Sustainable Biofuel Production Research Articles

Recent advancements in harnessing biodiesel from microalgae through attached growth systems

Conventional microalgal cultivation method relied on suspended growth or attached growth that uses inert solid surfaces, requiring the addition of external carbon sources to promote microalgal growth. The inert solid substrates for attached microalgae pose constraints on the maximal capabilities of these microorganisms as it does not possess the ability to provide the necessary nutritional support required for optimal microalgal growth. As a result, microalgae are obliged to only rely on the solid substrate as a means of attachment, without any further nutritional provisions. Slower growth rates, larger resource requirements, and exorbitant operating expenses had prompted the exploration of an alternative strategy, i.e; the innovative use of solid organic waste as nutritional source cum attachment platform. The findings of this review unveil key insights into microalgae cultivation system, offering a solution to economic challenges in the production phase. This innovation not only promotes economic feasibility and sustainability, but also contributes to economically viable and environmentally sustainable biofuel production.

Sustainable biofuel production in Sub‐Saharan Africa: Exploring transesterification process, nonedible feedstocks, and policy implications

AbstractThe world is currently dealing with an energy crisis, primarily due to heavy reliance on finite fossil fuels and the associated rise in energy demand. In response to this crisis, replacing heavy reliance on finite fossil fuels with biodiesel has gained attention as an alternative solution. Sub‐Saharan African (SSA) biodiesel studies have traditionally focused on improving transesterification but overlook socio‐economic, policy, and institutional impacts on production sustainability. To address this gap, this study comprehensively reviews the sustainability of transesterification‐based biodiesel production from nonedible feedstocks in SSA. The study's incorporation of socio‐economic factors, policy considerations, sustainability concerns, and institutional frameworks reveals the complex prospects and challenges facing biodiesel production in SSA. The findings reveal that sustainability challenges in SSA stem from a lack of an integrated approach, resulting in conflicting local and global policies. The study determines that neglecting socio‐economic factors, policy considerations, sustainability concerns, and institutional frameworks weakens regional biodiesel production sustainability. Evidence from the study emphasizes the role of an integrated approach in promoting biofuel production, establishing markets, and improving the livelihoods of the region's population. Furthermore, the review shows that transesterification can yield biodiesel with comparable physical properties to conventional diesel, making it a wide region's favored option.This article is categorized under: Human and Social Dimensions > Energy and Climate Justice Sustainable Energy > Bioenergy Sustainable Development > Goals

Exploring the synergy of nanomaterials and microbial cell factories during biohydrogen and biobutanol production from different carbon sources

The escalating environmental concerns and burgeoning energy crises, coupled with the desire to achieve net-zero carbon emissions by 2050 to avert potential climate change disaster, highlight the urgent need for sustainable production methods, and innovative technological advancements. Microbial fermentation plays a pivotal role in the pursuit of sustainable production of biofuels and chemicals with properties similar to those of fossil-derived fuels and chemicals. Biofuels, such as biohydrogen and biobutanol derived from renewable sources, offer a promising, cost-effective, and eco-friendly solution for mitigating long-term carbon footprint. Currently, there is a surge of interest in biohydrogen (bioH2) and biobutanol production through dark fermentation using diverse biomass feedstocks, due to its capacity to simultaneously generate gas and liquid fuels from renewable nonfood resources. Furthermore, the incorporation of nanoparticles in fermentation process has demonstrated considerable potential in enhancing bioH2 yields. Similarly, biobutanol emerges as an eco-friendly, less polluting, and potentially commercially viable substitute for conventional gasoline. This review emphasizes the diverse spectrum of nanoparticle applications in biomanufacturing, with a particular focus on their role in enhancing microbial activities to boost bioH2 and biobutanol yields. Additionally, the review highlights potential underlying mechanisms of nano-catalyzed fermentation pathways, future trends, and the prospects of nano-catalysts in advancing biofuel technology.

