Professional Documents
Culture Documents
Urbana, Illinois
AOCS Mission Statement
To be a global forum to promote the exchange of ideas, information, and experience, to enhance personal
excellence, and to provide high standards ofquality among those with a professional interest in the science
and technology of fats, oils, surfactants, and related materials.
TP248.B57B557 2009
668’.1--dc22
2008055036
The paper used in this book is acid-free and falls within the guidelines established to ensure permanence
and durability.
Contents
Introduction ............................................................................................................. .vii
V
12 Synthesis of Saccharide Fatty Acid Ester Biosurfactants Catalyzed by Lipase
Sang-Hyun Pyo and Douglas G. Hayes .................................................................. 323
13 Synthesis, Aggregation Properties, and Applications of Biosurfactants Derived
from Arginine
Ma Rosa Infante, Lourder Pirez, Carmen Mordn, Ramon Pons,
andAurora Pinazo ............................................................................................ ..35 1
14 Design of Vegetable Oil Metalworking Fluid Microemulsions Using Biobased
Surfactants
Fu Zhao, Kim Hayes, Steven J. Skerlos ................................................................... 389
15 Polyol and Amino Acid-based Biosurkctants, Builders, and Hydrogels
Kenneth M. Doll and Sevim Z. Erhan .................................................................. 425
16 Interfacial Properties of Sugar-basedSurfactants
OrlandoJ. Rojas, Cosima Stubenrauch, Lucian A. Lucia, and YOussefHabibi..........449
vi
For the majority of the 2OChcentury, petroleum was utilized as the main feedstock for
transportation and other fuels, chemical intermediates, and many co-products. The
Surfactants and Detergents industrial sector, like many others, relied heavily upon
petroleum as its main feedstock.
It is now clear that the relative abundance of petroleum is decreasing, leading to
increased prices, as experienced during 2008. In addition, it is generally accepted that
the heavy utilization of petroleum results in an increased emission of carbon dioxide
into earth‘s atmosphere, which may lead to severe climate change throughout our
planet. More sustainable feedstocks for fuels and chemical intermediates are necessary.
The consumer is becoming aware of the potential danger of climate change and has
interest in products derived from more sustainable starting materials and processing
methods.
Due to the reasons discussed above, Surfactants and Detergents manufacturers have
increased interest in the utilization of biobased feedstocks. The primary motivation
is the rising costs of petroleum relative to oleochemical feedstocks, with the gap in
the price of the two anticipated to further narrow in the upcoming years. Of note,
the manufacturing costs associated with petrochemical and biobased feedstocks are
generally equal.
This bookwas prepared to highlight for the community ofsurfactant and Detergent
developers, formulators, and designers in industry, academia, and government-
sponsored laboratories throughout the world on the current industrial status of
biobased surfactants, new biobased surfactants being developed, the potential for
the “green” manufacturing of biobased surfactants via a biocatalytic route, and novel
applications for biobased surfactants. We hope this book will further the employment
of biobased surfactants in commercial products and inspire scientists worldwide to
develop new biobased surfactants and applications for their use.
The authors who prepared the chapters of this book are experts in their respective
fields, and thus are supplying the reader with a full, current, and critical evaluation of
biobased surfactant preparation and applications. We recognize that several research
groups are active in the research and development of biobased surfactants in addition
to those represented by this book‘s authorship.
The book has been divided into four sections. The first section, “Introduction,
Importance, and Relevance of Biobased Surfactants,” consisting of Chapter 1,
reports on the motivation and current use for biobased surfactants, particularly in
the industrial sector, and to serve as an introductory chapter to the remainder of the
book, moreover, to highlight the other chapters in relation to biobased surfactant
research and development. The second section, “Biosurfactants: Biosynthesis and
vii
0 D.G.Hayes et al.
Douglas G. Hayes
Dai Kitamoto
Daniel K. Y. Solaiman
Richard D. Ashby
February 13,2009
viii
0. PART1
and cosmetics products (discussed below). In addition, glycerine can be converted into
other “head group” moieties: 1,2-propanediol or propylene glycol (1,3-propanediol),
the former via a catalytic process-a technological approach under development by
Dow, Huntsman, and others-and the latter, 1,3-propanediol, through a fermentation
process developed by DuPont Tate and Lyle LLC (Loudon, TN) (Shelley, 2007); and,
polyglycerol, through homogeneous or heterogeneous catalysis (Barrault et al., 2004).
Amino acids also serve as a feedstock for the hydrophilic moiety (Xia et al., 2001). In
addition, amino acids can be converted into ethanolamine and isopropylamine (from
serine and threonine, respectively), important intermediates for cationic surfactants
(Scott et al., 2007). (See Rosa Infante et al., “Synthesis, Aggregation Properties
and Applications of Biosurfactants Derived From Arginine,” and Otero, “Selective
Enzymatic Synthesis of Alcoholamine Emulsifiers,” in this book.) Surfactants with
DNA hydrophiles were recently reported (Bilalov et al., 2004; Leal et al., 2006; Xu et
al., 200.5). Alternatively, renewable resources serve as surfactant feedstocks indirectly
by serving as the carbon-energy source of biosurfactant synthesis from microorganisms
(Lang, 2003; Lu et al., 2007). (See “Production and Modification of Sophorolipids
from Agricultural Feedstocks,” Ashby et al., in this book.) Biosurfactants include
trehalose esters, rhamnolipids and sophorolipids (see chapters written by Ashby et
al. and Pinzon et al. in this book), mannosylerythritol lipids (chapter by Rau and
Kitamoto, “Mannosylerythritol Lipids: Production and Downstream Processing”),
other glycolipids, phospholipids, and lipopeptides (chapter by McInerney et al.)
(Lang, 2003; Lu et al., 2007). For synthetic nonionic surfactants employed today,
the major source of hydrophile is polyethylene glycol (or equivalently, polyethylene
oxide, or “ethoxylate”),which is derived from petroleum; however, one can potentially
derive it from bioethanol through the synthesis of ethylene as an intermediate (Gielen
et al., 2008). Ethylene oxide, the starting material, is carcinogenic, mutagenic, highly
flammable, volatile, and reactive. Therefore, for the reasons given above, biobased
hydrophiles for surfactants and detergents will become more commonly employed.
Perhaps polymerization products of bioderived 1,2- or 1,3-propanediol or polyglycerol
may become future substitutes.
habitat loss, which have already occurred in Malaysia and Indonesia as a result of
palm-oil production-and a loss of biodiversity for oilseed-bearing plants ( McCoy,
2007a,b; Patel, 2004). However, sustainability certification is also being addressed for
feedstocks to prevent the occurrence of such events. The Roundtable for Sustainable
Palm Oil serves as an example (Roundtable for Sustainable Palm Oil-RSPO,
2008).
An important aspect of eco-friendliness is reduced environmental impact. Several
environmentally-related issues are facing the surfactants and detergents industry that
may be effectively addressed by using biobased surfactants (Table 1.2). The need
to replace alkylphenyl ethoxylates (APEs), nonionic surfactants commonly used in
cleaning products, was accelerated by the recent decision of Walmart to place APEs
on a list of "chemicals of concern" that its laundry product suppliers must replace
before 2010 (McCoy, 2007a) and by the request of the Sierra Club (Washington,
DC) to the U.S. EPA (Environmental Protection Agency) to ban the use of APES
(Sissell, 2007). Linear and branched alkyl ethoxylates, which one can manufacture
from petroleum and/or biobased feedstocks, are the leading substitution candidates
(McCoy, 2007a). The increased demand for cold water laundry detergents will provide
an opportunity for the use of methyl ester sulfonates (MESS), which are derived
from fatty acid methyl esters (FAMEs) (described below) (Roberts et al., 2008). For
Table 1.2. Environmental Goals for the Surfactants and Detergents Industry
Environmental goal Rationale
Reduce carbon-dioxide emissions Reduce climate change
Replacement of alkylphenyl ethoxylates (APES) Produce toxic biodegradation products;
may promote reproductive problems
Use of cold-water-compatiblelaundry detergents Reduce household-energy consumption
Increased concentration of liquid laundry Reduce packaging and shipping costs
detergents
Replacement of phosphatesand zeolites as Reduce phosphate eutrophication
builders for dishwasher detergent in lakes and rivers; zeolites are
nondearadablesolids
Replacement of formaldehyde-releasing biocides Nasopharyngealcancer agent
Replacement of solvents (e.g., ethylene glycol Decreasevolatile organic compounds;
butvl ether in mrav cleaners) oossible asthma aaent
Replacement of quaternary ammonium Possible asthma agent
compounds
McCoy, 2007a,b; Watkins, 2007.
Biobased Surfactants: Overview and Industrial State-of-the-Art0 9
Fig. 1.1) have many uses as surfactants in foods, cosmetics, and pharmaceuticals, as
described in Table 1.3 (Nelen & Cooper, 2004). (See also “Basic Properties of Sucrose
Fatty Acid Esters and their Application,” Otomo, and “Synthesis of Saccharide-Fatty
Acid Ester Biosurfactants via Lipase,” Pyo and Hayes in this book.) Rhamnolipids,
sophorolipids, trehalose lipids, mannosylerythritol lipids (not depicted in Fig. 1. I),
and other polysaccharide-based biosurfactants derived from fermentation have many
potential applications in the petroleum, pharmaceutical, food-processing, pesticide,
and environmental-remediation industries. (See chapters written by Ashby et al.., Rau
and Kitamoto, Nguyen and Sabatini, Kitamoto et al., and Pinzon et al. in this book.)
Amino acid- and peptide- or protein-based surfactants, encountered in nature (e.g.,
lipoproteins; see McInerney et al., “Lipopeptide Surfactants and Their Use in Oil
Recovery” in this book) and synthesized chemically or chemoenzymatically (see Rosa
Infante et al., “Synthesis, Aggregation Properties and Applications of Biosurfactants
Derived From Arginine,” and Otero, “Selective Enzymatic Synthesis of Alcoholamine
Emulsifiers” in this book), have several current and potential applications in cosmetics
(e.g., thickeners, emollients, and conditioners), foods, and cleaners (Nnanna et al.,
2001; Sakamoto, 2001; Xia et al., 2001); and one can employ them in oil recovery
(McInerney et al.$ chapter). In addition, amphiphilic polypeptides (e.g., copolymers
of leucine, tryptophan, and lysine) are useful lung surfactants. (See Nakahara et al.’s
“Influence of Pulmonary Surfactant Protein Mimics on Model Lung Surfactant” in
this book.)
MES, produced from FAMEs, derived primarily from coconut and palm oils
[with a C,, FAME-rich fraction, a possible by-product stream from a palm oil-
based biorefinery produced by thermal fractionation due to its undesirability in
biodiesel formulations (Foster, 2006d), being of particular interest as an economic
feedstock], is a common and inexpensive anionic surfactant in powder laundry
detergent products (e.g., Nl/Surf powder detergent from Unilever). The process
for synthesizing MES, developed in the 1950s from USDA research, involves the
reaction of FAME with a slight stoichiometric excess of SO, in the presence of NaOH
(Ahmad et al., 2007; Edser, 2006; Foster, 2006a; Maurad et al., 2006; Roberts et
al., 2008). MES is produced commercially in Japan by Lion (Johor, Malaysia; 40
ktodyear) and in the United States by Stepan (Northfield, IL; 50 ktondyear) and
Huish Detergents (Salt Lake City, UT; 80 ktondyear) (Bognolo, 2008). Currently,
linear alkylbenzene sulfonates (LASs) are the most commonly employed anionic
surfactants for detergents and other cleaners, and have been so for the past 30 years
(Ahmad et al., 2007). Until recently, the manufacture of MESS was more expensive
than for LASs due to the slower reaction rate of sulfonation, the need to control
hydrolysis during the reaction, and the need to perform bleaching on the reaction
product (Edser, 2006). However, the higher rate of the price increase for petroleum
relative to palm and coconut oils is making MES production more cost-competitive
with that of LAS (Edser, 2006), and the product quality has improved as well
(Bognolo, 2008; Foster, 2006~).Moreover, the development of biodiesel facilities
Biobased Surfactants: Overview and Industrial State-of-the-Art 0 11
-
B
Sophorolipid
n,
HO OH 0
in Malaysia and Southeast Asia has increased the availability of FAME feedstock.
An additional economic advantage for MES versus fatty alcohol-based surfactants
such as alcohol (and alkyl ester) sulfates and alkyl ethoxylates is that the former does
not require high-temperature and high-pressure hydrogenation for the conversion
of FAME into fatty alcohol (Ahmad et al., 2007). MESs perform well compared
to other anionic surfactants for both hot- and cold- water formulations due to its
stability in hard water and its inertness to enzymes contained in laundry formulations
(Cohen et al., 2008; Foster, 2006c; Maurad et al., 2006; Roberts et al., 2008). They
rapidly undergo biodegradation aerobically (a-oxidation) to produce monomethyl
a-sulfosuccinate, which then undergoes desulfonation to produce succinic acid and
inorganic sulfate as its ultimate products, with its biodegradation reported to be at
least equal to, and in some reports, faster than that of LAS (Ahmad et al., 2007;
Edser, 2006; Masuda et al., 1993; Roberts et al., 2008). The latter's biodegradability
was investigated thoroughly, and performed well, yielding degradation products that
benignly undergo mineralization (HEM, 2008). However, MES does not undergo
mineralization effectively under anaerobic conditions (Garcia et al., 2008). Although
MESs present unique advantages, deficiencies in their performance were noted. They
possess low foam characteristics, limiting their utility in hand-washing and other
foam-based products, and are unstable in high-pH liquid formulations (Bognolo,
2008; Foster, 2006b). However, one can address these deficiencies by proper product
formulation (Foster, 20OGb,c). But as an overall assessment, MES surfactants are well-
suited to further increase their market share of anionic detergents at the expense of
petroleum-derived MS. Ahmad et al. (2007) suggest a goal for MES-replace 30%
of the 3.4 MMT LAS market by 2010, resulting in a 1 MMT production capability.
Esterquats are cationic, quaternary ammonium surfactants derived from animal
fats or vegetable oils (in the form of FAMEs) that are important rinse-added fabric
softeners and ingredients of hair conditioners, dyes, and bacterial and sanitizer
products, including swimming-pool biocides (Ahmad et al., 2007; Mishra & Tyagi,
2007; Overkempe et al., 2003). Gemini esterquats were developed that are useful
flotation agents, for example, in the mining of calcite (Mishra & Tyagi, 2007).
O n a molecular level, they adsorb via an electrostatic attractive driving force to the
negatively charged surfaces of cotton, collagen, and other fibrous biopolymers. The
hydrophobic moieties of the surfactants extend outward and prevent friction between
adjacent fibers. They typically are formed by first esterifying methyldiethanolamine
(or other amine derivative) with two mole equivalents of fatty acyl groups at ~ 2 5 0 ° C
for a few hours under vacuum to remove the product water and then quaternizing
the molecule (e.g., with methylethylsulfate at c1Oo"C for a few hours) to form the
derivative depicted in Fig. 1.1 (Mishra &Tyagi, 2007; Overkempe et al., 2003). They
are readily biodegradable and possess an excellent environmental profile. Although one
can consider the traditionally used fatty acid feedstock, tallow, biobased, the medium-
chain fatty acyl groups derived from palm oil, for example, are becoming increasingly
popular and provide improved performance (Ahmad et al., 2007; Overkempe et al.,
2003).
Biobased Surfactants:Overview and Industrial State-of-the-Art 0 13
Many of the products described above are synthesized directly from FAMEs;
e.g., saccharide- and polyol-fatty-acid esters, ethoxylated fatty acids, amino acid
surfactants, esterquats, and MESS. The majority of the other biobased surfactants
are indirectly manufactured from FAMEs. AGs and biobased alkyl ethoxylates are
synthesized from fatty alcohols, which are derived from FAMEs. Fatty amines are also
derived from FAMEs. Since FAMEs are primary targets of oleochemical processing
for the manufacture of biodiesel, biobased surfactant production should integrate
well into an oleochemical “biorefinery.” The latter term refers to the utilization
of biobased feedstock by a chemical plant, which through various processing and
separation schemes produces electrical power and yields an array of hels and
products/co-products/intermediateproducts, similar to a petrochemical refinery,
where petroleum is fractionated to produce home and transportation fuels, chemical
intermediates, lubricants, asphalt, etcetera. Moreover, FAME will serve as a key
intermediate oleochemical biorefinery product stream that can be utilized as biodiesel
he1 or as a chemical intermediate for the production of biobased products, including
biobased surfactants. Glycerine, a key by-product stream, will be readily utilized as
an intermediate for biobased surfactants as well, as described above. Other biobased
surfactant intermediates such as saccharides, proteins, and lactic acid (employed for
the synthesis of monoglyceride esters) would occur as biorefinery by-product streams
that utilize certain seed oils, starch, or lignocellulosic biomass as feedstock. A further
discussion of the oleochemical biorefinery concept is given elsewhere (Hatti-Kaul et
al., 2007; Hill, 2007; Johansson & Svensson, 2001).
Table 1.4 lists several biobased surfactants that are available commercially and
their manufacturers. This list also includes commercial products which incorporate
biobased surfactants, many of which have achieved one of the eco-friendly or biobased
certifications listed in Table 1.1. This list truly reflects the global nature of biobased
surfactants, with manufacturers based in several different countries represented.
Japan)
MitsubishiTanabe Pharma Corporation SURFACTEN lung surfactant (obtained from
(Tokyo, Japan) finely ground bovine-lung tissue; contains
84% of DhosDholiDidsl
Polyoxyethylene-sorbitanesters, ethylene-
glycol and propylene-glycol esters,
monoglycerides,and sorbitan esters
Nippon Fine Chemicals (Tokyo, Japan) Sucraph AC-8 (caprylylglucoside)
ONY Inc. (Amherst, NY) lnfasurf lung surfactant (obtained from calf-
Biobased Surfactants: Overview and Industrial State-of-the-Art 19
Conclusions
The ideal surfactant was recently described as possessing low surface and interficial
tension, low critical micelle concentration, low Krafi-point temperature, high
solubility in cold and hot water (or in alkanes, squalene, aliphatic esters, or other
lipophilic solvents), fast kinetics for their self-assembly, high biodegradability and
biocompatibility, an excellent environmental profile, and a low cost-to-performance
ratio (Scheibel, 2007). Up to recent years, biobased surfactants competed favorably
with, and in some cases outperformed, synthetic, petroleum-derived surfactants (e.g.,
for biodegradability and biocompatibility), except in terms of the cost-to-performance
ratio. However, with the recent surge of petroleum prices, biobased surfactants are
becoming a more economically viable choice. In addition, the increased consumer
desire to purchase eco-friendly and biobased products favors biobased surfactants.
Furthermore, the development of oleochemical biorefineries based on FAMEs for
biodiesel will lead to a more abundant and consistent feedstock supply for surfactant
synthesis. Therefore, biobased feedstocks will become increasingly popular for
surfactants and detergents manufacture in the near-term. The utilization of biobased
feedstock and sustainable manufacturing by the surfactants and detergents industry
20 0 D.G.Hayes
Acknowledgments
?he author thanks the U.S. Department of Agriculture, Grant 2006-35504-17262,
and the National Science Foundation, Grant BES-0437507, for supporting his
research in the biobased surfactant and microemulsions areas; Dr. Warren Schmidt,
Shell Global Solutions (U.S.) Inc., Houston,TX, and Mr. William J. Schalitz, Spartan
Chemical, Maumee, OH, who provided technical assistance.
References
Ahmad, S.; I? Siwayanan; Z. Abd Murad; H. Abd Aziz; H. Seng Soi. Beyond biodiesel: Methyl
Esters as the Route for the Production of Surfactants Feedstock. INFORM2007, 18,216-220.
Allef, I?; W. Berkels; H. Foetsch; J. Meyer. Partial Esters of Polyglycerol with PolyricinoleicAcid
and Multifunctional Carboxylic Acids for Use in the Preparation of Emulsions and Dispersions.
European Patent EP 2006-243 1683781 (2006).
Andrysek, T. Excipients and their role in absorption: influencing bioavailability of cyclosporine by
triglycerides and polyglycerol esters. Biomed. Pap. 2006, 150, 227-233.
Anonymous. Surfactants and detergents: Cleaning the green way. Fonu on Su$2006,2,6.
Balzer, D.; H. Luders (Eds.) Nonionic Su$mtants: Alkyl Polyglucosides. Surfactant Science Series.
Marcel Dekker: New York, 2000; Vol. 91.
Barrault, J.; J.M. Clacens; Y. Pouillow. Selective Oligomerization of Glycerol Over Mesoporous
Catalysts. Top. Catal. 2004,27, 137-142.
Baum, R.M. Sustainability: Learning to Live Off the Sun. Chem. Eng. News 2008,86(33), 42-46.
Biobased Surfactants:Overview and Industrial State-of-the-Art0 2 1
Bilalov, A.; C. Leal; B. Lindman. Mixing Oil and Water by a DNA-based Surfactant. J. Pbys.
Chem. B 2004,108,15408-15414.
Biobased US. What Are Bio-Based Products?2007; http://www.biobased,us/biobased.html.
Bognolo, G. Surfactants Market Trends in Europe. CESIO 7th World Surfactants Conzrence, Euro-
pean Committee of Organic Surfactants and their Internaliates, Brossels, Paris, 2008.
Brasher, I? Biodiesel Comes Under Fire as Next Target of Fuel Costs. 7he Des Moines (L4) Regizter
2008, July 13, 2008, 1 .
Carter, C.; W. Finley; J. Fry; D. Jackson; L. Willis. Palm Oil Markets and Future Supply. Eur. /.
Lipid Sci. Technol. 2007, 109, 307-314.
Chemistry in Britain. Surfactants: The Ubiquitous Arnphiphiles. Chem. World 2003, (Iuuly), 1-7.
Chetri, A.B.; M.S. Tango; S.M. Budge; K.C. Watts; M.R. Islam. Non-edible Plant Oils as New
Sources for Biodiesel Production. Int.J. Mol. Sci. 2008,9, 169-180.
Chisti, Y. Biodiesel From Microalgae. Biotechnol Adv. 2007,25,294-306.
Cohen, L.; F. Soto; A. Melgarejo; D.W Roberts. Performance of @-sulfa Fatty Methyl Ester
Sulfonate versus Linear Alkylbenzene Sulfonate, SecondaryAlkane Sulfonate and a-sulfo Fatty
Methyl Ester Sulfonate.J. Su$ Deterg. 2008, 11, 18 1-1 86.
Coons, R.; K. Phillips. Soapers Awash in Green Issues and Feedstock Concerns. Chem. Week
2008, 170(5), 22.
cosmeticinfo.org. Cosmetic Ingredients Search Database, 2008; http://www.cosmeticsinfo.org/
ingredient-details.php?ingredient-id= 1 186.
Cottrell, T.; J. van Peij. Sorbitan Esters and Polysorbates.Emuls$ers in Food Zchnolau; R.J.
Whitehurst, Ed.; Blackwell Publishing: Oxford, UK, 2004; 162-185.
Cowan, D.; K.M. Oxenbell; H.C. Holm. Enzymatic Bioprocessing of Oils and Fats. INFORM
2008,19,210-212.
Ducret, A.; M. Trani; R. Lortie. Comparison Berween Various Commercial Sources of Almond
P-glucosidase for the Production of Alkyl Glucosides.J. Mol. Catal B: Enzym. 2006,38, 9 1-94.
Edser, C. Growing Use of Oleochemicals. F o w on Su$2004, (Nov), 1-2.
Edser, C. Is the Time Ripe for Methyl Ester Sulfonates?Focus on Su62006, (Sept.), 1-2.
Flora Health Manufacturing & Distributing Ltd. (Burnaby, Canada). Herb & Supplement Ency-
clopedia: Glycerol Stearate Supplement; 2008, http://www.florahealth.com/flordhome/Canada/
HealthInformation/Encyclopedias/GlycerolStearate.htm.
Foster, N. Commercial Applications of the Chemithon MES Process. INFORM2006a, (‘Methyl
Ester Sulfonates-A Guide to Biorenewabk Resources: A Special Supplement,” Biorenewable Re-
sources #3), 7-8.
Foster, N. Gimme a Break! (Price, that is). INFORM2006b, (‘Metbylfiter Sulfonates-A Guide to
Biorenewable Resources: A Special Supplement, ” Biorenewable Resources #3), 3.
Foster, N. Methyl Ester Sulfonates Industry Poll. INFORM 2 0 0 6 ~(‘Methyl Bter Su&nates-A
Guide to Biorenewable Resources: A Special Supplement,” Biorenewabk Resources #3), 4-6.
Foster, N. Questions About MES. INFORM 20064 (“Methyl firer Sulfonates-A Guide to Biore-
newable Resources: A Special Supplement,” Biorenewable Resources #3), 9-1 0 .
22 0 D.G.Hayes
Lu, S.; l? Somasundaran. Intermolecular Packing of Sugar-based Surfactant and Phenol in a Micel-
lar Phase. Langmuir 2007,23,9960-9966.
Lu, J.R.; X.B. Zhao; M. Yaseen. Biomimetic Amphiphiles: Biosurfactants. Curr. Opin. Colloid
Interface Sci. 2007, 12, 60-67.
Marti-Mestres, G.; F. Nielloud. Main Surfactants Used in the Pharmaceutical Field. Drugs Pharm.
Sci. 2000, 105, 1-18.
Masuda, M.; H. Odake; K. Miura; K. Ito; K. Yamada; K. Oba. Biodegradation of 2-sulfonato
Fatty Acid Methyl Ester (a-SFMe). 11. Biodegradation pathways of a-SFMe. J. Jpn. Oil Chem.
SOC.1993,42, 905-909.
Maurad, Z.A.; R. Ghazali; I? Siwayanan; Z. Ismail; S. Ahmad. Alpha-sulfonated Methyl Ester as
an Active Ingredient in Palm-based Powder Detergents.J. Surf: Deterg. 2006,9, 161-167.
McCoy, M. Going Green. Chem. Eng. News 2007a, 85(5), 13-19.
McCoy, M. A New Kind of Clean. Chem. Eng. News 2007b, 85 (12), 29-3 1.
McCoy, M. Converging Pathways: Chemical Companies and Environmentalists Edge Closer
Together in the Pursuit of Sustainability. Chem. Eng. News 2008a, 86(33), 47-46.
McCoy, M. Greener Cleaners. Chem. Eng. News 2008b, 86(3), 15-23.
Mishra, S.; V.K. Tyagi. Ester Quats: The Novel Class of Cationic Fabric Softeners. J. Oleo Sci.
2007,56269-276,
Miyamoto, Y.; M. Sakamoto; T. Maeda; N. Morita. Application of Polyglycerol Mono-fatty Acid
Esters to Improve Breadmaking. FoodSci. Technol. Res. 2005, 11, 19-25.
Morflex Inc. (Greensboro, NC). Pharmaceutical Coatings Bulletin 102-3: Influence of Plasticizers
on the Dissolution and Physical Properties of Ethyl Cellulose Films and Coated Beads, 2008;
http://www.morflex.com/pdf/bul102.3.pdf.
Nelen, B.A.P.; J.M. Cooper. Sucrose Esters. Emuh$ers in Food Technology; R.J. Whitehurst, Ed.;
Plackwell Publishing: Oxford, UK, 2004; pp. 131-161.
Nnanna, LA.; G.Y. Cheng; J. Xia. Potential Applications of Protein-based Surfactants. Protein-
Based Sut.factants:Synthesis, PbysicochemicalProperties, and Applications; I.A. Nnanna; J. Xia,
Eds.; Surfactant Science Series; Marcel Dekker: New York, 2001; Vol. 101, pp. 227-260.
Norn, V. Polyglycerol Esters. Emuh$en in Food Technology; R.J. Whitehurst, Ed.; Blackwell Pub-
lishing: Oxford, UK, 2004; pp. 110-130.
Overkempe, C.; A. Annerling; C.G. Van Ginkel; l?C. Thomas; D. Boltersdorf; J. Speelman.
Esterquats. Novel Surfactants: Preparation, Applications, and Biodegradability: K. Holmberg, Ed.;
Surfactant Science Series; Marcel Dekker: New York, 2003; Vol. 114, pp. 347-384.
Pandey, V.; R. Banerji; B.S. Dixit; M. Singh; S. Shukla; S.l?Singh. Cuphea, a Rich Source of
Medium-chain Triglycerides: Fatty Acid Composition and Oil Diversity in Cupheaprocumbens.
Eur. J. Lipid Sci. Technol. 2000, 102, 463-466.
Patel, M. Surfactants Based On Renewable Raw Materials: Carbon Dioxide Reduction Potential
and Policies and Measure for the European Union. J. Znd. Ecol. 2004, 7,47-62.
Perkins, W.S. Surfactants-A Primer. Am. Tmtil. Znt. 1998, Mug), 5 1-53.
Phillips, K.; R. Coons; A. Scott; K. Walsh. Soaps and Detergents: Producers Race to Lower Costs
and Go Green. Chem. Week 2008, 170(3),23-25.
24 0 D.G. Hayes
Phillips, K.; E. D’Amico; K. Walsh. Soaps and Detergents: Measuring Out Costs. Chem. Week
2007,167(3),23-28.
Plasman, V.; T. Caulier; N. Boulos. Polyglycerols. Versatile Ingredients for Personal Care. House-
hold Pers. Prod. Ind. 2004,41,94-97.
Plasman, V.;T. Caulier; N. Boulos. Polyglycerol Esters Demonstrate Superior Antifogging Proper-
ties for Films. P h t . Addit. Compd. 2005,7, 30-33.
Quadri, G.; X. Chen; J.W. Jawia; F. Tambone; I? Genevini; F. Faoro; F. Adani. Biobased Surfac-
tant-Like Molecules From Organic Wastes: The Effect of Waste Composition and Composting
Process on Surfactant Properties and on the Ability to Solubilize Tetrachloroethene (PCE).
Environ. Sci. Zchnol. 2008,42,261 8-2623.
Rahman, EK.S.M.; E. Gakpe. Production, Characterization and Applications of Biosurfactants-
Review. Biotechnohgy (Faisalabad, Pakistan) 2008,7, 360-370.
Roberts, D.W.; L. Giusti; A. Forcella. Chemistry of Methyl Ester Sulfonates. INFORM2008,
(“Surfactantsj?om Biorenewable Sources: A Special Supplement,” Biorenewable Resources #5), 2-9.
Roundtable for Sustainable Palm Oil (RSPO). Promoting the Growth and Use of Sustainable
Palm Oil, 2008;http://www.rspo.org/.
Sagalowicz, L.; M.E. Leser; H.J. Watzke; M. Michel. Monoglyceride Self-assembly Structures as
Delivery Vehicles. Trends Food Sci. Technol. 2006,17, 204-214.
Sakamoto, K. Current Market Developments and Trends in Amino Acid- and Protein-Based
Surfactants. Protein-Baed Surfactants: Synthesis, Physicochemical Propedes, and Applications; LA.
Nnanna; J. Xia, Eds.; Surfactant Science Series; Marcel Dekker: New York, 2001;Vol. 101,pp.
261-280.
Santosa, S.J. Palm Oil Boom in Indonesia: From Plantation to Downstream Products and Biodie-
sel. Clean: Soii Air, Water 2008,36453-465.
Schalitz, W.J. The Role Awaiting Bio Based Surfactants in the Industrial Cleaning Market. 78th
American Oil Chemists’ Society Annual Meeting, Quebec City, Canada, 2007.
Scheibel, J. The Impact of Feedstocks on Future Innovation in Surfactant Technology for the
Detergent Market. 78th American Oil Chemists’ Society Annual Meeting, Quebec City, Canada,
2007.
Scott, E.; F. Peter; J. Sanders. Biomass in the Manufacture of Industrid Products-The Use of
Proteins and Amino Acids. Appl. Microbiol. Biotechnol. 2007,75,751-762.
Shelley, S. A Renewable Route to Propylene Glycol. Chem. Eng. Prog. 2007,103,G-9.
Sissell, K. Sierra Club Asks EPA to Ban NPEs in Commercial and Household Detergents. Chem.
Week 2007,167(20), 45.
Smith, J.; L. Hong. FoodAdditives Databook; Blackwell Publishing: Oxford, UK, 2003.
Solvay Chemicals (Deer Park, TX). Polyglycerol in Personal Care (Technical Bulletin
PGL-03-00 l), 2004;http://www.solvaychemicals.us/static/wma/pdf/6/6/9/2/PGL-03-001 .p&.
Solvay Chemicals (Deer Park, TX). Polyglycerols in Industrial Applications (Technical Bulletin
TDS-PGL-05-00 1), 2005;http://www.solvaypolyglycerol.com/docroot/glycerol/static-files/at-
tachments/polyglycerol-in_industrial-applications-li~t.pd~
Sparso, F.V.; N. Krog. Propylene Glycol Fatty Acid Esters. Emulsifiers in Food Technology; R.J.
Biobased Surfactants:Overview and IndustrialState-of-the-Art 0 25
Introduction
Surfactants or “Surhce Acting Agents” are molecules that are made up of hydrophobic
and hydrophilic components allowing them to either concentrate at interfaces such
as aidwater or oil/water and act to reduce surface and/or interfacial tension or self-
associate to form micelles or other nanostructured aggregates. Generally, surfactants
are classified as either ionic (e.g., alkyl sulfonates, anionic, phospholipids, zwitterionic;
or quaternary ammonium compounds, cationic) or nonionic (e.g., alkyl glucosides or
alkyl ethoxylates) based on their chemical structure. Regardless of their classification,
surfactant effectiveness tends to be concentration-dependent; increasing surfactant
concentrations beyond a critical value, unique to each surfactant, known as the critical
micelle concentration (CMC), causes micellar aggregation to occur. Thus above the
CMC many surfactants do not exhibit additional changes in surface and/or interfacial
tension.
Many microorganisms have the capability to naturally synthesize surfactant-
type molecules. Many of these so-called “biosurfactants” express a broad range of
functional properties including emulsification, phase partitioning, wetting, foaming,
surface activity, and in some cases potential clinical applications and as such (along
with the mounting concern for the environment and the persistent rise in petroleum
prices) are drawing increased industrial interest for various applications traditionally
occupied by petroleum-based surfactants. Many different biosurfactants have been
identified. Some, such as glycolipids, neutral lipids, and lipopeptides, are of relatively
small molar mass while others may form polymeric structures or be created through
complex chemical interactions that result in larger molar mass materials. Examples of
such large molar mass biosurfactant materials include lipoproteins, lipopolysaccharide-
29
30 0 R.D. Ashby et at.
Sophorolipid Biosynthesis
Introduction and Applications of Sophorolipid Biosurfactants
One of the proposed hnctions of sophorolipids in the producing organisms is to
assist in accessing lipophilic substrates (It0 & Inoue, 1982), but their large production
capacity and amphiphilic character have increased awareness for promising industrial
applications (Solaiman et al., 2004a). The industrial potential of these molecules is
dependent on their surface-active properties, which are dictated by the molecular
distribution of the open-chain and lactone structures. While a large capability
application has yet to be discovered, sophorolipids do have the capacity to lower
surface and interfacial tension (Cooper & Paddock, 1983) thus providing a potential
Production and Modification of Sophorolipidsfrom Agricultural Feedstocks0 3 1
HO
0
A B
mechanism for use in many diverse applications (Banat et al., 2002). In fact,
sophorolipids have been reported to be active as primary surfactants for shampoos,
body washes, and detergents (Hall et al., 1995; Inoue et al., 1980) and as emulsifiers
for skin care products (Mager et al., 1987). In addition, they have been reported to
have applications as food encapsulants (Allingham, 1971), as degreasing agents (Hall et
al., 1995), and to enhance soil bioremediation and waste water treatment (Makkar &
Cameotra, 2002; Mulligan et al., 2001). Some reports have suggested that the lactone
form ofsophorolipids can be utilized as a bacteriostatic agent (Mager et al., 1987), has
anticancer properties (Scholz et al., 1998; Isoda et al., 1997), can be used to stimulate
skin fibroblast metabolism (Boneix, 2003), and treat skin diseases (Maingault, 1997).
In contrast, the acidic form of sophorolipids has been shown to be therapeutically
active for skin treatment, particularly as agents for fibrinolysis (promoting healing),
desquamation, depigmenting, macrophage activation (Maingault, 1999), and
as moisturizing agents (Abe et al., 1981; Tsutsumi et al., 1981). In addition, the
unique sophorolipid structure has increased interest in their use as a precursor for
the production of specialty chemicals such as sophorose, a known inducer of hngal
cellulase enzymes (Sternberg & Mandels, 1980), monohydroxy fatty acids (Rau et
al. 2OO1), and other derivatives (Zerkowski & Solaiman, 2006, 2007; Zerkowski et
al. 2008). To a limited extent, structural variation (and hence control over physical
properties) can be achieved by changing the lipidic carbon source, which alters the
sophorolipid fatty acid content (see below).
Saccharide/Lipidic Feedstocks
Since the 1980s research has focused on the use ofsimple monosaccharides (primarily
glucose) and lipids as substrates for sophorolipid synthesis. In order to minimize
production costs, low cost substrates, such as triacylglycerols and free fatty acids, were
32 0 R.D. Ashby et al.
Co-product Feedstocks
In addition to the lipid sources described above, inexpensive co-product streams such
as crude glycerine from biodiesel manufacturing (Ashby et al., 2005), soy molasses
from soybean processing (Solaiman et al., 2004b), and oil refinery waste including
soapstock and post-refinery fatty acids (Bednarski et al., 2004) have also been reported
as co-substrates in the production of sophorolipids. Biodiesel is synthesized by the
chemical transesterification of triacylglycerols with short chain alcohols, as shown in
Fig. 2.3.
Over the past few years the biodiesel industry has advanced rapidly throughout
the world because it is an attractive alternative to petroleum diesel as it burns cleaner
Productionand Modificationof Sophorolipidsfrom Agricultural Feedstocks 0 33
Table 2.1. Maximum Reported Sophorolipid Yields Produced by C. bombicola from Various
Lipidic Substrates (Unless Otherwise Noted the Carbohydrate Source Was Glucose)
~
Min. ST = 35 mN/m
% 7 CMC = 35 mg/L
t o i , , ~ , ,) , , ) i , ~ , ? l ? * ~ , l & , t , , , ,
Min. ST = 36 mN/m
Yo : CMC = 250 mg/L
i.L
0 - - ~ . l ~ ~ ~ ~ I I * “ . 9
Time (min)
Fig. 2.2. Liquid chromatograms of the region corresponding to the diacetylated lactonic forms of the
sophorolipids derived from glucose and palmitic acid (SL-PA, 92% lactone; A), stearic acid (SL-SA, 99%
lactone; B), oleic acid (SL-OA, 100% lactone; C), and linoleic acid (SL-LA, 98% lactone; D). Minimum
surface tensions (Min. ST) and critical micelle concentrations (CMC)were measured in a waterhapeseed
oil system (Ashby et al., 2008).
32% free fatty acid/fatty acid methyl ester, 2% partial glycerides, and 26% water. The
difference in sophorolipid productivity between pure glycerine and the crude material
was at least partially due to the presence of a relatively large exogenous fatty acid
source and the decreased osmotic stress put on the organism when crude glycerine was
used. In addition, by using crude glycerine that contained an appreciable amount of
fatty acid methyl esters, the conformational equilibrium was shifted from the lactonic
form towards the open-chain form. This was brought about by the presence of the
methyl group on the carboxyl end of the fatty acid which inhibited lactonization,
resulting in a larger concentration of open-chain sophorolipids, thereby facilitating
property control and permitting post-synthetic modification of the fatty acid side-
chain without the need to chemically break the lactone ring. The chemical structures
of the sophorolipid products derived from the glycerine samples were determined
by LC/APCI-MS (Ashby et al., 2005). The results proved that the structures of the
sophorolipids derived from pure and crude glycerine were indeed different (Fig. 2.4).
When the yeast was grown with pure glycerine, LC/APCI-MS analysis revealed the
formation of the sophorolipid lactone forms with the majority of the fatty acids being
either palmitic, stearic, or oleic as the main products (99%) with negligible amounts
of open-chain form. In contrast, the use of crude glycerine resulted in sophorolipids
that favored the open-chain form, accounting for GO% (24% free acid and 36%
methyl esters) of the total sophorolipid content with oleic acid and linoleic acid
predominating.
Soy molasses is aco-product stream from soybean processing (Fig. 2.5).It is derived
from the whole bean after flaking and removal of the crude soy oil through solvent
x
a
8
-
aL
Time (min.)
Fig. 2.4.The total ion current (TIC) chromatogramsof the sophorolipids derived from pure glycerine (A)
and crude glycerine (co-product of biodieselsynthesis), B).The peaks eluting above 17 min. correspond
to lactonic sophorolipids while the peaks eluting between 2 min. and 17 min. correspond to open-chain
sophorolipids (specifically,peaks eluting prior to 10 min. are free acid, open-chain moleculeswhile those
eluting between 10 and 17 min. correspond to the methyl esterified, open-chain forms) (Ashby, 2005).
Production and Modification of Sophorolipidsfrom Agricultural Feedstocks0 37
extraction. Then, after a grinding and sizing step to produce soy flour and soy protein/
concentrate, the remaining material is soy molasses. Soy molasses is high in potentially
fermentable carbohydrates (accounting for approximately 30% of the total solids) and
therefore has the potential to be a valuable feedstock in microbial fermentations. In
addition, the cost of this material can be as low as 20% of the commonly used glucose
substrate. Since feedstock costs are generally the driving force behind the economics
of biobased production systems, the inexpensive soy molasses could potentially result
in a large cost-saving measure for sophorolipid biosynthesis. Soy molasses is primarily
composed of sucrose, raffinose, and stachyose as soluble carbohydrates. When using
soy molasses in place of glucose in sophorolipid fermentations, we found that soy
molasses could indeed function as a substitute for glucose (Solaiman et al., 2004b)
Whole
Soybeans
Cleaning
Cracking
Dehulling
Flaking
Soy Flakes
I
Solvent Extraction
Spent Crude
Flakes Soy Oil
Grinding Refining
Soy Flour Sizing Soy Protein Soy Lecithin Refined
Concentrate Soy Oil
Isolate
Coproduct
Soy Molasses
Fig. 2.5. Soybean processing and the generation and carbohydrate content of soy molasses.
38 0 R.D. Ashby et at.
but only the sucrose fraction of the soluble carbohydrates was actually utilized by the
organism (Solaiman et al., 2007). The raffinose and stachyose remained unutilized
throughout the fermentations. The inability of the organism to utilize all the available
soluble carbohydrates resulted in diminished yields (Table 2.2) which were further
diminished when yeast extract and urea were completely omitted from the growth
media. A solution to this dilemma is currently being explored by our efforts to express
a-galactosidase activity in C. bombicokz, thus providing the organism a means to liberate
the a-D-galactose monosaccharides from the stachyose and raffinose molecules, thus
making more of the soluble carbohydrates utilizable to the organism. While yields
were affected by the change from glucose to soy molasses, sophorolipid structural
composition remained relatively constant. By using soy molasses in conjunction
with oleic acid as the lipid co-substrate, 97% of the sophorolipids were synthesized
in the lactone form. In comparison, glucose/oleic acid fermentations resulted in
sophorolipids that were greater than 99% lactonic. In addition, the distribution of
sophorolipid lactones between the two systems containing yeast extract and urea was
also comparable. As expected, C18:1 lactones predominated in both the glucose/oleic
acid and soy molasses/oleic acid fermentations with relative abundances of 80 and
8 I%, respectively. However, when yeast extract and urea were omitted from the growth
media the lactone distribution did show variability. From this data, we concluded that
soy molasses can be an effective substitute for glucose for sophorolipid fermentations
without sacrificing structural continuity. Also,when using soy molasses, it is possible
to omit the yeast extract and urea, thus reducing media cost, and still obtaining the
desired sophorolipid products. Success in the utilization of these abundant, low cost
co-product streams provides a new application for these materials and helps reduce
the cost of sophorolipid synthesis and co-product disposal.
Table 2.2. Maximum Volumetric Yields and Lactone Distribution for Sophorolipids
Produced by C. bombicolu from the Fermentation of Glucose or Soy Molasses with Oleic
Acid as the Lipid Source According to Hydroxy Fatty Acid Content
Substrates Hydroxy Fatty Acid Content (96)'
Carbohydrate Lipid Yield (Q/L)~ C16:O C18:O C181 C182 Others'
Glucose Oleic acid 100 8(3)d 2(2) 80(71) 3 1
Soy molasses Oleicacid 75 4(3) 6(6) 81(68) 5 4
Soy molasses* Oleic acid 53 17(17) 12(11) 57(38) 7 7
'Percentages were determined based on peak integration of the total ion current (TIC) of LC/
APCI-MS ( Nuiiez et al., 2001).
bExpressedas g purified sophorolipid/L (starting volume) of culture broth. Values are the
averages of two determinations with standard deviations of ~ 1 0 % .
'These include the C170, C18l-monoacetylated, and C18:3 sophorolipid species.
dAll parentheticalvalues under"Hydroxy fatty acid content" correspond to the % of that
sophorolipid that was in the w-1 conformation. Those entries without parentheticalvalues
were unresolvableunder the LC/APCI-MS method used.
'Yeast extract and urea were completely omitted from the growth media.
Production and Modificationof Sophorolipidsfrom Agricultural Feedstocks 0 39
Biochemistry/Enzymology
Research on sophorolipids in the past decades has largely focused on the development
of fermentation processes to generate large amounts of these “ecofriendly” materials
and bioengineering methods to achieve an overall lowering of the production costs.
Comparatively, fundamental research on the biochemical pathways for the biosynthesis
of sophorolipids from C. bombicokz is limited even though production from numerous
feedstocks has been reported. Earlier biochemical studies on the enzymes putatively
involved in the biosynthesis of sophorolipid in Rbodotorukz bogoriensis (formerly
Cundidu bogoriensis) have nevertheless provided valuable information important to
the understanding of the biosynthesis pathway for this glycolipid in related yeast
strains. The production of an extracellular sophorolipid by R. bogoriensis was first
reported by Tulloch et al. (1968a). These investigators determined from chemical and
NMRstudies that the structure of the primary sophorolipid produced by R. bogoriensis
was 13-[(2’-0-[~]-D-glucopyranosyl-[~]-D-glucopyranosyl)-oxy]docosanoic acid
in its 6’-mono- and 6’,6”-diacetylated forms. Ruinen (1963) and Ruinen and
Deinema (1 964) in fact had earlier described the production of extracellular lipids
in R. bogoriensis,but the definitive chemical structures of those compounds were not
elucidated.
Very recently, Nuhez et al. (2004) employed a previously developed LC/APCI-
MS method (Nuhez et al., 2001) to confirm the finding of Tulloch et al. (1968a)
that sophorolipids produced by R. bogoriensis contained a 22-carbon hydroxy fatty
acid (C22). In that same study, Nuhez et al. further reported that a small quantity
(< 10%) of the sophorolipids isolated from R. bogoriensh actually contained a hydroxy
fatty acid with 24 carbons (Nuiiez et al., 2004). Esders and Light (1972a) reported
that the sophorolipids of R. bogoriensis exist in di-, mono-, and un-acetylated forms,
in which the primary alcohol functions of the sophorose moiety were variously
esterified to an acetyl group. These investigators then pioneered the biochemistry
of sophorolipid biosynthesis in R. bogoriensis. The first enzymes they characterized
were the glycosyl- and acetyl-transferase activities of this yeast (Esders & Light,
1972b). With a combination of various column chromatographic techniques
(i.e., DFAE-Sephadex anion exchange, hydroxyapetite, and gel filtration) and a
disc electrophoretic separation step, these authors described the identification of
glucosyltransferases 1 and 2, which catalyze the addition of a glucose molecule in its
activated form (i.e., UDP-glucose) to the 13-hydroxydocosanoic acid (13-HDA) and
methyl 13-glucopyranosyloxydocosanoate,respectively, to form the mono- and di-
glucopyranosyl-13-HDA (i.e., sophorolipid). The two glucosyltransferases, however,
were co-purified throughout all the procedures and could not be individually separated
and/or isolated. The same study also described the presence of acetyltransferaseactivity
in the organism which was capable of catalyzing the transfer of an acetyl group from
acetyl coenzyme A (acetyl-CoA) to a mono- or un-acetylated sophorolipid. The
mono-glucopyranosyl- 13-hydroxydocosanote, however, was a poor substrate for this
acetyltransferase activity. The temporal appearance of these enzyme activities during
40 0 R.D. Ashby et al.
Post-synthetic EnzyrnaticKhemical
Modifications of Sophorolipids
In an effort to expand the applications of sophorolipids, many chemical and enzymatic
processes have been reported that seek to modify the sugar or fatty acid moieties of
sophorolipids. Enzymes have been used to alter the structure of sophorolipid in two
principal ways, either to modify the carbohydrate moieties or to modify the terminal
carboxyl group on the fatty acid moieties. The former approach was the first to be
investigated in a degradative sense. The earliest report used acetylesterase (EC 3.1 .I .6)
to remove both the 6‘ and 6” acetyl groups while maintaining the macrolactone ring
(Asmer et al., 1988). The analytical yield was reported to be only 30%, but the product
was not isolated. The authors suggested that hrther degradation andlor concurrent
opening of the lactone ring occurred, limiting yield. By contrast, later reports used
a hngal cutinase expressed in E. coli (de Koster et al., 1995) or immobilized C
rugosa lipase (Otto et al., 1999) and found that the 6’ acetyl group was removed
preferentially, again keeping the lactone intact. Molecular modeling concurred with
this observation: a structural model derived from NMR data (de Koster et al., 1995)
42 0 R.D. Ashby et al.
showed that the 6” acetyl group was hidden between the lipid chain and the sophorose
unit and so should be less accessible to enzymatic cleavage.
Proceeding in the synthetic direction, Bisht et al. (1999) showed that Novozyme
435” (immobilized lipase from Candida antarctica) could be used to add additional
acyl groups, either acetyl, acryloyl, or succinoyl, to the carbohydrates of acyclic
sophorolipid esters. With an excess of acylating reagent, both the 6’ and 6” hydroxyl
groups were modified to give the diacyl compounds. In the absence of an external
acylating group, or with only one equivalent, the terminal fatty acid carboxyl group
reacted intramolecularly to form a macrolactone different from the native one. NMR
data showed that the new lactone had formed at the 6” position (Fig. 2.6a). This
compound was then further reacted with Novozyme 435” and an excess of vinyl
acrylate to give the monoacryloyl derivative at 6’. The same non-native macrolactone
was observed by Nuhez, although in that case both the 6’ acetylated and non-acetylated
versions were formed (Nuhez et al., 2003). These by-products were isolated from a
reaction where the 6’, 6” diacetyl sophorolipid methyl ester was reacted with galactose
diacetonide using Novozyme 435”. The intended product was the transesterified
galactose ester, linking the sophorolipid carboxylate to the primary 6-hydroxyl group
of galactose, which was obtained, although deacetylation at the sophorolipid 6‘ and
6” groups had occurred to varying extents. This one-pot reaction with immobilized
lipase therefore produced sophorolipid that was modified at both the carbohydrate
head and the carboxylic tail. A similar macrolactonization, again using Novozyme
435”, was performed on a similar sophorolipid, namely one with a C22 lipid tail
(NuAa et al., 2004). These results were significant since the C22 sophorolipid is not
originally isolated in macrolactone form, but only as the acyclic free acid. It could not
be determined from mass spectral data whether lactonization had occurred at the 6’ or
6” site, although the latter was proposed by analogy with results in Bisht et al. (1999)
and Nuhez et al. (2003).
The next advance came in determining that monoacylation could be performed
regioselectively (Singh et al., 2003). Novozyme 435”, which, as described above,
gave diacetylated products, could be used to prepare 6’ monoacetyl derivatives of
sophorolipid ester when shorter reaction times and less vinyl acetate were used.
Alternatively, Lipase PS (isolated from Burkholderia cepacia; Amano Enzyme USA,
Elgin, IL) on a ceramic support gave completely opposite regioselectivity, producing
the 6” monoacetylated products. Amides of sophorolipid were also suitable substrates
for these reactions. The amides themselves (five analogs from para-substituted
phenethylamines) were prepared by Novozyme 435”-catalyzed amidation of the
desacetyl sophorolipid ethyl ester. A different set of substrates was used by Carr and
Bisht (2003) who employed per-acylated sophorolipid esters with four different lipases.
Here, the reaction was entirely at the carboxyl group to give either the n-butyl or isobutyl
ester; no de-acylation was observed. It was proposed that only when the macrolactone
was present could binding in the enzyme active site occur so that deacylation could
occur. With acyclic substrates, the carboxyl terminus was preferentially attacked. A
Production and Modification of Sophorolipidsfrom Agricultural Feedstocks 0 43
possible item for hture study would be to use the hexa-acetylated macrolactone.
The deacylation reaction was put to use for in situ polymerization by Hu and Ju
(2003). Any of four lipases could be used to first generate the 6’-hydroxy-”’-acetyl
sophorolipid macrolactone, which then underwent self-oligomerization by enzymatic
ring-opening, forming esters between the 6’ hydroxyl group and the C18 carboxyl
group. Unfortunately, the amount of oligomer extractable into solvents such as ethyl
acetate or tetrahydrofuran was small, reaction times were lengthy (10 or 20 days),
and the insoluble material could only tentatively be identified as polymer (a peak
corresponding to the nonamer was found by mass spectrometry). Finally, a more
extensive modification to the sophorolipid structure was effected by hesperidinase
(EC 3.2.1.40) or several other glycosidases (Rau et al., 1999, 2001). These enzymes
operated on the desacetyl acyclic free acid sophorolipid to remove the “outer” glucose,
leaving a monosaccharide glycolipid in 80% yield.
To the best of our knowledge, the only acyl groups that have been appended
enzymatically to the sophorolipid carbohydrates are acetyl, acryloyl, methacryloyl,
and succinoyl. It would be interesting to test how structurally variable is the set of
acyl donors tolerated by the lipases in conjunction with the sophorose headgroup
(more general studies on lipase utility have of course been performed). Branched
and aromatic acyl groups could afford novel and usehl sophorolipids. While the
methacryloyl group is a-branched, its vinyl unit is small, so it may be an exception.
Some of the earliest work on chemical modification to the sophorolipid skeleton
was performed in the course of initially determining the structure of the macrolactone
by Tulloch et al. (1968b).The techniques involved are essentially classical degradations
used in carbohydrate chemistry, such as peracetylation with HBr/acetic acid. Although
these methods apparently were applied afier these pioneering studies, they may bear
hrther investigation, since some interesting structural changes can be accomplished.
One intriguing derivative, for example, consists of a ring-opened form where the
C18 fatty acid remains esterified at the 4” hydroxy site, but has been cleaved at the
glycosidic 1’ site.
The more common method of ring opening, which has been employed in a
number of studies, is alkaline hydrolysis or alcoholysis. Sodium alkoxide solutions
remove the acetyl groups and concurrently form the acyclic alkyl ester of the fatty
acid chain. Methyl through hexyl (although not, apparently, pentyl) esters have been
prepared in this way (Bisht et al., 1999; Nuhez et al., 2003; Singh et al., 2003; Carr
& Bisht, 2003; Zhang et al., 2004). Sodium or potassium hydroxide generates the
sophorolipid free acid, after acidification. A related method uses primary amines to
form the sophorolipid amides (Rau et al., 2001), although ammonium chloride was
apparently necessary as a catalyst, since other workers reported that refluxing the
sophorolipid lactone with amines, presumably without ammonium chloride, did not
work (Singh et al., 2003). More hindered amines, such as diethylamine, required
prior deprotonation with n-butyl lithium (Rau et al., 2001). A milder method of
converting the carboxylic acid tail to an amide employed dicyclohexylcarbodiimideas
44 0 R.D. Ashby et al.
a coupling reagent with ethyl or benzyl esters ofa-amino acids (Azim et al., 2006). The
amino acids used included nonpolar (leucine and phenylalanine) and polar (aspartic
and glutamic acids) types. The sophorolipid variants were assayed for antimicrobial
activity against seven species, with mixed results: minimal inhibitory concentrations
ranged from 5 mglmL to 10-4 mglmL. Anti-HIV and antispermicidal effects were
also investigated.
Chemical methods have also been used to add groups to the carbohydrate units.
Peracetylation is readily accomplishedwith acyl anhydrides anddimethylaminopyridine
catalysis (Carr & Bisht, 2003). Selective acylation through non-enzymatic means has
not yet been demonstrated. Our own work used carbodiimide-mediated esterification
to affix amino acids to the carbohydrate hydroxyl groups of sophorolipid, but
a mixture of two major and one minor isomer was obtained (Zerkowski et al.,
2006). We hypothesized that the two major isomers are the 6’ and 6” derivatives.
An aminobenzoate linker unit was used (Fig. 2.6b) at the point of attachment to
the hydroxyl groups, since we found that aliphatic acyl groups (e.g., N-protected
glycine) were readily cleaved in the presence of weak bases and protic solvents, while
benzoates resisted such decomposition. The amino acids were primarily polar ones
such as hydroxyproline, glutamic acid, and lysine, which were chosen to increase
water solubility of the sophorolipid. We also used carbodiimides to prepare esters of
the fatty acid chain from the sophorolipid free acid and an alcohol, particularly benzyl
alcohol. The sophorolipid skeleton was not decomposed by treatment with 100%
trifluoroacetic acid or by catalytic hydrogenation over palladium on carbon (Azim et
al., 2006, Zerkowski et al., 2006).
Polymers of sophorolipid have been prepared through chemical means by two
routes. First was free-radical polymerization of the 6’ monoacryloyl 6” macrolactone
(Bisht et al., 2000). Co-polymers with acrylic acid and acrylamide were also prepared.
Molecular weights were on the order of lo4 glmol, and the material was soluble in
polar aprotic solvents dimethylformamide and dimethyl sulfoxide but not in water.
A B
Fig. 2.6. The non-natural macrolactone linking the carboxyl group to the 6 hydroxy group that is
produced enzymatically (Bisht et al., 1999) when a substoichiometric amount of external acylating
reagent is used (A). An example of a head-group-modified sophorolipid where a polar unit (here,
hydroxyproline) is attached via a linker unit (para-aminobenzoic acid) to one of the sophorose hydroxy
groups (6).
Production and Modification of Sophorolipidsfrom Agricultural Feedstocks 45
Concl usions
Many of the glycolipid biosurfactants, including sophorolipids, are making inroads
for potential industrial application simply because they can be synthesized biologically
in relatively large concentrations and they also exhibit properties that lend themselves
favorably as substitutes for or additives to currently used petroleum-based surfactants.
Reports suggest that sophorolipid concentrations on the order of hundreds of grams
per liter can be attained under suitable fermentation conditions and that some of
those sophorolipids (depending on their predominant fatty acid content and structural
form) possess surface active properties that approximate those shown by commonly
used surfactants. As further work continues to elucidate the biochemical pathways
involved in sophorolipid synthesis and to synthesize new structural variants through
post-synthetic chemical/enzymatic modifications, new products with improved
properties will be produced with diminished costs. This will make these molecules
even more favorable for industrial application and may also help stimulate the search
for new methods to utilize abundant, low value co-product materials such as crude
glycerine and soy molasses.
Mention of trade names or commercial products in this artick is soklyfor the purpose of
providing specifc information and does not imply recommendation or endorsement by the
US.Department OfAgricultUre.
References
Abe, Y.; S. Inoue; A. Ishida. Cosmetic Composition for Skin and Hair Treatment. US.Patent
M 2 9 Z 3 4 0 . 1 9 8 1.
Allingham, R. Sophoroside Esters in Prepared Food Products. US.Patent #3,622,344. 1971.
Ashby, R.; A. Nufiez; D. Solaiman;T. Foglia. Sophorolipid Biosynthesis from a Biodiesel Co-
Product Stream.J Am. Oil Cbem. Soc., 2005, 82,625-630.
Ashby, R.; D. Solaiman;T. Foglia. Property Control of Sophorolipids: Influence of Fatty Acid
Substrate and Blending. Biotecbnol. Lett., 2008,30, 1093-1 100.
Asmer, H-J.; S. Lang; F, Wagner; V. Wray. Microbial Production, Structure Elucidation and Bio-
conversion of Sophorose Lipids.J. Am. Oil Cbem. Soc., 1988, 65, 1460-1466.
h i m , A.; V. Shah; G. Doncel; N.Peterson; W. Gao; R. Gross. Amino Acid Conjugated Sophoro-
46 0 R.D. Ashby et al.
lipids: A New Family of Biologically Active Functionalized Glycolipids. Bioconj. Chem., 2006,
17, 1523-1529.
Banat, I.; R Makkar; S. Cameotra. Potential Commercial Applications of Microbial Surfactants.
Appl. Microbiol. Biotechnof., 2002, 53, 495-508.
Bednarski, W.; M. Adamczak; J. Tomasik; M. Plaszczyk. Application of Oil Refinery Waste in the
Biosynthesis of Glycolipids by Yeast. Biores. Technof.,2004,95, 15-18.
Bisht, K.; R. Gross; D. Kaplan. Enzyme-Mediated Regioselective Acylations of Sophorolipids.
J. Org. Chem., 1999, G4, 780-789.
Bisht, K.; W. Gao; R. Gross. Glycolipids from Candid? bombicola: Polymerization of a 6-0-Acry-
loyl Sophorolipid Derivative. Macromolecules, 2000,33,6208-6210.
Borzeix, C. Use of Sophorolipids Comprising Diacetyl Lactone as Agent for Stimulating Skin
Fibroblast Metabolism. US.Patent #6,596,21;5. 2003.
Brakemeier, A.; D. Wullbrandt; S. Lang. Candida bombicola: Production of Novel AIkyl Gluco-
sides Based on Glucosel2-Dodecanol. Appf. Microbiol. Biotechnol., 1998,50, 161-166.
Breithaupt, T.; R. Light. Atfinity Chromatography and Further Characterization of the Glucosyl-
transferases Involved in Hydroxydocosanoic Acid Sophoroside Production in Candida bogorien-
sis. J. Biol. Chem., 1982,257, 9622-9628.
Bucholtz, M.; R. Light. Acetylation of 13 Sophorosyloxydocosanoic Acid by an Acetyltransferase
Purified from Candid? bogoriensis. J. Biol. Chem., 1976a, 251,424-430.
Bucholtz, M.; R. Light. Hydrolysis of 13 Sophorosyloxydocosanoic Acid Esters by Acetyl and
Carboxyesterases Isolated from Candida bogoriensis. J. Biol. Chem., 1976b, 251,431-437.
Carr, J.; K. Bisht. Enzyme Catalyzed Regioselective Transesterification of Peracetylated Sophoro-
lipids. Tetrahedron, 2003,59, 7713-7724.
Cooper, D.; D. Paddock. Torubpsis petrophilum and Surface Activity. Appl. Environ. Microbiol.,
1983,4G, 1426-1429.
Cutler, A.; R. Light. Regulation of Hydroxydocosanoic Acid Sophoroside Production in Candiuk
bogoriensis by the Levels of Glucose and Yeast Extract in the Growth Medium. J. Biol. Chem.,
1979,254, 1944-1950.
Cutler, A.; R. Light. Effect of Glucose Concentration in the Growth Medium on the Synthesis of
Fatty Acids by the Yeast Candida bogoriensis. Can. J. Microbiol., 1982, 28, 223-230.
Daniel, H-J.; M. Reuss; C. Syldatk. Production of Sophorolipids in High Concentration from
Deproteinized Whey and Rapeseed Oil in a Two Stage Fed Batch Process Using Candid?
bombicoh ATCC 222 14 and Ctyptococcus curvatus ATCC 20509. Biotecbnol. Lett., 1998,20,
1153-1 156.
Davila, A-M.; R. Marchal; J-I? Vandercasteele. Sophorose Lipid Production from Lipidic Precur-
sors: Predictive Evaluation of Industrial Substrates. J. Ind. Mirrobiol., 1994, 13, 249-257.
de Koster, C.; W. Heerma; H. Pepermans; A. Groenewegen; H. Peters; J. Haverkamp. Tandem
Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy Studies of Candida bombi-
cola Sophorolipids and Product Formed on Hydrolysis by Cutinase. Anal. Biochem., 1995,230,
135-148.
Deshpande, M.; L. Daniels. Evaluation of Sophorolipid Biosurfactant Production by Candida
Productionand Modification of Sophorolipids from Agricultural Feedstocks 0 47
Yeast Candida apicokz: Heterogeneity and Polymerase Chain Reaction-Mediated Cloning of Two
Genes. Yeart, 1996, 12, 565-575.
Mager, H.; R. Rothlisberger; E Wagner. Cosmetic Composition Containing One Sophorose
Lipid-Lactone and Its Use. European Patent EP0207783. 1987.
Maingault, M. Pharmaceutical and Cosmetic Compositions Containing Sophorolipids. Canadian
Patent cAN126242874.1997.
Maingault, M. Utilization of Sophorolipids as TherapeuticallyActive Substances or Cosmetic
Products, in Particular for the Treatment of the Skin. US.Patent #5,981,477,1999.
Makkar, R.; R. Cameotra. An Update on the Use of Unconventional Substrates for Biosurfactant
Production and Their Applications. Appl. Microbiol. Biotecbnol., 2002, 58, 428-434.
Mulligan, C.; R. Yong; B. Gibbs. Heavy Metal Removal from Sediments by Biosurfactants.
J Hamrd. Mater., 2001, 85, 1 1 1-125.
Muthusamy, K.; S . Gopalakrishnan;T. Ravi; I? Sivachidambaram. Biosurfactants: Properties,
Commercial Production and Application. Cum Sci., 2008,74,736-747.
National Biodiesel Board. Ektimated US. Biodiesel Production by Fiscal Year, http://www.biodie-
sel.org/pdf_files/fuelfactsheets/production-graph-slide.pdf, , 2007.
Nufiez, A,; R. Ashby; T. Foglia; D. Solaiman. Analysis and Characterization of Sophorolipids by
Liquid Chromatography with Atmospheric Pressure Chemical Ionization. Chromatographia,
2001,53,673-677.
Nufiez, A,; T. Foglia; R. Ashby. Enzymatic Synthesis of a GalactopyranoseSophorolipid Fatty
Acid-Ester. Biotechnol. Lett., 2003, 25, 1291-1297.
Nufiez, A,; R Ashby; T. Foglia. LC/MS Analysis and Lipase Modification of the Sophorolipids
Produced by Rbodotorukz bogoriensis. Biotechnol. Lett., 2004,26, 1087-1093.
Otto, R.; H. Daniel; G. Pekin; K. Miiller-Decker; G. Fiirstenberger; M. Reuss; C. Syldatk.
Production of Sophorolipids from Whey. 11. Product Composition, Surface Active Properties,
Cytotoxicity and Stability Against Hydrolases by Enzymatic Treatment. Appl. Microbiol. Biotecb-
nol., 1999,52495-501.
Pekin, G.; F. Vardar-Sukan; N. Kosaric. Production of Sophorolipids from Candida bombicokz
ATCC 22214 Using Turkish Corn Oil and Honey. En5 Life Sci., 20055,357-362.
Rau, U.; C. Manzke; F. Wagner. Influence of Substrate Supply on the Production of Sophorose
Lipids by Candida bombicokz ATCC 22214. Biotecbnol. Lett., 1996, 18, 149-154.
Rau, U.; R. Heckmann; V. Wray; S. Lang. Enzymatic Conversion of a Sophorolipid into a Glu-
cose Lipid. Biotecbnol. Lett., 1999,21,973-977.
Rau, U.; S. Hammen; R. Heckmann; V. Wray; S. Lang. Sophorolipids:A Source for Novel Com-
pounds. Ind. Crop Prod., 2001, 13, 85-92.
Ruinen, J. The Phyllosphere 11. Yeasts from the Phyllosphere of Tropical Foliage. Antonie van
Leeuwenhoek, 1963,27,425-438.
Ruinen, J.; M. Deinema. Composition and Properties of the Extracellular Lipids of Yeast Species
from the Phyllosphere. Antonie van Leeuwenhoek, 1964,30, 377-384.
Scholz, C.; S. Mehta; K. Bisht; V. Guilmanov; D. Kaplan; R. Nicolosi; R. Gross. Bioacrivity of
Extracellular Glycolipids-Investigation of Potential Anti-Cancer Activity of Sophorolipids and
Production and Modification of Sophorolipidsfrom Agricultural Feedstocks 0 49
Introduction
Biosurfactants are synthesized by several microorganisms and/or by enzymatic
treatment (Banat et al., 2000) and are assigned to three natural roles in the microbial
environment: (a) increasing the surface area of hydrophobic water-insoluble growth
substrates; (b) increasing the bioavailability of hydrophobic substrates by enhancing
their apparent solubility or desorbing them from surfaces; (c) regulating the attachment
and detachment of microorganisms to and from surfaces.
Microbial surfactants are used in a broad range of industrial applications, including
enhanced oil recovery, crude oil drilling, lubrication, surfactant-aided bioremediation,
health care, cosmetics, and food (Fiechter, 1992; Lin, 1996; Dossat et al., 2002;
Eliora & Rosenberg, 2002; Kitamoto et al., 2002; Lang & Trowitzsch-Kienast,
2002; Sekhon, 2006). The high-molecular-mass bioemulsifiers are amphipathic
polysaccharides, proteins, lipopolysaccharides, lipoproteins, or complex mixtures of
these biopolymers, which are effective at stabilizing oil-in-water emulsions. The low-
molecular-mass microbial surfactants are generally lipopeptides, such as surfactin,
gramicidin S, and polymyxin or glycolipids, such as mannosylerythritol lipids,
sophorolipids, and rhamnolipids, which primarily reduce surface and interfacial
tensions (Rosenberg & Ron, 1999). Biosurfactants possess important advantages
compared to chemical surfactants. These include mild production conditions, lower
toxicity, higher biodegradability, and environmental compatibility. Recently, common
reviews about biosurfactants were published by Van Hamme et al. (2006), Lu et al.
(2007) and Singh et al. (2007).
Among these amphiphilic molecules, the glycolipids represent a potent group
of biosurfactants. They are low-molecular-weight products ( ~ 1 , 5 0 0g mol-I). The
constituent mono-, di-, or oligosaccharides include glucose, mannose, galactose,
-51 9
52 0 U. Rau and D. Kitamoto
glucuronic acid, rhamnose, and galactose sulphate. The lipophilic moieties contain
saturated, unsaturated, and/or hydroxylated fatty acids or fatty alcohols (Karanth et
al., 1999; Lang, 2002). Mannosylerythritol lipid (MEL) is one of the most promising
glycolipids for widespread applications in the future. Beside mannose and fatty acids/
alcohols of different chain lengths, it also contains the sugar alcohol erythritol.
One of the reasons for increase interest in MEL is due to their pharmaceutical
applications (Shibahara et al., 2000; Zhao et al., 2000). For example, Aventis
Pharma Deutschland GmbH published a patent about a MEL, named Ustilipid,
that is produced by Ustilago maydis DSM 11494 and can be used in the treatment
of schizophrenia or diseases caused by dopamine metabolic dysfunction (Vertesy et
al., 2002). However, other applications, such as chemical tools for purification of
proteins (Im et al., 2003; Kitamoto et al., 2000) or anti-agglomeration agents of ice-
slurry (Kitamoto et al., 2OO1b) are also known. Kitamoto et al. (2002), reviewed the
functions and potential applications of MEL.
Despite the feasible use of MEL in such manifold areas, a real breakthrough with
this component has not yet occurred. One reason could be that the development and
scale-up ofa biotechnical production plant including thecost-intensive downstreamwill
require more money and time effort compared to a chemical surfactant manufacturing
process if there is no outstanding characteristic which justifies the higher investment.
The different applications for MEL are covered in another chapter of this book and
should stimulate commercial interests when combined with this chapter concerning
recent findings of bioreactor production and downstream processing of MEL.
Fig. 3.1. Molecular structure of mannosylerythritol lipid. The length and saturation of the fatty acid
residues (R2,R3)depend on the substrate and microorganismused. R4,R6 = acetyl or H. Ball-stick-model:
Diacetylated MEL-A with two C100 fatty acid esters at position C-2’and C-3:
DSM 4500 in a shake flask with sunflower oil fatty acids and glucose as substrates.
However, the production of an additional glycolipid (cellobiose lipid or ustilagic acid)
could only be lowered by using a suitable ratio of these substrates but not hlly avoided.
Geotrichum candidum ST 0025 15 also secretes a mixture of both cellobiose lipid and
MEL (Kurz et al., 2003). Deml et al. (1980) used Schizonella melanogramma for the
production of MEL that was termed Schizonellin. Candida sp. B-7 (Kawashima et
al., 1983) and Kurtzmanomycessp. 1-1 1 (Kakugawa et al., 2002) are also known to be
producers of MEL (Table 3.1).
Pseudozyma (formerly Candidz) is the most potent genus for the secretion of
MEL. Primarily in the 1990s, Kitamoto and co-workers thoroughly investigated
the production characteristics and substrate influence on MEL structure by
using Pseudozyma (Candidz) antarctica T-34 (Table 3.2). Pseudozyma strains are
usitilaginomycetous anamorphic yeasts, which are classified into Ustilagininales and
show a close relationship (97% identity) from the sequence of ITS1, 5,8S rRNA
gene, and ITS2 (Morita et al., 2006a). Based on this sequence homology, analysis of
ribosomal DNA on yeast strains of the genus Pseudozyma was undertaken and yielded
I! ruplosa NBRC 10877 as a novel MEL producer (Morita et al., 2006b).
The results of Tables 3.1 and 3.2 obviously show that a prediction of the fatty
acids introduced into the MEL is not possible when employing the same substrate
(soybean oil) since it depends on the strain used. Related to the microorganisms listed
in Table 3.1, a broader spectrum of fatty acids was detected compared to the genus
Pseudozyma. However, all substrates were degraded by the release of one C2-unit at
minimum. In the past, this partial &oxidation was only known in mammalian cells
and was first described for the yeast I! antarctica by Kitamoto et al. (1998) as a chain-
shortening pathway.
Table 3.1.Variation of the Fatty Acid Moiety in MEL Depending on Microorganism and Substrate
Schizonella
Ustilago maydis melanogramma Kurtzmanomyces
Microorganism Candida sp. 6-7 Candidasp. 6-7 Candida sp. SY 16 DSM 4500 GD 325 sp.1-1 1
Substrate Mixture of n-alkanes Soybean oil Soybean oil Sunflower fatty acidsa Glucose Soybean oil
Reference Kawashima et al. 1983 Kawashima et al. 1983 Kim et al. 1999 Spoeckner et al. 1999 Deml et al. 1980 Kakuqawa et al. 2002
MELb A SchizonellinA/B
Fatty acid
(%, WIW)
c40 9.1
c5:o 1.5
C69 49 19.6 1.4
c79 4.2
C80 18 19.2 0.7 36.4
C81 1.1
c99 21.6
c10 0 1.9 9.7 0.7 1.2
C10l 0.5
c102 2.0
c1 l:o 13.7
c120 24 25 23 3.0 ~
c130 142
c140 2.4 45.7 19 3.8 6.2
0 c141 9 42.9
z
c)
u_
c10 71.3 58.6 72 - 5 67 55.7 14.8 59 6 57 12 53 16 50 61.5 32 22.7 51.3 51.9 1
c101 - 6.2 46.4 - _ _ - _ - _ - - 3
-
c102 - - - - - _ - - - - - - 1.
o_
c11 10.1 13.3 - 40 40 1 1.2 0.2 - 45 <1 42 5 40 11 - 23.1 4.4 0.7 r:
C11:l - - _ _ _ _ - _ _* - -
~
P
c12 - 6 - - 18 1.9 2.0 15 3 14 2 8 2 9 - - 1.6 2.5 2.7 E
c131
C14 -
-
3
-
0.2 0.8 -
-
-
-
_
-
-
-
-
-
-
-
-
-
-
-
-
z-
0
c141 - - - - - - - - - - - - 3
Q
c142 - - _ - - - - - - - 0
"A mixture of MEL was analysed if not indicatedotherwise 2
b2-dodecanol, 2-dodecanone yielded only slightly different data 2
'3-dodecanone
1
3
d2-tridecanol,2-tridecanone yielded only slightly different data -u
'3-tridecanone a
n
'2-tetradecanol, 2-tetradecanone yielded only slightly different data a5.
3
gfatty acid methylester were analysed by GC after hydrogenation 0
0
Ln
Ln
56 0 U. Rau and D. Kitamoto
The fatty acid distribution in MEL derived from the same genus but a different
strain Pseudozyma aphidis DSM 70725 is shown in Table 3.3. Odd fatty acid esters
were primarily shortened to C7, C9, and C11 acids. The amount of the C7 acid
increased with the chain length of the substrate, but generally only small quantities
of the C7 acid were present. Even fatty acid esters were degraded to C8, C10, and
C12 with a preference for CIO, particularly for the unsaturated ones. The double
bond in the C18:l ester completely disappeared. The C18:2 ester was transformed
predominately to C1O:l acid. Soybean oil contains about 50% (w/w) C18:2 and
25% (wlw) C18:l fatty acids. The degradation pattern of the soybean oil fatty acids
was similar to that of the even fatty acid methyl esters, although the quantities were
different. The sum of C1O:O and C1O:l made up approximately 70% (w/w) of all
esters with the exception of stearic acid methyl ester as substrate, which yielded lower
amounts. Fatty acids larger than C14 or smaller than C7 were detected only in trace
amounts. Likewise C6 fatty acids were also not detected. These findings are in contrast
to the composition of MEL secreted by Candida sp. B-7 (Kawashima et al., 1983) and
Candida sp. SYl6 (Candida antarctica KCTC 7804) (Kim et al., 1999), respectively.
Both microorganisms were also cultivated using soybean oil as substrate (Table 3.1).
MEL B-7 and MEL SYl6 contained 45.7 and 19.0% (w/w), respectively, C14:O fatty
acids. The last MEL also contained 9.0% (wlw) C14:l and notable amounts of C6:O
(49.0% (w/w)). The MEL of Kurtzmanomyces sp. 1-1 1 (Kakugawa et al., 2002) even
contained 25.9% (w/w) C14:2. However, all these other MEL showed a relative low
content ~ 1 0 % (w/w) of C10 fatty acids. MEL-T34 (Table 3.2) was similar to the
MEL of I? aphidis but differed in the fatty-acid content by an average of 5 1 0 % (w/w)
depending on the substrate used (Kitamoto et al., 1995,2001a). However, some clear
distinctions exist. For example, when using oleic acid methyl ester as a substrate the
resulting MEL differed by 10-20% (w/w) with respect to the concentrations of C8:O,
CIO:O, and C1O:l fatty acids (Kitamoto et al., 1993). Additionally, when soybean oil
was used Kitamoto et al. (1990a) did not find C10:l fatty acids, but the fatty acids
were subsequently detected as methyl esters by GC after a hydrogenation step.
Among the high-level MEL producers, such as I? antarctica and I? apbidis, MEL-A
is formed in largest amount. In contrast, Pseudozyma tsukubaensisJCM 10324 (Morita
et al., 2007c) and Pseudozyma sp. KM-160 (Konishi et al., 2007) selectively produce
only a single glycolipid product, which corresponds to MEL-B, in high yield (more
than 25 g L ’ from 4% (w/w) of soybean oil). A detailed inspection of the ’H and
I3C-NMR data derived from the I? tsukubaensis product showed that beside the known
MEL-B one diastereomer having a different chirality is also formed. The diastereomer
was identified as 1-0-(6’-0-acetyl-2’,3’-di-O-alka(e)noyl-i3-D-mannopyranosyl)-D-
erythritol, indicating that erythritol is bound to mannose in a manner different from
all known MEL (Fukuoka et al., 2008).
Among the other MEL homologs, the more hydrophilic MEL C can be produced
in increased amounts of 65% (w/w) and 85% (w/w) by Pseudozyma hubeiensis KM-59
(Konishi et al., 2008) and Pseudozymagraminicokz CBS 10092 (Morita et al., 2008).
Mannosylerythritol Lipids: Productionand Downstream Processing 0 57
Table 3.3. Influence of the Substrate (Soybean Oil or Fatty Acid Methyl Ester with Different
Chain Length) on the Fatty Acid Compositionof the MEL Secreted by Pseudozymaaphidis
DSM 70725"
Fatty acid content (96,w/w) of MELs and MEL A after cultivation on
Soybean oil Fatty acid methyl ester
Fatty acid type C15:O C160 C17:O C180 C181 C182
C7:O O.lZb 0.04 2.69 - 6.84 0.44 0.04 0.06
C80 18.54b 12.96 0.20 15.52 0.27 15-96 20.66 12.65
C81 2.65b 2.38 - - - - - -
c90 O.lOb 0.20 44.63 0.22 41.18 1.68 0.53 0.07
c9: 1 - - 0.08 - 0.12 - - -
c100 36.18b 55.83 1.18 71.55 1.48 57.10 74.81 17.49
c101 31.46b 16.81 0.31 0.26 1.86 0.99 56.69
c102 0.51b 0.10 - - - - - 0.48
c1 l:o 0.23b 0.39 48.50 0.52 45.36 3.41 0.16 0.19
C11:l - - 0.94 - 0.19 - - -
c12:o 2.53b 2.78 0.16 10.42 0.15 21.76 1.74 1.24
C12:l 0.94b 0.92 0.07 0.73 0.05 4.67
C13:O - - 0.73 - 0.35 0.48 - -
C13:l - - 0.09 - - - - -
C14:O 0.05b 0.06 0.02 0.16 - 1.12 - 0.36
c141 - 0.15 - - - - - -
c142 - 0.25 - - - - - 2.45
'Related to soybean oil, isolated me1 a (left column) was additionally analyzed beside the
mixture of mels.
bpureMEL A
Rau et al., 2005c.
MEL, mono-acylated at R3 (Fig. 3.1) without any acetyl groups at the mannose
moiety, can be produced by using I? antarctica T-34 and JCM 10317 as well as Z?
parantarctica JCM 11752 and glucose as sole substrate. Due to its increased hydrophilic
character and higher water solubility, the CMC (critical rnicelle concentration) value
o f 3 . 6 ~ 1 0M
. ~ is 100-fold higher compared to the MEL hitherto reported and therefore
Edvors the use as oil-in-water type emulsifiers and washing detergents. However, the
yields are low (1.1-1.3 g 1-') and the di-acylated MEL are formed likewise (Fukuoka
et al., 2007b).
Recently, a more hydrophobic tri-acylated MEL was produced from cultures
using I? antarcticaT-34 and I? ruplosa NBRC 10877. Soybean oil served as substrate.
R2 and R3 carry the common medium-chain fatty acids (C8-Cl4). The third, long-
chain fatty acid (Cl6 and C18) is linked to the primary hydroxyl group of erythritol.
