Industrial uses of vegetable oils

Editor
Sevim Z. Erhan
Food and Industrial Oil Unit
National Center for Agricultural Utilization Research
Agricultural Research Service
United States Department of Agriculture
Peoria, IL 61804
Champaign, Illinois
Industrial Uses of
Vegetable Oils
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Copyright © 2005 AOCS Press
AOCS Mission Statement
To be the global forum for professionals interested in lipids and related materials
through the exchange of ideas, information science, and technology.
AOCS Books and Special Publications Committee
M. Mossoba, Chairperson, U.S. Food and Drug Administration, College Park, Maryland
R. Adlof, USDA, ARS, NCAUR, Peoria, Illinois
P. Dutta, Swedish University of Agricultural Sciences, Uppsala, Sweden
T. Foglia, ARS, USDA, ERRC, Wyndmoor, Pennsylvania
V. Huang, Abbott Labs, Columbus, Ohio
L. Johnson, Iowa State University, Ames, Iowa
H. Knapp, Deanconess Billings Clinic, Billings, Montana
D. Kodali, Global Agritech, Inc., Plymouth, Minnesota
T. McKeon, USDA, ARS, WRRC, Albany, California
R. Moreau, USDA, ARS, ERRC, Wyndoor, Pennsylvania
A. Sinclair, RMIT University, Melbourne, Victoria, Australia
P. White, Iowa State University, Ames, Iowa
R. Wilson, USDA, REE, ARS, NPS, CPPVS, Beltsville, Maryland
Copyright (c) 2005 by AOCS Press. All rights reserved. No part of this book may be reproduced
or transmitted in any form or by any means without written permission of the publisher.

The paper used in this book is acid-free and falls within the guidelines established to ensure
permanence and durability.
Library of Congress Cataloging-in-Publication Data
Industrial uses of vegetable oils / editor, Sevim Z. Erhan.
p. cm.
Includes index.
ISBN 1-893997-84-7
1. Vegetable oils Industrial applications. I. Erhan, Sevim Z.
TP680.I555 2005
665'.384 dc22 2005007927
Printed in the United States of America.
08 07 06 05 04 5 4 3 2 1
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Copyright © 2005 AOCS Press
iii
Preface
Vegetable oils are used in various industrial applications such as emulsifiers, lubri-
cants, plasticizers, surfactants, plastics, solvents and resins. Research and develop-
ment approaches take advantage of the natural properties of these oils. Vegetable
oils have superb environmental credentials, such as being inherently biodegradable,
having low ecotoxicity and low toxicity towards humans, being derived from
renewable resources, and contributing no volatile organic chemicals.
United States agriculture produces over 25 billion pounds of vegetable oils
annually. These domestic oils are extracted from the seeds of soybean, corn, cotton,
sunflower, flax, and rape. Although a major part of these oils are used for food prod-
ucts such as shortenings, salad and cooking oils and margarines, large quantities
serve feed and industrial applications. Other vegetable oils widely used industrially
include palm, palm kernel, coconut, castor, and tung. However, these are not of
domestic origin. The three domestic oils most widely used industrially are soybean,
linseed from flax, and rapeseed.

Nonfood uses of vegetable oils have grown little during the past 40 years.
Although some markets have expanded or new ones added, other markets have been
lost to competitive petroleum products. Development of new industrial products or
commercial processes is the objective of continued research in both public and pri-
vate interests. The following selected examples illustrate progress in identifying and
developing new technologies based on vegetable oils.
Great progress has been made in understanding of the biochemical basis for
biosynthesis of oils containing fatty acids. This biochemical information is in turn
used to identify and isolate genes that are needed to make these oils. By genetical-
ly engineering the introduction and expression of these genes, domesticated crops
that can produce these potentially useful fatty acids have been engineered and are
continuing to be developed to produce an ever wider range of novel oils.
Chapter 1 explains the biochemical changes that can be introduced to alter fatty
acid composition. It also discusses industrial oils that have been developed through
genetic engineering, as well as some that have been developed on the laboratory
scale, but have not yet been introduced commercially.
Recent environmental awareness and depletion of world fossil fuel reserves
have forced to look a substitute for mineral oils with the biodegradable fluids such
as vegetable base oils and certain synthetic fluids in grease formulations. The non-
toxic and readily biodegradable characteristics of vegetable oil based greases pose
less danger to soil, water, flora, and fauna in case of accidental spillage or during
disposal. Biodegradable greases are particularly useful in open lubrication systems
where the lubricant is in direct contact with environment, and total loss lubricants
like railroads, where immediate contact with the environment is anticipated. Chapter
2 discusses the various components (base oils, thickeners and additives), functional
properties, and characteristics of biodegradable greases. The base oils included syn-
thetic esters, castor, rapeseed, and soybean oil.
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Copyright © 2005 AOCS Press
Chapter 3 reviews some of the advantages and disadvantages of using vegetable

