MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS,
`0146-0749/97/$04.0010
`Copyright q 1997, American Society for Microbiology
`
`Mar. 1997, p. 47–64
`
`Vol. 61, No. 1
`
`Microbial Production of Surfactants and Their Commercial Potential
`JITENDRA D. DESAI1* AND IBRAHIM M. BANAT2
`Applied Biology and Environmental Sciences Division, Research Center, Indian Petrochemicals Corporation Limited,
`Vadodara 391 346, India,1 and Department of Biology, Faculty of Science, United Arab Emirates University,
`Al-Ain, Abu-Dhabi, United Arab Emirates2
`
`INTRODUCTION .........................................................................................................................................................47
`RECENT ANALYTICAL METHODS ........................................................................................................................48
`Screening of Potential Biosurfactant-Producing Microorganisms.....................................................................48
`Estimation of Biosurfactant Activity......................................................................................................................48
`BIOSURFACTANT CLASSIFICATION AND THEIR MICROBIAL ORIGIN ...................................................48
`Glycolipids .................................................................................................................................................................48
`Rhamnolipids ........................................................................................................................................................48
`Trehalolipids..........................................................................................................................................................50
`Sophorolipids.........................................................................................................................................................50
`Lipopeptides and Lipoproteins ...............................................................................................................................50
`Fatty Acids, Phospholipids, and Neutral Lipids ..................................................................................................50
`Polymeric Biosurfactants .........................................................................................................................................51
`Particulate Biosurfactants .......................................................................................................................................51
`PHYSIOLOGY AND GENETICS...............................................................................................................................51
`Physiological Role.....................................................................................................................................................51
`Biosynthesis ...............................................................................................................................................................52
`Regulation ..................................................................................................................................................................52
`Genetic Characterization .........................................................................................................................................52
`Genetics of rhamnolipid synthesis .....................................................................................................................52
`Genetics of surfactin synthesis ...........................................................................................................................53
`KINETICS OF FERMENTATIVE PRODUCTION .................................................................................................53
`Growth-Associated Production................................................................................................................................53
`Production under Growth-Limiting Conditions ...................................................................................................53
`Production by Resting or Immobilized Cells ........................................................................................................54
`Production with Precursor Supplementation........................................................................................................54
`FACTORS AFFECTING BIOSURFACTANT PRODUCTION ..............................................................................54
`Carbon Source...........................................................................................................................................................54
`Nitrogen Source ........................................................................................................................................................55
`Environmental Factors.............................................................................................................................................55
`BIOSURFACTANT PRODUCTION BY BIOTRANSFORMATION .....................................................................55
`RECOVERY OF BIOSURFACTANTS.......................................................................................................................56
`POTENTIAL COMMERCIAL APPLICATIONS .....................................................................................................57
`CONCLUDING REMARKS........................................................................................................................................58
`ACKNOWLEDGMENTS .............................................................................................................................................59
`REFERENCES ..............................................................................................................................................................59
`
`“By which one sees an unperishable entity in all beings
`and undivided among the divided then that knowledge is pure.
`But if one merely sees the diversity of things with their divisions and
`limitations, without the truth, then that knowledge is merely an igno-
`rance.”
`
`The Bhagavad Gita, chapter XVIII
`
`INTRODUCTION
`Surfactants are amphipathic molecules with both hydro-
`philic and hydrophobic (generally hydrocarbon) moieties that
`partition preferentially at the interface between fluid phases
`with different degrees of polarity and hydrogen bonding such
`as oil/water or air/water interfaces. These properties render
`surfactants capable of reducing surface and interfacial tension
`and forming microemulsion where hydrocarbons can solubilize
`in water or where water can solubilize in hydrocarbons. Such
`
`* Corresponding author. Fax: 91-265-372098.
`
`characteristics confer excellent detergency, emulsifying, foam-
`ing, and dispersing traits, which makes surfactants some of the
`most versatile process chemicals (71, 72).
