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J Resource Requirements for Production of Microbial Biomass P roducing biodiesel or other fuels from algae would require large-scale pro- duction of algae. This appendix discusses the resource requirements for pro- ducing algal feedstocks for production of transportation fuel. PRODUCTION SYSTEMS Two primary types of systems have been developed for large-scale cultivation of photosynthetic microorganisms: open systems (for example, ponds and “race- ways”) and closed systems (often referred to as photobioreactors). Open Production Systems Open production systems have been used successfully for many years for the com- mercial production of algae and cyanobacteria for the nutraceutical industry and have been incorporated into various fish-farming operations and wastewater-treat- ment facilities. The open systems use “low technology” and typically have an oval raceway configuration with a paddlewheel that mixes the culture. Data are avail- able from numerous sources regarding the productivity of these systems, which tends to fall in the range of 25–35 g/m2 per day during periods of maximum pro- ductivity (Sheehan et al., 1998; Lee, 2001; Huntley and Redalje, 2007). Simpler designs have been used for beta-carotene production; these designs feature large wind-mixed ponds and have rather low productivity. The primary advantages of open pond systems are lower capital and operat- ing costs. The main disadvantages of open systems are poor control of culture 

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0 Liquid Transportation Fuels from Coal and Biomass conditions (for example, higher susceptibility to contamination by undesired algae and predators, dilution by rain, and fouling by windborne dust and debris), high evaporative water loss, requirement for large expanses of level terrain, and high regulatory hurdles with respect to containment of recombinant strains. Closed Production Systems Different kinds of closed photobioreactor systems have been designed and tested. Generally, these systems are in two categories: tubular systems made of rigid or flexible plastic, and flat plate or annular reactors made of rigid materials and typically placed at upright angles to maximize use of light by the cultures. Pho- tobioreactor design is a subject of active research in several algal-biotechnology companies. Because of high capital costs associated with rigid plastics, many of the designs being pursued are focused on tubes manufactured from flexible films. Some press releases have reported the achievement of productivity as high as 170 g/m2 per day in novel photobioreactors (for example, GreenFuel Technologies Corporation, 2007), but it will be important to increase understanding of how the calculations were conducted to ensure valid comparisons between the various systems. The primary advantages of closed photobioreactors are a higher degree of control over some culture conditions (for example, protection from the elements, less water evaporation and outgassing of carbon dioxide [CO2], and delayed onset of contamination by undesired species and predators), potentially higher productivity as a result of improved use of light, and containment of recombinant strains. The overriding disadvantage of closed photobioreactors is the high capital cost associated with the construction materials, circulation pumps, and nutrient- loading systems. There are other disadvantages: • Fouling of interior surfaces and difficulty of cleaning them. • Accumulation of high concentrations of photosynthetically generated oxygen, which leads to photooxidative cell damage. • Absence of evaporative cooling, which can lead to very high temperatures. Comparison of the Two Types of Systems Both types of systems have inherent advantages and disadvantages. It is highly unlikely that one standard system will be applicable for all strains, products, or

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Appendix J  sites, and research is being conducted on various designs. A combination of closed and open systems will probably be used in many cases—enclosed bioreactors for inoculum generation and open ponds as final production units. To reduce the volume of water handled during cell harvesting, a flocculent (such as alum or various ionic polymers) is typically added to the cells to facilitate their concentration in a settling tank; the biomass is concentrated further with continuous centrifugation, which is an expensive process because of the capital and operating costs. For some filamentous strains, strainers or filters can be used to collect the cells. Clearly, additional research and development to improve the biomass-harvesting process will have a great effect on production costs. Harvesting of Algae from Natural Bodies of Water Harvesting of naturally occurring algae would eliminate the need for a photobio- reactor, but harvesting appreciable amounts of biomass would require filtering large quantities of water and extensive operating-expense outlays. In addition, environmental groups strongly resist this approach because of potential unpredict- able environmental consequences of ocean and lake fertilization. STRAINS OF MICROORGANISMS FOR BIOMASS PRODUCTION Naturally Occurring Strains Cultivation of the dominant strains of photosynthetic organisms in the locale of the installed system might be the easiest way to maximize the productivity of the system. However, those strains might not be optimal for biofuel production. Most fuels under development require the use of microorganisms that have high lipid content, which might not be an attribute of random local strains. Many research- ers in the field therefore believe that commercial production strains will be initially selected on the basis of superior product formation and processing attributes and then developed through dedicated strain-improvement programs. Genetically Modified Strains Various information and tools are available for the genetic modification of pho- tosynthetic microorganisms, including whole genome sequences, systems for gene introduction, and protocols for random and directed mutagenesis.