oil lubricants and their availability. Some of the history in the development of veg-
etable-based engine oils and their current status is described. The requirements for
further development and penetration of the petroleum based engine oil market are
discussed.
Besides transesterification to alkyl esters, three other approaches—dilution with
conventional, petroleum-based diesel fuel, microemulsions (co-solvent blending),
and pyrolysis—have been explored for utilizing vegetable oils as fuel. However, as
the mono-alkyl esters of vegetable oils and animal fats—biodiesel—are the only
approach that has found widespread use (and, accordingly, the vast majority of
research papers deal with this approach), Chapter 4 focuses on such mono-alkyl
esters in terms of use, properties, economies, and regulatory issues.
Chapter 5 presents a background on home heating systems and highlights recent
research to develop renewable biofuels for home heating applications. Petroleum-
based liquid home heating oil is used to heat over 8 million homes in the U.S., pre-
dominantly in the northeastern U.S. This comprises approximately 6.6 billion gal-
lons of fuel oil annually. With recent rises in petroleum prices to over $50 per bar-
rel and anticipated future price increases as petroleum resources become less avail-
able, many applications that depend on petroleum are searching for alternatives.
Additional concerns over environmental issues involving sulfur and nitrogen oxide
emissions from oil-based home heating systems have sparked a search for alterna-
tive fuels to supply this market.
Polyurethanes are the most versatile group of polymers which can be used in the
form of foams, cast resins, coatings, adhesives and sealants. Polyols used in the
polyurethane industry currently exceed 2.4 million tons/year in the U.S. To use nat-
ural oils as raw materials for polyurethane production, multiple hydroxyl function-
ality is required. Castor oil has hydroxyl functionality naturally built in, thus it has
received extensive exploration as polyurethane building blocks, such as casting
resins, elastomers, urethane foams, and interpenetrating networks. Hydroxyl func-
tionality can be introduced synthetically in other natural oils. This process involves
a number of approaches and has been studied extensively by scientists around the

world, but commercial production of oil-based polyols has been scarce. Chapter 6
discusses the four main approaches for the hydroxylation of vegetable oils.
In Chapter 7, the authors summarize the type of natural composites reinforced
with different fibers along with different composite molding methods. The Solid
Freeform Fabrication Method and its advantages are included in the discussion.
Technologies that have improved the use of oils in coatings are highlighted in
Chapter 8.
The petroleum shortage in the 1970s stimulated research on vegetable oil-based
inks as a substitute for petroleum based products. Vegetable oils are mainly used in
paste inks; therefore the role of vegetable oils in the paste ink formulations and their
environmental properties are the main subject of Chapter 9.
Chapter 10 explains that vegetable oils provide a renewable source of fatty
acids that can serve as raw materials for the production of numerous surfactant com-
pounds. Structural modification of the fatty acids can impart unique physical prop-
iv Preface
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Preface v
erties that alter the performance of the product in a predictable manner. Chemical
functionality can be introduced at the carbonyl carbon or along the carbon chain by
appropriate selection of reactants, catalysts, and reaction conditions. A tremendous
diversity of products is available with these oleochemical substrates. In addition,
vegetable oils provide a favorable alternative to petrochemical feedstocks.
The editor of this timely publication thanks the authors and their organizations
for their technical contributions in the chapters of this book. A special thanks goes
to Brittney Mernick for her assistance in the preparation of chapters for publication.
Sevim Z. Erhan
February 14, 2005
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Copyright © 2005 AOCS Press

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Chapter 1 Genetic Modification of Seed Oils for Industrial
Applications
Thomas A. McKeon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Chapter 2 Current Developments of Biodegradable Grease
Atanu Adhvaryu, Brajendra K. Sharma, and Sevim Z. Erhan . . 14
Chapter 3 Vegetable Oil-Based Engine Oils: Are They Practical?
Joseph M. Perez . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Chapter 4 Biodiesel: An Alternative Diesel Fuel from Vegetable
Oils or Animal Fats
Gerhard Knothe and Robert O. Dunn . . . . . . . . . . . . . . . . . . . 42
Chapter 5 Biofuels for Home Heating Oils
Bernard Y. Tao . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Chapter 6 Vegetable Oils-Based Polyols
Andrew Guo and Zoran Petrovic . . . . . . . . . . . . . . . . . . . . . . . . 110
Chapter 7 Development of Soy Composites by Direct Deposition
Zengshe S. Liu and Sevim Z. Erhan . . . . . . . . . . . . . . . . . . . . . 131
Chapter 8 Vegetable Oils in Paint and Coatings
Michael R. Van De Mark and Kathryn Sandefur . . . . . . . . . . . 143
Chapter 9 Printing Inks
Sevim Z. Erhan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Chapter 10 Synthesis of Surfactants from Vegetable Oil Feedstocks
Ronald A. Holser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Contents vii
vii
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1

Chapter 1
Genetic Modification of Seed Oils for
Industrial Applications
Thomas A. McKeon
USDA, ARS, WRRC, Albany, CA 94710
Introduction
While most vegetable oils are produced for food and feed uses, up to 15% of soy
(as well as other food oils) and up to 100% of certain commodity oils are used for
industrial purposes. Most food oils, such as soybean or canola, are composed pri-
marily of five fatty acids (FA): palmitic, stearic, oleic, linoleic, and linolenic; these
oils are used to produce surfactants, lubricants, inks, coatings, and polymers.
Commodity oils containing uncommon FA, such as castor (90% 12-hydroxy-
oleate) and tung (up to 80% conjugated FA), have no nutritive value, but due to the
unusual properties of the FA, they prove very useful for industrial applications. It
is the chemical functionality of a vegetable oil that can make it useful to industry;
chemical functionality can alter physical properties or allow chemical precursors
or useful derivatives to be made. For example, ricinoleate, the FA from castor oil,
has a mid-chain hydroxyl group that enhances its viscous properties for use as
grease and also enables production of an extensive range of chemical derivatives
(1). Coconut oil contains laurate (12:0) which has excellent foaming properties and
is used to make anionic surfactants. Hydroformylation of petroleum provides an
equivalent surfactant (2). The possibility of replacing such petroleum products
with plant-derived FA is a major goal of seed oil utilization research.
There are hundreds of FA with unusual functionalities, at least some of which
would have immediate application if readily available from a suitable crop. To the
extent that uncommon FA are produced in a given plant, these are a result of evolu-
tion, perhaps providing selective advantage as a result of toxic or other protective
effects of the FA on pathogens. Though it operates on a long time scale, evolution
has provided an unusual array of genetic material for production of useful FA.
However, many of these FA are produced in plants that are unsuitable as crops.

