WO2001030997A2 - Microbe, microbial exopolysaccharide, and uses thereof - Google Patents
Microbe, microbial exopolysaccharide, and uses thereof Download PDFInfo
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- WO2001030997A2 WO2001030997A2 PCT/US2000/029456 US0029456W WO0130997A2 WO 2001030997 A2 WO2001030997 A2 WO 2001030997A2 US 0029456 W US0029456 W US 0029456W WO 0130997 A2 WO0130997 A2 WO 0130997A2
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
- C08J5/18—Manufacture of films or sheets
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the present invention relates in general to a novel non-pathogenic microbe that produces a nontoxic, non-antigenic exopolysaccharide.
- the use of the microbe and exopolysaccharide in environmental engineering, agricultural, geologic, consumer and medical applications is described. Inhibition and control of mucoidy exopolysaccharide is also described.
- the invention pertains to a novel non-pathogenic microbe that produces a non-toxic, non-antigenic exopolysaccharide.
- the exopolysaccharide can be used as a biofilm in environmental engineering and agricultural applications and as a filler or polymer in consumer and medical applications. Biofilm applications are described first, then particular medical applications are described.
- biofilm is used to describe an organic material that includes microorganisms embedded in a polymer matrix of their own making.
- the matrix consists largely of exopolysaccharides and is a tough, elastic, mucoidal material that adheres strongly to soil particles. Growth of a biofilm in a sandy soil is achieved by injecting a bacterial and nutrient solution into soil specimens. The resulting biofilm treatment is used to clog soil pores, thereby reducing the ability of the soil to transmit fluids.
- biofilms are produced by certain strains of Klebsiella pneumoniae and Pseudomonas species.
- a problem with the use of K. pneumoniae is that Klebsiella is a genus that includes a number of human pathogens.
- the pathogenicity of K. pneumoniae itself is associated with its ability to create a mucoidal exopolysaccharide used in attachment and colonization that helps the pathogen evade both the non-specific and specific immune clearing defensive mechanisms .
- U.S. Patent No. 4,800,959 discloses the use of a microbial process for selectively plugging a subterranean formation.
- a highly permeable stratum or zone in a subterranean reservoir is plugged using Klebsiella or Pseudomonas bacteria that were starved to reduce their size prior to being injected into the target zone.
- Klebsiella or Pseudomonas bacteria that were starved to reduce their size prior to being injected into the target zone.
- the bacteria regain full cell size, proliferate and commence production of biofilm-forming exopolysaccharides upon exposure to minimal nutrient containing media.
- the biofilm produced by these bacteria selectively seal off the high permeability zones of a formation and reduce aqueous flow through the zone.
- 5,130,230 discloses a blood substitute that may be used as a system of solutions in which a number of solutions, are used sequentially to completely replace the blood of living subjects.
- U.S. Patent No. 5,130,230 discloses that the blood substitute comprises "an aqueous solution of electrolytes at physiological concentration, a macromolecular oncotic agent, a biological buffer having a buffering capacity in the range of physiological pH, simple nutritive sugar or sugars, and magnesium ion in a concentration sufficient to substitute for the flux of calcium across cell membranes."
- a number of commercially available products have been used for the treatment of hypovolemic patients.
- HESPANTM 6% hetastarch in 0.9% sodium chloride injection
- PENTASPANTM 10% pentastarch in 0.9% sodium chloride injection [both by DUPONT PHARMACEUTICALSTM, Wilmington Del.]
- MACRODEXTM 6% dextran 70 in 5% dextrose injection or 6% dextran 70 in 0.9% sodium chloride injection
- RHEOMACRODEXTM % dextran 40 in 5% dextrose injection or 10% dextran 40 in 0.9% sodium chloride injection [PHARMACIA, INC.TM, Piscataway, N.J.]).
- the newly discovered bacterium LAB-1 deposited at ATCC No. PTA-2500, possesses a number of potential commercial biofilm applications. These include, but are not limited to: (1) subsurface biofilm cutoff wall formation; (2) subsurface liners that include compacted, biofilm treated soil; (3) in-situ biofilm liners,- (4) barriers made by treating geotextiles with biofilm materials; (5) improved ability of sand to retain moisture; (6) reclamation of poor soils and conversion into agriculture land; (7) significantly increased soil biomass in the form of polymers that function as a nutrient supply for plant growth and/or help retain nutrients and water; and (8) providing cohesion to otherwise cohesionless soils (such as sand dunes) , thus making the soil more resistant to erosion by wind and/or water.
