WO2022229225A1 - Method of treating aquatic or marine animals using polymer supports - Google Patents

Method of treating aquatic or marine animals using polymer supports Download PDF

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Publication number
WO2022229225A1
WO2022229225A1 PCT/EP2022/061110 EP2022061110W WO2022229225A1 WO 2022229225 A1 WO2022229225 A1 WO 2022229225A1 EP 2022061110 W EP2022061110 W EP 2022061110W WO 2022229225 A1 WO2022229225 A1 WO 2022229225A1
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microorganisms
polymer support
spp
immobilized
polymer
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PCT/EP2022/061110
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French (fr)
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Nicholas Bagger GURIEFF
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Novozymes A/S
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Publication of WO2022229225A1 publication Critical patent/WO2022229225A1/en

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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/30—Aerobic and anaerobic processes
    • C02F3/302—Nitrification and denitrification treatment
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/084—Polymers containing vinyl alcohol units
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/098—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer formed in the presence of the enzymes or microbial cells
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/02—Aerobic processes
    • C02F3/08—Aerobic processes using moving contact bodies
    • C02F3/085—Fluidized beds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/02—Aerobic processes
    • C02F3/10—Packings; Fillings; Grids
    • C02F3/105—Characterized by the chemical composition
    • C02F3/108—Immobilising gels, polymers or the like
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/341—Consortia of bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

  • the present invention relates to polymer supports of immobilized microorganisms for use in a method of treating aquatic or marine animals, including treating the water body of aquatic or ma rine animals.
  • Nitrifying bacteria are chemoautotrophs that convert ammonia into nitrite then nitrate.
  • a biofilter provides a substrate for the bacterial community, which results in thick biofilm growing within the filter. Water is passed through the biofilter, and ammonia is utilized by the bacteria for energy. Nitrate is less toxic than ammonia and therefore does not impact the health of the fish as acutely as ammonia.
  • the nitrate in the system can be removed by a denitrifying biofilter or by water replacement. Stable environmental conditions and regular maintenance are required to ensure the biofilter is operating efficiently, both of which are not always available in an operating RAS process.
  • H2S hydrogen sulfide
  • chemical oxidizers such as ozone, chlorine dioxide, and hydrogen, calcium, or magnesium peroxide
  • Biofilters are difficult to start up, operate and restart after disinfection because there is little chance of con trolling the identity, abundance or activity of native nitrifying bacteria in the competitive environ ment that is an open biofilter unit process.
  • the invention provides a polymer support of immobilized microorganisms for use in a method of treating aquatic or marine animals wherein the method comprises contacting a water body with a polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydro gel comprises a cross-linked polymeric material and water or an aqueous medium; and wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
  • the polyvinyl alcohol of the polymer support of immobilized microorganisms is in one embodiment cross linked with glutaraldehyde. In another or further embodiment, the polymer support of immo bilized microorganisms is porous.
  • the polymer support of immobilized microorganisms are selected from the group consisting of one or more sulfur oxidizing microorganisms, one or more ammonium oxidizing microorganisms, one or more nitrite oxidizing microorganisms, one or more denitrifying microorganisms, one or more anammox microorganisms and any mixtures thereof.
  • the invention furthermore provides a method of treating aquatic or marine animals or treating a water body comprising aquatic or marine animals, comprising contacting the water body with a polymer support of immobilized microorganisms, wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; and typically wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; and wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
  • the invention furthermore is directed to the use of a polymer support of immobilized microorganisms for treating aquatic or marine animals, wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; and wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
  • a further aspect of the invention is directed to a method of reducing the ammonia, nitrate, or nitrite levels in a water body comprising the use of the polymer support of immobilized microorganisms of the invention.
  • a further aspect is related to the denitrification of a water body comprising the addition of a polymer support of immobilized microorganisms of the invention.
  • Figure 1 illustrates, as shown in Example 3, NH4-N concentrations in the influent (NH4 in) and effluent for Blank and Seeded biobead reactors over a period of 27 days of continuous operation.
  • the chlorine disinfection soaking is indicated with a dotted line.
  • Figure 2 illustrates, as shown in Example 3, N02-N concentrations in the influent (N02 in) and effluent for Blank and Seeded biobead reactors over 27 days of continuous operation.
  • the chlo rine disinfection soaking is indicated with a dotted line.
  • Figure 3 illustrates, as shown in Example 4, TN concentrations in the influent (N03 in) and effluent for reactors R37 (blank biobeads) and R38 (seeded biobeads) over a period of 21 days of contin uous operation. The period of chlorine addition is shown with dotted and dashed lines.
  • Figure 4 illustrates, as shown in Example 4, Specific TN removal activity for the biobeads in re actors R37 and R38 over 21 days of continuous operation. The period of chlorine addition is shown with dotted and dashed lines.
  • Figure 5 illustrates, as shown in Example 5, TN concentrations in the influent (feed) and effluent for reactors R37 (blank biobeads) and R38 (seeded biobeads) over a period of 129 days of con tinuous operation.
  • Figure 6 illustrates, as shown in Example 5, Specific TN loading rate and removal activity for the biobeads in reactors R37 and R38 over 129 days of continuous operation.
  • Figure 7 illustrates, as shown in Example 5, Nitrite production as a proportion of the nitrate re moved in each reactor over a period of 129 days of continuous operation.
  • FIG. 8 illustrates, as shown in Example 5, Nitrate and nitrite effluent concentrations after pass ing through a biobead plug-flow reactor system.
  • the performance of the biobeads in a plug-flow system is similar to that achieved in the previous completely mixed reactor system, but with only 1 ⁇ 4 of the HRT.
  • Figure 9 illustrates, as shown in Example 6, (A) TN concentrations in the influent (feed) and effluent for reactors R38 and R45 over a period of 25 days of continuous operation; (B) Specific TN removal activity for the biobeads in reactors R38 and R45 over 25 days of continuous opera tion.
  • Figure 10 illustrates, as shown in Example 6, Nitrite production as a proportion of the nitrate re moved in each reactor over a period of 25 days of continuous operation.
  • Figure 11 illustrates, as shown in Example 7, Specific NH4 loading rate and removal activity for the biobeads in reactors M06 and M07 over 21 days of continuous operation.
  • Figure 12 illustrates, as shown in Example 7, NH4 concentrations in the influent (feed) and effluent for reactors M06 (blank biobeads) and M07 (seeded biobeads) over a period of 21 days of con tinuous operation.
  • Figure 13 illustrates, as shown in Example 7, Nitrate production as a proportion of the ammonia removed in each reactor over a period of 25 days of continuous operation.
  • Alginate is the salt of alginic acid and is a polysaccharide naturally occurring in brown algae.
  • an alginate may be any salt of alginic acid such as e.g. sodium alginate or calcium alginate.
  • Anammox is an abbreviation for ANaerobic AMMonium Oxidation and is a microbial process of the nitrogen cycle where nitrite and ammonium ions are converted directly into nitrogen (N2) and water.
  • Anammox microorganisms or bacteria are thus microorganisms and bacteria mediating the anammox process of converting nitrite and ammonium ions directly into nitrogen and water.
  • Non-limiting examples of anammox microorganisms and bacteria include bacteria from the genera Brocadia, Kuenenia, Anammoxoglobus, Jettenia or Scalindua.
  • Aqueous medium is a solution comprising water as a solvent and one or more other components such as e.g. sodium chloride.
  • Bead load The mass of beads applied to a reactor volume. For example, if 100 kg of beads was applied in a reactor with a volume of 1 cubic meter, the bead load is 10% w/v.
  • Denitrifying The term is herein used for removal or reduction of nitrite and nitrate from a water body through the process of biological denitrification where the final product is nitrogen gas (N2).
  • Footprint The spatial area that a building or installation occupies. It is measured in square meters.
  • High activity refers to the specific ammonium or nitrate treatment rates in a biofilter and is defined as mg Nitrogen/m3.d.
  • Baseline activity is defined as the activity that can be expected from a fixed film type of biofilter, including fixed and moving bed bioreactors.
  • High activity is de fined a specific nitrogen treatment activity that is in excess of the activity general expected from fixed film biofilters. In one aspect, the activity is high and is at least 400 g-N/m3.d.
  • Self-cleaning in relation with biobeads means that the movement of the beads in the reactor and the constant resultant collisions between beads, other beads and the walls of the reactor, prevents any significant biofilm from forming in the reactor (either on the beads or any surfaces). Therefore, the beads and the reactor they are applied in is considered self-cleaning as no action is required by operators.
  • Solids As used herein, the term “solids” in relation with biobeads means solid materials evolving on or inside the biobeads such as e.g.
  • start-up and restart As used in relation to nitrification and denitrification biofilters, “start-up” is the period of time immediately after a biofilter is taken into use for the first time. During this period, activity in the biofilter increases over time, until the biofilter reaches a level of activity (nitrification, denitrification and/or sulfur removal) that the biofilter was designed to operate with. A similar pe riod is recognised after disinfection or cleaning of a biofilter, and this is referred to as a biofilter “restart”.
  • Water body is herein understood as a compilation of water forming a reservoir.
  • Non-limiting examples of water bodies e.g. include an aquaculture farm, tank, raceway, creek, river, pool, pond, waste lagoon, paddy, lake, estuary or ocean.
  • the present invention relates to a method of treating aquatic or marine animals using a recirculating aquaculture system (RAS) comprising the step of contacting a water body with a polymer support of immobilized microorganisms, wherein the polymer support comprises said microorganisms immobilized within a polymer hydrogel, wherein the polymer hy drogel comprises a cross-linked polymeric material and water or an aqueous medium, and wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol (PVA) and alginate.
  • a polymer support of immobilized microorganisms is herein used inter changeably with the term biobeads.
  • the polyvinyl alcohol may be cross linked with any cross- linking agent such as e.g. glutaraldehyde.
  • the polymer support of immobilized microorganisms when used in the method of the invention uses competitive exclusion activity to actively deter most unwanted bacteria from establishing themselves on or in the beads.
  • the polymer support of immobilized microorganisms used in the present method are self-cleaning, safe and easy to handle.
  • the in ventors also found that the method of the invention resulted in a surprisingly effective reduction in ammonium (NH4+).
  • the ammonium is defined as being removed when it is either consumed by the microbes present to support the growth of the microbes, or converted by nitrifying microor ganism to nitrate.
  • the level of ammonium in the water is reduced compared to the level of ammonium without using the claimed method. In a further aspect, the level of ammonium in the water is reduced by 50-66% compared to the level of ammonium without using the claimed method. In an aspect of the invention, the method results in reduction in am monium without increasing the level of nitrogen dioxide substantially. In another or further aspect of the invention the level of ammonium in the water is reduced to reach a maximum level of ammonia of 2 mg NH4-N/I water.
  • the level of ammonium in the water is reduced and at the same time the level of nitrogen dioxide is substantially remaining at the same level as the level of nitrogen dioxide compared to the level of ammonium and nitrogen dioxide without using the claimed method.
  • Another by-product of ammonium removal can be the formation of dinitrogen oxide.
  • the method results in a reduction of ammonium without leading to the production of dinitrogen oxide due to partial, interrupted or poorly controlled nitrification.
  • the method of the invention furthermore provides a fast start-up or restart, robust performance despite the presence of inhibitors, toxins or process changes, high activity, low footprint and low production of solids.
  • the polymer support used in the method of the invention is a polymer support of immobilized microorganisms wherein the polymer support comprises said microorganisms immobilized within a polymer hydrogel, wherein the polymer hydrogel comprises polyvinyl alcohol polymeric chains cross linked with glutaraldehyde, and water or an aqueous solution.
  • the polymer support of immobilized microorganisms comprises said microorganisms immobilized within a polymer hydrogel, wherein the polymer hydrogel comprises cross-linked polymeric material, and water or aqueous medium, wherein the cross- linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and glutaraldehyde.
  • the polymer support of immobilized microorganisms comprises a covalent bond between glutaraldehyde and polyvinyl alcohol polymeric chains. The covalent bond crosslinks the polyvinyl alcohol polymeric chains.
  • the polyvinyl alcohol polymeric chains will vary in length. Typically, the polyvinyl alcohol polymeric chains consist of 500-3.000 monomer units and a molecular weight of 22.000 g/mol to 130.000 g/mol.
  • the polyvinyl alcohol polymeric chains are cross-linked with glutaraldehyde.
  • the crosslink may comprise monomeric, dimeric, oligomeric, or polymeric forms of glutaraldehyde.
  • the crosslinking density may affect morphological properties of the polymer support.
  • the crosslinking density, determined as the number of glutaraldehyde crosslinking units is 8 to 25%, such as 10 to 20%, such as 12-20%, such as about 12%, 13%, 15%, 16%, 17%, 18%, 19% or 20%.
  • the polymer support of immobilized microorganisms is, as stated, a polymer hydrogel.
  • the hydrogel comprises from 60 to 99 wt% cross-linked polymeric material and 1 to 40 wt% water or aqueous medium, such as from 60 to 98 wt% cross-linked polymeric material and 2 to 40 wt% water or aqueous medium, such as from 65 to 95 wt% cross-linked polymeric material and 5 to 35 wt% water or aqueous medium, such as from 65 to 90 wt% cross-linked polymeric material and 10 to 35 wt% water or aqueous medium, such as from 65 to 85 wt% cross-linked polymeric material and 15 to 35 wt% water or aqueous medium, such as from 65 to 80 wt% cross-linked polymeric material and 20 to 35 wt% water or aqueous medium, or such as from 65 to 75 wt% cross-linked polymeric material and 25 to 35 wt% water or aqueous medium.
  • the polymer support of immobilized microorganisms further comprises alginate in monomer or polymeric form, entangled with cross-linked polyvinyl alcohol polymeric chains, such as so as to form an interpenetrating polymer network or semi-interpenetrating polymer network.
  • the polymer support comprises pores.
  • the polymer support is a porous structure.
  • the immobi lized microorganisms are immobilized within the polymer support on the surface of the pores. Entrapment of the microorganisms in the inner matrix provides an advantage of the method in that it serves to physically protected immobilized cells.
  • Cell attachment or adsorption of microor ganisms to the inner matrix of polymer support may be by weak (non-covalent), generally non specific interactions such as electrostatic interactions.
  • the polymer support is adequately porous to allow the substrate to diffuse into the polymer hydrogel support and the products or metabolites to diffuse out.
  • a central volume of the polymer support comprises one or more macropores, and major volume of the polymer support comprises micropores.
  • the central macropores allows for convection, which is an efficient method of mass transfer with out applying pressure.
  • the pores of the polymer support are non-uniform in size.
  • an inner central fraction of the bead volume comprises macropores, whereas an outer fraction of the bead volume comprises micropores.
  • macropores is intended to mean pores with an average size of at least 100 microns.
  • micropore is intended to mean pores with an average pore size of less than 100 microns.
  • the core of polymer support is devoid of polymer, such as a core of at least 100 microns in longest diameter, such as at least 200 microns, typically from 100-2.000 microns, such as 200-2.000 microns, such as 200-1.500 microns, such as 100-1.000 microns, such as 200-1.000 microns
  • the polymer support comprises pores having a gradient pore size in that inner portion of the volume of the polymer support has a larger pore size than average pore size if the remaining volume of the polymer support.
  • the polymer support comprises pores having a gradient pore size in that the outer one-third of the polymer support has pores of a smaller average diameter than the pore size of the middle third of the polymer support, which in turn has a smaller average diameter than the pore size of the inner one-third of the polymer support.
  • the outer one-third of the polymer support has an average pore diameter from 5 to 20 microns.
  • the middle third of the polymer support has a larger average pore diameter than the outer one- third. It a suitable embodiment, it has an average pore diameter from 10 to 100 microns.
  • the inner one-third of the polymer support has, in an embodiment, a larger average pore diameter than the middle third of the polymer support.
  • the inner third of the polymer support has, in one embodi ment, an average pore diameter from 100- 2.000 microns. In one embodiment, at least 50% of the volume of the inner third is a cavity.
  • the cavity is a volume within the polymer support that is substantially free from cross-linked polyvinyl alcohol.
  • the center of the polymer support may com prise, in its center, a cavity having volume comprising 50-100% of the volume of the inner one third of the polymer support.
  • a cavity is a volume free from cross-linked polyvinyl alcohol. Without being bound to a particular theory, the cavity serves as a central distribution center for nutrients, metabolites and substrate for the microorganisms, allowing for flow and distribution within the polymer support.
  • the immobilized microorganisms grow within the polymer hydrogel. That is to say that the micro organisms grow on the surface of the pores.
  • the inert polymer hydrogel retains the microorgan ism, albeit not irreversibly in that a fraction of the population of the microorganisms may leak out of the polymer support by detaching from the inner matrix of the polymer support and leaking through the pores to exit the polymer support.
  • the outer surface of the polymer support does not comprise a skin or shell, which may serve to retain the cells or metabolites by means of having a smaller pore diameter than the outer third of the polymer sup port.
  • the polymer support of the invention is resistant to dissolution in water. It is reusable.
  • the polymeric support is chemically substantially uniform in that the surface, body and core of the carrier is made of the same chemical components.
  • some physio-chemical properties on the surface may differ with the physio-chemical properties within the core and throughout the hydrogel. Accordingly, there may be different degrees of crosslinking at the surface.
  • these differences do not constitute a shell or coating or fibrous network on the surface.
  • the invention is directed to a polymer support of immobilized microorganisms wherein the polymer support comprises said microorganisms immobilized within a polymer hy drogel; wherein polymer hydrogel comprises cross-linked polymeric material, alginate and water or aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer com prising polyvinyl alcohol and glutaraldehyde.
  • at least some alginate may remain within the polymer support of immobilized microorganisms. The alginate entrapped within the polymer support of immobilized microorganisms does not negatively impact performance of the polymer support.
  • the polymer support of immobilized microorganisms, after multiple washing, may comprise alginate.
  • the polymer support of immo bilized microorganisms does not comprise alginate.
  • the polymer support comprises glutaraldehyde cross-linking polyvinyl alcohol chains.
  • An increased content of glutaraldehyde results in more PVA hydroxyl groups consumed and more acetal rings and ether linkages formed as a result of the crosslink formation.
  • an acid catalyst in the preparation of the polymer support such as sulfuric acid, also increases crosslink formation.
  • Cross-link formation increases mechanical strength of the polymer support. Increased mechanical strength is observed with increased degree of crosslinking.
  • the polymer support rigidity increases.
  • the polymer support is elastic and malleable with excellent mechanical properties.
  • the elasticity modulus is typically between 1.4 and 2.2 GPa, such as between 1.5 and 2 GPa.
  • the tensile strength is typically between 3 and 6 MPa.
  • the polymer support used in the method of the invention comprises said microorganisms immobilized within a polymer hydrogel.
  • concentration of microorganisms within the polymer hydrogel known as the microbial load, is typically in the range of 5 g/kg bead to 250 g/kg, typically 10 g/kg to 150 g/kg.
  • the cell density of the microorganisms can be tailored to the type of microorganisms, intended metabolic activity.
  • a high microbial load of immobilized microorganisms within the polymer support may be used, in some embodiments, to improve the product yield and the volumetric productivity of the bioreactors.
  • the microbial load in the support is suitably at a concentration of at least about 5 grams, such as at least 10 grams/kg, such as at least 20 grams/kg, such as at least 50 grams/kg.
  • the polymer support of immobilized microorganisms may be of any shape but is typically according to spherical, oval, elliptical, bead-shaped, oblong, cylindrical, or capsule-like in shape.
  • the polymer support is typically 1 to 10 mm long at its longest axis, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm. In the axis perpendicular to the longest axis, the polymer support may be 1 to 10 mm, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm.
  • the aspect ratio is from 0.5 to 1, such as 0.5, 0.6, 0.7, 0.8, 0.9 or 1.
  • the polymer support of immobilized microorganisms is typically spherical or bead-shaped having a diameter of 1 to 10 mm, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm with an aspect ratio from 0.6 to 1 , typically from 0.8 to 1.
  • the polymeric support of immobilized microorganism (the biobeads) used in the method of the invention may typically be prepared in a process comprising a first step comprising a pre-bead formation and second step comprising polyvinyl alcohol linking.
  • the polymer material comprises polyvinyl alcohol wherein the polyvinyl alcohol (PVA) is a blend of polyvinyl alcohol of different molecular weights (MW), such as a blend of 2, 3, 4 or 5 PVA types, each with a MW from approximately 75.000 to approximately 225.000, such as a MW of approximately 95.000 to approximately 205.000, such as a PVA blend comprising a PVA selected from the group consisting of a PVA with a MW of approximately 125.000, PVA with a MW of approximately 145.000, and PVA with a MW of approximately 195.000.
  • PVA polyvinyl alcohol
  • MW molecular weights
  • the pre-bead formation step comprises combining sodium alginate, polyvinyl alcohol (PVA) and the microorganisms.
  • PVA polyvinyl alcohol
  • the mixture is then added to a divalent or trivalent cation-containing solution, such as a divalent-containing solution, such as a Ca 2+ -containing solution.
  • a divalent-containing solution such as a Ca 2+ -containing solution.
  • the mixture is added in a dropwise fashion to the divalent or trivalent cation-containing solution, such as to the divalent cation-containing, e.g. a Ca 2+ -containing solution.
  • the divalent or trivalent cation- containing solution such as the Ca 2+ -containing solution typically comprises a dissolved salt such as CaCh, SrCh, BaCh or AhiSC
  • the divalent or trivalent cation containing-solution, such as the Ca 2+ -containing solution may have a cation concentration, (wt/wt) such as calcium concentra tion (wt/wt) ranging from 0.1% to 10%, typically from 0.5% to 5%, such as 0.5% to 2%, such as 0.5%, 1%, 1.5%, or 2%.
  • wt/wt calcium concentra tion
  • the alginate and the divalent or trivalent cation such as e.g.
  • the heterogenous solution comprises a gelate structure of an alginate-diva- lent/trivalent complex within the divalent or trivalent-containing solution and with microorganisms and PVA loosely entrapped within the gelate structure.
  • the divalent or trivalent-containing solu tion typically further comprises PVA and microorganisms.
  • the alginate- divalent or trivalent cation complex comprises physical cross-linking, which relies on divalent or trivalent cation cross-linking between alginate chains.
  • the pre-bead formation step comprises combining sodium alginate, polyvinyl alcohol (PVA) and the microorganisms to form a mixture to be dripped into the divalent or trivalent-containing solu tion.
  • the polyvinyl alcohol (PVA) is suitably present at a concentration 3% - 15% wt/wt, such as 5-10% wt/wt, such as 5%, 6%, 7%, 8%, 9% or 10%.
  • the alginate is typically present at a concentration of 0.25-5% wt/wt, such as 0.5-2%, such as 0.5%, 1%, 1.5% or 2%.
