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`148
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`Biosurfactants: Research Trends and Applications
`
`Surfactants are classified as cationic, anionic, zwitterionic, and nonionic and are
`
`Some surfactants, known as biosurfactants, are biologically produced from yeast
`or bacteria (Lin, 1996). They can be potentially as effective with some distinct
`advantages over the highly used synthetic surfactants due to high specificity, biode-
`gradability, and biocompatibility (Cooper, 1986).
`Biosurfactants are grouped as glycolipids, lipopeptides, phospholipids, fatty acids,
`and neutral lipids (Bierman et al., 1987). Most of these compounds are either anionic
`or neutral, with only a few cationic ones. The hydrophobic parts of the molecule are
`based on long-chain fatty acids, hydroxy fatty acids, or or-alkyl—p-hydroxy fatty acids.
`The hydrophilic portion can be a carbohydrate, amino acid, cyclic peptide, phosphate,
`carboxylic acid, or alcohol. A wide variety of microorganisms can produce these
`compounds. The CMCs of the biosurfactants generally range from 1 to 200 mg/L
`and their moiecular weights (MWS) from 500 to 1500 amu (Lang and Wagner, 1987).
`
`made synthetically from hydrocarbons, lignosulfonates, or triglycerides. Some com—
`mon synthetic surfactants include linear alkyl benzenesulfonates, alcohol sulfates,
`alcohol ether sulfates, alcohol glyceryl ether suifonates, oc~olefin sulfonates, alcohol
`ethoxylates, and alkylphenol ethoxylates (Layman, 1985). Surfactants have many
`applications industrially with multiphasic systems. Sodium dodecyl sulfate (SDS,
`CI2H25~SO4' Nat) is a widely used anionic surfactant. The effectiveness of a surfac-
`tant is determined by surface tension lowering, which is a measure of the surface free
`energy per unit area or the work required to bring a molecule from the bulk phase to
`the surface (Rosen, 1978). These amphiphilic compounds (containing hydrophobic
`and hydrophilic portions) concentrate at solid—liquid, liquid—liquid, or vapor—liquid
`interfaces. An interfacial boundary exists between two immiscible phases. The
`hydrophobic portion concentrates at the surface while the hydrophilic is oriented
`toward the solution. A good surfactant can lower the surface tension of water from 72
`to 35 mN/m and the interfacial tension (tension between nonpolar and polar liquids)
`for water against n-hexadecane from 40 to l mN/m. Efficient surfactants have a
`low CMC (i.e., less surfactant is necessary to decrease the surface tension} as the
`CMC is defined as the minimum concentration necessary to initiate micelle forma—
`tion (Becher, 1965). In practice, the CMC is also the maximum concentration of sur-
`factant monomers in water and is influenced by pH, temperature, and ionic strength.
`An important factor in the choice of surfactant is the product cost (Mulligan and
`Gibbs, 1993). In general, surfactants are used to save energy and consequently energy
`costs (such as the energy required for pumping or mixing). Charge type, physico—
`chemical behavior, solubility, and adsorption behavior are some important selection
`criteria for surfactants.
`
`etal., 2006), D (Zhao et al., 2010), bacillomycin (Roongsawang et al., 2003), fengycin
`
`LIPOPEPTIDE BIOSURFACTANTS
`
`Lipopeptides are produced by a variety of microorganisms, including Bacillus,
`Lacrobacilins, Srrepromyces, Pseudomonas, and Serratia (Cameotra and Makkar,
`
`2004; Georgiou et al., 1992). The lipopeptides are cyclic peptides with a fatty acyl
`chain. Various lipopeptides include surfactin (Roongsawang et al., 2003; Youssef
`et al., 2007),1ichenysin A (Yakimov et al., 1995) or C (Jenny et al., 1991), B (Folmsbee
`
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`Characterization, Production, and Applications of Lipopeptides
`
`149
`
`Surfactin A
`
`CH3“ CH (CH2)? — CH — CH2 — CO * Glu — Leu — Leo \
`CH3
`
`Leu — Leu Asp
`
`Surfactin B
`
`CHai CH (CH2)S — CH7 CH2 — CO 7 Glu - Leu — Leu
`
`CH3
`
`SurfactinC
`
`Leu e Leo fl Asp
`
`(Vanittanakom and Loeffier, 1986), and iturin (Bonrnatin et al., 2003). Surfactin is a
`cyclic heptapeptide, with antibacterial, antifungal, antiviral, and antitumor activities
`(Folrnsbee et al., 2006; Zhao et a1... 2010).