- subsurface biofilm cutoff wall formation include, but are not limited to: (1) subsurface biofilm cutoff wall formation; (2) subsurface liners that include compacted, biofilm treated soil; (3) in-situ biofilm liners,- (4) barriers
- the present invention is directed to a non-pathogenic bacterium that produces a biofilm made of exopolysaccharide that is essentially made of neutral sugars that migrate at the same rate as: mannose, fucose, fructose and galactose, acidic sugars that migrate at the same rate as fucose and amine sugars that migrate at the same rate as glucose and fucose.
- the bacterium is a LAB-1 strain.
- the biofilm producing bacterium may be further defined as being capable of growth between about pH 4 and 11 and between about 15° and 45°C.
- the LAB-1 strain is capable of growth in minimal growth media, or may be grown in an aqueous nutrient medium that includes yeast, peptone and mineral salt ingredients.
- LAB-1 is a gram-negative, rod-shaped bacterium of about 0.2 X 0.8 ⁇ m that secretes the exopolysaccharide described herein.
- the LAB-1 strain is used in plugging a permeable subterranean stratum by providing LAB-1 bacteria in a nutrient-containing solution into the target stratum.
- the nutrient-containing solution is generally adapted to provide substantial and uniform growth conditions for the LAB-1.
- Sufficient biofilm is produced under these conditions to effectively plug the stratum.
- the bacterium in si tu can yield a saturated hydraulic conductivity equal to or less than 1.5 x IO "5 cm/sec, equal to or less than 1.0 x IO "7 cm/sec or even equal to or less than 1.5 x 10 "8 cm/sec.
- the bacteria may be preincubated in culture in an aqueous suspension medium with agitation for an incubation period sufficient to initiate bacterial exopolysaccharide production before injection into the stratum.
- the method of plugging the subterranean stratum may also include draining nutrient deficient suspension medium from the reservoir, and recharging the reservoir with aqueous nutrient medium to maintain bacterial growth for an elapsed time period sufficient to establish a biofilm of prescribed saturated hydraulic conductivity.
- the draining and recharging steps with aqueous nutrient medium may be conducted at least once every 48 hours of elapsed time period.
- the step of pre-incubating the bacteria may be, e.g., for at least about 72 hours.
- the biofilm may be used to plug open conduits, deposited in a subsurface biofilm cutoff wall, used to enhance the water retaining ability of subsurface liners or even for improving the water retention capabilities of compacted, semi -compacted or loosened biofilm treated soil.
- the biofilm When used in a liner, the biofilm may be deposited in -si tu .
- the biofilm may also be used along with and/or to enhance environmental barriers by treating geotextiles with the biofilm.
- Another important aspect of this polymer is its lack of antigenicity and toxicity in an animal system. This suggests several consumer/medical applications, including: (1) use a food additive or food thickening or filler agent; (2) use as plasma expander; (3) use in polymer industry; (4) use as chromatography matrix support for purification of chemicals; (5) use in scientific research as suspension solution instead of ficoll and the like; (6) use in determining the gene content of the organism, especially those coding for the biosynthesis of the exopolysaccharide polymer;
- (11) for use as an additive in toothpaste, ointments, creams and lotions; (12) for mixing with dyes, stains, paints and varnishes; (13) for inclusion in dialysis; (14) for use in composite materials, e.g., bricks, tile, mortars; (15) for use as part of a sealant; (16) viscosity modifier for oils, waxes & greases; (17) use as a filler, thickener or extender in pharmaceutical preparations; (18) use of the polymer in bioscaffolding applications, including wound-healing applications; and (19) use as a bacteriostatic (biostat) agent to inhibit or at least fail to support bacterial growth, and even possibly as a biocide.
- biostat bacteriostatic
- a compound is needed for use as a plasma extender that serves to increase blood volume and that is impermeable at blood capillaries.
- the compound must not readily dissociate or be rapidly broken down by natural physiologic enzymes with time.
- the compound and its use as a plasma expander must not provide bacteria with an exogenous nutrient source, which may lead to accentuating already severe septicemia in patients that are infected by pathogens at the time of the injury that is causing hypovolemia.
- the present invention is an exopolysaccharide produced by the LAB-1 bacterial stain.
- the exopolysaccharaide does not appear to easily support bacterial growth. This was determined by testing the ability of E. coli or B . indica to grow on the exopolysaccharide and no growth was observed. Further, the exopolysaccharide is not antigenic as tested by injection into mice. Thus, the product appears to satisfy some of the basic parameters required for a plasma expander.
- the exopolysaccharide is secreted into the cell culture medium and collected for use in, e.g., a plasma expander.
- a plasma expander When used as a plasma expander alone, or in combination with other elements, the exopolysaccharide will be provided in an isotonic solution.