  • the pre-bead formation step comprises combining sodium alginate, polyvinyl alcohol (PVA) and the microorganisms to form a mixture.
  • a stock broth solution of microorganisms comprising from 200 g of cells/L to 1000 g of cells/L, such as 100 to 500 g of cells/L is typically used.
  • the stock broth solution is diluted, said diluted solution comprising to 100 to 500 g of cells/L, such as 50 to 250 g of cells/L, such as 50 g/L, 100 g/L, 125 g/L, 150 g/L, 200 g/L, and 250 g/L.
  • This diluted stock solution is used to form a mixture comprising of sodium alginate, polyvinyl al cohol (PVA) and the microorganisms.
  • the microorganisms are suitably present in the mixture at a concentration of 10g/L to 500 g/L, such as 20g/L to 80g/L, typically from 20 g/L to 60 g/L.
  • the second step is a cross-linking step and comprises adding the gelate structure of the alginate- divalent or trivalent-complex to a cross-linking solution comprising glutaraldehyde so as to provide a cross-linked polymer support of immobilized microorganisms.
  • the cross-linking solu tion further comprises an acid, such as sulfuric acid, H 2 SO 4 , or hydrochloric acid, HCI, adjusting to same pH as the physical properties like elasticity, visual appearance or sphericity of the beads change along.
  • the cross-linking solution has an acidic pH, such as a pH of 1-5, typically 1.5 to
  • the cross-linking solution when comprising sulfuric acid typically has a pH of
  • the cross-linking solution further comprises a catalytic agent such as a sulphate, typically sodium sulphate, ammonium sulphate or potassium sulphate.
  • a catalytic agent such as sodium sulphate performs at least two functions. It forms a complex with the hydroxyl groups of PVA, thereby interlinking PVA strands and/or two different positions within a PVA strand. This function supports the cross-linking with glutaraldehyde. It is furthermore thought that the catalytic agent catalyses the reaction of the aldehyde units of glutaraldehyde with the alcohol groups.
  • the alginate is hydrolysed under acidic pH conditions.
  • the hydrolysed alginate washes out, at least in part from the cross- linked polymer support of immobilized microorganisms.
  • the molar ratio of glutaraldehyde to polyvinyl alcohol is suitably from 1 : 10 5 to 1 : 10 10 , such as from 1 : 10 6 to 1 : 10 9 , such as 1 : 10 7 to 1 : 10 9 , such as in the order of 1 : 10 7 , 1 : 10 s or 1 : 10 9 , such as in the order 1 : 10 8 .
  • the concentration of glutaraldehyde in the cross-linking step is less than 0.3%, such as from 0.02% to 0.25%, preferably from 0.02% to 0.2%, such as 0.05%, 0.10%, 0.15% and 0.20%.
  • the concentration of sulfuric acid (H2SO4) is from 0.2% to 1.0% (g/g), such as 0.3% to 0.9%, such as 0.3%, 0.4%, 0.5%, 0.6%. 0.7%, or 0.8%, typically 0.4% to 0.6%, such as 0.4%, 0.5% or 0.6%.
  • the reaction time of the cross-linking step is typically from 1 to 6 hours, typically 2 to 5 hours, such as 2 to 4 hours, such as 2.5 to 3.5 hours, such as 3 hours.
  • the polymer support of immobilized microorganisms is washed in a wash ing step in aqueous medium, such as an alkaline buffer, such as a carbonate buffer or a phos phate buffer.
  • aqueous medium such as an alkaline buffer, such as a carbonate buffer or a phos phate buffer.
  • the buffer is typically at a pH of 7 to 10, typically pH 7.5 to 10, such as pH 8 to 10, such as 8.5, 9, 9.5 or 10, such as 8.5, 9 or 9.5, such as pH 9.
  • the washing step may comprise the use of an alkaline buffer or the use of an amine-rich solution such as a poly-ethyleneimine solution.
  • the washing step removes, at least in part the unreacted glutaraldehyde.
  • the washing step may further wash out, at least in part, the alginate.
  • the washing step may be repeated 1 to 5 times, typically 3 times.
  • At least some alginate may remain within the polymer support of immobi lized microorganisms.
  • the alginate entrapped within the polymer support of immobilized microor ganisms does not impact performance.
  • the polymer support of immobilized microorganisms, after multiple washing, may comprise alginate.
  • the polymer support of immobilized microorganisms does not comprise alginate.
  • polymer support of immobi lized microorganisms comprises polyvinyl alcohol polymeric chains covalently cross-linked via glutaraldehyde linkages.
  • the microorganisms are the microorganisms.
  • microorganisms immobilized within the polymer hydrogel used in the method of the invention may be selected from the group consisting of ammonia oxidizing microorganisms, nitrite oxidising microorganisms, sulfur oxidizing microorganisms, heterotrophic microorganisms and anaerobic ammonium-oxidizing microorganisms.
  • the microorganisms immobilized within the polymer hydrogel may be selected from the group consisting of Nitrosomonas spp., Nitrobacter spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp., Nitrotoga spp., Nitrospira spp.
  • the microorganisms immobilized within the polymer hydrogel may be selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkyl phenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomonas
  • Candidatus Kuenenia Candidatus Brocadia, Candidatus Anammoxoglobus,
  • Paracoccus pantotrophus was formerly named Paracoccus denitrificans or Thiosphaera pantotropha.
  • an advantageous feature of the microorganisms of the invention are microorganisms having a feature selected from the group consisting of a robust performance, activity at low temperatures, activity with low levels of additional carbon, selectivity for specific carbon sources.
  • the invention is directed to a polymer support of immobilized microorganisms wherein the polymer support comprises said microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises polyvinyl alcohol polymeric chains cross linked with glutaraldehyde; and water or an aqueous solution, wherein has at least 80% its maximum activity at below 20°C, such as below 15°C, such as below 10°C.
  • Polymeric support of immobilized microorganisms can be applied in multiple ways and e.g. in several configurations for RAS processes.
  • One method of application is in a mixed reactor where the beads are added with a bead fill of 10-30% w/v.
  • the beads are constantly mixed in the reactor together with the influent water and any other additives required to achieve the desired outcome such as e.g. alkalinity and dissolved oxygen for nitrification and Biochemical Oxygen Demand (BOD) for denitrification.
  • BOD Biochemical Oxygen Demand
  • Another method of application is in a fluidized bed reactor.
  • the beads are applied to a column with a bead fill of 40-80% w/v.
  • the influent water is pumped into the base of the column reactor and distributed so as to maintain a plug flow up through the layer of beads in the reactor.
  • the water is passed through the column with such a velocity so as to overcome the settling velocity of the beads and allow the beads to move slightly within the reactor.
  • Nitrogen removal from a water body typically involves the processes known as nitrification (oxidation of ammonium (NhV) to nitrite (NCV) and then nitrate (NO 3 ) by a community of nitrifying microorganisms) and denitrification (reduction of nitrate and/or nitrite to elemental nitrogen (N2) by a community of denitrifying microorganisms), thereby removing the nitrogen from the water.
  • nitrification oxidation of ammonium (NhV) to nitrite (NCV) and then nitrate (NO 3 ) by a community of nitrifying microorganisms
  • denitrification reduction of nitrate and/or nitrite to elemental nitrogen (N2) by a community of denitrifying microorganisms
  • the polymer support of immobilized microorganisms is a blend of robust, highly-active strains of nitrifying bacteria. This accelerates biofilter start up and restart in recirculating aquaculture systems (RAS).
  • RAS recirculating aquaculture systems
  • the polymer support of immobilized microorganisms of the invention were able to demonstrate the ability to achieve a high removal of ammonia within a few days, such as less than 100 days, or less than 90 days, while at the same time achieving a high conversion of ammonia to nitrate.
  • polymer support with immobilized microorganisms provides significant advantages over current technologies applied in biological nitrification processes due to the ability of the biobeads to achieve high levels of ammonia removal and conversion to nitrate within a very short period and under challenging environmental conditions. Total nitrogen concentrations were significantly reduced while using the polymer support of immobilized microorganisms of the invention.
  • the efficacy of biological nitrogen removal processes is herein enhanced through the application of high concentrations of nitrifying/denitrifying organisms in a biological treatment process.
  • the microroganisms of the invention may be ammonia-oxidizing bacteria and/or nitrite-oxidizing bacteria and may be combined with other bacteria, e.g., Bacillus such as a combination of the commercial product Prawn Bac PB-628 (product of Novozymes Biologicals), together with Enterobacter or Pseudomonas.
  • the nitrifying consortium may be formulated as a liquid, a lyophilized powder, or a biofilm, e.g., on bran or corn gluten.
  • the invention is furthermore directed to a method of denitrifying a water body comprising adding to the water body a polymer support as defined herein. Denitrification is the reduction of nitrate and/or nitrite to elemental nitrogen N 2 .
  • Nitrogen removal from a water body typically involves the processes known as nitrification (oxidation of ammonium (NhV) to nitrite (NO 2 ) and then nitrate (NO 3 ) by a community of nitrifying microorganisms) and denitrification (reduction of nitrate and/or nitrite to elemental nitrogen (N 2 ) by a community of denitrifying microorganisms), thereby removing the nitrogen from the water.
  • nitrification oxidation of ammonium (NhV) to nitrite (NO 2 ) and then nitrate (NO 3 ) by a community of nitrifying microorganisms
  • denitrification reduction of nitrate and/or nitrite to elemental nitrogen (N 2 ) by a community of denitrifying microorganisms
  • a further aspect is directed to a method of denitrifying a water body, or reducing the ammonium, nitrite and/or nitrate levels in a water body, comprising adding a polymer support of immobilized microorganisms, wherein the polymer support comprises said microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises polyvinyl alcohol polymeric chains cross linked with glutaraldehyde; and water or an aqueous solution, typically wherein the microorganism is selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus versutus and combinations thereof.
  • Denitrification occurs under anoxic/anaerobic conditions. Denitrification is the sequential process involving the dissimilatory reduction of one or both the ionic nitrogen oxides, nitrate (NO 3 ) and nitrite (NO 2 ) to gaseous nitrogen oxides, nitric oxide (NO), nitrous oxide (N 2 O) and finally reduce to the ultimate product, dinitrogen (N 2 ) thus removing biologically available nitrogen and returning it to the atmosphere.
  • Both nitrate and nitrite are fully converted to atmospheric nitrogen.
  • insufficient carbon sources, low dissolved oxygen (DO) concentrations and or environmental conditions may lead to improper denitrification and N 2 O accumulation and emissions.
  • One pathway to reduce N 2 O production is to select the right organisms for the denitrification process.
  • Denitrifiers with their facultative anaerobic traits perform denitrifying activities under the presence of oxygen driving an increase in N 2 O as a denitrification intermediate.
  • Many heterotrophic nitrifiers along with the oxidation of NH 3 can simultaneously perform aerobic denitrification. N 2 O is then generated. Accordingly, the judicious selection of denitrifiers is an important aspect to commercial success.
  • Polymer hydrogels comprising Paracoccus are a preferred embodiment in the denitrification of a water body.
  • Paracoccus pantotrophus grows aerobically with a large variety of carbon sources and with molecular hydrogen or thiosulfate as an energy source, and nitrate serves as electron acceptor under anaerobic conditions.
  • the denitrification properties of Paracoccus denitrificans render it a preferred microorganism.
  • Paracoccus denitrificans reduces nitrite to nitrogen gas while either Nitrosomonas eutropha or Nitrosomonas europaea oxidizes ammonia to nitrite, thus fuelling the former metabolism.
  • one embodiment comprises the combined use of Paracoccus denitrificans and either Nitrosomonas eutropha or Nitrosomonas europaea.
  • the denitrification properties of Paracoccus versutus such as Paracoccus versutus NN066467, also render it a preferred microorganism.
  • the denitrification process may suitably require the addition of a carbon source.
  • the microorganism is a denitrifier and denitrification is accompanied by the addition of a carbon source.
  • Suitable embodiments of this aspect of the invention comprise a carbon source selected from the group consisting of methanol, ethanol, acetate, acetic acid, glycerol, glycol, molasses, corn syrup, sucrose solutions, commercially available carbon sources, fer mented organic wastes, industrial wastewaters.
  • methanol, ethanol, acetate, acetic acid, glycerol commercially available carbon sources.
  • the carbon source is selected from the group consisting of methanol, glycerol or commercially available carbon sources.
  • a typical “water body” is an aquaculture farm, tank, raceway, creek, river, pool, pond, waste lagoon, paddy, lake, estuary or ocean. Accordingly, the water of the water body may be fresh water or salt water. Other stated, the water body may be of non saline or saline water. Otherwise stated, the water body may have low to high salinity, such as 0 to 10% salinity, as determined by level of NaCI.
  • the salinity of the water body is from 0% to 7%, such as 0.1% to 5%.
  • the salinity if the water body may be from 0% to 15%, such as 0.1% to 15%, such as 0.1% to 12%, such as 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12%.
  • Suitable microorganisms when the salinity of the water body is from 0% to 15%, such as 0.1% to 15%, such as 0.1% to 12%, such as 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12% may typically be selected from any of the microorganisms of the invention, are typically se lected the group consisting of a Paracoccus, Pseudomonas and Nitrosomas, such as Paracoccus pantotrophus, Paracoccus versutus.
  • the invention is, in one embodiment, directed to nitrogen removal, such as denitrification, in a water body with salinity from 0% to 15%, such as from 0.1% to 12%.
  • the water body typically has less than 50 ppt salinity, such as less than 30 ppt salinity, such as less than 25 ppt salinity, such as less than 20 ppt salinity, such as 20 ppt salinity, 19 ppt salinity, 18 ppt salinity, 17 ppt salinity, 16 ppt salinity, 15 ppt salinity, 14 ppt salinity, 13 ppt salinity, 12 ppt salinity, 11 ppt salinity, 10 ppt salinity, 9 ppt salinity, 8 ppt salinity, 7 ppt salinity, 6 ppt salinity, 5 ppt salinity, 4 ppt salinity, 3 ppt salinity, 2 ppt salinity, and 1 ppt salinity.
  • ppt salinity such as less than 30 ppt salinity, such as less than 25 ppt salinity, such as less than 20 ppt
  • the water body may be warm water body or cold water body.
  • the cold water temperature is 15°C or less, such as 12°C or less such as 10°C or less.
  • the warm water temperature is at least 15°C, such as at least 17°C, such as at least 20°C.
  • a method of treating aquatic or marine animals comprising: contacting a water body with a polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
  • polyvinyl alcohol polymeric chains consist of 500-3.000 monomer units and a molecular weight of 22.000 g/mol to 130.000 g/mol.
  • crosslink comprises monomeric, dimeric, oligomeric, or polymeric glutaraldehyde.
  • crosslinking density determined as the number of glutaraldehyde crosslinking units, is 8 to 25%, such as 10 to 20%, such as 12-20%, such as about 12%, 13%, 15%, 16%, 17%, 18%, 19% or 20%.
  • polymer hydrogel comprises from 60 to 99 wt% cross-linked polymeric material and 1 to 40 wt% water or aqueous medium, such as from 60 to 98 wt% cross-linked polymeric material and 2 to 40 wt% water or aqueous medium, such as from 65 to 95 wt% cross-linked polymeric material and 5 to 35 wt% water or aqueous medium, such as from 65 to 90 wt% cross-linked polymeric material and 10 to 35 wt% water or aqueous medium, such as from 65 to 85 wt% cross-linked polymeric material and 15 to 35 wt% water or aqueous medium, such as from 65 to 80 wt% cross-linked polymeric material and 20 to 35 wt% water or aqueous medium, or such as from 65 to 75 wt% cross-linked polymeric material and 25 to 35 wt% water or aqueous medium.
  • alginate is in monomer or polymeric form, entangled with cross-linked polyvinyl alcohol polymeric chains so as to form an interpenetrating polymer network.
  • the polymer sup port is porous.
  • the polymer support comprises micropores.
  • the method according to embodiment 10 or 11 wherein a portion of the polymer support comprises micropores with an average pore diameter of 5 to 40 microns, such as 5 to 30 microns, such as 5 to 20 microns.
  • a portion of the polymer support comprises micropores with an average pore diameter of 10 to 40 microns, such as 20 to 40 microns.
  • the polymeric support is spherical, oval, elliptical bead-shaped, oblong, cylindrical, or capsule-like in shape.
  • the polymeric support is spherical or bead-shaped having a diameter of 1 to 10 mm, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm.
  • the microbial load is 5 g/kg bead to 250 g/kg bead, typically 10 g/kg bead to 150 g/kg bead.
  • the immobilized microorganisms are selected from the group consisting of one or more sulfur oxidizing microorganisms, one or more ammonium oxidizing microorganisms, one or more nitrite oxidizing microorganisms, one or more denitrifying microorganisms, one or more anammox microorganisms and any mixtures thereof.
  • the immobilized microorganisms are selected from the group consisting of a mixed or pure culture of sulfur oxidizing microorganisms, a mixed or pure culture of ammonium oxidizing microor ganisms, a mixed or pure culture of nitrite oxidizing microorganisms, a mixed or pure culture of denitrifying microorganisms, a mixed or pure culture of anammox microorganisms and any mixtures thereof.
  • the immobilized microorganisms are a combination of two or more of the microorganisms selected from the group consisting of: ammonium oxidizing microorganisms, nitrite oxidizing microorganisms, denitrifying microorganisms and sulfur oxidizing microorganisms.
  • ammonia oxidizing microorganisms are selected from Nitrosomonas spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp
  • Paracoccus spp. is used as denitrifying and/or sulfur oxidizing microorganisms.
  • Paracoccus spp. is selected from the group consisting of: P. alcaliphilus, P. alkenifer, P. aminophilus, P. aminovorans, P. carotinifaciens, P. denitrificans, P. kocurii, P. marcusii, P. methylutens, P. pantotrophus, P. solventivorans, P. thiocyanatus, P. versutus, and any combination thereof.
  • the immobilized microorganisms are a combination of the ammonia oxidizing microorganisms Nitrosomonas spp., the nitrite oxidizing microorganisms Nitrobacter spp. and optionally the sulfur oxidizing microorganisms Paracoccus spp.
  • the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and a Nitrobacter, Nitrosomonas eutropha and a Paracoccus, Nitrosomonas eutropha and a Nictrobacter and a Paracoccus, Nitrosomonas europaea and a Nitrobacter, Nitrosomonas europaea and a Paracoccus, and of Nitrosomonas europaea and a Nitrobacter and a Paracoccus.
  • the immobilized microorganisms are selected from the group consisting of a combination of a Nitrosomonas and Nitrobacter winogradskyi, a Paracoccus and Nitrobacter winogradskyi, and, a Nitrosomonas and a Paracoccus and Nitrobacter winogradskyi.
  • the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus pantotrophus, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus Versutus, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus pantotrophus, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus Versutus, preferably a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi.
  • the immobilized microorganisms are selected from the group consisting of Nitrosomonas spp., Nitrobacter spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp., Nitrotoga spp., Nitrospira spp.
  • Pseudomonas spp. ;, Paracoccus spp., Hyphomicrobium spp., Castellaniella spp., Janthinobacterium spp., Acidovorax spp., Aeromonas spp., Cellulomonas spp., Buttiauxella spp., Microvirgula spp., Klebsiella spp., Shewanella spp., Pelosinus spp., Variovorax spp., Hydrogenophaga spp., Raoultella spp., Bacillus spp., Achromobacter spp., Ochrobactrum spp., Flavobacterium spp., and Delftia and combinations thereof.
  • the immobilized microorganisms are selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkylphenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomonas
  • Flavobacterium banpakuense, Buttiauxella agrestis, Buttiauxella noackiae,
  • Achromobacter denitrificans Pelosinus fermentans, Variovorax dokdonensis, Hydrogenophaga bisanensis, Raoultella terrigena, Delftia lacustris, Shewanella putrefaciens, Acidovorax soli, Hyphomicrobium denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, Microvirgula aerodenitrificans, Candidatus Kuenenia, Candidatus Brocadia, Candidatus Anammoxoglobus,
  • Candidatus Jettenia Candidatus Scalindua, and combinations thereof.
  • the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus pandtodrophus, Castellaniella defragans, Pseudomonas proteolytica, Paracoccus versutus, Paracoccus denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, and combinations thereof.
  • the immobilized microorganisms are selected from the group consisting of Nitrosomonas eutropha , Nitrobacter winogradskyi and combinations thereof.
  • the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus versutus and combinations thereof.
  • the immobilized microorgansims comprise Castellaniella defragans.
  • the im mobilized microorgansims comprise Pseudomonas proteolytica.
  • the method comprises mixing the polymer support into the water of an aquaculture pond such as aquaculture, farm, pool, pond, paddy, lake, estuary, ocean, or combinations thereof.
  • an aquaculture pond such as aquaculture, farm, pool, pond, paddy, lake, estuary, ocean, or combinations thereof.
  • the polymer support is combined with the water in the water body at a bead load of 60% to 80% w/v, such as 65% to 75% w/v, such as 70% to 75% w/v of the water.
  • the polymer support is combined with the water in the water body at a bead load of 5% to 30% w/v or 60% to 80% w/v of the water.
  • the water body comprises from 0.5 to 10 mg/I of ammonia, such as 1 to 5 mg/I ammonia, such as 1 to 2 mg/I of ammonia.
  • a polymer support of immobilized microorganisms for treating aquatic or ma rine animals comprising: contacting a water body with said polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
  • crosslink comprises monomeric, dimeric, oligomeric, or polymeric glutaraldehyde.
  • crosslinking density determined as the number of glutaraldehyde crosslinking units, is 8 to 25%, such as 10 to 20%, such as 12-20%, such as about 12%, 13%, 15%, 16%, 17%, 18%, 19% or 20%.
  • polymer hydrogel comprises from 60 to 99 wt% cross-linked polymeric material and 1 to 40 wt% water or aqueous medium, such as from 60 to 98 wt% cross-linked polymeric material and 2 to 40 wt% water or aqueous medium, such as from 65 to 95 wt% cross-linked polymeric material and 5 to 35 wt% water or aqueous medium, such as from 65 to 90 wt% cross-linked polymeric material and 10 to 35 wt% water or aqueous medium, such as from 65 to 85 wt% cross-linked polymeric material and 15 to 35 wt% water or aqueous medium, such as from 65 to 80 wt% cross-linked polymeric material and 20 to 35 wt% water or aqueous medium, or such as from 65 to 75 wt% cross-linked polymeric material and 25 to 35 wt% water or aqueous medium.
  • alginate is in monomer or polymeric form, entangled with cross-linked polyvinyl alcohol polymeric chains so as to form an interpenetrating polymer network.
  • a portion of the polymer support comprises micropores with an average pore diameter of 5 to 40 microns, such as 5 to 30 microns, such as 5 to 20 microns.
  • a portion of the polymer support comprises micropores with an average pore diameter of 10 to 40 microns, such as 20 to 40 microns.
