Evaluation of seed oil from Hura crepitans, Trichosanthes cucumerina and Thevetia nerifolia.

The oil contents and fatty acid composition of three non-edible seed oils extracted using Soxhlet extraction with hexane as the solvent were presented. The physical and chemical properties of the oils were determined from which cetane number, biofuel potential, higher heating values, and antimicrobial activities were assessed. The dominant fatty acids were 49 % linoleic acid, 37 % pentadecenoic acid, and 38 % cis-10-heptadecenenoic acid for Hura crepitans (HC), Thevetia nerifolia (TN) and Trichosanthes cucumerina (TC), respectively. The seed oils were majorly unsaturated, with HC having the highest degree of unsaturation. Acid value, saponification value, iodine value, and free fatty acids were low compared to many reported values in literature. The cetane values were generally high because the oils have a reasonable amount of saturated fatty acid, with TN having the highest cetane number. The low iodine value and saponification value make the biofuel potential and higher heating value to be high with TN having the highest in both and thus the best seed oil for biofuel. However, TN and HC have no antimicrobial activity to Klebsiella pneumoniae (gram -ve), Staphylococcus aureus (gram +ve), Escherichia coli (gram -ve), Bacillus subtilis, Enterobacter aerogenes, Candida albican, Rhizopus stolonifer, Fusarium Solani, Aspergillus flavus and Candida tropicalis, while TC has broad spectrum of activity against all tested bacteria and fungi, except Klebsiella pneumoniae.

Fine-scale genomic analysis of the tree endophyte Annulohypoxylon sp. FPYF3050 producing monoterpene 1,8-cineole.

1,8-Cineole has a potential in control crop pests and biofuels. The endophytic fungus, Annulohypoxylon sp. FPYF3050 (Neolitsea pulchella), can produce over 90% 1,8-cineole of relative area in its natural volatiles, possessed nematicide properties. The annotated genome of this strain will provide insights into potential application in biofumigation and terpene-based advanced biofuel.

Process scale-up simulation and techno-economic assessment of ethanol fermentation from cheese whey.

BACKGROUND: Production of cheese whey in the EU exceeded 55 million tons in 2022, resulting in lactose-rich effluents that pose significant environmental challenges. To address this issue, the present study investigated cheese-whey treatment via membrane filtration and the utilization of its components as fermentation feedstock. A simulation model was developed for an industrial-scale facility located in Italy's Apulia region, designed to process 539 m3/day of untreated cheese-whey. The model integrated experimental data from ethanolic fermentation using a selected strain of Kluyveromyces marxianus in lactose-supplemented media, along with relevant published data. RESULTS: The simulation was divided into three different sections. The first section focused on cheese-whey pretreatment through membrane filtration, enabling the recovery of 56%w/w whey protein concentrate, process water recirculation, and lactose concentration. In the second section, the recovered lactose was directed towards fermentation and downstream anhydrous ethanol production. The third section encompassed anaerobic digestion of organic residue, sludge handling, and combined heat and power production. Moreover, three different scenarios were produced based on ethanol yield on lactose (YE/L), biomass yield on lactose, and final lactose concentration in the medium. A techno-economic assessment based on the collected data was performed as well as a sensitivity analysis focused on economic parameters, encompassing considerations on cheese-whey by assessing its economical impact as a credit for the simulated facility, dictated by a gate fee, or as a cost by considering it a raw material. The techno-economic analysis revealed different minimum ethanol selling prices across the three scenarios. The best performance was obtained in the scenario presenting a YE/L = 0.45 g/g, with a minimum selling price of 1.43 €/kg. Finally, sensitivity analysis highlighted the model's dependence on the price or credit associated with cheese-whey handling. CONCLUSIONS: This work highlighted the importance of policy implementation in this kind of study, demonstrating how a gate fee approach applied to cheese-whey procurement positively impacted the final minimum selling price for ethanol across all scenarios. Additionally, considerations should be made about the implementation of the simulated process as a plug-in addition in to existing processes dealing with dairy products or handling multiple biomasses to produce ethanol.