- a blood-free plasma expander and blood substitute for use in a subject in need thereof includes a single solution with at least two water soluble oncotic agents, one of which is a water soluble polysaccharide oncotic agent and one of which is serum albumin, wherein the exopolysaccharide consisting essentially of mannose, fucose, fructose and galactose, acidic fucose and amine containing glucose and fucose.
- the plasma expander and blood substitute may have a ratio of water soluble exopolysaccharide oncotic agent to serum albumin between 1:1 and 1:2, weight to weight.
- the combined percentage of water-soluble exopolysaccharide oncotic agent and serum albumin in a solution of the plasma expander and blood substitute may be in the range of between about 4%-6% weight to volume .
- the plasma expander and blood substitute may also include a number of cations, alone or in combination.
- the cations may be provided in the following concentrations: Na + at 110 to 120 mEq/1, Ca ++ at about 5 mEq/l, K + at 0 to 3 mEq/1, and Mg ++ at 0 to 0.9 mEq/1. These cations may be supplied as dissolved chloride salts.
- the plasma expander and blood substitute may also include at least one buffer, for example, a lactate and/or bicarbonate buffer. When buffered, the plasma expander will generally be a biological buffer having a buffering capacity in the pH range of about 6.8 to 7.8.
- agents When used in hypovolemic patients, e.g., those that have lost a large volume of blood due to trauma, additional agents may be included in the plasma expander to aid in recovery.
- agents may include, Vitamin K in a concentration of about 1-4 mg/1, amylase, clotting factors, t-PA or even erythropoietin.
- the exopolysaccharide of the present invention may be used as a chromatography matrix support for purification of chemicals.
- One such use will be as a suspension solution for use in centrifugation.
- the exopolysaccharide may even be used in solution as a suspension solution for use in size separation.
- the present invention may also be used as a biologically stable, non-toxic material for use in coated plates for a number of biological and analytical uses.
- uses include the coating of tissue culture plates for maintaining the growth, in vitro, of cells.
- Cells that may be grown on the surface of the exopolysaccharide include prokaryotic and eukaryotic cells.
- the exopolysaccharide disclosed herein may be used as a coating for instrumentation, such as biosensors, that require the maintenance of a biologically compatible environment .
- compositions containing propionic acid and ibuprofen when incorporated into liquid or solid growth media of the LAB-1 strain at a concentration range of 0.1-1.0 % (w/v) differentially inhibits its growth, development, cell attachment and biofilm production.
- Growth of the newly discovered LAB-1 strain as well as its production of mucoidal exopolysaccharide and biofilm may be inhibited or controlled by propionic acid, derivatives of propionic acid, compounds with related chemical structures or backbones such as 2- (4-isobutylphenyl) -propionic acid, otherwise known as ibuprofen, and solutions, mixtures, suspensions and other kinds of preparations comprising such compounds singly or in combination with other materials and compounds. It would be apparent to one of ordinary skill in the art to apply the above methods to inhibit the production of mucoid compounds and biofilm in any mucoid organism.
- FIGURE 1 is a photograph of Gram stained LAB-1 at 100X magnification.
- FIGURE 2 is a Coomassie stained SDS-PAGE gel of the total protein content of LAB-1 and B . indicia grown on solid culture.
- FIGURE 3 is a Coomassie stained SDS-PAGE gel of the total protein content of LAB-1 and B . indicia grown in liquid culture.
- FIGURE 4 is the data from gas chromatography of fatty acids in LAB-1.
- FIGURE 5 is a FACE gel showing the sugars identified in the exopolysaccharide produced by LAB-1.
- Lanes 1 - MONO Ladder Standard 2 (100 pmol ea. monosaccharide) ; 2 - Amine hydrolysis reaction products; 3 [S] - MONO Ladder Standard 2 (100 pmol ea . monosaccharide; scanned for trace shown in [S] Scan) ; 4 - Neutral hydrolysis reaction products; 5 - Sialic acid hydrolysis reaction products; 6 - NANA labeling control 1 (100 pmol) ; 7 - MONO composition control; 8 - MONO Ladder Standard 2 (100 pmol ea . monosaccharide) .
- FIGURE 6 is a MALDI trace of the exopolysaccharide produced by LAB-1.
- FIGURE 7 is a drawing of a cross sectional view of a barrier created with the biofilm of the present invention.
- FIGURE 8 is a graph of the hydraulic conductivity versus time of a LAB-1 containing biofilm.
- LAB-1 is a Gram-negative, rod-shaped bacterium isolated by the present inventor from constructed soil samples in the state of Wyoming.