  • any one of embodiments 50 to 64 wherein the microbial load is 5 g/kg bead to 250 g/kg bead, typically 10 g/kg bead to 150 g/kg bead.
  • the immobilized microor ganisms are selected from the group consisting of a mixed or pure culture of sulfur oxi dizing microorganisms, a mixed or pure culture of ammonium oxidizing microorganisms, a mixed or pure culture of nitrite oxidizing microorganisms, a mixed or pure culture of denitrifying microorganisms, a mixed or pure culture of anammox microorganisms and any mixtures thereof.
  • the immobilized microorganisms are a combination of two or more of the microorganisms selected from the group consisting of: ammonium oxidizing microorganisms, nitrite oxidizing microorganisms, denitrifying microorganisms and sulfur oxidizing microorganisms.
  • ammonia oxidizing microorganisms are selected from Nitrosomonas spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp
  • the nitrite oxidizing microorganisms are selected from Nitrobacter spp., Nitrococcus spp., Nitrospira spp., and Nitrospina spp.
  • Paracoccus spp. is selected from the group consisting of: P. alcaliphilus, P. alkenifer, P. aminophilus, P. aminovorans, P. carotinifaciens, P. denitrificans, P. kocurii, P. marcusii, P. methylutens, P. pantotrophus, P. solventivorans, P. thiocyanatus, P. versutus, and any combination thereof.
  • the immobilized microorganisms are a combination of the ammonia oxidizing microorganisms Nitrosomonas spp., the nitrite oxidizing microorganisms Nitrobacter spp. and optionally the sulfur oxidizing microorganisms Paracoccus spp.
  • any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and a Nitrobacter, Nitrosomonas eutropha and a Paracoccus, Nitrosomonas eutropha and a Nictrobacter and a Paracoccus, Nitrosomonas europaea and a Nitrobacter, Nitrosomonas europaea and a Paracoccus, and of Nitrosomonas europaea and a Nitrobacter and a Paracoccus.
  • the immobilized microorganisms are selected from the group consisting of a combination of a Nitrosomonas and Nitrobacter winogradskyi, a Paracoccus and Nitrobacter winogradskyi, and, a Nitrosomonas and a Paracoccus and Nitrobacter winogradskyi.
  • any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus pantotrophus, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus Versutus, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus pantotrophus, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus Versutus, preferably a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi.
  • any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas spp., Nitrobacter spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp., Nitrotoga spp., Nitrospira spp.
  • Pseudomonas spp. ;, Paracoccus spp., Hyphomicrobium spp., Castellaniella spp., Janthinobacterium spp., Acidovorax spp., Aeromonas spp., Cellulomonas spp., Buttiauxella spp., Microvirgula spp., Klebsiella spp., Shewanella spp., Pelosinus spp., Variovorax spp., Hydrogenophaga spp., Raoultella spp., Bacillus spp., Achromobacter spp., Ochrobactrum spp., Flavobacterium spp., and Delftia and combinations thereof.
  • the immobilized microorganisms are selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkylphenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomon
  • Flavobacterium banpakuense, Buttiauxella agrestis, Buttiauxella noackiae,
  • Achromobacter denitrificans Pelosinus fermentans, Variovorax dokdonensis, Hydrogenophaga bisanensis, Raoultella terrigena, Delftia lacustris, Shewanella putrefaciens, Acidovorax soli, Hyphomicrobium denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, Microvirgula aerodenitrificans, Candidatus Kuenenia, Candidatus Brocadia, Candidatus Anammoxoglobus,
  • Candidatus Jettenia Candidatus Scalindua, and combinations thereof.
  • the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus pandtodrophus, Castellaniella defragans, Pseudomonas proteolytica, Paracoccus versutus, Paracoccus denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, and combinations thereof.
  • any one of embodiments 66 to 70, wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas eutropha , Nitrobacter winogradskyi and combinations thereof.
  • the use according to any one of embodiments 66 to 70, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, preferably a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi, and combinations thereof.
  • the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus versutus and combinations thereof.
  • the immobi lized microorgansims comprise Pseudomonas lini.
  • the immobi lized microorgansims comprise Paracoccus pantotrophus.
  • the immobilized microorgansims comprise Castellaniella defragans.
  • the immobi lized microorgansims comprise Pseudomonas proteolytica.
  • the immobilized microorgansims comprise Paracoccus versutus.
  • the immobi lized microorgansims comprise Paracoccus denitrificans.
  • the immobi lized microorgansims comprise Nitrosomonas eutropha.
  • the water body comprises from 0.5 to 10 mg/I of ammonia, such as 1 to 5 mg/I ammonia, such as 1 to 2 mg/I of ammonia.
  • a polymer support of immobilized microorganisms for use in a method of treating aquatic or marine animals comprising: contacting a water body with a polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
  • the polymer support of immobilized microorganisms according to embodiment 99 wherein the polyvinyl alcohol is cross linked with glutaraldehyde.
  • the polymer support of immobilized microorganisms according to any one of embodiments 99 to 102, wherein the crosslink comprises monomeric, dimeric, oligomeric, or polymeric glutaraldehyde.
  • polymer hydrogel comprises from 60 to 99 wt% cross-linked polymeric material and 1 to 40 wt% water or aqueous medium, such as from 60 to 98 wt% cross-linked polymeric material and 2 to 40 wt% water or aqueous medium, such as from 65 to 95 wt% cross-linked polymeric material and 5 to 35 wt% water or aqueous medium, such as from 65 to 90 wt% cross-linked polymeric material and 10 to 35 wt% water or aqueous medium, such as from 65 to 85 wt% cross-linked polymeric material and 15 to 35 wt% water or aqueous medium, such as from 65 to 80 wt% cross-linked polymeric material and 20 to 35 wt% water or aqueous medium, or such as from 65 to 75 wt% cross-linked polymeric material and 25 to 35 wt% water or aqueous medium
  • the polymer support of immobilized microorganisms according to any one of embodiments 99 to 105 wherein alginate is in monomer or polymeric form, entangled with cross-linked polyvinyl alcohol polymeric chains so as to form an interpenetrating polymer network.
  • the polymer support of immobilized microorganisms according to embodiment 108 comprising one or more macropores in the central volume of support and micropores.
  • the polymer support of immobilized microorganisms according to any one of embodiments 99 to 113, wherein the microbial load is 5 g/kg bead to 250 g/kg bead, typically 10 g/kg bead to 150 g/kg bead.
  • the polymer support of immobilized microorganisms according to any one of embodiments 99 to 115, wherein the immobilized microorganisms are selected from the group consist ing of a mixed or pure culture of sulfur oxidizing microorganisms, a mixed or pure culture of ammonium oxidizing microorganisms, a mixed or pure culture of nitrite oxidizing micro organisms, a mixed or pure culture of denitrifying microorganisms, a mixed or pure culture of anammox microorganisms and any mixtures thereof.
  • the polymer support of immobilized microorganisms according to any one of embodiments 99 to 116, wherein the immobilized microorganisms are a combination of two or more of the microorganisms selected from the group consisting of: ammonium oxidizing microorganisms, nitrite oxidizing microorganisms, denitrifying microorganisms and sulfur oxidizing microorganisms.
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 117, wherein the ammonia oxidizing microorganisms are selected from Nitrosomonas spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp,
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 119, wherein Paracoccus spp. is used as denitrifying and/or sulfur oxidizing microorganisms.
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 121, wherein the immobilized microorganisms are a combination of the ammonia oxidizing microorganisms Nitrosomonas spp., the nitrite oxidizing microorganisms Nitrobacter spp. and optionally the sulfur oxidizing microorganisms Paracoccus spp.
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and a Nitrobacter, Nitrosomonas eutropha and a Paracoccus, Nitrosomonas eutropha and a Nictrobacter and a Paracoccus, Nitrosomonas europaea and a Nitrobacter, Nitrosomonas europaea and a Paracoccus, and of Nitrosomonas europaea and a Nitrobacter and a Paracoccus.
  • the polymer support of immobilized microorganisms according to embodiments 115 to 121, wherein the immobilized microorganisms are selected from the group consisting of a combination of a Nitrosomonas and Nitrobacter winogradskyi, a Paracoccus and Nitrobacter winogradskyi, and, a Nitrosomonas and a Paracoccus and Nitrobacter winogradskyi.
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 121, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus pantotrophus , a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus Versutus, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus pantotrophus, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus Versutus, preferably a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi.
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas spp., Nitrobacter spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp., Nitrotoga spp., Nitrospira spp.
  • Pseudomonas spp. ;, Paracoccus spp., Hyphomicrobium spp., Castellaniella spp., Janthinobacterium spp., Acidovorax spp., Aeromonas spp., Cellulomonas spp., Buttiauxella spp., Microvirgula spp., Klebsiella spp., Shewanella spp., Pelosinus spp., Variovorax spp., Hydrogenophaga spp., Raoultella spp., Bacillus spp., Achromobacter spp., Ochrobactrum spp., Flavobacterium spp., and Delftia and combinations thereof.
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkylphenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica,
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus pandtodrophus, Castellaniella defragans, Pseudomonas proteolytica, Paracoccus versutus, Paracoccus denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, and combinations thereof.
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 wherein the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus versutus and combinations thereof.
  • the polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 127 to 128, wherein the immobilized microorgansims comprise Nitrobacter Winogradsky.
  • the polymer support of immobilized microorganisms according to any one of embodiments 99 to 140, wherein the polymer support of immobilized microorganisms comprises mixing the polymer support into the water of an aquaculture pond such as aquaculture, farm, pool, pond, paddy, lake, estuary, ocean, or combinations thereof.
  • an aquaculture pond such as aquaculture, farm, pool, pond, paddy, lake, estuary, ocean, or combinations thereof.
  • the polymer support of immobilized microorganisms according to any one of embodiments 99 to 141, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 80% w/v, such as 10% to 80% w/v, such as 15% to 80% w/v of the water.
  • the polymer support of immobilized microorganisms according to embodiment 142 wherein the polymer support is combined with the water in the water body at a bead load of 60% to 80% w/v, such as 65% to 75% w/v, such as 70% to 75% w/v of the water.
  • the polymer support of immobilized microorganisms according to embodiment 142 wherein the polymer support is combined with the water in the water body at a bead load of 5% to 30% w/v or 60% to 80% w/v of the water.
  • a method for the denitrification of a water body comprising adding the polymer support of immobilized microorganisms of the invention.
  • Example 1 Paracoccus containing polyvinyl alcohol-glutaraldehyde polymer support
  • the dropped polymer/microbe mixture immediately gelated when hitting the CaCh bath.
  • the spherical products were separated using a small sieve and washed lightly with tap water before transferring the preformed product back into a glass beaker.
  • a second cross link solution was added containing 0.25g glutaraldehyde (10% solution), 1.7g sulfuric acid (30% solution), 10g of Na 2 S0 4 and 38.05g water.
  • This second cross link solution was heated to 40°C and was kept at this temperature during bead cross linking. After 3 hours of curing the beads were separated from the cross-link solution and washed with a tris buffer for 30 min. After washing, the beads were transferred into cell free water for storage.
  • Example 2 Nitrosomonas eutropha and Nitrobacter winogradskyi- containing polyvinyl alcohol-glutaraldehyde polymer support
  • the dropped polymer/microbe mixture immediately gelated when hitting the CaCI2 bath.
  • the spherical products were separated using a small sieve and washed lightly with tap water before transferring the preformed product back into a glass beaker.
  • a second cross link solution was added containing 1g glutaraldehyde (10% solution), 1.7g sulfuric acid (30% solution), 10g of Na 2 S0 4 and 37.3g water.
  • This second cross link solution was heated to 40°C and was kept at this temperature during Bead cross linking. After 3 hours of curing the beads were separated from the cross-link solution and washed with a tris buffer for 30 min. After washing, the Beads were transferred into cell free water for storage.
  • Example 3 Effect of microbe encapsulation on protecting nitrifying microbes from the effects of chemical disinfection
  • a key reason for encapsulating microbes in a polymeric biobead is the expec tation that the encapsulation in a polymeric bead will provide an increased level of protection to the microbes within from potentially toxic components in the water being treated when compared to microbes in suspended or fixed film growth in the water.
  • Biofilters in RAS processes are regu larly exposed to chemical disinfectants in order to ensure the biofilter does not become a biosecurity hazard as batches of fish are transferred in and out of the process.
  • testing the effect of a chemical disinfectant on the activity of nitrifying biobeads and their ability to recover after exposure to this disinfectant will provide a suitable model for demonstrating the efficacy of biobeads at protecting the microbes housed within.
  • Biobeads applied o The biobeads utilized in the experiment were of two types
  • Alkalinity was supplied in a ratio of 10:1 compared to NH4-N. Alkalinity was supplied as CaC03
  • Table 1 Operational conditions for the reactors from Day 1 - 78 o On day 63, the reactors were emptied of water. The reactor was then refilled with a 25 mg/L solution of sodium hypochlorite. The beads were then soaked in this solution for 4 hours. Afterwards, the reactors were drained again and normal op eration was restarted - Analytical methods o Sampling was undertaken two (2) to three (3) times per week by a certified ana lytical laboratory o Standard methods known to one skilled in the art were used to measure the com ponents of the feed water and the water coming from the reactors listed in Table 2
  • Table 2 List of components analysed in the feed and effluent water from the reactors
  • the NH4-N concentration had fallen to 1.2 mg NH4-N/L while in the blank bi obead reactor it had fallen to only 2.6 mg NH4-N/L.
  • the blank biobeads showed some level of NH4-N removal after disinfection, most of the NH4-N was only partially nitrified and converted to N02-N rather than N03-N. Such a peak in N02-N post-disinfection was not meas ured in the seeded biobead reactor.
  • microbeads were able to provide a protec tive environment for microbes that were integrated as part of the production process of the bi obeads. While microbes were able to colonize the outer surfaces of the blank biobeads and pro vide nitrification activity, these microbes were not protected against exposure to disinfectants, toxins or inhibitors as those microbes that were integrated into the biobeads.
  • RAS aquaculture processes often apply chemical disinfection as a means of preventing biosecu rity risks during the production of the fish in the process.
  • the ability of the seeded biobead reactor to re-start after disinfection and deliver water treatment that would allow the RAS to be re-stocked with fish presents a significant benefit for the producer as the lag time between production batches could be reduced to 24 hours. This is in contrast to the blank biobeads (or other biofilter technology that uses biofilms) where the nitrification process did not recover within the 14 days after disinfection and therefore not allowing the RAS system to restart fish production.
  • Example 4 Effect microbe encapsulation on protecting denitrification microbes from the effects of exposure to free chlorine
  • a key reason for encapsulating microbes in a polymeric biobead is the expectation that the en capsulation in a polymeric bead will provide an increased level of protection to the microbes within from potentially toxic components in the water being treated when compared to microbes in sus pended or fixed film growth in the water.
  • Biofilters in RAS processes are regularly exposed to chemical disinfectants in order to ensure the biofilter does not become a biosecurity hazard as batches of fish are transferred in and out of the process. Therefore, testing the effect of a chemical disinfectant on the activity of nitrifying biobeads and their ability to recover after exposure to this disinfectant, will provide a suitable model for demonstrating the efficacy of biobeads at protecting the microbes housed within.
  • biobeads seeded with the denitrifying micro-organism Paracoccus Pantotrophus can recover activity to pre-exposre levels within a short period of time Compare the performance of biobeads seeded with microbes to blank biobeads where denitrification activity comes only from microbes on the surface of the biobeads
  • Biobeads applied o The biobeads utilized in the experiment were of two types
  • ⁇ BOD was supplied as a 50:50 (on a BOD basis) mixture of sodium ace tate and acetic acid
  • Table 4 List of components analysed in the feed and effluent water from the reactors
  • FIG. 3 illustrates the concentration of total ni trogen (nitrate + nitrite) in both the influent and effluent of both R37 and R38.
  • Figure 4 demon strates the specific total nitrogen (TN) removal rate for the biobeads in both R37 and R38.
  • the dotted lines at day 14 indicates where the sodium hypochlorite started being added to the reactor feed, with the second dashed lines at day 17 indicating where the addition of sodium hypochlorite stopped.
  • Biobeads in both reactors were negatively impacted by the addition of the chlorine, with increased levels of total nitrogen (TN) in the effluent of both reactors (Figure 3).
  • TN total nitrogen
  • the blank beads in R37 lost all denitrification activity, with this activity not recovering even after stopping the exposure to chlorine at day 17.
  • the seeded beads only lost approximately 12% of their TN removal activity after one day of exposure, and 37% of activity after three days of exposure.
  • the blank biobeads in R37 did not recover any activity after 4 days.
  • the seeded biobeads in R38 recovered a significant proportion of the activity lost during the exposure to chlo rine, with and expectation that a full recovery to pre-exposure levels of activity would occur within seven days.
  • biobeads were able to provide a protec tive environment for microbes that had been integrated as part of the production process of the biobeads. While microbes were able to colonize the outer surfaces of blank (or not seeded) bi obeads and provide denitrification activity, these microbes were not protected against exposure to toxins or inhibitors like those that were integrated into the biobeads.
  • Biobeads applied o The biobeads utilized in the experiment were of two types
  • ⁇ BOD was supplied as a 50:50 (on a BOD basis) mixture of sodium ace tate and acetic acid
  • Table 7 List of components analysed in the feed and effluent water from the reactors Compound Name Unit of measurement
  • FIG. 5 illustrates the concentration of total ni trogen (nitrate + nitrite) in both the influent and effluent of both R37 and R38.
  • the dotted line at day 45 indicates where the biobead loading (% w/v) was increased from 10% to 20%.
  • the seeded biobeads demonstrated an ability to remove TN immediately, while it took the blank beads at least 10 days to demonstrate any TN removal activity. However, after 27 days of operation, both the blank and seeded biobead reactors were achieving similar results.
  • the effluent concentration was approximately 6 mg-N/L, which was at least 3x higher than was the objective.
  • more biobeads were added to each reactor to increase the bead load from 10% to 20% w/v. This increase in bead load in the reactors led to an immediate drop in the effluent concen tration of TN, most markedly in the seeded biobead reactor (R38).
  • the seeded biobead reactor (R38) had achieved an effluent concentration of ⁇ 2 mg-N/L, and had largely maintained this level for the following 65 days of operation, illustrating how stable a biobead process can operate in contrast to a conventional biofilm type biofilter (represented by R37).
  • the specific activity (mg-N removed per kilogram of biobeads per hour(mg-N/kg.hr)) of the bi obeads closely reflects the effluent TN concentrations achieved and is illustrated in Figure 6.
  • the specific TN loading rate and biobeads activities for R37 and R38 are illustrated in Figure 7. Again, it is clear that the seeded biobeads in R38 demonstrated a sharply increasing rate of TN removal from day 1. This rate of TN removal stabilized after approximately 20 days and remained consistent at 70-80 mg-N/kg.hr from day 31 to day 129.
  • the increase in biobead load in the reac tor did not impact the specific activity of the biobeads in R38 as the amount of TN removed in the reactor increased sharply with the addition of the extra biobeads (as shown in Figure 6).
  • the blank biobeads in R37 did not demonstrate significant activity until approximately day 20, and then remained steady at that level of 20 mg-N/kg.hr until day 66. At this point, the activity in creased steadily to a maximum of 60 mg-N/kg.hr on day 90. This increasing level of activity is likely caused by the increased surface area that adding the extra biobeads on day 45 provided to colonizing dentifiers in the process.
  • the lag in increasing activity is due to the time taken for the colonizing denitrifiers to grow and populate the new beads.
  • the increased bi obead load also leads to less turbulence in the reactor, which means the biobeads were not con tacting each other with the same energy as when the biobead load was only 10% w/v. This results in more stable growth conditions for surface colonizing biofilms.
  • nitrate removal is the primary objective of denitrification biofilter processes, this must be achieved with minimal production of nitrite (N02) during the process.
  • N02 nitrite
  • fish can tolerate very high concentrations of nitrate, nitrite should be maintained at a level of ⁇ 0.5 mg N02-N/L to avoid impacting fish health. Therefore, the formation of nitrite as a by-product in the nitrate removal process was monitored in both reactors throughout the trial. The proportion of the N03 removed that was converted to nitrite and left the reactor in the effluent is illustrated in Figure 7. The results indicate that initially in both reactors the denitrification process often stalled at nitrite.
  • Table 8 Average TSS concentrations in the reactors over the 129 day period of continuous op eration
  • the biobeads in both reactors were sampled for fluorescent in-situ hybridization (FISH) analysis to identify whether the microbes originally seeded in the biobeads were still present and dominant and whether other microbes had been able to colonise the biobeads during operation.
  • FISH fluorescent in-situ hybridization
  • biobeads in a completely mixed reactor results in first order kinetics of reaction between the increasingly low concentration of nitrate and the microbes in the biobeads limiting the HRT that can be applied. This results in longer HRTs to achieve lower nitrate effluent concen trations, thereby increasing the reactor size and therefore capital and operational costs for apply ing biofilters.
  • One pathway to overcome the first order kinetics limitations is to apply the biobeads in a plug-flow type reactor. At the end of the trial outlined above, the biobeads from R38 were transferred to a fluidized bed type reactor. The operational specifics of this reactor is shown in Table 10.
  • the bead load in the reactors was required to be at least 20% w/v in order to overcome the first order kinetics of the continuously stirred reactor system.
  • the limitations of the first order kinetics could be overcome, and similar levels of performance were achieved with only 1 ⁇ 4 of the HRT used in the completely mixed reactors.
  • the biobeads produced TSS, indicating that the biomass seeded into the biobeads is not irreversibly retained in the biobeads, although the mass of biomass produced was significantly lower than could be expected from an activated sludge process treating the same water.
  • TN total nitrogen
  • the process of denitrification requires the oxidation of a carbon source to act as an electron donor to drive the conversion of nitrate (N03) and nitrate (N02) to nitrogen gas (N2).
  • Common carbon sources used for denitrification are organic materials in the RAS water, and external carbon sources such as methanol, ethanol, glycol, molasses and other easily bio-degradable substances. However, the use of these external carbon sources rep resents a potentially significant cost for TN removal. Therefore, the integration of a denitrifying microbe that can consume less carbon per unit of TN removed represents a significant commer cial opportunity as it could significantly reduce the operational expenses of a biological TN re moval system in a RAS process.
  • Biobeads applied o The biobeads utilized in the experiment were of two types
  • Nitrate was supplied as NaN03 ⁇ BOD was supplied as a 50:50 (on a BOD basis) mixture of sodium ace tate and acetic acid
  • Table 12 List of components analysed in the feed and effluent water from the reactors
  • the reactors were started up using identical operational conditions as outlined in Table 13.
  • the concentration of total nitrogen (TN) was monitored in the influent and effluent of the reactors (Figure 9A).