`Lipopeptides have been tested in enhanced oil recovery and the transportation of
`crude oils (Hayes et al., 1986). They were demonstrated to be effective for antimi-
`crobial activity and in the reduction of the interfacial tension of oil and water and the
`viscosity of the oil, the removal of water from the emulsions prior to processing, and
`the release of bitumen from oil sands. Although most biosurfactant—producing organ-
`isms are aerobic, a few anaerobic producers exist. Bacillus iichemformis JFu2 is an
`example, which would be well suited for in situ studies for enhanced oil recovery or
`soil decontamination (Javaheri et al., 1985).
`Surfactin is the most studied lipopeptide and consists of a seven-amino acid
`sequence in a cyclical structure with a 13wl6 carbon fatty acid (Kakinurna et al., 1969)
`and has two charged amino acids (glutamic and aspartic acids). In addition to sur-
`factin, iturins and fengycins are also produced (Deleu et al., 1999). Their structures
`are shown in Figures 6.1 through 6.3. Iturins are cyclic peptides with seven amino
`acids and a B«amino closure. Fengycin lipopeptides are B-hydroxy fatty acids with
`an eight-member ring in an N-terminal decapeptide. At the therrninal end, there is a
`tyrosine residue at position 3. This forms an eight-member lactone ring. Fengycin A
`and B vary at position 6. The A form has an Ala compared to the B form of a valine.
`
`Bioresomr. Techno!” 101, 6118, ZOlU.)
`
`CH3— CH (CH2er —' CH "- CH2 W CO — Glu — Leu — Leu
`
`lU
`
`wH—mu e Leu — Asp
`
`Surfactin D
`
`CH3— CH (Cl-12)1U e CH- CHZ— C0 * Giu - Leo — Leu
`
`CH3
`
`OW"— Leu — Lea ~ Asp
`
`FIGURE 6.1
`
`Structures of various forms of surfactin. (Adapted from Janeir, T. et £11.,
`
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`Peng et al. (2008)
`
`The fatty acid normally varies from 14 to 18 carbons in length (Arirna et al., 1968;
`Matsuyama et al., 1992; Roongsawang et al., 2003). Fengycin has three charged
`amino acids (two glutamic acids and an ornithine). The CMC is 6.25 mglL.
`Other lipopeptides have also been studied. They include lichenysin from
`B. lichem'formis (Grangemard et al., 1999; Horowitz et al., 1990), arthrofactin from
`Arthmbacter sp. (now known as Pseudomonas sp. M1838) (Morikawa et al., 1993;
`Roongsawang et al., 2003), pumilacidin from P. pumilus (Naruse et al., 1990), and
`serrawettin from Serratia marcescens (Matsuyarna et al., 1992). Others include mas-
`setolide A (Sen and Swaminathan, 1997), putisolvins I and II (Kuiper et al., 2004).
`and pumilacidin (Naruse et al., 1990). As can be seen, there is no generally accepted
`nomenclature based on the structure. Pseudomonas lipopeptides include viscosin,
`amphisin, tolassin, and syringomycin (Raaijmakers et al., 2006). Viscosin has nine
`amino acids with a 3-hydroxy fatty acid, whereas amphisin has 11 amino acids
`linked to a similar fatty acid (Soresen et al., 2001). A comparison of the CMC of
`some lipopeptides is shown in Table 6.1.
`
`— CH (CH2)12_1S — co — Glu — Orn
`
`CH3
`
`Ile — O—rw Tyr — AlloThr
`
`/
`Tyr
`\ Gln —— Pro w Ala — Glu
`
`FIGURE 6.3 Structure fengycin.
`
`W T
`
`ABLE 6.1
`
`CMC Values of Various Isolated Lipopeptides
`
`Lipopeptide
`Fengycin
`
`Microbial Source
`B. circulans DMS-2
`
`CMC (mg/L)
`10—13
`
`Surfactin
`Surfactin and fengycin
`Biosurfactant
`Lipopeptide
`
`B. subtilis
`B. subriiis
`B. Iichenifonnis
`Rhodocaccus 5p.
`
`17
`l0 and 11
`0.6
`23.?
`
`Reference
`Sivapathasekaran et al.
`(2009, 2050)
`
`Deleu et al. {1999)
`Lin et a1. (1998)
`Barres et al. (2008)
`
`Biosurfactants: Research Trends and Applications
`
`Iturin
`
`CH3- CH (CH2}9_14 r CH- CH2 — CO -’ ASn — Tyr 7 Asn \
`1
`/
`
`Set — Asn ‘- Pro
`
`FIGURE 6.2
`
`Structure of iturin.
