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Applied and Environmental Microbiology, January 2004, p. 114-120, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.114-120.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Structure and Characterization of Flavolipids, a Novel Class of Biosurfactants Produced by Flavobacterium sp. Strain MTN11

Adria A. Bodour,1 Claudia Guerrero-Barajas,2 Beth V. Jiorle,3 Mark E. Malcomson,3 Amanda K. Paull,3 Arpad Somogyi,3 Long N. Trinh,3 Robert B. Bates,3 and Raina M. Maier4*

Department of Earth and Environmental Science, The University of Texas—San Antonio, San Antonio, Texas 78249,1 Department of Chemical and Environmental Engineering,2 Department of Chemistry,3 Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona 857214

Received 20 June 2003/ Accepted 2 October 2003


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ABSTRACT
 
Herein we report the structure and selected properties of a new class of biosurfactants that we have named the flavolipids. The flavolipids exhibit a unique polar moiety that features citric acid and two cadaverine molecules. Flavolipids were produced by a soil isolate, Flavobacterium sp. strain MTN11 (accession number AY162137), during growth in mineral salts medium, with 2% glucose as the sole carbon and energy source. MTN11 produced a mixture of at least 37 flavolipids ranging from 584 to 686 in molecular weight (MW). The structure of the major component (23%; MW = 668) was determined to be 4-[[5-(7-methyl-(E)-2-octenoylhydroxyamino)pentyl]amino]-2-[2-[[5-(7-methyl-(E)-2-octenoylhydroxyamino)pentyl]amino]-2-oxoethyl]-2-hydroxy-4-oxobutanoic acid. The partially purified flavolipid mixture isolated from strain MTN11 exhibited a critical micelle concentration of 300 mg/liter and reduced surface tension to 26.0 mN/m, indicating strong surfactant activity. The flavolipid mixture was a strong and stable emulsifier even at concentrations as low as 19 mg/liter. It was also an effective solubilizing agent, and in a biodegradation study, it enhanced hexadecane mineralization by two isolates, MTN11 (100-fold) and Pseudomonas aeruginosa ATCC 9027 (2.5-fold), over an 8-day period. The flavolipid-cadmium stability constant was measured to be 3.61, which is comparable to that for organic ligands such as oxalic acid and acetic acid. In summary, the flavolipids represent a new class of biosurfactants that have potential for use in a variety of biotechnological and industrial applications.


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INTRODUCTION
 
Biosurfactants are a group of natural products of interest for biotechnological and industrial applications (6, 13). In addition, the importance of biosurfactants to cellular architecture has recently been reported for a number of bacteria. These include the role of surfactin in Bacillus subtilis fruiting body formation (8), the role of rhamnolipid in Pseudomonas aeruginosa biofilm formation (10), and the role of streptofactin in the formation of Streptomyces tendae aerial mycelia (46). In terms of structure, biosurfactants are amphiphilic molecules, containing distinct polar and nonpolar moieties that confer the ability to accumulate at surfaces and interfaces, hence the term surface active.

Biosurfactants are produced by diverse bacterial genera (6, 13). Biosynthetic genes appear to be entirely unrelated (26), and as a result, the associated molecular structure of each surfactant class is quite varied, with attendant differences in physicochemical properties. The nonpolar "tail" groups are generally similar among biosurfactants, so that differences are related to the polar "head" groups. In fact, the structure of the head group has been used to categorize biosurfactants into one of the following classes: glycolipids, lipoproteins, phospholipids, fatty acid salts, and polymeric biosurfactants.

We have recently reported that the genus Flavobacterium produces a biosurfactant (7). Flavobacterium is an aerobic, nonfermenting, gram-negative, rod-shaped microorganism that exhibits gliding motility. It belongs to the Flavobacteriaceae family in the Cytophaga-Flavobacterium-Bacteroides phylum. The small-subunit rRNA of Flavobacterium suggests that it is closely related to the sulfur bacteria (14, 56). This organism is ubiquitous in the environment. Some species are opportunistic pathogens known to cause disease in humans (27) and other animals, such as fish (53). For example, Flavobacterium psychrophilum is a fish pathogen that has shown marked resistance to agents used to protect fish farming in Denmark (47). Flavobacterium is known to produce pigments ranging in color from yellow to orange, pink, red, and brown. Some species of Flavobacterium degrade organic contaminants, such as pentachlorophenol, nylon oligomers, polyaromatics, and pesticides (22, 25, 34, 39, 52).