  • the two reactors had an effluent TN concentration that was decreasing at a similar rate over time.
  • the biobeads in R45 were able to achieve a lower effluent TN concentration compared to the biobeads in R38.
  • the biobeads in R45 demonstrated from an early stage a significantly higher rate of TN removal, eventually achieving a rate that was approximately 50% higher than the biobeads in R38.
  • Table 13 Average consumption of COD per unit of TN removed in each of the reactors R38 and R45 over a period of 25 days of continuous operation
  • Ammonia is a highly problematic pollutant in RAS as it is toxic to fish and aquatic animals.
  • a key objective of a RAS biofilter is the removal of ammonia from the water to maintain a high level of fish health and productivity in the RAS. Normally this is done using biological water treatment methods, where nitrifying bacteria are exploited to convert ammonia to nitrite and then nitrate.
  • Nitrification bacteria are generally recognized as being sensitive to process changes and relatively slow growing compared to other bacteria in the water treatment process. For example, in an MBBR RAS process, it can take up to 100 days to achieve enough nitrification activity to allow for fish stocking in the RAS. This represents a significant loss in potential productivity.
  • nitrification processes can be relatively easily impacted negatively by tox ins, changing process conditions or poor operation leading to a loss of nitrification capacity.
  • the recovery time after such an upset may be over a period of weeks rather than days.
  • the only biomass in the process would be nitrifiers as this is what is being added to biobeads, allowing for a significant increase in the concentration of nitrifiers in the treatment process and increasing the oxygen utilization efficiency of the process as only nitrifiers would be consuming dissolved oxygen.
  • the biobeads also provide an environment where the nitrifiers are protected from process changes, toxins and other shocks that would normally have a strong neg ative impact on the nitrification capacity of the treatment process.
  • Biobeads applied o The biobeads utilized in the experiment were of two types ⁇ M06 - Blank biobeads without microbes
  • Alkalinity was supplied in a ratio of 10:1 compared to NH4-N. Alkalinity was supplied as CaC03
  • Table 15 List of components analysed in the feed and effluent water from the reactors
  • the seeded biobeads were able to demonstrate an ability to achieve 60% removal of ammonia within 5 days of operation, while at the same time achieving over 85% conversion of ammonia to nitrate compared to a system relying solely on a naturally occurring nitrification biofilm.

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Abstract

The present invention relates to polymer supports of immobilized microorganisms for use in a method of treating aquatic or marine animals.

Description

METHOD OF TREATING AQUATIC OR MARINE ANIMALS USING POLYMER SUPPORTS
FIELD OF THE INVENTION
The present invention relates to polymer supports of immobilized microorganisms for use in a method of treating aquatic or marine animals, including treating the water body of aquatic or ma rine animals.
BACKGROUND OF THE INVENTION
In aquaculture systems, accumulation of high concentrations of ammonia and nitrite is toxic to aquatic organisms such as fish and pose a problem. The problem may be prevented using recir culating aquaculture systems (RAS) that use biofiltration to convert ammonia (NH4+ and NH3) excreted by the fish into nitrate and then nitrate into nitrogen gas.
Nitrifying bacteria are chemoautotrophs that convert ammonia into nitrite then nitrate. A biofilter provides a substrate for the bacterial community, which results in thick biofilm growing within the filter. Water is passed through the biofilter, and ammonia is utilized by the bacteria for energy. Nitrate is less toxic than ammonia and therefore does not impact the health of the fish as acutely as ammonia. The nitrate in the system can be removed by a denitrifying biofilter or by water replacement. Stable environmental conditions and regular maintenance are required to ensure the biofilter is operating efficiently, both of which are not always available in an operating RAS process.
Also high concentrations of hydrogen sulfide (H2S), that typically may be generated in anaerobic zones in aquaculture systems together with e.g. ammonia and methane, is toxic to aquatic organ isms. This problem may be prevented by chemical oxidizers, such as ozone, chlorine dioxide, and hydrogen, calcium, or magnesium peroxide, by biological sulfide oxidation by bacteria or by water flushing.
For RAS suppliers and producers, existing biofilter technology comes with limitations. Biofilters are difficult to start up, operate and restart after disinfection because there is little chance of con trolling the identity, abundance or activity of native nitrifying bacteria in the competitive environ ment that is an open biofilter unit process.
Most wild-type bacteria normally found in biofilters do not contribute to nitrification performance, and they sometimes create off-flavors in the fish. While off-flavors can be purged from fish before processing, this results in the fish losing mass in the purge tanks, thereby negatively impacting the profitability of the producer. For producers, this lack of control results in slow start-ups and restarts, headaches for operators, and sometimes off-flavor fish which impacts production plan ning, predictability and profitability. There is still a need for an optimized method of treating aquatic or marine animals that can main tain an optimal water body for the aquaculture systems while decreasing the risk of biofilter failure and increasing the profitability of the business.
SUMMARY OF THE INVENTION
The invention provides a polymer support of immobilized microorganisms for use in a method of treating aquatic or marine animals wherein the method comprises contacting a water body with a polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydro gel comprises a cross-linked polymeric material and water or an aqueous medium; and wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
The polyvinyl alcohol of the polymer support of immobilized microorganisms is in one embodiment cross linked with glutaraldehyde. In another or further embodiment, the polymer support of immo bilized microorganisms is porous.
In a further embodiment, the polymer support of immobilized microorganisms are selected from the group consisting of one or more sulfur oxidizing microorganisms, one or more ammonium oxidizing microorganisms, one or more nitrite oxidizing microorganisms, one or more denitrifying microorganisms, one or more anammox microorganisms and any mixtures thereof.
The invention furthermore provides a method of treating aquatic or marine animals or treating a water body comprising aquatic or marine animals, comprising contacting the water body with a polymer support of immobilized microorganisms, wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; and typically wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; and wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate. The invention furthermore is directed to the use of a polymer support of immobilized microorganisms for treating aquatic or marine animals, wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; and wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
A further aspect of the invention is directed to a method of reducing the ammonia, nitrate, or nitrite levels in a water body comprising the use of the polymer support of immobilized microorganisms of the invention. A further aspect is related to the denitrification of a water body comprising the addition of a polymer support of immobilized microorganisms of the invention. BRIEF DESCRIPTION OF DRAWINGS
Figure 1 illustrates, as shown in Example 3, NH4-N concentrations in the influent (NH4 in) and effluent for Blank and Seeded biobead reactors over a period of 27 days of continuous operation. The chlorine disinfection soaking is indicated with a dotted line.
Figure 2 illustrates, as shown in Example 3, N02-N concentrations in the influent (N02 in) and effluent for Blank and Seeded biobead reactors over 27 days of continuous operation. The chlo rine disinfection soaking is indicated with a dotted line.
Figure 3 illustrates, as shown in Example 4, TN concentrations in the influent (N03 in) and effluent for reactors R37 (blank biobeads) and R38 (seeded biobeads) over a period of 21 days of contin uous operation. The period of chlorine addition is shown with dotted and dashed lines.
Figure 4 illustrates, as shown in Example 4, Specific TN removal activity for the biobeads in re actors R37 and R38 over 21 days of continuous operation. The period of chlorine addition is shown with dotted and dashed lines.
Figure 5 illustrates, as shown in Example 5, TN concentrations in the influent (feed) and effluent for reactors R37 (blank biobeads) and R38 (seeded biobeads) over a period of 129 days of con tinuous operation.
Figure 6 illustrates, as shown in Example 5, Specific TN loading rate and removal activity for the biobeads in reactors R37 and R38 over 129 days of continuous operation.
Figure 7 illustrates, as shown in Example 5, Nitrite production as a proportion of the nitrate re moved in each reactor over a period of 129 days of continuous operation.
Figure 8 illustrates, as shown in Example 5, Nitrate and nitrite effluent concentrations after pass ing through a biobead plug-flow reactor system. The performance of the biobeads in a plug-flow system is similar to that achieved in the previous completely mixed reactor system, but with only ¼ of the HRT.
Figure 9 illustrates, as shown in Example 6, (A) TN concentrations in the influent (feed) and effluent for reactors R38 and R45 over a period of 25 days of continuous operation; (B) Specific TN removal activity for the biobeads in reactors R38 and R45 over 25 days of continuous opera tion.
Figure 10 illustrates, as shown in Example 6, Nitrite production as a proportion of the nitrate re moved in each reactor over a period of 25 days of continuous operation.
Figure 11 illustrates, as shown in Example 7, Specific NH4 loading rate and removal activity for the biobeads in reactors M06 and M07 over 21 days of continuous operation. Figure 12 illustrates, as shown in Example 7, NH4 concentrations in the influent (feed) and effluent for reactors M06 (blank biobeads) and M07 (seeded biobeads) over a period of 21 days of con tinuous operation.
Figure 13 illustrates, as shown in Example 7, Nitrate production as a proportion of the ammonia removed in each reactor over a period of 25 days of continuous operation.
Definitions
Alginate: Alginate is the salt of alginic acid and is a polysaccharide naturally occurring in brown algae. When used herein, an alginate may be any salt of alginic acid such as e.g. sodium alginate or calcium alginate.
Anammox: Anammox is an abbreviation for ANaerobic AMMonium Oxidation and is a microbial process of the nitrogen cycle where nitrite and ammonium ions are converted directly into nitrogen (N2) and water. Anammox microorganisms or bacteria are thus microorganisms and bacteria mediating the anammox process of converting nitrite and ammonium ions directly into nitrogen and water. Non-limiting examples of anammox microorganisms and bacteria include bacteria from the genera Brocadia, Kuenenia, Anammoxoglobus, Jettenia or Scalindua.
Aqueous medium: An aqueous medium is a solution comprising water as a solvent and one or more other components such as e.g. sodium chloride.
Bead load: The mass of beads applied to a reactor volume. For example, if 100 kg of beads was applied in a reactor with a volume of 1 cubic meter, the bead load is 10% w/v.
Denitrifying: The term is herein used for removal or reduction of nitrite and nitrate from a water body through the process of biological denitrification where the final product is nitrogen gas (N2).
Footprint: The spatial area that a building or installation occupies. It is measured in square meters.
High activity: Activity refers to the specific ammonium or nitrate treatment rates in a biofilter and is defined as mg Nitrogen/m3.d. Baseline activity is defined as the activity that can be expected from a fixed film type of biofilter, including fixed and moving bed bioreactors. High activity is de fined a specific nitrogen treatment activity that is in excess of the activity general expected from fixed film biofilters. In one aspect, the activity is high and is at least 400 g-N/m3.d.
Self-cleaning: As used herein, the term “self-cleaning” in relation with biobeads means that the movement of the beads in the reactor and the constant resultant collisions between beads, other beads and the walls of the reactor, prevents any significant biofilm from forming in the reactor (either on the beads or any surfaces). Therefore, the beads and the reactor they are applied in is considered self-cleaning as no action is required by operators. Solids: As used herein, the term “solids” in relation with biobeads means solid materials evolving on or inside the biobeads such as e.g. growth of encapsulated bacteria in the biobeads, uncon trolled bacteria populating the outside of the biobeads and/or material sloughed from the bi obeads.. Herein, low production of solids thus means low build-up of any bacteria and other un wanted materials in or on the biobeads.
Start-up and restart: As used in relation to nitrification and denitrification biofilters, “start-up” is the period of time immediately after a biofilter is taken into use for the first time. During this period, activity in the biofilter increases over time, until the biofilter reaches a level of activity (nitrification, denitrification and/or sulfur removal) that the biofilter was designed to operate with. A similar pe riod is recognised after disinfection or cleaning of a biofilter, and this is referred to as a biofilter “restart”.
Water body: A “water body” is herein understood as a compilation of water forming a reservoir. Non-limiting examples of water bodies e.g. include an aquaculture farm, tank, raceway, creek, river, pool, pond, waste lagoon, paddy, lake, estuary or ocean.
DETAILED DESCRIPTION OF THE INVENTION
Herein is described an optimized method of treating aquatic or marine animals using a recirculat ing aquaculture system (RAS). The present invention relates to a method of treating aquatic or marine animals using a recirculating aquaculture system (RAS) comprising the step of contacting a water body with a polymer support of immobilized microorganisms, wherein the polymer support comprises said microorganisms immobilized within a polymer hydrogel, wherein the polymer hy drogel comprises a cross-linked polymeric material and water or an aqueous medium, and wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol (PVA) and alginate. A polymer support of immobilized microorganisms is herein used inter changeably with the term biobeads. The polyvinyl alcohol may be cross linked with any cross- linking agent such as e.g. glutaraldehyde.
With the present invention, optimal conditions for microbial performance and retention is provided. It has surprisingly been found that the polymer support of immobilized microorganisms when used in the method of the invention uses competitive exclusion activity to actively deter most unwanted bacteria from establishing themselves on or in the beads. The polymer support of immobilized microorganisms used in the present method are self-cleaning, safe and easy to handle. The in ventors also found that the method of the invention resulted in a surprisingly effective reduction in ammonium (NH4+). The ammonium is defined as being removed when it is either consumed by the microbes present to support the growth of the microbes, or converted by nitrifying microor ganism to nitrate. In an aspect of the invention, the level of ammonium in the water is reduced compared to the level of ammonium without using the claimed method. In a further aspect, the level of ammonium in the water is reduced by 50-66% compared to the level of ammonium without using the claimed method. In an aspect of the invention, the method results in reduction in am monium without increasing the level of nitrogen dioxide substantially. In another or further aspect of the invention the level of ammonium in the water is reduced to reach a maximum level of ammonia of 2 mg NH4-N/I water. In an aspect of the invention, the level of ammonium in the water is reduced and at the same time the level of nitrogen dioxide is substantially remaining at the same level as the level of nitrogen dioxide compared to the level of ammonium and nitrogen dioxide without using the claimed method. Another by-product of ammonium removal can be the formation of dinitrogen oxide. In an aspect of the invention, the method results in a reduction of ammonium without leading to the production of dinitrogen oxide due to partial, interrupted or poorly controlled nitrification. The method of the invention furthermore provides a fast start-up or restart, robust performance despite the presence of inhibitors, toxins or process changes, high activity, low footprint and low production of solids.
The polymer support
The polymer support used in the method of the invention is a polymer support of immobilized microorganisms wherein the polymer support comprises said microorganisms immobilized within a polymer hydrogel, wherein the polymer hydrogel comprises polyvinyl alcohol polymeric chains cross linked with glutaraldehyde, and water or an aqueous solution.
In one embodiment, the polymer support of immobilized microorganisms comprises said microorganisms immobilized within a polymer hydrogel, wherein the polymer hydrogel comprises cross-linked polymeric material, and water or aqueous medium, wherein the cross- linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and glutaraldehyde. The polymer support of immobilized microorganisms comprises a covalent bond between glutaraldehyde and polyvinyl alcohol polymeric chains. The covalent bond crosslinks the polyvinyl alcohol polymeric chains.
The polyvinyl alcohol polymeric chains will vary in length. Typically, the polyvinyl alcohol polymeric chains consist of 500-3.000 monomer units and a molecular weight of 22.000 g/mol to 130.000 g/mol. The polyvinyl alcohol polymeric chains are cross-linked with glutaraldehyde. The crosslink may comprise monomeric, dimeric, oligomeric, or polymeric forms of glutaraldehyde.
The crosslinking density, determined as the number of glutaraldehyde crosslinking units, may affect morphological properties of the polymer support. In one embodiment, the crosslinking density, determined as the number of glutaraldehyde crosslinking units, is 8 to 25%, such as 10 to 20%, such as 12-20%, such as about 12%, 13%, 15%, 16%, 17%, 18%, 19% or 20%. The polymer support of immobilized microorganisms is, as stated, a polymer hydrogel. Typically, the hydrogel comprises from 60 to 99 wt% cross-linked polymeric material and 1 to 40 wt% water or aqueous medium, such as from 60 to 98 wt% cross-linked polymeric material and 2 to 40 wt% water or aqueous medium, such as from 65 to 95 wt% cross-linked polymeric material and 5 to 35 wt% water or aqueous medium, such as from 65 to 90 wt% cross-linked polymeric material and 10 to 35 wt% water or aqueous medium, such as from 65 to 85 wt% cross-linked polymeric material and 15 to 35 wt% water or aqueous medium, such as from 65 to 80 wt% cross-linked polymeric material and 20 to 35 wt% water or aqueous medium, or such as from 65 to 75 wt% cross-linked polymeric material and 25 to 35 wt% water or aqueous medium.
In a further embodiment, the polymer support of immobilized microorganisms further comprises alginate in monomer or polymeric form, entangled with cross-linked polyvinyl alcohol polymeric chains, such as so as to form an interpenetrating polymer network or semi-interpenetrating polymer network.
The polymer support comprises pores. The polymer support is a porous structure. The immobi lized microorganisms are immobilized within the polymer support on the surface of the pores. Entrapment of the microorganisms in the inner matrix provides an advantage of the method in that it serves to physically protected immobilized cells. Cell attachment or adsorption of microor ganisms to the inner matrix of polymer support may be by weak (non-covalent), generally non specific interactions such as electrostatic interactions.
In a typical embodiment, the polymer support is adequately porous to allow the substrate to diffuse into the polymer hydrogel support and the products or metabolites to diffuse out. In a typical em bodiment, a central volume of the polymer support comprises one or more macropores, and major volume of the polymer support comprises micropores. Without being bound to a particular theory, the central macropores allows for convection, which is an efficient method of mass transfer with out applying pressure.
In a typical embodiment, the pores of the polymer support are non-uniform in size. In one embod iment, an inner central fraction of the bead volume comprises macropores, whereas an outer fraction of the bead volume comprises micropores. The term macropores is intended to mean pores with an average size of at least 100 microns. The term micropore is intended to mean pores with an average pore size of less than 100 microns.
Defined alternatively, in an embodiment, the core of polymer support is devoid of polymer, such as a core of at least 100 microns in longest diameter, such as at least 200 microns, typically from 100-2.000 microns, such as 200-2.000 microns, such as 200-1.500 microns, such as 100-1.000 microns, such as 200-1.000 microns In one embodiment, the polymer support comprises pores having a gradient pore size in that inner portion of the volume of the polymer support has a larger pore size than average pore size if the remaining volume of the polymer support.
In one embodiment of the invention, the polymer support comprises pores having a gradient pore size in that the outer one-third of the polymer support has pores of a smaller average diameter than the pore size of the middle third of the polymer support, which in turn has a smaller average diameter than the pore size of the inner one-third of the polymer support. Within this embodiment, the outer one-third of the polymer support has an average pore diameter from 5 to 20 microns. The middle third of the polymer support has a larger average pore diameter than the outer one- third. It a suitable embodiment, it has an average pore diameter from 10 to 100 microns. The inner one-third of the polymer support has, in an embodiment, a larger average pore diameter than the middle third of the polymer support. The inner third of the polymer support has, in one embodi ment, an average pore diameter from 100- 2.000 microns. In one embodiment, at least 50% of the volume of the inner third is a cavity. The cavity is a volume within the polymer support that is substantially free from cross-linked polyvinyl alcohol. The center of the polymer support may com prise, in its center, a cavity having volume comprising 50-100% of the volume of the inner one third of the polymer support. A cavity is a volume free from cross-linked polyvinyl alcohol. Without being bound to a particular theory, the cavity serves as a central distribution center for nutrients, metabolites and substrate for the microorganisms, allowing for flow and distribution within the polymer support.
The immobilized microorganisms grow within the polymer hydrogel. That is to say that the micro organisms grow on the surface of the pores. The inert polymer hydrogel retains the microorgan ism, albeit not irreversibly in that a fraction of the population of the microorganisms may leak out of the polymer support by detaching from the inner matrix of the polymer support and leaking through the pores to exit the polymer support. In an embodiment of the invention, the outer surface of the polymer support does not comprise a skin or shell, which may serve to retain the cells or metabolites by means of having a smaller pore diameter than the outer third of the polymer sup port.
The polymer support of the invention is resistant to dissolution in water. It is reusable.
In one embodiment, the polymeric support is chemically substantially uniform in that the surface, body and core of the carrier is made of the same chemical components. However, as known to the person skilled in the art, due to different rates and extent of curing at the surface compared to within the body of the carrier, during the preparation of the polymer, some physio-chemical properties on the surface may differ with the physio-chemical properties within the core and throughout the hydrogel. Accordingly, there may be different degrees of crosslinking at the surface. However, according to the invention, these differences do not constitute a shell or coating or fibrous network on the surface.
In one embodiment, the invention is directed to a polymer support of immobilized microorganisms wherein the polymer support comprises said microorganisms immobilized within a polymer hy drogel; wherein polymer hydrogel comprises cross-linked polymeric material, alginate and water or aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer com prising polyvinyl alcohol and glutaraldehyde. In some embodiments, at least some alginate may remain within the polymer support of immobilized microorganisms. The alginate entrapped within the polymer support of immobilized microorganisms does not negatively impact performance of the polymer support. In an embodiment, the polymer support of immobilized microorganisms, after multiple washing, may comprise alginate. In an embodiment, the polymer support of immo bilized microorganisms does not comprise alginate.
The polymer support comprises glutaraldehyde cross-linking polyvinyl alcohol chains. An increased content of glutaraldehyde results in more PVA hydroxyl groups consumed and more acetal rings and ether linkages formed as a result of the crosslink formation. Furthermore, the presence of an acid catalyst in the preparation of the polymer support, such as sulfuric acid, also increases crosslink formation. Cross-link formation increases mechanical strength of the polymer support. Increased mechanical strength is observed with increased degree of crosslinking. With the increasing content of glutaraldehyde, the polymer support rigidity increases. The polymer support is elastic and malleable with excellent mechanical properties. The elasticity modulus is typically between 1.4 and 2.2 GPa, such as between 1.5 and 2 GPa. The tensile strength is typically between 3 and 6 MPa.
In one aspect, the polymer support used in the method of the invention comprises said microorganisms immobilized within a polymer hydrogel. The concentration of microorganisms within the polymer hydrogel, known as the microbial load, is typically in the range of 5 g/kg bead to 250 g/kg, typically 10 g/kg to 150 g/kg.
The cell density of the microorganisms can be tailored to the type of microorganisms, intended metabolic activity. In one embodiment of the invention, a high microbial load of immobilized microorganisms within the polymer support may be used, in some embodiments, to improve the product yield and the volumetric productivity of the bioreactors. The microbial load in the support is suitably at a concentration of at least about 5 grams, such as at least 10 grams/kg, such as at least 20 grams/kg, such as at least 50 grams/kg.