Integrated biorefinery approach for sustainable biofuel production from algal biomass

Integrated biorefinery approach for sustainable biofuel production from algal biomass

Lignosulfonate-based Carbon-supported Pellets Catalyst to Enhance Sustainable Biofuel Production from Waste Cooking Oil.

In this study, a cost-effective and stable heterogeneous acidic carbocatalyst (CZnLS950) derived from Na-lignosulfonate (LS), a side product of the paper industry, was employed to produce hydrocarbon fuels through the pyrolysis of waste cooking oil (WCO) and crude natural-oil extracted from sunflower seeds, aligning with the principles of the circular economy. To enhance its practicality in industrial settings, the catalyst was synthesized in pellet form, enabling easy separation from the biofuel produced during the reaction. CZnLS950 exhibited remarkable catalytic efficiency in the pyrolysis of WCO, resulting in a 71 wt.% liquid biofuel yield under mild conditions. This performance is attributed to the unique synthesis procedure of acidic carbocatalyst, which utilizes LS and nano ZnO (20 nm) to create a hierarchical pore structure with acidic properties (1.1 mmol of NH3 g-1). Stability and reusability of the carbocatalyst were evaluated, and the results showed excellent stability with small catalytic deactivation (~5 wt.%) after the fourth use. Attempts at distinct catalytic mechanisms for WCO and sunflower seeds crude natural-oil pyrolysis were provided to understand the processes involved in obtaining the two different biofuels produced. Overall, this study sets the stage for exploring Lignosulfonate-based materials to achieve renewable biofuel from recycling streams.

Sustainable production of biofuels using different microbial consortia: the state of the art

AbstractPopulation growth has generated an enormous increase in demand for energy and chemicals to supply goods and services and to maintain quality of life. Oil, coal, and natural gas are the primary sources of the chemicals and energy that directly impact the economy. However, the reserves of these resources are continuously reducing. New alternatives have emerged, including biotechnological tools such as microbial consortia (MC) to produce biofuels, chemicals such as organic acids, and processed foods. The diversity of MC as a biotechnological tool allows it to be used in complex substrates, opening the possibility of using other solid waste to implement consolidated bioprocesses (CPs). This review presents an overview of the uses of MC and CP for producing biofuels using different feedstocks.

Carboxysomes: Green Solutions for Sustainable Agriculture and Renewable Energy

Carboxyomes are emerging as potential tools in the sustainable agriculture and biofuel production. They are proteinaceous organelles that are distributed in different species of photosynthetic bacteria and archaea and are significantly involved in carbon fixation. They enable the organisms to thrive in diverse environmental conditions and enhance the efficacy of photosynthesis. They have shown promising potential to address challenging regarding global food security, climate change and renewable energy.  These nanocompartments encapsulate Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), an enzyme that catalyzes the first step of the Calvin-Benson-Bassham cycle that is a key pathway in carbon fixation. These specialized structures concentrate CO2 around RuBisCO ad shield it from the competing oxygen, thereby optimizing the efficiency of photosynthetic process and minimize wasteful photorespiration. The photosynthetic organisms having carboxysomes are therefore ideal candidates for sustainable agriculture as they are able to thrive in the harsh conditions such as low CO2 concentrations, water scarcity and high temperatures.   They are recently being studied for enhancing the productivity and yield of crops. Efforts are being exerted to introduce carboxysome shell proteins and RuBisCO enzymes into crop genomes. Researchers are aiming to create plants with the ability to efficiently capture and utilize CO2 for photosynthesis. These genetically modified crops can potentially increase crop yields, improve water and nutrient efficiency significantly. Moreover, these carboxysomal transgenes are also able to enhance the resilience of GM plants to different environmental stressors such as drought and heat, and ultimately contribute to global food security and sustainable agriculture.  In addition to their applications in agriculture, carboxysomes hold promise for biofuel production as well, as they are capable of carbon fixation. They have enabled researchers to engineer photosynthetic organisms to produce renewable fuels such as ethanol, biodiesel and hydrogen. Furthermore, nanobioreactors that are based on carboxysomes can contribute protectively against climate change as they have the potential to mitigate greenhouse gas emissions by capturing CO2 from industrial emissions and converting it into valuable bioproducts.  This approach represents a promising avenue to address global challenges of food insecurity, climate crisis and rapidly declining non-renewable energy sources. If leveraged appropriately, these nanocompartments can significantly contribute in paving a way for more sustainable and resilient future.      

