IRON-FUNCTIONALIZED ALGINATE FOR PHOSPHATE AND OTHER CONTAMINANT REMOVAL AND RECOVERY FROM AQUEOUS SOLUTIONS

This application claims the benefit of U.S. Provisional Application Serial No.

61/791,202, filed March 15, 2013, which is incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Grant No. CMMI-1125674 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates, generally, to biodegradable materials, including iron- crosslinked alginate, that are useful as a remediation agent for eutrophication caused by environmental contaminants such as phosphate.

BACKGROUND

Eutrophication of lakes and other natural bodies of water, caused by the presence of excess nutrients, is a growing problem. Phosphate is delivered to surface and ground water as a result of agricultural and feedlot run-offs, and municipal and industrial wastewaters.

Treatment of domestic and agro-industrial wastewater often releases large amounts of phosphorus and nitrogen into water. Excess phosphorous concentration (>1.0 mg/L P) in water bodies causes eutrophication of aquatic ecosystems, which results in deterioration of water quality (Smith, 2003, Environ Sci Poll Res 10:126-139). Therefore, it is important to reduce phosphorous concentrations in water to improve water quality.

On the other hand, phosphorus is essential for plant growth and is an important constituent of agricultural fertilizers. Phosphorous is typically obtained by mining inorganic phosphate rock, such as apatite, followed by chemical treatment to produce phosphoric acid, thereby generating phosphate. These natural supplies of inorganic phosphate are, however, diminishing. With increasing world population the demand of phosphorous for food production is estimated to peak sometime between 2030 and 2040. It is predicted that world phosphorous production will begin to decline around 2035. The consequent possible shortfall of phosphorous fertilizers is a major concern for global food security.

Methods for removing phosphate from agricultural, municipal and industrial wastewater are known, but they often result in the production of a solid, insoluble phosphate fraction that is not amenable to recycling or reuse (de-Bashan et al., 2004, Water Res 38 :4222-4246). SUMMARY OF THE INVENTION

Biodegradable materials, including iron-crosslinked alginate, are useful as a remediation agent for eutrophication caused by environmental contaminants such as phosphate. The biodegradable material is used in a form that allows contact with and absorption of aqueous and dissolved ions, removing them from the aqueous medium such as a eutrophic lake or wastewater treatment plant effluent. After absorbing the dissolved ions, for example phosphorous, the biodegradable material can be used as an agricultural fertilizer.

The present invention is directed to a biodegradable material comprising iron- functionalized alginate. The iron-functionalized alginate can be Fe(II)-functionalized alginate or Fe(III)-functionalized alginate. The biodegradable material can be formulated as a bead. The biodegradable material may include an entrapped nanoparticle. The

biodegradable material can include a phosphorous-containing compound or a selenium containing compound.

The present invention is also directed to a method for making a biodegradable material comprising iron-functionalized alginate. The method includes contacting sodium alginate with FeCl ₂ or FeCl ₃ under conditions and for a time effective to yield iron- functionalized alginate. The method may further include treating the iron-functionalized alginate with a hardening solution. The hardening solution may contain FeCl ₂ , FeCl ₃ , CaCl ₂ , or any combination thereof. When the method involves a crosslinking process followed by a hardening process, at least one of the processes, and optionally both processes, involves contacting the alginate with at least one iron-containing ionic compound, such as FeCl ₂ , FeCl ₃ or both. Optionally, the alginate can be contacted during cross-linking or hardening with one or more additional divalent or polyvalent cations. The method may also include drying the iron-functionalized alginate after manufacture. The iron-functionalized alginate may be air dried or dried in an oven.

The present invention is also directed to a method for removing a contaminant from an aqueous medium where the aqueous medium is contacted with a biodegradable material comprising iron-functionalized alginate under conditions and for a time effective to sorb the contaminant. The method can further include collecting the used biodegradable material, wherein the used biodegradable material comprises a sorbed contaminant. The method can also include applying the used biodegradable material to soil as a fertilizer, wherein the used biodegradable material comprises a sorbed contaminant, and wherein the sorbed contaminant comprises a nutrient. The nutrient can include a phosphorous containing compound, or a selenium containing compound, or a combination thereof.

The contaminant can include a phosphorous-containing compound, a selenium containing compound, or arsenic. The contaminant can also include orthophosphate (P0 ₄ ^3~ ), hydrogen phosphate (HP0 ₄ ^2~ ), dihydrogen phosphate, (H ₂ P0 ₄ ^~ ), magnesium ammonium phosphate (MgNH ₄ P0 ₄ · 6H ₂ 0, struvite), hydroxyapatite, a polyphosphate, an organic phosphate, a selenate, Se(VI), Se0 ₄ ^~2 , a selenite, Se(IV) , HSe0 ₃ , elemental selenium, a selenide, (Se-II), Se ^2~ , or HSe ^" , or any combination thereof. If the contaminant is phosphate, the concentration of the contaminant may be in the range of 20 μg P0 ₄ ^3" -P /L to 1000 mg P0 ₄ ^3" -P /L.

The aqueous medium can be a eutrophic lake, municipal and industrial wastewater, agricultural runoff, effluent from water or sewer treatment plants, acid mine drainage, sludge, groundwater, a reservoir, well water, a marsh, swamp, a bay, an estuary, a river, a stream, an aquifer, a tidal or intertidal area, a sea or an ocean. The pH of the aqueous environment can be higher than pH 7.5. Alternatively, the pH of the aqueous environment can be between pH 3 and pH 9. The aqueous medium can be surface water, ground water, and aquifer, or well water.

The biodegradable material may be disposed within a stationary treatment medium, and the stationary treatment medium can include a permeable reactive barrier, a slurry wall, a filtration bed, or a filter.

The present invention is further directed to a method for increasing the nutrient content of a soil. The method includes applying a biodegradable material including iron- functionalized alginate and at least one sorbed nutrient, to a soil. The method may further include transporting the biodegradable material to the soil application site. At the application site, a plant disposed in the soil can take up at least one nutrient from the biodegradable material. The nutrient can include a phosphorous-containing compound, a selenium- containing compound, phosphorus, selenium, or iron, or a combination thereof. The nutrient can be released slowly over time as the biodegradable material degrades. The nutrient may be released over a twenty-four hours or over a year or over any period in between.

The present invention is also directed to a method for making a fertilizer. The method includes collecting from a remediation site a biodegradable material. The biodegradable material includes iron-functionalized alginate and at least one sorbed contaminant, where the contaminant includes a nutrient.

