Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/02—Membranes; Filters

Definitions

• the present disclosure relates to a bioreactor in which microorganisms are maintained to produce desirable products.
• the microorganisms are immobilized in an artificial or synthetic biofilm.
• bioreactors that use such biofilms.
• the bioreactor designs of the present disclosure are based on artificially immobilizing microorganisms in various configurations and shapes.
• the unique shape and structure of each synthetic biofilm can be tailored to the specific reactor design.
• the bioreactors can be used to produce desirable or valuable products from microorganisms using specified inputs.
• an synthetic biofilm comprising microorganisms immobilized within a matrix, wherein the matrix is water permeable and gas permeable.
• the matrix may be made from or contain alginate, sol-gel silica, carrageenan, latex, polyvinyl alcohol, polystyrene sulfonate, or mixtures thereof.
• the biofilm can further comprise a strengthening base dispersed throughout the biofilm. Such strengthening base may be cloth, metal or a foam.
• the biofilm may be in the shape of a sphere, a sheet, or a tube.
• the biofilm can be coated on or embedded within a supporting substrate.
• the supporting substrate can be in the shape of a tube or a sheet.
• the supporting substrate can be a porous metal or ceramic tube.
• the supporting substrate is a porous foam in which the biofilm is embedded as a distinct layer.
• the microorganisms in the biofilm can be bacteria.
• a bioreactor comprising an synthetic biofilm and a supporting substrate.
• the synthetic biofilm comprises bacteria immobilized in an alginate matrix and a porous foam strengthening base.
• the synthetic biofilm may be in the shape of a tube.
• a membrane may be located on a surface of the synthetic biofilm.
• a liquid flow is provided on one side of the synthetic biofilm, and a gas flow is provided on an opposite side of the synthetic biofilm.
• FIG. 1 is a diagram illustrating a synthetic biofilm coated on a supporting substrate.
• FIG. 2 is a diagram illustrating a synthetic biofilm embedded in a supporting substrate.
• FIG. 3 is a picture of a porous metal tube, which has been coated with a synthetic biofilm, mounted in a bioreactor.
• FIG. 4 is a picture of asynthetic biofilm that has a porous foam strengthening base, in the shape of a tube.
• FIG. 5 is a picture showing the foam reinforced alginate tube of FIG. 4 with water flowing through the interior.
• FIG. 6 is a picture of other synthetic biofilm tubes where the synthetic biofilm includes a foam strengthening base and surrounds a metal screen tube.
• FIG. 7 is a picture of a synthetic biofilm that has a cloth strengthening base.
• FIG. 8 is a picture showing the synthetic biofilm in the form of spheres.
• the spheres are made from microorganisms and alginate.
• FIG. 9 is a picture showing the alginate spheres of FIG. 8 being used in a fluidized bed reactor.
• FIG. 10 is a diagram illustrating the configuration of the fluidized bed reactor.
• approximating language may be applied to modify any quantitative representation that may vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language may correspond to the precision of an instrument for measuring the value.
• the modifier "about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4" also discloses the range "from 2 to 4.”
• the present disclosure relates to artificial or synthetic biofilms that can be used in a bioreactor.
• microorganisms such as bacteria, algae, or fungi
• the artificial biofilm can be placed in various configurations, shapes, or structures tailored to the specific design of the bioreactor.
• the bioreactor is used for the production of a valuable/desirable product from the microorganisms.
• the microorganisms may be able to produce butanol from hydrogen gas.
• biofilm refers to an aggregate of microorganisms that are embedded within a self-produced matrix of an extracellular polymeric substance.
• This extracellular polymeric substance generally contains extracellular DNA, proteins, and polysaccharides.
• the present disclosure relates to an artificial biofilm composed of microorganisms immobilized in a matrix.
• the terms “artificial” and “synthetic” are used interchangeably to indicate that the matrix in which the microorganisms are embedded or immobilized is made of a material that is not naturally produced by the microorganisms.
• the microorganisms in the artificial biofilm may be bacteria, algae, or fungi.
• a suitable bacterium is Ralstonia eutropha.
• R. eutropha is a hydrogen- oxidizing bacterium that can grow in both anaerobic and aerobic environments.
• eutropha can perform aerobic respiration or anaerobic respiration by denitrification of nitrate and/or nitrite to nitrogen gas. It can also easily adapt between a heterotrophic lifestyle and an autotrophic lifestyle. Under autotrophic conditions, R. eutropha fixes carbon through the pentose phosphate pathway. R. eutropha can also produce and sequester polyhydroxyalkanoate (PHA) plastics when exposed to excess amounts of sugar substrate. PHA can accumulate to levels of approximately 90% of the cell's dry weight.
