SUPPLEMENTAL TEXT
S1
BACKGROUND
Worldwide, the availability of fossil feedstocks for fuel production is predicted to decrease, and there is continued
concern about the release of carbon dioxide from the use of fossil fuels.The use of biomass feedstocks for fuel
production is an intriguing alternative because of their greater price stability and the positive effects they offer in
mitigating environmental impacts such as climate change. Bioenergy challenges include

meeting the ambitious goals of the U.S. Energy Independence and Security Act of 2007

responding to uncertain economics and policies

reducing risk for deployment of novel technologies

feedstocks and products gaining social, political, and environmental acceptability from national and
international communities.
S1.1
Feedstocks
The potential feedstock resource base consists of a wide variety of components, including agricultural and forestry
resources, residues from industrial processes, and municipal solid and urban wood residues. First-generation
biofuels have typically used oil crops, animal fats, or sugar or starch from agricultural crops such as sugarcane and
corn. 70 Bioethanol, the most common first-generation biofuel, is produced by the fermentation of sugars derived
from starch (e.g., corn) or sugar (e.g. sugarcane) crops. Biodiesel produced from oil crops (soybeans) or fats is also
considered a first-generation biofuel. The next generation of biofuels is expected to use cellulosic biomass as the
feedstocks. The advantage of cellulosic feedstocks is their extensive spatial extent, lack of land competition with
food crops, and large potential supply. The near-term source of this biomass will likely include dedicated energy
crops, forest and agricultural residues, and municipal solid wastes. There is some longer-term potential for algal
feedstocks.
Energy crops (e.g. grasses or fast rotation woody species) are projected to constitute more of biofuel feedstocks
beyond 2022. Switchgrass (Panicum virgatum), a fast-growing perennial grass, is a dedicated energy crop that has
received increased attention in the past 5 years from the genomics community. Candidate genes identified through
these techniques are beginning to be validated as genetically controlling lignin content and composition, and they
may enable modification of the composition to facilitate subsequent conversion processes. 71 , 72 , 73 , 74 , 75 Similar
efforts are proceeding for other grasses like Miscanthus.
S1.2
Logistics and Land Use
A variety of land types are available for growing bioenergy feedstocks. First-generation biofuels derived from plant
starches, sugars, and oils are currently grown on cropland. However, concern over competing demands on cropland
for fuels, food, and feed production has highlighted the need to develop second-generation biofuels that do not rely
on feedstocks that displace food and feed production. Logistics components include planting, harvesting, storage,
transport, and size reduction operations.
S1.3
Conversion Technologies
A number of large-scale industrial processes are coming online for next-generation biofuels, using a variety of
approaches, including thermochemical and biochemical conversion. Among the biochemical conversion
approaches, enzymatic hydrolysis of plant biomass coupled with microbial fermentation remains the predominant
technology currently being deployed, either sequentially in a process referred to as “separate hydrolysis and
fermentation” or simultaneously in a process called SSF. While enzyme production represents a significant expense,
it allows for ethanol production by proven industrial organisms (e.g. Saccharomyces cerevisiae or Zymomonas
mobilis).Companies including Abengoa, Beta Renewables, DuPont, and POET have commercial facilities that are
expected to come online within the next year. The primary target product is currently ethanol.
Thermochemical conversion uses pressure, heat, and catalysts to convert biomass into a variety of fuels and
products. Direct liquefaction by pyrolysis and indirect liquefaction by gasification are the two types of processes
that can be used to convert food and nonfood biomass to fuels. 76 , 77 Gasification converts the biomass to syngas by
partial oxidation at high temperatures. Pyrolysis also creates pyrolysis oils or ‘biocrude’ from biomass but does so
at temperatures around 1000°C in the absence of oxygen.72
S1.4
Products
Products are both fuels produced from biomass feedstocks and high value co-products that can be economically
generated through a selected conversion technology. Fuels include currently manufactured products, such as
bioethanol and biodiesel, and new fuels, such as butanol and hydrocarbons. Examples of possible co-products are
fine chemicals and biopolymers that can serve as feedstocks for other products.
S1.5
Utilization
Products from the supply chain can be used in a variety of ways, including blended and stand-alone vehicle fuels,
fuels for next-generation engines, and, in the case of co-products, feedstocks for other supply chains that generate
additional products such as plastics.
S2
ALGAL FEEDSTOCKS
Selected species of microalgae may also serve as biofuel feedstocks; 78 for example,