`Current worldwide surfactant markets are around $9.4 bil-
`lion per annum (226), and their demand is expected to increase
`at a rate of 35% toward the end of the century (71). Almost all
`surfactants currently in use are chemically derived from petro-
`leum; however,
`interest in microbial surfactants has been
`steadily increasing in recent years due to their diversity, envi-
`ronmentally friendly nature, the possibility of their production
`through fermentation, and their potential applications in the
`environmental protection, crude oil recovery, health care, and
`food-processing industries (10, 11, 60, 118, 155, 257).
`Biosurfactants are a structurally diverse group of surface-
`active molecules synthesized by microorganisms. These mole-
`cules reduce surface and interfacial tensions in both aqueous
`solutions and hydrocarbon mixtures, which makes them poten-
`tial candidates for enhancing oil recovery (219, 227, 234) and
`
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`48
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`DESAI AND BANAT
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`MICROBIOL. MOL. BIOL. REV.
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`deemulsification processes (28). Biosurfactants have several
`advantages over the chemical surfactants, such as lower toxic-
`ity; higher biodegradability (266); better environmental com-
`patibility (65); higher foaming (203); high selectivity and spe-
`cific activity at extreme temperatures, pH, and salinity (126,
`257); and the ability to be synthesized from renewable feed-
`stocks. Earlier work on biosurfactants centered mainly on the
`properties, biosynthesis, and chemistry and has been reviewed
`by many workers (34, 49, 50, 209a, 244). However, in the last
`few years, significant work on the fermentative production,
`genetics, and commercial applications of biosurfactants has
`been done; this, along with a brief account on the recent
`developments in microbial screening for biosurfactants, forms
`the subject matter of the present review.
`
`RECENT ANALYTICAL METHODS
`
`Screening of Potential Biosurfactant-Producing
`Microorganisms
`Recent advances in the field of microbial surfactants are
`largely attributed to the development of quick, reliable meth-
`ods for screening biosurfactant-producing microbes and assess-
`ing their potential. Van der Vegt et al. (254) developed an
`axisymmetric drop shape analysis (ADSA) by profile for the
`assessment of potential biosurfactant-producing bacteria. In
`this technique, drops of culture broth are placed on a fluoro-
`ethylene-propylene surface and the profile of the droplet is
`determined with a contour monitor. Surface tensions are cal-
`culated from the droplet profiles by ADSA. Only biosurfac-
`tant-producing bacterial suspensions show reduction in surface
`tensions. Shulga et al. (231) described a colorimetric estima-
`tion of biosurfactants based on the ability of the anionic sur-
`factants to react with the cationic indicator to form a colored
`complex. Development of other simple methods include the
`following: (i) a rapid drop-collapsing test (105), in which a drop
`of a cell suspension is placed on an oil-coated surface, and
`drops containing biosurfactants collapse whereas non-surfac-
`tant-containing drops remain stable; (ii) a direct thin-layer
`chromatographic technique for rapid characterization of bio-
`surfactant-producing bacterial colonies as described by Mat-
`suyama et al. (143); (iii) colorimetric methods described by
`Siegmund and Wagner (232) and Hansen et al. (79) for screen-
`ing of rhamnolipid-producing and hydrocarbon-degrading bac-
`teria, respectively; and (iv) estimation of the emulsification
`index value (E-24) by vigorously shaking culture broth samples
`with an equal volume of kerosene and measuring the percent
`emulsification after 24 h by the method of Cooper and Gold-
`enberg (35), which is most suitable for emulsifying biosurfac-
`tants.
