Aktien Forum › Usa



N-acetylated derivatives of the amino sugars were never detected. Next Patent Method for producing As a control, 0.

Aktien Forum


Finally, that the emulsans and their deproteinized and deacylated derivatives are strongly adsorbed onto aluminosilicate ion-exchanges and are unusually efficient bioflocculents which may be used to mediate flocculation of various types of aluminosilicate clays, such as kaolin and bentonite.

Based on some of these discoveries, the invention provides a process for producing extracellular microbial lipopolysaccharides which comprises A inoculating an aqueous fermentation medium containing a growth-sustaining amount of one or more fatty acids with a culture of Acinetobacter Sp. Using the data contained herein, these emulsifying agents produced by the process of the invention may be used, among other things, 1 for cleaning hydrocarbonaceous residues, including residual petroleum, from tankers, barges, storage tanks, tank cars and trucks, pipelines and other containers; 2 for cleaning oil spills which are floating on the sea or which have been washed ashore or which are deposited on land; and 3 for the enhanced recovery of oil by chemical flooding techniques, particularly with respect to those petroleum reservoirs located in sand or sandstone or limestone formations.

A new lexicon has been used herein to identify and refer to the various types of extracellullar microbial polysaccharides and their semi-synthetic derivatives which are derived from Actinetobacter Sp. ATCC and its mutants.

The name "emulsans", which reflects the polysaccharide structure of these compounds and the exceptional emulsifying activity of the biologically produced materials, has been created to identify generically those extracellular microbial protein-associated lipoheteropolysaccharides produced by Acinetobacter Sp. The name "apoemulsans", the prefix of which is derived from the Greek word meaning "from", has been created to identify generically those deproteinized lipopolysaccharides obtained from the emulsans.

ATCC and its mutants in which the lipoploysaccharide components, i. The proemulsans have no emulsifying activity under the standard assay techniques described below. As used herein, the term "Acinetobacter Sp. ATCC or its mutants" refers not only to the organism i. To more fully comprehend the invention, reference should be made to the accompanying drawings, in which. ATCC on an ethanol medium, showing the relationship of the growth of the organism in such medium, the production of the bioemulsifier during such growth, and the change of pH during such growth, all as a function of time;.

ATCC on a hexadecane medium, showing the relationship of the growth of the organism in such medium and the production of the bioemulsifier during such growth, both as a function of time;.

The fermentation process may be conducted with automatic or manual control in batch or continuous fermenters, using either fresh water or sea water media. Selection of suitable fermentation equipment may be made from designs engineered to give the most efficient oxygen transfer to the biomass at lowest operating cost. In addition to the stirred tank fermenters, other types of fermenters may be used, such as thin channel fermenters, tubular loop fermenters, film fermenters, recirculating tower fermenters, deep shaft fermenters, and jet fermenters, the most important criteria being efficiency in the fermentation process, especially with respect to oxygen transfer and power consumption.

The microorganism used to produce both neoemulsans and protoemulsans from utilizable carbon sources is Acinetobacter Sp. This organism, which has been described by A. During the exponential growth phase the cells appear mostly as irregular short rods, 0.

The cells occur often as V-shaped pairs, indicating snapping division. Occasionally, the rods are slightly bent or swollen. Coccoid cells, approximately 1. The cocci are gram-positive; the rods are gram-negative.

The amount of inoculum used to initiate the fermentation will be dependent upon the type of fermentation equipment used.. Even though it has previously been reported by A. For example, growth of Acinetobacter Sp. ATCC or its mutants on an aqueous fermentation medium in which one or more fatty acid salts are the primary assimilable carbon source.

Such fatty acids salts include the assimilable saturated fatty acids, such as decanoic acid capric acid , dodecanoic acid lauric acid , tetradecanoic acid myristic acid , hexadecanoic acid palmitic acid and octadecanoic acid stearic acid ; unsaturated C 10 to C 18 fatty acids, including monoethenoid and diethenoid fatty acids; hydroxysubstituted fatty acids, such as 2-hydroxydodecanoic acid, 3-hydroxydodecanoic acid and hydroxyoleic acid ricinoleic acid.

In addition, mixtures of fatty acids may be used, such as the mixed fatty acids derived from saponification of lard, soybean oil, peanut oil, cottonseed oil, sunflower oil, coconut oil, castor oil, palm oil, linseed oil, and various fish oils or marine mammal oils.

ATCC on media containing fatty acid salts are unusually efficient bioemulsifiers, exhibiting a high degree of specificity in emulsifying those hydrocarbon substrates such as crude oils, gas-oils and Bunker C fuel oils that contain both aliphatic and aromatic or cyclic components.

Maximum growth of Acinetobacter Sp. Additionally, phosphorus-containing compounds are also essential nutrients. Suitable source of available nitrogen include ammonium salts, such as ammonium sulfate or ammonium chloride; nitrates, such as ammonium nitrate or sodium nitrate; or organic sources of available nitrogen, such as urea or soybean meal.

Suitable sources of available phosphorous include dibasic potassium phosphate, monobasic potassium phosphate and the like. In addition, liquid fertilizers, such as or , may serve as a source of nitrogen and phosphorous nutrients for the growth of Acinetobacter Sp.

As shown below in the data set forth in Section 8. These divalent cations are present in sea water or "hard" water when fermentation media are prepared from such sources.

When "soft" fresh water or distilled water are used to prepare the fermentation media, then small amounts of one or more salts of a divalent cation should be added to the fermentation media, the concentration being such that the resultant culture media will contain from about 1 to about mM and preferably from about 5 to about 40 mM of at least one divalent cation.

Discussed below are the best conditions which have been found for consistently producing high yields of emulsans from sodium palmitate media in conventional liter stirred fermenters. These conditions probably will undergo subtle or pronounced changes to obtain higher yields upon large-scale production in fermenters specifically designed or adapted to give more efficient oxygen transfer at the lowest power consumption.

Subsequent work on optimizing the process will, of course, focus on a consumption of the substrate, which is a fucntion of the physiology of Acinetobacter Sp. ATCC and its mutants; b consumption of oxygen, which is a function of oxygen diffusion to the cells which, in turn, will be influenced i by making the surface through which the diffusion occurs as large as possible i.