Morita et al. (2007b) assumed that this component could be directly introduced
into the MEL from soybean oil by mediation of extracellular lipase and/or esterase,
58 0 U. Rau and D. Kitamoto
which are present during growth on triglycerides. 'The assumption was confirmed by
successful esterification of purified di-acylated MEL-A with methyl oleate by using
the culture supernatant of I! mgulosa and Novozyme 435 (lipase).
Also a new MEL type, with shorter hydrocarbon chain (C2 or C4) at R2 but again
di-acylated, was found (Fukuoka et al., 2007a) by cultivation of the basidiomycetous
yeast Pseudozyma shanxiensir CBS 10075 (Wang et al., 2006) on soybean oil. Due to
shorter ester, an enhanced hydrophilicity of the molecule is assumed.
A nal yses
In order to quantitatively detect and separate MEL A-D, triglycerides and fatty acids,
a 3 mL culture suspension was acidified with two drops of 5 N HCI to p H 2 and was
subsequently extracted three times with 3 mL methyl tertiary butyl ether (MTBE). The
mixture was vortexed for 1 min and centrifuged for 5 min at 6000 rpm. The organic
phases were combined and analyzed either by TLC or HPLC. The distinct separation
of single spots for qualitative TLC analysis can be carried out by the development of
Silica gel 60 F254 (Merck, Germany) plates with the ternary solvent system CHCI,/
MeOH/H,O (65:152). HPLC is performable on a silica gel column (Nucleosil 100-5;
CS-Chromatographie Service GmbH, Langenvehe, Germany) with an evaporative
light scattering detector (ACS Mass Detector model 750/14; Houston, TX, USA)
using a gradient solvent program consisting of various proportions of CHCl, and
CH,OH (from 99:l to 0:lOO (v/v)) at a flow rate of 1 mL min-' (Fig. 3.2).
A qualitative and rapid method to determine MEL or other biosurfactants formed
throughout a cultivation can be attained by the drop collapse method (Kuiper et al.,
2004; Hewald et al., 2005; Morita et al., 2007b), which estimates the surface tension
of the culture medium (Fig. 3.3).
The size of a constant volume drop which spreads on a surface is a measure for the
content of MEL.
Biosynthesis
The biosynthetic route of MEL formation was thoroughly examined with Pseudozyma
antarctica T-34 (Kitamoto et al., 1993., 1998) and Ustilago maydis (Hewald et al.,
2006) and is depicted in Fig. 3.4.
Both strains are able to utilize triglycerides as soybean oil. Therefore, extracellular
action of a lipase/esterase is necessary for the cleavage into free fatty acid and glycerol.
Lipase activity from l? antarctica was also noted when the strain was grown on glucose,
and the activities reached levels corresponding to approximately 50% of that obtained
with soybean oil (Morita et al., 2007b). After ester cleavage, glycerol is consumed by
the cells for the production of energy and growing but not for MEL formation (Rau
et al., 2005b). Likewise the released fatty acid is transported into the cell and coupled
to CoA. One part of the activated fatty acid must be &oxidized to acetyl-CoA to
supply energy and precursors for anabolic pathways. Subsequent, mannose is de novo
synthesized by gluconeogenesis. Erythritol is formed through the gluconeogenesis and
MannosylerythritolLipids: Production and Downstream Processing 0 59
Fig. 3.2. HPLC and TLC of MEL containing culture suspension extracted with MTBE. FA=Fatty acid; SO=
Soybean oil.
Fig. 3.3. Secretion of MELs reduce the surface tension of culture supernatant (Morita et al. 2007b).
The effects of MELs on the surface tension of the culture medium were visualized by placing 50 pI of
culture supernatant of fseudozyma antarctica T-34 grown with glucose, glycerol, or soybean oil (40 g I.’
respectively), as the sole carbon source on the hydrophobic surface of Parafilm-M. The pictures of the
droplets formed were taken from side (upper panel) and top view (lower panel).
phosphogluconate pathways yielding erythrose which can be converted into the sugar
alcohol by action of erythrose reductase which was verified in Candida magnoliae
(Lee et al., 2003). However, this enzyme has not yet been found in I! antarctica or
1/. maydis. Enzymes indicated with asterisks in Fig. 3.4 were verified in 1/. maydis
(Hewald et al., 2005, 2006) and should be also present in I! antarctica. The activated
GDP-mannose is linked to erythritol by a glucosyltransferase. The transcription of
60 0 U. Rau and D. Kitamoto
Soybean oil
Lipase +H,O
Esterase
Acyl-CoA
Chain shortenin &-Oxidation
pathway
Mannose Erythritol
GDP-Mannose
*GIUCQS~I-
transferase
GDP
Mannosylerythritol
CoA
'Acyltransferase
C2-,CS-acylated MEL
2 Acetyl-COA
"Acetyltransferase
MEL A
C4-.Cf%acetvlated
*Major facilitator
Fig. 3.4. Presumptive biosynthesis of MEL-A in U. rnuydis and f! unturcticu based on soybean oil as
substrate. Enzymes indicated with an asterisk were verified so far in U. rnuydis only. The scheme was
adopted from Kitamoto et al. 1998 and Hewald et al. 2006.
Mannosylerythritol Lipids: Production and Downstream Processing 0 61
the related gene is strongly induced under conditions of nitrogen starvation. ‘The
intermediate mannosylerythritol has been isolated in significant amounts from MEL-
producing cells (Boothroyd et al., 1956). The shortening of acyl-CoA was in the past
only found in mammalian peroxisomes until Kitamoto et al. (1998) could verify this
pathway in I? antarctica.
Even fatty acids, as contained in soybean oil, are primary degraded down to
C8-, C10-, and C12-acids. Odd chain substrates yield predominantly C7-, C9-,
and C1 1-acids. ‘The regulation of this partial &oxidation is still unknown. ‘There is
no preference for fatty acids connected at position C-2’ or C-3’ in I? antarctica. In
contrast, U. maydis links shorter fatty acids (C2-C8) at position C-2’ and medium or
longer fatty acids at C-3’ (Spoeckner et al., 1999; Kun. et al., 2003). The final step
of biosynthesis of MEL A proceeds with an acetyl-CoA dependent acetyltransferase
which displays relaxed regioselectivity and is able to transfer acetyl groups to both
positions C-4’ and C-6’ of the mannosyl moiety. Wild-type cells of U. maydis secrete
large amounts of MEL which are acetylated at only one position (MEL-B/ MEL-C).
This could indicate that the second acetylation reaction is slower than the first one
since partially acetylated glycolipids are secreted before the second acetylation occurs.
Secretion of MEL in U. maydis is supposed to be catalyzed by the major facilitator
Mmfl because the suppression mutant for mmfr is unable to produce extracellular
MEL. ‘The exporter Mmfl appears to display only limited specificity for its substrates,
since deacetylated MEL are secreted as efficiently as the acetylated ones. Furthermore,
the observed spectrum of MEL carrying acyl groups of different lengths suggests that
the putative exporter does not distinguish between these derivatives.
Production
Substratesand Shake Flask Cultivation
Depending on the microorganism used (Table 3. l), manifold substrates (Tables 3.2
and 3.3) can be applied for MEL production as pentoses, hexoses, sugar alcohols,
soluble starch, alkanes, alkanols, alkanones, fatty acids, and their methyl or ethyl esters
as well as triglycerides (Kawashima et al., 1983; Kitamoto et al., 2001a; Kim et al.,
2002b; Sugita et al., 2003). In principle, glucose can be used for MEL formation but
always with reduced yields compared to hydrophobic substrates (Kim et al., 2002a;
Morita et al., 2007b). Therefore, vegetable oils (in particular soybean oil), fatty acids,
and their esters are the most suitable carbon sources for MEL production although a
further increase of yield can be attained by addition of glucose as co-substrate (Rau
et al., 2005b; Konishi et al., 2008). A variety of common triglycerides and fatty acids
used for MEL production by I? aphidis shows Fig. 3.5.
Beside soybean oil, oleic acid (containing 70% (vh) pure C18:l fatty acids),
and mixtures of different fatty acids derived from sunflower oil are also suitable
carbon sources. Even biodiesel (rapeseed oil methyl ester) from a petrol station was
convertible. O n an average, 250% (vh) of substrate is converted into the product
except for glycerol, which is not a proper carbon source for MEL formation using
62 0 U. Rau and D. Kitamoto
Soybesn Sunflower Rapeseed Corn oil Biodiesel Oleic scld Sunflower Glycerol
oil oil oil fatty acids
Fig. 3.5. MEL formation of R aphidis depending on the carbon source used (medium A). The cultivations
were terminated after 10 days (Rau et al. 2005~).Biodiesel=rapeseed oil methyl ester. Medium A (per liter
deionised water): 80 ml carbon source, 2 g NaNO,, 0.2 g KH,PO, 0.2 g MgS0,x7H20, 1 g yeast extract, pH
6.2 not adjusted, 27°C.
M6
Fig. 3.6. Ten days shake flask cultivation (medium see Fig. 3.5) of R aphidis using soybean oil (80 ml I-’)
and erythritol (E, 40 g I-’) or mannose (M, 40 g k’) as second carbon source added at day 0,2,4 or 6 (Rau
et al. 200%). Ref.: 10 days reference cultivation only with soybean oil.
obtained (Morita et al., 2006b). This high concentration of MEL resulted in increased
viscosity of the culture suspension with paste-like characteristics which prevents
hrther shaking and sufficient oxygen supply and, therefore, probably constitutes the
limit of MEL yield in shake flasks. Likewise, 140 g L-’ MEL were attained using
resting cells of I! unturcticu T34 during a 28 day 30 mL fed-batch shake cultivation
with a total of 156.6 g L ’n-octadecane as the carbon source. The cultivation started
with 46.2 g L ’n-octadecane and afier every 7 days the same amount of substrate was
added (Kitamoto et al., 2001a).
As a secondary metabolite, most of the MEL is secreted during the stationary
growth phase under nitrogen-limited conditions. However, the nitrogen source is
essential for prepended growing and subsequent maintenance purposes; it also
influences the MEL formation as well as the grade of acetylation (Fig. 3.7).
Sodium nitrate was the best nitrogen source followed by ammonium nitrate when
pH was not controlled. The drastic decrease to pH 2 was clearly the reason for the
decreased MEL formation observed when ammonium salts were used as a nitrogen
source. The consumption of ammonium led to acidification of the medium. When
only ammonium nitrate was used, the ammonium was initially consumed. Afier 4
64 0 U. Rau and D. Kitamoto
30 - = MELA
MEL B
= MELC
20 -
10 -
n
"-
day 7 11 21 7 11 21 7 11 21 7 11 21
NaNO, NH,CI NH,NO, (NH,),SO,
Fig. 3.7. MEL formation by k! aphidis depending on the nitrogen source used. Each flask contained
medium A (Fig. 3.5) with 23.5 mM nitrogen. MEL was quantified after 7, 11 and 21 days (Rau et al.
2005c).
days the total ammonium was assimilated and the nitrate was consumed, accompanied
by simultaneous increase of pH. The concentration of MEL C was highest at the
beginning and but decreased in favor of MEL B at later stages. A deacetylation at C-6'
with subsequent acetylation at C-4' took place. A preference for sodium nitrate was
also reported in studies of Candida sp. S-10 (Kawashima et al., 1983), I? antarctica
T34 (Kitamoto et al., 1990b), and I? rugulosa NBRC 10877 (Morita et a]., 2006b). In
contrast, Candida sp. SYl6 produced higher amounts of MEL using NH,NO, than
NaNO, (Kim eta]., 2002a).
Bioreactor
Only limited information about bioreactor production of MEL is available. Kim et
al. (1999) used Candida sp. SYl6 with soybean oil as substrate in a 5-L bioreactor for
the formation of 100 g L-'crude MEL. However, the crude product contained only
4% (w/w) pure MEL. One year later, Adamczak and Bednarski (2000) succeeded in
the production of 46 g L-' MEL equivalent to a productivity of 7.4 g L-' d' achieved
by using I? antarctica ATCC 20509 in a 5-L bioreactor with 80 g L' soybean oil and
p02-control at 50%. Two-stage culturing avoided foaming but decreased MEL yield
to 28 g L'.
The MEL are metabolites that are primarily secreted if the nitrogen source is
depleted and cells have entered the stationary phase. One method to increase the yield
MannosylerythritolLipids: Productionand Downstream Processing0 65
for resting cells is to raise the amount of product-secreting biomass. Results from l?
aphidis in shake-flask cultivations (Rau et al., 2005b) showed that glucose is a better
substrate for growth compared with soybean oil and was therefore added at the start.
The amount of sodium nitrate was increased to 3 g L ' . The initial concentration
of soybean oil was essentially reduced from 80 mL L' to 20 mL L ' but could not
be fully avoided due to foaming. Foam formation is a serious problem during the
production of MEL which can lead to essential discharge of culture suspension
accompanied with loss of product and risk of infection. Conventional anti-foam
agents, such as polypropylene, polyethylene glycol, or silicone oil, have to be avoided
because the microorganisms do not consume them and, therefore the agents pollute
the hydrophobic MEL fraction during the downstream process. However, soybean
oil can simultaneously act as substrate and anti-foam agent. As a result, a medium
with two carbon sources and intermittent as well as controlled feeding was used for
cultivation (Fig. 3.8)
The cells first consumed nitrate and attained a short period of resting conditions
after about 1 day. At this time, glucose was still not completely consumed. In
order to initiate regrowth, a concentrated solution of glucose (285 g L'), sodium
nitrate (16 g L'), and yeast extract (14 g L') was added at a feed rate of 125 mL
h' after 1.75 days. Substrate feeding was stopped after 2.8 days. The feed rate and
substrate concentrations resulted from a detailed analysis of the preculture maximum
consumption rates of glucose (1.25 g L' h'), sodium nitrate (0.07 g L-' h'), and yeast
extract (0.06g L-'hl). Cell protein and bio dry mass were qualitatively determined
to be in accordance with the period of substrate and soybean oil supply. However,
during the first growth phase and late stages of cultivation essential differences are
apparent due to the accumulation of storage material, probably lipids, inside the cells
(Fig. 3.9). Therefore, cell protein was chosen as an indirect measure of biomass.
A conductivity sensor triggered the addition of soybean oil, which was primarily
emulsified in the culture suspension while glucose was present. Between 0.75 and
3.75 days, depending on the actual foam formation, a total volume of 6.1 L soybean
oil was added. Most of the consumption of soybean oil took place after glucose was
consumed. At the end of cultivation, soybean oil and released fatty acids were totally
converted by the cells. If the addition of soybean oil was stopped at the proper time,
the microorganisms were able to convert the total substrate which would facilitate the
subsequent downstream process.
MEL was secreted during the substrate feed and after the added glucose was
consumed, in spite of excess nitrate being present up to a cultivation time of 6.8
days. This observation is in contrast to the accumulation process in oleaginous yeast
(Ratledge, 1997) where nitrogen limitation is a prerequisite for lipid biosynthesis.
Potentially, the following explanations could be argued. l? aphidis is not a mere
oleaginous yeast, because the MEL is secreted in addition to the accumulation of
lipids inside the cells (Fig.3. 9). Exhaustion of nutrients other than nitrogen can also
lead to the onset of lipid formation (Granger et al., 1993). The biosynthesis of lipids
66 0 U. Rau and D. Kitamoto
-
r
P)
Y
UI
I
E
f
U
0
ijj
+ 4
Time (days)
Fig. 3.8. Fed-batch cultivation of i? aphidis DSM 14930 in 30-L medium B with soybean oil and glucose
as carbon sources. Three Rushton turbine impellers, 300 rpm, aeration rate: 540 L h-1. After 1.75 days,
a concentrated substrate solution 5 (glucose 285 g I-', sodium nitrate 16 g I-],yeast extract 14 gl-') was
Fed with 125 ml h.l. Due to Foam control, 6.14 soybean oil (SBO) was added between 0.75 and 3.75 days.
Insert: Formation of MEL-beads. Medium B (per liter deionised water): 30 g glucose, 20 ml soybean oil,
3 g NaNO,, 0.2 g KH,PO, 0.2 g MgS0,x7H20, 1 g yeast extract, pH=6.2 not controlled, T=27"C (Rau et al.
2005b).
could also be initiated by the first nitrate limitation after 1 day; and is not shut down
in the hrther course of cultivation.
After 5.2 days, high concentrated MEL beads appeared at a concentration of
about 50 g L' MEL. HPLC analysis of these beads revealed MEL content greater
than 80% (w/w) beside soybean oil and fatty acids. Occasionally, these beads were
also noticed during shake-flask cultivation. Due to the fact that these beads only
occurred at concentrations greater than 40 g L-l MEL, they could be regarded as
indicators for enhanced MEL production. After 11.8 days, 3.8 g L-I cell protein and
Mannosylerythritol Lipids: Productionand Downstream Processing 0 67
Fig. 3.9. Storage substances in f! aphidis observable as refractive globules during the assimilation of
soybean oil (Rau et al. 2005b).
110 g L-I MEL resulted from the last sample. After the cultivation was finished, a
huge amount of sticky MEL adhered to the cap and internal wall of the vessel. This
MEL was collected, resuspended into the culture fluid; the final result was 165 g Ll.
Kim et al. (2006)used NH,NO, as the N-source, which led to drastic variation
of pH due to consumption of first ammonia (pH decrease) and subsequent nitrate
(pH increase). Therefore, batch cultivation of Cundidu sp. SYl6 under pH control
at 4.0 yielded 48 g L ' MEL after 200 h. A two-stage culture system, similar to Fig.
3.8,under suitable growth-limiting conditions and subsequent fed-batch technique
during the stationary production phase, raised the MEL yield to 95 g L' after 200 h.
The cultivation was started with a combination of glucose and soybean oil (each 15
g L')as the initial carbon sources during the cell growth phase. The first and second
addition of 70 g L'and 100 g L' soybean oil, during the N-limiting MEL production
phase, occurred after 44 h and 104 h. The self-decreasing pH from 7.5 was controlled
at 4.0 after 24 h. With this microorganism, the foam-stat strategy, using soybean oil
as both an antifoam agent and carbon source, was not successhl and did not lead to
increased yield. The authors suppose that a higher substrate pressure is necessary for
enhanced MEL formation, which was not present due to antifoam sensor controlled
addition of soybean oil. As a result, Table 3.4 summarizes maximum MEL yields
hitherto attained in shake-flask and bioreactor cultivation.
Downstream Processing
To date, in spite of interesting applications, a real breakthrough of MEL is hampered
due to their high production cost. Beside a high yield and productivity of the bioreactor
process, the subsequent downstream handling of the product is economically crucial.
Therefore, an easy to handle procedure is required for economic isolation and
purification of MEL.
68 0 U. Rau and D.Kitamoto
Table 3.4. Maximum MELYields Achieved by Various Strains and Culture Systems
Culture Yield Productivity .Y,?,
Microorqanism system (q L-’1 (g L-’ d’) (q q’ ) Substrates Reference
RrugulosaNBRC SF 143 5 0.5 Soybean oil/ Morita et al. 2006b
10877 Erythritol
R antarcticaT34 SF 140 5 0.89 n-octadecane Kitamoto et al. 2001a
Rantarctica ATCC BR 46 7.4 0.57 Soybean oil Adamuak & Bednarski
20509 2000
Candidasp.SY16 BR 95 11.4 0.45 Soybean oil/ Kim et al. 2006
Glucose
RaphidisDSM BR 165 13.9 0.92 Soybean oil/ Rau et al. 2005b
14930 Glucose
SF = Shake flask
BR = Bioreactor
.Yield coefficient = g MEL produced/ g substrate consumed
IOO%(w/w) MEL
Recovery 8% (w/w)
Fig. 3.10. Scheme of the stepwise extraction procedure by using different solvents for isolation and
purification of MEL. The TLC represents the repeated extraction steps by n-hexane. Lanes 1-6, organic
phase; lanes 7-12, aqueous phase; lanes 1,2 + 7,8, first extraction; lanes 3,4 + 9,10, second extraction;
lanes 5,6 + 11,12, third extraction. MTBE, methyl tertiary butyl ether; FA, fatty acid; SO, soybean oil (Rau
et al. 2005a).
70 0 U. Rau and D. Kitamoto
increases the manufacturing costs so that an acceptance of this bioprocess for industry
is further inhibited.
The MEL can also be isolated by preparative HPLC equipped with silica gel
columns (Kim et al., 1999; Kitamoto et a]., 2000; Rau et al., 2005b). 'This is a superior
method to produce a very pure MEL mixture or even to separate the individual MEL
A-D. However, the loss ofproduct is substantial, and so this is not a beneficial solution
in order to decrease the costs for the downstream process.
When the bioreactor production process is finished and the MEL-containing
culture suspension is transferred into a glass bottle, the separation of aggregated MEL
beads can be observed at the bottom as highly viscous fluid (Fig.3.11, left picture).
'This viscous MEL phase and the MTBE extract (Fig. 3.10) of the whole culture
suspension possessed a similar composition (Fig. 3.1 1). After sterilization of the
MEL-containing culture suspension at 121°C for 20 min, two MEL-containing
phases, a solid sticky and an aqueous one, were formed, both fatty acid enriched as
well as soybean oil depleted (Fig. 3.1 I, right picture). A small volume of a primary
v)
9
U
m
Fig. 3.1 1. Composition of different MEL phases from a culture suspension before (left) and after (right)
heat treatment at 121°C for 20 min (Rau et al. 2005a).
Mannosylerythritol Lipids:Production and Downstream Processing 0 71
soybean oil-containing top phase was also observed. After heating, the MEL extracted
by MTBE were distributed into the solid and aqueous phases at 89% and 11%
(w/v), respectively. This solid phase was easy to separate by pouring off the cell
debris-containing supernatant. About 11% (v/v) of MEL remained suspended in the
aqueous cell debris phase and could also be recovered by extraction with ethanol,
centrifugation, rotary evaporation of the solvent, and vacuum drying.
A comparison of known MEL downstream procedures is given in Table 3.5.
Unfortunately, to the best knowledge of the authors, only two references are available
with a quantitative description of the different methods. The heat treatment attained
the highest yield and is the fastest method by far, but only on average 87% (w/w)
MEL is contained in the precipitated fraction. However, this solid-enriched MEL
phase should be pure enough for most industrial applications (Kitamoto et al., 2001 b;
Hua et al., 2003, 2004; Im et al., 2003). For example, pure MEL-A, purified 100%
(w/w) MEL A-D, and a mixture of 88.3% MEL, 6.6% soybean oil and 5.1% (w/w)
fatty acids reduced the surface tension of water/air to similar data of 34.7, 26.7, and
31mN m-', respectively (Rau et al., 2005b).
Table 3.5. Different Quantitatively Described Methods for the
Downstream Processing of MEL
Method bYield Purity Reference
% (w/w) % (w/w)
Ethyl acetate extraction + preparative aHPLC 79 1 00 Kitamoto et al. 2000
Ethyl acetate extraction + preparativeaHPLC 4 100 Kim et al. 1999
Stepwise extraction with different solvents 8 100 Rau et al. 2OOSa
Heat treatment 93 a7 Rau et al. 2005a
aPreparativeHPLC was performed with silica gel column.
gMEL recovered
x 100
byield= g MEL before downstream
Conclusions
The recent results of MEL bioreactor production and downstream research show that
yields far over 100 g L' are possible by the use of the proper microorganism and
cultivation technique. In combination with facilitated heat treatment as a downstream
process, these findings should stimulate the industrial production of MEL.
References
Adamczak, M.; W. Bednarski. Influence of Medium Composition and Aeration on the Synthesis
of Biosurfactant Produced by Cundida unturcticu. Biotechnol. Lett. 2000,22, 3 13-3 16.
Banat, I.M.; RS. Makkar; S.S. Cameotra. Potential Commercial Applications of Microbial Surfac-
rants. Appl Microbiol Biotechnol2000,53,495-508.
Boothroyd, B.; J.A. Thorn; R.H.Haskins. Biochemistry of the Ustilaginales.XI. Characterization
of Extracellular Glycolipids Produced by Ustilago sp. Can. /. Biochem. Physiol. 1956,34, 10-14.
Crich, D.; M.A. Mora; R. Cruz. Synthesis of the Mannosyl Erythritol Lipid MEL A; Confirma-
72 0 U. Rau and D. Kitamoto
Karanth, N.G.K.; EG. Deo; N.K. Veenanadig. Microbial Production of Biosurfactants and Their
Importance. Cum Sci. 1999, 77, 116126.
Kawashima, H.; T. Nakahara; M. Oogaki; T. Tabuchi. Extracellular Production of a Mannosy-
lerythritollipid by a Mutant of Candida sp. from n-Alkanes and Triacylglycerols. J. Ferment.
Zchnol. 1983, GI,143-149.
Kim, H.-S.; J.-W. Jeon; B.-H. Kim; C.-Y. Ahn; H.-M. Oh; B.-D. Yoon. Extracellular Production
of a Glycolipid Biosurfactant, Mannosylerythritol Lipid, by Candidz sp. SYl 6 Using Fed-Batch
Fermentation. App.! Microbiol. Biotechn. 2006, 70,39 1-396.
Kim, H.-S.; B.D. Yoon; D.H. Choung; H.-M. Oh; T. Katsuragi; Y. Tani. Characterization of a
Biosurfactant, MEL, Produced from Candida sp. SYl 6. Appl. Microbiol. Biotechnol. 1999,52,
71 3-721.
Kim, H.S.; J.W. Jeon; H.W. Lee; Y.I. Park; W.T. Seo; H.M. Oh; T. Katsuragi; Y.Tani; B.D. Yoon.
Extracellular Production of a Glycolipid Biosurfactant, Mannosylerythritol Lipid, from Candida
antarctica. Biotechn. Lett. 2002a, 24, 225-229.
Kim, S.W.; H.J. Hwang; J.P Park; Y.J. Cho; C.H. Song; J.W Yun. Mycelial Growth and Exo-
Biopolymer Production by Submerged Culture of Various Edible Mushrooms Under Different
Media. Lett. Appl. Microbiol. 2002b, 34, 5G-61.
Kitamoto, D.; S. Akiba; C. Hioki; T. Tabuchi. Extracellular Accumulation of Mannosylerythritol
Lipids by a Strain of Candida antarctica.Agric. BioL Chem. 199Oa, 54, 31-36.
Kitamoto, D.; K. Haneishi; T Nakahara; T. Tabuchi. Production of Mannosylerythritol Lipids by
Candida antarctica from Vegetable Oils. Agri. Bio. Chem. 1990b, 54, 37-40.
Kitamoto, D.; T. Yokoshima; H. Yanagishita; K. Haraya; H.K. Kitamoto Formation of glycolipid
biosurfactant, mannosylerythritol lipid, by Candida antarctica from aliphatic hydrocarbons via
subterminal oxidation pathway. JJpn Oil Chem SOC1999,48, 1377-1384.
Kitamoto, D.; S . Ghosh; 0. G.; Y. Nakatani. Formation of Giant Vesicle from Diacylmannosy-
lerythritols and Their Binding to ConcanavalinA. Chem. Comm. 2000, 10, 861-862.
Kitamoto, D.; T. Ikegami; G. Suzuki; A. Sasaki; Y. Takeyama; Y. Idemoto; N. Koura; H. Yanag-
ishita. Microbial Conversion of n-Alkanes into Glycolipid Biosurfactants, Mannosylerythritol
Lipids, by Pseudozyma (Candida) antarctica. Biotechnol. Lett. 2001a, 23, 1709-1714.
Kitamoto, D.; H. Isoda; T. Nakahara. Functions and Potential Applications of Glycolipid Biosur-
factants - from Energy-Saving Materials to Gene Delivery Carriers./. Biosci. Bioeng, 2002, 94,
187-20 1.
Kitamoto, D.; T. Nemoto; H. Yagishita; T. Nakane; H. Kitamoto; T. Nakahara. Fatty Acid
Metabolism of Mannosylerythritol Lipids as Biosurfactants Produced by Candida antarctica
-Incorporation of Medium-Chain Fatty Acids as Beta-Oxidation Intermediates into Glycolipids.
J. Jap. Oil Chem. Soc. 1993, 42, 346358.
Kitamoto, D.; H. Yanagashita; K. Haraya; H.K. Kitamoto. Effect of Cerulenin on the Production
of Mannosyl-Erythritol Lipids as Biosurfactants by Candida antarctica. Biotechnol. Lett. 1995,
17,25-30.
Kitamoto, D.; H. Yanagishita; A. Endo; M. Nakaiwa; M. Nakane; T.Akiya. Remarkable Antiag-
glomeration Effect of a Yeast Biosurfactant, Diacylmannosylerythritol, on Ice-Water Slurry for
Cold Thermal Storage. BiotechnoL Progress 2001b, 17, 362-365.
74 0 U. Rau and D. Kitamoto
Ratledge, C. Microbial Lipids. Biotechnology. Rehm, H.J., G. Reed, Eds.; VCH Verlagsgesellschaft:
Weinheim, 1997; Vol7, 133-197.
Rau, U.; L.A. Nguyen; H. Roeper; H. Koch; S. Lang. Downstream Processing of Mannosyleryth-
ritol Lipids Produced by Pseudozyrna aphidis. Eur. /. Lipid Sci. Technol. 2005a, 107, 373-380.
Rau, U.; L.A. Nguyen; S. Schulz; V. Wray; M. Nimtz; H. Roeper; H. Koch; S. Lang. Formation
and Analysis of Mannosylerythritol Lipids Secreted by Pseuhzyma aphidis. Appl. Microbiol.
Biotechnol. 2005b, 66, 551-559.
Rau, U.; L.A. Nguyen; S. Schulz; V. Wray; M. Nimtz; H. Roeper; H. Koch; S. Lang Formation
and analysis of mannosylerythritol lipids secreted by Pseudozyma aphidis. Appl Microbiol Bio-
techno1 2005c, 66, 551-559.
Rosenberg, E.; E.Z. Ron. High- and Low-Molecular-MassMicrobial Surfactants.Appl. Microbiol.
Biotechn. 1999,52, 154-162.
Sekhon, B.S. Biosurfactants: An Overview. Nut. Acad. Sci. Lett. 2006,29, 317-332.
Shibahara, M.; X. Zhao; Y. Wakamatsu; N. Nomura; T. Nakahara; C. Jin; H. Nagaso; T, Murata;
K.K. Yokoyama. Mannosylerythritol Lipid Increases Levels of Galactoceramide in and Neurite
Outgrowth from PC12 Pheochromocytoma Cells. Cytotechnol. 2OOO,33,247-25 1.
Singh, A.; J.D. Van Hamme; 0.P.Ward. Surfactants in Microbiology and Biotechnology: Part 2.
Application Aspects. Biotechn. Adv. 2007,25, 99-121.
Spoeckner, S.; V. Wray; M. Nimtz; S. Lang. Glycolipids of the Smut Fungus Ustikzgo maydb from
Cultivation on Renewable Resources. Appl. Microbiol. Biotechnol. 1999,51, 33-39.
Sugita, T.; M. Takashima; N. Poonwan; N. Mekha; K. Malaithao; B. Thungmuthasawat; S.
Prasarn; l? Luangsook; T. Kudo. The First Isolation of Ustilaginornycetous Anamorphic Yeasts,
Pseudozyma Species, from Patients’ Blood and a Description of Two New Species: I!parantarctica
and I! thailandica. Microbiol. Immunol. 2003, 47, 183-1 190.
Syldatk, C.; F. Wagner. Production of Biosurfactants. Biosu8actants and Biotechnology. Kosaric, N.,
W.L. Cairns, N.C.C. Gray, Eds; Marcell Dekker, Inc.: New York, 1987; Vol25, 89-120.
Van Hamme, J.D.; A. Singh; 0.P Ward. Physiological Aspects: Part 1 in a Series of Papers De-
voted to Surfactants in Microbiology and Biotechnology. Biotechn. Adv. 2006,24, 604-620.
Vertesy, L.; M. Kurz; J. Wink; G. Noelken. Ustilipides, Method for the Production and the Use
Thereof, US 6,472,158, 2002.
Wang, Q.-M.; J.-H. Jia; F.-Y. Bai. Pseudozyma hubeiensis sp. nov. and Psetufozyma shanxiensis sp.
nov., Novel Ustilaginomycetous Anamorphic Yeast Species from Plant Leaves. Int. /. Syst. Evol.
Microbiol. 2006,56,289-293.
Zhao, X.; X. Geltinger; S. Kishikawa; S. Ohshima; S. Murata; S. Nomura; S. Nakahara; K.K.
Yokoyarna. Treatment of Mouse Melanoma Cells with Phorbol 12-Myristate 13-Acetate Coun-
teracts Mannosylerythritollipid-Induced Growth Arrest and Apoptosis. Cytotechnol. 2000,33,
123-130.
Advances in Bioprocess
Development of Rhamnolipid and
Sophorolipid Production
Neissa M. Pinzon, Qin Zhang, Srujana Koganti, and Lu-Kwang Ju
Department of Chemical and BiomolecularEngineering,The Universityof Akron, Akron, OH 44325-3906
Introduction
Rhamnolipids and sophorolipids are two major glycolipid biosurfactants that have
attracted significant attention because of their potential medical, cosmetic, hygienic,
environmental, and other industrial applications. The focus of this chapter is on the
microbial-fermentation processes for the industrial production of these glycolipids.
Some brief descriptions about their structures, properties, and applications are
included, particularly those affecting the designs and operations of the fermentation
processes.
d
Fig. 4.1. Common types of rhamnolipids found in the Pseudornonas species: (a) Rha-C10-C10, (b)
Rha-C10, (c) Rha-Rha-C10-C10,and (d) Rha-Rha-00.
Advances in Bioprocess Development of Rhamnolipid and Sophorolipid Production 0 79
COOH
Lactonic SL Acidic SL
Fig. 4.2. Structures of lactonic and acidic sophorolipids(SLs) (Hu & Ju, 2001a).
Properties
As their synthetic-surfactant counterparts, rhamnolipids and sophorolipids can reduce
the surface and interfacial tensions (IFTs), resulting in excellent detergent, emulsifying,
foaming, and/or dispersing properties. For instance, rhamnolipids reduce the IFT of
watedkerosene systems from 43 to <1 mN/m (Parra et al., 1989), and the surface
tension of water from 72 to <30 mN/m (Nitschke et al., 2005). Low critical micelle
concentration (CMC) properties were reported for different rhamnolipid molecules:
5 mg/L for the di-rhamnolipid Rha-Rha-C10-C10, and 40 mg/L for the mono-
rhamnolipid Rha-C1 0-C10 (Nitschke et al., 2005). Rhamnolipids with shorter fatty-
acid chains, such as Rha-C10 and Rha-Rha-C10 (Fig. 4,1), have larger CMC values
of about 200 mg/L. The pKa of a mono-rhamnolipid mixture in water measured
by potentiometric titration was determined to be 4.28 f 0.16 and 5.50 f 0.06 for
concentrations below and above the CMC of this rhamnolipid, respectively (Lebron-
Paler et al., 2006). In this same study, 'H nuclear magnetic resonance (NMR) titration
and spectrochemical titration using attenuated total reflectance Fourier transform-
80 0 N.M. Pinzon et al.
infrared (FT-IR) were also used to measure the pKa of the mono-rhamnolipid.
These analyses resulted in a value of 4.39 f 0.06 and 4.84 f 0.05, respectively. The
first value was measured at a concentration close to the CMC, and the latter at a
concentration higher than the CMC. The hydrophilicllipophilic balance (HLB) of a
rhamnolipid produced by l? aeiuginosa UG2 was estimated to be 24.1 using group
contributions and 17.0 using a correlation of HLB with CMC for sodium carboxylic
acids (Noordman et al., 2002). Another study using l? aeruginosa AT10 reported an
HLB value of 10.07 (Abalos et al., 2004).
Sophorolipids have diverse physicochemical properties because of their different
molecular structures, particularly the presence of both lactonic and acidic forms.
Properties such as lowering surface tension, foam-formation capacity, solubility in
organic solvents and water, and antimicrobial activity are affected by the degree of
lactone formation. In general, lactonic sophorolipids are more effective in lowering
the surface tension of water, and have stronger antimicrobial activity, whereas the
acidic sophorolipids at pH >pKa are more foaming and more soluble in aqueous
solutions. The presence of acetyl groups also affects the properties of sophorolipids:
for example, acetyl groups lower the hydrophilicity and, reportedly, enhance the
antiviral- and cytokine-stimulating effects of the glycolipids (Shah et al., 2005).
Accordingly, the diacetylated lactonic sophorolipids have oily property, and are
not readily soluble in water, whereas the nonacetylated acidic sophorolipids are readily
soluble in water. Purified diacetylated lactonic sophorolipids have solubility no more
than 70 mg/L (Otto et al., 1999). The properties of acidic sophorolipids are expected
to vary with pH. For example, the solubility of acidic sophorolipids in water (pH
uncontrolled) was reported to be 0.4 g/L (Rau et al., 1999), but was found to be in
the range of 12-15 g/L in phosphate buffers at a pH of 6-8 (Hu & Ju, 2001a). The
reported values of lowered surface and IFTs as well as the CMC in aqueous solutions
also varied substantially, presumably due to the different composition and purity of
the samples and the measurement methods employed. Davila et al. (1993) reported
that sophorolipids could lower the surface tension ofwater from 72.8 mN/m to 40-30
mN/m, with a C M C of 40 to 100 mg/L. A mixture of purified lactonic sophorolipids
lowered the surface tension of water to 36 mN/m, with a CMC of 10 mg/L (Otto
et al., 1999). Purified lactonic sophorolipids (10 mg/L) reduced the IFT between
n-hexadecane and water from 40 mN/m to about 5 mN/m, relatively independent
of a pH (in the range of 6-9) and a temperature (in the range of 20-90°C). O n the
other hand, a mixture of acidic sophorolipids could reduce the IFT of hexadecane or
several vegetable oils to 1-2 mN/m (Rosenberg & Ron, 1999). Most recently, Ashby
et al. (2008) reported the following properties for four sophorolipid mixtures (with
292% being lactonic): minimal surface tension-35-36 mN/m, CMC-35 to >200
mg/L, and canola (rapeseed) oil IFT-3-7 mN/m.
Similarly, a wide range of HLB values was reported: lower for acetylated lactonic
sophorolipids and higher for acidic sophorolipids (pH-dependent). For example, the
HLB was given as 3-8 for a commercially available mixture of acidic and lactonic
Advances in Bioprocess Development of Rhamnolipid and Sophorolipid Production 0 8 1
Applications
Both rhamnolipids and sophorolipids were reported as biosurfactants that are
biodegradable, nontoxic to humans, and able to be produced from renewable
resources (Desai & Banat, 1997; Furuta et al., 2003). These properties make these
glycolipids attractive for more environment-friendly usages of surfactants. Many
applications were also proposed and/or demonstrated in the food, cosmetic, and
pharmaceutical industries. Excellent reviews on the applications of these glycolipids
are available elsewhere (Maier & Soberon-Chavez, 2000; Mulligan, 2004; Nitschke et
al., 2005; Singh et al., 2007; Van Bogaert et al., 2007). Only some of them are briefly
mentioned here.
Due to their excellent effectiveness in solubilizing and emulsifying hydrocarbons,
rhamnolipids are proven to be one of the most effective surfactants for bioremediation
and enhanced oil recovery (Nguyen et al., 2008; Wang et al., 2007). They also have
antibacterial, antifungal, mycoplasmacidal, and antiviral activities that promote
their potential applications in the pesticide industry (Nitschke & Costa, 2007).
In addition, one can obtain rhamnose from rhamnolipids. Rhamnose is currently
Table 4.1. Comparison of Some Propertiesof Glycolipid Biosurfactants and
Synthetic Surfactants
Surfactant Min. SurfaceTension (mN/m) CMC (mg/L)
Sophorolipids 33-37 10->200
Rhamnolipids 26-29 5-200
Detergent alkylate dodecylbenzene 47 590
Sodium dodecyl sulfate 37 2000-3000
Abbreviation: CMC, critical micelle concentration.
82 0 N.M. Pinzon et al.
produced by extracting quercitrin from oak bark, naringin from citrus peels, or rutin
from oak bark or other plants. This process generates considerable amounts of toxic
wastes, and involves corrosive materials (Chayabutra et al., 2001). O n the other hand,
rhamnolipids can be easily hydrolyzed to produce rhamnose.
Sophorolipids are used as a cleaning agent in dishwashers (Furuta et al., 2003).
They also were proposed to be used in the petroleum industry because their emulsifying
properties and surface and IFT-lowering properties are not significantly affected even
at high salt concentrations. They are useful in washing the drill-cutting polluted by
hydrocarbons and in regenerating hydrocarbons from mud (Baviere et al., 1994).
Also,sophorolipids have properties such as inhibiting free radical and elastase activity,
stimulating the dermal fibroblast metabolism and collagen neosynthesis, and the
fibrinolytic property (Hillion et al., 1998; Maingault, 1999). These properties make
sophorolipids attractive for cosmetic formulations. One can also use the antibacterial
properties of sophorolipids to fight against infectious diseases (Napolitano Lena,
2006). In addition, purified sophorolipids showed cytotoxic effects on cancer cells,
and therefore, had potential applications in liver-cancer treatment. The anticancer
activity was effected by triggering apoptosis in the H7402 human liver-cancer cell
(Chen et al., 2006a).
Rhamnolipid Production
Producing Microorganisms and Characteristics
The production of rhamnolipids is a unique characteristic of the opportunistic
pathogen I? aeruginosa. While some isolates of the nonpathogenic I? rlororaphis
(Gunther et al., 2005) and the pathogenic Burkholderiapseudomallei (Haussler et al.,
2003) also produce rhamnolipids, the product concentrations (or yields) obtained in
the fermentations using I? aeruginosa are, so far, significantly higher than those from
the other species. Various I? aeruginosa strains-for example, DSM 2874 (Trummler
et al., 2003), ATCC 9027 (Chen et al., 2004), ATCC 10145 (Chayabutra & Ju,
2001), and UG2 (Mata-Sandoval et al., 2001)-are reportedly good producers of
rhamnolipids.
Rhamnolipids can be obtained with batch fermentation (Chayabutra et al.,
2001; Chen et al., 2007) and continuous culture (Chen et al., 2003; Gruber et al.,
1993), and in the processes using immobilized cells (Wagner et al., 1984; Wilson
& Bradley, 1996) and/or resting cells (Wagner et al., 1984). Since rhamnolipids are
predominantly reported as the secondary metabolites overproduced by cultures under
certain nutrient limitation, the fermentation strategies for rhamnolipid production
commonly involve designs of growth-limiting conditions or employ resting cells. In
these cases, rhamnolipid levels sharply increase as a consequence of the limitation of
one or more components in the medium.
We demonstrated the production of rhamnolipids when the culture reaches the
stationaryphase ofgrowth due to thelimitation ofnitrogen or another essential nutrient
(Chayabutra et al., 2001). Optimal p H and temperature for rhamnolipid production
Advances in Bioprocess Development of Rhamnolipid and Sophorolipid Production 0 83
under aerobic conditions and in continuous culture were 6.2-6.4 and 32"C-34"C,
respectively (Guerra-Santos et al., 1986). We confirmed that rhamnolipid production
is sensitive to pH. At a lower pH of 6.0, the specific rhamnolipid production rate
was approximately 4 mg of rhamnose per gram of cells (dry weight) per hour [mg
of rhamnose/(g CDW-h)], while at a higher pH of 6.5, it increased to 12.5 mg of
rhamnose/(g CDW-h).
Rhamnolipid production is not only affected by the bacterial strain selected, but
also by the carbon source used; the nitrogen and other nutrient concentrations in
the medium; the culture conditions such as pH, temperature, and dissolved oxygen
concentration; and the design or approach of the fermentation process. We can use
various carbon sources for rhamnolipid production, for example, glycerol, ethanol,
glucose, and corn oil (Nitschke et al., 2005). The Pseudomonus species can also use
hydrocarbons for growth in aerobic conditions. The rhamnolipid concentrations
obtained are usually higher when n-alkanes and vegetable oils are used as the carbon
sources as compared to when water-soluble carbon substrates are used (Nitschke et al.,
2005). However, in the absence of oxygen, hydrocarbons cannot be degraded, at least
not effectively, for industrial-production purposes, by these microorganisms. Recent
efforts to improve the yield of rhamnolipids were also directed toward genetically
engineered strains and the use of low-cost feedstock or agricultural by-products, such as
vegetable oils and the residue from the vegetable-oil refinery, as substrates (Benincasa,
2007; Mercade & Manresa, 1994). For example, the rhamnolipid production from
different carbon sources varies significantly: ~ 1 . 2g/L using corn oil (Anna et al.,
2002), 1.5 g/L using glucose (Guerra-Santos et al., 1984), 6.9 g/L using glycerol
(Anna et al., 2002), and 45 g/L using rapeseed oil (Trummler et al., 2003).
Currently, the industrial production of rhamnolipids faces significant obstacles
in high production costs and low production rates as compared to the production of
synthetic surfactants and certain other microbial products, including sophorolipids.
One particular process challenge is caused by the fast and stable foam generated
in aerobic I! ueruginosu fermentations (Wu & Ju, 1998). Rhamnolipids, with their
free carboxylic-acid group (Fig. 4.1), and other lipidic metabolites produced by Iz
ueruginosu cause extreme foaming in the fermentation broth at the near neutral pH
optimal for the bacterial fermentation. (The foaming was much more subdued at a
pH lower than 5.5.)
Foaming is common in aerobic fermentations. Mechanical foam breakers
and chemical antifoaming agents were developed for controlling the foaming in
many fermentation processes. Unfortunately, the foaming in aerobic rhamnolipid
fermentation appears extremely fast, and is too stable to be handled by these methods
(Siemann &Wagner, 1993). In addition, chemical antifoams can have adverse effects
on the downstream recovery and purification processes besides the considerable
extra costs associated with the large amounts of antifoams required to match the
rhamnolipids produced along the fermentation process. Different strategies were
attempted to break the foams. For example, we used an external vessel to hold and
84 0 N.M. Pinzon et al.
collapse the overflowed foams, with an automatic acid addition if necessary (Wu
& Ju, 1998). The obtained broth was pumped back into the fermentor after the
foam was broken down. Siemann and Wagner (1993) used a similar setup for their
process employing immobilized cells. However, even with this setup, the foaming
was still difficult to control, and the aeration rate had to be significantly lowered.
This caused oxygen limitation, and compromised cell viability as well as rhamnolipid
productivity.
We proposed an alternative to the conventional aerobic fermentation approach
by taking advantage of the capability of l? aeruginosa to perform denitrification as
a complementary or alternative respiration route. The denitrification approach can
completely eliminate the problems associated with the severe foaming nature of
rhamnolipid broth (Chayabutra et al., 2001; Chen et al., 2003). We describe the
progress in the development of the denitrification-based fermentation technology in
more detail in a later section.
PA12
Fig. 4.3. Two known quorum-sensing systems (/as and rho in f! aeruginosa involved in rhamnolipid
synthesis (Chen et al., 2004).
Advances in Bioprocess Development of Rhamnolipid and Sophorolipid Production 0 85
medium. Each A1 binds to its specific recipient protein (LasR or RhlR), forming
an AI-protein complex that activates and/or regulates various genes, including the
rhlAB and rhlG genes for rhamnolipid synthesis (Chen et al., 2005). Note that the
AI-protein complexes also activate the rhll and lad genes, thus “auto-inducing” the
A1 syntheses themselves.
In the kzs system, PA11 [N-(3-oxododecanoyl) homoserine lactone] is first
synthesized by Lad (an AI synthase). Subsequently, PA1 1 binds with the hR-encoded
transcriptional activator, LasR (Chen et al., 2004). In the same way, the rhl system
controls the synthesis of the second AI, PA12 (N-butyryl homoserine lactone).
?he enzyme that catalyzes this reaction is the AI synthase RhlI. PA12 binds to the
transcriptional activator protein RhlR, forming the RhlR-PAI2 complex, which
controls the expression of rhlAB and rhlG. ?he two quorum-sensing systems are not
independent (Fig. 4.3). For instance, the production of PAI2 is not only promoted by
the RhlR-PAI2 complex but also by the LasR-PAI1 complex from the h quorum-
sensing system (Chen et al., 2005).
We studied the kinetics of PA12 synthesis and degradation in the batch
fermentation of I! aeruginosa using the wild-type PA01 and its rhll(-) and rhlR(-)
mutants (Chen et al., 2005). The rhll(-) and rhlR(-) mutants were first found to
produce insignificant amounts of rhamnolipids as compared to the wild-type I?
aeruginosa PAOl. Then, to study the PA12 degradation kinetics, the AI-bearing
supernatant was collected from a batch culture of PAO1, and added to two systems
for comparison: one system contained the washed cells of rhlZ(-) mutant collected
from a continuous culture maintained at the dilution rate of 0.025 hour-’; the other
system was cell-free. The use of rhll(-) cells was to ensure that no PAI2 synthesis
occurred during the experiment so that the degradation could be examined apart
from biosynthesis. The results are shown in Fig. 4.4. They indicated that the PA12
degradation was predominantly cell-associated. While the A1 concentration decreased
insignificantly in the cell-free systems (especially when considering the range of data
fluctuation caused by the sensitive bioassay), clear degradation was observed in the
systems containing rhll(-) cells.
PA12 concentrations were next followed along the batch fermentation of the
wild-type PAOl (Fig. 4.5) and the rhlR(-) mutant (Fig. 4.6). Both AI synthesis and
degradation were expected to take place in the two systems, but the synthesis kinetics
might differ: the synthesis would be autoinduced by the RhlR-PA12 complex in the
wild-type culture but not in the rhlR(-) mutant (without RhlR production in the
latter). The stationary-phase decay of PAI2 in the wild type was slower than that in
the rhll(-) mutant (Fig. 4.5). Assuming that the mutant had the same degradation
kinetics as the wild type, the observed difference in stationary-phase decay rates could
be attributed to the PA12 synthesis occurring with the wild type [but, as expected,
not with the rhll(-) mutant]. Nonetheless, the synthesis rate was slower than the
degradation rate, resulting in the net decay profile seen in Fig. 4.5. Furthermore,
we observed different PAI2- concentration profiles during the growth phase of the
86 0 N.M. Pinzon et al.
120
110
8 100
50
40
30
0 50 100 150 200 250 300 350
Time (Min)
Fig. 4.4. Degradation profiles of Al PA12 in media with ( 0 )and without (A) rh//(-) mutant cells. The
smooth lines correspond to exponential degradation with the labeled best-fit values of the degradation
constant, k, (Chen et al., 2005).
p 200 10
C
2
C
'3 160
-m
0
e
K
120
.-
0
I
C 80
-
8C
8 40 2
3
p!
Q
a 0 . 0
0 2 4 6 a 10 12 14 16 18
Time (Day)
Fig. 4.5. Profiles of autoinducer PA12 concentration in a batch cultivation of F! aeruginosa PAO1. The
empirically exponential decay profile, with the best-fit "net" decay constant (k,-k) taking into account
both the degradation and synthesis of PA12 in the culture, was shown for the stationary phase (Chen et
al., 2005).
Advances in Bioprocess Development of Rhamnolipid and Sophorolipid Production 0 87
wild type (Fig. 4.5) and the rhlR(-) mutant (Fig. 4.6). 'The mutant's growth-phase
AI production was slower, yielding a maximal PAI2 concentration of about one-third
of those from the wild-type PAO1. As the two cultures had similar profiles of cell
growth, the results implied that the autoinduction mechanism enabled a threefold
higher rate of AI synthesis.
We extended this work to evaluate the effect of PAI2 on specific rhamnolipid
productivity, using the rhll(-) mutant cultures added with various A1 concentrations
(Chen et al., 2004). We developed a model accordingly to link the essential features
of the rhl quorum-sensing system to the observed rhamnolipid-production profiles.
As expected, higher added PAI2 concentrations gave not only higher initial synthesis
rates but also longer induced synthesis (Fig. 4.7). Interested readers are referred to
the reference (Chen et al., 2004) for more information on the mathematical model
developed, the best-fit empirical constants obtained, and the potential physical
significance of the quorum-sensing systems on rhamnolipid synthesis.
Time (Day)
Fig. 4.6. Profiles of autoinducer PA12 concentration in a batch cultivation of the rh/R(-) mutant of P.
aeruginosa PA01 (Chen et al., 2005).
88 0 N.M. Pinzon et al.
Fig. 4.7. Experimental results and model profiles for the rhamnolipid production by a rh//(-) mutant of
PAO1, effected by the addition of autoinducers at different initial concentrations (Chen et al., 2004).
of the fermentor that bubbles up through the fermentation broth. By breaking the
large bubbles into fine ones, the agitation also increases the gas-liquid interfacial area
available for oxygen transfer to take place.
Unfortunately, these methods cannot be readily employed for rhamnolipid
production because of the highly foaming nature of the broth. To solve the
problems associated with severe foaming, we are developing the denitrification-based
fermentation technology for rhamnolipid production. I? aeruginosa is a facultative
aerobe, capable of performing aerobic and anaerobic respiration, for the latter, in
the presence of nitrate and/or nitrite as alternative electron acceptors. The anaerobic
nitrate-based respiration is known as denitrification. Nitrate is highly water-soluble.
It can be easily supplied to the growth media, thus, eliminating the vigorous, high-
shear agitation required in conventional fermentors for generating fine bubbles. Since
aeration is also not essential in denitrification-based fermentation, the aeration rate
can be adjusted to avoid uncontrollable foaming problems.
In the denitrification process, nitrate is first converted to nitrite, which is then
reduced to nitrogen (N,). This process is energetically favorable, and provides N,
gas as the principal end product (Ferguson, 1994; Hernandez et al., 1991). Four
reduction steps are involved in the denitrification mechanism:
The catalytic site of nitrate reductase is oriented toward the cytoplasm, and generates
nitrite at the inner face of the membrane (Zumfi, 1997). Once nitrite is produced,
another enzyme called nitrite reductase is responsible for the next reducing step to
nitric oxide. Unlike nitrate reductases, nitrite reductases are periplasmic enzymes.
After nitric oxide is produced, the nitric reductase and nitrous-oxide reductase further
reduce the nitricoxide and nitrous oxide to nitrous and nitrogen (NJ, respectively. The
enzymes involved in the dissimilative nitrate reduction process are generally repressed
by 0, and are synthesized only under anaerobic or anoxic conditions. Nonetheless,
denitrification under aerobic conditions was reported (Ka et al., 1997; Zhao et al.,
1999). We observed this with I? aeruginosa ATCC 9027 culture, which performed
denitrification even at dissolved oxygen concentrations higher than 1 mg/L when
grown in continuous culture using glucose as the carbon source (Chen et al., 2003).
Besides the effect of oxygen on the mechanism of denitrification, Thomas et al.
(1994) studied the effect of pH on denitrification using Pseudomonus species. A p H
range from 4.0-8.8 was examined, and the maximal denitrification rate took place at
a p H around 7.0-7.5. At low p H values, such as 4.0, the nitrogen-oxide reductases,
especially the N,O reductases, were progressively inhibited, resulting in a decrease of
the overall denitrification rate.
We examined the effect of different carbon sources (i.e., palmitic acid, stearic
acid, oleic acid, linoleic acid, glycerol, vegetable oil, and glucose) on rhamnolipid
production (Chayabutra et al., 2001). All of these substrates were able to support the
growth of I? aeruginosa ATCC 10145 under both aerobic and denitrifying (anaerobic)
conditions (Fig. 4.8). However, under magnesium-limited conditions, rhamnolipid
production was only detected when hexadecane, palmitic acid, or stearic acid was
used as the carbon substrate. O n palmitic acid and stearic acid, the growth of the
bacterium was slower probably because these were solid substrates that were not
readily available to the cells.
Rhamnolipid production in aerobic fermentationswas typically done in nitrogen-
source-limited media. Since I? aeruginosa can use nitrate as the N-source for growth,
the media may need to be designed with other limiting nutrients. We investigated the
rhamnolipid production in systems limited by nitrogen (N), iron (Fe), magnesium
(Mg), phosphorus (P), sulfur (S), and calcium (Ca) sources, respectively (Chayabutra
et al., 2001). P limitation gave four to five times higher specific rhamnolipid
productivity than N limitation. S limitation was comparable to N limitation in
supporting rhamnolipid production, while Mg limitation gave much poorer results.
Ca and Fe were not effective for limiting cell growth.
Thevalues ofspecific rhamnolipid productivity obtainedwith different C substrates
(hexadecane versus palmitic acid) and limiting nutrients (N versus P) under aerobic
versus denitrihing conditions are summarized in Fig. 4.9. The productivity with
palmitic acid as substrate was about three times higher under the aerobic conditions
than under the denitrifying conditions. As for the effect of C substrates, the aerobic
productivity from palmitic acid was lower than that from hexadecane. The former was
about 72% of the latter under N limitation and 82% under P limitation. Furthermore,
90 0 N.M. Pinzon et al.
0.4
h
.-5
c,
0.3
2
c,
C
8
C
0.2
0
V
-aJ
0.0
0 10 20 30 40 50 60 70 80 90 100
Time (h)
Fig. 4.8. Growth of F! aeruginosa under denitrificationwith different carbon substrates:(A)palmitic acid;
(0) linoleic acid; (m) glycerol; and (A)glucose (Chayabutraet al., 2001).
stearic acid; (V)oleic acid; (0)
P limitation was about four to five times more effective than N limitation, with either
hexadecane or palmitic acid as the substrate.
We also demonstrated the feasibility of rhamnolipid production under denitrifying
conditions by using a phosphorus-limited medium with palmitic acid as the carbon
substrate (Fig. 4.10) (Chayabutra et al., 2001). Although the specific rhamnolipid
productivity under denitrification was about one-third of that under aerobic
conditions, the process did not encounter the problems of foaming and respiration
limitation.
Time (h)
Fig. 4.9. Comparison of specific rhamnolipid productivity obtained with different carbon substrates
(hexadecane versus palmitic acid) and limiting nutrients(N versus P) under aerobic versus denitrifying
conditions (Chayabutra et al., 2001).
Respiration: NO3- 02 02 02 02
Palmitic Palmitic Hexa- Hexa- Palmitic
Substrate: Acid Acid decane decane Acid
Limiting Nutrient: P P P N N
Fig. 4.10. Experimentalresults of P-limitedfermentation with palmitic acid as C substrate, made under
denitrifying and then aerobic conditions (Chayabutra et at., 2001).
92 0 N.M. Pinzon et at.
Time (h)
Fig. 4.1 1. Effect of nitrate and/or nitrite concentrations on the growth oft? aeruginosa under anaerobic
denitrifying conditions using glucose as thecarbon source (OD,,,:optical density at 460 nm) (Chayabutra
& Ju, 2000).
change of the cellular electron-accepting state, including, for example, the start and
end points of denitrification in I! aeruginosa cultures (Chen et al., 2003; Ju et al.,
2005). We are working on developing a robust control strategy, based on the online
NAD(P)H fluorescence monitoring, for industrial rhamnolipid production using the
denitrification-based fermentation technology.
Sophorolipid Production
Producing Microorganisms and Characteristics
Sophorolipids were first identified as the extracellular glycolipids produced by the yeast
Tordopsis magnoliae, originally isolated from sow-thistle petals (Gorin et al., 1961).
The species was later identified as 7:apicola, currently known as Candida bombicokz.
The novel yeast species Starmella bombicola (isolated from flowers Calystegiasepium and
the associated sap beetles of genus Conotelus) identified in 1998 was the teleomorph of
C. bombicokz because of their high ability to mate with each other to form ascospores
(Rosa & Lachance, 1998). More recently, a new strain of Wickerhamiella domericgiae
synthesized sophorolipids. This strain was isolated from oil-containing wastewater
samples by using the enrichment techniques, with the measurements of the oil film-
collapsing activity and surface tension (Chen et al., 2006b).
Sophorolipid
Fig. 4.1 2.The possible biosynthetic pathway of sophorolipidsby Candida sp. when using carbohydrates
as the substrate (Lang & Wagner, 1993).
94 N.M. Pinzon et aI.
Sophoroiipid
Fig. 4.13. The possible biosynthetic pathway of sophorolipids by Candido sp. when using triglycerides
as the substrate (Lang &Wagner, 1993).
hydroxylation of fatty acids to form the hydroxyl fatty acids required for sophorolipid
synthesis. Consequently, fatty-acid accumulation would inhibit sophorolipid
production.
The effect of dissolved oxygen concentration on the sophorolipid synthesis by C
bombicokz was fully investigated. Sufficiently high dissolved oxygen concentrations,
25-1 5% ofair saturation, are necessary to achieve high sophorolipid yields (Guilmanov
et al., 2002; Lang et al., 2003; Spencer et al., 1979). Garcia-Ochoa and Casas (1999)
used an unstructured kinetic model to demonstrate the effect of the dissolved oxygen
concentration. The results also confirmed the beneficial effects of higher dissolved
oxygen concentrations on sophorolipid production.
Fermentation temperature affects cell growth and product formation. For
sophorolipid production by C. bombicokz, the optimal temperature is around 2 1°C
(Goebbert et al., 1984). C bombicokz grew well in the temperature range from
22-37"C, but the sophorolipid formation dropped significantly at temperatures
above 30°C (Spencer et al., 1979).
Stuewer and co-workers (1987) have thoroughly studied the effect of pH on the
sophorolipid formation by C bombicokz.Different types ofsophorolipids, such as water-
soluble (acidic) or crude crystalline (lactonic), are formed at different cultivation pH
values. Water-soluble sophorolipids are formed at a pH below 2, while the crystalline
sophorolipids form at a pH between 3 and 5. Sophorolipids are not significantly
produced at a pH higher than 5 (Spencer et al., 1979; Stuewer et al., 1987). Similar
results were obtained by Davila et al. (1997) and Goebbert et al. (1984). The optimal
pH for sophorolipid formation with resting cells was 3.5, while that with the batch
fermentation was in the range of 3.5-4.5. The higher yield at lower pH was attributed
to the heightened end-product inhibition resulting from the increased solubility of
sophorolipids in the aqueous medium when pH was raised from 3.5 to 5 (Davila et
al., 1997). Stuewer et al. (1987) further observed that C. bombicokz could convert
the water-soluble sophorolipids into the crystalline sophorolipids by adjusting the
pH but not vice versa. O n the other hand, Goebbert et al. (1984) and Spencer et al.
(1979) did not observe the change in product nature (water-soluble versus crystalline)
with the changed cultivation pH, although the observations on the effect of the pH
on product yield were largely consistent among these researchers.
Other factors also affect the sophorolipid formation. For example, the inclusion
of sodium citrate in the culture medium, in the concentration range of 2.5-10 g/L,
increased the biomass yield slightly, but the sophorolipid yield significantly (Stuewer
et al., 1987). The optimal concentration of sodium citrate was 5 g/L, and some other
organic acids, such as succinate, tartrate, or malonate, can be used as alternatives.
With the addition of these organic acids, the exolipid pattern was also altered,
and more hydrophobic (lactonic) sophorolipids were formed. Evans and Ratledge
(1985) reported that citric acid was one of the factors involved in the regulation of
lipid accumulation with oleaginous yeast. Stuewer et al. (1987), on the other hand,
proposed that the main effect of the organic acids was caused by their influence on the
cultivation pH.
96 0 N.M. Pinzon et al.
Fig. 4.14. Structures and mass spectra of sophorolipids produced by C. bombicolo in media containing
the following C-source(s):(a) glucose only, (b) glucose plus hexadecane as the second C-source, and (c)
glucose plus soybean oil as the second C-source.
98 0 N.M. Pinzon et al.
phosphate bufferswere more effective for purifying lactonic sophorolipids than ethanol,
phthalate buffers gave rise to a slightly more loss of the lactonic sophorolipids than
phosphate buffers. The phosphate buffers with higher pH were particularly promising
because they would keep more acidic sophorolipids soluble while precipitating out
the very poorly soluble lactonic sophorolipids. As a result, the loss of the lactonic
sophorolipids in the phosphate buffer (2.4-3.1 % of the lactonic sophorolipids
originally present in the crude product, shown in Fig. 4.17a) was much less than the
loss in ethanol (47-77%), and the purity of lactonic sophorolipids in the crystallites
improved. For example, the purity reached up to 99.6% (Fig. 4.17b) in the phosphate
buffer of pH 7.5. ?he FTIR spectroscopy confirmed that indeed significantly less
acidic sophorolipids were in the purified product than in the crude product (spectra
not shown).
According to the study results, we designed a two-step process for purifying the
lactonic sophorolipids. The first step was a wash by phosphate buffer (pH 26.5) at
a low (room) temperature to remove the more soluble acidic sophorolipids while
a
f?
0 i3ms (h)
Fig. 4.15.The changes of sophorolipid composition when using hexadecane (a and b) or soybean oil (c
and d) as the second C-source during the fermentation (Hu& Ju,2001b).
Advances in Bioprocess Development of Rhamnolipidand Sophorolipid Production 0 99
Fig. 4.16. (a) Solubility of different sophorolipids in ethanol at various temperatures. (b) Solubility of
different sophorolipids in aqueous buffers at various pHs at 25°C (Hu & Ju, 2001a).
3.5000
3 3.0000
100.00
2.5000 99.50
‘E g 2.0000 2
8 1.5000 ,g 99.00 Purity
3
23i .I1.0000
i Lost Lactonic n
SLS 98.50
2:s 05000
’
$ 0.0000 98.00
6 6.5 7 7.5 8 6 6.5 7 7.5 8
PH PH
Fig.4.17. (a) Lossof lactonicsophorolipids in the phosphatebuffer at adifferent pH during crystallization;
(b) Purity of lactonic sophorolipids in the crystallites (Hu & Ju, 2001a).
Conclusions
Rhamnolipids and sophorolipids are two major groups of glycolipid biosurfactants that
have attracted much attention for various potential applications. The two groups have
different properties primarily because of their different hydroxyl fatty-acid moieties:
rhamnolipids’ lipid moiety is P-hydroxylated and has a free carboxylic- acid end group;
sophorolipids’ lipid moiety is primarily hydroxylated at the (O-1) position, and the
carboxylic acid group is either free (acidic sophorolipids) or intraesterified with the
sugar (lactonic sophorolipids). The above differences affect not only their properties
100 0 N.M. Pinzon et al.
Acknowledgments
The original work from the group of Lu-Kwang Ju reported in this chapter was
supported by National Science Foundation (BES-9900694 and 01 04122), U.S.
Department ofTransportation (Office of the Secretary, DTOS59-07-G-00050), Ohio
Board of Regents (for Ohio Bioprocessing Research Consortium), and The University
of Akron (Faculty Research Grant).
Advances in Bioprocess Developmentof Rhamnolipid and Sophorolipid Production 0 101
References
Abalos, A.; M. Vinas; J. Sabate; M.A. Manresa; A.M. Solanas. Enhanced biodegradation of Casa-
blanca crude oil by a microbial consortium in presence of a rharnnolipid produced by Pseudomo-
nas aeruginosa AT10. Biodegradation 2004, 15(4), 249-260.
Anna, L.M.S.; G.V. Sebastian; E.P. Menezes; T.L.M. Alves; AS. Santos; N. Pereira, Jr.; D.M.G.
Freire. Production of biosurfactants from Psdomonas aeruginosa PA1 isolated in oil environ-
ments. Brazilian/. Chem. Eng. 2002, 17(2), 159-166.
Ashby, R.D.; D.K.Y. Solaiman; T.A. Foglia. Property control of sophorolipids: influence of fatty
acid substrate and blending. Biotechnol. Lett. 2008,30(6), 1093-1 100.
Baviere, M.; D. Degouy; J. Lecourtier. Process for Washing Solid Particles Comprising a Sophoro-
side Solution. U.S.Patent 5,326,407 (1994).
Benincasa, M. Rhamnolipid produced from agroindustrial wastes enhances hydrocarbon biodegra-
dation in contaminated soil. Curr. Microbiol. 2007,54(@, 445-449.
Campos-Garcia, J.; A.D. Caro; R. Najera; R.M. Miller-Maier; R.A. Al-Tahhan; G. Soberon-
Chavez. The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent j3-ketoacyl
reductase which is specifically involved in rhamnolipid synthesis.J. Bacteriol. 1998, 180(17),
4442-4451.
Casas, J.A.; F. Garcia-Ochoa. Sophorolipid production by Candidz bombicola: Medium composi-
tion and culture methods. J. Biosci. Bioeng. 1999, 88(5), 488-494.
Chayabutra, C.; L.-K. Ju. Degradation of n-hexadecane and its metabolites by Pseudomonas
aeruginosa under microaerobic and anaerobic denitrifying conditions. Appl. Environ. Microbiol.
2000, GG(2), 493-498.
Chayabutra, C.; L.-K. Ju. Polyhydroxyalkanoicacids and rharnnolipids are synthesized sequen-
tially in hexadecane fermentation by Pseudomonas aeruginosa ATCC 10145. Biotechnol. Prog.
2001, 17(3), 419-423.
Chayabutra, C.; J. Wu; L.-K. Ju. Rharnnolipid production by Pseudomonas aeruginosa under deni-
trification: Effects of limiting nutrients and carbon substrates. Biotechnol.Bioeng. 2001, 72(1),
25-33.
Chen, F.; C.-C. Chen; L. Riadi; L.-K. Ju. Modeling rhl quorum-sensing regulation on rharnno-
lipid production by Pseudomonas aeruginosa. Biotechnol. Prog. 2004,20(5), 1325-1331.
Chen, C.-C.; L. Riadi; S.-J.Suh; D.E. Ohman; L.-K. Ju. Degradation and synthesis kinetics of
quorum-sensing autoinducer in Pseudomonas aeruginosa cultivation. JBiotechnol. 2005, 11 7(1),
1-10.
Chen, J.; X. Song; H. Zhang; Y.-B. Qu; J.-Y Miao. Sophorolipid produced from the new yeast
strain Wickerhamieh domercqiae induces apoptosis in H7402 human liver cancer cells. Appl.
MicrobioL Biotechnol. 2006a, 72(1), 52-59.
Chen, J.; X. Song; H. Zhang; Y Qu. Production, structure elucidation and anticancer proper-
ties of sophorolipid from Wickerhamiella domercqiae. Enzyme Microb. Technol. 2006b, 37(3),
50 1-506.
Chen, S.-Y.; Y-H. Wei; J.-S. Chang. Repeated pH-stat fed-batch fermentation for rhamnolipid
production with indigenous Pseudomonas aeruginosa S2. Appl. Microbiol. Biotechnol. 2007,
7G(l), 67-74.
102 0 N.M. Pinzon et al.
Chen, F.; Q. Xia; L.-K. Ju. Aerobic denitrification of Pseudomonas aeruginosa monitored by online
NAD(P)H fluorescence.Appl. Environ. Microbiol. 2003, 69(11), 671 5-6722.
Davila, A.M.; R. Marchal; N. Monin; J.l? Vandecasteele. Identification and determination of indi-
vidual sophorolipids in fermentation products by gradient elution high-performance liquid chro-
matography with evaporative light-scattering detection. J. Chromatogx 1993, 648(1), 137-147.
Davila, A.-M.; R. Marchal; J.-I? Vandescasteele. Sophorose lipid production from lipidic precur-
sors:Predictive evaluation of industrial substrates.J. Indus. Microbiol. 1994, 13(4), 247-257.
Davila, A.M.; R. Marchal; J.I? Vandecasteele. Kinetics and balance of a fermentation free from
product inhibition: Sophorose lipid production by Candida bombicola. Appl. Microbiol. Biotech-
no!. 1992,38(1), 6-1 1.
Davila, A.M.; R. Marchal; J.l? Vandecasteele. Sophorose lipid fermentation with differentiated
substrate supply for growth and production phases. Appl. Microbiol. BiotechnoL 1997, 47(5),
496-501.
Desai, J.D.; I.M. Banat. Microbial production of surfactants and their commercial potential.
Microbiol. Mol. BioLReu. 1997, 61 (1). 47-64.
Daiel, E.; F. Lepine; S. Milot; R. Villemur. Mass spectrometry monitoring of rhamnolipids from
a growing culture of Pseudomonas aeruginosa strain 57W. Biochim. BiophysActa Mol. Cell Biol.
Lipids 2000,1485(2-3), 145-152.
Evans, C.T.; C. Ratledge. The physiological significance of citric acid in the control of metabolism
in lipid-accumulating yeasts. Biotechnol. Genetic Engin. Rev. 1985,3. 347-375.
Ferguson, S.J. Denitrification and its control. Antonie van Leeuwenhoek 1994, 66(1-3), 87-1 10.
Fiehler, K.; A. Albrecht; D. Rasch; U. Rau. Continuous production of sophorolipids by Cundida
bombicola ATCC 22214. Fett/Lipid 1997,99(1), 19-24.
Furuta, T.; K. Igarashi; Y. Hirata. Biodegradable Low-Foaming Detergent Compositions Contain-
ing Sophorolipid. International Patent 2002-JP6457 (2003).
Garcia-Ochoa, F.; J.A. Casas. Unstructured kinetic model for sophorolipid production by Candida
bombicola. Enqme Microb. Echnol. 1 9 9 9 , 2 5 ( 3 , 613-621.
Goebbert, U.; S. Lang; F. Wagner. Sophorose lipid formation by resting cells of Tordopsis bombi-
cola. Biotechnol. Lett. 1984, 6(4), 225-230.
Gorin, P.A.J.; J.ET Spencer; A.R Tulloch. Hydroxy fatty acid glycosides of sophorose from Toru-
lopsis magnoliae. Can. J. Chem. 1961,39(4), 846-855.
Gruber, T.; H. Chmiel; 0. Kaeppeli; I? Sticher; A. Fiechter. Integrated process for continuous
rhamnolipid biosynthesis. Su$ Sci. Series 1993,48(Biosu~actants),157-173.
Guerra-Santos, L.; 0. Kaeppeli;A. Fiechter. Pseudomonas aeruginosu biosurfactant production
in continuous culture with glucose as carbon source. Appl. Environ. Microbiol. 1984, 48(2),
301-305.
Guerra-Santos, L.H.; 0. Kaeppeli; A. Fiechter. Dependence of Pseudomonds aeruginosu continu-
ous culture biosurfactant production on nutritional and environmental factors. Appl. Microbiol.
Biotechnol. 1986, 24(6), 443-448.
Guilmanov, V.; A. Ballistreri; G. Impallomeni; R.A. Gross. Oxygen transfer rate and sophorose
lipid production by Candida bombicola. Biotechnol. Bioens 2002, 77(5),,: 487-474.
Advances in Bioprocess Developmentof Rhamnolipid and Sophorolipid Production 0 103
Products, in Particular for the Treatment of the Skin. U.S. Patent 5,981,497 (1999).
Mata-Sandoval, J.C.; J. Karns; A. Torrents. Effect of nutritional and environmental conditions on
the production and composition of rhamnolipids by I? aeruginosa UG2. Microbiol. Res. 2001,
155(4), 249-256.
Mercade, M.E.; M.A. Manresa. The use of agroindustrid byproducts for biosurfactant production.
J. Am. Oil Chem. SOC.1994,71(1), 61-64.
Mulligan, C.N. Environmentd applications for biosurfactants. Environ. Pollut. (Amsterdam, Neth-
erlandt) 2004,133(2), 183-198.
Napolitano Lena, M. Sophorolipids in sepsis: Antiinflammatory or antibacterial? Crit. Care Med.
2006,34(1), 258-259.
Nguyen, T.T.; N.H. Youssef; M.J. Mcinerney; D.A. Sabatini. Rhamnolipid biosurfactant mixtures
for environmental remediation. Water Res. 2008, 42(G-7), 1735-1743.
Nitschke, M.; S.G.V.A.O. Costa. Biosurfactants in food industry. Tzendt Food Sci. Technol. 2007,
I8(5), 252-259.
Nitschke, M.; S.G.V.A.O. Costa; J. Contiero. Rhamnolipid surfactants: an update on the general
aspects of these remarkable biomolecules. Biotechnol. Prog. 2005, .?I(@, 1 593-1600.
Noordman, W.H.; J.H.J. Wachter; G.J. De Boer; D.B. Janssen. The enhancement by surfactants
of hexadecane degradation by Pseudomonas acruginosa varies with substrate availability.J. Bio-
technol. 2002, 94(2), 195-212.
Otto, R.T.; H.J. Daniel; G. Pekin; K. Muller-Decker; G. Furstenberger; M. Reuss; C. Syldatk.
Production of sophorolipids from whey. 11. Product composition, surface active properties,
cytotoxicity and stability against hydrolases by enzymatic treatment. Appl. Microbiol. Biotechnol.
1999,52(4), 495-501.
Parra, J.L.; J. Guinea; M A . Manresa; M. Robert; M.E. Mercade; E Comelles; M.P Bosch.
Chemicd characterization and physicochemical behavior of biosurfactants.].Am. Oil Chem. SOC.
1989, &(I), 141-145.
Rau, U.; R. Heckmann; V. Wray; S. Lang. Enzymatic conversion of a sophorolipid into a glucose
lipid. Biotechnol. Lett. 1 9 9 9 , 2 1(I I), 973-977.
Rau, U.; H. Manzke; E Wagner. Influence of substrate supply on the production of sophorose
lipids by Candida 6ombicolaATCC 22214. Biotechnol. Lett. 1996, 18(2), 149-154.
Rosa, C.A.; M.A. Lachance. The yeast genus Starmerella gen. nov. and Starmerelh bombicola sp.
nov., the teleomorph of Candida bombicola (Spencer, Gorin & Tullock) Meyer & Yarrow. Znt. J.
Syst. Evol. Microbiol. 1998,48(4), 1413-1417.
Rosenberg, E.; E.Z. Ron. High- and low-molecular-mass microbid surfactants.Appl. Microbiol.
Biotechnol. 1999, 52(2), 154-162.
Shah, V.;G.F. Doncel; T. Seyoum; K.M. Eaton; I. Zalenskaya; R. Hagver; A. h i m ; R. Gross.
Sophorolipids, microbial glycolipids with anti-human immunodeficiency virus and sperm-
immobilizing activities. Antimicrob. Agents Chemother. 2005, 49(10), 4093-4 100.
Siemann, M.; F. Wagner. Prospects and limits for the production of biosurfactants using immobi-
lized biocatdysts. Su$ Sci. Series. 1993, 48(Biosufactants), 99-133.
Singh, A,; J.D. Van Hamme; 0.P Ward. Surfactants in microbiology and biotechnology: part 2.
Advances in Bioprocess Developmentof Rhamnolipid and Sophorolipid Production 0 105
Introduction
Microemulsions have received significant attention during the last several decades
due to their thermodynamic stability, favorable phase behavior, ultralow interfacial
tension (IFT), and high solubilization capacity of both organic and aqueous phases
(Kumar & Mittal, 1999). These features of microemulsions make them valuable for
a wide range of applications.
Microemulsions produce ultralow IFT (less than 0.1 mN/m) between the
microemulsion phase and the excess oil and/or water phases, thus overcoming the
capillary forces that trap oil in porous media. In addition, microemulsions have
the ability to solubilize both oil and water. These two features promote the use of
microemulsions in enhanced oil recovery and detergency (Uchiyama et al., 2000;
Acosta et al., 2002b., 2003). The transparency of microemulsions makes them attractive
in cosmetic formulations. The ultralow IFT between oil and water also facilitates
the penetration of the formulations into human skin, making microemulsions of
interest in personal care products and drug delivery applications (Acosta et al., 2005;
Komesvarakul et al., 2006).
While only a limited number of studies have evaluated the use of biosurfactants
in microemulsion formulations, several researchers have shown that glycolipid
biosurfactants, the most common class of biosurfactants composed of carbohydrate
heads and fatty acid tails (e.g., rhamnolipid and sophorolipid), can be used in a wide
range of applications (Gautam & Tyagi, 2006). The rhamnolipid biosurfactant has
been evaluated for the remediation of petroleum-contaminated sites due to its ability
to solubilize, mobilize, and stimulate the biodegradation of petroleum hydrocarbons
(Georgiou et al., 1992; Zhang & Miller, 1992 , 1995; Herman et al., 1997; Maier &
Soberh-Chivez, 2000; Kitamoto et al., 2002; Whang et al., 2008). The rhamnolipid
biosurfactant has also been used in applications such as removal of heavy metals from
contaminated soil (Kitamoto et al., 2002; Maier & Sober&-Chivez, 2000; Mulligan
et al., 2001) and has antimicrobial and antiviral activities (Kitamota et al., 2002;
Cameotra & Makkar, 2004). The sophorolipid biosurfactant, on the other hand, has
107
108 0T.T. Nguyen and D.A. Sabatini
been evaluated for pharmaceutical application (Shah et al., 2005; Rodrigues et al.,
2006) and for cosmetic and personal care applications (Van Bogaert et al., 2007) due to
its good surface active properties and excellent skin compatibility. Replacing synthetic
(petroleum-based) surfactants with biosurfactants in product formulations is of great
interest in many applications due to their lower toxicity, higher biodegradability, and
biorenewable nature.
Although microemulsions using a single surfactant have been reported (Holmberg
& Osterberg, 1986; Sunwoo &Wade, 1992), consumer product formulations are most
often prepared with surfactant mixtures (Rosen, 1989). The purpose of combining
surfactants/co-surfactants is to obtain the proper hydrophilic/lipophilic balance for
the required oil and water phases under a specific condition, which is quite difficult
to achieve in a single-surfactant system. In most of the studies where mixtures of
surfactantdco-surfactantswere used, the surfactant mixtures show apparent advantages
and synergism over the use of a single surfactant (Huibers & Shah, 1997; Li et al.,
2005; Nguyen et al., 2008). This is especially true for biosurfactants which are not
available with the wide range of properties found in synthetic surfactants. Single
biosurfactant systems are thus less likely to provide a proper hydrophilic/lipophilic
balance for a required oil while mixtures of two or more biosurfactants with different
hydrophilicity/lipophilicity can. This chapter thus focuses on the use of biosurfactant
mixtures to achieve microemulsion formulations and improve system properties.
Microemulsions in General
Phase Behavior and interfacial Tension
Microemulsions are thermodynamically stable dispersions of two immiscible liquids
(oil and water) stabilized by surfactant films (Rosen, 1989). Microemulsions can be
distinguished from macroemulsions and miniemulsions in different ways. First of all,
microemulsions have much smaller droplets (less than 100 nm) than macroemulsions
and miniemulsions (100-400 nm or greater). Secondly, microemulsion formation
is spontaneous while macroemulsion formation requires intense agitation and
miniemulsion formation also requires prolonged mixing. Finally, and most
importantly, microemulsions are thermodynamically stable while macroemulsions
and miniemulsions are not.
