Feedstock For Biofuel Production Research Articles

Pretreatment optimization for microalgae oil yield enhancement and residual biomass characterization for sustainable biofuel feedstock production

Pretreatment optimization for microalgae oil yield enhancement and residual biomass characterization for sustainable biofuel feedstock production

Evaluating black wattle bark industrial residue as a new feedstock for bioenergy via pyrolysis and multicomponent kinetic modeling

This work presents an evaluation of the industrial residue from exhausted black wattle bark after the extraction of tannin for use as a new feedstock for biofuel production, without competing with food production. The physicochemical and thermodynamic properties, the influence of temperature on pyrolysis products and multicomponent kinetics were evaluated. Principal component analysis showed that sugars, carboxylic acids, ethers/esters, furans and aldehydes were formed as primary products of pyrolysis at 450 °C. At 550 °C, phenols and mainly ketones were favored, especially acetone and 2,3-butanedione. Pyrolysis at 650 °C favored the production of aldehydes, alcohols, and 1-alkene, polyene and alkane hydrocarbons. After an upgrade, the series of 1-alkenes produced, with carbons C7 to C16, could be suitable for a range of gasoline and jet fuels. Kinetic modeling was based on the deconvolution of the mass loss of three events of the pseudo-components: hemicellulose (DE-HC), cellulose (DE-CL) and lignin (DE-LG). The activation energy for kinetic models Friedman, Flynn-Wall-Ozawa, Kissinger-Akahira-Sunose and Starink presented average values of ∼141.4–149.4 for DE-HC, ∼190.5–200.9 for DE-CL and ∼237.2–252.3 kJ mol−1 for DE-LG. This study encourages using agro-industrial waste to produce second-generation drop-in biofuels and bio-based chemicals, aiming at industrial decarbonization.

Intensifying outdoor cultivation of oleaginous microalgae in photobioreactors for simultaneous wastewater bioremediation and low-cost production of renewable biodiesel feedstocks

Oleaginous microalgae are gaining increasing attention for simultaneous wastewater bioremediation and biofuel feedstock production. In this study, four oleaginous species of microalgae including Chlamydomonas sp. WWP, Scenedesmus sp. HP56, Haematococcus sp., and Nannochloropsis sp., were screened for their growth and lipid production at 30, 35, and 40 °C, under light intensity at 3, 4, and 5 klux. Among the strains screened, Haematococcus sp. grew best and gave comparable high growth at 30 °C and 35 °C (0.7–0.8 g/L) indicating its thermotolerant characteristic, and the optimal light intensity was found at 4 klux. Haematococcus sp. also grew well using unsterile secondary effluent from the seafood processing plant and produced high biomass (0.53–0.57 g/L) and lipids (0.17–0.19 g/L), especially in the column photobioreactor (CP). The repeated-batch outdoor cultivation mode showed that CP equipped with four-blade baffles was most efficient for the production of biomass (0.40–0.63 g/L) and lipids (0.15–0.30 g/L) during 4 cycles. The microalga also removed total nitrogen and total phosphorus from secondary effluent by 70.59% and 94.97%, respectively. The fatty acid compositions in microalgal lipids were C16-C18, especially palmitic acid, oleic acid, and linolenic acid, which show superior fuel properties, suggesting their promising use as biodiesel feedstocks. These strategies may have a significant impact not only on bioremediation of industrial effluent but also low-cost production of microalgae-based biodiesel feedstocks.

An overview of biodiesel, a fuel blend made from palm oil.

With the increasing global demand and rising fossil fuel prices, the oil industry is turning to alternative fuels from renewable sources like biodiesel or vegetable oils. To compensate for the scarcity of fossil fuels, fuel from vegetable oils is one of the alternatives to meet energy needs. Biodiesel is considered a suitable and better choice due to its environmentally friendly features, reduced carbon dioxide emissions, and functional properties similar to fossil fuels. The diesel blend is a renewable fuel derived from a mixture of hydrocarbons (diesel) and fatty acid methyl esters (FAME). This article focuses on biodiesel produced in Indonesia from palm oil as a feedstock for biofuel production. As one of the world’s largest palm oil producers, the production of biodiesel in Indonesia, especially biodiesel from palm oil, has many advantages and disadvantages in terms of economic, social and economic aspects. environment. Use specifically as a fuel is sometimes problematic because biodiesel is hygroscopic and can corrode steel. The scope of this study is based on a brief overview of the progress and challenges in developing palm oil biodiesel feedstock to pursue in the future.