`
`Estimation of Biosurfactant Activity
`Biosurfactant activities can be determined by measuring the
`changes in surface and interfacial tensions, stabilization or
`destabilization of emulsions, and hydrophilic-lipophilic bal-
`ance (HLB). Surface tension at the air/water and oil/water
`interfaces can easily be measured with a tensiometer. The
`surface tension of distilled water is 72 mN/m, and addition of
`surfactant lowers this value to 30 mN/m. When a surfactant is
`added to air/water or oil/water systems at increasing concen-
`trations, a reduction of surface tension is observed up to a
`critical
`level, above which amphiphilic molecules associate
`readily to form supramolecular structures like micelles, bilay-
`ers, and vesicle. This value is known as the critical micelle
`concentration (CMC). CMC is defined by the solubility of a
`
`surfactant within an aqueous phase and is commonly used to
`measure the efficiency of a surfactant. Microbial culture broth
`or biosurfactants are diluted severalfold, surface tension is
`measured for each dilution, and the CMC is calculated from
`this value. The values of the surface tension, interfacial ten-
`sion, and CMC of some known biosurfactants are listed in
`Table 1.
`An emulsion is formed when one liquid phase is dispersed as
`microscopic droplets in another liquid continuous phase. Bio-
`surfactants may stabilize (emulsifiers) or destabilize (deemul-
`sifiers) the emulsion. The emulsification activity is assayed by
`the ability of the surfactant to generate turbidity due to sus-
`pended hydrocarbons such as a hexadecane–2-methylnaphtha-
`lene mixture (47, 210) or kerosene (35), etc., in an aqueous
`assay system. The deemulsification activity is derived by deter-
`mining the effect of surfactants on a standard emulsion by
`using a synthetic surfactant (209a, 266).
`The HLB value indicates whether a surfactant will promote
`water-in-oil or oil-in-water emulsion by comparing it with sur-
`factants with known HLB values and properties. The HLB
`scale can be constructed by assigning a value of 1 for oleic acid
`and a value of 20 for sodium oleate and using a range of
`mixtures of these two components in different proportions to
`obtain the intermediate values. Emulsifiers with HLB values
`less than 6 favor stabilization of water-in-oil emulsification,
`whereas emulsifiers with HLB values between 10 and 18 have
`the opposite effect and favor oil-in-water emulsification.
`
`BIOSURFACTANT CLASSIFICATION AND THEIR
`MICROBIAL ORIGIN
`
`Unlike chemically synthesized surfactants, which are classi-
`fied according to the nature of their polar grouping, biosurfac-
`tants are categorized mainly by their chemical composition and
`their microbial origin. In general, their structure includes a
`hydrophilic moiety consisting of amino acids or peptides an-
`ions or cations; mono-, di-, or polysaccharides; and a hydro-
`phobic moiety consisting of unsaturated, saturated, or fatty
`acids. Accordingly, the major classes of biosurfactants include
`glycolipids, lipopeptides and lipoproteins, phospholipids and
`fatty acids, polymeric surfactants, and particulate surfactants.
`Although there are a number of reports on the synthesis of
`biosurfactants by hydrocarbon-degrading microorganisms,
`some biosurfactants have been reported to be produced on
`water-soluble compounds such as glucose, sucrose, glycerol, or
`ethanol (35, 74, 92, 184, 186). The biosurfactant-producing
`microbes are distributed among a wide variety of genera. The
`major types of biosurfactants, with their properties and micro-
`bial species of origin, are listed in Table 1 and are described
`briefly in the following section. For more details, readers are
`referred to Desai and Desai (50), Rosenberg (209a), Kosaric et
`al. (124), and Banat (11).
`
`Glycolipids
`
`Most known biosurfactants are glycolipids. They are carbo-
`hydrates in combination with long-chain aliphatic acids or hy-
`droxyaliphatic acids. Among the glycolipids, the best known
`are rhamnolipids, trehalolipids, and sophorolipids.
`Rhamnolipids. Rhamnolipids, in which one or two molecules
`of rhamnose are linked to one or two molecules of b-hydroxy-
`decanoic acid, are the best-studied glycolipids. Production of
`rhamnose-containing glycolipids was first described in Pseudo-
`monas aeruginosa by Jarvis and Johnson (108). L-Rhamnosyl-
`L-rhamnosyl-b-hydroxydecanoyl-b-hydroxydecanoate (Fig.
