Next-generation Biofuel Production Research Articles

Heterologous expression of a yeast lipid transporter promotes accumulation of storage compounds in Chlamydomonas

Microalgae offer renewable and sustainable sources for the bioindustrial production of next-generation biofuels and biomaterials. In this work, we heterologously expressed a lipid transporter gene (MMF1) from the major facilitator superfamily in the model green microalga Chlamydomonas reinhardtii, under the influence of a strong light and temperature-inducible promoter. The full-length cDNA sequence of MMF1 from Moesziomyces antarcticus T-34 (MaMMF1) encodes for a 542 amino acid polypeptide with transmembrane efflux pump activity. M. antarcticus is a fungus that can produce and secrete large amounts of glycolipid biosurfactants known as mannosylerythritol lipids (MELs) under nitrogen starvation conditions. Heterologous expression of MaMMF1 in C. reinhardtii resulted in a slight increase in cell biomass, with mutants exhibiting on average about 1.8-fold larger cell sizes. Total lipid content almost doubled in cells expressing MaMMF1 relative to the control strain when cultures were grown for two days under nitrogen starvation. Moreover, average starch content was over 2-fold higher in the mutants. Our results showed that algal strains engineered with the MaMMF1 MEL transporter were capable of possibly redirecting the carbon flux, which resulted in improved growth and accumulation of high-value storage compounds.

Potential of Straw for Energy Purposes in Poland—Forecasts Based on Trend and Causal Models

The mitigation of climate change poses a major challenge to the legal framework which aims to stimulate the development of renewable energy sources. The European Union’s direction for the use of renewable energy is distributed generation and an increased use of by-products and organic waste, especially in the production of next-generation biofuels. The main aim of this study is to evaluate the production potential of straw in Poland and the possibility of its use for energy purposes, including a forecast for 2030, on the assumption that the management of this resource is in accordance with the provisions of the Polish Code for Good Agriculture Practice. In Poland, in the years 1999–2018, the average annual surplus of straw harvested over agricultural consumption equalled 12.5 million tons (4.2 Mtoe). Its largest surpluses were in the Dolnośląskie, Kujawsko-Pomorskie, Lubelskie, Wielkopolskie, and Zachodniopomorskie voivodeships (NUTS2). Based on the developed panel models, forecasts for straw surpluses in Poland are presented in three perspectives: realistic, pessimistic, and optimistic. The forecasts show regional differentiation until 2030. Each of the three perspectives indicate a slow increase in these surpluses, and depending on the adopted version, it will range from 10.6% to 21.9%.

Isobutanol production freed from biological limits using synthetic biochemistry

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L−1 h−1, a titer of 275 g L−1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.

Enhanced bioconversion of hemicellulosic biomass by microbial consortium for biobutanol production with bioaugmentation strategy

As a renewable and sustainable source for next-generation biofuel production, lignocellulosic biomass can be effectively utilized in environmentally friendly manner. In this study, a stable, xylan-utilizing, anaerobic microbial consortium MC1 enriched from mangrove sediments was established, and it was taxonomically identified that the genera Ruminococcus and Clostridium from this community played a crucial role in the substrate utilization. In addition, a butanol-producing Clostridium sp. strain WST was introduced via the bioaugmentation process, which resulted in the conversion of xylan to biobutanol up to 10.8 g/L, significantly improving the butanol yield up to 0.54 g/g by 98-fold. When this system was further applied to other xylan-rich biomass, 1.09 g/L of butanol could be achieved from 20 g/L of corn cob. These results provide another new method to efficiently convert xylan, the main hemicellulose from lignocellulosic biomass into biofuels through a low-cost and eco-friendly manner.