Traditional breeding techniques can alter levels of FA present in the oil and,
with suitable germ plasm, can reduce or eliminate one or more of the FA normally
present, as was the case in the development of canola (low-erucic acid rapeseed)
(3,4). Breeding has been used to develop plant selections with a high proportion of
a single component, e.g., such as high oleic safflower. High enrichment of a single
component such as oleate represents another industrially useful feature, as it
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2 T.A. McKeon
reduces the expense of purifying the desired component. But breeding cannot be used
to introduce a FA not already present in one of the crossed plants. Random mutagene-
sis using chemical or radiation agents to alter the genome followed by screening and
breeding has also produced varieties with altered FA composition in oil (5). Genetic
identification and chemical characterization of FA biosynthetic mutants in mutated
Arabidopsis thaliana has provided an extensive genetic map of FA and lipid biosyn-
thetic steps during plant growth and development (6), in many cases providing null
mutants lacking a specific enzymatic activity. Since the mutagenic approach is
geared toward eliminating genes, this approach has been used as part of breeding
programs to reduce levels of undesirable FA components such as high polyunsatu-
rates from linseed oil (7) or to increase levels of a desired FA, e.g., oleate in sun-
flower by eliminating the enzyme that normally converts it to linoleate (8). A
recent innovation in this approach is TILLING (Targeting Induced Local Lesions
IN Genomes), which uses a mutagenic approach, but introduces high-throughput
screening of the M
2
generation (the second generation of self-pollinated, mutated
lines) in order to identify specific genes that have been altered or inactivated by
mutagenic events (9). Plant selections carrying these mutated genes can then be
screened directly for desired characteristics. The TILLING process thus moves
most of the screening effort into the laboratory, considerably reducing the popula-

tion that would otherwise have to be grown in the field for phenotypic screening.
With the advent of genetic engineering, the technology needed to introduce novel
traits became available to breeders. A driving force behind development of genetically
engineered oils is the perennial surplus of oils produced. The unused inventory of soy-
bean oil may reach nearly two billion pounds in any year. Crops with altered oil com-
position hold the promise of reducing or preventing annual inventory carryover, thus
stabilizing or improving farm income. This chapter will explain the biochemistry
underlying the alteration of FA composition, briefly describe some oils that have been
developed through genetic engineering and mention some of the “target” FA of inter-
est for production in transgenic oilseed crops.
FA Biosynthesis
FA biosynthesis in plants proceeds from acetylCoA, which initiates a set of condensa-
tion reactions with malonyl-ACP through six or seven additional condensations with
malonyl-ACP. This yields the saturated FA palmitate or stearate, respectively, as
depicted in Figure 1.1, which depicts the pathway of FA biosynthesis to linoleic acid,
with the reactions leading to palmitate, stearate, and oleate occurring in the plastid,
separate from reactions leading to oil biosynthesis. Given the dependence of FA pro-
duction on malonyl-CoA production (to provide malonyl-ACP), the acetyl-CoA car-
boxylase (ACCase) is generally thought to play a regulatory role in FA production
and oil biosynthesis (10). This hypothesis is supported by research in which ACCase
from Arabidopsis was overexpressed in potato, leading to an increase in FA produc-
tion and a fivefold increase in triacylglycerol levels in the tuber (11).
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Genetic Modification of Seed Oils 3
Fig. 1.1. The pathway of fatty acid biosynthesis to linoleic acid.
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4 T.A. McKeon
Medium Chain-Length FA Biosynthesis

In seeds of certain plants such as coconut, palm kernel, bay laurel, and cuphea, the
flow of carbon to the long-chain saturated FA is disrupted, and this occurs as the
result of an acyl-ACP thioesterase (product of the FAT B gene), which removes
the ACP from the elongating FA chain prior to achieving full length. This produces
a medium chain-length FA which is transported from the plastid and enters the oil
biosynthetic pathway. This approach copied from nature led to the development of
the first transgenic oilseed modified to produce an industrial oil product, namely
Laurate Canola (12). By inserting into Canola the cDNA for a medium-chain spe-
cific acyl-ACP thioesterase (13) from California bay laurel, a plant which produces
seeds containing >60% laurate(dodecanoate) in its oil, plastidial FA synthesis was
diverted to the production of laurate, which was incorporated into the seed oil (14).
Although this achievement was a key early success in the contribution of genetic
engineering to agriculture, the underlying science also pointed to a number of tech-
nical problems that have since been widely recognized. The production of a FA not
normally produced by the seed may trigger a “counter-reaction.” In the case of lau-
rate, considerable amounts of the laurate were β-oxidized, since the cytoplasmic
lauroyl-CoA used to acylate glycerolipid is also an intermediate in β-oxidation
(15,16). While increased carbon flux through the FA biosynthetic pathway
enhanced laurate production, the overall outcome was a canola cultivar with
reduced oil yield, since some of the carbon incorporated into laurate production
was oxidized through the futile cycle.
The laurate canola oil produced also lacked laurate in the sn-2 position of the
triacylglycerol (TAG) (17). The canola seed lacked a lyso-phosphatidic acid acyl-
transferase (LPAAT) that could use lauroyl-CoA as an acyl donor for the sn-2
position of glycerolipid. Researchers at Calgene solved this problem by crossing a
canola plant containing an LPAAT gene from coconut (17), with a laurate canola
plant (18). The resulting plant produced an oilseed in which laurate is distributed
among all three positions of the TG. The resulting “High-Laurate Canola” had a
laurate content of up to 70%. The successful design of a novel, temperate-climate
industrial crop provided a great impetus to follow this approach for other industri-

