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J
Resource Requirements for Production of Microbial Biomass
Producing biodiesel or other fuels from algae would require large-scale
production of algae. This appendix discusses the resource requirements for producing
algal feedstocks for production of transportation fuel.
PRODUCTION SYSTEMS
Two primary types of systems have been developed for large-scale cultivation of
photosynthetic microbes: open systems (for example, ponds and “raceways”) and closed
systems (often referred to as photobioreactors).
Open Production Systems
Open production systems have been used successfully for many years for the
commercial production of algae and cyanobacteria for the nutraceutical industry and have
been incorporated into various fish-farming operations and wastewater-treatment
facilities. The open systems use “low technology” and typically have an oval raceway
configuration with a paddlewheel that mixes the culture. Data are available 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 productivity (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 operating
costs. The main disadvantages of open systems are poor control of culture 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.
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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
plasti, and flat plate or annular reactors made of rigid materials and typically placed at
upright angles to maximize use of light by the cultures. Photobioreactor 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 systms.
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 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.
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Harvesting of Algae from Natural Bodies of Water
Harvesting of naturally occurring algae would eliminate the need for a
photobioreactor, 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 unpredictable
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 researchers 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
photosynthetic microorganisms, including whole genome sequences, systems for gene
introduction, and protocols for random and directed mutagenesis.
Genome-Sequence Information
Genome-sequence information is extremely useful for developing metabolic
engineering 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, electroporation, 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
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integrated at specific locations in the genome via homologous recombination. A variety
of selectable marker genes have been successfully 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 generated in some species by transposon insertion or by chemical- or
radiation-mediated 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 integration 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 introduction can be accomplished via particle bombardment,
electroporation, or agitation with abrasive materials. A number of selectable markers
have been used for various 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.
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 production of small
quantities of ethanol in the strains (Deng and Coleman, 1999; Fu and Dexter, 2007).
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 laboratories 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 temperatures
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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 historically been considered the
preferred site for implementation of large-scale production 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.
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 southwestern 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 vs 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 pipelines 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
processes 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 potential sources are fermentation
facilities (such as ethanol plants), cement factories, ammonia-production plants, and oil
refineries. Those sources are not all equivalent 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 contaminants (such as heavy metals, sulfur oxides, and nitrogen oxides)
that can have an adverse effect on cell growth.
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The CO2 sources listed above are all point sources, so it would be highly
advantageous to colocate biofuel-production facilities with CO2-generating plants. 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 concentrated 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 concentration of
CO2 from the atmosphere are under way. Success could have a substantial effect on the
economics of biofuel production by photosynthetic microorganisms because colocation
of CO2 and biofuel-production facilities would not be necessary.
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REFERENCES
Deng, M. D. and J. R. Coleman. 1999. Ethanol synthesis by genetic engineering in
cyanobacteria. Applied and Environmental Microbiology 65:523-528.
Feinberg, D. and M. Karpuk. 1990. CO2 sources for microalgae-based liquid fuel
production. Golden: Solar Energy Research Institute and National Renewable
Energy LaboratoryI.
Fu, P. and J. Dexter, inventors. 2007. Methods and compositions for ethanol producing
cyanobacteria.
GreenFuel Technologies Corporation. 2007. Growth Rates of Emission-Fed Algae Show
Viability of New Biomass Crop. GreenFuel Technologies Corporation Press
Release. Accessed on April 21, 2008 at
http://www.greenfuelonline.com/gf_files/GreenFuel%20Growth%20Rates.pdf
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:573-608.
Lee, Y.K. 2001. Microalgal mass culture systems and methods: Their limitation and
potential. Journal of Applied Phycology: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:
National Renewable Energy Laboratory.
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