The Impact of Sulfur-Containing Inorganic Compounds during the Depolymerization of Lignin by Hydrothermal Liquefaction of Black Liquor.

Lignin is a promising resource for the sustainable production of platform chemicals and biofuels. The paper industry produces large quantities of lignin every year, mostly dissolved in a black liquor. With the help of hydrothermal liquefaction, black liquor can be used directly as a feedstock to depolymerize the lignin to desired products. However, because various cooking chemicals (e.g., NaHS, NaOH) used in the Kraft process, dominant in the paper industry, are also dissolved in the black liquor, it is necessary to study in detail their influence on the process as well as their fate. In this work, the focus was on the fate of sulfur and the influence of sulfide (HS-). For this purpose, hydrothermal liquefaction experiments (250-400 °C) were carried out with black liquor and self-prepared model black liquor with different sulfide concentrations (0-3 g·L-1 HS-) in batch reactors (V = 25 mL), and the products were analyzed to understand the chemical pathways involving sulfur. It was found that the inorganic sulfur compounds react with organic matter to produce organic sulfur compounds. Dimethyl sulfide is the most abundant of these products. The HS- concentration correlates with the amount of dimethyl sulfide produced. Because methanethiol has also been qualitatively detected, the reaction mechanism of Karnofski et al. for the formation of dimethyl sulfide in the Kraft process also applies to the hydrothermal liquefaction of black liquor. Increased sulfide concentration in the feed leads to an accelerated depolymerization of lignin. In contrast, the yields of some aromatic monomers decrease slightly, possibly as a result of repolymerization reactions also occurring more quickly.

Mid-Long Chain Dicarboxylic Acid Production via Systems Metabolic Engineering: Progress and Prospects.

Mid-to-long-chain dicarboxylic acids (DCAi, i ≥ 6) are organic compounds in which two carboxylic acid functional groups are present at the terminal position of the carbon chain. These acids find important applications as structural components and intermediates across various industrial sectors, including organic compound synthesis, food production, pharmaceutical development, and agricultural manufacturing. However, conventional petroleum-based DCA production methods cause environmental pollution, making sustainable development challenging. Hence, the demand for eco-friendly processes and renewable raw materials for DCA production is rising. Owing to advances in systems metabolic engineering, new tools from systems biology, synthetic biology, and evolutionary engineering can now be used for the sustainable production of energy-dense biofuels. Here, we explore systems metabolic engineering strategies for DCA synthesis in various chassis via the conversion of different raw materials into mid-to-long-chain DCAs. Subsequently, we discuss the future challenges in this field and propose synthetic biology approaches for the efficient production and successful commercialization of these acids.

Environmentally-friendly preparation of Sn(II)-BDC supported heteropolyacid as a stable and highly efficient catalyst for esterification reaction

Facilitating energy resource deficiency and environmental contamination, this work focuses on sustainable biodiesel production through the esterification reactions of oleic acid (OA) with methanol. To address the reaction, a novel heterogeneous acid catalyst, 12-tungstophosphoric acid (TPA) immobilized on Sn-based MOFs (Sn(II)-BDC) was synthesized via a simple, green solvent, and easy-to-implement synthesis strategy for the first time, and applied effectively for esterification process of OA to produce biodiesel. The structure and composition of as-obtained catalyst have been verified using XRD, FTIR, N2 physisorption, SEM, EDX, TG, Py-FTIR, TPD-NH3, and XPS techniques. The obtained TPA/Sn(II)-BDC catalyst was found to be the best with 60 wt% of TPA loading, which resulted in an OA conversion of 91.7 % at optimized conditions of 0.15 g catalyst loading and methanol to OA molar ratio of 20:1 at temperature of 120 °C in 4 h, and the excellent performance arises from available pores structure, large amounts of acidic sites, good stability and the synergistic catalytic effect of TPA and Sn(II)-BDC. Furthermore, the composite catalyst reusability has been studied for five cycles, and it exhibits an acceptable conversion. This research provides a green and large-scale synthesis route for the sustainable production of biofuels by constructing heteropolyacids/Sn-based MOFs synergistic catalysts.