ally useful products, especially oils. It also provided a foreshadowing of the diffi-
culties to be encountered in engineering production of uncommon FA in oilseeds.
Monounsaturated FA Biosynthesis
In general, once saturated FA are released from acyl-ACP, they are incorporated
into oil without any apparent modification except, to a minor extent, elongation. In
the plastid, though, the saturated fatty acyl-ACP can be desaturated by the ∆9-
desaturase, a class of soluble enzymes (as opposed to membrane-bound) formerly
identified as the stearoyl-ACP desaturase, which is the type present in most
oilseeds. These enzymes share a considerable degree of amino acid sequence
homology and the same type of active site in which the desaturation is carried out.
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Genetic Modification of Seed Oils 5
In most plants, the ∆9-desaturase produces oleate which, for purposes of oil
biosynthesis, is transported from the plastid to the endoplasmic reticulum and
incorporated into CoA, phospholipid, and acylipid. Oils high in oleic acid content
have been considered desirable both for food and nonfood uses. A high-oleate soy-
bean oil containing greater than 80% oleic acid was developed by suppressing
expression of the desaturase enzyme that converts oleate to linoleate in soybean.
This oilseed has been commercialized and for industrial purposes find applications
as a stable, biodegradeable hydraulic oil and is likely useful for developing other
bio-based lubricant applications (19).
Some plants produce monounsaturated FA of differing chain-length or with
the double-bond in a different position on the carbon chain, or both. Many such
desaturases have been cloned, the crystal structure of the soluble desaturase from
castor (Ricinus communis) has been determined, and considerable insight on fac-
tors involved in chain-length and positional specificity of the desaturase reaction
have been revealed (20,21). The ability to engineer this type of enzyme to intro-
duce a cis-double bond at a specific position on a selected chain-length represents
a bench chemist’s dream for saturated hydrocarbon chemistry. However, despite

the apparent similarity of some products to oleate, e.g., 18:1 ∆6 (petroselenate),
their production can differ from that of oleate, resulting in limited amounts of the
product when introduced into a transgenic plant (10). It has been shown that, in
some cases, co-factors such as ferredoxin and ACP isoforms that interact specifi-
cally with the enzyme are required. Moreover, the FA may also require altered
lipid metabolism to be suitably incorporated into TAG (10). Thus, further under-
standing of lipid biochemistry leading to TAG production will underly successful
attempts to engineer oil composition.
Modification of Oleate
In most temperate climate oilseeds, the oleate may be further desaturated to
linoleate and α-linolenate. In rapeseed, crambe and na-sturtium, the oleate may be
elongated to erucic acid by the action of an acylCoA based elongation reaction,
mediated in part and possibly regulated by expression of a keto-acyl synthase
(KAS) specific to elongation of long-chain FA. The products of elongation, usually
20:1 ∆11 and 22:1 ∆13 are incorporated into the TAG fraction (oil). In some
plants, the oleate is oxidized to uncommon FA. For example, in Vernonia, 18:1 ∆9,
12-13 epoxy (vernolate) is formed and then incorporated into TAG (21,22). The
possibilities resulting from oleate production provided the basis for the original
concept of oleate as the central substrate in plant FA biosynthesis (23). The set of
modification reactions that can alter oleate is unusual, in that it comprises a family
of homologous enzymes that have evolved from the FAD2 genes, which encode
the oleoyl desaturase in oilseeds. Enzymes that have evolved from the FAD2 have
been found to carry out an unusual array of conversions, using an oleoyl-phospho-
choline (oleoylPC)-based substrate. These reactions include hydroxylation, epoxi-
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6 T.A. McKeon
dation, desaturation-conjugation and desaturation to a triple bond (22,24,25). In the
case of hydroxylation and desaturation, changes in as few as 4–6 amino acid
residues result in an interchange of the two types of activity (24,26). In fact, the

oleate 12-hydroxylase from Lesquerella has mixed functionality, and can introduce
a hydroxyl group or double bond (27). Interestingly, as with some other uncom-
mon FA described in previous sections, introduction of genes with suitable production
of these uncommon FA in the oil of a transgenic plant has also proven difficult.
The next section will elaborate on this theme by describing the biochemistry of
castor oil, an important commodity oil with numerous applications (1).
Castor Oil Biosynthesis
Castor oil is a product of great interest to plant lipid scientists. It is an established
commercial product with a significant market and a cost of 45–50 cents per pound
versus soybean oil at 15–25 cents, yet is entirely imported by most industrialized
nations. Because castor seed contains noxious proteins, it is problematic as a crop.
Therefore, producing castor oil transgenically represents an enticing target and a
long-term challenge. Understanding the basis for the regulation of seed oil yield is
also a major research goal and castor, at 60% oil, has served as a benchmark for
high oil content.
Interest in castor oil biochemistry precedes the genetic engineering revolution.
In the 1960s, both the Stumpf research group at University of California, Davis,
and the Morris group at Unilever Research in Great Britain, carried out basic
research investigating the hydroxylation reaction that converts oleate to ricinoleate
(28,29). These early biochemical developments were followed by the research
groups of Stymne at Uppsala and Somerville and colleagues from MSU. These
groups contributed greatly to current understanding of ricinoleate production, and
the latter two groups elucidated the genetic basis for castor oil production by iden-
tifying and cloning two of the key genes (30,31)
The oleoyl-12-hydroxylase enzyme proved challenging to purify (32–36).
Although the enzyme has not been purified to date, the cDNA for its gene was
cloned by a genomics approach (30). Based on the hypothesis that the hydroxyla-
tion reaction is analogous to, or the first step in, the desaturation reaction, this
research group proposed that the hydroxylase would share sequence elements in
common with FA desaturases. Using this approach, hundreds of cDNAs from

