The use of fossil fuels is now widely accepted as unsustainable due to depleting resources and the accumulation of greenhouse gases in the environment that have already exceeded the “dangerously high” threshold of 450 ppm CO2-e. To achieve environmental and economic sustainability, fuel production processes are required that are not only renewable, but also capable of sequestering atmospheric CO2. Currently, nearly all renewable energy sources (e.g. hydroelectric, solar, wind, tidal, geothermal) target the electricity market, while fuels make up a much larger share of the global energy demand (∼66%). Biofuels are therefore rapidly being developed. Second generation microalgal systems have the advantage that they can produce a wide range of feedstocks for the production of biodiesel, bioethanol, biomethane and biohydrogen. Biodiesel is currently produced from oil synthesized by conventional fuel crops that harvest the sun’s energy and store it as chemical energy. This presents a route for renewable and carbon-neutral fuel production. However, current supplies from oil crops and animal fats account for only approximately 0.3% of the current demand for transport fuels. Increasing biofuel production on arable land could have severe consequences for global food supply. In contrast, producing biodiesel from algae is widely regarded as one of the most efficient ways of generating biofuels and also appears to represent the only current renewable source of oil that could meet the global demand for transport fuels. The main advantages of second generation microalgal systems are that they: (1) Have a higher photon conversion efficiency (as evidenced by increased biomass yields per hectare): (2) Can be harvested batch-wise nearly all-year-round, providing a reliable and continuous supply of oil: (3) Can utilize salt and waste water streams, thereby greatly reducing freshwater use: (4) Can couple CO2-neutral fuel production with CO2 sequestration: (5) Produce non-toxic and highly biodegradable biofuels. Current limitations exist mainly in the harvesting process and in the supply of CO2 for high efficiency production. This review provides a brief overview of second generation biodiesel production systems using microalgae.

https://www.springer.com/cda/content/document/cda_downloaddocument/BioEnergyResearch_Second+Generation+Biofuels+High-Efficiency+Microalgae+for+Biodiesel+Production.pdf?SGWID=0-0-45-1347846-0

Second generation biofuels: high-efficiency microalgae for biodiesel production

1. Light and Energy. 2. Organization and Structure of Photosynthetic Systems. 3. History and Development of Photosynthesis. 4. Photosynthetic Pigments-Structure and Spectroscopy. 5. Antenna Complexes and Energy Transfer Processes. 6. Reaction Center Complexes. 7. Electron Transfer Pathways and Components. 8. Chemiosmotic Coupling and ATP Synthesis. 9. Carbon Metabolism. 10. Genetics, Assembly and Regulation of Photosynthetic. Systems. 11. Origin and Evolution of Photosynthesis. Appendix 1. Light, Energy and Kinetics

Molecular mechanisms of photosynthesis

There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H2 yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H2 production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.

Genetic Engineering of Algae for Enhanced Biofuel Production

Diatoms are the world's largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research.

Numerous studies have characterized parameters of silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of genes encoding the transporter proteins. Multiple types of silicic acid transporter gene have been identified in a single diatom species, and multiple types appear to be present in all diatom species. The controlled expression and perhaps localization of the transporters in the cell may be factors in the overall regulation of silicic acid uptake. Transport can also be regulated by the rate of silica incorporation into the cell wall, suggesting that an intracellular sensing and control mechanism couples transport with incorporation. Sizable intracellular pools of soluble silicon have been identified in diatoms, at levels well above saturation for silica solubility, yet the mechanism for maintenance of supersaturated levels has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be an important part of the silicification process because of the close coupling between silica incorporation and uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about the molecular details of this process. However, proteins occluded within silica that promote silicification in vitro have recently been characterized, and the application of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy. As such, diatom silicon metabolism differs from that of other major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport are tightly coupled to the cell cycle, which results in a dependency in the extent of silicification on growth rate. Silica dissolution is an important part of diatom cellular silicon metabolism, because dissolution must be prevented in the living cell, and because much of the raw material for mineralization in natural assemblages is supplied by dissolution of dead cells. Perhaps part of the reason for the ecological success of diatoms is due to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms and other siliceous organisms has depleted the oceans of silicon, such that silicon availability is now a major factor in the control of primary productivity. Much new progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques. Such insight is likely to lead to a greater understanding of the role of silicon in controlling diatom growth, and hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.

Silicon metabolism in diatoms: implications for growth

Algae biofuels may provide a viable alternative to fossil fuels; however, this technology must overcome a number of hurdles before it can compete in the fuel market and be broadly deployed. These challenges include strain identification and improvement, both in terms of oil productivity and crop protection, nutrient and resource allocation and use, and the production of co-products to improve the economics of the entire system. Although there is much excitement about the potential of algae biofuels, much work is still required in the field. In this article, we attempt to elucidate the major challenges to economic algal biofuels at scale, and improve the focus of the scientific community to address these challenges and move algal biofuels from promise to reality.

/pdf/biofuels-from-algae-challenges-and-potential-596xth9xzn.pdf

Biofuels from algae: challenges and potential.

A nuclear transformation system has been developed for the diatomPhaeodactylum tricornutum using microparticle bombardment to introduce thesh ble gene fromStreptoalloteichus hindustanus into cells. Thesh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from theP. tricornutum fcp genes were used to drive expression ofsh ble. Between 10–100 transformants were recovered/108 cells. Transformants were able to grow on at least 500 µg/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations thesh ble gene was present in 1–3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1–2.0 µg/mg total protein). Thecat reporter gene fused to afcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed inP. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.

Stable nuclear transformation of the diatom Phaeodactylum tricornutum.

Evolution of the phycobiliproteins.

Six members of a multigene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex from female gametophytes of the brown alga Macrocystis pyrifera have been cloned and characterized. The deduced amino acid sequences are very similar to those of fucoxanthin chlorophyll binding proteins (Fcp) from the diatom Phaeodactylum tricornutum and exhibit limited homology to chlorophyll a/b binding (Cab) polypeptides from higher plants. The primary translation products from the M. pyrifera fcp genes are synthesized as higher molecular weight precursors that are processed prior to their assembly into the Fcp complex. The presumed N-terminal 40-amino acid presequence of the Fcp precursor polypeptide has features resembling that of a signal sequence. This presequence may be required for the protein to transverse the endoplasmic reticulum that surrounds the plastid in brown algae. A subsequent targeting step would be required for the protein to cross the double membrane of the plastid envelope. M. pyrifera fcp transcripts are of two sizes, 1.2 and 1.6 kb. The size difference is accounted for by the length of the 3′ untranslated region, which can be up to 1000 bases. Transcript abundance's of members of the fcp gene family are dependent on light quantity, light quality, or both. Transcript levels of one gene increased approximately five- to tenfold in thalli grown in low intensity relative to high intensity white or blue light. Transcripts from this gene also significantly increase in red light relative to blue light at equivalent light intensities.

Kirk E. Apt

Papers

Stable nuclear transformation of the diatom Phaeodactylum tricornutum.

Evolution of the phycobiliproteins.

The gene family encoding the fucoxanthin chlorophyll proteins from the brown alga Macrocystis pyrifera

Stable nuclear transformation of the diatom

The gamma subunit of R-phycoerythrin and its possible mode of transport into the plastid of red algae