The present invention is further directed to a fertilizer composition including a biodegradable material. The biodegradable material includes iron-functionalized alginate and at least one sorbed contaminant, and the contaminant includes a nutrient. The nutrient can be a phosphorous containing compound, or a selenium containing compound, or a combination thereof.

The present invention is also directed to a method for increasing the amount of bioavailable phosphorus, selenium or iron, or any combination thereof, in a soil. The method includes contacting the soil with a fertilizer composition comprising a biodegradable material comprising iron-functionalized alginate and at least one sorbed contaminant, wherein the contaminant comprises a nutrient. The nutrient can be a phosphorous containing compound or selenium containing compound, or combination thereof

The present invention is further directed to a method for making a fertilizer. The method includes identifying an aqueous medium that includes a nutrient comprising phosphorus-containing compound or a selenium-containing compound, or both. The method further includes contacting the aqueous medium with a biodegradable material comprising iron-functionalized alginate. The aqueous medium and the biodegradable material are contacted under conditions and for a time effective to sorb the nutrient onto the

biodegradable material, yielding a charged biodegradable material. The method also includes incorporating the charged biodegradable material into a fertilizer composition.

The present invention is also directed an iron-functionalized alginate prepared by a process including adding a solution including sodium alginate dropwise to a second solution containing FeCl ₃ under conditions and for a time effective to yield alginate beads. The process may further include treating the alginate beads by submersion in a solution including FeCl ₂ .

The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, "a," "an," "the," and "at least one" are used

interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows (a) air-dried Fe-cross-linked alginate (FCA) beads and (b) FCA beads after batch studies.

FIG. 2 shows (a) pouch without FCA beads and (b) FCA beads in pouches.

FIG. 3 shows a schematic of an FCA beads column study set-up

FIG. 4 shows synthesized FCA beads

FIG. 5 shows (a) a scanning electron microscopy (SEM) image of the surface of a fresh dry FCA bead, (b) a light microscope image of a used FCA bead, (c and e) SEM images of the cross-section of the center of a fresh dry FCA bead, and (d and f) SEM images of the cross-section of the center of an used dry FCA bead.

FIG. 6 shows (a) an energy-dispersive spectroscopy (EDS) spectrum of one point of a fresh FCA bead, and (b) an EDS spectrum of one point of a used FCA bead.

FIG. 7 shows (a) zero, (b) first, and (c) second order rate equations for 5 mg P0 ₄ ^3~ -

P/L removal by wet FCA beads.

FIG. 8 shows (a) zero, (b) first, and (c) second order rate equations for 50 mg P0 ₄ ^3" - P/L removal by wet FCA beads.

FIG. 9 shows (a) zero, (b) first, and (c) second order rate equations for 100 mg P0 ₄ ^3" - P/L removal by wet FCA beads.

FIG. 10 shows Phosphate removal by wet FCA beads from solutions with different initial P0 ₄ ^3" concentrations {diamonds: 5 mg P0 ₄ ^3" -P/L, squares: 50 mg P0 ₄ ^3" -P/L, and triangles: 100 mg P0 ₄ ^3" -P/L).

FIG. 11 shows Phosphate removal by dry FCA beads from a solution with 5 mg P0 ₄ ³ -P/L as initial P0 ₄ ^3" concentration.

FIG. 12 shows (a) zero, (b) first, and (c) second order rate equations for 5 mg P0 ₄ ^3" - P/L removal by dry FCA beads.

FIG. 13 shows Freundlich and Langmuir isotherms models for the P0 ₄ ^3" removal by wet FCA beads.

FIG. 14 shows Phosphate removal by FCA beads from solutions with 100 μg P0 ₄ ^3" -

P/L initial P0 ₄ ^3" concentration.

FIG. 15 shows the comparative ability of different beads for phosphate removal over

24 h; initial concentration = 50 mg P0 ₄ ^3" -P/L. The use of FeCl ₃ and FeCl ₂ at different stages of synthesis created the most efficient beads.

FIG. 16 shows the effects of competing compounds on phosphate removal by FCA beads (Co = 5 mg/L, contact time = 24 h). The control is P0 ₄ ^3" solution prepared in DI water and treated with FCA beads.

FIG. 17 shows removal of phosphate (50 mg P0 ₄ ^3" -P/L) by FCA beads in pouches under (a) shaking condition (24 h) and (b) static condition (24 h).

FIG. 18 shows P0 ₄ ^3" removal using wet FCA beads at pH 4, 7, 8 and 9 (Co = 5 mg

P0 ₄ ^3" -P/L). pH 4.5 is the typical pH at which the batch studies were conducted. FIG. 19 shows FCA bead column study results (C ₀ = 15 and 30 mg P0 ₄ ^3~ -P/L, circles 15 mg/L, squares 30 mg/L).

FIG. 20 shows desorption of phosphate from FCA beads over time.

FIG. 21 shows an SEM/EDS image of cross sections of new dry Fe-functionalized alginate (FFA) beads prior to use.

FIG. 22 shows an SEM/EDS image of cross sections of spent dry FFA beads after use.

FIG. 23 shows an SEM image of fresh dry FCA beads.

FIG. 24 shows removal of P0 ₄ ^3~ from wastewater treatment plant effluent (WTPE) using bare NZVI and FCA beads.

FIG. 25 shows removal of P0 ₄ ^3" from animal feedlot effluent/runoff (AFLE) using NZVI and FCA beads.

FIG. 26 shows removal of P0 ₄ ^3" from AFLE using NZVI and FCA beads over a 24 h period.

FIG. 27 shows phosphate sorption over a 24 hour period as a function of phosphate concentration.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides a biodegradable material for the collection and sequestration of phosphates and other contaminants, particularly anionic contaminants, from contaminated aqueous environments. Advantageously, the phosphate and other compounds thereby collected can be recycled as an agricultural fertilizer. The invention thus provides for both environmental remediation/decontamination, as well as reuse of the recovered phosphate, thereby reducing the need for production of phosphate from apatite mining. This is a green technology that follows the principles of "reduce, reuse and recycle." It represents a sustainable practice that facilitates efficient recovery of used or wasted phosphates, and is well suited to the needs of the fertilizer industry, municipalities and pollution control agencies.