The surfactant concentration at which surfactant monomers start to aggregate
to form micelles is defined as the critical micelle concentration (CMC). Above the
CMC, many system properties remain unchanged since additional surfactant forms
micelles rather than increasing the surfactant aqueous activity (Rosen, 1989). The
surfactant concentration at which a microemulsion first forms is defined as the critical
microemulsion concentration (CpC).
Winsor introduced four types of microemulsions, known as Winsor Type I, 11,
111, and IV microemulsions (Winsor, 1948; Rosen, 1989). Winsor Type I and Type
I1 microemulsions are two-phase systems. Winsor Type I (oil-in-water or Onxr)
microemulsions solubilize oil (the excess phase) in spherical normal micelles within
Microemulsionsof Rhamnolipid and Sophorolipid Biosurfactants0 109
the water phase while Type I1 (water-in-oil or W/O) microemulsions solubilize water
(the excess phase) in reverse micelles within the oil phase. Type I11 microemulsions,
on the other hand, are three-phase systems which contain the excess oil and excess
water phases in equilibrium with the bilayer middle phase microemulsion. Type I11
microemulsions are formed when lamellar micelles exist. With increasing surfactant
concentration, the volume of the middle phase microemulsion increases and
eventually incorporates all the excess oil and excess water phase into a single phase
microemulsion, known as a Winsor Type IV microemulsion.
The phase change or microemulsion transition from Winsor Type I + I11 +
I1 is often produced by the change in a tuning parameter, such as temperature for
alkyl ethoxylate (AE) surfactants or electrolyte concentration for ionic surfactants.
For AE nonionic surfactants, increasing temperature causes the ethoxylate groups
to dehydrate, which increases the lipophilicity of the surfactant. As a result, the
surfactant can solubilize more oil and the micelles change from the normal spherical
shape (Type I microemulsion) to a rod-like shape and form a bilayer phase (Type I11
microemulsion) that solubilizes both oil and water in a thermodynamically stable
phase. As the temperature continues to increase, the surfactant becomes more and
more lipophilic until a point is reached where the micelles start to invert into reverse
micelles which solubilizes water into the excess oil phase (Type I1 microemulsion).
Therefore, the microemulsion transitions from Type I to Type I11 to Type I1 with
increasing temperature for AE surfactants. For ionic surfactants, adding electrolyte
reduces the repulsion between the ionic head groups, thus increasing the surfactant
lipophilicity. As the electrolyte concentration increases, the shape of the micelles
changes from normal spherical to rod-like to reverse micelles. Similar to the change in
temperature of AE surfactant systems, the microemulsion transitions from Type I to
111to I1 with increasing electrolyte concentration for ionic surfactant systems (Rosen,
1989). As seen in Fig. 5.1, with increasing lipophilicity of the surfactant, the micelles
not only change in shape, but also in size (Komesvarakul et al., 2006). R, is the radius
of the Type I micelles and R, is the radius of the Type I1 micelles. The curvature (H)
of the micelles is equal to 1/R, and 1/R, respectively. At low salinity, the surfactant is
hydrophilic and the micelle curvature is large and positive, andType I microemulsions
are formed. As the salinity increases, the net curvature decreases and approaches a net
value of zero with Type I11 microemulsion formation. When the surfactant becomes
more hydrophobic at higher salinity, the curvature decreases further and becomes
negative. At this point, Type I1 microemulsions are formed (Bourrel & Schechter,
1988; Rosen, 1989). Figure 5.1 also demonstrates that at a given temperature/salinity,
as the surfactant concentration increases the Winsor Type IV system is approached.
The IFT ofwatedoil systems y(o), is closely related to microemulsion formation
(Bourrel & Schechter, 1988). Ultralow IFT (< 0.1 mN/m) is achieved in Type I11
middle phase microemulsions (Rosen, 1989). Kunieda and Shinoda (1982) plotted
the IFT of an AE surfactant/oil/water system as a function of temperature as shown
in Fig. 5.2. As the temperature increases, the surfactant becomes more hydrophobic.
1 10 OT.T. Nguyen and D.A. Sabatini
Type ZI:
Reverse
miceller
GwMtUn(H)=l/&
Reducing Curvature
Fig. 5.1. Phase behavior and changes in curvature with surfactant concentration and hydrophobicity
as adjusted by a scanning variable (salinity for anionic surfactant (AIS); temperature for alkyl ethoxylate
surfactant (NISI) (Reprinted from Komesvarakul et al., 2006. Reprinted with permissionfrom Society of
Cosmetic Chemists).
As a result, the interaction between the surfactant and the water decreases while
the interaction between the surfactant and the oil increases. Consequently, the IFT
between surfactant-water increases and that between surfactant-oil decreases. This
occurs with the phase change from the Type I O W microemulsion to the Type I1
W/O microemulsions. 7he point when the IFT values yo-D and yD-wcross is the
optimum middle phase microemulsion where the IFT between the excess oil and
excess water (yo,) is minimum (Bourrel & Schechter, 1988).
Fig. 5.2. Interfacial tension values between oil and microemulsion phase ,)y(, microemulsion phase
and water (yBw) and oil and water ) ,7( as a function of temperature for an alkyl ethoxylate surfactant
(Adapted from Kunieda and Shinoda, 1982).
where SPoand SP,are solubilization parameters for oil and water, respectively; V, V,
and V, are the volume of oil, water, and surfactant in the micellar phase, respectively.
These solubilization parameters are usually calculated excluding additives even
though they are present in the system (Bourrel & Schechter, 1988). As the salinity
concentration increases, the surfactant micelle size grows and thus can solubilize more
oil and less water. As a result, the SP, increases and the SP, decreases with the scan
of the tuning parameter (increasing temperature for AE surfactants and increasing
electrolyte concentration for ionic surfactants). Similar to the optimum formulation
obtained by evaluating IFT values, optimum solubilization can also be identified as
the point where SP, and SP, intercept. At this point, the volumes of oil and water
solubilized in the micellar phase are equal and we have the optimum solubilization
parameter (SP' = SP, = SP,; Graciaa et al., 1993). SP* can also be calculated by
equation 2 (Bourrel & Schechter, 1988):
sp* =-v*
m,
Eq. 5.2.
1 12 OT.T. Nguyen and D.A. Sabatini
NaCI, wt%
Fig. 5.3. Phase behavior showing the transition for Winsor type I to 111 to II with corresponding interfacial
tensions and solubilization level as a function of salinity of an ionic surfactant (Adapted from Acosta et
al., 2002a).
Microemulsions of Rharnnolipid and Sophorolipid Biosurfactants0 113
whereAco is the interaction between the surfactant and the oil, Acw is the interaction
between the surfactant and the water phase; A,, andA,,are the interactions between
oil molecules and water molecules, respectively; A,, and A , are the interactions
between the lipophilic tails and hydrophilic heads, respectively, of the surfactant
molecules at the interface. In equation 4, the numerator and denominator represent
the net interactions of oil-surfactant and water-surfactant, respectively. As seen in Fig.
5.4, for R < 1, the water-surfactant interaction (Acw)is stronger than the oil-surfactant
interaction (AcJ and the surfactant resides in the water phase making normal micelles
(Type I) while the opposite is true for R > 1. As a result, when R c I , the characteristic
system is Type I and when R > 1, it is Type 11. At optimum formulation, R = 1, which
indicates that the oil-surfactant and water-surfactant interactions are balanced and
the surfactant prefers to be at the interface. As both the numerator and denominator
increase, in other words, as the oil-surfactant and water-surfactant interactions at the
optimum both increase while maintaining an Rvalue of 1, the optimum solubilization
(SPJis enhanced (Winsor, 1954; Bourrel & Schechter, 1988).
PLCO
Fig. 5.4. Microemulsiontransition with changes in Winsor's R-ratio (Adapted from Winsor, 1954).
1 14 0T.T. Nguyen and DA. Sabatini
WA HLBA + WBHLBB
= HLB,,
WA +WB
Eq. 5.5.
where W,and W, are the amounts (weight) of the first emulsifier (A) and the second
emulsifier (B) used, respectively; HLB, and HLB, are the assigned HLB values for
emulsifiers A and B; and HLB0,, is the “required HLB” of the oil for the desired
type of emulsion. The one Caveat to this approach is that if the two surfactants have
drastically different HLB values, they may not act together but rather segregate into
separate phases; a third surfactant with intermediate HLB can prevent this segregation
(Tongcumpou et al., 2006). This equation can also be applied for a surfactant system
composed of more than two components with the expansion of the left side of the
equation.
In the following sections we will discuss the surfactant mixtures in formulating
microemulsions and the improved microemulsifying power of surfactant mixtures
over single surhctant systems. All the surfactant mixture results we present contain at
least one biosurfactant in the mixture.
Rhamnolipid-based Microemulsions
To date only limited studies have evaluated microemulsion formulations with
biosurhctants. Most of the microemulsion studies have evaluated rhamnolipid
biosurhctants with the addition of alcohol to enhance the microemulsion formation
(Xie et al., 2005). Xie et al. (2005) studied system of rhamnolipids + alcohol/n-
heptane/water. When n-propanol was used as the co-surfactant, only Winsor Type
Microemulsionsof Rhamnolipid and Sophorolipid Biosurfactants 0 115
Rhamnolipid-aloneMicroemulsions
Rhamnolipid biosurfactants with two head groups-the anionic carboxylated head and
the bulky rhamnosyl head and two identical tails of C8 hydrocarbon (Fig. 5.5) are
known to have high HLB values of 22-24 (Xie et al., 2005). Based on the guideline
of formulating microemulsions from the Winsor R-ratio and the HLB method,
rhamnolipid is expected to be a good emulsifying agent for extremely hydrophilic
oils. Nguyen et al. (2008) studied the interfacial properties and phase behavior of
rhamnolipid biosurfactant with oils of different equivalent alkane carbon number
(EACN). The IFT between the oil phase and the aqueous phase was measured using
glass capillary tubes and a spinning drop tensiometer (Model 500 purchased from the
University ofTexas). The capillary tube is 2 mm in diameter and has a volume of 300
pL. The tube was filled with the aqueous phase (the denser phase), and then 1 pL of
the oil phase (the less dense phase) was injected into the aqueous solution to form
a droplet (Childs et al., 2004). The filled tube was then placed in the spinning drop
tensiometer and the oil droplet size was measured.
(b)
Fig. 5.5. Molecular structures of the rhamnolipids: (a) monorhamnolipid, (b) dirhamnolipid (Adapted
from Helvaci et al., 2004).
116 0T.T. Nguyen and D.A. Sabatini
where k is a constant (often between 0.1 to 0.17), ACN is the alkane carbon number
(for nonlinear hydrocarbons, the ACN becomes the EACN), AA) represents the
effect of alcohol, 0 is a function of surfactant type, aTis a constant, and AT is the
temperature difference between the studied temperature and the reference temperature
(25°C). This equation indicates that as the ACN (EACN) value increases, 5" is also
expected to increase, all other variables being held constant. This is consistent with the
rhamnolipid microemulsion results shown in Fig. 5.6. As the electrolyte was added
to the ionic rhamnolipid solution, it reduced the electrical repulsion between the
ionic head groups, causing the system net curvature to decrease towards zero where
E
c
3-
EA( "
Fig. 5.6. Relationship between optimal salinity (53 (0) and optimal interfacial tension (/FT*) ( 0 )versus
oil EACN (toluene (1). hexane (6),decane (lo),and hexadecane(16)).The rhamnolipidconcentration was
0.1 wt% and IFT was measuredat 25 f 1°C for all oils (Reprinted from Nguyen et al., 2008. Reprinted with
permission from Elsevier).
Microemulsionsof Rhamnolipid and Sophorolipid Biosurfactants 0 1 17
the optimal middle-phase microemulsion is achieved (the lowest IFT and highest SP)
(Rosen, 1989). The S'increases with increasing EACN value, suggesting that more salt
was required to force the ionic surfactant into the oil/water interface. At neutral pH,
rhamnolipid is anionic in character due to the presence of the carboxylate head group.
The lowest IFT was observed for the lowest optimum salinity (i.e., toluene with the
lowest EACN value of I), indicating that rhamnolipid is best matched to toluene, and
thus requires the least salt addition to achieve the lowest IFT and generates the lowest
IFT observed for all the oils.
Nguyen et al. (2008) also developed a "fish" phase diagram of rhamnolipid
biosurfactant with toluene (Fig. 5.7), which is similar to the diagram illustrated in Fig.
5.1. The phase diagram was generated by varying the salt concentration for a series
of surfactant concentrations. The phase boundary is shown as a line that connects all
the points where a transition in microemulsion type was observed. The phase diagram
was reported at 23°C (solid line) and 55°C (dashed line). For a fixed rhamnolipid
concentration (e.g., 1 w/w%), the microemulsion transitions from Winsor Type I to
111 to I1 as the NaCl concentration increased. At a fixed electrolye concentration (e.g.,
12 w/w?/o), the volume of the middle phase increased with increasing rhamnolipid
concentration. As the concentration of rhamnolipid reached 10 w/w?h, the middle
phase incorporated all the excess oil and excess water into a single phase microemulsion.
Thus above 10 w/w?h rhamnolipid concentration, a single phase microemulsion Winsor
Type IV phase was observed. Figure 5.7 also demonstrates that temperature has only
a slight effect on the phase behavior of rhamnolipid biosurfactant; the system requires
slightly less salinity to form middle phase at higher temperature, indicating that the
Fig. 5.7. Phase diagram of rhamnolipid with toluene (CMC of 0.001 wt%). The boundary lines (solid for
23°C; dashed for 55°C) connect all the points where a transition for microemulsion type was observed.
The line labeled optimum salinity which cuts through the interior of the phase diagram corresponds to
the optimum salinity at each surfactant concentration (Reprinted from Nguyen et al., 2008. Reprinted
with permission from Elsevier).
118 0 T.T. Nguyen and D.A. Sabatini
sugar groups are less soluble at elevated temperature and thus requires lower NaCl
concentration to form Type 111 systems.
Corresponding to the fish diagram in Fig. 5.7, the IFT was plotted as a hnction
of rhamnolipid concentration at optimum formulations (Fig. 5.8). By the definition
of CMC and CpC, rhamnolipid was reported to have the CMC and CpC of 0.001
w/w% (or 1 . 9 ~ 1 0M)~ and 0.01 w/wYo (or 1 . 9 ~ 1 0M),
~ respectively, at a salinity of
14.5 w/w%. This high optimum salinity indicates that rhamnolipid is very hydrophilic
even for the extremely hydrophilic toluene and thus is much too hydrophilic for
higher EACN oils (hexane, decane, and hexadecane).
Rhamnolipid-Co-surfactant Microemulsions
Due to its hydrophilic nature, rhamnolipid was able to form middle phase
microemulsions with only toluene, and even then the optimum salinity was very
high, ranging from 11 to 17 w/w %. In order to increase the hydrophobicity of a
system containing rhamnolipid (decrease its HLB), a surfactant with lower HLB was
mixed with rhamnolipid. When mixed with a lower HLB surfictant, the rhamnolipid
surfictant system should be able to form microemulsions with more hydrophobic oils
and requires less salinity to achieve optimum conditions.
Nguyen et al. (2008) studied the effect of mixing synthetic (petroleum-based)
surhctants with lower HLB values with rhamnolipid to formulate microemulsions
for different oils. Figure 5.9 shows the synergistic effect of using surfictant mixtures
Riiamnoiipid, wt%
Fig. 5.8. Optimum equilibrium IFT for toluene versus rhamnolipid concentration (following vertical line
in Fig. 5.7). IFTwas measuredat 25 f 1°C. (Reprinted from Nguyen et al., 2008. Reprinted with permission
from Elsevier).
Microemulsionsof Rhamnolipid and Sophorolipid Biosurfactants 0 1 19
E
2
E
Fig. 5.9. IFT of mixtures of rhamnolipid and Cl,,l,-8PO-S0,Na for toluene (01,hexane (01 ,
decane (V),
and hexadecane (A)at optimum salinity (S)for each system. Fractionis by mass and the total surfactant
concentration isO.l wt%.The IFTshown in this figure was the optimum IFTforeach formulation and was
measuredat 25 f 1°C. (Reprinted from Nguyen et al., 2008. Reprinted with permission from Elsevier).
in reducing the IFT for toluene and hexane; the surfactant mixture was rhamnolipid
and a more hydrophobic co-surfactant alkyl propoxylated (PO) sulfate (C,2,,3-8PO-
SO,Na, MW = 713), which was provided by Sasol Chemical Company. This co-
surfactant has a hydrophobic characteristic due to the long hydrocarbon chain length
(CIz,13 versus C, for rhamnolipid) and the presence of the hydrophobic PO groups.
The two surfactants were mixed at different ratios that produce lower IFT as well as
required less salt at optimum formulations for toluene than rhamnolipid alone (within
the range of 3-8 w/w % of salt, compared to 1 1-1 7 w/w Yo for toluene microemulsion
when only rhamnolipid was used). We also see synergism for hexane, with IFT values of
the mixture dropping below 0.1 mN/m (middle phase formation), albeit with a higher
fraction of the hydrophobic C,z,,3-8PO-S0,Na in the mixture (higher value on the
x-axis or C,2,,3-8PO-S0,Naconcentration in the mixture-less rhamnolipid and more
CIz,,,-8PO-SO,Na in the optimum mixture than for toluene). The synthetic surfactant
alone (x value of 1 in Fig. 5.9) still generates the lowest IFT for decane and hexadecane,
demonstrating the more hydrophobic nature of this surfactant. The IFT for decane and
hexadecane increases with the increasing fraction of rhamnolipid in the mixture. This
indicates that this synthetic surhctant is not hydrophobic enough to balance the HLB
of the rhamnolipid surfactant system for the more hydrophobic decane and hexadecane.
Figure 5.9 thus not only illustrates the synergism of using surfactant mixtures, but also
120 0T.T. Nguyen and D.A. Sabatini
demonstrates the importance of having the right surfactant combination to match the
hydrophobicity of the target oil.
The results in Fig. 5.9 suggest that we should use a surfactant even more lipophilic
than C,, ,,-8PO-S04Na to achieve a low IFT for decane and hexadecane. We thus
evaluated the more lipophilic alkyl polypropylene oxide ether sulfates (C,6-18PO-
2EO-S04Na, MW = 1590.7) provided by Huntsman Chemical Company; its longer
alkyl group and greater number of lipophilic PO groups makes it more lipophilic than
the C,,,,3-8PO-S0,Na discussed above. The resulting data are shown in Fig. 5.10 for
an equal molar mixture of rhamnolipid and C,,-18PO-2EO-SO4Na. The optimum
salinity decreases for all oils to the range between 2 to 8 w/w% and the relationship
between optimum salinity and EACN follows the trend described in equation 6. In
contrast to the results in Fig. 5.9, the IFT for decane and hcxadecane shown in Fig.
5.10 are observed to be ultralow (< 0.1 mN/m) and about 10 times lower than the IFT
for toluene and hexane. Thus C,,-18PO-2EO-S04Na is lipophilic enough to counter
the hydrophilicity of rhamnolipid and provide an appropriate HLB to the surfactant
mixture to form middle phase microemulsions with decane and hexadecane. The
results also indicate that C,6-18PO-2EO-SO4Na is too hydrophobic for toluene and
hexane (the IFT values are larger for these oils).
Nguyen and Sabatini (2008) hrther studied the microemulsion formation of
rhamnolipid biosurfactant in mixtures with conventional synthetic surfactant sodium
EACN
Fig. 5.10. Optimum IFT ( 0 )and optimum salinity (5’)(0) of a mixture of rhamnolipid and C16-18PO-2E0
sulfate versus EACN (EACN values of 1 for toluene, 6 for hexane, 10 for decane, and 16 for hexadecane).
The total surfactant concentration was 0.1 w/w%. The ratio of rhamnolipid to Cl6-?8PO-2K)sulfate was
fixed at 3 7 by weight basis (or 1:l by molar basis). IFT was measured at 25 f 1°C for all oils. (Reprinted
from Nguyen et al., 2008. Reprinted with permission from Elsevier).
Microemulsionsof Rhamnolipid and Sophorolipid Biosurfactants 0 121
bis(2-ethyl) dihexyl sulfosuccinate (AOT; AOT has lower HLB, more soluble in oil) and
lipophilic linker oleyl alcohol for limonene and diesel oils. Limonene has a moderate
hydrophilicity (EACN value of =6 behaves similar to hexane from a microemulsion
perspective) and diesel is relatively hydrophobic (EACN value of 12 to 14); thus,
mixing rhamnolipid with the more hydrophobic surhctant AOT is required to form
microemulsions with these oils. Rhamnolipid is too hydrophilic while AOT is too
hydrophobic when used alone for both limonene and diesel. Therefore, neither of
these surfactants alone was able to form microemulsions with these oils. However,
mixing the two surfactants at an equal molar ratio was able to produce all three types
of microemulsions (Winsor Type I, 111, and 11) for both oils (Fig. 5.1 1). With the same
surfactant system, diesel microemulsion has higher optimum salinity (=8wt%) than
limonene ( ~ 2 . 5wt%) microemulsions, which agrees with the relationship between
optimum salinity and oil EACN proposed in equation 6. These results demonstrate
the advantage of using surfactant mixtures to promote microemulsion formation.
A "fish" phase diagram was also developed for the surfactant system containing soy
methyl ester ethoxylate (SMEE3EO), rhamnolipid biosurfactant UBR) and lipophilic
linker oleyl alcohol (OA) at a fixed ratio of SMEE3EO/JBR/OA = 4/1.75/2.5 by
weight percent with limonene oil (Nguyen & Sabatini, 2008). As can be seen in
Fig. 5.12, at 12.4 wt% total surfactant and linker concentration, the salinity of
middle phase microemulsions ranges from 7.5 to 10.5 wt% while at 2.1 wt% total
concentration, the salinity range is larger, from 11.5 to 17.5 wt%. Also, we can see
Ka('l, ?Vl%
Fig. 5.1 1. Microemulsionswith limonene and diesel oils using surfactant mixtures of AOThhamnolipid
= 0.05/0.05M. IFT was measured at 25 f 1°C for both oils.Three types of microemulsions (WinsorType I,
111, and II) were observed for both oils.
122 0 T.T. Nguyen and D.A. Sabatini
1
e
z
E
NVuCl, w t v o
Fig. 5.12. Phase behavior of surfactact and linker system containing SMEEKO/JBWOA at ratio of
4/1.75/2.5wt96with limoneneoil.lnterfacialtensionsweremeasuredatlow(2.1 wt96)and high(12.4wt96)
total surfactant and linker concentrations.IFTwas measuredat 25 f 1"C.Three types of microemulsions
(WinsorType I, 111, and II)were observed at both total concentration.
Sophorolipid-based Microemulsions
Sophorolipid biosurfactant (Fig. 5.13), donated by the United State Department
of Agriculture (USDA) with high purity (~100%active), was a diacetylated open
ring with two heads-sucrose head attached to two acetyl groups and carboxylic
acid head. The hydrophilicity/lipophilicity of sophorolipid was assessed based on its
Microemulsionsof Rhamnolipid and Sophorolipid Biosurfactants 0 123
K d l , Wt%l
Fig. 5.14. Interfacial tensions of limonene microemulsions with and without sophorolipid in the
formulation, measured at 25 f 1°C.
adjusting this ratio to 5/5.25/5 wt%, the SP'value increased to 3.4 mL oil/surfactant.
Both systems have low optimum salinity, 1.2 and 3 wt%, respectively, which is
appropriate for use in cosmetics and pharmaceutical applications.
The results shown in Table 5.1 demonstrate the effectiveness of rhamnolipid
and sophorolipid biosurfactants in formulating microemulsions with limonene as
compared to conventional synthetic surfactants. Comparing the SP' of the first two
surfactant systems, we observe that at the same surfactant molar concentration, the
surfactant system consisting of rhamnolipid has an SP' value about two times higher
than that of the other surfactant system that does not contain rhamnolipid (8.1
versus 3.4 mL oil/g surfactant). Looking at the SP' values of the last two surfactant
systems, we observe that the formulation that has sophorolipid achieves the SP' value
of 5.2 mL oil/g surfactant at total surfactant concentration of 15.25 wt% while the
surfactant system that uses oleyl alcohol instead of sophorolipid has the SP' value of
5 mL oil/g surfactant at total surfactant concentration of 24.75 mL oil/g surfactant).
Thus, replacing the lipophilic linker oleyl alcohol with the hydrophobic biosurfactant
sophorolipid achieves similar SP' values but at lower total surfactant concentration.
Conclusions
Results presented here show that biosurfactants can be as effective as conventional
surfactants when used in mixtures that tailor the surfactant system hydrophilicity/
lipophilicity to the EACN of the oil. We found that rhamnolipid works best for
very hydrophilic oils (low EACN values) while sophorolipid works best for more
hydrophobic oils (high EACN oils). Mixtures of these and other surfactants produce
Microemulsionsof Rhamnolipidand Sophorolipid Biosurfactants0 125
Table 5.1. Optimum Solubilization Parameter (SP*) for Different Surfactant Formulations
with Limonene Microemulsions
Formulationa Total Concentration SP‘ (mL Oil/g Surfactant)
AOT/JBR
(0.05/0.05 M or 0.1 M 8.1
2.2/2.0 wt%) (4.2 wt%)
AOT/AMA/SMDNS
(0.025/0.025/0.05 M or 0.1 M 3.4
1.1/1.0/1.25 wt%) (3.35 wt%)
SMEE3EO/SPL/JBR
(5/5.25/5 wt%) 15.25 wt% 5.2
SMEE3EO/OA/JBR
(12/7.5/5.25 wt%) 24.75 wt% 5.0
“AOT, Sodium bis(2-ethyl)dihexyl sulfosuccinate; JBR, Rhamnolipid; AMA, Sodium dihexyl
sulfosuccinate; SMDNS, Sodium mono- and dimethyl naphthalene; SMEE3E0, Soy methyl ester
ethoxylate; SPL, Sophorolipid; OA, Oleyl alcohol.
high efficiency systems for a wide range of oils (from toluene (EACN of 1) to
hexadecane (EACN of 16)).Future research should develop additional biosurfactants
with a range of hydrophilicity/lipophilicityvalues to hrther expand our ability to
formulate with biosurfactant mixtures and replace petroleum-based surfactants in
pursuit of economical and sustainable products and processes.
References
Acosta, E.; H. Uchiyama; D.A. Sabatini; J.H. Hanvell. The Role of Hydrophilic Linkers.]. Sufac-
t a m Deterg. 2002a, 5, 1 5 1- 157.
Acosta, E.; S. Tran; H. Uchiyama; D.A. Sabatini; J.H. Hanvell. Formulating Chlorinated Hydro-
carbon Microemulsions Using Linker Molecules. Environ. Sci. Technol. 2002b, 36, 4618-4624.
Acosta, E.; PD. Mai; J.H. Harwell; D.A. Sabatini. Linker-Modified Microemulsions for a Variety
of Oils and Surfactants./. Sufactants Deterg. 2003, 6, 353-363.
Acosta, E.J.; T. Nguyen; A. Witthayapanyanon; J.H. Harwell; D.A. Sabatini. Linker-Based Bio-
Compatible Microemulsions. Environ. Sci. Tecbnol. 2005,39, 1275-1282.
Bourrel, M.; and R.S. Schechter. Microemulsions and Related Systems: Formuhion, Solvenq and
Physical Properties. Surfactant Science Series 30; Marcel Dekker, 229-302, 1998.
Cameotra, S.S.; R.S. Makkar. Recent Applications of Biosurfactants as Biological and Immuno-
logical Molecules. Curr. Opin. MicrobioL 2004, 7, 262-266.
Childs, J.D.; E. Acosta; R. Knox; J.H. Hanvell; D.A. Sabatini. Improving the Extraction of Tetra-
chloroethylene from Soil Columns Using Surfactant Gradient Systems.]. Contam. Hydrol. 2004,
71,27-45.
Gautam, K.K.; V.K. Tyagi. Microbial Surfactants:A Review.]. Oleo Sci. 2006,55, 155-166.
Georgiou, G.; S.C.Lin; M.M. Sharma. Surface-Active Compounds from Microorganisms. Biol
Tecbnol. 1992,10, 60-65.
1260T.T. Nguyen and D.A. Sabatini
Graciaa A.; J. Lachaise; C. Cucuphat; M. Bourrel; J.L. Salager. Improving Solubilization in Micro-
emulsions with Additives. 1. The Lipophilic Linker Role. Lungmuir 1993,9, 669-672.
Griffin, W.C. Classification of Surface-Active Agents by "HLB".J. SOC.Cosmct. Chemkts 1949, I,
31 1-326.
Helvaci, $$.; S. Pecker; G. m e m i r . Effect of Electrolytes on the Surface Behavior of Rhamnolip-
ids R1 and R2. CoUoidr Surf B 2004,35, 225-233.
Herman, D.C.; Y. Zhang; R.M. Miller. Rhamnolipid (Biosurfactant) Effects on Cell Aggregation
and Biodegradation of Residual Hexadecane under Saturated Flow Conditions. AppL Environ.
MicrobioL 1997,63, 3622-3627.
Holmberg, K.; E. h e r b e r g . Structure of a Cosurfactant-Free Microemulsion.J Dkpersion Sci.
TecbnoL 1986, 7, 299-306.
Huh, C. Interfacial Tensions and SolubilizingAbility of a Microemulsion Phase That Coexists
with Oil and Brine. J. Colloid Interface Sci. 1979, 71,408-426.
Huibers, P.D.T.; D.O. Shah. Evidence for Synergism in Nonionic Surfactant Mixtures: Enhance-
ment of Solubilization in Water-in-Oil Microemulsions. Langmuir 1997, 13, 5762-5765.
Kitamoto, D.; H. Isoda; T. Nakahara. Functions and Potential Applications of Glycolipid
Biosurfactants-from Energy-SavingMaterials to Gene Delivery Carries. J Biosci. Bioeng. 2002,
94, 187-201.
Komesvarakul, N.; M.D. Sander; E. Szekeres; E.J. Acosta; J.F. Faller; T.Mendik; L.B. Fisher; G.
Nicoll; DA. Sabatini; J.E Scamehorn. Microemulsions of Triglyceride-Based Oils: The Effect of
Co-Oil and Salinity on Phase Diagrams.J. Cormet. Sci. 2006,57,309-325.
Kumar, I?; K.L. Mittal. Handbook ofMicroemulsion Science and Tecbnohn. Marcel Dekker, Inc.:
New York, 1999, pp 13-44.
Kunieda, H.; K. Shinoda. Correlation between Critical Solution Phenomena and Ultralow Inter-
facial Tensions in a SurfactantIWaterlOil System. BufL Cbem. SOC.Jpn. 1982,55, 1777-1781.
Li, I?; A. Ghosh; RF. Wagner; S. Krill; Y.M. Joshi; A.T.M. Serajuddin. Effect of Combined Use of
Nonionic Surfactant on Formation of Oil-in-Water Microemulsions. Int. J. Pbarm. 2005,288,
27-34.
Maier, R.M.; G. Sober6n-Chiva. Prcudomanus ueruginosa Rhamnolipids: Biosynthesisand Poten-
tial Applications. Appf. MicrobioL Biotecbnol. 2000,54, 625-633.
Mulligan, C.N.; R N . Yong; B.F. Gibbs. Heavy Metal Removal from Sediments by Biosurfactants.
J. Hazard. Matez 2001,85, 111-125.
Nguyen, T.T.; N.H. Youssefi M.J. McInerney D.A. Sabatini. Rhamnolipid Biosurfactant Mixtures
for Environmental Remediation. Water Rcr. 2008,42, 1735-1743.
Nguyen, T.T.; DA. Sabatini. Formulating Alcohol-Free Microemulsions Using Rhamnolipid
Biosurfactant and Rhamnolipid Mixtures. J Sufm. Detrrg. 2008, DO1 10.1007/sl1743-008-
1098-y
Ozdemir, G.; U. Malayoglu. Wetting Characteristicsof Aqueous Rhamnolipids Solutions. Cofhidr
Surf B 2004,39, 1-7.
&emir. G; S. Peker; S.S. Helvaci. Effect of pH on the Surface and Interfacial Behavior of Rham-
nolipids R1 and R2. CoUoidr Surf A 2004,234, 135-143.
Microemulsionsof Rharnnolipidand Sophorolipid Biosurfactants 0 127
Rodrigues, L.; I.M. Banat; J. Teixeira; R. Oliveira. Biosurfactants: Potentid Applications in Medi-
cine. J. Antimicrob. Chemother. 2006,57, 609-61 8.
Rosen, M.J. Surfactants andIntefuciul Phenomena. 2nd ed. John Wiley & Sons, Inc., 1989. pp.
304-336393-420.
Sdager, J.L.; M. Borrel; R.S. Schechter; W.H. Wade. Mixing Rules for Optimum Phase-Behavior
Formulations of Surfactant/Oil/Water Systems. SOC.Petro. Eng. /. 1979, 19, 271-278.
Shah, V.; G.E Doncel; T Seyoum; K.M. Eaton; I. Zdenskaya; R. Hagver; A. h i m ; R. Gross.
Sophorolipids, Microbial Glycolipids with Anti-Human ImmunodeficiencyVirus and Sperm-
Immobilizing Activities.Antimicrob. Agents Chemother. 2005, 49, 4093-4 100.
Sunwoo, C.K.; W.H. Wade. Optimal Surfactant Structures for Cosurfactant-Free Microemulsion
Systems I. C16 and C14 Guebert Alcohol Hydrophobes.J. Dispersion Sci. Technol. 1992, 13,
491-5 14.
Tongcumpou, C.; E.J. Acosta; L.B. Quencer; A.F. Joseph; J.F. Scamehorn; D.A. Sabatini; S.
Chavadej; N. Yanumet. Microemulsion Formation and Detergency with Oily Soils: I. Phase
Behavior and Interfacial Tension. J. Sufuctunts Deterg. 2006, 6 191-203.
Uchiyama, H.; E. Acosta; S. Tran; D.A. Sabatini; J.H. Hanvell. Supersolubilizationin Chlorinated
Hydrocarbon Microemulsions: Solubilization Enhancement by Lipophilic and Hydrophilic
Linkers. Ind. Eng. Chem. Res. 2000,39, 2704-2708.
Van Bogaen, I.N.A.; K. Saerens; C.D. Muynck; D. Develter; W. Soetaert; E. Vandamme. Micro-
bial Production and Applications of Sophorolipids.Appl. Microbiol. BiotechnoL 2007, 76 23-34.
Whang, L.M.; P.W.G. Liu; C.C. Ma; S.S. Cheng. Application of Biosurfactants, Rhamnolipid,
and Surfactin, for Enhanced Biodegradation of Diesel-Contaminated Water and Soil. J. Huazrd.
Muter. 2008, 151, 155-163.
Winsor, I? Hydrotrophy, Solubilisation and Related Emulsification Processes. Trans. Furday SOC.
1948944, 376-389.
Winsor, I? Solvent Properties ofAmphiphilic Compounak Butterworth, London, 1954, pp. 1-10.
Xie, Y.; Y. Li; R. Ye. Effect of Alcohols on the Phase Behavior of Microemulsions Formed by a
Biosurfactant-Rhamnolipid. J. Dicpersion Sci. Technol. 2005,26 455-461.
Zhang, Y.; RM. Miller. Enhanced Octadecane Dispersion and Biodegradation by a Pseudomonar
Rhamnolipid Surfactant (Biosurfactant).Appl. Environ. Microbiol. 1992, 58, 3276-3282.
Zhang, Y.; R.M. Miller. Effect of Rhamnolipid (Biosurfactant) Structure on Solubilization and
Biodegradation of n-Alkanes. Appl. Environ. Microbiol. 1995, GI,2247-225 1.
Lipopeptide Biosurfactants and
‘Their Use in Oil Recovery
MichaelJ. Mclnerneyl, Noha YousseP, David P. Naglel
- ’Departmentof Botany and Microbiology, Universityof Oklahoma,770 Van Vleet Oval, Norman, OK73019;
‘Department of Microbiology and Molecular Genetics, Oklahoma State University, I 1 10s. Innovation Way,
Stillwater, OK 74074
Introduction
The standard of living that many nations enjoy is highly correlated to energy
consumption (Hall et al., 2003). The desire of individuals to improve their standard
of living coupled with the predicted increase in the world’s population will place large
demands on the world’s energy resources in the future. How will we meet the future
demand for more energy? Historically, we use fossil fuels as our main energy sources
with oil, coal, and natural gas supplying about 85% of world’s energy needs (Energy
Information Administration, 2006). The reliance on fossil fuels increased CO,
emissions and fostered global climate change. The use of more carbon-neutral energy
sources is desired, but difficult to achieve at the magnitude required. Even the most
optimistic projections suggest that renewable energy sources will meet less than 10%
of the world’s requirement for energy by 2030 (Energy Information Administration,
2006). The most difficult energy demand to meet is liquid transportation fuel. The
use of biofuels such as ethanol will increase substantially, but will only account for
~10% of the demand by 2030 (Energy Information Administration, 2007). Most
likely, crude oil will continue to be the dominant source of transportation fuels until
adequate supplies of more carbon-friendly fuels can be developed.
Are sufficient crude-oil reserves available to meet future energy demands?
The answer to this question is controversial, and answers range from “Global oil
production has peaked because the discovery of additional resources is unlikely” to
“Global oil production may increase because new resources may be discovered or
new technologies may be developed (Witze, 2007). Current technologies recover
only about one-third to one-half of the oil contained in the reservoirs. Globally, this
means that about 1 trillion barrels (0.16 Tm3) of oil were recovered to date and that
about 2 to 4 trillion barrels (0.3 - 0.6 Tm3) of oil remain entrapped in reservoirs
(Hall et al., 2003). In the United States, about 600 billion barrels (=96 Gm3) of oil
remain in U.S. reservoirs after conventional technologies reach their economic limit
(Energy Information Administration, 2007; Witze, 2007). A critical feature of U.S.
129
130 0 MJ. Mclnerney et al.
Lipopeptide Biosurfactants
Lipopeptide biosurfactants are surface-active agents produced mostly by members of
the Bacillus and Pseudomonas genera (Banat, 1995a; Bodour & Miller-Maier, 2002;
Van Dyke et al., 1991). Types of lipopeptides include surfactins, iturins, mycosubtilin,
bacillomycin, lichenysins, arthrofactin, putisolvin, and others. Besides their surface-
active properties, lipopeptide biosurfactants have biomedical applications as
therapeutic agents. Surfactins and iturins produced by Bacillus subtilis have antifungal
and antimicrobial properties as well as some antiviral activity (Kleinkauf & H. von
Dohren, 1990; Rodrigues et al., 2006; Singh & Cameotra, 2004; von Dohren,
1995). Surfactin also inhibits fibrin-clot formation, making it a good candidate in
thrombolytic therapy (Arima et al., 1968).
R-~H-CH,-CO-Lglu-LIm-Dlm-Lval-LaspDleu
0 I
R-~H-CH,-CO-Lglu-L1eu-Dlcu-Lval-Lasp-Dlcu-Lval
surfactin
R=CC,o-C,,
n,iso, anteiso
R-~H-CH,-CO-Lglu-LIm-Dleu-Lval-Lasp-D1-L11~
0 I
MycoinbtUln
R-~H-CH,-CO-Lasn-Dtyr-Dasn-tgln-Lgln-Lpro-Dser-~n
Nu 1 R=C,*-C,'
R-~€i-CH,-CO-Lglu-Lleu-Dleu-Lval-Lasn-Dle~-~~~l
S u r f s c t ~ 86
t
0 1 R=C I o-cy
n,iso, anteiso
R-~H-CH,-CO-Lgln-Lle-Dleu-Lval-Lasp-Dleu-Lval
Surfsctsnt *6
0 1 R=CI,-C,,
n,iso, anteno
3
R-CO-Lleu-Lglu-Llm-Lile-Lgln-Lser-Lval-Lile-La
-7
-Llm Lval L r €'StisoMn I
R=C,
Lleu Ls
R-CO-Llm-Lglu-Lleu-Lile-Lgln-Lscr-Lval-~Lleu
r PuthM~
R=C,
11
4 2
-Lleu-Lile-L
R-CO-Lleu-Lglu-Lleu-Lile-Lgln-Lser-Lval-Lib PutLoMn 11 vsr
R=C,
Fig. 6.1 .Types of lipopeptide biosurfactants produced by Bacillus and Pseudomonas species.The lactone
ring is formed between the carboxyl group of t h e C-terminal amino acid and t h e p-hydroxyl o r p-amino
group of t h e fatty acid.
Lipopeptide Biosurfactants and Their Use in Oil Recovery 0 133
Biosynthesis
Nonribsomal biosynthesis is an important mechanism for the production of natural
products. These include a variety of compounds: glutathiones, antibiotics (p-lactams,
gramicidins, tyrocidins, surfactins, etc.), and lipopeptide biosurfactants (Desai &
Banat, 1997; Kleinkauf & H. von Dohren, 1990; von Dohren, 1995). A molecular
analysis of the genetic sequences of the nonribosomal peptide synthetases shows very
high levels of homology between organisms, even for peptide products of significantly
different properties (see von Dohren, 1995).
Nonribosomal peptide synthetases are modular proteins that catalyze the selective
binding, activation, and condensation of the amino acids in the peptide polar head
of lipopeptides via specific domains of each module according to the multiple-carrier
134 0 MJ. Mclnerney et al.
thiotemplate mechanism (Berti et al., 2007; Galli et al., 1994; Menkhaus et al., 1993;
Stein et al., 1996; Vater et al., 1997). The groups of Zuber, Marahiel, and Grandi
developed the understanding of this process, which was exploited to engineer strains
of B. subtilis that produced altered surfactins (Peypoux et al., 1999; Schneider et al.,
1998; Stachelhaus et al., 1995). Figure 6.2 represents the functional gene organization
of the surfactin synthetase operon. Each module consists of at least three domains:
(i) the adenylation or amino- acid activation domain where the amino-acid residue is
recognized and activated to its acyl adenylate derivative by a reaction with adenosine
triphosphate (ATP), (ii) the thiolation domain where the activated amino-acid ester is
covalently linked as its thioester to the enzyme-bound 4-phosphopantothiene group
(transferred to a conserved serine residue in the thiolation domain by the action of
4-phosphopantothienyl transferase, Sfp in case of surfactin synthetase), and (iii) the
condensation domain catalyzing the direct transfer of the amino-acid residue from
the 4-phosphopantothiene to an adjacent acylamino acid covalently linked to the
downstream module via peptide bond formation.
Additional domains within the module catalyze modifications to the structure
(e.g., epimerization of L-leu to D-leu). Thioesterase domains at the C-terminal end
of the module that add the C-terminal amino acid to the peptide chain (Schneider &
Marahiel, 1998) and distinct thioesterases encoded by genes present downstream of the
nonribosomal peptide synthetase operon catalyze the release of the peptide from the
enzyme complex and the lactonization or cyclization step (Bruner et al., 2002; Kohli
et al., 2001). SrfXD is an external thioesterase associated with surfactin synthetase
that stimulates the initiation of surfactin synthesis by transferring P-hydroxy fatty
acyl-CoA to glutamate to form P-hydroxymyristoyl-glutamate(Steller et al., 2004).
Regulation
Tratucriptional Regulation of Sur&tin Bioryntbesh
Biosurhctant biosynthesis has complex regulation. The genes encoding nonribosomal
peptide synthetases are organized in operons that are transcriptionally induced in
Fig. 6.2. Surfactin synthetase gene organization. Each module consists of at least three domains:
adenylation, thiolation, and condensation. Modules 3 and 6 also have an epimerization domain for
D-leu formation. Module 7 has an additional thioesterasedomain at the 3’end. Adenylation domains are
labeled by the amino acid activated by that domain.
Lipopeptide Biosurfactants and Their Use in Oil Recovery 0 135
the hydrophobic domain (Bonmatin et al., 1995) (Table 6.2).Morikawa et al. (1993)
argued that the acidic residues- Asp and Glu-are most important for activity. But,
the monoanionic biosurfactant, lichenysin A, which has asparagine in position 5 , has
better surface activity than the dianionic biosurfactant, surfactin, which has aspartate
in position 5 (Grangemard et al., 1999; Yakimov et al., 1995).
Formulating Biosut$actant M h m s
An optimal balance between the hydrophilic-hydrophobic interactions between the
displacing fluid and the crude oil is critical to obtain very low IFTs (<0.1 mN/m).
Table 6.3 shows several approaches to alter hydrophilic-hydrophobic interactions to
achieve low IFTs. One approach is to mix biosurfictants, either lipopeptides with
different fatty-acid compositions or lipopeptides with rhamnolipid. In both cases, the
mixtures became more hydrophilic, and had IFT values <0.1 mN/m against toluene
(Youssef et al., 2007a). Low IFT values against hydrophobic hydrocarbons (decane or
hexadecane) require the addition of a hydrophobic, synthetic surhctant.
Lastly, the salt concentration and the pH of the displacement fluid can be
manipulated to enhance interficial activity (Nguyen et al., 2008).
Wat&oding
Biosurfactant-enhanced water flooding differs from well treatments in that the goal
of biosurfactant-enhanced water flooding is to mobilize entrapped oil deep within
the reservoir rather than to increase the production of a single well. Nutrients with
or without a bacterial inoculum are injected into the reservoir to stimulate microbial
activity and biosurfactant production throughout the reservoir (Fig. 6.3). While
laboratory studies indicate that this approach is very effective, only two field tests of
biosurfactant-enhanced waterflooding were performed (Bryant & Burchfield, 1991;
Bryant et al., 1990, 1993, 1994). In both tests, the investigators used a mixed culture
with the B. mojavensis strain JF-2 and molasses as the nutrient. After inoculation,
the molasses was periodically added to each injection well into the first test. The oil-
production rate of the field increased by 14%, and the ratio of water-to-oil produced
from the field decreased after the microbial treatment (Bryant & Burchfield, 1991;
Bryant et al., 1990). The only evidence for in situ biosurfictant production was the
lowering of the surfice tension of produced fluids 6 weeks after inoculation. The
presence ofa lipopeptide biosurfactant was not confirmed by hrther chemical analyses.
In the second test, the inoculum was added, and then molasses was continuously
injected along with the injected brine (Bryant et al., 1993, 1994). The oil-production
rate increased in the second field by about 19% for 3 years. In each case, the change
in oil production and the total incremental oil recoveries, 88 and 400 m3in 2 years,
were low, which raised skepticism as to whether the microbial treatment improved oil
recovery above pretreatment levels (Bryant & Lockhart, 2002).
Conclusions
We must develop new, cost-effective technologies to recover the large amounts of
entrapped oil that exist in oil reservoirs to meet the growing demand for energy.
Laboratory studies show that lipopeptide biosurfactants are promising oil-recovery
agents. Lipopeptides have CMCs that are much lower than synthetic surfactants,
they generate low IFT between the hydrocarbon and aqueous phases (cO.1 mN/m),
and they remain active over a wide range of environmental conditions. Genetic and
144 0 MJ. Mclnerney et al.
I P
Biosurfactant
Acid
Solvent
Acknowledgments
We thank the U.S.Department of Energy (contracts DE-FC26-02NT15321 and
DE-FC26-04NT15522) for support of our work.
References
Abhati, N.; R. Roostaazad; F. Ghadiri. Biosurfactant production in MEOR for improvement of
Iran’s oil reservoirs production experimental approach. SPE 84907. SPE International Improved
Oil Recovery Conference in Asia Pacgc; Kuala Lumpur, Malaysia, 2003; Society of Petroleum
Engineers, Richardson, TX.
Abu-Ruwaida, A.S.; I. M. Banat; S. Haditiro; A. Salem; M. Kadri. Isolation of biosurfactant-pro-
ducing bacteria: Product characterization and evaluation. Acta Biotechnol. 1991, 11, 31 5-324.
Adkins, J.I?; RS. Tanner; E.O. Udegbunam; M.J. McInerney; R.M. Knapp. Microbially enhanced
oil recovery from unconsolidated limestone cores. Geomicrobiol.1.1992, 10, 77-86.
Akpa, E.J.P.; B. Wathelet; M. Paquot; R. Fuchs; H. Buzikiewicz; I? Thornart. Influence of culture
conditions on lipopeptide production by Bacillw subtilis. Appl. Microbiol. Biotechnol. 2001,
91-93, 551-561.
146 0 MJ. Mclnerney et al.
1991,31,399-420.
Bryant, RS.; T.E. Burchfield; D.M. Dennis; D.O. Hinman. Microbial-enhanced waterflooding:
Mink Unit project. SOC.Pet. Eng. Reservoir Eng. 1990,5,9-13.
Bryant, RS.; T.E. Burchfield; M.D. Dennis; D.O. Himman. Microbial enhanced waterflooding
Mink Unit Project. SPE 17341. SPE/DOE Enhanced oilRecovmy Symposium 1988; Society of
Petroleum Engineers, Richardson, Tx
Bryant, RS.; J. Douglas. Evaluation of microbial systems in porous media for E O R SOC.Pet. Eng.
Reservoir Eng. 1988,3,489-495.
Bryant, S.L.; TI?Lockhart. Reservoir engineering analysis of microbial enhanced oil recovery. SOC.
Pet. Eng. Reservoir EvaL Eng. 2002,5, 365-374.
Bryant, RS.; A.K. Stepp; KM. Bertus; T.E. Burchfield; M. Dennis. Microbial-enhanced water-
flooding field pilots. Dcv. Pet. Sci. 1993,39,289-306.
Bryant, RS.; A.K. Stepp; K.M. Bertus; T.E. Burchfield; M. Dennis. Microbial enhanced water-
flooding field tests. Be 9th Symposium on improved Oil Recourry, Tulsa, OK, USA, 1994; Society
of Petroleum Engineers (SPE): Richardson, TX, USA, pp. 159-170.
Buciak, J.M.; A.E. Vazqua; RJ. Frydman; J.A. Mediavilla; RS. Bryant. Enhanced oil recovery
by means of microorganisms: pilot test. Proceedings of the 3tdLatin American and Caribbean Pe-
t r o h m Engineering Confirenre,Buenos Ares, Argentina, 1994; Society of Petroleum Engineers
(SPE): Richardson, TX, USA, pp. 279-287.
Cameotra, S.S.; RS. Malckar. Synthesis of biosurfactants in extreme conditions. AppL Microbiol.
Biotecbnol. 1998,50, 520-529.
Chang, Y. Preliminary studies assessing sodium pyrophosphate effects on microbially mediated oil
recovery. Ann. N . Y A c d . Sci. 1987,506,296-307.
D’Souza, C.; M.M. Nakano; I? Zuber. Identification of comS, a gene of the s@ operon that
regulates the establishment of genetic competence in Baiflw subtilis. Prof. NatL A c d . Sci. USA
1994,91,9397-940 1.
Das, K.; A.K. Mukherjee. Characterization of biochemical properties and biological activities of
biosurfactants produced by Pscudomonac amginosa mucoid and non-mucoid strains isolated
from hydrocarbon-contaminated soil samples. Appl. MicrobioL BiotecbnoL 2005,69, 192-1 99.
Das, K.; A.K. Mukherjee. Comparison of lipopeptide biosurfactants production by Bacillw subtilis
strains in submerged and solid state fermentation systems using a cheap carbon source: Some
industrial applications of biosurfactants. Proccss Biocbem. 2007,42, 1191-1 199.
Davis, D.A.; H.C. Lynch; J. Varley. The production of surfactin in batch culture by Baillw
subtilis ATCC 21332 is strongly influenced by the conditions of nitrogen metabolism. Enyme
Microb. Techno1 1999,25, 322-329.
Deleu, M.; H. Radindralambo; Y. Popineau; I? Jacques; I? Thonart; M. Paquot. Interfacial and
emulsiFying properties of lipopeptides from Bacillw subtilis. Colloidc Su$, A. Pbysicocbem. Eng.
Aspects 1999,152,3-10.
Desai, J.; I.M. Banat. Microbial production of surktants and their commercial potential. Micro-
biol. MoL BioL &. 1997,61,47-64.
Energy Information Administration. international Energy OutlooR, U.S.Department of Energy:
Washington, D.C., 2006.
148 0 M.J. Mclnerney et at.
Lin, S.-C.; K.S. Carswell; M.M. Sharma; G. Georgiou. Continuous production of the lipopeptide
biosurfactant of Bacillus licbeniformis JF-2. Appl. Microbiol. Biotecbnol. 1994a, 41,281-285.
Lin, S.-C.; M.A. Minton; M.M. Sharma; G. Georgiou. Structural and immunological charac-
terization of a biosurfactant produced by Bucilh licbeniformis JF-2. AppL Environ. Microbiol.
1994b, GO, 31-38.
Lin, S.-C.; M.M. Sharrna; G. Georgiou. Production and deactivation of biosurfactant by Bacillus
licbenijirmis JF-2. Biotecbnol. Prog. 1993,9, 138-145.
Macquaire, F.; F. Baleux; E. Giaccobi; T. Huynh-Dinh; J.-M. Neumann; A. Sanson. Peptide sec-
ondary structure induced by a micellar phospholipidic interface: proton NMR conformational
study of a lipopeptide. Biochemistry 1992,31,25762582.
Maier, R.M.; G. Soberon-Chavez.Pseudomonac ueruginosu rhamnolipids: biosynthesis and poten-
tial apllications. Appl. Microbiol. Biotechnol. 2000,54, 625-633.
Makkar, R.S.; S.S. Cameotra. Utilization of molasses for biosurfactant production by two Bacillus
strains at therrnophilic conditions./. Am. Oil. Cbem. SOC.1997, 74, 887-889.
Makkar, R.S.; S.S. Cameotra. Production of biosurfactant at mesophilic and thermophilic condi-
tions by a strain of'Bucillus subtilis. /. Ind. Microbiol. Biotechnol. 1998, 20, 48-52.
Marahiel, M.A.; M.M. Nakano; l? Zuber. Regulation of peptide antibiotic production in Bacillus.
Mol. Microbiol. 1993, 7, 631-636.
Matsuyama, T.; K. Kaneda; Y. Nakagawa; K. Isa; H. Hara-Hotta; I. Yano. A novel extracellular
cyclic lipopeptide which promotes flagellum-dependentand -independent spreading growth of
Serrutiu murcescens.J. Bucteriol. 1992, 174, 1769-1 776.
Maudgalya, S.; R.M. Knapp; M.J. McInerney; D.l? Nagle; M.J. Folsmbee. Development of a
bio-surfactant-based enhanced oil recovery procedure. SPE 89473. ?he SPEIDOE Improved Oil
Recovery Symposium, Tulsa, OK, 2004; Society of Petroleum Engineers, Richardson, TX.
Maudgalya, S.; M.J. McInerney; R.M. Knapp; D.l? Nagle; M.M. Folmsbee. Tertiary oil recovery
with microbial biosurfactant treatment of low-permeability Berea sandstone cores. SPE 94213.
Society of Petroleum Engineers Production and Operutions Symposium, Oklahoma City, OK, 2005;
Society of Petroleum Engineers: Richardson, TX.
McInerney, M.J.; K.E. Duncan; N. Youssef; T. Fincher; S.K. Maudgalya; M.J. Folrnsbee; R.
Knapp; D.R. Simpson; N. Ravi; D.l? Nagle. Development ofMicroorgunisms with Improved Trans-
port and Biosurfactant Activityfor Enhunced Oil Recouep Department of Energy, ASM Press,
Inc., Washington, D.C.: 2005a.
McInerney, M.J.; M. Javaheri; D.l? Nagle. Properties of the biosurfactant produced by Bacillw
licbenzjirmis strain JF-2. /. Ind. Microbiol. 1990,5, 95-102.
McInerney, M.J.; G.E. Jenneman; R.M. Knapp; D. E. Menzie. Biosurfactant and enhanced oil
recovery. 1985.
McInerney, M.J.; D.l? Nagle; R.M. Knapp. Microbially enhanced oil recovery: Past, present,
and future. Petrokum Microbiology; B. Ollivier, M. Magot, Eds.; ASM Press: Washington, DC,
2005b; pp. 215-238.
McInerney, M.J.; K.L. Sublette. Petroleum microbiology: Biofouling, souring, and improved oil
recovery. Manual ofEnvironmentul Microbiohgy; C.J. Hurst, G.R. Knudsen, M.J. McInerney,
L.D. Stetzenbach, M.V. Water, Eds.; 1997; pp. 600-607.cPublisher and location?>
LipopeptideBiosurfactants and Their Use in Oil Recovery 0 15 1
McInerney, M.J.; G. Voordouw; G.E. Jenneman; K L. Sublette. Oil field microbiology. Manual of
Environmental Microbiology; C.J. Hurst, RL. Crawford, J.L. Garland, D.A. Lipson, A.L. Mills,
L.D. Stetzenbach, Eds.; ASM Press, Inc.: Washington, D.C, 2007; pp. 898-91 1.
Menkhaus, M.; C. Ullrich; B. Kluge; J. Vater; D. Vollenbroich; R.M. Kamp. Structural and func-
tional organization of the surfactin synthetase multienzyme system./. BioL Chcm. 1993,2G8,
7678-7684.
Morikawa, M.; H. Daido; T. Takao; S. Murata; Y. Shimonishi; T. Imanaka. A new lipopeptide
biosurfactant produced by Arthrobacter sp. strain MIS38. J. Bacteriol 1993, 175,01594466.
Morikawa, M.; Y. Hirata; T. Imanaka. A study on the structure-function relationship of lipopep-
tide biosurfactants. Biocbim. Biopbys. Acta 2000, 1488,211-218.
Mukherjee, S.; I? Das; R. Sen. Towards commercial production of microbial surfactants. Trcndc
Biotcchnol. 2006,24, 509-5 15.
Nguyen, T.; N. Youssef; M.J. McInerney; D. Sabatini. Rhamnolipid biosurfactant mixtures for
environmental remediation. Water &s. 2008,42, 1735-1743.
Oka, K.; T. Hirano; M. Homma; H. Ishii; K. Murakami; S. Mogami; A. Motizuki; H. Morita; K.
Takeya; H. Itokawa. Satisfactory separation and MS-MS spectrometry of six surfactins isolated
from Bacillw subtilis natto. Chcm. Pharm. BuU. 1993,41, 1000-1002.
Ollivier, B.; M. Magot. Pctrohm Microbiology. ASM Press: Washington, D.C., 2005.
Peypoux, F.; J.M. Bonmatin; J. Wallach. Recent trends in the biochemistry of surfactin. Appl
MicrobioL BiotecbnoL 1999,5I, 553-563.
Peypoux, F.; J-M. Bonmatin; H. Labbe; I. Grangemard; B.C. Das; M. Ptak; M. Wallach; G. Mi-
chel. [Ala4]Surfactin, a novel isoform from Bacillw subtilis studied by mass and NMR spectro-
copies. Eur, /. Biocbem. 1994,224,89-96.
Peypoux, F.; 1.-M. Bonmatin; H. Labbe; B.C. Das; M. Ptak; G. Michel. Isolation and character-
ization of a new variant of surfactin, the [Val7]-surfactin.Eur. /. Biocbem. 1991,202, 101-106.
Peypoux, F,; G. Michel. Controled biosynthesis of Vd7- and Leu7-surfactins. Appl. Microbiol.
Biotecbnol. 1992,36, 5 15-5 17.
Planckaert, M. Oil reservoirs and oil production. Pctrohm Microbiolou; 8. Ollivier, M. Magot,
Eds.; ASM Press: Washington, D.C., 2005; pp. 3-20.
Reed, RL.; R. Healy. Some physical aspects of microemulsion flooding: a review. Improved Oil
Rccovrry by Surfacklnt and Polymer Flooding; D.O. Shah, RS. Scheckter, Eds.; Academic Press,
Inc.: New York, 1977; pp. 383437.
Rodrigues, L.; I.M. Banat; J. Teixeira; R Oliveira. Biosurfactants: potential applications in medi-
cine. /. Antimicrob. Chcmotber. 2006,57,609-618.
Rosenberg, E.; E.Z. Ron. High- and low-molecular-mass microbial surfactants.AppL Microbiol.
Biotecbnol. 1999,52, 154-162.
Schaller, K.D.; S.L. Fox; D.F. Bruhn; KS. Noah; GA. Bala. Characterization of surfactin from
Bacillw subtilis for application as an agent for enhanced oil recovery. AppL Biocbm. BiotecbnoL
2004,113-116,827-836.
Schneider,A.; M.A. Marahiel. Genetic evidence for a role of thioesterase domains, integrated in
or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Baciffzasubtilis.
152 0 M.J. Mclnerney et al.
Wang, Q.; X Fang; 8. Bai; X. Liang; RJ. Shuler; W. A. Goddard; Y. Tang. Engineering bacteria for
production of rhamnolipid as an agent for enhanced oil recovery. Biotecbnol. Bioeng. 2007,98,
842-853.
Weihong, Q.D.; D. Liangjun; 2. Zhongkui; Y. Jie; L. Huamin; L. Zongshi. Interfacial behavior of
pure surfactants for enhanced oil recovery-Pan 1: A study on the adsorption and distribution
of cetylbenzene sulfonate. Tensidc Surfactants DeteTents 2003, 40, 87-89.
Wine, A. That's oil, folks... Nature 2007, 445, 14-17.
Yakimov, M.M.; M.M. Amro; M. Bock; K. Boseker; H.L. Fredrickson; D.G. Kessel; K.N. Tim-
mis. The potential of Bacillw Licbenz$tmis strains for in situ enhanced oil recovery. /. Pet. Sci.
Ens 1997,18, 147-160.
Yakimov, M.M.; H.L. Fredrickson; K.N. limmis. Effect of heterogeneity of hydrophobic moities
on surface activity of lichenysin A, a lipopeptide biosurfictant from Bacillw licbenz$rmis BASSO.
Biotecbnol. Appl Biocbem. 1996,23, 13-18.
Yakimov, M.M.; K.N. Timmis; V. Wray; H.L. Fredrickson. Characterization of a new lipopeptide
surfactant produced by thermotolerant and halotolerant subsurface Bacillw licbenz$rmis BASSO.
Appl Environ. Mirrobiol. 1995, 61, 1706- 17 13.
Youssef, N.; K. Duncan; M. McInerney. Importance of the 3-hydroxy fatty acid composition of
lipopeptides for biosurfactant activity. Appl. Environ. Microbiol. 2005, 71,7690-7695.
Youssef, N.; K. Duncan; D. Nagle; K. Savage; R Knapp; M. McInerney. Comparison of methods
to detect biosurfactant production by diverse microorganisms. /. Mimbiol Metboa? 2004,56,
339-347.
Youssef, N.; T. Nguyen; DA. Sabatini; M.J. McInerney. Basis for fbrmulating biosurfactant
mixtures to achieve ultra low interfacial tension values against hydrocarbons./. Znd Microbiol.
Biotecbnol. 2007% 34,497-507.
Youssef, N.; D.R. Simpson; K.E. Duncan; M.J. McInerney; M. Folmsbee; T Fincher; RM.
Knapp. In situ biosurfactant production by Baciflw Strains injected into a limestone petroleum
reservoir. Appf. Environ. Mirrobiol 2007b, 73, 1239-1247.
Zaijic, E.J. Scale up of microbes for single well injection. Proceedings of the First International
MEOR Workshop; 1987; J. King, J.; D. Stevens, Eds.; U.S.Department of Energy, Bartlesville
Project Office: Bartlesville, OK, 1987, pp. 241-246.
Zekri, A.Y.; R Almehaideb; 0. Chaalal. Project of increasing oil recovery from UAE reservoirs
using bacteria flooding. SPE 56827. SPEAnnual Technical Conjkence, Richardson, TX,1999;
Society of Petroleum Engineers.
Zhang, X.; R M . Knapp; M.J. McInerney. A mathematical model for microbially enhanced oil
recovery process. Dcy. Pet. Sci. 1993,39, 171-186.
Employment of Phospholipids
and their Mimics in Biomedical
Applications
Influence of Pulmonary Surfactant
Protein Mimics on Model Lung
Surfactant
Hiromichi Nakahara', Sannamu Leez, and Osamu Shibata'
'Departmentof Biophysical Chemistry,Facub of PharmaceuticalSciences, Nagasaki lnternational
University,2825-7Huis Ten Bosch, Sasebo, Nagasaki 859-3298,Japan, 'Department of Chemistry,Faculty of
Science,Fukuoka University,8- 19- 1 Nanakuma,Johnan-ku, Fukuoka814-0180, Japan,
157
158 0 H. Nakaharaet al.
also are tightly associated with surfactant lipids. SP-D is less abundant than SP-A and
is found largely in the lipid-depleted supernatant of centrifuged lavage rather than
sediments with the majority of surfactant lipids and the other proteins (Kuroki et al.,
1991). SP-D is absent from packaged intracellular surfactant in type I1 cell lamellar
bodies (Crouch et al., 1991; Voorhout et al., 1992).
DPPC is known to form tightly packed and solid surface films and has properties
that lower surface tension to near zero in the multilayer state during Z-A (surface
pressure-molecular area) cycling. Disaturated phospholipids in PS are thought to
become enriched in the surface films at the expense of fluid components during
compression to low interfacial area, or high surface concentration. However, the
resultant rigidity of the condensed films at physiological conditions causes it to
adsorb slowly from the alveolar fluid and to respread poorly from multiple materials
(Fleming & Keough, 1988). The unsaturated and anionic PG is thought to help
DPPC molecules adsorb and respread rapidly despite the fact that it does not
lower surface tension as effectively as DPPC. In addition, the potential for specific
molecular biophysical associations between anionic phospholipids and one or more
of the surfactant apoproteins has also been widely suggested (Baatz et al., 1990;,
Johansson et al., 1991; Chang et al., 1998). PA is present in a relatively low amount
in comparison with DPPC, but it is used as a very important additive for the proper
Lnctioning of both natural and synthetic PS replacement formulations. The addition
of 510 wt% PA to natural PS extracts obtained from animal sources has induced a
significant improvement in their properties both in vitro and in vivo (Cockshutt et
al., 1991; Gorree et al., 1991).
Besides lipids, the four proteins (SP-A, -B, -C, and -D) are associated with PS
functions in many cases (Hawgood & Shiffer, 1991; Johansson & Curstedt, 1997).
SP-A and SP-D, which contain hydrophilic proteins, play an important role in the
first-line defenses against inhaled pathogens (Miyamura et al., 1994) and in the
storage and transport of PS (Goerke, 1974; Voorhout et al., 1991). O n the contrary,
SP-B and SP-C are hydrophobic proteins that dominate the surface activity of PS.
They promote considerable adsorption of PS from the hypophase to the surface
at the air-water interface. Furthermore, they trigger a reversible exclusion of fluid
materials from the multicomponent monolayers when they are compressed beyond
their collapse pressures (Taneva & Keough, 1994a, 1994b, 1994c). SP-A is the most
abundant apoprotein in endogenous lung surfactants and has an amino acid sequence
that is highly conserved among animal species (Benson et al., 1985; White et al.,
1985; Floros et al., 1986; Boggaram et al., 1988). The N-terminal portion of the
molecule is collagen-like, while its C-terminal carbohydrate-binding sequence places
it in the family of mammalian C-type lectins and the collectin family. The human
SP-A (35 kDa) contains 228 amino acids with a mix of hydrophilic and hydrophobic
residues. This monomer has a short N-terminal domain of 7 amino acids followed
by a collagen-like region with 23 proline-rich Gly-X-Y repeats broken with a Pro-
Cys-Pro-Pro sequence between the lYh and 14th residues. SP-A also contains an
Influenceof Pulmonary Surfaaant Protein Mimics on Model Lung Surfactant0 159
than SP-B for particular phospholipids (Pkrez-Gil et al., 1995). Both SP-B and SP-C
participate in the selective refining process during expiration. Fluid lipids with less
ability to sustain high surface pressures are preferentially ejected to enrich the surface
films in rigid phospholipids (mainly DPPC) (Nahmen et al., 1997; Lipp et al., 1998).
In general, this behavior has been known as the “extraction” phenomenon. It has been
suggested that the extracted materials form and occupy a “surface-associated reservoir”
at the adjacent interface (Schiirch et al., 1998; Takamoto et al., 2001; Ding et al.,
2003). This extraction is indispensable for PS function. However, its mechanism has
not been made clear yet.
preparation with KL, peptide (consisting of novel 21-amino acid residues) has been
approved for clinical use (Revak et al., 1996; Ma et al., 1998; Cai et al., 2003).
Moreover, the preparation containing recombinant SP-C (rSP-C; 34 amino acid
residues) has been under active investigation for clinical efficacy (Amrein et al., 1997;
Krol et al., 2000; Grigoriev et al., 2003). Both preparations are composed of the
DPPC/PG/PA (= 68:22:9 by weight) mixture with the exception of minor differences
in the PG species. Although a few medicines containing new protein analogs have
been developed (Ma et al., 1998; Veldhuizen et al., 2000), no replacement surfactant
has been found comparable to the complete native surfactant with SP-B and -C.
Therefore, the development of such synthetic-type medicines is strongly desired.
Number of
residue 1-10 1 1-20 21-30 31-34
He1 13-5 KLLKLLLKLW LKLLKLLL
KL. KLLLLKLLLL KLLLLKLLLL K
rSP-C GIPFFPVHLK RLLIWWW LlVWlVGAL LlGL
Fig. 7.1. Amino acid sequences of synthetic peptides (He1 13-5, KL,, and rSP-C) mimicking native
SP-B and SP-C. Abbreviations: K, lysine; L leucine; W, tryptophan; G, glycine; I, isoleucine; P, proline; F,
phenylalanine;V, valine; H, histidine; R, arginine; A, alanine.
162 0 H.Nakahara et al.
G
Fig. 7.2. Helical wheel representations of He1 13-5, KL,, and rSP-C. The abbreviations with open cycles
denote hydrophilic amino acids.
(12 Leu and 1 Trp) and 5 hydrophilic residues (5 Lys) (Fig. 7.1 and 7.2). The
hydrophobic-hydrophilic balance was estimated both theoretically from the calculated
hydrophobicity value (or the magnitude of hydrophobic faces) and experimentally
from the retention times in reverse phase high-performance liquid chromatography
(RP-HPLC). For this modulation (hydrophobicity of He1 13-5 is 0.07) (Kiyota et
al., 1996), it is supposed that He1 13-5 mimics SP-B of the amphipathic helical
structure rather than SP-C of the transmembrane helical structure at the alveolar
air-liquid interface. In addition, when it forms an ideal a-helical structure, it has the
amphiphilic structure with a 260" hydrophobic sector region as shown in the helical
wheel representation (Fig. 7.2). This means that the hydrophobic and the hydrophilic
parts are completely separated in the a-helical structure. KL, peptides have been used
as an element in synthetic-type RDS medicines (or Surfaxin) to mimic the native
SP-B (Gustahon et al., 1996; Ma et al., 1998; Cai et al., 2003), whereas rSP-C
peptides are in clinical trials (Phase 111) as Venticute and are based on recombinant
SP-C (Amrein et al., 1997; Nahmen et al., 1997; Krol et al., 2000; Grigoriev et al.,
2003). Although rSP-C forms a transmembrane a-helix across the air-water interface,
He1 13-5 and KL, take amphiphilic a-helical structures at the interface. Notice that
the a-helical representations for KL, and rSP-C reveal a disheveled configuration of
hydrophobic and hydrophilic amino acids (Fig. 7.2).
Adrotpion Isotberms
Surface pressure (z)-time (t) isotherms of pure DPPC and the representative DPPC/
He1 13-5 mixture (XHe, ,3-5 = 0.1) formed by adsorption from their vesicle solutions
are shown in Fig. 7.3. It indicated a slow adsorption of DPPC molecules from
0.15 M NaCl subphase as reported previously (Serrano et al., 2005). For X,,,3-5 =
0.1, on the contrary, the z-t isotherm had a shoulder at 5 15 min and reached the
equilibrium surface pressure of 537 mN/m. SP-B accelerated the adsorption rate of
vesicular phospholipids from the subphase to the air-water interface (Oosterlaken-
Dijksterhuis et al., 1991). Similarly to native SP-B,our mimicking peptide, He1 13-5,
facilitated the interfacial adsorption of DPPC at the air-water interface (Nakahara et
al., 2006b).
70
60 DPPC
DPPC/Hel13-5(XHe1 13-5=o,1)
- 50
k
20
10
0
0 20 40 60 80 100
t l min
Fig. 7.3. n-t isotherms of pure DPPC and the representative DPPC/Hel 13-5 system (X,,,, ,3-5 = 0.1)
adsorbing from their vesicular solutions in 0.15 M NaCl at 298.2 f 1.0 K.
13-5 from surface monolayers into the subphase upon compression. The value of the
extrapolated area reflects the molecular conformation of peptides. The value of =2.6
nm2 supports a-helical structure rather than Fsheet, random, and other structures.
Further investigation of its conformation at the interface was referred to in a previous
paper (Nakahara et al., 20OGb). The surface potential (AV) of He1 13-5 always showed
a positive variation under compression (Fig. 7.4). The surface potential of He1 13-5
monotonically increased to =380 mV, =350 mV, and =310 mV on compression
at 298.2, 303.2, and 310.2 K, respectively. The reduction of AVvalues reflects an
aggravation of molecular orientation due to the increase in molecular motions.
>
.
a
a
E
.
z
E
k
Fig. 7.4. n-A and AV-A isothermsof pure He1 13-5 monolayer in a 0.02MTris buffer solution (pH 7.4) with
0.13MNaClat298.2,303.2,and310.2K.
provides the evidence that He1 13-5 forms the disordered film miscible with the FM
probe, independent of surface pressure. The results also correspond to those of the
PA isotherms for He1 13-5 (Fig. 7.4).
10
Fig. 7.5. Fluorescence microscopy (FM)images of pure He1 13-5 spread in 0.02 M Tris buffer with 0.13 M
NaCl at 298.2 K for various surface pressures.The monolayers contain 1 mol% fluorescent probe (R18).
The scale bar in the lower right represents 100 pm.
is much smaller than the diameter of a-helical He1 13-5 (=I nm) as estimated by a
computer simulation (CS ChemOffice Ultra 5.0). After the collapse of He1 13-5 at 45
mN/m, many protrusions are observed over the whole range as shown in Fig. 7.6~. They
are located in a line, implying that the collapse of He1 13-5 promotes another collapse
beside it. Zasadzinski and co-workers (Ding et al., 2001) reported that above the
plateau pressure many protrusions appeared in the natural system of Survanta, which
is a commercial R D S medicine containing a natural bovine PS. The resultant patches
were also observed in the DPPC/POPG (palmitoyloleoyl-phosphatidylglycerol)films
containing low amounts of protein analogs (Diemel et al., 2002), indicating that
they were induced by the extraction of fluid compositions (one of PS functions). In
the topography image (Fig. 7.6c), the protrusions (bright) and monolayers (dark) of
single-species He1 13-5 molecules coexist. The height of these protrusions is -2.0-3.0
nm, suggesting that He1 13-5 are excluded from the monolayer after plateau regions
on z-A isotherms (Fig. 7.4) and then a three-dimensional folding made of two or
three He1 13-5 molecules is formed. Notice that two kinds of domains are observed:
the domain with a bright midpoint (indicated by an arrow) and a domina without
it (indicated by a dashed arrow). The former shows a central higher protrusion, as
shown in the phase contrast image of Fig. 7.6d. These protrusions are found to be
disk like in shape with a typical diameter of =33 nm, corresponding to aggregations
containing =340 molecules of He1 13-5.
(a)
constant value. Judging from the above phenomena, if the surface pressure goes
beyond the collapse pressure, the A V-A isotherms become almost parallel to the area
axis. Namely, the rising of A Vbeyond the second sharp bend means that the packing
state or orientation of the molecule is still developing. At least, these results support
the extraction phenomenon of He1 13-5 from the monolayer surface, However, the
value of AV became almost similar ( ~ 5 0 0mV) at =55 mN/m, indicating that He1
13-5 molecules are not extracted completely from the monolayer.
2
\
E
k
FM Obsmations
A series of FM images for the DPPC/Hel 13-5 system at 15 and 20 mN/m is
presented in Fig. 7.8, which show the disordered/ordered coexistence states; that is,
the dark domains reflect the ordered phase of DPPC, whereas the bright regions
reflect the disordered phases of DPPC and He1 13-5. The ordered domains of all
molar fractions grow in size with increasing surface pressure from 15 to 20 mN/m.
When the films were compressed further, they formed dark homogeneous images
consisting of almost the ordered phase up to the collapse pressure except for X,,,3-5
= 0.025. Note that the addition of a small amount of He1 13-5 to DPPC induced
the “moth-eaten” aggregation (shown by an arrow) of LC domains of DPPC. ‘This
moth-eaten aggregation occurred only when a small amount of He1 13-5 coexists
with DPPC. In addition, its aggregation enlarged and expanded the regions of each
LC domain. ‘Therefore, the disordered phases of He1 13-5 penetrated into the DPPC
ordered domains and promoted their nucleation.
Fig. 7.8. FM images of the DPPC/Hel 13-5 mixture system at 298.2 K for surface pressures of 15 and 20
mN/m: (a) pure DPPC; (b)XMII3.+= 0.005; (c) X, , ~ =
+ 0.025. In the co-existencephase, percentagerefers
to the ordered domains in the micrcgraph.The monolayerscontain 1 mol%fluorexent probe (R18).The
arrow shows the’moth-eaten”aggregation of LC domains made of pure DPPC.The scale bar in the lower
right represents 100pm.
170 0 H. Nakahara et al.
M M Obsewations
AFM images (500 nm x 500 nm) ofa LB film ofDPPC films containingsmall amounts
of He1 13-5 (XHe, 13-5 = 0.1) transferred onto mica at 35,45, and 5 5 mN/m are shown
in Fig. 7.9. Pure DPPC monolayers provided a homogeneous AFM image (Nakahara
et al., 2005a). In contrast, the monolayer containing He1 13-5 (XHe, 13-5 = 0.1) at 35
mN/m shows two different phases as shown in Fig. 7.9a. Because diacyl chain lengths
in DPPC molecules are ~ 2 . 5nm, bright phases represent DPPC monolayer while
dark phases indicate He1 13-5 monolayer. The height difference of these phases is = 1.O
nm, demonstrating that experimental values agree with theoretical ones.
Fig. 7.9. AFM images of the DPPC monolayer containing moderately low amounts of He1 13-5 (at X,,
,3.5 = 0.1) in a tapping mode at the scan area of 500 x 500 nm: topography (a) and corresponding phase
contrast image (b) transferred onto mica at 35 mN/m; topography (c) and corresponding phase contrast
image (d)transferred onto mica at 45 mN/m; topography (e) and correspondingphase contrast image (f)
transferred onto mica at 55 mN/m.The ratio (percent)of occupied areas by DPPC monolayersto whole
images is shown in (a), (c), and (e). The arrow (c) shows the brightest lobes by the extracted particles.
Influence of Pulmonary Surfactant Protein Mimicson Model Lung Surfactant 0 171
The corresponding phase contrast image (Fig. 7.9b) shows the morphological
character more clearly due to the difference in the surface physicochemical property
between DPPC and He1 13-5. The behavior shown in these AFM images agrees with
the FM images that the way to disperse the DPPC-ordered domains is to add a small
amount of He1 13-5. O n the other hand, three different phases appear at 45 mN/m,
where He1 13-5 is extracted from binary DPPC/Hel 13-5 monolayers (Fig. 7.9~).The
brightest lobes (indicated by an arrow) by the extracted particles appear. Interestingly,
most of the He1 13-5 protrusions sit on DPPC monolayers. The height of protrusions
from the DPPC monolayer is sl.5 nm, which corresponds to that of one and one-half
a-helical He1 13-5 molecules (= 1 nm). The corresponding phase contrast image (Fig.
7.9d) clearly shows morphological changes between respective monolayers.
Upon further compressionto 55 mN/m, the topography image (Fig. 7.9e) indicates
that DPPC regions and the protrusions increase in number, while the He1 13-5 rich
regions decrease in comparison with those in Fig. 7.9a and 7.9~.This indicates that the
ratio of DPPC-occupied areas increases as the surface pressure increases from 35-55
mN/m. That is, the content of ordered domain is increased by compression. The
percentage of the ordered domain becomes 46,66, and 78%, respectively, for surface
pressures of 35, 45, and 55 mN/m. This result demonstrates that only He1 13-5 is
extracted from binary monolayers beyond the plateau regions on the rr-A isotherms
and that the extracted He1 13-5 molecules occupy 3-D surface-associated reservoirs.
As a result, the surface is refined to a DPPC monolayer. There is no difference in
the height and size of the protrusions between 45 and 55 mN/m. The diameter of
disk-like protrusions is found to be 4 . 6 nm, corresponding to 10 molecules of He1
13-5. Note that all of the protrusions are located on DPPC monolayers, whereas
the He1 13-5 molecules that are still not extracted exist as a monolayer regardless
of the high surface pressure ( 5 5 mN/m), indicating that all He1 13-5 molecules are
not completely extracted from the binary system. This supports the result from the
quantitative analysis based on rr-A isotherms. In the phase contrast image (Fig. 7.90,
the protrusions become indistinct. This reason and mechanism have been discussed
by others (Krol et al., 2000; Nakahara et al., 2006b).
shifted to smaller areas in the repeated cycling processes at high surface pressures.
Figure 7.10 indicates that the easy re-spreading behavior displays the ability for
desorbed molecules to re-enter into the interface and works for a highly reproducible
hysteresis loop. 'These results demonstrate that He1 13-5 can accelerate the spreading
of DPPC and induce good re-spreading. Furthermore, these hysteresis curves resemble
those of the DPPC/SP-B and DPPC/SP-C mixtures that were reported previously
(Wustneck, eta]., 2001,2002).
70
60
7 50
E
z 40
E
-%
k
30
20
10
0
0.4 0.6 0.8 1 1.2
Area per molecule I mi2
Fig. 7.10. Cyclic compression and expansion isotherms (or hysteresis curves) of the DPPC/Hel 13-5 (X,,,,
13-5 = 0.1) mixture on a 0.02 MTris buffer solution (pH 7.4) with 0.13 M NaCl at 298.2 K.The compression
and expansion cycle was repeated five times at a compression rate of 0.1-0.2 nm2molecule-' m i d .