- the soil sample was constructed by J. Turner at the University Wyoming and contained a contaminant in a background of a Beijerinckia indica .
- the contaminant, LAB-1 was isolated and studied because of its excessive slime production.
- biochemical tests were performed in order to identify the species and the genus of organism. The tests included: assessment of growth conditions, culture appearance, cell appearance and staining characteristics, optimal temperature and pH growth range, oxygen requirements, antibiotic sensitivity testing, tests for catalase and oxidase
- exopolysaccharide was further analyzed by GLYKOTM (NOVATOTM, CA) fluorophore-assisted carbohydrate electrophoresis (FACE) , and matrix-assisted laser desorption/ionization mass spectrometry (MALDI) . Immunogenicity of the exopolysaccharide was also tested in mice.
- GLYKOTM NOVATOTM, CA
- FACE fluorophore-assisted carbohydrate electrophoresis
- MALDI matrix-assisted laser desorption/ionization mass spectrometry
- Colonies of LAB-1 on solid medium were irregular with an undulate edge. They exhibited convex elevation, a smooth glistening surface, were white in color and translucent to opaque. The consistency of the colonies was that of a very tenacious and elastic slime.
- the LAB-1 exopolysaccharide exhibits tremendous tenacity, extending without breakage when pulled with a glass rod over a foot. Due to the production of this exopolysaccharide, it was found difficult to lift the colonies from agar plates.
- AB13 culture medium may be used to grow the LAB-1 strain and is made as follows: Per liter of water add 20 g glucose, 1 g NaCI, 1 g yeast extract or 2 g NaN0 3 , 8 g K 2 HP0 4 , 0.2 g KH 2 P0 4 , 0.5 g MgS0 4 , and 150 ⁇ l 5% FeCl 2 . To make solid medium, 15 g of agar are added to the medium. This simplified end medium makes large- scale production of the polysaccharide polymer affordable .
- FIGURE 1 shows the Gram stained cells at 100X.
- the spore stain showed only red vegetative cells, no spores were observed.
- the capsule stain showed no capsules, but rather an indefinite exopolysaccharide surrounding the cells. Observation of the stab culture showed that the organism was motile.
- Colony morphology on plates grown at 26°, 30°, 37°C and 45°C was the same, although growth was optimal at 37°C. Elasticity of the slime layer was also unchanged. Growth was unchanged in liquid AB13 cultures ranging from pH 4 , 9 and 11. Growth was not observed at pH 2. Colony morphology on plates was also unchanged over this pH range. The LAB-1 organism was determined to be a facultative anaerobe.
- Samples used for FIGURE 3 were grown in liquid AB13 culture. One ml was harvested by pelleting for five minutes, washed with 1% NaCI and again with dH 2 0. Pellets were resuspended in 180 ⁇ l of 50 mM Tris, and 10 mM EDTA, pH 8.0. Sixty ⁇ l of PSS (4X PSS is 700 ⁇ l of 1 M Tris-HCl, pH 6.8, 4.3 g sucrose, 4 mg bromophenol blue, 1.45 ⁇ l of 20% SDS, 44 ⁇ l of 0.5 M EDTA, 10 mg DTT and dH 2 0 to 10 ml) were added to the cells and the mixture was boiled for five minutes. Samples were overlaid with powdered sucrose to remove cell debris and run as above .
- the next logical step was to determine the 16S rRNA gene sequence. This technique currently is the method of choice for identification purposes.
- the gene sequence is shown in SEQ ID NO: 1.
- the identification based on the 16S rRNA gene sequence was determined to be Leclercia adecarboxylata .
- the difference in sequence homology between LAB-1 and L. adecarboxylata was only 0.59%.
- Stackebrandt & Goebel, INT ' L J. SYSTEM. BACTERIOL. 44: 846 (1994) would consider this a species level match, however the confidence limits of the data obtained by MIDI LABSTM allowed identification only at the genus level.
- biochemical characteristics of LAB-1 and of L . adecarboxylata (9 th edition Bergey's Manual of Determinative Bacteriology 1994) are compared, there are yet again, numerous differences. The differences led to the questions regarding this method.
- results of the MALDI study of the exopolysaccharide are shown in FIGURE 6.
- the data indicates that polymerization and depolymerization of the polysaccharide occurred readily as evidenced by the large range of molecular weights found.
- All monosaccharides identified were neutral sugars that migrate at the same rate as: mannose, fucose, fructose and galactose, acidic sugars that migrate at the same rate as fucose and amine sugars that migrate at the same rate as glucose and fucose. All are six-carbon sugars. This fact makes it impossible to determine the composition of the polymer when only the molecular weight is known. Those substances with molecular weights below 180 are likely breakdown products of the polymer. The largest polymer, molecular weight of 1066.38, was comprised of approximately six 6C sugars.