Cell-type-specific transcriptomics uncovers spatial regulatory networks in bioenergy sorghum stems.

Bioenergy sorghum is a low-input, drought-resilient, deep-rooting annual crop that has high biomass yield potential enabling the sustainable production of biofuels, biopower, and bioproducts. Bioenergy sorghum's 4-5 m stems account for ~80% of the harvested biomass. Stems accumulate high levels of sucrose that could be used to synthesize bioethanol and useful biopolymers if information about cell-type gene expression and regulation in stems was available to enable engineering. To obtain this information, laser capture microdissection was used to isolate and collect transcriptome profiles from five major cell types that are present in stems of the sweet sorghum Wray. Transcriptome analysis identified genes with cell-type-specific and cell-preferred expression patterns that reflect the distinct metabolic, transport, and regulatory functions of each cell type. Analysis of cell-type-specific gene regulatory networks (GRNs) revealed that unique transcription factor families contribute to distinct regulatory landscapes, where regulation is organized through various modes and identifiable network motifs. Cell-specific transcriptome data was combined with known secondary cell wall (SCW) networks to identify the GRNs that differentially activate SCW formation in vascular sclerenchyma and epidermal cells. The spatial transcriptomic dataset provides a valuable source of information about the function of different sorghum cell types and GRNs that will enable the engineering of bioenergy sorghum stems, and an interactive web application developed during this project will allow easy access and exploration of the data (https://mc-lab.shinyapps.io/lcm-dataset/).

Advancements, Challenges, and Pathways to Sustainable Biofuel Production

AbstractThe discovery of new energy sources plays a vital role to fulfil the needs of energy and depletion of fossil fuel sources. An inclusive review of all available technologies for the synthesis of biofuels from biomass is presented. Amongst the innovative biomass conversion technologies, thermochemical technology encompasses significant potential practices and is required for expansion. Universally, biofuels are gaining importance due to the depletion of non‐renewable fuel sources in transport and industries by equivalent work proficiencies. The reaction mechanism thoroughly explores all aspects associated with reaction conditions, types of reactor, and products. Biomass thermochemical conversion technologies will be influential in producing sustainable biofuels.

Lipid Yields and Fatty Acid Composition if Indigenous Microalgae, Isolated from the Beira, and Diyawanna Urban Lakes, for Sustainable Biofuel Production in Sri Lanka