A global analysis of gene expression in Fibrobacter succinogenes S85 grown on cellulose and soluble sugars at different growth rates

BackgroundCellulose is the most abundant biological polymer on earth, making it an attractive substrate for the production of next-generation biofuels and commodity chemicals. However, the economics of cellulose utilization are currently unfavorable due to a lack of efficient methods for its hydrolysis. Fibrobacter succinogenes strain S85, originally isolated from the bovine rumen, is among the most actively cellulolytic mesophilic bacteria known, producing succinate as its major fermentation product. In this study, we examined the transcriptome of F. succinogenes S85 grown in continuous culture at several dilution rates on cellulose, cellobiose, or glucose to gain a system-level understanding of cellulose degradation by this bacterium.ResultsSeveral patterns of gene expression were observed for the major cellulases produced by F. succinogenes S85. A large proportion of cellulase genes were constitutively expressed, including the gene encoding for Cel51A, the major cellulose-binding endoglucanase produced by this bacterium. Moreover, other cellulase genes displayed elevated expression during growth on cellulose relative to growth on soluble sugars. Growth rate had a strong effect on global gene expression, particularly with regard to genes predicted to encode carbohydrate-binding modules and glycoside hydrolases implicated in hemicellulose degradation. Expression of hemicellulase genes was tightly regulated, with these genes displaying elevated expression only during slow growth on soluble sugars. Clear differences in gene expression were also observed between adherent and planktonic populations within continuous cultures growing on cellulose.ConclusionsThis work emphasizes the complexity of the fiber-degrading system utilized by F. succinogenes S85, and reinforces the complementary role of hemicellulases for accessing cellulose by these bacteria. We report for the first time evidence of global differences in gene expression between adherent and planktonic populations of an anaerobic bacterium growing on cellulose at steady state during continuous cultivation. Finally, our results also highlight the importance of controlling for growth rate in investigations of gene expression.

A Pseudomonas putida efflux pump acts on short-chain alcohols

BackgroundThe microbial production of biofuels is complicated by a tradeoff between yield and toxicity of many fuels. Efflux pumps enable bacteria to tolerate toxic substances by their removal from the cells while bypassing the periplasm. Their use for the microbial production of biofuels can help to improve cell survival, product recovery, and productivity. However, no native efflux pump is known to act on the class of short-chain alcohols, important next-generation biofuels, and it was considered unlikely that such an efflux pump exists.ResultsWe report that controlled expression of the RND-type efflux pump TtgABC from Pseudomonas putida DOT-T1E strongly improved cell survival in highly toxic levels of the next-generation biofuels n-butanol, isobutanol, isoprenol, and isopentanol. GC-FID measurements indicated active efflux of n-butanol when the pump is expressed. Conversely, pump expression did not lead to faster growth in media supplemented with low concentrations of n-butanol and isopentanol.ConclusionsTtgABC is the first native efflux pump shown to act on multiple short-chain alcohols. Its controlled expression can be used to improve cell survival and increase production of biofuels as an orthogonal approach to metabolic engineering. Together with the increased interest in P. putida for metabolic engineering due to its flexible metabolism, high native tolerance to toxic substances, and various applications of engineering its metabolism, our findings endorse the strain as an excellent biocatalyst for the high-yield production of next-generation biofuels.

Producing next-generation biofuels from filamentous cyanobacteria: An economic feasibility analysis

The need for renewable, sustainable sources of biofuels continues to increase as the world’s population continues to grow. Using microorganisms as biofuel producers is one area that is being researched extensively for this purpose. Anabaena sp. PCC 7120 is a filamentous strain of cyanobacteria capable of fixing atmospheric nitrogen, and has been genetically engineered to produce limonene, a cyclic hydrocarbon which has potential as a next-generation biofuel. This study analyzed the economic feasibility of a theoretical next-generation production facility that uses genetically engineered Anabaena 7120 to produce limonene. The economic feasibility of a limonene production facility was analyzed using the Farm-level Algae Risk Model (FARM). This model is an integrated systems compilation of numerous technoeconomic models that has been used previously in several algal production scenarios. FARM simulated 10 years of operation for the production facility for two scenarios. The 1st scenario used actual limonene productivity data (0.018mg/L/d) from a genetically engineered strain of filamentous cyanobacteria, while the 2nd scenario used a ‘best case’ assumption that limonene productivity can be increased 100-fold (1.8mg/L/d). It was determined that the average probability of economic success of the 1st scenario at year 5 was 0%, while the average probability of success of the 2nd scenario was 100%. Assuming no fractional reductions in OPEX and CAPEX, the average net present value (NPV) at year 5 of the 1st scenario was −$588 million, compared to $392 million for the 2nd scenario. Further analysis determined that a limonene productivity of 1.02mg/L/d is needed to yield an NPV of 0 dollars at year 5. This study shows strong evidence that a next-generation biofuel production facility utilizing genetically engineered strains of filamentous cyanobacteria could become economically feasible in the future if strains are developed with increased biofuel productivities.