Using liter stirred fermenters with the fermentation medium and process conditions described below in Section This oxygen flow rate is not limiting but can, if necessary, be increased to as high as millimoles per liter per hour, or even higher, with the more efficiently designed fermenters.

To promote maximum oxygen diffusion to the cell mass, the fermentation media must be agitated either by stirring or circulating the media through the fermenter, depending upon the type of fermentation equipment employed. Using liter stirred fermenters with the fermentation medium and other process conditions described below in Section This value is not limiting but will be varied in the more efficiently designed fermenters to achieve maximum oxygen transfer at the lowest power consumption.

The pH of the fermentation medium should be maintained between 6 and 7, and preferably between 6. Stirred-tank fermentations of Acinetobacter Sp. Although many types of chemical defoamers may be used in the fermentation media, great care must be taken when adding chemical defoaming agents to keep the diffusion constant as high as possible.

Using the liter stirred fermenters with the fermentation medium and other process conditions described below in Section ATCC so that the similarities as well as differences between these biopolymers may be understood.

In order to study the kinetics of bioemulsifier production by Acinetobacter Sp. These assays were based upon the large increase in turbidity of a mixture of oil and water arising from the emulsion of the hydrocarbon in the aqueous phase.

The first assay involved the emulsification of gas-oil in sea water under standardized conditions and subsequent measurement of turbidity. Each assay technique consisted of adding hydrocarbon 0. Appropriate dilutions were made in water so that the final readings were between 30 and Klett units, and values for Klett units reported as final readings times the dilution.

Values for controls containing no bioemulsifier 5 to 20 Klett units were subtracted. One unit of bioemulsifier per ml is defined as that amount of activity which yields Klett units using 0. Specific Emulsification Activity or specific activity is units per mg of bioemulsifier, dry weight basis.

Curve 1-A represents the relationship between the amount of emulsification between 0. Each point in FIG. Measurement of extracellular emulsifying activity was determined at different stages of growth of Acinetobacter Sp. Growth was estimated by turbidity using a Klett-Summerson colorimeter fitted with a green filter or a Gilford Spectrophotometer Model One hundred Klett units of exponentially growing Acinetobacter Sp. ATCC correspond to an absorbance at nm 1-cm light path of 0.

Similar data have been obtained on the growth of Acinetobacter Sp. ATCC on a sodium palmitate medium. Measurement of extracellular emulsifying activity was also determined at different stages of growth of Acinetobacter Sp. Viable cell number was determined by spreading 0. The data contained in FIG. After 40 hours of incubation of Acinetobacter Sp. ATCC in the ethanol medium and in the hexadecane medium as described above in Sections 6. The pellicle formed during centrifugation of the hexadecane culture was removed, washed twice with growth medium before assaying for activity.

Emulsifying activity in each fraction for the ethanol and hexadecane growth cultures was assayed by the standard assay technique described above in Section 6.

The results of such assays are summarized in Table I. ATCC was grown on hexadecane medium. The small amount of activity associated with the pellet fraction was variable; in certain cases no measureable cell-bound activity could be found.

Disruption of the pellet fractions by sonic oscillation did not release additional emulsifying activity. Apoemulsans may be prepared by deproteinization of the particular emulsans, which technique was used to isolate and purify samples for the chemical characterization of both Acinetobacter bioemulsifiers described below.

The associated protein may be separated from both bioemulsifiers by the hot phenol extraction technique described by O.

Alternatively, the protein may be removed enzymatically by proteolytic digestion. The extracellular protein-associated lipopolysaccharides produced by Acinetobacter Sp. ATCC and their respective deproteinized derivatives may be isolated and purified by various procedures, including selective precipitation, selective solvent extraction or partitioning or selective adsorption onto a solid adsorbant followed by subsequent elution or extraction. For many industrial uses, isolation and purification of the Acinetobacter bioemulsifiers is not necessary, since the cell-free growth media may be used directly.

ATCC have been isolated and purified. Three different procedures have been followed, including a heptane partitioning of the crude extracellular lipopolysaccharide from the fermentation medium, followed by extraction of impurities from the heptane-partitioned biopolymer and subsequent work-up; b precipitation of the extracellular lipopolysaccharide by ammonium sulfate, followed by work-up of the precipitate; and c precipitation of the extracellular lipopolysaccharide by a detergent quaternary ammonium cation followed by work-up of the precipitate.

The addition of ammonium sulfate to the fermentation broth has been used to fractionally precipitate the extracellular lipopolysaccharides from the culture medium, from which the concentrate may be recovered and further treated to remove impurities.

The resulting precipitate, which may be collected by centrifugation, has been extracted by ether to remove impurities, dialyzed against water and lyophilized, yielding the purified extracellular lipopolysaccharide. Because the extracellular lipopolysaccharides produced by Acinetobacter Sp.

ATCC were found to be anionic biopolymers, a procedure was developed to precipitate the anionic biopolymer with a cationic detergent, such as cetyltrimethyl ammonium bromide, from which precipitate the detergent cation could be separated while leaving the purified extracellular lipopolysaccharide. This precipitate is soluble in 0. Chemical and physical characterization of emulsans and apoemulsans were measured on samples which had been purified to apparent homogeneity, from which characterization conclusions were reached on the structure of these unique extracellular lipopolysaccharides.

Such information is necessary to give a better understanding of the relationship between the molecular structure of this class of bioemulsifiers and their specificity in emulsifying various hydrocarbon substrates. The emulsan samples used for chemical and physical characterization were prepared by aerobically growing acinetobacter Sp. The apoemulsan samples used for chemical and physical characterization were prepared by hot phenol extraction of the associated protein moiety from the emulsan samples.

The deproteinization procedure, which is described more fully in the example set forth below in Sections The resulting emulsion was then centrifuged to separate the denatured protein in the phenol phase from the apoemulsan in the aqueous phase. All of the emulsifying activity was in the recovered emulsan.

None of the emulsifying activity was in the denatured protein fraction. After three phenol extractions, the combined water extracts were extracted four fimes with an equal volume of ether to remove residual phenol.

The procedure was repeated to obtain a slightly turbid second precipitate between These two fractions contained similar specific activities and exhibited substantially the same chemical composition.