developing castor seed were sequenced, prospective hydroxylase cDNAs
expressed in tobacco seed, and the seed oil assayed for hydroxy FA. Although rici-
noleate production was low, 0.1%, it was sufficient to show that the hydroxylase
had been cloned and successfully expressed in a transgenic plant. However, to
date, oilseeds transformed to express the gene for oleoyl-12-hydroxylase produce
much less than the 90% present in castor oil, with most transgenes producing less
than 20% hydroxy FA content in oil (37). It has been hypothesized that the rici-
noleate incorporated in lipid inhibits membrane function in most plants, so it may
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Genetic Modification of Seed Oils 7
be eliminated from the membrane by endogenous phospholipases (38) and beta-
oxidized (39) by analogy to laurate. On the other hand, castor has evolved bio-
chemically to produce and incorporate ricinoleate into oil. This led to the approach
of identifying additional enzyme components in castor that enable it to produce an
oil with 90% ricinoleate. Based on a considerable body of research
(31,33,38,40–43), a number of enzymes have been identified that appear to be
involved in high ricinoleate production, ricinoleate incorporation into oil, or maxi-
mizing oleate conversion to ricinoleate (44). The latter role is clearly fundamental,
since the final content of oleate in castor oil is less than 4%, and the castor oil
biosynthetic pathway is 96% efficient in converting oleate.
This research has been aided by development of methods for “metabolic pro-
filing” castor oil biosynthesis. In an effort to develop an alternative oilseed that
could produce castor oil, a microsomal system that carries out the biosynthesis of
castor oil in microsomes prepared from immature castor seed endosperm and
embryo has been developed (36,42). The microsomal system is effective in synthe-
sizing the TAG produced by the intact seed and provides a realistic model system
for investigating castor oil biosynthesis. Using this system and analysis of lipid
metabolites by high-performance liquid chromatography with selected columns
and solvent conditions, intermediates that accumulate during castor oil biosynthe-

sis can be separated and identified (44). This approach has enabled the identifica-
tion of additional enzymes that provide the unique basis for biosynthesis of castor
oil, since the gene for FA hydroxylation by itself is not sufficient to produce high
levels of ricinoleate in other oilseeds (37). Based on these research results and
other published research, the pathway in Figure 1.2 has been proposed.
The following narrative of the pathway summarizes these findings, with key
reactions and their role described briefly:
(i) The lyso-phosphatidylcholine acyltransferase (LPCAT) transfers the oleoyl-
moiety from oleoyl CoA into the sn-2 position of PC for hydroxylation.
(ii) The oleoyl-12-hydroxylase hydroxylates the sn-2 oleate to form sn-2 rici-
noleoyl-PC.
(iii) The phospholipase A
2
preferentially removes ricinoleate from the sn-2 posi-
tion of PC and releases lyso-PC for reincorporation of oleate by LPCAT.
(iv) The free ricinoleate is preferentially incorporated into ricinoleoyl-containing
diacylglycerols by the diacylglycerol acyltransferase (DGAT) to form dirici-
noleins and triricinolein, which make up castor oil.
(v) The phospholipid-diacylglycerol acyltransferase (PDAT) incorporates the
sn-2 ricinoleate directly from the ricinoleoyl-PC product of the hydroxylase
reaction into the TAG end product.
The final step in oil biosynthesis (Fig. 1.2) shows a high degree of selectivity for
incorporating ricinoleate preferentially. Based on in vitro results, both the DGAT
and PDAT (45) appear to be active in carrying out the incorporation of ricinoleate
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8 T.A. McKeon
into castor oil. The DGAT cloned from castor shows a preference for using dirici-
nolein as a substrate in comparison to the DGAT from Arabidopsis, a plant that
does not produce hydroxy FA in its seed oil (46).

New and Improved Crops
The production of industrially useful FA in transgenic crops is complicated by the
need for greater understanding of how such FA are efficiently made in the plants
that make them, and how their incorporation into oil is directed. Table 1.1 lists a
number of FA and related products that are of interest to researchers seeking to
expand the role of seed oils in the “hydrocarbon economy.” The plants developed
would be renewable resources, enhance opportunities for rural development, and
contribute to the improvement of the environment. Current research efforts are on
the appropriate control of gene expression, elucidating the synthesis of the FA, and
controlling its “destiny”—assuring its incorporation in oil and preventing it from
being further metabolized.
Another application of transgenic technology is the development of oilseeds with
improved agronomic characteristics. In fact, this has been the primary goal of agricul-
tural chemical producers that have initiated programs to produce GM crops.
Currently, the four genetically engineered crops that have been adopted are all oilseed
crops: soy, corn, cotton and canola. They account for 99% of transgenic crops planted
Fig. 1.2. Castor oil pathway.
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Genetic Modification of Seed Oils 9
worldwide. Over 70% of the soy grown in the U.S, 50% of the corn and 70% of the
cotton are genetically engineered. Most of the canola grown in Canada, a leading
producer, is transgenic. An increasing number of countries have adopted the tech-
nology. The U.S., Argentina, Canada, Brazil, China, and South Africa account for
99% of the transgenic crops produced, with an additional 12 countries adopting the
technology (47). The growth in planting of transgenic crops is remarkable in that it
has all occurred in the last eight years, from the time the first transgenic crops were
introduced in 1996. At this time, each of these crops has been modified for “input”
traits, reducing or eliminating the need for chemical applications by the introduc-
tion of genes encoding herbicide tolerance (soy, canola), insect resistance (corn,