Overcoming genetic paucity of Camelina sativa: possibilities for interspecific hybridization conditioned by the genus evolution pathway.

Camelina or false flax (Camelina sativa) is an emerging oilseed crop and a feedstock for biofuel production. This species is believed to originate from Western Asian and Eastern European regions, where the center of diversity of the Camelina genus is located. Cultivated Camelina species arose via a series of polyploidization events, serving as bottlenecks narrowing genetic diversity of the species. The genetic paucity of C. sativa is foreseen as the most crucial limitation for successful breeding and improvement of this crop. A potential solution to this challenge could be gene introgression from Camelina wild species or from resynthesized allohexaploid C. sativa. However, both approaches would require a complete comprehension of the evolutionary trajectories that led to the C. sativa origin. Although there are some studies discussing the origin and evolution of Camelina hexaploid species, final conclusions have not been made yet. Here, we propose the most complete integrated evolutionary model for the Camelina genus based on the most recently described findings, which enables efficient improvement of C. sativa via the interspecific hybridization with its wild relatives. We also discuss issues of interspecific and intergeneric hybridization, aimed on improving C. sativa and overcoming the genetic paucity of this crop. The proposed comprehensive evolutionary model of Camelina species indicates that a newly described species Camelina neglecta has a key role in origin of tetra- and hexaploids, all of which have two C. neglecta-based subgenomes. Understanding of species evolution within the Camelina genus provides insights into further research on C. sativa improvements via gene introgression from wild species, and a potential resynthesis of this emerging oilseed crop.

Food, biofuels or cosmetics? Land-use, deforestation and CO2 emissions embodied in the palm oil consumption of four European countries: a biophysical accounting approach

Around 75% of tropical deforestation in the XXI century has been driven by the expansion of agriculture and forest plantations. Since 1990s, palm oil has been standing for a critical global traded product in terms of embodied deforestation. The European Union (EU) is one of the major players in terms of embodied deforestation linked to palm oil consumption. By adopting a biophysical accounting approach, the study: (1) investigated the palm oil imports between 2000 and 2020 by four EU countries (Germany, France, Italy, and Spain) from Indonesia, Malaysia, and Papua New Guinea, (2) estimated the share of imports driven by the demand from the food, oleochemicals, and energy sectors, and (3) quantified land, deforestation, and CO2 emissions associated with Land-Use Change (LUC) embodied in trade. Different trade profiles have emerged among the four importing countries. Italy and Spain showed a major direct trade link with producing countries, while France and Germany have significant connections with non-producing countries (i.e. intermediate trade partners). Overall, our results show that, following different trends, leading consumption sectors have shifted from the food towards the energy sector. Consequently, the growing demand for palm oil as a feedstock for biofuel production has determined increased environmental impacts in South-East Asia. Since 2000, the total embodied land footprint has increased four-fold, while, over the period considered, according to our second and the third attribution approaches (i.e. historical and rapid-conversion), between 5–78 m2 of deforestation and 28–445 kg CO2 emissions associated with LUC activities have been incorporated in the per-capita consumption of palm oil and its co-products in the leading European economies. Moreover, according to the first attribution approach (i.e. concession-level) and the allocation by sector, we concluded that, between 2004 and 2016, the German food sector is the one that embodied the larger deforestation footprint, followed by the Italian and Spanish energy sectors.

Lemnaceae as Novel Crop Candidates for CO2 Sequestration and Additional Applications.

Atmospheric carbon dioxide (CO2) is projected to be twice as high as the pre-industrial level by 2050. This review briefly highlights key responses of terrestrial plants to elevated CO2 and compares these with the responses of aquatic floating plants of the family Lemnaceae (duckweeds). Duckweeds are efficient at removing CO2 from the atmosphere, which we discuss in the context of their exceptionally high growth rates and capacity for starch storage in green tissue. In contrast to cultivation of terrestrial crops, duckweeds do not contribute to CO2 release from soils. We briefly review how this potential for contributions to stabilizing atmospheric CO2 levels is paired with multiple additional applications and services of duckweeds. These additional roles include wastewater phytoremediation, feedstock for biofuel production, and superior nutritional quality (for humans and livestock), while requiring minimal space and input of light and fertilizer. We, furthermore, elaborate on other environmental factors, such as nutrient availability, light supply, and the presence of a microbiome, that impact the response of duckweed to elevated CO2. Under a combination of elevated CO2 with low nutrient availability and moderate light supply, duckweeds' microbiome helps maintain CO2 sequestration and relative growth rate. When incident light intensity increases (in the presence of elevated CO2), the microbiome minimizes negative feedback on photosynthesis from increased sugar accumulation. In addition, duckweed shows a clear propensity for absorption of ammonium over nitrate, accepting ammonium from their endogenous N2-fixing Rhizobium symbionts, and production of large amounts of vegetative storage protein. Finally, cultivation of duckweed could be further optimized using hydroponic vertical farms where nutrients and water are recirculated, saving both resources, space, and energy to produce high-value products.

