Enhancing xylose-fermentation capacity of engineered Saccharomyces cerevisiae by multistep evolutionary engineering in inhibitor-rich lignocellulose hydrolysate.

Major progress in developing Saccharomyces cerevisiae strains that utilize the pentose sugar xylose has been achieved. However, the high inhibitor content of lignocellulose hydrolysates still hinders efficient xylose fermentation, which remains a major obstacle for commercially viable second-generation bioethanol production. Further improvement of xylose utilization in inhibitor-rich lignocellulose hydrolysates remains highly challenging. In this work, we have developed a robust industrial S. cerevisiae strain able to efficiently ferment xylose in concentrated undetoxified lignocellulose hydrolysates. This was accomplished with novel multistep evolutionary engineering. First, a tetraploid strain was generated and evolved in xylose-enriched pretreated spruce biomass. The best evolved strain was sporulated to obtain a genetically diverse diploid population. The diploid strains were then screened in industrially relevant conditions. The best performing strain, MDS130, showed superior fermentation performance in three different lignocellulose hydrolysates. In concentrated corncob hydrolysate, with initial cell density of 1 g DW/l, at 35°C, MDS130 completely coconsumed glucose and xylose, producing ± 7% v/v ethanol with a yield of 91% of the maximum theoretical value and an overall productivity of 1.22 g/l/h. MDS130 has been developed from previous industrial yeast strains without applying external mutagenesis, minimizing the risk of negative side-effects on other commercially important properties and maximizing its potential for industrial application.

Comprehensive network of stress-induced responses in Zymomonas mobilis during bioethanol production: from physiological and molecular responses to the effects of system metabolic engineering.

Nowadays, biofuels, especially bioethanol, are becoming increasingly popular as an alternative to fossil fuels. Zymomonas mobilis is a desirable species for bioethanol production due to its unique characteristics, such as low biomass production and high-rate glucose metabolism. However, several factors can interfere with the fermentation process and hinder microbial activity, including lignocellulosic hydrolysate inhibitors, high temperatures, an osmotic environment, and high ethanol concentration. Overcoming these limitations is critical for effective bioethanol production. In this review, the stress response mechanisms of Z. mobilis are discussed in comparison to other ethanol-producing microbes. The mechanism of stress response is divided into physiological (changes in growth, metabolism, intracellular components, and cell membrane structures) and molecular (up and down-regulation of specific genes and elements of the regulatory system and their role in expression of specific proteins and control of metabolic fluxes) changes. Systemic metabolic engineering approaches, such as gene manipulation, overexpression, and silencing, are successful methods for building new metabolic pathways. Therefore, this review discusses systems metabolic engineering in conjunction with systems biology and synthetic biology as an important method for developing new strains with an effective response mechanism to fermentation stresses during bioethanol production. Overall, understanding the stress response mechanisms of Z. mobilis can lead to more efficient and effective bioethanol production.

Sustainability assessment of biofuel and value-added product from organic fraction of municipal solid waste.

The current study aims to examine the techno-economic and environmental assessment of biorefinery development within a circular bioeconomy context by using an organic fraction of municipal solid waste (OFMSW) by extraction of lipids, carbohydrates, and proteins with 98, 51 and 62 % by mass of conversion efficiency and yield recovery, and value-added fractions production as well. Fatty acid methyl ester (biodiesel) and glycerol (biofuel) were produced by applying transesterification process, and the remaining biomass was converted into biocrude oil by thermal liquefication. The biorefinery using 613 kg of OFMSW produced biodiesel, glycerol, and bioethanol with 126 litter, 14.3 kg, and 172 litter respectively, as well as value-added fractions, such as biocrude oil with 78 kg. The environmental impact was assessed using the life cycle assessment (LCA) framework, ReCiPe2016 Mid-point (H) approach, through 18 different environmental categories. The key findings elucidate that Terrestrial ecotoxicity, Climate change, Fossil depletion and Human toxicity were the main impact categories which are potentially contributed 9.81E+02 kg 1,4-DB eq., 1.43E+03 kg CO2 eq., 2.04E+02 kg oil eq., and 8.08E+01 kg 1,4-DB eq. The normalization (person per equivalent) analysis revealed that only categories of resource reduction (fossil and metal depletion) are the key contributors to environmental degradation. The biorefinery system's total revenue was estimated at USD 6.817,509 million annually. The calculated revenue was USD 0.026 million daily in a shift of 8 h. The Net present worth (NPW) was calculated at USD 499.97 million by assuming a discount factor of 10 % and inflation rate of 5 % for 15 years. The project is considered feasible by demonstrating 7.15 payback year. This research showcased the efficient portrayal of the biorefinery system and succinctly conveyed the significant circular bioeconomy for a greener future. Thus, it could be helpful to the stakeholder's context towards techno-economic and environmental evaluation.