Functional group tests were positive for carboxyl and ester groups and negative for methoxy and ethoxy groups. The polymer contained less than 0. The nonreducing polymer was resistant to high temperatures in neutral and alkaline conditions. As shown in FIG. After hour hydrolysis in 0. In addition, there were considerable amounts of incompletely hydrolyzed material remaining near the origin. After 5-hour hydrolysis in 0. N-acetylated derivatives of the amino sugars were never detected. Even under these conditions significant amounts of emulsifying agent were incompletely hydrolyzed.

Longer periods of hydrolysis resulted in further destruction of the sugars. The relative amount of amino sugars to glucose increased with time of hydrolysis due both to the slower release of amino sugars from the polymer and faster destruction of free glucose. Unknown compound A did not separate from glucose in solvents A or B and yielded a positive D-glucose reaction directly on the paper.

Unknown compound C gave positive reactions for reducing sugar, amino sugar and carboxylate ion. Moreover, it was similar both in chromatographic behavior and in its reaction with the nitrous acid-indole test to 2-aminodeoxyhexuronic acids.

Spot tests were determined directly on the chromatograms. EM is the modified Elson and Morgan reagent [R. Wheat in the monograph edited by E. Any glucose present is probably an impurity. The NMR spectrum in CDCl 3 indicated that the mixture consisted mainly of saturated and hydroxy-substituted fatty acids. After removal of the methanol in vacuo, ml of water were added. The clear alkaline solution was washed three times with ml of ether, the ether discarded, and the aqueous solution acidified to pH 2 with hydrochloric acid.

The acid solution was then extracted five times with ml ether, the interphase in each extraction being set aside. The combined interphase fractions were treated with acetone to precipitate protein and polysaccharide. After removal of the precipitate by filtration and the acetone by distillation in vacuo, the aqueous phase was again extracted with ether.

The combined ether extracts were dried over magnesium sulfate. The methyl esters of the fatty acid mixture were prepared with diazomethane by standard techniques. Gas liquid chromatography of the methyl esters of the fatty acid mixture led to the separation of eleven peaks, nine of which were identified by comparison of retention volumes of pure samples of known structure.

Table IV sets forth the relative retention volumes of the methyl esters of the fatty acids obtained from emulsan. The aqueous hydrolysate was extracted with ether and the ether extract was treated by diazomethane to convert to methyl esters whatever fatty acids remained after such strong acid hydrolysis.

Gas chromatographic analysis of this material revealed the presence of methyl 3-hydroxydodecanoate as the only fatty acid. Additional data on physical characterization is set forth below: With all three samples, reduced viscosity was independent of concentration between 0. Exposure for an additional 20 minutes did not further reduce the viscosity.

The large increase in reduced viscosity at low ionic strengths is characteristic of polyelectrolytes and has been attributed to dilution of counterions. Specific viscosity was also measured as a function of pH using 0. Throughout the entire range pH The diffusion coefficient, D, also determined in the analytical centrifuge was 5. The partial specific volume of the material, V, was 0. Alternatively, the molecular weight can be estimated using the determined values for intrinsic viscosity, sedimentation constant, S, and partial molar volume, V, according to the equation of Scheraga and Mandelkern [J.

Molecular weight determination from sedimentation and diffusion data closely fit the value obtained from a consideration of sedimentation and viscosity measurements. In both cases the determined value for the partial molar volume of 0. Using Simha's factor [C. Assuming the aminouronic acid to be an N-acetylhexosamine uronic acid M. Rough estimates from intensities of reducing and ninhydrin positive materials on chromatograms indicate that the amount of D-galactosamine is similar to the quantity of aminouronic acid.

Growth of Acinetobacter Sp. ATCC on an ethanol medium. Table VIII shows the differences between the emulsans prepared by growing the organism on ethanol and on sodium palmitate, respectively. In each case, the fermentation media and conditions were identical except for the carbon source.

To immunologically characterize the Acinetobacter bioemulsifiers produced by Acinetobacter Sp. The rabbits were bled 11 to 14 days later, from which sera a crude immunoglobulin fraction was obtained by ammonium sulfate fractionation. ATCC so that similarities as well as differences between these biopolymers may be understood. As before, unless the particular type of extracellular lipopolysaccharide produced by the organism is identified by name, the phrase "Acinetobacter bioemulsifier" refers collectively to both classes of emulsans.

Unless otherwise indicated, emulsifying activity was assayed in accordance with the standard assay technique described above in Section 6. The rate of emulsification of gas-oil by purified Acinetobacter bioemulsifier is summarized in FIG. At fixed concentrations of bioemulsifier 0. Emulsion formation as a function of gas-oil concentration is shown in FIG. Each mixture was reciprocally shaken for 60 minutes at strokes per minute, and emulsion formation then measured.

Emulsins were formed over the entire gas-oil concentration range studied, 0. Between 8 to 30 mg gas-oil per ml, turbidity increased about 5 Klett units per mg gas-oil.

Acinetobacter bioemulsifier-induced emulsification of gas-oil as a function of pH is shown in FIG. The data shown in FIG. In sea water, near maximum emulsions were obtained from pH 5 to at least pH 9. Above pH 9 precipitation of salts prevented accurate measurements of emulsion. In aqueous solutions containing Tris buffer, citrate-phosphate buffer, or diluted saline, a sharp maximum was obtained between pH Above pH 7, activity was completely lost. In order better to understand the different results obtained in sea water and fresh water, the effect of salts on bioemulsifier-induced emulsification was measured at pH 7.

Maximum activity was obtained with mM magnesium sulfate or magnesium chloride. Half maximum activity was achieved with 1. Calcium chloride 10 mM and manganese chloride 10 mM could be substituted for magnesium sulfate. On the other hand, sodium chloride mM had little effect on emulsion formation, either in the presence or absence of magnesium ions.

Consequently, the ability of Acinetobacter bioemulsifiers to emulsify hydrocarbons above pH 6 is dependent upon divalent cations and appears to be independent of sodium chloride concentration.

Because of this property, these bioemulsifiers are capable of functioning in the presence of high concentrations of sodium chloride found in sea water or connate water.

Gas-oil emulsions formed in the presence of Acinetobacter bioemulsifier slowly separate into two phases when allowed to stand undisturbed; namely, a lower clear aqueous phase and a turbid upper phase containing concentrated oil droplets, bound bioemulsifier and water. As observed with a phase microscope, emulsion breakage demulsification was a result of "creaming" due to density differences between the two phases and was not accompanied by droplet coalescence or aggregation.