The polymer support of immobilized microorganisms may be of any shape but is typically according to spherical, oval, elliptical, bead-shaped, oblong, cylindrical, or capsule-like in shape. The polymer support is typically 1 to 10 mm long at its longest axis, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm. In the axis perpendicular to the longest axis, the polymer support may be 1 to 10 mm, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm. Typically, the aspect ratio is from 0.5 to 1, such as 0.5, 0.6, 0.7, 0.8, 0.9 or 1. The polymer support of immobilized microorganisms is typically spherical or bead-shaped having a diameter of 1 to 10 mm, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm with an aspect ratio from 0.6 to 1 , typically from 0.8 to 1.
Preparation of polymeric support of immobilized microorganisms
The polymeric support of immobilized microorganism (the biobeads) used in the method of the invention may typically be prepared in a process comprising a first step comprising a pre-bead formation and second step comprising polyvinyl alcohol linking.
In a suitable embodiment, the polymer material comprises polyvinyl alcohol wherein the polyvinyl alcohol (PVA) is a blend of polyvinyl alcohol of different molecular weights (MW), such as a blend of 2, 3, 4 or 5 PVA types, each with a MW from approximately 75.000 to approximately 225.000, such as a MW of approximately 95.000 to approximately 205.000, such as a PVA blend comprising a PVA selected from the group consisting of a PVA with a MW of approximately 125.000, PVA with a MW of approximately 145.000, and PVA with a MW of approximately 195.000.
The pre-bead formation step comprises combining sodium alginate, polyvinyl alcohol (PVA) and the microorganisms. The mixture is then added to a divalent or trivalent cation-containing solution, such as a divalent-containing solution, such as a Ca2+-containing solution. Typically, the mixture is added in a dropwise fashion to the divalent or trivalent cation-containing solution, such as to the divalent cation-containing, e.g. a Ca2+-containing solution. The divalent or trivalent cation- containing solution, such as the Ca2+-containing solution typically comprises a dissolved salt such as CaCh, SrCh, BaCh or AhiSC The divalent or trivalent cation containing-solution, such as the Ca2+-containing solution may have a cation concentration, (wt/wt) such as calcium concentra tion (wt/wt) ranging from 0.1% to 10%, typically from 0.5% to 5%, such as 0.5% to 2%, such as 0.5%, 1%, 1.5%, or 2%. Without being bound to particular theory, it is believed that the alginate and the divalent or trivalent cation such as e.g. Ca2+ rapidly form a complex resulting in a gelate structure dispersed within the divalent or trivalent cation-containing solution forming a hetero genous solution. Still within the theory, it is believed that the non-crosslinked PVA and microor ganisms are temporarily trapped within said gelate structure but leach out of gel and into the divalent or trivalent-containing solution. The PVA is thought to leach out at a higher rate than the microorganisms. The heterogenous solution comprises a gelate structure of an alginate-diva- lent/trivalent complex within the divalent or trivalent-containing solution and with microorganisms and PVA loosely entrapped within the gelate structure. The divalent or trivalent-containing solu tion typically further comprises PVA and microorganisms.
The alginate- divalent or trivalent cation complex comprises physical cross-linking, which relies on divalent or trivalent cation cross-linking between alginate chains. The pre-bead formation step comprises combining sodium alginate, polyvinyl alcohol (PVA) and the microorganisms to form a mixture to be dripped into the divalent or trivalent-containing solu tion. In the sodium alginate, polyvinyl alcohol (PVA) and microorganism-containing solution, the polyvinyl alcohol (PVA) is suitably present at a concentration 3% - 15% wt/wt, such as 5-10% wt/wt, such as 5%, 6%, 7%, 8%, 9% or 10%. The alginate is typically present at a concentration of 0.25-5% wt/wt, such as 0.5-2%, such as 0.5%, 1%, 1.5% or 2%.
The pre-bead formation step comprises combining sodium alginate, polyvinyl alcohol (PVA) and the microorganisms to form a mixture. A stock broth solution of microorganisms comprising from 200 g of cells/L to 1000 g of cells/L, such as 100 to 500 g of cells/L is typically used. Typically, the stock broth solution is diluted, said diluted solution comprising to 100 to 500 g of cells/L, such as 50 to 250 g of cells/L, such as 50 g/L, 100 g/L, 125 g/L, 150 g/L, 200 g/L, and 250 g/L.
This diluted stock solution is used to form a mixture comprising of sodium alginate, polyvinyl al cohol (PVA) and the microorganisms. The microorganisms are suitably present in the mixture at a concentration of 10g/L to 500 g/L, such as 20g/L to 80g/L, typically from 20 g/L to 60 g/L.
The second step is a cross-linking step and comprises adding the gelate structure of the alginate- divalent or trivalent-complex to a cross-linking solution comprising glutaraldehyde so as to provide a cross-linked polymer support of immobilized microorganisms. Suitably, the cross-linking solu tion further comprises an acid, such as sulfuric acid, H2SO4, or hydrochloric acid, HCI, adjusting to same pH as the physical properties like elasticity, visual appearance or sphericity of the beads change along. The cross-linking solution has an acidic pH, such as a pH of 1-5, typically 1.5 to
3.5 such as 2 or 3. The cross-linking solution when comprising sulfuric acid typically has a pH of
1.5 to 3.5 such as 2 or 3. In this step, PVA and glutaraldehyde form covalent bonds. Typically, the cross-linking solution further comprises a catalytic agent such as a sulphate, typically sodium sulphate, ammonium sulphate or potassium sulphate. Without being bound to a particular theory, the catalytic agent such as sodium sulphate performs at least two functions. It forms a complex with the hydroxyl groups of PVA, thereby interlinking PVA strands and/or two different positions within a PVA strand. This function supports the cross-linking with glutaraldehyde. It is furthermore thought that the catalytic agent catalyses the reaction of the aldehyde units of glutaraldehyde with the alcohol groups.
Wthout being bound to a particular theory, during the cross-linking step, the alginate is hydrolysed under acidic pH conditions. The hydrolysed alginate washes out, at least in part from the cross- linked polymer support of immobilized microorganisms.
Wthout being bound to a particular theory, it is thought that the microorganisms are protected from the harsh conditions of the cross-linking solution, namely the low pH and the presence of high amounts of the biocide glutaraldehyde by being trapped within the gelated alginate-divalent or trivalent complex such as a gelated alginate-Ca2+ complex. The molar ratio of glutaraldehyde to polyvinyl alcohol plays a role in the cross-linking density of the polymer. Typically, the molar ratio of glutaraldehyde to polyvinyl alcohol is suitably from 1 : 105 to 1 : 1010, such as from 1 : 106 to 1 : 109, such as 1 : 107 to 1 : 109, such as in the order of 1 : 107, 1 : 10s or 1 : 109, such as in the order 1 : 108.
In a suitable embodiment, the concentration of glutaraldehyde in the cross-linking step is less than 0.3%, such as from 0.02% to 0.25%, preferably from 0.02% to 0.2%, such as 0.05%, 0.10%, 0.15% and 0.20%.
In a suitable embodiment, the concentration of sulfuric acid (H2SO4) is from 0.2% to 1.0% (g/g), such as 0.3% to 0.9%, such as 0.3%, 0.4%, 0.5%, 0.6%. 0.7%, or 0.8%, typically 0.4% to 0.6%, such as 0.4%, 0.5% or 0.6%.
The reaction time of the cross-linking step is typically from 1 to 6 hours, typically 2 to 5 hours, such as 2 to 4 hours, such as 2.5 to 3.5 hours, such as 3 hours.
After the reaction time, the polymer support of immobilized microorganisms is washed in a wash ing step in aqueous medium, such as an alkaline buffer, such as a carbonate buffer or a phos phate buffer. The buffer is typically at a pH of 7 to 10, typically pH 7.5 to 10, such as pH 8 to 10, such as 8.5, 9, 9.5 or 10, such as 8.5, 9 or 9.5, such as pH 9. The washing step may comprise the use of an alkaline buffer or the use of an amine-rich solution such as a poly-ethyleneimine solution.
The washing step removes, at least in part the unreacted glutaraldehyde. The washing step may further wash out, at least in part, the alginate. The washing step may be repeated 1 to 5 times, typically 3 times.
In some embodiments, at least some alginate may remain within the polymer support of immobi lized microorganisms. The alginate entrapped within the polymer support of immobilized microor ganisms does not impact performance. In an embodiment, the polymer support of immobilized microorganisms, after multiple washing, may comprise alginate. In an embodiment, the polymer support of immobilized microorganisms does not comprise alginate.
Unlike the alginate-divalent or trivalent complex which comprised physical cross-linking relying on divalent or trivalent cation cross-linking between alginate chains, polymer support of immobi lized microorganisms comprises polyvinyl alcohol polymeric chains covalently cross-linked via glutaraldehyde linkages.
The microorganisms
The microorganisms immobilized within the polymer hydrogel used in the method of the invention may be selected from the group consisting of ammonia oxidizing microorganisms, nitrite oxidising microorganisms, sulfur oxidizing microorganisms, heterotrophic microorganisms and anaerobic ammonium-oxidizing microorganisms.
In a typical embodiment, the microorganisms immobilized within the polymer hydrogel may be selected from the group consisting of Nitrosomonas spp., Nitrobacter spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp., Nitrotoga spp., Nitrospira spp. Pseu domonas spp., Paracoccus spp., Hyphomicrobium spp., Castellaniella spp., Janthinobacterium spp., Acidovorax spp., Aeromonas spp., Cellulomonas spp., Buttiauxella spp., Microvirgula spp., Klebsiella spp., Shewanella spp., Pelosinus spp., Variovorax spp., Hydrogenophaga spp., Ra- oultella spp., Bacillus spp., Achromobacter spp., Ochrobactrum spp., Flavobacterium spp., and Delftia.
In a suitable embodiment, the microorganisms immobilized within the polymer hydrogel may be selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkyl phenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomonas hominis, Flavimobis soli, Flavobacterium banpa uense, Buttiauxella agrestis, Buttiauxella noackiae, Achromobacter denitrificans, Pelosinus fermentans, Variovorax dokdonensis, Hydrogenophaga bisanensis, Raoultella terrigena, Delftia lacustris, Shewanella putrefaciens, Acidovorax soli, Hyphomicrobium denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, Microvirgula aerodenitrificans,
Candidatus Kuenenia, Candidatus Brocadia, Candidatus Anammoxoglobus,
Candidatus Jettenia, and Candidatus Scalindua
Paracoccus pantotrophus was formerly named Paracoccus denitrificans or Thiosphaera pantotropha.
An advantageous feature of the microorganisms of the invention are microorganisms having a feature selected from the group consisting of a robust performance, activity at low temperatures, activity with low levels of additional carbon, selectivity for specific carbon sources. In one aspect, the invention is directed to a polymer support of immobilized microorganisms wherein the polymer support comprises said microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises polyvinyl alcohol polymeric chains cross linked with glutaraldehyde; and water or an aqueous solution, wherein has at least 80% its maximum activity at below 20°C, such as below 15°C, such as below 10°C. Application of the polymeric support of immobilized microorganisms
Polymeric support of immobilized microorganisms (biobeads or beads) can be applied in multiple ways and e.g. in several configurations for RAS processes. One method of application is in a mixed reactor where the beads are added with a bead fill of 10-30% w/v. The beads are constantly mixed in the reactor together with the influent water and any other additives required to achieve the desired outcome such as e.g. alkalinity and dissolved oxygen for nitrification and Biochemical Oxygen Demand (BOD) for denitrification. The beads are then separated from the treated water and retained in the reactor. Another method of application is in a fluidized bed reactor. In such a reactor, the beads are applied to a column with a bead fill of 40-80% w/v. The influent water is pumped into the base of the column reactor and distributed so as to maintain a plug flow up through the layer of beads in the reactor. The water is passed through the column with such a velocity so as to overcome the settling velocity of the beads and allow the beads to move slightly within the reactor.
Nitrification
Nitrogen removal from a water body typically involves the processes known as nitrification (oxidation of ammonium (NhV) to nitrite (NCV) and then nitrate (NO3 ) by a community of nitrifying microorganisms) and denitrification (reduction of nitrate and/or nitrite to elemental nitrogen (N2) by a community of denitrifying microorganisms), thereby removing the nitrogen from the water.
In one aspect of the invention, the polymer support of immobilized microorganisms is a blend of robust, highly-active strains of nitrifying bacteria. This accelerates biofilter start up and restart in recirculating aquaculture systems (RAS).
The polymer support of immobilized microorganisms of the invention were able to demonstrate the ability to achieve a high removal of ammonia within a few days, such as less than 100 days, or less than 90 days, while at the same time achieving a high conversion of ammonia to nitrate. This indicates that polymer support with immobilized microorganisms provides significant advantages over current technologies applied in biological nitrification processes due to the ability of the biobeads to achieve high levels of ammonia removal and conversion to nitrate within a very short period and under challenging environmental conditions. Total nitrogen concentrations were significantly reduced while using the polymer support of immobilized microorganisms of the invention.
The efficacy of biological nitrogen removal processes is herein enhanced through the application of high concentrations of nitrifying/denitrifying organisms in a biological treatment process. The microroganisms of the invention may be ammonia-oxidizing bacteria and/or nitrite-oxidizing bacteria and may be combined with other bacteria, e.g., Bacillus such as a combination of the commercial product Prawn Bac PB-628 (product of Novozymes Biologicals), together with Enterobacter or Pseudomonas. The nitrifying consortium may be formulated as a liquid, a lyophilized powder, or a biofilm, e.g., on bran or corn gluten.
Denitrification
The invention is furthermore directed to a method of denitrifying a water body comprising adding to the water body a polymer support as defined herein. Denitrification is the reduction of nitrate and/or nitrite to elemental nitrogen N2.
Nitrogen removal from a water body typically involves the processes known as nitrification (oxidation of ammonium (NhV) to nitrite (NO2 ) and then nitrate (NO3 ) by a community of nitrifying microorganisms) and denitrification (reduction of nitrate and/or nitrite to elemental nitrogen (N2) by a community of denitrifying microorganisms), thereby removing the nitrogen from the water.
A further aspect is directed to a method of denitrifying a water body, or reducing the ammonium, nitrite and/or nitrate levels in a water body, comprising adding a polymer support of immobilized microorganisms, wherein the polymer support comprises said microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises polyvinyl alcohol polymeric chains cross linked with glutaraldehyde; and water or an aqueous solution, typically wherein the microorganism is selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus versutus and combinations thereof.
Denitrification occurs under anoxic/anaerobic conditions. Denitrification is the sequential process involving the dissimilatory reduction of one or both the ionic nitrogen oxides, nitrate (NO3 ) and nitrite (NO2 ) to gaseous nitrogen oxides, nitric oxide (NO), nitrous oxide (N2O) and finally reduce to the ultimate product, dinitrogen (N2) thus removing biologically available nitrogen and returning it to the atmosphere.
Both nitrate and nitrite are fully converted to atmospheric nitrogen. However, insufficient carbon sources, low dissolved oxygen (DO) concentrations and or environmental conditions may lead to improper denitrification and N2O accumulation and emissions. One pathway to reduce N2O production is to select the right organisms for the denitrification process. Denitrifiers with their facultative anaerobic traits perform denitrifying activities under the presence of oxygen driving an increase in N2O as a denitrification intermediate. Many heterotrophic nitrifiers along with the oxidation of NH3 can simultaneously perform aerobic denitrification. N2O is then generated. Accordingly, the judicious selection of denitrifiers is an important aspect to commercial success.
Polymer hydrogels comprising Paracoccus are a preferred embodiment in the denitrification of a water body. Paracoccus pantotrophus grows aerobically with a large variety of carbon sources and with molecular hydrogen or thiosulfate as an energy source, and nitrate serves as electron acceptor under anaerobic conditions. The denitrification properties of Paracoccus denitrificans render it a preferred microorganism. Paracoccus denitrificans reduces nitrite to nitrogen gas while either Nitrosomonas eutropha or Nitrosomonas europaea oxidizes ammonia to nitrite, thus fuelling the former metabolism. Accordingly, one embodiment comprises the combined use of Paracoccus denitrificans and either Nitrosomonas eutropha or Nitrosomonas europaea. The denitrification properties of Paracoccus versutus, such as Paracoccus versutus NN066467, also render it a preferred microorganism.
The denitrification process may suitably require the addition of a carbon source. In one aspect of the invention, the microorganism is a denitrifier and denitrification is accompanied by the addition of a carbon source. Suitable embodiments of this aspect of the invention comprise a carbon source selected from the group consisting of methanol, ethanol, acetate, acetic acid, glycerol, glycol, molasses, corn syrup, sucrose solutions, commercially available carbon sources, fer mented organic wastes, industrial wastewaters. In a more preferable embodiment consisting of methanol, ethanol, acetate, acetic acid, glycerol, commercially available carbon sources. In a most preferred embodiment, the carbon source is selected from the group consisting of methanol, glycerol or commercially available carbon sources.
Water body and Salinity
As stated, a typical “water body” according to the invention is an aquaculture farm, tank, raceway, creek, river, pool, pond, waste lagoon, paddy, lake, estuary or ocean. Accordingly, the water of the water body may be fresh water or salt water. Other stated, the water body may be of non saline or saline water. Otherwise stated, the water body may have low to high salinity, such as 0 to 10% salinity, as determined by level of NaCI.
In a suitable embodiment, the salinity of the water body is from 0% to 7%, such as 0.1% to 5%. The salinity if the water body may be from 0% to 15%, such as 0.1% to 15%, such as 0.1% to 12%, such as 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12%. Suitable microorganisms when the salinity of the water body is from 0% to 15%, such as 0.1% to 15%, such as 0.1% to 12%, such as 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12% may typically be selected from any of the microorganisms of the invention, are typically se lected the group consisting of a Paracoccus, Pseudomonas and Nitrosomas, such as Paracoccus pantotrophus, Paracoccus versutus. The invention is, in one embodiment, directed to nitrogen removal, such as denitrification, in a water body with salinity from 0% to 15%, such as from 0.1% to 12%.
The water body typically has less than 50 ppt salinity, such as less than 30 ppt salinity, such as less than 25 ppt salinity, such as less than 20 ppt salinity, such as 20 ppt salinity, 19 ppt salinity, 18 ppt salinity, 17 ppt salinity, 16 ppt salinity, 15 ppt salinity, 14 ppt salinity, 13 ppt salinity, 12 ppt salinity, 11 ppt salinity, 10 ppt salinity, 9 ppt salinity, 8 ppt salinity, 7 ppt salinity, 6 ppt salinity, 5 ppt salinity, 4 ppt salinity, 3 ppt salinity, 2 ppt salinity, and 1 ppt salinity.
The water body may be warm water body or cold water body. In one embodiment the cold water temperature is 15°C or less, such as 12°C or less such as 10°C or less. In another embodiment, the warm water temperature is at least 15°C, such as at least 17°C, such as at least 20°C.
PREFERRED EMBODIMENTS
The invention is further described in the following list of embodiments:
1. A method of treating aquatic or marine animals comprising: contacting a water body with a polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
2. The method according to embodiment 1 , wherein the polyvinyl alcohol is cross linked with glutaraldehyde.
3. The method according to embodiment 1 or 2, wherein a covalent bond between glutaraldehyde and polyvinyl alcohol polymeric chains crosslinks the polyvinyl alcohol polymeric chains.
4. The method according to any one of the preceding embodiments, wherein the polyvinyl alcohol polymeric chains consist of 500-3.000 monomer units and a molecular weight of 22.000 g/mol to 130.000 g/mol.
5. The method according to any one of the preceding embodiments, wherein the crosslink comprises monomeric, dimeric, oligomeric, or polymeric glutaraldehyde.
6. The method according to any one of the preceding embodiments, wherein the crosslinking density, determined as the number of glutaraldehyde crosslinking units, is 8 to 25%, such as 10 to 20%, such as 12-20%, such as about 12%, 13%, 15%, 16%, 17%, 18%, 19% or 20%.