Short-term adaptation as a tool to improve bioethanol production using grass press-juice as fermentation medium.

Grass raw materials collected from grasslands cover more than 30% of Europe's agricultural area. They are considered very attractive for the production of different biochemicals and biofuels due to their high availability and renewability. In this study, a perennial ryegrass (Lolium perenne) was exploited for second-generation bioethanol production. Grass press-cake and grass press-juice were separated using mechanical pretreatment, and the obtained juice was used as a fermentation medium. In this work, Saccharomyces cerevisiae was utilized for bioethanol production using the grass press-juice as the sole fermentation medium. The yeast was able to release about 11 g/L of ethanol in 72 h, with a total production yield of 0.38 ± 0.2 gEthanol/gsugars. It was assessed to improve the fermentation ability of Saccharomyces cerevisiae by using the short-term adaptation. For this purpose, the yeast was initially propagated in increasing the concentration of press-juice. Then, the yeast cells were re-cultivated in 100%(v/v) fresh juice to verify if it had improved the fermentation efficiency. The fructose conversion increased from 79 to 90%, and the ethanol titers reached 18 g/L resulting in a final yield of 0.50 ± 0.06 gEthanol/gsugars with a volumetric productivity of 0.44 ± 0.00 g/Lh. The overall results proved that short-term adaptation was successfully used to improve bioethanol production with S. cerevisiae using grass press-juice as fermentation medium. KEY POINTS: • Mechanical pretreatment of grass raw materials • Production of bioethanol using grass press-juice as fermentation medium • Short-term adaptation as a tool to improve the bioethanol production.

Exploring xylose metabolism in non-conventional yeasts: kinetic characterization and product accumulation under different aeration conditions.

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.

Simultaneous production of biofuel, and removal of heavy metals using marine alga Turbinaria turbinata as a feedstock in NEOM Region, Tabuk.

Depletion of fossil fuel and pollution by heavy metals are two major global issues. The cell wall of algae consists of polymers of polysaccharides such as cellulose, hemicellulose, alginate, starch, and many others that are readily hydrolyzed to monosaccharides and hence are amenable to fermentation into bioethanol. Moreover, algae contain lipids that may undergo trans-esterification to biodiesel, and can be absorbed by heavy metals. In this study, extraction of lipids from Turbinaria turbinata (common brown alga) from the beach of Sharma, NEOM, Tabuk, Saudi Arabia by different solvents hexane, methanol, and hexane: methanol (1:1), and trans-esterification was performed to obtain biodiesel and investigated by GC.MS. The alga residue after fats extractions by different solvents was used in bioremediation synthetic wastewater containing 50 ppm of As-3, Co+2, Cu+2, Fe+2, Mn+2, and Zn+2. The residue of defatted alga was hydrolyzed by 2% H2SO4 and then fermented to obtain bioethanol. The combination of hexane: methanol (1:1) gave the greatest amount of petroleum hydrocarbons, which contain Tetradecane, 5-methyl, Octacosane, Pentatriacontane, and a small amount of Cyclotrisiloxane, Hexamethyl. The most effective removal % was obtained with alga residue defatted by hexane: methanol (1:1), and methanol, 100% removal of As-3, 83% Co+2, 95% Cu+2, 97.25% Fe+2, Mn+2 79.69%, Zn+2 90.15% with 2 g alga /L at 3 hours. The lowest value of sugar was obtained with hexane: methanol residue but gave the highest bioethanol efficiency. Thus, it is possible to use Turbinaria turbinata, or brown alga as a feedstock to produce bio-diesel, and bioethanol, and to remove heavy metals from wastewater, which may have a great economic and environmental significance.