The objective of this study was to describe the surfactant, flavolipid, that was isolated from Flavobacterium sp. strain MTN11. This description includes the growth conditions for flavolipid production, the purification process, structural characterization, and some associated physicochemical properties, including critical micelle concentration (CMC), surface activity, emulsification index, the ability to solubilize and facilitate biodegradation of a model organic compound, hexadecane, and the ability to complex a model metal, cadmium. As will be discussed, flavolipid represents a new class of biosurfactants, molecules that have been explored for their roles in bioremediation, biofilm formation, biological control, and antibiotic activity.


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MATERIALS AND METHODS
 
Flavolipid production and partial purification.
Flavobacterium sp. strain MTN11 (accession number AY162137) was isolated from a sandy loam soil collected from Mount Lemmon in the Coronado National Forest near Tucson, Ariz., during a screening study for biosurfactant-producing bacteria in soil (7). The entire 16S rRNA gene sequence from MTN11 showed 97% homology to Flavobacterium sp. (accession number AF388029). MTN11 was maintained on R2A plates and transferred monthly. A preculture was prepared by inoculating MTN11 into 25 ml of mineral salts medium (MSM) amended with 2.0% glucose as the sole carbon and energy source in a 125-ml flask. MSM is composed of solution A, containing the following per liter: 2.5 g of NaNO3, 0.4 g of MgSO4 · 7H2O, 1.0 g of NaCl, 1.0 g of KCl, 0.05 g of CaCl2 · 2H2O, 10 ml of H3PO4 (85.0%), adjusted to pH 7.2 with KOH pellets. One milliliter of solution B was added, containing the following per 100 ml: 50 mg of FeSO4 · 7H2O, 150 mg of ZnSO4 · 7H2O, 150 mg of MnSO4 · H2O, 30 mg of H3BO3, 15 mg of CoCl2 · 6H2O, 15 mg of CuSO4 · 5H2O, and 10 mg of NaMo2O4 · 2H2O. The preculture was incubated with gyratory shaking at 200 rpm for 36 h at 26°C, and then 3 ml was used to inoculate 300 ml of MSM-2.0% glucose in a 1-liter flask. This flask was incubated under the same conditions for 6 days.

The culture supernatant containing flavolipid was separated from the cells by centrifugation at 15,300 x g for 10 min. The flavolipid was then partially purified by the following procedure. Methanol (1.0%) was added to the supernatant and then subjected to solid-phase extraction. A series of solid-phase extraction columns were prepared, using 5 g of bulk C8 Isolute sorbent (International Sorbent Technology Ltd., Hengoed Mid Glam, United Kingdom) which was sandwiched between 27-mm polyethylene frits (International Sorbent Technology Ltd.) inside a 60-ml plastic syringe. The columns were conditioned with 100% methanol, and then approximately 60 ml of the supernatant solution was added to each column. Following elution of the supernatant, the columns were rinsed with 60 ml of water containing 1% methanol to remove salts and hydrophilic pigments. These two steps (application of supernatant and rinsing) were repeated four times for each column. Then each column was rinsed with 150 ml of an 8:2 water-methanol mixture to remove further pigments and impurities. Finally, the surfactant was eluted with 100 ml of a 2:8 water-methanol mixture. Elution steps used a vacuum manifold (Burdick & Jackson, VWR Scientific Products, Phoenix, Ariz.) to maintain a constant drip. The pressure applied varied but was <-15 kPa.

The eluted surfactant fraction was rotary evaporated to remove methanol (Rotavapor RE-140/EL141; Brinkmann Instruments, Inc., Westbury, N.Y.). Finally, the aqueous surfactant solution was oven dried (50 to 60°C) to remove water. After obtaining the oven-dry mass, standard solutions of surfactant were prepared in nanopure water for further analysis.