7. The method according to any one of the preceding embodiments, wherein polymer hydrogel comprises from 60 to 99 wt% cross-linked polymeric material and 1 to 40 wt% water or aqueous medium, such as from 60 to 98 wt% cross-linked polymeric material and 2 to 40 wt% water or aqueous medium, such as from 65 to 95 wt% cross-linked polymeric material and 5 to 35 wt% water or aqueous medium, such as from 65 to 90 wt% cross-linked polymeric material and 10 to 35 wt% water or aqueous medium, such as from 65 to 85 wt% cross-linked polymeric material and 15 to 35 wt% water or aqueous medium, such as from 65 to 80 wt% cross-linked polymeric material and 20 to 35 wt% water or aqueous medium, or such as from 65 to 75 wt% cross-linked polymeric material and 25 to 35 wt% water or aqueous medium. The method according to any one of the preceding embodiments, wherein alginate is in monomer or polymeric form, entangled with cross-linked polyvinyl alcohol polymeric chains so as to form an interpenetrating polymer network. The method according to any one of the preceding embodiments, wherein the polymer sup port is porous. The method according to embodiment 9, wherein the polymer support comprises micropores. The method according to embodiment 10, comprising one or more macropores in the central volume of support and micropores. The method according to embodiment 10 or 11 , wherein a portion of the polymer support comprises micropores with an average pore diameter of 5 to 40 microns, such as 5 to 30 microns, such as 5 to 20 microns. The method according to anyone of embodiments 10 to 12, wherein a portion of the polymer support comprises micropores with an average pore diameter of 10 to 40 microns, such as 20 to 40 microns. The method according to any one of the preceding embodiments, wherein the polymeric support is spherical, oval, elliptical bead-shaped, oblong, cylindrical, or capsule-like in shape. The method according to any one of the preceding embodiments, wherein the polymeric support is spherical or bead-shaped having a diameter of 1 to 10 mm, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm. The method according to any one of the preceding embodiments, wherein the microbial load is 5 g/kg bead to 250 g/kg bead, typically 10 g/kg bead to 150 g/kg bead. The method according to any one of the preceding embodiments, wherein the immobilized microorganisms are selected from the group consisting of one or more sulfur oxidizing microorganisms, one or more ammonium oxidizing microorganisms, one or more nitrite oxidizing microorganisms, one or more denitrifying microorganisms, one or more anammox microorganisms and any mixtures thereof. The method according to any one of the preceding embodiments, wherein the immobilized microorganisms are selected from the group consisting of a mixed or pure culture of sulfur oxidizing microorganisms, a mixed or pure culture of ammonium oxidizing microor ganisms, a mixed or pure culture of nitrite oxidizing microorganisms, a mixed or pure culture of denitrifying microorganisms, a mixed or pure culture of anammox microorganisms and any mixtures thereof. The method according to any one of the preceding embodiments, wherein the immobilized microorganisms are a combination of two or more of the microorganisms selected from the group consisting of: ammonium oxidizing microorganisms, nitrite oxidizing microorganisms, denitrifying microorganisms and sulfur oxidizing microorganisms. The method according to any one of embodiments 17 to 19, wherein the ammonia oxidizing microorganisms are selected from Nitrosomonas spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp, The method according to any one of embodiments 17 to 20, wherein the nitrite oxidizing microorganisms are selected from Nitrobacter spp., Nitrococcus spp., Nitrospira spp., and Nitrospina spp. The method according to any one of embodiments 17 to 21, wherein Paracoccus spp. is used as denitrifying and/or sulfur oxidizing microorganisms. The method according to embodiment 22, wherein Paracoccus spp. is selected from the group consisting of: P. alcaliphilus, P. alkenifer, P. aminophilus, P. aminovorans, P. carotinifaciens, P. denitrificans, P. kocurii, P. marcusii, P. methylutens, P. pantotrophus, P. solventivorans, P. thiocyanatus, P. versutus, and any combination thereof. The method according to any one of embodiments 17 to 23, wherein the immobilized microorganisms are a combination of the ammonia oxidizing microorganisms Nitrosomonas spp., the nitrite oxidizing microorganisms Nitrobacter spp. and optionally the sulfur oxidizing microorganisms Paracoccus spp. The method according to any one of embodiments 17 to 23, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and a Nitrobacter, Nitrosomonas eutropha and a Paracoccus, Nitrosomonas eutropha and a Nictrobacter and a Paracoccus, Nitrosomonas europaea and a Nitrobacter, Nitrosomonas europaea and a Paracoccus, and of Nitrosomonas europaea and a Nitrobacter and a Paracoccus. The method according to embodiments 17 to 23, wherein the immobilized microorganisms are selected from the group consisting of a combination of a Nitrosomonas and Nitrobacter winogradskyi, a Paracoccus and Nitrobacter winogradskyi, and, a Nitrosomonas and a Paracoccus and Nitrobacter winogradskyi. The method according to any one of embodiments 17 to 23, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus pantotrophus, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus Versutus, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus pantotrophus, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus Versutus, preferably a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi. The method according to any one of embodiments 17 to 23, wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas spp., Nitrobacter spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp., Nitrotoga spp., Nitrospira spp. Pseudomonas spp.;, Paracoccus spp., Hyphomicrobium spp., Castellaniella spp., Janthinobacterium spp., Acidovorax spp., Aeromonas spp., Cellulomonas spp., Buttiauxella spp., Microvirgula spp., Klebsiella spp., Shewanella spp., Pelosinus spp., Variovorax spp., Hydrogenophaga spp., Raoultella spp., Bacillus spp., Achromobacter spp., Ochrobactrum spp., Flavobacterium spp., and Delftia and combinations thereof. The method according to any one of embodiments 17 to 23, wherein the immobilized microorganisms are selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkylphenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomonas hominis, Flavimobis soli,
Flavobacterium banpakuense, Buttiauxella agrestis, Buttiauxella noackiae,
Achromobacter denitrificans, Pelosinus fermentans, Variovorax dokdonensis, Hydrogenophaga bisanensis, Raoultella terrigena, Delftia lacustris, Shewanella putrefaciens, Acidovorax soli, Hyphomicrobium denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, Microvirgula aerodenitrificans, Candidatus Kuenenia, Candidatus Brocadia, Candidatus Anammoxoglobus,
Candidatus Jettenia, Candidatus Scalindua, and combinations thereof. The method according to any one of embodiments 17 to 23, wherein the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus pandtodrophus, Castellaniella defragans, Pseudomonas proteolytica, Paracoccus versutus, Paracoccus denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, and combinations thereof. The method according to any one of embodiments 17 to 21, wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas eutropha , Nitrobacter winogradskyi and combinations thereof. The method according to any one of embodiments 17 to 21, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, preferably a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi, and combinations thereof. The method according to any one of embodiments 17 to 23, wherein the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus versutus and combinations thereof. The method according to any one of embodiments 17 to 23 and 29 to 30, wherein the im mobilized microorgansims comprise Pseudomonas lini. The method according to any one of embodiments 17 to 23 and 29 to 30, wherein the im mobilized microorgansims comprise Paracoccus pantotrophus. The method according to any one of embodiments 17 to 23 and 30, wherein the immobilized microorgansims comprise Castellaniella defragans. The method according to any one of embodiments 17 to 23 and 29 to 30, wherein the im mobilized microorgansims comprise Pseudomonas proteolytica. The method according to any one of embodiments 17 to 23, 27 and 29 to 30, wherein the immobilized microorgansims comprise Paracoccus versutus. The method according to any one of embodiments 17 to 23 and 29 to 30, wherein the im mobilized microorgansims comprise Paracoccus denitrificans. The method according to any one of embodiments 17 to 23 and 29 to 30, wherein the im mobilized microorgansims comprise Nitrosomonas eutropha. The method according to any one of embodiments 17 to 23 and 29 to 30, wherein the im mobilized microorgansims comprise Nitrosomonas europaea. The method according to any one of embodiments 17 to 23 and 29 to 30, wherein the im mobilized microorgansims comprise Nitrobacter Winogradsky. The method according to any one of the preceding embodiments, wherein the method comprises mixing the polymer support into the water of an aquaculture pond such as aquaculture, farm, pool, pond, paddy, lake, estuary, ocean, or combinations thereof. The method according to any one of the preceding embodiments, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 80% w/v, such as 10% to 80% w/v, such as 15% to 80% w/v of the water. The method according to embodiment 44, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 30% w/v, such as 10% to 30% w/v, such as 15% to 30% w/v of the water. The method according to embodiment 44, wherein the polymer support is combined with the water in the water body at a bead load of 60% to 80% w/v, such as 65% to 75% w/v, such as 70% to 75% w/v of the water. The method according to embodiment 44, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 30% w/v or 60% to 80% w/v of the water. The method according to any one of the preceding embodiments, wherein the water body comprises from 0.5 to 10 mg/I of ammonia, such as 1 to 5 mg/I ammonia, such as 1 to 2 mg/I of ammonia. The method according to any one of the preceding embodiments, wherein the method is a method of reducing the amount of ammonia in a water body and/or a method of denitrifying water in a water body. 50. Use of a polymer support of immobilized microorganisms for treating aquatic or ma rine animals comprising: contacting a water body with said polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
51. The use according to embodiment 50, wherein the polyvinyl alcohol is cross linked with glutaraldehyde.
52. The use according to embodiment 50 or 51, wherein a covalent bond between glutaraldehyde and polyvinyl alcohol polymeric chains crosslinks the polyvinyl alcohol polymeric chains.
53. The use according to any one of embodiments 50 to 52, wherein the polyvinyl alcohol polymeric chains consist of 500-3000 monomer units and a molecular weight of 22.000 g/mol to 130.000 g/mol.
54. The use according to any one of embodiments 50 to 53, wherein the crosslink comprises monomeric, dimeric, oligomeric, or polymeric glutaraldehyde.
55. The use according to any one of embodiments 50 to 54, wherein the crosslinking density, determined as the number of glutaraldehyde crosslinking units, is 8 to 25%, such as 10 to 20%, such as 12-20%, such as about 12%, 13%, 15%, 16%, 17%, 18%, 19% or 20%.
56. The use according to any one of embodiments 50 to 55, wherein polymer hydrogel comprises from 60 to 99 wt% cross-linked polymeric material and 1 to 40 wt% water or aqueous medium, such as from 60 to 98 wt% cross-linked polymeric material and 2 to 40 wt% water or aqueous medium, such as from 65 to 95 wt% cross-linked polymeric material and 5 to 35 wt% water or aqueous medium, such as from 65 to 90 wt% cross-linked polymeric material and 10 to 35 wt% water or aqueous medium, such as from 65 to 85 wt% cross-linked polymeric material and 15 to 35 wt% water or aqueous medium, such as from 65 to 80 wt% cross-linked polymeric material and 20 to 35 wt% water or aqueous medium, or such as from 65 to 75 wt% cross-linked polymeric material and 25 to 35 wt% water or aqueous medium. The use according to any one of embodiments 50 to 56, wherein alginate is in monomer or polymeric form, entangled with cross-linked polyvinyl alcohol polymeric chains so as to form an interpenetrating polymer network. The use according to any one of embodiments 50 to 57, wherein the polymer support is porous. The use according to embodiment 58, wherein the polymer support comprises micropores. The use according to embodiment 59, comprising one or more macropores in the central volume of support and micropores. The use according to embodiment 59 or 60, wherein a portion of the polymer support comprises micropores with an average pore diameter of 5 to 40 microns, such as 5 to 30 microns, such as 5 to 20 microns. The use according to any one of embodiments 59 to 61, wherein a portion of the polymer support comprises micropores with an average pore diameter of 10 to 40 microns, such as 20 to 40 microns. The use according to any one of embodiments 50 to 62, wherein the polymeric support is spherical, oval, elliptical bead-shaped, oblong, cylindrical, or capsule-like in shape. The use according to any one of embodiments 50 to 63, wherein the polymeric support is spherical or bead-shaped having a diameter of 1 to 10 mm, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm. The use according to any one of embodiments 50 to 64, wherein the microbial load is 5 g/kg bead to 250 g/kg bead, typically 10 g/kg bead to 150 g/kg bead. The use according to any one of embodiments 50 to 65, wherein the immobilized microorganisms are selected from the group consisting of one or more sulfur oxidizing microorganisms, one or more ammonium oxidizing microorganisms, one or more nitrite oxidizing microorganisms, one or more denitrifying microorganisms, one or more anammox microorganisms and any mixtures thereof. The use according to any one of embodiments 50 to 66, wherein the immobilized microor ganisms are selected from the group consisting of a mixed or pure culture of sulfur oxi dizing microorganisms, a mixed or pure culture of ammonium oxidizing microorganisms, a mixed or pure culture of nitrite oxidizing microorganisms, a mixed or pure culture of denitrifying microorganisms, a mixed or pure culture of anammox microorganisms and any mixtures thereof. The use according to any one of embodiments 50 to 67, wherein the immobilized microorganisms are a combination of two or more of the microorganisms selected from the group consisting of: ammonium oxidizing microorganisms, nitrite oxidizing microorganisms, denitrifying microorganisms and sulfur oxidizing microorganisms. The use according to any one of embodiments 66 to 68, wherein the ammonia oxidizing microorganisms are selected from Nitrosomonas spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp, The use according to any one of embodiments 66 to 69, wherein the nitrite oxidizing microorganisms are selected from Nitrobacter spp., Nitrococcus spp., Nitrospira spp., and Nitrospina spp. The use according to any one of embodiments 66 to 70, wherein Paracoccus spp. is used as denitrifying and/or sulfur oxidizing microorganisms. The use according to embodiment 71 , wherein Paracoccus spp. is selected from the group consisting of: P. alcaliphilus, P. alkenifer, P. aminophilus, P. aminovorans, P. carotinifaciens, P. denitrificans, P. kocurii, P. marcusii, P. methylutens, P. pantotrophus, P. solventivorans, P. thiocyanatus, P. versutus, and any combination thereof. The use according to any one of embodiments 66 to 72, wherein the immobilized microorganisms are a combination of the ammonia oxidizing microorganisms Nitrosomonas spp., the nitrite oxidizing microorganisms Nitrobacter spp. and optionally the sulfur oxidizing microorganisms Paracoccus spp. The use according to any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and a Nitrobacter, Nitrosomonas eutropha and a Paracoccus, Nitrosomonas eutropha and a Nictrobacter and a Paracoccus, Nitrosomonas europaea and a Nitrobacter, Nitrosomonas europaea and a Paracoccus, and of Nitrosomonas europaea and a Nitrobacter and a Paracoccus. The use according to embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of a combination of a Nitrosomonas and Nitrobacter winogradskyi, a Paracoccus and Nitrobacter winogradskyi, and, a Nitrosomonas and a Paracoccus and Nitrobacter winogradskyi. The use according to any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus pantotrophus, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus Versutus, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus pantotrophus, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus Versutus, preferably a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi. The use according to any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas spp., Nitrobacter spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp., Nitrotoga spp., Nitrospira spp. Pseudomonas spp.;, Paracoccus spp., Hyphomicrobium spp., Castellaniella spp., Janthinobacterium spp., Acidovorax spp., Aeromonas spp., Cellulomonas spp., Buttiauxella spp., Microvirgula spp., Klebsiella spp., Shewanella spp., Pelosinus spp., Variovorax spp., Hydrogenophaga spp., Raoultella spp., Bacillus spp., Achromobacter spp., Ochrobactrum spp., Flavobacterium spp., and Delftia and combinations thereof. The use according to any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkylphenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomonas hominis, Flavimobis soli,
Flavobacterium banpakuense, Buttiauxella agrestis, Buttiauxella noackiae,
Achromobacter denitrificans, Pelosinus fermentans, Variovorax dokdonensis, Hydrogenophaga bisanensis, Raoultella terrigena, Delftia lacustris, Shewanella putrefaciens, Acidovorax soli, Hyphomicrobium denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, Microvirgula aerodenitrificans, Candidatus Kuenenia, Candidatus Brocadia, Candidatus Anammoxoglobus,
Candidatus Jettenia, Candidatus Scalindua, and combinations thereof. The use according to any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus pandtodrophus, Castellaniella defragans, Pseudomonas proteolytica, Paracoccus versutus, Paracoccus denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, and combinations thereof. The use according to any one of embodiments 66 to 70, wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas eutropha , Nitrobacter winogradskyi and combinations thereof. The use according to any one of embodiments 66 to 70, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, preferably a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi, and combinations thereof. The use according to any one of embodiments 66 to 72, wherein the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus versutus and combinations thereof. The use according to any one of embodiments 66 to 72 and 78 to 79, wherein the immobi lized microorgansims comprise Pseudomonas lini. The use according to any one of embodiments 66 to 72 and 78 to 79, wherein the immobi lized microorgansims comprise Paracoccus pantotrophus. The use according to any one of embodiments 66 to 72 and 79, wherein the immobilized microorgansims comprise Castellaniella defragans. The use according to any one of embodiments 66 to 72 and 78 to 79, wherein the immobi lized microorgansims comprise Pseudomonas proteolytica. The use according to any one of embodiments 66 to 72, 27 and 78 to 79, wherein the immobilized microorgansims comprise Paracoccus versutus. The use according to any one of embodiments 66 to 72 and 78 to 79, wherein the immobi lized microorgansims comprise Paracoccus denitrificans. The use according to any one of embodiments 66 to 72 and 78 to 79, wherein the immobi lized microorgansims comprise Nitrosomonas eutropha. The use according to any one of embodiments 66 to 72 and 78 to 79, wherein the immobi lized microorgansims comprise Nitrosomonas europaea. 91. The use according to any one of embodiments 66 to 72 and 78 to 79, wherein the immobi lized microorgansims comprise Nitrobacter Winogradsky.
92. The use according to any one of embodiments 50 to 91 , wherein the use comprises mixing the polymer support into the water of an aquaculture pond such as aquaculture, farm, pool, pond, paddy, lake, estuary, ocean, or combinations thereof.
93. The use according to any one of embodiments 50 to 92, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 80% w/v, such as 10% to 80% w/v, such as 15% to 80% w/v of the water.
94. The use according to embodiment 93, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 30% w/v, such as 10% to 30% w/v, such as 15% to 30% w/v of the water.
95. The use according to embodiment 93, wherein the polymer support is combined with the water in the water body at a bead load of 60% to 80% w/v, such as 65% to 75% w/v, such as 70% to 75% w/v of the water.
96. The use according to embodiment 93, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 30% w/v or 60% to 80% w/v of the water.
97. The use according to any one of embodiments 50 to 96, wherein the water body comprises from 0.5 to 10 mg/I of ammonia, such as 1 to 5 mg/I ammonia, such as 1 to 2 mg/I of ammonia.
98. The use according to any one of embodiments 50 to 97, wherein the use is a use of reducing the amount of ammonia in a water body and/or a use of denitrifying water in a water body.
99. A polymer support of immobilized microorganisms for use in a method of treating aquatic or marine animals wherein the method comprises: contacting a water body with a polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
100. The polymer support of immobilized microorganisms according to embodiment 99, wherein the polyvinyl alcohol is cross linked with glutaraldehyde. The polymer support of immobilized microorganisms according to embodiment 99 or 100, wherein a covalent bond between glutaraldehyde and polyvinyl alcohol polymeric chains crosslinks the polyvinyl alcohol polymeric chains. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 101, wherein the polyvinyl alcohol polymeric chains consist of 500-3000 monomer units and a molecular weight of 22.000 g/mol to 130.000 g/mol. The polymer support of immobilized microorganismsaccording to any one of embodiments 99 to 102, wherein the crosslink comprises monomeric, dimeric, oligomeric, or polymeric glutaraldehyde. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 103, wherein the crosslinking density, determined as the number of glutaraldehyde crosslinking units, is 8 to 25%, such as 10 to 20%, such as 12-20%, such as about 12%, 13%, 15%, 16%, 17%, 18%, 19% or 20%. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 104, wherein polymer hydrogel comprises from 60 to 99 wt% cross-linked polymeric material and 1 to 40 wt% water or aqueous medium, such as from 60 to 98 wt% cross-linked polymeric material and 2 to 40 wt% water or aqueous medium, such as from 65 to 95 wt% cross-linked polymeric material and 5 to 35 wt% water or aqueous medium, such as from 65 to 90 wt% cross-linked polymeric material and 10 to 35 wt% water or aqueous medium, such as from 65 to 85 wt% cross-linked polymeric material and 15 to 35 wt% water or aqueous medium, such as from 65 to 80 wt% cross-linked polymeric material and 20 to 35 wt% water or aqueous medium, or such as from 65 to 75 wt% cross-linked polymeric material and 25 to 35 wt% water or aqueous medium. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 105, wherein alginate is in monomer or polymeric form, entangled with cross-linked polyvinyl alcohol polymeric chains so as to form an interpenetrating polymer network. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 106, wherein the polymer support is porous. The polymer support of immobilized microorganisms according to embodiment 107, wherein the polymer support comprises micropores. The polymer support of immobilized microorganisms according to embodiment 108, comprising one or more macropores in the central volume of support and micropores. The polymer support of immobilized microorganisms according to embodiment 108 or 109, wherein a portion of the polymer support comprises micropores with an average pore diameter of 5 to 40 microns, such as 5 to 30 microns, such as 5 to 20 microns. The polymer support of immobilized microorganisms according to any one of embodiments 108 to 110, wherein a portion of the polymer support comprises micropores with an average pore diameter of 10 to 40 microns, such as 20 to 40 microns. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 111, wherein the polymeric support is spherical, oval, elliptical bead-shaped, oblong, cylindrical, or capsule-like in shape. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 112, wherein the polymeric support is spherical or bead-shaped having a diameter of 1 to 10 mm, typically 2 to 8 mm, such as 3 to 7 mm or 3 to 6 mm. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 113, wherein the microbial load is 5 g/kg bead to 250 g/kg bead, typically 10 g/kg bead to 150 g/kg bead. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 114, wherein the immobilized microorganisms are selected from the group consisting of one or more sulfur oxidizing microorganisms, one or more ammonium oxidizing microorganisms, one or more nitrite oxidizing microorganisms, one or more denitrifying microorganisms, one or more anammox microorganisms and any mixtures thereof. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 115, wherein the immobilized microorganisms are selected from the group consist ing of a mixed or pure culture of sulfur oxidizing microorganisms, a mixed or pure culture of ammonium oxidizing microorganisms, a mixed or pure culture of nitrite oxidizing micro organisms, a mixed or pure culture of denitrifying microorganisms, a mixed or pure culture of anammox microorganisms and any mixtures thereof. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 116, wherein the immobilized microorganisms are a combination of two or more of the microorganisms selected from the group consisting of: ammonium oxidizing microorganisms, nitrite oxidizing microorganisms, denitrifying microorganisms and sulfur oxidizing microorganisms. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 117, wherein the ammonia oxidizing microorganisms are selected from Nitrosomonas spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp, The polymer support of immobilized microorganisms according to any one of embodiments 115 to 118, wherein the nitrite oxidizing microorganisms are selected from Nitrobacterspp., Nitrococcus spp., Nitrospira spp., and Nitrospina spp. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 119, wherein Paracoccus spp. is used as denitrifying and/or sulfur oxidizing microorganisms. The polymer support of immobilized microorganisms according to embodiment 120, wherein Paracoccus spp. is selected from the group consisting of: P. alcaliphilus, P. alkenifer, P. aminophilus, P. aminovorans, P. carotini facie ns, P. denitrificans, P. kocurii, P. marcusii, P. methylutens, P. pantotrophus, P. solventivorans, P. thiocyanatus, P. versutus, and any combination thereof. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121, wherein the immobilized microorganisms are a combination of the ammonia oxidizing microorganisms Nitrosomonas spp., the nitrite oxidizing microorganisms Nitrobacter spp. and optionally the sulfur oxidizing microorganisms Paracoccus spp. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and a Nitrobacter, Nitrosomonas eutropha and a Paracoccus, Nitrosomonas eutropha and a Nictrobacter and a Paracoccus, Nitrosomonas europaea and a Nitrobacter, Nitrosomonas europaea and a Paracoccus, and of Nitrosomonas europaea and a Nitrobacter and a Paracoccus. The polymer support of immobilized microorganisms according to embodiments 115 to 121, wherein the immobilized microorganisms are selected from the group consisting of a combination of a Nitrosomonas and Nitrobacter winogradskyi, a Paracoccus and Nitrobacter winogradskyi, and, a Nitrosomonas and a Paracoccus and Nitrobacter winogradskyi. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus pantotrophus , a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi and Paracoccus Versutus, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus pantotrophus, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi and Paracoccus Versutus, preferably a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas spp., Nitrobacter spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp., Nitrotoga spp., Nitrospira spp. Pseudomonas spp.;, Paracoccus spp., Hyphomicrobium spp., Castellaniella spp., Janthinobacterium spp., Acidovorax spp., Aeromonas spp., Cellulomonas spp., Buttiauxella spp., Microvirgula spp., Klebsiella spp., Shewanella spp., Pelosinus spp., Variovorax spp., Hydrogenophaga spp., Raoultella spp., Bacillus spp., Achromobacter spp., Ochrobactrum spp., Flavobacterium spp., and Delftia and combinations thereof. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkylphenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomonas hominis, Flavimobis soli, Flavobacterium banpakuense, Buttiauxella agrestis, Buttiauxella noackiae, Achromobacter denitrificans, Pelosinus fermentans, Variovorax dokdonensis, Hydrogenophaga bisanensis, Raoultella terrigena, Delftia lacustris, Shewanella putrefaciens, Acidovorax soli, Hyphomicrobium denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, Microvirgula aerodenitrificans, Candidatus Kuenenia, Candidatus Brocadia,
Candidatus Anammoxoglobus, Candidatus Jettenia, Candidatus Scalindua, and combinations thereof. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus pandtodrophus, Castellaniella defragans, Pseudomonas proteolytica, Paracoccus versutus, Paracoccus denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, and combinations thereof. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 119, wherein the immobilized microorganisms are selected from the group consisting of Nitrosomonas eutropha , Nitrobacter winogradskyi and combinations thereof. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 119, wherein the immobilized microorganisms are selected from the group consisting of a combination of Nitrosomonas eutropha and Nitrobacter winogradskyi, and a combination of Nitrosomonas europaea and Nitrobacter winogradskyi, preferably a combination of Nitrosomonas eutropha, Nitrobacter winogradskyi, and combinations thereof. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , wherein the immobilized microorganisms are selected from the group consisting of Pseudomonas lini, Paracoccus pantotrophus, Paracoccus versutus and combinations thereof. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 127 to 128, wherein the immobilized microorgansims comprise Pseudomo nas lini. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 127 to 128, wherein the immobilized microorgansims comprise Paracoccus pantotrophus. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 128, wherein the immobilized microorgansims comprise Castellaniella defra- gans. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 127 to 128, wherein the immobilized microorgansims comprise Pseudomo nas proteolytica. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 , 27 and 127 to 128, wherein the immobilized microorgansims comprise Para coccus versutus. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 127 to 128, wherein the immobilized microorgansims comprise Paracoccus denitrificans. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 127 to 128, wherein the immobilized microorgansims comprise Nitrosomo- nas eutropha. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 127 to 128, wherein the immobilized microorgansims comprise Nitrosomo- nas europaea. The polymer support of immobilized microorganisms according to any one of embodiments 115 to 121 and 127 to 128, wherein the immobilized microorgansims comprise Nitrobacter Winogradsky. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 140, wherein the polymer support of immobilized microorganisms comprises mixing the polymer support into the water of an aquaculture pond such as aquaculture, farm, pool, pond, paddy, lake, estuary, ocean, or combinations thereof. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 141, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 80% w/v, such as 10% to 80% w/v, such as 15% to 80% w/v of the water. The polymer support of immobilized microorganisms according to embodiment 142, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 30% w/v, such as 10% to 30% w/v, such as 15% to 30% w/v of the water. The polymer support of immobilized microorganisms according to embodiment 142, wherein the polymer support is combined with the water in the water body at a bead load of 60% to 80% w/v, such as 65% to 75% w/v, such as 70% to 75% w/v of the water. The polymer support of immobilized microorganisms according to embodiment 142, wherein the polymer support is combined with the water in the water body at a bead load of 5% to 30% w/v or 60% to 80% w/v of the water. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 145, wherein the water body comprises from 0.5 to 10 mg/I of ammonia, such as 1 to 5 mg/I ammonia, such as 1 to 2 mg/I of ammonia. 147. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 146, wherein the polymer support of immobilized microorganisms is a polymer support of immobilized microorganisms of reducing the amount of ammonia in a water body and/or a polymer support of immobilized microorganisms of denitrifying water in a water body.
