Microbial exopolysaccharide and uses thereof

Details Microbial exopolysaccharide and uses thereof Microbial exopolysaccharide and uses thereof

2000-10-25

2004-02-17

Prats, Francisco (Department: 1651)

Wells

Processes

Cementing, plugging or consolidating

C166S246000, C435S101000, C536S123000, C536S114000

Reexamination Certificate

active

06691783

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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.
The term “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.
Examples of 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. Furthermore, 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.
Another example of a biofilm is described in U.S. Pat. No. 4,800,959, by Costerton, which discloses the use of a microbial process for selectively plugging a subterranean formation. In the process taught, 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. 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.
In addition to the above described biofilm uses, there has been a need for perfusion solutions and blood substitutes. Currently available and approved compounds, however, have so far failed to meet the increasing demands on our blood provider system. A number of blood substitutes have been developed over the last few years to attempt to meet the increasing demand for blood, blood substitutes and plasma expanders. Unfortunately, many of the plasma expanders that are currently in use fail as the small molecules on which they depend to provide osmotic pressure readily traverse capillary beds as a consequence of the negative osmotic pressure found in post-arterial capillary beds. The loss of osmotic potential, makes the long-term use of current plasma expanders for maintaining proper ionic or fluid balance or plasma volume in a mammalian subject unsatisfactory.
Those blood substitutes that have an impermeable substance to maintain volume use human serum albumin or a mixture of plasma proteins as the oncotic agent. These substitute plasma proteins depend on the same blood and plasma supply as our current blood provider system, therefore failing to meet the increased demand for these products.
A number of patents have issued to Segall that are directed to blood and plasma substitutes. U.S. Pat. No. 4,923,442, and the reissue thereof, discloses a number of solutions used in blood substitution of living subjects all of which include at least some concentration of a cardioplegia agent, usually potassium ion. U.S. Pat. No. 4,923,442 discloses surgical methods, particularly in respect to instrument placement and the control of pulmonary wedge pressure generally applicable to perfusion of subjects. U.S. Pat. No. 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. Pat. 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.”
In addition to the patented inventions described above, a number of commercially available products have been used for the treatment of hypovolemic patients. These include: H
ESPAN
™ (6% hetastarch in 0.9% sodium chloride injection, P
ENTASPAN
™ (10% pentastarch in 0.9% sodium chloride injection [both by D
UPONT
P
HARMACEUTICALS
™, Wilmington Del.]), M
ACRODEX
™ (6% dextran 70 in 5% dextrose injection or 6% dextran 70 in 0.9% sodium chloride injection [P
HARMCIA
, I
NC
.™, Piscataway, N.J.]) and R
HEOMACRODEX
™ (10% dextran 40 in 5% dextrose injection or 10% dextran 40 in 0.9% sodium chloride injection [P
HARMACIA
, I
NC
.™, Piscataway, N.J.]). All of these products, however, depend on compounds that are polymeric and that often dissociate or are broken down by natural physiologic enzymes with time. Alternatively, bacteria may take advantage of these newly supplied nutrient sources, causing severe septicemia in patients that are infected by pathogens at the time of injury. Thus, a need remains for a better oncotic agent.
SUMMARY OF THE INVENTION
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.
It has been found that the prior art methods and biofilms fail to provide biologically and environmentally safe and efficacious water, soil and waste retention characteristics. A significant problem with existing technology is the pathogenicity of the bacteria used to produce the biofilms. The present invention, therefore, 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.
More particularly, 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 0.2×0.8 &mgr;m that secretes the exopolysaccharide described herein.
In one embodiment of the present invention, 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 b

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