- the initial exopolysaccharide sample analyzed for immunogenicity in mice contained residual amino acids.
- the small amount of protein present was from the yeast extract in the media and from dead cells. Even with these amino acids present, it was determined that the exopolysaccharide was non-immunogenic, although a very small immunogenic reaction was observed. This reaction made it necessary to further purify the exopolysaccharide so that it was protein-free.
- the monosaccharide linkages and branching of the polysaccharides should be determined. Also, it would be very useful to determine the nature of its overall polymerization.
- the localization of the gene(s) coding for the polysaccharides may be determined, as will be known to those of skill in the art of molecular biology. Even further studies may be conducted to identify LAB-1, and may include: chromosomal DNA fingerprinting, random primer PCR profiling, rRNA or other gene sequencing, determination of the G+C % content, lipid analysis and BIOLOGTM analysis (a more comprehensive biochemical analysis) . Also, detailed studies regarding the chemistry of the polysaccharide will be completed.
- the LAB-1 strain may be used to construct environmental biofilm barriers for containment and treatment of contaminated soil and groundwater.
- containment barriers is to control the transport of chemical contaminants from waste disposal facilities or from areas which have become contaminated by spills, industrial processes, illegal dumping or other sources.
- Several different types of barriers are possible, including the following: (1) subsurface biofilm cutoff wall; (2) subsurface liners consisting of compacted, biofilm treated soil; (3) in-si tu biofilm liners; and (4) barriers made by treating geotextiles with biofilm.
- soil hydraulic conductivity may be reduced by several orders of magnitude by the addition of the biofilm- producing bacterium disclosed herein.
- the reductions of k obtained using the LAB-1 strain are sufficient to meet Environmental Protection Agency (EPA) criteria for barrier materials, defined as a k value of IO "7 cm/sec or less.
- EPA Environmental Protection Agency
- the low hydraulic conductivity persists when the soil is permeated with a variety of chemical solutions, suggesting that a biofilm barrier may be compatible with a wide range of contaminants.
- the biofilm disclosed herein may also be useful for controlling contaminant transport mechanisms, such as diffusion, adsorption and biodegradation .
- FIGURE 7 shows one use of the present invention for the formation of subsurface liners for the containment of wastes in engineered disposal facilities, such as landfills.
- a landfill 10 is depicted in cross-sectional view.
- Waste 12 is disposed within a subsurface liner 14. If the liner 14 is being placed during the creation of the landfill 10, a biofilm liner may be used prior to deposition of the liner 14. In addition, a containment wall may be erected that surrounds the waste site, and additional layers of decontaminating biofilm barriers may be included.
- waste may leach in the form of a leachate 16 into subsurface strata 18 and 20.
- a biofilm barrier wall 28 is created that surrounds the waste 12 and captures the leachate 16.
- the biofilm barrier wall 28 is constructed so as to reach into strata 22, 24 into which the waste 12 does not leach.
- One advantage of the biofilm of the present invention is that it permits such remedial application to existing landfills that may be leaking and even prevents leachate 16 from reaching a subterranean water layer 26.
- One specific field of use for the LAB-1 biofilms is creating subsurface biofilm liners that include spreading untreated soil in loose (uncompacted) lifts using conventional soil spreading equipment (e.g., bulldozers) .
- Loose lifts will generally be 150 to 225 cm thick.
- a solution that includes water, LAB-1, and nutrients are applied to the soil, using, e.g., conventional equipment used to apply water to soil (e.g., a truck-mounted water tank with sprinkler hoses) .
- the soil is then compacted using conventional equipment (e.g., sheeps foot rollers) to achieve the specified density, typically resulting in a compacted lift thickness of 100 to 150 cm.
- the required number of lifts and liner total thickness are site-specific design parameters which are determined by analysis of contaminant transport and regulatory requirements for containment.
- Compacted clay liners typically range from 0.6 to 1.3 meters thick.
- the proposed procedure is similar to field construction of clay liners, except that the soil is treated with a biofilm-producing solution.
- solutions of strain LAB-1 and nutrients are injected into the ground at a specified depth to create in si tu biofilm liners. This type of liner is particularly useful at sites contaminated by accidental spills.
- previously grown biofilm may be mixed directly into or onto the soil.
- Subsurface liners may also be constructed by treating geotextiles with biofilm.