148. The method according to any one of embodiments 1 to 49, wherein the water body is contacted with a polymer support of immobilized microorganisms in a recirculating aquaculture system (RAS).
149. The use according to any one of embodiments 50 to 98, wherein the water body is contacted with a polymer support of immobilized microorganisms in a recirculating aquaculture system (RAS).
150. The polymer support of immobilized microorganisms according to any one of embodiments 99 to 147, wherein the water body is contacted with a polymer support of immobilized microorganisms in a recirculating aquaculture system (RAS).
151. A method for the denitrification of a water body comprising adding the polymer support of immobilized microorganisms of the invention.
EXAMPLES
Example 1 - Paracoccus containing polyvinyl alcohol-glutaraldehyde polymer support
13g polyvinyl alcohol (PVA, Poval 15-99) was filled up with 87g tap water and autoclaved for 1h to dissolve the PVA. Then the solution was cooled to room temperature. Additionally, an 8% so dium alginate (Satialgine S60NS) solution was prepared.
40g of the PVA solution, 8g of the sodium alginate solution and 16g of a “Paracoccus” microbe suspension (50g centrifuged cells per L) was mixed and subsequently dropped into a 1% CaCh solution via a peristaltic pump with a pumping speed of 200 g/h. The outlet of the tubes coming from the peristaltic pump had an inner diameter of 1.8mm and the tips were positioned 5-10 cm from the liquid surface.
The dropped polymer/microbe mixture immediately gelated when hitting the CaCh bath. After adding 50g of the mix into 50g of the CaCh bath, the spherical products were separated using a small sieve and washed lightly with tap water before transferring the preformed product back into a glass beaker. On top of these spheres a second cross link solution was added containing 0.25g glutaraldehyde (10% solution), 1.7g sulfuric acid (30% solution), 10g of Na2S04 and 38.05g water. This second cross link solution was heated to 40°C and was kept at this temperature during bead cross linking. After 3 hours of curing the beads were separated from the cross-link solution and washed with a tris buffer for 30 min. After washing, the beads were transferred into cell free water for storage.
Example 2 - Nitrosomonas eutropha and Nitrobacter winogradskyi- containing polyvinyl alcohol-glutaraldehyde polymer support
13g polyvinyl alcohol (PVA, Poval 15-99) was filled up with 87g tap water and autoclaved for 1h to dissolve the PVA. Then the solution was cooled to room temperature. Additionally, an 8% so dium alginate (Satialgine S60NS) solution was prepared.
40g of the PVA solution, 8g of the sodium alginate solution and 16g of a Prawnbac (Novozymes product comprising Nitrosomonas eutropha and Nitrobacter winogradskyi) microbe suspension (500g centrifuged cells per L) was mixed and subsequently dropped into a 1% CaCI2 solution via a peristaltic pump with a pumping speed of 200 g/h. The outlet of the tubes coming from the peristaltic pump had an inner diameter of 1.8mm and the tips were positioned 5-10 cm from the liquid surface.
The dropped polymer/microbe mixture immediately gelated when hitting the CaCI2 bath. After adding 50g of the mix into 50g of the CaCh bath, the spherical products were separated using a small sieve and washed lightly with tap water before transferring the preformed product back into a glass beaker.
On top of these spheres a second cross link solution was added containing 1g glutaraldehyde (10% solution), 1.7g sulfuric acid (30% solution), 10g of Na2S04 and 37.3g water. This second cross link solution was heated to 40°C and was kept at this temperature during Bead cross linking. After 3 hours of curing the beads were separated from the cross-link solution and washed with a tris buffer for 30 min. After washing, the Beads were transferred into cell free water for storage.
Example 3 - Effect of microbe encapsulation on protecting nitrifying microbes from the effects of chemical disinfection
Background:
A key reason for encapsulating microbes in a polymeric biobead (polymer support) is the expec tation that the encapsulation in a polymeric bead will provide an increased level of protection to the microbes within from potentially toxic components in the water being treated when compared to microbes in suspended or fixed film growth in the water. Biofilters in RAS processes are regu larly exposed to chemical disinfectants in order to ensure the biofilter does not become a biosecurity hazard as batches of fish are transferred in and out of the process. Therefore, testing the effect of a chemical disinfectant on the activity of nitrifying biobeads and their ability to recover after exposure to this disinfectant, will provide a suitable model for demonstrating the efficacy of biobeads at protecting the microbes housed within.
Experimental aims:
Demonstrate the efficacy of biobeads to protect the microbes within from prolonged ex posure to 25 mg/L of free chlorine
Demonstrate that biobeads seeded with nitrifying micro-organisms can recover activity to pre-exposure levels within a short period of time
Compare the performance of biobeads seeded with microbes to blank biobeads where nitrification activity comes only from microbes on the surface of the biobeads
Methods:
Biobeads applied o The biobeads utilized in the experiment were of two types
■ Blank biobeads - no microbes were included in the production process
■ Seeded biobeads - microbes from the nitrifying product Prawnbac (Novo- zymes AS) were included in the production
Lab scale reactor operation o Two reactors were operated under identical conditions for a period of 78 days o In the Bead-Blank reactor, blank biobeads were used. In Bead-Seeded reactor, seeded biobeads were used o General operational conditions during trials are outlined in Table 1
■ Tap water was used in the experiments
■ Ammonia was supplied as NH4CI
■ P was supplied as KH2P04
■ Alkalinity was supplied in a ratio of 10:1 compared to NH4-N. Alkalinity was supplied as CaC03
■ pH was adjusted as required by addition of 10% HCI or 10% NaOH
■ A trace elements solution was applied at a concentration of 60 mg/L
Table 1 : Operational conditions for the reactors from Day 1 - 78
Figure imgf000038_0001
Figure imgf000039_0001
o On day 63, the reactors were emptied of water. The reactor was then refilled with a 25 mg/L solution of sodium hypochlorite. The beads were then soaked in this solution for 4 hours. Afterwards, the reactors were drained again and normal op eration was restarted - Analytical methods o Sampling was undertaken two (2) to three (3) times per week by a certified ana lytical laboratory o Standard methods known to one skilled in the art were used to measure the com ponents of the feed water and the water coming from the reactors listed in Table 2
Table 2: List of components analysed in the feed and effluent water from the reactors
Compound Name Unit of measurement
N03 Nitrate mg-N/L
N02 Nitrite mg-N/L
NH4 Ammonia mg-N/L
TN Total Nitrogen mg-N/L
P04 Ortho-phosphate mg-P/L
SCOD Soluble chemical oxygen de mg-02/L mand
TSS Total suspended solids mg/L
Results and discussion:
The concentration of ammonia and nitrite in the influent (feed) and effluent from the reactors was measured frequently throughout the trial period. Figure 1 illustrates the concentration of ammonia in both the influent and effluent of both the blank and seeded reactors. Figure 2 demonstrates the concentration of nitrite in each of the reactors used in this experiment. The dotted line at day 63 indicates where the sodium hypochlorite soaking of the biobeads was undertaken. Biobeads in both reactors were negatively impacted by the soaking in chlorine, with increased levels of am monia and nitrite in each reactor after the soaking on day 63 (Figure 1 and 2). On day 64 (24 hours after exposure to chlorine), the effluent from each reactor was analyzed. In the reactor with seeded biobeads, the NH4-N concentration had fallen to 1.2 mg NH4-N/L while in the blank bi obead reactor it had fallen to only 2.6 mg NH4-N/L. Interestingly, while the blank biobeads showed some level of NH4-N removal after disinfection, most of the NH4-N was only partially nitrified and converted to N02-N rather than N03-N. Such a peak in N02-N post-disinfection was not meas ured in the seeded biobead reactor.
The results show that the seeded biobead reactor was able to recover sufficient activity that it would be able to safely support any fish in the RAS process, while the blank biobead reactor would not be able to. Surprisingly, over the next 14 days, the blank biobeads never recovered the same level of nitrification activity they had before disinfection, with only partial nitrification taking place and increasingly high levels of N02-N being measured in the effluent. Again, no such issues with excessive N02-N generation were seen in the seeded biobead reactor, with over 97% of the NH4-N removed in the reactor converted to N03-N rather than N02-N. The results from this experiment confirm that the nitrifiers in the seeded biobeads were protected from the chlorine soaking and remained active and able to support fish populations in a RAS. However, the uncon trolled growth on the surface of the biobeads that supported the nitrification activity of these beads was severely adversely impacted by the chlorine soaking and would not be able to support any fish in the RAS for at least 14 days after disinfection.
The exposure to the chlorine did not impact the physical stability of the biobeads (blank or seeded) in any way.
Conclusions:
The experiment presented clearly demonstrates that the biobeads were able to provide a protec tive environment for microbes that were integrated as part of the production process of the bi obeads. While microbes were able to colonize the outer surfaces of the blank biobeads and pro vide nitrification activity, these microbes were not protected against exposure to disinfectants, toxins or inhibitors as those microbes that were integrated into the biobeads.
RAS aquaculture processes often apply chemical disinfection as a means of preventing biosecu rity risks during the production of the fish in the process. The ability of the seeded biobead reactor to re-start after disinfection and deliver water treatment that would allow the RAS to be re-stocked with fish presents a significant benefit for the producer as the lag time between production batches could be reduced to 24 hours. This is in contrast to the blank biobeads (or other biofilter technology that uses biofilms) where the nitrification process did not recover within the 14 days after disinfection and therefore not allowing the RAS system to restart fish production.
Example 4 - Effect microbe encapsulation on protecting denitrification microbes from the effects of exposure to free chlorine
Background:
A key reason for encapsulating microbes in a polymeric biobead is the expectation that the en capsulation in a polymeric bead will provide an increased level of protection to the microbes within from potentially toxic components in the water being treated when compared to microbes in sus pended or fixed film growth in the water. Biofilters in RAS processes are regularly exposed to chemical disinfectants in order to ensure the biofilter does not become a biosecurity hazard as batches of fish are transferred in and out of the process. Therefore, testing the effect of a chemical disinfectant on the activity of nitrifying biobeads and their ability to recover after exposure to this disinfectant, will provide a suitable model for demonstrating the efficacy of biobeads at protecting the microbes housed within.
Experimental aims:
Demonstrate the efficacy of biobeads to protect the microbes within from prolonged ex posure to 5 mg/L of free chlorine
Demonstrate that biobeads seeded with the denitrifying micro-organism Paracoccus Pantotrophus can recover activity to pre-exposre levels within a short period of time Compare the performance of biobeads seeded with microbes to blank biobeads where denitrification activity comes only from microbes on the surface of the biobeads
Methods:
Biobeads applied o The biobeads utilized in the experiment were of two types
■ Blank biobeads (R37) - no microbes were included in the production pro cess
■ Seeded biobeads (R38) - Paracoccus Pantotrophus microbes were in cluded in the production process
Lab scale reactor operation o Two reactors were operated under identical conditions for a period of 129 days o In R37, blank biobeads were used. In R38, seeded biobeads were used o General operational conditions during trials are outlined in Table 3
■ Tap water was used in the experiments
■ Nitrate was supplied as NaN03
■ BOD was supplied as a 50:50 (on a BOD basis) mixture of sodium ace tate and acetic acid
■ P was supplied as KH2P04
■ pH was adjusted as required by addition of 10% HCI
■ A trace elements solution was applied at a concentration of 60 mg/L
Table 3: Operational conditions for the reactors from Day 1 - 21
Figure imgf000042_0001
o Chlorine was added as part of the reactor feed from day 14 - 17 as sodium hypo chlorite at a concentration of 5 mg/L of chlorine - Analytical methods o Sampling was undertaken two (2) to three (3) times per week by a certified ana lytical laboratory o Standard methods known to one skilled in the art were used to measure the com ponents of the feed water and the water coming from the reactors listed in Table 4
Table 4: List of components analysed in the feed and effluent water from the reactors
Compound Name Unit of measurement
N03 Nitrate mg-N/L
N02 Nitrite mg-N/L
NH4 Ammonia mg-N/L
TN Total Nitrogen mg-N/L
P04 Ortho-phosphate mg-P/L
SCOD Soluble chemical oxygen de mg-02/L mand
TSS Total suspended solids mg/L Results and discussion:
The concentration of nitrate and nitrite in the influent (feed) and effluent from the reactors was measured frequently throughout the trial period. Figure 3 illustrates the concentration of total ni trogen (nitrate + nitrite) in both the influent and effluent of both R37 and R38. Figure 4 demon strates the specific total nitrogen (TN) removal rate for the biobeads in both R37 and R38. The dotted lines at day 14 indicates where the sodium hypochlorite started being added to the reactor feed, with the second dashed lines at day 17 indicating where the addition of sodium hypochlorite stopped. Biobeads in both reactors were negatively impacted by the addition of the chlorine, with increased levels of total nitrogen (TN) in the effluent of both reactors (Figure 3). After one day of exposure to the chlorine, the blank beads in R37 lost all denitrification activity, with this activity not recovering even after stopping the exposure to chlorine at day 17. In R38, the seeded beads only lost approximately 12% of their TN removal activity after one day of exposure, and 37% of activity after three days of exposure. After the addition of chlorine to the reactors was stopped at day 17, the blank biobeads in R37 did not recover any activity after 4 days. However, the seeded biobeads in R38 recovered a significant proportion of the activity lost during the exposure to chlo rine, with and expectation that a full recovery to pre-exposure levels of activity would occur within seven days.
The exposure to the chlorine did not impact the physical stability of the biobeads (blank or seeded) in any way.
Conclusions:
The experiment presented clearly demonstrates that the biobeads were able to provide a protec tive environment for microbes that had been integrated as part of the production process of the biobeads. While microbes were able to colonize the outer surfaces of blank (or not seeded) bi obeads and provide denitrification activity, these microbes were not protected against exposure to toxins or inhibitors like those that were integrated into the biobeads.
Biological nitrogen removal processes were often exposed to toxins and inhibitors, with chlorine being one of the most effective microbial contamination control chemicals used, with chlorine used to ensure the disinfection of water by killing any suspended microbes in the water. This experiment has demonstrated that a biological nitrogen removal process utilizing biobeads with integrated microbes would be resistant to the impact of chlorine (or other toxin) exposure and would recover its nitrogen removal activity in a very short period of time. This would have signifi cant value for any operator using a biobead system as the biobeads would ensure toxic exposure would have a minimal impact on TN removal compared to a comparable suspended or fixed film system. Example 5 - Efficacy of biobeads for total nitrogen (TN) removal from water at low hydraulic retention time (HRT) conditions
Background:
While the focus for RAS systems is the generally the removal of ammonia in the recirculated water, nitrification of the ammonia leads to the production of nitrate. While nitrate is significantly less toxic to fish and other aquatic life compared to ammonia, it can still pose a fish health and environmental challenge for RAS installations. With increasing pressure on RAS systems to be neutral net water consumers, there is an increasing focus on integrating denitrification biofilters into RAS systems in order to remove the nitrate and allow for the closing of the water cycle. Such a denitrification biofilter would need to operate at low hydraulic retention times (HRT) and effi ciently convert the nitrate to nitrogen gas without producing toxic nitrite as a by-product. This study is looking to investigate whether a biobead denitrification biofilter could fulfil the demands that a RAS process would have for such a biofilter.
Experimental aims:
Demonstrate the efficacy of biobeads seeded with a known denitrifying micro-organism ( Paracoccus pantrophus) to achieve an effluent concentration of Total Nitrogen (TN) < 2.0 mg-N/L at an operational hydraulic retention time (HRT) of 30 minutes Demonstrate that biobeads seeded with the known denitrifying micro-organism have su perior TN treatment capabilities and lower nitrite production compared to biobeads that have not been seeded with the denitrifying microbes during production
Methods:
Biobeads applied o The biobeads utilized in the experiment were of two types
■ Blank biobeads (R37) - no microbes were included in the production pro cess
■ Seeded biobeads (R38) - Paracoccus Pantotrophus microbes were in cluded in the production process
Lab scale reactor operation o Two reactors were operated under identical conditions for a period of 129 days o In R37, blank biobeads were used. In R38, seeded biobeads were used o General operational conditions at the start of the trials are outlined in Table 5
■ Tap water was used in the experiments
■ Nitrate was supplied as NaN03
■ BOD was supplied as a 50:50 (on a BOD basis) mixture of sodium ace tate and acetic acid
■ P was supplied as KH2P04
■ pH was adjusted as required by addition of 10% HCI
■ A trace elements solution was applied at a concentration of 60 mg/L
Table 5: Operational conditions for the reactors from Day 1 - 45
Figure imgf000045_0001
o The operational conditions were adjusted after 45 days of continual operation as shown in Table 6
Table 6: Operational conditions for the reactors from Day 46 - 129
Figure imgf000045_0002
- Analytical methods o Sampling was undertaken three (3) times per week by a certified analytical labor atory o Standard methods known to one skilled in the art were used to measure the com ponents of the feed water and the water coming from the reactors listed in Table 7
Table 7: List of components analysed in the feed and effluent water from the reactors Compound Name Unit of measurement
N03 Nitrate mg-N/L N02 Nitrite mg-N/L
NH4 Ammonia mg-N/L
TN Total Nitrogen mg-N/L
P04 Ortho-phosphate mg-P/L
SCOD Soluble chemical oxygen de- mg-02/L mand
TSS Total suspended solids mg/L
Results and discussion:
The concentration of nitrate and nitrite in the influent (feed) and effluent from the reactors was measured frequently throughout the trial period. Figure 5 illustrates the concentration of total ni trogen (nitrate + nitrite) in both the influent and effluent of both R37 and R38. The dotted line at day 45 indicates where the biobead loading (% w/v) was increased from 10% to 20%. The seeded biobeads demonstrated an ability to remove TN immediately, while it took the blank beads at least 10 days to demonstrate any TN removal activity. However, after 27 days of operation, both the blank and seeded biobead reactors were achieving similar results. In both reactors, the effluent concentration was approximately 6 mg-N/L, which was at least 3x higher than was the objective. At day 45, more biobeads were added to each reactor to increase the bead load from 10% to 20% w/v. This increase in bead load in the reactors led to an immediate drop in the effluent concen tration of TN, most markedly in the seeded biobead reactor (R38). By day 64, the seeded biobead reactor (R38) had achieved an effluent concentration of < 2 mg-N/L, and had largely maintained this level for the following 65 days of operation, illustrating how stable a biobead process can operate in contrast to a conventional biofilm type biofilter (represented by R37). This decrease in TN concentration achieved in R38 after the increase in biobead load in the reactor is due to the increase in active denitrifying microbes in the reactor due to the addition of the extra biobeads. This then allows the biobeads to overcome the limitations of first order kinetics in the continuously stirred reactor and remove more TN from the system. This positive effect of adding more active denitrifying microbes in the biobeads to the reactor is highlighted by the fact that the reactor with blank biobeads (R37) have never demonstrated an effluent concentration of < 2 mg-N/L. After an initial sharp decrease in effluent TN concentration after day 45, R37 had maintained an effluent TN concentration that is generally 2x higher than the effluent TN concentration achieved by the seeded biobeads in R38.
The specific activity (mg-N removed per kilogram of biobeads per hour(mg-N/kg.hr)) of the bi obeads closely reflects the effluent TN concentrations achieved and is illustrated in Figure 6. The specific TN loading rate and biobeads activities for R37 and R38 are illustrated in Figure 7. Again, it is clear that the seeded biobeads in R38 demonstrated a sharply increasing rate of TN removal from day 1. This rate of TN removal stabilized after approximately 20 days and remained consistent at 70-80 mg-N/kg.hr from day 31 to day 129. The increase in biobead load in the reac tor did not impact the specific activity of the biobeads in R38 as the amount of TN removed in the reactor increased sharply with the addition of the extra biobeads (as shown in Figure 6). The blank biobeads in R37 did not demonstrate significant activity until approximately day 20, and then remained steady at that level of 20 mg-N/kg.hr until day 66. At this point, the activity in creased steadily to a maximum of 60 mg-N/kg.hr on day 90. This increasing level of activity is likely caused by the increased surface area that adding the extra biobeads on day 45 provided to colonizing dentifiers in the process. The lag in increasing activity is due to the time taken for the colonizing denitrifiers to grow and populate the new beads. At the same time, the increased bi obead load also leads to less turbulence in the reactor, which means the biobeads were not con tacting each other with the same energy as when the biobead load was only 10% w/v. This results in more stable growth conditions for surface colonizing biofilms.