• suitable bacteria include Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodospirillum rubrum. These bacteria contain bacteriochlorophyll a/b.
• the microorganisms are immobilized in a matrix (i.e. encapsulated) to form the artificial biofilm.
• the matrix is made of a material that is both gas-permeable and water-permeable.
• the matrix can be described as a gel.
• Exemplary materials suitable for the matrix include alginate, sol-gel silica, carrageenan, latex, polyvinyl alcohol, polystyrene sulfonate, and mixtures of these materials.
• Alginate is also known as algin or alginic acid (CAS# 9005-32-7), is commercially available, and can absorb a large quantity of water.
• Sol-gel silica refers to using silica in a sol-gel procedure to obtain a three-dimensional network containing both a liquid phase and a solid phase.
• exemplary silicates include tetraethyl orthosilicate (TEOS).
• TEOS tetraethyl orthosilicate
• the microorganisms can be contained within the network.
• Carrageenan refers to polysaccharides which can gel, and which can be obtained from seaweed. Other materials, which are both gas-permeable and water-permeable, may also be used to immobilize the microorganisms.
• gases and liquids will be provided to the microorganisms on different sides of the artificial biofilm, e.g. liquids will pass on one side and gases will pass on the other side. Maintaining gas-liquid separation can be achieved in several different ways.
• pressure differences and gas solubility in water at running conditions maintain the separation.
• the gas can be provided at a pressure level that is sufficient to permit the liquid to contact the artificial biofilm, but high enough to prevent the liquid from entering the gas pathway.
• one or more membranes are provided to maintain the desired separation of gas and liquid flows, limiting their free interaction, while allowing the exchange of the required constituents to support metabolism and valuable/desirable product production within the organism-containing matrix.
• Such membranes can be micro-porous and hydrophobic, for example, which might be placed to prevent the liquid from entering the gas flow.
• the membranes could alternatively be micro-porous and hydrophilic.
• a strengthening base may be added to the artificial biofilm.
• the strengthening base (and the microorganisms) is in the matrix of the artificial biofilm, and could alternatively be described as being generally dispersed throughout the biofilm.
• the strengthening base increases the structural stability of the artificial biofilm.
• An exemplary strengthening base is cloth.
• Another exemplary strengthening base is a metal screen.
• Yet another exemplary strengthening base is an open celled foam. In such foams, the microorganisms and their matrix could be described as being dispersed throughout the foam. For example, bacteria and alginate could fill the holes within the open celled foam.
• the artificial biofilm can be shaped into any desired configuration or shape.
• the artificial biofilm may be in the shape of a sphere, a sheet, or a tube.
• Spheres can be used in a packed bed or fluidized bed reactor format.
• Sheets, which are flat and planar, can be used in a reactor as a series of stacked sheets.
• a tube is a hollow cylinder, and can also be described as a rolled-up sheet. Tubes can be used to deliver one growth medium on the exterior of the tube, and to deliver a second different growth medium on the interior of the tube while keeping the two growth mediums from mixing.
• the sphere When the artificial biofilm is in the shape of a sphere, the sphere may have a diameter of from about 0.1 millimeter (mm) to about 20 mm.
• the tube When the artificial biofilm is in the shape of a tube, the tube may have an inner diameter of from about 0.1 mm to about 30 mm, an outer diameter of from about 0.1 mm to about 30 mm (the outer diameter being greater than the inner diameter), and may have any length appropriate for the desired design.
• the artificial biofilm can retain structural integrity and bacterial viability in different shapes, which allows it to be incorporated into many different types of reactors. The cell density, cell placement, and consistency of the artificial biofilm can also be controlled.
• the artificial biofilm can be formed quickly, without the need for a long incubation period while waiting for a natural biofilm to be created.
• the microorganisms are immobilized in the biofilm instead of living in the growth medium that typically circulates within the bioreactor vessel.
• the artificial biofilm may be coated on or embedded within a supporting substrate.
• the supporting substrate may be in any desired shape, such as a sphere, a sheet, or a tube.