Structural analysis.
Mass spectrometry (MS) was performed by the fast atom bombardment (FAB) and electrospray ionization (ESI) methods. The FAB analyses were performed on an HX-110A model magnetic sector instrument (JEOL USA, Inc., Peabody, Mass.) using a xenon FAB source and a matrix consisting of 50% glycerol, 25% thioglycerol, 25% m-nitrobenzyl alcohol, and 0.1% trifluoroacetic acid. The low-resolution ESI measurements were performed on an LCQ model system (Thermo Finnigan, San Jose, Calif.) with direct infusion, using a 50:50 water-methanol solution. Samples were run in both positive and negative ion modes. MS/MS analyses were performed on selected anions. High-resolution ESI+ spectra were run on a HiResESI instrument (IonSpec Corp., Irvine, Calif.).

Samples were prepared for nuclear magnetic resonance (NMR) as follows: flavolipid (55 mg) in 0.5 ml of CD3OD with 0.1 ml of D2O was centrifuged to remove undissolved solids. Spectra were acquired on the following Bruker instruments (Bruker Biospin Corporation, Billerica, Mass.): an AVANCE DRX-500 model instrument, operating at a proton frequency of 500 MHz with a 5-mm triple-resonance triple-axis gradient probe (Nalorac Corporation, Martinez, Calif.) or a 5-mm direct 13C observe probe (13C DEPT), or an AVANCE DRX-600 spectrometer, operating at a proton frequency of 600 MHz with a 5-mm TXI triple-resonance triple-axis gradient probe. The edited gradient HSQC spectrum (54) was acquired in TPPI mode (29, 45, 51). The HMBC spectrum (3) was acquired using echo-antiecho selection. The gradient-selected DQF-COSY spectrum (43) was acquired in echo-antiecho mode. The NOESY spectrum (4, 20) was acquired in TPPI mode, with a mixing time of 0.35 s.

Surface tension and emulsification index.
Surface tension measurements were performed on 20-ml flavolipid samples ranging in concentration from 0 to 3,100 mg/liter by the du Nouy ring method (Surface Tensiomat, Model 21; Fisher Scientific Pittsburgh, Pa.) (5).

The flavolipid emulsification index was determined by using a modification of a method described by Willumsen and Karlson (55). Surfactant solutions were prepared at a range of concentrations: 0, 18.75, 37.5, 75, 150, and 300 mg/liter. A 10% (vol/vol) Pennzoil 10W-40 oil was overlaid on 6 ml of each surfactant solution in a screw-top glass test tube (16 by 150 mm). The height of the oil layer in each tube was measured, and the tube was vortexed for 1 min and then shaken manually for 1 min to create an emulsion. The height of the emulsion layer was measured 2 h, 24 h, and 1 week later and an emulsification index (EI) was calculated using the equation EI (%) = [(height of emulsion layer)/(height of the oil plus emulsion layer)] x 100. Both the surface tension and EI measurements were performed in triplicate and each experiment was repeated twice.

Solubilization and biodegradation experiments.
The ability of flavolipid to facilitate the solubilization and biodegradation of a model hydrocarbon, hexadecane, was determined as previously described (59, 60). In brief, to determine solubilization, a 1-ml flavolipid solution (0, 25, 75, 100, 150, 200, 300, 400, 500, 600, 900, 1,050, and 1,200 mg/liter) was dispensed into 4-ml screw-top glass vials (15 by 45 mm) with caps containing Teflon-faced silicone septa (Kimble, VWR Scientific Products). Ten microliters of a mixture of hexadecane and hexadecane-1[14C] (specific activity, 0.74 mCi/mmol; 98.0% pure) (Sigma, St. Louis, Mo.) was added to each vial. For the no surfactant treatment, only [14C]hexadecane (specific activity, 12 mCi/mmol) was used to achieve a higher specific activity. The vials were incubated on a rotary shaker at 300 rpm at 23°C for 24 h and then allowed to stand for 24 h. A 100-µl aliquot was carefully removed from the bottom of the vials to avoid both floating hexadecane on the top of the surfactant solution and the emulsification layer. The 100-µl aliquot was added to 5 ml of Scintiverse BD (Fisher) and assayed for radioactivity in a Packard Tri-Carb liquid scintillation counter (model 1600 TR) (Meriden, Conn.).