- Geotextiles are generally made of synthetic fibers that are either woven or matted together, yielding a porous fabric that is used for soil separation, reinforcement, filtration or drainage. Containment barriers can be created by spraying bitumen, rubber-bitumen or other polymeric mixtures into a properly deployed geotextile that contains the LAB-1 produced biofilm disclosed herein.
- One particular example for use of the LAB-1 biofilm is in the application of a liquid solution containing strain LAB-1 and nutrients to geotextiles to clog the pore spaces and reduce permeability, creating a barrier to flow.
- Soil used by the present inventors to analyze waste containment capability is a naturally occurring, easily attainable sand. Based on its grain size distribution and Atterberg limits, this soil is classified as SM, or silty sand of low plasticity, in the Unified Soil Classification System. Permeability tests yield a saturated hydraulic conductivity (k) of approximately 1.5 x IO "5 cm/sec when compacted to maximum dry density. This value of k would make the soil unsuitable for use as a waste containment barrier. Initial studies indicated that k could be reduced to values on the order of 10 "8 to IO "7 cm/sec, which is in the range required for waste containment, by treating this soil with the biofilm-producing bacterial strain LAB-1.
- the operational procedure for use of the LAB-1 bacterium to form a biofilm that may be used to test water permeability may include the following steps: (1) compacting soil into a cylindrical specimen which is placed in a flexible wall permeameter, (2) permeating the specimen with a solution containing LAB-1, and
- FIGURE 8 is a graph that shows hydraulic conductivity versus time for a specimen treated with LAB-
- the present invention includes the use of the LAB-1 derived biofilm for the treatment of agricultural soils to improve the following soil agricultural properties: (1) improved water retention characteristics; (2) enhanced ability to establish and support plant growth; and (3) improved erosion resistance.
- These improvements may be obtained by adding complete or dried and pulverized biofilm, or by the application of LAB-1 strain in bacterial/nutrient solutions using conventional soil watering equipment (e.g., a truck-mounted tank with sprinkler hoses or conventional irrigation systems) .
- the biofilm of the present invention has been used for the treatment of soil.
- the biofilm altered the soil's properties in many ways that enhanced the soil' s ability to support agriculture. These include the following: (1) an improved ability of sand to retain moisture; (2) an increased biomass in the form of polysaccharides that function as a nutrient supply for plant growth; (3) improved soil cohesion; and (4) increased resistance of soil to erosion.
- the biofilm of bacterium LAB-1 possesses a number of characteristics that are of potential commercial application in medical devices and treatment, including its low antigenicity, non-toxicity, and its biodegradable nature.
- the exopolysaccharide may be purified, e.g., by the addition of concentrated NaOH to the cell culture at a final concentration of 0.2 M, followed by the addition of 3 volumes of ethanol to precipitate the polymer and other materials. The precipitate is collected and redissolved in half the original volume of water. Protein is removed by either extracting twice with phenol or by ultra- filtration. The aqueous phase is dialyzed, lyophilized and ground to yield a fine white powder as will be known to those skilled in the art.
- the exopolysaccharide produced by LAB-1 may, alternatively, be purified without using the alkali treatment or the phenol extraction. Not only is the purification process thereby simplified, it may prevent the removal of alkali- labile acetyl moieties from the purified LAB-1 exopolysaccharide.
- Total carbohydrate concentration in culture broths and polymer solutions may be determined by the phenol reaction, described by Gerhardt in Manual of Methods for General Bacteriol . ( Amer. Soc . Microbiol . , Washington, DC, 1991) . Glucose, galactose and xanthan gum (SIGMA CHEMICAL CO.TM, St. Louis, MO) may be used as standards.
- Total protein concentration in culture broths and polymer solutions may be determined using the BIO-RADTM protein assay (BIO-RAD LABORATORIESTM, Richmond, CA) . Lysozyme may be used as the standard. Cellular protein may be released by boiling the cells in 0.2 M NaOH.
- Purified polysaccharide may be further hydrolyzed in 1 M trifluoroacetic acid at 120°C for times varying between 30 minutes and 2 hours.
- Monosaccharides in the polysaccharide hydrolysate may be separated using, e.g., a WATERSTM HPLC equipped with a BROWNLEETM polypore PB, lead loaded cation exchange column, operated at 85°C, with water as the eluent.
- Detection may be performed by refractive index using, e.g., a WATERSTM Model 401 Differential Refractometer .
- the polysaccharide may be further characterized by proton NMR spectroscopy and infrared spectroscopy. Infrared analysis, along with the monosaccharide composition data, may be compared to the composition and IR scans of polysaccharides from mutant or genetically manipulated strains to detect changes in structure.