Chlorella (ethanol)

Chlorococcum (ethanol)

Nannochloropsis (biodiesel)

Chlamydomonas (biohydrogen)

Botryococcus (jet fuel)

Spirulina (high protein)
Many challenges remain in making algal biofuels a reality. The use of brackish water is being pursued; however,
evaporation is expected to lead to hypersalinity unless facilities are located where the more-concentrated saline
water can be easily exchanged and replaced, as in a tidal region. Advanced biotechnology may be able to improve
the salt tolerance of algae, but the mechanisms of salt tolerance are not well understood; therefore, it would be
challenging to engineer rationally a salt-tolerant strain of algae within the next decade. Traditional biology, using
screening and evolution, may have a greater opportunity to improve algae to make them suitable for cultivation in
brackish water. The open ponds used for algal aquaculture have very high rates of evaporation, even relative to
other irrigated soils for crop systems. Most research targets the improvement of eukaryotic algae, especially those
that naturally accumulate significant quantities of lipids (>10 wt %) under stress. The bacterial ‘algae’ such as
cyanobacteria can also produce chemicals such as sugar, cellulose, hydrogen, or other fuels from sunlight and
CO2.These microbes may be more amenable to manipulation than the eukaryotic algae; however, there are robust
genetic tools for only a few of the cyanobacteria.
Recent advances suggest that a combination of metabolic engineering and process control can help alleviate these
algal challenges. Heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase II genes in
Synechococcus created a novel pathway to channel fixed CO2 toward synthesis of ethanol that diffused into the
culture medium. 79 It has been shown that sulfur deprivation can lead to increased photolysis of water or indirect
conversion from starch to biohydrogen. With nitrogen deprivation, an increased carbon flux to lipid biosynthesis
pathways has been shown. This positive impact on a pathway relevant to biodiesel synthesis can be more easily
controlled using advanced biotechnological tools—such as manipulating the expression of the malic enzyme, a key
regulator of oil content—than with traditional approaches to vary the nutrient content of growth media. 80 Although
most -omics, systemsbiology, and geneticmanipulation studies have used Chlamydomonas and Volvox as models,
the presence of multiple isoforms and organelles complicates the precise genetic engineering of eukaryotic algae for
enhanced lipid or carbohydrate content. The recent report identifying 80 independent mutants with altered fatty-acid
synthesis activity highlights the complexity of lipid metabolism. 81 An improved microRNA-mediated genesilencing technique (miRNA or RNAi) has been successfully demonstrated in C. reinhardtii. 82 , 83 In addition, the
use of GMO algae in open ponds is a matter of active debate and potential regulation because of the perceived high
potential for escape and survival of these algae outside the pond.
In any algal system, the primary goal of most research is to increase the production of a directly usable fuel;
however, in any of these systems, the residual algal biomass (cellulose and other polysaccharides in the cell walls)
will need to be converted by methods such as those described in this report for cellulosic biomass (see Section 3.6).
S3
LOGISTICS
The logistics (considered here to be harvest, storage, transport, and size-reduction operations) of sustainably
supplying such large volumes of biomass are challenging but are considered achievable with adequate investments
in research, development, demonstration, and deployment and with input from stakeholders across the supply chain
(e.g., biorefineries, biomass producers, neighbors, equipment manufacturers).4Such systems must address the
following unique challenges associated with biomass supply operations:

Loss of dry matter must be minimized and undesirable quality changes during storage must be avoided for
herbaceous biomass (e.g. agricultural residues, perennial grasses, energy sorghum) that is harvested
seasonally.

Planting and harvesting delays may occur as a result of inclement weather.

The low bulk density of biomass makes it more difficult and expensive to handle, transport, and store.

Distributed biomass sources require large supply areas and transportation distances.