`
`Fengycin
`
`H o
`
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`tiitaracterization, Production, and Applications of Lipopeptides
`
`- 151
`
`Synthesis of the lipopeptides is performed based on a series of enzymes for each
`W'or amino acid additioa, ring closure, and acylation (Peypoux et al., 1999). This
`makes genetic manipulation difficult for enhanced production. Most studies are con-
`;cntrated on grewth optimization and isolation of overproducers (Peypoux et al.,
`1099') With the exception of a few studies (Gu et al., 2007', Nakayama et al., 1997;
`Uhno et al., 1995; Peypoux et al., 1999).
`Most of the fecus has been on higher-priced applications due to the low yields and high
`gust of the media. Higher-volume low applications including environmental remediation,
`enhanced oil rec0very, laundry soaps, and polymerization of emulsions, need the develop-
`ment of low-cost substrates such as agroindustrial wastes (Makkar and Cameotra, 1999;
`N-Iukherjee et al., 2006) and isolation techniques, bioreactor design, and higher yields.
`
`SURFACTIN
`
`B. subtilis produces surfactin, one of the first lipopeptides found in 1968 (Figure 6.1).
`It has seven amino acids bonded to the carboxyl and hydroxyl groups of a l4~carbon
`acid (Kakinuma et al., 1969) and has blood clotting properties. Surfactin concentra-
`tions as low as 0.005% reduce the surface tension to 27 mN/m, making it a powerful
`hiosurfactant. The CMC can be as low as 10 mg/L (Dae et al., 2006). The interfacial
`tension for hydrocarbon—water interfaces can be less than 1 rnN/rn.
`The primary structure of surfactin was determined many years ago by Kaldnurna et al.
`(1969). It is a heptapeptide with a B—hydroxy fatty acid within a lactone ring structure. The
`seven amino acids are bonded to the carboxyl and hydroxyl groups of a 14—carbon acid.
`More recently, the three-dimensional structure was determined by 1H NMR techniques
`(Bonrnatin et al., 1995). Surfactin folds into a b—sheet structure, which resembles a horse
`saddle both in aqueous solutions and at the air/water interface (Ishigami et al., 1995). The
`solubility and surface-active properties of the surfactin are dependent on the orientation
`of the residues. The fatty acyl chain with the hydrophobic residues formed one face,
`while the two carboxylic acid side chains form a claw structure enabling the chelation of
`heavy metals (Bonmatin et al., 1994; Gallet et al., 1999; Magetdana and Ptak, 1992). This
`property has been evaluated in soil remediation studies (Mulligan et al., 1999).
`Mixtures of surfactin produced by B. subtilis have been characterized by mass
`spectrometry (Hue et al., 2001). A combination of liquid—secondary ion mass spec-
`trometry (LSI-MS) and high—energy tandem mass spectrometry (MS/MS) showed
`that the amino acid composition or length of the acyl chain can vary from 12 to
`16 carbons. Leucine and isoleucine can also be differentiated. Data obtained from
`protonated and cationized fragments were also useful for structural characterization.
`They are known as A, B, C, and D forms (Figure 6.1).
`
`
`
`SURFACTIN PRODUCTION
`
`Most biosurfactants are produced from hydrocarbon substrates (Syldatk and Wagner,
`1987). Production can be growth associated. In this case, they can either use the emulsi-
`ficaticn of the substrate (extracellular) or facilitate the passage of the substrate through
`the membrane (cell membrane associated). Biosurfactants, however, are also pro-
`duced from carbohydrates, which are very soluble. Gram-positive and gram-negative
`
`m a! u;
`
`nun-cunt In)Mimi; '
`
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`1152
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`Biosurfactants: Research Trends and Applications
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`bacteria can produce cyclic lipopeptides. The different structures can lead to different
`properties. The biosurfactants have been postulated to enhance the growth on hydro-
`carbons and, in this case, may influence the ecology of the host sponge.
`Solid-state fermentation using okara, a soybean curd residue, has been performed
`by Ohno et al. (1995). Other substrates studied have included starch (Sandrin et al.,
`1990), cassava waste (Santos et al., 2000), molasses (Makkar and Cameotra, 1997b),
`soybean (Kim et al., 2009), and potato wastes (Fox and Bala, 2000). Surfactin yields
`from an autoclaved purified starch were 0.154 g/g. Low solid potato effluents exhib-
`ited a 66% lower surfactin yield than the purified starch (Thompson et al., 2001).
`It has been postulated that higher yields result from nutritional limitations.