cotton), or both (cotton). As plant genomics and proteomics programs identify
other agronomically useful genes, other transgenic traits will also be incorporated.
These can range from elimination of noxious components (48) to introduction of
dwarfing genes for greater plant efficiency. Small volume crops, such as papaya
and squash, have already been genetically modified for viral resistance. Crop
genetic engineering holds great promise as a means for developing oilseed crops
with unique characteristics that add both commercial and nutritive value, increase
utilization, and benefit the environment.
TABLE 1.1
Industrially Useful Fatty Acids for Transgenic Plant Production
Fatty acid Functionality Source Use
Eleostearic Conjugated Tung, bitter Drying oil
Octadeca-9c,11t,13t-trienoic double bonds melon
Erucic Very long-chain Rapeseed, Lubricants, anti-
Docosa-13c-enoic (VLC) crambe slip agent
γ-Linolenic Polyunsaturate Borage, Nutraceutical
Octadeca-6c,9c,12c-trienoic blackberry
Caproic to Myristate Medium chain-length Cuphea, coconut, Detergents
6 to 14 carbons bay laurel
Oleic Monounsaturate Many Hydraulic oil,
Octadeca-9c-enoic oleochemicals
Petroselenic Monounsaturate Coriander Nylon 6,6
Octadeca-6c-enoic isomer
Ricinoleic Hydroxylated Castor Lubricants,
Octadeca-9c,12-OH-enoic polymers
Vernolic Epoxy Vernonia, Coatings,
Octadeca-9c, 12,13-O-enoic Euphorbia plasticizer
lagascae
Docosahexaenoic VLC polyunsaturated Algae Nutraceutical
Nervonyl Erucate VLC wax ester Jojoba High-temperature

lubricant
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Copyright © 2005 AOCS Press
10 T.A. McKeon
Summary
Oilseeds are an important source of chemicals for industry. Most temperate climate
oilseeds produce oils containing the same five FA (palmitate, stearate, oleate,
linoleate, and α-linolenate) in different proportions. In addition to nutritive uses,
these FA are used to produce soaps and detergents, coatings, lubricants, cosmetics,
plastics, plasticizers, and numerous chemical derivatives. For specific uses, certain
FA are more desirable. For example, the conjugated double bond system present in
FA of tung oil gives it excellent properties as a drying oil. Lauric acid from coconut
provides a chemical feedstock for producing detergents. Laurate canola was the first
commercial crop that was genetically designed to produce an industrial FA. The abil-
ity to manipulate FA composition in oilseeds resulted from a combination of three
approaches. First, biochemical characterisation has identified most of the steps in FA
biosynthesis. Secondly, genetic identification and chemical characterization of
Arabidopsis thaliana mutants has provided an extensive genetic map of FA and lipid
biosynthetic steps during plant growth and development. Finally, the additional
information needed to broaden the spectrum of FA available from oilseeds has been
provided by the identification, characterization, and cloning of unusual enzyme
activities from plants that produce uncommon, industrially useful FA.
Hundreds of uncommon FA, with unusual chemical functionalities, are pro-
duced by one or more oilseed plants. A considerable amount of research has gone
into elucidating the biosynthetic process by which such FA are made; much of the
enzymology underlying the introduction of unsaturation, conjugated unsaturation,
and hydroxyl, acetylenic, and epoxy functionality is now understood. As knowl-
edge of the mechanistic and structural knowledge of these enzymes expands, there
is potential for engineering production of FA that are not yet known. The specifici-
ty of the chemistry carried out on what is essentially a straight hydrocarbon chain

is unprecedented for the bench chemist, and presents the possibility of “green”
chemistry carried out in green plants to produce a wide array of chemicals
designed for industrial applications.
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14

Chapter 2
Current Developments of Biodegradable Grease
Atanu Adhvaryu
a,b
, Brajendra K. Sharma
a,b
, and Sevim Z. Erhan
b
a
Department of Chemical Engineering, Pennsylvania State University, University Park, PA
16802, USA;
b
USDA, Food and Industrial Oil Research, 1815 N. University Street, Peoria,
IL 61604, USA
Introduction
The modern definition of lubricating grease, according to the American Society for
Testing and Materials (ASTM), is a solid or semi-solid product obtained by the
dispersion of a thickening agent in a liquid lubricant. This system may also include
other ingredients that impart special properties (see the American Society for
Testing and Materials, Standard Definition of Terms Relating to Petroleum
Products, 2000). This definition was further extended by the National Lubricating
Grease Institute (NLGI): “The material we disperse in a liquid lubricant is usually
a solid. The dispersion . . . will not settle out when left standing. In order to devel-
op thickening, the solid and the lubricating liquid had best have some affinity for
each other. This affinity also helps keep the dispersion stable” (1).
Lubricating greases are semi-solid colloidal dispersions of a thickening agent
in a liquid lubricant matrix. They owe their consistency to a gel-forming network
where the thickening agent is dispersed in the lubricating base fluid. The fluid
lubricant that performs the actual lubrication can be petroleum (mineral) oil, syn-
thetic oil, or vegetable oil. The thickener gives grease its characteristic consistency

(hardness) that is sometimes thought of as a “three-dimensional fibrous network”
or “sponge” that holds the oil in place. Therefore, the base fluid imparts lubricating
properties to the grease while the thickener, essentially the gelling agent, holds the
matrix together. This is a two-stage process. First, the absorption and adhesion of
base oil in the soap structure results, and secondly, there is a swelling of the soap
structure when the remaining oil is added to the reaction mixture.
A typical grease composition contains 60–95% base fluid (mineral, synthetic,
or vegetable oil), 5–25% thickener (common thickeners are fatty acid soaps and
organic or inorganic non-soap thickeners), and 0–10% additives (antioxidants, cor-
rosion inhibitors, anti-wear/extreme pressure, antifoam, tackiness agents, etc.) (2)
(Fig. 2.1). Additives enhance performance and protect the grease and lubricated
surfaces (3).
Grease has been described as a temperature-regulated feeding device: when
the lubricant film between wearing surfaces thins, the resulting heat softens the
adjacent grease, which expands and releases oil to restore film thickness. The
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Biodegradable Grease Developments 15
semi-solid nature of lubricating grease has several advantages over lubricating oils.
Oxidative stability and consistency of the grease matrix controls a wide variety of
performance properties in grease lubrication. Some of these properties are the abil-
ity to flow under force and subsequently lubricate hard-to-reach points; lower fric-
tion coefficient through adhesion on surface (4); wide temperature range effective-
ness; water stability; the ability to seal out contaminants as a physical barrier;
decreased dripping, spattering, and frequency of relubrication (act as sink for lubri-
cating oils). It is important to note at this point that grease structure and composi-
tion undergoes significant modification while working by shearing and oxidation.
The usefulness of grease in a particular application is controlled to a large extent
by the ability of the grease to sustain change in temperature, pressure, operating
environment, and shearing force.