The inherent variability of biomass between crops and within fields makes it difficult to design reliable,
consistent commercial-scale conversion processes.
Building a commercial biofuels industry capable of achieving US goals to offset fossil fuel consumption while
competing for a land base will require higher levels of biomass production, through both improved production and
biotechnology practices. Thus a major goal of feedstock biotechnology efforts is sustainably increasing yields of
dedicated bioenergy crops while minimizing nutrient inputs. Higher yields are generally advantageous in reducing
the costs of crop production and logistics. Sokhansanj et al. 84 showed that increasing switchgrass yield from 10 to
30 dry Mg/ha (4.5–13.4 ton/ac) decreased production costs by more than50%, from $41.50/Mg ($37.66/ton) to
$19.14/Mg ($17.37/ton).Similarly, although larger, more robust machinery may be needed for harvesting higheryielding crops, harvest and collection costs tend to decrease with increasing yield.8However, field studies have
shown that conventional forage harvest equipment (e.g., mowers, balers, and forage harvesters) is designed for
forage cropswith yields averaging 4.5 to 7.6 dry Mg/ha (2to 3.4 dry tons/ac) per year13and harvested two or three
times per year. It will need to be modified to operate efficiently in bioenergy crops optimistically projected to
achieve yields up to 45 dry Mg/ha (20 dry tons/ac) over the next 10 years ([based on 4% annual yield increases
considered by participants in a 2009 DOE-sponsored workshop to be possible with adequate research funding).5,8, 85
All else being equal, transportation costs also decrease with higher yields as the area required to supply a
conversion facility decreases.81
Increased feedstock yield (discussed above and in S2) will have a major impact on logistics. Since herbaceous
biomass is harvested seasonally and is stored for several months to supply conversion facilities operating yearround, strategies for avoiding biomass losses are critical.The economic tradeoffs between biomass value and the
storage facility costs are important parameters in designing biomass supply chains. One question yet to be explored
experimentally is the stability of low-lignin biomass during storage. Easier degradation of this material may justify
investment in storage facilities that offer more protection from the ambient environment, or it may be that some
degradation of biomass fractions is desirable if it makes sugars more readily available for conversion. For example,
Agrivida 86 is developing plants with specially designed enzymes that accelerate cell wall degradation following
harvest.
Lignin has been shown to play a role in plant shear strength. A study by Yu et al. 87 found that shear is the “weakest
mode of failure” and that grinders and harvesters designed to apply shear forces on biomass are much more energy
efficient. Based on this, and given that reduced-lignin plants tend to have lower shear resistance, it is hypothesized
that new lower-lignin bioenergy crops will require less energy for size reduction. As adequate quantities of
modified bioenergy crop material become available in field trials, experiments are needed to test the relationship
between lignin content and grinding energy.
For thermal processes such as burning and gasification, increasing density (mass per unit volume) and lignin
content of woody biomass is beneficial. Reversing the processes used to reduce lignin content and to improve the
digestibility of biomass can, theoretically, improve the thermal energy of wood. Hinchee et al. 88 estimate that a
25% to 35% increase in lignin content can increase the calorific value of wood by 1046 kJ/kg (450 BTU/lb).
To date, most assessments of biofuel feedstocks have focused on supplying quantities capable of achieving US
biofuel production goals at minimal cost. However, as the industry begins to emerge, there is a realization that the
quality of the feedstock is critical to maintaining a large-scale industry. The inherent variability of biomass is a
barrier to developing reliable conversion processes. Advanced biomass supply systems and practices are needed to
reduce the variability of biomass quality parameters such as ash (the residue following biomass conversion) and
moisture. 89 The content of ash is especially problematic, and genetic improvements to reduce ash content would be
particularly desirable. 90 Improved microbial robustness against inconsistent plant biomass will also be an important
component of reducing the impact of feedstock variability.
S4
LAND AND WATER USE
Biotechnology will have an indirect impact on land and water use, primarily in the areas of feedstocks, as described,
but also from more sustainable conversion processes. The current use of food and feed crops, namely corn, to
produce ethanol has created much concern over the competition between food, feed, and fuel for natural resources
such as land. Envisioning feedstock supply systems capable of meeting the demands of an expanded bioenergy
industry is troubling to some; they worry about producing adequate food supplies to meet the needs of the rapidly