The ability of flavolipid to facilitate biodegradation of hexadecane was evaluated for both Flavobacterium sp. strain MTN11 and a known hexadecane degrader, P. aeruginosa ATCC 9027. Precultures were prepared by inoculating each bacterium into a 250-ml Erlenmeyer flask with 10 ml of basal salts medium (BSM) amended with 2% glucose. BSM contained the following per liter: 0.4% Na2HPO4, 0.15% KH2PO4, 0.1% NH4Cl, 0.02% MgSO4 · 7H2O, 0.0005% iron ammonium citrate, 0.001% CaCl2, and 2% glucose. The precultures were incubated at 23°C for 24 h, with gyratory shaking at 200 rpm. Biodegradation experiments were performed in modified 250-ml screw-top Erlenmeyer flasks as described by Marinucci and Bartha (28). A mixture of hexadecane and [14C]hexadecane was added to each flask to achieve a specific activity of 0.2 µCi/flask and a mass of 10 mg. Ten milliliters of BSM containing 0, 100, 300, or 1,000 mg of flavolipid per liter was added to each flask, and finally, each flask was inoculated with 0.5 ml of one of the precultures. The flasks were sealed with specially designed caps to allow purging and collection of 14CO2 and 14C-volatile organic compounds and then were incubated at 23°C and 200 rpm. Flasks were purged periodically through a train of six scintillation vials containing either Oxosol for 14CO2 or Scintiverse BD for 14C-volatiles (Fisher), and the radioactivity in each sample was assayed by liquid scintillation counting. For both the solubilization and biodegradation experiments, three replicates were performed for each surfactant concentration and the experiment was performed twice.

Cadmium complex formation.
The ability of an organic ligand to bind a metal is often expressed as a conditional stability constant, log K. An ion-exchange technique was used to determine the conditional stability constant for flavolipid and cadmium, as previously described (41). In brief, 0.1 g of sodium-saturated SP Sephadex C-25 (Pharmacia Biotech AB, Uppsala, Sweden) was placed into a 7-ml polypropylene scintillation vial. Test solutions were made with various concentrations of surfactant (0, 130, 330, 1,300, 2,000, and 3,300 mg/liter) and 0.5 mM cadmium in a 0.01 M PIPES [piperazine-N,N'-bis-(2-ethanesulfonic acid)] buffer solution adjusted to pH 7.0 (Sigma). Five milliliters of each test solution was placed into a 7-ml vial containing Sephadex resin. These vials were shaken at 100 rpm for 2 h at 23°C. The vials were removed and allowed to settle for a minimum of 1 h. Then 3 ml of the supernatant was removed and analyzed for cadmium by atomic absorption (AA) (Instrument Laboratory Video 12 aa/ae spectrophotometer; Allied Analytical Systems, Waltham, Mass.). Standard cadmium solutions were prepared and used to obtain a calibration curve for the AA. Four replicates were performed for each test solution.


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RESULTS AND DISCUSSION
 
Flavolipid production, surface tension, and CMC.
The flavolipid yield from the MSM-glucose medium was 0.05 to 0.1 g/liter. This yield is relatively low but presumably could be improved with optimization. The culture supernatant of Flavobacterium sp. strain MTN11 reduced surface tension to 40.7 mN/m, while the partially purified surfactant reduced surface tension to a minimum of 26.0 mN/m. This indicates strong surfactant activity comparable to that of other biosurfactants (6). A plot of surface tension versus the log of biosurfactant concentration was used to estimate a CMC of 300 mg/liter (Fig. 1). This is high compared to those of other biosurfactants. For example, glycolipid CMC values range from 27 to 40 mg/liter for rhamnolipid (5, 59) and from 4 to 15 mg/liter for trehalose lipid (19, 44), depending on the surfactant components present. For the lipoproteins, CMC values for lichenysin range from 10 to 30 mg/liter (18, 23, 58), and for surfactin, iturin, and fengycin, reported CMC values are 10, 20, and 11 mg/liter, respectively (12).