In one embodiment, the biodegradable material is, or includes, an iron-functionalized polymer. Preferably, in order to increase the green content and provide biodegradability, a natural polymer such as alginate, collagen, carboxymethylchitin, chitin, cellulose, pectin, agarose, chitosan, carrageenan and plant-derived gums. Synthetic polymers can be employed as well, in addition to or in place of natural polymers. Examples of suitable synthetic polymers include polyacrylate, poly(methyl methacrylate) (PMMA), polyvinyl acetate, and polyvinyl alcohol. In a preferred embodiment, the polymer contains at least one functional group, such as a carboxyl group or a hydroxyl group, to which a cation can bind. If the polymer does not contain a functional group then a desired functional group can be added chemically.

Alginates, natural polysaccharides obtained from brown marine algae and/or seaweed by collection, extraction, or otherwise, are particularly preferred for use in the invention because of their abundance, ease of use, and biocompatibility. Alginates are useful for water remediation because they are inexpensive, non-toxic, porous and biodegradable (Bezbaruah et al, J. Haz. Mat. 2009, 166: 1339-1343; Bezbaruah et al, J. Nanopart. Res., 2011, 13:6673- 6681). Alginates readily form complexes with cations such as sodium and calcium. In the presence of multivalent cations (e.g., calcium and iron) alginate undergoes a sol-gel transition due to the presence of carboxyl groups.

In a preferred embodiment, the biodegradable material is, or includes, an iron- functionalized alginate (referred to herein as Fe-alginate, Fe-functionalized alginate, or FFA). The iron-functionalized polymer of the invention is prepared by reacting a source of ionic iron (e.g., Fe ²⁺ or Fe ³⁺ ), such as ferrous chloride or ferric chloride, with the polymer, such as sodium alginate, to yield an iron-containing polymer. Preferably, Fe ²⁺ and/or Fe ³⁺ is used to functionalize the polymer. In this regard it should be noted that the examples refer to the iron-functionalized alginate of the invention as "iron-crosslinked alginate" (or FC alginate, or FCA). The term "crosslinked" is commonly used in the art to describe cation- or metal- functionalized polymers, such as alginates, particularly those functionalized with a polyvalent cation like Ca ²⁺ , because of the presumed or observed interaction between the cation or metal and, for example, one or more acid or hydroxyl groups of the polymer. It is to be understood, however, that the iron-functionalized polymer of the invention is not limited by the type of association between the iron and the polymer. For example, the iron can be ionically bound to one or more negatively charged or partially negatively charged groups on the polymer, and/or it may be chelated, internalized, entrapped, encapsulated, covalently bound, or otherwise associated with the polymer. The iron (particularly Fe ²⁺ or

Fe ³⁺ ) is preferably, but not necessarily, bound to or chelated with one or more carboxylic acid and/or hydroxyl groups of the polymer such that multiple polymer acid groups are linked together through the cation. Thus, unless otherwise stated to the contrary, the term "iron-crosslinked" as used in the examples and elsewhere herein is intended to be inclusive of any type of interaction between the iron and the polymer, such as alginate, that results from the synthesis of the iron-functionalized polymer according to methods described or referenced herein.

The iron-containing polymer of the invention can be Fe(II)-functionalized (using ferrous iron) or Fe(III)-functionalized (using ferric iron). In one embodiment, the iron- functionalized polymer is Fe(II)-functionalized alginate. In another embodiment, the iron- functionalized polymer is Fe(III)-functionalized alginate. In yet another embodiment, the iron-functionalized polymer is alginate functionalized with both Fe(II) and Fe(III). The invention relates primarily to iron-functionalized alginate but it should be understood that other iron-functionalized polymers can be used in the same applications. Likewise, the invention relates primarily to Fe(II)- and Fe(III) -functionalized iron but it should be understood that iron of different valencies can be utilized for functionalizing the polymer if and when desired.

The biodegradable material can take the form of a sol, a gel, a hydrogel, a bead, a capsule, a particle or nanoparticle, a slurry, a matrix, or any other form that can be used in an aqueous environment to contact aqueous or dissolved ions.

In a preferred embodiment, the iron-functionalized alginate (FFA) is in the form of a bead. The FFA bead can be nanometer scale or millimeter scale; typically, the FFA bead (freshly synthesized) is millimeter scale, between 1 and 5 mm in diameter, more typically between about 2 and 4 mm in diameter. The diameter of the FFA bead decreases when the bead is dried. In some embodiments, the functionalized bead can entrap or encapsulate a remediation or decontamination agent, in which case it is optionally referred to as a capsule. In one embodiment, for example, an FFA bead synthesized in accordance with the methods described herein contains, within it, nanoparticles. The entrapped or encapsulated nanoparticles typically average between 20 and 200 nm in diameter, more typically between 50 and 100 nm in diameter. Without being bound by theory, it is believed that the nanoparticles found within the iron-functionalized alginate beads contain iron.

A preferred method for synthesizing the FFA beads of the invention involves an aqueous crosslinking process followed by an optional "hardening" process. A solution used in the crosslinking process may be referred to as a "crosslinking solution" and a solution used in the hardening process may be referred to herein as a "hardening solution." Iron in the form of Fe ²⁺ and/or Fe ³⁺ can be introduced into the alginate or other substrate during the crosslinking process, and/or later in an optional hardening process. The cation or cations used to dope or crosslink the alginate are typically supplied in the form of an ionic solution, such as a solution containing one or more of the ionic compounds CaCl ₂ , FeCl ₂ and/or FeCl ₃ . Suitable ionic compounds can generally include ionic compounds that contain a divalent or polyvalent cation. The aqueous ionic compound or compounds are contacted with the alginate in an initial crosslinking process. Preferred crosslinking solutions include but are not limited to solutions that contain CaCl ₂ , FeCl ₂ and/or FeCl ₃ . The crosslinking solution can contain one ionic compound or it can contain more than one ionic compound. A preferred crosslinking solution contains one, two or all three of CaCl ₂ , FeCl ₂ and FeCl ₃ .

After the bead is formed in a crosslinking process, it is optionally further treated in a hardening process contacting the beads in a solution sometimes referred to as a "hardening solution." Typically the beads are submerged or immersed in the hardening solution. The hardening solution can contain the same ionic compounds as the crosslinking solution, and/or can contain different ionic compounds. At least one of the crosslinking or hardening solutions preferably contains an ionic compound containing Fe ²⁺ , Fe ³⁺ , or both Fe ²⁺ and Fe ³⁺ , typically supplied in the form of an iron halide such as an iron chloride. Additionally or alternatively, Fe ²⁺ and/or Fe ³⁺ can be supplied in the form of an iron sulfate. Preferred hardening solutions include but are not limited to solutions that contain CaCl ₂ , FeCl ₂ and/or FeCl ₃ . The hardening solution can contain one ionic compound, or it can contain more than one ionic compound. For example, the hardening solution can contain one, two or all three of CaCl ₂ , FeCl ₂ and FeCl ₃ .