Liquid lubricants possess certain shortcomings and are not able to cope with an
exponential rise in performance requirements in automotive and industrial sectors.
Technology is constantly being challenged to develop multifunctional lubricants to
operate at higher temperatures, higher pressures, and with a variety of contact sur-
faces to minimize friction and increase system efficiency. This has triggered a
steady rise in the development and application of greases in elastohydrodynamic
regimes. Thickness and stability of lubricant film is largely dependent on the
unique chemistry and composition delivered by greases. The function of grease is
to remain in contact with and lubricate moving surfaces without leaking out under
gravity or centrifugal action, or be squeezed out under applied pressure.
Development of vegetable oil-based greases has been an area of active
research for several decades (5,6). Technical progress taking place in industry and
Fig. 2.1. Grease composition.
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16 A. Adhvaryu et al.
agriculture has caused an intensive exploitation of natural resources like mineral
oil. The search for environmentally friendly materials to replace mineral oil is cur-
rently being considered a top priority research in the fuel and energy sector. This
emphasis is largely due to the rapid depletion of world fossil fuel reserves and
increasing concern for environmental pollution from excessive mineral oil use and
disposal. Renewable resources like seed oils and their derivatives are being consid-
ered as potential replacements for mineral oil base stocks in certain lubricant appli-
cations, where immediate contact with the environment is anticipated. The nontox-
ic and readily biodegradable characteristics of vegetable oil based lubricants pose
less danger to soil, water, flora and fauna in case of accidental spillage or during
disposal (7).
Environmentally friendly lubricants and greases are already in market (8). These
products are highly desired in total loss lubricants like railroads, as their accidental
spillage doesn’t invoke alarm and cause any harm to environment. Dwivedi et al.

described the preparation of total vegetable oil-based grease using castor oil (9).
Florea et al. have studied the effect of different base fluids on the properties of
biodegradable greases (10). A suitable composition of grease is desired with good
performance properties capable of use in multifunctional products. Despite the over-
whelming importance of biodegradable greases, very little is known about the rela-
tionship between their composition and performance properties.
Biodegradable Grease
Base Oils
Base fluids make up to 75 to 95% of the total composition of grease. Generally, the
base oils can be divided into two main categories: (i) water miscible, and (ii) non-
water miscible. Glycols are exclusively water soluble; the most frequently used are
monopropylene glycol or polyethylene glycol with an average molecular weight of
200–1500. The advantages of these compounds lies in their resistance to aging and
hydrolysis, while the major disadvantages are solubility in water and incompatibility
with mineral base oils. Non-water soluble base oils can be subdivided into two groups:
(i) vegetable oils, and (ii) synthetic esters (11,12). This class of compounds basically
has the same structure, and therefore, similar physical and chemical properties.
The search for bio-based material as industrial and automotive lubricants has
accelerated in recent years. This trend is primarily due to the nontoxic and
biodegradable characteristics of seed oils and esters (13) that can substitute mineral
oil as base fluid in grease making. The performance properties of grease are pri-
marily dependent on their ability to provide lubrication to mechanically operating
moving parts by supplying base oil as a thin film separating the metallic surfaces,
and also removing heat and wear debris from the friction zone. Today, greases are
expected to work under extreme operating conditions, including shock load, wide
temperature range, varying pressure, surface material and environment. As
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Biodegradable Grease Developments 17
mechanical systems become more complex in operation, eco-friendly base oils are