Functional conservation and divergence of Miscanthus lutarioriparius GT43 gene family in xylan biosynthesis.

BackgroundXylan is the most abundant un-cellulosic polysaccharides of plant cell walls. Much progress in xylan biosynthesis has been gained in the model plant species Arabidopsis. Two homologous pairs Irregular Xylem 9 (IRX9)/9L and IRX14/14L from glycosyltransferase (GT) family 43 have been proved to play crucial roles in xylan backbone biosynthesis. However, xylan biosynthesis in grass such as Miscanthus remains poorly understood.ResultsWe characterized seven GT43 members in M. lutarioriparius, a promising bioenergy crop. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of MlGT43 genes was ubiquitously detected in the tissues examined. In-situ hybridization demonstrated that MlGT43A-B and MlGT43F-G were specifically expressed in sclerenchyma, while MlGT43C-E were expressed in both sclerenchyma and parenchyma. All seven MlGT43 proteins were localized to Golgi apparatus. Overexpression of MlGT43A-E but not MlGT43F and MlGT43G in Arabidopsis irx9 fully or partially rescued the mutant defects, including morphological changes, collapsed xylem and increased xylan contents, whereas overexpression of MlGT43F and MlGT43G but not MlGT43A-E complemented the defects of irx14, indicating that MlGT43A-E are functional orthologues of IRX9, while MlGT43F and MlGT43G are functional orthologues of IRX14. However, overexpression of all seven MlGT43 genes could not rescue the mucilage defects of irx14 seeds. Furthermore, transient transactivation analyses of MlGT43A-E reporters demonstrated that MlGT43A and MlGT43B but not MlGT43C-E were differentially activated by MlSND1, MlMYB46 or MlVND7.ConclusionThe results demonstrated that all seven MlGT43s are functionally conserved in xylan biosynthesis during secondary cell wall formation but diversify in seed coat mucilage xylan biosynthesis. The results obtained provide deeper insight into xylan biosynthesis in grass, which lay the foundation for genetic modification of grass cell wall components and structure to better suit for next-generation biofuel production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0793-5) contains supplementary material, which is available to authorized users.

An Unusual Iron-Dependent Oxidative Deformylation Reaction Providing Insight into Hydrocarbon Biosynthesis in Nature

The development of new pathways for next-generation biofuel production has spurred recent investigations into the mechanisms of enzymes that biosynthesize hydrocarbons. One widely distributed group of enzymes, aldehyde decarbonylases, catalyze unusual deformylation reactions in which long-chain fatty aldehydes are converted to alkanes. These enzymes are all iron-dependent and in insects are represented by a cytochrome P450 enzyme that releases the aldehyde carbon as CO2. Here we describe a novel nonenzymatic reaction of an α-cyclopropyl-substituted aldehyde that mimics the enzyme reaction. This aldehyde is oxidatively deformylated in the presence of aqueous iron(II) salts and oxygen to yield an alkyl-substituted cyclopropane and CO2, in a reaction that competes with the more conventional oxidation of the aldehyde to the carboxylic acid. Like the enzymatic reaction, the reaction occurs with retention of the aldehyde proton in the alkane product and probably proceeds through an iron–peroxo species. Computat...

Fuelling the future: microbial engineering for the production of sustainable biofuels.

Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

Laccases for biorefinery applications: a critical review on challenges and perspectives.

