Fuel Processing Technology 86 (2005) 1137 1147

www.elsevier.com/locate/fuproc

Modifying soybean oil for enhanced performance in

biodiesel blends
A.J. Kinneya,*, T.E. Clementeb
a
DuPont Experimental Station, Wilmington, DE 19880-0402, USA
b
Plant Science Initiative, Center for Biotechnology, Department of Agronomy and Horticulture,
University of Nebraska-Lincoln, Lincoln, NE 68588-0665, USA

Abstract

Soybean (Glycine max Merr.) oil is primarily composed of five fatty acids; palmitic acid
(~13%), stearic acid (~4%), oleic acid (~18%), linoleic acid (~55%) and linolenic acid (~10%). The
average U.S. production of soybean oil from 1993 to 1995 was 6.8 billion kg and in 2002 soybeans
were harvested from more than 30 million ha across the U.S., which accounts for 40% of the total
world soybean output. This production capacity accounts for more than 50% of the total available
biobased oil for industrial applications. A useful industrial application of soybean oil is in biodiesel
blends. On a liquid basis, the total soybean oil production capacity would be equivalent to 1.9
billion gal of diesel, about 6.9% of the diesel fuel consumed in the United States for transportation
in 1996. A number of positive attributes are realized with the use of soybean oil-derived biodiesel,
including enhanced biodegradation, increased flashpoint, reduced toxicity, lower emissions and
increased lubricity. However, the two parameters that have limited usefulness of a soybean oil-
derived biodiesel as a fuel are oxidative instability and cold flow in northern climates. The latter is
not an issue in warmer environments, and thus soybean oil modifications designed to maximize
engine performance should be targeted with marketplace locale considerations in mind.
Implementing the tools of biotechnology to modify the fatty acid profile of soybean for locale
performance enhancement may increase the attractiveness of biodiesel derived from this
commodity crop.
D 2004 Elsevier B.V. All rights reserved.

* Corresponding author.
E-mail address: anthony.kinney@usa.dupont.com (A.J. Kinney).

0378-3820/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.fuproc.2004.11.008
1138 A.J. Kinney, T.E. Clemente / Fuel Processing Technology 86 (2005) 11371147

1. Introduction

Biodiesel is currently used in primarily three markets, mass transit, marine industry and
on farm. Biodiesel is made from vegetable and animal oils. These oils typically consist of
long-chain fatty acids, predominantly 16 and 18 carbons in length, esterified to
triacylglycerol. In theory, these oils can be used directly as fuel without any prior
treatment. However, because of their high viscosity, this practice can lead to engine
degradation, and hence most vegetable and animal oils are usually transesterified to free
alkyl monoesters for biodiesel use. It is thought that oils composed of triglycerides
harboring shorter chain fatty acids may circumvent the need for transesterfication, thereby
reducing cost. Short-chain saturated fatty acids, however, are only found in their seed oils of
a few, relatively exotic plant species, such as those within the genus Cuphea [18]. Geller et
al [16] monitored the fuel performance in a rapid screening procedure of short-chain
triglycerides using simulated mixtures of triglycerides containing approximately 40%
caprylic acid and 37% capric acid on the triglyceride backbone, which closely reflected the
oil profile of one such Cuphea species, Cuphea wrightii. The data revealed similar
performance measures to that of no. 2 petroleum diesel suggesting that if such commodity
oil was available, it could be used without transesterification. However, since the melting
point of biodiesel derived from these short-chain saturated fatty acids is fairly high,
additional winterization steps would be required to improve cold flow properties [16].
Biodiesel offers a number of advantages over standard fossil fuels, such as enhanced
biodegradation, increased flashpoint, reduced toxicity, lower emissions and increased
lubricity. Of these, the acceleration of degradation of biodiesel is the most attractive
property for environmentally sensitive areas, such as aquatic habitats. For example, Zhang
et al. [46] addressed the fate of various biodiesel formulations and found that degradation
of all formulations tested was significantly faster when compared with 2-D low-sulfur
petroleum diesel fuel. Moreover, in blended formulations, the degradation data led to the
conclusion that the biodiesel component actually promotes breakdown of petroleum
diesel [46].
Flashpoint is the temperature at which the fuels vapor will ignite. This parameter
reflects a fuels safety, the higher the flashpoint the less likelihood the fuel will
accidentally ignite. Number 2 diesel fuel typically has a flashpoint of 71 8C, while
biodiesel blends generally possess greater flashpoint values (N100 8C) [17].
Reduced toxicity of biodiesel per se and the derived emissions are both especially
important when engines are being run in confined environments, such as in the mining
industry. Bagley et al. [2] monitored exhaust from soybean oil-derived biodiesel in engines
coupled with an oxidation catalytic converter. The data revealed a reduced content of
mutagenic molecules in the exhaust compared with fossil fuel. However, the authors did
observe an increase in the soluble organic fraction within the biodiesel exhaust, thus
requiring the use of the oxidation catalytic converter when engines are being used in
confined spaces [2]. Mutagenic capacity of a diesel bus engine exhaust monitored by
implementing a bioassay demonstrated significant reduction in mutagen emission rates of
biodiesel fuels derived from a variety of feedstocks, including soybean [21]. Thus, there is
a correlation between reduction in known chemical mutagens in exhaust fumes [2] with
lessened mutagenic rates in a biological assay [21].
 A.J. Kinney, T.E. Clemente / Fuel Processing Technology 86 (2005) 11371147 1139