 
 Biofuels are carbon-neutral combustibles produced from renewable biomass. Third generation biofuels, derived from microalgal biomass, are distinctly superior to their preceding generations, due to higher productivity and resource use efficiency of microalgae compared to conventional energy crops. It is a viable source of sustainable energy for our country due to the availability of water, ambient temperatures and year-round sunlight needed for microalgae cultivation, and most importantly, the abundance of diverse microalgae species. However, these native microalgae are relatively unexplored for their productivities and thereby, greatly underutilized. This research was aimed to investigate the lipid yields and fatty acid compositions of local freshwater microalgae, and to identify their potential for biodiesel production. This was carried out by (1) microalgae isolation and morphological identification (2) microalgae cultivation (3) biomass harvesting, lipid extraction, gravimetric measurement of extracted lipids and (4) acid-catalysed transesterification, followed by GCMS analysis of the biodiesel product. Chlorella sp., Chlorococcum sp. and Chlamydomonas sp. were isolated from Beira/ Diyawanna Lakes using streak plate technique in solid standard BBM media. They were next cultivated under axenic conditions in liquid standard BBM media under 16: 8 (light: dark), at 25°C for 20 days in duplicates. The biomass was then harvested using alum, dried to reach a constant weight and the Bligh and Dyer lipid extraction was done in triplicate. The extracted lipids were trans-esterified with methanol, using sulfuric acid catalyst at 90° C for 60 minutes. The separated product was analysed by GCMS. Chlorella sp., Chlorococcum sp. and Chlamydomonas sp. cultivated under standard conditions gave average lipid yields of 11.34±1.01%, 15.00±1.40% and 2.10±0.50% respectively. Average lipid yield of Chlamydomonas sp. was significantly lower than the other two species at 95% confidence level (Two-sample T test on SPSS Version 21). GCMS analysis of the biodiesel product from Chlorella sp. indicated significant levels of saturated fatty acids including palmitic and stearic acids, monounsaturated fatty acids like cis-11- eicosenoic acid and polyunsaturated fatty acids including linoleic and linolenic acids. The fatty acid ratios were used to calculate the saturation value, cetane number, density, iodine value and pour point of the produced biodiesel. These quality parameters were in accordance with the ASTM D6751- 20a standards. Therefore, with a substantial yield and fatty acid composition of intracellular lipids, this local species of Chlorella can be a prospective candidate for sustainable bioenergy production in Sri Lanka.
  
 Keywords: Chlorella, Chlorococcum, Chlamydomonas, Microalgal lipids, Biodiesel

Sustainable Production of Biofuels and Biochemicals via Electro-Fermentation Technology.

The energy crisis and climate change are two of the most concerning issues for human beings nowadays. For that reason, the scientific community is focused on the search for alternative biofuels to conventional fossil fuels as well as the development of sustainable processes to develop a circular economy. Bioelectrochemical processes have been demonstrated to be useful for producing bioenergy and value-added products from several types of waste. Electro-fermentation has gained great attention in the last few years due to its potential contribution to biofuel and biochemical production, e.g., hydrogen, methane, biopolymers, etc. Conventional fermentation processes pose several limitations in terms of their practical and economic feasibility. The introduction of two electrodes in a bioreactor allows the regulation of redox instabilities that occur in conventional fermentation, boosting the overall process towards a high biomass yield and enhanced product formation. In this regard, key parameters such as the type of culture, the nature of the electrodes as well as the operating conditions are crucial in order to maximize the production of biofuels and biochemicals via electro-fermentation technology. This article comprises a critical overview of the benefits and limitations of this emerging bio-electrochemical technology and its contribution to the circular economy.

Impact of Synergy Partner Cel7B on Cel7A Binding Rates: Insights from Single-Molecule Data.

Enzymatic degradation of cellulosic biomass is a well-established route for the sustainable production of biofuels, chemicals, and materials. A strategy employed by nature and industry to achieve an efficient degradation of cellulose is that cellobiohydrolases (or exocellulases), such as Cel7A, work synergistically with endoglucanases, such as Cel7B, to achieve the complete degradation of cellulose. However, a complete mechanistic understanding of this exo-endo synergy is still lacking. Here, we used single-molecule fluorescence microscopy to quantify the binding kinetics of Cel7A on cellulose when it is acting alone on the cellulose fibrils and in the presence of its synergy partner, the endoglucanase Cel7B. To this end, we used a fluorescently tagged Cel7A and studied its binding in the presence of the unlabeled Cel7B. This provided the single-molecule data necessary for the estimation of the rate constants of association kON and dissociation kOFF of Cel7A for the substrate. We show that the presence of Cel7B does not impact the dissociation rate constant, kOFF. But, the association rate of Cel7A decreases by a factor of 2 when Cel7B is present at a molar proportion of 10:1. This ratio has previously been shown to lead to synergy. This decrease in association rate is observed in a wide range of total enzyme concentrations, from sub nM to μM concentrations. This decrease in kON is consistent with the formation of cellulase clusters recently observed by others using atomic force microscopy.