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  		FIG. 1. Effect of flavolipid concentration on surface tension. The surface tension of the partially purified Flavobacterium sp. strain MTN11 flavolipid was measured in solutions of nanopure water at pH 6.8 and 23°C. The CMC was determined from the intersection of regression lines that describe two parts of the curve, below CMC (•) and above CMC ({circ}). Error bars represent the standard deviations of the averages of three replicate samples.

Flavolipid structure.
The structures of flavolipids are shown in Fig. 2. The polar moiety in all of the flavolipids was previously found for the bacterial iron-chelating agent arthrobactin (15, 21) and is closely related to that in aerobactin (24, 33). This moiety arises from a citric acid and two cadaverine molecules. The nonpolar moiety is composed of two branched chain acyl groups, R and R', ranging from 6 to 10 carbons. These acyl groups replace the acetyl groups found in arthrobactin and aerobactin and increase the chain length, from the carboxylate head to the methyl tails, to 17 to 21 carbons. As will be discussed further, this makes salts of flavolipids effective solubilizing and emulsifying agents. Fatty acids with one methyl branch at the {omega} end, like those in flavolipids, have been employed by other bacteria for the synthesis of surfactants, including surfactin and lichenysin (6).



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  		FIG. 2. Structures of flavolipids isolated from Flavobacterium sp. strain MTN11, arthrobactin (15, 21), and aerobactin (24, 35). The structural analysis is described in Materials and Methods.

A combination of 1H, 13C, COSY, HSQC, HMBC, and NOESY NMR and ESI-, ESI+, and FAB+ mass spectral studies identified five unsaturated (U) and five saturated (S) acyl groups distributed to give the 37 compounds shown in Table 1. Analysis of the major peaks in the NMR spectra coupled with the largest peak in the ESI- mass spectrum, m/z 667 for m-1, led to the structure 9U,9U (mass, 668) (4-[[5-(7-methyl-(E)-2-octenoylhydroxyamino)pentyl]amino]-2-[2-[[5-(7-methyl-(E)-2-octenoylhydroxyamino)pentyl]amino]-2-oxoethyl]-2-hydroxy-4-oxobutanoic acid) for the most abundant flavolipid (23%). The NMR spectra of arthrobactin (24) and aerobactin (15) served as excellent models for the NMR spectra of all but the R and R' groups of the flavolipid. The double bonds in the U forms are clearly trans because of the large coupling constant (15 Hz) between the vinyl protons and also because of their chemical shifts. The {alpha},ß-unsaturated nine-carbon acid giving 9U units has not been described previously, but the saturated nine-carbon branched acyl group giving 9S units (present in nine of the flavolipids) has been reported attached in an amide linkage to a lysine nitrogen for a natural product from a marine bacterium (11). Esters of the seven- and eight-carbon saturated acids giving 7S and 8S units have been found as natural products (48).


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  		TABLE 1. Molecular formulas, masses, designations and relative abundances of Flavobacterium sp. strain MTN11 flavolipid components

Analysis of the cation mass spectra was complicated by the presence of a mixture of metal cations (Na+, K+, Al3+, and Fe3+) picked up from the medium and from instruments. This is not surprising, since arthrobactin and aerobactin are important iron chelators, and the latter has even been suggested as a corrosion inhibitor for iron and aluminum (35). As a result, the distribution of the various acyl groups was seen most clearly from the ESI- spectra. A high-resolution ESI+ mass spectrum with an angiotensin II standard strongly supported the molecular formulas shown in Table 1. This spectrum gave four sets of peaks with different cations: the main set contained aluminum, the second strongest contained aluminum and sodium, the third strongest contained iron, and the fourth strongest contained an extra proton. In this spectrum, 20 of the largest peaks representing the 10 most abundant flavolipids were within 13 ppm of the molecular formulas shown in Table 1. Since the signal-to-noise ratio and resolution of this spectrum were much better than those of the ESI- spectrum, the percentages of flavolipids with each mass were determined from the ESI+ spectrum.