`1A) and L-rhamnosyl-b-hydroxydecanoyl-b-hydroxydecanoate,
`
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`VOL. 61, 1997
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`MICROBIAL PRODUCTION OF SURFACTANTS
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`49
`
`TABLE 1. Microbial source and properties of important types of microbial surfactants
`
`Biosurfactant
`
`Organisms
`
`Surface tension
`(mN/m)
`
`CMC
`
`Interfacial tension
`(mN/m)
`
`Reference(s)
`
`Glycolipids
`Rhamnolipids
`
`Trehalolipids
`
`Sophorolipids
`
`Cellobiolipids
`
`Lipopeptides and lipoproteins
`Peptide-lipid
`Serrawettin
`Viscosin
`Surfactin
`Subtilisin
`Gramicidins
`Polymyxins
`
`Fatty acids, neutral lipids, and phospholipids
`Fatty acids
`Neutral lipids
`Phospholipids
`
`Polymeric surfactants
`Emulsan
`Biodispersan
`Mannan-lipid-protein
`Liposan
`Carbohydrate-protein-lipid
`
`Protein PA
`
`Particulate biosurfactants
`Vesicles and fimbriae
`Whole cells
`
`P. aeruginosa
`Pseudomonas sp.
`R. erythropolis
`N. erythropolis
`Mycobacterium sp.
`T. bombicola
`T. apicola
`T. petrophilum
`U. zeae, U. maydis
`
`B. licheniformis
`S. marcescens
`P. fluorescens
`B. subtilis
`B. subtilis
`B. brevis
`B. polymyxa
`
`C. lepus
`N. erythropolis
`T. thiooxidans
`
`A. calcoaceticus
`A. calcoaceticus
`C. tropicalis
`C. lipolytica
`P. fluorescens
`D. polymorphis
`P. aeruginosa
`
`A. calcoaceticus
`Variety of bacteria
`
`0.25
`1
`14–17
`3.5
`15
`1.8
`0.9
`
`0.1–0.3
`
`1
`
`2
`3
`
`29
`25–30
`32–36
`30
`38
`33
`30
`
`27
`28–33
`26.5
`27–32
`
`30
`32
`
`27
`
`0.1–10
`4
`20
`0.3
`
`12–20
`
`150
`23–160
`
`150
`
`10
`
`74, 208
`88, 128, 185
`200
`140, 142
`40
`40, 68
`93, 250
`38
`24, 242
`
`109, 263
`143
`176
`3, 20
`20
`139
`240
`
`40, 43
`136
`17, 121
`
`210, 270
`211, 213
`112
`32, 33
`47, 189
`236
`87, 89
`
`76, 113
`58, 209a
`
`referred to as rhamnolipid 1 and 2, respectively, are the prin-
`cipal glycolipids produced by P. aeruginosa (57, 88, 102, 103).
`The formation of rhamnolipid types 3 and 4 containing one
`b-hydroxydecanoic acid with one and two rhamnose units, re-
`spectively (242), methyl ester derivatives of rhamnolipids 1 and
`
`2 (86), and rhamnolipids with alternative fatty acid chains (128,
`185, 206) has also been reported. Rhamnolipids from Pseudo-
`monas spp. have been demonstrated to lower the interfacial
`tension against n-hexadecane to 1 mN/m and the surface ten-
`sion to 25 to 30 mN/m (74, 128, 185). They also emulsify
`
`FIG. 1. Structure of some common glycolipid biosurfactants. (A) Rhamnolipid type 1 from Pseudomonas aeruginosa in which two rhamnose subunits are linked to
`two b-hydroxydecanoic acids in a side chain. (B) Trehalose dimycolate from Rhodococcus erythropolis, in which disaccharide trehalose is linked to two long-chain
`a-branched b-hydroxy fatty acids. (C) Sophorolipid from Torulopsis bombicola in which dimeric sophorose is linked to a long-chain (C18) hydroxy fatty acid.