`, Surfactin yields during production are low (0.02 g/g glucose) (de Roubin et al., 1989).
`Addition of iron and manganese can enhance concentrations to 0.7 g/L (Rosenberg, 1986).
`Further work on iron addition performed by Wei and Chu (1998) determined that addition
`of 1.7 mM of iron can lead to the production of up to 3.5 g/L of surfactin and enhanced
`biomass production. Alkaline addition is required to overcome the decrease in pH to
`below 5 due to acid formation. Further studies by Wei and Chu (2002) showed the effect
`of manganese on nitrogen utilization and subsequently surfactin production. A 0.1 mM
`magnesium sulfate concentration increased almost ninefold the surfactin level to 2.6 g/L.
`Wei et a1. (2007) subsequently used the Taguchi method to optimize surfactin production
`with regard to the presence of Mg, K, Mn, and Fe. They found that K and Mg were criti-
`cal. Kinsinger et al. (2003, 2005) further determined that concentrations of the four ions
`
`high concentrations of NaCl (up to 4%) and pH values from 4.5 to 10.5.
`
`could be optimized and allowed the production of 3.34 g/L of surfactin.
`Yields of 0.14 g/g sugar have been obtained using peat as a substrate after hydro-
`lysis with 0.5% sulfuric acid for 1 h at 120°C (Sheppard and Mulligan, 1987). Citric
`acid addition to glucose media could also enhance production (de Roubin et al., 1989).
`In attempts to influence the metabolic pathway, glutamic acid, leucine, aspartic acid,
`and valine were added to the media but did not enhance production. Nitrogen, how-
`ever, was a significant factor in surfactin production. Doubling anunonium nitrate
`concentrations from 0.4% to 0.8% increased yields by a factor of 1.6, while organic
`nitrogen addition did not have any benefit.
`Other investigators (Davis et al., 1999) found that surfactin yields were high-
`est in nitrate~1imited oxygen-depleted conditions, followed by ammonium-limited
`(0.075 g surfactin per g biomass), oxygen-depleted conditions (0.012 g/g biomass),
`and carbon-limited, oxygen—depleted conditions (0.0069 g/g biomass).
`A strain of B. subrilis was able to produce biosurfactant at 45°C at high NaCl con—
`centrations (4%) and a wide pH range (45—105) (Makkar and Cameotra, 1997a,b).
`It was able to remove 62% of the oil in a sand pack saturated with kerosene and thus
`could be used for in situ oil removal and cleaning sludge from sludge tanks.
`Makkar and Cameotra (2002) studied another strain of B. subtilis MTCC2423.
`They found it preferred sodium or potassium nitrate (3 g/L) or urea (1 g/L).
`Magnesium concentrations of 2.43 mM and calcium concentrations of 0.36 mM
`were optimal for biosurfactant yield. Unlike for previous studies for B. sabtilis by
`de Roubin et al. (1989), aspartic acid, asparagine, glutamic acid, valine, and lysine
`increased biosurfactant production by 60%. While glycine and leucine addition had
`no affect, alanine and arginine decreased production. Production was good even at
`
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`Characterization, Production, and Applications of Lipopeptides
`
`153
`
`Solid carriers have also been evaluated for surfactin yield enhancement (Yeh et a1.,
`2005). Activated carbon and expanded clay were added at concentrations of 133 g/L.
`Surfactin at concentrations of 2150 and 3300 mgi’L were obtained for each carrier,
`resPectively. Activated carbon was more appropriate for the fermentation process and
`seemed to increase cell growth and thus yield. A summary of the yields of surfactin
`can be seen in Table 6.2.
`
`Das et a1. (2009) determined that antimicrobial activity was obtained from a
`glucose substrate, instead of sucrose, starch. and glycerol. Emulsifying lipopeptide
`biosurfactants from Azotobacter chroocaccum can be produced from oil (crude,
`waste motor lubricant) and peanut oil cake (Thavasi et 211., 2009).
`Production of another lipopeptide, brevifactin, was characterized and opti—
`mized by the marine strain Brevibacterium anreum MSA13 (Kiran et al., 2010).