Modern biorefinery concepts focus on lignocellulosic biomass as a feedstock for the production of next generation biofuels and platform chemicals. Lignocellulose is a recalcitrant composite consisting of several tightly packed components which are stuck together by the phenolic polymer lignin hampering the access to the carbohydrate compounds of biomass. Certain saprophytic organisms are able to degrade lignin by the use of an enzymatic cocktail. Laccases have been found to play a major role during lignin degradation and have therefore been intensively researched with regard to potential applications for biomass processing. Within this review, we go along the process chain of a third generation biorefinery and highlight the process steps which could benefit from laccase applications. Laccases can assist the pretreatment of biomass and promote the subsequent enzymatic hydrolysis of cellulose by the oxidative modification of residual lignin on the biomass surface. In combination with mediator molecules laccases are often reported being able to catalyze the depolymerization of lignin. Studies with lignin model compounds confirm the chemical possibility of a laccase-catalyzed cleavage of lignin bonds, but the strong polymerization activity of laccase counters the decomposition of lignin by repolymerizing the degradation products. Therefore, it is a key challenge to shift the catalytic performance of laccase towards lignin cleavage by optimizing the process conditions. Another field of application for laccases is the detoxification of biomass hydrolyzates by the oxidative elimination of lignin-derived phenolics which inhibit hydrolytic enzymes and are toxic for fermentation organisms. This review critically discusses the potential applications for laccases in biorefinery processes and emphasizes the challenges and perspectives which go along with the use of this enzyme for the technical utilization of lignocellulose.

Integration of next-generation biofuel production in the Swedish forest industry – A geographically explicit approach

The geographic locations of biofuel production facilities should be strategically chosen in order to minimise the total cost of using biofuels. Proximity to biomass resources, possibilities for integration, and distance to biofuel users are aspects that need to be considered. In this paper, the geographically explicit optimisation model BeWhere Sweden was used to investigate the future production of next-generation biofuels from forest biomass in Sweden. A focus was placed on the integration of biofuel production with the existing forest industry, as well as on how different parameters affect biofuel production costs, the choice of technologies and biofuels, and the localisation of new biofuel plants. Six examples of different biofuel routes were considered. A methodology was developed considering detailed, site-specific conditions for potential host industries. The results show that the cost of biomass and the biofuel plant capital cost generally dominate the biofuel cost, but the cost for biomass transportation and biofuel distribution can also have a significant impact. DME produced via black liquor gasification (naturally integrated with chemical pulp mills) and SNG produced via solid biomass gasification (mainly integrated with sawmills), dominate the solutions. The distribution of these technology cases varies depending on a number of parameters, including criteria for sizing biofuel plants, the electricity price, the biofuel distribution cost and the cost of biomass, and is sensitive to changes in these parameters. Generally, plants with low specific investment costs (i.e., high biofuel production) and/or plants with low specific biomass transportation costs occur most frequently in the solutions. Because these properties often vary significantly among biofuel production facilities at different host industry sites of the same type, the results show the advantage of including site-specific data in this type of model. (C) 2015 Elsevier Ltd. All rights reserved.

Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels.

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production.

A gene stacking approach leads to engineered plants with highly increased galactan levels in Arabidopsis.

BackgroundEngineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production. Lignocellulosic biomass contains a high proportion of pentose residues, which are more difficult to convert into fuels than hexoses. Therefore, increasing the hexose/pentose ratio in biomass is one approach for biomass improvement. A genetic engineering approach was used to investigate whether the amount of pectic galactan can be specifically increased in cell walls of Arabidopsis fiber cells, which in turn could provide a potential source of readily fermentable galactose.ResultsFirst it was tested if overexpression of various plant UDP-glucose 4-epimerases (UGEs) could increase the availability of UDP-galactose and thereby increase the biosynthesis of galactan. Constitutive and tissue-specific expression of a poplar UGE and three Arabidopsis UGEs in Arabidopsis plants could not significantly increase the amount of cell wall bound galactose. We then investigated co-overexpression of AtUGE2 together with the β-1,4-galactan synthase GalS1. Co-overexpression of AtUGE2 and GalS1 led to over 80% increase in cell wall galactose levels in Arabidopsis stems, providing evidence that these proteins work synergistically. Furthermore, AtUGE2 and GalS1 overexpression in combination with overexpression of the NST1 master regulator for secondary cell wall biosynthesis resulted in increased thickness of fiber cell walls in addition to the high cell wall galactose levels. Immunofluorescence microscopy confirmed that the increased galactose was present as β-1,4-galactan in secondary cell walls.ConclusionsThis approach clearly indicates that simultaneous overexpression of AtUGE2 and GalS1 increases the cell wall galactose to much higher levels than can be achieved by overexpressing either one of these proteins alone. Moreover, the increased galactan content in fiber cells while improving the biomass composition had no impact on plant growth and development and hence on the overall biomass amount. Thus, we could show that the gene stacking approach described here is a promising method to engineer advanced feedstocks for biofuel production.