`
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`50
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`DESAI AND BANAT
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`MICROBIOL. MOL. BIOL. REV.
`
`alkanes and stimulate the growth of P. aeruginosa on hexade-
`cane (88). Itoh and Suzuki (102) isolated two mutants of P.
`aeruginosa, PU-1 and PU-2, which grew poorly on alkanes due
`to their inability to produce rhamnolipids. These mutants grew
`normally when the growth medium was supplemented with
`rhamnolipid.
`Trehalolipids. Several structural types of microbial trehalo-
`lipid biosurfactants have been reported (128, 133). Disaccha-
`ride trehalose linked at C-6 and C-69 to mycolic acids is asso-
`ciated with most species of Mycobacterium, Nocardia, and
`Corynebacterium. Mycolic acids are long-chain, a-branched-b-
`hydroxy fatty acids. Trehalolipids from different organisms dif-
`fer in the size and structure of mycolic acid, the number of
`carbon atoms, and the degree of unsaturation (5, 40, 128, 244).
`Trehalose dimycolate produced by Rhodococcus erythropolis
`(Fig. 1B) has been extensively studied (126, 200). R. erythropo-
`lis also synthesizes a novel anionic trehalose lipid (207). Tre-
`halose lipids from R. erythropolis and Arthrobacter sp. lowered
`the surface and interfacial tensions in the culture broth to 25 to
`40 and 1 to 5 mN/m, respectively (128, 133, 200).
`Sophorolipids. Sophorolipids, which are produced mainly by
`yeasts such as Torulopsis bombicola (39, 68, 99), T. petrophilum
`(38), and T. apicola (250), consist of a dimeric carbohydrate
`sophorose linked to a long-chain hydroxy fatty acid (Fig. 1C).
`These biosurfactants are a mixture of at least six to nine dif-
`ferent hydrophobic sophorosides. Similar mixtures of water-
`soluble sophorolipids from several yeasts have also been report-
`ed (93). Cutler and Light (44) showed that Candida bogoriensis
`produces glycolipids in which sophorose is linked to doco-
`sanoic acid diacetate. T. petrophilum produced sophorolipids
`on water-insoluble substrates such as alkanes and vegetable
`oils (38). These sophorolipids, which were chemically identical
`to those produced by T. bombicola, did not emulsify alkanes or
`vegetable oils. When T. petrophilum was grown on a glucose-
`yeast extract medium, however, sophorolipids were not pro-
`duced, but an effective protein-containing alkane emulsifying
`agent was formed (38). These results appear to contradict the
`conventional belief that microbial emulsifiers and surfactants
`are produced to facilitate the uptake of water-insoluble sub-
`strates. Although sophorolipids can lower surface and interfa-
`cial tension, they are not effective emulsifying agents (39).
`Both lactonic and acidic sophorolipids lowered the interfacial
`tension between n-hexadecane and water from 40 to 5 mN/m
`and showed remarkable stability toward pH and temperature
`changes (38, 128).
`
`Lipopeptides and Lipoproteins
`
`A large number of cyclic lipopetides including decapeptide
`antibiotics (gramicidins) and lipopeptide antibiotics (polymyx-
`ins), produced by Bacillus brevis (139) and B. polymyxa (240),
`respectively, possess remarkable surface-active properties. Or-
`nithine-containing lipids from P. rubescens (265) and Thioba-
`cillus thiooxidans (120), cerilipin, an ornithine- and taurine-
`containing lipid from Gluconobacter cerinus IFO 3267 (246),
`and lysine-containing lipids from Agrobacterium tumefaciens
`IFO 3058 (245) also exhibit excellent biosurfactant activity. An
`aminolipid biosurfactant called serratamolide has been iso-
`lated from Serratia marcescens NS.38 (164). Studies on serrata-
`molide-negative mutants showed that the biosurfactant in-
`creased cell hydrophilicity by blocking the hydrophobic sites on
`the cell surface (14).
`The cyclic lipopeptide surfactin (Fig. 2), produced by B.