Evaluation of highly adsorptive Guefoams (multifunctional guest-containing foams) as a potential sorbent for determination of volatile organic compounds (VOCs) by means of thermal desorption.

The present work delves into the feasibility of employing a novel structured sorbent referred to as GFAD (Guefoam Adsorption Device) for the determination of volatile organic compounds (VOCs) in liquid samples. The chosen method has been static headspace sorptive extraction-thermal desorption gas chromatography mass spectrometry (HSSE-TD-GC-MS). The GFAD comprises an aluminum cellular material with a distinct replication structure and a solid guest phase consisting of activated carbon particles dispersed within the cavities of the cellular aluminum. The extensive specific surface area, robustness, and exceptional thermal conductivity of this pioneering material offer distinct advantages over commercially available polydimethylsiloxane-based Twister® devices. Therefore, the trapping efficiency for volatile organic compounds is enhanced, and it is possible to perform the analysis of concentrated samples. According to computational simulations, it has been demonstrated that GFAD has a high heat conductivity. As a result, the desorption efficiency is improved, and minimal temperature gradients are generated throughout the GFAD during the heating process. Besides, the energy consumption is significantly lowered, thus aligning with environmentally conscientious and sustainable analytical practices.The experimental results give a proof of the suitability of the GFAD for determining gaseous compounds in liquid samples through HSSE-TD-GC-MS. For volatile species, the new material provides higher peak areas and lower limits of detection than a commercially available Twister® device. Furthermore, the GFAD is reusable, its adsorbing properties remaining unchanged during, at least, 100 consecutive analyses. In addition, unlike to the Twister®, no intense siloxane peaks are observed in the chromatograms obtained with the GFAD. The feasibility of qualitative and semi-quantitative analysis with the new accessory has been demonstrated with both standards and a cereal bioethanol real sample.

Valorization of coffee bean processing waste for bioethanol production: comparison and evaluation of mass transfer effects in fermentations using free and encapsulated cells of Saccharomyces cerevisiae.

Coffee husk, an agricultural waste abundant in carbohydrates and nutrients, is typically discarded through landfills, mixed with animal fodder, or incinerated. However, in alignment with sustainable development principles, researchers worldwide are exploring innovative methods to harness the value of coffee husk, transforming it into profitable products. One such avenue is the biotechnological approach to bioethanol production from agricultural wastes, offering an eco-friendly alternative to mitigate the adverse effects of fossil fuels. This study delves into the feasibility of utilizing coffee husk as a substrate for bioethanol production, employing and comparing various hydrolysis methods. The enzymatic hydrolysis method outshone thermochemical and thermal approaches, yielding 1.84 and 3.07 times more reducing sugars in the hydrolysate, respectively. In examining bioethanol production, a comparison between free and encapsulated cells in enzyme hydrolysate revealed that free-cell fermentation faced challenges due to cell viability issues. Under specific fermentation conditions, bioethanol yield (0.59 and 0.83 g of bioethanol/g of reducing sugar) and productivity (0.1 and 0.12 g/L h) were achieved for free and encapsulated cells, respectively. However, it was noted that bioethanol production by encapsulated cells was more significantly influenced by internal mass transfer effects, as indicated by the Thiele modulus and effectiveness factor. In conclusion, our findings underscore the potential of coffee husk as a valuable substrate for bioethanol production, showcasing its viability in contributing to sustainable and eco-friendly practices.

Phytostabilization of fly ash from a coalmine in Botswana and biovalorisation of the recovered Napier grass (Pennisetum purpureum Schumach.).

The disposal of fly ash (FA) from coal power plants polluting the air, soil, and groundwater is a major environmental concern. Phytoremediation to rehabilitate fly ash dumpsites is a promising alternative but has practical concerns about the disposal of harvested biomass. This study investigated the effect of supplementing fly ash with fresh sewage sludge (FSS), aged sewage sludge, food waste, and compost (COM) to enhance the phytoremediation potential of Napier grass and its subsequent utilization for ethanol production. The highest removal of Mn (1196.12 g ha-1) and Ni (128.06 g ha-1) from FA could be obtained when Napier is grown in the presence of FSS and inorganic fertilizer (NPK). In addition, the highest bioethanol yield (19.31 g L-1) was obtained from Napier grown in fly ash with COM + NPK, thus providing additional economic benefits aside from the remediation process. Given the significant levels of heavy metals present in the pulp and bio-slurry after ethanol production, further research is required in this area to determine the best ways to utilize this waste such as converting it into biochar.