Influence of Pulmonary Surfactant Protein Mimics on Model Lung Surfactant 0 173
measured at various He1 13-5 molar fractions (XHCI 13-5) in Fig. 7.1 1 and 7.12. For the
DPPCIPGIHel 13-5 system, as shown in Fig. 7.1 1, the n-A isotherms exhibit two
plateau regions at =21 and =42 mN/m; they are indicated by arrows at X,, 13-5 = 0.1
(curve f). All of the n-A isotherms have the first plateau at the same surface pressure
(transition pressure, f l = 521 mN/m), and the sharp bend become less visible with
increasing X,, The second sharp bend at X,, I E 5 = 0.1 appears at -42 mN/m
(or the collapse pressure of He1 13-5), and the second plateau range expands as the
amount of He1 13-5 increases. The DPPC/PG monolayer forms a stable film up to
=45 mN/m. O n the other hand, the ternary DPPClPGlHel 13-5 monolayers (0 <
XHC1 1E5s 0.1) can retain stable states up to 155 mN/m (see the inset in Fig. 7.1 1). This
is the first evidence that He1 13-5 interacts with the fluid component (PG), and the
two fluid components begin to be extracted from the ternary mixtures at 542 mN/m.
Then the degree of extraction is gradually increased upon Further compression, and
the interface is ultimately refined to DPPC-rich monolayers at =55 mN/m because
the collapse pressure of DPPC monolayers is =55 mN/m (Fig. 7.7). At =42 mN/m,
the AV-A isotherm at XHe, 13-5 = 0.1 shows a sharp bend corresponding to the second
plateau on its n-A isotherm, as indicated by an arrow, and then the AVvalue gradually
rises with Further compression, indicating that monolayer orientations change due to
the exclusion of fluid materials. Finally, it becomes constant above 55 mN/m. This
also supports the extraction phenomena of He1 13-5.
For the DPPC/PA/Hel 13-5 system shown in Fig. 7.12, n-A isotherms also have
two plateau regions at 910-14 mN/m and =42 mN/m. The n-A isotherm at X,, 13-5
= 0.005 has only the first plateau. On the other hand, two plateaus are observed in
the n-A isotherms of 0.01 c X,,,13-5 c 0.1. The transition pressures of the first plateau
change from = 11 to = 14 mN/m and become less visible with increasingXHd which
is the same as the DPPC/PG/Hel 13-5 system. The second sharp bend in 0.01 < X,, 13-5
cO.1 also appears at 242 rnN/m, and the second plateau range elongates as the amount
of He1 13-5 increases. This behavior is the same as that of the DPPC/PG/Hel 13-5
system. However, the DPPC/PA/Hel 13-5 system displays a jagged second plateau,
revealing that the He1 13-5 species is not easily extracted from the ternary monolayers
because of the absence of fluid components. In contrast to the collapse behavior of the
DPPC/PG/Hel 13-5 system, the DPPC/PA monolayer forms a stable film up to 4 0
mN/m, and all of the DPPC/PA/Hel 13-5 monolayers (0 < X,, c 0.1) also remain
stable up to 560 mN/m, despite adding He1 13-5 to the DPPC/PA monolayer (see
the inset in Fig. 7.12). This also reveals that He1 13-5 interacts with rigid components
(DPPC and PA), even at high surface pressures, and only He1 13-5 is gradually
extracted from the ternary monolayers above =42 mN/m. That is, the extraction
slowly starts upon hrther compression, and the interface becomes rich in the DPPC/
PA monolayer at =60 mN/m. At more than 42 mN/m, the AV-A isotherms within
0.01 c X,, c 0.1 indicate the same behavior as that of the previously mentioned
DPPC/PG/Hel 13-5 system.
174 0 H. Nakahara et al.
z,
\
E
k
Hystmsir Curves
The repeated compression-expansion cycling z-A and A V-A isotherms of ternary
DPPC/PG/Hel 13-5 (A) and DPPC/PA/Hel 13-5 (B) systems at X,, ,3-5 = 0.1 are
shown for the first and fifth rounds in Fig. 7.14. These two systems were compressed
up to a surface pressure of =55 mN/m and then expanded to the respective starting
areas. In Fig. 7.14a, the second sharp bend clearly appears, despite the repeated
cycling processes, indicating that He1 13-5 is reversibly desorbed into the subphase
in the z-A isotherms. In Fig. 7.14b, for z-A isotherms, the second kink shoulder for
repeated hysteresis curves clearly appears without becoming unclear as it does in Fig.
7.14a The surhce pressure decreases sharply like a solid film in the expansion process.
Consequently, Fig. 7.14 indicates good re-spreading behavior from the first to fifth
rounds, showing the ability of the desorbed molecules to relocate to the interface.
These results demonstrate that small amounts of He1 13-5 peptides can accelerate the
good re-spreading of rigid components (DPPC and PA).
z
E
z
E
z
6
Fig. 7.14. Cyclic compression and expansion isotherms of (A) DPPC/PG/Hel 13-5 and (B) DPPC/PA/Hel
13-5 at&-, ,)-5 = 0.1 on a 0.02 M Tris buffer solution (pH 8.4) with 0.1 3 M NaCl at 298.2 K.The compression
and expansion cycle was performed five times at the compression rate of ~ 0 . 1nm2molecule-' min-'.
Influence of Pulmonary Surfactant Protein Mimics on Model Lung Surfactant0 179
FM Observations
Shown in Fig. 7.16 are the selected FM micrographs of the DPPC/PG/PA/Hel
13-5 preparations. For XHe,
13-5 = 0.01 (Fig. 7.lba), where there is no plateau region
corresponding to the extraction of the P A isotherm, two-phase coexistence states
180 0 H.Nakahara et al.
‘E
z
E
Fig. 7.15. n- and AV-A isotherms of the DPPC/PG/PA/Hel 13-5 preparations with a fixed DPPC/PG/PA
ratio on a 0.02 M Tris buffer solution (pH 7.4) with 0.1 3 M NaCl at 298.2 K. The transition pressure for X,,
13.5 = 0.1 is indicated by a straight-line arrow. The plateau regions displaying the extraction are shown by
dashed arrows on the n-A isotherms of X,, 13.5 = 0.05 and 0.1. (Inset) Enlarged x-A isotherms show the
plateau region.
Influence of Pulmonary Surfactant Protein Mimics on Model Lung Surfactant 0 181
similar to the resultant images in the lipid mixture (Nakahara et al., 2008) are
observed. The FM image at 30 mN/m indicates a clear contrast between ordered
and disordered domains. With an increase in surface pressure, however, the images
become slightly unclear due to the quenching of FM probes. Moreover, the percentage
of ordered domains doess not vary in spite of an increase in surface pressure and show
the formation of phase-separated films at high surface pressures. For X,, ,s5 = 0.05
(Fig. 7.16b), at which the n-A isotherm has a plateau, FM images indicate a different
behavior from those of X,, ,3-5 = 0.01, Although a completely dark image appears
at 40 mN/m due to the quenching, the image at 50 mN/m no longer suffers from
quenching and shows better contrast. The recovery of FM contrast results from the
extraction of He1 13-5 with fluid components including PG and FM probes in the
plateau region, with the surface concentration of FM probes decreasing. This behavior
was previously observed in the DPPC/PA/Hel 13-5 systems as a “refluorescence”
phenomenon, and provides novel morphological evidence of He1 13-5extraction
events (Nakahara et al., 200Ga).
HysteresJrjcurves
Repeated cycling x- and AV-A isotherms of the selected DPPC/PG/PA/Hel 13-5
preparations for first and fifth rounds are shown in Fig. 7.17. These monolayers are
compressed to a surface pressure of =62 mN/m and then expanded to the respective
Fig. 7.16. FM micrographs of the DPPC/PG/PA/Hell3-5 preparations at X,,,,, = 0.01 (a)and 0.05 (b)on a
0.02 MTris buffer solution (pH 7.4) with 0.13 M NaCl at 298.2 K. In the coexistencephases,the percentage
(96)refers to ordered domains in the micrograph. The monolayers contain 1 mol %of fluorescent probe
(R18).The scale bar in the lower right represents 100gm.
182 0 H. Nakaharaet al.
starting molecular areas. Both of z-A isotherms have large hysteresis in area and
indicate good reproducibility during cycling. Afier reaching the maximum surface
pressure on compression, the surface pressure rapidly decreases by 120 mN/m by a
small expansion, suggesting that the extracted He1 13-5 instantly reenters the surface
and then disperses the close-packed monolayers (DPPC/PA) during expansion. The
ability of He1 13-5 to respread with PG in the present preparations was improved
in terms of the rapid reentry to the surface in comparison with that in the previous
systems (Nakahara et al., 2005b, 2006b). Furthermore, reproducible plateaus at 42
mN/m demonstrate that the exclusion and the successive inclusion of He1 13-5 are
a reversible process during cycling. Consequently, these actions during compression-
expansion cycling demonstrate that the preparations have sufficient pulmonary
functions in the basic in vitro level (Notter, 2000).
Concl usions
The present work is meant to investigate the interfacial behavior of a synthetic peptide
(He1 13-5), a mimic peptide of human surfactant protein B, in a phospholipid
and various lipid mixtures. He1 13-5 itself is an amphiphilic peptide and can form
an a-helical structure at the air-water interface. DPPC is a main phospholipid
component in PS and contributes to lowering surface tension during exhalation.
DPPC alone has some defects in pulmonary functions such as rapid respreading,
adsorption, and large hysteresis. However, addition of a small amount of He1 13-5
to DPPC led to improved adsorption of DPPC. Moreover, the addition resulted in
enlargement of reversible hysteresis loops of z-A curves for DPPC. These results
support the possibility of He1 13-5 as a substitute for SP-B (Nakahara et al., 2005b,
2006b). In addition, both PG and PA components prevented the DPPC monolayer
from collapsing after the extraction of He1 13-5. PG is thought to interact selectively
and electrostatically with cationic He1 13-5 during the extraction process; then 3-D
aggregates containing He1 13-5 and PG could be formed just below the surface
Influence of Pulmonary Surfactant Protein Mimics on Model Lung Surfactant 0 183
'E
z
E
2:
E
Fig. 7.17. Cyclic compression and expansion isotherms of the DPWPG/PA/Hel 13-5 preparations at
X,, 13-5 = 0.05 (A) and 0.1 (B) on a 0.02 M Tris buffer solution (pH 7.4) with 0.13 M NaCl at 298.2 K. The
compression-expansioncycle was performed five times at the compression rate of 50.17 nml molecule-
lmin-l.
184 0 H. Nakahara et al.
z
E
5-r
k
Trough area I YO
Fig. 7.18. Cyclic compression and expansion isotherms of Surfacten (A) on a 0.02 M Tris buffer solution
(pH 7.4) with 0.1 3 M NaCl at 298.2 K. Hysteresis curves for the first round for the DPPC/PG/PA/Hel 13-5
preparations at X,,,,,, =0.05 and 0.1, and Surfacten (B).The compression-expansioncycle was performed
five times at the compression rate of 390 cm2min-’. The area (abscissa axis) represents the relative area
(%) to the initial surface area prior to compression.
Influence of Pulmonary Surfactant Protein Mimics on Model Lung Surfactant 0 185
monolayer (Diemel et al., 2002; Ding et al., 2003; Alonso et al., 2004; Nakahara et
al., 2006b). This formation contributes to the stability of DPPC monolayers. On the
other hand, PA components are not mainly extracted from surface monolayers, and
form a close packed monolayer with DPPC at the interface (Nakahara et al., 2006a).
Finally, various kinds of measurements for surface chemistry (Langmuir isotherms,
fluorescent images, temperature dependence, and hysteresis curves) have been made
for the comparison between the DPPCIPGIPAIHd 13-5 preparations and Surfacten,
which is commercially used for NRDS patients in Japan (Nakahara et al., 2008).
These results suggested that the preparations are comparable to Surfacten in terms
of surface activity and the interfacial behavior. The present work on PS substitutes
containing a synthetic peptide provides a deep insight into the clarification of
functional mechanisms of PS near the air/alveolar liquid interface. At the same time,
it will contribute to “tailoring” surfactant preparations to various states of surfactant
deficiency, insufficiency, and inactivation.
Acknowledgment
This work was supported by a Grant-in-Aid for Scientific Research 20500414 from
the Japan Society for the Promotion of Science (JSPS).
References
Alonso, C.; T. Alig J. Yoon; F, Bringezu; H. Warriner; J.A. Zasadzinski, More than a Monolayer:
Relating Lung Surfactant Structure and Mechanics to Composition. Biophys. J 2004,87,
4188-4202.
Amrein, M.; A. von Nahmen; M. Sieber, A Scanning Force- and Fluorescence Light Microscopy
Study of the Structure and Function of a Model Pulmonary Surfictant. Eur. Biophys. /. 1997,
26,349-357.
Anderson, M.; T.Curstedt; H. Jornvall; J. Johansson, Amphipathic Helical Motif Common to
Tumourolytic Polypeptide NK-Lysin and Pulmonary Surfactant Polypeptide SP-B. FEBS Len.
1995,362,328-332.
Avery, M.E.; J. Mead, Surface Properties in Relation to Atelectasis and Hyaline Membrane Dis-
ease. Am. J. Dis. Cbiki. 1959,97,517-526.
Baatz, J.E.; B. Elledge; J.A. Whitsett, Surfictant Protein SP-B Induces Ordering at the Surfice of
Model Membrane Bilayers. Biocbem. 1990,29,6714-6720.
Bastacky, J.; C.Y. Lee; J. Goerke; H. Koushafir; D. Yager; T.P. Speed; Y. Chen; J.A. Clements, Al-
veolar Lining Layer Is ‘Thin and Continuous: Low-Temperature Scanning Electron Microscopy
of Rat LungJ AppL PbysioL 1995,79,1615-1628.
Benson, B.; S. Hawgood; J. Schilling; J. Clements; D. Damm; B. Cordell; RT. White, Structure
of Canine Pulmonary Surfactant Apoprotein: cDNA and Complete Amino Acid Sequence. Proc.
NatL Acad Sci. USA 1985,82,63794383.
Boggaram, V.;K. Qing; C.R Mendelson, The Major Apoprotein of Rabbit Pulmonary Surfac-
tant. Elucidation of Primary Sequence and Cyclic AMP and Developmental Regulation. J Biol.
Cbem. 1988,263,2939-2947.
186 0 H. Nakaharaet al.
Brown, E.S., Isolation and Assay of Dipalmityl Lecithin in Lung Extracts. Am. J Pbysiol. 1964,
207,402-406.
Cai, I?; C.R. Flach; R. Mendelsohn, An Infrared Reflection-Absorption Spectroscopy Study of
the Secondary Structure in (KLJ4K, a Therapeutic Agent for Respiratory Distress Syndrome, in
Aqueous Monolayers with Phospholipids. Biochem. 2003,42,9446-9452.
Cajal, Y.; C. Dodia; A.B. Fisher; M.K. Jain, Calcium-Triggered Selective Intermembrane Exchange
of Phospholipids by the Lung Surfactant Protein SP-A. Biochem. 1998,37, 12178-12188.
Chang, R.; S. Nir; F.R. Poulain, Analysis of Binding and Membrane Destabilization of Phospho-
lipid Membranes by Surfactant Apoprotein B. Biochim. Biopbys. Acta 1998, 1371,254-264.
Cockshutt, A.M.; D.R. Absolom; F. Possmayer, The Role of Palmitic Acid in Pulmonary Surfac-
tant: Enhancement of Surface Activity and Prevention of Inhibition by Blood Proteins. Biochim.
Biopbys. Acta 1991, 1085, 248-256.
Crouch, E.; A. Persson; D. Chang; D. Parghi, Surfactant Protein D. Increased Accumulation in
Silica-Induced Pulmonary Lipoproteinosis. Am. /.Pathol. 1991, 139, 765-776.
Crouch, E.C., Collectins and Pulmonary Host Defense. Am. J Respir. CellMol. Biol. 1998, 19,
177-20 1.
Curstedt, T.; J. Johansson; I? Persson; A. Eklund; B. Robertson; B. Uwenadler; H. Jornvall,
Hydrophobic Surfactant-Associated Polypeptides: SP-C Is a Lipopeptide with Two Palrnitoylated
Cysteine Residues, Whereas SP-B Lacks Covalently Linked Fatty Acyl Groups. Proc. Natl. Acad
Sci. US4 1990,87, 2985-2989.
Diemel, R.V.; M.M.E. Snel; A.J. Waring; EJ. Walther; L.M.G. van Golde; G. Putz; H.I? Haags-
man; J.J. Batenburg, Multilayer Formation upon Compression of Surfactant Monolayers
Depends on Protein Concentration as Well as Lipid Composition. An Atomic Force Microscopy
Study.j. Biol. Chem. 2002,277, 21 179-21 188.
Ding, J.; D.Y. Takamoto; A. Von Nahmen; M.M. Lipp; K.Y.C. Lee; A.J. Waring; J.A. Zasadzinski,
Effects of Lung Surfactant Proteins, SP-B and SP-C, and Palmitic Acid on Monolayer Stability.
Biopbys. J 2001,80,2262-2272.
Ding, J.; I. Doudevski; H.E. Warriner; T. Alig; J.A. Zasadzinski; A.J. Waring; M.A. Sherman,
Nanostructure Changes in Lung Surfactant Monolayers Induced by Interactions between Palmi-
toyloleoylphosphatidylglyceroland Surfactant Protein B. Langmuir 2003,19, 1539-1 550.
Fleming, B.; K.M.W. Keough, Surface Respreading after Collapse of Monolayers Containing
Major Lipids of Pulmonary Surfactant. Chem. Phys. Lipid 1988,49, 81-86.
Floros, J.; R. Steinbrink; K. Jacobs; etc., Isolation and Characterization of cDNA Clones for the
35-kDa Pulmonary Surfactant-Associated Protein. /. Biol. Chem. 1986,261, 9029-9033.
Furuya, T.; T. Kiyota; S. Lee; T. Inoue; G. Sugihara; A. Logvinova; I? Goldsmith; H.M. Ellerby,
Nanotubules Formed by Highly Hydrophobic Amphiphilic a-Helical Peptides and NaturaJ
Phospholipids. Biopbys. J 2003,84, 1950-1959.
Glasser, S.W.; T.R. Korfhagen; T Weaver; T. Pilot-Matias; J.L. Fox; J.A. Whitsett, cDNA and De-
duced Amino Acid Sequence of Human Pulmonary Surfactant-Associated Proteolipid SPL(Phe).
Proc. Natl. Acad. Sci. USA 1987,84,4007-40 1 1 .
Goerke, J., Lung Surfactant. Biochim. Biophys. Acta 1974,344, 241-261.
Gorree, G.; J. Egberts; G. Bakker; A. Beintema; M. Top, Development of a Human Lung Surfac-
influenceof Pulmonary Surfactant Protein Mimics on Model Lung Surfactant 0 187
tant, Derived from Extracted Amniotic Fluid. Biochim. Biopbys. Acta 1991, 1086,209-216.
Grigoriev, D.O.; J. Kragel; A.V. Akentiev; B.A. Noskov; R. Miller; U. Pison, Relation Between
Rheological Properties and Structural Changes in Monolayers of Model Lung Surfactant under
Compression. Biophys. Chcm. 2003, 104, 633-642.
Gustafsson, M.; G. Vandenbussche; T. Curstedt; J.M. Ruysschaert;J. Johansson, The 21-Residue
Surfactant Peptide (LysLeu,),Lys(KL,) is a Transmembrane Alpha-helix with a Mixed Nonpolar/
Polar Surface. FEBS Letten 1996,384, 185-188.
Gustafsson, M.; M. Palmblad; T. Curstedt; J. Johansson; S. Schurch, Palmitoylation of a Pulmo-
nary Surfactant Protein C Analogue Affects the Surface Associated Lipid Reservoir and Film
Stability. Biochim. Biophys. Acta 2000, 1466, 169-178.
Hawgood, S.; B.J. Benson; J. Schilling; D. Damm; J.A. Clements; R.T. White, Nucleotide and
Amino Acid Sequences of Pulmonary Surfactant Protein SP 18 and Evidence for Cooperation
between SP 18 and SP 28-36 in Surfactant Lipid Adsorption. Proc. Natl. Acad. Sci. USA 1987,
84, 66-70.
Hawgood, S.; K. Shiffer, Structures and Properties of the Surfactant-Associated Proteins. Ann. Rev.
Pbysioi. 1991,53,375-394.
Johansson, J.; H. Jornvall; A. Eklund; N. Christensen; B. Robertson; T. Curstedt, Hydrophobic
3.7 kDa Surfactant Polypeptide: Structural Characterization of the Human and Bovine Forms.
FEBS Len. 1988,232,6144,
Johansson, J.; T Curstedt; H. Jornvall, Surfactant Protein B: Disulfide Bridges, Structural Proper-
ties, and Kringle Similarities. Biochcm. 1991,30, 6917-6921.
Johansson, J.; T. Curstedt, Molecular Structures and Interactions of Pulmonary Surfactant Com-
ponents. Eur. J. Biochcm. 1997,244,675-693.
Johansson, J., Structure and Properties of Surfactant Protein C. Biochim. Biopbys. Acta 1998,
1408, 161-172.
Kirkness, J.I?; I?R. Eastwood; I. Szollosi; RR Platt; J.R. Wheatley; T.C. Amis; D.R Hillman,
Effect of Surface Tension of Mucosal Lining Liquid on Upper Airway Mechanics in Anesthetized
Humans. J. AppL Pbysio.! 2003a, 95,357-363.
Kirkness, J.I?; M. Madronio; R. Stavrinou; J.R. Wheatley; T.C. Amis, Relationship Between Sur-
face Tension of Upper Airway Lining Liquid and Upper Airway Collapsibilityduring Sleep in
Obstructive Sleep Apnea Hypopnea Syndrome.J. Appl. Pbysiot! 2003b, 95, 1761-1766.
Kitamura, A.; T. Kiyota; M. Tomohiro; A. Umeda; S. Lee; T.Inoue; G. Sugihara, Morphological
Behavior of Acidic and Neutral Liposomes Induced by Basic Amphiphilic a-helical Peptides
with SystematicallyVaried Hydrophobic-Hydrophilic Balance. Biopbys. J. 1999, 76, 1457-1468.
Kiyota, T.; S. Lee; G. Sugihara, Design and Synthesis of Amphiphilic a-Helical Model Peptides
with SystematicallyVaried Hydrophobic-Hydrophilic Balance and Their Interaction with Lipid-
and Bio-Membranes. Biochcm. 1996,35, 13196-13204.
Krol, S.; M. Ross; M. Sieber; S. Kunneke; H.-J. Galla; A. Janshoff, Formation of Three-Dimen-
sional Protein-Lipid ®ates in Monolayer Films Induced by Surfactant Protein B. Biophs. 1.
2O00,79,904-9 18.
Kriiger, I?; J.E. Baatz; RA. Dluhy; M. Usche, Effect of Hydrophobic Surfactant Protein SP-C
on Binary Phospholipid Monolayers. Molecular Machinery at the Airwater Interface. Biopbys.
188 0 H. Nakahara et al.
Chem. 2002,99,209-228.
Kuroki, Y.;M. Shiratori; Y. Ogasawara; A. Tsuzuki; T. &no, Characterization of Pulmonary Sur-
factant Protein D: Its Copurification with Lipids. Biochim. Biophys. Acta 1991, 1086, 185-190.
Lee, S.; T. Furuya; T. Kiyota; N. Takami; K. Murata; Y. Niidome; D.E. Bredesen; H.M. Ellerby;
G. Sugihara, De Novo-Designed Peptide Transforms Golgi-SpecificLipids into Golgi-Like
Nanotubu1es.J Biol. Chem. 2001,276,4122441228.
Lee, S.; G. Sugihara; 0. Shibata; H. Yukitake JP Patent Appl. P 2004-305006A,2004.
Lipp, M.M.; K.Y.C. Lee; D.Y. Takamoto; J.A. Zasadzinski; A.J. Waring, Coexistence of Buckled
and Flat Monolayers. Phys. Rev. Lett. 1998,81,1650-1653.
Losche, H.; H. Mohwald, Fluorescence Microscopy on Monomolecular Films at an Aidwater
Interface. Colbidc Surf: 1984, 10,217-224.
Ma, J.; S. Koppenol; H. Yu; G. Zografi, Effects of a Cationic and Hydrophobic Peptide, KL,, on
Model Lung Surfactant Lipid Monolayers. Biophys. J 1998, 74,1899-1907.
Magoon, M.W.; J.R. Wright; A. Baritussio; M.C. Williams; J. Goerke; B.J. Benson; R.L. Ham-
ilton; J.A. Clements, Subfractionation of Lung Surfactant. Implications for Metabolism and
Surface Activity. Biochim. Biophys. Acta 1983, 750,18-3 1.
Mason, R.J.; K. Greene; D.R. Voelker, Surfactant Protein A and Surfactant Protein D in Health
and Disease. Am. J PhysioL 1998,275,Ll-Ll3.
Miyamura, K.;L.E. Leigh; J. Lu; J. Hopkin; A. Lopez Bernal; K.B. Reid, Surfactant Protein D
Binding to Alveolar Macrophages. Biochem. J 1994,300,237-242.
Nahmen, A.V.; M. Schenk; M. Sieber; M. Amrein, The Structure of a Model Pulmonary Surfac-
rant as Revealed by Scanning Force Microscopy. Biophys. J 1997, 72,463-469.
Nakahara, H.; S. Nakamura; H. Kawasaki; 0. Shibata, Properties ofTwo-Component Langmuir
Monolayer of Single Chain Perfluorinated Carboxylic Acids with Dipalmitoylphosphatidylcho-
line (DPPC). Colloidr Surf: B 2005% 41,285-298.
Nakahara, H.; S. Nakamura; S. Lee; G. Sugihara; 0. Shibata, Influence of a New Amphiphilic
Peptide with Phospholipid Monolayers at the Air-Water Interface. Colloidr Surf: A 2005b,
270-271, 52-60.
Nakahara, H.; S. Lee; G. Sugihara; 0. Shibata, Mode of Interaction of Hydrophobic Amphiphilic
a-Helical Peptide/Dipalmitoylphosphatidylcholinewith Phosphatidylglycerol or Palmitic Acid
at the Air-Water Interface. Langmuir 200Ga,22,5792-5803.
Nakahara, H.; S. Nakamura; T. Hiranita; H. Kawasaki; S. Lee; G. Sugihara; 0. Shibata, Mode
of Interaction of Amphiphilic a-Helical Peptide with Phosphatidylcholinesat the Air-Water
Interface. Langmuir 200Gb,22,1182-1 192.
Nakahara, H.; S. Lee; G. Sugihara; C.-H. Chang; 0. Shibata, Langmuir Monolayer of Artificial
Pulmonary Surfactant Mixtures with an Amphiphilic Peptide at the Aidwater Interface: Com-
parison of New Preparations with Surfacten (Surfactant TA). Langmuir 2008,24,3370-3379.
Notter, R.H.; D.P. Penney; J.N. Finkelstein; D.L. Shapiro, Adsorption of Natural Lung Surfac-
tant and Phospholipid Extracts Related to Tubular Myelin Formation. Pediatr. Res. 198G,20,
97-101.
Notter, R.H., Lung Surfactants: Basic Science and ClinicalApplications. Marcel Dekker, Inc.: New
Influence of Pulmonary Surfactant Protein Mimics on Model Lung Surfactant 0 189
Taneva, S.; K.M. Keough, Pulmonary Surfactant Proteins SP-B and SP-C in Spread Monolayers at
the Air-Water Interface: I. Monolayers of Pulmonary Surfactant Protein SP-B and Phospholip-
ids. Biopbys. /. 1994c, GG, 1137-1 148.
Veldhuizen, E.J.A.; A.J. Waring; F.J. Walther; J.J. Batenburg; L.M.G. van Golde; H.P. Haagsman,
Dimeric N-Terminal Segment of Human Surfactant Protein B (dSP-B1-25) Has Enhanced
Surface Properties Compared to Monomeric SP-B1-25. Biopbys. 1.2000, 79,377-384.
Veldhuizen, R.; K. Nag; S. Orgeig; F. Possmayer, The Role of Lipids in Pulmonary Surfactant.
Biocbim. Bioplys. Acta 1998, 1408,90-108.
Voorhout, W.F.; T. Veenendaal; H.P. Haagsman; A.J. Verkleij; L.M.G. van Golde; H.J. Geuze,
Surfactant Protein A Is Localized at the Corners of the Pulmonary Tubular Myelin Lattice. 1.
Histocbem. Cytochem. 1991,39, 1331-1336.
Voorhout, WX; T. Veenendaal; Y. Kuroki; Y. Ogasawara; L.M. van Golde; H.J. Geuze, Immu-
nocytochemical Localization of Surfactant Protein D (SP-D) in Type I1 Cells, Clara Cells, and
Alveolar Macrophages of Rat Lung. J. Histocbem. Cytocbem. 1992, 40,1589-1 597.
White, R.T.; D. Darnm; J. Miller; K. Spratt; J. Schilling; S. Hawgood; B. Benson; B. Cordell,
Isolation and Characterization of the Human Pulmonary Surfactant Apoprotein Gene. Nature
1985,317,361-363.
Wiistneck, N.; R. Wiistneck; V.B. Fainerman; R. Miller; U. Pison, Interfacial Behaviour and
Mechanical Properties of Spread Lung Surfactant Protein/Lipid Layers. Colloids Surj B 2001,
21, 191-205.
Wiistneck, R.; N. Wiistneck; B. Moser; U. Pison, Surface Dilatational Behavior of Pulmonary
Surfactant Components Spread on the Surface of a Pendant Drop. 2. Dipalmitoyl Phosphatidyl-
choline and Surfactant Protein B. Langmuir 2002, 18,1125-1 130.
Yu, S.-H.; F. Possmayer, Lipid Compositional Analysis of Pulmonary Surfactant Monolayers and
Monolayer-Associated Reservoirs.1.Lipid Res. 2003, 44,62 1-629.
Lung Surfactants : Formulation,
Evaluation, and Polymeric Additives
Edgar J. Acostalez, Sameh M.I. Saad2,Ningxi Kangl, Zdenka Policova',
Michael L. Hairz,and A. Wilhelm Neumann'
'Departmentof Chemical Engineeringand Applied Chemistv Universityof Toronto, 200 CollegeStreet,
Toronto,Ontario, Canada M5S 3€5; 'Deportment ofMechanical and lndustrial Engineering,Universityof
Toronto,5 King's CollegeRoad, Toronto,Ontario, Canada MSS 3G8
Introduction
This chapter opens with a brief historical review of the development of lung surfactant
therapy for the treatment of neonatal respiratory distress syndrome (nRDS), which
is now a standard therapy that has increased the survival of preterm neonates from
50% to over 80%. The most successhl formulations are extracts from lung lavages
of mammals, in particular from bovine and porcine sources. The current methods of
extraction and evaluation are also reviewed and discussed. The challenges for these
preparations include the variability of their performance, and their susceptibility to
inhibition by fluidizing lipids and proteins. To address these limitations, numerous
additives were proposed including neutral, anionic, and cationic polyelectrolytes.
Among these additives, cationic polyelectrolytes and amphiphilic cationic peptides
were among the most effective in resisting inhibitory factors. The mechanism of
action of these cationic additives on extract lung surfactants is discussed.
191
192 0 EJ. Acosta et al.
Before lung surfactant therapy became commonly used, nRDS was the leading
cause of death (>50% mortality) of premature babies with less than 29 weeks of
gestation (McDorman & Rosenberg, 1993). The development of therapeutical
surfactant formulas was a slow process. Avery and Mead, in 1959, were the first to show
that RDS is linked to an abnormal function of lung surfactant, and they proposed the
need for lung surfactant replacement therapy. The development of various formulas
is described in the next section. With the introduction of exogenous lung surfactant
formulations, the rate of the mortality of nRDS patients was reduced to approximately
20% (Hoekstra et al., 1991; Jacobs et al., 2000; McMillan et al., 1995; Petty, 2003).
Unfortunately, lung surfactant therapy was not effective in reducing the mortality of
ARDS patients (=40%), where surfactant inhibition (rather than lack of surfactant) is
associated with the pathology (Adhikari et al., 2004; Lewis et al., 2003; Notter, 2000;
Petty, 2003; Taeusch, 2000; Zasadzinski et al., 2001).
Alveolar lining
layer
Lung rurfaclant
components
Fig. 8.1. Schematic of the alveoli and the distribution of lung surfactants in the alveolus (not to scale).
0
II
0 - IP - x
I
I n -
0
Zwitterionic (net zero charae) DhospholiDids:
Phosphatidylcholine (PC) => X = -(CH 2)z(N+)(CH3)3
Phosphatidylethanolamine (PE) => X= -(CH2)z(N+)H3
Anionic phospholipids:
Phosphatidylglycerol (PG) => X= -CH2 -CH(OH) -CH2 -OH
Phosphatidylinositol (PI) => X= -C 6H1105
Phosphatidylserine (PSI => X= -CH2 -CH(NH2) -COO-
Saturated DhosDholiDids:
R1 ,R 2 :e.g. palmitic (P)(C16), steric (Cl8), etc.
Unsaturated phospholipids:
R 1,R2: e.g. oleyl (0) (C18, one double bond), etc.
2004). The general structure for the most common PLS found in lung surfactant is
presented in Fig. 8.2.
At neutral pH, PCs, PEs, and SPHs are zwitterionic, and have both negative
and positive charges within the molecule. O n the other hand, PGs, PSs, and PIS
are anionic. Dipalmitoyl-phosphatidylcholine (DPPC) accounts for nearly one-half
of PC components. When DPPC films are compressed, they form a closely packed
monolayer at the air-water interface, reducing the surface tension to values of 1 mJ1
m2 or less (Notter et al., 1980). The reduction in surface area per molecule and in
surbce tension is accompanied by a phase transition of the surfactant film from a
“gaseous” phase to a liquid-expanded (LE) phase, and finally to a liquid-condensed
(LC) phase (Nag et al., 1998; Taneva & Keough, 2000). In general, saturated PLs,
such as DPPC, form films capable of resisting larger compressions and reaching
lower surface tensions, and this is typically correlated with a higher melting point
of the PL (=41.5”C for DPPC). According to one of the mechanisms that explain
lung- surfactant behavior, after a series of compression cycles, surfactant molecules
that are “less surface active” are squeezed out of the film, thus producing DPPC-
rich films that yield lower minimal surface tensions (Goerke, 1998; Notter et al.,
2000; Possmayer et al., 2001). Surfactant proteins have an important influence on the
physicochemical properties of the surfactant film. Four types of proteins are in lung
surfactants: SP-A, SP-B, SP-C, and SP-D (Persson et al., 1989; Possmayer, 1988).
SP-A and SP-D are hydrophilic, whereas SP-B and SP-C are hydrophobic. SP-A is the
most abundant of these proteins. It has a large molecular weight (25-38 KDa), and is
relatively acidic. SP-A contributes to rapid film formation, the formation of tubular
myelin, and it interacts mostly with the hydrophilic groups of the PLs (Creuwels
et al., 1997; McCormack, 1998; Morrow et al., 2003; Notter, 2000; Weaver &
Whitsett, 1991). SP-B has a small molecular weight (=9KDa), and it contributes
to the formation of tubular myelin and other large aggregates, contributes to film
refining, increases respreading, and interacts with the heads and tails of the PLs
(Creuwels et al., 1997; Morrow et al., 2004; Notter, 2000; Weaver &Whitsett, 1991;
Withsett et al., 1995). SP-C is the most hydrophobic of all surfactant proteins, and
contributes to “softening lipid bilayers which is important for fast film formation
and film respreading. SP-D does not seem to have any impact on the physicochemical
properties of lung- surfactant films, but it plays an important role (along with SP-A)
in the immunological system (Creuwels et al., 1997; Notter et al., 2000; Weaver &
Whitsett, 1991). An important note to highlight is that SP-A and SP-D are absent
in exogenous lung surfactant formulations. The role of SP-A in exogenous lung
surfactant preparations was imitated, at least in principle, with nonionic and anionic
additives. SP-A has a collagenous domain in its “tail” and a carbohydrate-recognition
domain (CRD) at its “head” (McCormack, 2001). The CRD groups are known for
their binding capacity to PL monolayers through electrostatic interactions. This often
occurs through calcium ions (McCormack, 2001; Nag et al., 2004; Worthman et al.,
2000).
Lung Surfactants:Formulation, Evaluation, and Polymeric Additives 0 195
Four types of exogenous lung surfictant preparations are used in treating RDS:
synthetic PL mixtures (e.g., Exosurf), extracts from lung tissue (e.g., Survanta),
extracts from lung lavages (e.g., BLES, C U E ) , and whole surfactant from amniotic
fluid (Notter, 2000). The first lung surfactant formulations to be developed were
based on synthetic PL mixtures, in part to avoid an adverse immunological response to
surfactant proteins that are present in complete lung surfactants (e.g., from amniotic
fluid). In clinical trials, these formulations failed and were eventually phased out.
Assumably, lung surfactants obtained from a chloroform extraction of lung lavages
would be free of proteins; however, later it was found that, in the case of BLES they
are free of surfactant proteins SP-A and SP-D, but they still contain SP-B and SP-C
(Smyth et al., 1983).Current therapeutical formulations are based on lung extract
from large mammals including bovine and porcine. The typical composition and
method of extraction of BLES and other surfactant formulations are discussed in the
next section.
While no concrete numbers exist on the market for lung surfictants, the estimate
from one of the manufacturers (Discovery Laboratories, Inc.) suggests that the
global market for these kinds of formulations is about U.S.$90 million for NRDS
(lung deficiency) and U.S. $95 million for meconium induced lung deficiency
(Capetole, 1998).The larger market, for which lung surfictant therapy is yet to be
proven effective, is for the treatment of ARDS, and is estimated to be U.S.$4 billion
worldwide (Capetole, 1998).
t
LI-4 Natural
Lung Surfactant formations
I Synthetic
1
Fig. 8.3. Classification of lung surfactant formulations and their commercial name.
I*
extract with Acetone
(deplete Cholesterol and NL)
1 Alveofact 1 lnfasurf
Fig. 8.4. Methods of extraction of natural lung surfactants from lung lavages.
Lung Surfactants: Formulation, Evaluation, and Polymeric Additives 0 197
from animal (mammals) lung tissue (Curosurf). The third group is similar to the
second, except that the natural surfactants are supplemented with synthetic additives
(Surfacten, Survanta) to improve the surface activity of the formulation. The general
extraction procedure of the second and third groups (categories) is summarized in Fig.
8.5. A comparison of the composition of the six natural surfactants’ extracts is shown
in Fig. 8.6. More details about the extraction, composition, and surface activity of
natural lung surfactants are found in a recent review by Blanco and Pkrez-Gil (2007).
In the following paragraphs, we concentrate on providing the essential information
about these different surfactants.
Alveofact
Alveofact (bovactant) is a clinical exogenous lung surfactant obtained from the
bronchoalveolar lavage of bovine lung surfactant (Bartmann et al., 1992; Gortner
et al., 1990, 1992). The lavage is formed into a pellet by centrifugation, followed by
extraction with chloroform and methanol to remove hydrophilic surfactant protein
(Fig. 8.5). It contains a weight distribution of 99% of PLs and NLs (including 4% of
cholesterol), and 1% of hydrophobic surfactant proteins SP-B and SP-C as shown in
Fig. 8.6. It is produced as a sterile suspension in 0.15 M of NaCl at 45 mg/mL, and
is dosed at about 1.2 mL/kg. Alveofact is produced by Boehringer Ingelheim Co.,
Ingelheim, Germany.
BLES
BLES is a clinical exogenous lung surfactant obtained from the lung lavages of
benefited cows (Dunn et al., 1991; Enhorning et al., 1985; Smyth et al., 1983). The
lavage is pelleted by centrifugation, followed by extraction with chloroform methanol
to give BLSE (bovine lung surfactant extract). According to a recent composition
analysis of BLES by thin layer chromatography (TLC), this clinical surfactant
contains nearly 40% of disaturated PC (29.7% of DPPC), ~ 4 0 %of unsaturated
PCS, 13.6-14.5% of PGs, 0.9% of PIS, 2.8% of PEs, 0.3% of PSs, 1.4% of SPHs,
and 0.7% of lysophosphatidycholine (LPCs) (Nag et al., 2007). A range of 1-2% of
proteins SP-B and SP-C is contained in BLES, close to their natural amount NLs .
(3%, mainly of cholesterol and diacylglycerol) are removed from BLES by acetone
washing, because this provides better surface activity in vitro. BLES is delivered as
a sterile suspension in 0.15 M of NaCl and 1.5 m M of CaCl at 25 mg/mL, and
its recommended dose for NRDS is 5 mL/kg (135 mg/kg), repeated every 8 hours.
BLES is produced by BLES Biochemicals Inc., London, Ontario, Canada.
Infasutf
Infasurf (CLSE or calfactant) is a clinical exogenous lung surfactant obtained from
calf lung lavage (CLSE) (Kendig et al., 1998; Kwong et al., 1985; Willson et al.,
1999). The lavage is pelleted by centrifugation at 10,000-12,000 g, followed by
extraction with 2:1 chloroform/methanol to give CLSE or calf lung surfactant extract
198 0 EJ. Acosta et al.
I
Bovine Lung Tissue
I
I I Bovine LungTissue
I
homogenizeor mince homogenizeor mince homogenizeor mince
(in 0.1 5 M NaCL) (in 0.15 M NaCL) (in 0.15 M NaCL)
wash and ientrifuge differential cLntrifugation differential cLntrifugation
(1,000-3,OOO~) and flotation steps and flotation steps
extract with'Chloroform- extract with ;thy1 acetate extract with kthyl acetate
Methanol (21) (reduce neutral lipids) (reduce neutral lipids)
-r
i I I
liquid-gel affinity extract with Chloroform- extract with Chloroform-
chromatography Methanol (remove ethyl acetate) Methanol (remove ethyl acetate)
f
Lung Tissue Extract Lung Tissue Extract
supplement h t h synthetic
I
supplement with synthetic
DPPC, Palmitic Acid, and DPPC, Palmitic Acid, and
Tripalmitin Tripalmitin
J J
LI Curosurf Surfacten I I Survanta
I
Fig. 8.5. Methods of extraction of natural lung surfactants from lung tissue.
Survanta
Fig. 8.6. Dry base composition (wt%) of lung surfactant extracts from lung lavages and lung tissue.
PL: phospholipids, NL: neutral lipids, P C phosphatidylcholines, P G phosphatidylglycerols, PI/PS
phosphaidylinositoVserine, PE: phosphatidyl ethanolamine, SPH: sphingolipids. SP-B/SP-C surfactant
proteins B/C.
Lung Surfactants: Formulation, Evaluation,and Polymeric Additives 0 199
(Fig. 8.5). It contains a weight distribution of 93% of PLs (that include 83% of PC,
6% of PG, 4% of PI and PS, 3% of PE, and 2% of SPH), 5% of cholesterol and NLs,
and 1.5% of hydrophobic surfactant proteins SP-B and SP-C as shown in Fig. 8.6.
Disaturated PC is approximately 50% of the total PLs. I n h u r f is produced as a heat-
sterilized suspension in 0.15 M of NaCl at 35 mg/mL. The recommended dose for
NRDS is 3 mL/kg (105 mg/kg). Infasurf is produced by O N Y Inc., USA.
cumsu?f
Curosurf (poractant alpha) is a clinical exogenous lung surfactant obtained from
minced porcine lung tissue (Bevilacqua et al., 1996; Noack et al., 1987; Robertson,
1991). The process includes washing, centrihgation at 1,000-3,000 g, extraction
with 2: 1 chloroform/methanol, and liquid-gel affinity chromatography (see Fig. 8.5).
The average composition of Curosurf contains 93% of PLs (including 67-75% of
PC, 12-22% of PE, and SPH), and 1% of hydrophobic surfactant proteins SP-B
and SP-C, as shown in Fig. 8.6. Curosurf is produced as a suspension sterilized by
ultrafiltration (0.45- and 0.2-pm filters). Curosurf's recommended dose is 2.5 mL/kg
(200 mg/kg). Curosurf is produced by Chiesi Farmaceutici, Parma, Italy.
su?j2cten
Surfacten (Surfactant TA) is a clinical exogenous lung surfactant obtained after the
organic solvent extraction of finely ground bovine lung tissue. The final product is
supplemented with synthetic DPPC, palmitic acid, and tripalmitin (Fujiwara et al.,
1990; Konishi et al., 1992; Taeusch et al., 1986). The lung tissue goes through several
centrihgation and flotation processes, extraction with ethyl acetate to reduce NLs,
and additional extraction with chloroform-methanol to remove ethyl acetate. The
extract is then supplemented with the aforementioned synthetic additives (Fig. 8.5).
The final product contains a weight distribution of 84% of DLs, 8% of palmitic acid,
7% of tripalmitin, and 1% of hydrophobic surfactant proteins SP-B and SP-C as
shown in Fig. 8.6. Surfacten is produced as a suspension sterilized by high-pressure
filtration, lyophilized, stored in vials, and then suspended in sterile 0.15 M of NaCl
at 30 mg/mL prior to, and its recommended use is 4 mL/kg (100 mg/kg), 1-4 doses
every 6 hours. Surfacten is produced by Mitsubishi Tanabe Pharma Corporation,
Tokyo, Japan.
Survanta
Survanta (beractant) is a clinical exogenous lung surfactant made by organic solvent
extraction of processed, supplemented bovine lung tissue (Bloom et al., 1997; Horbar
et al., 1989; Speer et al., 1995). Survanta is prepared using similar methods and
identical synthetic additives as in Surfactant TA (Fig. 8.5). The final product contains
slightly less than 1% of hydrophobic surfactant proteins (including a small amount of
SP-B) as shown in Fig. 8.6. Survanta is produced as an autoclave sterilized suspension
in 0.15 M of NaCl at 25 mg/mL. The recommended dosage is 4 mL/kg (100 mg/kg),
200 0 EJ. Acosta et al.
1-4 doses every 6 hours. Survanta is produced by Abbott Laboratories, Abbott Park,
Illinois, United States.
Synthetic Surfactan ts
One can also subdivide synthetic surfactants (Bengt-Robertson et al., 2000; Seurynck-
Servoss et al., 2006; Wu et al., 2003) into various categories. The first involves synthetic
exogenous surfactants with no proteins (ALEC, Exosurf); the second utilizes synthetic
peptide analogs (Surfaxin); and the third contains recombinant human apoproteins
(Venticute). A comparison of the composition of the four synthetic surfactants is
shown in Fig. 8.7 ; hrther details are found in recent reviews (Curstedt & Johansson,
2006; Mingarro et al., 2008; Walther et a]., 2007).
ALEC
ALEC (artificial lung expanding compound) is a clinical exogenous lung surfactant
containing synthetic DPPC and egg PG in a 7:3 molar ratio (Bangham et al.,
1979; Morley, 1987; Wilkinson et al., 1985) as shown in Fig. 8.7. DPPC is mainly
responsible for lowering surface tension; the egg PG is added to improve adsorption
and spreading. ALEC was produced by Britannia Pharmaceuticals Ltd., United
Kingdom, but was withdrawn from the market.
Hexadecanol: 9%
Fig. 8.7. Dry-base composition (wt%) of synthetic lung surfactant formulations. DPPC: dipalmitoyl
phosphatidylcholine, POPG palmitoyloleyl phosphatidyl glycerol, rPC-: recombinant surfactant protein C.
Lung Surfactants: Formulation, Evaluation,and Polymeric Additives 0 20 1
Suflm'n
Surfaxin (KL4 or lucinactant) is a clinical exogenous lung surfactant containing
synthetic DPPC, palmitoyl-oleoyl-phosphatidyl-glycerol(POPG), palmitic acid, and
a 21 amino-acid synthetic hydrophobic peptide with repeated sequences of one lysine
(K) and four leucine (L) residues (Cochrane & Revak, 1991; Moya et al., 2005;
Saleem et al., 2008). DPPC and POPG are present in a 3:l weight ratio. Palmitic
acid is present at 15% by weight relative to PLs, while the hydrophobic (KL) peptide
is present at 3% by weight as shown in Fig. 8.7. Surfkin is produced by Discovery
Laboratories Inc., Pennsylvania , USA. Recently, an aerosol version of Surfkin was
developed by the same producer, and named Aerosurf. Surfkin and Aerosurf are still
in their developmental stages, and are not yet approved for clinical use.
Vmticute
Venticute (recombinant SP-C or lusupultide) is a clinical exogenous lung surfactant
containing synthetic DPPC, POPG, palmitic acid, and human sequence or modified
human-sequence recombinant surfactant apoprotein (rSP-C) (Benson, 1993; Markart
et al., 2007; Spragg et al., 2004). DPPC and POPG are present in a 7:3 weight ratio.
Palmitic acid is present at 5% by weight relative to PLs, and the rSP-C protein is
present at 2% by weight (Fig. 8.7). It is produced by Altana Pharma AG, Germany.
Venticute is not yet approved for clinical use.
Eq. 8.1.
where W and T are the width and thickness length of the slide, 8 is the contact angle,
AF is the difference in force (measured by a balance) between the case where the slide
in pure solvent (no surfactant) and the case where the slide is inmersed in the solvent
containing the surfactant film. Equation 8.1 is usually hrther simplified by assuming
the slide thickness T is neglected with respect to the slide width W , and assuming
complete wetting so that the contact angle is zero, giving cose = 1.
The Langmuir trough with the associated Wilhelmy balance is the standard
method to study insoluble surface active films. The surface pressure isotherms of
fatty acids, PLs, proteins, and other surface active material were studied using this
methodology. To produce these isotherms, the surface active material is dissolved in an
organic volatile solvent like hexane. This solution is then placed on the surface of the
aqueous (subphase) solution in the trough. Once the organic solvent has evaporated,
the surface coverage (r)
of the insoluble surfactant is calculated based on the mass of
(r
the surfactant deposited divided by the area of the trough = masdarea). The mobile
Film collapse
Phase
transitions
a, -1 /r
Fig. 8.8. Schematic of a Langmuir trough (cross-sectionalview), and a typical surface-pressureisotherm
obtained from these experiments.
Lung Surfactants: Formulation,Evaluation, and Polymeric Additives 0 203
barrier(s) of the trough is(are) slowly moved to gradually reduce the area of the trough
and compress the surfactant film. Upon compression, the surfactant film excludes
water molecules from the interface, producing lower surface tensions (higher surface
pressures). The experiment produces a set of data of surface pressure (n)versus the
area per molecule of surfactant (u, -l/r). This is used to study the phase transitions
of the film, and the ultimate surface pressure and area at collapse. This output is
illustrated in Fig. 8.8.
A major disadvantage ofthe LWB is that it requires a large volume of the subphase.
Also, only slow compression rates (dcl/dt)can be used to avoid the formation ofwaves.
Furthermore, unless special measures are taken, most LWBs experience film “leakage”
(spreading over the boundaries of the trough), and this prevents the surfactant film
from reaching the low surface tension values of 0-5 mJ/m2 (or surface pressures of
67-72 mJ/m2) required to confirm proper lung surfactant function.
Pulsating-Bubbk Sulfitetometer
In a pulsating-bubble surfactometer (PBS) (Enhorning, 1977; Hall et al., 1993;
Seurynck et al., 2005), an air bubble is formed in a surfactant suspension by drawing
air from the atmosphere through a capillary tube. After surfactant adsorption reaches
equilibrium, the bubble is pulsated between two set positions. The radius of the
bubble is usually monitored, and the pressure in the subphase is measured using a
pressure transducer. The surface tension, y, is calculated from the measured values of
the pressure drop, AP , and the bubble radius, R , using the Laplace equation and
assuming a sherical shape,
Y =z Eq. 8.2.
This method has several advantages including simplicity of use. It requires only a
small volume of subphase, it can be operated at physiologically relevant cycling rates,
and it is commercially available. However, still two major disadvantages remain
to be accounted for. The design is prone to film leakage into the capillary at low
surface tensions, and the spherical shape assumption is not appropriate at low surface
tensions. The output from these experiments is the minimal and maximal dynamic
surface tensions during a cycle with given compression ratio (reduction in surface
area) and compression period (duration of the compression-expansion cycle).
Grptive-bubbk Suflactometer
In a captive-bubble surfactometer (CBS) (Codd et al., 2002; Prokop et al., 1998;
Schurch et al., 1989), an air bubble is formed inside a closed chamber filled with a
surfactant suspension. The air bubble floats against a hydrophobic ceiling coated with
1% of agarose gel (similar to the ADSA-CB setup in Fig. 8.9). After the surfactant
film is formed and it reaches its equilibrium surface tension, the bubble is cycled
204 0 EJ. Acosta et al.
Eq. 8.3.
where R, and R, are the principal radii of curvature at a point on the interface, R,
is the symmetric radius ofcurvature at the apex point of the interface, Ap is the density
difference across the interface, g is the local gravitational acceleration, and z is the
vertical distance between the apex point and the point on the interface. Therefore, the
surface tension value, y, can be determined from the shape of the interface. In CBS,
the height-to-diameter ratio of the bubble is used to calculate surface tension, bubble
area, and volume (Schoel et al., 1994).
The CBS has all the advantages of the PBS setup, but the confined bubble
solves the problem of leakage, and does not assume a spherical drop. The CBS does,
however, introduce another set of challenges-is difficult to use and can only handle
dilute surfactant solutions (c2 mg/mL) due to the obscuration of the optical path. No
way is available to appropriately control the composition of the air entrapped in the
bubble, and the large liquid-to-air volume ratios are not compatible with physiological
conditions.
Stirring bar
Fig. 8.9. Schematic diagram of the ADSA setup (top)and the three different drop configurationsthat can
be used (bottom):captive bubble (ADSA-CB), pendant drop (ADSA-PD),and constrained sessile droplet
(ADSA-CSD).
configuration are that it is a simple setup and easy to use. It requires only small
subphase volumes (1-3 mL), and can simulate physiologically relevant conditions
including the cycling rate and compression ratio. The environmental conditions of the
surrounding atmosphere (humidity, CO, content, 0, content) and temperature are
readily controlled. A major advantage of this method is that no.optica1 limits exist to
the opacity of the liquid, and therefore no limitation exists as to the concentration of
the surfactant in the preparation. One can use ADSA-CSD for adsorbed and spread
films, and it produces accurate and fast measurements of surface tensions (especially
at values below 5 mJ/m’). The main disadvantage of the technique is that it requires
more sophisticated hardware that is not commercially available, and so it is still in a
developmental mode.
Also important is to highlight this: by controlling the humidity in ADSA-CSD to
100% of RH (relative humidity), we have determined that tests carried out in LWB,
PB, CB, ADSA-CB, and ADSA-PD may not represent the physiological condition
in the alveoli. Particularly, we observed that in saturated ( I 00% of RH) air at 37°C
numerous lung surfactant preparations fail to reach low surface tensions. However,
the same preparations evaluated at room temperature or at 37°C but in unsaturated air
( ~ 1 0 0 %of RH) produce low surface tensions and are considered “good formulations
(Acosta et al., 2007; Zuo et al., 2005). The discussion on lung- surfactant additives
in the last part of this chapter concentrates on experiments carried out using ADSA-
CSD in an environment of 100% of RH at 37°C.
In Situ Evaluation
In situ techniques are used to estimate the alveolar surface tension in excised lungs
(i.e., in its natural place). In situ techniques are usually considered intermediate
benveen in vitro and in vivo.
Microdroplet Method
In the microdroplet method (Craster & Matar, 2006; Im Hofet al., 1997; Schiirch et
al., 1976), a test fluid droplet is formed on the tip of a micropipette inside an alveolus.
After deposition onto the alveolar surface of an excised lung, the drop spreads to a
diameter determined by the surface tension of the alveolar fluid, the surface tension
of the test liquid, and the volume of the test liquid. The diameter of the liquid lens
is usually monitored via microscopy, The film surface tension is determined from a
calibration of the liquid lens diameter (for a constant volume) of various test liquids
versus the surface tension of these liquids. This technique is difficult to implement,
but it was essential in demonstrating that the surface tension of the alveolar film at the
end of expiration approaches a near-zero value.
of excised lungs ventilated dynamically. In this method, the excised lung is filled with
air, and is ventilated (dynamic compression-expansion cycles) to obtain P-V data.
The P-V work necessary for this cycling has two components-the work required to
overcome tissue forces and the work required to overcome surface tension forces. The
same excised lung is later filled with a saline solution, and is cycled using the same
protocol. In this case, the P-V work only accounts for the tissue forces. The difference
in the P-V isotherms of air-filled and saline-filled lungs indicates the contribution of
surface tension forces. The P-V curve associated with surface tension forces can be
related to surface tension using an energy balance approach. In its simple form, the
equation used to estimate the surface tension is (Bachofen et al., 1970):
Q. 8.4.
where Vis the volume of the air in the lung; P, is the difference between the pressure
required to achieve P for an air-filled lung minus the pressure required to fill the
saline-filled lung; R is a constant that must be determined by using the maximal
inflation pressure and volume and assuming a value of surface tension for that
maximal surface tension. The overall accuracy of the method depends on the accuracy
of that assumption.
In Vivo Evaluation
In vivo techniques involve experimentation done on a living animal. Contrary to the
surface tension as an output from in vitro and in situ techniques, the main outputs
in this approach are usually the lung compliance (the ability of the lungs to stretch
their volume-AV- in response to an applied pressure-AP; C =AVlAP) and blood
oxygenation (the partial pressure of oxygen in blood sample). Higher compliances
and blood oxygen levels are indicative of the success of the preparation. Different
animal models were used to evaluate the efficiency of lung surfactant preparations on
premature and adult animals.
lmMtunAnimal Modcl
The premature animal model (Kobayashi et al., 1989; Pinkerton et al., 2000; Sun et
al., 1997) is usually used to evaluate surfactant efficacy in premature animals (e.g.,
premature rabbits or lambs) with natural surfactant deficiency. In these experiments,
the animal fetuses are delivered at an early gestational age to ensure that the lungs are
still surfactant deficient. The premature animal is connected to a ventilator system, and
an exogenous surfactant preparation is administered to the lungs through an injection
via an endotracheal tube. The tidal volume (V)and the peak inspiratory pressure
(PIP) are continuously monitored during ventilation. Dynamic lung compliance is
the main output from this model.
208 0 EJ. Acosta et at.
Saline-lungLavage Model
This model (Bailey et al., 2004; Hafner et al., 1998; Lewis et al., 1996) is also used to
evaluate surfactant efficacy in dealing with a natural surfactant deficiency. However,
in this model, adult animals (e.g., rats, rabbits, pigs, or sheep) are used. To ensure
that the lungs are surfactant-deficient, the endogenous lung surfactant is removed by
repeatedly lavaging the lungs with a saline solution. During this process, the adult
animal is connected to a mechanical ventilator. After a complete depletion of the
endogenous surfactant, an exogenous surfactant preparation is administered to the
lungs through a bolus injection via an endotracheal tube. Throughout the experiment,
the arterial partial pressure of oxygen (PaO) is measured in arterial-blood samples.
The main output from this model is the blood-oxygen level.
In addition to the methods testing surfactant deficiency, other methods are used
to study surfactant dysfunction or inactivation. In these cases, the objective is to
investigate the efficacy of an exogenous surfactant in the therapy of lung injury. One
can induce a lung injury by using methods such as meconium aspiration (Lu et al.,
2005b; Moses et al., 1991; Sun et al., 1993), acid aspiration (Davidson et al., 2005;
Eijking et al., 1992; Lamm et al., 1990), and high-stretch ventilation (Bailey et al.,
2006; Nakamura et al., 2002; Wakabayashi et al., 2006).
Anionic Polymers
K. Lu et al. (2005 ) and Taeusch et al. (2005) introduced the use of the anionic
glucosamine and hyaluronan as an alternative to improving the surface activity of
lung surfactants. Hyaluronan consists of the N-acetyl-glucosamine conjugated by a
2 10 0 EJ. Acosta et al.
Fig. 8.10. Schematic of the depletion-attraction mechanism. The polymers in solution (with a given
gyration radius, Rg) cannot penetrate the excluded volume between the surfactant aggregates. This
difference in concentration induces an osmotic pressure difference that "pushes" the surfactant
aggregates together.
' (2-b)
08 10 0.8 1 .o
Relative Area Relative Area
Fig. 8.1 1. Surface tension (y)-relative-area compression (open symbol)-expansion (solid symbol)
cyclic isotherms for lung surfactant systems containing adult serum or albumin.The arrows indicate film
collapse. Fig. (1-a)and (1-b) are adapted from Zuo et al. (2006).Cycling conditions: 37"C, 100% of RH, 20%
of compression, 3 skycle compression.
Lung Surfactants: Formulation, Evaluation,and Polymeric Additives 0 2 11
p-1,4 linkage to glucuronic acid (See Fig. 8.12 for its dissociated form). The original
inspiration for its use is its presence in various body fluids, where it helps control
the colloids stability. In keeping with the idea of using nonadsorbing polymers
to promote the depletion-attraction of surfactant aggregates, since the soluble
hyaluronan molecule and lung surhctant aggregates are negatively charged, polymer
adsorption should be minimized and the depletion-attraction mechanism enhanced.
This strategy produced formulations that are 20 times more effective than PEG-based
formulations in albumin-inhibited systems (Taeusch et al., 2005). Hyaluronan was
successfully used in vivo to recover the injury produced by meconium instillation in
rats (K. Lu et al., 2005b).
More recent work on the use of hyaluronan as a lung surfactant additive
corroborated that this polymer is about one order of magnitude more active than
PEG in producing fist-adsorbing formulations (Taeusch et al., 2008). The authors
propose that the lower concentration of hyaluronan should decrease the concerns
of the high viscosity and cytotoxicity observed in formulations containing a high
concentration of PEG. Furthermore, because hyaluronan is a polymer synthesized in
the body, no concerns exist of undesirable interactions between hyaluronan and the
alveolar tissue.
To develop potential low-cost alternatives to hyaluronan, we conducted
preliminary studies using porcine mucine (an anionic glycoprotein that mimics the
mucosal secretion in lungs) to evaluate its potential impact on the dynamic surface
tension of lung surfactant preparations. Figure 8.13 presents one experiment where
a preparation of 2.0 mg/mL of BLES containing 10 mg/mL of porcine mucin was
exposed to 50 pL/mL of serum (a ten-times higher inhibitor content to that evaluated
by Taeusch et al., 2005). As shown in Fig. 8.13, the minimal surface tension during
the dynamic compression (conducted at 3 slcycle, 20% of compression, 100% of
RH, and 37°C using the ADSA-CSD device) is 2 mJ/m*, which is an exceptionally
low value for this level of serum content. The actual concentration of mucine is about
eight times that of hyaluronan used in the work ofTaeusch et al. (2005). Quite likely,
at this concentration, porcine mucine also follows the depletion-attraction model
suggested for hyaluronan.
Anionic polymeric additives appear to offer a valid option for the improvement of
the surface activity of lung surfactant preparations, and they continue to be considered
by various research groups.
Cutionic Polymers
Recently, our group introduced the use of a cationic polymer chitosan, and showed
that it can produce positive effects similar to PEG, but uses 1000- times less polymer
(Zuo et al., 2006). Chitosan is a polymer of p(l+4)-linked D-glucosamine (Fig.
8.14), usually derived from chitin (the skeletal material of invertebrates) by progressive
deacetylation in alkaline suspension. Chitosan’s amino (-NH,’) groups, with a pKa
of 6.5, become fully protonated when dissolved in an acidic environment (pH <6).
212 0EJ.Acostaetal.
n
Fig. 8.12. Molecular structure of the repeating unit in hyaluronan (dissociated hyaluronic acid).
50
-
N
._
E
40
3 30
E
W
* 20
10
0
0.80 0.90 1.oo
Relative Area
Fig. 8.13. Surface tension (y)-relative-area cyclic compression isotherms for 2 mg/mL BLES and 10
mg/mL of porcine mucin (extractedfrom intestine lining, and used as an anionic polymeric additive) in
the presence of a high-serum concentration (50VVmL). Cycling conditions: 37T, 100% of RH,20% of
compression, 3 s/cycle compression.
Lung Surfactants: Formulation, Evaluation,and Polymeric Additives 0 21 3
Fig. 8.14. Molecular structure of chitin (left) and chitosan-deacetylated chitin (right).
2 14 0 EJ. Acosta et al.
et al., 2006 ). Another important finding in that article is that morphological changes
occur in the lung surfactant preparation after chitosan treatment. In the presence of
chitosan, numerous small vesicles flocculate to form large aggregates. The presence of
these larger aggregates seems, once more, to be correlated with the ability to obtain
low surface tensions, even in the presence of inhibitors.
Kang et al. (2008) studied the mechanism of action of chitosan in BLES
formulations. The dynamic surface tension of these preparations in 100% of RH at
37°C was evaluated as a function of BLES-chitosan ratios using 0.5 and 2.0 mg/mL
of BLES. These studies were carried out using the ADSA-CSD, employing a period
of 3 s/cycle, and a compression ratio of 20%. Kang et al. (2008) also determined
the <-potential of the aggregates in these formulations in an attempt to evaluate the
binding of chitosan to BLES lipids.
Figure 8.15a presents the <-potential of the aggregates of formulations prepared
with 0.5 mg/mL and 2.0 mg/mL of BLES as a function of the total chitosan
concentration used in the preparation. The <-potential of the aggregates increased
from -15 mV to +25 mV as the chitosan concentration increased. The pH of these
preparations was approximately 5.5. 'This change in <-potential reflects the binding of
the positively charged chitosan to the anionic lipids, in particular PG, in BLES.
The binding isotherm for BLES and chitosan has remained elusive to our group
due to the analytical challenges in measuring the unbound chitosan in mixtures with
BLES. However, the binding isotherm can be estimated using the c-potential values
in Fig. 8.15a and the method of Mezei and MCszdros (2006). The principle of the
method is that the binding between surfactants and oppositely charged macromolecules
produces a shift in the <-potential, and that two systems with the same <-potential (if
they depart from approximately the same <-potential value) have the same amount
of bound surfactant. Three important assumptions are built into this analysis: (i)
the system is at equilibrium; (ii) all the ionic charges of the polymer are available
for binding; and (iii) the excess unbound solute does not affect the (-potential of
the surfactant-polymer aggregates. The method has worked well (i.e., less than 20%
of difference with respect to the mass-balance data) for polyethyleneimine -sodium
dodecyl sulfate systems (Mezei & Mtszdros, 2006).
The method involves producing two <-potential curves in the systems with two
concentrations of the substrate-Cs. In this case, the substrate is BLES, and the
actual binding (n+/n-)is the moles' positively charged glucosamine groups of chitosan
<-
bound to one mole of anionic lipid (PGs) in BLES. The shift of potential (A<) is
proportional to the value of an actual binding ratio:
where C1 and CL are the initial (dose) concentrations of the polymer (chitosan)
expressed in terms of moles of cationic groups for a given A<, and Csl and Cs2 are
Lung Surfactants: Formulation, Evaluation,and Polymeric Additives 0 2 15
u)
s?
(0
Pw (32-2.0mg/ml BLES
w
lu
-lu
*.a
c Total chitosan (mg/ml)
s?
0
P
.-0
U
e
F
5
aC
ir 0.5 mg/ml BLES
2.0mg/ml BLES
the molar ionic lipid concentrations in the BLES preparations (0.5 mg/mL and 2.0
mg/mL). These points are illustrated, for example, in Fig. 8.1 5a. Ce is the equilibrium
concentration of the unbound polymer in the solution, and is assumed to be the
same in two mixtures with the same & potential (Demarger-Andre & Domard, 1994;
Mezei & Mtszdros, 2006). To estimate the actual binding isotherm, the last equality
of Equation 8.5 is used to determine Ce (for a given A&), and then n+/n-is calculated
using either one of the last two terms of Equation 8.5. The change from mass units
(used in Fig. 8.15a) to molar units requires knowledge of the anionic lipid content
in BLES, an assumption regarding their molecular weight, and knowledge of the
degree of deacetylation and dissociation of chitosan. Further details in this change of
mass to mole ratio are found elsewhere (Kang et al., 2008). Figure 8.15b presents the
estimated binding isotherm for the BLES-chitosan system.
The estimated binding curve is consistent with the hypothesis of the binding
of the cationic groups in chitosan to the anionic lipids in BLES, and we directed
our efforts to determine the effect that different chitosan/BLES ratios have on the
properties of the formulation in the presence and absence of serum. Figure 8.16
presents the surface tension-relative compression area curves for systems formulated
with 2.0 mg/mL of BLES and chitosan (0,0.1, and 0.25 mg/mL) in the absence (top
row) and in the presence (bottom row) of 50 pL/mL of serum. Figure 8.16 presents
three main features of a dynamic compression cycle. The first feature is film relaxation
in which the surface tension of the film at the end of the compression stage increases
while holding the area constant. Film relaxation is linked to the hydration of some
of the compressed lipid molecules in the surfactant film (Acosta et al., 2007). The
second feature of the compression cycles in Fig. 8.16 is the dilatational elasticity of the
film ( E = (RA0)[4/d(RA0)]), which is proportional to the slope of the compression
stage of the graphs in Fig. 8.16. The higher the elasticity (the more pronounced the
slope), the more solid-like (better) is the surfactant film, and the lower the surface
tensions can be reached for a given compression. The concept of dilatational elasticity
is related to that of lung compliance introduced in the discussion of the in vivo tests.
The last feature observed in some cycles of Fig. 8.16 is film collapse, in which case the
progressive reduction in surface area (compression) does not result in further reduction
of surface tension. Film collapse indicates the onset of film inhibition when it occurs
at relatively high surface tensions (e.g., 10 to 20 mJ/m’). In summary, the desired film
properties are: no relaxation, high elasticity, and film collapse (if it happens at all) at
near-zero surface tensions.
Figure 8.16 shows that the addition of chitosan, up to a certain concentration,
helps improve the properties of BLES, and achieves low minimal surface tensions
even in the presence of serum. However, a large dose of chitosan induces surfactant
inhibition. To understand the behaviors observed in Fig. 8.16, the elasticity ( E ) and
relaxation rate (dyldt at the end of the compression stage) were plotted as a function
of the binding ratio (nil#-) between the cationic groups in chitosan and the negatively
charged lipids in BLES (Fig. 8.17) for BLES-chitosan formulations (no serum).
In both cases, the maximal elasticity and minimal relaxation are found at the n+/n-
Lung Surfactants: Formulation, Evaluation,and Polymeric Additives 0 2 17
binding ratio of 1.5 to 2.0. For binding ratios larger than 2.0, the surfactant film
collapses at high surface tensions. Similar trends were observed in the presence of
serum. Kang et al. (2008) also observed that although batch-to-batch performance of
manufacturer-supplied BLES samples was variable in 3TC, 100% of RH air, when all
these systems were formulated at the optimal BLES-chitosan ratio, they all reached
the same desirable performance.
To understand the optimal chitosan-BLES ratio, Kang et al. (2008) proposed
the "patch model" illustrated in Fig. 8.18. The patch model implies that, due to the
presence of the cationic polymer, anionic lipids form patches as they bind to these
polymers on the surface of the vesicles and on the multilayer lipid structures adsorbed
at the air-water interface. In that case, the area occupied by the anionic charges of
the lipid (n-) should be equal to the area of the cationic groups in chitosan (n').
Therefore, n+(a+)= n-(a-), where a+ and a- are the area per molecule of the cationic
groups in the polymer and a- is the area per molecule of the anionic li id. In the case
of chitosan, the area per each ionized glucosamine group is about 25 R,
and the area
of the most abundant anionic lipid in BLES, PG, is 40 A2/molecule when it is hlly
compressed, just before film collapse (Kang et al., 2008). This predicts that at optimal
conditions (full compression of the anionic lipid) the ratio of chitosan to the anionic
lipid is n+/n- = 40 A2/25A2 3 1 . 6 This is in agreement with the data presented in Fig.
1 .o
Fig. 8.16. Surface tension (y)-relative-area cyclic compression isotherms for 2.0 mg/mL of BLES
formulationscontainingdifferent levelsofchitosan(0,0.l.and0.25 mg/mL) in theabsence/presence of 50
pUmL of serum. Cycling conditions:37"C, 10096 of RH, 20% of compression, 3 sec/cycle compression.
2 18 0 EJ. Acosta et al.
200
N
-
E Film collapse
:
150
w
=
S
100
.-c0
m
-am
c
50
0 0.5 1 1.5 2 2.5
Actual n+/n- binding ratio
18
B
N
E
3 12
E
v
Film collapse
0
0 0.5 1 1.5 2 2.5
Actual n+/n- binding ratio
Fig. 8.1 7. (A) Dilatational elasticity ( E ) and (6) relaxation rate at the end of compression (dy/dt) for BLES-
chitosan systems (no added serum, cycled at 100% of RH, 37"C,20% of compression, and 3 sedcycle
period) as a function of the actual n+/n-binding ratio.The dashed line corresponds to 0.5 mg/rnL of BLES
systems, and the solid lines correspond to 2.0 mg/mL of BLES systems.
Lung Surfactants: Formulation, Evaluation,and Polymeric Additives 0 2 19
8.17. The same model was successhlly used to formulate BLES-cationic polymers by
using different polyelectrolytes and cationic peptides (articles in preparation).
Air
Liquid
Chitosan - anionic lipid patches
Chitosan
Fig. 8.18. Schematic of the binding between the ionized glucosamine groups of chitosan and the
anionic lipids in BLES to produce a patch on the surface of the vesicles and on the multilayer structures
adsorbed at the air-water interface.
220 0 EJ. Acosta et al.
three possible additives presented in this chapter, increasing agreement exists in the
surfactant community that nonionic polymers are not effective enough to produce
surfactant preparations that can counteract the action of potential inhibitors found
in the alveolar fluid of ARDS patients. Anionic polymers are a promising alternative.
The potential breakthrough change may come from some form of cationic additive
whether that is chitosan or recombinant SP-C or SP-B, o r peptide analogs of SP-B and
SP-C. As we continue to understand the interaction between these cationic additives
with the lipids in the surfactant preparation, we will be in better shape to formulate
more effective surfactant preparations.
References
Acosta, E.J.; R. Gitiafroz; Y.Y. Zuo; Z. Policova; PN. Cox; M.L. Hair; A.W. Neumann. Effect of
humidity on lung surfactant films subjected to dynamic compression/expansion cycles. Resp.
Physiol. Neurobiol. 2007, 155, 255-267.
Adhikari, N.; K.E.A. Burns; M.O. Meade. Pharrnacologic therapies for adults with acute lung
injury and acute respiratory distress syndrome. Cochrane Database Rev. 2004, 4. Art. No.
CD004477.
Anzueto, A.; R.I? Baughman; K.K. Guntupalli; J.G. Weg; H.I? Wiedemann; A.A. Ravent6s; F.
Lemaire; W. Long; D.S. Zaccardelli; E.N. Pattishall. Aerosolized surfactant in adults with sepsis-
induced acute respiratory distress syndrome. N. Engl. /. Med. 1996,334, 1417-1421.
Avery, M.E.; J. Mead. Surface properties in relation to atelectasis and hyaline membrane disease.
Am. /. Dis. Chikf. 1959,97, 517-523.
Asakura, S.; F. Oosawa. On interaction between two bodies immersed in a solution of macromol-
ecu1es.J. Chem. Phys. 1954,22, 1255-1256.
Bachofen, H.; J. Hildebrandt; M. Bachofen. Pressure-volume curves of air- and liquid-filled
excised lungs-surface tension in situ. /. Appl. Physiol. 1970, 29, 422-431.
Bachofen, H.; S. Schiirch; M. Urbinelli; E.R. Weibel. Relations among alveolar surface tension,
surface area, volume, and recoil pressure. J. Appl. Physiol. 1987, G2(5), 1878-1887.
Bailey, T.C.; A.A. Maruscak; A. Petersen; S. White; J.F. Lewis; R.A.W. Veldhuizen. Physiologi-
cal effects of oxidized exogenous surfactant in vivo: effects of high tidal volume and surfactant
protein a. Am. J. Physiol. 2006, 291, L703-L709.
Bailey, T.C.; K.A.D. Silva; J.F. Lewis; K. Rodriguez-Capote; F. Possmayer; R.A.W. Veldhuizen.
Physiological and inflammatory response to instillation of an oxidized surfactant in a rat model
of surfactant deficiency./. Appl. Physiol. 2004, 96, 1674-1680.
Bangham, A.D.; C.J. Morley; M.C. Phillips. The physical properties of an effective lung surfac-
tant. Biochim. Biopbys. Acta 1979, 573, 552-556.
Bartmann, I?; U. Bamberger; F. Pohlandt; L. Gortner. Immunogenicity and immunomodulatory
activity of bovine surfactant (sf-ri 1). Acta Paediatrica, Int. J, Paediatr. 1992, 81, 383-388.
Been, J.V.; L.J.I. Zimmermann. What’s new in surfactant? A clinical view on recent developments
in neonatology and paediatrics. Eur. J. Pediatr. 2007, IG6, 889-899.
Bengt-Robertson, B.R.; T. Curstedt; J. Johansson. Prospects for a new synthetic surfactant. Acta
Biomedica de PAteneo Parmense 2000, 71, 409-4 12.
Lung Surfactants:Formulation, Evaluation, and Polymeric Additives 0 221
Benson, B.J. Genetically engineered human pulmonary surfactant. Clin. PerinutoL 1993,20(4),
791-8 1 1.
Bernhard, W.; J. Mottaghian; A. Gebert; G.A. Rau; H. von der Hardt; C.F. Poets. Commerical
versus native surfactants. Am. /. Crit. Cure Med. 2000, 162, 1524-1 533.
Bevilacqua, G. et al . Prophylaxis of respiratory distress syndrome by treatment with modified
porcine surfactant at birth: A multicentre prospective randomized trial. J Perinutul Med. 1996,
24(6), 609-620.
Blanco, 0.;J. PCra-Gil. Biochemical and pharmacological difference between preparations of
exogenous natural surfactant used to treat respiratory distress syndrome: Role of the different
components in an efficient pulmonary surfactant. Eur. J. Pburmucol. 2007,568(1-3), 1-1 5.
Bloom, B.T. et al . Comparison of infasurf (calf lung surfactant extract) to survanta (beractant) in
the treatment and prevention of respiratory distress syndrome. Pediutrics. 1997, 100(1), 31-38.
Cabrerizo-Vflcha, MA.; HA. Wege; ].A. Holgado-Terriza;A.W. Neumann. Axisymmetric drop
shape analysis as penetration langmuir balance. &. Scientifir Instr. 1999, 70(5), 2438-2444.
Campbell, H.; K. Bosma; A. Brackenbury; L. McCaig; Li-J. Yao; R Veldhuizen; J. Lewis. Polyeth-
ylene glycol (PEG) attenuates exogenous surfactant in lung-injured adult rabbits. Am. J Rrspir.
Crit. Cure Med. 2002, 165, 475-480.
Capetole, R. EX-99.1 Interim report. (http://www.secinfo.com/duwTa.733c.d.htm), 1998.
Cheng, I?; D.Li; L. Boruvka; Y.Rotenberg; A.W. Neumann. Automation of axisymmetric drop
shape analysis for measurements of interfacial tensions and contact angles. Colloids Surj 1990,
43, 151-167.
Clements, J.A. Surface tension oflungextracts. Proc. Sor. Etp. Biol. Med, 1957,95, 170-172.
Cochrane, C.G.; S.D. Rev&. Pulmonary surfactant protein B (sp-B): Structure-function relation-
ships. Science 1991,254,566-568.
Codd, J.R; S.Schurch; C.B. Daniels; S. Orgeig. Torpor-associated fluctuations in surfactant activ-
iry in gould’s wattled bat. Biocbim. Biopbs. Arta 2002, 1580, 57-66.
Craster, RV.; O.K.Matar. On the dynamics of liquid lenses. J Cohidlntefuce Sci. 2006,303,
503-516.
Creuwels L.; L. van Golde; H. Haagsman. ‘The pulmonary surfactant system-Biochemical and
clinical aspects. Lung 1997, 175, 1-39.
Curstedt, T.; J. Johansson. New synthetic surfactant-how and when? Biol. Neonate 2006,89,
336-339.
Dargaville, PA.; J.F. Mills. Surfactant therapy for meconium aspiration syndrome: Current status.
Drug 200565,2569-2591.
Davidson, B.A.; ER. Knight; Z. Wang; E R Chess;’ BA. Holm; T.A. Russo; A. Hutson; 0.1. Del
No;A.W. Neumann. hisymmetric drop shape analysis: Computational methods for the mea-
surement of interfacial properties from the shape and dimensions of pendant and sessile drops. 1.
CoUoidlntefaeSci. 1997, 196, 136-147.
Demarger-Andre, S.; A. Domard. Chitosan behaviours in a dispersion of undecylenic acid. Struc-
tural parameters. Curbobydx Polym. 1994, 24, 177-184.
Dunn, M.S.; A.T. Shennan; D. Zayack; E Possmayer. Bovine surfactant replacement therapy in
222 0 EJ. Acosta et al.
neonates of less than 30 weeks’ gestation: A randomized controlled trial of prophylaxis versus
treatment. Pediatrics 1991,87, 377-386.
Durand, D. J.; R.I. Clyman; M.A. Heymann. Effects of a protein-free, synthetic surfactant on
survival and pulmonary function in preterm lambs. /. Pediatr 1985, 107, 775-780.
Dynarowicz-Latka, I?; A. Dhanabalan; O.N. Oliveira, J . Modern physicochemical research on
langmuir monolayers. Adv. ColloidZnterface Sci. 2001,91, 221-293.
Eijking, E.E; D. Gommers; J. Kullander; E.D. Buijzer; R.W. Beukenholdt; B. Lachmann. Differ-
ent surfactant treatment strategies for respiratory failure induced by hydrochloric acid aspiration
in rats. Adv. Exp. Med. Biol. 1992,317, 349-355.
Enhorning, G. Pulsating bubble technique for evaluating pulmonary surfactant. J Appl. Physiol.
1977,43, 198-203.
Enhorning, G.; A.T. Shennan; E Possmayer. Prevention of neonatal respiratory distress syndrome
by tracheal instillation of surfactant: A randomized clinical trial. Pediatrics 1985, 76 145-153.
Fisher, M.J.; M.F. Wilson; K.C. Weber. Determination of alveolar surface area and tension from in
situ pressure-volume data. Respir. Physiol. 1970, 10,159-171.
Frerking, I.; A. Giinther; W. Seeger; U. Pison. Pulmonary surfactant: functions, abnormalities and
therapeutic options. Intensive Care Med. 2001,27, 1699-1717.
Fujiwara, T.; M. Konishi; S. Chida; K. Okuyama; Y. Ogawa; Y.Takeuchi; H. Nishida; H. Kito;
M. Fujimura; H. Nakamura; T. Hashimoto . Surfactant replacement therapy with a single post-
ventilatory dose of a reconstituted bovine surfactant in preterm neonates with respiratory distress
syndrome: Final analysis of a multicenter, double-blind, randomized trial and comparison with
similar trials. Pediatrics 1990, 86, 753-764.
Goerke, J. Pulmonary surfactants: functions and molecular composition. Biochim. Biophys. Acta
1998,1408,79-89.