Another fuel property that neat biodiesel, or blends with petroleum diesel, tends to
enhance is lubricity. The lubricity of a fuel will ultimately impact engine wear and the
limited lubricity of sulfur-free petroleum diesel fuels can accelerate engine deterioration.
Drown et al. [11] evaluated a series of transesterified vegetable oil feedstocks as biodiesel
additives for their impact on lubricity. All of these feedstocks significantly reduced engine
wear compared with neat petroleum diesel and most showed a similar improvement in
lubricity. The exception was castor oil, which had an even better lubricity than the other
plant oils, including soybean, rapeseed, and coconut [11].
Although the above attributes of biodiesel have drawn attention to this renewable
resource as an alternative fuel, there exist some significant drawbacks, in addition to cost,
that have limited its commercial application. These include the oxidative instability of
biodiesel derived from vegetable oils, a significant high viscosity of unblended biodiesel
and reduced cold flow properties when compared with petroleum diesel. The limitations of
biodiesel are further compounded by the inverse relationship between cold flow and
oxidative stability [7]. Alterations in the fatty acid profile that increase the saturated fatty
acid content will augment oxidative stability but worsen cold flow. Moreover, as biodiesel
oxidizes, it becomes more viscous which can result in gumming of the fuel. The cetane
number of a fuel reflects on the time of ignition, the lower the cetane value, the longer
ignition delay time. The presence of double bonds in fatty acids will lower the cetane
value; hence, strategies to shift the fatty pool of a vegetable oil towards saturated moieties
will improve ignition quality of the derived biodiesel, but as with oxidative stability may
compromise cold flow properties [27].
Given the inverse relation between cold flow and oxidative stability (and cetane
number), it may be difficult to design an optimal fuel for all environments. However,
information acquired using simulated mixtures of oil can provide insight for the ideal
fatty acid composition for fuel. With the available tools of biotechnology, it is now
possible to translate these data on simulated fatty acids mixtures into targeted oil
modification in commodity crops. The development of modified oils that possess
enhanced performance in biodiesel blends can ultimately impact the economy,
environment and augment energy supplies. This in turn may heighten the allure of
biodiesel, providing opportunities for market expansion of commodity oils and job
creation in rural agriculture sectors.
There are a number of potential feedstocks available for use in biodiesel. These
include vegetable oils such as soybean, corn, sunflower, cottonseed, peanuts, canola and
rapeseed, in addition to yellow grease and recycled restaurant oils. Moreover, a number
animal-derived fats, tallow (cow) and lard (swine), can also be used. Expanding the use
and production of a feedstock for biodiesel must be evaluated in terms of the
environmental impacts of broadening production of the feedstock. To this end, soybean
is one of the few oilseed crops that acquire nitrogen through biological nitrogen fixation.
Hence, the energetics of producing soybean biobased fuels and other industrial
compounds is highly favorable when compared with other crops. Furthermore, soybean
accounts for the majority of the available potential feedstock for use for industrial
applications. Approximately 3.6% of the U.S. soybean oil production is targeted for
industrial applications (approximately 288 million kg), of which 1% (4.5 million kg) is
used for biodiesel (http://www.unitedsoybean.org). Soybean oil-derived biodiesel is
1140 A.J. Kinney, T.E. Clemente / Fuel Processing Technology 86 (2005) 11371147