Using energy crops as a phytoremediation agent for fly ash dumpsites has the potential to remediate heavy metal contamination and provide additional economic benefits. Napier grass was able to tolerate high concentrations of heavy metals and yield high biomass in fly ash in the presence of organic amendments. The harvested biomass was successfully converted into substrate for bioethanol production using heavy metal-tolerant yeast. This is the first report on the production of ethanol from the phytoremediation biomass of Napier grass.

Regional bioethanol supply chain optimization with the integration of GIS-MCDM method and quantile-based scenario analysis.

This study presents a novel decision-support framework for the bioethanol supply chain network planning and management under uncertainties. Under the holistic framework, the most suitable sites for biorefineries are first screened out by adopting a GIS-based multi-criteria decision-making approach. Then, a mixed-integer linear programming model combined with quantile-based scenario analysis is developed to determine the strategic planning (i.e. locations and size of biorefineries) and tactical management (i.e. biomass purchasing, feedstock transportation, bioethanol production, and product delivery) under uncertainties. The model can effectively search for reliable solutions under uncertainties and achieve tradeoff solutions with the consideration of decision makers' risk tolerance. The proposed framework is demonstrated through a case study in China. It is suggested to build seven biorefineries with a capacity of 100 million liters in Zhumadian city. Utilizing 41% of local agricultural residues could satisfy the bioethanol requirement in the transportation sector under the E20 policy. However, the estimated production cost of bioethanol in Zhumadian is very high, about 1.11 $/L, which makes it lose cost advantage in the fuel market. Thus, currently, effective subsidies, mandatory energy substitution policies, along other environmental regulatory measures are desired to promote the bioethanol industry development.

Renewable Energy Potential: Second-Generation Biomass as Feedstock for Bioethanol Production.

Biofuels are clean and renewable energy resources gaining increased attention as a potential replacement for non-renewable petroleum-based fuels. They are derived from biomass that could either be animal-based or belong to any of the three generations of plant biomass (agricultural crops, lignocellulosic materials, or algae). Over 130 studies including experimental research, case studies, literature reviews, and website publications related to bioethanol production were evaluated; different methods and techniques have been tested by scientists and researchers in this field, and the most optimal conditions have been adopted for the generation of biofuels from biomass. This has ultimately led to a subsequent scale-up of procedures and the establishment of pilot, demo, and large-scale plants/biorefineries in some regions of the world. Nevertheless, there are still challenges associated with the production of bioethanol from lignocellulosic biomass, such as recalcitrance of the cell wall, multiple pretreatment steps, prolonged hydrolysis time, degradation product formation, cost, etc., which have impeded the implementation of its large-scale production, which needs to be addressed. This review gives an overview of biomass and bioenergy, the structure and composition of lignocellulosic biomass, biofuel classification, bioethanol as an energy source, bioethanol production processes, different pretreatment and hydrolysis techniques, inhibitory product formation, fermentation strategies/process, the microorganisms used for fermentation, distillation, legislation in support of advanced biofuel, and industrial projects on advanced bioethanol. The ultimate objective is still to find the best conditions and technology possible to sustainably and inexpensively produce a high bioethanol yield.

Using Extracted Sugars from Spoiled Date Fruits as a Sustainable Feedstock for Ethanol Production by New Yeast Isolates.

This study focuses on investigating sugar recovery from spoiled date fruits (SDF) for sustainable ethanol production using newly isolated yeasts. Upon their isolation from different food products, yeast strains were identified through PCR amplification of the D1/D2 region and subsequent comparison with the GenBank database, confirming isolates KKU30, KKU32, and KKU33 as Saccharomyces cerevisiae; KKU21 as Zygosaccharomyces rouxii; and KKU35m as Meyerozyma guilliermondii. Optimization of sugar extraction from SDF pulp employed response surface methodology (RSM), varying solid loading (20-40%), temperature (20-40 °C), and extraction time (10-30 min). Linear models for sugar concentration (R1) and extraction efficiency (R2) showed relatively high R2 values, indicating a good model fit. Statistical analysis revealed significant effects of temperature and extraction time on extraction efficiency. The results of batch ethanol production from SDF extracts using mono-cultures indicated varying consumption rates of sugars, biomass production, and ethanol yields among strains. Notably, S. cerevisiae strains exhibited rapid sugar consumption and high ethanol productivity, outperforming Z. rouxii and M. guilliermondii, and they were selected for scaling up the process at fed-batch mode in a co-culture. Co-cultivation resulted in complete sugar consumption and higher ethanol yields compared to mono-cultures, whereas the ethanol titer reached 46.8 ± 0.2 g/L.