Chromium and Lead Phytoremediation with Water Hyacinth and Use of Contaminated Biomass as Feedstock for Biofuel Production

Chromium and Lead Phytoremediation with Water Hyacinth and Use of Contaminated Biomass as Feedstock for Biofuel Production

Production and characterization of Cymbopogon citratus biofuel and its optimization study for efficient and cleaner production blended with water and cetane improver: A response surface methodology approach

The use of low viscous biofuel derived from grasses has a better prospect of turning into a renewable source of diesel fuel. Cymbopogon citratus, a profoundly unsaturated fatty acid grass, is proposed as feedstock for biofuel production as none of the assessments tried to explore the Cymbopogon citratus bio-oil (CCB) as an alternative source for diesel fuel with reasonable fuel change. The current study intends to extract low viscous biofuel from Cymbopogon citratus grass and work on its energy and environmental features by adding an optimum amount of water and Di-tert-butyl peroxide (DTBP). A steam refining process was employed to extract the CCB, and the fuel characterizations were assessed and quantified by FTIR and GC–MS investigations. Since the individual impact of CCB, water, and DTBP on energy and environmental boundaries is unique, a response surface methodology was applied to achieve the optimum level of individuals for overall efficient and cleaner production. The fuel characteristics report that the extricated CCB's chemical substances are tantamount to ordinary diesel and can be suggested as an alternative source for fuel applications. The optimization results demonstrate that the coefficient of determination is significantly closer to one, and the chance of error is less than 0.1%. The optimum value of CCB, water, and DTBP is determined as 22.08%, 12.23%, and 1.31%, respectively, and the energy and environmental parameters agree well with the predicted values. The progress in the combustion parameters is also documented for the test fuel while the fuel modification parameters approach their optimal state. The proposed fuel blend would lessen the demand for fossil fuel energy sources by around 35%.

Co-Fermentation of Chlorella vulgaris with Oleaginous Yeast in Starch Processing Effluent as a Carbon-Reducing Strategy for Wastewater Treatment and Biofuel Feedstock Production

Low biomass yield and nutrient removal efficiency are problems challenging the employment of microorganisms for wastewater remediation. Starch processing effluent (SPE) was used as a fermentation substrate to co-culture Chlorella vulgaris and Rhodotorula glutinis for biofuel feedstock production. Co-culture options were compared, and the optimal conditions were identified. The result shows that microalgae and yeast should be inoculated simultaneously at the beginning of SPE-based fermentation to achieve high biomass yield and the optimal inoculation ratio, light intensity, and temperature should be 2:1, 150 μmol/m2/s, and 25 °C, respectively. Under the optimal conditions, the lipid yield of microorganisms was 1.81 g/L and the carbon–conversion ratio reached 82.53% while lipid yield and the carbon–conversion ratio in a monoculture fell in the range of 0.79–0.81 g/L and 55.93–62.61%, respectively. Therefore, compared to the monoculture model, the co-fermentation of Chlorella vulgaris and Rhodotorula glutinis in starch processing effluent could convert nutrients to single-cell oil in a more efficient way. It should be noted that with the reduced concentration of residual organic carbon in effluent and the increased carbon–conversion ratio, co-fermentation of microalgae and yeast can be regarded as a promising and applicable strategy for starch processing effluent remediation and low-cost biofuel feedstock production.