As shown in FIGS. With ratios between and , half-maximum turbidities were reached in hours and minutes, respectively. In all cases, the upper "cream" immediately dispersed in aqueous media.

Emulsion breakage was enhanced by divalent cations. The calculated droplet sizes were in good agreement with measurement of droplet size by phase microscopy using a calibrated eye-piece micrometer. The ability of Acinetobacter bioemulsifiers to lower the interfacial tensions between a series of n-alkanes and sea water is shown in FIG.

Using similar techniques, the interfacial tensions between Prudhoe Bay crude oil and sea water were measured using 1 and 10 mg of bioemulsifier per ml. Apart from classification as anionic, cationic or nonionic, most emulsifiers are described in terms of their HLB numbers, which is a measure of the hydrophile-lipophile balance of the emulsifier.

Very often, emulsifiers with similar HLB numbers interact differently with hydrocarbon substrates. Because biologically produced polymers often exhibit specificities not found in chemically synthesized materials, the hydrocarbon substrate specificity for Acinetobacter bioemulsifier-induced emulsion formation was studied using a wide variety of pure hydrocarbons, binary mixtures of hydrocarbons, crude oils, fractions of crude oils and mixtures of crude oil fractions and pure hydrocarbons.

All crude oils tested were emulsified by both types of Acinetobacter bioemulsifiers. In addition to the crude oils shown in Table XIV, various crude oils from Alaska, Louisiana and Texas were emulsified by both Acinetobacter bioemulsifiers. Gas-oil was a better substrate for Acinetobacter bioemulsifier-induced emulsification than kerosene or gasoline, both of which formed somewhat unstable emulsions. Straight and branch chain aliphatic hydrocarbons from heptane to octadecane were emulsified only to a slight extent by the Acinetobacter bioemulsifier as illustrated by the data in FIG.

The data summarized in FIG. Increasing or decreasing the hydrocarbon concentration by a factor of five did not improve emulsification. Pentane and hexane were also not emulsified effectively; however, quantitative data for these two paraffins were not obtained because of extensive evaporation during incubation.

The solid hydrocarbons, nondecane, n-octacosane and hexatriacontane, were not dispersed by Acinetobacter bioemulsifier. Emulsification of n-alkyl cyclohexane derivatives ranging from propylcyclohexane to tridecylcyclohexane by Acinetobacter bioemulsifier are summarized in FIG. The data for octyl, nonyl and decylcyclohexanes were obtained from redistilled materials which contained no ultraviolet-absorbing impurities.

Nonylcyclohexane did not contain any apparent inhibitors of emulsification, since mixtures of octyl and nonylcyclohexane were emulsified to about the same extent as octylcyclohexane alone.

Bicyclohexane and decalin were not emulsified significantly. Emulsification of n-alkylbenzene derivatives by Acinetobacter bioemulsifier are summarized in FIG.

Maximum activity was obtained with hexyl and heptylbenzenes. The total number of carbon atoms in the side chains may be more crucial than the chain length since p-diisopropylbenzene behaved like hexylbenzene. The low molecular weight benzene derivatives, toluene, p-xylene, m-xylene, ethyl-benzene and 1,2,3,4-tetramethylbenzene, were not emulsified significantly. Aromatic compounds containing more than one ring, naphthalene, biphenyl, phenanthrene, anthracene, 3-phenyltoluene, 1-methylnaphthalene and 2-methylnaphthalene were also not emulsified significantly by the Acinetobacter bioemulsifier.

Table IX summarizes a number of experiments in which the Acinetobacter bioemulsifier-induced emulsification of aliphatic, aromatic and cyclic hydrocarbons were measured in the presence of hexadecane or 1-methylnaphthalene. Athough neither the aliphatic compounds nor 1-methylnaphthalene were emulsified by themselves, all mixtures containing the aromatic compound and one of the aliphatic hydrocarbons were excellent substrates for emulsification by the bioemulsifier.

The ability of aromatic compounds to stimulate emulsification of aliphatics was not limited to 1-methylnaphthalene, but occurred with toluene, p-xylene, 3-phenyltoluene and 2-methylnaphthalene. Addition of hexadecane to the aliphatic compounds did not stimulate emulsification, that is, only an additive effect was observed. The minor exception to this finding was nonadecane which became liquid when mixed with hexadecane.

As mentioned above, the only aromatic compounds that served as substrates for emulsification by Acinetobacter bioemulsifier were alkylbenzene derivatives containing six or seven carbon atoms on the side chain s. Aromatic compounds containing less than six carbon atoms on the side chain were converted into good substrates for emulsification by addition of hexadecane.

Hexylbenzene and diiopropylbenzene were converted into even better substrates for emulsification by addition of hexadecane. On the other hand, heptyl, decyl and pentadecylbenzene were emulsified more poorly in the presence of hexadecane than by themselves. Only alkylbenzene derivatives containing side chains of five or more carbon atoms were activated by 1-methylnaphthalene. With few exceptions, cycloparaffin derivatives were converted into better substrates for Acinetobacter bioemulsifer-mediated emulsification by addition of either hexadecane or 1-methylnaphthalene.

In general, cyclohexane derivatives with short side chains e. Dicyclohexane behaved like an aromatic compound in that it was emulsified by the bioemulsifier in the presence of hexadecane but not 1-methylnaphthalene.

The fused dicylic compound decalin could not be emulsified by the bioemulsifier even by addition of hexadecane or 1-methylnaphthalene. Acinetobacter bioemulsifier-induced emulsion formation as a function of the relative concentrations of aliphatic hexadecane and aromatic methylnaphthalene compounds is shown in FIG. Using either 1-methylnaphthalene or 2-methylnaphthalene, maximum emulsion was obtained with 25 vol.

An identical experiment using decane in place of hexadecane yielded similar curves except that the peak of emulsion activity was obtained with 33 vol. To verify this conclusion, experiments were designed to determine whether or not addition of hexadecane or methylnaphthalene could enhance Acinetobacter bioemulsifier-induced emulsification of petroleum fractions which had been fractionated to separate a fraction rich in aliphatics Fraction 1 from two fractions Fractions 2 and 3 rich in aromatics.

These experiments, which are more fully described below in Section Addition of even one part of the aromatic compound to ten parts of gasoline or kerosene resulted in a much improved substrate for emulsification.