used that can deliver performance properties similar to mineral base fluids and yet
are nontoxic to the environment.
Synthetic esters are generally obtained from branched alcohols and long-chain
fatty acids (e.g., oleic acids) for better properties. Long-chain esters with several
branching sites exhibit good low-temperature properties and resistance to hydrolyt-
ic degradation (14). Among the esters used for grease making are trimethylol-
propane, pentaerythritol, and neopentylpolyol. Compared to vegetable oils, these
fluids deliver good thermal stability, solvency, low temperature fluidity, sub-ambi-
ent storage stability, lubricity, compatibility with mineral oil, biodegradability, and
longer service life. Diesters of a number of fatty acids like oleic and stearic acid or
dibasic acids like adipic, azelaic, phthalic and sebacaic acids are widely used for
grease making.
A real need exists for research and development of new technologies for pro-
duction of lubricants according to the most advanced, “ecological” trends. The best
approach seems to focus on alternative, renewable, widely available, natural
resources, such as vegetable oils. They are naturally occurring triacylglycerols that
are formed by the reaction of one mole of glycerol with three moles of fatty acids
or a mixture of fatty acids (Fig. 2.2). Preferably the fatty acids are oleic acid,
linoleic acid and linolenic acid or mixtures thereof. Vegetable oils are a potential
source of environmentally friendly base oils that have the additional advantage of
not disturbing the global carbon dioxide equilibrium. They exhibit excellent lubri-
cation properties due to unbalanced electrical charges which make them attach to
metal surfaces. Vegetable oils that are extensively used for biodegradable grease
preparations are soybean, rapeseed, sunflower, and castor oil. Other vegetable oils
used are olive, peanut, palm, corn, cottonseed, safflower, lesquerella, coconut and
linseed. Genetically modified vegetable oils typically contain higher than normal
oleic acid content. For example, normal sunflower oil has an oleic acid content of
20–30% which can be up to 60–90% in genetically modified high oleic sunflower
Fig. 2.2. Typical vegetable oil structure.
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Copyright © 2005 AOCS Press
18 A. Adhvaryu et al.
oil. It may be noted that genetically modified vegetable oils have a high oleic acid
content at the expense of the di- and tri-unsaturated acids.
The presence of a polar group with a long hydrocarbon chain makes vegetable
oil amphiphilic in nature, allowing it to be used as a boundary lubricant. The mole-
cules have strong affinity for and interact strongly with metal surfaces. The long
hydrocarbon chain is oriented away from the metal surface to form a monomolecu-
lar layer with excellent boundary lubrication properties. When the molecule is
adsorbed on the metal surface, two types of interactions occur. The adhesive inter-
action between the ester group and metal is very sensitive to the type and number
of functional groups. The lateral interaction caused by dipole-dipole and dispersive
interaction between the hydrocarbon chains is sensitive to structural properties
including chain length, unsaturation, and stereochemistry (15).
Castor Oil. Castor oil consists of triacylglycerols with the major fatty acid compo-
nent being ricinoleic acid (~89 wt%) (16). It is a nondrying oil with high viscosity
and is quite suitable for various lubricant applications. It can be mixed with other
vegetable oils to obtain various viscosity grades (17) and offer excellent viscosity-
temperature characteristics. Phoronic acid (having shorter chain length as com-
pared to 12-hydroxystearic acid) derived from castor oil is superior in making
greases since it has a higher metal content, delivering long grease life at higher
temperatures. The shorter chain of phoronic acid is less subject to shear degrada-
tion when used in a grease matrix (18). Castor oil has also been used to prepare
total vegetable oil based grease with sodium and lithium gallants. Vegetable oil,
alcohol, and alkali are taken in such a ratio as to give a predetermined ratio of soap
and ester in the product. The alkali is selected based on the type of grease to be
formed (Li, Na or Ca) and alcohol selection controls the viscosity of the lubricant.
Higher carbon number and molecular weight of the alcohol produces lubricants
with higher viscosity (19). The residual hydroxyl group in the ricinoleic acid chain
offers an active site for adherence to metal surfaces. It is therefore expected that

greases prepared from castor oil will have better extreme pressure characteristics.
Rapeseed Oil. Rapeseed oil has a high viscosity and is often used as a lubricant
base oil mixed with other seed and mineral oils. Lithium greases prepared with
soap made from rapeseed oil and lithium hydroxide had better mechanical stability
if some calcium hydroxide was used in the mixture (20).
Soybean Oil. Soybean is the second highest value cash crop in the United States.
The farm value of soybean production in the crop year 2000 was $13 billion. The
3.1 billion gallons of soybean oil produced in the United States is half of the 6.2
billion gallons produced worldwide. Soy oil (typically 18% of the weight of the
soybean) can be used in its raw or refined form in a variety of industrial products
(fuels, inks, paints, industrial fluids, etc.). This oil is a good source if a high unsat-
uration in the triacylglycerol is desired for grease formulation. Current develop-
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Copyright © 2005 AOCS Press
Biodegradable Grease Developments 19
ments on bioengineered (high oleic and/or low linoleic) soybean oil may provide
highly desirable improvements for fuels and other industrial products. Unlike
petroleum-based grease that takes 12 to 18 months to decompose, soy-based prod-
ucts are less toxic than traditional products and are less likely to catch fire. The use
of oils from genetically modified seeds has opened up several possibilities in the
field of nonfood uses of vegetable oils. DuPont has developed a genetically modi-
fied soybean that would produce soy oil with enhanced stability for a variety of
industrial uses including application in grease making (21).
Soap Thickeners
Vegetable oil-based greases are semi-solid colloidal dispersions of a thickening
agent (a metal soap), in a liquid lubricant matrix (vegetable oil). The thickener is a
reaction product of a metal (alkali or alkaline earth metal) based material (oxide,
hydroxide, carbonate or bicarbonate) and carboxylic acid or its ester. Acids can be
derived from animal fat such as beef tallow, lard, butter, fish oil, or from vegetable
fat such as olive, castor, soybean, or peanut oils. The most common alkalies used

are the hydroxides from earth metals such as aluminum, calcium, lithium, sodium,
and titanium. Soap is created when a long-carbon-chain fatty acid reacts with the
metal hydroxide. This reaction often produces some amount of water. For certain
types of grease, the water assists in forming the soap structure. The metal is incor-
porated into the carbon chain and the resultant compound develops a polarity. The
polar molecules form a fibrous network that holds a certain amount of base fluid
by interaction forces. The soap structure is very important to the performance of
the grease and will vary in thickness, length and oil solubility, depending on the
type of metal hydroxide used. These variations are ultimately displayed in the final
properties of the grease. Listed in Table 2.1 are some of the important physical
properties of grease affected by the structure of fatty acids.
Vegetable oil-based grease thickened with polyurea is environmentally friendly
and biodegradable in nature (22). Polyurea is the most important organic nonsoap
thickener and has excellent oxidation resistance due to the absence of metal soaps
(which tend to initiate oxidation). It effectively lubricates over a wide temperature
range (–20 to 177°C) and has a long service life that makes it suitable in sealed-for-
life bearing applications. Polyurea complex grease is produced when a complexing
agent, most commonly calcium acetate or calcium phosphate, is incorporated into the
polymer chain. Such greases showed good shear stability when subjected to the roll
stability test.
Organic clay, though readily biodegradable, is a naturally occurring nontoxic
material, so its carbon content is not counted in the determination of ready bio-
degradability (23). Thickeners based on organic clay pose the least manufacturing
challenges for biodegradable greases. When vegetable oil is used, the required con-
centration of organo-clay is typically 14%, which may be higher for NLGI No. 2
consistency. Organo-clay thickeners have amorphous gel-like structure rather than
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Copyright © 2005 AOCS Press