Helium-collision-induced fragmentations of selected prominent anions in the ESI- spectra (and some further fragmentations of selected ions in these spectra) were used to determine the distribution of the acyl groups shown in Table 1 for flavolipids with the same mass. The major fragmentations of the anions, other than the loss of a water(s) molecule, are shown in Fig. 3, using 9U,9U (m/z 667) as an example. The negative charge is primarily on the carboxylate ion. Ready loss of two water molecules from the hydroxamic acid units gives the base peak [m/z 631 (100)]. Loss of the remaining water molecule to give an anion with m/z 613 is rapidly followed by a McLafferty-type cleavage (Fig. 3A) involving loss of an isocyanate to give the second strongest peak at m/z 349 (33% of the base). The next strongest four peaks, at m/z 529(2), 511(4), 493(7), and 475(14), can result from loss of a ketene of mass 138 before or after the loss of up to three water molecules. Fig. 3B and C show mechanisms that result in the m/z 529 peak, which still retains all three water molecules (Fig. 3B), and show how the m/z 631 anion gives m/z 493 and 475 when it is selected and fragmented further (3C).



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  		FIG. 3. Major fragmentations of anions, other than the loss of water, from 9U,9U (m/z 667) in ESI- mass spectra. (A) Following the loss of three water molecules, a McLafferty-type cleavage results in the loss of an isocyanate to give a peak at m/z 349. (B) Loss of a ketene of mass 138 results in a peak at m/z 529. (C) Loss of a ketene, following the loss of one, two, or three water molecules, results in peaks at m/z 511, 493, and 475.

Fragmentation of the dihydro compound 9U,9S (m/z 669) gave a base peak at m/z 633, peaks at 349, 529, 511, 493, and 475 (just as for 9U,9U) from losses of the saturated chain, and a new set of peaks at m/z 351, 531, 513, 495, and 477 from losses of the unsaturated chain. When a U,S compound undergoes 2B or 2C fragmentation, the saturated group is lost about twice as often as the unsaturated group, probably because the latter cleavage gives a more strained cumulene-type ketene.

Minor NMR peaks supported the structures shown for the minor components. About 25% of the acid chains were saturated, and all of these added to the {alpha}-methylene absorptions observed at about {delta}1.59 (1H) and 25.7 (13C). The NMR shifts of the two methylenes in the cadaverine chain nearest to the fatty acid were also different when saturated acids were present: {delta}3.59 (1H) and 48.6 (13C) for the nearest methylenes and {delta}1.62 for the next nearest methylene protons (Table 2).


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  		TABLE 2. NMR shifts and coupling constants for 9U,9U and a 9S acyl chain in methanol-d4 containing a few drops of D2O

A complex mixture of sugars (apparently mostly disaccharides from 3,6-dideoxyhexoses and 6-deoxyhexoses) which accompanied the flavolipids, as shown by the NMR spectra, was not completely analyzed. These sugars were present during all of the analyses performed with the flavolipids.

The biosynthesis of flavolipids has not been studied, but as mentioned earlier, flavolipids are closely related to the bacterial iron-chelating agents aerobactin and arthrobactin. Aerobactin is a siderophore first isolated from Aerobacter aerogenes but since found in a variety of gram-negative bacteria, including Citrobacter, Enterobacter, Escherichia coli, Salmonella, Shigella, and Vibrio (9, 38, 40, 42). The biosynthesis of aerobactin has been elucidated, and the aerobactin operon is known to be plasmid associated in E. coli (40) and has also been reported to be associated with phage genes and mobile elements in Shigella (42). In contrast, much less information is available concerning the closely related arthrobactin, although a recent report suggests that it is present in Bacillus (57).