The requirement for both aliphatic and aromatic constituents was further supported by studying emulsification of column fractionated crude oil. Although crude oil itself is emulsified by the Acinetobacter bioemulsifier, none of the fractions were good substrates by themselves.

However, mixtures containing one fraction Fraction 1 rich in aliphatics and the other Fractions 2 or 3 rich in aromatics were efficiently emulsified. ATCC , may be subdivided into a differences in yield; b differences in structure; and c differences in emulsifying activity. EM is the modified Elson and Morgan reagent [R. Wheat in the monograph edited by E. Any glucose present is probably an impurity. The NMR spectrum in CDCl 3 indicated that the mixture consisted mainly of saturated and hydroxy-substituted fatty acids.

After removal of the methanol in vacuo, ml of water were added. The clear alkaline solution was washed three times with ml of ether, the ether discarded, and the aqueous solution acidified to pH 2 with hydrochloric acid.

The acid solution was then extracted five times with ml ether, the interphase in each extraction being set aside. The combined interphase fractions were treated with acetone to precipitate protein and polysaccharide. After removal of the precipitate by filtration and the acetone by distillation in vacuo, the aqueous phase was again extracted with ether.

The combined ether extracts were dried over magnesium sulfate. The methyl esters of the fatty acid mixture were prepared with diazomethane by standard techniques. Gas liquid chromatography of the methyl esters of the fatty acid mixture led to the separation of eleven peaks, nine of which were identified by comparison of retention volumes of pure samples of known structure.

Table IV sets forth the relative retention volumes of the methyl esters of the fatty acids obtained from emulsan. The aqueous hydrolysate was extracted with ether and the ether extract was treated by diazomethane to convert to methyl esters whatever fatty acids remained after such strong acid hydrolysis.

Gas chromatographic analysis of this material revealed the presence of methyl 3-hydroxydodecanoate as the only fatty acid. Additional data on physical characterization is set forth below: With all three samples, reduced viscosity was independent of concentration between 0.

Exposure for an additional 20 minutes did not further reduce the viscosity. The large increase in reduced viscosity at low ionic strengths is characteristic of polyelectrolytes and has been attributed to dilution of counterions.

Reduced viscosity was also measured as a function of pH using 0. Throughout the entire range pH The diffusion coefficient, D, also determined in the analytical centrifuge was 5. The partial specific volume of the material, V, was 0.

The calculated viscosity average molecular weight for apoemulsan-WA was 9. X-ray diffraction analysis of apo- -emulsan, which was performed on a film formed by evaporation of a water solution of apoemulsan, showed crystallinity.

Molecular weight determination from sedimentation and diffusion data closely fit the value obtained from a consideration of sedimentation and viscosity measurements. In both cases the determined value for the partial molar volume of 0. Using Simha's factor [C. Assuming the aminouronic acid to be an N-acetylhexosamine uronic acid M. Rough estimates from intensities of reducing and ninhydrin positive materials on chromatograms indicate that the amount of D-galactosamine is similar to the quantity of aminouronic acid.

Growth of Arthrobacter Sp. To immunologically characterize the Arthrobacter bioemulsifiers produced by Arthrobacter Sp. The rabbits were bled 11 to 14 days later, from which sera a crude immunoglobulin fraction was obtained by ammonium sulfate fractionation.

ATCC so that similarities as well as differences between these biopolymers may be understood. As before, unless the particular type of extracellular lipopolysaccharide produced by the organism is identified by name, the phrase "Arthrobacter bioemulsifier" refers collectively to both classes of emulsans. Unless otherwise indicated, emulsifying activity was assayed in accordance with the standard assay technique described above in Section 6.

The rate of emulsification of gas-oil by purified Arthrobacter bioemulsifier is summarized in FIG. At fixed concentrations of bioemulsifier 0. Emulsion formation as a function of gas-oil concentration is shown in FIG. Each mixture was reciprocally shaken for 60 minutes at strokes per minute, and emulsion formation then measured.

Emulsions were formed over the entire gas-oil concentration range studied, 0. Between 8 to 30 mg gas-oil per ml, turbidity increased about 5 Klett units per mg gas-oil. Arthrobacter bioemulsifier-induced emulsification of gas-oil as a function of pH is shown in FIG. The data shown in FIG. In sea water, near maximum emulsions were obtained from pH 5 to at least pH 9. Above pH 9 precipitation of salts prevented accurate measurements of emulsion.

In aqueous solutions containing Tris buffer, citratephosphate buffer, or diluted saline, a sharp maximum was obtained between pH Above pH 7, activity was completely lost. In order better to understand the different results obtained in sea water and fresh water, the effect of salts on bioemulsifier-induced emulsification was measured at pH 7. Maximum activity was obtained with mM magnesium sulfate or magnesium chloride.

Half maximum activity was achieved with 1. Calcium chloride 10 mM and manganese chloride 10 mM could be substituted for magnesium sulfate. On the other hand, sodium chloride mM had little effect on emulsion formation, either in the presence or absence of magnesium ions.

Consequently, the ability of Arthrobacter bioemulsifiers to emulsify hydrocarbons above pH 6 is dependent upon divalent cations and appears to be independent of sodium chloride concentration. Because of this property, these bioemulsifiers are capable of functioning in the presence of high concentrations of sodium chloride found in sea water or connate water.

Gas-oil emulsions formed in the presence of Arthrobacter bioemulsifier slowly separate into two phases when allowed to stand undisturbed; namely, a lower clear aqueous phase and a turbid upper phase containing concentrated oil droplets, bound bioemulsifier and water. As observed with a phase microscope, emulsion breakage demulsification was a result of "creaming" due to density differences between the two phases and was not accompanied by droplet coalescence or aggregation.

As shown in FIGS. With ratios between and , half-maximum turbidities were reached in hours and minutes, respectively. In all cases, the upper "cream" immediately dispersed in aqueous media. Emulsion breakage was enhanced by divalent cations. The calculated droplet sizes were in good agreement with measurement of droplet size by phase microscopy using a calibrated eye-piece micrometer.

The ability of Arthrobacter bioemulsifiers to lower the interfacial tensions between a series of n-alkanes and sea water is shown in FIG. Using similar techniques, the interfacial tensions between Prudhoe Bay crude oil and sea water were measured using 1 and 10 mg of bioemulsifier per ml, yielding 8.