commonly generated by transesterification of the extracted oil in the presence of a

catalyst. The derived methyl or ethyl esters can be used in neat form (100%) or blended
with petroleum diesel and used in diesel engines without modification of the engine
design. A recent overview of the various feedstocks, corresponding fuel characteristics
and targeted oil modifications of vegetable biodiesel [12], discusses potential oil
modifications directed at generating two feedstocks. The first, an oil profile with
elevated oleic acid (C18:1) coupled with reduced polyunsaturated and saturated fatty
acids, would possess increased oxidative stability and be useful under most environ-
mental conditions. The second oil suggested by the authors would couple elevated oleic
acid with an increase in stearic acid (C18:0), thus targeting enhance fuel performance,
while compromising cold flow. A fuel with such a oil profile would be more suitable in
warmer climates [12].
Designing specialty oils in soybean for targeted usage in biodiesel is facilitated by well-
developed genetic engineering methodologies for soy, as well as the wealth of genomics
and biochemical information pertaining to oil biosynthesis in oilseed plants. Herein, we
will provide an overview of the approaches for modifying the oil metabolism of soy seeds
that could lead to the production of designer soybean oil-derived biodiesel. We will focus
the discussion on the oil modifications suggested by Duffield et al. [12] and, in addition,
we will outline a potential strategy to further improve upon the lubricity of soybean oil-
derived biodiesel.

2. Genetic engineering of soybean

The ability to introduce foreign genes into soybean was first demonstrated in the mid-
1980s [20,32]. Transformation protocols for soybean rely on either Agrobacterium
tumefaciens or microprojectile bombardment coupled with organogenic or embryogenic in
vitro regeneration systems. Recent advances in genetic engineering of soybean have been
reviewed by others [35,42]. This section will highlight two methods currently used to
deliver transgenes in to the crop, Agrobacterium-mediated transformation using cotyledon
explants [10,20,34,47] and microprojectile-mediated transformation using somatic
embryos as the target for transgene introduction [13,39,41].
The axillary meristem at hypocotyl/cotyledon junction in young soybean seedlings is
the target for Agrobacterium-mediated delivery with the cotyledon explant tissue. A
number of selection systems have been effectively employed to identify transgenic
plants during in vitro culture using this system, including herbicidal molecules,
glyphosate [9] and glufosinate [47] in addition to the antibiotic hygromycin [34]. This
protocol requires multiple steps, explant preparation from young seedlings, inoculation
and co-cultivation with A. tumefaciens, followed by three in vitro regeneration steps,
shoot initiation, shoot elongation and root initiation. Rooted plantlets are subsequently
acclimated to soil and matured under greenhouse conditions. Typically, the time frame
from Agrobacterium-mediated DNA delivery to establishing a transformant in the
greenhouse is 35 months.
Another reliable means to introduce transgenes in to soybean is via microprojectile-
mediated [26] DNA delivery to somatic embryos. Somatic embryos undergo the same
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anatomical stages of zygotic embryos, except they are induced from somatic cells from
immature cotyledon segments. As with the organogenic in vitro culture regime,
embryogenesis requires a multi-step process. Embryos are induced by exposure to high
levels of auxin; once induced, the embryogenesis proceeds by reducing the exogenous
auxin levels. Once the cotyledonary stage of embryogenesis has been reached, maturation
and desiccation steps are carried out. Conversion to plantlets proceeds following
imbibition of the mature, desiccated somatic embryo. The most widely used selection
agent for this system is the antibiotic hygromycin. With this system, the time frame from
DNA delivery into somatic embryo culture to recovery of a transformant in the greenhouse
ranges from 6 to 9 months. A major advantage of the soybean somatic embryo system is
the fact that protein and oil deposition during somatic embryogenesis mirrors that of the
soybean zygotic embryogenesis [31]. This permits rapid monitoring of the metabolic
perturbations imparted by transgene(s) expression, without waiting for whole plant
development.
When considering strategies to modulate oil composition, it is imperative to ensure the
fatty acid profile of non-seed (or vegetative) tissue is not altered since this may lead to
developmental abnormalities, which in turn will have a negative impact on agronomic
performance. Employing a genetic engineering approach for fatty acid modification in
plants permits metabolic perturbations in a cell-specific manner. Moreover, with genetic
engineering approaches the inheritance of the resultant phenotype will behave as a single
dominant allele, which facilitates breeding.
Expression of transgene(s), downregulation of endogenous gene(s) or a combination
thereof can be used as a means to alter metabolic pathways in cells. Ectopic expression
of a transgene usually involves the placing of the coding sequence into a proper genetic
context to permit the desired transcriptional regulation. However, in some cases it may
be necessary to optimize codon usage to maximize translation efficiency [36].
Downregulating endogenous gene expression in plant cells is typically carried out by
inducing post-transcriptional gene silencing (PTGS) [8,44]. PTGS can be induced via a
number of strategies, all of which involve the introduction into the plant cell all or part
of a gene homologous to the endogenous gene targeted for silencing. The introduced
gene may be in a sense, antisense or inverted repeat (RNAi) configuration. Coupling
these strategies with tissue-specific promoter elements, for example, soybean h-
conglycinin [15] or common bean h-phaseolin [14], permits targeted perturbation of
fatty acid biosynthesis in the seed, without changing the lipid composition of root or leaf
tissues.