The beads may be contacted with the hardening solution for short or long periods of time, including but not limited to 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or any period in between. In a preferred embodiment, the beads are contacted with the hardening solution for between 3 and 10 hours, preferably between 5 and 7 hours. In one embodiment, the beads are contacted with the hardening solution for about 6 hours.

In a one embodiment, FeCl ₃ is used in the crosslinking solution and FeCl ₂ is used in the hardening solution. In another embodiment, CaCl ₂ is used in the crosslinking solution and FeCl ₂ and/or FeCl ₃ used in the hardening solution. Alternatively, any one or

combination of CaCl ₂ , FeCl ₂ and FeCl ₃ solutions may be used for either the crosslinking solution or the hardening solution. For example, FeCl ₂ may be used for both the crosslinking solution and the hardening solution; FeCl ₃ may be used for both the crosslinking solution and the hardening solution; CaCl ₂ may be used for the crosslinking solution and FeCl ₂ for the hardening solution; CaCl ₂ may be used for the crosslinking solution and FeCl ₃ for the hardening solution; FeCl ₂ may be used for the crosslinking solution and CaCl ₂ for the hardening solution; FeCl ₂ may be used for the crosslinking solution and FeCl ₃ for the hardening solution; FeCl ₃ may be used for the crosslinking solution and CaCl ₂ for the hardening solution; FeCl ₃ may be used for the crosslinking solution and FeCl ₂ for the hardening solution.

Combinations of cations may optionally be used in any of the synthetic steps.

The beads may be used immediately after they are formed, immediately after they are removed from the hardening solution. Optionally, the beads may be dried after formation or hardening. The synthesized beads may be allowed to air dry for a period of time including 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or any period in between. In a preferred embodiment, the beads are air dried for 48 hours. Alternatively, the beads may be dried in an oven for a period of time. The beads may be dried at a range of temperatures, including but not limited to 30°C, 35°C, 40°C, 45°C, 50°C, or higher. The beads may be dried for a range of times, including but not limited to 10 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 24 hours, and 48 hours, and every time in between. In a preferred embodiment, the beads were oven dried at 40°C for 4 hours.

The use of dried beads may allow greater compactability or easier transport as compared with wet beads. The dried beads are smaller than the wet beads and may be lighter weight than a comparable number or volume of wet beads. The dried beads can tolerate higher flow rates than a comparable number or volume of wet beads. Additionally, the dried beads may be better able to withstand handling during shipping, use, or both than wet beads.

Prior to use, the FFA bead optionally contains chloride ions, derived from the synthesis using ferrous or ferric chloride. After use in a remediation or decontamination process, the FFA bead contains the contaminant, such as a phosphate. FFA beads can be used directly as a remediation or decontamination agent, or they can be further modified to entrap or encapsulate other remediating or decontaminating agents including biological agents such as bioremediating microorganisms.

In addition to providing a biodegradable material such as an iron-functionalized polymer, more particularly iron-functionalized alginate, the invention provides compositions containing the iron-functionalized polymer and methods of making and using the iron- functionalized polymer.

The invention provides a method for making the iron-functionalized polymer of the invention. Sodium alginate is readily available as starting material for the synthesis of iron- functionalized alginate. In one embodiment of the method, sodium alginate is combined with ferrous chloride, FeCl ₂ . Sodium alginate can be added dropwise to a solution of FeCl ₂ under conditions to produce Fe(II)-functionalized alginate according to methods described in the examples herein, as well as methods described in Kroll et al. (1996). FFA beads may contain carbon in an amount, for example, of 15-50 wt %, or 20-40 wt %, or 24-38 wt%. FFA beads may contain iron in an amount, for example, of 5-50 wt%, or 10-40 wt%. In some embodiments, the ratio of iron to carbon (Fe:C, w/w) in the FFA beads can be between 1 : 10 and 5: 1. In some embodiments, for example, Fe:C (w/w) can be at least 1 : 10, or at least 1 :5, or at least 1 :2, or at least 1 : 1 ; in some embodiments, for example, Fe:C (w/w) can be less than 5 : 1 , or less than 3: 1.

Optionally, the process can be reversed, such that of FeCl ₂ is added dropwise to a solution of sodium alginate. In the reverse process, oxygen is controlled in order to prevent further oxidation of the iron. The reverse process can be used to form alginate structures such as capsules, for example, that entrap the iron. Sodium alginate is typically provided an aqueous solution, for example between 0.5% and 5% w/v, more preferably between 1% and 3% w/v). Ferrous chloride is also typically provided as an aqueous solution, for example between 0.5% and 5%> w/v, more preferably between 1%> and 3%> w/v), although other solvents can be included such as methanol. Additionally, in any method effective to make calcium-crosslinked alginate (Ca-crosslinked alginate), FeCl ₂ can be substituted for CaCl ₂ in order to produce Fe(II)-functionalized alginate. More generally, the procedures that are known to the art for production of calcium alginate from sodium alginate can be readily adapted for the production of iron-functionalized alginate. Ferric chloride (FeCl ₃ ) can be used in place of ferrous chloride to produce Fe(III)- functionalized alginate, if desired.

Iron-functionalized alginate can be formulated as beads using, for example a variable flow minipump (VWR; see, e.g., Bezbaruah et al, J. Nanopart. Res., 2011, 13:6673-6681). Beads provide a convenient medium for recovery and reuse of the phosphate ion, because they can be readily stored, transferred, measured, dispersed, etc., although any desired formulation can be used, such as a gel, hydrogel, sol, matrix, slurry, etc. Advantageously, iron-functionalized alginate beads can be used as freshly produced or dried. Synthesis and production of the iron-functionalized alginate beads can be readily scaled up for industrial, municipal or commercial applications.