Effect of physiographic type and harvesting age on biomass yield, chemical composition, and carbon sequestration of mangrove plantations for biofuel feedstock production

Many coastal communities of SE-Asia, including Thailand have managed mangrove forests for charcoal production. However, understanding and managing the harvesting age of mangrove plantations in different ecotypes is limited for sustainable biofuel production. Therefore, the objectives of this research were to determine the effect of physiographic type and harvesting age on the biomass yield, chemical composition, CO2 adsorption of aboveground mangrove biomass. The data were collected during 2018–2019 from two geographical types including the riverine and basin mangrove plantations in Samut-Songkhram, Thailand. The experimental plots were selected at different ages (5, 10, and 15 years). The riverine mangrove forest had higher dry aboveground woody biomass, calorific value, and CO2 adsorption than did the basin mangrove forest. For both mangrove ecotypes, there were no significant differences in chemical elements. Extending harvesting age from 5 to 15 years tended to increase the woody biomass yield and quality, and CO2 adsorption of mangrove forests. With increasing harvesting age, the dry aboveground woody biomass, calorific value, and CO2 adsorption of mangroves increased 34.4–283.9 t ha−1, 3841.1- 3924.9 kcal kg−1, and 55.6–453.4 t CO2 ha−1, respectively, but ash and N content decreased 4.8–3.4% and 0.2–0.1%, respectively. Compared with other ages, extending harvest until 15 years yielded the highest potential biomass for biofuel feedstock production and carbon sequestration in both mangrove ecotypes.

Economic and environmental impacts of biofuels in Indian context

Carbon dioxide (CO2), one of the major greenhouse gases, is released when fossil fuels are burned. Over the past decade, there has been a significant progress in global energy recovery from biomass resources and the use of biofuels as a renewable and sustainable alternative to fossil fuels. Biofuels are considered a carbon–neutral energy source, due to the fact that the CO2 through photosynthesis is captured by the plants during their growing period. The commonly used feedstocks for the production of biofuels are sugar cane, corn and vegetable oils etc. However, in order to minimize the impact on land use, food and feed prices, and other environmental factors; advanced feedstocks such as agriculture residue, algae, aquatic biomass, and waste derived feedstock are strongly suggested. The aim of this work is to review about different available feedstock for biofuel production, generations of biofuels, and its application as automotive fuels. The study concludes with recent advancements in modern biofuel production technology, significant policy implications, challenges and the prospects of biofuel in India.

Biomass to Biofuels: A sure Sustainable Energy Strategy in Nigeria

The utilization of biomass resources as feedstocks for biofuel production is gaining traction globally leading to increasing quantity of biofuel from 187 thousand barrels of oil equivalent per day in 2000 to 1.75 million barrels of oil equivalent per day in 2021. This increase is attributed to high demand, and favourable policy initaited by various government of the world including tax credit and subsidy in the quest to mitigate climate change. Nigeria government in 2007, gazetted the Nigerian Biofuels Policy aimed at promoting the utilization, importation and production of biofues in Nigeria. It encourages investment in biofuels through tax waiver including importation and exportation, waiver on import and customs duties, and insurance. The biomass resources of Nigeria include agricultural and residues, forest products and residues, and municipal solid wastes amounting to over 200 billion kg/yr with energy potential greater than 61 Mtoe. To realize this energy potential, there is a need for the Nigerian government to implement its biofuel policy completely, and to expand the current approved biomass (cassava, sugarcane, palm oil, jatroph, and cellulosic materials) for biofuel production to include the use of lignocellulosic biomass, non-edible oil other jatropha, and algae. The production and utilization of the biofuel will help to increase energy reserve, improve the quality of automotive fossil-based fuels, develop the agricultural sector, mitigate climate change, reduce importation of fuels and create jobs.

Modeling the microbial pretreatment of camelina straw and switchgrass by Trametes versicolor and Phanerochaete chrysosporium via solid-state fermentation process: A growth kinetic sub-model in the context of biomass-based biorefineries.

Advancing microbial pretreatment of lignocellulose has the potential not only to reduce the carbon footprint and environmental impacts of the pretreatment processes from cradle-to-grave, but also increase biomass valorization, support agricultural growers, and boost the bioeconomy. Mathematical modeling of microbial pretreatment of lignocellulose provides insights into the metabolic activities of the microorganisms as responses to substrate and environment and provides baseline targets for the design, development, and optimization of solid-state-fermentation (SSF) bioreactors, including substrate concentrations, heat and mass transfer. In this study, the growth of Trametes versicolor 52J (TV52J), Trametes versicolor m4D (TVm4D), and Phanerochaete chrysosporium (PC) on camelina straw (CS) and switchgrass (SG) during an SSF process was examined. While TV52J illustrated the highest specific growth rate and maximum cell concentration, a mutant strain deficient in cellulose catabolism, TVm4D, performed best in terms of holocellulose preservation and delignification. The hybrid logistic-Monod equation along with holocellulose consumption and delignification models described well the growth kinetics. The oxygen uptake rate and carbon dioxide production rate were directly correlated to the fungal biomass concentration; however, a more sophisticated non-linear relationship might explain those correlations better than a linear model. This study provides an informative baseline for developing SSF systems to integrate fungal pretreatment into a large-scale, on-farm, wet-storage process for the utilization of agricultural residues as feedstocks for biofuel production.