Development of an Integrated Bioprocess System for Bioethanol and Arabitol Production from Sugar Beet Cossettes.

Research background: An innovative integrated bioprocess system for bioethanol production from raw sugar beet cossettes (SBC) and arabitol from remaining exhausted sugar beet cossettes (ESBC) was studied. This integrated three-stage bioprocess system is an example of the biorefinery concept to maximise the use of raw SBC for the production of high value-added products such as sugar alcohols and bioethanol. Experimental approach: The first stage of the integrated bioprocess system was simultaneous sugar extraction from SBC and its alcoholic fermentation to produce bioethanol in an integrated bioreactor system (vertical column bioreactor and stirred tank bioreactor) containing a high-density suspension of yeast Saccharomyces cerevisiae (30 g/L). The second stage was the pretreatment of ESBC with dilute sulfuric acid to release fermentable sugars. The resulting liquid hydrolysate of ESBC was used in the third stage as a nutrient medium for arabitol production by non-Saccharomyces yeasts (Spathaspora passalidarum CBS 10155 and Spathaspora arborariae CBS 11463). Results and conclusions: The obtained results show that the efficiency of bioethanol production increased with increasing temperature and prolonged residence time in the integrated bioreactor system. The maximum bioethanol production efficiency (87.22 %) was observed at a time of 60 min and a temperature of 36 °C. Further increase in residence time (above 60 min) did not result in the significant increase of bioethanol production efficiency. Weak acid hydrolysis was used for ESBC pretreatment and the highest sugar yield was reached at 200 °C and residence time of 1 min. The inhibitors of the weak acid pretreatment were produced below bioprocess inhibition threshold. The use of the obtained liqiud phase of ESBC hydrolysate for the production of arabitol in the stirred tank bioreactor under constant aeration clearly showed that S. passalidarum CBS 10155 with 8.48 g/L of arabitol (YP/S=0.603 g/g and bioprocess productivity of 0.176 g/(L.h)) is a better arabitol producer than Spathaspora arborariae CBS 10155. Novelty and scientific contribution: An innovative integrated bioprocess system for the production of bioethanol and arabitol was developed based on the biorefinery concept. This three-stage bioprocess system shows great potential for maximum use of SBC as a feedstock for bioethanol and arabitol production and it could be an example of a sustainable 'zero waste' production system.

Characteristics and kinetics of thermophilic actinomycetes' amylase production on agro-wastes and its application for ethanol fermentation.

Thermophilic actinomycetes are commonly found in extreme environments and can thrive and adapt to extreme conditions. These organisms exhibit substantial variation and garnered significant interest due to their remarkable enzymatic activities. This study evaluated the potential of Streptomyces griseorubens NBR14 and Nocardiopsis synnemataformans NBRM9 strains to produce thermo-stable amylase via submerged fermentation using wheat and bean straw. The Box-Behnken design was utilized to determine the optimum parameters for amylase biosynthesis. Subsequently, amylase underwent partial purification and characterization. Furthermore, the obtained hydrolysate was applied for ethanol fermentation using Saccharomyces cerevisiae. The optimal parameters for obtaining the highest amylase activity by NBR14 (7.72 U/mL) and NBRM9 (26.54 U/mL) strains were found to be 40 and 30 °C, pH values of 7, incubation time of 7 days, and substrate concentration (3 and 2 g/100 mL), respectively. The NBR14 and NBRM9 amylase were partially purified, resulting in specific activities of 251.15 and 144.84 U/mg, as well as purification factors of 3.91 and 2.69-fold, respectively. After partial purification, the amylase extracted from NBR14 and NBRM9 showed the highest activity level at pH values of 9 and 7 and temperatures of 50 and 60 °C, respectively. The findings also indicated that the maximum velocity (Vmax) for NBR14 and NBRM9 amylase were 57.80 and 59.88 U/mL, respectively, with Km constants of 1.39 and 1.479 mM. After 48 h, bioethanol was produced at concentrations of 5.95 mg/mL and 9.29 mg/mL from hydrolyzed wheat and bean straw, respectively, through fermentation with S. cerevisiae. Thermophilic actinomycetes and their α-amylase yield demonstrated promising potential for sustainable bio-ethanol production from agro-byproducts.