Removal of phosphate and other contaminants with iron-functionalized alginate

The biodegradable polymer of the invention, preferably iron-functionalized alginate, is used for removal of a contaminant from an aqueous medium. The contaminant, such as a phosphate or other anion, is sorbed onto the iron-functionalized alginate material, such as beads, and removed from the aqueous medium. In a preferred embodiment, the contaminant is a phosphorus (P)-containing compound, preferably a phosphorous-containing anion, such as orthophosphate (P0 ₄ ^3~ ), hydrogen phosphate (HP0 ₄ ^2~ ), dihydrogen phosphate, (H ₂ P0 ₄ ^~ ), magnesium ammonium phosphate (MgNH ₄ P0 ₄ · 6H ₂ 0, struvite), hydroxyapatite, a polyphosphate and/or an organic phosphate. An example of another contaminant that can be removed is selenium, including selenate, Se(VI) (e.g., Se0 ₄ ^~2 ); selenite, Se(IV) (e.g., HSe0 ₃ ); elemental selenium, Se; or selenide (-II), (e.g., Se ^2~ , HSe ^" ). In yet another embodiment, the contaminant that can be removed is arsenic, particularly As (III) or As (IV).

Iron-functionalized alginate is contacted with a contaminated aqueous medium to remove phosphates and other contaminants, preferably other anionic contaminants.

Phosphates that can be removed include, but are not limited to, orthophosphates,

polyphosphates and organic phosphates. Contact can be static, as in a batch process, or can involve the flow of the aqueous media over or through the iron-functionalized alginate.

Aqueous media can flow naturally over or through the iron-functionalized polymer of the invention, or the aqueous media to be decontaminated can be pumped over or through the iron-functionalized polymer. Exemplary flow rates are can be 1 gallon/minute or slower, 2 gal/min, 3 gal/min, 4 gal/min, 5 gal/min, 6 gal/min, 7 gal/min, 8 gal/min, 9 gal/min, 10 gal/min, or faster, such as 15 gal/min or 20 gal/min. For higher flow rates, dry beads are preferred over wet (nondried) beads. Iron-functionalized alginate can be used as an environmental remediation agent in remediation methods that are well -known to the art. For example, it can be injected into the soil, groundwater or a well, used as a matrix for a filtration mechanism, such as a cylinder, canister, or the like, applied as groundcover or into a trench, and/or utilized as a component of a deposit, layer, treatment zone, permeable or slurry wall or barrier, such as a permeable reactive barrier or slurry wall, filtration bed, or the like. Examples of aqueous media that can be decontaminated with the iron-functionalized alginate of the invention include eutrophic lakes, municipal and industrial wastewater, agricultural runoff, effluent from water or sewer treatment plants, mine waste including acid mine drainage (AMD), sludge, groundwater, reservoirs, well water, marshes, swamps, bays, estuaries, rivers, streams, aquifers, tidal or intertidal areas, seas, oceans and the like.

Examples of aqueous media that may be high in phosphate and/or selenium include wastewater treatment plant effluent (WTPE) and animal feedlot effluent/runoff (AFLE).

The iron-functionalized alginate of the invention can be exposed to the aqueous solution for a range of times. The iron-functionalized alginate of the invention can be used in both static and nonstationary aqueous media. Depending on the form of iron-functionalized alginate used, the motion of the aqueous solution, and the desired proportion of phosphate to be removed, the time the iron-functionalized alginate of the invention must be exposed to the aqueous solution will vary. For example, the iron-functionalized alginate may be exposed to the aqueous solution for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 72 hours, 96 hours or any period of time in between. Advantageously, the iron-functionalized alginate exhibits good phosphate removal in relatively short contact times (e.g., exposure times of under 4 hours, under 2 hours, under 1 hour, under 45 minutes, under 30 minutes, or shorter times) with the contaminated aqueous solution.

The iron-functionalized alginate of the invention is well-suited for use in both large and small scale treatment facilities, as well as in field operations. In some embodiments, most (e.g., over 80%, or over 90%, or over 95%>) of the phosphate is removed with the first

10-30 minutes of contact with the iron-functionalized alginate. When employed in or as a filter or in a filtration system, the detention or hydraulic retention time is therefore short (e.g., less than an hour, more preferably, less than 30 minutes), making the iron-functionalized beads of the invention well-suited for use in high flow systems (e.g., with high flow rate pumps). The high flow rates that can be used with the iron-functionalized polymers of the invention, together with their biodegradability and usefulness in both removal and recovery/reuse of contaminants, position these materials as ideally suited for use in any and all existing water and wastewater treatment applications.

The iron-functionalized alginate of the invention is well-suited to remove phosphate from aqueous solutions with various phosphate concentrations. Advantageously, it has been shown to remove phosphate at very low concentrations, such as those found in eutrophic lakes. For example, the iron-functionalized alginate has been shown to remove about 60% of phosphate from a 100 μg P0 ₄ ^3~ -P/L medium in about 10 minutes; after 2 hours, it has been shown to remove 80% of phosphate. It should be noted that phosphate concentrations are expressed herein in terms of "P0 ₄ ^3" -P /L", using the industry recognized standard

terminology for contaminated aqueous environments or media. Any designations of "P/L", "P0 ₄ ^3" P" and "P0 ₄ -P/L" appearing herein are interchangeable with P0 ₄ ^3" -P/L.

The iron functionalized alginate of the invention can remove phosphate from eutrophic lakes that typically have concentrations of up to 100 μg P0 ₄ ^3" -P/L, as well as wastewater that typically has concentrations of 10 mg P0 ₄ ^3" -P /L or higher. More specifically, the iron-functionalized alginate can remove phosphates from aqueous solutions with concentrations between 5 μg P0 ₄ ^3" -P /L and 1000 mg P0 ₄ ^3" -P /L, including but not limited to aqueous solutions with phosphate concentrations of 5 μg P0 ₄ ^3" -P /L, 10 μg P0 ₄ ^3" - P /L, 15 μg P0 ₄ ^3~ -P /L, 20 μg P0 ₄ ^3~ -P /L, 25 μg P0 ₄ ^3~ -P /L, 30 μg P0 ₄ ^3~ -P /L, 35 μg P0 ₄ ^3~ - P /L, 40 μg P0 ₄ ^3~ -P /L, 45 μg P0 ₄ ^3~ -P /L, 50 μg P0 ₄ ^3~ -P /L, 60 μg P0 ₄ ^3~ -P /L, 70 μg P0 ₄ ^3~ - P /L, 80 μg P0 ₄ ^3~ -P /L, 90 μg P0 ₄ ^3~ -P /L, 100 μg P0 ₄ ^3~ -P /L, 500 μg P0 ₄ ^3~ -P /L, 1 mg P0 ₄ ^3~ -P /L, 2 mg P0 ₄ ^3~ -P /L, 5 mg P0 ₄ ^3~ -P /L, 10 mg P0 ₄ ^3~ -P /L, 15 mg P0 ₄ ^3~ -P /L, 20 mg P0 ₄ ^3~ -P /L, 50 mg P0 ₄ ^3~ -P /L, 100 mg P0 ₄ ^3~ -P /L, 250 mg P0 ₄ ^3~ -P /L, 500 mg P0 ₄ ^3~ -P /L, 750 mg P0 ₄ ^3" -P /L, 1000 mg P0 ₄ ^3" -P /L, and aqueous solutions with concentrations of phosphate in between those listed. In a preferred embodiment, the iron-functionalized alginate removes phosphates from aqueous solutions with concentrations between 20 μg P0 ₄ ^3" -P /L and 30 mg P0 ₄ ^3~ -P /L.