Flavolipid as an emulsifier.
The flavolipid was a strong and stable emulsifier. Flavolipid concentrations as low as 19 mg/liter exhibited an emulsification index of 100%, indicating complete emulsification of the oil layer. Emulsions were stable even after 1 week.

Remediation applications.
Biosurfactants have been intensively studied for application in remediation of organic chemical- and metal-contaminated sites (6, 13). Therefore, the flavolipid was subjected to a series of tests to begin evaluation of its ability to enhance solubilization and biodegradation of hydrocarbons and to determine whether it has the ability to complex metals.

(i) Solubilization and biodegradation of hexadecane.
The flavolipid was effective at increasing the apparent aqueous solubility of hexadecane (Fig. 4). In comparison to the measured solubility of hexadecane in water, 0.003 ± 0.0002 mg/liter, the addition of sub-CMC amounts of flavolipid increased the apparent solubility of hexadecane by several orders of magnitude. For instance, in the presence of 75 mg of flavolipid per liter, the solubility of hexadecane was 113 ± 66 mg/liter, an increase of more than 4 orders of magnitude. After this initial increase in solubility, no further increase took place until 900 mg of flavolipid per liter was added, which is well above the CMC. The solubilization power of a surfactant is evaluated by use of the molar solubilization ratio (MSR), which is defined as the moles of organic compound solubilized per mole of surfactant at concentrations above the CMC. A hexadecane/flavolipid MSR of 1.78 was determined from the slope of a linear regression of the data shown in Fig. 4. This value is very comparable to MSR values for the following hydrocarbon-surfactant mixtures for which the surfactant is considered an effective solubilizing agent: hexadecane/dirhamnolipid methyl ester, 5.2 (60); hexadecane/dirhamnolipid acid, 0.13 (60); octadecane/Triton X-114, 0.013 (49); octadecane/Corexit 0600, 0.034 (49); octadecane/phosphatidylcholine, 3.1 (36).



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  		FIG. 4. Effect of biosurfactant on the apparent aqueous solubility of hexadecane above the CMC ({circ}) and below the CMC (•). A regression line was plotted from the CMC value (300 mg/liter) to 1,200 mg/liter, the slope of which was used to determine the MSR. Error bars represent the standard deviations of the averages of three replicate samples.

The flavolipid was also tested to determine its ability to enhance hexadecane biodegradation. Two isolates, Flavobacterium sp. strain MTN11 and P. aeruginosa ATCC 9027, were examined. Isolate MTN11 produces flavolipid, so a stimulating effect was hypothesized. Isolate ATCC 9027 is a well-studied hexadecane degrader and produces its own surfactant, rhamnolipid. This isolate was used to determine whether the flavolipid would have an inhibitory effect on a genus unrelated to Flavobacterium.

Hexadecane mineralization was evaluated for each isolate with hexadecane-flavolipid at four mass ratios (1:0, 10:1, 3.3:1, and 1:1). These ratios correspond to the following flavolipid concentrations: none, sub-CMC (100 mg/liter), CMC (300 mg/liter), and super-CMC (1,000 mg/liter). The results showed that strain MTN11 mineralized hexadecane very slowly in the absence of flavolipid, with only 0.5% of the added hexadecane mineralized in 18 days (Fig. 5A). However, the addition of a sub-CMC concentration of flavolipid, 100 mg/liter, enhanced mineralization to 26% in 18 days. Flavolipid added at the higher concentrations, 300 and 1,000 mg/liter, also enhanced mineralization, but to a lesser degree. Strain ATCC 9027 had a higher inherent ability to mineralize hexadecane, with 5.6% mineralized in the absence of flavolipid in 6 days (Fig. 5B). The addition of 100-mg/liter flavolipid increased mineralization to 27.4%, almost a fivefold increase. However, by the end of the experiment, 13 days, the difference was less pronounced, at 18.9% without flavolipid compared to 35.1% with flavolipid. For ATCC 9027, the addition of higher flavolipid concentrations did not have a pronounced effect on mineralization. Specifically, the addition of 300-mg/liter flavolipid had a stimulating effect for the first 6 days but thereafter was not different from the control. The 1,000-mg/liter treatment was not different from the control.