Enzymatic hydrolysate of water hyacinth with NaOH pretreatment for biobutanol production via ABE fermentation by Clostridium beijerinckii JCM 8026

Water hyacinth (WH; Eichhornia crassipes) is an invasive and free-floating aquatic plant that is rapidly spreading over the world causing various environmental problems. The utilization of WH as a feedstock for biofuel production is a challenging solution to overcome the environmental problem as well as address the energy shortage. Due to the complex structure of lignocellulosic materials, pretreatment is an inevitable step to maximize the sugar yield before being used as a substrate in fermentation. The aim of this study was to investigate the utilization of WH for biobutanol production via acetone-butanol-ethanol fermentation by Clostridium beijerinckii JCM 8026. The WH was pretreated with sodium hydroxide (NaOH) at various concentrations [0, 2, and 4% (w/v)], reaction times (30, 60, and 90 min), and temperatures (80, 100, and 120 °C). The pretreatment results were optimized using response surface methodology with a Box-Behnken design and Minitab software analysis. The maximum total sugar concentration (21.05 g/L) following cellulose digestion was obtained after a 2% (w/v) NaOH for 90 min at 100 °C. This hydrolysate slurry gave a subsequent butanol concentration, yield, and productivity of 2.09 g/L, 0.15 g/g, and 0.012 g/L/h, respectively, following C. beijerinckii JCM 8026 fermentation. To help understand the effect of the pretreatments, changes in the pretreated samples were observed by surface area analyses, scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and thermogravimetric analysis.

Breeding sugar-fodder beet hybrids for biofuel production

Topicality. The development of biofuel engineering in Ukraine demands to cultivate the agrofuel crops as renewable energy sources. The sugar-fodder hybrids created using high-value sterile forms of sugar beet and fertile forms of fodder beet may hold promise for biofuel feedstock production. Purpose. Develop experimental sugar-fodder beet hybrids with high energy yield, suitable for alternative biofuels production, and select the best ones. Materials and Methods. 6 pollen-sterile breeding lines of sugar beet and 23 selection numbers of fertile fodder beet were used to create the sugar-fodder hybrids. The breeding process comprised the following steps: selection of initial parental lines, evaluation them by combining ability, and, based on the best, formation of the hybrids. Method of multi-tester top crossing was applied and field trials of the experimental hybrids held. Results. The parent components varied significantly in energy yield, which ranged within 53.5–61.5 GJ/ha (for the MS-samples) and from 80.0 to 87.8 GJ/ha for the top 4 pollinators. On completion of the tester crosses, the following have been selected: MS-samples of the sugar beet lines MS Iv. 24869 and MS Iv. 2484 with proven significant effects of general combining ability at 6.7 GJ/ha and 4.0 GJ/ha; and pollinators of the fodder beet varieties Halytskyi and Lvivskyi zhovtyi – at 8.0 GJ/ha and 6.5 GJ/ha, respectively. In the genotypic structure of variability of the “energy output” trait, the total share of the influence of valuable additive gene complexes of parental samples (51 %) was almost equivalent to the share of non-additive interaction of components (49 %). Conclusions. By the method of combinative selection, three new genotypes of sugar-fodder hybrids have been developed, with energy output rate higher than 120 GJ/ha, suitable for the biofuel production from renewable sources. These are the genotypes MS Iv. 24869/k 41/42; MS Iv. 24869/k 13/14; and MS Iv. 24869/s. Lvivskyi zhovtyi, with indicators exceeding the standard by 14.3 %, 15.4 % and 8.5 %, respectively. Keywords: bioenergy, pollen-sterile forms, pollinators, sugar-fodder hybrids, combining ability, energy yield

Evaluation of Babassu Cake Generated in the Extraction of the Oil as Feedstock for Biofuel Production