`
`
`
`TABLE 6.2
`
`Production of Surfactin
`
`B. subfilis Strain
`
`ATCC 21332
`
`R1314
`
`Substrate
`
`Synthetic or scmisynthetic
`peat hydrolysate
`Aqueous two phase
`
`M1113 (pc12)
`M1113 (pC12)d
`ATCC 55033
`
`Mutant strain of
`ATCC 21332
`
`C9 (KCTC 370113)
`ATCC 21332
`ATCC 21332
`
`ATCC 21332
`
`MTCC 2423
`SD 901
`ATCC 21332
`Isolate
`
`Semisynthetic
`
`Solid-state okara
`
`Semisynthetic
`Solid-state okara
`
`Semisynthetic
`
`Sy nthetic
`
`Glucose with modified salts
`and oxygen limitation
`Glucose and mineral salts
`with iron
`
`Glucose with oxygen and
`nitrogen depletion
`Purified starch
`Sucrose with mineral salts
`
`Bean extract
`Solid carriers
`Sucrose with foam collection
`
`—Lu__,—___~_—___H__—_
`
`Surfactin Yield 0r
`Concentration
`
`100—250 rug/L
`
`160 ingi'L
`350 mg/L
`250 mg/L
`
`200-250 rug/kg
`wet mass
`
`350 mg/L
`2000 mgi'kg wet mass
`
`3500—4300 mgi‘L
`2000—4000 mg/L
`550 IngfL
`7.0 gi'L
`
`3.5 gi'L
`
`0.44 g/L
`
`0.154 gig
`1.23 glL
`3000750000 mglL
`
`21503300 mg/L
`0,25 gig
`
`Reference
`
`Arirna et a1. (1968):
`Cooper et a1. (1981)
`Sheppard and
`Mulligan (1987)
`Drouin and
`
`Cooper (1992)
`Ohno et al. (1992)
`Ohno et a1. (1992)
`
`Ohno et a1. (1992)
`Ohno et a1. (1992)
`Carrera et a]. (1992)
`Camera et al. (1993a,b)
`Mulligan et a]. (1989)
`Kim et all (1997)
`
`Wei and)ChLl (1998)
`
`Davis et a]. (1999)
`
`Fox and Bela (2000)
`Maidtar and
`
`Cameotra (2002)
`Yoneda et a1, (2005)
`Yeh et al. (2005)
`Amani et a1. (2010)
`
`Source: Adapted from Shaligram, NS. and Singhal, R.S. Food Tbchnol. Biotechnol.. 48: 119—134. 2010.
`
`HHIIILIJK
`
`091‘ I I I H I It‘ll. [I] U. mun Ii-
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`EXHIBIT NO. 1019 Page 8 of 35
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`

`LIPOPEPTIDE PRODUCTION REACTOR DESlGN
`AND OPTIMIZATiON
`
`Free and immobilized cells of B. .mbti'lis ATCC 21332 were grown to produce
`surfactin and fengycin (Chtioui et al., 2010). Although the production of both
`biosurfactants was enhanced by two to four times, fengycin was particularly
`improved. N-heptane was used for extracting the biosurfactant. A continuous
`extraction with a liquid membrane called petraction was used, but the stripping
`was too slow. Further optimization is needed. Petraction was also employed by
`Dimitrov et al. (60) for surfactin. At pH 5.65, 97% recovery was achieved com-
`pared to 83% at pH 6.05 in 4 h. However, approximately 90% was removed in
`30 min.
`
`Further studies were performed using a rotating disk bioreactor (Chtioui et a1.,
`2012). Cells were immobilized on the rotating disks. Foaming did not occur
`as the aeration was bubbleless. Fengycin production was favored (838 mg/L)
`compared to surfactin (212 mg/L). Increasing the number of disks improved
`the production of both products. Surfactin production was more correlated with
`improved oxygenation, while the fengycin production was related to more bio-
`film formation.
`
`1154
`
`Biosurfactants: Research Trends and Applications
`
`Various agro and industrial solid waste substrates including molasses, olive oil, and
`acrylarnide were evaluated. The biosurfactant was stable over the pH range of 5—9,
`and up to 5% NaCl and a temperature of 121°C. The surface tension was 28.6 mN/m.
`The lipopeptide was characterized as an octadecanoic acid methyl ester with four
`amino acids profileuigly-gly. This lipopeptide, thus, could have potential for micro-
`bial enhanced oil recovery and oil spill remediation.
`
`and aeration of 0.75 vvm. Primary inoculum age (55-57 h) of 5%—6% by
`
`A two-phase reactor with polyethylene glycol and dextran (D-40) was evaluated for
`surfactin production by B. subtilis ATCC 21332 (Drouin and Cooper, 1992) in a cyclone
`reactor. Cells accumulated in the dextran phase and surfactin in the other phase. This
`enabled the separation of surfactin from the cells to decrease cell inhibition.