Engineered Chlorella vulgaris improves bioethanol production and promises prebiotic application.

Microalgal biomass for biofuel production, integration into functional food, and feed supplementation has generated substantial interest worldwide due to its high growth rate, non-competitiveness for agronomic land, ease of cultivation in containments, and presence of several bioactive molecules. In this study, genetic engineering tools were employed to develop transgenic lines of freshwater microalga Chlorella vulgaris with a higher starch content, by up-regulating ADP-glucose pyrophosphorylase (AGPase), which is a rate-limiting enzyme in starch biosynthesis. Expression of the Escherichia coli glgC (AGPase homolog) gene in C. vulgaris led to an increase in total carbohydrate content up to 45.1% (dry cell weight, DCW) in the transgenic line as compared to 34.2% (DCW) in the untransformed control. The starch content improved up to 16% (DCW) in the transgenic alga compared to 10% (DCW) in the control. However, the content of total lipid, carotenoid, and chlorophyll decreased differentially in the transgenic lines. The carbohydrate-rich biomass from the transgenic algal line was used to produce bioethanol via yeast fermentation, which resulted in a higher ethanol yield of 82.82 mg/L as compared to 54.41 mg/L from the untransformed control. The in vitro digestibility of the transgenic algal starch revealed a resistant starch content of up to 7% of total starch. Faster growth of four probiotic bacterial species along with a lowering of the pH of the growth medium indicated transgenic alga to exert a positive prebiotic effect. Taken together, the study documents the utilization of genetically engineered C. vulgaris with enriched carbohydrates as bioethanol feedstock and functional food ingredients.

Axenic green microalgae for the treatment of textile effluent and the production of biofuel: a promising sustainable approach.

An integrated approach to nutrient recycling utilizing microalgae could provide feasible solutions for both environmental control and energy production. In this study, an axenic microalgae strain, Chlorella sorokiniana ASK25 was evaluated for its potential as a biofuel feedstock and textile wastewater (TWW) treatment. The microalgae isolate was grown on TWW supplemented with different proportions of standard BG-11 medium varying from 0 to 100% (v/v). The results showed that TWW supplemented with 20% (v/v) BG11 medium demonstrated promising results in terms of Chlorella sorokiniana ASK25 biomass (3.80 g L-1), lipid production (1.24 g L-1), nutrients (N/P, > 99%) and pollutant removal (chemical oxygen demand (COD), 99.05%). The COD level dropped by 90% after 4 days of cultivation, from 2,593.33 mg L-1 to 215 mg L-1; however, after day 6, the nitrogen (-NO3-1) and total phosphorus (TP) levels were reduced by more than 95%. The biomass-, total lipid- and carbohydrate- production, after 6 days of cultivation were 3.80 g L-1, 1.24 g L-1, and 1.09 g L-1, respectively, which were 2.15-, 2.95- and 3.30-fold higher than Chlorella sorokiniana ASK25 grown in standard BG-11 medium (control). In addition, as per the theoretical mass balances, 1 tonne biomass of Chlorella sorokiniana ASK25 might yield 294.5 kg of biodiesel and 135.7 kg of bioethanol. Palmitic acid, stearic acid, and oleic acid were the dominant fatty acids found in the Chlorella sorokiniana ASK25 lipid. This study illustrates the potential use of TWW as a microalgae feedstock with reduced nutrient supplementation (20% of TWW). Thus, it can be considered a promising feedstock for economical biofuel production.

Exploring Seaweed-Associated Marine Microbes: Growth Impacts and Enzymatic Potential for Sustainable Resource Utilization.