We have shown that FFA beads are effective to remove phosphate from water after several hours of treatment, with little or no observed major interference from chloride, bicarbonate, sulfate, nitrate and natural organic matter. Iron-functionalized alginate (FFA) beads performed significantly better than calcium-crosslinked alginate (CCA) beads (with no iron). Moreover, FFA beads were shown to perform substantially equally well at pH 7, pH 8 and pH 9. This is important because the pH in eutrophic lakes is typically somewhat basic, ranging from 7.5 to 8.5 (Michaud JP (1991) A citizen's guide to understanding and monitoring lakes and streams. Publ. # 94-149. Washington State Department of Ecology, Publications Office, Olympia, WA, USA 360-407-7472) FFA is thus well suited for use in phosphate removal from eutrophic lakes. The adsorption capacity of FFA is also good, especially for batch treatment, but also for flow through treatments. Isotherm studies indicate that high amount of phosphate (typically, about 15 mg/g of dry FFA beads) can be removed using FFA (with up to 100% removal efficiency). Importantly the presence of other ions and materials/compounds (e.g., sulfate, chloride, nitrate, bicarbonate, natural organic matters) does not affect phosphate removal. With respect to pH, phosphate removal is also not impacted until pH reaches 9; this is significant because many eutrophic water bodies have high pH (e.g., 7.5 -8.5). These results indicate that the FFA phosphate removal is very robust and can be used for most waters and wastewaters.

Advantageously, the iron-functionalized alginate of the invention is effective for removing phosphorus from aqueous media over wide range of pHs, from acidic to basic pH. Exemplary pH values at which the iron-functionalized alginate can be used include but are not limited to pH values of 3, 3.5, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.5 and values in between.. In one embodiment, the iron-functionalized alginate is used to remove phosphate ions from eutrophic lakes with a pH of up to pH 8.5, up to pH 9.0 or higher. The method represents an advance in the art because it works at a broad range of pHs and can be used in aqueous media with a range of pHs, ranging from eutrophic lakes to industrial wastewaters.

Recovery of phosphate and other nutrients for further use

Iron-functionalized alginate that has been used to remove contaminants such as phosphorus and selenium that function as nutrients, i.e., which has sorbed the nutrient and thus is "charged" with phosphate or other nutrients, can be used as an agricultural fertilizer.

Advantageously, as noted above, alginate is biodegradable; thus, used or spent FFA beads can be used directly as fertilizer, without the need for extracting the phosphate or other nutrient that has been sorbed onto the bead (although the invention also encompasses extraction or desorption of phosphate or other nutrient from the bead for use as or

incorporation into a fertilizer, if desired). The phosphate or other nutrient bound by the FFA is bioavailable and accessible without further modification for plant and microbial uptake. The biodegradable beads release the nutrient, such as phosphate, as they degrade, making the nutrient bioavailable on a time release basis for plants and other organisms. Moreover, the iron present in iron-functionalized alginate is also bioavailable and accessible without further modification for plant and microbial uptake. As the beads degrade, iron is made bioavailable for plants and other organisms on a time release basis as well. Thus, charged FFA beads are expected to have utility as possible materials for slow release fertilizers. FFA beads can be applied in the field using any convenient method, either in the spring prior to planting, during the growing season, or in the fall. The FFA beads can be mixed with other nutrients, fertilizers, or additives, or applied separately. Illustrative compositions and methods for using recovered phosphate as fertilizer are described in de-Bashan et al., First International Meeting on Microbial Phosphate Solubilization, Developments in Plant and Soil Sciences Volume 102, 2007, pp 179-184.

The contaminants or nutrients and the iron in the iron-functionalized alginate of the invention may be made bioavailable for plants and other organisms on a time release basis. For example, the release may occur after hours, days, weeks, or months. In one embodiment, all of the phosphate or other nutrient may be released from the iron-functionalized alginate after, for example, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 6 months, or 12 months or any period in between. In an alternative embodiment, the phosphate may be released slowly with, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the phosphate or other nutrient being released over, for example, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3, months, 6 months, 12 months or any period in between.

Additionally, the iron from the iron-functionalized alginate may be fully after, for example, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 6 months, or 12 months or any period in between. Alternatively, the iron from the iron-functionalized alginate may be released slowly with, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the iron being released over, for example, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3, months, 6 months, 12 months or any period in between.

The dual functionality of the iron-functionalized alginate beads of the invention makes them especially valuable. Not only are they useful in environmental remediation, decontamination, pollution control and the like but, when charged with a nutrient such as phosphate or selenium, they can be used as fertilizer. Advantageously, the iron present in the iron-functionalized alginate is also in bioavailable form. The new technology can thus be used to recover phosphates or other nutrients from eutrophic lakes, agricultural run-offs, and municipal and industrial wastewaters, and the like as detailed herein; after which the iron- functionalized beads with phosphate and/or other nutrients can be used as a fertilizer in agricultural fields as a source of phosphate/phosphorus.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Example 1. Aqueous Phosphate Removal Using Iron-Functionalized Alginate