Apart from classification as anionic, cationic or nonionic, most emulsifiers are described in terms of their HLB numbers, which is a measure of the hydrophile-lipophile balance of the emulsifier. Very often, emulsifiers with similar HLB numbers interact differently with hydrocarbon substrates. Because biologically produced polymers often exhibit specificities not found in chemically synthesized materials, the hydrocarbon substrate specificity for Arthrobacter bioemulsifier-induced emulsion formation was studied using a wide variety of pure hydrocarbons, binary mixtures of hydrocarbons, crude oils, fractions of crude oils and mixtures of crude oil fractions and pure hydrocarbons.

All crude oils tested were emulsified by both types of Arthrobacter bioemulsifiers. Gas-oil was a better substrate for Arthrobacter bioemulsifier-induced emulsification than kerosene or gasoline, both of which formed somewhat unstable emulsions.

Straight and branch chain aliphatic hydrocarbons from heptane to octadecane were emulsified only to a slight extent by the Arthrobacter bioemulsifier as illustrated by the data in FIG.

The data summarized in FIG. Increasing or decreasing the hydrocarbon concentration by a factor of five did not improve emulsification. Pentane and hexane were also not emulsified effectively; however, quantitative data for these two paraffins were not obtained because of extensive evaporation during incubation.

The solid hydrocarbons, nondecane, n-octacosane and hexatriacontane, were not dispersed by Arthrobacter bioemulsifier. Emulsification of n-alkyl cyclohexane derivatives ranging from propylcyclohexane to tridecyclohexane by Arthrobacter bioemulsifier are summarized in FIG. The data for octyl, nonyl and decylcyclohexanes were obtained from redistilled materials which contained no ultraviolet-absorbing impurities.

Nonylcyclohexane did not contain any apparent inhibitors of emulsification, since mixtures of octyl and nonylcyclohexane were emulsified to about the same extent as octylcyclohexane alone. Bicyclohexane and decalin were not emulsified significantly. Emulsification of n-alkylbenzene derivatives by Arthrobacter bioemulsifier are summarized in FIG.

Maximum activity was obtained with hexyl and heptylbenzenes. The total number of carbon atoms in the side chains may be more crucial than the chain length since p-diisopropylbenzene behaved like hexylbenzene. The low molecular weight benzene derivatives, toluene, p-xylene, m-xylene, ethyl-benzene and 1,2,3,4-tetramethylbenzene, were not emulsified significantly.

Aromatic compounds containing more than one ring, naphthalene, biphenyl, phenanthrene, anthracene, 3-phenyltoluene, 1-methylnaphthalene and 2-methylnaphthalene were also not emulsified significantly by the Arthrobacter bioemulsifier.

Table VIII summarizes a number of experiments in which the Arthrobacter bioemulsifier-induced emulsification of aliphatic, aromatic and cyclic hydrocarbons were measured in the presence of hexadecane or 1-methylnaphthalene. Although neither the aliphatic compounds nor 1-methylnaphthalene were emulsified by themselves, all mixtures containing the aromatic compound and one of the aliphatic hydrocarbons were excellent substrates for emulsification by the bioemulsifier.

The ability of aromatic compounds to stimulate emulsification of aliphatics was not limited to 1-methylnaphthalene, but occurred with toluene, p-xylene, 3-phenyltoluene and 2-methylnaphthalene. Addition of hexadecane to the aliphatic compounds did not stimulate emulsification, that is, only an additive effect was observed. The minor exception to this finding was nonadecane which became liquid when mixed with hexadecane. As mentioned above, the only aromatic compounds that served as substrates for emulsification by Arthrobacter bioemulsifier were alkylbenzene derivatives containing six or seven carbon atoms on the side chain s.

Aromatic compounds containing less than six carbon atoms on the side chain were converted into good substrates for emulsifiction by addition of hexadecane. Hexylbenzene and diiopropylbenzene were converted into even better substrates for emulsification by addition of hexadecane. On the other hand, heptyl, decyl and pentadecylbenzene were emulsified more poorly in the presence of hexadecane than by themselves.

Only alkylbenzene derivatives containing side chains of five or more carbon atoms were activated by 1-methylnaphthalene. With few exceptions, cycloparaffin derivatives were converted into better substrates for Arthrobacter bioemulsifier-mediated emulsification by addition of either hexadecane or 1-methylnaphthalene. In general, cyclohexane derivatives with short side chains e. Dicyclohexane behaved like an aromatic compound in that it was emulsified by the bioemulsifier in the presence of hexadecane but not 1-methylnaphthalene.

The fused dicylic compound decalin could not be emulsified by the bioemulsifier even by addition of hexadecane or 1-methylnaphthalene. Arthrobacter bioemulsifier-induced emulsion formation as a function of the relative concentrations of aliphatic hexadecane and aromatic methylnaphthalene compounds is shown in FIG.

Using either 1-methylnaphthalene or 2-methylnaphthalene, maximum emulsion was obtained with 25 vol. An identical experiment using decane in place of hexadecane yielded similar curves except that the peak of emulsion activity was obtained with 33 vol. To verify this conclusion, experiments were designed to determine whether or not addition of hexadecane or methylnaphthalene could enhance Arthrobacter bioemulsifier-induced emulsification of petroleum fractions which had been fractionated to separate a fraction rich in aliphatics Fraction 1 from two fractions Fractions 2 and 3 rich in aromatics.

These experiments, which are more fully described below in Section Addition of even one part of the aromatic compound to ten parts of gasoline or kerosene resulted in a much improved substrate for emulsification.

The requirement for both aliphatic and aromatic constituents was further supported by studying emulsification of column fractionated crude oil. Although crude oil itself is emulsified by the Arthrobacter bioemulsifier, none of the fractions were good substrates by themselves. However, mixtures containing one fraction Fraction 1 rich in aliphatics and the other Fractions 2 or 3 rich in aromatics were efficiently emulsified.

ATCC , may be subdivided into a differences in yield; b differences in structure; and c differences in emulsifying activity. Both bioemulsifiers were purified by ammonium sulfate fractionation, and the deproteinized derivative of each bioemulsifier was prepared by hot phenol extraction and further purified prior to analysis. Total fatty acids content was determined using the hydroxamic acid test, taking the average equivalent weight of the fatty acid esters to be Both bioemulsifiers were purified by ammonium sulfate fractionation.