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 Liquid Transportation Fuels from Coal and Biomass Genome-Sequence Information Genome-sequence information is extremely useful for developing metabolic engi- neering strategies, including pathway modeling, gene-knockout strategies, and expression vector construction. Complete genome sequences are available for 11 eukaryotic microalgal species and more than 20 cyanobacterial strains. Additional genome-sequencing projects are under way. Genetic-Engineering Tools for Cyanobacteria The materials and methods available for genetic modification of cyanobacteria are substantially more advanced than those available for eukaryotic microalgae. DNA can be introduced into cyanobacteria via natural transformation, electro- poration, or conjugation, but different strains require different methods. In some transformation systems, transgenes are included on replicating plasmids; in other cases, the foreign DNA becomes integrated at specific locations in the genome via homologous recombination. A variety of selectable marker genes have been suc- cessfully used to enable introduction of multiple foreign genes or inactivation of endogenous genes in separate steps. In addition to site-specific gene inactivation by double-crossover homologous recombination, random mutations can be gener- ated in some species by transposon insertion or by chemical- or radiation-medi- ated mutagenesis. Genetic-Engineering Tools for Eukaryotic Microalgae Genetic engineering has been reported for a few microalgal species, including green algae, diatoms, red algae, and dinoflagellates. The limited success can be attributed in part to the small number of laboratories working in this field. In some cases, nuclear transformation was achieved, typically via random integra- tion of the entire delivery vector into one or more chromosomes and sometimes in the form of tandem repeats. In other cases, transgenes have been successfully targeted to the chloroplast genome by the use of vectors that contain flanking regions of DNA identical with sequences found in the chloroplast. DNA introduc- tion can be accomplished via particle bombardment, electroporation, or agitation with abrasive materials. A number of selectable markers have been used for vari- ous microalgae, including several antibiotic-resistance genes and native genes used to complement some mutations. Mutation of nuclear genes is currently limited to classical chemical- or radiation-mediated random mutation, which can be difficult with diploid organisms, such as diatoms.

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Appendix J  Application of Genetic Tools to Production of Liquid Biofuel There have been few efforts to use genetic-engineering tools to enhance biofuel production by photosynthetic microorganisms. In one example, the introduction of pyruvate decarboxylase and alcohol dehydrogenase genes from Zymomonas into the cyanobacteria Synechococcus and Synechocystis resulted in the produc- tion of small quantities of ethanol in the strains (Deng and Coleman, 1999; Fu and Dexter, 2009). Attempts have been made to enhance lipid production in the diatom Cyclotella cryptica by overexpressing the native acetyl-CoA carboxylase gene, but little effect was observed (Sheehan et al., 1998). A number of labora- tories and companies have initiated programs to enhance biofuel production by photosynthetic microorganisms via metabolic engineering. A key goal will be to develop strains that produce large quantities of storage lipids even during periods of rapid cell division. OTHER REQUIREMENTS FOR PRODUCTION OF MICROBIAL BIOMASS Land High productivity of algal or cyanobacterial cultures depends on high levels of solar radiation and an extended growing season (that is, more days with tempera- tures conducive to rapid culture growth). But use of land that cannot readily be used for production of food or feed crops provides cost and social advantages. Consequently, the desert regions of the southwestern United States have histori- cally been considered the preferred site for implementation of large-scale produc- tion systems. The culture depth of large-scale open-pond systems is typically only 20–30 cm, so precise leveling of the ground is necessary during pond construction. Because level land is needed, many regions in the United States are not suitable as production sites. Level terrain is not as important for some photobioreactor systems, however, because the growth modules tend to be less dependent on level ground and in some cases can actually benefit from the gravitational potential energy inherent in sloped land.