Gortner, L.; I? Bartmann; F. Pohlandt; U. Bernsau; F. Porz; H.H. Hellwege; R.C. Seitz; G.
Hieronimi; C. Bremer; G. Jorch. Early treatment of respiratory distress syndrome with bovine
surfactant in very preterm infants: a multicenter controlled clinical trial. Pediatr. PulmonoL
1992, 14, 4-9.
Gortner, L.; U. Bernsau; H.H. Hellwege; G. Hieronimi; G. Jorch; H. L. Reiter. A multicenter
randomized controlled clinical trial of bovine surfactant for prevention of respiratory distress
syndrome. Lung 1990, 168, 864-869.
Hgner, D.; EG. Germann; D. Hauschke. Effects of rSP-C surfactant on oxygenation and histol-
ogy in a rat-lung-lavage model of acute lung injury. Am. /. Respir. Crit. Care Med. 1998, 158,
270-278.
Hall, S.B.; M.S. Bermel; Y.T. KO; H.J. Palmer; R.H. Notter. Approximation in the measurement
of surface tension with the oscillating bubble surfactometer. 1.Appl. Physiol. 1993, 75, 468-477.
Halliday, H.L. Overview of clinical trials comparing natural and synthetic surfactants. Biol. Neo-
nate. 1995, 67, 32-47.
Henriksen, I.; G. Smistad; J. Karlsen. Interactions between liposomes and chitosan. Znt. /. Pharm.
1994,101,227-236.
Hoekstra, R.E.; J.C. Jackson; T.F. Myers; I.D. Frantz; M.E. Stern, W.F. Powers; M. Maurer; J.R.
Raye; S.T Carrier; J.H. Gunkel; A.J. Gold . Improved neonatal survival following multiple
Lung Surfactants:Formulation, Evaluation,and Polymeric Additives 0 223
doses of bovine surfactant in very premature infants at risk for respiratory distress syndrome,
Pediatrics 1991, 88, 1&18.
Horbar, J.D. et al . A multicenter randomized, placebo-controlledtrial of surfactant therapy for
respiratory distress syndrome. New Eng. J. Med. 1989,320, 959-965.
Hudak, M.L. et al . A multicenter randomized masked comparison trial of synthetic surfactant
versus calf lung surfactant extract in the prevention of neonatal respiratory distress syndrome.
Pediatrics 1997, 100, 39-50.
Im Hof, V.; I? Gehr; V. Gerber; M.M. Lee; S. Schurch. In vivo determination of surface tension in
the horse trachea and in vitro model studies. Rrspir. Pbysiol. 1997, 107, 81-93.
Jacobs, S.E.; K. O'Brien; S. Inwood; E.N. Kelly; H.E. Whyte. Outcome of infants 23-26 weeks'
gestation pre and post surfactant. h a Pacdatr. 2000, 89, 1149-1 151.
Kang, N.; Z. Policova; G. Bankian; M.L. Hair; Y.Y. Zuo;A.W. Neumann; E.J. Acosta. Interac-
tion between chitosan and bovine lung extract surfactants. Biochim. Biopbys. h a 2008, 1778,
291-302.
Kendig, J.W. et al . Comparison of rwo strategies for surfictant prophylaxis in very premature
infants: A multicenter randomized trial. Pediatrics 1998, 101, 1006-1012.
Khanala, D.R; Y. Okamotoa; K. Miyatakea; T. Shinobub; Y. Shigemasab; S. Tokurac; S. Minamia.
Protective effects of phosphated chitin (P-chitin) in a mice model of acute respiratory distress
syndrome (ARDS). Carbohydr. Polym. 2001,44, 99-106.
Kobayashi, T.; T.Curstedt; G. Grossmann; B. Robertson. Inhibition of exogenous surfactant in
ventilated immature newborn rabbits. Rcspir. Pbysiol. 1989, 76 1-12.
Kobayashi, T.; K. Ohta; K. Tashiro; K. Nishizuka; W. Chen; S. Ohmura. Dextran restores
albumin-inhibited surfice activity of pulmonary surfactant extract./. Appl. Pbysiol. 1999,86
1778-1 784.
Konishi, M.; T. Fujiwara; S. Chida; H. Maeta; S. Shimada; T. Kasai; Y. Fuji; Y. Murakami. A pro-
spective, randomized trial of early versus late administration of a single dose of Surfictant-TA.
Earb Human DmcIop. 1992,29, 275-282.
Kumar, M.N.; RA. Muzzarelli; C. Muzzarelli; H.Sashiwa; A.J. Domb. Chitosan chemistry and
pharmaceutical perspectives. Chem. Rev. 2004, 104, 6017-6084.
Kwok, D.Y.; D. Vollhardt; R. Miller; D. Li; A.W. Neumann. Axisymmetric drop shape analysis as
a film balance. Col Su$ A Pbysicocbem. Aspects. 1994, 88, 51-58.
Kwong, M.S.; E.A. Egan; R H . Notter; D.L. Shapiro. Double-blind clinical trial of calf lung sur-
factant extract for the prevention of hyaline membrane disease in extremely premature infants.
Pediatrics 1985, 76 585-592.
Lamm, W.J.E.; RK. Albert. Surfactant replacement improves lung recoil in rabbit lungs after acid
aspiration. Am. Rev. Rcspir. Dis. 1990, 142, 1279-1283.
Langmuir, 1. The constitution and fundamental properties of solids and liquids. ii. Liquids. J.Am.
Chcm. SOC.1917,37, 1848-1906.
Lewis, J.F.; A. Brackenbury. Role of exogenous surfactant in acute lung injury. Crit. Care Med.
2003,31, S324-328,
Lewis, J.E; J. Goffin; I? Yue; L.A. McCaig; D. Bjameson; R.A.W. Veldhuizen. Evaluation of
224 0 EJ. Acosta et al.
exogenous surfactant treatment strategies in an adult model of acute lung injury. /. Appl. Physiol.
1996,80,11561164.
Lu, J.J.; WW.Y Cheung; L.M.Y. Yu; Z. Policova; D. Li; M.L. Hair; A.W. Neumann. The effect
of dextran to restore dynamic surface tension properties of pulmonary surfactant inhibited by
albumin. Resp. Physiol. Neurobiol. 2002, 130, 169-179.
Lu, K.; J. Goerke; J. Clements; H. Taeusch. Hyaluronan decreases surfactant inactivation in vitro.
Pediatz Res. 2005a, 55, 237-241.
Lu, K.W.; J. Goerke; J.A. Clements; H.W. Taeusch. Hyaluronan reduces surfactant inhibition and
improves rat lung function after meconium injury. Pediatz Res. 2005b, 58, 206210.
Lu, K.W.; B. Robertson; H. Taeusch. Dextran or polyethylene glycol added to curosurf for treat-
ment of meconium lung injury in rats. Biol Neonate. 2005c, 88, 46-53.
Lu, K.W.; H.W. Taeusch; B. Robertson; J. Goerke; J.A. Clements. Polymer-surfactant treatment
of meconium-induced acute lung injury. Am. 1.Respir. Crit. Care Med. 2000, 162, 623428.
Lu, K.; H. Taeusch; B. Robertson; J. Goerke; J. Clements. Polyethylene glycol/surfactant mixtures
improve lung function after HCI and endotoxin lung injuries. Am. /. Respir. Crit. Care Med.
2001,164, 1531-1536.
Lu, J.J.; L.M.Y.Yu; W.W.Y. Cheung; LA. Goldthrope; Y.Y. Zuo; Z. Policova; I?N. Cox; A.W.
Neumann. Polyethylene glycol (PEG) enhances dynamic surface activity of a bovine lipid extract
surfactant (BLES). Colloidc Surf: B, 2005d, 41, 145-151 .
Lu, J.J.; L.M.Y. Yu; W.W.Y. Cheung; Z. Policova; D. Li; M.L. Hair; A.W. Neumann. The effect
of concentration on the bulk adsorption of bovine lipid extract surfactant (BLES). Coll. Surf: B
Biointefaces. 2003,29, 1 19-1 30.
Markart, I?; C. Ruppert; M. Wygrecka; T. Colaris; B. Dahal; D. Walmrath; H . Harbach; J.
Wilhelm; W. Seeger; R. Schmidt; A. Guenther. Patients with ARDS show improvement but
not normalisation of alveolar surface activity with surfactant treatment: Putative role of neutral
lipids. Borax 2007,62, 588-594.
McCormack, EX. Structure, properties and processing of surfactant protein-A. Biochim. Biopbys.
Acta 1998,1408, 109-131.
McCormack, EX. Functional mapping of surfactant protein A. Pediatr. Pathol. Mol. Med. 2001,
20,293-318.
McDorman, M.F.; H.M. Rosenberg. Trends in infant mortality by cause of death and other char-
acteristics, 1960-1989. Etaland Health Statistics ‘(DDHS publication PHS 93-1857); National
Center for Health Statistics. 1993; pp. 1-51.
McIntyre, R.C.; E.J. Pulido; D.D. Bensard; B.D. Shames; E. Abraham. Thirty years of clinical
trials in acute respiratory distress syndrome. Crit. Care Med. 2000,28, 33 14-333 1.
McMillan, D.; V. Chernick; N. Finer; D. Schiff; H. Bard; J. Watts; R Krzeski; W. Long. Effects
of two rescue doses of synthetic surfactant in 344 infants with respiratory distress syndrome
weighing 750 to 1249 grams: a double-blind, placebo-controlled multicenter Canadian trial. /.
Pediatr. 1995,126; S90-98.
Mezei, A.; R. MkszPros. Novel method for the estimation of the binding isotherms of ionic surfac-
tants on oppositely charged polyelectrolytes. Langmuir 2006,22, 7148-71 5 1.
Mingarro, I.; D. Lukovic; M. Vilar; J. Pkra-Gil. Synthetic pulmonary surfactant preparations:
Lung Surfactants:Formulation, Evaluation,and Polymeric Additives 0 225
new developments and future trends. Cum Med Cbem. 2008, 15,393-403.
Morley, C.J. Ten centre trial of artificial surfactant (artificial lung expanding compound) in very
premature babies. Brit. Med 1.1987,294, 991-996.
Morrow, M.R.; N. Abu-Libdeh; J. Stewart; K.M.W. Keough. Interaction of pulmonary surfactant
protein SP-A with DPPClegg-PG bilayers. Biophys./. 2003,85, 2397-2405.
Morrow, M.R.; J. Stewart; S. Taneva; A. Dico; K.M. Keough. Perturbation of DPPC bilayers by
high concentrations of pulmonary surfactant protein SP-8. Eur. Biophys.J. 2004,33, 285-290.
Moses, D.; B.A. Holm; I? Spitale; M. Liu; G. Enhorning. Inhibition of pulmonary surfactant
function by meconium. Am. J. Obstet. Gynecot! 1991, 164, 477481.
Moya, ER.; J. Gadzinowski; E. Bancalari; V. Salinas; B. Kopelman; A. Bancalari; M.K. Kor-
nacka; TA. Merritt; R segal;C.J. Schaber; H. Tsai; J. Massaro; RD’Agostino. A multicenter,
randomized, masked, comparison trial of lucinactant, colfosceril palmitate, and beractant for the
prevention of respiratory distress syndrome among very preterm infants. Pediutrin 2005, 115,
1018-1029.
Moya, F.; A. Maturana. Animal-derived surfactants versus past and current synthetic surfactants:
Current status. Clin. PerinatoL 2007,34, 145-177.
Nag, K.; M. Frinen-Garcia; R Devraj; A.K. Panda. Interfacial organizations of gel phospholipid
and cholesterol in bovine lung surfactant films. Langmuir 2007,23, 44214431.
Nag, K; J. Perez-Gil; M.L. Ruano; LA. Worthman; J. Stewart; C. Casals; K.M. Keough.
Phase transitions in films of lung surfactant at the air-water interface. BiopLys. 1.1998, 74,
2983-2995.
Nag, K.; K. Rodriguez-Capote;A.K. Panda; L. Frederick: S.A. Hearn; N.O. Petersen; S. Schiirch;
F. Possmayer. Disparate effects of two phosphatidylcholine binding proteins, C-reactive protein
and surfactant protein A, on pulmonary surfactant structure and function. Lung Cell MoL
PbysioL 2004,287, L1145-1153.
Nakamura, T.; M. Tamura. Partial liquid ventilation with low dose of perflubron and a low stretch
ventilation strategy improves oxygenation in a rabbit model of surfactant depletion. BioL Neo-
nate 2002, 82, 66-69.
Noack, G.; I? Berggren; T. Cuntedt; G. Grossmann; I? Herin; W. Mortensson; R Nilsson; B.
Robertson. Severe neonatal respiratory distress syndrome treated with the isolated phospholipid
fraction of natural surfactant. Actu Puediutr Scund 1987, 76 697-705.
Notter, R H . Lung Surfactant, Batic Science and CfinicufApplication; Marcel Dekker, New York,
2000.
Notter, RH.; S.A. Tabak; R D . Marvis. Surface properties of binary mixtures of some pulmonary
surfactants components. J. Lipid Res. 1980,2I, 10-22.
Pattle, RE. Properties, function and origin of the alveoli lining layer. Natun 1955, 175,
1125-1 126.
Pattle, RE.; L.C. Thomas. Lipoprotein composition of the film lining the lung. Natun 1%1,
189, 844.
Persson, A.; D. Chang; K. Rust; M. Moxley; W. Longmore; E. Crouch. Purification and biochem-
ical characterization of CP4(SP-D), a collagenous surfactant-associated protein. Biocbem. 1989,
28,6361-6367.
226 EJ. Acosta et al.
Petty, T.L. Overview. Acute Respiratory Distress Syndrome; M. Mattay, Ed.; Marcel Dekker: New
York, 2003; pp. 1-6.
Pinkerton, K.E.; M. Ikegami; L.M. Dillard; A.H. Jobe. Surfactant treatment effects on lung struc-
ture and type I1 cells of preterm ventilated lambs. Biol. Neonate 2000, 77, 243-252.
Possmayer, F.A. Proposed nomenclature for pulmonary surfactant-associated proteins. Am. Rev.
Respir. Dis. 1988,138, 990-998.
Possmayer, E; K. Nag; K. Rodriguez; R. Qanbar; S. Schiirch. Surface activity in vitro: role of
surfactant proteins. Comp. Biochem. Physiol. A. 2001, 129, 209-220.
Prokop, R. M.; A. Jyoti; M. Eslamian; A. Garg; M. Mihaila; 0.1. del No; S.S. Susnar; Z. Policova;
A.W. Neumann. A study of captive bubbles with axisymmetric drop shape analysis. Coll. Surf: A
Physicochem. Aspects. 1998, 131, 231-247.
Robertson, B. Factors influencing the clinical response to surfactant replacement therapy in babies
with severe respiratory distress syndrome. Eur. 1.Pediatr. 1991, 150, 433-439.
Robertson, B.; H.L. Halliday. Principles of surfactant replacement. Biochim. Biophys. Actu 1998,
1408,346361.
Rotenberg, Y.; L. Boruvka; A.W. Neumann. Determination of surface tension and contact angle
from the shapes of axisymmetric fluid interfaces.]. ColloidZntel.faceSci. 1983,93, 169-183.
Rubenfield, G.D.; M.J. Neff. Epidemiology of acute lung injury. A public health perspective.
Acute Respiratory Distress Syndrome; M. Mattay, Ed.; Marcel Dekker: New York, 2003; pp.
37-54.
Riidiger, M.; A. Tolle; W. Meier; B. Riistow. Naturally derived commercial surfactants differ in
composition of surfactant lipids and in surface viscosity. Am. 1.Physiol. 2005,288, L379-L383.
Saleem, M.; M.C. Meyer; D. Breitenstein; H.J. Galla. The surfactant peptide KL4 in lipid
monolayers: phase behavior, topography, and chemical distribution. J Biol. Chem. 2008,283,
5 195-5207.
Sarin, I?; H. Taeusch; K. Lu; J. Clements; J. Goerke. Polyethylene glycol results in concentration-
dependent sedimentation of Survanta and improvement of its function. Pediatr. Res. 1999, 45,
3 19A.
Schoel, W.M.; S. Schiirch; J. Goerke. The captive bubble method for the evaluation of pulmonary
surfactant: surface tension, area, and volume calculations. Biochim. Biophys. Actu 1994, 1200,
281-290.
Schiirch, S.; H. Bachofen; J. Goerke; F. Possmayer. A captive bubble method reproduces the in
situ behavior of lung surfactant monolayers. 1.Appl. Physiol. 1989, 67,2389-2396.
Schiirch, S.; J. Goerke; J.A. Clements. Direct determination of surface tension in the lung. Proc.
Nut. Acad. Sci. 1976, 73, 4698-4702.
Schiirch, S.; F.H.Y. Green; H. Bachofen. Formation and structure of surface films: Captive bubble
surfactometry. Biochim. Biophys. Acta 1998, 1408, 180-202.
Scott, J.E. Phosphatidylcholine synthesis, secretion, and reutilization during differentiation of
the surfactant-producing type I1 alveolar cell from fetal rabbit lungs. Eup. Lung Res. 1992, 18,
563-580.
Seeger, W.; C. Grube; A. Giinther; R. Schmidt. Surfactant inhibition by plasma proteins: differen-
Lung Surfactants:Formulation, Evaluation,and Polymeric Additives 0 227
quency, clinical course, and costs of care. Crit. Care Med. 1999,27, 2367-2374.
Veldhuizen, R.; F. Possmayer. Phospholipid metabolism in lung surfactant. Su6-cell. Biochem.
2004337,359-388.
Von Neergaard, K. Neue Auffassungen iiber einen Grundbegriff der Atemmechanik. Z. Ges. Eup.
Med. 1929, 66,1.
Wakabayashi, T.; M. Tamura; T Nakamura. Partial liquid ventilation with low-dose
perfluorochemical and high-frequency oscillation improves oxygenation and lung compliance in
a rabbit model of surfactant depletion. Biol. Neonate 2006, 89, 177-182.
Walther, F.J.; A.J. Waring; M.A. Sherman; J.A. Zasadzinski; L.M. Gordon. Hydrophobic surfac-
tant proteins and their analogues. Neonatol. 2007,9I, 303-310.
Weaver, T.; J. Whitsett. Function and regulation of expression of pulmonary surfactant-associated
protein. Biochem. J. 1991,273, 249-264.
Wilkinson, A.; PA. Jenkins; J.A. Jeffrey. Two controlled trials of dry artificial surfactant: Early ef-
fects and later outcome in babies with surfactant deficiency. Lancet. 1985,2, 287-291.
Willson, D.F.; A. Zaritsky; L.A. Bauman; K. Dockery; R.L. James; D. Conrad; H. Craft; W.E.
Novotny; E.A. Egan; H. Dalton. Instillation of calf lung surfactant extract (calfactant) is
beneficial in pediatric acute hypoxemic respiratory failure. Crit. Cure Med. 1999, 27, 188-195.
Wilson, T.A. Relations among recoil pressure, surface area, and surface tension in the lung. J. Appl.
Physiol. 1981,50, 921-926.
Withsett, J.; L. Nogee; T. Weaver; A. Horowitz. Human surfactant protein B: structure, function,
regulation and genetic disease. Physiol. Rev. 1995, 75, 749-757.
Worthman, L.A.; K. Nag; N. Rich; M.L. Ruano; C. Casals; J. Perez-Gil; K.M. Keough. Pulmo-
nary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant
lipid extract. Biophys. /. 2000, 79, 2657-2666.
Wu, C.W.; S.L. Seurynck; K.Y.C. Lee; A.E. Barron. Helical peptoid mimics of lung surfactant
protein C. Chem. Biol. 2003, 10, 1057-1063.
Wulf, M.; S . Michel; K. Grundke; 0.1. del Rio; D.Y. Kwok; A.W. Neumann. Simultaneous
determination of surface tension and density of polymer melts using axisymmetric drop shape
analysis. J. Colloid Interfce Sci. 1999,210, 172-1 8 1.
Yu, L.M.Y.; J.J. Lu; Y.W. Chan; A. Ng; L. Zhang; M. Hoorfar; Z. Policova; K. Grundke; A.W.
Neumann. Constrained sessile drop as a new configuration to measure low surface tension in
lung surfactant systems. J. Appl. Physiol. 2004a, 97, 704-71 5.
Yu, L.M.Y.; J.J. Lu; I.W.Y. Chiu; K.S. h u n g ; Y.W. Chan; L. Zhang; Z. Policova; M.L. Hair; A.W.
Neumann. Poly(ethy1ene glycol) enhances the surface activity of a pulmonary surfactant. Coll.
SuI$ B. Biointerfaces 2004b, 36, 167-176.
Zasadzinski, J.A.; J. Ding; H.E. Warriner; F. Bringezu; A.J. Waring. The physics and physiology of
lung surfactants. Cur% Opin. CollOidInterf:Sci. 2001,6, 5 0 6 5 13.
Zuo, Y.Y. Biophysical and Gas Transfer Properties of Lung Surfactant and Polymer-Enhanced
Lung Surfactant Films. Ph.D .Dissertation. University ofToronto, 2006.
Zuo, Y.Y.; H. Alolabi; A. Shafiei; N. Kang; Z. Policova; PN. Cox; E. Acosta; M.L. Hair; A.W.
Neumann. Chitosan enhances the in vitro surface activity of dilute lung surfactant preparations
Lung Surfactants:Formulation, Evaluation, and Polymeric Additives 0 229
Introduction
A variety of amphiphilic compounds, such as complex lipids and proteins, are known
in living organisms. ‘These compounds play an important role in creating a well-
ordered biological system by relating to the exchange of energies, substances, and
signals at difference interfaces (Holmberg, 2001; Dembitsky, 2005). For example,
phospholipids self-assemble to form the basic structure of biomembranes, where
membrane proteins and glycolipids work as an essential platform for molecular
recognition, signal transduction, transmembrane transportation, cell adhesion, and
so on (Hakomori, 2002; Vankar & Schmidt, 2000). The unique properties of these
amphiphilic compounds arise from the co-existence of a hydrophobic headgroup and
a hydrophilic part, which often prompt the formation of highly organized structures
via self-assembly.
Amphiphilic compounds are generally called surfactants in the industry, and
more than 10 million tons of surfactants are produced each year for the world
market. Surfactants are used in various industries like textile, paper, polymer, plastic,
cosmetics, pharmaceuticals, food, and machinery manufacture (Luk & Abot, 2002).
They are definitely one of the most frequently used chemicals in our daily lives. The
development of the chemical industry provided a wide variety of petroleum-based
chemical surfactants. In addition to chemical surfactants, some biologically based
amphiphilic compounds, such as soaps (fatty acid salt), lecithin (phospholipid),
and saponin (glycolipid) have long been consumed for home and industrial use
before chemical surfactants were produced and became widespread. With increasing
environmental awareness and emphasis on a sustainable society in harmony with
the global environment, these “biobased surfactants” are becoming much more
important.
Among biobascd surfactants, ones of microbial origin are especially classified
into “biosurfactants” (BS) (Kitamoto et al., 2002; Banat et al., 2000). BS were first
231
232 0 D. Kitamoto et al.
but sophisticated structures, (iii) higher biodegradability and lower toxicity, (iv) lower
critical micelle concentration and higher surfice activity, (v) gradual adsorption and
continuing activity, (vi) superior ability to form molecular assembly and liquid crystal,
and (vii) versatile biological activity (antimicrobial and antitumor actions, etc.).
The structures ofsurfactants are largely reflected in that ofinterfacial characteristics.
Typical hydrophilic groups of BS are carbohydrates, peptides, etcetera, while typical
hydrophobic groups are various fitty acids, such as saturated, unsaturated, branched,
or hydroxylated ones.
The combinations of these hydrophilic and hydrophobic groups are very
efficient and elegant in BS. The entire BS structures have probably been refined and
optimized during the integration process of “biological evolution” in microorganisms.
Moreover, BS are regio- and stereo-selectively synthesized by enzymatic reactions in
microorganisms from simple biomolecules, most of which are chiral compounds
having a unified molecular configuration. BS are thus able to exhibit excellent
orientation and packing properties in their interfaces. These structural features allow
BS to perform more surprising actions than conventional chemical surfactants.
BS are mainly classified into four categories, (i) glycolipid type (Kitamoto et al.,
2002; Lang, 20021, (ii) fatty acid type (Zhang et al., 2004a), (iii) lipopeptide type
(Ongena &Jacques, 2007) and (iv) polymer type (Ron & Rosenberg, 2002), based
on the structure of their hydrophilic part. The chemical structures of representative
glycolipid BS and their producers are presented in Figures 9.1 to 9.7 and Table 9.1,
respectively.
1 2
MEL-A: R = R = A c
1 2
MEL-B: R = A c , R = H
1 2
MEL-C: R = H, R = AC
(n = 6 to 10)
Fig. 9.1. Chemical structures of mannosyletythritollipids.
234 0 D. Kitamoto et al.
Acidic forms
R 1 =R2 =HorAc
R2=xcooH
R4=Tc0 CH3 CH 3
roR OH
TL-1: RI =R2=
H3
H
H 3C
TL-2: RI =
CH3 R 2 = H
(m + n = 27 to 31)
Fig. 9.4. Chemical structures of trehaiose lipids.
HO
OR3 OR1 OH
H : !OH
4, " O
&
$
HHO OH
R4= M ( C H 2 l n C H 3 (n = 2 or 4)
.OH
OH
sophorose can be used as the sole carbon source by C. bombicokz (Van Bogaert, 2007).
The proposed physiological roles of BS are illustrated in Fig. 9.8 according to the
previous studies (Kitamoto et al., 2002; Van Bogaert, 2007; Nitschke, 2005). These
roles very likely originate from their unique amphiphilic structures.
PhysicochemicalAspects of Biosurfactants
Interfacial Properties of Glycolipid Biosurfactants
BS are surface-active compounds capable of reducing surface and interfacial tension
at the interfaces between liquids, solids, and gases, thereby allowing them to mix or
disperse readily as emulsions in water or other liquids. BS, especially glycolipid BS,
show versatile surface-active properties including emulsiGing, dispersing, solubilizing,
foaming, penetrating, and wetting actions. Table 9.2 shows some examples of surface
and interfacial tension-lowering activities of glycolipid BS.
Mannosykrythritol Lipid
MEL-Aand MEL-B (Fig. 9.1) exhibit excellent surface and interfacial tension-lowering
actions and low critical aggregate concentrations (CAC), although the hydrophobic
parts consist of medium-chain fatty acids ranging from C, to C , , (Kitamoto et al.,
2002; Imura et al., 2006). O n the Wilhelmy method, MEL-A shows a CAC at 2.7
x lo4 M and can reduce the aqueous surface tension and interfacial tension against
n-hexadecane to about 28 and 2 mN/m, respectively. The CAC of MEL-A and
MEL-B obtained by the pyrene fluorescence method are 4.0 x 104 and 6.0 x 104 M,
respectively (Imura et al., 2006).
SoDhoroliDid
SL-1 (C.bomibocola) 40d 35 - Lang et al., 2002
2-tetradecyl-SL 40 30 - Lang et al., 2002
-
n-butvlamide-SL 80 45 - Lanq et al., 2002
n-decylamide-SL 5d 60 - Lang et al., 2002
SL-5 (C. batistae) 138d 39.3 - Konishi et al., 2008b
SL-mixture (C.batistae) 366d 40.2 - Konishi et al., 2008b
Rhamnolipid
RL-A (Na salt) 6.2~ 28.0 O.Zb (0.1%) lshigami et al., 1987
RL-B (Na salt) 1.5 x lo4 28.0 3.2 (0.05%) lshigami et al., 1987
RL-1 20d 26 4= Lang & Wullbrandt, 1999
RL-2 lod 27 <1C Lanq & Wullbrandt, 1999
RL-3 200d 25 <1 Lang & Wullbrandt, 1999
RL-4 ZOOd 30 <lC Lang & Wullbrandt, 1999
Trehalose lipid
TL- 1 4d 36 17< Lang & Philp, 1999
TL-2 4 d 32 14 Lanq & Philp, 1999
Trehalose-2,2:3,4-tetraester 15 26 <1 Lang & Philp, 1999
STL-1 + STL-2 (Na salt) 9 . 6 ~ 1 0 30
~ 8b(0.2%) Lang & Philp, 1999
Oligosaccharide lipid
GL-1 1 . 5 ~ 1 0 35
~ <lo' Vollbrecht et al., 1999
GL-2 1.1 x i 0 4 23 <lo' Vollbrecht et al., 1999
SophotvIipircs
Sophorolipids produced by Candida bombicola, which are inevitably a mixture of
acid- and lactone-form sophorolipids (Fig. 9.2), lower the aqueous surface tension
below 40 mN/m with a critical micelle concentration of 40 - 100 mg/L; they are not
so effective at stabilizing oil-in-water emulsions. They can be modified into various
derivatives having a different hydrophilic-hydrophobic balance. Their derivatives
show a wide range of surface activities, such as emulsifling, wetting, cleaning, and
solubilizing (Shete et al., 2006). In particular, the propylene glycol adduct shows
an excellent hygroscopic activity, and is commercialized as a skin moisturizer and
softener in cosmetics.
Zhang et al. (2004b) recently synthesized different sophorolipid alkyl (methyl,
ethyl, propyl, and butyl) esters, and examined the effect of n-alkyl ester chain length
on the interfacial properties of the corresponding analogs. The CMC of the esters
decreases to about 1/2 per additional CH, group to the alkyl ester moiety. Interestingly,
these surfactants absorb strongly on alumina but weakly on silica. O n these esters, the
average area per molecule at the aidwater interface is around 80 A*, and is larger
than those of other surfactants with structure similar to sugar-based n-dodecyl-P-D-
maltoside of 50 A2.
242 0 D. Kitamoto et al.
100
c
80
-
E
E
L.
0)
*,
60
40
m
E
20
0
0 3 6 9 12 16
100 nm
Fig.9.9. Mean diameter of colloidal structures obtained from ternary mannosylerythritollipid-A (MEL-A)/
waterln-decane system (n-decanelMEL-A molar ratio was 4.8) as a function of water-to-surfactant mole
ratio (W,) at 25°C (upper figure). Freeze fracture electron micrographs of colloidal structures obtained
from the above ternary MEL-A/water/n-decane system (lower figure). Bar length is 100 nm. (a) W, = 1.5,
(b) W, = 8.3, and (c) W, = 14.3.
Self-assernbling Propertiesof Glycolipid Biosurfactants and Their Functional Developments 0 243
Rhamnofipith
Rhamnolipids (Fig. 9.3) show good surface activities, including emulsifying,
dispersing, foaming, and penetrating actions. They display a lower CMC (10' to lov5
M) despite being an anionic surfactant (Lang & Wullbrandt, 1998). They reduce the
surface tension of water from 72 mN/m to values below 30 mNlm and the interfacial
tension of waterloil systems from 43 mN/m to values below 1 mNlm. Sodium salts
of RL-A and RL-B have the micellar weight of 38,000 and 7,000 in phosphate buffer
saline (pH 7.35) at 3VC, respectively. ?he molecular occupation area of sodium salt
of RL-B is 79.1 AVmolecule at the air-water interface (30°C). These data show that
the lipids have an excellent packing property despite their bulky and complicated
structure (Ishigami et al., 1987).
TmbaloseLip&
Trehalosc lipids (Fig. 9.4) are chemically stable and their surface activities are
independent over a wide range of temperature, pH values, and salt concentrations.
Trehalose di-corynomycolate (TL-1) and mono-corynomycolate (TL-2) reduce the
aqueous surface tension from 72 mN/m to 36 and 32 mN/m with a CMC of 4 mg/L,
and reduce the interhcial tension of waterln-hexadecane system from 43 mN/m to
17 and 14 mN/m, respectively.
Succinoyl-trehaloselipids (STL- 1 and -2), which are produced from n-hexadecane
by R. clythrupulfi SD-74 (Uchida et al., 1989), have both carboxyl and hydroxyl
244 0 0.Kitarnoto et al.
groups in the molecule, and thus exhibit high surface activities; especially dispersing
and dispersion-stabilizing activities towards solid particles such as red iron oxide
(a-Fe,O,), carbon black, and a-copper phthalocyanine blue (Lang & Philp, 1998).
water systems start to form a range of liquid crystalline phases. In particular, glycolipid
BS spontaneously self-assemble into a variety of molecular assemblies with well-
defined and/or unique structures: sponge (coacervate, LJ, cubic (V,), hexagonal (HJ,
or lamellar (La) to name a few (Imura et al., 2007a). These structures are restricted
by a glycolipid’s need to keep its hydrophobic and hydrophilic parts surrounded in a
favorable way, thus minimizing the free energy of the system (Hato, 2001).
Among these molecular assemblies, vesicles are one of the most intensively
studied and are prepared from natural glycolipids such as macrocyclic tetraether
glycolipids in archaebacteria and digalactosyldiacylglycerols in plants. Vesicles made
from phospholipids (liposomes) have been studied over the last three decades. In
contrast to liposomes prepared from natural phospholipids, investigations on vesicles
made from glycolipids, both naturally occurring and synthetic, have been relatively
rare. This appears due to the limited amount and heterogeneity of natural glycolipids,
and to the high values of 7K for synthetic glycolipids (Kitamoto et al., 2005).
As mentioned previously, glycolipid BS are easy to prepare in large amounts by
microbial processes compared to membrane glycolipids or complicated synthetic
surfactants. In addition, the hydrophobic part of glycolipid BS constitutes a variety of
fatty acids, such as short or medium-chain, unsaturated, and/or branched ones. This
enables the BS to lower the 7K and to be always in a fluid state, and then makes it
easier to control their liquid crystal structures at low temperatures (Hato et al., 1999).
Accordingly, glycolipid BS-based vesicles or bilayer membranes appear to be very
promising and may open new avenues for exploiting usehl nanostructured materials
and/or systems.
Mannosyhythritol Lipid
The self-assembling manner of MEL-A and MEL-B is illustrated in Fig. 9.10.
Kitamoto et al. recently reported that MEL-B and -C (Fig. 9.1), which possess alkyl
chains at the C-2 and C-3 positions, spontaneously form giant unilamellar vesicles
of diameters larger than 10 pm (Kitamoto et al., 2000). Giant vesicles can serve
as biophysical model systems; they have been used as cell models for studying the
dynamic and structural features of many cellular processes, including endcytosis,
exocytosis, cell fusion, viral infection, and transport phenomena (Dobereiner, 2000).
However, it is generally difficult to obtain giant vesicles from glycolipids, because
the vesicle structure requires strictly balanced hydrophobic and hydrophilic groups.
This indicates that these MEL have an excellent molecular orientation property
and superior balance between hydrophilic and hydrophobic groups. As described
below, vesicle-forming lipids such as MEL-B or MEL-C can be applied to various
kinds of drug- and gene-delivery systems, coupling with other carrier materials like
phospholipids and polymers.
In contrast, MEL-A (Fig. 9.1) self-assembles to form a “sponge phase” at
concentrations above 1 mM, which has been often called “L3 phase,” “plumber‘s
nightmare,” or “blue I phase” (Imura et al., 2004, 2006). Menger et al. (2000)
246 0 D. Kitarnoto et al.
MEL-A
MEL-B
recently demonstrated the spontaneous formation of the sponge phase from a single
compound of a synthetic gemini surfactant, and they proposed that the surfactant
phase of sponge morphology should be redefined as “coacervates.” Coacervates are
well known as the origin of life, from which all present-day cells bearing ordered
bilayer membranes are generated. The study on the structure-property interaction of
coacervates is thus of great interest, considering how the ordered bilayer membranes
of cells have been constructed from coacervates. However, coacervates now known
are generally prepared from complicated multicomponent systems such as surfactants
with salt/co-solvent or two oppositely charged polyelectrolytes (De Kruif et al., 2004;
Maldonado et al., 2007). This makes their structural characterization difficult.
Nevertheless, MEL-A is the first “natural” compound to display the formation of
coacervates by themselves, while MEL-B and -C form vesicles. The manner reveals
that that only a slight decrease in “spontaneous curvature” resulting from the absence
of the 4’-0- or 6’-O-acetyl group induces a drastic morphological change in the self-
assembled structure from coacervates to vesicles, namely ordered bilayer membranes
(Imura et al., 2004). The FF-TEM observation on the MEL-A assemblies clearly
shows the typical coacervate morphology of a sponge phase composed of a randomly
connected 3D network of the bilayers. In many parts of the micrograph, we can
Self-assembling Propertiesof Glycolipid Biosurfactants and Their Functional Developments 0 247
follow the bilayer over a range of several micrometers, indicating the bilayer is indeed
continuous.
The spontaneous curvature (HJ is a usehl parameter to characterize the lipid-
assembled structures. It is given by H,= 114,where H, is the curvature of one
monolayer which forms a bilayer and 4 is the spontaneous radius of curvature.
When the spontaneous curvature is nearly zero, the lipids self-assemble into the
lamella phase (La) (Imura et al., 2006). The spontaneous curvature of MEL-B or
MEL-C assemblies is thus nearly zero since vesicles are obtained by the dispersion of
the La phase. These La phases seem to be stabilized by the hydrogen-bonding network
between the headgroups resulting from the C-4’ or C-6’ hydroxyl group because
La phase is less dynamic than L, phase (Imura et al., 2004). O n the other hand,
the presence of an acetyl group on the mannose moiety is likely to induce a slightly
negative spontaneous curvature of the glycolipid assemblies; this should lead to the
formation of the L, phase.
L, phase obtained from MEL-A would be a potential host for water-soluble
proteins and DNA using the interior water channels of the phase, because the L, phase
resembles the bicontinuous cubic phase. Although the cubic phase acts as a better
solvent for membrane protein bacteriorhodopsin crystallization, it is a stiff liquid
crystal, making it difficult to handle in high throughput screening systems (Ridell et
al., 2003). In contrast, the L, phase is fluid, giving it the potential to overcome this
problem.
Another interesting feature of sponge phase (LJ is the basis of thermodynamically
stable vesicles. Watanabe et al. (2001) reported that the thermodynamically stable
vesicle phase (La,) exists next to the sponge phase (L,) by the phase diagram
determination study of surhctant mixed systems. Imura et al. (2005) recently
demonstrated the formation of thermodynamically stable vesicles with a high
dispersibility from the above-mentioned MEL-A sponge phase by adding L-a-D-
dilauroyl-phosphatidylcholines (DLPC). They investigated the mixed system of
MEL-A and DLPC and confirmed the existence of three regions of self-assembly; (1)
droplets with a sponge phase (L,) with diameters from 2 to 20 pm & , I O.I), (2)
thermodynamically stable vesicles (La,) (0.2 I %LK 5 0.7), (3) multilamellar vesicles
(La)with diameters from 2 to 10 pm KLPc 2 0.8). Figure 9.1 1 shows the trapping
efficiency for calcein and visual observation of MEL-NDLPC mixed self-assemblies.
Interestingly, the average size of the vesicles at the composition of qLpC
= 0.3 was
633.2 nm, which is remarkably small compared to other compositions (Fig. 9.1 1).
Moreover, the obtained vesicle solution was slightly bluish and turbid and kept its
dispersion stability at 25°C for more than 3 months. The asymmetric distribution
of MEL-A and DLPC in the two vesicle monolayers caused by the difference in
geometrical structures is very likely to have changed their self-assembled structure
from a sponge phase (L,) to a thermodynamically stable vesicle (La,) (Imura et al.,
2005).
Recently, the self-assemblingproperties of MEL-A and -Bhave been characterized
in more detail using the fluorescence probe method, DLS analysis and synchrotron
248 0 D. Kitamoto et al.
FF-fEM
Fig. 9.1 1. Thermodynamic vesicle formation from mannosylerythritol lipid-A and a-D-dilauroyl-
phosphatidylcholine.
small/wide- angle X-ray scattering (SMWAXS) (Imura et al., 2006). Figure 9.12
shows the schematic diagram for the self-assembled nanostructures prepared from
MEL-A and -B in aqueous solutions.
Surfactants generally form spherical micelles above CMC, and the radius of micelle
is several nanometers (almost equal to its hydrophobic chain length). Surprisingly,
both MEL-A and -B exhibit excellent self-assembling properties at extremely low
concentrations; they self-assemble into large unilamellar vesicles ( L W ) of diameter
lager than 160 nm, just above their critical aggregation concentration (CAC,). The
CAC, value is 4.0 x l o 6 M for MEL-A and 6.0 x lo4 M for MEL-B, respectively.
Moreover, the self-assembled structure of MEL-A over CAC,, of 2.0 x l o 5 M
drastically changes into sponge structures (L,) composed of randomly connected
bilayers network as indicated above. The average water channel diameter (6) of the
sponge structure is approximately 100 nm and is relatively large compared with those
obtained from “synthetic” surfactant multi-component systems (24 nm), that is,
cetylpyridinium/hexanoI/dextrose/brinesystem (Imura et al., 2006).
Meanwhile, MEL-B which has a hydroxyl group at the C-4’ position on mannose
instead of an acetyl group gives only one CAC; the self-assembled structure of MEL-B
Self-assembling Propertiesof Glycolipid Biosurfactants and Their Functional Developments 0 249
MEL-B @EL-A
Fig. 9.13. Formation of lyotropic liquid crystals; water penetration scans of mannosylerythritol lipid-A
and -B.
Sponge
(L3)
E3 Bicontinuous
c cubic
E
; (V,)
Lamella
(La)
MEL-A concentration (wt%)
Fig. 9.14. Temperature dependence of the binary phase diagram of mannosylerythritol lipid-A/water
system.
L;, sponge phase, V;, bicontinuous cubic phase, La; lamellar phase, FI; isotropic phase.
80
W
65
c
3
f 50
Q
5 35
I-
20
0 20 40 60 80 100
MEL -B concentration (wt%)
Fig. 9.1 5. a) Temperature dependence of the binary phase diagram of rnannosylerythritol lipid-8 (R
tsukubaensis)/water system.
La; lamellar phase, FI; isotropic phase.
b) Confocal laser scanning micrograph of mannosylerythritol lipid-6 (f?tsukubaensis) vesicles (1
wt% in water).
Bar length is 5 pm.
252 0 D. Kitamoto et al.
Sophorolipid
Vesicle forming glycolipids often give ribbon and tube structures. Several groups
reported formation of ribbon, tube, myelin figure and gel structures, using acyclic
single chain glycolipids (Kitamoto et al., 2005; Shimizu et al., 2005). The resultant
morphology depends on the carbohydrate headgroup structure. From the analysis
of crystallographic structure and other spectral data, the hydrogen bonding of the
linkage amide bond is shown to stabilize the formation of the unidirectional three-
dimensional structures. A ribbon structure is known to often be a precursor of
tubules.
As described previously, acidic sophorolipid (Fig. 9.2) molecules represent a novel
type of asymmetrical bolaamphiphile due to their unique structural features that
include an asymmetrical polar head size (disaccharide vs. carboxylic acid), a nicked
hydrophobic core (oleic acid), and a non-amide polar-nonpolar linkage. Zhou et al.
(2004)recently investigated the supramolecular structures of self-assembled aggregates
of the BS using light microscopy, DLS, SAXS/W’S, and FT-IR spectroscopy.
Figure 9.16 shows the self-assembling manner of sophorolipids under different
conditions. Interestingly, acidic sophorolipids self-assemble into giant twisted and
helical ribbons of 5-11 pm width and several hundreds of micrometers length in
acidic conditions (pH c 5.5). An increase in the solution pH decreases the yield of
solidlike ribbon products. It also slows the formation of giant ribbons, and increases
the helicity and entanglements of the giant ribbons. The sophorolipid molecules form
highly crystallized lamellar structures at room temperature but with a much shorter
long period of 2.78 nm compared to oleic acid molecules. This lamellar spacing
excludes the possibility that a similar bilayer molecular packing is formed in which
the sophorolipid carboxylic acid groups form hydrogen-bond-stabilizing dimers.
The layered structures with a long period of 2.78 nm within the giant ribbons
are likely to be formed by interdigitated packing, which is stabilized by both the
strong hydrogen bonding between the intermolecular disaccharide headgroups
and the strong hydrophobic-hydrophobic interactions between the closely packed
hydrocarbon chains (Zhou et al., 2004). Neutralization of acidic sophorolipids with
dilute NaOH to pH >5.9 produces clear solutions with the formation of short range
ordered micelles. The self-assembly of the sodium salt molecules strongly depends on
the solution concentration. At dilute concentrations, the size of the micellar aggregates
increases with increasing concentration. When the concentration of the sodium salt
is above 1.O mg/mL, narrowly distributed large micellar aggregates with a constant
hydrodynamic radius of about 100 nm are formed.
These data clearly indicate that the supramolecular assemblies arise from the
unique structure of both hydrophobic and hydrophobic parts, which are elegantly
designed and refined by yeasts through a long period of evolution.
Rbamnolipid
Rhamnolipids also self-assemble into variety of structures, and their assembled
structures drastically change with slight variation in the headgroup. Because of the
Self-assembling Propertiesof Glycolipid Biosurfactants and Their Functional Developments 0 253
lnterdigitated
lamellar packing
Acidic sophorolipid
Carboxylic group
Acidic conditions
( < pH 3.6)
Sophrose group neutralization
With NaOH
Giant ribbons
Higher concentrations
(> .OImglmL)
A few hundred
micrometers
5 to 10 pm
Large micelles
(100 nm)
Helical ribbons
Rhamnolipid
Carboxylic group
Rhamnosyl group
Vesicles
pH 4.3-5.8
Micelles
pH > 6.8
Here the authors would like to focus special attention on the application of BS in
biomedical area.
Compound
Microorqanism MEL-Aa MEL-Ba SLb RLc SEafd Span20a
MIC (mg/L)
Gram-positives
Arthrobacter oxidans -* - - 16 - -
Azo tobacter chroococcum - 1.95 - - -
Bacillus subtilis 6.2 25 0.12 64 800 400
Micrococcus luteus 3.1 12.5 0.48 32 400 400
Mycobacrerim phlei - - - 16 - -
Mycobacterium rhodochrous 25 25 - - >800 400
Mycobacterium rubrum - - 0.12 - - -
StaDhv/ococcusaureus 12.5 25 >800 128 >800 ~ ~
-
~~~
Staphylococcus edipermidis - - - 8 - -
Streptococcus faecalis - - - 64 - -
Gram-negatives
Alcaligenes faecalis - - - 32 - -
Borderella bronchiseotica - - - 128 - -
Escherichia coli >400 >400 7.8 32 >800 >800
Proteus vulgaris - - >31.3 - - -
Pseudomonas aeruginosa >400 >400 7.8 >256 >800 S O 0
Pseudomonas rivoflavina 12.5 25 - - >800 S O 0
STL-1 STL-1
induce neurite outgrowth even in the presence of an anti-nerve growth factor (NGF)
receptor antibody that obstructs NGF action. These results indicate that MEL and
NGF induce neurite outgrowth from PC-12 cells by different signal transduction
pathways (Wakamatsu et al., 2001).
MEL-A has been recently demonstrated to inhibit the growth of mouse melanoma
B 16 cells in a dose-dependent manner. Exposure of B 16 cells to the lipid (10 pM)
causes the condensation of chromatin, DNA fragmentation, and sub-GI arrest, all of
which are hallmarks of cells that are undergoing apoptosis. The lipid treatment also
enhances expression of PKC,, suggesting that the lipid triggers the differentiation of
the cells through a signal pathway that involves PKC, (Zhao et al., 2001).
It is truly surprising that many glycolipid BS show certain activities against various
organisms ranging from prokaryotes to mammalians. The complicated but naturally
engineered structures of the BS allow them to display versatility over conventional
chemical surfactants. Therefore, the functional development of the BS should also be
carried out from the viewpoints of biochemistry and molecular biology.
Imm unoligan ds
A large variety of glycolipids can be found in all species from bacteria to mammals as
constituents of cell membranes (Kitamoto et al., 2005), where they are positioned to
interact with extracellular signal transducers such as immunoglobulins, lectins, and
enzymes or receptors on the surfaces of other cells. For example, glycosphingolipids,
Self-assembling Propertiesof Glycolipid Biosurfactants and Their Functional Developments 0 259
120
n 100
.-caJ,
8
80
sPI
0
0 2 4 6 8 10 12
IgG solution concentration (mg / mi)
Fig. 9.19. Binding of human immunoglobulin G to MEL-polymer composite. The polymer composite
(0.33f 0.08 g) bearing MEL-A (7.1 pmol/g composite) was suspended in 50 mM phosphate buffer (pH
4.6, 1 M Na,SO, ) in a polypropylene tube. Different amounts of human immunoglobulin G were added
to the tube, and then the tube was incubated for 1 hr. Ka,apparent binding constant.
MEL-A monolayer was estimated to be the Fab region. Imura et al. (2007b) also
demonstrated a large amount of HIgG and HIgM bind to the monolayer with a
markedly high density using atomic force microscopy.
Ito et al. (2007) recently reported kinetic studies on the interactions between the
monolayers of MEL (MEL-A, -B, and -C) and various classes of immunoglobulins
(HIgG, IgA, and IgM) using SPR. Interestingly, the monolayer of MEL-A gives high
affinity (K, = 1.7 x 104 M) toward HIgG, while those of MEL-B or MEL-C give
little affinity. The MEL-A monolayer also gives high affinity toward HIgA (K, = 2.4
x l o 7M) and HIgM (K, = 2.2 x l o 7M). Interestingly, the binding manner between
MEL-A and these immunoglobulins was estimated as not being the “monovalent
Self-assembling Propertiesof Glycolipid Biosurfactants and Their Functional Developments0 261
Lectins
Immunoglobulins
MEL-A
self-assembled
monolayer .Octadecanethiol (C1
HPA chip
(Biacore)
800
n
3
QC 600 MEL-A
.-c
c
3
400
0
3
2 200
Protein-A
mode” but the “bivalent mode.” Figure 9.21 shows the images for the binding of
these immunoglobulins to the surface of the MEL-A monolayer. These results clearly
demonstrate that the yeast glycolipid monolayers would be useful as noble affinity
ligand system for various immunoglobulins.
MEL-A shows binding affinity not only towards immunoglobulins but also
towards lectins, which are one of the most important signal transducers (Konishi
et al., 2007). O n SPR (Fig. 9.20), the monolayer of MEL-A exhibits high binding
affinity to Concanavalin A (Cod)and Maackia amurensis lectin-I (MAL-I). The
observed affinity constants for C o d and MAL-I were estimated to be 9.48 f 1.31
x 106and 3.13 f 0.274 x 10‘ Me’, respectively; the value was comparable to that of
a-Man-( 1-3)-[a-Man( l-G)]-Man, which is the most specific probe to Cod.
More significantly, a-methyl-D-mannopyranoside (1 mM) exhibited no binding
inhibition between MEL-A and C o d . MEL-A is thus likely to self-assemble to give
a high affinity surface, where C o d binds to the hydrophilic headgroup in a different
262 0 D. Kitamoto et al.
Fig. 9.21. Tapping mode of atomic force microscopy images of different immunoglobulins bound to
the self-assembled monolayers of mannosylerythritol lipid-A (MEL-A). IgG, immunoglobulin G; DPPC,
a-dipalmitoyl-phosphatidylcholine.
Gene Carriers
Gene delivery across cell membranes into the nucleus, that is, gene transfection,
is a fundamental technology not only for bioscience, but also for the clinical gene
therapy of generic and acquired diseases like cancer. The success of gene therapy, in
particular, is highly dependent on the development of transfection vectors that are
safe and efficient. Although the most efficient methods for gene transfection involve
the use of viral vectors, there are still arguments about risks regarding propagation
and immunogenicity. A variety of nonviral gene-delivery systems have thus been
investigated (Kawakami et al., 2008).
Among nonviral vectors hitherto reported, “cationic liposomes” are one of the
most efficient vectors for the delivery of plasmid D N A and antisense oligonucleotides
into mammalian cells (Dass & Choong, 2006; Nakanishi, 2003). However, cationic
liposomes alone nonspecifically interact with cellular membranes by electrostatic
interactions; targeted gene transfection systems have been strongly desired. Recently,
glycolipids have received much attention and frequently been employed as key
materials for targeted gene delivery using liposomes, providing liposomes with
enhanced colloidal stability in blood circulation, and specific affinity towards targeting
Self-assembling Propertiesof Glycolipid Biosurfactants and Their Functional Developments0 263
Cationic liposornes
Cationic cholesteKEi
t Gene (DNA)
Lipowme-DNA complexes
efficiently bind to cell surface
High efficiency
70-foM higher than with
convsntind liposwnes
Gene expression
Fig. 9.22. Scheme of gene delivery into mammalian cells using liposomes prepared from cationic
cholesterol and mannosylerythritol lipid-A.
nonviral vectors. Together with the unique mechanism for the gene transfection,
glycolipid BS are a critical lipid formulation of the desired vectors, and will facilitate
the development of novel gene delivery systems in terms of efficiency, time, and
toxicity.
Conclusions
As described in the above sections, BS show unique properties compared to their
chemical counterparts. The numerous advantages of BS have prompted applications
not only in the food, cosmetic, and pharmaceutical industries but in environmental
protection and energy-saving technology as well. Among known BS, glycolipid types
are the most promising, due to high productivity from renewable resources and
versatile biochemical properties.
Development of nanotechnology requires nanoscale materials, which should be
easy to prepare and handle. To this end, the molecular self-assembly process is one of
the most promising routes for creating a new class of nanomaterials (Shimizu et al.,
2005; Ariga et al., 2007). ‘There have been a lot of natural and synthetic amphiphiles
that are able to form self-assembled nanostructures. However, the advantages of
using glycolipid BS for novel nanomaterials are their ability to “spontaneously” form
molecular assemblies, as well as their environmentally friendly features.
During the past decade, the research and development of glycolipids has
undoubtedly rushed into a new era. Glycolipid BS display versatile performance over
that attained by conventional chemical surfactants. In addition, recent advances in
molecular biology and biotechnology have enabled processes that drastically improve
the productivity of the BS and control their chemical structures (Mukherjee at al.,
2006). Total syntheses of mannosylerythritol lipid (Crich et al., 2002), sophorolipids
(Furstner et al., 2000), and rhamnolipid (Duynstee et al., 1998) have already been
attained. These synthetic approaches will also facilitate the functional development of
glycolipid BS along with the progress of biotechnology.
266 0 D. Kitamotoet al.
Acknowledgments
The authors would like to thank Professor Yoichi Nakatani at the University of Louis
Pasteur (Strasbourg, France) and Professor Mamoru Nakanishi at Aichi Gakuin
University (Nagoya, Japan) as excellent collaborators. We also thank Dr. Masaru
Kitagawa of TOYOBO Co., Ltd. (Osaka, Japan) for providing information on the
use of glycolipid BS in cosmetics.
Fermentation orocesses
Biobased materials
Fig. 9.23. Self-assembled nanostructuresof glycolipids biosurfactants and their potential applications.
Self-assembling Propertiesof Glycolipid Biosurfactants and Their Functional Developments0 267
References
Abalos, A,; A. Pinazo; M.R. Infante; M. Casals; F. Garcia; A. Manresa. Physicochemid and
Antimicrobial Properties of New Rhamnolipids Produced by Pseuhmonas aeruginosa AT10 from
Soybean Oil Refinery Wastes. L a n p u i r . 2001,17,1367-1371.
Ariga, K.; T. Nakanishi; J.I? Hill. Self-Assembled Microstructures of Functional Molecules. Curr.
Opin. Colloid Interface Sci. 2007,12,106120.
Banat, I.M.; R.S. Makkar; S.S. Cameotra. Potential Commercial Applications of Microbial Surfac-
tants. Appl. MicrobioL Biotechnol. 2000,53,495-508.
Cameotra, S.S.; R.S. Makkar. Recent Applications of Biosurfactants as Biological and Immuno-
logical Molecules. Curr. Opin. Microbiol. 2004,7,262-266.
Crich, D.; M.A. de la Mora; R. Cruz. Synthesis of the Mannosylerythritol Lipid MEL A; Confir-
mation of the Configuration of the Meso-Erychritol Moiety. Gtrahedron. 2002,58,35-44.
Dass, C.R.; P.M.F. Choong. Selective Gene Delivery for Cancer Therapy Using Cationic Lipo-
somes: In Vivo Proof of Applicability.J. Control. Rehase. 2006,113,155-163.
De Kruif, C.G.; E Weinbreck; R. de Vries. Complex Coacervation of Proteins and Anionic Poly-
saccharides. Cum Opin. Colloid Interface Sci. 2004,9,340-349.
Dembitsky, V.M. Astonishing Diversity of Natural Surfactants. Lipid. 2005,39,933-953.
Dobereiner, H.-G. Properties of Giant Vesicles. Cum Opin. ColbidInterfce. Sci. 2000,5,
256263.
Duynstee, H.; M.J. van Vliet; G.A. van der Marel; J.H. van Boom. An Efficient Synthesis of
(R)-3-{(R)-3-[2-O-(a-L-Rhamnosyl)- a-L-Rhamnosyl] Oxydecanoyl}Oxydecanoic Acid, a
Rhamnolipid from Pseudomonas aeruginosa. Eur. J. Org. Chem., 1998,303-307.
Evans, S.V.; C.R. MacKenzie. Characterization of Protein-Glycolipid Recognition at the Mem-
brane Bi1ayer.J. Mol. Recognit. 1999,12,155-168.
Fukuoka, T.; T. Morita; M. Konishi; T. Imura; D. Kitamoto. Characterization of New Types of
Mannosylerythritol Lipids as Biosurfactants Produced from Soybean Oil by a Basidiomycetous
Yeast, Pseudozyma shanxiensis. J. Oleo Sci. 2007% 56,435-442.
Fukuoka, T.; T. Morita; M. Konishi; T. Imura; D. Kitamoto. A BasidiomycetousYeast, Pseudozyma
tsukubaensis, Efficiently Produces a Glycolipid Biosurfactant; Identification as a New Diastere-
omer of Conventional Mannosylerythritol Lipid-8. Carbohydrate Res. 2008,343,555-560.
Fukuoka, T.; T. Morita; M. Konishi; T. Imura; D. Kitamoto. Development of Highly Functional
Biosurfactants Produced by Yeasts. Home. Pers. Care Today. 2007b,1,74-76.
Fiirstner, A.; K. Radkowski;J. Grabowski; C. Wirtz; R. Mynott. Ring-Closing Alkyne Me-
tathesis. Application to the Total Synthesis of Sophorolipid Lactone.J. Org. Chem. 2000,65,
8758-8762.
Goodby, J.W.; V. Gortz; S.J. Cowling; G. Mackenzie; I? Martin; D. Plusquellec;T. Benvegnu; I?
Boullanger; D. Lafont; Y.Queneau; S. Chamberte; J. Fitremannf. Thermotropic Liquid Crystal-
line Glycolipids. Chem. SOC.Rev. 2007,36,1845-2128.
Hakomori, S. The Glycosynapse.Proc. NatL Acaa! Sci. USA.2002,99,225-232.
Hashida, M.; M. Nishikawa; F. Yamashita; Y. Tikakura. Cell-Specific Delivery of Genes with
Glycosylated Carriers. Adv. Drug Delivery Rev. 2001,52,187-196.
268 0 0.Kitarnoto et at.
Introduction
Sucrose fatty acid esters (SEs) are recognized as very functional emulsifiers. They
are mainly used in the food industry because they are safe and environmentally
friendly. Cosmetic, pharmaceutical, and industrial uses are also growing steadily. The
environmental “friendliness”is a consequence of the naturalness of the raw materials,
namely sucrose from sugar cane and fatty acids derived from vegetable fats.
The production of SEs was first commercialized by Dai-Nippon Sugar
Manufacturing Co. Ltd. in Japan in 1959, and the business was subsequently
developed by Mitsubishi Chemical Co. and Dai-Ichi Kogyo Seiyaku Co., both also
Japanese companies. These two companies dominate the worldwide manufacture of
SEs, and the total production capacity was estimated as more than 5500 tonnes in
2007.
This chapter describes the application of commercial SE products manufactured
by Mitsubishi-Kagaku Foods Corporation (Tokyo, Japan) with the brand name
Ryoto’ Sugar Ester. Table 10.1 shows the list of them and their ester compositions.
275
276 0 N. Otomo
Table 10.1, Typical Grades of Ryoto Sugar Ester Manufactured by Mitsubishi Kagaku-
Foods CorDoration
Auuroximate ester comuosition, wt%
Fattyacid Type HLB mono- di- tri- tetra- and higher
Lauric (C12) L-595 5 30 40 25 5
L-1695 16 80 18 2 0
Mvristic(C14) M-1695 16 83 15 2 0
Palmitic (C16) P-170 1 0 3 10 87
P-1670 16 80 18 2 0
Stearic (C18) 5-170 1 0 5 10 85
5-370 3 20 30 30 20
5-570 5 30 35 25 10
5-1170 11 55 30 10 5
5-1670 16 77 20 3 0
Behenic (C22) 8-370 3 15 25 25 35
Oleic IC18:l) 0-170 1 0 5 10 85
0-1570 15 75 22 3 0
Erucic (C22:l) ER-290 2 1 7 20 72
Source: Mitsubishi Chemical, unpublished data.
0 C6
C6'
P -D-Fructofuransy1 a -D-glucopyranoside 6- octadecanoate
Fig. 10.1. Chemical structure of sucrose monostearate.
Basic Propertiesof Sucrose Fatty Acid Esters and Their Applications 0 277
HLB
16
Mono
11 Di
Tri
7 Tetra
Penta
3 Hexa
Hepta
1 Octa
0% 50% 100%
Fig. 10.2. Relationship between HLB and ester composition of sucrose stearates (Mitsubishi Kagaku
Foods Corp.,Technical information brochure, 2000).
and contain a small percentage of filly esterified octaester. Fig. 10.3 shows how the
combination of different esterified fatty acids and HLBs creates esters suitable for
different applications.
Hydrophilicity is derived from various groups (e.g., -OH, -0-,and -COOH),
and is affected by many environmental factors, like temperature, salt concentration,
pH, and so on. Also, hydrophilicity is affected by these factors to different extents.
For example, hydrophilicity of the -OH group (SE) is not affected by temperature,
while that of the -0-group (polyoxyethylene) becomes lower at a high temperature.
Historically, the HLB system was developed for linear alkyl ethoxylates. It is a useful
index for an approximate classification of these emulsifiers. However, if one mixes an
SE with an HLB of 16 and a linear alkyl ethoxylate (AE) with an HLB of 10 at the
ratio of 1:I (w/w), the behavior of the surfactant mixture would not necessarily reflect
that of a surfactant with an HLB value of 13.
In the production of SEs, the use of solvents such as dimethylformamide (DMF)
or dimethylsulfoxide (DMSO) is necessary to mix the raw materials uniformly. Since
both solvents have high boiling points, very sophisticated purification processes are
required to reduce the content of solvent to conform with the F A O M 0 (Food and
Agriculture Organization/World Health Organization) limits (DMF: not more than
1 mg/kg; DMSO: not more than 2 mg/kg).
The primary -OH group on C6 of the glucose moiety is the first to be esterified.
In monoesters, more than 70 mol% of ester is that of C6. After the C6 hydroxyl
278 0 N. Otomo
HLB
Fig. 10.3. Application map of sucrose fatty-acid esters. (Mitsubishi-Kagaku Foods Corp., Technical
information brochure, 2000).
group of the glucose moiety, the C6’ and C1’ hydroxyls of the fructose group are the
next to be esterified. Thus, sucrose diesters are composed mainly of C6 and CG’-ester
linkages. Because of the chemical structure, pure diester is easily crystallized. The
melting point of sucrose distearate is over 100°C (Fig. 10.4; Otomo, 2003). Food-
process temperatures are rarely above this temperature, except for heat sterilization
processes. Therefore, the pure ester, having such a high melting point, is not suitable
for use in foods. However, commercial sucrose stearate is a mixture of esters with
different DE (see Table 10.1). The resultant melting point is around 55-65”C, and
therefore is suitable for applications in food processing.
+
o 3.0
w
3 2.0
E
1.o
Fig. 10.4. Differential scanning calorimetry (DSC) thermogram of sucrose stearates: (a)puredistearate,
and (b)monostearate/distearate l/lg/g.Temperature program: 0 to 150°C, 5"C/min; sample weight of
2mg. Apparatus: Seiko Instruments Inc. EXSTAR 6000DSC. Otomo (2003)(Reprintedwith permission
from the Japan Society for Food Engineering).
Fig. 10.5. Schematic illustration of a Langmuir film balance with a Wilhelmy plate electrobalance
measuring the surface pressure,x , and barriers for controlling the available surface area, A.
shapes of these esters. The relationship between the average DE and molecular area is
shown in Fig. 10.7 (Otomo, 2003).
The results are shown in Fig. 10.6. In this figure, the top of each structure is
the hydrophilic part. As shown in Fig. 10.6, sucrose monoester is conical, and the
di- and tri-esters are cylindrical in shape. As mentioned above, commercial SEs are
a mixture of esters with different DE. SEs with a high content of monoester tend to
form micelle structures. These SEs are used for oil in water ( O W ) emulsions, and
solubilization of oil in water.
SE mixtures containing mono-, di-, and triesters tend to form lamellar structures.
A lamellar structure is very important for the stability of 0" emulsions. SEs with an
average DE around 2.7 are most suitable for the formation of lamellar structures. The
280 0 N. Otomo
monolaurate monooleate
Fig. 10.6. Schematic illustration of molecular shapes of sucrose esters at 293 K.The top of each
structure is the hydrophile, sucrose. Otomo (2003)(Reprinted with permission from the Japan Society
for Food Engineering).
thickness of the internal water layer of multilayer lamellar systems can be increased
by adding a small amount (5-10 wt% to total SE) of ionic surfactant. Fig. 10.8 is a
schematic representation of the effect on the internal water layer of adding an ionic
surfactant (e.g., monoglyceride citrate or succinyl monoglyceride) to a system using
sucrose stearate as the principal emulsifier. Using SAXS (small-angle x-ray scattering)
analysis, we observed the expansion of the water layer to 25 nm (Mitsubishi Chemical
Corp., unpublished data). In this test, we used S-1170 (seeTable 10.1), and the total
emulsifier concentration was 30% in water. The test was done at 25 and 60°C by
using X-ray apparatus of 5OkV, 200 mA, Ni-filter (Anton-Paar) with a Kratky camera
U-slit.
This expansion of the water layer helps to prevent coalescence of the oil droplets.
This is comparable to the thickness of protein film formed when using caseinate. That
is why ionic surfactants, especially anionic, play a significant role in emulsion stability.
SEs with an average DE higher than 4 tend to form reverse micelle structures.
Surface Activity of Sucrose Fatty Acid Esters
The sucrose moiety of SEs is the key factor in their unique interfacial properties. Fig.
10.9 shows the surface-tension measurement of sucrose monopalmitate (Takagi et al.,
sucrose stearate
only
sucrose stearate +
anionic surfactant
Fig. 10.8. Schematic illustration of the expansion of internal water layer thickness in a multilamellar
system by the addition of an anionic surfactant to an aqueous sucrose stearate solution.
282 0 N. Otomo
1995). This shows the quite unique behavior of SEs when compared with conventional
linear AEs (Ueno et a]., 1981). The surface tension of sucrose-monopalmitate
solutions equals that of pure water at a low concentration, and then drops sharply
near the critical micelle concentration (CMC), whereas with AEs, the surface tension
decreases gradually as the concentration increases.
This behavior means that in the region between a and b of Fig. 10.9, SE mol-
ecules are not adsorbed at the interface. It is more thermodynamically stable for the
SE molecules to exist as monomers rather than as components of aggregates. In other
words, SE molecules have a strong affinity to the water structure. Above point b, SE
molecules begin to form pre-micelles, lose their affinity to water structure quickly,
and begin to be adsorbed at the interface. This explains the strong hydrophilicity of
SE molecules that leads to the high efficiency of adsorption at interfaces.
We also found this property of high adsorption efficiency in water-in-oil (W/O)
emulsion formation (Fig. 10.10, Otomo et al., 2000). We made a W / O high internal
phase emulsion (HIPE), which had an extremely high-volume fraction of dispersed
water, by using a hydrophobic sucrose erucate in the oil phase (i.e., ER-290) (see
Table 10.1). Also a small amount of a hydrophilic sucrose stearate S-1670 (see Table
10.1) was added to the water phase. This combination of two types of emulsifiers is
especially useful to improve the stability at lower temperatures. We assume that the
combination makes a molecular assembly with a stiffer interfacial membrane. This
synergistic effect is because sucrose stearate is adsorbed at the interface very efficiently.
When we used polyoxyethylene sorbitan monolaurate (Tween 20) in place of the
sucrose stearate , the emulsion became very unstable and collapsed in the course of
preparation. We observed the same instability by using polyoxyethylene sorbitan
monopalmitate (Tween 40). The hydrophilicity of the Tween 20 and 40 decreases at
a higher temperature in aqueous solutions, and the surfactant phase separates as an
oily liquid from water due to the dehydration of the ether group. This temperature
is called the cloud point. This means ethoxylates can dissolve in the oil phase, and
explains why the polyoxyethylene sorbitan fatty acid ester did not stabilize the
interfacial membrane.
This strong hydrophilicity remains, even in oil- soluble SEs having DE of around
5. Oil-soluble SEs can be dissolved in oil, but if an O/W interface is present, the SEs
will readily orientate at the interface due to the lipophobicity of the remaining -OH
groups on the sucrose moiety.
Lipophobicity is a relatively new concept when considering surfactant properties.
A surfactant is considered efficient when it has a low saturation concentration of its
monomeric form in water or oil solvents. Hydrophilicity is strongly dependent on
alkyl chain length, while lipophobicity is mainly related to the length and type of
hydrophile group. Fukuda and Shinoda (1999; Fukuda, 2005) tried to measure the
lipophobicity of different surfactants. They concluded that the lipophobicity of each
-OCH,CH(OH)CH,- unit (one secondary hydroxyl group) corresponds to about 5.6
times greater than that of -OCH,CH,- (oxyethylene group). Because oil-soluble SEs
Basic Properties of Sucrose Fatty Acid Esters and Their Applications 0 283
80
-b 60
20
0
-8 -7 -6 -5 -4 -3
logC(mo1 L-l)
Fig. 10.9. Measurement of surface tension, y, for (0)sucrose monopalmitate and (4octaethyleneglycol
monotetradecyl ether as a function of concentration at 303.1 K. Positionsa and bare referred in the
text.Takagi et al. (1995) and Ueno et al. (1981) (Reprintedwith permission from Japan Oil Chemists’
Society and Springer).
Fig. 10.10. Stability test for water in oil emulsions formed by the system water/oil-soluble surfactant/
water soluble surfactanthqualene 89.9/1.0/0.1/9.0 w/w/w/w at 5°C. From left to right: ER-290 1% only,
+ +
ER-290 1% 5-570 0.1%; ER-290 P-1670 0.1%; ER-290 1% +Tween 20 (polyoxyethylene(20) sorbitan
monolaurate)0.1%, respectively. Stored at 5°C for 5 weeks. ER-290,5-570 and P-1670 are sucros ester
products identified inTable 10.1. Otomo et at. (2000) (Reprinted with permission from the Society of
Cosmetic Chemists of Japan).
284 0 N. Otomo
have many hydroxyl groups, they are strongly lipophobic. Thus, they are very efficient
in many emulsion systems. Furthermore, the lipophobicity of SEs is not affected by
temperature change, because the hydration of the' hydroxyl group is much stronger
than that of an ether group.
Fig. 10.1 1 shows the temperature dependence of the interfacial tension between oil
and water (Otomo, 2007). In the case of glycerol monostearate (GMS), the molecule
is dissolved in the bulk oil phase at a high temperature due to the structural similarity
to the triglycerides, and thus interfacial tension is not low. As the temperature drops,
its solubility decreases and begins to adsorb at the interface.
The interfacial tension of sucrose pentastearate S- 170 and pentaoleate 0-170
(see Table 10.1) is not temperature-dependent because of the sugar esters' strong
lipophobicity. Fig. 10.12 shows the concentration dependence of interfacial tension
(Otomo, 2007). GMS (0.1%) is necessary to decrease the interfacial tension; but in
the case of sucrose pentastearate, 0.01% is sufficient for a temperature less than 20°C.
Again, this is due to the strong lipophobicity and high efficiency of SEs.
The reduction of interfacial tension in ONV emulsions has great importance in
food systems because proteins and emulsifier molecules show competitive adsorption
at the interface (Dickinson et al., 1993). Consequently, they influence emulsion
stability and rheology.
%
f
GMS:Glycerol monostearate
Y 20
0-170 : Sucrose pentaoleate
S-170 : Sucrose pentastearate
$8 15
5
b
s?
0 S-170
0 10 20 30 40 50
Temperature ["CI
Fig. 10.1 1.Temperature dependence of interfacial tension between canola oil and water For three
different nonionic surfactants. Emulsifier concentrations: 0.1% in oiLTemperature programming: 50°C -+
5°C by -O.Ol"C/s.
Apparatus: model K100MK2 surface tensiometer from Kruss, Helsinki, Finland. Otomo
(2007) (Reprintedwith permission from Science &Technology, Inc., Tokyo, Japan).
Basic Propertiesof Sucrose Fatty Acid Esters and Their Applications 0 285
s
h c
Blank 3 Blank
Y
E 20 uZE
C
,E I5
C
z
3 10
s
E
3
5
0 0
0 10 20 30 40 50 0 10 20 30 40 50
Temperature [TI Temperature [TI
Fig. 10.12. Concentration dependence of interfacial tension between canola oil and water for two
nonionic surfactants. Temperature programming and instrumentation given in Fig.lO.11. Otomo (2007)
(Reprinted with permission from Science &Technology, Inc., Tokyo, Japan).
sold in Japan, Korea, and other Asian countries. In winter, the cans are bought hot
from vending machines. In the vending machine, the canned coffee is held hot, about
55°C (131"F), until sold. This temperature is ideal for the growth of thermophilic
bacteria; also these beverages contain high levels of sugar, proteins, oils and other
nutrients, providing an ideal environment for bacterial growth (Fig. 10.13).
These canned beverages are sterilized under very severe conditions, such as 121"C
(250°F) for 30 - 40 minutes. Table 10.2 shows the heat resistance of the spores of
thermophilic bacteria. D-value is defined as the time necessary to reduce the number
of bacteria to one-tenth of the original value. In the example, the temperature is
121°C. For commercial sterilization, at least four times the D-value is required. Table
10.2 shows the sterilization times for six times the D-value for a range of microbial
organisms and spores. Some spores are extremely heat- resistant, and they can survive
the sterilization conditions. For example, if we use Moorefa thermoacetica as a target
bacterium of a canned food, it requires a heat treatment of 121°C (250°F) for 270
minutes for commercial sterilization. Such a long sterilization process results in bad-
quality food, poor production efficiency, and big energy consumption.
SE inhibits the germination process at a very low dosage. Suwa et al. (1989)
showed that sucrose palmitate was the most effective of the different fatty acid esters
(Fig. 10.14) (Otomo, 2007). Also, they showed that monoesters were more effective
than more highly substituted esters. In canned coffee, 300 to 600 ppm of Ryoto' SE
P-1670 (80% of monoester content) is the standard dosage. However, this depends on
the ingredients (fadoil and protein content), homogenization pressure, sterilization
conditions, and so on. In a pure buffer system, sucrose monopalmitate inhibits spore
germination at as low as 10 ppm.
286 0 N. Otomo
Sucrose monopalmitate
Without SE 500ppm
Fig. 10.13. Flat sour spoilage of milk coffee stored at 40°C for 2 months. UHT, ultra high temperature
sterilization, condition; 137°C for 60s. Inoculated with Geobacillus stearothermophilus spore (Mitsubishi
Chemical, Unpublished data).
Starch forms complexes with fatty acid esters such as monoglycerides. Similarly
SEs also form this type of complex with starch. Thus SE’s effectiveness against
bacterial spores is reduced in starch-containing foods. Recently, Sashida et al. (2007)
reported that a mixture of sucrose dicaprylate and tricaprylate (C8 fatty acid) had
high activity toward thermophilic bacterial spores, and the activity was not inhibited
by the presence of starch. We assume that di- and tricaprylate are more sterically
hindered than monopalmitate, and do not form complexes with starch.
Noteworthy is that this activity of SEs is, unlike that of sorbic-acid or benzoic-
acid esters, not bactericidal but is bacteriostatic. Sucrose monopalmitate inhibits the
germination process of the life cycle of spore-forming bacteria (Fig. 10.15). If the
spores are washed and transferred to an ester-free culture broth, they will germinate
and grow. Vegetative growth of these bacteria is only slightly affected by the ester.
Moriyama et al. (1996) tried to establish the mechanism of this activity. They found
that esters became bound to the spore coat; but this does not fully explain the mode
of action.
Basic Propertiesof Sucrose Fatty Acid Esters and Their Applications 0 287
L- 1695
m 250PPm
M- 1695 200ppm
P- 1695
S- 1695
0-1695
CONTROL] ,
0 5 10 15 20 25
Apparent D,,,.c value ( min.)
Fig. 10.14. Reduction of apparent D value of Moorela thermoacetico in milk coffee by sucrose fatty acid
esters. Formulation: whole milk powder: 1.896, skim milk powder: 1.696, coffee extract: 2.096, sucrose:
8.0%. P-1695,5-1695 and 0-1695 are laboratory scale products made with highly purified fatty acids
with monoester content approximately equal to 8096, with nomenclaturelisted inTable 10.1. Otomo
(2007) (Reprinted with permission from Science &Technology ,Inc., Tokyo, Japan).
Q;mination
Sporulation ( (23
*‘I-*‘,
+:7‘
&L(
--
J Outgrowth
Overbeek who developed it in the 1940s). In this figure, the x-axis represents the
distance between two droplets, and the y-axis is the electrostatic potential, where
“+” represents a repulsive force. The bold line is the resultant potential which is the
cumulative effect of the repulsive potential and the van der Waals attractive potential.
The resultant potentid is mostly negative (i.e., attractive over small distances), but
may be positive (repulsive) at intermediate distances, leading to a stable colloidal
system (Hiemenz, 1986).
Although sucrose monostearate is a nonionic emulsifier, it confers a negative
charge to particles and stabilizes the dispersion. SE forms a bilayer around calcium-
carbonate particles. Fig. 10.17 shows the results of an experiment that can help
illustrate the nature of this adsorption (Otomo, 2003). We added an equal volume
of hexane to aqueous dispersions containing various concentrations of SE, from 0.2
to 1.5%, and containing 10% of solid calcium carbonate. At a low level of SE, SE
molecules are adsorbed to form a monolayer. The hydrophobic “tails” faced the bulk
water phase. Thus the surface becomes hydrophobic, and particles migrate into the
hexane phase. As the concentration of SE increases, a double layer is formed around
the particles, and the surface becomes hydrophilic.
We found that the &-potential of untreated calcium-carbonate particles was
positive. After adding sucrose stearate, it turned negative. The subsequent addition of
anionic surfactants (e.g., succinyl monoglyceride and monoglyceride citrate) enhanced
the negative charge (Fig. 10.18). The difference in the absolute value of the potential
did not appear to be significant, but the effect on the stability of the dispersion was
remarkable (Fig. 10.19). This is because the &-potential is the electrical potential of
the sliding surfaces of the particles (Microtec, 2009), and does not reflect the charge
of particles directly. Assumably, the addition of anionic surfactants increased the
hydration radius of the particles leading to the much greater stability of the dispersion
(Otomo, 2007).
PL
P
(f
Electrostatic Repulsive 4
Potential
+ repulsive - The resultant potential +
Distance
Distance between
two dropleis
Fig. 10.16. An electrostatic repulsion force leads to stable colloidal suspensions (DLVOTheory).
Hexane layer
Aqueous layer
Fig. 10.1 7. Effect of sucrose ester content on the phase behavior of an aqueous suspension of calcium
carbonate particles (10 ml) mixed with hexane (10 ml) at room temperature. Concentrationof 5-1670
(sucrose monostearate; seeTable 10.1) in original aqueous solution were: 0.2,0.6,1.0, and 1.5 wt.%
from left to right. Otomo (2003) (Reprinted with permission from Japan Society for Food Engineering),
Basic Properties of Sucrose Fatty Acid Esters and Their Applications 0 29 1
h
>
E CHzO C-
::- (CHz)iCH3
v
I
CH-OH
I
CHz-O-$-(Oi Zh-COOH
0
Main component of
succinyl monoglyceride
-E
3.
30
.-paEg 15
aJ
.%
I
10
0
L S-16701 Succinyl Monoglyceride
$ 5 = 111 wlw
i
a
5 0
0 6 12 18 24 30 36 42 48 54 60 66
Storage period (days)
Fig. 10.19. Stability of an aqueous dispersion of calcium carbonate (10% w/w) in the presence of
Iwt. % total emulsifier during storage at 5°C. Emulsifiers described in Fig. 10.18. Apparent increase
of diameter was due to aggregation of calcium carbonate particles. Otomo (2007) (Reprinted with
permission from Science &Technology Inc., Tokyo, Japan).
292 0 N.Otomo
Yield value
_- Plastic viscositv
(The force required to initiate flow) (The force required to maintain constant flow)
300
Lecithin
- PGPR
Q 20 Lecithin
ER-290
ER-29U
i
'$ 100
PGPR
0 0
0 0.5 0 0.5 I
Added emulsifier % Added emulsifier %
Fig. 10.21.The effect of emulsifiers on the plastic viscosity and yield value of semi-sweet chocolate
(cocoa butter 32%, Lecithin 0.45%). ER-290: Sucrose pentaerucate (seeTable 10.1); PGPR: polyglycerin
polyricinoleate. (Mitsubishi Chemical, Unpublished data.)
Basic Propertiesof Sucrose Fatty Acid Esters and Their Applications 0 293
viscosity without a great decrease of yield value (see Fig. 10.21). This property is
greatly advantageous to chocolate manufacturers, especially when they want to make
a low-calorie chocolate. The practical difference between sucrose pentaerucate and
PGPR emulsifiers is shown in Fig. 10.22 (Otomo, 2007). Some works were produced
on the effect of emulsifiers on chocolate viscosity (Babin et al., 2005; Rousset et al.,
2002); however, the mechanism of emulsifiers is not fully understood. Consequently,
manufacturers should select emulsifiers according to their practical requirements.
Fig. 10.22. Chocolate coating of biscuits. Left chocolate with 0.5% of ER-290 (sucrose pentaerucate: see
Table 10.1). Right: chocolate with 0.5% of PGPR. Otomo (2007) (Reprinted with permission from Science
&Technology Inc.,Tokyo, Japan).
294 0 N. Otomo
Pharmaceutical Applications
SEs are also widely used in pharmaceutical applications in solid and liquid products.