`An airlift reactor in batch mode was employed to enhance aeration with a potato
`process effluent as the substrate (Noah et al., 2002). A 0.5 vvm air flow rate enabled
`surfactin removal. Conditions of a large inoculum, pH control, and the use of a
`pressurized reactor optimized the growth of B.
`.mbtilis over indigenous bacteria.
`Noah et a1. (2005) subsequently used a chemostat and low solid potato effluents.
`At 0.5 vvm, a surfactin concentration increased to 1.1 g/L was obtained at high agita—
`tion rates (400 rpm).
`Martinov et a1. (2008) studied aeration in a stirred tank reactor with foaming.
`Different agitators were tested due to the decrease in aeration in the presence of sur-
`factin. A low shear impeller Narcissus maintained stable kLa values while reducing
`foaming. Studies by Yeh et a1. (2006) indicated however that agitation rates above
`350 rpm and aeration above 2 vvrn lead to higher foaming levels that caused low
`surfactin production and low of cells. A he of 0.012/5 was optimal.
`Sen and Swaminathan (1997, 2004) studied surfactin production by B. subtilis
`3256. Maximal production (1.1 g/L) was at 314°C, pH 6.75. agitation of 140 rpm.
`
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`Characterization, Production, and Applications of Lipopeptides
`
`155
`
`MEASUREMENT AND CHARACTERIZATION TECHNIQUES
`
`Enhanced surfactin production can be determined by blood agar plate screening
`due to hemolysis by surfactin (Mulligan et al., 1984). To verify that the isolates are
`biosurfactant producers, then the cultures must be grown and the surfactin levels
`determined. The most common technique for determining surfactant concentration
`is surface tension measurement and CMC determination. HPLC is also frequently
`used. An assay based on hemolysis was used for the analysis of surfactin in the
`fermentation broth. It was determined that the method could be used as a quick low-
`
`volume and secondary inoculation of (4—6 h) 9.5% by volume were also impor-
`tant for optimizing surfactin production.
`Gancel et a1. (2009) investigated lipopeptide production during cell immobili-
`zation on iron-enriched polypropylene particles. Immobilization improved biosurv
`factant production by up to 4.3 times. The amount of fengycin to surfactin varied
`depending on the iron content of the pellets. Highest surfactin (390 mg/L) and fengy-
`cin (680 mgfL) production was at 0.35% iron.
`Guez et a1. (2008) evaluated the influence of oxygen transfer rate on the produc«
`tion of the Iipopeptide mycolysin by B. subrilis ATCC6633. A respiratory activity
`monitoring system used for the study showed that oxygen metabolism has an effect
`on the homologue production and that the regulatory system is complex. Chenikher
`et al. (2010) examined the ability to control the specific growth rate for the produc-
`tion of surfactin and mycosubtilin. Most feeding strategies do not take into account
`the loss of the biomass with the foam. This must be taken into account to enable the
`maintenance of the specific growth rate and subsequently production. The growth
`rate of 0.05/11 was maintained.
`An integrated foam collector was integrated for biosurfactant production to study
`parameters for scale—up (Amani et al., 2010). The best conditions were 300 rpm and
`1.5 vvm for a surfactant yield on sucrose of 0.25 g/g. KLa of 0.011% was achievable in
`shake flasks and bioreactors, and this could potentially be used for scale-up.
`
`from 12 to 15 carbons. Leucine and isoleucine could be differentiated.
`
`technology method of surfactin analysis.
`Huang et al. (2009) compared blood plate hemolysis, surface tension, oil spread-
`ing, and demulsification. Surface tension measurement followed by demulsification
`tests allowed isolation of a demulsification strain Alcaligenes sp. S—Xj-l, which pro—
`duced a lipopeptide that was able to break O/W and W/O emulsions.
`,
`Knoblich et al. (1995) studied surfactin micelles by ice embedding and trans-
`mission electron cryomicroscopy. The micelles found were ellipsoidal with dimen—
`sions of 19, and 11 nm in width and length,respectively or spherical with a 5—9 nm
`in diameter, at pH 7. However, at pH 9.5, the micelles were more cylindrical with
`width and length dimensions of 10—14, and 40—160, or spherical with diameters of
`10—20 nm. Addition of 100 mM NaCl and 20 mM CaCl2 at pH 9.5 formed small
`
`spheres instead of the cylindrical micelles.