Novel iron cross-linked alginate beads (FCA) were successfully used for aqueous phosphate removal. Batch experiments were conducted with wet beads using three different concentrations of phosphate (5, 50 and 100 mg P0 ₄ ^3" -P/L) with 0.11812 gm (dry weight) iron cross-linked beads. Kinetic studies were also done with dry beads using 5 mg P0 ₄ ^3" -P/L. About 94% phosphate was removed in 6 h from the aqueous solution by wet FCA beads having an initial phosphate concentration of 5 mg P0 ₄ ^3" -P/L. With 50 mg P0 ₄ ^3" -P/L, the beads were found to remove only -41% in 6 but achieved 89% phosphate removal in 96 h. The second order reaction model fitted better for all the concentrations and observed reaction rates were found to be 0.2979, 0.083 and 0.0181 per h for 5, 50, and 100 mg P0 ₄ ^3" -P/L, respectively. Dry FCA beads were found to remove phosphate steadily and gradually from the solution. Removal percentages by FCA beads were -42%, ~56%> and ~62%> after 12 h, 18 h and 24 h, respectively. Interference of CI ^" , HC0 ₃ ^~ , S0 ₄ ^2~ , N0 ₃ ^" , natural organic matter (NOM) and humic acid was investigated and no change in the removal efficiency of phosphate was observed. To investigate the feasibility of using these FCA beads in real life situation (e.g., eutrophic lakes), "pouch experiments" were conducted in two conditions, namely shaking and static where FCA beads were introduced into the water in pouches. The efficacy of beads in pouches was found to be the same as free beads (used in earlier batch experiments). Desorption studies showed that the sorbed phosphate desorb over time at a rate that is sufficient to satisfy the demand of plants. The successful sorption of phosphate by FCA beads and subsequent desorption is expected to have enormous implications for nutrient removal and recovery for possible use in agriculture.

Phosphorus (P) is important for the growth of plants and microorganisms in most ecosystems. However, excess phosphorus becomes problematic when present in aquatic bodies leading to the overgrowth of algae and plant species (common symptoms of eutrophication of the water bodies). The usual forms of aqueous P are orthophosphates (dissolved P), polyphosphates (particulate P), and organic phosphates (Mezenner and Bensmaili, 2009). Increase in P leads to eutrophication and eutrophication results in the depletion of oxygen leading to fish death and affecting other aquatic life forms adversely. While eutrophication is a natural process, it is accelerated through anthropogenic activities. Municipal and industrial wastewaters are the major point sources that contribute to P buildup in aquatic environment. P-based fertilizers are extensively used in food crops constituting the major non-point source for P. Animal agriculture is another non-point source that adds to the problem. Accelerated eutrophication not only impacts the aquatic life but indirectly hampers the economic progress of communities that rely on aquatic food and other resources (Cleary et al, 2009). Dissolved phosphate of -0.02 mg/L is known to cause profuse algal growth in waters, thereby posing a host of problems (USEPA, 1995). The EPA water quality criteria state that phosphates should not exceed 0.05 mg/L if streams discharge into lakes or reservoirs, 0.025 mg/L within a lake or reservoir, and 0.1 mg/L in streams or flowing waters not discharging into lakes or reservoirs to control algal growth (USEPA, 1986). Surface waters that are maintained at 0.01 to 0.03 mg/L of total phosphorus tend to remain uncontaminated by algal blooms. Therefore, the amount of P compounds in waters should be curtailed to prevent eutrophication in lakes and other surface waters. It is imperative to devise effective methods to remove excessive phosphate from water and wastewater. There is a significant gap in technology to remove low concentrations P from waters, specifically from eutrophic lakes.

There is another aspect to the P issue. Phosphorus for fertilizer production is mined chiefly from select mines from Morocco, Western Saharan region, and China (Cordell et al., 2009). Phosphorus is a nonrenewable resource and a recent assessment indicated that natural phosphate (P0 ₄ ^3~ ) deposits will last for approximately 60-240 years (Cornel and Schaum, 2009). World phosphorus production rate is predicted to decline from around 2033 and the humankind will certainly be hard put to keep up with the cumulative demand for phosphate fertilizer (Cordell et al, 2011). Municipal wastewater, runoff from animal feedlot, agricultural runoff, and eutrophic lakes rich in phosphates can serve as nonconventional "mines" for P. The phosphates present in these aquatic sources are otherwise considered pollutants (causing eutrophication). Mining phosphates from these sources will, thus, offer viable solutions to both pollution and global food security issues.

Typical methods for phosphate removal from wastewater include biological treatments (de-Bashan and Bashan, 2004), chemical precipitation (with aluminum, iron and calcium salts) (Tchobanoglous et al, 2003), adsorption (Liu and Hesterberg, 2011), and reverse osmosis (Dolar et al., 2011) (Table 1). Chemical treatment methods for aqueous phosphate removal are widely practiced using chemicals like lime (Ahn and Speece, 2006), alum (Babatunde and Zhao, 2010), and ferric chloride (Caravelli et al., 2010). However, biological treatments and chemical precipitation are generally not suitable for low

concentration phosphate removal, and reverse osmosis is capital intensive. In addition, there is no accepted method for phosphate removal from eutrophic lakes.

Table 1. Phosphorus removal and recovery technologies (adapted from Morse et al, 1998)

Adsorption is one of the most attractive options for phosphate removal from aqueous media. In the recent years considerable amount of research has been done on the use of low cost (ad)sorbents. Sorption has edge over other technologies because of its effectiveness even at low phosphate concentrations. Cost effectiveness is identified as the main criterion in the selection of a sorption technology whether it uses synthetic or natural sorbents (Mishra et al., 2010). Phosphate can be removed from water using sorbents such as oxides of iron, natural ores like calcite, and goethite (FeOOH), active red mud, and activated carbon. A number of novel adsorbents have also been investigated for phosphate removal. NZVI (Almeelbi and Bezbaruah, 2012), fly ash (Cheung and Venkitachalam, 2000), red mud (Huang et al, 2008), iron oxide tailing (Zeng et al., 2008, Environ Sci Technol 42: 147-152), iron based compounds (Zeng et al, 2008, Environ Sci Technol 42: 147-152), aluminum based compounds (Tchobanoglous et al, 2003), and layered double hydroxides have been tried for phosphate removal by scientists.

Orthophosphate is the most readily removable form of phosphate. Physical, chemical, and biological methods, and combination of these methods have been utilized to remove phosphorus from water (de-Bashan and Bashan 2004; Gouider et al., 2011; Mishra et al.,

2010) . While most of the methods can remove phosphate to reasonable degree, adsorption is getting more attention in recent years as it is cost effective and the adsorbed phosphate can be recovered under the right environmental conditions. Different adsorbents have been used for aqueous phosphate removal which include oxides of iron, natural ores like calcite, and goethite (FeOOH), active red mud, and activated carbon (Chitrakar et al., 2006, Cordary 2008, Hussain et al, 2011, Karageorgiou et al, 2007, Yan et al, 2010, J Haz Mat 179:244- 250).