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 Liquid Transportation Fuels from Coal and Biomass Water Some strains of microalgae and cyanobacteria are able to grow in a wide variety of water types, including freshwater, saline water, brackish water, and alkaline water. Large quantities of saline groundwater that are available in the southwest- ern United States could be used to support the mass culture of photosynthetic microorganisms; saline water is unsuitable for crop irrigation or consumption by humans or livestock, so use of this water largely eliminates “food versus fuel” concerns that have been raised for some crop-based biofuels. It will be important to ensure that withdrawal of water from saline aquifers does not interfere with the hydrodynamics of freshwater aquifers and that the aquifers are shallow enough to avoid prohibitive pumping costs. For open-pond systems, it might be necessary to have access to freshwater for dilution of the culture medium when it becomes too saline because of evaporative water loss. Another potential option for production facilities in coastal areas is a seawater-based culture medium. This option is probably more viable for foreign countries because much of the United States is not suitable. If cost-effective pipe- lines can be constructed, the number of suitable facility sites would probably increase. Recycling of nutrients and the eventual return of spent water to the ocean would probably be necessary for seawater-based production systems and would require review for regulatory compliance. Carbon Dioxide Large quantities of CO2 required for biofuel production via photosynthetic pro- cesses have to be delivered to the production facility in a concentrated form. The two largest sources of CO2 that could be tapped are coal-fired and gas-fired electric-power plants and oil wells that have been flooded with CO2 as part of previous enhanced oil-recovery efforts (Feinberg and Karpuk, 1990). Other poten- tial sources are fermentation facilities (such as ethanol plants), cement factories, ammonia-production plants, and oil refineries. Those sources are not all equiva- lent in that CO2 is present at varied concentrations and the sources can contain different types of contaminating compounds. Purification and pressurization of the CO2 would be necessary to reduce transportation costs and reduce contami- nants (such as heavy metals, sulfur oxides, and nitrogen oxides) that can have an adverse effect on cell growth. The CO2 sources listed above are all point sources, so it would be highly advantageous to colocate biofuel-production facilities with CO2-generating plants.

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Appendix J  That will not always be possible because of unsuitable terrain or the lack of a sufficient water supply, so it will be necessary in some cases to transport concen- trated CO2 by pipeline or rail car to the production facility. Allowable transport distances will be dictated by process economics and existing market conditions for CO2 and fuel but are not expected to exceed a few hundred miles. Efforts to develop technology to enable cost-effective uptake and concentra- tion of CO2 from the atmosphere are under way. Success could have a substantial effect on the economics of biofuel production by photosynthetic microorgan- isms because colocation of CO2 and biofuel-production facilities would not be necessary. REFERENCES Deng, M.D., and J.R. Coleman. 1999. Ethanol synthesis by genetic engineering in cyano- bacteria. Applied and Environmental Microbiology 65:523-528. Feinberg, D., and M. Karpuk. 1990. CO2 Sources for Microalgae-based Liquid Fuel Production. Golden, Colo.: Solar Energy Research Institute and National Renewable Energy Laboratory. Fu, P., and J. Dexter, inventors. 2009. Methods and Compositions for Ethanol Producing Cyanobacteria. USPTO Application #: 2009015587. GreenFuel Technologies Corporation. 2007. Growth Rates of Emission-Fed Algae Show Viability of New Biomass Crop. GreenFuel Technologies Corporation Press Release. Available at http://www.greenfuelonline.com/gf_files/GreenFuel%20Growth%20Rates. pdf. Accessed April 21, 2008. Huntley, M.E., and D.G. Redalje. 2007. Global-scale CO2 mitigation and renewable energy from photosynthetic microbes: A new appraisal. Mitigation and Adaptation Strategies for Global Change 12:573-608. Lee, Y.K. 2001. Microalgal mass culture systems and methods: Their limitation and poten- tial. Journal of Applied Phycology 13:307-315. Sheehan, J., T. Dunahay, J. Benemann, and P. Roessler. 1998. A Look Back at the U.S. Department of Energy’s Aquatic Species Program: Biodiesel from Algae. Golden, Colo.: National Renewable Energy Laboratory.

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