Millet as a promising C4 model crop for sustainable biofuel production.

The rapid depletion of conventional fuel resources and rising energy demand has accelerated the search for alternative energy sources. Further, the expanding need to use bioenergy crops for sustainable fuel production has enhanced the competition for agricultural land, raising the "food vs. fuel" competition. Considering this, producing bioenergy crops on marginal land has a great perspective for achieving sustainable bioenergy production and mitigating the negative impacts of climate change. C4 crops are dual-purpose crops with better efficiency to fix atmospheric CO2 and convert solar energy into lignocellulosic biomass. Of these, millets have gained worldwide attention due to their climate resilience and nutraceutical properties. Due to close synteny with contemporary C4 bioenergy crops, millets are being considered a model crop for studying diverse agronomically important traits associated with biomass production. Millets can be cultivated on marginal land with minimum fertilizer inputs and maximum biomass production. In this regard, advanced molecular approaches, including marker-assisted breeding, multi-omics approaches, and gene-editing technologies, can be employed to genetically engineer these crops for enhanced biofuel production efficiency. The current study aims to provide an overview of millets as a sustainable bioenergy source and underlines the significance of millets as a C4 model to elucidate the genes and pathways involved in lignocellulosic biomass production using advanced molecular biology approaches.

Signal amplification strategy based on target-controlled release of mediator for ultrasensitive self-powered biosensing of acetamiprid.

Self-powered biosensors with high sensitivity have garnered significant interest for their potential applications in the realm of portable sensing. Herein, a self-powered biosensor with a novel signal amplification strategy was developed by integrating target-controlled release of mediator with an enzyme biofuel cell for the ultrasensitive detection of acetamiprid (ACE). Zeolitic imidazolate framework-67 was utilized as both a nanocontainer for capturing the electron mediator 2,2'-azidobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and a precursor for the synthesis of cobalt nanoparticles/nitrogen, sulfur-codoped carbon nanotubes (Co NPs/NS-CNTs), which were employed as the electrode material for constructing both the glucose oxidase-based bioanode and the laccase-based biocathode. The target analyte ACE can specifically bind to its aptamer, leading to the release of ABTS, which cyclically participates in the catalytic reaction of the biocathode, thereby amplifying the electrochemical signal. By leveraging the benefits of ABTS cyclic catalysis and the effective electrocatalysis of bioelectrodes based on Co NPs/NS-CNTs, the self-powered biosensor has a broad detection range of 0.1-1000 fM and a low detection limit of 25 aM toward ACE. The proposed signal amplification approach presents a promising strategy for enhancing sensitivity and enabling portable analysis in applications of food safety, environmental monitoring, and medical diagnostics.

A Self-Powered Enzymatic Glucose Sensor Utilizing Bimetallic Nanoparticle Composites Modified Pencil Graphite Electrodes as Cathode.