3. Enhancing oxidative stability of soybean oil

Commodity soybean oil has a relatively high oxidative reactivity and this compromises
the storage life of soybean oil-derived biodiesel. Fuel oxidation is usually manifested as
the formation of gums and sediment in the fuel which in turn can clog the fuel filter [7,33].
This high oxidative instability is due to polyunsaturated fatty acid content of soybean oil.
Reducing the relative abundance of linoleic and linolenic acids has a profound impact on
oxidative reactivity of the oil. In developing seeds the biosynthesis of the majority of
1142 A.J. Kinney, T.E. Clemente / Fuel Processing Technology 86 (2005) 11371147

polyunsaturated fatty acids channels through a seed-specific D12 desaturase designated

Fad2-1 [19,22]. In theory, downregulation of Fad2-1 should block the flux from oleic acid
into polyunsaturated fatty acids, thus enhancing the monounsaturated fatty acid content at
the expense of oxidatively reactive polyunsaturated fatty acids. This concept was first
tested in the soybean somatic embryogenic system by introducing a plus sense copy of the
soybean Fad2-1 gene under the control of the seed-specific promoter h-conglycinin [15] in
order to trigger PTGS [30]. The data revealed a drastic reduction in polyunsaturated fatty
acids and a concomitant enhancement in oleic acid in the soybean somatic embryos
[24,31]. Development of a high oleic acid transgenic soybean line followed, using a
similar sense Fad2-1 cassette introduced into an elite soybean genotype, A2396, via
microprojectile bombardment [22,25]. Three breeding populations were generated, derived
from one initial event designated 260-05, which consistently displayed elevated oleic acid
content ranging from 8488% across multiple environmental conditions [25]. Importantly,
yield trials on the high oleic populations revealed no agronomic penalty associated with
the novel trait.
Mutational breeding approaches have demonstrated that decreasing the saturated fatty
acid content of soybean oil also results in an elevation of the oleic acid content [5,38].
Using the transgenic soybean somatic embryo model it was determined that the pools of
16 and some 18 carbon-length acyl-ACPs synthesized in the plastid and subsequently
exported to the cytoplasm are substrates of a palmitoyl-ACP thioesterase, encoded by the
FatB gene [23,24]. Downregulation of FatB resulted in a reduced level of palmitate and
elevated oleate phenotype [24]. It is noteworthy that the high oleic acid phenotype
imparted by silencing Fad2-1 also generates a reduction in palmitic acid. This is probably
due to the enhanced availability of oleoyl-CoA vs. palmitoyl-CoA for the acyltransferase
reactions, resulting in less saturates on the glycerol backbone. These observations led to
the design of soybean transformation experiments targeting dual downregulation of both
the FatB and Fad2-1 genes in a seed-specific manner. Soybean transformants in which
these genes were both silenced had an oleic acid content of up to 91% and palmitic acid
contents as low as 2.2% in the seed storage lipids [6]. Populations derived from one of
these events designated 335-13 have been evaluated under field conditions across two
environments, in Puerto Rico and Nebraska. The fatty acid profile observed across the
locations among the populations derived from 335-13 is 4.1% palmitic acid, 2.3% stearic
acid, 86.4% oleic acid, 2,7% linoleic acid and 3.5% linolenic acid. Preliminary yield trials
conducted on this material, under irrigated and non-irrigated conditions, in Nebraska
revealed no significant changes from the parental line, Asgrow A3237, under the watering
regimes tested (Clemente unpublished).
Standard oxidation measurements demonstrate that high oleic and high oleic, low
palmitic oils are substantially more oxidatively stable than commodity soybean oils [25].
In preliminary engine emission tests, this increased stability results in substantially
reduced NOx emissions from high oleic oils when compared with standard soy biodiesel