The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC–MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.

Endophytic Fungi: Prospects in Biofuel Production

Endophytic fungi are now being recognized as a yet unexplored source of volatile low molecular mass hydrocarbons and lipids for the production of next generation biofuels. These next generation biofuels are to be produced by more efficient and lesser number of biosynthetic steps directly from the microorganisms responsible and would have the preferred adaptability for immediate use in the current internal combustion engines without bringing about a radical change in automobile design. Research groups working across the world have reported isolation of endophytes having optimum lipid profiles for use as precursors for biofuel generation. Fungal endophytes have also been found to produce volatile organic compounds having similarity with conventional diesel fuel. The recent publication of the genome sequence of one such biofuel producing endophytic fungi Ascocoryne sarcoides will increase the understanding of the biosynthetic machinery by which endophytes are able achieve the complex feat of generating volatile hydrocarbons. Research on this important area of alternative bio-energy for ensuring India’s energy security in the near future has slowly started gaining momentum. The present article therefore aims to review the current status of research in the Indian perspective, in order to encourage and generate interest among research groups across India and the world for initiating and undertaking more vigorous and focused research activity on endophytic fungi from the Indian sub-continent having the potential to make fuel-related hydrocarbons.

Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels

The idea of renewable and regenerative resources has inspired research for more than a hundred years. Ideally, the only spent energy will replenish itself, like plant material, sunlight, thermal energy or wind. Biodiesel or ethanol are examples, since their production relies mainly on plant material. However, it has become apparent that crop derived biofuels will not be sufficient to satisfy future energy demands. Thus, especially in the last decade a lot of research has focused on the production of next generation biofuels. A major subject of these investigations has been the microbial fatty acid biosynthesis with the aim to produce fatty acids or derivatives for substitution of diesel. As an industrially important organism and with the best studied microbial fatty acid biosynthesis, Escherichia coli has been chosen as producer in many of these studies and several reviews have been published in the fields of E. coli fatty acid biosynthesis or biofuels. However, most reviews discuss only one of these topics in detail, despite the fact, that a profound understanding of the involved enzymes and their regulation is necessary for efficient genetic engineering of the entire pathway. The first part of this review aims at summarizing the knowledge about fatty acid biosynthesis of E. coli and its regulation, and it provides the connection towards the production of fatty acids and related biofuels. The second part gives an overview about the achievements by genetic engineering of the fatty acid biosynthesis towards the production of next generation biofuels. Finally, the actual importance and potential of fatty acid-based biofuels will be discussed.

Production of advanced biofuels in engineered E. coli

Commercial fermentation processes have long taken advantage of the synthetic power of living systems to rapidly and efficiently transform simple carbon sources into complex molecules. In this regard, the ability of yeasts to produce ethanol from glucose at exceptionally high yields has served as a key feature in its use as a fuel, but is also limited by the poor molecular properties of ethanol as a fuel such as high water miscibility and low energy density. Advances in metabolic engineering and synthetic biology allow us to begin constructing new high-flux pathways for production of next generation biofuels that are key to building a sustainable pipeline for liquid transportation fuels.

Production of xylitol by recombinant microalgae

Microalgae have received significant attention recently as a potential low-cost host for the production of next-generation biofuels and natural products. Here we show that the chloroplast genome of the eukaryotic green microalga Chlamydomonas reinhardtii can be genetically engineered to produce xylitol through the introduction of a gene encoding a xylose reductase (XR) from the fungi Neurospora crassa. Increased levels of heterologous protein accumulation and xylitol production were achieved by synthesizing the XR gene in the chloroplast codon bias and by driving expression of the codon-optimized XR gene using a 16S/atpA promoter/5′-UTR fusion. These results demonstrate the feasibility of engineering microalgae to produce xylitol, and show the importance of codon optimizing the XR gene and using the 16S/atpA promoter/5′-UTR fusion to express XR in the chloroplast of C. reinhardtii.

Association with an Ammonium-Excreting Bacterium Allows Diazotrophic Culture of Oil-Rich Eukaryotic Microalgae

Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.