growing population. However, if handled strategically, it has been shown that large-scale cellulosic biomass
production can coexist alongside food production systems by incorporating strategies such as double cropping. 91
Double-cropping strategies can be seasonal (i.e., planting a winter cover crop) or spatial (i.e., planting grasses
between the rows of a tree plantation). Analyses show that expanded use of dedicated perennial energy crops to
meet bioenergy demands provides sustainability benefits such as increased soil fertility and reduced greenhouse gas
emissions over annual crops.88, 92
The two most basic resources required for biomass production, whether for food, feed, or fuel, are land and water.
As the world’s population grows, so do the demands for food, feed, and fuel, which result in competition among
producers for land and water resources. The rapidly growing bioenergy industries offer new technologies and
practices that may improve the sustainability of biomass production. The IEA Task 43 Bioenergy committee 93
pointed out that crop selection and management decisions must be made at a watershed scale with careful
consideration of local weather and soil conditions. For example, analysis by Zhuang et al. 94 showed that
Miscanthus, which can serve as a substitute for corn in ethanol production, requires much less land and water than
corn.Of course, there are many other factors to be considered in selecting crops, including local markets,
infrastructure, and producer acceptability. However, this analysis shows that carefully selecting and transforming
crops for higher yields and lower water use is necessary for achieving sustainability goals.
Work is under way to produce crops that are suitable for soils with high salt concentrations. Some tree species in
particular (e.g., willow) appear to be able to tolerate saline conditions. Hangs et al.34 and Wicke et al.35 estimated
that up to 1.1 Gha of land worldwide are “salt-affected.”They concluded that producing trees selected or designed
for saline conditions on salt-affected forest lands could supply up to 8% of global primary energy consumption.
Further investigation is needed to optimize the performance of salt-tolerant trees in saline soils, to test their growth
in field conditions, and to test microbial bioconversion of the resulting plant biomass, but the preliminary results are
promising.
S5
POLICY ISSUES
Policy and economic issues will also affect biofuel production and use. Broad bioenergy policy issues include the
stability of the Renewable Fuel Standard (RFS) and other government policies that directly support the growing
biofuels industry. Uncertainty in the long-term stability of these policies influences investment in this area. The
recent increase in domestic natural gas production has increased pressure against policies such as the RFS.
Biotechnological advances do not directly influence these issues of policy stability, but they do offer hope to be
able to move to a point at which the policies are no longer needed because of economic improvements; however,
stability in these policies is seen as essential for the near-term deployment and for developers to gain access to the
capital funds (more than one hundred million dollars) required for a first-generation biorefinery. 95
Policies regarding GMOs and the public perception of them will have a more direct bearing on the use and
implementation of biotechnological solutions. The use of GMOs within the confines of a biorefinery has generally
been well accepted within the United States and even in Europe. Reasonable biocontainment is practiced, but
assessments are made of the viability of these GMO industrial strains if they were to escape. Generally, the viability
of the modified industrial strains is considered lower outside the biorefinery. While the risk must still be assessed
for each improved microbe, the use of GMOs in a biorefinery is not considered to be a barrier to adoption—unlike
the concerns for feedstocks and algae.
The use of GMO plants or GMO algae is a matter of active debate worldwide. The United States is generally
accepting of GMO plants after increased regulation and study. This can add several years to both their field testing
and the final deployment to a new biofeedstock plant. There are biotechnological advances that can assist these
determinations, such as pollen sterility to prevent gene spread.
GMO issues and the science behind understanding the potential escape of GMOs and competition with natural algae
are still being evaluated and researched. Most current algal bioenergy developers presume that GMOs will be
contained within closed photobioreactors and only non-GMO algae will be used in open systems.
Another important and active area of policy debate is the perceived competition between food and fuel. 96 , 97
Technically, this debate is covered under issues in land use change (LUC) and indirect land use change (ILUC).
Biotechnological advances will lower both LUC and ILUC, in particular by increasing yields and thus lowering
land requirements. However, ILUC is becoming part of policies in some areas of the world even though it is
difficult to define or measure.
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