Utilization of microalgae for agricultural runoff remediation and sustainable biofuel production through an integrated biorefinery approach

Generally wastewater such agricultural runoff is considered a nuisance; however, it could be harnessed as a potential source of nutrients like nitrates and phosphates in integrated biorefinery context. In the current study, microalgae Chlorella sp. S5 was used for bioremediation of agricultural runoff and the leftover algal biomass was used as a potential source for production of biofuels in an integrated biorefinery context. The microalgae Chlorella sp. S5 was cultivated on Blue Green (BG 11) medium and a comprehensive optimization of different parameters including phosphates, nitrates, and pH was carried out to acquire maximum algal biomass enriched with high lipids content. Dry biomass was quantified using the solvent extraction technique, while the identification of nitrates and phosphates in agricultural runoff was carried out using commercial kits. The algal extracted lipids (oils) were employed in enzymatic trans-esterification for biodiesel production using whole-cell biomass of Bacillus subtilis Q4 MZ841642. The resultant fatty acid methyl esters (FAMEs) were analyzed using Fourier transform infrared (FTIR) spectroscopy and gas chromatography coupled with mass spectrometry (GC–MS). Subsequently, both the intact algal biomass and its lipid-depleted algal biomass were used for biogas production within a batch anaerobic digestion setup. Interestingly, Chlorella sp. S5 demonstrated a substantial reduction of 95% in nitrate and 91% in phosphate from agricultural runoff. The biodiesel derived from algal biomass exhibited a noteworthy total FAME content of 98.2%, meeting the quality standards set by American Society for Testing and Materials (ASTM) and European union (EU) standards. Furthermore, the biomethane yields obtained from whole biomass and lipid-depleted biomass were 330.34 NmL/g VSadded and 364.34 NmL/g VSadded, respectively. In conclusion, the findings underscore the potent utility of Chlorella sp. S5 as a multi-faceted resource, proficiently employed in a sequential cascade for treating agricultural runoff, producing biodiesel, and generating biogas within the integrated biorefinery concept.Graphical

Increased production of isobutanol from xylose through metabolic engineering of Saccharomyces cerevisiae overexpressing transcription factor Znf1 and exogenous genes.

Only trace amount of isobutanol is produced by the native Saccharomyces cerevisiae via degradation of amino acids. Despite several attempts using engineered yeast strains expressing exogenous genes, catabolite repression of glucose must be maintained together with high activity of downstream enzymes, involving iron-sulfur assimilation and isobutanol production. Here, we examined novel roles of nonfermentable carbon transcription factor Znf1 in isobutanol production during xylose utilization. RNA-seq analysis showed that Znf1 activates genes in valine biosynthesis, Ehrlich pathway and iron-sulfur assimilation while coupled deletion or downregulated expression of BUD21 further increased isobutanol biosynthesis from xylose. Overexpression of ZNF1 and xylose-reductase/dehydrogenase (XR-XDH) variants, a xylose-specific sugar transporter, xylulokinase, and enzymes of isobutanol pathway in the engineered S. cerevisiae pho13gre3Δ strain resulted in the superb ZNXISO strain, capable of producing high levels of isobutanol from xylose. The isobutanol titer of 14.809±0.400g/L was achieved, following addition of 0.05g/L FeSO4.7H2O in 5L bioreactor. It corresponded to 155.88mg/g xylose consumed and+264.75% improvement in isobutanol yield. This work highlights a new regulatory control of alternative carbon sources by Znf1 on various metabolic pathways. Importantly, we provide a foundational step toward more sustainable production of advanced biofuels from the second most abundant carbon source xylose.