Seaweed, a valuable marine resource widely cultivated worldwide, can be vulnerable to stress and microbiome alterations, resulting in the decay of seaweeds and substantial economic losses. To investigate the seaweed-microbiome interaction, our study aimed to isolate marine bacteria and fungi that can cause Ice-Ice disease and evaluate their enzymatic characteristics for potential application in bioethanol production from seaweed biomass. Three red seaweed species (Gracilaria edulis, Kappaphycus alvarezii, and Eucheuma cottonii) were obtained for our study and placed in separate culture tanks. Among the 18 isolated marine microbial species, 12 tested positive for agar and carrageenan activity: six exhibited both activities, three displayed only agar activity, and three only carrageenan activity. DNA sequencing of the positive microbes identified ten bacteria and two yeast species. The 3,5-Dinitrosalicylic acid (DNSA) assay results revealed that the identified bacterial Caldibacillus kokeshiiformis strain FJAT-47861 exhibited the highest carrageenase activity (0.76 units/ml), while the yeast Pichia fermentans strain PM79 demonstrated the highest agarase activity (0.52 units/ml). Notably, Pichia fermentans strain PM79 exhibited the highest overall agarase and carrageenase activity, averaging 0.63 units/ml. The average carrageenase activity of all six positive microbes was 1.5 times higher than their agarase activity. These findings suggest that the 12 isolated microbes hold potential for bioethanol production from macroalgae, as their agarase and carrageenase activity indicates their ability to break down seaweed cell wall carbohydrates, causing ice-ice disease. Moreover, these results provide exciting prospects for harnessing the bioconversion capabilities of these microbes, paving the way for sustainable and efficient bioethanol production from seaweed resources. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01205-w.

Air-Promoted Light-Driven Hydrogen Production from Bioethanol over Core/Shell Cr2O3@GaN Nanoarchitecture.

Light-driven hydrogen production from biomass derivatives offers a path towards carbon neutrality. It is often however operated with the limitations of sluggish kinetics and severe coking. Herein, a disruptive air-promoted strategy is explored for efficient and durable light-driven hydrogen production from ethanol over a core/shell Cr2O3@GaN nanoarchitecture. The correlative computational and experimental investigations show ethanol is energetically favorable to be adsorbed on the Cr2O3@GaN interface, followed by dehydrogenation toward acetaldehyde and protons by photoexcited holes. The released protons are then consumed for H2 evolution by photogenerated electrons. Afterward, O2 can be evolved into active oxygen species and promote the deprotonation and C-C cleavage of the key C2 intermediate, thus significantly lowering the reaction energy barrier of hydrogen evolution and removing the carbon residual with inhibited overoxidation. Consequently, hydrogen is produced at a high rate of 76.9 mole H2 per gram Cr2O3@GaN per hour by only feeding ethanol, air, and light, leading to the achievement of a turnover number of 266,943,000 mole H2 per mole Cr2O3 over a long-term operation of 180 hours. Notably, an unprecedented light-to-hydrogen efficiency of 17.6 % is achieved under concentrated light illumination. The simultaneous generation of aldehyde from ethanol dehydrogenation enables the process more economically promising.

Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield.

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.

Consolidated bioprocessing for bioethanol production by metabolically engineered cellulolytic fungus Myceliophthora thermophila.

Using cellulosic ethanol as fuel is one way to help achieve the world's decarbonization goals. However, the economics of the present technology are unfavorable, especially the cost of cellulose degradation. Here, we reprogram the thermophilic cellulosic fungus Myceliophthora thermophila to directly ferment cellulose into ethanol by mimicking the aerobic ethanol fermentation of yeast (the Crabtree effect), including optimizing the synthetic pathway, enhancing the glycolytic rate, inhibiting mitochondrial NADH shuttles, and knocking out ethanol consumption pathway. The final engineered strain produced 52.8 g/L ethanol directly from cellulose, and 39.8 g/L from corncob, without the need for any added cellulase, while the starting strain produced almost no ethanol. We also demonstrate that as the ethanol fermentation by engineered M. thermophila increases, the composition and expression of cellulases that facilitate the degradation of cellulose, especially cellobiohydrolases, changes. The simplified production process and significantly increased ethanol yield indicate that the fungal consolidated bioprocessing technology that we develop here (one-step, one-strain ethanol production) is promising for fueling sustainable carbon-neutral biomanufacturing in the future.