`Hue et al. (2001) examined the use of a combination of LSI-MS and MS/MS for
`the characterization of the mixtures of surfactin produced by B. subtih‘s. Amino acid
`composition was determined, and the length of the acyl chain was shown to vary
`
`lM‘illHlIk‘li- I
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`PETITIONERS
`
`EXHIBIT NO. 1019 Page 10 of 35
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`156
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`Biosurfactants: Research Trends and Applications
`
`Biosurfactant proteins produced by Lactobact‘llus fermentum RC—I4 have also
`been identified by a ProteinChip-interfaced mass spectrometer (Reid et al., 2002).
`Five tryptic peptide sequences by collision—induced dissociation tandem mass spec—
`trometry were identified following on-chip digestion of collagen—binding proteins.
`This may lead to the determination of the factors that are responsible for antistaphy~
`lococcal activity.
`lH—NMR was used by Bonmatin et a1. (1994) to show that surfactin can have two
`conformations depending on the pH. The saddle-like structure is bidentate with the
`two charged amino acids as sites for cation binding.
`SANS studies Were performed to study the characteristics of surfactin (Shen
`et al., 2009). At pH 7.5, the aggregation number was only 20, and the diameter of the
`micelles was 50 A with a hydrophobic core of 22 A radius. It is postulated that the
`leucines are in the hydrophobic core, which is consistent with its foaming characten
`istics. Further work (Shen et al., 2010) showed the solubilization of diphenylcarba~
`myl chloride.
`Pecci et al. (2010) characterized the biosurfactants produced by B. licheniformis
`V9T14 strain. This strain exhibited antimicrobial activity that inhibited biofilm for—
`mation of human pathogens. LC-ESI—MS/MS analyses were used, and fengycin and
`surfactin homologues were determined. Fractionation was further performed by
`silica gel chromatography. C13, C14. and C15 surfactin homologues were found plus
`C17 fengycins A and B. Other Cl4eC16 fengycin homologues were also confirmed.
`Most of the surfactin (61.3%) was in the C15 form with an MW of 1035. The two
`most common forms of fengycin A and B, respectively, were the C17 of MW 1477
`(25.1%) and 1505 (55.1%). The LC-ESIeMS/MS proved useful for the characteriza—
`tion of the lipopeptides.
`An oil emulsification test was used to screen for biosurfactants and bioemulsi-
`fiers for strains from a sea mud (Liu et al., 2010). A B. velezensr's H3 strain was
`isolated and could produce biosurfactants on starch and ammonium sulfate. C14 and
`C15 surfactins were discovered, which could lower the surface tension to 25.7 and
`27.0 mN/m, respectively, from pH 4 to 10. CMCs were in the order of 10'5 Incl/L.
`Antimicrobial properties were shown. The yield however was only 0.49 g/L. Highest
`yields of up to 50 g/L have been previously found by a strain on maltose and soybean
`flour (Yoneda et al., 2006).
`
`Pseudomonas sp. M1838. Arthrofactin are cyclic lipopeptides that function as
`
`GENETICS OF UPOPEPTIDE PRODUCTION
`
`Ultraviolet radiation mutation betWeen argC4 and hisAl on the genetic map led
`to a strain that produced 3.5 times surfactin (Mulligan et al., 1989). Another tech-
`nique included random mutagenesis by N—methyLN’ nitro-N-nitrosoguanidine of
`B. lichem'formis, where an increase in surfactin production of 12—fold was obtained
`(Lin et al., 1998). Tsuge et al. (2001) not only found that the yerP gene is involved in
`surfactin resistance in the strain but also evaluated if this gene was involved in sun
`factin production. Although the sfp gene was inserted into the strain. production was
`low. Therefore, it did not appear that the yer-P gene was linked to surfactin production.
`Washio et al.
`(2010) analyzed the genetics of arthrofactin production by
`
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`

`Chelardi et al. (2012) also studied the motility of B. subrilis. They confirmed that
`.ver gene is needed for swarming, and surfactin increases surface wettability to
`allow swarming on low humidity surfaces.
`Various oil reservoirs of salinities from 2.1% to 15.9% were examined to deter-
`
`mine the presence of biosurfactant-producing strains (Simpson et al., 2011). The
`presence of surfactin (srfAj‘) and lichenysin (iicAj’) genes to evaluate the potential
`for biosurfactant production potential was confirmed. Subsequently, nutrient addi—
`tion was performed to stimulate production. This confirmed the ability to biostimu-
`lute biosurfactant production in an oil reservoir for oil recovery.
`
`EXTRACTION OF LIPOPEPTIDES
`
`Characterization, Production, and Applications of Lipopeptides
`
`157
`
`antibiotics, immunosuppressants, antitumor agents, siderophores, and surfactants.