Magnesium stearate is a widely used lubricant for tabletting. SEs are also effective
tabletting lubricants. In addition SEs are nonionic, and very little danger of them
reacting with medicinal chemicals is likely, unlike magnesium stearate. ‘Therefore, SEs
are increasingly used in tablet production as a lubricant and sometimes as a release-
controlling agent. Also, because these low HLB stearates are tasteless, one can use
them in chewable tablets (Shibata et al., 2002).
Basic Propertiesof Sucrose Fatty Acid Esters and Their Applications 0 295
Conclusions
In conclusion, I hope that I demonstrated that SEs are powerful emulsifiers with
a wide range of applications in foods and nonfood systems. The sucrose molecule,
having eight hydroxyl groups as an acyl acceptor, enables the production of a wide
range of compounds. These range from the monosubstituted esters that are extremely
hydrophilic and are used to produce oil in water, to highly substituted esters that
facilitate water-in-oil systems. In addition SEs have unpredictable properties such as
being bacteriostatic against spore-formingorganisms and having the ability to facilitate
the suspension of calcium-carbonate particles. I hope the information contained in
this chapter is useful to readers who work in many industrial fields.
.Donor Phase
D w Drug
Skin
Sampling Sampling
PBS PBS
Stirrc
Receptor Phase
Fig. 10.24. Flow-throughdiffusion cell used to investigate the penetration of skin.”PBS”refers to phos-
phate buffer saline. Okamoto (2001) (Reprinted with permission).
296 0 N. Otomo
1. 2
1
n
W
8 0.8
0.4
0. 2
0
0 1 2 3 4 5 6
Time (hr)
Fig. 10.25. Effect of sucrose esters on lidocaine penetration through a propylene glycol matrix at 37°C.
Conditions: lidocaine(5% in propylene glycol)with 1.5%emulsifier: (0) sucrose L-595 (seeTable10.1);
(A)L-1695; or (0)no emulsifier (control). Okamoto (2001)(Reprintedwith permission).
References
Arima, S.; T. Ueji; S. Ueno; A. Ogawa; K. Sato. Retardation of crystallization-induceddestabi-
lization of pmf-in-water emulsion with emulsifier additives. Colloids and Surfaces B 2007,55,
98-106.
Babin, H.; E. Dickinson; H. Chisholm; S. Beckertt. Interactions in dispersions of sugar particles
in food oils: influence of emulsifier. Food Hydrocolloids 2005, 19,513-520.
Dang, H.T.; 0. Oriana; D.G. Hayes. Feed batch addition of saccharide during saccharide-fatty
acid esterification catalyazed by immobilized lipase: time course, water activity, and kinetic
model./. Am. Oil Chem. SOC.2005,82(7), 487-493.
Dickinson, E.; G. Iveson; S. Tanai. Competitive adsorption in protein-stabilized emulsions
containing oil-soluble and water-soluble surfactants. Food Colloids and Polymer4 E. Dickinson, I?
Walstra, Eds.; The Royal Society of Chemistry: Cambridge, UK, 1993;p. 312.
Fukuda, M. 'The importance of lipophobicity in surfactants: methods for measuring lipophobicity
and its effect on the properties of two types of nonionic surfactant. J Colloid and Interface Sci.
2005,289,512-520.
Basic Propertiesof Sucrose Fatty Acid Esters and Their Applications 0 297
Fukuda M.; K. Shinoda. Importance of the lipophobicity of hydrophilic groups of surfactants and
evaluation. Nippon Yukagaku ffiishi, 1999,48(6), 587-594.
Hiemenz, I?C. Principles of Colloid and Surface Chemisty, 2”ded.; Marcel Dekker Inc.: New York,
1986.
Kaneko, N.; T. Horie; S. Ueno; J. Yano; T. Katsuragi; K.Sato. Impurity effects on crystallization
rates of n-hexadecane in oil-in-water emulsions. J. C y t a l Growth 1999 197,263-270.
Katsuragi, T; N. Kaneko; K. Sato. DSC study of effects of addtion of sucrose fatty acid esters on
oil phase crystallization of oil in water emulsion. Nippon Yukagaku ffiishi 2000 49(3), 255-262.
KSV Instruments USA (Monroe, CT, USA) “Langmuir and Langmuir-Blodgett Films: WHAT
and HOW? AN#107”; 2009, http://www.ksvinc.com/LB.htm
Lee, S.; M. Heuberger; I? Rousset; N.D. Spencer. Chocolate at a Sliding 1nterface.J. Food
Sci.2002 G7(7), 2712-2717.
Microtec Co., Ltd. (Chiba, Japan) “What is zeta potential?”, 2009, http://nition.com/en/prod-
ucdzeecom-s.htm
Moriyama, R.; K. Sugimoto; S. Miyata; T. Katsuragi; S. Makino. Antimicrobial action of sucrose
esters of fatty acids on bacterial spores.J. Antibact. Antt$ng. Agents 1996 24(I). 3-8.
Okamoto, H. Meijo University, Faculty of Pharmacy, Nagoya, Japan, Personal Communication,
2001.
Okamoto, H.; T. Sakai; K. Danjo. Effect of sucrose fatty acid esters on transdermal permeation of
lidocaine and ketoprofen. Biol. Pharm. Bull. 2005 28(9), 1689-1694.
Otomo, N. Self-organizedstructure of food emulsifiers and their applications. Nippon Shokuhin
Kougaku Kaishi 2003, 4(I), 1-9.
Otomo, N. Applications in food industry. Selertions andApplications of Surfactants; Science &
Technology Inc.: Tokyo, Japan, 2007; pp. 346-368.
Otomo, N.; Y. Watanabe; M. Tsuruta. A novel method for the stabilization of high internal phase
water-in-oil emulsions. J. SOC.Cosrnet. Chem. Jpn. 2000,34(3),,299-306.
Rousset, I?; I? Sellappan; I? Daoud. Effect of emulsifiers on surface properties of sucrose by inverse
gas chromatography.]. Chromatop A. 2002,9G9,97-101.
Sashida, R.; M. Tomida; K. Motoda; N. Matsumoto;T Kaji. Growth inhibitory effect of sucrose
fatty acid esters in starch containing foods. Kitnzume-Jihou 2007, 8G(10), 1162-1 163.
Sato, K. Hiroshima University, Faculty of Applied Biological Science, Hiroshima, Japan, Personal
Communication, 2007.
Servais, C.; H. Ranc; I.D. Roberts. Determination of chocolate viscosity.J. Terture Stuu‘’, 2004,
34,467-497.
Shibata, D.; Y. Shimada; Y. Yonaawa; H. Sunada; N. Otomo; K. Kasahara. Application and
evaluation of sucrose fatty acid esters as lubricants in the production of pharmaceuticals. Yaku-
zaigaku 2002,G2(4), ,133-1 45.
Suwa, N.; H. Machida; A. Nishimura. Effect of sucrose fatty acid esters on spoilage of canned
coffee caused by thermophilic spore forming bacteria. Mitsubishi k k e i R6.0 Rev. 1989 3(2),
2633.
Takagi, K.; S. Hirai; Y. Fujinuma; H. Fujimatsu; H. Usami; S. Ogasawara; Y. Kasahara; A. Yuki.
278 0 N. Otomo
Introduction
Biodegradable surfactants containing amide functional groups are of great interest
for applications requiring relatively stable emulsifiers because amide linkages are very
stable chemically and are not easily degraded in alkaline media. These emulsifiers are
nonionic and are completely biodegradable. They are a particularly attractive class
of compounds that are potential substitutes for emulsifiers derived from petroleum.
They are characterized by their skin tolerance, good biological degradability, and low
toxicity (Maag, 1984; Garrison, 1968).
The skin tolerance of surfactant formulations can be improved by adding fatty
acid alkanolamides.
Esters of amides have excellent antimicrobial activities. They are employed in
the manufacture of foods, cosmetics, pharmaceutical products, and a wide variety of
specialty chemicals that exploit the surface active properties of these materials. The
biodegradability and the relatively nonallergenic character of mono- and diethanol
amides of fatty acids depend on both the chemical nature of the substituents on the
nitrogen atom and the chain length of the fatty acid residue.
Table 1I. 1 summarizes the applications of alkanolamines whose constituent fatty
acid residues have different chain lengths. Amides with short-chain fatty acid groups
may be used as foam stabilizers, while other amines may be used as conditioning agents
or other purposes. Alkanolamines are frequently employed in formulating personal
care products to serve as foam boosters and viscosity enhancers in aqueous-based
cleansers. In addition, alkanolamines are utilized commercially in the manufacture
of textiles and in the formulation of industrial grade detergents. These compounds
perform a variety of functions including viscosity enhancement, foam stabilization,
emulsification, corrosion inhibition, and detergency.
Routes for the chemical synthesis of these compounds are not very specific and
require multiple steps for protection/deprotection of the hydroxyl groups (Maag, 1984).
9 299
300 0 c. Otero
0bjectives
This report describes the potential of enzymatic technology for selective N-acylation
of alkanolamine and dialkanolamine, namely ethanolamine and diethanolamine.
The strategies developed to accomplish this goal also consider and/or minimize
problems associated with the high viscosity and/or low solubility of the precursor
alkanolamine.
In this work we have employed different lipolytic enzymes as biocatalysts for
the acylation of alkanolamines with fatty acids, and explored the associated reaction
engineering to determine the optimum conditions for N-acylation ofethanolamine and
diethanolamine so as to accomplish these reactions in a short time while maximizing
both yield and selectivity. This work focuses on the lipase-catalyzed synthesis of
N-acylated alkanolamines of different fatty acids at low and high substrate loads.
The authors wanted to maximize the volumetric productivity of the process without
significant loss in yield or selectivity. To achieve this goal the authors employed high
substrate loads and explored the synthesis reactions using both commercial and tailor-
made immobilized lipases as biocatalysts.
Enzymatic Reactions
For trials involving a 1:1 mole ratio of reactants, 0.1 mmol of both the ethanolamine
(6.1 mg) and the fatty acid (17 mg of capric acid, 20 mg of lauric acid, 22.8 mg of
myristic acid, or 25.6 mg of palmitic acid) were mixed with 0.5 mL of solvent in
stoppered glass bottles. Precipitation of the reactant mixture was observed. Additional
experiments were conducted with different mole ratios of lauric acid to ethanolamine.
The solution was placed in a thermoconstanter orbital shaker (Stuart Scientific, Surrey,
England) and held at the reaction temperature for 5 min. Subsequently 25 mg of
Novozym 435 were added and the mixture was shaken at 200 rpm for the indicated
time in the corresponding experiment. Molar yields were calculated with respect to
the limiting reactant.
Screening of Biocatalysts
Of the native and immobilized lipases investigated (Novozym 435, Pseudomonas
sp.lcelite, C. antarctica A, Mucor mieheilduolite 568, Pseudomona sp., C. rugosa,
Penicillium rp., C. antarctica Alcelite, Penicillium sp./celite, C. antarctica Alsilica,
and Penicillium sp./silica ), only Novozym 435 produced an appreciable amount of
reaction within 24 hours. This enzyme is an immobilized C. antarctica B lipase that
was used in subsequent experiments.
n)
Fig. 11.1. Effect of the molar ratio of lauric acid to ethanolamine. Conditions:4PC, 0.5 ml of acetonitrile
and 25 mgof Novozym435.A).Excessofacid:6.1mg (0.1 mmo1)ethanolamine.B) Excessofethanolamine:
20 mg (0.1 mmol) lauric acid. A/E = amide-ester. Results expressed as percentage molar yield with
respect to the limiting reagent. Experimental values correspond to the mean of at least 2 independent
experiments. Experimental errors were between 0.4 and 4%.
Triethylamine (mmol)
Fig. 11.2. Inhibition by non reactive triethylamine. Conditions: 6.1 mg (0.1 mmol) ethanolamine, 20 mg
(0.1 mmol) lauric acid, 0.5 ml acetonitrile, 40% and 0.1-0.5 mmol triethylamine.
I SYNTHETIC ROUTES I
I 1
\r.
DIRECT ACYLATION TRANSACYLATION
ETHANOLAMINE ETHANOLAMINE +
+ FATTY ACID ETHYL-ESTER
F=-=?
Cprecipitat4
Fig. 11.3. Scheme of reaction systems corresponding to direct acylation of ethanolamine with a free
fatty acid and to the transacylation of the amide with the correspondingfatty acid ethyl ester.
306 0 C. Otero
case. The two consecutive acylation steps generate products of different polarities.
Consequently, the solvent could determine the product distribution in the final
mixture. Three solvents of different polarities [i.e., dioxane (Log P = -1.1), acetonitrile
(Log P = -0.33), and n-hexane (Log P = 3.5)] were compared. Log P i s the logarithm
of the partition coefficient of the indicated solvent in the octanol-water two-phase
system.
For this particular biotransformation the two synthetic routes differ not only with
respect to the type of the precursor reagent employed, but also with respect to the
nature of the intermediates formed from the original reagents. For direct acylation
in dioxane, acetonitrile, and hexane, rapid precipitation of a significant fraction of
the precursor reagents was observed when the liquid ethanolamine was added to
the solution of the fatty acid. Figure 11.4 depicts the IR spectra of lauric acid and
the corresponding ion pair formed with the amine. Interpretation of the infrared
spectrum of the mixture of the two precursor reagents indicates the formation of an
ionic complex between these reactants. The carboxylic acid band that appears in the
spectrum of lauric acid at 1700-1715 cm-' has disappeared from the spectrum of the
mixture, whereas a band corresponding to a carboxylate ion appeared at 1558-1560
cm-I.
In some solvents the solubility of the ion pair complex of reagents formed
when the reaction proceeds via the direct acylation route and the solubility of free
ethanolamine present when transacylation is the route of choice are very small (Table
11.3). The solubilities of the precursor reagents determined in this work permit one
to identify whether or not partial solubilization of the precursor reagents affects the
reaction rates associated with these two alternative routes to synthesize the desired
products. In polar solvents, such as acetonitrile and dioxane, free ethanolamine is 1-2
orders of magnitude more soluble than the corresponding ion complex of reagents
(Table 1 1.3, see scheme depicted in Fig. 11.3).
The selectivity of the reaction and the time required to achieve the maximum yield
of the desired intermediate product depend on both the solvent and the temperature
employed. More precisely, the selectivities of these reactions depend very much on
the solubility of the desired product (Table 11.4). The solubility of the N-acylated
alkanolamine is 1-2 orders of magnitude lower in n-hexane than in acetonitrile and
dioxane (Table 11.4). Hence, at 40°C in n-hexane continuous precipitation of this
intermediate product prevents subsequent acylation to the ester of alkylamine (Fig.
11.5).
Although the selectivity of the reaction is best in n-hexane, use of this solvent
suffers from two disadvantages, namely: the reaction rate is slow because of the low
solubility of the reagents, and elimination of hexane from the final product is more
difficult.
To increase the selectivity of reaction in polar solvents, one needs to achieve
maximum precipitation of the intermediate N-acylated alkanolamine. In polar
solvents the selectivity for formation of the N-acylated alkanolamine can be increased
Selective Enzymatic Synthesisof N-Acylated Alkanolamine Emulsifiers 0 307
Wavelength (ern-')
Absorbance
Wavelength (ern-')
Fig. 11.4. infrared spectra of (A) free lauric acid and (6) the ionic complex formed by ethanolamine and
lauric acid at 40°C.
308 0 c. Otero
Table 11.3. Solubility of Ethanolamine and the Ion-pair Formed by this Reagent with
Lauric Acid in Different Solvents at 40°C
Solubility
Product Solvent mg/mL mol/L
Ethanolamine Dioxane >250 4.20
Ethanolamine Acetonitrile >250 >4.20
Ethanolamine n- Hexane 1.25 0.02
Ion complex Dioxane 53 0.205
ion complex Acetonitrile 6.5 0.025
Ion complex n-Hexane 4.7 0.01 8
0
0 1 2 3 4
ea
0
0 1 2 3 4
Time (houls)
Fig. 11.5. Selective mono-acylation of ethanolamine in n-hexane at 400C. Conditions: 25 mg Novozym
435, 0.5 ml n-hexane, 0.1 mmol ethanolamine, equimolar ratio of reagents. Results expressed as
percentage molar yield.
Selective Enzymatic Synthesisof N-Acylated Alkanolamine Emulsifiers 0 309
by carrying out the reaction at relatively low temperatures (namely 2O-3O0C, Fig.
11A). However, this method markedly increases the time required to achieve high
levels of reaction. Hence, alternative reaction conditions were further explored in
efforts to increase the selectivity to the intermediate N-acylated alkanolamine in polar
solvents.
Solubility
40°C 60°C
Product Solvent mg/mL mol/L mg/mL mol/L
N-Caproylethanolamine Acetonitrile 77 0.356 - -
N- Lauroylethanolamine Dioxane >48 >0.200 >82 >0.340
N- Lauroylethanolamine Acetonitrile" 23 0.095 >80 >0.330
N-Lauroylethanolamine n-Hexane 1.o 0.004 1.5 0.006
N-Mvristovlethanolamine Acetonitrile 8.0 0.030 - -
N-palmitoy let hanolamine Acetonitrile 3.5 0.011 - -
LI Solubility a t 3OoC = 9.6 mg/mL (0.039 mol/L).
Tm (hours)
Tm (hours)
Fig. 11.6. Effect of the temperature in direct acylation ofethanolaminewith lauric acid in a polar solvent.
Conditions: 25 mg Novozym 435,6.1 mg (0.1 mmol) ethanolamine, equimolar ratio of reagents. Results
expressed as percentagemolar yield. 5°C (*), 10°C(W), 20°C (O), 30°C(A),40°C (A)~60°C (O).The y-axis
labels areexplained in Fig. 11.1.
Table 11.5. Comparative study of the two synthetic routes using different charges of substrates in equimolar concentrations of
reactants (ethanolamine and lauric acid acyl groups) and lipase at 40°C"
Total
Substrate Precursor Dissolvedb Amide Dissolved' ReactionTime Conversion Amide-Ester
Reaction Route Solvent (mol/L) (mole%) (mole %) (hr) (mole %) (mole %)
Direct Acylation Acetonitrile O.Zd - 20 24 95 0
0.2 12 50 4 96 6
0.4 6 24 4 93 4 wl
%
0.6 4 16 15 50 4 2
Transacylation Acetonitrile 0.2 100 50 1.5 97 4
a.
m
rn
0.6 100 16 1.5 96 0 2
.c
N
1.o 100 9 1.5 94 0
2.0 100 5 1.5 92 0
E.3
In
Y
The amount of C. anrafcrica lipase was increased in proportionto the amount of reagents employed (e.g., 25 mg biocatalyst for 0.2 mol/L of
substrate). 5
n
bMolepercent of ethanolamine or the corresponding ion-pair.
Mole percent of N-acyl ethanolamine dissolved in the reaction medium when the ethanolamine is completely transformed to this product. 2.
Reactionat 30°C. T
R
2
h
0 )
3
0
0)
3.
3
n
m
3 12 0 C.Otero
mol/L, without having any significant decrease in the reaction conversion. But, the
reaction conversion dramatically diminishes when the presence of solids (the reagents
and N-acylated ethanolamine) becomes excessively large at reagent loads higher than
3 mol/L.
In reactions with reagent loads above 0.4 mol/L, the percentage of the desired
product (N-acylated ethanolamine) precipitated in the reaction medium, is sufficiently
high for obtaining maximum selective formation of this intermediate product
(92-95%, Table 11.6). Note that the selectivity towards the intermediate product
(N-acylated ethanolamine) remains constant independent of the load of reagents
utilized in the range of loadings studied (0.4-3 mol/L).
Use of a higher temperature (60°C) permitted the author to increase the
concentrations of reagents from 0.4 mol/L to 3 mol/L while minimizing problems
associated with the solubilities of reagents and intermediates, thereby minimizing the
limitations on the rate (Table 11.6). Consequently, the increase of temperature from
40 - 60°C permits one to increase the volumetric productivity of these reaction routes
by one order of magnitude.
the author has developed for selective synthesis of N-acylated diethanolamines in two
different media. These strategies also consider and/or minimize problems associated
with the high viscosity and low solubility of the precursor diethanolamine.
Experimental
Materaah
Diethanolamine (> 98% pure), lauric acid (> 99% pure), myristic acid (98% pure),
and dioxane were purchased from Merck (Darmstadt, Germany). Other reagents
were the same as those used in the ethanolamine study. Before use, all solvents were
dried with molecular sieves with an effective pore diameter of 4A.
Enzymatic Reactions
For trials involving a 1/1 mole ratio of reactants, 0.1 mmol (0.2 M) of both the
diethanolamine (10.5 mg) and the fatty acid (20 rng of lauric acid, 22.8 mg of
rnyristic acid, or 25.6 mg of palmitic acid) were mixed with 0.5 mL of solvent in
stoppered glass bottles. Additional experiments were conducted using 2/1 or 3/1 mole
ratios of lauric acid (40 mg or 60 mg, respectively) to diethanolamine (10.5 mg). In
experiments in which the mole ratio of lauric acid to diethanolamine was 1/2 or 1/3,
the quantity of diethanolamine that was employed was increased to 21 mg or 31.5
mg, respectively. For reactions involving solutions which were intended to be 0.5,
0.8, 1.2, 1.5, or 2 M for both reagents, the amounts of lauric acid added were 50,
80, 120, 150, and 200 mg while the corresponding amounts of diethanolamine were
26.2, 42, 63, 78.7, and 100.5 mg. For the transacylation reactions, the quantities of
lauric acid ethyl ester used to produce 0.2, 0.5, 0.8, and 1.2 M solutions were 22.8,
57, 91.2, and 136.8 mg, respectively. The resulting solution was equilibrated at the
reaction temperature for a few minutes prior to addition of the enzyme. Subsequently
2 5 ,6 2 5 , 100, 150, 187.5, and 250 mg of Novozym 435 were added to the solutions
of the acidic precursor (corresponding to concentrations of 0.2,0.5,0.8, 1.2, 1.5, and
2 M, respectively). Then the mixture was shaken at 200 rpm for 5 min to 96 hr at the
temperature of interest.
At predetermined reaction times, dimethylformamide (DMF) was added until
precipitated reagents and/or products were dissolved and a transparent solution was
obtained. The immobilized enzyme was then rapidly removed by filtration with a 0.1
mm sieve, and the filtrate was centrifuged. DMF increased the volume of final solution
to 10 mL (when the reactant concentration was 0.2 M). For reactant concentrations
of 0.5, 0.8, 1.2, and 2 M, DMF increased the final volume to 25, 50, 50, and 100
mL, respectively. An HPLC system consisting of an autosampler L-7200, a L-7100
pump, connected to a Kromasil C18 250 x 4.6 mm column (Eka Chemicals AB,
Bohus, Sweden) and a SEDEX 55 light-scattering detector (Sedere Sa., Alfortville,
France) was used to analyze the product mixture. Molar yields were calculated with
respect to the limiting reactant.
Results and Discussion
Elucidation of the Structures of the Reaction Products
FTIR, NMR, and molecular mass data for the reaction products are given ..I Table 1 7.
A) Dioxane B) Hexane
Fig. 11.7. Reaction courses of direct acylation of diethanolamine with lauric acid at 4PC in polar (A) and
apolar (B) solvents. Conditions: 25 rng Novozyrn 435,O.l mmol of both diethanolamine and lauric acid
and 0.5 ml solvent.
formed from the reactants does not seem to be a limiting factor in n-hexane, and
the direct acylation reaction in n-hexane remains more selective to the N-acylated
alkanolamine intermediate than is the case in dioxane (86 and 75 % selectivity to the
amide intermediate at 60°C, respectively) (Ferndndez-Pkrez & Otero, 2003).
11.8). The rate decreases when amounts of enzyme greater than 50 mg are employed.
A similar effect was reported by the author's group for biotransformations of
heterogeneous mixtures of sugar esters and a-hydroxy acids. This effect is explained
by the failure of the shaker to maintain uniform suspensions of the solids (ion pair of
reagents + biocatalyst) in the reaction mixtures with high reagent loads (Arcos et al.,
1998a, 1998b; Torres & Otero, 1999; Torres et al., 1999).
60 25
n
sW
20
n
40 I?
W
E 15
E
.-0 6
W
G2 20
10 .E
b
3 5
0 0
12.5 25 50 75 100 150 200
Loading of Biocatalyst (mg)
Fig. 11.8. Effect of the amount of the biocatalyst on direct acylation of diethanolamine in n-hexane.
Conditions:0.1 mmol of both diethanolamine and lauric acid, 0.5 ml n-hexane, 60°C.
3 18 c. ~ t e r o
viscosity of the reaction mixture (Table 11.8). In the case of dioxane these results
can not be attributed to solubility problems, because neither precipitation nor phase
separation of the precursor reagents was observed in the more concentrated dioxane
solutions.
Table 11.8. Conversion, Selectivity, and Viscosity of Reaction System of Direct Acylation
of Diethanolamine with Lauric Acid in Equimolar Concentrations at 60°C with Higher
Concentrations of Substrates in Both, Polar, and Apolar Media
Reactant C. antarctica Amide Conversion’ Selectivitp Viscosity
Solvent
Concentration (M) Lipase (mole Yo) (mole %) (%) (CSt)
0.2 25 56 74 76 0.87 (* 0.01)
0.5 62.5 71 73 97 -
0.8 100 67 73 92 1.38 (* 0.01)
Dioxane
1.2 150 51 55 93 1.87(* 0.01)
1 .5 187.5 45 50 90 -
2 250 41 46 89 -
0.2 25 59 69 86 0.90 (* 0.01)
0.5 62.5 64 68 94 -
n-Hexane 0.8 100 69 73 94 1.40 (k 0.08)
1.2 150 35 39 90 2.00 (* 0.011
1.5 187.5 36 39 92 -
Reaction time: 24 hours.
(IConversion of the limiting reactant.
Selectivity to the amide.
Selective Enzymatic Synthesis of N-AcylatedAlkanolamine Emulsifiers 0 3 19
ratio of enzyme to support). One must also consider that the viscosity of the reagent
mixtures can increase significantly when increased substrate loads are employed.
The author has prepared different tailor-made immobilized lipases according to
the requirements of the reaction. Namely, the author employed an immobilization
procedure using a support with a high porosity, an appropriate mean pore size to get
a high specific surface area (surface area 300-320 m2/g, average pore diameter 30-40
nm), a chemically modified matrix surface to facilitate adsorption of the enzyme in
an activated form that is accessible to the substrate, and a mesoporous silica modified
with octyltriethoxysilane (Blanco et al., 2004). The author selected a lipase with
high specificity for the substrates. More precisely, Candida antarctica B lipase was
immobilized on the aforementioned relatively hydrophobic silica.
The hydrolytic activities of both commercial (Novozym 435) and tailor-made
(Silica 600) immobilized derivatives were 2634 and 9200 unidg, respectively. The
activity units of these two solid biocatalysts employed in biotransformations of
ethanolamine were similar, namely 197.5 and 197.9 activity units, respectively. The
amount of grams of solid biocatalyst added to the reaction mixture was lower in the
case of the tailor-made catalyst than in the case of the commercial one (Table 11.6).
The new biocatalyst gives results comparable to those of the commercial biocatalyst
at an intermediate substrate load (0.6 mol/L) and temperature (50°C). The new
biocatalyst also gives excellent reaction conversions and selectivities to the N-acylated
alkanolamine at the highest substrate load studied (namely 2.5-4 mol/L; Table 11.6).
These values are much better than those of the best commercial catalyst.
The results of the comparative study of transacylation of diethanolamine with
ethyl laurate catalyzed by Novozym 435 and the new tailor-made biocatalysts at
60°C and 1.2 M reagents concentration are summarized in Table 11.10. In this
study, the amount of activity units of the immobilized catalysts were approximately
the same, but the reaction mediated by the most efficient lipase contained a lower
amount of the solid enzyme preparation (Table 11.10). The conversion obtained at
24 hr with new biocatalyst (81%) is significant higher than that obtained with the
commercial immobilized lipase (65%). These results demonstrate that use of a more
efficient biocatalyst permits the reduction of the amount of solids in reaction systems
with high substrate loads. Consequently, higher conversions are obtained with the
more efficient biocatalyst when high substrate loads are employed. This study also
Selective Enzymatic Synthesis of N-Acylated Alkanolamine Emulsifiers0 32 1
demonstrates that the presence of high amount of solids in the medium limits the
reaction conversion that can be achieved. The more efficient biocatalyst permits to
work at much higher reagent concentrations. Under these conditions the reaction
selectivity can be increased (from GO% to 75% amide, Table 11.10).
Concl usions
A general strategy has been developed for optimization of biotransformations at
high substrate concentrations, especially, for reactions that involve species with low
solubilities and relatively high viscosity of the reaction medium. Optimal reaction
conditions for the synthesis of N-acylated ethanolamine and diethanolamine
emulsifiers have been identified. The optimum corresponds to maximum conversion
of substrate and maximum selectivity to the desired product. Factors which limit the
conversion and the selectivity of this reaction have been identified. These limitations
have been overcome at high substrate loads. The new immobilization procedure
permits one to increase the volumetric productivity of ethanolamine reaction to 4
moles/L. The high catalytic efficiency of the new biocatalyst permits a reduction in
the amount of solids present in the reaction system, and increases the selectivity and
reaction conversion of the transacylation of diethanolamine. Under these conditions
the volumetric productivity is also increased.
References
Arcos, J.A.; M. Bernabt; C. Otero. Quantitative Enzymatic Production of 6-O-Acylglucose Esters.
Biotechnol. Bioeng. 1998a,37, 505-509.
Arcos, J.A.; M. Bernabe; C. Otero. Quantitative Enzymatic Production of 1,6-Diacyl Sorbitol
Esters. Biotechnol. Bioeng. 1998b, GO, 53-60.
Blanco, RM.; I!Terreros; M. Ferndndez-Perez; C. Otero. Functionalization of Mesoporous Silica
for Lipase Immobilization. Characterization of the Support and the Catalysts./. Mol Catal.
2004,30, 83-93.
Ferninda-Perez, M; C. Otero. Enzymatic Synthesis of Amide Surfactants from Ethanolamine.
Enzyme Micro&. Zchnol. 2001,28, 527-536.
Ferndnda-Perez, M.; C. Otero. Selective Enzymatic Synthesis of h i d e Surfactants from Dieth-
anolamine. Enzyme Micro&. Technol. 2003,33,650-660.
Furutani, T.; M. Furui; H. Ooshima; J. Kato. N-Acylation of P-Amino Alcohol by Acyl Migration
Following Enzyme Catalyzed Esterification. Enzyme Micro&. Technol. 1996, 19, 578-584.
Garrison, J. Monoethanolamides. Detergent Age 1968, 27-29.
Gotor, V.; R. Brieva; F. Rebolledo. EnantioselectiveAcylation of Amino Alcohols by Porcine Pan-
creatic Lipase. /. Chem. Soc. Chem. Commun. 1988, 957-58.
Khemelnitsky, Y.U.; A.V. Levashov; N.L. Klyachko; K. Martinek. Engineering Biocatalytic Sys-
tems in Organic Media With Low Water Content. EnzymeMicrob. Zchnol. 1988, 10,710-724.
Kanerva, L.T.; M. Kosonen; E. Mnttinen; T. Huuhtanen; M. Dahlqvist. Studies of the Chemo-
and Enantio- Selectivity of the Enzymatic Monoacylations of Amino Alcohols. Acta Chemica
322 0 c. Otero
Saccharide-fatty acid esters are amphiphilic and mainly non-ionic type surfactants
having the saccharide serve as polar head group and one or more fatty acyl group are
apolar tail moieties (Fig. 12.1; Sabeder et al., 2006; Cs6ka et al., 2007).
?he chemical properties of the saccharide and fatty acyl groups control the surface
energy related properties of the esters. Saccharides, such as glucose, sucrose, fructose
and xylose, contain more than 5 hydroxyl groups as possible reactive sites with acyl
donor. The reaction mainly occurs at the more reactive primary hydroxyl groups
among them (typically 1-2 primary hydroxyl per molecule) in accordance with lipase’s
regioselectivity.
Taking sucrose as an example, the enzymic approach appears to be functionally
distinct from chemical acylation in at least two respects (Rich et al., 1995). First,
chemical acylation with acid chlorides, acid anhydrides, acyl azides, acyl cyanides,
and N-acylimidazoles results in an acylation preference of 6-OH 2 6’-OH > 1’-OH >
secondary-OH. In contrast, enzymes nearly universally acylate with 1’-OH = 6-OH
> secondary-OH >> 6’-OH. Second, enzymes often differ in their regioselectivities.
For example, subtilisin Carlsberg (alkaline serine protease from B. lichenifuormis)
preferentially catalyzes acylation of sucrose at the 1’-OH (with small yields of 6-OH)
in pyridine with short-chain ester donors, whereas lipase from Tbermomyces lanuginusus
(Novozymes Inc., Franklinton, NC USA) is selective toward the 6-OH (Rich et al.,
1995, Ferrer et al., 2005). Resultant products possess various HLB values with a wide
range according to the properties and number of saccharide and fatty acyl groups.
Sucrose-fatty acid esters have been produced commercially by Mitsubishi-Kagaku
Foods Corporation (Tokyo, Japan) and Sisterna B.V. (Roosendaal, Netherlands)
by chemical processing. Sucrose has eight free hydroxyl groups, which allow the
formation of mono- to octa-esters using different fatty acids (stearic, lauric, myristic,
and oleic acid) ( C d ka et al., 2007). The commercial sucrose fatty acid esters contain
mixtures of the previously mentioned fatty acyl groups and partial esters, to obtain an
HLB range from 1 to 16 HLB with the HLB decreasing as the chain length of fatty
acyl group and degree of esterification increase. (See Table 10.1 of Otomo’s Chapter
10, in this book.)
Fig. 12.1. Lipase-catalyzed synthesis of fructose-fatty acid esters (R = acyl group) Sabeder et al. (2006).
Reproduced with permission from Elsevier.
Synthesisof SaccharideFatty Acid Ester BiosurfactantsCatalyzed by Lipase 0 325
Table 12.1. Activity of Lipase (Novozym SP435*, Novozymes, Inc, Franklinton, NC USA),
Glucose Solubility, and Enzyme Stability for the Esterification of Glucose and Myristic
Acid as a Function of Solvent Hydrophobicity
Enzyme
activity Glucose Solvent hydrophobicitf
Solvents OmoVmin g) solubilityb (mM) Residual activity (%) (log P)
DMSO 0 29 0 -1.3
Dioxane 1.1 7.5 53 -1.1
DMF 0 12 0 -1 .o
Acetonitrile 0 1.1 27 -0.33
Acetone 3.O 2.6 46 0.23
THF 1.6 2.1 46 0.49
Pvridine 0 134 0 0.69
tert-8utanol 3.7 12 75 0.80
tert -Pentanol 3.6 10 71 1.4
Toluene 0 0.6 54 2.5
Hexane 0 0 80 3.5
Measured by reacting 150 mg glucose, 1 H,O and 750 mg myristic acid in 5 mL of solvent in
the presence of 35 mg lipase and molecular sieves (3A; 0.5 g) at 45°C and rotary shaking (250
rpm) for 24 h.The reaction was carried out at 45°C (250 rpm).
bDeterminedafter the initial 24 h of incubation at 45"C, before enzyme addition.
The enzymes were recoveredafter 24 h of reaction to determine their residual activity. After
decanting the solvent, the immobilized enzyme preparation and the molecular sieves were
washed 4 times with warm (45°C) ten-butanol followed by drying in vacuo for 2 h. Additional
molecular sieve (500 mg) was added before reuse.
dFromLaane et al. (1987)
Degn & Zimmermann (2001). Reproduced with permissionJohn Wiley and Sons.
Yahya et al., 1998; Kim et al., 2004; Kennedy et al., 2006). The highest enzyme
activities are found when using nonpolar solvents (Degn & Zimmermann, 2001);
for example, solvents with log Pvalues >3 in Table 12.1 are widely used in reactions
such as the lipase-catalyzed interesterification of oils and fats to obtain new fats with
improved physical and/or nutritional properties (Villeneuve, 2007). However, to
synthesize fatty acid esters of saccharide and other polyols, acyl acceptor substrates are
poorly soluble in these solvents, which makes them unsuitable.
The stability of the enzyme is usually higher in the more hydrophobic solvents.
In contrast, solvents with lower log P values (e.g., pyridine, dimethylformamide
(DMF), tert-butanol, and acetone) can partially co-solubilize acyl donors and many
polar saccharide molecules (Table 12.2) and thus can be employed for polyol ester
synthesis (Villeneuve, 2007). However, these solvents often inactivate the enzyme by
their ability to remove water molecules of hydration, promote accumulation of water
in the reaction media, leading to hydrolysis and hence reduced product yield and/
or the formation of by-product and are incompatible with food applications (Degn
& Zimmermann, 2001; Ganske & Bornscheuer, 2005a; Hayes, 2004). For example,
Table 12.2. RegioselectiveSynthesisofFatty Acid Esters of Monosaccharidesand RelatedCompounds Using Lipases and Proteases
Reaction Yield
S no. Acyl Acceptor Solvent Time Enzyme" Product (%) Ref.
1 Arabinose Diisopropylether 72 h PPL 1-0-Acetylarabinofuranoside 68 Sharma & Chattopadhyay, 1993
2 D-Glucose Pyridine 18 h Subtilisin 6-0-butyrylglucose 64 Riva et al., 1988
3 DGlucose Pyridine 5d Subtilisin 6-0-butyrylglucose 60 Riva et al., 1988
4 D-Glucose Heptane 46h LipozymeIM 1- or 6-0-stearate glucose 9.32 Oguntimein et al., 1993 Y
3
5 D-Glucose Diisopropylether 72 h PPL 6-OAcetylglucopyranoside 62 Sharma & Chattopadhyay, 1993 3.
2
6 D-Glucose Pyridine l h Subtilisin 6-Omonoacylglucose 96 Park et al., 1996 Gi.
4
7 D-Glucose Dimethylformamide 7h Proteases 6-0-Vinyladipoylglucose 56 Shibataniet al., 1997 m
W
8 D-Fructose tert-Butanol 46h SP382 1- or 6-0-stearate glucose 8.6 Oguntimein et al., 1993 n
s-
9 D-Fructose 2-Methyl-2-butanol 26 h Lipozyme IM Fructoseoleate 83 Khaled et al, 1991 s.
P
b
10 D-Fructose n-Heptane 12 h Lipozyme IM Fructosemonostearate 40 Schlotterbecket al., 1993
11 D-Fructose Diisopropylether 72 h PPL 1-0-Acetylarabinofuranoside 70 Sharma & Chattopadhyay, 1993 ,$
12 D-Fructose 2-Methyl-2-butanol 24 h Lipozyme IM 1-0-Monoacylfructose
6-0-monoacylfructose
39 Scheckermann et al., 1995 g
rn
3
13 D-Fructose n-Hexane 12h LipozymeIM 1-0-Monoacylfructose 40 Scheckermann et al., 1995 I
m
14 D-Fructose tert-Butanol ByssOChlumys'lVu Mixture of esters 71.3 Ku & Hang, 1995 a
24h lipase %
15 D-Fructose
3
Dimethylformamide 3 h Pseudomonus sp 1-0-Laurylfructose 80 Sin et al., 1998 2
W
Chmobacteriurn viscosurn 3
60-Butyryl-
D-glucose
Tetrahydrofuran 40 h ,ipase 3.6-Di-0-butyrylglucose 50 Therisod & Klibanov, 1987 in
x
J
17 Sorbitol 2-Pyrrolidone 74 h C. viscosum lipase Mixture of sorbitol esters 80 Janssen et al., 1991 5
Methyl a-D- Benzene/pyridine
l8 glucopyranoside (21 v/v)
48h SP382 Mixture of esters 56 Akoh & Mutua, 1994 a
Y
CT
r
l9 lsopropylidenc
D-xylofuranose Dimethylformamide 30 h Novozym 435 Xylose-5-arachidonate 83 Ward et al., 1997 2
t
aPPL = porcine pancreatic lipase, Lipozyme IM = immobilized Rhizopmucormiehei lipase (Novozymes, Inc, Frankllinton, NC), SP 382 and Novozym 435 = 0
immobilized CundiduuntarcticuA+B and B lipases, respectively(Novozymes) w
h,
Kennedy et al. (2006). Reproducedwith permission by John Wiley and Sons. w
328 0 5-H. Pyo and D.G. Hayes
(FFA acyl donor) or methanol (FAME acyl donor), by creating an azeotropic mixture
that was subsequently treated by distillation.
Mixtures of high and low hg P solvents, which represent a compromise between
high enzyme activity and saccharide solubility, can be employed for lipase-catalyzed
polyol ester synthesis (Degn & Zimmermann, 2001; Ferrer et al., 2005). The highest
activity for the Novozyme 435'-catalyzed synthesis of saccharide-myristic acid esters
was observed for the relatively nonpolarlpolar solvent mixture tert-butano1:pyridine
55:45 v/v (Degn & Zimmermann, 2001). For solvent systems that yielded low glucose
solubility (< 60 mM), the measured enzyme activity retention was generally high;
in contrast, solvent systems that achieved high glucose solubility led to low activity
retention, reflecting a loss of enzyme stability due to the relatively polar solvent, and/
or to substrate inhibition. The effect of the addition of dimethyl sulfoxide (DMSO),
a polar solvent known to inactivate enzymes, on the activity of the lipase was also
investigated for a ternary (tert-butano1:pyridine:DMSO) solvent system. With > 2%
(v/v) DMSO in the reaction mixture the enzyme activity and stability decreased,
indicating a denaturing effect of DMSO on the enzyme (Degn & Zimmermann,
2001).
Ferrer et al. (2005) employed apolar/polar solvent systems (2-methyl-2-propanol/
DMSO, 4/1 v/v) as reaction media for the acylation of sucrose, maltose, and glucose
catalyzed by granulated I: kznuginosus lipase (Novozymes, Inc.), which is selective
toward the 6-OH group of sucrose. In this solvent system, over 80% of the initial
activity was retained after 20 successive 6-h batch reactions (Ferrer et al., 2005). Ferrer
et al. (1999) demonstrated with I: kznuginosus lipase that the DMSO percentage
in the solvent mixture substantially modified the final esterification degree. Thus,
at DMSO concentrations 510% the formation of diesters was favored, whereas at
percentages higher than 15% the formation of diesters was minimized (Ferrer et al.,
1999). Moreover, the increased polarity of the reaction medium promoted by the
higher concentration of DMSO led to an increased proportion of the more polar
monoester product.
Although there have been many successes on a laboratory scale, use of organic
solvents for the synthesis of sugar fatty acid esters has certain limitations for large-
scale synthesis. There are only a few solvents, such as acetone, acetonitrile, tert-
butanol, pyridine, DMF, or DMSO, which can solubilize saccharide to a significant
extent; but, the toxicity and environmental safety of these solvents is a concern.
Also, many enzymes do not retain their catalytic activity in these solvents. Therefore,
alternate methods that reduce the solvent amount or eliminate their need have been
recommended recently.
and inexpensive. Since enzymes are insoluble in supercritical CO,, the catalyst can
be easily separated from the reaction mixture (Habulin et al., 2008). The Novozym
435"-catalyzed synthesis of fructose palmitate, fructose laurate, sucrose palmitate
and sucrose laurate were carried out at 60°C in 2-methyl-2-butanol at atmospheric
pressure and in supercritical CO, at lOMPa (Habulin et al., 2008). The highest
conversions for fructose palmitate synthesis were obtained in 2-methyl-2-butanol
and in supercritical CO, (65% and 61%, respectively). Tsitsimpikou et al. (1998)
investigated the acylation of glucose with lauric acid at a mole ratio of 1:5 catalyzed
by Novozym 435" (12.5 mg/cm3) in supercritical CO,. Glucose conversions up to
60% at 60°C have been observed. Since the only substrate or product that is soluble
in the supercritical phase is lauric acid, the system offers the potential advantage of
easy separation of the glucose laurate product from remaining substrate and enzyme
upon completion of the reaction.
Although supercritical CO, is a promising alternative to organic solvents, its
employment has limitations: only non-polar compounds are soluble at an acceptable
level (Tsitsimpikou et al., 1998), and capital costs for systems that include a high
pressure reactor and controller are high.
0 0
II II
HzC-OH H~C-OC(CH~)~OCHS H~C-OC(CH~)~OCH~
$G
HO Vinyl laurate
Novozym435
" ~
Acetic acid
_____e) H~~~ H ~
H3C 0 H3C 0 90% HO H
H3CXO-CH2 H3CXO--CH2 HO-CH2
Fig. 12.2. Synthesis of 1-0-lauroyl-D-mannitol. Pinna et al. (2004). Reproduced with permission from
Elsevier.
Acetalization
I OH I b'CH3
0
II Lipase Fatty acids
H&-C-CH3 Methyl esters
t
Deacetalization
'.0 XH3
c,
CH3
Fig. 12.3. Synthesis of xylose -fatty acid esters via isopropylidene derivatives. R represents alkyl group.
Fregapane et al. (19911. Reproducedwith permission from Elsevier.
332 0 5-H. Pyo and D.G. Hayes
0
II ::
CH20CCH=CH2
Fructose
1. Phenylboronicacid OH
Lipozyme, 60°C
+ *
2. Mild hydrolysis OH
Stearic
acid
OH 0
Fig. 12.5. Reaction scheme for the enzymatic monoacylation of fructose with stearic acid in a one-pot-
process. Schlotterbeck et al. (1993).Reproducedwith permission from Springer.
polar solvents reducing stability. In the solid-phase system, the polarity of the liquid
phase is strongly influenced by the dissolved fatty acid; however, the influence of the
sugar or sugar fatty acid ester on lipase stability can be neglected due to their low
solubility (Cao et al., 1999). The lower conversion and lipase stability observed using
short-chain htty acid can be explained by the higher polarity (i.e., lower log P) of the
short-chain FFA, producing a lower overall logPvalue for the liquid phase (Cao et al.,
1999).
In summary, a solid-phase system results in quantitative saccharide fatty acid
ester yield and a relatively high reaction rate, provides an enhanced selectivity toward
monoesters, and can utilize near-ambient temperatures. However, this approach
possesses disadvantages, such as the requirement of organic solvent and its inherently
batch nature, and its translation into a continuous process is difficult (Ganske &
Bornscheuer, 2005a). The separation of the monoester, unreacted saccharide, and
immobilized biocatalyst from the solid phase may also be challenging.
+ x
CH3
P N N R =CnH~n+i
X = PF6, BF4,MeS04, octylsulfate(OS04),
dimethylphosphate(DMP), etc
Fig. 12.6. Structures of ionic liquids based on methylimidazolium salts.
l2.l), and an acceptable conversion rate could only be achieved for the solid-phase
approach. Ionic liquids (IL) yield several-fold higher values of solubility (Table 12.3);
thus, their addition to tert-butanol results in an increased liquid-phase saccharide
concentration, In addition, the same authors also successhlly employed FFA as acyl
donor under the same conditions to achieve a high yield and reaction rate (Ganske &
Bornscheuer, 2005a).
Novozym 435" catalyzed synthesis of 6-0-lauroyl-D-glucose using supersaturated
glucose solution in IL was investigated (Table 12.3; Lee et al., 2008a). The highest
lipase activity was obtained in water-miscible 1-butyl-3-methylimidazolium
trifluoromethanesulfonate, [bmim] [TfO], which can dissolve high concentrations
of glucose, while the highest stability of lipase was shown in hydrophobic l-butyl-
3-methylimidazolium bistrifluoromethylsulfonylimide, [bmim] [TQN] . The
supersaturated solution is usually prepared by dissolving excess solute at high
temperature and then slowly cooling to low temperature. However, Lee et al. recently
developed an alternate dissolution process that uses water as a mediator (Lee et al.,
2008b). First, saccharide (50 mg) was dissolved in water (0.3 mL) and mixed with IL (1
mL) at room temperature. After clear solutions were obtained, the water in the mixtures
was removed by vacuum evaporation for 12 h at 60°C. The saturated sugar solutions
were slowly cooled to 25°C and then incubated for 2 h at 25°C. After centrifugation,
the supernatant was obtained and its sugar concentration measured. Supersaturated
solutions prepared by this method yielded apparent glucose concentrations of 113
and 46.3 g/L at 25°C for [emim][TfD] and [bmim][TfO], respectively, 19 and 10
times higher than the true solubility (6.1 and 4.8 g/L, respectively). A supersaturated
[bmim] [TfD]:[bmim] [TON] (1: 1 v/v) mixture produced an even higher solubility
at 25"C, 222 mM. Furthermore, the supersaturated glucose solutions in IL were
maintained over several hours without any significant precipitation of glucose.
The optimal activity and stability of lipase were obtained in a [bmim][TfO]/
[bmim][TBN] (1:1, v/v) mixture. Specifically, the activity of lipase was increased
from 1.1 to 2.9 pmol/min g by using a supersaturated glucose solution in this mixture,
compared with reaction using a saturated glucose solution (Table 12.3). To measure
enzyme stability, lipase (Novozym 435") was employed and then subsequently
recovered from 5 subsequent batch reactions. After the fifth batch reaction, Novozym
retained 86% of initial activity when the [bmim] [TfO]/[bmim] [TON] (1: 1, v/v)
mixture was employed; in contrast, the residual activity using the pure polar IL [bmim]
[TfO] was only 36%. Therefore, the productivity obtained by using IL mixtures was
Synthesis of Saccharide Fatty Acid Ester BiosurfactantsCatalyzed by Lipase 0 337
Table 12.3. Dissolved Glucose Concentration, Enzyme Activity, and Residual Enzyme
Activity in Ionic Liquids (IL) and IL Mixtures
Residual
Dissolved Concentration Enzyme Activity" enzyme
of Glucose at 40°C (mM) (pmol/min/g) Activitpb(%)
Saturated Supersaturated Saturated Supersaturated Supersaturated
IL' Solutionc Solutiond Solution Solution Solution
[bmiml[BFJ 6.3 43.4 0.65 1.29 66.1
higher than those in pure IL. Figure 12.7 plots the time course for the enzymatic
synthesis of 6-0-lauroyl-D-glucose in pure IL and the IL mixtures. Lauroyl glucose
concentrations of 190 and 153 mM were obtained after 11 h of incubation in the
pure IL and the IL mixture, respectively (Lee et al., 2008a).
Biocatalytic conversions in IL can be beneficial with regard to activity, selectivity,
and stability in comparison with more classical organic solvents (Sheldon et al., 2002;
Villeneuve, 2007). Indeed, the use of enzymes in IL opens up new possibilities for
nonaqueousenzymology (Sheldon et al., 2002). However, there remainseveralproblems
to overcome, such as enzyme inactivation, poor solvent toxicity, and biocompatibility
for many IL. To date, the use of IL on an industrial scale for biocatalyzed reactions is
limited due to the high price of such compounds and can be seen as a realistic solution
only for the production of value-added products (Villeneuve, 2007). However,
material costs for ILs are predicted to decline as new applications for IL continue to
develop.
100
80
20
0
0 2 4 6 8 10 12
Time (h)
Fig. 12.7.Time courses for the lipase-catalyzed esterificationof glucose with vinyl laurate in pure IL and
.
IL mixtures Reaction conditions: 222 mM glucose, 444 mM vinyl laurate, 0.5 mL IL, 50 rng Novozym
435,4OoC(+, [bmiml[TfOl: @,SO% [bmiml[TfOl with 50% [bmimltTf,Nl: A,[bmiml[Tf,NI). Filled symbols
represent the reaction with supersaturated glucose solution. Unfilled symbols represent the reaction
with saturated solution with undissolved glucose crystals present. Lee et al. (2008a) Reproduced with
permission from Elsevier.
Synthesis of SaccharideFatty Acid Ester Biosurfactants Catalyzed by Lipase 0 339
Oleic Acid
Fructose Monoester
Fig. 12.8.Ternary phase diagram for fructose/oleic acidhechnical-grade monoester (5% diester and 95%
monoester) at 60°C.An apparent one-phase liquid mixture exists to the right of the phase boundary,
two-phase media to the left of the boundary. Dang et al. (2005). Reproduced with permission from
Springer.
the initial period to enhance fructose solubility, and (along with the product, water)
was allowed to evaporate away, with 100% solvent removal achieved upon reaching
~ 2 5 %conversion. The acyl acceptor was added in fed-batch mode. A conversion of
80-93% was achieved (Fig. 12.9), with the product consisting of mono- and di-ester
at a ratio of 9: 1 g/g. Due to the high conversion and the use of a stoichiometric feed, a
technical-grade bio-based surfactant product was formed, with the only downstream
processing required being the simple centrifugation or filtration-based removal of
immobilized lipase. Lipozyme IM" exhibited excellent operational stability within 1
month of continuous use (Dang et al., 2005).
These results provide the basis for the development of bioreactor system design
for Lipozyme 1M"-catalyzed solvent-free synthesis of saccharide fatty acid esters,
Synthesisof Saccharide Fatty Acid Ester Biosurfactants Catalyzed by Lipase 0 341
Time, h
Fig. 12.9.Time course of lipase-catalyzedfructose ester synthesis ( A conversion of oleic acid, 6 product
distribution, and C fructose concentration on a tert-butanol-free basis) using a reaction medium
saturated with fructose and nearly stoichiometrically equal amounts of the two substrates. Initial
conditions: 100 mmol(28.247 g) oleic acid, 25.00 g (32.05 mL) tert-butanol, 1.00 g Lipozyme IM, fructose
at saturation (y-intercept of panel C), 65T, and a 400-rpm stir rate.The reaction medium was resaturated
with fructose periodically (indicated in panel C by increases of fructose concentration). t-BuOH and
water were allowed to freely evaporate during the time course of esterification. The curve in panel A
represents the prediction by the Ping-Pong Bi Bi kinetic model. Dang et al. (2005). Reproduced with
permission from Springer.
under investigation in the authors’ laboratory (Fig. 12.10). The bioreactor system’s
main components are the bioreactor and a packed column containing saccharide as a
major component of its stationary phase to deliver saccharide at saturated conditions.
The desorption of saccharide from a fructose/silica gel packed column to a solvent-
free oleic acid/fructose oleic acid ester liquid solution at 65°C increases as the fructose
oleate concentration in the liquid phase increases and the saccharide mass fraction
342 0 S.-H. Pyo and D.G. Hayes
F
C
E
B
D
Fig. 12.1 0. Bioreactor System Designs for Solventless Synthesis of Fructose-Oleic Acid Ester, FOE. A.
Reservoir tank "(no lipase) or Stirred Tank Bioreactor (STBR, with lipase, designated as A'); B. Peristaltic
Pump (0.1 mL/rnin); C. Fructose desorption column (DC; 100 x 10 mm ID); D. Molecular sieves column
(MSC; lg, 50 x 10 mm ID); E. Packed-Bed Bioreactor (PBBR; 50 x 10 mm ID, Lipozyme RM IM.); F.Temp-
controlled Oven; 1. STBR /fed-batch addition of fructose: A'; 2. STBR / DC: A' B C A'; 3. PBBR/ DC: A B C
E A ;4. PBBR / DC / MSC: A B C D E A. Pyo & Hayes, submitted 2008. Reproduced with permission from
Springer.
for the stationary phase increases (up to 70 wt Yo), and follows the well-known
Freundlich isotherm (Pyo & Hayes, 2008). Preliminary results demonstrate that
the reaction rate and conversion are maximized with use of a packed-bed bioreactor
(80285% conversion), and that control of water activity, most readily achieved using
free evaporation, is a critical parameter in the bioreactor system's performance. (Pyo
& Hayes, submitted 2008).
However, the resultant rates of reaction for the bioreactor systems were slower by
a factor of 2-3 compared to the batch-mode reactions, presumably due to the fructose
concentration produced by the packed fructose/silica gel column being a factor of
2-3 times smaller than that produced in the batch-mode reactions (Pyo & Hayes, in
preparation). Through analysis using dynamic light scattering and optical density it
was discovered that under magnetic stirring above 200 rpm suspensions of fructose
crystals formed in oleic acid/fructose oleate mixtures (Pyo & Hayes, 2008), akin to
the supersaturated solutions of saccharides formed in IL as discussed previously. Such
Synthesisof Saccharide Fatty Acid Ester BiosurfactantsCatalyzed by Lipase 0 343
conditions mirror those used for the batch-mode reaction that yielded the results of
Fig. 12.9. In contrast, light scattering of the packed column effluent demonstrated
the absence of aggregates, suggesting this approach yielded saccharide concentrations
corresponding to the true solubilization limit.
lipase-catalyzed synthesis was carried out using an equimolar mixture of fructose and
palmitic acid in 2-methyl 2-butanol at 60°C. The initial water activity strongly affected
the time course of reaction with the lowest water activity (a, = 0.07) leading to the
highest conversion yield (28.5%) and initial rate (4.9 glL h). The use of molecular
sieves enhanced yield, however the selectivity of the enzyme was affected. Whereas only
fructose monopalmitate was produced in the absence of molecular sieves, the addition
of molecular sieves as a drying agent promoted the synthesis of fructose dipalmitate,
by conversion of up to 18.3% of the acyl acceptor. The change of selectivity may be
explained by the reduction of the hydration layer surrounding the proteins. 'The lack of
water near the enzyme promotes an increase of the hydrophobicity and consequently
decreases the local fructose solubility. In these conditions, monopalmitate becomes
a better substrate than fructose for the nucleophilic attack on the acyl-enzyme
intermediate (Chamouleau et al., 2001). The increase of conversion yield and initial
rate due to the reduction of water activity can be explained by the adsorption curve
of Novozym 435", which indicates that a small increase ofwater amount from 0 - 20
mg water/g dry catalyst promotes an increase of water activity, aw,from 0.0 - 0.8, the
latter value promoting hydrolysis over esterification (Fig. 12.1 1; Chamouleau et al.,
200 1).
100
80
60
40
20
0
0 0.2 0.4 0.6 0.8 1
measured wate r activity
Fig. 12.11. Water adsorption curve for Novozym 435'at 20°C.Chamouleau et al. (2001). Reproduced
with permission from Elsevier.
Synthesisof SaccharideFatty Acid Ester BiosurfactantsCatalyzed by Lipase 0 345
Although the proper amount of water for a given enzymatic reaction depends
on many factors (the selected enzyme, support, solvent system, and polarity and
quantities of substrates), some authors have proposed the existence of an optimum
water content, generally in the range of 0 . 2 4 % (Foresti et al., 2007; Rocha et al.,
1999; Manj6n et al., 1991; Yadav & Piyush, 2003; Is0 et al., 2001). This supposition
is based on two opposing trends: an increase of enzyme activity as water content is
increased (and the essential water molecules of hydration for the enzyme are restored)
versus the increased equilibrium conversion of the reaction as the water content is
decreased (Rocha et al., 1999).
In summary, water, which helps maintain the enzyme activity when at the
proper amount and results from the synthesis reaction of saccharide fatty acid esters,
should be removed to maintain the increased equilibrium conversion of the reaction.
Although several different approaches have been successfully implemented, it is
necessary for new methods to be developed that are applicable to bioreactor systems
upon scaleup.
Conclusio ns
Saccharide-fatty acid esters are value-added products derived from natural feedstocks
that have recently been employed in the foods, cosmetics, and pharmaceutical industry
as green, biodegradable, and biocompatible nonionic surhctants or emulsifiers. Most
commercial saccharide fatty acid esters are produced by chemical methods that require
costly and environmentally unsafe conditions. In contrast, the biocatalytic approach
using immobilized lipase provides milder and more environmentally friendly operating
conditions, lower operating costs, reduced downstream separation requirements, and
enhanced regioselectivity of reactions. Thus lipases have been employed as tools for
organic reactions due to their catalytic behavior for numerous applications in various
areas of industrial microbiology and biotechnology. In particular, their catalytic
behavior has been very effective for the esterification of saccharide and fatty acids.
Various parameters, such as solubility of saccharide in reaction media, control of
water activity and content, reaction temperature, nature of acyl donor, and substrate
ratio, have been varied in a systematic fashion to improve reaction conversion yields
and rates. Although there are still difficulties for their employment at an industrial
scale and in establishing cost-effective scale-up and downstream processing protocols,
additional research will yield improvements in these area and more widespread use of
the biocatalytic approach is anticipated.
Acknowledgment
The authors thank the U.S. Department of Agriculture, Grant 2006-35504-1 7262,
for supporting their research in the bio-based surfactant area.
346 0 5-H. Pyo and D.G. Hayes
References
Akoh, C.C.; L.N. Mutua. Synthesis of Alkyl Glucoside Fatty Acid Esters: Effect of Reaction Pa-
rameters and Incorporation of n-3 Polyunsaturated Fatty Acids. Enzyme Microb. Techno1 1994,
16, 115-119.
Allen, D.; B. Tao. Carbohydrate-Alkyl Ester Derivatives as Biosurfactants. /. Sufact. Deters 1999,
2, 383.
Ballesteros, A.; F.J. Plou; M. Alcalde; M. Ferrer; H. Garcia-Arellano; D. Reyes-Duarte; I. Ghazi.
Enzymatic Synthesis of Sugar Esters and Oligosaccharides from Renewable resources. In Bio-
catalysis in the Pharmaceutical and Biotechnology Indwtries, Patel, R.N., Ed.; CRC Press: Boca
Raton, FL, 2007,463-488.
Bjorkling, F,; S.E. Godtfredsen; 0. Kirk. A Highly Selective Enzyme-Catalysed Esterification of
Simple Glucosides. J. Chem. Soc. Chem. Commun. 1989, 934-935.
Cao, L.; U.T. Bornscheuer; R.D. Schmid. Lipase-catalyzed solid-phase synthesis of sugar esters.
Influence of immobilization on productivity and stability of the enzyme. J. Mol. Catal. B: Enz.
1999,6,279-285.
Cao, L.; U.T. Bornscheuer; R.D. Schmid. Lipase-Catalyzed Solid Phase Synthesis of Sugar Esters.
Fett. Lipid 1996, 98, 332-335.
Carta, G.; J.L. Gainer; A.H. Benton. Enzymatic Synthesis of Esters Using an Immobilized Lipase.
Biotechnol. Bioeng. 1991, 37, 1004-1009.
Cauglia, F.; l? Canepa. The Enzymatic Synthesis of Glucosylmyristate as a Reaction Model for
General Considerations on ‘Sugar Esters’ Production. Bioresource 2chnol. 2008, 99, 4065-4072.
Chamouleau, F.; D. Coulon; M. Girardin; M. Ghoul. Influence of Water Activity and Water Con-
tent on Sugar Esters Lipase-Catalyzed Synthesis in Organic Media. J. Mol. Catal. B: Enz. 2001,
1 1 , 949-954.
Chittenden, G.J.F. Isopropylidenation of D-Mannitol under Neutral Condition. Carbohyd. Res.
1980, 87, 219-226.
Compton, D.L.; K.E. Vermillion; J.A. Laszlo. Acyl Migration Kinetics of 2-Monoacylglycerols
from Soybean Oil via ‘H NMR. J. Amer. Oil Chem. Soc. 2007, 84,343-348.
Chopineau, J.; ED. McCaffery; M. Therisod; A.M. Klibanov. Production of Biosurfactants from
Sugar Alcohols and Vegetable Oils Catalyzed by Lipases in a Non Aqueous Medium. Biotechnol.
Bioeng. 1998, 31, 208-214.
Coulon, D.; M. Girardin; M. Ghoul. Enzymic Synthesis of Fructose Monooleate in a Reduced
Pressure Pilot Scale Reactor Using Various Acyl Donors. Process Biochem. 1999, 34, 913-918.
Csbka, G.; S . Marton; R. Zelko; N. Otomo; I. Antal. Application of Sucrose Fatty Acid Esters in
Transdermal Therapeutic Systems. Eur. J. Pharrn. Biopharm. 2007,65,233-237.
Dang, H.T.; 0.0.Obiri; D.G. Hayes. Feed Batch Addition of Saccharide During Sacharide-Fatty
Acid Esterification Catalyzed by Immobilized Lipase: Time Course, Water Activity, and Kinetic
Model. J. Am. Oil Chem. Sor. 2 0 0 5 8 2 , 4 8 7 - 4 9 3 .
Degn, l?; W. Zimmermann. Optimization of Carbohydrate Fatty Acid Ester Synthesis in Organic
Media by a Lipase from Candida antarctica. Biotechnol. Bioeng. 2001, 7 4 , 483-491.
Ferrer, M.; M.A. Cruces; M. Bernabt; A. Ballesteros; F.J. Plou. Lipase-Catalyzed Regioselective
Synthesis of Saccharide Fatty Acid Ester Biosurfactants Catalyzed by Lipase 0 347
Kim, J.; S. Haarn; D.-W. Park; I.-S. Ahn; T. G. Lee; H.-S. Kim; W.-S. Kim. Biocatalytic Esterifica-
tion of P-Methylglucoside for Synthesis of Biocompatible Sugar-Containing Vinyl Esters. Cbem.
Eng. J. 2004,99, 15-22.
Kohya, H.; F. Ishii; S. Takano; T Katori; T. Ebina; N. Ishida. Antitumor Effect of a Synthetic
Cord Factor, 6,6’-Di-0 Decanoyl-a, a-Trehalose (SS 554) in Mice. ]pn. J. Cancer Res. 1986,
77,602-609.
Ku, M.A.; Y.D. Hang. Enzymatic Synthesis of Esters in Organic Medium with Lipase from Bys-
socblamysfilva. Biotecbnol. Lett. 1995, 17, 1081-1084.
Laane, C.; K.V. Boeren; C. Veeger. Rules for Optimization of Biocatalysis in Organic Solvent.
Biotecbnol. Bioeng. 1987,30, 81-87.
Lee, S.H.; S.H. Ha; N.M. Hiep; W.-J. Chang; Y.-M. Koo. Lipase-Catalyzed Synthesis of Glucose
Fatty Acid Ester Using Ionic Liquids Mixtures. J. Biotecbnol. 2008a, 133, 486-489.
Lee, S.H.; D.T. Dang; S.H. Ha; W.-J. Chang; Y.-M. Koo. Lipase-Catalyzed Synthesis of Fatty
Acid Sugar Ester Using Extremely Supersaturated Sugar Solution in Ionic Liquids. Biotecbnol.
Bioeng. 2008b,99, 1-8.
Manjbn, A.; J.L. Iborra; A. Arocas. Short-Chain Flavour Ester Synthesis by Immobilised Lipase in
Organic Media. Biotecbnol. Lett. 1991, 13, 339-344.
Mutua, L.N.; C.C. Akoh. Synthesis of Alkyl Glycoside Fatty Acid Esters in Non-Aqueous Media
by Candida sp. Lipase. J. Am. Oil Cbem. Soc. 1993,70,43-46.
Nakarnura, S. Using Sucrose Esters as Food Additives. INFORM 1997, 8, 866-874.
Napier, I?E.; H.M. Lacerda; C.M. Rosell; R.H. Valivety; A.M. Vaidya; PJ. Halling. Enhanced
Organic-Phase Enzymatic Esterification with Continuous Water Removal in a Controlled Air-
Bleed Evacuated-Headspace Reactor. Biotecbnol. Prog. 1996, 12,47-50.
Ntawukulilyayo, D.; C. Demuynck; J.I? Remon. Microcrystalline Cellulose-Sucrose Esters as
Tablet Matrix Forming Agents. h t . J. Pbarm. 1995, 121, 205-221.
Ntawukulilyayo, J.D.; S.C. De Smedt; J. Demeester; J.l? Remon. Stabilisation of Suspensions Us-
ing Sucrose Esters and Low Substituted n-Octenylsuccinate Starch-Xanthan Gum Associations.
Int. J. Pbarm. 1996, 128,73-79.
Oguntimein, G.B.; H. Erdmann; R.D. Schmid. Lipase Catalysed Synthesis of Sugar Ester in
Organic Solvents. Biotecbnol. Lett. 1993, 15, 175-180.
Park, O.J.; H.G. Park; J.W. Yang. Enzymatic trans-Esterification of Monosaccharides and Amino
Acid Esters in Organic Solvents. Biotecbnol. Lett. 1996, 18, 473-478.
P a d , I?; A. Guerrero; M. Berjano; C. Gallegos. Transient Flow of O W Sucrose Palmitate Emul-
sions. J. Food Eng. 1999,4 1, 33-4 1.
Pinna, M.C.; A. Salis; M. Monduzzi. Novel Mannitol Based Non-Ionic Surfactants from Bioca-
talysis Part Two: Improved Synthesis. J. Mol. Catal. B: Enz. 2004, 27,233-236.
Pyo, S.-H.; D.G. Hayes. Desorption of Fructose from a Packed Column to an Oleic AcidFruc-
tose Oleate Mixture for Employment in a Bioreactor System. J. Am. Oil Cbem. SOC.2008, 85,
1033-1 040.
Rich, J.O.; B.A. Bedell; J.S. Dordick. Controlling Enzyme-Catalyzed Regioselectivity in Sugar
Ester Synthesis. Biotecbnol. Bioeng. 1995,45, 426-434.
Synthesis of Saccharide Fatty Acid Ester Biosurfactants Catalyzed by Lipase 0 349
Riva, S.; J. Chopineau; A.P.G. Kiebomm; A.M. Klibanov. Selective Enzymatic Acylations and
Deacylations of Carbohydrates and Related Compounds. /. Am. Chem. SOC.1988, 110,
584-589.
Rocha, J.M.S.; M.H Gil; F.A.I? Garcia. Optimisation of the Enzymatic Synthesis of n-Octyl
OIeate with Immobilised Lipase in the Absence of Solvents. /. Chem. Technol. Biotechnol. 1999,
74,607-612.
Sabeder, S.; M. Habulin; 2. Knez. Lipase-Catalyzed Synthesis of Fatty Acid Fructose Esters. /.
Food Eng. 2006, 77, 880-886.
Sakaki, K.; A. Aoyama; T. Nakane; T. Ikegami; H. Negishi; K. Watanabe; H.,Yanagishita. Enzy-
matic Synthesis of Sugar Esters in Organic Solvent Coupled with Pervaporation. Desalination
2006, 193, 260-266.
Sarney, D.B.; E.N. Vulfson. Enzymatic Synthesis of Sugar Fatty Acid Esters in Solvent-Free Me-
dia. In Enzymes in Nonaqueous Solvents: Methods and Protocols (Method in Biotechnology GL.15),
Vulfson, E.N., PJ. Halling, H.L. Holland, Eds.; Humana Press: Totawa, NJ, 2001, 531-543.
Scheckermann, C.; A. Schlotterbeck; M. Schmidt; V. Wray; S. Lang. Enzymatic Monoacylation of
Fructose by Two Procedures. Enzyme Microb. Technol. 1995, 17, 157-162.
Schlotterbeck, A.; S. Lang; V. Wray; F. Wagner. Lipase-Catalyzed Monoacylation of Fructose.
Biotechnol. Lett. 1993, 15,61-64.
Sharma A.; S. Chattopadhyay. Lipase Catalysed Acetylation of Carbohydrates. Biotechnol. Lett.
1993, 15, 1145-1 146.
Sheldon, R.A.; R.M. Lau; M.J. Sorgedrager; F. van Rantwijk; K.R. Seddon. Biocatalysis in Ionic
Liquids. Green Chem. 2002,4, 147-1 5 1 .
Shibatani, S.; M. Kitagawa; Y. Tokiwa. Enzymatic Synthesis ofvinyl Sugar Ester in Dimethylfor-
mamide. Enzyme Microb. Technol. 1997, 19, 5 1 1-5 14.
Sin, Y.M.; K.W. Cho; T.H. Lee. Synthesis of Fructose Esters by Pseudomonm sp. Lipase in Anhy-
drous Pyridine. Biotechnol. Lett. 1998, 20, 91-94.
Szuts, A.; E. Pallagi; G. Regdon; Jr., 2. Aigner; I? Szabo-Revesz. Study of Thermal Behaviour of
Sugar Esters. Int. J. Pharm. 2007, 336, 199-207.
Tarahomjoo, S.; I. Alemzadeh. Surfactant Production by an Enzymatic Method. Enzyme Microb.
Echnol. 2003, 33, 33-37.
Therisod, M.; A.M. Klibanov. Facile Enzymic Preparation of Monoacylated Sugars in Pyridine. J.
Am. Chem. SOC.1986, 108, 5638-5640.
Therisod, M.; AM. Klibanov. Regioselective Acylation of Secondary Hydroxyl Groups in Sugars
Catalyzed by Lipases in Organic Solvents. /. Am. Chem. SOC.1987, 109, 3977-398 1 .
Thevenin, M.A.; J.L. Grossiord; M.C. Poelman. Sucrose EsterslCosurfactant Microemulsion
Systems for Transdermal Delivery: Assessment of Bicontinuous Structures. Int. /. Pharm. 1336,
137, 177-186.
TriantaFyllou, A.O.; E. Wehtje; I? Adlercreutz; B. Mattiasson. Effects of Sorbitol Addition on the
Action of Free and Immobilized Hydrolytic Enzymes in Organic Media. Biotechnol. Bioeng.
1995,45,406-414.
Tsavas, I?; S. Polydorou; I. Faflia; E.C. Voutsas; D. Tassios; M.V. Flores; K. Naraghi; I?J. Halling;
350 0 5-H. Pyo and D.G. Hayes
Introduction
Surfactants are amphiphilic molecules composed of a polar group (ionic or non-ionic)
and one or more hydrophobic chains (usually hydrocarbon). This duality confers a
unique range of properties to surfactants, which are used in a variety of processes.
These compounds have garnered ever increasing interest owing to their interfacial
activity, ability to self-aggregate into myriad supramolecular motifs on the nano scale,
biological activities, and diverse applications. Nonetheless, research and development
of new surfactants has shifted towards compounds with multifunctional benefits, and
must be carried out in accordance with acting regulations for human health and the
environment (Holmberg, 2003).
One of the key strategies to minimize the toxicity and environmental impact of
synthetic surfactants is to mimic natural surfactant structures, such as lipoaminoacids
or their analogs (i.e., surfactant-like peptides) all of which are found in cell membranes
(Epand et al., 1998;Yasuhara et al., 2005; Adams et al., 2007). Lipoaminoacids constitute
an important class of natural surface active bio-molecules of great interest to organic and
physical chemists as well as to biologists; these molecules have an unpredictable number
of basic and industrial applications (Xia & Nnanna, 200 1). Structurally, lipoaminoacids
are a very heterogeneous group of compounds but with a common advantage, in
that they are relatively easy to design and synthesize. Often these molecules combine
charged, or noncharged residues [i.e., glutamic acid (Glu), lysine (Lys), arginine (Arg),
serine (Ser), leucine (Leu); phenylalanine (Phe), and alanine (Ala)] as the hydrophilic
head group with an hydrophobic tail of different structure, length, and number [i.e.,
fatty acids, fatty alcohols, and fatty amines] as synthons for the amphiphilic structure
(Takehara 1989; Boyat et al., 2000; Zhang et al., 2005; Gerova et al., 2008; Vijay et al.,
2008). This fact explains the diversity of amino acid/peptide-based surfactants and the
variety of their physicochemical and biological properties (Roy & Dey, 2006; Das et al.,
2006; Varka et al., 2006; Ohta et al., 2008; Capone et al., 2008).
351
352 0 M.R. Infante et al.
........_ . .. ....
............................
...... _,..
~ arginine ...............
\,___.. ._-
'... .
..I
........
........
......... ../
0
' C _-- -
0 0 , arginine ,
-\
- - __
0 -i
c o
d
3
Fig. 13.1. Schematic structure of arginine-based surfactants. Reproduced from (Moran et a1.2004~)with
permission of the Royal Society of Chemistry (RSC).
Synthesis,Aggregation Properties, and Applications of Biosurfactants Derived from Arginine 0 353
In terms of surfactant behavior, the authors have evaluated and compared their
physicochemical properties of adsorption and self aggregation in aqueous media at a
range of concentrations and in the presence and absence of other components.
The presence of the positive charged arginine in such amphiphilic structures gives
an extensive series of compounds with rich phase behavior properties and strong
antimicrobial activity (Mordn et al., 2004b).
These compounds are water soluble, nontoxic if orally administered, nonirritating,
biodegradable, and have a minimal aquatic impact, thus guaranteeing their ultimate
commercial development in the food and cosmetic sector and highlighting their
potential for biochemical applications (Pkrez et al., 2002; Benavides et al., 2004;
Pkrez et al; 2005; Martinez et al., 2006). They can be regarded as an alternative to
conventional synthetic surfactants in which fundamental requirements for industrial
development are present: biodegradability, low toxicity, multifunctional performance,
and renewable sources of raw materials. The authors' work in this field contains basic
science as well as technology elements, and has been of the most interdisciplinary
nature possible, drawing on the expertise of researchers from chemical synthesis,
biocatalysis, physical chemistry of colloids and surfaces, ecotoxicity, toxicology,
microbiology, and has relied on numerous industrial collaborators. In this chapter
the authors present and analyse the data obtained from our group on the synthesis,
physicochemical properties, and some potential applications of these three types
of biosurfactants derived from arginine with an emphasis on the self-aggregation
properties.
Synthesis
Monocatenary Arginine Surfactants
Amino acids are linked to long aliphatic chains through the a-amino, a - C O O H ,
or side chain groups. Thus, fatty acids or alkyl halides can react with amino groups
yielding the corresponding N-acyl and N-alkyl derivatives, respectively. Alternatively,
the carboxyl group of the amino acid can be condensed with alkyl amines or aliphatic
alcohols to give N-alkyl amides or 0-alkyl esters. Among the different types of linkages
between the long aliphatic chain and the amino acid, the N a -acyl, 0 a - alkyl esters,
and 0" -alkyl amides of arginine have attracted much interest in order to prepare
low toxic, biodegradable antimicrobial cationic surfactants. They selectively disrupt
bacterial membranes at submicellar concentrations, but not erythrocytes or skin cell
membranes. It has been demonstrated that the incorporation of ester functionality
accelerates biodegradation. (Infante et al., 1984, 1992a; Mordn et al., 2001 b; Seguer
et al., 1994).
Thus Nu-acyl-arginine-methyl ester hydrochloride (Fig. 13.2), arginine-Oa-alkyl
ester dihydrochloride (Fig. 13.3) and arginine-Oa-alkyl amide dihydrochloride (Fig.
13.4) salts of different alkyl chain lengths have been synthesized by our group using
chemical and biotechnological methodologies. Fig. 13.2-1 3.4 indicate the structure
and acronyms of the different homologs for each series.
354 M.R.Infante et a\.
H2N
Fig. 13.2. Nn-acyl-arginine-methylester hydrochloride.CAM n = 8; LAM n = 1 0 M A M n = 12; PAM n = 14.
C1'
H2N
Fig. 13.4. Arginine-0 a-alkyl amide dihydrochloride.ACA n = 8; ALA n = 10; AMA n = 12.
CH3 CH3
I I
c1- c1-
Fig. 13.5. Structure of bis(Args) gemini cationic surfactants Cs(XA)2 where s refers to the length of the
spacer chain, 2 refers to the presence of two symmetrical long-chain N a-acyl-arginine residues, X refers
tothelengthofthefattyacylchains;X=O,C,andLrefertofattyacylchainsoflength8(n=6), 10(n = 8 ) ,
and 12 (n = lo), respectively. C3 (OA)2 n = 6, s = 1; C3 (CAI2 n = 8,s = 1; C4 (CA)2: n = 8,s = 2; C6 (CA)2: n =
8 , ~ = 4 ; C 9 ( C A ) 2 n = 8 , ~ = 7 ; C l O ( C A ) Z : n = 8 , ~ =C3(LA)2n=
8; 1O,s= l;C4(LA)Z:n= 1O,s=2; C6(LA)2
n = 10,s =4;C9(LA)2 n = 10, s = 7; ClO(LA)2: n = lO,s=8C12(LA)2: n =8, s = 10.
the melting point of the corresponding fatty acid. The 1,2-diacyl-3-aminoacyl glycerol
derivatives were in fact a mixture of two regioisomers: 1,2-diacyl-racglycero-3-(amino
acid) derivative as the major one, and 1,3-diacyl-glycero-2-(aminoacid) derivative.
With this methodology a series of mono and dilauroylated glycerol derivatives
of acetyl-arginine, aspartic acid, glutamic acid, asparagine, glutamine, and tyrosine
were prepared. Purification of the amino acid glyceride conjugates was achieved
by preparative HPLC on a C4 reversed-phase column eluted with gradients of
acetonitrile in aqueous trifluoroacetic acid or with flash chromatography on silica.
Both methodologies provided similar recovery yields. Reversed-phase HPLC on a C4
column was better suited for ionic derivatives while silica was preferred for neutral
products. Both methodologies provided highly pure products.
?he Na-arginine compounds (Fig. 13.7a,b) were prepared using chemical
methodologies. The synthesis of these surfactants consists of three steps: Step 1
corresponded to the preparation of 1-0-(N-Cbz-L-arginyl) rac-glycerol monochloride
(OORZ) by chemical esterification of the a-carboxyl group of N-Cbz-L-arginine
oHC1 with the primary hydroxyl function of glycerol using boron trifluoroetherate as
catalyst. The overall reaction yield was 95%. Step 2 consisted of the synthesis of the
1-acyl-3-O-(N-Cbz-L-arginyl)rac-glycerol~HCl (XORZ) and 1,2-diacyl-3-O-(N-Cbz-
L-arginyl)rac-glycerol.HCl (XXRZ) by acylation of one hydroxyl group of (OORZ)
b 0
II CI-
Physicochemical Properties
Molecular self-assembly of biomimetic molecules has recently attracted considerable
attention for its use in the design and fabrication of advanced biocompatible materials
with a wide range of applications in nanotechnology, medicine, and drug delivery
systems (Gorbitz, 2007). The aggregation morphologies of the acyl amino acids in
water and especially the effect of the molecular structure of the amphiphile on the
formation and type ofself-aggregatesis a fascinating task. It has been extensivelystudied
by different groups, particularly during the last fav years, for their potential uses in
advanced industrial technologies (Imae et al., 2000;Yamashita et al., 2007; Roy &
Dey, 2007;Oshimura et al., 2007; Gerova et al., 2008, Lee et al., 2008). In this
section the authors review the surface and aggregation properties of the three types of
arginine-based surfactants whose acronyms are provided in Table 13.1,
Table 13.1. Acronyms and Chemical Names of the Three Types of Arginine-Based
Surfactants Described in this Chapter
Acronym Chemical Name Figure
CAM N a-caproyl-arginine-methylester.HCI (n = 8)
LAM N a-lauroyl-arginine-methylester.HCI (n= 10)
13.2
MAM N "-myristoyl-arginine-methyl ester.HC1 (n = 12)
PAM N "-palmitoyl-arginine-methylester.HCI (n = 14)
As expected, the surfactant activity of all these compounds was similar to those
with conventional quaternary cations, CMC values in the range 1-30 mM and ycMc
values being in the range 30-37 mN/m. The CMC values of the three families of
monocatenary surfactants depend of the alkyl chain length; the more hydrophobic
the molecule, the lower the CMC value (Fig. 13.8). Contrarily, the nature of the
hydrophilic group does not significantly affect the CMC and ycMc.