`subtilis ATCC 21332, is one of the most powerful biosurfac-
`tants. It lowers the surface tension from 72 to 27.9 mN/m at
`concentrations as low as 0.005% (3). B. licheniformis produces
`
`FIG. 2. Structure of cyclic lipopeptide surfactin produced by Bacillus subtilis.
`
`several biosurfactants which act synergistically and exhibit ex-
`cellent temperature, pH, and salt stability (147, 263). The sur-
`factant BL-86, produced by B. licheniformis 86, is capable of
`lowering the surface tension of water to 27 mN/m and the
`interfacial tension between water and n-hexadecane to 0.36
`mN/m and promoting excellent dispersion of colloidal b-silicon
`carbide and aluminum nitride slurries (94, 95). Recent struc-
`tural analysis revealed that it is a mixture of lipopeptides with
`major components ranging in size from 979 to 1,091 Da. Each
`molecule contains seven amino acids and a lipid portion which
`is composed of 8 to 9 methylene groups and a mixture of linear
`and branched tails (96). Another important characteristic of
`this compound is its ability to lyse mammalian erythrocytes and
`to form spheroplasts (3, 20); this property has been used to
`detect surfactin production through hemolysis on blood agar.
`Recently, Yakimov et al. (263) have shown production of a
`new lipopetide surfactant, lichenysin A, by B. licheniformis BAS-
`50 containing long-chain b-hydroxy fatty acids. Lichenysin A
`reduces the surface tension of water from 72 to 28 mN/m with
`a CMC of as little as 12 mM, comparing favorably with surfac-
`tin (24 mM). The detailed characterization of lichenysin A
`showed that isoleucine was the C-terminal amino acid instead
`of leucine and an asparagine residue was present instead of
`aspartic acid as in the surfactin peptide. Addition of branched-
`chain a-amino acids to the medium caused similar changes in
`lipophilic moieties of lichenysin-A and lowering of surface
`tension activity (263a).
`
`Fatty Acids, Phospholipids, and Neutral Lipids
`
`Several bacteria and yeasts produce large quantities of fatty
`acid and phospholipid surfactants during growth on n-alkanes
`(5, 33, 43, 208). The HLB is directly related to the length of the
`hydrocarbon chain in their structures. In Acinetobacter sp.
`strain HO1-N phosphatidylethanolamine (Fig. 3), rich vesicles
`are produced (113), which form optically clear microemulsions
`of alkanes in water. The quantitative production of phospho-
`lipids has also been detected in some Aspergillus spp. (113) and
`Thiobacillus thiooxidans (17). Arthrobacter strain AK-19 (259)
`and P. aeruginosa 44T1 (208) accumulate up to 40 to 80%
`(wt/wt) of such lipids when cultivated on hexadecane and olive
`oil, respectively. Phosphatidylethanolamine produced by R.
`erythropolis grown on n-alkane caused a lowering of interfacial
`
`FIG. 3. Structure of phosphatidylethanolamine, a potent biosurfactant pro-
`duced by Acinetobacter sp. R1 and R2 are hydrocarbon chains of fatty acids.
`
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`MICROBIAL PRODUCTION OF SURFACTANTS
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`51
`
`large amounts of mannoprotein by Saccharomyces cerevisiae;
`this protein showed excellent emulsifier activity toward several
`oils, alkanes, and organic solvents. The purified emulsifier con-
`tains 44% mannose and 17% protein. Kappeli et al. (111, 112)
`have isolated a mannan-fatty acid complex from alkane-grown
`Candida tropicalis; this complex stabilized hexadecane-in-water
`emulsions. Schizonella malanogramma and Ustilago maydis
`produce biosurfactant which has been characterized as eryth-
`ritol- and mannose-containing lipid (59). Recently, Kitamoto
`et al. (117) demonstrated the production of two kinds of man-
`nosylerythritol lipids in Candida antarctica T-34. Hisatsuka et
`al. (87, 89) described the isolation from P. aeruginosa of a
`protein-like activator that was involved in emulsification of
`hydrocarbons. It has a molecular weight of 14,300 and contains
`147 amino acids, of which 51 are serine and threonine (89).