- the biofilm of the present invention may be used as a vaccine adjuvant or carrier that provides a reservoir for antigens.
- An adjuvant may be prepared using the LAB-1 derived biofilm of the present invention.
- Adjuvants may be synthesized by any one of a number of established methods, as has been described by M. Bodansky, et al . , "Peptide Synthesis,” second edition, Wiley, New York 1976 and R. W. Roeske, Peptides (N.Y.) 3, 102 (1981).
- a particularly useful method is the methanesulfonic acid catalyzed esterification procedure described by C. Penney, et al . , J. Organic Chemistry 50, 1457-1459 (1985) .
- the adjuvant may be purified by any of the techniques described previously.
- One such purification technique is silica gel chromatography, in particular the "flash" (rapid) chromatographic technique, as described by W. Clark Still, et al . , J. Organic Chemistry, 43, 2923-2925 (1978) .
- Other chromatographic methods may be used for purification of the adjuvant.
- Crystallization may also be used to purify the adjuvant. In some cases, no purification is required as a product of analytical purity is obtained directly from the synthesis. Methods of formulating the adjuvant into vaccine preparations are well known in the art, and not detailed herein.
- the base bacterium of the present invention does not appear to be a human pathogen, and its exopolysaccharide is not toxic or antigenic and does not readily support bacterial growth, the exopolysaccharide provides an ideal source for oncotic plasma expanders. Furthermore, the exopolysaccharaide ' s lack of intrinsic antigenicity makes it an ideal candidate for use in, e.g., all mammals.
- the present invention includes a mixture of components that, when placed in aqueous solution, may be used to expand the plasma volume of a subject in need thereof.
- the mixture of plasma expanding components according to the invention will be discussed as an aqueous solution. From the following description of the invention it is expected that one ordinarily skilled in the art would be enabled to provide the mixture as a dry mixture and make the adjustments to amounts of sodium chloride, fluid and/or dextrose, etc. as necessary.
- the exopolysaccharide oncotic agents of the foregoing mixture of components are ones that are generally water semi-soluble.
- water semi-soluble oncotic agent is meant partially-water soluble molecules that when dissolved in the fluid phase of circulating plasma in a living subject are of a size sufficient to prevent their immediate loss from the circulation by traversing the fenestration of the capillary bed into the interstitial spaces of the tissues of the body.
- polysaccharide oncotic agent thus does not include such polysaccharides as chitin, because chitin is not soluble in water.
- the plasma expander is made by solubilizing the purified exopolysaccharide in a biocompatible fluid.
- Plasma expander solutions according to the invention may also contain additional ingredients, including but not limited to, sodium ions (110-120 mEq/1) , calcium ions (5 mEq/1) , potassium ions (0-3 mEq/1) , magnesium ions (0-0.9 mEq/1) , and vitamin K (0-10 mg per subject or 0-3 mg/1) .
- the expander may be buffered to about pH 7.4 and may provide assimilable sugar (e.g., dextrose or glucose at 5-10 mM) .
- the expander is provided as a sterile solution, but it may also be provided as a powder that can be reconstituted in a sterile manner, or sterilized after reconstitution.
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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JP2001533979A JP2004500806A (en) | 1999-10-26 | 2000-10-25 | Microorganisms, microbial exopolysaccharides and their uses |
CA002388203A CA2388203A1 (en) | 1999-10-26 | 2000-10-25 | Microbe, microbial exopolysaccharide, and uses thereof |
AU12322/01A AU784594B2 (en) | 1999-10-26 | 2000-10-25 | Microbe, microbial exopolysaccharide, and uses thereof |
EP00973866A EP1224279A2 (en) | 1999-10-26 | 2000-10-25 | Microbe, microbial exopolysaccharide, and uses thereof |
IL14929500A IL149295A0 (en) | 1999-10-26 | 2000-10-25 | Microbe, microbial exopolysaccharide and uses thereof |
HK03100671.3A HK1050214A1 (en) | 1999-10-26 | 2003-01-24 | Microbe, microbial exopolysaccharide, and uses thereof |
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US16139199P | 1999-10-26 | 1999-10-26 | |
US16158899P | 1999-10-26 | 1999-10-26 | |
US60/161,391 | 1999-10-26 | ||
US60/161,588 | 1999-10-26 |