The deproteinized derivatives of each bioemulsifier were prepared by hot phenol extraction and further purified prior to analysis. ATCC are used as innocula on ethanol and hexadecane media, respectively.

The adsorption or non-adsorption of emulsans and apoemulsans on various types of solid substrates, such as sand, limestone or clay minerals, were measured to determine whether these anionic lipopolysaccharides could function as bioemulsifiers in the presence of such solid substrates.

Neither emulsans nor apoemulsans are adsorbed to any significant extent on sand or on limestone over the pH range in which these bioemulsifiers will be used to form oil-in-water emulsions. When oil is present on the sand or limestone, such as in sand or sandstone reservoir formations or in limestone reservoir formations, the oil may be recovered by enhanced oil recovery using chemical flooding with dilute concentrations of emulsan, since bench scale experiments have shown that when oil-saturated sand or oil-saturated limestone are treated with dilute solutions i.

Comparable results may be obtained using sea water solutions of emulsans, since the presence of sodium chloride in the concentrations found in sea water or in connate water do not affect the ability of emulsans to emulsify crude oils, including crude oils which are quite viscous or tarry, which are found in sand or sandstone formations or in limestone formations or which remain in such formations after secondary recovery techniques such as steam stripping are employed.

Emulsans and their deproteinized derivatives, the apoemulsans, both of which are strongly anionic, are adsorbed on aluminosilicate ion-exchangers, such as kaolin, bentonite and other clay minerals which have ion-exchange capacity. Moreover, the supernatant fluid obtained using emulsan-modified flocculation was clear, while the sedimentation of bentonite without the emulsan yielded an upper layer which remained opalescent even after prolonged standing. Similar results may be obtained with other clay minerals with ion-exchange capacity.

The flocculated aluminosilicate clays now have certain fluid and flow properties which suggest an enormous number of uses for emulsans and apoemulsans and their derivatives in flocculation, including a the use of emulsans and apoemulsans as a clay particle flocculent in drilling muds; b the prevention of clogging in sewage treatment systems; c enhancing the porosity of clay solids to structure poor soils for uses in agriculture; d the inclusion of emulsans in coatings and aerosol sprays containing such clays; and e the use of emulsans and apoemulsans as a general flocculating agent for recovery and settling processes.

By way of illustration, emulsification of 1 ml Agha Jari crude oil in 10 ml sea water containing about 0. The addition of 1 g of preswelled bentonite to this stable emulsion, followed by intense shaking for about 20 seconds, resulted in breakage of the emulsion in 15 minutes.

After 20 hours, there were two separated layers, namely an upper clear liquid and a lower gel-like sediment which occupied about one-half of the prior volume of the emulsion. These sorptive properties of emulsans and apoemulsans with respect to aluminosilicate clay ion-exchangers may also be utilized to remove oil and hydrocarbonaceous sludge from oily ballast water or other oily water, either by filtering such oily waters through an aluminosilicate clay such as kaolin or bentonite on which an emulsan or apoemlusan had been adsorbed or, alternatively, by adding the emulsan or apoemulsan to the oily water and then filtering the mixture through an aluminosilicate clay.

In both cases, the filtrate will be clear and the oily residue will remain in the clay filter. Moreover, the resultant oil-in-water emulsions can be broken by physical or chemical techniques, and the oil recovered for fuel values or for refining.

Oil spill management is another environmentally important use for the emulsifying agents produced by the process of the invention. In most processes for cleaning oil spills, an aqueous solution of a detergent or surfactant is brought into contact with the oil slick, which is floating on the sea or which has been washed ashore or deposited on land to emulsify the oil so that it may be dispersed and either removed or biodegraded. Most of the detergents or surfactants commonly used are somewhat toxic to marine life and are not biodegradable.

This technique is especially useful in cleaning beaches contaminated with oil. All processes in the enhanced recovery of oil by chemical flooding involve the injection of a chemically-augmented "slug" comprising water and one or more added chemicals into a petroleum reservoir followed by displacement of the "slug" through the reservoir to recover crude oil from the injected reservoir. Moreover, these anionic lipopolysaccharides may be used as the sole emulsifier or in conjunction with other emulsifying agents such as the nonionic surfactants used for tertiary oil recovery , as well as in conjunction with the mobility control polymers used in such processes.

To a liter fermenter fitted with four baffles and a variable-speed agitator were added The final pH of the medium was 6. ATCC grown under similar fermentation conditions. The pH of the fermentation broth was maintained between pH 6. Throughout the fermentation, foam was controlled by automatic pulse additions of a silicone defoamer Dow-Corning , sterlizable, diluted 1: Commencing at the 11th hour of fermentation, ethanol was continuously added to the fermentation broth at the rate of 40 ml per hour.

Ammonium sulfate was periodically added to the fermentation broth at the rate of 2 g per hour for the first 30 hours. Maximum growth was obtained between 24 to 30 hours after inoculation. Using substantially identical conditions, as much as 5. ATCC was grown in a ml flask containing 40 ml filtered sea water, 0. The medium was inoculated with 2 ml of a late exponential culture of Arthrobacter Sp. ATCC grown under similar conditions.

Using an aqueous medium containing As before, growth was initiated by inoculating 0. ATCC on an ethanol medium was deproteinized by the hot phenol method described by O. After transferring the viscous aqueous phase to a flask, the remaining phenol layer and interface were extracted three more times with ml water.

The ability of each of these fractions to emulsify gas-oil was then determined using the standard assay technique. Emulsion formation was measured in ml flasks containing 7. Contents of the flasks were then transferred to Klett tubes for measurement of turbidity in a Klett-Summerson colorimeter fitted with a green filter. The results of these tests are summarized in Table XI, the specific activity reported in units per mg dry weight having been determined from the standard curve Curve 1-B shown in FIG.

The data contained in Table XI show that all of the emulsifying activity is in the O-lipoacyl heteropolysaccharide and that none of the activity is associated with the denatured protein fraction. The hot phenol method of O. Using the experimental method described above in Section All of the emulsifying activity was found to be in the O-lipoacyl heteropolysaccharide and none of such activity was found to be associated with the denatured protein fraction.