`The production by P. aeruginosa P-20 of a peptidoglycolipid
`bearing 52 amino acids, 11 fatty acids, and a sugar unit has
`been described previously (123). An emulsifying and solubiliz-
`ing factor containing protein and carbohydrate from hexade-
`cane-grown Pseudomonas spp. has also been reported (17,
`153). Desai et al. (47) demonstrated the production of bio-
`emulsifier by P. fluorescens during growth on gasoline. This
`bioemulsifier is composed of 50% carbohydrate, 19.6% pro-
`tein, and 10% lipid. Trehalose and lipid-o-dialkyl monoglycer-
`ides were the major components of the carbohydrate and lipid,
`respectively. Similarly, an extracellular bioemulsifier composed
`of carbohydrate, protein, and lipids was isolated from C. tropi-
`calis (236) and Phormidium strain J1 (58).
`
`Particulate Biosurfactants
`Extracellular membrane vesicles partition hydrocarbons to
`form a microemulsion which plays an important role in alkane
`uptake by microbial cells. Vesicles of Acinetobacter sp. strain
`HO1-N with a diameter of 20 to 50 nm and a buoyant density
`of 1.158 g/cm3 are composed of protein, phospholipid, and
`lipopolysaccharide (113). The membrane vesicles contain
`about 5 times as much phospholipid and about 350 times as
`much polysaccharide as does the outer membrane of the same
`organism.
`Surfactant activity in most hydrocarbon-degrading and
`pathogenic bacteria is attributed to several cell surface com-
`ponents, which include structures such as M protein and lipo-
`teichoic acid in the case of group A streptococci, protein A in
`Staphylococcus aureus, layer A in Aeromonas salmonicida, pro-
`digiosin in Serratia spp., gramicidins in Bacillus brevis spores,
`and thin fimbriae in A. calcoaceticus RAG-1 (49, 58, 128, 209a,
`262).
`
`PHYSIOLOGY AND GENETICS
`
`Physiological Role
`Biosurfactants are produced by a variety of microbes, se-
`creted either extracellularly or attached to parts of cells, pre-
`dominantly during growth on water-immiscible substrates (17,
`88, 114, 122, 210, 235). Biosurfactant-negative mutants of
`P. aeruginosa KY-4025 (102) and P. aeruginosa PG-201 (122)
`showed poor growth compared to the parent strains on n-
`paraffin and hexadecane, respectively, and addition of rhamno-
`lipid to the medium restored growth on these hydrocarbons.
`From a physiological point of view, production of such a large
`amount of polymer will be a waste if it has no function. The
`genetic system also loses the expression of the gene over a long
`period by mutation and selection if the gene product has no
`specific advantage for survival. The function of biosurfactant in
`
`FIG. 4. Structure of emulsan, produced by Acinetobacter calcoaceticus, in
`which fatty acids are linked to a heteropolysaccharide backbone.
`
`tension between water and hexadecane to less than 1 mN/m
`and a CMC of 30 mg/liter (126).
`
`Polymeric Biosurfactants
`The best-studied polymeric biosurfactants are emulsan, li-
`posan, mannoprotein, and other polysaccharide-protein com-
`plexes. Acinetobacter calcoaceticus RAG-1 produces a potent
`polyanionic amphipathic heteropolysaccharide bioemulsifier
`(Fig. 4) called emulsan (210). The heteropolysaccharide back-
`bone contains a repeating trisaccharide of N-acetyl-D-galac-
`tosamine, N-acetylgalactosamine uronic acid, and an uniden-
`tified N-acetyl amino sugar (271). Fatty acids are covalently
`linked to the polysaccharide through o-ester linkages (18, 225,
`271). Emulsan is a very effective emulsifying agent for hydro-
`carbons in water even at a concentration as low as 0.001 to
`0.01%. It is one of the most powerful emulsion stabilizers
`known today and resists inversion even at a water-to-oil ratio
`of 1:4 (18, 76, 270). On long standing, this emulsion separates
`into two layers. The upper cream layer, which is known as
`emulsanosol, contains 70 to 75% oil (270). Biodispersan is an
`extracellular, nondialyzable dispersing agent produced by A.