The growing demand for energy and the concern about environmental impacts reinforce the necessity for renewable energy sources such as biofuels. In this study, cake generated in the babassu oil extraction was evaluated as a potential feedstock for solid biofuel production, and it contains a blend of cashew nutshell, sugarcane bagasse, carnauba straw, and carnauba stalk. All samples were characterized by proximate analysis and Higher Heating Value. Carbonization was used to improve energy performance and compaction to understand the mechanism and the characteristics of the biomasses compacted. In the extraction of babassu oil, fresh and aged (90 days) kernel samples were used. The fresh samples reached a yield of 59.8%, and the aged samples reached a yield of 70.66%. The carbonization of babassu cake was carried out in a Muffle furnace at temperatures of 250, 300, 350, and 400 °C. The fresh babassu cake showed an HHV of 23.06 MJ kg−1 and after carbonization of 28.07 (250 °C), 30.69 (300 °C), 28.24 (350 °C), and 18.27 MJ kg−1 (400 °C). At 400 °C, a decrease in HHV of 20.8% occurred, and an increase in Ash (%) of 195% occurred. Proximate analysis showed that biomasses are compatible, with some having a higher compatibility than other biological materials already used as fuels in the industry.

Valorization of Hemp-Based Packaging Waste with One-Pot Ionic Liquid Technology.

The range of applications for industrial hemp has consistently increased in various sectors over the years. For example, hemp hurd can be used as a resource to produce biodegradable packaging materials when incorporated into a fungal mycelium composite, a process that has been commercialized. Although these packaging materials can be composted after usage, they may present an opportunity for valorization in a biorefinery setting. Here, we demonstrate the potential of using this type of discarded packaging composite as a feedstock for biofuel production. A one-pot ionic liquid-based biomass deconstruction and conversion process was implemented, and the results from the packaging material were compared with those obtained from untreated hemp hurd. At a 120 °C reaction temperature, 7.5% ionic liquid loading, and 2 h reaction time, the packaging materials showed a higher lignocellulosic sugar yield and sugar concentrations than hemp hurd. Hydrolysates prepared from packaging materials also promoted production of higher titers (1400 mg/L) of the jet-fuel precursor bisabolene when used to cultivate an engineered strain of the yeast Rhodosporidium toruloides. Box-Behnken experiments revealed that pretreatment parameters affected the hemp hurd and packaging materials differently, evidencing different degrees of recalcitrance. This study demonstrated that a hemp hurd-based packaging material can be valorized a second time once it reaches the end of its primary use by supplying it as a feedstock to produce biofuels.

Comprehensive insights into conversion of microalgae to feed, food, and biofuels: Current status and key challenges towards implementation of sustainable biorefineries

Microalgae have been promoted as important feedstocks for producing biofuels and bioproducts. However, their production on a large scale would require a large amount of water and nutrients. Considering the importance of food security and the high economic value of microalgal biomass, food and feed applications should be prioritized if microalgae are grown using conventional water resources. Nevertheless, microalgae can also be grown in nutrient-rich wastewater streams while producing valuable biomass and treating harmful effluents. Because of health issues, food and feed applications of microalgal biomass grown using waste resources are not allowed; hence, it can be used as a feedstock for biofuel production. In line with that, this review first briefly discusses the applications of microalgae in food, feed, and wastewater treatment and then summarizes the efforts put into utilizing microalgal biomass to produce biofuels through different pathways. These technologies, including thermochemical (200–1300 °C), biochemical (25–55 °C), and chemical (25–200 °C) conversion methods, are critically discussed. The pros and cons of using different microalgal conversion technologies are outlined to identify possible future directions for the field. Different microalgal biomass harvesting methods are also reviewed, and the process conditions are summarized. Parameters affecting technology selection, such as energy costs, reaction time, and technological sophistication, are also explored and presented. In general, microalgal biofuels still face many challenges in replacing traditional fossil fuels. Future work should focus on maximizing the yield and quality of microalgal biofuels while enhancing their economic viability.

Engineering triacylglycerol accumulation in duckweed (Lemna japonica).

Duckweeds are amongst the fastest growing of higher plants, making them attractive high-biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol-inducible cyan fluorescent protein-Arabidopsis WRINKLED1 fusion protein (CFP-AtWRI1), strong constitutive expression of a mouse diacylglycerol:acyl-CoA acyltransferase2 (MmDGAT), and a sesame oleosin variant (SiOLE(*)). Individual expression of each gene increased TAG accumulation by 1-to 7-fold relative to controls, while expression of pairs of these genes increased TAG by 7- to 45-fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45-fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108-fold to 8.7% of DW after incubation on medium containing 100 μm estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three-fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY-stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven-fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.