• the artificial biofilm can be applied as a layer upon a surface of the supporting substrate.
• the artificial biofilm could be applied as a coating on the outer surface of a porous metal or ceramic tube.
• the artificial biofilm could be embedded within the supporting substrate.
• the artificial biofilm can be surrounded by a porous foam.
• the supporting substrate and the strengthening base can be made from the same material. The distinction is that the strengthening base is dispersed within the artificial biofilm and generally cannot / could not be separated.
• the artificial biofilm maintains itself as a separate layer.
• FIG. 1 is a diagram illustrating the artificial biofilm coated upon a supporting substrate.
• the supporting substrate 100 has a surface 102 upon which the artificial biofilm 110 is coated. One side of the artificial biofilm contacts the supporting substrate, while the other side 114 of the artificial biofilm is exposed.
• the supporting substrate here may have a thickness such that the microorganisms in the artificial biofilm have access to all the necessary nutrients, delivered from both sides, to support metabolism and valuable/desirable product production.
• FIG. 2 is a diagram illustrating an artificial biofilm embedded within a supporting substrate.
• the supporting substrate is illustrated in the form of a tube 200 formed from a porous foam.
• the artificial biofilm is embedded within the tube as a distinct layer 210. Both inner surface 212 and outer surface 214 of the artificial biofilm contact the supporting substrate.
• the tube could also be described as an inner annulus 202 and an outer annulus 204. It should be noted that the microorganisms are not dispersed within the porous foam, but are rather within a distinct layer.
• the tube may have an inner diameter of from about 0.1 mm to about 30 mm, an outer diameter of from about 0.1 mm to about 30 mm (the outer diameter being greater than the inner diameter), and may have any length appropriate for the desired design.
• the combination of the artificial biofilm with a supporting substrate is referred to herein as a bioreactor.
• the purpose of the bioreactor is to control the location of the microorganisms and permit the collection of products secreted by the microorganisms.
• the shape of the artificial biofilm may aid in collecting the product. For example, if the artificial biofilm is in the shape of a sphere, the products may accumulate within the sphere, and could be collected by removal of the spheres. Alternatively, the artificial biofilm could be made into a thin sheet so that the product could migrate or otherwise collected in a liquid growth medium that is flowed past the sheet.
• FIG. 3 is a picture of a porous metal or ceramic tube, which has been coated with an artificial biofilm in a glass bioreactor.
• FIG. 4 is a picture of a an artificial biofilm in the shape of a tube.
• the microorganisms were immobilized using an alginate matrix, with an open celled foam used as a strengthening base.
• the microorganisms and alginate were embedded in the holes within the open cell foam.
• FIG. 5 is a picture showing the foam reinforced alginate tube of FIG. 4 (i.e. an artificial biofilm) with water flowing through the interior.
• FIG. 6 is a picture of other synthetic biofilms in the shape of tubes. Again, the microorganisms were immobilized using an alginate matrix and a foam strengthening base. This artificial biofilm surrounds a porous metal screen tube which is used as a supporting substrate.
• FIG. 7 is a picture of an artificial biofilm that includes a strengthening base.
• the microorganisms are in an alginate matrix with cloth serving as the strengthening base.
• FIG. 8 is a picture showing the artificial biofilm in the form of spheres.
• the spheres are made from microorganisms and alginate.
• FIG. 9 is a picture showing the alginate spheres of FIG. 8 being used in a fluidized bed reactor.
• FIG. 10 is a diagram illustrating the configuration of the fluidized bed reactor.
• Liquid medium flows into the bioreactor through an inlet tube (labeled "Media In”), carrying the liquid medium to the bottom of the bioreactor vessel.
• the medium outlet tube (labeled Media Out”) has foam around the opening to assist in retaining the spheres within the reactor.
• the outlet tube is located at a higher elevation compared to the inlet tube.
• the gas enters the bioreactor (labeled "Gas In”) through a frit in the bottom of the reactor vessel and exits via the medium outlet tube.
• the buoyancy of the gas bubbles, the flow of the liquid medium, and the density of the spheres induce motion of the spheres, ensuring sufficient mixing and opportunity for nutrient/waste exchange between the spheres, the gas, and the medium.

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Citations (1)

• 2013
  • 2013-08-02 WO PCT/US2013/053339 patent/WO2014022734A1/fr not_active Ceased

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