Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.13 mM and V(max) 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K(m) 27 mM and V(max) 3.8 nM/mg/min, and PgAxt1p has K(m) 65 mM and V(max) 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K(m) 371 mM and V(max) 341 nM/mg/min for L-arabinose, and K(m) 25 mM and V(max) 76 nM/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering.

Oleaginous microalgae contain a high level of lipids, which can be extracted and converted to biofuel. The lipid-extracted residue can then be further utilized through anaerobic digestion to produce biogas. However, long-chain fatty acids (LCFAs) have been identified as the main inhibitory factor on microbial activity of anaerobic consortium. In this study, the mechanism of LCFA inhibition on anaerobic digestion of whole and lipid-extracted algal biomass was investigated with a range of calcium concentrations against various inoculum to substrate ratios as a means to alleviate the LCFA inhibition.

The primary aim of this study was to completely investigate extensive biological methane potential (BMP) on both whole microalgae and its lipid-extracted biomass residues with various degrees of biomass pretreatment. Specific methane productivities (SMP) under batch conditions for non-lipid extracted biomass were better than lipid-extracted biomass residues and exhibited no signs of ammonia or carbon/nitrogen (C/N) ratio inhibition when digested at high I/S ratio (I/S ratio ⩾ 1.0). SMP for suitably extracted biomass ranged from 0.30 to 0.38 L CH4/g VS (volatile solids). For both whole and lipid-extracted biomass, overall organic conversion ranged from 59.33 to 78.50 as a measure of %VS reduction with greater percentage biodegradability in general found within the lipid-extracted biomass. Higher production levels correlated to lipid content with a linear relationship between SMP and ash-free lipid content being developed at a R2 of 0.814.

To identify algal strains with altered starch metabolism from a large pool of candidates of growing algal colonies, we have developed a novel, high-throughput screening tool by combining gaseous bleaching of replica transferred colonies and subsequent iodine staining to visualize starch. Screening of healthy growing colonies of microalgae has not been possible previously because high levels of chlorophyll make the detection of starch with an iodine stain impossible. We demonstrated that chlorine dioxide (ClO2) removes essentially all chlorophyll from the colonies and enables high-throughput screening of, for example, a population of mutagenized cells or a culture collection isolated in a bioprospecting project.

Microalgae are photosynthetic microorganisms that can use CO2 and sunlight to generate the complex biomolecules necessary for their survival. These biomolecules include energy-rich lipid compounds that can be converted using existing refinery equipment into valuable bio-derived fuels, including jet fuel for military and commercial use. Through a dedicated and thorough collaborative research,development and deployment program, the team of the National Renewable Energy Laboratory (NREL) and Chevron will identify a suitable algae strain that will surpass the per-acre biomass productivity of terrestrial plant crops.

Most people consider algae a nuisance. But it turns out that one man's pond scum is another man's gold, to draw on an old expression. An algal species with the right properties could be immensely valuable. That's why we and others at the National Renewable Energy Laboratory, in Golden, Colo., have been busy "bioprospecting" for promising scoops of slime.

Background One of the activities of IEA Bioenergy Task 39 is to commission state-of-the-art reports on some of the most important relevant clean energy, liquid biofuels technology topics. You can access many Task 39 past reports at www.Task39.org One area that has received considerable recent attention is the potential of algae to produce low carbon energy dense liquid biofuels suitable for uses such as aviation, or as petrol/gasoline and diesel replacements. IEA Bioenergy Task 39 is fortunate to have, within its extensive network, colleagues who have had long experience with algae technologies, both in terms of commercial growth of algae (as has occurred in Australia over many years of operating high-productivity open ponds) and in assessing the technical status and potential of algal biofuels (as carried out by the United States' National Renewable Energy Laboratory (NREL) during the Aquatic Species Program). We want to thank the authors, Al Darzins and Philip Pienkos (NREL, US) and Les Edye (BioIndustry Partners, Australia) for their hard work in writing this report! Contributions of text and figures were provided by We also want to thank Don O'Connor of (S&T) 2 Consultants Inc. for the final editing and layout of this report as well all the Task 39 member Country Representatives and IEA Bioenergy Executive Committee members for providing excellent constructive feedback on initial drafts of the report. With algal biofuels research and development evolving rapidly, we are confident that areas such as technical approach, process scale-up/commercial demonstration, life cycle/sustainability analysis, etc., of algal systems for liquid biofuels production will warrant further extended examination in the future. Such work will likely become one focus of future IEA Bioenergy Task 39 activities. Executive Summary This IEA Bioenergy report, ―Current Status and Potential for Algal Biofuels Production,‖ seeks to examine the technical and economic feasibility of generating algal biomass for the production of liquid biofuels. As worldwide petroleum reserves diminish due to consumption exceeding discoveries, many countries are becoming increasingly dependent upon imported sources of oil. The United States, for example, currently imports a full two-thirds of its petroleum from only a few countries around the world. The demand for energy is growing worldwide especially in many of the rapidly developing countries such as in China and India. Furthermore, the continued combustion of fossil fuels has created serious environmental concerns over global warming due to the increased release of greenhouse gases (GHG). Biofuels are one of the …

Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20-50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.

Genetic transformation of two species of diatoms has been accomplished by introducing chimeric plasmid vectors containing a bacterial antibiotic resistance gene driven by regulatory sequences from the acetyl-CoA carboxylase (ACCase) gene from the diatom Cyclotella cryptica. The recombinant DNA integrated into one or more random sites within the algal genome and the foreign protein was produced by the algal transformants. This is the first report of genetic transformation of any chlorophyll c-containing microalgal strain. We are using this system to introduce additional copies of the ACCase gene into diatoms in an attempt to manipulate lipid accumulation in transformed strains.

Significant progress has been made toward the successful genetic engineering of microalgal species with high potential for fuel production. Foreign DNA has been transferred into a green alga,Chlorella ellipsoidea, and has been successfully expressed in this heterologous system. In addition, electroporation has shown promise as a means of introducing DNA into intact algal cells. We have analyzed the composition of DNA from several algal species and demonstrated the presence of elevated GC contents in several green algal species. These results suggest that homologous selectable markers may be required for the development of stable transformation systems for the algae; the development of nitrate reductase and orotidine-5′-phosphate decarboxylase as homologous selectable markers forMonoraphidium minutum and forCyclotella cryptica is in progress. We have constructed a cDNA expression library forCyclotella cryptica, and are constructing cDNA and genomic libraries for several other algal species. The libraries will be screened with heterologous or homologous probes for nitrate reductase and acetyl-CoA carboxylase in order to clone these genes, which appear to influence lipid accumulation in the algae. This work represents important steps toward the genetic improvement of micro-algae for fuel production.

We have examined the relative contributions of MCM1 and STE12 to the transcription of the a-specific STE2 gene by using a 367 bp fragment from the STE2 5'-noncoding region to drive expression of a reporter lacZ gene. Mutation of the MCM1 binding site destroyed MCM1.alpha 2-mediated repression in alpha cells and dramatically reduced expression in a cells. The residual expression was highly stimulated by exposure of cells to pheromone. Likewise, the loss of STE12 function reduced lacZ expression driven by the wild-type STE2 fragment. In the absence of both MCM1 and STE12 functions, no residual expression was observed. Thus, the STE2 fragment appears to contain two distinct upstream activation sequences (UASs), one that is responsible for the majority of expression in cells not stimulated by pheromone, and one that is responsible for increased expression upon pheromone stimulation. In further support of this idea, a chemically synthesized version of the STE2 MCM1 binding site had UAS activity, but the activity was neither stimulated by pheromone nor reduced in ste12 mutants. Although transcription of alpha-specific genes also requires both MCM1 and STE12, these genes differ from a-specific genes in that they have a single, MCM1-dependent UAS system. The activity of the minimal 26 bp UAS from the alpha-specific STE3 gene was both stimulated by pheromone and reduced in ste12 mutants. These data suggest that at alpha-specific genes STE12 and MCM1 exert their effects through a single UAS.

We report here on the development of a transient expression system for Chlorella ellipsoidea using a heterologous gene, firefly luciferase. Cells of this unicellular green alga were converted to protoplasts and treated with plasmid pDO432, which bears luciferase under the control of the CaMV 35S promoter. This treatment resulted in detectable luciferase activity in cell extracts. Expression required Cellulysin treatment, active cell metabolism, and the addition of carrier DNA and polyethylene glycol. Linearization of the luciferase plasmid did not significantly alter the activity. A time course of expression showed that luciferase is made rapidly, within about 7 h after addition of DNA, but that the activity disappears over the course of a few days. These experiments represent an important first step in the development of a Chlorella transformation system.