`calcoaceticus A2 (213). It is an anionic heteropolysaccharide,
`with an average molecular weight of 51,400 and contains four
`reducing sugars, namely, glucosamine, 6-methylaminohexose,
`galactosamine uronic acid, and an unidentified amino sugar
`(211). Recently, Navonvenezia et al. (173) described the iso-
`lation of alasan, an anionic alanine-containing heteropolysac-
`charide-protein biosurfactant from Acinetobacter radioresistens
`KA-53, which was found to be 2.5 to 3 times more active after
`being heated at 1008C under neutral or alkaline condition.
`Liposan is an extracellular water-soluble emulsifier synthe-
`sized by Candida lipolytica (32, 110) and is composed of 83%
`carbohydrate and 17% protein (32). The carbohydrate portion
`is a heteropolysaccharide consisting of glucose, galactose,
`galactosamine, and galacturonic acid. Palejwala and Desai
`(184) reported the production by a gram-negative bacterium of
`a potent bioemulsifier with carbohydrate as a major compo-
`nent. Sar and Rosenberg (218) demonstrated that polysaccha-
`ride had no emulsification activity alone but became a potent
`emulsifier when combined with some proteins released during
`growth on ethanol.
`Cameron et al. (29) recently reported the production of
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`MICROBIOL. MOL. BIOL. REV.
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`a producing microbial cell is not fully understood. However,
`there has been speculation about their involvement in emulsi-
`fication of water-insoluble substrates (64, 81, 178, 267). Direct
`contact of cells with hydrocarbon droplets and their interaction
`with emulsified droplets have been described (64, 209a, 235).
`In addition, biosurfactants have been shown to be involved in
`cell adherence which imparts greater stability under hostile
`environmental conditions and virulence (209a, 214), in cell
`desorption to find new habitats for survival (214), in antago-
`nastic effects toward other microbes in the environment (117,
`129, 251), etc.
`
`Biosynthesis
`In their amphiphilic structure, the hydrophobic moiety is
`either a long-chain fatty acid, a hydroxy fatty acid, or a-alkyl-
`b-hydroxy fatty acid, and the hydrophilic moiety may be a
`carbohydrate, carboxylic acid, phosphate, amino acid, cyclic
`peptide, or alcohol. Two primary metabolic pathways, namely,
`hydrocarbon and carbohydrate, are involved in the synthesis of
`their hydrophobic and hydrophilic moieties, respectively. The
`pathways for the synthesis of these two groups of precursors
`are diverse and utilize specific sets of enzymes. In many cases,
`the first enzymes for the synthesis of these precursors are
`regulatory enzymes; therefore, in spite of the diversity, there
`are some common features of their synthesis and regulation.
`The detailed biosynthetic pathways for the major hydrophobic
`and hydrophilic moieties have been extensively investigated
`and are well documented; however, a brief account by Hommel
`and Ratledge (91) may be useful. According to Syldatk and
`Wagner (244), the following possibilities exist for the synthesis
`of different moieties of biosurfactants and their linkage: (i) the
`hydrophilic and hydrophobic moieties are synthesized de novo
`by two independent pathways; (ii) the hydrophilic moiety is
`synthesized de novo while the synthesis of the hydrophobic
`moiety is induced by substrate; (iii) the hydrophobic moiety is
`synthesized de novo, while the synthesis of the hydrophilic
`moiety is substrate dependent; and (iv) the synthesis of both
`the hydrophobic and hydrophilic moieties is substrate depen-
`dent.
`Examples of all the above possibilities have been well doc-
`umented by Syldatk and W