Ten milliliters of an aqueous solution containing 2. The solution was then cooled in an ice bath and carefully neutralized to pH 7. The resultant products are the proemulsans. After removal of the methanol at low pressure, 15 ml of water were added and the pH adjusted to pH2. The ester content of the proemulsan, as assayed by the hydroxamic acid test, was zero. Moreover, the product has no emulsification activity when assayed by the standard emulsification test.

A late exponentialculture 1: The fermentation was conducted using aeration at about 15 liters per minute and agitation at rpm without baffles, adding ethanol as required. After standing overnight, the supernatant fluid was collected by decantation. The combined supernatant fluids were further clarified by passage through a thin layer of Kieselgel. The pellet fraction, which contained all the emulsifying activity, was washed once with distilled water. The washed cetyltrimethyl ammonium bromide precipitate was dissolved in ml of 0.

One gram of potassium iodide was then added to the clear solution with mixing. The remaining supernatant fluid was dialyzed extensively against distilled water and lypohilized to yield a white solid. This material had a Specific Emulsification Activity of units per mg. The hydrolyzed material was then analyzed by thin layer chromatography on a cellulose-F plate; silver nitrate staining showed only a trace of glucose, probably as an impurity.

Using the medium described above in Section Twenty-seven liters of the hexadecane-grown culture were cooled and the cells removed by centrifugation in a Sorvall KSB continuous flow centrifuge. Residual ether in the aqueous phase was removed by bubbling with filtered nitrogen gas. The ether phase contained no measurable emulsifying activity and was discarded. The aqueous phase was filtered successively through 3, 1. The heptane fractions were combined and evaporated to a yellow syrup in vacuo.

The resulting viscous solution was dialyzed against several changes of distilled water and lyophilized. After removal of the methanol in vacuo, addition of water and acidification to pH 1, the fatty acids were extracted with ether, methylated with diazomethane and were then subjected to gas chromatographic analysis.

The phenol extraction method described above in Section Each of the resulting solutions remaining after such purification was freeze-dried and analyzed. The results of such analyses are set forth in Table XII. Moreover, both of the active fractions had high specific viscosities. None of the fractions contained significant quantities of protein. This contaminating material was a lipopolysaccharide which contained glucose.

It had no emulsifying activity when assayed by the standard emulsification technique. This conclusion was demonstrated by a series of tests which were conducted to determine the effect of both bioemulsifiers on various types of petroleum fractions which are widely used within and sold by the oil industry. Contents of the flask were then transferred to Klett tubes for measurement of turbidity in a Klett-Summerson colorimeter fitted with a green filter.

Readings were taken after standing undisturbed for 10 minutes. Controls lacking either the particular Arthrobacter emulsifier or hydrocarbon yielded readings of less than 5 Klett units. In fact, emulsions of gas-oils were as stable as crude oil emulsions, the major reason for the higher Klett readings of crude oil emulsions than those for gas-oil emulsions being the dark color of crude oil compared to gas-oil. All mixtures of hydrocarbons were 1: In some of the tests, fractions of Agha Jari crude oil were used, the fractions having been prepared by the procedure of A.

ATCC on an ethanol medium in accordance with the invention and was purified by the ammonium sulfate fractionation technique. The results of these tests are summarized in Table XIV. The requirement that the hydrocarbon substrate contain both aliphatic and aromatic or cyclic components was further supported by the results obtained in the emulsification of mixtures of column fractionated crude oil. Mixtures containing one fraction rich in aliphatics Fraction 1 and the other rich in aromatics Fractions 2 or 3 were efficiently emulsified.

ATCC on an ethanol medium can be used directly or after suitable dilution. Using the data which is set forth above in Sections 8 and 9, processes can be designed to clean any oil-contaminated vessel and to recover the hydrocarbonaceous residue from the resultant oil-in-water emulsion, either by breaking the emulsion physically or chemically.

To show the use of the cell-free fermentation broth as an emulsifying agent for such cleaning, Arthrobacter Sp. The final pH of the ethanol-salts medium was 7. During the course of fermentation the pH dropped to 6.

Throughout the fermentation, foam was controlled by periodic addition of silicone defoamer in the form of a spray. After removal of the cells by centrifugation or filtration, the resultant cell-free fermentation broth could be used to wash crude oil from the oil-contaminated surface of a steel container which simulated the inner wall of a tank which had been emptied of crude oil.

In each of these tests, 0. By itself, however, this mobility control polymer had no ability to emulsify the hydrocarbon. Because of the importance of aluminosilicate clays, such as kaolin and bentonite, in many industrial and petroleum production and refining processes, a series of tests was conducted to determine whether emulsans adsorbed onto the surface of such aluminosilicate clays.

The theoretical treatment of adsorption from a mixed solution is somewhat complicated, since it involves competition between solutes and solvents for the solid surface. In these tests, adsorption from solution was analyzed by the Freundlich equation: ATCC on an ethanol medium and purified in accordance with the ammonium sulfate fractionation technique described above in Section Nonactivated, technical grade bentonite was used as the sorbent.

The equilibrium solutions were separated from the bentonite by centrifugation or filtration. Emulsan assays were performed by the standard assay technique. The results of the tests are summarized in Table XVI. Adsorption of emulsan on bentonite results in flocculation of suspended particles of the clay, with sedimentation occurring about 5 to 10 times faster than in the absence of emulsan. The results of these tests, which are graphically illustrated in FIG.

More importantly, the supernatant fluid obtained following the emulsan-mediated flocculation was clear, while the supernatant fluid obtained in the control remained opalescent even after prolonged standing. Proemulsans are even more effective than emulsans in the flocculation of suspended particles of bentonite. Table XVII summarizes an experiment in which the flocculating of 0.

After vigorous shaking for 2 minutes, the suspension was centrifuged at 2, rpm for 60 minutes. Since emulsans form stable oil-in-water emulsions and, moreover, since emulsans are adsorbed onto bentonite, a series of tests were conducted to determine the behavior of such emulsan-induced emulsions in the presence of bentonite.

In one test, an emulsion of Agha Jari crude oil 1 ml in 10 ml sea water containing about 0. After 2 days, 1 g of preswelled bentonite was added to the stable emulsion and the dispersion was shaken intensively for about 20 seconds, after which it was transferred to a tube and allowed to settle.

After 15 minutes, a breakage of the emulsion was observed.