While nitrate removal is the primary objective of denitrification biofilter processes, this must be achieved with minimal production of nitrite (N02) during the process. While fish can tolerate very high concentrations of nitrate, nitrite should be maintained at a level of < 0.5 mg N02-N/L to avoid impacting fish health. Therefore, the formation of nitrite as a by-product in the nitrate removal process was monitored in both reactors throughout the trial. The proportion of the N03 removed that was converted to nitrite and left the reactor in the effluent is illustrated in Figure 7. The results indicate that initially in both reactors the denitrification process often stalled at nitrite. This is likely due to the initial very high loading of nitrate the biobeads were exposed to in both reactors over whelmed the microbial mass that was present. This is illustrated clearly as the relative production of nitrite from nitrate was significantly higher in the blank biobeads (R37) where the denitrifying biomass was much lower than in the seeded biobeads (R38). The seeded biobeads were also able to recover much more quickly than the blank biobeads, achieving less than 10% incomplete denitification after only 31 days, while the blank biobeads were first able to achieve this after 70 days. The benefits of seeding the biobeads with specific denitrifying biomass was clearly demon strated by these results, especially when looking towards RAS denitrification biofilter applications.
In both reactors, biomass production was evidenced and measured. The results are presented in Table 8. The results indicate that the biobeads did not irreversibly withhold the biomass that was immobilized within the beads. The seeded biobeads in R38 had a TSS production that was on average 50% higher than the blank biobeads in R37. This indicates that the presence of a signif icantly amount of biomass that has a high activity will lead to increased biomass production. How ever, the biomass production was significantly lower than what would be expected in a compara ble activated sludge or even moving bed biofilm reactor (MBBR) process, indicating that the im mobilization of the denitrifiying microbes in the biobeads may have lead to a lower specific bio mass yield compared to competing biological denitrification processes. The biomass production measured was highly variable, with significant standard deviations in both reactors. This is most likely caused by the difficulties in measuring such low concentrations of TSS in lab scale systems and changes in the biobeads themselves over time (for example, as the seeded microbes grow in the seeded biobeads, they will eventually fill the biobead and therefore the biobeads will excrete more biomass after the biobeads are filled).
Table 8: Average TSS concentrations in the reactors over the 129 day period of continuous op eration
Reactor Average TSS St Dev.
(mg/L) (mg/L)
R37 - Blank 4 3
R38 - Seeded 6 4
The biobeads in both reactors were sampled for fluorescent in-situ hybridization (FISH) analysis to identify whether the microbes originally seeded in the biobeads were still present and dominant and whether other microbes had been able to colonise the biobeads during operation. A qualita tive assessment of the presence of Paracoccus pantrophus was performed on the beads in reac tors R37 and R38 on day 44, and the results are presented in Table 9.
Table 9: Qualitative FISH results indicating the dominance of Paracoccus pantrophus in the bi obeads from R37 and R38 after 44 days of continuous operation
R37 - Blank R38 - seeded
Paracoccus pan- x xxx trophus x = 0 - 10% of visible bacteria; xx = 11-20% of visible bacteria; xxx = 20-50% of visible bacteria; xxxx = >50% of visible bacteria
The application of biobeads in a completely mixed reactor results in first order kinetics of reaction between the increasingly low concentration of nitrate and the microbes in the biobeads limiting the HRT that can be applied. This results in longer HRTs to achieve lower nitrate effluent concen trations, thereby increasing the reactor size and therefore capital and operational costs for apply ing biofilters. One pathway to overcome the first order kinetics limitations is to apply the biobeads in a plug-flow type reactor. At the end of the trial outlined above, the biobeads from R38 were transferred to a fluidized bed type reactor. The operational specifics of this reactor is shown in Table 10.
Table 10: Operational conditions for the denitrifying biobead plug-flow reactor
Figure imgf000048_0001
Figure imgf000049_0001
The results from the trials are presented in Figure 8. After some initial challenges operating the plug-flow reactor, after 6 days of the operation, the level of nitrate removal was similar to that achieved in the completely mixed reactors trialed previously although the plug-flow reactor was operating with a HRT that was 4x shorter than the completely mixed reactors. Further optimization is required due to the higher production of nitrite seen the reactor, but the concept of applying biobeads in a plug-flow system shows merit and has the potential to further increase the compet itiveness of the biobeads against existing RAS biofilter technologies.
Conclusions:
The experiments clearly demonstrated the advantages of applying seeded biobeads in a biologi cal nitrate removal system for water recirculating in a RAS process. Under commercially applica ble conditions, the seeded biobeads in R38 were able to achieve the goal of an effluent concen tration of < 2 mg-N/L of total nitrogen (nitrate and nitrite), while limiting the production of nitrite to a level where it is acceptable for the safe use of the process in a RAS process. The performance of the beads demonstrated that a biobead denitrification biofilter would be a preferable process for nitrate removal in a RAS, thereby reducing significantly the water demand for RAS processes.
The bead load in the reactors was required to be at least 20% w/v in order to overcome the first order kinetics of the continuously stirred reactor system. However, through the application of the biobeads in a novel plug-flow reactor system, the limitations of the first order kinetics could be overcome, and similar levels of performance were achieved with only ¼ of the HRT used in the completely mixed reactors. The biobeads produced TSS, indicating that the biomass seeded into the biobeads is not irreversibly retained in the biobeads, although the mass of biomass produced was significantly lower than could be expected from an activated sludge process treating the same water. And although the biobeads did not demonstrate that the seeded biomass was irreversibly retained in the biobeads, FISH analysis confirmed that the seeded microbes still dominated the microbial community in the biobeads after 44 days of continuous operation. Example 6 - Carbon source utilization in a TN removal biobead reactor through the application of unique biotechnology
Background:
The removal of total nitrogen (TN) is required in RAS operators want to decrease the level of make up water required to operate a RAS process. This can be achieved through a biological process utilizing denitrifying microbes. The process of denitrification requires the oxidation of a carbon source to act as an electron donor to drive the conversion of nitrate (N03) and nitrate (N02) to nitrogen gas (N2). Common carbon sources used for denitrification are organic materials in the RAS water, and external carbon sources such as methanol, ethanol, glycol, molasses and other easily bio-degradable substances. However, the use of these external carbon sources rep resents a potentially significant cost for TN removal. Therefore, the integration of a denitrifying microbe that can consume less carbon per unit of TN removed represents a significant commer cial opportunity as it could significantly reduce the operational expenses of a biological TN re moval system in a RAS process.
Experimental aims:
Demonstrate the ability to immobilize a carbon source efficient denitrifier ( Pseudomonas lini) into a biobead while maintaining low carbon source consumption denitrification ca pability
Compare the denitrification capabilities of the carbon source efficient denitrifier with a more traditional denitrifying microbe (Paracoccus pantrophus)
Method:
Biobeads applied o The biobeads utilized in the experiment were of two types
■ R45 - Seeded biobeads containing P. lini microbes
■ R38 - Seeded biobeads containing Paracoccus Pantotrophus microbes Lab scale reactor operation o Two reactors were operated under identical conditions for a period of 25 days o In R38, biobeads seeded with Paracoccus pantrophus were used. In R45, bi obeads seeded with were Pseudomonas lini used o General operational conditions at the start of the trials are outlined in Table 11
■ Tap water was used in the experiments
■ Nitrate was supplied as NaN03 ■ BOD was supplied as a 50:50 (on a BOD basis) mixture of sodium ace tate and acetic acid
■ P was supplied as KH2P04
■ pH was adjusted as required by addition of 10% HCI
■ A trace elements solution was applied at a concentration of 60 mg/L
Table 11 : Operational conditions for the reactors from Day 1 - 25
Figure imgf000051_0001
- Analytical methods o Sampling was undertaken three (3) times per week by a certified analytical labor atory o Standard methods known to one skilled in the art were used to measure the com ponents of the feed water and the water coming from the reactors listed in Table 12
Table 12: List of components analysed in the feed and effluent water from the reactors
Compound Name Unit of measurement
N03 Nitrate mg-N/L
N02 Nitrite mg-N/L
NH4 Ammonia mg-N/L
TN Total Nitrogen mg-N/L
P04 Ortho-phosphate mg-P/L
SCOD Soluble chemical oxygen de mg-02/L mand
TSS Total suspended solids mg/L
Results and discussion:
The reactors were started up using identical operational conditions as outlined in Table 13. The concentration of total nitrogen (TN) was monitored in the influent and effluent of the reactors (Figure 9A). Initially, the two reactors had an effluent TN concentration that was decreasing at a similar rate over time. However, from day 6 onwards, the biobeads in R45 were able to achieve a lower effluent TN concentration compared to the biobeads in R38. This was also reflected in the specific activity of the biobeads (mg-N removed per kg of biobeads per hr (mg-N/kg.hr)) as illustrated in Figure 9B. The biobeads in R45 demonstrated from an early stage a significantly higher rate of TN removal, eventually achieving a rate that was approximately 50% higher than the biobeads in R38.
The significantly higher TN removal activity evidenced in R45 was due to the higher conversion of nitrate to nitrogen gas (complete denitrification) by the biobeads seeded with Pseudomonas Uni (R45) compared to those seeded with Paracoccus pantrophus (R38). This is illustrated in Figure 10, where it is clear the biobeads in R45 have a higher complete denitrification capability compared to the biobeads in R38. This is important, as nitrite is a component of TN measure ments and is toxic to fish, and therefore converting nitrate to nitrite is not useful for meeting TN treatment objectives in a RAS process. This result further illustrates that the biobeads in R45 have a denitrification microbe that is significantly more efficient at utilizing carbon sources for denitrifi cation than the denitrifiers integrated into the biobeads in R38.
It would be expected that the lower rates of TN removal and lower conversion of nitrate to nitrogen gas would mean the carbon source consumption in R38 would be significantly lower than that in R45. However, surprisingly, this was not the case. In Table 13, the average consumption of COD per unit of TN removed is shown. The results show that the biobeads with the Pseudomonas lini (R45) were on average 28% more efficient at removing TN compared to the biobeads with Para coccus pantrophus (R38). This represents a potential saving of 28% on external carbon source required by a TN treatment plant to achieve denitrification, which has significant commercial ad vantages for the biobead system operator.
Table 13: Average consumption of COD per unit of TN removed in each of the reactors R38 and R45 over a period of 25 days of continuous operation
Figure imgf000052_0001
Conclusions:
The experiment demonstrated that unique microbes with specific commercial advantageous ca pabilities could be successfully integrated into the biobeads while maintaining the specific advan tageous capabilities. In this case, the Pseudomonas lini denitrifying microorganism was integrated into the biobeads. The expected advantages of high denitrification activity and significantly lower carbon source consumption were realized. The average 28% lower carbon source consumption while achieving higher rates of denitrification can have significant financial and competitive ad vantages for RAS biofilter operators that utilize Pseudomonas Uni in a biobead.
Example 7 - Efficacy of biobeads for total ammonia nitrogen (TAN) treatment from water
Background:
Ammonia is a highly problematic pollutant in RAS as it is toxic to fish and aquatic animals. There fore, a key objective of a RAS biofilter is the removal of ammonia from the water to maintain a high level of fish health and productivity in the RAS. Normally this is done using biological water treatment methods, where nitrifying bacteria are exploited to convert ammonia to nitrite and then nitrate. Nitrification bacteria are generally recognized as being sensitive to process changes and relatively slow growing compared to other bacteria in the water treatment process. For example, in an MBBR RAS process, it can take up to 100 days to achieve enough nitrification activity to allow for fish stocking in the RAS. This represents a significant loss in potential productivity. Also, in general the total biomass that could be classified as nitrifiers is generally less than 10% in such a process. Therefore, nitrification processes can be relatively easily impacted negatively by tox ins, changing process conditions or poor operation leading to a loss of nitrification capacity. Given the relatively slow growth rate of nitrifiers, the recovery time after such an upset may be over a period of weeks rather than days. With this knowledge, it is clear that there would be clear ad vantages to developing a nitrification process that utilized encapsulated nitrifiers (biobeads). In such a system, the only biomass in the process would be nitrifiers as this is what is being added to biobeads, allowing for a significant increase in the concentration of nitrifiers in the treatment process and increasing the oxygen utilization efficiency of the process as only nitrifiers would be consuming dissolved oxygen. The biobeads also provide an environment where the nitrifiers are protected from process changes, toxins and other shocks that would normally have a strong neg ative impact on the nitrification capacity of the treatment process.
Experimental aims:
Demonstrate the efficacy of biobeads seeded with a known nitrifying consortia (Prawn- bac, Novozymes AS) in a system treating water with a loading rate of ammonia that rep resents a high loading rate for a RAS process (720 mg-N/L.d)
Method:
Biobeads applied o The biobeads utilized in the experiment were of two types ■ M06 - Blank biobeads without microbes
■ M07 - Seeded biobeads containing Prawnbac nitrifier product (Novo- zymes A/S)
Lab scale reactor operation o Two reactors were operated under identical conditions for a period of 21 days o In M06, blank biobeads were used. In M07, biobeads seeded with Prawnbac (Novozymes AS) nitrification product were used o General operational conditions at the start of the trials are outlined in Table 14
■ Tap water was used in the experiments
■ Ammonia was supplied as NH4CI
■ P was supplied as KH2P04
■ Alkalinity was supplied in a ratio of 10:1 compared to NH4-N. Alkalinity was supplied as CaC03
■ pH was adjusted as required by addition of 10% HCI or 10% NaOH
■ A trace elements solution was applied at a concentration of 60 mg/L
Table 14: Operational conditions for the reactors from Day 1 - 21
Figure imgf000054_0001
- Analytical methods o Sampling was undertaken three (3) times per week by a certified analytical labor atory o Standard methods known to one skilled in the art were used to measure the com ponents of the feed water and the water coming from the reactors listed in Table
15
Table 15: List of components analysed in the feed and effluent water from the reactors
Compound Name Unit of measurement
N03 Nitrate mg-N/L
N02 Nitrite mg-N/L
NH4 Ammonia mg-N/L
TN Total Nitrogen mg-N/L P04 Ortho-phosphate mg-P/L
SCOD Soluble chemical oxygen de mg-02/L mand
TSS Total suspended solids mg/L
Results and discussion:
The reactors were started up using identical conditions. There was a significant lag period for both reactors with relatively similar and stable levels of specific ammonia treatment rates (mg- N/kg.hr) (Figure 11) and ammonia effluent concentration (mg-N/L) (Figure 12). From day 9, there was a divergence in performance, with the seeded biobead reactor M07 exhibiting increasingly high specific ammonia treatment rates and a decreasing ammonia concentration in the effluent compared to the blank biobeads in reactor M06. After 21 days of continuous operation, the seeded biobeads in M07 delivered 40% higher specific ammonia treatment rate compared to the blank beads in M06. Similarly, the seeded biobeads were removing 73% of the ammonia fed to the reactor, while the blank biobeads were only treating 48% of the influent ammonia.
While the performance of the two reactors was similar with regards to ammonia treatment, it was clear from the start of the trials that the seeded biobeads had a significantly greater capacity to complete the nitrification process (convert ammonia to nitrate) as illustrated in Figure 13. After 9 days of operation, the difference between the nitrification capacity of the seeded and blank bi obeads was reduced. This is likely due to the ability of the blank beads to build up a more balanced colonizing community of nitrifiers that were able to more effectively convert ammonia to nitrate. However, at no time during the trial were the blank biobeads able to demonstrate an ability to achieve full nitrification of the ammonia as effectively as the seeded biobeads. This clearly demon strated the value of utilizing seeded biobeads compared to blank biobeads or carriers to achieve an efficient nitrification process.
Conclusions:
The results indicate that there is a clear advantage to integrating a strong nitrification microbial community into a biobead system. The seeded biobeads were able to demonstrate an ability to achieve 60% removal of ammonia within 5 days of operation, while at the same time achieving over 85% conversion of ammonia to nitrate compared to a system relying solely on a naturally occurring nitrification biofilm. This indicates that the seeding of biobeads with nitrifiers has the potential to provide significant advantages over current technologies applied in biological nitrifi cation processes due to the ability of the biobeads to achieve high levels of ammonia removal and conversion to nitrate within a very short period and under challenging operational conditions (high ammonia loading on the reactor). At no time during the trials, were the blank beads able to achieve the same results or performance as the seeded biobeads, further demonstrating the ad vantage the seeding of biobeads provides compared to traditional biofilm carrier based technolo gies that rely on colonization of the surface of carriers (in this case the blank biobeads) by indig enous nitrifying communities. The conditions in the current trials were not optimized for RAS op erations, but the results do illustrate that there is significant potential for seeded biobeads to be a commercially attractive alternative for biological nitrification in RAS biofilters compared to tradi tional, existing processes and technologies.

Claims

1. A polymer support of immobilized microorganisms for use in a method of treating aquatic or marine animals wherein the method comprises: contacting a water body with a polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
2. The polymer support of immobilized microorganisms according to claim 1, wherein the polyvinyl alcohol is cross linked with glutaraldehyde.
3. The polymer support of immobilized microorganisms according to claim 1 or 2, wherein a covalent bond between glutaraldehyde and polyvinyl alcohol polymeric chains crosslinks the polyvinyl alcohol polymeric chains.
4. The polymer support of immobilized microorganisms according to any one of claims 1 to 3, wherein the polymer support is porous.
5. The polymer support of immobilized microorganisms according to any one of claims 1 to 4, wherein the immobilized microorganisms are selected from the group consisting of one or more sulfur oxidizing microorganisms, one or more ammonium oxidizing microorganisms, one or more nitrite oxidizing microorganisms, one or more denitrifying microorganisms, one or more anammox microorganisms and any mixtures thereof.
6. The polymer support of immobilized microorganisms according to claim 5, wherein the ammonia oxidizing microorganisms are selected from Nitrosomonas spp., Nitrosococcus spp., Nitrosospira spp., Nitrosolobus spp., and Nitrosovibrio spp,
7. The polymer support of immobilized microorganisms according to claim 5 or 6, wherein the nitrite oxidizing microorganisms are selected from Nitrobacter spp., Nitrococcus spp., Nitrospira spp., and Nitrospina spp.
8. The polymer support of immobilized microorganisms according to any one of claims 5 to 7, wherein Paracoccus spp. is used as denitrifying and/or sulfur oxidizing microorganisms.
9. The polymer support of immobilized microorganisms according to any one of claims 5 to 8, wherein the immobilized microorganisms are selected from the group consisting of Par acoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlorora- phis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkylphenolica, Pseudo monas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum in termedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hy- drophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomonas hominis, Fla- vimobis soli, Flavobacterium banpakuense, Buttiauxella agrestis, Buttiauxella noackiae, Achromobacter denitrificans, Pelosinus fermentans, Variovorax dokdonensis, Hy- drogenophaga bisanensis, Raoultella terrigena, Delftia lacustris, Shewanella putrefa- ciens, Acidovorax soli, Hyphomicrobium denitrificans, Nitrosomonas eutropha, Nitrosomo- nas europaea, Nitrobacter Winogradsky, Microvirgula aerodenitrificans, Candida- tus Kuenenia, Candidatus Brocadia, Candidatus Anammoxoglobus, Candidatus Jettenia, Candidatus Scalindua, and combinations thereof.
10. A method of treating aquatic or marine animals comprising: contacting a water body with a polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
11. The method according to claim 10, wherein the polyvinyl alcohol is cross linked with glutaraldehyde.
12. The method according to claim 10 or 11 , wherein a covalent bond between glutaraldehyde and polyvinyl alcohol polymeric chains crosslinks the polyvinyl alcohol polymeric chains.
13. The method according to any one of claims 10 to 12, wherein the immobilized microorganisms are selected from the group consisting of one or more sulfur oxidizing microorganisms, one or more ammonium oxidizing microorganisms, one or more nitrite oxidizing microorganisms, one or more denitrifying microorganisms, one or more anammox microorganisms and any mixtures thereof.
14. The method according to claim 13, wherein the immobilized microorganisms are selected from the group consisting of Paracoccus pantotrophus, Paracoccus versutus, Paracoccus denitrificans, Castellaniella defragrans, Pseudomonas proteolytica, Pseudomonas alcaliphila, Pseudomonas chlororaphis, Pseudomonas monteilii, Pseudomonas lini, Pseudomonas alkylphenolica, Pseudomonas nitroreducens, Pseudomonas stutzeri, Ochrobactrum anthropic, Ochrobactrum intermedium, Aeromonas media, Aeromonas veronii, Aeromonas jandaei, Aeromonas hydrophila, Cellulomonas chitinilytica, Cellulomonas cellasea, Cellulomonas hominis, Flavimobis soli, Flavobacterium banpakuense, Buttiauxella agrestis, Buttiauxella noackiae, Achromobacter denitrificans, Pelosinus fermentans, Variovorax dokdonensis, Hydrogenophaga bisanensis, Raoultella terrigena, Delftia lacustris, Shewanella putrefaciens, Acidovorax soli, Hyphomicrobium denitrificans, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrobacter Winogradsky, Microvirgula aerodenitrificans, Candidatus Kuenenia, Candidatus Brocadia,
Candidatus Anammoxoglobus, Candidatus Jettenia, Candidatus Scalindua, and combinations thereof.
15. Use of a polymer support of immobilized microorganisms for treating aquatic or ma rine animals comprising: contacting a water body with said polymer support of immobilized microorganisms; wherein the polymer support comprises said immobilized microorganisms immobilized within a polymer hydrogel; wherein the polymer hydrogel comprises a cross-linked polymeric material and water or an aqueous medium; wherein the cross-linked polymeric material is a cross-linked polymer comprising polyvinyl alcohol and alginate.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0861808A2 (en) * 1997-02-28 1998-09-02 Kuraray Co., Ltd. Waste water treatment apparatus
CN108217936A (en) * 2018-02-26 2018-06-29 扬州市职业大学 A kind of nitrosation-anaerobic ammoxidation immobilization and cultivating wastewater purification technique
CN109402105A (en) * 2018-10-15 2019-03-01 山东交通学院 A kind of microbe carrier that floatability is degradable and preparation method and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0861808A2 (en) * 1997-02-28 1998-09-02 Kuraray Co., Ltd. Waste water treatment apparatus
CN108217936A (en) * 2018-02-26 2018-06-29 扬州市职业大学 A kind of nitrosation-anaerobic ammoxidation immobilization and cultivating wastewater purification technique
CN109402105A (en) * 2018-10-15 2019-03-01 山东交通学院 A kind of microbe carrier that floatability is degradable and preparation method and application

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