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WO2001030997A2 true WO2001030997A2 (en) | 2001-05-03 |
WO2001030997A3 WO2001030997A3 (en) | 2002-01-17 |
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PCT/US2000/029414 WO2001030996A2 (en) | 1999-10-26 | 2000-10-25 | Microbial exopolysaccharide and uses thereof |
PCT/US2000/029456 WO2001030997A2 (en) | 1999-10-26 | 2000-10-25 | Microbe, microbial exopolysaccharide, and uses thereof |
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EP (1) | EP1224279A2 (en) |
JP (1) | JP2004500806A (en) |
AU (2) | AU784594B2 (en) |
CA (1) | CA2388203A1 (en) |
HK (1) | HK1050214A1 (en) |
IL (1) | IL149295A0 (en) |
WO (2) | WO2001030996A2 (en) |
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JP2005313159A (en) * | 2004-03-31 | 2005-11-10 | Rom:Kk | Method for cleaning polluted soil or polluted water, and apparatus for cleaning polluted soil or polluted water |
GB2430198B (en) * | 2005-09-19 | 2008-10-15 | Charles Michael De Franca Hedges | Device for producing a contaminant barrier |
CN104642734B (en) * | 2013-11-15 | 2018-05-22 | 中国科学院过程工程研究所 | A kind of broiler chicken feed additive and its application |
CN105519518A (en) * | 2015-04-08 | 2016-04-27 | 上海蓝盎电子科技发展有限公司 | Chinese herbal medicine bacterial fermentation product-containing eco-friendly harmless pathological tissue preservation and treatment solution |
CN113999660B (en) * | 2020-07-28 | 2023-05-26 | 中国石油天然气股份有限公司 | Plugging material, plugging agent and plugging method |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US4806636A (en) * | 1985-03-20 | 1989-02-21 | The Dow Chemical Company | Heteropolysaccharide produced by Enterobacter sakazakii |
US5229277A (en) * | 1991-03-05 | 1993-07-20 | Louisiana State University Board Of Supervisors | Process for the production of dextran polymers of controlled molecular size and molecular size distributions |
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US4800959A (en) * | 1987-11-19 | 1989-01-31 | Alberta Oil Sands Technology And Research Authority | Microbial process for selectively plugging a subterranean formation |
CA2284671A1 (en) * | 1997-03-26 | 1998-10-01 | Kevin P. Killeen | Carbohydrate antigens which immunoreact with polyclonal antisera against h. pylori |
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2000
- 2000-10-25 IL IL14929500A patent/IL149295A0/en unknown
- 2000-10-25 WO PCT/US2000/029414 patent/WO2001030996A2/en active Application Filing
- 2000-10-25 JP JP2001533979A patent/JP2004500806A/en active Pending
- 2000-10-25 CA CA002388203A patent/CA2388203A1/en not_active Abandoned
- 2000-10-25 AU AU12322/01A patent/AU784594B2/en not_active Ceased
- 2000-10-25 WO PCT/US2000/029456 patent/WO2001030997A2/en active Application Filing
- 2000-10-25 EP EP00973866A patent/EP1224279A2/en not_active Withdrawn
- 2000-10-25 AU AU15751/01A patent/AU1575101A/en not_active Abandoned
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4806636A (en) * | 1985-03-20 | 1989-02-21 | The Dow Chemical Company | Heteropolysaccharide produced by Enterobacter sakazakii |
US5229277A (en) * | 1991-03-05 | 1993-07-20 | Louisiana State University Board Of Supervisors | Process for the production of dextran polymers of controlled molecular size and molecular size distributions |
Non-Patent Citations (4)
Title |
---|
EL-FALAHA B M A ET AL: "Antimicrobial activity of some anti-inflammatory compounds." EGYPTIAN JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 39, no. 1-3, 1998, pages 1-17, XP001009850 ISSN: 0301-5068 * |
OHTANI KIMIKO ET AL: "Characterization of an extracellular polysaccharide elaborated by TX-1, a new strain of Beijerinckia indica." BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, vol. 59, no. 9, 1995, pages 1628-1631, XP002167918 ISSN: 0916-8451 * |
SCAMPARINI ADILMA ET AL: "Structural studies of CV-70 polysaccharide." INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 21, no. 1-2, 1997, pages 115-121, XP001002091 ISSN: 0141-8130 * |
See also references of EP1224279A2 * |
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CA2388203A1 (en) | 2001-05-03 |
AU1232201A (en) | 2001-05-08 |
JP2004500806A (en) | 2004-01-15 |
EP1224279A2 (en) | 2002-07-24 |
HK1050214A1 (en) | 2003-06-13 |
WO2001030996A3 (en) | 2002-01-17 |
AU1575101A (en) | 2001-05-08 |
IL149295A0 (en) | 2002-11-10 |
AU784594B2 (en) | 2006-05-11 |
WO2001030997A3 (en) | 2002-01-17 |
WO2001030996A2 (en) | 2001-05-03 |
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