase Pccbgl1 of Phanerochaete chrysosporium. The most efficient recombinant, M2n[pBKD2- Pccbgl1 ]-C1, was compared to the laboratory S. cerevisiae Y294[ Pccbgl1 ] strain, expressing Pccbgl1 from episomal plasmids, in terms of cellobiose fermentation in a steam exploded sugarcane bagasse pre-hydrolysate. Saccharomyces cerevisiae Y294[ Pccbgl1 ] was severely hampered by the pre-hydrolysate. The industrial M2n[pBKD2- Pccbgl1 ]-C1 could tolerate high inhibitors-loading in pre-hydrolysate under aerobic conditions. However, in oxygen limited environment, the engineered industrial strain displayed ethanol yield higher than the laboratory Y294[ Pccbgl1 ] only when supplemented with supernatant containing further recombinant β-glucosidase. This study showed that the choice of the host strain is crucial to ensure bioethanol production from lignocellulose. A novel cellobiose-to-ethanol route has been developed and the recombinant industrial yeast could be a promising platform towards the future consolidated bioprocessing of lignocellulose into ethanol. [ABSTRACT FROM AUTHOR]

The exploitation of agri-food wastes is of great importance for environmental and economic reasons. Date wastes are attractive biomasses that could be used as a carbon source for the growth of microorganisms to obtain added-value products. In this work, spoilage date syrup, containing 102.01 and 101.00 g/L of glucose and fructose, respectively, was assessed as a feedstock for the production of bioethanol and polyhydroxyalkanoates (PHAs) by Saccharomyces cerevisiae MEL2 and Cupriavidus necator DSM 545, respectively. The waste date syrup was first evaluated as a carbon source for microbial growth and resulted to sustain the growth of both strains. 47.95 g/L of ethanol, corresponding to the 93.52% of the theoretical yield, were obtained from the fermentation of date syrup by S. cerevisia e MEL2, here adopted as a proficient bioethanol yeast strain. Furthermore, C. necator DSM 545, a well-known PHAs-producer, was able to accumulate up to 79.20 % (w/w on dry mass) of PHAs. This study demonstrates that bioethanol and PHAs can be obtained from date wastes, contributing to developing cost-effective exploitation of these residues with economic and environmental advantages. [Display omitted] • Spoilage dates are suitable substrate for bioethanol and PHAs production. • A simple hot water extraction allows to obtain a date syrup containing simple sugars. • Simple sugars in syrup were converted by Saccharomyces cerevisiae MEL2 into ethanol. • A one step fermentation mode appears efficient in terms of sugars to PHAs conversion. • Cupriavidus necator DSM 545 is an effective PHAs‐ accumulator from date syrup. [ABSTRACT FROM AUTHOR]

Increased environmental concern over climate change due to higher oil usage has made human being to shift to cleaner and greener alternatives. The utilization of abundant agricultural waste streams as renewable feedstock for biofuels production can be a pivotal strategy. Among others, rice is one of the most largely grown crops, and more than 4.8% of the total production goes to waste. Although previous reviews are related to biofuels obtained from some rice waste, most of those are focused on lignocellulosic rice residues with much attention to thermo-chemical processes. The present paper, instead, reviews for the first time the biotechnological approaches to convert all rice wastes, like rice husk, rice straw, broken rice, discolored rice, unripe rice, into liquid (bioethanol, biobutanol, biodiesel), and gaseous (biogas, biohydrogen) biofuels through the use of pure or mixed microbial cultures. The global availability of each rice byproduct has been also investigated and the potential of rice waste as a 'fuel farm' has been estimated for bioethanol. The physical, chemical, enzymatic, or microbial pretreatments, which play a key role in making carbon available for hydrolysis and fermentation, are here discussed and evaluated. Despite the great promise of technologies so far developed, further research is still required for their up-scale and industrial commercialization. Moreover, future process integrations will open the landscape to biorefinery schemes where rice waste streams can be processed into biofuels and other added-value products, towards the full exploitation of the feedstocks and the economic and environmental sustainability of the overall process. [Display omitted] • Rice waste has the potential to produce many gigalitres of biofuels worldwide. • Selection of pretreatment is the key to obtain the highest biofuel production. • Different biotechnological approaches for biofuel production are elucidated. • Complete rice waste valorization using a biorefinery approach is underscored. [ABSTRACT FROM AUTHOR]