Enzyme Cocktails Catalyzed Bioconversion of Lignocellulosic Biomass into Bioethanol

Abstract: Sustainable production of biofuels from lignocellulosic biomass (LBM) is considered one of the promising solutions to solve the current energy crisis caused by overreliance on fossil fuels. Enzymatic hydrolysis is a crucial step in determining the efficient conversion of biomass cellulose into biofuels as well as high value-added chemicals, and it has the characteristics of efficient reaction, economy, and environmental friendliness. With the advancement of enzyme engineering technology, enzymes have been endowed with more functions, such as mixed substrate utilization, inhibitor resistance and specific metabolism, called enzyme cocktails. This Paper introduces the typical procedure of enzymatic hydrolysis of LBM and discusses their applications in hybrid LBM hydrolysis. In addition, the challenges of enzymatic LBM hydrolysis and possible development directions are outlined to guide integrated enzymatic strategies in biorefinery processes.

Enhanced isobutanol recovery from fermentation broth for sustainable biofuels production

Isobutanol is a highly attractive renewable alternative to conventional fossil fuels, with superior fuel properties as compared to ethanol and 1-butanol. Even though the isobutanol production by fermentation has significant potential, complex downstream processing is limiting the wide-spreading of this technology. Accordingly, this original research significantly contributes to the advancement in industrial biofuel production by developing two eco-efficient downstream processes for the industrial-scale recovery of isobutanol (production capacity 50 ktonneIBUT/y), from a highly dilute fermentation broth (>98 wt% water). Vacuum distillation and a novel hybrid combination of gas stripping and vacuum evaporation were coupled with atmospheric azeotropic distillation to recover over 99.9 % of isobutanol as a high-purity product (100 wt%). Advanced heat pumping and heat integration techniques were further implemented to allow the complete electrification of these recovery processes. Furthermore, implementation of these techniques significantly decreased total annual costs (0.131–0.161 $/kgIBUT), reduced energy requirements (0.488–0.807 kWeh/kgIBUT) and lowered CO2 emissions (0.303–0.449 kgCO2/kgIBUT), resulting in highly competitive purification processes. In addition to efficiently recovering isobutanol, the designed downstream processes provide the potential to enhance the fermentation process by recycling all present microorganisms and reducing water demand. Therefore, the results of this original research substantially contribute to the advancement in industrial biotechnology and the wide-spreading of biofuel production.

Overexpression of gamma-glutamyl cyclotransferase 2;1 (CsGGCT2;1) reduces arsenic toxicity and accumulation in Camelina sativa (L.).

Overexpressing CsGGCT2;1 in Camelina enhances arsenic tolerance, reducing arsenic accumulation by 40-60%. Genetically modified Camelina can potentially thrive on contaminated lands and help safeguard food quality and sustainable food and biofuel production. Environmental arsenic contamination is a serious global issue that adversely affects human health and diminishes the quality of harvested produce. Glutathione (GSH) is known to bind and detoxify arsenic and other toxic metals. A steady level of GSH is maintained within cells via the γ-glutamyl cycle. The γ-glutamyl cyclotransferases (GGCTs) have previously been shown to be involved in GSH degradation and increased tolerance to toxic metals in plants. In this study, we characterized the GGCT2;1 homolog from Camelina sativa for its role in arsenic tolerance and accumulation. Overexpression of CsGGCT2;1 in Camelina under CaMV35S constitutive promoter resulted in strong tolerance to arsenite (AsIII). The overexpression (OE) lines had 2.6-3.5-fold higher shoots and sevenfold to tenfold enhanced root biomass on media supplemented with AsIII, relative to wild-type plants. The CsGGCT2;1 OE lines accumulated 40-60% less arsenic in root and shoot tissues compared to wild-type plants. Further, the OE lines had ~ twofold higher chlorophyll content and 35% lesser levels of malondialdehyde (MDA), an indicator of membrane damage via lipid peroxidation. There was a slight but non-significant increase in 5-oxoproline (5-OP), a product of GSH degradation, in OE lines. However, the transcript levels of Oxoprolinase 1 (OXP1) were upregulated, indicating the accelerated conversion of 5-OP to glutamate, which is further utilized for the resynthesis of GSH to maintain GSH homeostasis. Overall, this research suggests that genetically modified Camelina may have the potential for cultivation on contaminated marginal lands to reduce As accumulation; thereby could help in addressing food safety issues as well as future food and biofuel needs.