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  		FIG. 5. Effect of flavolipid on mineralization of [14C]hexadecane by Flavobacterium sp. strain MTN11 (A) and P. aeruginosa ATCC 9027 (B). Each flask contained 10 mg of hexadecane and either 0 ({circ}), 1 ({triangledown}), 3 ({square}), or 10 ({diamond}) mg of flavolipid in 10 ml of BSM. Error bars represent the standard deviations of the averages of three replicate samples.

The mineralization results indicate that the flavolipid is highly effective with the producing organism, strain MTN11. The impact of the flavolipid on ATCC 9027 was not as strong as that on MTN11, but it was comparable to the effect of rhamnolipid on ATCC 9027, as reported previously (60). In that study, a sub-CMC concentration of rhamnolipid enhanced hexadecane mineralization by ATCC 9027 from 4.9 ± 1.5% in the absence of rhamnolipid to 12.4 ± 0.5% (with dirhamnolipid) and 28.6 ± 0.6% (with dirhamnolipid methyl ester) in 3 days. In comparison, the present study showed that the flavolipid enhanced hexadecane mineralization by ATCC 9027 from 1.9 ± 0.7% in the absence of flavolipid to 16.1 ± 3.1% in the presence of sub-CMC flavolipid in 4 days (Fig. 5A). These results are significant in two respects. First, the flavolipid worked effectively with a microbe that represents a different genus, which is important for successful environmental application. Second, these data support recent studies that suggest that sub-CMC surfactant concentrations are more effective than super-CMC concentrations for enhancing hydrocarbon biodegradation (2, 16, 17, 60). The impact of sub-CMC surfactant concentrations appears to come from the interaction of the surfactant with the cell surface rather than from solubilization effects. For rhamnolipid, this interaction results in the removal of lipopolysaccharide, causing an increase in cell surface hydrophobicity that facilitates hydrocarbon uptake (1). It is not yet known how the flavolipid interacts with the cell surface.

(ii) Effect of biosurfactant on complex formation with cadmium.
The ability of biosurfactants, including rhamnolipid and surfactin, to complex metals and remove them from contaminated soil or mitigate metal toxicity during organic biodegradation has recently been reported (32, 37, 50). We were therefore interested in determining the capacity for flavolipid to complex a model metal, cadmium. The measured conditional stability constant for flavolipid and cadmium was 3.61. This can be compared to the reported stability constants for the following organic ligands and cadmium: rhamnolipid, 6.89; acetic acid, 1.2 to 3.2; oxalic acid, 4.1; and citric acid, 4.5 (30, 31, 41). Flavolipid very likely has a much higher stability constant for Fe3+ due to its structural similarity to arthrobactin and aerobactin, which were selected to complex Fe3+.

Conclusion.
The flavolipids described herein represent a new class of biosurfactants with strong surface activity and emulsifying ability. The polar moiety of flavolipid features citric acid and two cadaverine molecules, which is quite different from the polar moieties found in any of the currently reported classes of biosurfactants, which are glycolipids, lipoproteins, phospholipids, fatty acid salts, and polymeric biosurfactants. This new class of biosurfactants will be of interest for potential use in a wide variety of industrial and biotechnology applications. We are currently synthesizing the major flavolipid, 9U,9U, to see to what extent the measured properties of the surfactant are due to this single component.


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ACKNOWLEDGMENTS
 
This work was supported by grant number 2 P42 ESO4940-11 from the National Institute of Environmental Health Sciences, NIH, by grant CHE-0133237 from the National Science Foundation, and by a Camille and Henry Dreyfus Foundation Senior Scientist Mentor Initiative Award.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Soil, Water and Environmental Science, University of Arizona, 429 Shantz Building #38, Tucson, AZ 85721. Phone: (520) 621-7231. Fax: (520) 626-6782. E-mail: rmaier{at}ag.arizona.edu. Back


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Applied and Environmental Microbiology, January 2004, p. 114-120, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.114-120.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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