Enzymatic biofuel cells (EBFC) are promising sources of green energy owing to the benefits of using renewable biofuels, eco-friendly biocatalysts, and moderate operating conditions. In this study, a simple and effective EBFC was presented using an enzymatic composite material-based anode and a nonenzymatic bimetallic nanoparticle-based cathode respectively. The anode was constructed from a glassy carbon electrode (GCE) modified with a multi-walled carbon nanotube (MWCNT) and ferrocene (Fc) as a conductive layer coupled with the enzyme glucose oxidase (GOx) as a sensitive detection layer for glucose. A chitosan layer was also applied to the electrode as a protective layer to complete the composite anode. Chronoamperometry (CA) results show that the MWCNT-Fc-GOx/GCE electrode has a linear relationship between current and glucose concentration, which varied from 1 to 10 mM. The LOD and LOQ were calculated for anode as 0.26 mM and 0.87 mM glucose, respectively. Also the sensitivity of the proposed sensor was calculated as 25.71 µ A/mM. Moreover, the studies of some potential interferants show that there is no significant interference for anode in the determination of glucose except ascorbic acid (AA), uric acid (UA), and dopamine (DA). On the other hand, the cathode consisted of a disposable pencil graphite electrode (PGE) modified with platinum-palladium bimetallic nanoparticles (Nps) which exhibit excellent conductivity and electron transfer rate for the oxygen reduction reaction (ORR). The constructed EBFC was optimized and characterized using various electroanalytical techniques. The EBFC consisting of MWCNT-Fc-GOx/GCE anode and Pt-PdNps/PGE cathode exhibits an open circuit potential of 285.0 mV and a maximum power density of 32.25 µW cm-2 under optimized conditions. The results show that the proposed EBFC consisting of an enzymatic composite-based anode and bimetallic nanozyme-based cathode is a unique design and a promising candidate for detecting glucose while harvesting power from glucose-containing natural or artificial fluids.

Carbon dioxide capture, sequestration, and utilization models for carbon management and transformation.

The elevated level of carbon dioxide in the atmosphere has become a pressing concern for environmental health due to its contribution to climate change and global warming. Simultaneously, the energy crisis is a significant issue for both developed and developing nations. In response to these challenges, carbon capture, sequestration, and utilization (CCSU) have emerged as promising solutions within the carbon-neutral bioenergy sector. Numerous technologies are available for CCSU including physical, chemical, and biological routes. The aim of this study is to explore the potential of CCSU technologies, specifically focusing on the use of microorganisms based on their well-established metabolic part. By investigating these biological pathways, we aim to develop sustainable strategies for climate management and biofuel production. One of the key novelties of this study lies in the utilization of microorganisms for CO2 fixation and conversion, offering a renewable and efficient method for addressing carbon emissions. Algae, with its high growth rate and lipid contents, exhibits CO2 fixation capabilities during photosynthesis. Similarly, methanogens have shown efficiency in converting CO2 to methane by methanogenesis, offering a viable pathway for carbon sequestration and energy production. In conclusion, our study highlights the importance of exploring biological pathways, which significantly reduce carbon emissions and move towards a more environmentally friendly future. The output of this review highlights the significant potential of CCSU models for future sustainability. Furthermore, this review has been intensified in the current agenda for reduction of CO2 at considerable extends with biofuel upgrading by the microbial-shift reaction.

Dynamic changes in carbohydrate components and the bacterial community during the ensiling of wilted and unwilted sweet sorghum.

Sweet sorghum can be used to produce a substantial quantity of biofuel due to its high biological yield and high carbohydrate content. In this study, we investigated the dynamic changes in fermentation characteristics, carbohydrate components, and the bacterial community during the ensiling of wilted and unwilted sweet sorghum. The results revealed a rapid fermentation pattern and high-quality fermentation quality in wilted and unwilted sweet sorghum, wherein lactic acid, and acetic acid accumulated and stabilized during the initial 9 days of ensiling, with the pH values less than 4.2, until 60 days of ensiling. We found that the ensiling of sweet sorghum involved the degradation (5% ~ 10%) of neutral detergent fiber (NDF) and hemicellulose and that the degradation of NDF fit a first-order exponential decay model. A shift in dominance from Lactococcus to Lactobacillus occurred before the first 9 days of ensiling, and the abundance of Lactobacillus (r = -0.68, p < 0.001) was negatively correlated with the NDF content. The relative abundances of Lactobacillus in wilted and unwilted sweet sorghum after ensiling for 60 days were 76.30 and 93.49%, respectively, and relatively high fermentation quality was obtained. In summary, ensiling is proposed as a biological pretreatment for sweet sorghum for subsequent biofuel production, and unlike other materials, sweet sorghum quickly achieves good fermentation quality and has great potential for bioresource production.