(Kinney and Clemente, unpublished results).
Duffield et al. [12] suggested an oil with elevated oleic acid combined with increased
stearic acid as a means to couple oxidative stability with increased cetane number of the
derived fuel. Soybean oil with such a profile would provide a useful fuel in warmer rather
than cooler climates because of the decreased cold flow associated with an increased
 A.J. Kinney, T.E. Clemente / Fuel Processing Technology 86 (2005) 11371147 1143

saturated fatty acid content. Mutational breeding has been successful in generating
soybean germplasm with elevated stearic acid content (20%) [37]. Moreover, genetic
engineering approaches have also been successful in increasing stearic acid content of the
seed storage lipids in both canola [28] and soybean [24] by seedspecifically
downregulating a stearoyl-ACP desaturase. This enzyme carries out the first desaturation
step in the fatty acid pathway, stearic acid to oleic acid. A reduction in enzymatic activity
of this protein reduces the carbon flow through the desaturation steps of fatty acid
biosynthesis, thereby increasing stearic acid. Soybean germplasm with increased stearic
acid content was sexually crossed to the Fad2-1 downregulated high oleic acid germplasm,
the resultant progeny produced an oil with stearic acid and oleic acid at 30% and 60%,
respectively [22]. Such oils have not been tested yet in biodiesel applications.

4. Combining oxidative stability with enhanced lubricity

A number of plant species are capable of producing hydroxy fatty acids [43], with the
most extensively studied being ricinoleic acid. Castor bean (Ricinus communis)
accumulates significant levels of ricinoleic acid in the seed. Castor oil has been used
for numerous industrial products including plastics, foams, surfactants, cosmetics and
lubricants. Castor bean, like other plant species in which hydroxy fatty acids are found, has
not been fully domesticated. The meal of castor contains some very highly toxic
compounds, including ricin, and the yield potential of many genotypes being grown for
industrial uses is severely limited. These attributes make the cost-effective and reliable
production of this renewable oil problematic.
Ricinoleic acid is produced from oleic acid via an oleate 12-hydroxylase [1]. Oleate 12-
hydroxylase genes have been cloned from castor bean [29,43] and Lesquerella fendleri
[3]. Seed-specific expression of the castor-bean oleate 12-hydroxylase gene in Arabidopsis
thaliana resulted in ricinoleic acid accumulation of up to 7.8% in seeds [4]. Interestingly,
expression of the castor-bean oleate 12-hydroxylase in A. thaliana led to a concomitant
enhancement in oleic acid in the seed oil, from 14.7% to 24.1% [4]. This observation was
probably not caused by PTGS of the Fad2 gene, even though the A. thaliana Fad2 gene
and castor-bean oleate 12-hydroxylase gene share a relatively high degree of homology.
RNA transcript levels of Fad 2 were not altered in the oleic acid elevated lines expressing
the castor-bean oleate 12-hydroxylase, the increased oleate was probably the result of
altered oleate metabolism [4]. Introducing the castor-bean oleate 12-hydroxylase into a
Fad2 mutant (deficient in D12 desaturase activity, therefore elevated in oleic acid content)
of Arabidopsis led to an approximate threefold increase in ricinoleic acid content over the
comparable control [4]. This last observation is presumably due to increased availability of
substrates for hydroxylase action as a result of the lack of competitive interference by Fad2
desaturase activity.
With these data in mind, we are now poised to deliver the combined benefits of
enhanced oxidative stability and lubricity of high oleic acid and ricinoleic acid,
respectively, in soybean. To this end, designing a soybean oil with 1020% ricinoleic
acid coupled with 7080% oleic acid is a reasonable target for an enhanced performance
biodiesel fuel. Experiments are currently in progress to generate soybeans with such an
1144 A.J. Kinney, T.E. Clemente / Fuel Processing Technology 86 (2005) 11371147