`Schwartzer et a1. (2003) postulated it to be superior as a biosurfactant to surfactin
`and is necessary for the swarming and biofiim formation by the bacteria. Mutants
`from gene insertion were isolated that did not produce the biosurfactant, gaining
`some info on the synthesis.
`Ion beam implantation has also been utilized for generating high surfactine
`producing mutant of B. subrilis (Liu et al., 2006). N* is implanted by this method.
`Gong et a1. (2009) indicated that a mutant using this technique on the concentra-
`tion of a crude surfactin of 12.2 g/L could be produced from 6.5 g/L of biomass.
`It is not known what the effect of the implantation has on the metabolism of the
`microorganisms.
`
`Makkar and Cameotra (2001) also used foarn fractionation to rec0ver surfactin
`
`(‘rude extraction of lipopeptides is summarized in Table 6.3 but is usually by
`chemical extraction. For example, surfactin can be extracted by acid precipita~
`tion (pH 2) followed by solvent extraction by methanol (Vater et al., 2002). For
`Pxendomonns lipopeptides, multiple extraction by the ethyl acetate can be used
`{Kuiper et al., 2004).
`Purification of tipopeptides is important for subsequent industrial application. Thin
`layer chromatography, HPLC, gel permeation chromatography, ion exchange chroma—
`tography, and ultrafiltration have been used. HPLC by reverse phase chromatography
`in particular is often used with the detection by UV absorbance or mass spectrometry
`to provrde some information on the molecular mass of the components. Ultrafiltration
`with 30 kDa (UP-1) and 10 kDa (UF—II) cutoff membranes was employed in a single
`Step (Sivapathasekaran et al., 2011). The recovery was higher with the 10 cha mem_
`home (89%) compared to 73% with the 30 kDa. Purity was also higher (83% come
`Pared to 78%). The product was a mixture of surfactin and fengycin.
`The foaming characteristic of surfactin can be used to remove it during fer~
`mentation (Figure 6.4). At a concentration of 0.05 trig/L,
`it is comparable to
`SDS and bovine serum albumin (Razafindralambo et al., 1996). Low agitation
`3Foods in the fermentor were beneficial for the removal of high concentrations of
`surfactin in the collected foam (Davis et al., 2001). Between 10 and 30 h, stirrer
`SPEBdS of i46 and 166 rpm ted to surfactin concentrations of 1.67 and 1.22 g/L,
`re5-‘133Ctivc.=:ly, and agitation rates of 269 rpm produced concentrations of only
`75 mg/L. Overall recovery of the produced surfactin in the foam was over 90%.
`
`klilfh_ fl" 'IliiI-HKOIKIIU-m'n‘i
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`EXHIBIT NO. 1019 Page 12 of 35
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`tion (Montastruc et at, 2008). Adsorption capacities were about 30 mg of surfactin
`
`by B. sabrilis MTCC 2423. Using sucrose as the substrate, yields of 4.5 g/L were
`obtained at 45°C. Other studies are shown in Table 6.4.
`Liu et a1. (2007) evaluated different conditions for adsorption for surfactin on
`activated carbon. pH values from 6.5 to 8.5 and 30°C were optimal. Adsorption
`onto activated carbon was studied to incorporate surfactin removal with produc-
`
`Source: Modified from Winterburn, LB. and Martin. R]. Biotecitnol. Letters, 34(2): 187—l 95.
`
`a Recovery = (CfVIIClV’i). where Cr, Ci are the foam and initial surfactin concentrations and V}. V[ are the
`initial liquid and foam volumes.
`
`5 Enrichment: Cfl'Cv
`M__w
`
`Air exhaust line
`
`Air outlet
`
`_
`Biosurfactant
`production
`
`Foam collection
`
`FIGURE 6.4 Recovery of surfactin by foam collection.
`
`Biosurfactants: Research Trends and Applications
`
`W T
`
`ABLE 6.3
`
`Recovery of Surfactin by Foam Collection
`Producing
`Foam
`
`Enrichment
`
`Generation
`Not controlled
`
`Recovery "/u “
`92.3
`
`Factorb
`55.0
`
`Not controlled
`
`28.7
`
`Controlled
`
`.
`
`50.1
`
`2.9
`
`Referen co
`Chen et al.
`(2006a)
`Chen et al.
`(2006b)
`Davis ct a1.
`(200!)
`
`Controlled
`
`Not controlled
`
`Organism
`B. subrilis
`BBK 006
`B. snbrilis
`BBK 006
`B. subtilis
`ATCC
`2133]
`
`Description
`Recovery during batch
`operation
`Recovery during
`continuous operation
`Separate foam
`fractionat