Clostridium carboxidivorans and Rhodosporidium toruloides as a platform for the valorization of carbon dioxide to microbial oils.

There is growing scientific interest in oleaginous yeasts producing microbial oils as precursors of biofuels and potential substitutes for fossil fuels. Due to the high cost of substrates commonly metabolized by yeasts, volatile fatty acids (VFAs) are gaining interest as alternative cheap and sustainable carbon sources, which can be obtained from solid, liquid and gas pollutants. In this research, Rhodosporidium toruloides was proven to be able to accumulate microbial oils from VFAs obtained from the fermentation of syngas by Clostridium carboxidivorans. Using CO2 and CO as carbon sources from the syngas mixture and H2 as energy source, this acetogen produced, via the Wood-Ljungdahl pathway, a mixture of acetic, butyric and caproic acids. It was first revealed that R. toruloides exhibited minimal inhibition at concentrations below 12 g/L when exposed to a mixture of VFAs, which included acetic, butyric and even hexanoic acids. The yeast was then grown on the culture medium derived from the acetogenic fermentation of syngas. Between the two yeast strains tested of the same species, R. toruloides DSM 4444 reached a total VFAs consumption of 69.1 g/L, supplied by successive additions of acids to the reactor, yielding a maximum lipid content of 29.7% w/w cell. The lipid profile obtained in this case, in terms of abundance followed the order C18:1 > C16:0 ≥ C18:0 > C18:2>others; in which the dominant compound (C18:1), represented approximately 50% of the total. This research opens new possibilities in the cultivation of oleaginous yeasts for the production of biofuels and bioproducts from C1 gases.

Toward More Realistic Estimates of Product Displacement in Life Cycle Assessment.

Life-cycle assessment (LCA) is one of the most widely applied methods for sustainability assessment. A main application of LCA is to compare alternative products to identify and promote those that are more environmentally friendly. Such comparative LCA studies often rest on, explicitly or implicitly, an idealized assumption, namely, 1:1 displacement between functionally equivalent products. However, product displacement in the real world is much more complicated, affected by various factors such as the rebound effect and policy schemes. Here, we quantitatively review studies that have considered these aspects to evaluate the magnitude and distribution of realistic displacement estimates across several major product categories (biofuels, electricity, electric vehicles, and recycled products). Results show that displacement ratios concentrate around 40-60%, suggesting considerable overestimation of the benefits of alternative products if the 1:1 displacement assumption was used. Overall, there have been a small number of modeling studies on realistic product displacement and their scopes were limited. Additional research is needed to cover more product categories and geographies and improve the modeling of market and policy complexities. As such research accumulates, their displacement estimates can form a database that can be drawn upon by comparative LCA studies to more accurately determine the environmental impacts of alternative products.

Advancements in sustainable production of biofuel by microalgae: Recent insights and future directions.

Microalgae is considered as sustainable and viable feedstock for biofuel production due to its significant advantages over terrestrial plants. Algal biofuels have received significant attention among researchers and energy experts owing to an upsurge in global energy issues emanating from depletion in fossil fuel reserves increasing greenhouse gases emission conflict among agricultural crops, traditional biomass feedstock, and potential futuristic energy security. Further, the exploration of value-added microalgae as sustainable and viable feedstock for the production of variety of biofuels such as biogas, bio-hydrogen, bioethanol, and biodiesel are addressed. Moreover, the assessment of life-cycle, energy balance, and environmental impacts of biofuel production from microalgae are briefly discussed. The present study focused on recent advancements in synthetic biology, metabolic engineering tools, algal bio refinery, and the optimization of algae growth conditions. This paper also elucidates the function of microalgae as bio refineries, the conditions of algae-based cultures, and other operational factors that must be adjusted to produce biofuels that are price-competitive with fossil fuels.

Antioxidants alleviated low-temperature stress in microalgae by modulating reactive oxygen species to improve lipid production and antioxidant defense.