The effect of the hydrophilic group on Amis most interesting. The authors found
that the Am values for the arginine-Oa-alkyl ester dihydrochlorides (Fig. 13.3) and
arginine-Oa-alkyl amides dihydrochloride salts (Fig. 13.4) (0.62-1.14 nm', and
0.96- 1.22 nm'. respectively) were higher than that for Na -acyl-arginine-methyl
ester compounds with the same alkyl chain length (0.67-0.62 nm') (Mordn et al.,
2001 b). These results indicate that the arginine-Oa-alkyl ester and arginine-Oa-alkyl
amides structures are packed less densely at the interface. The two charged groups
contained in their molecular structure tend to spread them out on the interface due
to electrostatic repulsion forces.
362 0 M.R. Infante et al.
Arginyl amides
Arginyl esters
P q i arginines
Carbon number
Fig. 13.8. Relationship between CMC and the alkyl chain length for the arginine-alkyl amides (Fig. 13.4),
arginine-alkyl esters (Fig. 13.31, and acyl-arginine (Fig. 13.2) surfactants. CMC were determined in water
at 25°C using Wilhelmy plate technique. Taken from MorBn et al., 2001 b. (Reprinted with permission
from ACS Press)
and geometrical dimensions) have been evaluated from the analysis of the SAXS data
of LAM solutions at different concentrations above the CMC (Table 13.2).
The area per molecule value, 0.65 f 0.01 nm2, agrees roughly with those obtained
from surface tension. The polar head and hydrophobic core electron densities are
consistent with the molecular structure of the surfactant. Moreover, hydration per
*
molecule obtained from the fits, 27 5 moles of water per mole of surfactant, is also
reasonable because in the molecular structure of the head group several hydrogen
bonding groups are present, the guanidinium cationic group and the chloride anion.
The PGSE-NMR was used to determine the self-difision coefficients of LAM at
25°C using standard procedures (Stilbs, 1987), Table 13.3.
Fitting the two-site exchange model (Lindman et al., 1984) to the diffusion/
concentration curve we calculated the CMC, the intradiffusion coefficient of free
monomers, Djcc,and that of the micellized molecules, D,,.
The CMC values obtained by NMR agree with those obtained previously by
surface tension and the structural micelar parameters determined by N M R are in
accordance with those calculated from the SAXS data.
Phase behavior, including structural characterization of the monocatenaries, Na-
acyl-arginine-methyl esters hydrochloride salts CAM, LAM, MAM, and PAM (Fig.
13.2), in binary watedsurfactant and ternary water/surfactant/co-surfactant systems
have been systematically investigated as a function of the alkyl chain length of the
surfactants, and co-surfactants. These studies were carried out using phase diagrams,
optical microscopy observations, light scattering, spectrofluorimetry, Fourier
transform PGSE-NMR, and dielectric spectroscopy (Solans et al., 1989,1990; Infante
et al., 1992b; Fordedal et al., 1993). Solans showed by polarized light microscopy
that lyotropic liquid crystals of hexagonal (CAM, LAM, MAM, PAM), cubic (LAM,
MAM, PAM), and lamellar (MAM, PAM) structure appear as a function of the chain
length along with the hexagonal liquid crystal the more favored structure in LAM
in binary systems that exceed the solubility limit of micelles (Krafft temperature) at
25°C.
The phase behavior of monocatenary arginine surfactants in multicomponent
systems was also investigated. Thus, the ternary systems were studied using n-alkanols
of different lengths (C5-Cl6) with the four N-acyl arginine homologs. Generally
in all systems three monophasic regions were identified: micelles, reversed micelles,
and lamellar liquid crystals. Microemulsion formation in presence of hydrocarbon
Table 13.3. Critical Micelle Concentration (CMC), Free Monomer Diffusion Coefficient
(DJ, Micelle Diffusion Coefficient (DmJ and Derived Parameters, Obtained from
PGS-NMR at 25 " C for N "-Lauroyl-Arginine-MethylEster HCI, LAM, and 6is(Args)Gemini
Surfactants
components (hexadecane, squalane, and toluene) was also studied by Solans (Solans
et al., 1990; P&, 1993). More interestingly, reversed vesicles in a system containing
lecithin-LAM/squalene/water were also described by Kunieda as biocompatible
systems to be applied in cosmetic and pharmaceutical formulations (Kunieda et al.,
1992).
Cationic liposomes are now recognized as potent vehicles for the delivery of genes
and other nucleic acids to cells. Cationic liposomes are formed from either a double
chain cationic surfactant in water or from a combination of a single cationic and
single anionic amphiphile, (catanionic systems) which at a critical concentration and
ratio can form cationic vesicles (dispersions of a lamellar phase). Combinations of
different mixtures of ALA, the hydrochloride salt of argininyl O-lauryl amide (Fig.
13.4, n = lo), and LAM with different anionic surfactants in water were studied. The
ability of these systems to spontaneously form vesicles, cubosomes (dispersions of a
cubic liquid crystalline phase), and hexosomes (dispersed particles with a hexagonal
internal structure) has recently been described. (Rosa et al., 2006).
play a role in the solution behavior and the practical properties of these surfactants.
For practical applications an important consideration is the performance of bis(Args)
under dynamic conditions. The dynamic surface tension was studied under constant
area and pulsating area for three bis(Args): C,(LA),, C,(LA),, and C,(LA),, and
compared to that of their monomer, LAM (Pinazo et al., 2001). Below the CMC,,
adsorption is quite slow, but becomes faster for thegemini surfactants between the first
and second CMC, and even faster above the CMC,. The time parameter ty the time
needed for the surface tension to drop 50% from the initial value (i.e., tl% of pure
water) compared to the equilibrium value, decreases with increasing concentration of
each surfactant, and correlates best with the dimensionless concentration, c/CMC,,
or c/CMC for LAM. The tension equilibration slows down significantly at longer
times, with ty95 being much larger than tys0 compared to what would be expected
for diffusion-controlled adsorption. Dynamic surface tensions at pulsating area are
consistent with the above inferences.
For new surfactants, one of the most studied properties to determine the possible
application scope is the foaming power. Foam stability and break up depend on a
series of complex phenomena, including hydrodynamic liquid drainage, aqueous
film thinning, and bubble coalescence. These phenomena are affected by equilibrium
surface tension, the equilibrium surface equation of state, Gibbs elasticity, surface
charge, film disjoining pressure, and also complex surface deformation and dynamic
surface tension properties (Prud’homme, 1996; Langevin, 1999). When surfactant
solutions are shaken, a large surface area free of surfactant is created. Then, the
molecules adsorb from the bulk solution, and the foam film surface is covered by
monolayers which protect against film rupture.
The ability of C (LA), C6(LA),,and C9(LA)*surfactants and their monomeric
counterpart LAM to form stable foams was measured from the maximum foam height
with solutions confined in glass vials and shaken vigorously (Pinazo et al., 2001). The
data correlate best when plotted as dimensionless foam height, H/H,, where H, is the
maximum possible height, versus the dimensionless concentration C/CMC, for LAM
or C/CMC, for bis(Args). Gemini surfactants were more efficient foam stabilizers
than LAM.
The effect of the spacer length on the shape and size of the micelles formed by C,
(LA),, C6(LA),, and C9(LA), has been studied by SAXS and PGSE-NMR and their
geometries compared with those obtained for their single chain homolog LAM (Pkrez
et al., 2007). Figure 13.9 shows the self-diffusion coefficient versus concentration
curve for the C,(LA),.
From the PGSE-NMR data the CMC, the monomer diffusion coefficient (Dmon)
and the micelle diffusion coefficient (DmiJwere obtained (Table 13.3). Deuterated
water (95%) was used to prepare these samples in order to have comparable signals
from water and surfactant. For LAM the “theoretical” value of the monomer size
Rmon(T), calculated based on the surfactant’s molecular geometry (see Table 13.3 for
further details), is similar to R,,,, the hydrodynamic radius calculated from Dmon; in
Synthesis, Aggregation Properties, and Applications of Biosurfactants Derived from Arginine 0 367
contrast, for the gemini compounds, Rmonis higher than Rmo,(T).This suggests the
presence of small aggregates, dimers, and trimers for bis(Args) concentrations between
CMC, and CMC,. These observations agree with those obtained by surface tension,
fluorescence, and conductivity (Pkrez, et al., 1998, Pinam et al., 1999).
The aggregation number has been calculated using Rmon(T),as shown in Table
13.3. The high aggregation number obtained for the bis(Args) indicates the formation
of large aggregates. This compares well with the electronic microscopy results
published previously (Weihs et al., 2005). The authors obtained electron micrographs
of these four surfactants. It was observed that at low concentrations the single chain
LAM forms spheroidal micelles while the gemini surfactants form twisted-ribbons,
flat-ribbons, and thread-like ribbons. PGSE-NMR showed that for the C9(LA)*the
intensity of the signals of the simple proton experiment decreases as a function of time.
The main peak of the spectrum, corresponding to the CH, groups of the alkyl chain,
loses one-half of the intensity after 1 day and about 80% in 6 days. This reduction in
C,( LA),Concentration
Fig. 13.9. Self-diffusioncoefficients obtained using PGSE-NMR versus concentrationfor C3(LA)2.The line
corresponds to the best fit of the two-site exchange model (equation 3 of Perez et al., 2007) Reproduced
from Perez et al., 2007 with permission of the American Chemical Society (ACS). Nomenclature of
surfactant is referred to in Table 13.1.
368 0 M.R.Infante et al.
intensity can be explained by the formation of large aggregates and the subsequent
broadening of the PGSE-NMR peaks due to the slow local anisotropic motions of the
alkyl chain, as it is usually observed in vesicular systems (Cocquyt et al., 2004).
The analysis of the SAXS data indicates that while the LAM compound forms
spherical micelles, thegemini surfactants C3(LA)2and C9(LA)2 form cylinders. The area
per molecule and water molecules per head group obtained for the gemini surfactants
are consistent with those obtained for the single chain surfactant (Table 13.2). In both
cases the value obtained for the gemini surfactants is approximately twice that of the
single chain surfactant.
For the C, (Q) a change with time of the S A X S scattering curves in the region
of the structure factor was detected; moreover, the intensity at small scattering vector,
q, decreases with increasing time. Concurrently, an increase of the viscosity of the
solution is observed with increasing time to give, at long times, a gel appearance
of the aqueous samples. These results could indicate a development of a long-range
order with cylindrical aggregates. This agrees with the PGSE-NMR observation
of a reduction of the signal intensity with the time due to the formation of large
aggregates.
In general the aggregation behavior shown by PGSE-NMR agrees with those
obtained by SAXS. Classical spherical micelles have been observed for LAM. Moreover,
larger micelles, which can be considered of cylindrical shape, have been observed for
the three gemini surfactants. This type of aggregation has been also described for bis
quaternary ammonium surfactants or bis(Quats) with short spacer chain (Danino et
al., 1995).
The microstructures formed in aqueous solutions of bis(Args) have been studied
by cryogenic-temperature-transmission electron microscopy (cryo-TEM) (Weihs al.,
2005). The effect of a perturbation of the local arrangement of polar heads, by the
spacer length, on the micellar and mesomorphic properties of surfactants in aqueous
solutions were examined. Cryo-TEM was employed to image microstructures appearing
as a hnction of spacer length and concentration. The results show that bis(Args) tend
to form aggregates of lower curvature than the corresponding monomeric surfactant
LAM. For the bis(Args) molecule with a short spacer, C,(LA), (Fig. 13.5, s = l),
the microstructures observed were spheroidal micelles that changed to thread-like
micelles and disk-like structures as the concentration was increased. The bis(Args)
molecules with longer spacers exhibited lower-curvature microstructures, mainly flat
and twisted-ribbons. Those microstructures appeared at lower concentrations as the
spacer became longer and more hydrophobic. When the spacer no longer inhibits
head-group separation (s > I), low curvature and twisted ribbons structure occur. In
all likelihood, ribbons form because of chirality of the amphiphiles and enhanced
hydrogen bonding of the spacer with the surrounding water that leads to rigid
filament-like structures.
Synthesis,Aggregation Properties,and Applications of BiosurfactantsDerived from Arginine 0 369
4ov
the aggregate type formed for these compounds at these low concentrations. The
IOlOR compound in particular showed a vesicle to ribbon transition as a function of
concentration. The scattered intensity of lOlORat concentrations as low as 0.001mM
showed that aggregates were already present in the solution. The form and size of
the aggregates was evaluated to be polydisperse vesicles at low concentrations and
ribbons at millimolar concentrations. This change in form was interpreted in terms
of charging of the bilayer. At low concentration the surfactant molecules dissociate
giving a chloride ion and the cationic species. The latter can further dissociate, with
a proton being released. The highly charged surface that forms is unfavorable and a
pK, shift as large as 7 units is observed (Hagslatt et al., 1991) producing a more acidic
behavior than that typically observed for arginine. As the concentration increases the
proportion of charged species increases with a concomitant increase in preferred area
per molecule, thus inducing a change in the curvature of the aggregates. (Pinazo et
al., 2004). This change induced by the intrinsic pH change was independently proven
by acidification of a vesicle-forming concentration. This is exemplified in Fig. 13.1 1
Synthesis, Aggregation Properties, and Applications of Biosurfactants Derived from Arginine 0 371
where the scattered intensity as a function of squared scattering vector q is shown. The
upper curve corresponds to a 0.05mM concentration in the vesicle-forming regime
while the lower curve corresponds to the same sample afier acidification with 0.04M
HCI. The evident decrease in intensity implies a reduction in the overall size of the
aggregates.
The physicochemical properties of diacyl glycerides from acetyl-arginine 12 12RAc
and 1414RAc, Fig. 13.6b, have been studied based on their ability to form monolayers
and multilayers. Because they have two hydrocarbon chains, their aggregation in
aqueous solutions starts at very low concentrations. A change in the slope of specific
conductivity versus concentration was observed for 1212RAc and 1414RAc around
0.1 mM. Because there was no significant difference between the two compounds,
it was considered that this change did not correspond to a true CMC but to some
change in the aggregate form (Pkrez et al., 2004a), probably a transformation from
vesicles to ribbons (Pinazo et al., 2004). Dilauroyl glycerol acetyl-arginine conjugates
can be considered analogs of partial glycerides and phospholipids. During their
preparation, spontaneous intramolecular acyl-migration reactions were observed and
both possible regioisomers were obtained: 1,2-dilauroyl rac- glycero-3-(Nu-acetyl-L-
arginine) (1212RAc) and 1,3-dilauroylglycero-2-(Nu-acetyl-L-arginine) (12RAc12).
1XI o-~
1X I 0"
0 2XIOl4 4~10'~ 6~10'~ 8~10'~ IxIO'~
Fig. 13.1 1. Scattered light intensity measured using Static Light Scattering as a function of the square of
the scattering vector, q, for a O.05mM aqueous solution of 1,2-di-0-decyl-glycero-3-0-(Na-arginine) 2HCl
(1010R) at 2S'C. Full symbols: aqueous solution prior to acidification. Open symbols: aqueous solution
.
acidified with 0.04M HCI The lines correspond to a vesicular model and ribbon model respectively.
Adapted from Pinazo et al., 2004. Nomenclature of surfactant is referred to in Table 13.1. (Reproduced
with permission of the PCCP Owner Societies)
372 0 M.R. infante et al.
The presence of both regioisomers influences the phase behavior. The study of the
thermotropic phase behavior in the dry state of pure 1,2-dilauroyl-rac-glycero-3-(N
a-acetyl-L-arginine) and two mixtures of both regioisomers showed that they arrange
in multilamellar stacks. When observed by optical polarized microscopy the typical
texture for smectic systems was found to coincide with a characteristic peak ordering
of the small angle x-ray scattering curves. At low temperature, the lamellar distance
of the pure 1,2-compound was compatible with that of fully extended, all-trans, alkyl
chains. For the mixtures, the difference in bilayer thickness was associated with a
tilting of the hydrocarbons chains away from the bilayer normal caused by difficulties
of packing. The higher the temperature, the shorter the lamellar distance. This change
was associated with the introduction of a kink into the hydrocarbon chain that marks
the onset of melting. Above the transition temperatures, all the samples show the
same repeat distance that would correspond to the melted hydrocarbon chains in the
lamellar liquid crystal phase (Mordn et al., 2004~ ).When comparing this behavior
with that of the monoacyl derivative, the immediate difference corresponds to the
repeat distance of the corresponding smectic gel phases. While the diacyl derivatives
present repeat distances around 4.5nm, the monoacyl derivative (Fig. 13.6a) presents
a repeat distance of only 3.8nm. This is shown in Fig. 13.12. The coexistence of two
lamellar orders is seen for the diacylated derivative, curve a. This was attributed to the
coexistence of two lamellar phases caused by the crystallization from the solvent of the
product that had two enantiomers.
The difference in repeat distances for the monoacyl and diacyl compounds is
due to the preservation of a large area per molecule while there is a strong reduction
in hydrophobic volume. Therefore, the monoacyl derivative self-assembles into a
hydrocarbon interdigitated lamellar phase while the diacylated counterpart forms a
normal bilayer arrangement. The self-assembly structures influence the headgroup
conformation that in the diacylated compound expands to a shorter length (0.8 nm)
than in the monoacylated derivative (1.1 nm). (Mordn et al., 2005). Monoacylated
derivatives melt (80°C) only slightly above the formation of the liquid crystalline phase
(70°C) while the diacylated derivatives form liquid crystals at a lower temperature
(19°C) and melt at a higher temperature (93°C). This is probably due to the more
compact packing of the chains in the monoacylated derivative compared to the
diacylated derivative in which pairs of hydrophobic chains are restricted by their polar
head binding.
Concerning the monolayer behavior, it was shown that 1,2-diacyl glycerol argin-
ine-based surfactants exhibits a behavior similar to that found in natural phospholip-
ids. The use of BAh4 (Brewster Angle Microscopy) image analysis of the inner textures
revealed that condensed phases of the dimyristoyl glycerol compounds exhibit hexatic
order. Variations in the chain length introduce similar changes as those commonly
found in lipid monolayers (Abalat et al., 2003).
The compatibility studies of the diacylated surfactants 1212RAc and 1414RAc
with phospolipids in water surface monolayers show that the behavior of 1414RAc is
Synthesis, Aggregation Properties, and Applications of Biosurfactants Derived from Arginine 0 373
Fig. 13.12. SAXS scattering intensity as a function of scattering vector modulus for a) l212RAc and b)
12ORAc dry products at 25°C. Arrows and double arrows show the position of the reflectionsattributed
to the two coexisting lamellar phases of 1212RAc. From (MorBn et al., 2004~).Nomenclature is referred
to inTable 13.1. (Reprinted with permission from the American Chemical Society (ACS))
of droplets is not usual and could be related to the ability to form lamellar type liquid
crystalline phases.
A pplicat ions
Due to their interesting properties, research on arginine-based surfactants has moved
in the last years from fundamental research to first applications in life sciences. In
this section three different biological applications are discussed antimicrobial activity,
sequestration of membrane lipopolysaccharide,and DNA compaction.
An timicrobial Properties
After years of overuse for antibiotics and biocides, Gram-positive and Gram-negative
organisms have developed a broad range of mechanisms to evade antimicrobial agents,
resulting in a potential global health crisis (Heir et al., 1995). In order to overcome
this rapid development of drug resistance, development of new classes of antimicrobial
compounds has stimulated substantial research interest (Tan el al., 2008; Ozdemir
et al., 2007). Available microbicides and formulations differ considerably in their
physical and chemical properties, effectiveness, and spectra of activity although all
these variables have to be compatible with the toxicological demands of the society.
One important milestone in our research activity is the design and development
of amino-acid-based surfactants with a low toxicity profile and antimicrobial activity.
(Infante et al., 1985, 1992). Arginine derivatives present the best activity against
bacteria due to the presence of the protonated guanidine group at a wide range of
pH. The antimicrobial activity of all type of arginine derivatives was systematically
determined in vitro on the basis of the minimum inhibitory concentration (MIC)
values (Jones et al., 1980), defined as the lowest concentration of antimicrobial agent
which inhibits the development of visible growth of microorganisms after 24 h of
incubation at 37°C. The results obtained for monocatenary arginine surfactants,
gemini arginine surfactants, and glycerolipid arginine surfactants, are summarized in
Tables 13.4, 13.5a and 13.5b, and 13.6, respectively.
Data show that all the cationic surfactants from arginine showed inhibition
activities against a wide range of microorganisms. These substances showed a
moderate activity level against bacteria with MIC values of 4-256 mglL. Structure-
activity relationships indicated that the higher the antimicrobial activity the higher
the toxicity against the Daphnia magna and the Photobacteriumphosphoreum bacteria,
determined using the procedures described by Pdrez el al. (2002b). It is worth nothing
that a consequence of these MIC values is the low toxic and ecotoxic activity of these
compounds. In general, Gram-negative bacteria were more resistant than the Gram-
positive bacteria, which can make them suitable for the subsequent biodegradability
process of these surfactants (Pirez et al., 2002b, 2005; Morin et al., 2001b). It is
known that some Gram-negative bacteria are relatively insensitive because their outer
membranes are impermeable to some hydrophobic-hydrophilic compounds (Rosen
et al., 1999) the antibacterial activity of these being higher against Gram-positive
Table 13.4. Minimum Inhibitory Concentration (mg/L) of Monocatenary Arginine Surfactants
Microorganisms ACA' ALA' AMA' AOE' ACE' ALE' CAM' LAM' MAM'
than against Gram-negative bacteria. Moreover, given that the MIC values occur at
concentrations below the CMC of surfactants in water, it may be inferred that the
surfactant monomers and not the aggregates are the species that interact with the cells
(Rosen et al., 1999). These compounds are particularly interesting as preservatives for
food and pharmaceutical formulations as well as active ingredients in dermatology and
personal care products. The antimicrobial action of cationic surfactants is based on
their ability to disrupt the integral bacterial membrane by a combined hydrophobic
and electrostatic adsorption phenomenon at the membranelwater interface followed
by membrane disorganization. The antimicrobial activity of the arginine surfactants
depends on their structure and size, the chain length being a critical structural
parameter for their effectiveness.
Gemini Sugactants
Results in Table 13.5a and 13.5b demonstrate that thegemini surfactants with spacer
chain lengths of 3-9 and alkyl chains of 10 carbon atoms, exhibit higher antibacterial
activity than the corresponding single chain homolog (CAM) whereas the bis(Args)
with alkyl chains of 12 carbon atoms exert lower activity than their homolog LAM
(Pirez et al., 2002b). When keeping the alkyl chain length constant, activity seems
to decrease with values of s25. When keeping the spacer chain length constant at 5
= 1, the relationship between the alkyl chain length and the activity is not linear,
showing a maximum for the hornologs C,(CA),. (Pirez et al., 1996, 2002b; Piera et
al., 2000).
of all the microorganisms tested (Table 13.6) (Pirez et al., 2004a, 2004b, 2005). The
power of monoglycerides from arginine does not change drastically with the alkyl
chain. For diglyceride compounds, the antimicrobial activity decreases when the alkyl
chain increases. Nevertheless, the activity of the shorter alkyl chain diglycerides, 88R
and lOlOR (average MIC value of 0.081 mM), is considerably superior to the activity
of monoglycerides (average MIC value of 0.235 mM). The 88R and lOlOR (Pkrez
et al., 2OO2a, 2005) were able to inhibit bacterial growth and even kill some bacteria
at concentration as low as 4 mg/L which is comparable to the activity showed by the
benzalkonium chloride, a well known biocide compound, and that of new analogs
of benzalkonium chlorides (Pernak et al., 1999).This demonstrates that the presence
of two alkyl chain of 8-10 carbon atoms is effective in improving the intensity of the
antimicrobial activity.
Since the perturbation of the cell membrane by these compounds is directed
primarily by physicochemical processes, model membrane systems can provide
valuable information for understanding the mechanism of action of these molecules.
The relationship between the antimicrobial activity of bis(Args), LAM, and other
agents and the physicochemical process involved in the perturbation of the cell
membrane, has been studied (Castillo et al.,2004). To this end, the interaction of
these surfactants with two biomembrane models, DPPC multilamellar lipid vesicles
(MLV) and monolayers of DPPC, 1,2 dipalmitoyl phosphatidylglycerol sodium salt
(DPPG), and ficherichia coli total lipid extract was investigated using Differential
Scanning Calorimetry (DSC) and Langmuir monolayers. DSC results show that
variations in both the transition temperature and the transition width at one-half of
the height of the head absorption peak were consistent with the antimicrobial activity
of the compounds. Penetration kinetics and compression isotherm studies indicated
that both steric hindrance effects and electrostatic forces explained the antimicrobial
agent-lipid interactions.
Sequestration of Lipopolysaccharide
Gram-negative sepsis is a common clinical problem (Gasche et al., 1995) and the
mortality due to septic shock reflects the absence of specific therapy aimed at the
underlying pathogenetic mechanisms. Cationic hydrophobic compounds can interact
with the toxic portion of the lipopolysaccaride (constituent of the outer membranes
of the gram-negative bacteria) and reduce this serious problem. Bis(Args) bind to
lipopolysaccharide and neutralize endotoxic activity in in vitro tumor necrosis factor-a
and nitric oxide release assays (David et al., 2002). The presence in thegemini structure
of two highly basic protonatable guanidinium hnctionalities separated by a spacer chain
provides for excellent recognition of the bis-phosphates on the lipopolysaccharide. In
spite of these results, bis(Args) are themselves unlikely to be of therapeutic d u e due to
their cytotoxicity. However, this class of compound offers an excellent point of departure
with which refine the design and development of less toxic analogs for the treatment of
Gram-negative sepsis.
Synthesis, Aggregation Properties,and Applications of Biosurfactants Derived from Arginine 0 38 1
DNA Compaction
DNA packaging in the living cellular environment is a very important phenomenon.
DNA compaction by polyamines, like spermidine and spermine, are examples of
events that occur in cells and are believed to be important in the regulation of cell
proliferation and differentiation. In the literature one can find many studies of DNA
compaction in aqueous solution. DNA molecules are known to undergo a discrete
conformational transition from an extended to a collapsed state by interacting with
single or double chain cationic amphiphiles. Cationic surfactants associate strongly to
DNA and produce compaction but are often toxic. Since one of our main motivations
consists of the development of new nontoxic biocompatible and biodegradable
systems, we studied the interaction of the single chain arginine-based surfactant ALA
(Fig. 13.4, n = 10) with DNA (Rosa et al., 2007). The ability of this surfactant alone
to compact DNA is compared by fluorescence microscopy studies to classical cationic
surfactants. Furthermore, toxicity studies revealed that the incorporation of ALA in
catanionic vesicle systems transformed them into cell viable systems, extending their
use to drug and gene delivery systems.
A precondensation step of DNA as a viable approach for liposome-based gene
delivery has been also addressed (Rosa et al., 2008). To the best of our knowledge,
ALA is the first cationic amphiphile based on an amino acid structure used in gene
delivery. This approach consists in both the precondensation of plasmid DNA with
an arginine-based cationic surfactant, ALA, and the incorporation of the blood
protein transferrin (Tf) into the formulations. Two cationic liposome formulations
were used, one composed of a mixture of dioleoyl trimethylammoniopropane and
cholesterol (D0TAP:Chol) and the other a pH sensitive formulation constituted of
DOTAP, Chol, 1,2-Dioleoyl phosphatidylethanolamine (DOPE), and cholesteryl
hemisuccinate (CHEMS). The lipidic composition played an extremely relevant role in
transfection efficiency. The pair D0PE:CHEMS enhanced transfection in comparison
with the complexes composed of D0TAP:Chol liposomes. Remarkable transfection
results were obtained for ALA-CatpH-complexes. Correlation between formulations
that transfect poorly and large mean sizes was made. Overall, we demonstrate that the
presence of ALA improves the transfection efficiency when conjugated with cationic
liposome systems.
Conclusions
The proposed biobased surfactants will contribute to the field of biocompatible
surfactant research and ultimately lead to the commercial advancement of biochemical
products for industrial use. Moreover, we believe that the results on the membrane
interaction studies will elucidate new functions of the surfactants, thus expanding
their list of potential applications in biochemistry. Finally, the surfactants proposed in
this review could be of great interest to field of biology, specifically as substitutes for
natural phospholipids.
382 0 M.R. Infante et al.
References
Adams, D.J.; K. Holtzmann; C. Schneider; F. Michael. Self-Assembly of Surfactant-Like Peptides.
Langmuir 2007,23, 12729-12736.
Albalat R.; J. Claret; J. IgnCs-Mullol; F. SaguCs; C. Morin, L. Ptrez; I? ClapCs, A. Pinazo. Lang-
muir monolayers of Diacyl Glycerol Amino Acid-Based Surfactants. Effect of Substitution Pat-
tern of the Glycerol Backbone, Langmuir 2003, 19, 10878-10884.
Allouch, M; J. Seguer; M.R Infante; C. Selve. Nonionic Amphiphile Compounds from Aspartic
and Glutamic Acids as Molecular Mimics of Lecithins.]. Am. Oil Chem. Soc. 1996, 7 3 , 8 7 4 6 .
Benavides, T.; V. Martinez; M. Mitjans; M.R. Infante; M.C. Morin; I? ClapCs; R. Clothier; M.I?
Vinardell. Assessment of the Potential Irritation and Photoirritation of Novel Amino Acid-Based
Surfactants by in vitro Methods as Alternative to the Animal Tests. Toxicol. 2004,201,87-93.
Birnie, C.R., D. Malamud, R.L. Schnaare, Antimicrobial Evaluation of N-Alkyl Betaines and
N-Alkyl-N,N-Dimethylamine Oxides with Variations in Chain Length. Antimicrob. Agents
Chemother. 2000, 44, 2514- 2517.
Boyat, C.; V. Rolland-Fulcrand; M.-L. Roumestant; P.H. Viallefont; J. Martinez. Chemo-Enzy-
matic Synthesis of New Nonionic Surfactants from Unprotected Carbohydrates. Prep. Biochem.
Biotechnol, 2000,30, 28 1-294.
Capone, S ; I? Walde; D. Seebach; T. Ishikawa; R. Caputo. pH-Sensitive Vesicles Containing a
Lipidic Beta-Amino Acid with Two Hydrophobic Chains. Chem. Biodivers. 2008,5, 16-30.
Castillo, J.A.; A. Pinazo; J. Carilla; M.R. Infante; M.A. Alsina; I. Haro; I? Clapes. Interaction
of Antimicrobial Arginine-Based Cationic Surfactants with Liposomes and Lipid Monolayers.
Langmuir 2004,20,3379-3387.
Clap&, E; M.R. Infante. Amino Acid-Based Surfactants: Enzymatic Synthesis, Properties and
Potential Applications. Biocatal. Biotranrform., 2002,20, 2 1 5-233.
Clapts, I?; M.C. Morin; M.R Infante. Enzymatic Synthesis of Arginine-Based Cationic Surfac-
tants. Biotechnol. Bioeng., 1999, 63, 333-343.
Cocquyt, J.; U. Olsson; G. Olofsson; l? Van der Meeren. Temperature Quenched DODAB Dis-
persions: Fluid and Solid State Coexistence and Complex Formation with Oppositely Charged
Surfactant. Langmuir 2004,20,3906-3912.
Danino, D.; Y.Talmon; R. Zana. Alkanediyl-Alpha,Omega-bis(Dimethylalkyylammonium
Bromide) Surfactants (Dimeric Surfactants).5. Aggregation and Microstructure in Aqueous-
Solutions. Langmuir, 1995, 11, 1448-1456.
Das, D.; A. Dasgupta; S. Roy; R.N. Mitra; S . Debnath; I?K. Das. Water Gelation of an Amino
Acid-Based Amphiphile. Chem-Eur J. 2006, 12, 5068-5074.
David, S . , L. Ptrez; M . R Infante. Sequestration of Bacterial Lipopolysaccharide by bis(Args) Gemini
Compounds. Bioorg. Med. Chem. Lett. 2001, 12,357-360.
Epand, R E ; M.R. Infante; T.D. Flanagan; R.M. Epand. Properties of Lipoarnino Acids Incorpo-
rated into Membrane Bilayers. Biochim. Biophys, Acta Biomembr. 1998, 1373, 67-75.
Fordedal, H.; S . Sjoblolm; M.R. Infante. Lipoaminoacid Association in the System Na-Lauroyl-
I-Arginine Methyl Ester/l-PentanollWater as Studied by Dielectric Spectroscopy. Coll. Su$ A
Physicochem. Eng. Aspect, 1993, 79,81-88.
Gasche, Y.;D. Pittet; I? Sutter. In Clinical Eiahrfor Treatment ofsepsis, Sibbald, W.J., J.L. Vincent,
Synthesis, Aggregation Properties,and Applicationsof Biosurfactants Derived from Arginine 0 383
Assembled Helical Ribbon and Tubes of Alanine-Based Amphiphiles Induced by Two Different
Formation Mechanisms. Tetrahedron 2008,G4,1301-1308.
Lindman, B.; M.C. Puyal; N. Kamenka; R. Rymden; l? Stilbs, Micelle Formation ofAnionic and
Cationic Surfactants from Fourier-Transform H- 1 and Li-7 Nuclear Magnetic-Resonance and
Tracer Self-Diffusion Studies, J Phys. Chem., 1984,88, 5048-5057.
Lozano, N:, L. PCrez; R. Pons; J.R. Luque-Ortega; M. Ferninda-Reyes; L. Rivas; A. Pinazo.
Interaction Studies of Diacyl Glycerol Arginine-Based Surfactants with DPPC and DMPC
Monolayers, Relation with Antimicrobial Activity. Cod Su$ A Physicochem. Eng. Aspect 2008,
319,196-203.
Martinez V.; E. Corsini; M. Mitjans; A. Pinazo; M.I? Vinardell. Evaluation of Eye and Skin Irrita-
tion of Arginine-Derivative Surfactants Using Different in vitro Endpoints as Alternatives to the
in vivo Assays. Toxicol. Lett. 2006,lG4,259-267.
Morin, M. C.; M.R. Infante; I? ClapCs. Synthesis of Glycerol Amino Acid-Based Surfactants. Part
1. Enzymatic Preparation of rac-1-O-(N“-Acetyl-LAminoacyI)Glycerol Derivatives. J Chem.
SOC.,Perkin Trans.I . 2001a,2063-2070.
Morin, M.C.; M.R. Infante; I? ClapCs. Synthesis of Glycerol Amino Acid-Based Surfactants. Part
2. Lipase-Catalysed Cynthesis of 1 -0-Lauroyl-rac-Glycero-3-(Nu-Acetyl-L-Amino Acid) and
1 ,2-Di-0-Lauroyl-rac-Glycero-3-(Nu-Acetyl-L-Amino Acid) Derivatives. J Chem. SOC.,Perkin
Trans.I.2002,1124-1 134.
Morin, M.C.; A. Pinazo; L. PCrez; I? ClapCs; R. Pons; M.R. Infante. Enzymatic Synthesis and
Physicochemical Characterization of Glycerol Arginine-Based Surfactants. C R Chim. 2004a,7,
169-176.
Morin, M.C.; l? ClapCs; F. Comelles; M.T Garcia; L. Perez; M.l? Vinardell; M.R. Infante. Chemi-
cal Structure/Properties Relationship in Single-Chain Arginine Surfactants. Langmuir 2001b,17,
507 1-5075.
Morin, M.C; A. Pinazo; L. Pkrez; l? ClapCs; M. Angelet; M.T. Garcia; M.l? Vinardell; M.R.
Infante. “Green” Amino Acid-Based Surfactants. Green Chem. 2004b,G,233-240.
Morin, M.C.; A. Pinazo; I? ClapCs; M.R. Infante; R. Pons. The Effect of Molecular Shape on the
Thermotropic Liquid Crystal Behavior of Monolauroylated Amino Acid Glyceride Conjugates.
J PhyJ. Chem. B, 2005,109,22899-22908.
Morin, M.C.; A. Pinazo; I? ClapCs; M.R. Infante; R. Pons. Investigation of the Thermotropic Be-
havior of Isomer Mixtures of Diacyl Arginine-Based Surfactants. Comparison of Polarized Light
Microscopy, DSC, and SAXS Observations, J Phys. Chem. B,2004c, 108, 11080-1 1088.
Ohta, A; K. Toda; Y. Morimoto; T. Asakawa; S. Miyagishi. Effect of the Side Chain of N-Acyl
Amino, Acid Surfactants on Micelle Formation: An Isothermal Titration Calorimetry Study.
CoL! Su$ B Biointerfaces, 2008,317,316-322.
Oshimura, E.; Y. Yuji; S. Kazutami. Conformational Behavior of N-Acylamino Acid Oil and N-
Acylamino Acid Surfactant in Aqueous Solution. J OIpo. Sci. 2007,5G,1 1 5-21.
Ozdemir, A.;G. Turan-Zitouni; Z.A. Kaplancikli; G. Revial; K. Guven. Sintesis and Antimicrobi-
Derivatives, Eur. J Med.
al Activity of 1-(4-Aryl-2-?hiazoyl)-3-(2-Thienyl)-5-Aryl-2-Pyrazoline
Chem., 2007,42,403-409.
PCrez, L.; A. Pinazo; M.J. Rosen; M.R. Infante. Surface Activity Properties at Equilibrium of
Synthesis, Aggregation Properties, and Applications of Biosurfactants Derived from Arginine 0 385
Novel Gemini Cationic Amphiphilic Compounds from Arginine bis (Args). Langmuir 1998, 14,
2307-23 15.
Pkrez, L.; A. Pinazo; M.R. Infante; R. Pons. Investigation of the Micellization Process of Single
and Gemini Surfactants from Arginine by SAXS, NMR Self-Diffusion, and Light Scattering. ].
Pbys. Cbem. B, 2007, I l l , 11379-11378.
Ptrez, L.; J.L.Torres; A. Manresa; C. Solans; M.R. Infante. Synthesis, Aggregation, and Biologi-
cal Properties of a New Class of Gemini Cationic Amphiphilic Compounds from Arginine,
bis(Args). L a n p u i r , 1996, 12, 5296-5301.
Pkrez, L.; M.R Infante; R. Pons; C. Mordn; M.I? Vinardell; M. Mitjans; A. Pinazo. A Synthetic
Alternative to Natural Lecithins with Antimicrobial Properties. Coll. Su$ B Biointefaces, 2004a,
35,235-242.
Pkrez, L.; I? Clap&; A. Pinazo; M. Angelet; M.I? Vinardell; M.R. Infante. Synthesis and Biological
Properties of Dicationic Arginine-Diglycerides. Nau]. Cbem. 2002a, 26, 1221-1227.
Pkrez, L.; A. Pinazo; M.T. Garcia; C. Mordn; M.R. Infante. Monoglyceride Surfactants from Argi-
nine: Synthesis and Biological Properties. New J. Cbem., 2004b, 28, 1326-1334.
Pkrez, L; M.T. Garcia; I. Ribosa; M.I? Vinardell; A. Manresa; M . R Infante. Biological Properties of
Arginine-Based Gemini Cationic Surfactants. Environ. Toxicol. Cbem. 2002b, 21, 1279-1285.
Ptrez, N.; L. Perez; M.R. Infante; M.T. Garcia. Biological Properties of Arginine-Based Glyceroli-
pidic Cationic Surfactants. Green Chem 2005, 7, 540-546.
Pernak, J.; I. Mirska; R. Kmiecik. Antimicrobial Activities of New Analogues of Benzalkonium
Chloride. Eur. J. Med. Cbem., 1999, 34,765-771.
Pes, A. Ph.D Thesis. Estudio del Comportamiento Fhico de Sistemas con Tensioactivos Deriva-
dos de Arninoicidos y Caracterizaci6n Structural de las Diversas Fases Is6tropas y Anis6tropas,
Universidad de Barcelona 1993.
Piera, E.; M.R. Infante; I? Clapts. Chemo-Enzymatic Synthesis of Arg-Based Gemini Surfactants.
Biotecbnol. Bioeng. 2000, 70, 323-33 1.
Pinazo, A,; X. Wen; L Ptrez; M.R. Infante; E.I. Franses. Aggregation Behaviour in Water of
Monomeric and Gemini Cationic Surfactants Derived from Arginine. Langmuir 1999, 15,
3 134-3142.
Pinazo, A,; L. Ptrez; M.R. Infante; E.I. Franses. Relation of Foam Stability to Solution and Sur-
face Properties of Gemini Cationic Surfactants Derived from Arginine. Coil. SuTf:A Physicorhem.
Eng. Aspect 2001,183,225-235.
Pinazo, A.; L. Ptrez; M.R. Infante; R. Pons. Unconventional Vesicle-to-Ribbon Transition Behav-
iour of Diacyl Glycerol Amino Acid Based Surfactants in Extremely Diluted Systems Induced by
pH-Concentration Effects. Pbys . Chem . Cbem . Pbs, 2004, 6, 1475-1481.
Pinazo, A.; M. Diz; A. Pts; I? Erra; M.R Infante. Synthesis and Properties of Cationic Surfactants
Containing a Disulfide Bond.]. Am. Oil Cbem. SOC.1993, 70,37-42.
Prud'homme, R.K.; S.A. Khan. In Foams: Beory, Measurements, andApplications; Prud'homme,
R.K.; S.A. Khan; Eds.; Marcel Dekker: New York, 1996, pp. 276-279.
Rosa, M.; M.R. Infante; M.G. Miguel; B.Lindman. Spontaneous Formation of Vesicles and
Dispersed Cubic and Hexagonal Particles in Amino Acid-Based Catanionic Surfactant Systems.
Langmuir, 2006; 22, 5588-5596.
386 0 M.R.Infante e t al.
Rosa, M.; M.C. Morin; M.G. Miguel; B. Lindman. The Association of DNA and Stable Catani-
onic Amino Acid-Based Vesicles. ColL Su$ A: Pbysicocbem. Eng. Aspects, 2007,301, 361-375.
Rosa, M:, N. Penacho; S. Simoes; M.C.I? Lima; B. Lindman; M.G. Miguel. DNA Pre-Condensa-
tion with an Amino Acid-Based Cationic Amphiphile. A Viable Approach for Liposome-Based
Gene Delivery. Mol. Membt. BioL, 2008,25,23-34.
Rosen, M.J. In Sufactants and Intefacial Phenomena, 2nd Edn.; Wiley-Interscience Publication:
New York, 1988, pp. 65-67.
Rosen, M.J.; L. Fei; Y.l?Zhu; S.W. Morral. The Relationship of the Environmental Effect of Sur-
factants to Their Interfacial Properties.J. Su$ Deterg., 1999, 2, 343-347.
Roy, S.; J. Dey. Effect of Hydrogen-Bonding Interactions on the Self-Assembly Formation of Sodi-
um N-( 1 1-Acrylamidoundecanoy1)-L-Serinate,LAsparaginate, and L-Glutaminate in Aqueous
Solution. J. Colloid Interjace Sci. 2007,307,229-234.
Roy, S; J. Dey. Self-organization Properties and Microstructures of Sodium N-( 1 1-
Acrylamidoundecanoy1)-L-Valinateand -L-Threoninate in Water. Bull. G e m . SOC. Japan, 2006,
79, 59-66.
Seguer, J.; J. Molinero; A. Manresa; J. CaeUes; M.R. Infante. Physicochemical and Antimicrobial
Properties of N-Alpha-Acyl-I-Arginine Dipeptides from Acid-Hydrolyzed Collagen. JSoc. Cosm.
Cbem., 1994,45, 53-63.
Solans, C.; M.R. Infante; N. Azemar; T. Warnheim. Phase Behaviour of Cationic Lipoaminoacid
Surfactant Systems. Prog. Colloid Polym.Sci. 1989, 79,70-75.
Solans, C.; M.A. Pks; N. Azemar; M.A. Infante. Lipoaminoacid Surfactants: Phase Behaviour of
Long Na'pha-AcylArginine Methyl Esters. Prog. Colloid Polym. Sci. 1990, 81, 144-150.
Stilbs, l? Fourier Transform Pulsed-Gradient Spin-Echo Studies of Molecular Diffusion. Prog.
NMRSpectrosc., 1987, 19,1-45.
Takehara, M. Properties and Applications of Amino-Acid Based Surfactants. Coll. Su$, 1989,38,
149-167.
Tausk, R.J.M.; J. Karmiggelt; C. Oudshoorn; J.T.G. Overbeek. Physical Chemical Studies of
Short-Chain Lecithin Homologues. I. Influence of the Chain Length of the Fatty Acid Ester and
of Electrolytes on the Critical Micelle Concentration. Biopbys Cbem, 1974, I , 175-183.
Tan, H.; H. Xiao. Synthesis and Antimicrobial Properties of Novel L-Lysine Gemini Surfactants
Pended with Reactive Groups. Tetrahedron Lett., 2008,49, 1759-1761.
ThorsteinssonT; M. Masson; K.G. Kristinsson; M.A. Hjalmarsdottir; H. Hilmarsson; T. Loftsson.
Soft Antimicrobial Agents: Synthesis and Activity of Labile Environmentally Friendly Long
Chain Quaternary Ammonium Compounds. J. Med. Cbem., 2003,4154 173-4 18 1.
Torres, J.L.; E. Piera; M.R. Infante; l? Clapks. Purification of Non-Toxic, BiodegradableArginine-
Based Gemini Surfactants, Bis(Args), by Ion Exchange Chromatography. Prep. Biocbem. Biotecb-
nol. 2001,31,259-274.
Tsatsaroni, E.; S. Pegiadou; G. Dememis. Synthesis and Properties of New Cationic Surfactants,
11, Odd Homologous Members.]. Am. Oil G e m . SOC.,1987, G4, 1444-1447.
Varka, E.M.; M.G. Heli; E. Coutouli-Argyropoulou; S.A. Pegiadou. Synthesis and Characteriza-
tion of Nonconventional Surfactants of Aromatic Amino Acid-Glycerol Ethers: Effect of the
Amino Acid Moiety on the Orientation and Surface Properties of These Soap-TypeAmphiphiles.
Synthesis, Aggregation Properties,and Applications of Biosurfactants Derived from Arginine 0 387
Introduction
Metalworking fluids (MWF) are “engineering materials that optimize metalworking
processes” such as metal cutting and metal forming (McCoy, 2006). The primary
function of MWF is to provide lubrication and cooling. Additionally, MWF provide
some secondary functions such as chip transport, corrosion protection, and tool/
work-piece cleaning (Childers, 2006). Worldwide more than 2 billion liters of MWF
are consumed annually (Stanford et al., 2002).
389
390 0 F. Zhao et al.
sizes less than 100 nm. The selection of surfactants to disperse the oil and other
hydrophobic additives in water is critical for producing a stable microemulsion.
Recent efforts have reported on the use of vegetable oils to replace the petroleum oil
in MWF formulations (Belluco & De Chiffre, 2004; John et al., 2004; Abdalla &
Patel, 2006; Oliveira & Alves, 2006). However, most of these formulations contain
surfactants made from petroleum feedstock and therefore are not completely biobased.
According to the German Blue Angel label standard for environmentally friendly
lubricant packages, 70% (by weight) of the components must be biodegradable and
made from biobased feedstock (Bartz, 1998). With the goal of moving towards 100%
biobased MWF formulations, biobased surfactants should also be considered. Similar
to converting petroleum-based MWF to vegetable oil-based formulations, switching
to biobased surfactants would have negligible impact on world vegetable oil demand
since about 0.1% of the worldwide crude oil consumption (i,e,, about 3.5 million
tons) is used for the production of surfactants (Saouter, 2003). Although other
biobased constituents/additives of a fully hnctional MWF could also be targeted, in
this chapter only biobased oils and surfactants are considered.
The challenge of formulating vegetable oil MWF with biobased surfactants exists
because, even for traditional petroleum-based fluids, the creation of new formulations
has long been carried out using guidelines developed through trial-and-error experience
(Childers, 2006). A systematic process to select biobased surfactants with appropriate
concentrations to disperse vegetable oil into water to form MWF microemulsion
systems with maximum stability has yet to be developed using current advances in
emulsion science. As a result this chapter develops a set of structure-based surfactant
selection guidelines and a model-based tool for optimizing surfactant concentrations
that can be used to design highly stable biobased semi-synthetic metalworking fluids
without extensive trial-and-error experiments.
Constituent w/w
Petroleum oil 15%
Sodium Detroleum sulfonate (anionic surfactant) 6.0%
Diisoprooanolamfne (co-surfactant) 8.0%
Corrosion inhibitor 6.0%
Coupler 1.5%
Fattv acids 3.7%
-- __ -
Monoethanolamine 1.9Yo
Chelatinq aqent 0.15%
Biocides 2.0Yo
water balance
392 0 F. Zhao et al.
Hl.B Method
Despite the long history in developing M W , formulating petroleum-based MWF
is still highly empirical (Childers, 2006). Consequently, it is often unclear why a
particular surfactant mixture has been selected for a given MWF, and why certain
combinations of surfactants at specific concentrations form more stable MWF than
others. Among the approaches used to quantitatively correlate surfactant structures
with their emulsification effectiveness, the HLB (hydrophile-lipophile balance)
method has achieved some degree of acceptance for selecting surfactants by M W
formulators (Myers, 2006; Canter, 2005). This method was proposed by Griffin in
1949 for the design of emulsion systems using nonionic surfactants, and has been
extended to systems with anionic and nonionic surfactants (Griffin, 1949; Myers,
2006).
In the HLB method, a number (0-40) indicative of emulsification behavior is
assigned to a surfactant. The HLB number can be calculated from the structure of the
surfactant molecules or it can be determined experimentally. For a binary surfactant
mixture, the composite HLB can be determined by (Myers, 2006):
(Eq 14.1)
wheref;, is the wt% of the first surfactant. In addition, commonly used hydrophobic
substances such as benzene, castor oil, and soybean oil are assigned nominal HLB
numbers based on emulsification experiments. For example, benzene has a nominal
HLB of 15 and paraffin has a required HLB of 10 (Myers, 2006). In theory, any
surfactant or surfactant combination with an HLB number that matches the nominal
HLB should be able to emulsify the hydrophilic substance (Rosen, 2004).
While the HLB method is widely used in industrial applications as an initial guide
for surfactant selection, it has serious limitations when applied to MWF, especially
vegetable oil M W . For instance, Zimmerman et al. (2003b) designed both mineral
oil- and canola oil-based semi-synthetic MWF using surfactant mixtures consisting
of a nonionic ethoxylated alcohol and either an anionic alkylbenzene sulfonate or
an alkyldiphenyl oxide disulfonate. The data revealed that stable canola oil-based
formulations could be achieved with a wider range of HLB values (6-18) than for
petroleum oil-based formulations (6-12). Also, at a given HLB value, it was found
that both stable and unstable formulations could be created. This is consistent with
industry experience suggesting that a single indicator of surfactant properties, such as
HLB, is not predictive ofstable MWF formulations. O n the other hand, Zimmerman
et al. (2003b) suggested that other physical characteristics, such as head and tail group
surfactant structures, could be considered when trying to predict the stability of an
emulsifier system during the design of M W formulations as discussed h r t h e r in the
next section.
Design of Vegetable Oil Metalworking Fluid MicroemulsionsUsing Biobased Surfactants 0 395
Fig. 14.1. Progressionof a micelle to a swollen micelle in oil-in-water microemulsion upon the addition
of oil to surfactant aqueous solution.
for many other microemulsion systems, in which the phase separation is more complex
(e.g., lamellar or bicontinuous phases), the ratio can exceed 100. In semi-synthetic
MWF dilutions, the molar ratio of oil to surfactant is typically around 1:1 and the
volume fraction of oil can be as low as 0.5% (Childers, 2006). Therefore, semi-
synthetic MWF microemulsions can be thought of as swollen micelles, and as shown
below, developing surfactant selection guidelines consistent with this perspective is
useful.
Experimental Method
A protocol was developed to establish concentration conditions under which a given
surfactant system was able to produce a stable MWF. First a set of standard oils was
used that included a canola oil (AgriPure 75, Cargill, Inc), a soybean oil (Technical
Grade, Cargill, Inc.), and a trimethylolpropane (TMP) tetra-ester (Priolube 1427,
Uniqema, Inc.). Canola oil, soybean oil, and synthetic esters made from vegetable
oils are representatives of three biobased feedstocks that can be used as major base
lubricant substitutes for petroleum oil. As seen in Table 14.3, all three oils have a fatty
acid composition predominantly in the 16- to 18-carbon chain length range. Since
the focus here is on surfactant selection, for convenience all MWF were formulated
to have a fixed oil concentration of 0.019 mole/L, which is a representative oil
concentration found in semi-synthetic MWF.
For each formulation of surfactants selected (see single and binary system
descriptions below), theMWF fluidsampleswereagedfor 12-15 hoursat approximately
25°C and then subjected to stability measurements based on visual transparency, light
transmittance, and oil droplet size (Byers, 2006). The measurements were interpreted
as a score with possible values limited to 1, 3, or 9, with 9 corresponding to the
most stable. For light transmittance, 0-50% was assigned 9, 50-90% was assigned
3, and 90-100% was assigned 1. For particle size, a mean droplet size of 0-100
398 0 F. Zhao et al.
Table 14.3. Fatty Acid Composition of Canola Oil, Soybean Oil, and TMP Ester
Trimethylolpropane
Canola Oil Soybean Oil tetra-ester
C160 4% 5% 0%
C180 2% 5% 1%
C181 74% 61% 58%
Fatty Acid Composition*
C182 12% 7% 24%
C183 4% 3% 10%
Other 4% 1 9% 7%
Trade Name AgriPure 75 Technical Grade Priolube 1427
Vendor Cargill Industrial Oils & Lubricants, Uniqema, New
Minneapolis, MN Castle,. Delaware
*As provided by vendors.
Reprinted with permission from Zhao, F. et al, Environ.Sci.Technol., 40,24,7930-7937,2006.
Copyright 2006 American Chemical Society.
the polysorbitan esters with C=18 and EO=20. For all these systems, the required
surfactant to oil ratio was at least 3.51. However, this high surfactant to oil ratio
would be impractical for manufacturing operations due to the high cost of surfactants
and difficulties in controlling foam and treating the waste emulsions that are associated
with high concentration nonionic surfactant systems (Schott, 1988; Byers, 2006).
The high surfactant to oil ratio required to achieve stable h4WF microemulsions
when using only one nonionic surfactant was derived from the fact that there exists
an inherent trade-off between micelle size and micelle solubilization capacity. As
discussed previously, if the surfactant tail length is held constant, the head group of
the surfactant needs to be small enough so that large micelle size and high micelle
solubilization capacity can be achieved (Myers, 2006). This way the amount of
surfactant needed to emulsify a given amount of oil is reduced. However, a small
head group size leads to low micelle solubility in water and may not produce enough
micelles to achieve a stable system for the given oil concentration. O n the other hand,
a large head group can lead to high micelle solubility but produces smaller micelles
with lower and insufficient oil solubility. Even if the oil solubility is high enough,
it may be that an impractically large amount of surfactant will be needed in order
to maintain enough of the small micelles in the system. 'Therefore, for vegetable
oil MWF, the trade-off between micelle size and micelle solubility has to be taken
into consideration when determining the size of the surfactant head. To reduce the
limitations of a given surfactant chain length or head group size for oil solubilization,
it is customary to consider a binary mixture of surfactants.
Oil
100%
Co-Surfactant Primary
Surfactant Fraction Surfactant
Fig. 14.2. Formulation triangle representing different oil/surfactant molar ratios for a system of two
surfactants and one oil. (Reprinted with permission from Zhao, F. et al, Environ. Sci. Technol., 40, 24,
7930-7937,2006. Copyright 2006 American Chemical Society)
Design of Vegetable Oil Metalworking Fluid MicroemulsionsUsing Biobased Surfactants 0 401
Given the molecular weight of oil (MYi,),primary surfactant (MW ), and co-
surfactant ( M W c ) ,the weight fraction required to make the surfactantJoi1 mixture
can be determined by:
(Eq. 14.3)
Since in practice semi-synthetic MWF are sold as concentrates and diluted 10-20
times with water before use as metalworking fluids (Childers, 2006), the mixtures
of surfactants/oil have to be diluted before stability testing. Here ASTM I deionized
water adjusted to pH=9.5 with sodium hydroxide was used to make the resultant
diluted fluids with a pH consistent with a typical value found in commercial MWF.
Given that all MWF microemulsions were tested at a fixed oil concentration of 0.019
mole/L, the weight based dilution ratio R was calculated as:
W .
R= (Eq. 14.4)
M Y i l R'b.0 19 'lCrwF
where pMW is the specific weight of the diluted MWF microemulsions, which is close
to 1000 g/L.
Figure 14.3 shows the number of observed stable formulations (out of the ten
possible formulations indicated in Fig. 14.2) when using the selected binary surfactant
systems to emulsify canola oil. For some nonionic surfactants, no stable formulation
was achieved regardless ofwhich anionic was used in combination. This indicates that
the nonionic surfactant is more important than the anionic surfactant with respect
to microemulsion stability (which is why it serves as the primary surfactant). Figure
14.3 also indicates that in order to obtain at least one stable formulation, the tail
length of the nonionic surfactant should be at least 16, which is close to the carbon
chain length of the fatty acid compositions in canola oil. These observations are in
agreement with the principles of micelle solubilization.
From Fig. 14.3 it can be seen that ethoxylated glyceryl esters, ethoxylated alcohols,
and polysorbitan esters with tail lengths longer than 16 carbon atoms serve as effective
402 0 F. Zhao et al.
Co-Surfactant
sulfo-carboxylicester C=15
alkane sulfonate C=14
alcohol ether suifate C=12,EO=4
alcohol sulfate c=12
Fig. 14.3. Number of stable formulations out of ten achieved for each surfactant combination when
nonionic surfactants with different tail lengthsare usedto emulsify canola oil (Stabilitymetrics measured
at 25°C). (Reprinted with permission from Zhao, F. et al, Environ. Sci. Technol., 40,24, 7930-7937,2006.
Copyright 2006 American Chemical Society)
primary surfactants. Based on these results, surfactants with tail lengths of at least 16
carbon atoms but different head groups were investigated for the effect of head group
size. The results in Fig. 14.4 show that stable formulations were achieved when the
head group in the nonionic surfactant had an EO number of at least 10. One possible
explanation for this is that when the head group is very small (e.g., the number of
EO units is less than lo), the nonionic surfactant is too hydrophobic, leading to the
formation of a relatively low number of large micelles and low surfactant solubility.
As a result the total amount of oil solubilization is small and no stable formulation
can be achieved. When the size of the head group is too large, however, such as for
the ethoxylated glyceryl ester with C=18 and EO=40, stable formulations were only
observed when a very high concentration of nonionic surfactant was present (e.g.,
oi1:nonionic surfactant molar ratio of at least 1:3.5). As the head group size increases,
the micelle size decreases, leading in turn to a reduced micelle solubilization capacity,
?he micelles on the whole become more hydrophilic and surfactant solubility increases,
leading to stable formulations only if high surfactant concentrations are utilized.
Effect of Co-surfactant.
Figure 14.5 shows the number of stable formulations produced when the ethoxylated
glyceryl esters, ethoxylated alcohols, and polysorbitan esters with tail lengths longer
than 16 carbon atoms are paired with various co-surfactants (either anionic or
Design of Vegetable Oil Metalworking Fluid Microemulsions Using Biobased Surfactants 0 403
Co-Surfactant
illfo-carboxylic ester C=15
L
0)
D
alkane sutfonate C=14
E,C alcohol ether suffate C=12,EO=4
alcohol sulfate C=12
fattyacid soap C=12,EO=15
alkyi aryl disuifonate C=10
Fig. 14.4. Number of stable formulations out of ten achieved for each surfactant combination when
nonionic surfactants with different head group size are used to emulsify canola oil (Stability metrics
measured at 259). Only successful surfactant combinations from Fig. 14.3 are considered. (Reprinted
with permission from Zhao, F. et al, Environ. Sci. Technol., 40, 24, 7930-7937, 2006. Copyright 2006
American Chemical Society)
nonionic) representing different head structures and tail lengths. The results illustrated
in Fig. 14.5A reveal that at most one stable formulation out of ten is achieved when
the co-surfactant used has a small head group, corresponding to low water solubility.
The results illustrated in Fig. 14.5B reveal that with the same primary surfactant, the
number of stable formulations out of the ten defined in Fig. 14.2 is larger when the
co-surfactant tail length is close to that of the primary (e.g., the tail length difference
is less than 6) than when the tail length is significantly different from the tail length
of the primary (e.g., the difference is larger than 6).
The data suggest that when the oil-soluble surfactant is applied alone, the oil
solubility is limited by the surfactant (micelle) solubility in water. When the water-
soluble co-surfactant is applied alone, the oil solubility is limited by the micelle
solubilization capacity. When a mixture of one oil-soluble and one water-soluble
surfactant is applied, molecules of the two surfactants can form mixed micelles with
increased surfactant solubility and unchanged micelle size, which in turn leads to a
significant increase in the oil solubility.
404 0 F. Zhao et al.
A.
Fig. 14.5. Effect of co-surfactant on number of stable formulations achieved from each surfactant
combination (Stability metrics measuredat 25°C). (Reprinted with permissionfrom Zhao, F. et al, Environ.
Sci.Technol., 40,24,7930-7937,2006. Copyright 2006 American Chemical Society)
Design ofvegetable Oil Metalworking Fluid MicroemulsionsUsing Biobased Surfactants0 405
Since only the anionic surfactants of the sulfonate class are currently manufactured
exclusively from petroleum feedstock, one should avoid the use the sulfonates (currently
the most popular class) when selecting biobased surfactants for vegetable oil M W . To
replace sulfonates, an anionic surfactant from the classes of fatty acid soaps, alcohol
sulfates, alcohol ether sulfates, or sulfo-carboxylic esters can be used. Alternatively,
one can use a suitable nonionic surfactant available from any of the surfactant classes
considered here, taking into consideration that MWF microemulsions formulated with
only nonionic surfactants are difficult to demulsify at the end of life and can increase
wastewater treatment cost (Byers, 2006). Consequently, these results illustrate that the
combinations of vegetable oil and biobased surfactant systems investigated here can
serve as a starting point for the development of 100% petroleum-free formulations,
and help facilitate the transition from petroleum-based MWF to renewable biobased
MWF in the machine-tool industry.
Table 14.4. Similarity of MicroemulsionStability for Three Vegetable Oils Over 10 Formulation Points Identified in Fig. 14.2 A
0
Oils compared Soybean vs Canola Canola vs. TMP Soybean vs. TMP 9
nl
1
ethoxylated alcohol
I
# of combinations=20
I I 1 I I I
1
20% 20% 0% 70% 20% 10% 0%
1
25% 16% 0% 66% 17% 17% 0%
I
pobsorbitanester
# of combinations=16
total number of systems=60
/ / I / /
I
87% 13% 0% 0%
I I I I
62% 25% 13% 0% 75% 12% 13% 0%
250
200
n
S region to calculate
9 150
z
average torque
W
L
0
0
0 2 4 6 8 10 12 14 16
depth of cut (mm)
Fig. 14.6. Representative tapping torque curve vs. depth of cut.
To minimize the effect of variation on workpiece hardness and tool wear, the
MWF to be tested were randomized over the workpiece. 25 holes were tapped for
each fluid. MWF machining performance is reported as tapping torque efficiency
which is determined by normalizing the statistical average of tapping torque over all
the tapping torque data points collected in the plateau region from all the 25 tests to
that of the benchmark fluid. This protocol eliminates the wide variation in tapping
torque test results reported in the literature (Byers, 2006) and leads to statistically
significant results that are reasonably correlated with field data (Zimmerman et al.,
2003~).
Figure 14.7 shows the tapping torque efficiency of representative formulations
developed here using surfactant systems of ethoxylated glyceryl ester (C=18, EO=20)
and sulfated alcohol (C=12, EO=4) normalized to a representative petroleum semi-
synthetic MWF with same oil molar concentration. Interestingly, the vegetable
oil-based fluids have a small, but statistically significant improvement on tapping
performance relative to the high-performance petroleum-based MWF. This is
consistent with previous observations by Belluco and De Chiffre (2001 and 2004)
and Clarens et al. (2004) and follows from the higher lubricity of vegetable oils.
Design of Vegetable Oil Metalworking Fluid Microemulsions Using Biobased Surfactants 0 409
110
n
105
80
Petroleum Canola Soybean TMP Ester
Fig. 14.7. Tapping torque efficiency of vegetable oil MWFs with biobased surfactants. (Reprinted with
permission from Zhao, F. et al, Environ. Sci. Technol., 41, 3, 1016-1023, 2007. Copyright 2007 American
Chemical Society)
recycling can cost-effectively increase MWF lifetime significantly and nearly eliminate
manufacturing process contamination from particulates and microorganisms (Skerlos
& Zhao, 2003; Rajagopalan et al., 2004).
To measure the activation energy of microemulsions using microfiltration data, a
model that describes how the coalescence kinetics affects pore-blocking behavior and
eventually the microfiltration flow rate was developed (Zhao et al., 2004, 2007 ). In
short, the microfiltration flow rate at time t can be expressed as:
(Eq. 14.5)
where Jo is the microfiltration flow rate measured under the same trans-membrane
pressure when water (instead of MWF microemulsions) is applied, c (l/s) is a
lumped parameter dependent on queuing characteristics and micelle concentration
but independent of surfactant chemistry and concentrations, AG is the coalescence
activation energy, Rmais the maximum equivalent pore radius reduction when steady-
state is achieved between adsorption and desorption of surfactants, k, is the surfactant
adsorption rate (L/s/mole), kd is the surfactant desorption rate (l/s), and c is the
total molar surfactant concentration that can be calculated by dividing the oil molar
concentration co by the molar ratio of oil to total surfactants w.
For a binary surfactant mixture, Razavizadeh et al. (2004) suggest that AG can be
expressed as:
AG=G,- [LG,+
1+e
1+8
1 G,+ KRT --In-+-
e
i+e i+e
e
I l 1n-]I1
i+e 1+e
(Eq. 14.6)
where Go is the threshold free energy when droplet coalescence occurs, G, and G, are
the nominal free energy values when the surfactants are applied individually, and K is a
proportionality constant that determines the synergisticeffect between the surfactants.
When a surfactant mixture is used, the free energy decreases in accordance with lower
surface tension at the oil-water interface. This leads to a higher AG.
In the microfiltration flow rate model above (Equations 5 and 6) there are six
parameters that are dependent on surfactant concentrations. Since these parameters
are not known in advance for a given MWF formulation/membrane combination, six
microfiltration experiments using MWF microemulsion ofdifferent w-qcombinations
are required to calibrate the model. Although only four of the six parameters (G,
G,, G,, and K) are needed to estimate the activation energy using Equation 6, six
experiments are needed since all six parameters are presented in Equation 5, either
41 2 0 F. Zhao et al.
such as couplers, extreme pressure (EP) additives, corrosion inhibitors, and chelating
agents. Research is ongoing to develop biobased alternatives for these components
(Tandy et al., 2004; Pedisic, et al., 2003).
Even with the establishment of 100% biobased formulations, there are still
major challenges to be addressed. While in service, trap oils from leaking hydraulic
systems in machine tools, as well as particulates from the surrounding environment,
accumulate in the fluid (Foltz, 2006; Dick, 2006). In addition, microbial growth and
the chemicals (i.e., biocides) used to control microbial growth in MWF can present
significant occupational health risks to exposed workers (NIOSH, 1998; Mathias,
2006; Passman, 2006; Howell et al., 2006; Sondossi et al., 1999,2001; Skerlos et al.,
2003; Kleber et al., 2004). Also, increasing attention has been paid to bacterial by-
4.0
..
= 2.0
0
I.5
I.o
products in MWF, such as endotoxins present in MWF mists (Simpson et al., 2003;
Kreiss & Cox-Gamer, 1997). In total the accumulation of particulates, tramp oil,
microorganisms, heat, water hardness, and water evaporation is known to deteriorate
the quality of metalworking operations over time until the MWF can no longer be
used (Foltz, 2006). At this point, MWF must be disposed of, leading to significant
acquisition and disposal costs. Acquisition, maintenance, and disposal operations
create such high costs that one German study estimated that MWF systems account
for 7-17% of total metals manufacturing costs, an amount significantly higher than
tool costs (Klocke & Eisenblatter, 1997).
The aforementioned economic, environmental, and occupational health concerns
have created an interest to develop sustainable metalworking fluid systems. For aqueous
systems, sustainable metalworking fluids systems minimize life cycle environmental
impact by 1) minimizing the materials, energy, and toxicity of system inputs and
outputs, and 2) maximizing MWF lifespan by maintaining physical, biological, and
chemical parameters within limits appropriate to system function (Skerlos et al.,
2001 b). Figure 14.9 shows a conceptual approach to achieving a sustainable aqueous
MWF system. Basically, a sustainable MWF system can be thought of as the union of
an environmentally benign MWF chemical formulation and an appropriate control
system for this formulation that maximizes the MWF lifetime on the shop floor.
The research presented in this chapter has introduced biobased MWF formulations
that are designed for use with microfiltration, which is one approach to achieving
a more sustainable MWF systems. For the rest of this section, microfiltration as a
contaminant control strategy that maximizes MWF lifetime is discussed.
Although oil skimming, centrifugation, pasteurization, coalescence, settling,
depth filtration, magnetic separation, and flotation technologies have been used for
contaminant removal in the metalworking industry (Dick, 2006), these technologies
have yet to be shown to economically control the range of contaminants present in
MWF. Since the dimension of contaminants present in MWF is usually larger than
0.1 pm (Skerlos et al., 2000a, 2000b; Brandt, 2006), and 10-100 times larger (or
more) than microemulsion droplet sizes, the microfiltration approach shown above
for optimizing surfactant concentrations can also be used to cost effectively extend
the usable life of MWF by removing all major contaminants while allowing only the
hnctional MWF components to pass through (Skerlos et al., 2000a, 200Ob, 2OO1a,
2OO1b; Skerlos & Zhao, 2003; Rajagopalan et al., 2004). In this way microfiltration
can be viewed as a highly advanced form of recycling that features the additional
benefit of bacterial control without a heavy reliance on biocides.
Sustainable
MWF Formulation
Benign, Renewable Ingredients
Robust to destabilization
Physiochemical
Sensor Application Contaminant Control
Recycling
Treatment Systems
Bio-sensor
Application Data Acquisition,
Artificial Intelligence
Control Algorithms
System Optimization
Equation 5 with respect to w and q, and then solving the following system of
equations:
(Eq. 14.7)
The solution to this set of equations yields the surfactant concentrations that maximize
the microfiltration flux of a given microemulsion at a fixed oil concentration. The
solution to the system of equations in Equation 7 results in the following iteration
formulas that yield optimal values of anionic:nonionic molar ratio (q*) and
oihrfactant molar ratio (w*)for maximizing microfiltration flux:
(Eq. 14.8)
and,
- -
4k,RL, X c , ( l + O * )
RTIn *
.- x @ + k, l k , C , ) I G , + G , ) @ R ~ , c ,- a *- k, lk,c$
* 1+9'
0 =- t
9 w , +G,)
- -
(Eq. 14.9)
Again, six parameters (G, G,, G, , K, R",,, and k,/ k) are estimated from
microfiltration flow rate data collected in six microfiltration experiments using MWF
microemulsion of different w-q combinations. After estimating the six parameters
from the microfiltration flux data of the six different surfactant concentration
combinations within the stable region (Fig. 14.8), Equations 8 and 9 are used to
calculate the surfactant concentrations that lead to highest flux.
For MWF microemulsions based on disulfonate/glyceryl ester, the optimal
oi1:surfactant ratio (09 is 3.7 and the optimal anionic:nonionic ratio (ex) is 0.44
(this formulation is called YO in Fig. 14.10). For YO,the flux predicted by the model
Design of Vegetable Oil Metalworking Fluid MicroemulsionsUsing Biobased Surfactants0 41 7
4.0
-
.-
0
2.0
I.5
1.o
agrees with experimental results to within 7%, with flux of 3110 LMH (L/m2/
hr). This flux is higher than any of the six formulations originally selected within
the stable region, which have flux values ranging from 1800 LMH to 2810 LMH.
These values are well above the "rule-of-thumb" reference value of 100 LMH that
generally leads to the economical application of microfiltration to MWF for advanced
recycling and contaminant control (Skerlos & Zhao, 2003). Moreover, flux of the
optimized formulation YO is about three times higher than that of the benchmark
semi-synthetic MWF, which is petroleum oil based with the composition given in
Table 14.1. Economic analysis of general microfiltration implementation strategies
for MWF recycling has indicated that the increase of microfiltration flux can lead to
significant reductions of overall system cost (Skerlos & Zhao, 2003).
418 0 F. Zhaoet al.
Conclusions
While it would seem desirable to eliminate MWF in all manufacturing processes to
address their economic, environmental, and occupational health concerns, research
over the past two decades has demonstrated that this goal is extremely challenging and
perhaps impossible (Skerlos et al., 2008). Although machining under a completely
dry condition, or applying sprays of compressed air and a small amount of oil
(called minimum quantity lubrication), has proven successful in certain operations,
these approaches still face significant challenges in severe operations (Filipovic &
Stephenson, 2006; Li & Liang, 2007). For instance, in machining processes such
as titanium alloy milling, gun drilling, and compacted graphite iron boring, the
absence of cooling provided by MWF results in accelerated tool wear, residual stresses,
dimensional errors, and poor surface finish (Skerlos et al., 2008). While research
proceeds with the goal of addressing these concerns without the use of MWF, aqueous
MWF microemulsions will continue to be used all over the world. This chapter has
illustrated that combining biobased formulations with advanced recycling techniques
can prove to be an economically and environmentally sound approach until such
time as superior M W replacements with lower environmental and health costs are
found.
Acknowledgements
The authors would like to express their appreciation to the faculty, graduate students,
and undergraduate students who contributed to the results presented here: Professor
Julie Zimmerman (Yale, University), Professor Andres Clarens (University ofVirginia),
Ye Eun Park, Carlos Aguilar, Heather Landis, Ashley Murphree, Ashley Earle, David
Delind, and Marcy Urbance. The authors also appreciate the financial support from
the US National Science Foundation (DMII-0084796 and DMII-00935 14),the US
Environmental Protection Agency (R831457), and Ford Motor Company.
References
Abdalla, H.S.;S. Patel. The Performance and Oxidation Stability of Sustainable Metalworking
Fluid Derived from Vegetable Extracts. Proc. Inst. Mech. Eng. Part B. J Ens Man. 2006, 220,
2027-2040.
Adamson, A.W.; A.P. Gast. Physical Chemistry ofsurfaces, 6th edition, John Wiley & Sons, Inc.,
New York, 1997.
Axinte, D.; M. Axinte; J.D.T. Tannock. A Multicriteria Model for Cutting Fluid Evaluation. Proc.
Inst. Mech. Eng., Part B:]. Eng. Man. 2003,217, 1341-1353.
Ash, M.; E. Dohlrnan;. Oil Crops Situation and Outlook Yearbook 2005, Economic Research Ser-
vice, United States Department of Agriculture, Washington, DC, 2005.
Design of Vegetable Oil Metalworking Fluid Microemulsions Using Biobased Surfactants 0 4 19
Barn, W.J. Lubricants and the Environment. Trib. Internat. 1998,31, 35-47.
Belluco, W.; L. De Chiffre. Performance Evaluation of Vegetable-Based Oils in Drilling Austenitic
Stainless Steel. ].Mat. Proc. Tech. 2004, 148, 171-176.
Belluco, W.; L.De Chiffre. Testing of Vegetable-Based Cutting Fluids by Hole M&ing Operations,
Lub. Eng. 2001,57, 12-16.
Brandt, RH. Filtration Systems for Metalworking Fluids. Metalworking Fluids, 2nd Edn., Byers,
J.P Ed.; Taylor & Francis: New York, 2006; pp. 231-252.
Byers, JJ? Laboratory Evaluation of Metalworking Fluids. Metalworking Fluid, 2nd Edn., Byers,
JJ? Ed.; Taylor & Francis: New York, 2006; pp. 147-174.
Canter, N. H L B A New System for Water-Based Metalworking Fluids. Trib. Lubr. Tech. 2005,
GI,10-12.
Cheryan, M. Ultrafiration and Microf%ration Handbook, 2nd Edition, CRC Press, Boca Raton,
FL, 1998, Chapter 1, pp 1-30.
Childers, J.C. The Chemistry of Metalworking Fluids. Metalworking Fluid, 2nd Edn. Byers, JJ?
Ed., Taylor & Francis: New York, 2006; pp. 127-146.
Clarens, AX; J.B. Zimmerman; H.R. Landis; K.F. Hayes; S.J. Skerlos. Experimental Comparison
of Vegetable and Petroleum Base Oils in Metalworking Fluids Using the Tapping Torque Test.
Proceedings of the japan/USA Symposium on Flexible Manufacturing, Thomas Kurfess Ed.; Georgia
Institute ofTechnology. Denver, Colorado, July 19-21,2004.
Cohen, H.; E.M. White. Metalworking Fluid Mist Occupational Exposure Limits: A Discussion
of Alternative Methods. /. Occ. Environ. Hyg. 2006,3, 501-507.
De Chiffre, L.; W. Belluco. Comparison of Methods for Cutting Fluid Performance Testing. Ann.
CIRP 2000,49, 5 7 4 0 .
Dick, R.M. Recycling of Metalworking Fluids. Metalworking Fluids, 2nd Edn. Byers, J.E Ed.
Taylor & Francis: New York, 2006; pp. 279-300.
Filipovic, A.; D.A. Stephenson. Minimum Quantity Lubrication (MQL) Applications in Automo-
tive Power-Train Machining. Mach. Sci. Tech. 2006, IO,3-22.
Fletcher, ED.; J.S. Morris. Turbidity of Oil-in-Water Microemulsion Droplets Stabilized by Non-
ionic Surfactants. ColL Su$ A.: Physiocochem EngAspect 1995, 98, 147-154.
Fletcher, PD.; K. Suhling. Interactions Between Weakly Charged Oil-in-Water Microemulsion
Droplets. Langmuir. 1998, 14,4065-4069.
Foltz, G.J. Metalworking Fluid Management and Troubleshooting. Metalworking Fluid, 2nd
Edn., Byers, J.E Ed., Taylor & Francis: New York, 2006; pp. 253-278.
Griffin, W.C. Classification of Surface Active Agents by HLB. ]. SOC.Cosm. Chem. 1949, I ,
31 1-326
Hauthal, H.G. CESIO 2004-Dynamic Surfactants and Nanostructured Surfaces for an Innova-
tive Industry, Gth World Surfactants Congress, Berlin, 2004. SOW-J. 2004, 130, 3-17.
Hiemenz, EC.; R Rajagopalan. Principles ofcolloid and Sulface Chemistg grd Edn. Marcel
Dekker: New York, 1997. pp. 355-404.
Ho, W.S.W.; K.K. Sirkar. Membrane Handbook. Van Nostrand Reinhold: New York, 1992. pp.
1-16.
420 0 F. Zhao et al.
Houck, C., et al., “A Genetic Algorithm for Function Optimization: A Matlab Implementation,”
Technical Report No. NCSU-IE TR 95-09, published by North Carolina State University,
Raleigh, NC. 1995. pp 1-14
Howell, J.K.; WE. Lucke; E.M. White. Health and Safety Aspects in the Use of Metalworking
Fluids. Metalworking Fluid, 2nd Edn. Byers, J.l? Ed. Taylor & Francis: New York, 2006; pp.
337-376.
Institute of Advanced Manufacturing Sciences Incorporated, IAMS. Pollution Prevention Guide to
Using Metal Removal Fluid in Machining Operations. IAMS: Cincinnati, OH, 1995. pp. 46-58.
Iowa Waste Reduction Center. Cutting Fluid Management for Small Machining Operations: A Prac-
tical Pollution Prevention Guide, grd edn. University of Northern Iowa, 2003. pp. 5-13.
John, J.; M. Bhattacharya; PC. Raynor. Emulsions Containing Vegetable Oils for Cutting Fluid
Application. Coll. Sut$ A.: Physiocochem Eng Aspect 2004,237, 141-1 50.
Kibbey, T.; K.F. Hayes. Predictive Numerical Thermodynamic Model of Mixed Nonionic Surfac-
tant Sorption in Natural Systems. 2. Application to Broadly Distributed Mixtures.J Coll. Inter.
Sci. 1998, 197, 210-220.
Kleber, M.; W. Follmann; M. Blaszkewicz. Assessing the Genotoxicity of Industrial Cutting Fluids
under Conditions of Use. Toxicol. Lett. 2004, 151,211-217
Klocke, F,; G. Eisenblatter. Dry Cutting. Ann. CIPl? 1997, 46 1-8.
Kreiss, K.; G. Cox-Ganser. Metalworking Fluid-Associated Hypersensitivity Pneumonitis: A
Workshop Summary. Am. J Ind. Med. 1997,32,423-432.
Li, K-M; S.Y. Liang. Performance Profiling of Minimum Quantity Lubrication in Machining.
Internat. J Adv. Man. Techn. 2007, 35, 226-233.
Mang, T. Lubricants and Their Market. Lubricants and Lubrication, 2nd Edn. Mang, T.; W Dresel
Eds., Wiley-VCH: Berlin, 20071 pp. 1-6.
Mathias, C.G.T. Contact Dermatitis and Metalworking Fluids. Metalworking Fluid, 2nd Edn.
Byers, J.P Ed. Taylor & Francis: New York, 2006; pp. 325-336.
McCoy, J.S. Introduction: Tracing the Historical Development of Metalworking Fluids. Metal-
working Fluid, 2nd Edn. Byers, J.l? Ed. Taylor & Francis: New York, 2006; pp. 1-18.
Morrison, I.D.; S . Ross. Colloidal Dispersions: Swpensions, Emulsions, and Foams. John Wiley &
Sons: Inc., New York, 2002. pp. 420-455.
Myers, D. Surfctant Science and Tecbnologv, jrdEdn. Wiley-Interscience: Hoboken NJ, 2006; pp.
1-28.
NIOSH, National Institute of Occupational Safety and Health, Criteriafor a Recommended Stan-
dard: Occupationalfiposure to Metalworking Fluid. NIOSH: Cincinnati, OH, 1998; Chap.4-5.
http://www.cdc.gov/ nioshl98- 1O2.html
Oliveira, J.F.G.; S.M. Alves. Development of Environmentally Friendly Fluid for CBN Grinding.