oil profile. These soybean transformation studies mirror those that were conducted with
the model plant A. thalania [4]. We have fused the castor-bean oleate 12-hydroxylase
gene to the seed-specific h-phaseolin promoter [14] and the resultant plant gene
expression cassette was subcloned into a two T-DNA binary vector. This is a means to
recover transgenic soybean plants that only carry the gene of interest (oleate 12-
hydroxylase) and not the plant selectable marker gene [40,45]. Transgenic soybean
plants have been established in the greenhouse that carry castor-bean oleate 12-
hydroxylase gene. Once seeds are collected, the fatty acid profile will be monitored on a
portion of the cotyledon, while the remaining section of the seed, with embryonic axis,
will be planted. Those individuals in which ricinoleic acid production is observed will
be either self-pollinated or crossed to the high oleic acid/low palmitic acid population
derived from line 335-13 described above. The resulting population should yield beans
with an hydroxy-oleic oil.

5. Concluding remarks

Approximately 50% of the trade deficit of the United States is due to the import of
crude oil and fluctuations on crude oil prices have a direct effect on the U.S. economy.
Taking into the account military and foreign aid required to ensure an uninterrupted flow
of oil from the world reserves of petroleum, the cost per barrel of crude oil can be as
high as $100 (United States General Accounting Office). In 2000 the United States
produced 5 million gal of biodiesel fuel (National Biodiesel Board). This represents less
than 3% of the potential feedstock capacity of soybean. In the State of Minnesota, 550
million gal of petroleum diesel was consumed for on-highway use in 1999. An
economic analysis conducted by the Minnesota Department of Agriculture revealed that
if soybean oil-derived biodiesel was used at either 2% or 5% blend with petroleum
diesel, this in turn would generate a significant economic stimulus, including job
creation in rural areas.
Avenues to increase utilization of renewable energy resources have attracted significant
attention. Soybean oil represents the most widely available feedstock for biodiesel.
Soybean production can limit fertilizer-derived nitrogen runoff due to its nitrogen fixing
capacity. Moreover, recent improvements in soybean weed management through the
implementation of RoundUp Ready soybean permits low till practices, thereby reducing
soil erosion. These cropping system attributes enhance the value of soybean as a feedstock
for biodiesel. The major drawback associated with soybean oil, oxidative instability, has
limited its use in this regard. The enabling technologies developed through investments in
genomics and biotechnology have allowed for the direct perturbation of oil metabolism in
soybean that has led to the development of novel germplasm with significantly improved
oil profiles. In collaboration with Iowa States Biomass Energy Conversion Center,
soybean oil produced from populations derived from the 335-13 event, described above,
with an oil profile high in oleic acid and low palmitic acid, are currently being evaluated as
a biodiesel fuel. These types of interdisciplinary research endeavors will expedite progress
in development of a reliable and high-performance soybean oil for use as a renewable
biodiesel fuel across a range of environmental conditions.
 A.J. Kinney, T.E. Clemente / Fuel Processing Technology 86 (2005) 11371147 1145

Acknowledgements

This is journal series paper No. 14735 from the Nebraska Agriculture Research
Division.

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