The aim of this study was to investigate the effects of various concentrations of antioxidants, including butyl hydroxy anisd (BHA), butylated hydroxytoluene (BHT), fulvic acid (FA), melatonin (MT), glycine betaine (GB) and putrescine (Put), on growth and lipid synthesis of microalgae under low-temperature (15 ℃). Changes in biochemical indicators, reactive oxygen species (ROS) level, glutathione (GSH) content and antioxidant enzyme activities were also studied. The results indicated that the maximum biomass concentration (1.3 g/L) and lipid productivity (75.3 ± 5.8 mg/L d-1) were achieved under 100 µM MT and 1 µM GB, respectively. Moreover, antioxidants were able to increase the GSH and antioxidant enzymes activities in algal cells under low-temperature stress. This study was enlightening for the utilization of antioxidants to improve the resistance to low-temperature stress and lipid production in microalgae, and provided a theoretical basis for the application of microalgae for lipid accumulation in cold regions.

The genus Eutreptiella (Euglenophyceae/Euglenozoa) across its global distribution range.

The genus Eutreptiella (Euglenophyceae/Euglenozoa) comprises unicellular organisms known for their photosynthetic capacity and significant role in marine ecosystems. This review highlights the taxonomic, ecological, and biotechnological characteristics of Eutreptiella species, emphasizing their morphological and genomic adaptations. Eutreptiella species exhibit high phenotypic plasticity, enabling adaptation to various environmental conditions, from nutrient-rich waters to high-salinity conditions. They play a crucial role in primary production and nutrient cycling in marine ecosystems. Genetic and transcriptomic studies have revealed their complex regulatory mechanisms and potential for biofuel and nutraceutical production. Eutreptiella blooms significantly impact local ecosystems, influencing nutrient availability and community dynamics. Additionally, interactions with associated bacteria enhance their growth and metabolic capabilities. The genus shows substantial genetic variability, suggesting potential misidentifications or a polyphyletic nature. Further comprehensive studies are needed to clarify their taxonomy and evolutionary relationships. Understanding and managing Eutreptiella populations is essential to leverage their biotechnological potential and ensure the health of marine ecosystems.

Exploring microbial diversity in the rhizosphere: a comprehensive review of metagenomic approaches and their applications.

The rhizosphere, the soil region influenced by plant roots, represents a dynamic microenvironment where intricate interactions between plants and microorganisms shape soil health, nutrient cycling, and plant growth. Soil microorganisms are integral players in the transformation of materials, the dynamics of energy flows, and the intricate cycles of biogeochemistry. Considerable research has been dedicated to investigating the abundance, diversity, and intricacies of interactions among different microbes, as well as the relationships between plants and microbes present in the rhizosphere. Metagenomics, a powerful suite of techniques, has emerged as a transformative tool for dissecting the genetic repertoire of complex microbial communities inhabiting the rhizosphere. The review systematically navigates through various metagenomic approaches, ranging from shotgun metagenomics, enabling unbiased analysis of entire microbial genomes, to targeted sequencing of the 16S rRNA gene for taxonomic profiling. Each approach's strengths and limitations are critically evaluated, providing researchers with a nuanced understanding of their applicability in different research contexts. A central focus of the review lies in the practical applications of rhizosphere metagenomics in various fields including agriculture. By decoding the genomic content of rhizospheric microbes, researchers gain insights into their functional roles in nutrient acquisition, disease suppression, and overall plant health. The review also addresses the broader implications of metagenomic studies in advancing our understanding of microbial diversity and community dynamics in the rhizosphere. It serves as a comprehensive guide for researchers, agronomists, and policymakers, offering a roadmap for harnessing metagenomic approaches to unlock the full potential of the rhizosphere microbiome in promoting sustainable agriculture.

Lignin hydrogenolysis: Tuning the reaction by lignin chemistry.