CIRP Ann., 2006,55,343-346
Passman, F.J. Microbiology of Metalworking Fluids. Metalworking Fluid, 2nd Edn. Byers, J.l? Ed.
Taylor & Francis: New York, 2006; pp. 195-230.
Patel, M. Surfactants Based on Renewable Raw Materials: Carbon Dioxide Reduction Potential
and Policies and Measures for the European Union. J Ind. Ecol. 2004, 7, 47-62.
Design ofvegetable Oil Metalworking Fluid Microemulsions Using Biobased Surfactants 0 42 1
Paul, B.K.; S.P. Moulik. Microemulsions: An 0verview.J Dispers. Sci. Technol. 1997, 18,
30 1-367.
PediSiC, L.; M. Sarit; S. Bielen. Application Possibilities of New AW/EP Additive Types in Water-
miscible Metalworking Fluids. Ind. Lubr. Trib. 2003,55, 23-31.
Rajagopalan, N.; T. Rusk; M. Dianovsky. Purification of Semi-synthetic Metalworking Fluids by
Microfiltration. Eib. Lub. Tech. 2004, GO, 38-44.
Raynor, P.C.; S.W. Kim; M. Bhattacharya. Mist Generation from Metalworking Fluids Formu-
lated Using Vegetable Oils. Ann. Occ. Hyg.2005, 47,283-293.
Razavizadeh, B.M.; M. Mousavi-Khoshdel; H. Gharibi; R. Behjatmanesh-Ardakani; S. Javadian;
B. Sohrabi. Thermodynamic Studies of Mixed IoniclNonionic Surfactant Systems.]. Colloid
Interface Sci. 2004,276, 197-207.
Rose, B.; P. Rivera. Replacement ofPetroleum Based Hydraulic Fluid with a Soybean BasedAlterna-
tive. Sandia National Laboratory Technical Report DE-ACD4-94Al85000. Sandia National
Laboratory: Albuquerque, NM, 1998. pp. 1-10,
Rosen, M.J. Surfactants and Interfacial Phenomena. grd Edn. John Wiley & Sons, Inc.: Hoboken,
New Jersey, 2004. pp. 1-33.
Saouter, E. Natural and Synthetic Surfactants:Which One Is Better? P&G Technical Article,
2003.
Schott, H. Foaming of Nonionic Surfactant Solutions: Effect of Surfactant Concentration and
Temperature.]. Am. OilChem. SOC.1988,65, 816-819.
Shah, D.O.; R.S. Schechter. Improved Oil Recovery by Surfactant and Polymer Flooding. Academic
Press: New York, 1977. pp. 28-54.
Sheng, P.S.;S. Obenvalleney. Life-Cycle Planning of Cutting Fluids-A Review. ASMEJ Manuf:
Sci. Eng. 1997,119,791-800
Shinoda, K.; B. Lindman. Organized Surfactant Systems: Microemulsions. Langmiur 1987,3,
135-149.
Simpson, A.T.; M. Stear; J.A. Groves; M. Piney; S.D. Bradley; S. Stagg; B. Crook. Occupational
Exposure to Metalworking Fluid Mist and Sump Fluid Contaminants. Ann. Occ. Hyg.2003, 47,
17-30
Skerlos, S.J.; A.F. Clarens; K.E Hayes; E Zhao. Current Advances in Sustainable Metalworking
Fluids Research. Inter. J Swt. Man. 2008, in print.
Skerlos, S.J.; F. Zhao. An Optimization Approach to Implementing Microfiltration for Microbial
Control and Recycling in Metalworking Fluids.]. Man. Syst. 2003,22, 202-210.
Skerlos, S.J.; L.A. Skerlos; C.A. Aguilar; F. Zhao. Expeditious Identification and Quantification of
Mycobacteria Species in Metalworking Fluids Using Peptide Nucleic Acids. J Man. Syst. 2803,
22, 137-147.
Skerlos, S.J.; N. Rajagopalan; R.E. DeVor; S.G. Kapoor; V.D. Angspatt. Microfiltration of Poly-
oxyalkyleneMetalworking Fluid Additives Using Aluminum Oxide Membranes./. Man. Sci.
Eng. Trans. ASME. 2001a, 123,692-699.
Skerlos, S.J.; I! Adriaens; K. Hayes; A. Rihana; K. Kurabayashi; S. Takayama;J. Zimmerman; F.
Zhao. Challenges to Achieving SustainableAqueous Systems: A Case Study in Metalworking
422 0 F. Zhao et al.
Zimmerman, J.; K.F. Hayes; S.J.Skerlos. Influence of Ion Type and Concentration on the Emul-
sion Stability and Machining Performance of Two Semi-Synthetic Metalworking Fluids. Environ.
Sci. Technol. 2003%38, 242-2430.
Zimmerman, J.; A.F. Clarens; K.F. Hayes; S.J.Skerlos. Design of Hard Water Stable Emulsifier
Systems for Petroleum and Biobased Semi-Synthetic Metalworking Fluids. Environ. Sci. 2chnol.
2003b, 37, 5278-5288.
Zimmerman, J.; S . Takahashi; K.F. Hayes; S.J. Skerlos. Experimental and Statistical Design Con-
siderations for Economical Evaluation of Metalworking Fluids Using the Tapping Torque Test.
Lubr. Eng, 2 0 0 3 ~59, 17-24.
Polyol and Amino Acid-based
Biosurfactants, Builders, and
Hydrogels
Kenneth M. Doll and Sevim 2. Erhan
food and lndustrid Oil Research Unit; National Center for Agricultural UtilizationResearch, United States
Department ofAgriculture,Agricultural Research Service, 18 75 N. UniversitySt., Peoria, /L 6 1604 USA
Introduction
A modern detergent is a meticulously balanced blend of surfactants (to solubilize and
emulsify), builders (to remove hard water deposits), bleaches (to treat stains), and
other ingredients (Adami, 2004). According to the Soap and Detergent Association,
the combined global market of laundry, personal care, and dishwashing detergent was
valued at $70 billion in 2005 with growth projected to $78 billion by 2010 (SDA,
2007b). The United States alone spent over $12 billion on household detergents in
2005 (SDA, 2007b). With this market size comes tremendous competition where
over 200 companies are competing for consumer dollars (Spitz, 2004). Successful
products not only have to work, but they must have some innovative advantage over
competitive offerings (McCoy, 2006b).
In today’s world, the use of natural products in the detergent industry makes
sense for environmental, economic, and regulatory reasons (Hogue, 2006; McCoy,
2008; Short, 2007; Suszkiw, 2007). Originally, all surfactants were made from natural
materials (Rosen, 2004; Spitz, 2004), where these carboxylate-based soaps were used
for thousands of years. However, the 20rhcentury saw the rise of synthetic detergents
which gave superior performance in hard water due to their better foaming capability
and lower tendency to leave deposits. Other advantages of synthetic detergents
were favorable pH stability, ease of manufacture, and cost. Due to these advantages,
synthetic detergents had captured the majority of some markets before 1955. This
trend continued to the end of the 20th century, where the European production of
traditional soap dropped nearly in half between 1972 and 2000 (Berna et al., 2004).
However, there are reasons why the trend may be slowly changing.
425
426 0 K.M. Doll and S.Z. Erhan
Environmental Factors
Water Quality
Along with their success, synthetic detergents have had negative environmental
impacts. The contamination of major rivers in the United States and Europe was
a high profile example of this problem (Sullivan & Evans, 1968). At the height
of this problem in the early 1960s, the water of the Illinois and Mississippi rivers,
large rivers in the United States, was contaminated to the point where foaming was
becoming a serious problem (Barber et al., 1995). The mean value of the surfactant
concentration of samples taken from the Illinois River between 1959-1965 was 0.54
mg L'. Following a voluntary industry switch to more biodegradable surfactants in
1965, the surfactant level measured in the river went down to a more reasonable value
of 0.05 mg L-' by 1968 (Sullivan & Evans, 1968; Sullivan & Swisher, 1969). Further
improvements were mandated by the Federal Water Pollution Control Act of 1972
and the series of following amendments, Clean Water Acts, and Water Quality Acts.
Detergent builders, which function to remove metal ions from solution and inhibit
the formation ofprecipitates, have had negative high profile water quality issues as well.
The common builders in the detergents of the 1950s and 1960s were polyphosphate
compounds such as sodium diphosphate and sodium triphosphate. 'The problem with
these compounds was not that they couldn't be utilized by the environment, but that
they were too readily used by the environment. In many environmental systems, a low
level of inorganic phosphate is a key factor in regulating algal growth. When additional
phosphate is added to the environment, algal growth can increase dramatically. 'The
resultant algae decay causes low oxygen levels which can destroy desirable aquatic life
in a process called eutrophication. Because manmade sources contribute 75% of the
inorganic phosphate to the environment (Ferguson, 1968), elimination of phosphates
from synthetic detergents became a target of environmentalists and regulators.
Starting in 1972, major detergent manufactures reduced the level of phosphate used
(Knud-Hansen, 1994) and in 1994, they completely discontinued its use in laundry
detergent in the United States (Zini, 1995). Reduction of phosphate in dishwashing
detergents has been slower, but a recent policy of'The Soap and Detergent Association
will limit phosphate content to 0.5% or less, in all detergents made after July 2010
(SDA, 2007a, 2007b).
Because a detergent builder serves several functions, direct replacement of
phosphate with a single additive has not been achieved. Instead, a combination of
ion-sequestering chelators (Fig. 15.1) with crystallization-suppressing polymers, like
polyacrylic acid, are.used. However, these substitutes also have their own problems.
Polyacrylic acids are usually not biodegradable and some chelators, like nitrilotriacetic
acid, are suspected human carcinogens. There is clearly a further need for innovation
in this area.
Polyols and Amino Acids 0 427
Sodium tripolyphosphate
Sodium nitriloacetate (NTA)
Fig. 15.1. The structure of some common detergent builders. Note that effective detergent builders
usually have two or more metal bonding oxygen groups in close proximity to each other.
Biodegradation
Biodegradation can be classified as either primary biodegradation; which means that
the substance degrades to the point where its physical and chemical properties are
altered, or ultimate biodegradation; where the substance is reduced to CO,, H,O,
and inorganic minerals (Struijs & Stoltenkamp, 1994). Biodegradation of a material
will depend on several factors including its chemical structure, its molecular weight,
and its solubility. The poor biodegradability of surfactants which contain branching
in their alkyl chain, such as branched alkylbenzene sulfonates (ABS; Fig. 15.2), caused
the water quality problems in the early 1960s. This problem is made even worse if
the branching is adjacent to the terminal methyl groups in the hydrophobe (Rosen &
Dahanayake, 2000). Poor biodegradability caused an accumulation of the compounds
in the water which were not effectively removed by the water purification systems of
the era. A switch to linear compounds, such as linear alkylbenzene sulfonate (LAS; Fig.
15.2), helped alleviate this problem. It was shown to be is 5-8 times more biodegradable
than ABS (Sullivan & Evans, 1968), a finding which has been confirmed in recent
studies where LAS was found to have a high rate of primary biodegradability in sea
water and an even higher rate in river water (Perales et al., 2007).
The use of natural product based surfactants is an indicator, but not a guarantee,
that the resultant material will be biodegradable. Soybean oil itself is completely
biodegradable and shows greater than 90% biodegradation in less than 5 days under
soil-emulating conditions. Soybean oil which has been modified via heat bodying also
428 0 K.M. Doll and S.Z.Erhan
degrades, but at a slower rate (Erhan et al., 1997). However when larger polymeric
substances are made from soybean oil, the type of intramolecular covalent linkage
is important. Biodegradation studies of soybean oil based polymers show that
biodegradability is achieved if degradable linkages such as ester bonds occur, but not
if the linkages are poorly-degradable, such as amine linkages (Shogren et al., 2004).
This points to a strategy where soybean oil based hydrophobes for surfactant use
should be either linear, or have hydrolyzable bonds in their structure. It also suggests
that the biodegradation of a surfactant is a parameter which must be studied before
any material should be discharged into environment.
The polymeric materials used to replace functions of phosphate builders in
detergents also have biodegradability issues. For example, sodium polyacrylate is an
effective dispersing agent and metal ion chelator, with poor biodegradability, whereas
citric acid has rapid biodegradability but it is a poor dispersing agent and only a
fair chelator. The manufacturers must weigh the environmental cost of using a small
amount of a non-biodegradable compound with high performance properties, or
using a larger amount of a biodegradable compound with lower performance (McCoy,
2008).
Detergent builders are another product type where a biobased source of monomer
is suggestive of, but not necessarily a guarantee of, biodegradability. An interesting and
Polyols and Amino Acids 0 429
well studied example is polyaspartic acid. This polymer is formed by the condensation
of aspartic acid, a natural amino acid, which can form a simple linear peptide of
high biodegradability. Because it has two available carboxylate groups, aspartic acid
yields many polycondensation products (Scheme 15.1). When a simple acidic catalyst
like phosphoric acid is used (Chang & Swift, 1999; Swifc, 2002), both carboxylate
groups condense with the same amine group forming an imide structure which
can be hydrolyzed to form linear polyaspartic acid. However, when the synthesis is
conducted thermally without catalyst, the carboxylates can react with different amine
groups forming a branched structure. The branching density of this structure can be
high enough that the product loses biodegradability (Roweton et al., 1997; Swift,
2002). Because of the hidden complexity in this seemingly simple reaction, work on
these polymerization reactions is still being researched. In a recent development, a salt
of aspartic acid was incorporated into the polymerization. This change has resulted
in the production of more soluble products which eases further derivatization of the
polymer (Sikes, 1999; Swift & Redlich, 2005).
Toxicity
Both acute toxicity and environmental toxicity are important considerations for
the surfactant and detergent industries. Alcohol ethoxylates are common nonionic
surfactantsconsumed in the UnitedStatesataboutone-halftherateofanionicsurfactant
consumption. The United States consumed 0.380 MMT of anionic surfactants in
2005 (SDA, 2007b). Both alcohol ethoxylates and akylphenol ethoxylates, depicted
in Fig. 15.3, biodegrade in a relatively short time (Perales et al., 2007). However,
n+m
Scheme 15.1. The synthesis of either linear polyaspartic acid (top),or branched polyaspartic acid/
polysuccinimide (bottom).
430 0 K.M. Doll and S.Z. Erhan
Fig. 15.3. Common nonionic surfactants including alcohol ethoxylates and ester ethoxylates. Shown are
Brij 30. and Mryj 45*, products of Croda Industrial Specialties* formerly Uniqema. (top two structures,
respectively). A nonylphenol ethoxylate (bottom) which has a nonylphenol group that can be released
during biodegradation. R refers to an n-alkyl group.
Polyolsand Amino Acids 0 43 1
Energy Consumption
Another major environmental consideration for the detergent industry is to reduce the
amount of energy consumed by washing machines. 'The United States Department of
Energy estimates that about 90% of the energy consumed in washing is used to heat
water (DOE, 2008). Their Energy Star program recommends that consumers operate
washing machines using warm water rather than hot water and use high efficiency
detergents to significantly reduce energy consumption. Proctor and Gamble has
capitalized on this environmental concern by recently developing the Tide Coldwater"
brand. Other companies have patented similar products which may lead to market
growth in this area Achieving equivalent detergent performance using colder water
is difficult for two reasons. First, a surfactant's ability to solubilize oily material is
significantly increased with temperature, due to higher solubility and soil melting.
Second, the deposits which form from hard water are an increased problem in cold
water.
Economic Factors
Of course one needs to keep sight of the reasons which facilitated the rise of synthetic
detergents in the first place, high performance and traditionally low cost. However,
the large increase of oil prices have left a smaller and more costly supply of n-paraffins
for the industry resulting in price increases. For example, over the last year, the LAS
price has increased from = $0.50 per pound to over $0.71 per pound (Graff, 2008).
Although biobased feedstocks are also seeing cost increases in 2008 as well, the larger
increase for petrochemical cost has opened an opportunity for biobased surfactants.
The market has shown that consumer friendly green technologies can be
successful. For example, Seventh Generation (Burlington, Vermont, USA), a company
specializing in environmentally friendly cleaners, experienced 28% growth in 2006
and now commands nearly $100 million in annual sales (Case, 2007; Watkins, 2007).
More traditional detergent companies are also entering the green market. Clorox's
Green Works",and Proctor and Gamble's Pure Essentials" are product lines with big
potential value in 2008. Dow Chemical Company is also supporting the demand
by selling customers on its EcosurP surfactants, made from naturally derived palm
kernel oil.
The Soap and Detergent Association has backed sustainable development as
well, by maintaining a "Sustainability Central" online forum where companies can
432 0 K.M. Doll and S.Z. Erhan
Pressure transducer
Stainless steel .
reactor body
Fig. 15.4. A picture of the reactor used in the synthesis of polyaspartic acid in supercritical CO,.
sorbitan isosorbide
sorbitol
Table 15.1. The Extent of Reaction at Which an Insoluble Gel i s Expected t o Form in
Thermal Syntheses of L-Aspartic Acid:D-sorbitol Copolymersa
Extent of Reactionwhere Gelation is
Molar Ratio L-Aspartic Acid:D-Sorbitol Expectedb(%)
100
5:l 100
%e calculationswere done using the Carothers equation (Stevens, 1990).
bThepercent conversion of reactive groups at which an insoluble gel is expected to form. The
product a t this point will have a theoretical degree of polymerization approaching infinity.
436 0 K.M. Doll and S.Z. Erhan
1,4-anyhdrosorbitan
D-sorbitol
Scheme 15.4. A possible structure of the citric acid sorbitol copolymer. Anhydride structures of both
starting materials can form resulting in a complex final product.
438 0 K.M. Doll and S.Z. Erhan
Table 15.2.The Amount of Residual Acid and the Water Solubility and Water Absorbance
Indices o f Citric Acid-Sorbitol Copolymers, Synthesized under Vacuum at 150°C for 4 h
Remaining Water
Carboxylate Water Solubility Absorbance Water
Molar Ratio Citric Grows a at Room Index Without DH Absorbance Index
Acid:D-Sorbitol (meq. q-l) Temperature (W) Adjustmentb at pH 7
1:l 2.6 66 3.4 2.9
2:1 3.9 51 5.1 11.8
3:1 6.9 54 6.0 9.4
4:1 9.5 100 ND ND
5:l 10.2 100 ND ND
61 10.8 100 ND ND
"Carboxylategroups available per gram of polymer material as determined by titration of
samples with 0.2 M NaOH solution
bSamplesof the products were weighed and then suspended in water for 30 minutes.They
were then centrifuged, the unabsorbed water was removed with a pipette and the sample
was reweighed.The water absorbance index was calculated as the ration of the mass of wet
sample over the weight of the dry sample.
T h i s procedure was identical to the water absorbance index, except the solution was adjusted
to pH 7 with 1 .O M NaOH solution before stirring in the solution for 30 minutes.
pipette. The sample was weighed, dried under vacuum, and weighed again. The ratio
of these weights is the water absorbance index. This procedure was also repeated on
samples where the solution had been adjusted to pH 7 with a 1.O M sodium hydroxide
solution. The results (Table 15.2) show that the copolymer is capable of absorbing
almost 10 times its weight in water. Although this value is too low for application as a
superabsorbing polymer, it is of significant interest for agricultural or pharmaceutical
applications.
In another method (Shogren et al., 2007; Swift et al., 2007), reactive extrusion
was used in place of the vacuum oven synthesis. Citric acid, or sodium salts of
citric acid, were mixed with D-sorbitol and fed through a twin screw extruder.
Experimental parameters were varied including sodium:citrate ratio, sorbito1:citrate
ratio, temperature, feed rate, and extruder screw speed. Most of the samples were
soluble and had molecular weights, measured by light scattering, from 1,080-26,000
Daltons. These products were tested for their ability to inhibit CaCO, precipitation.
The best results were for samples of a molecular weight ~ 8 , 9 0 0Daltons, as determined
by light scattering. They were able to inhibit CaCO, precipitation, from a 0.003 M
CaCI, / 0.003 M NaCO, solution, for at least 10 min at a builder concentration of
only 5-6 ppm. ?his is sufficient for use in formulations which typically use polyacrylic
acid.
Thus natural polycarboxylic acids have potential use in detergent builders (Shogren,
2007), or a variety of other products. It also shows that a natural co-monomer, such
as a reduced sugar, can be used to enhance the properties of the polymer material.
Because they can sequester ions or inhibit crystallization, these types of systems have
a place in the environmentally friendly detergent products of the future.
Polyols and Amino Acids 0 439
'
)-. Catalyst I,* 0
Purification
Methyl oleate
Vegetable oil
I Formic Acid
0 0 "
-Methanol
Catalyst
Purification
H f "
,~
"C
NaOH
- H20
E M 0 polyglyceride surfactant
Scheme 15.6.The synthesisof a nonionic surfactant based on glycerol and epoxidized methyl oleate (EMO).
Sugactant Properties
Low concentration solutions of HPESO are soluble, and the resulting material is an
anionic surfactant. It was studied with sodium, potassium, or tetraethonalammoniom
counterions. All of the systems were able to reduce the surface tension of water to a
range of 20-24 mN m-' as measured by a pendant drop tensiometer at 23°C. The
interfacial tension of an aqueous solution of these surfactants with hexadecane were
found to be in the range of 12-17 mN m-'.
Polyols and Amino Acids 0 443
BF, Purification
NaOH - glycerol
after 2 h was nearly twice as high as in those treated with ordinary drug solutions. In
other words, the use of HPESO improved the quality on the solid lipid nanoparticle
drug delivery system resulting in an enhanced performance.
These results have shown that soybean oil can be used in both the high volume
surfactant market and also in the high value pharmaceutical market as well. This is
an area where natural materials not only match the performance of petrochemical
alternatives, but actually exceed them.
Conclusions
Natural materials are well suited for applications in the surfactants, detergents,
absorbents, and pharmaceutical delivery. Other industries, such as fuel and lubrication,
have already replaced a portion of their petroleum consumption with biobased
materials. Although they have many advantages from environmental and sustainability
standpoints, natural materials will only truly gain customer acceptance if they can also
compete on a cost and performance basis as well. Much work remains to be done and
innovative products, such as those discussed in this book, need to be developed before
there is a truly sustainable biobased economy for future generations.
Ihe use of trade, jirm, or corporation names in thispublication isfor the information and
convenience of the reader. Such use does not constitute an official endorsement or approval
by the United States Department OfAgricultureor the Agricultural Research Service of any
product or service to the exclusion of others that may be suitable.
References
Adami, I. Household Detergents Production: Technological Evolution and Trends. Sodeopec,
Soaps, Detergents, Oleochemicals, and Personal Care Products, L. Spitz, Ed.; AOCS Press, Cham-
paign, 2004; 10,288-323.
Agboola, S.O.; J.O. Akingbala; G.B. Oguntimein; 0. State Production of Low Substituted Cas-
sava Starch Acetates and Citrates. Starch-Sti!rke 1991 43, 13-15.
Ames, G.R. Long-Chain Derivative of Monosaccharides and Oligosaccharides. Chem. Rev. 1960
60,541-553.
Barber, I., B. Larry; J.A. Leenheer; W.E. Pereira; T.I. Noyes; G.K. Brown; C.F. Tabor; J.H. Writer
Organic Contamination of the Mississippi River from Municipal and Industrial Wastewater.
US.Geological Survey Circular 1133. http://water,er.usgs.gov/pubs/circ1133/organic.html. Ac-
cessed 2008.
Berna, J.L.; C. Bengoechea; A. Moreno. Laundry Products in Bar Form. Sodeopec, Soaps, Deter-
gents, Oleochemicals, and Personal Care Products, L. Spitz, Ed.; AOCS Press, Champaign, 2004;
6,189-211.
Biresaw, G.; Z.S.Liu; S.Z. Erhan. Investigation of the Surface Properties of Polymeric Soaps Ob-
tained by Ring-Opening Polymerization of Epoxidized Soybean Oil. /. Appl. Polym. Sci. 2008
108,1976-1985.
Case, F. The Emergence of Green Chemistry. INFORM2007 17,751-753.
Polyols and Amino Acids 0 445
Centolella, A.P.; B.G. Razor. Polyesters of Citric Acid and Sorbitol. US, 3661955, Polyesters of
Citric Acid and Sorbitol, May 9, 1972.
Chang, C.J.; G. Swift. Poly(AsparticAcid) Hydrogel. ]Macromol Sci Pure Appl Chem 1999 301,
963-970.
DOE, www.energystar.gov. Accessed 2008.
Doll, K.M.; S.Z. Erhan. Synthesis and Performance of Surfactants Based on Epoxidized Methyl
Oleate and Glycerol. /. Sufact, Deterg. 2006 9, 377-383.
Doll, K.M.; S.Z. Erhan. Synthesis of Carbonated Fatty Methyl Esters Using Supercritical Carbon
Dioxide. J Agric. Food Chem. 2005 53,9608-9614.
Doll, K.M.; RL. Shogren; R.A. Holser; J.L. Willett; G. Swift. Polymerization of L-Aspartic Acid
to Polysuccinimide and Copoly(Succinimide-Aspartate)in Supercritical Carbon Dioxide. Let.
Organ. Chem. 2005 2,689-689.
Doll, K.M.; R.L. Shogren; J.L. Willett; G. Swift. Solvent Free Polymerization of Citric Acid and
D-Sorbitol. /. Polym. Sci., Part A: Polym. Chem. 2006 44, 4259-4267.
Erhan, S.Z.; M.O. Bagby; T.C. Nelsen. Statistical Evaluation of Biodegradation of News Ink
Vehicles and Ink Formulations./. Amer. Oil Chem. Soc. 1997 74,707-712.
Ferguson, EA. Features. A Nonmyopic Approach to the Problem of Excess Algal Growths. Envi-
ron. Sci. Technol. 1968 2, 188-193.
Findley, T.W.; D. Swern; J.T. Scanlan. Epoxidation of Unsaturated Fatty Materials with Peracetic
Acid in Glacial Acetic Acid Solution.]. Am. Chem. Soc. 1945 67, 412-414.
Finfacts, Big Industrial Users of Energy in Huge Price Hikes; Dow Chemical to Also Idle Plants in
Europe and US. June 25, 2008., http://www.finfacts,ie/irishfinancenews/article-1014008.shtml
(2008).
Gaffar, M.A. Preparation and Utilization of New Carboxyl Group Containing Cation Exchangers
Based on Starch Using a Dry Reaction Method. Starch 2002 54, 185-192.
Giger, W.; EH. Brunner; C. Schdner. 4-Nonylphenol in Sewage Sludge: Accumulation of Toxic
Metabolites from Nonionic Surfactants. Science 1984 225, 623-625.
G r d , G., Petroleum, Plant Oils Send Surfactant Prices through the Roof. January 17, 2008,
http://www.purchasing.comlarticlelCA6518717.html (2008).
Griffin, W.C. Calculation of HLB Values of Non-Ionic Surfactants. /. SOC.Cosm. Chem. 1954 5,
249-256.
Griffin, W.C. Classification of Surface-Active Agents by HLB. .] SOC.Cosm. Chern. 1949 1,
312-326.
Hogue, C. Greener Cleaners. Chem. Eng. News 2006 84, 14.
Hogue, C. Surfactant Scrutiny. Chem. Eng. News 2007 85, 33-35.
Holser, R.A. Transesterification of Epoxidized Soybean Oil to Prepare Epoxy Methyl Esters. Ind.
Crops Prod. 2008 27, 130- 132.
Hooker, E. Final Report of the Amended Safety Assessment of PEG-5, -10, -16, -25, -30, and -40
Soy Sterol. Internat. J Toxicol. 2004 23, 23-47.
Johnson, W., J . Final Report of the Amended Safety Assessment of Glyceryl Laurate, Glyceryl
Laurate Se, Glyceryl Laurate/Oleate Etc. Internat. /. Toxicol. 2004 23, 55-94.
446 0 K.M. Doll and S.Z. Erhan
Introduction
Motivation
Interest is growing in the development of material technologies that are based on
renewable resources. The use of biomass as feedstock is of expanding industrial
significance, particularly in the energy and commodities sectors. Relative to these
arenas, mono- and polysaccharides, very flexible and tunable chemical starting
materials, are estimated to make up three-quarters of the world’s biomass. The efficient
use of this resource is now recognized as a major future objective in a wide range of
technology applications.
The need exists to identify secondary streams of saccharide by-products and to
use these materials as the basis for higher, value-added surfactant chemistries. Several
research groups in the world are creating a platform for advancing the knowledge
on the structure-performance properties and for facilitating the penetration of
sugar-based surfactants in traditional markets dominated by nonrenewable nonionic
surfactants. The general goals of the research work performed in these groups are to
propose and test new structures for targeted applications as well as to provide the basis
for future strategies aimed at enhancing cost-effectiveness. More specific aims involve
both identifying molecular factors that govern the surface activity and facilitating
the design and application of sugar-based surfactants as substitutes for conventional
poly(ethy1ene oxide) (EO) surfactants and others.
Significant groundwork was already done in this area (Balzer & Luders, 2000;
Kiraly & Findenegg, 2000; Kocherbitov et a]., 2002; Kumpulainen et al., 2004a,b,
2005; Liljekvist et al., 2001; Matsson et al., 2004; Muruganathan et al., 2003, 2004;
Nickel et al., 1996; Person et al., 2002, 2003a,b, 2004; Ruiz, 2009; Stubenrauch,
2001). Three classes of surfactants with sugar or a polyol derived from sugar as polar
head group were widely researched: alkyl polyglucosides (APGs) (also known as
alkylpolyglycosides), alkyl glucamides, and sugar esters (Holmberg, 200 1). Both these
surfactants and biosurfactants produced by microorganisms and other surfactants
derived from renewable raw materials are coming progressively onto the market.
’449
450 0 O.J. Rojas et al.
Deleu and Michel Paquot (2004) provided an interesting summary of trends, and
reported for APGs a share of ca. 3% of the total production. The reader is referred to
the book edited by Hill et al. (1 997) for more detailed information on this subject.
Overall, APGs have established themselves as natural surfactants of choice for diverse
applications-from emulsifiers for skin care to foaming agents.
The possibility of future development in the area of biofuels from cellulose may
open new opportunities, especially for secondary streams in related processes. We
1 believe that four critical work packages need pursuing to successfully expand the
potential of sugar-based surfactants from such sources:
Understanding and rationalizing the state-of-the-art in the area of saccharide-
based surfactants. The existing, highly fragmented activities need to be
systemized and analyzed to adequately respond to current changes in economic
and environmental aspects of surfactant production and use.
Identifying molecular and structural factors that govern the surface activity of
these materials so that we can facilitate their design and application in areas that
are currently utilizing petroleum-based surfactants. One can support this by
studies with model systems which will expand our understanding and allow us to
target specific applications.
Designing and characterizing new sugar surfactants based on natural products and
on new motifi (Blunk et al., 2006) to demonstrate the economic and technical
viabilities in different applications.
Exploring new synthetic approaches to utilize saccharides for the synthesis of new
surfactants. One could harvest saccharides from biomass processing (and related
secondary streams) or from agricultural/food by-products. One can then combine
this with an understanding of the current and projected economics ofsaccharide-
and petroleum-based surfactant technologies to facilitate the identification of key
future markets for these materials.
Recent research results show that glucose-based and sucrose-based surfactants have
a range of beneficial physical and performance properties, including high levels of
surfactancy (surface and interfacial activity), very rapid biodegradability, low human
and animal toxicity, effective emulsification properties, and surface interactions.
Therefore, we anticipate that we can rationally design saccharide-based surfactant
structures, centered on the combination of potentially low-cost (based on natural
starting materials), renewable saccharide components and appropriate renewable
(natural fatty acids) hydrophobic pendant groups that will allow the understanding
of the fundamental interactions governing their hnctionalities. Based on this
understanding as well as on the synthetic approaches to generating sugar-based
surfactants, we hope to promote interest in, and the prospects for, their utilization in a
number of surfactant markets, which would ultimately lead to a significant reduction
in the use of petroleum-based materials in these sectors.
Interfacial Properties of Sugar-based Surfactants 0 45 1
Nonionic Surfactants
Nonionic surfactants represent a major component material for applications ranging
from personal care to a wide range of industrial uses. Structurally based on the molecular
combination of hydrophilic and hydrophobic substructures, nonionic surfactants are
effective as wetting and spreading agents, as emulsifiers, and as foaming agents (while
having minimal skin and eye irritation effects). Furthermore, they are characterized
by a wide range of critical secondary-performance properties.
The hydrophilic component of these materials is currently based largely on EO,
which is petroleum-derived. In addition, a significant portion of the hydrophobic
components of these materials is also petroleum-derived. The production costs of
these commodity chemicals are closely linked to the highly volatile petroleum prices,
which one can regard as an additional motivation to search for substitutes. In view
of the large volume in the consumption of EO-based nonionic surfactants, further
replacement of the established market would be very attractive for a high-value
utilization of sugars from biomass.
However, extensive replacement of EO-based surfactants can be challenging,
and a market barrier may exist that prevents more widespread penetration of a new
generation of surfactants. Nevertheless, in our opinion, the time is right to further
advance efforts by starting with high-end applications such as drug emulsification,
pharmaceutical formulations, etcetera, which are industries that require these
technologies and can temporarily tolerate higher prices.
In summary, a need is evident to broaden the application of surfactants to replace
petroleum-based products toward new classes of highly biodegradable, low-irritating,
low-toxic, completely naturally-derived nonionic surfactants. As superior-performance
properties of current and novel sugar-based surfactants are demonstrated, they will
become further established in the future “green” market.
Before discussing these subjects, let us briefly outline the tools that were utilized in
these experiments since most of these are not widely available in typical laboratories.
Results from techniques used to measure surface tension, solution rheology, contact
angle, detergency, and emulsion properties are not covered in this chapter. Instead, we
describe the interfacial properties of adsorbed layers of sugar surfactants by using the
methods described below.
Techniques
Measurementsand Analysis of SurfaceInteractionsand Forces (MASIF)
Force measurements were conducted using the noninterferometric surface force
apparatus (Parker et al., 1989, 1994). This device, commonly known as MASIF
(Measurements and Analysis of Surface Interactions and Forces), employs a bimorph
force/deflection sensor that, after calibration of the spring (deflection) constant, yields
the interaction force. One of the surfaces (bottom surface) is mounted on the edge
of the bimorph, and the other (the top one) at the end of a piezoelectric tube. The
assembly is enclosed in a stainless-steel cell of ca. a 10-mL volume, and mounted on
a translation stage that is isolated from electrical and sound noise. During a typical
force measurement, the surfaces are driven closer until contact, and then they are
separated further apart. This is done by applying a triangular voltage wave to the piezo
crystal. Simultaneously, the charge produced upon any deflection of the bimorph,
due to repulsive or attractive forces, is recorded. Once the surfaces come into hard-
wall contact, the linear movement of the piezo deflects the bimorph, thus enabling
the force sensor to be calibrated against the known piezo-crystal expansion and
contraction as measured by a linear variable differential transformer (LVDT) sensor.
Provided the deflection and the spring constant of the bimorph are known, the data are
used to calculate the force-distance curves from Hooke’s law. The noninterferometric
surface force apparatus does not allow an absolute determination of the zero surface
separation; however, one can obtain the adsorbed surfactant-layer thickness from the
magnitude of inward “jumps” that typically occurs when a surfactant layer is pushed
out from the contact area upon compression.
Ederth et al. (1998) show that flame-polished glass surfaces are smooth enough
to enable accurate measurements of surface forces down to molecular separations. The
surfaces used in each experiment were prepared by melting one end of a borosilicate
glass rod (diameter 2 mm, length ca. 25 mm) in a butane-oxygen burner until the tip
formed into a sphere with a diameter of about 4 mm. The normal radii of curvature (rI
and r2)for each surface were determined more accurately at the end of the experiment
by using a micrometer, and the local harmonic mean radius of the interaction, R,
was then calculated as R = 2rlr2/(r,+ r2). The spring constant of the bimorph was
measured at the end of each experiment by placing known weights on the bimorph
spring and measuring the resulting deflection (usually about 100 N/m). The force was
then normalized by the local harmonic mean radius of interaction (flfl.
454 0 O.J. Rojas et al.
The hydrophobic surfaces on which the adsorption of the surfactants was studied
were obtained by silanization and thiolization, respectively, of the hydrophilic glass
surfaces. Despite being hydrophobic, the silanated glass carries a significant net
negative charge. This charge results from the dissociation of unreacted silanol groups
in the glass substrate that is not completely screened by the self-assembled silane
layer. The thiolated surfaces, however, are completely uncharged. One can find details
about the surface preparation in previous reports (Ederth et al., 1998; Stubenrauch et
al., 2004a).
All procedures for assembling the measuring chamber and preparing the
solutions were carried out in a laminar-flow cabinet. At the beginning of each set of
experiments, the interaction profiles were first determined in air to ensure that the
system showed no signs of contamination. Then a background electrolyte solution
was introduced into the measuring chamber, and the interaction profiles were again
determined. A stock surfictant solution was then introduced through a 0.2-pm poly
(tetrafluoroethylene) (PTFE) filter until the desired concentration inside the chamber
was attained. All measurements were carried out at 22 f 1°C.
& Neuman, 1987). From these operations, we obtained the parameters of the power
spectrum. The measured power spectrum was compared with the theoretical power
spectrum (Kramer, 1971) for capillary waves in the presence of air. ‘This theoretical
power spectrum is described, among others, in terms of the surface tension y, the
transverse viscosity p, the sum of interfacial shear elasticity and interfacial dilatational
elasticity E, and the sum of interfacial shear viscosity and interfacial dilatational
viscosity K.
‘The experimental data [i.e., the central frequency and the dampening coefficient
(after correction for instrumental broadening) together with the bulk properties
of the fluids] were used to calculate the viscoelasticity coefficients from the power-
spectrum equation. Hence, the sum of the real and imaginary parts of the complex
modulus (elasticity and viscosity) was obtained. We used polar diagrams for direct
interpretation of the rheological parameters E and K.These plots were constructed from
the dispersion equation for a given temperature, wave number, and surface tension
(or surface pressure) by using as parameters the normalized complex frequency (00/
ad slaw), where o0is the experimental central frequency, and a is the dampening
coefficient. Here the subscript “w” is used to denote the experimental values for a
film-free surface ( E = 0, K = 0) (i.e., water in our case).
HO -
Fig. 16.1 .The n-dodecyl-(3-Pmaltoside(P-C,,G,, top) and hexaoxyethylenedodecyl ether (C,E,
bottom)
are two nonionic surfactants with the same hydrophobic group that were compared regarding their
interfacial properties.
456 0 O.J. Rojas et al.
glass, and thiolated gold surfaces. The most important results with respect to the
comparison of different surfaces are summarized in this section.
AirMater Interfaces
The adsorption of nonionic surfactants at the airlwater surface leads to a decrease
of the surface charge of the interface (reviewed in Stubenrauch et al., 2003). This
decrease eventually results in a transition from an electrostatically stabilized common
black film (CBF) to a Newton black film (NBF) that is stabilized by short-range
repulsive forces. This phenomenon is illustrated in Fig. 16.2 with data obtained
for the nonionic surfactant hexaoxyethylene dodecyl ether (C,,E6). The formed
NBF consists of two densely packed monolayers, and creates a “force barrier” that
prevents the film from rupturing, thus stabilizing a foam. Apart from densely-packed
monolayers, a sufficient monolayer cohesion is required for the formation of a stable
NBF (Stubenrauch et al., 2004a,b). From the thickness of the NBF, one can estimate
the thickness of one monolayer.
Comparing Fig. 16.2 with the respective results obtained for n-dodecyl-P-D-
maltoside (P-C,,G,) shown in Fig. 16.3, apparently, the same general trend is observed.
In both cases, film thicknesses ranged from 930 nm to c5 nm, depending on the
surfactant concentration and the applied pressure, which ranges from 200-9000 Pa.
As was the case for C,,E6 (Fig. 16.2), two different kinds of films were observed: thick
CBFs stabilized by electrostatic repulsion, and thin NBFs stabilized by short-range
n
repulsion. The thicknesses of the CBFs decrease monotonically as increases. While
n
the slope d(1og )ldh is independent of the surfactant concentration, a significant
shik of the curves toward lower disjoining pressures is observed when increasing
the P-C,,G, concentration from 0.034-0.137 mM. Moreover, at the highest
concentration, no CBF is formed at all, but the foam film drains directly down to the
NBF.
Experimentally (reviewed in Stubenrauch et al., 2003) and only recently also
theoretically (Kudin & Car, 2008), the airlwater surface is negatively charged. This
charge is responsible for the long-range electrostatic repulsive forces observed in
foam films stabilized by nonionic surfactants. An increase in the nonionic surfactant
concentration leads to a decrease of the surface-charge density as more uncharged
molecules (i.e., nonionic C,,E6 and P-C,,G, surfactants) adsorb at an originally
charged surface. The electrostatic forces acting in foam films stabilized by P-C,,G,
were quantified by means of the DLVO theory by using constant-charge boundary
conditions and the theoretical Debye length of K-’= 30 nm. These calculations led
to surface-charge densities of qo = 1.55 mC rn-’ for the 0.035 mM solution and q,, =
0.95 mC rn-, for the 0.137 mM solution, respectively. The decrease in surface-charge
density destabilizes the CBF until no CBF is observed for c > CMC under the given
experimental conditions. At these concentrations, the immediate formation of an
NBF is observed. The NBFs are very thin (ca. 5 nm) with an aqueous core of 1-2 nm
assuming a length of 22 nm for the surfactant. In other words, these films consist of two
InterfacialProperties of Sugar-based Surfactants 0 457
100
hlnm
Fig. 16.2. (top) Pictures and schematic drawings of a common black film and a Newton black film,
respectively.Thepictureswere taken with an optical microscopeandthecircles represent the film formed
in a small hole with a diameter of 1-2 mm drilled through the disk in the TFPB. (bottom) Disjoining
pressure n as a function of film thickness h measured for three concentrationsof C,,E, in lo4 M of a NaCl
solution.The two lower concentrationsare below; the highest is above the critical micelle concentration
(CMC) (= 0.073 mM).The solid lines are calculated according to the Derjaguin-Landau-Verwey-Overbeek
(DLVO)theory assuming interactionsat constant charge (Stubenrauch et al., 2004a).
458 0 O.J. Rojas et al.
surfactant monolayers with only small amounts of water separating the head groups
(mainly hydration water). As is the case for the force barrier between nonpolar solid
surfaces (to be presented later), densely-packed monolayers are required to stabilize an
NBF and thus to prevent contact between the two bare surfaces (i-e., film rupture).
Hence, the presence of an NBF signifies the existence of a force barrier between two
aidwater surfaces analogous to the force barrier between two solid surfaces (see Figs.
16.4 and 16.5).
The remarkable similarities with regard to the n - h curves of &G, and C,,E,
are evident from the fact that the curves nearly lie on top of each other, which means
that the surface- charge densities qo are nearly equal. Indeed, surface-charge densities
of qo = 1.70 mC m-2 at c = 0.01 mM (0.12 CMC) for C,,E, and qo = 1.55 mC m-2
at c = 0.035 mM (0.25 CMC) for P-C,,G, were calculated from the experimental
10000
100
0 10 20 30 40 50 60 70 80 90 100
hlnm
Fig. 16.3. Disjoining pressure ll as a function of film thickness h measured for three concentrations of
P-C,,G, in lo4 M of a NaCl solution. The two lower concentrations are below; the highest is above the
critical rnicelte concentration (CMC) (= 0.14 mM). The solid lines are calculated according to the DLVO
theory assuming interactionsat constant charge(Stubenrauchet al., 2002) (Reproduced with permission
of the PCCP Owner Societies).
Interfacial Properties of Sugar-based Surfactants 0 459
0 10 20 30 40 50
D/nm
Fig. 16.4. Force F normalized by the radius R as a function of relative surface separation 0 between two
thiolated solid surfaces. The forces were measured across aqueous solutions containing 0.1 mM NaCl
in the absence (filled circle) and in the presence of 0.01 mM non-ionic surfactant C,,E, (open squares)
(Adapted with permission from Stubenrauch et al., 2004 and Rojas et at., 2005a. Copyright 2004 and
2005, American Chemical Society). Similar behavior was observed for p-Cl2G2 (see Fig. 16.5).
data. As the surface-charge density is a property of the bare aidwater surface, these
values mean that a surface concentration of 3.0 x 10" mol rn-, C,,E, (0.12 cmc)
reduces the surface-charge density to the same amount as a surface concentration
of 4.0 x lo4 mol m-2 P-C,,G, (0.25 cmc). Moreover, in both cases, the surface
concentration is enough to stabilize a foam film up to 10,000 Pa. We suggest that the
effective surface cross-section area covered per molecule determines the reduction in
surface-charge density. Since the head group of C,,E, is larger than that of P-C,,G,,
a smaller number of C,,E, molecules would be needed to achieve a given reduction
in the repulsive double-layer force. We conclude that, for each nonionic surfactant,
the surface- charge density is reduced with increasing adsorption. A more thorough
discussion regarding the influence of the head groups and the chain length of the
460 O.J. Rojas et al.
surfactant on film stability is found in Stubenrauch et al. (2002) and Schlarmann &
Stubenrauch (2003).
Solid/Liquid Interfaces
Silanated glass and thiolated gold surfaces were used as hydrophobic solid surfaces.
Upon adsorption, at the lowest surfactant concentration, we noted that the interaction
forces were dominated by a steric barrier at a short-surface separation. By increasing
the surfactant concentration, a more robust steric barrier was formed. Interestingly,
the NBF formation observed for foam films (see Figs. 16.2 and 16.3) corresponded
to the appearance of this force barrier between two solid surfaces (see Fig. 16.4 in the
case of thiolated gold).
The barrier shown in Fig. 16.4 is a measure of the force that is needed to remove
surfactant from between the two surfaces. Because this barrier is located at a distance
corresponding to the thickness of a surfactant bilayer, the analogy to the NBF
formation is obvious. Note that a “removal” of this bilayer results in film rupturing in
the case of foam films, whereas in the case of solid/liquid interfaces it leads to a direct
contact of the solid surfaces (Rojas et al., 2005a; Stubenrauch et al., 2004a).
Also concluded (data not shown) is that the nature of the surface at which the
surfactant adsorption takes place mainly influences the interaction forces at a low-
surface coverage. Once a densely-packed surfactant layer is formed, the surfactant
itself determines the interaction forces.
c
P'C12G2
\
c
k
rt 1 2E6
D/nm
0
0 5 10 15 20
Fig. 16.5. Steric force barriers for 0.035 mM of P-C,,G,and 0.01 mM of C,,E, respectively, between solid
thiolated surfaces. A stronger force barrier is observed for the sugar-based surfactant. This steric force
is responsible for the stabilization of dispersions, where the solid particles are separated by liquid films.
The stronger barrier seen in the case of the sugar surfactant is explained by a larger stiffness of its polar
group as compared to the EO units (Adapted with permission from Rojas, et al., 2005a. Copyright 2005
American Chemical Society).
Fig. 16.6. Isomeric sugar surfactants used to investigatethe effects of the molecularstructure on surface
viscoelasticty:n-octyl-0-glucoside (a), n-octyl-a-glucoside(b), and 2-ethylhexyl-a-glucoside (c).
Normalized Central Frequency ~ui$’ Normalized Central Frequency 1%’ Normalized Central Frequency w ~ ‘
Fig. 16.7. Helmet or polar plots showing the experimental surface viscosity and elasticity obtained from
the propagation (centralfrequency and damping)ofcapillarywavesprobed by surface light scatteringfor
n-octyl-&glucoside (left),n-octyl-a-glucoside (center), and 2-ethylhexyl-a-glucoside (right)surfactants.
The loci of the different curves show differences in the behavior of these isomeric surfactants (Adapted
with permission from Rojas et al., 2005b. Copyright 2005 American Chemical Society).
example, one can vary foamability and emulsion stabilization by changing the polar
group (position and stereochemistry), as well as the structure of the hydrophobic tail
(e.g., via branching).
Changes in the stereochemistry of the head group units are also expected to
influence the interaction with the ions present in solution. Such effects were made
clearer in the discussion presented in ‘Rir/Water Interfaces” on thin-film pressure-
balance experiments for other surfactants. One should stress, however, that dynamics’
effects (diffusion, relaxation, etcetera) play important roles as well. At this point, to
speculate further about the relationship between surfactant structure and properties
is meaningless until the nature of surface-charge density, surfactant packing, and
dynamic effects is better understood. An attempt to shed some light regarding the
effects of surfactant structure is made in the next sections.
Molecular Prospects
To obtain experimental information on the structure of water adjacent to solutes is
very difficult. The main reason for this is that in most cases the spectroscopic signal
from the hydration shell is swamped by that of bulk water. However, recently some
progress was made where an inherently surface-sensitive spectroscopic technique,
vibrational sum frequency spectroscopy (VSFS), was employed to investigate the
hydration of sugar groups and oligo(ethy1ene oxide) chains anchored to the aidwater
surface. Some very strong hydrogen bonds are formed between sugar and water,
whereas the hydration of the oligo(ethy1ene oxide) chain is dominated by a clathrate-
like structure (Claesson et al., 2006; Tyrode et al., 2005).
One can apply related arguments when considering short-range interactions
between surfactant layers. The fact that hydrogen bonds can form between polar
Interfacial Properties of Sugar-based Surfactants 0 465
head groups of, for example, sugar-based surfactants does not directly lead to the
conclusion that the short-range interaction across water should be attractive due to a
hydrogen-bond formation. To compile available data on the depth of the interaction-
force minimum is thus of some interest. In this case, the pull-off force needed to
separate surfaces that are in contact (for example, after bringing the surfaces together,
as shown in Fig. 16.4 and Fig. 16.5) is measured. This force is related to the adhesion
energy that, in our case, was measured between adsorbed nonionic surfactant layers.
This will allow us to learn if the possibility of forming hydrogen bonds between two
head groups has a bearing on the short-range attraction observed in these systems (see
Table 16.1).
We note that one cannot form direct hydrogen bonds between dodecyldimeth-
ylamine oxide (DDAO) molecules in uncharged form, decyldimethyl phosphine ox-
ide (DDPO), or surfactants with EO head groups (see Table 16.1). However, such
interactions are possible for surfactants with polyhydroxyl head groups and also for
amine head groups. By comparing the data compiled above, we note that the trend is
that head groups that are able to form interlayer hydrogen bonds display a somewhat
larger attractive short-range force component than layers that are unable to form such
bonds. This could indicate that a contribution from interlayer hydrogen-bond forma-
tion exists, but the extension of this effect is difficult to determine since the nature of
the head group also influences other short-range interactions, for example, van der
Waals forces.
Due to the lack of systematic data, we cannot compare quantitatively the
hydration numbers obtained for EO and glucose head groups. A qualitative evaluation,
however, might shed some light on the differences between these surfactants. From
extensive Small Angle Neutron Scattering ( S A N S ) measurements in bicontinuous
microemulsions, we know that the area per head group at the water/oil interface is
0.56 nm2 for a glucose unit (Kluge 2000), which is comparable to that of a surfactant
with four EO units which is 0.54 nm2 (Sottmann et al., 1777). 'The hydration number
of the glucose head group, as determined by nuclear magnetic resonance (NMR), is
6 at a surfactant concentration of 3 wt%, whereas NMR estimates the hydration
number to be 6 per an EO unit at a surfactant concentration of 10 wt% (i.e., 24 water
molecules per tetraoxyethylene head group). Keeping in mind that the hydration
number per EO unit is expected to be even larger at 3 wt%, one can conclude that
under similar conditions and for similar head group sizes the hydration of EO-based
surfactants is significantly higher than that of sugar-based surfactants (Claesson et al.,
2006).
The short-range interaction between surfactant layers exposing the polar part
toward solution is due to a complex interplay between van der Waals forces, hydration,
and steric effects. One can only measure the total force, and this force displays both
attractive and repulsive regimes. The van der Waals force is the main attractive force,
while hydrogen bonding within adsorbed layers, again directly or mediated by water
molecules, increases the packing density within adsorbed surfactant layers; this
increases the van der Waals force.
466 0 O.J. Rojas et al.
Table 16.1. Depth of Force Minimum between Various Nonlonic Surfactant Layers
Depth of force
minimum Substrate surface and
Compound Technique F/R (Mn/M) remarks
n-dodecyl amine oxide (DDAO) SFA 0.25 Bilayer on mica.
Unchargedform.
n-dodecyl phosphine oxide SFA 0.25 Hydrophobied mica. Force
(DDPO) minimum increases with
increasingtemperature.
tetraoxyethylenedodecyl MASIF 0 Silanatedglass surfaces.
ether (C,E,)
pentaoxyethylenedodecyl SFA 0.1 -0.2 Hydrophobized mica.
ether (C,E,) A t 20°C. Force minimum
increases with increasing
temperature.
hexaoxyethylenedodecyl MASIF 1-1.4 Silanatedglass surfaces.
ether (C,E,)
hexaoxyethylenedodecyl MASIF 0.2-0.4 Thiolated gold surfaces.
ether (C,E,)
tetraoxyethylenedodecyl SFA 0.3-0.4 Hydrophobizedmica and
amide silanated glass (Herder,
K,,ONHE,) 1991)
tetraoxyethylenedodecyl SFA 0.1 -0.2 Bilayer on mica, pH 9.4-9.9.
amine (C,,NHE,)
n-octyl-p-glucoside(P-C,G,) SFA 1.2-1.5 Hydrophobized mica.
n-decyl-j3-o-glucoside(j3-C,,G1) MASIF 1 Thiolated gold surfaces.
Estimated. (Waltermo, 1993)
n-decyl-P-o-maltoside(p-C,,G,) MASIF 0.9 Silanated glass surfaces.
n-dodecyl-j3-o-maltoside MASIF 1-2 Thiolated gold surfaces.
(P-C,,G,)
technical sugar-surfactant SFA 2 Hydrophobizedmica.
mixture Located on the repulsive
side.
n-dodecyl lactobion amide SFA 1.2 Hydrophobized mica
(LABA), inner sugar ring is
open
n-dodecyl lactobion amide MASIF 1.5 Silanated glass surfaces.
(LABA), inner sugar ring i s
open
maltose 6’-O-dodecanoate MASIF 1.o Silanatedglass surfaces
(C,,OGJ
n-dodecyl amine (C,,NH,) SFA 0.5-1 Bilayer on mica, pH 9.5-10.1
Most molecules in the outer
layer are uncharged.
Data correspond to aqueous surfactant solutions a t or above the respectiveCMC (i.e., to a
saturated monolayer).SFA = surface-forceapparatus, MASIF = measurementand analysis of
surface-interaction forces.
Reproduced with permission of the PCCP Owner Societies.
Claesson et al., 2006 and references therein.
Interfacial Properties of Sugar-based Surfactants 0 467
We note that both hydration and the mobility of surfactants are important for
short-range interactions. The steric interaction between surfactants with oligomeric
head groups also deserves further attention. As the head group size increases, the
short-range repulsion goes from a hard-wall repulsion, via compressible excluded
volume repulsion, to a steric repulsion described by polymer-brush theories (de
Gennes, 1987).
The experimental results discussed in this chapter help to demonstrate hndamental
features of nonionic surfactants based on sugar polar groups (mainly glucoside and
maltoside). These polar groups make them structurally more attractive in terms of
their capacity to stabilize interfaces (liquid-liquid, solid-liquid, and gas-liquid) as
compared to EO-based nonionic surfactants. Note that one can use the OH groups in
the sugar units for chemical reactions, and thus one has the possibility of engineering
the surfactant structure and its stereochemistry (which, as was demonstrated before,
play a leading role for the properties). Key molecular components (head group
configuration, identity, number, tail length, and saturation) lead to subsequent
packing aspects for “green” polysaccharide streams that one can ultimately use to
manufacture nonionic surfactants.
As mentioned in previous sections, nonionic, saccharide-based surfactants are
a veritable plethora of tangible benefits for state-of-the-art surface, colloid, and
condensed state research. Their solubility and phase behavior are typically insensitive
to temperature changes in contrast to EO-based surfactants. This allows much greater
amplitude in their synthetic design without compromising desirable physical and
chemical features such as small molecule (e.g., blood glucose) biosensor applications,
biophysical recognition (such as antigen-antibody mechanisms), host-guest chemistry,
and a number of other smart functions. One needs to use separations chemistries, and
chemically fine-tune the respective sugar surfactants with the appropriate physical
characteristics (functional groups and/or ligands) to achieve the “smart” functionality
we have described.
The performance of sugar surfactants (versus EO surfactants) in dispersed systems
(such as foams, emulsions, and dispersions) and their potential for the formation
of new structures in these systems (such as unilamellar vesicles, ribbons, spirals,
tubes, etcetera) depends on the detailed molecular structure. One may synthesize
new surfactants which may offer unique templates for more sophisticated electronic,
biomedical, and optical devices. Some strategies in the design of sugar surfactants to
provide greater functionalities include the introduction of both mono- (glucoside)
and di-saccharide (maltoside) moieties with various modes of attachments to the
head group of synthesized surfactants and also the introduction of different degrees
of hydrophobicity and crystallinity to the sugar surfactants by manipulating their
aliphatic tails.
Structure-Property Relationships
Efforts need to be devoted to the development of new surfactants based on the
structure-property heuristics. In the simplest form, a new sugar surfactant that
468 0 O.J. Rojas et al.
could be synthesized would consist of three discrete parts: (a) polar head group(s),
for example, one or two glucosides or one maltoside, with or without a (b) spacer
(that could display the versatility of allowing the attachment of a second head group
and two tails as well as two different tail placements for one group), and the (c)
aliphatic tail (for example, a fatty acid). How the changes in these components will
affect their performance in model systems (foams, emulsions, dispersions) is then the
key question. Some leads to answering such questions were presented in previous
sections.
What is exciting about all of the possible configurations is the inherent potential
for providing a number of different surface interactions based on the HLB (hydrophilic
and lipophilic balance) parameter (Griffin, 1954), the variation in the packing based
on steric interactions (Piispanen et al., 2004), and the packing variation based on
other interactions (John & Vemula, 2006).
The HLB is useful to characterize the balance between the hydrophilic and
hydrophobic parts of the surfactant molecule. One can vary the HLB by changing
the length of the tail of the surfactant. This can lead to different packing behaviors.
The molecules will have a pronounced ability to engage in discrete regular packing
motifs, as was shown in recent work (John et al., 2003). Typically, surfactant packing
is dominated by the hydrophobic interactions between the hydrophic chains, which,
in turn, influence the flexibility, the orientation, and the surface energy of molecular
assemblies.
A serious need is noted for a systematic study of the structure-property relations
that define sugar-based surfactants as the alternative of choice. Major issues in this
area are:
Development of new products, production practices, and business in the biomass
industry. This requires rapid changes in consumer demand for renewable products
and alternative market solutions.
Replacement (even small fractions) of petroleum-based nonionic surfactants with
sugar-based surfactants, which is important from both an environmental and
energetic point of view.
Synthetic strategies to reduce the costs of sugar-based surfactants since current
barriers for the acceptance of sugar-based surfactants are their costs. Linking
fundamental studies with economics and market analysis may promote this
goal.
Systematic studies of the properties of sugar-based surfactants as a lack of
information about their properties and performance benefits may be an obstacle
for their wide use.
Interfacial Properties of Sugar-based Surfactants 0 469
Conclusions
The use of nonbiodegradable, petroleum-based surfactants is a major drawback, and
therefore the development of biodegradable surfactants derived from agricultural
resources is justified. Sugar-based surfactants represent a proven candidate, but any
effort aimed at formulating dispersed systems by using the saccharide platform entails
that specific technical requirements in terms of their applications must be met. The
performance of (new) surfactants in any given application is expected to be closely
related to molecular factors, with the interfacial behaviors being some of the most
important ones.
Acknowledgments
O.J.R. greatly appreciates the financial support of the National Research Initiative
of the USDA Cooperative State Research, Education and Extension Service, grant
number #USDA 2008-01 5 19. C.S. is grateful for financial support of the European
Commission under the 6th Framework Program, contract No. MRTN-CT-2004-
5 12331-Project SOCON.
References
Balzer, D.; H. Luders (Eds.). Nonionic Sufactants:Alkyl Polygucosides; Surfactant Science; Marcel
Dekker: New York, 2000; Vol. 91.
Bellman, R.; R.H.Pennington. Effects of surface tension and viscosity on Taylor instability. Q.
Appl Math. 1954,12, 151-162.
Blunk, D.; I? Bierganns; N. Bongartz; R. Tessendorf; C. Stubenrauch. New speciality surfactants
with natural structural motifs. Newj. Chem. 2006,30, 1705-1717.
Bouchiat, M.A.; J. Meunier. Spectre des fluctuations thermiques de la surface libre d'un liquide
simp1e.J Phys. 1971,32, 561-571.
Claesson, EM.; T. Ederth; V. Bergeron; M.W. Rutland. Techniques for measuring surface forces.
Adv. Colloid Znteface Sci. 1996, G7, 1 19-1 84.
Claesson, EM.; M. Kjellin; O.J. Rojas; C. Stubenrauch. Short-range interactions between non-
ionic surfactant layers. Phys. Chem. Chem. Phys. 2006, 8(47), 5501-5514.
de Gennes, EG. Polymers at an interface, a simplified view. Adv. Colloid Znteface Sci. 1987,27,
189-209.
Deleu, M.; M. Michel Paquot. From renewable vegetables resources to microorganisms: New
trends in surfactants. Compt. Rendus Chem. 2004, 7(&7),641446.
Ederth, T.; I?M. Claesson; B. Lindberg. Self-assembled monolayers of alkanethiolates on thin gold
films as substrates for surface force measurements. long-range hydrophobic interactions and
electrostatic double-layer interactions. Langmuir 1998, 14, 4782-4789.
European Public Health Alliance, http://www.epha.org/a/926. Accessed August 22, 2005
Exerowa, D.;Kruglyakov. Foam and Foam Films- aeory, Experiment, Application [Studiesin
ZntefacialScience; Vol. 5, D. Mobius, R. Miller (Series Eds.)]; Elsevier: Amsterdam, 1998.
470 0 O.J. Rojas et at.
Exerowa, D.; A. Scheludko. Porous plate method for studying microscopic foam and emulsion
films. CR Acad Bulg. Sci. 1971,24,47-50.
Griffin, W.C. Calculation of HLB values of non-ionic surfactants./. Soc. Cosr.net. Chem. 1954,5,
249-258.
Hard, S.; R.D.J. Neuman. Accurate laser light-scattering measurements of capillary waves. /. Col-
loid Interface Sci. 1987, 120(1), 15-29.
Hill, K.; W. von Rybinski; G. Stoll (Eds.). Alkyl Polyglycosisides. Technolou, Properties andApplica-
tions; VCH Verlagsgesellschaft: Weinheim, 1997; 242 pp.
Holmberg, K. Natural surfactants. Cum Opin. ColloidInterj2te Sci. 2001, G, 148-159.
ILO (International Labour Office). Encyclopaedia ofOccupational Health and S a j g ; 4th ed.; Ge-
neva, Switzerland: International Labour Office, 1998; Vols. 1-4, p. 104.
John, G.; H. Minarnikawa; M. Masuda; T. Shimizu.Liquid crystalline cardanyl j3-D-
glucopyranosides. Liq. Cyst. 2003,30,747-749.
John, G.; I?K. Vemula. Design and development of softnanomaterials from biobased arnphiphiles.
So# Matter 2006,2,909-914.
Kiraly, Z.; G.H. Findenegg. Calorimetric study of the adsorption of short-chain nonionic
surfactants on silica glass and graphite: Dimethyldecylarnineoxide and octyl monoglucoside.
Langmuir 2000, IG,8842- 8849.
Kluge, K. Der Schlussel zum Verstandis von Mikroemulsionen aus Zuckertensiden: Die Interne
Grenzflache, Ph.D. thesis, Universidt zu Koln, 2000.
Kocherbitov, V.;0.Soderman; L.J. Wadso. Phase diagram and thermodynamics of the n-octyl
beta-D-glucoside/watersystem. Phys. Chem. B 2002, 106 2910- 2917.
Kramer, L. Theory of light scattering from fluctuations of membranes and monolayers. /. Chem.
Phys. 1971,55,2097-2105.
Kudin, K.N.; R. Car. Why are water-hydrophobic interfaces charged?/. Am. Chem. Soc. 2008,
130(12), 39 15-391 9.
Kumpulainen, A.J.; C.M. Persson; J.C. Eriksson. Head group and hydrocarbon tail effects on the
surface tension of sugar-based surfactant solutions. Langmuir 2004a, 20,10935-10942.
Kumpulainen, A.J.; C.M. Persson; J.C. Eriksson. n-Decyl-glucopyranoside and n-decyl-maltopy-
ranoside gibbs monolayers. Phase changes in the dilute liquid-expanded range. Langmuir 2004b,
20, 10534-10541.
Kumpulainen, A.J.; C.M. Persson; J.C. Eriksson; E.C. Tyrode; C.M. Johnson. Soluble monolayers
of n-decyl glucopyranoside and n-decyl maltopyranoside. Phase changes in the gaseous to the
liquid-expanded range. Langmuir 2005,21, 305-31 5 .
Lewis, R.J. Sr. (Ed.). Hawleys Condensed Chemical Dictionary; 13th ed.; John Wiley and Sons,
Inc.: New York, NY, 1997; 805pp.
Liljekvist, I?; M. Kjellin; J.C. Eriksson. The surface pressure effect of pentaoxyethylene and malto-
side surfactant head groups. Adv. ColloidInterfaceSci. 2001, 89, 293-302.
Lu, S.; I? Somasundaran. Tunable synergismlantagonismin a mixed nonionic/anionic surfactant
layer at the solidlliquid interface. Langmuir 2008,24(8), 3874-3879.
Lu, S.; Y. Bian; L. Zhang; I? Somasundaran. pH Dependence of adsorption of n-dodecyl-beta-D-
Interfacial Properties of Sugar-based Surfactants 0 471
11836-1 1843.
Rojas, O.J.; R.D. Neuman; PM. Claesson. Viscoelastic properties of isomeric alkylglucoside sur-
factants studied by surface light scattering. J. Phys. Cbem. B. 2005b, 107, 22440-22448.
Ruiz, C.C. (Ed.). Sugar-Bared Surfactants: Fundamentah andApplications; Taylor & Francis: Boca
Raton, 2009.
Schlarmann, J.; C. Stubenrauch. Stabilization of foam films with non-ionic surfactants: al-
kyl polyglycol ethers compared with alkyl polyglucosides. Tenside Su@ct. Deterg. 2003, 40,
190- 195.
Shrestha, L.K.; K. Aramaki; I. Johansson; PM. Claesson; G. Olanya; C. Stubenrauch. Aqueous
foams stabilized by n-dodecyl-P-D-maltoside, hexaethyleneglycol monododecyl ether and their
1 : 1 mixture (submitted to Soft Matter 2008).
Sottmann, T.; R. Strey; H.-S. Chen. A small-angle neutron scattering study of nonionic surfactant
molecules at the water-oil interface: area per molecule, microemulsion domain size, and rigidity.
J. Cbem. Pbys. 1997, 106,6483-649 1.
Stubenrauch, C. Sugar surfactants-aggregation, interfacial, and adsorption phenomena. Cur%
Opin. Colloid Inte$ace Sci. 2001, G, 160-170.
Stubenrauch, C.; O.J. Rojas; J. Schlarmann; PM. Claesson. Interactions between nonpolar sur-
faces coated with the nonionic surfactant hexaoxyethylene dodecyl ether C,,E, and the origin of
surface charges at the airlwater interface. Langmuir 2004a, 20, 4977-4988.
Stubenrauch, C.; J. Schlarmann; O.J. Rojas; PM. Claesson. Comparison between interaction
forces at aidliquid and solidlliquid interfaces in the presence of non-ionic surfactants. Tenside
S u ~ a c tDeterg.
. 2004b, 41, 174-1 79.
Tyrode, E.; M.C. Johnson; A. Kumpulainen; M.W. Rutland; PM. Claesson. Hydration state of
nonionic surfactant monolayers at the liquidlvapor interface: structure determination by vibra-
tional sum frequency spectroscopy. J. Am. Cbem. Soc. 2005,127, 16848-16859.
Waltermo, A,; EM. Claesson; S. Simonsson; E. Manev; I. Johansson; V. Bergeron. Foam and thin-
liquid-film studies of alkyl glucoside systems. Langmuir 1996, 12(22), 5271-5278.
INDEX
Arginine-Oα-alkyl ester
dihydrochloride 353 354
Arthrofactin 131 133
Aspartic acid 432
bulk polymerization of 433
Aspartic acid D-sorbitol copolymer builder 434
Atomic force microscopy 165 170
Autoinducers 84
Axisymmetric drop-shape analysis
(ADSA)-captive bubble (CB) 204
Bacillomycin 131
Bacillus Licheniformis, lipopeptides produced by 132
Bacillus megaterium 257
Bacillus mojavenesis JF-2
biosurfactant 135
Bacillus subtilis 131
inhibiting growth of 255
lipopeptides produced by 132
Bacteriostatic activity of sucrose
monopalmitate 284
BASF′s Pluronic® L43 440
Binary DPPC/Hel 13-5 preparations 166
AFM observations 170
cyclic compression and expansion isotherms 171
FM observations 169
Langmuir isotherms 166
Biofuels 3
Biological evolution,
integration process of, in
microorganisms 233
Biomass, use of, as feedstock 449
Biomaterials 3
Biomembranes 231
Bioreactor production process 70
Biosurfactant flooding experiments 140
Biosurfactant-mediated oil recovery,
field applications of 141
Biosurfactants (BS) 3 231
biological aspects 232
categories 233
functional properties 29
hydrophilic groups 233
nature 232
physicochemical aspects 239
physiological role 237
synthesis 51
synthesis, aggregation properties,
and applications of,
derived from arginine 351
BLES 195 197 209 211
213
binding isotherm for 214
BLES-chitosan system 216
BLES formulations, mechanism of
action of chitosan in 214
C7 acid 56 61
C8 acid 56 61
C9 acid 56 61
C10 acid 56 61
C11 acid 56 61
C12 acid 56 61
C14:0 fatty acids 56
C18:l lactones 38
C18:2 ester 56
Calcite, mining of 12
Calystegia sepium 92
Candida apicola 30 238
kinetics of cytochrome
P450 synthesis in 41
Candida bombicola 30 32 35 38
39 92 93 94
238
effect of dissolved oxygen
concentration on
sophorolipids synthesis by 95
sophorolipids production by 96 241
Candida magnoliae 59
Candida sp. B-7 53 56
Candida sp. SY16 56
batch cultivation of 67
Capillary number (NCA) 131
Captive-bubble surfactometer (CBS) 203
Carbodiimide-mediated esterification 44
Carbohydrate-recognition domain (CRD) 194
Carbon and energy sources,
storage of 238
Carbon dioxide, synthesis of
polyaspartic acid polymer
using supercritical 432
Carboxylate-based soaps 425
Cationic liposomes 262
Cationic polymers 211 213
Cell cycle regulation 254
Cellobiose lipids, chemical structures of 236
Centrifugation 68
Chitosan 211 213
binding isotherm for 214
Denitrification 88 89
advances in rhamnolipid
production by 87
Depletion attraction mechanism 208
Derivatization, synthesis of
saccharide fatty acid esters via 330
Detergency 299
Detergent builders
aspartic acid polymers as 434
biodegradability and 428
functions of 426
water quality and 426
Detergent industry vii
environmental goals for 8
market value within European Union 4
use of natural products in 425
Detergents
modern 425
synthetic 425 426
α-D-galactose monosaccharides 38
Diethanolamine 300 312
effect of type of solvent on
transacylation of, with
fatty acid ethyl ester 318
Diethylamine 43
Dilatational elasticity 216 218
L-α-D-Dilauroyl-
phosphatidylcholines (DLPC) 247
asymmetric distribution of 247
Dimethylformamide (DMF) in
production of SEs 277
Dimethylsulfoxide (DMSO)
effect of, on lipase 329
in production of SEs 277
Dimorphecolic acids 4
Dimorphotheca 4
α-D-Dioleoylphosphatidyletha-
nolamine (DOPE) 263
Dipalmitoylphosphatidylcholine
(DPPC) 157 182 194 201
Disaccharide 30
Disaturated phospholipids 158
Dishwashing detergent, global
market of 425
DLVO theory 287 289
DNA compaction 381
Dow Chemical Company 431
DPPC/PG/PA mixture 160
D-sorbitol 434
melting point of 436
Dynamic light scattering (DLS) 241
Eco-friendliness, environmental
impact and 7
Eco-friendly products, desire to
purchase 6
Emulsification 29 299
Energy, U.S. Department of, Energy
Star program 431
Energy consumption, standard of living and 129
Environmental and product
performance concerns 451
Environmental goals for surfactants
and detergents industry 8
Environmentally advanced
surfactants 244
Environmentally-sensitive
applications, surfactants for 3
Environmental remediation,
surfactants for 3
Enzymes, utilization of, for the
manufacture of biobased
surfactants 16 20
EO alkyl ethers 451
Epoxidized methyl oleate and
glycerol
synthesis of surfactant from 439
synthesis of surfactant from
glycerol and 439
Epoxidized seed oils 4
Equivalent alkane carbon number
(EACN) 115
Erythritol 58 62
ESO polymerization in forming
surfactant or hydrogel 441
Esterquats 12
Esters, critical micelle concentration of 241
Esters of amides, antimicrobial activities of 299
Ethanolamine 5 300
Ethanolamine and diethanolamine reactions 319
Ethoxylated fatty acids or
monoglycerides 13 15
Ethoxylated fatty amines 13
Ethoxylated sorbitan esters 13
Ethoxylates, biodegradation of 430
Ethylene-glycol esters 14
Ethylene oxide 5 9
Ethyoxysulfonates 430
Eutrophication 426
Exogenous lung surfactants
commercial 195
historical overview and medical
needs for 191
Exosurf (colfosceril palmitate) 160 201
Extraction phenomenon 160
Galactose 51
Galactose sulphate 52
-Galactosidase activity 38
Gangliosides 259
Gel permeation chromatography 433
Gemini arginine surfactants or bis (Args) 356
Gemini esterquats 12
Gemini surfactants 379
Gemini surfactants bis(Args) 365
Gene carriers 262
Gene delivery 254
Geotrichum candidum ST 002515 53
Global oil production 129
Global surfactant industry 3
Global warming 9
Glomerella cingulata 257
Gluconeogenesis 58
Glucosamine 209
Glucose 51
in MEL formation 61
Glucose/oleic acid fermentations 38
Glucosyltransferase 59
Glucuronic acid 52
Glutamic acid 432
Glycerine 4 16 35
Glycerol 238
synthesis of surfactant from
epoxidized methyl oleate and 439
Glycerolipid arginine surfactants 357
Glycol esters, applications of 13
Kurtzmanomyces sp I-11 53 56
Lipoproteins 29
Liquid laundry detergents, demand
for more concentrated 8
Long chain fatty acids, biosynthesis of 40
Lungs, deficiency symptoms in 160
Lung surfactant replacement therapy 192
Lung surfactants 191
additives
anionic polymers 209
cationic polymers 211 213
nonionic polymers 208
commercial exogenous 195
natural extracts 195
synthetic 200
evaluation
in situ 206
in vitro 201
in vivo 207
future trends and 219
historical overview and medical
needs for exogenous
discovery and physiology 192
respiratory distress syndrome
and lung surfactants 191
respiratory distress syndrome
(RDS) and 191
Lysophosphatidycholine (LPCs) 197
Microemulsions (Cont.)
sophorolipid-based 122
use of biosurfactants in 107
Microfiltration, coalescence
activation energy estimation based on 410
Microfiltration-based metalworking
fluids (MWF) recycling 414
Minimum inhibitory concentrations
(MIC) of MEL 255
Mississippi River, contamination of 426
Mitsubishi Chemical Co. 275
Mitsubishi-Kagaku Foods Corporation 275 324
grades of Ryoto Sugar ester manufactured by 276
Monoacylation 42
Mono- and diacylglycerols 4
Mono- and di-glyceride arginine surfactants 379
minimum inhibitory
concentration (mg/L) of 378
Monocatenaries, from arginine 360
Monocatenary arginine surfactants 353
minimum inhibitory
concentration (mg/L) of 375
Mono-corynomycolate (TL-2) 243
Mono-glucopyranosyl-13-hydroxydocosanote 39
Monoglycerides 14
applications of 13
Monohydroxy fatty acids 31
Monomethyl α-sulfosuccinate 12
Moorela thermoacetica 285
Nanotechnology 232
development of 265
Natural ceramides 265
The Natural Step 6
Natural surfactants' extracts 195
Neonatal respiratory distress
syndrome (NRDS) 160
Neutral lipids 29
Niche market 6
Nicotinamide adenine dinucleotide
phosphate (NADPH) 94
Nitrate reductase 88
Nitrilotriacetic acid 426
Nitrogen starvation 238
Nonionic polymers 208
Nonionic surfactants 4 392 451
Nonribosomal peptide synthetases 133
Rhamnolipids 5 10 30 77
107 243 252
applications 81
chemical structures of 234
production 82
advances in, by denitrifying
fermentation
technology 87
biosynthesis pathway and
regulations 84
ongoing and future work 90
producing microorganisms and
characteristics 82
properties 79
structures 77
Rhamnose 81
Rhamnosyl-β-hydroxydecanoyl-
β-hydroxydecanoate (Rha-C10-C10) 77
Rhamnosyl-rhamnosyl-β-
hydroxydecanoyl-β-
hydroxydecanoate (Rha-Rha-
C10-C10) 77
Rhizotecnia erythropolis 257
Rhizotecnia solani 257
Rhodotorula bogoriensis 30 39
Rhodotorula bogoriensis sophorolipids 40
Ricinoleic acid 4
Ring opening 43
Roundtable for Sustainable Palm Oil 7
Saccharide esters 14
Saccharide fatty acid esters
effects of water activity and
content in synthesis of 343
motivation for lipase-catalyzed
synthesis of 325
properties and applications 323
synthesis
in ionic liquid phase system 335
in organic solvents 325
in solid phase system 334
in solventless system 339
in supercritical CO2 329
using complexation agents 332
via derivatization 330
Saccharide-fatty acid esters 9
Saccharide/lipidic feedstocks 31
Saccharides 9 16
Saccharomyces cerevisiae,
trehalose synthesis of 238
Saline-lung Lavage Model 208
Saponins 13
SARAYA Co., Ltd. (Osaka, Japan) 243
Sasol Chemical Company 119
Schizonella melanogramma 53
Schizonellin 53
Sophorolipids 5 10 30 77
241 243 252
antiradical and anti-elastic
properties 264
applications 81 107
biosynthesis 30
chemical structures 234
hydrophilicity/lipophilicity 122
introduction and applications 30
post-synthetic enzymatic/
chemical modifications of 41
production 92
biosynthesis pathway 92
effects of culture conditions on formation 93
purification and 96
producing microorganisms and characteristics 92
production and modification
of, from agricultural feedstocks 29
biochemistry/enzymology 39
biosynthesis 30
post-synthetic enzymatic/chemical
modifications of 41
properties 79
structures 77
synthesis 30 238
Sophorose 31
Sorbitan derivatives, applications of 13
Sorbitan esters 15
ethoxylated 15
Sorbitol 4
Soybean oils 4 6 56 57
as biodegradable 427
Soybean processing 36
Soy molasses 36
SP-A 157 194 195
Span 13
SP-B 157 159 194 195
197 213
SP-C 157 159 161 194
195 197
SP-D 158 159 194 195
Sphingomyelins (SPHs) 192
SrfAD 134
Standard for Comparing Metal
Removal Fluids Using
the Tapping Torque Test
Machine (ASTM D5619) 407
Standard of living, energy
consumption and 129
Staphylococcus epidermidis, inhibiting
growth of 255
Starmella bombicola 92
Stepan 10
Sterols 4 13
Streptococcusfaecium, inhibiting
growth of 255
Structure based surfactant selection 395
Submerged aeration 87
Succinic acid 12
Succinoyl 43
Succinoyl-trehalose lipids (STL-1 and-2) 243
chemical structures of 235
Sucrose esters 14
applications
bacteriostatic activity of
sucrose monopalmitate 284
crystal control of fat in oil-in-water emulsion 293
dispersion of solid in aqueous media 287
pharmaceutical 294
chemical structure of 275
Sucrose fatty acid esters
molecular shape of 278
surface activity of 281
Sucrose monopalmitate 286
bacteriostatic activity of 284
effect of on heat stability of
microbes at 121°C 287
Sucrose monostearate 289
chemical structure 276
in dispersion of solid in aqueous media 287 289
Sugar-based surfactants 244
interfacial properties 449
environmental and product
performance concerns 451
fundamental studies on 455
air/water interfaces 456
Surfactants (Cont.)
classification 29
defined 29
environmentally advanced 244
ESO polymerization in forming 441
industrial uses 231
nonionic 392 451
optimization of concentrations
for maximum stability of
vegetable oil metalworking
fluids 409
poor biodegradability 427
selecting, based on molecular
structure and micelle
solubilization 397
structures 233
sugar-based 244
synthesis of, from epoxidized
methyl oleate and glycerol 439
used in metalworking fluids (MWF) 392
use of natural product based 427
Zwitterionic 392
Surfactant selection guidelines 405
Surfactant structure, effect of,
on micelle solubilization 396
Surfactin biosynthesis,
transcriptional regulation of 134
Surfaxin (KL, or lucinactant) 160 201
Tallow 4
Ternary DPPC/PG/Hel 13-5 and
DPPC/PA/Hel 13-5 systems 172
FM observations for DPPC/PA/
Hel 13-5 system 175
hysteresis curves 175
Langmuir isotherms 172
Tertiary oil recovery 3
Thermal synthesis 432
Thermodynamically stable vesicles 247
Thin-film pressure balance (TFPB) 454
Threonine 5
Top-down method 232
Torulopsis apicola 92
Torulopsis magnoliae 30 92
Toxicity, alcohol ethoxylate and 429
Transacylation
effect of type of solvent on, of
diethanolamine with fatty
acid ethyl ester 318
at high substrate loads 319
Transcriptional regulation of
surfactin biosynthesis 134
Trehalose di-corynomycolate (TL-1) 243
Trehalose esters 5
Trehalose lipids 10 238 243
chemical structures of 235
Triglycerides, sophorolipid
synthesis from 93
Tsukamurella sp. 257
Tubular myelin 157
Tween 13
Type II pneumocytes 192
Walmart 432
Water activity and content, effects of,
in synthesis of saccharide fatty acid esters 343
Xylitol 4