Replacing fossil resource with biomass is one of the promising approaches to reduce our carbon footprint. Lignin is one of the three major components of lignocellulosic biomass, accounting for 10-35 wt% of dried weight of the biomass. Hydrogenolytic depolymerization of lignin is attracting increasing attention because of its capacity of utilizing lignin in its uncondensed form and compatibility with the biomass fractionation processes. Lignin is a natural aromatic polymer composed of a variety of monolignols associated with a series of lignin linkage motifs. Hydrogenolysis cleaves various ether bonds in lignin and releases phenolic monomers which can be further upgraded into valuable products, i.e., drugs, terephthalic acid, phenol. This review provides an overview of the state-of-the-art advances of the reagent (lignin), products (hydrol lignin), mass balance, and mechanism of the lignin hydrogenolysis reaction. The chemical structure of lignin is reviewed associated with the free radical coupling of monolignols and the chemical reactions of lignin upon isolation processes. The reactions of lignin linkages upon hydrogenolysis are discussed. The components of hydrol lignin and the selectivity production of phenolic monomers are reviewed. Future challenges on hydrogenolysis of lignin are proposed. This article provides an overview of lignin hydrogenolysis reaction which shows light on the generation of optimized lignin ready for hydrogenolytic depolymerization.

Self-powered wearable biosensor based on stencil-printed carbon nanotube electrodes for ethanol detection in sweat.

Herein we introduce a novel water-based graphite ink modified with multiwalled carbon nanotubes, designed for the development of the first wearable self-powered biosensor enabling alcohol abuse detection through sweat analysis. The stencil-printed graphite (SPG) electrodes, printed onto a flexible substrate, were modified by casting multiwalled carbon nanotubes (MWCNTs), electrodepositing polymethylene blue (pMB) at the anode to serve as a catalyst for nicotinamide adenine dinucleotide (NADH) oxidation, and hemin at the cathode as a selective catalyst for H2O2 reduction. Notably, alcohol dehydrogenase (ADH) was additionally physisorbed onto the anodic electrode, and alcohol oxidase (AOx) onto the cathodic electrode. The self-powered biosensor was assembled using the ADH/pMB-MWCNTs/SPG||AOx/Hemin-MWCNTs/SPG configuration, enabling the detection of ethanol as an analytical target, both at the anodic and cathodic electrodes. Its performance was assessed by measuring polarization curves with gradually increasing ethanol concentrations ranging from 0 to 50 mM. The biosensor demonstrated a linear detection range from 0.01 to 0.3 mM, with a detection limit (LOD) of 3 ± 1 µM and a sensitivity of 64 ± 2 µW mM-1, with a correlation coefficient of 0.98 (RSD 8.1%, n = 10 electrode pairs). It exhibited robust operational stability (over 2800 s with continuous ethanol turnover) and excellent storage stability (approximately 93% of initial signal retained after 90 days). Finally, the biosensor array was integrated into a wristband and successfully evaluated for continuous alcohol abuse monitoring. This proposed system displays promising attributes for use as a flexible and wearable biosensor employing biocompatible water-based inks, offering potential applications in forensic contexts.

Identification and Analysis of the Mechanism of Stem Mechanical Strength Enhancement for Maize Inbred Lines QY1.

Enhancing stalk strength is a crucial strategy to reduce lodging. We identified a maize inbred line, QY1, with superior stalk mechanical strength. Comprehensive analyses of the microstructure, cell wall composition, and transcriptome of QY1 were performed to elucidate the underlying factors contributing to its increased strength. Notably, both the vascular bundle area and the thickness of the sclerenchyma cell walls in QY1 were significantly increased. Furthermore, analyses of cell wall components revealed a significant increase in cellulose content and a notable reduction in lignin content. RNA sequencing (RNA-seq) revealed changes in the expression of numerous genes involved in cell wall synthesis and modification, especially those encoding pectin methylesterase (PME). Variations in PME activity and the degree of methylesterification were noted. Additionally, glycolytic efficiency in QY1 was significantly enhanced. These findings indicate that QY1 could be a valuable resource for the development of maize varieties with enhanced stalk mechanical strength and for biofuel production.

Embracing the sustainable horizons through bioenergy innovations: a path to a sustainable energy future.

The urgent need for mitigating climate change necessitates a transformative shift in energy production and consumption paradigms. Amidst this challenge, bioenergy emerges as a pivotal contributor to the global energy transition, offering a diverse array of solid, liquid, and gaseous fuels derived from biomass. This mini review delves into the unique potential of bioenergy innovations, particularly renewable diesel, bio jet fuel, and ethanol, to reduce greenhouse gas emissions and transform various industries. The article highlights critical technological advancements, supportive policies, and cross-sector collaboration essential for a sustainable energy transition. Specific challenges such as ensuring a consistent biomass feedstock supply, decentralizing processing units, and navigating complex regulatory frameworks are examined. Innovative solutions like decentralized biomass processing and enhanced biomass logistics are discussed as pathways to overcome these barriers. The review provides specific recommendations for near-term policies and strategies to support decentralized facilities, showcasing bioenergy's role in achieving a sustainable future.

Biodiesel production through transesterification of waste Pistacia- Terebinthus Oil by pharmaceutical waste as a heterogeneous catalyst: A sustainable solution for reducing external costs.

A catalyst from the pharmaceutical waste of calcium and magnesium tablets was synthesized for biodiesel production from waste Pistacia-Terebinthus (PT) oil with the aim of creating added value and presenting a new approach for the management of such wastes. For this purpose, magnesium and calcium tablet wastes with a mass ratio of 70:30 (wt%) were calcined. The catalyst was investigated by several methods, such as thermal gravimetric analysis, X-ray diffraction, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, Brunauer-Emmett-Teller analysis, X-ray photoelectron spectroscopy, and CHNS/O elemental analysis. The high specific surface area of the catalyst confirms that the utilized synthesis method resulted in the formation of a high number of active sites in its structure, which allows it to function as a suitable catalyst for this reaction. Furthermore, the impact of effective parameters on the treansestrification reaction was optimized and investigated by designing the experiments and applying the RSM method. The maximum mass yield of 96 % was obtained in optimal conditions (temperature of 70 °C, catalyst loading of 4.498 wt%, methanol:oil ratio of 1.968 (vol:vol), and reaction time of 120 min). The reusability of the catalyst was investigated in four successive cycles. The mass yield of the last test declined from 96 % to 71.4 %. Gas chromatography-mass spectrometry analysis of the produced biofuel revealed that it comprises 91.37 % methyl ester compounds (64.28 % 12-Octadecenoic Acid, Methyl Ester). To evaluate the external costs of biofuel (B100) and compare it with diesel, combustion simulation was done with Diesel-RK software, which showed that its external costs were 0.05388 (€/Lit fuel) less than those of diesel.

Genome editing-based mutagenesis stably modifies composition of wax esters synthesized by Euglena gracilis under anaerobic conditions.

Microalgal oil production represents a promising renewable biofuel source. Metabolic engineering can enhance its utility, transforming it into an improved biofuel and expanding its applications as a feedstock for commodity chemicals, thereby increasing their value in biorefineries. This study focused on anaerobic wax ester production by the microalga Euglena gracilis, aiming to develop stable mutant strains with altered wax ester profiles through genome editing. Two enzymes in the fatty acid beta-oxidation pathway involved in wax ester production were targeted-3-ketoacyl-CoA thiolase and acyl-CoA dehydrogenase-using clustered regularly interspaced short palindromic repeats/Cas9. The results revealed one genetic mutation that lengthened and three that shortened the distribution of wax ester compositions compared to the wild-type (WT). The triple-knockout mutant, combining mutations that shorten wax ester chains, produced wax esters with acyl chains two carbons shorter than WT. This study established a methodology to stably modify wax ester composition in E. gracilis.