Of late, considerable attention has been focused on the study of biological materials including biopolymers as adsorbents for removing pollutants (Bezbaruah et al., 2009 and

2011) and they are promoted as cost-effective and eco-friendly. Sodium (Na) alginate, the salt of alginic acid, has been investigated as a sorbent for the removal of organic and inorganic pollutants from wastewaters (Li et al., 2013, Biores Technology 142:611-619). Na- alginate has drawn considerable attention owing to its biodegradability, hydrophilicity, presence of carboxyl and hydroxyl groups, low cost, natural and renewable nature (Li et al, 2013, Biores Technology 142:611-619). Alginates are linear copolymers composed of two monomeric units, β-Ι-4-linked D- mannuronic acid (M) and a-l,4-linked L-guluronic (G) acid.

β-D-Mannuronic Acid a-L-Guluronic Acid

The ion-binding properties of alginates are the basis for their gelling behaviour and have been extensively studied (Emmerichs et al., 2004). Alginate gels are most commonly formed by the addition of multivalent ions, with calcium being widely used for food applications and removal of heavy toxic metal cations. Although calcium alginate biopolymer is an efficient sorbent for the removal of metal ions, it is not capable of removing anionic species.

Pretreatment of Na-alginate with cations, such as Fe ³⁺ convert the biopolymer to anionic species absorber. Fe ³⁺ partially displaces loosely bound Ca ²⁺ ion to produce Fe-doped calcium-alginate beads (Min and Hering, 1998, Wat Res 32: 1544-1552). The iron-calcium gel beads are found to be effective in removing oxyanionic contaminants, specifically Se (IV), Cr (VI) and As (V) (Min and Hering, 1998, Wat Res 32: 1544-1552; Min and Hering, 1999). Keeping all these in mind, studies were carried out where novel biopolymer beads were synthesized for phosphate removal using sodium alginate and FeCl ₂ . To date, there has been no report on the removal of phosphate ions using alginate with Fe (II) as the crosslinking ions.

In the presence of multivalent cations (e.g., calcium, and iron) the polymer undergoes a sol-gel transition because of the reactive carboxylate groups (Kroll et al., 1996). When alginate reacts with metal ions it forms stable organic-inorganic hybrid composite. Alginate polymers are widely investigated for water remediation because they are inexpensive, nontoxic, porous, and biodegradable (Bezbaruah et al, 2009, 2011). It has been used in water remediation as an immobilizing agent for nanoparticles (Bezbaruah et al., 2009, 2011). Iron [Fe(III)] cross-linked alginate has been used as source of Fe catalyst for Fenton- enhanced decoloration/degradation of Orange II (Fernandez et al., 2000) and azo dyes (Dong et al., 2011). Sreeram et al. (2004) studied the interaction between iron (III) and alginate and suggested 'site binding model' where Fe(III) ions are bound to the binding sites in the alginate forming spatially separated iron(III) centers on the alginate backbone.

To determine if FCA beads can be used to remove aqueous phosphate, a series of batch experiments were conducted with the FCA beads to investigate the mechanisms of phosphate sorption onto FCA beads. The specific objectives of this work were as follows: (1) to investigate the phosphate sorption characteristics of FCA beads, (2) to determine the feasibility of using FCA beads in eutrophic lakes' reclamation.

Materials and Methods

Chemicals and reagents

Iron (II) chloride tetrahydrate (FeCl ₂ ^» 4H ₂ 0, reagent grade, J.T. Baker), calcium chloride (CaCl ₂ , ACS grade, BDH), monopotassium phosphate (KH ₂ P0 ₄ , 99% pure, EMD), sodium alginate (production grade, Pfaltz & Bauer), potassium nitrate (K O3, 99%, Alfa Aesar), sodium hydroxide (NaOH, ACS Grade, BDH), potassium sulfate (K ₂ S0 ₄ , ACS grade, HACH), natural organic matter (Suwannee River NOM, RO isolation, IHSS), and humic acid (HI 452, Spectrum) were used as received unless and otherwise specified.

Synthesis of Fe cross-linked alginate beads

Sodium alginate (20 g) was dissolved in 1L of deionized (DI) water to form a 2%> alginate solution. Fe cross-linked alginate (FCA) beads were synthesized by adding the alginate solution to ferrous chloride (FeCl ₂ ) solution (2%> w/v) at room temperature

(22±2°C). The alginate solution was added drop wise into FeCl ₂ solution using a peristaltic pump. FCA beads were prepared in batches using 5 mL alginate solution for each batch.

Alginate beads are formed immediately as the alginate comes into contact with the ferrous chloride solution. The beads from each batch were kept separately in a polypropylene tube fitted with a plastic cap. Enough FeCl ₂ solution was added to each tube to completely submerge the beads, and the beads in the tubes were allowed to harden in FeCl ₂ solution for an additional ~24 h. The hardened beads were then washed with DI water. The beads were blotted dry with tissue papers prior to their use in experiments.

Synthesis of dry beads

Dry FCA beads were synthesized in two ways. Synthesized wet beads were allowed to air dry for 48 h (FIG. 1(a)) after they were blotted dry with paper towel. Alternatively, they were oven dried at ~40 °C for 4 h. Thereafter, the FCA beads were used for batch studies (FIG. 1(b)). Batch studies

Adsorption kinetics: Adsorption kinetics was examined using wet FCA beads. For wet beads, beads produced in a single batch (0.11812 g dry alginate) were added into 50 mL phosphate solution (in multiple polypropylene plastic tubes fitted with plastic caps

(reactors)). The effect of initial P0 ₄ ^3~ -P concentration was carried out by choosing three initial P0 ₄ ^3" -P concentrations, namely 5, 50 and 100 mg P0 ₄ ^3" -P/L. The reactors and controls were then rotated at 28 rpm in a tailor-made end-over-end shaker to reduce mass transfer resistance. A set of sacrificial reactors was withdrawn at specific time intervals (0, 6, 12, 18, 24, 30, 36, 42, 48, 72 and 96 h). The phosphate concentration in the bulk solution was measured and reported as average (with standard deviations) of readings from three replicates. Ascorbic acid method (Eaton et al, 2005) was used for phosphate analysis. Blanks (no FCA beads but only P0 ₄ ^3" solution) were also run. Hach DR 5000 spectrophotometer was used for the analysis of orthophosphate in samples. The detection limit of the

spectrophotometer was 19 μg P0 ₄ ^3~ -P /L.

Adsorption isotherms: A set of experiments were conducted to understand the isotherm behavior of the FCA beads during P0 ₄ ^3" removal. One batch of FCA beads was used in each batch reactor and P0 ₄ ^3" in the bulk solution was analyzed after 24 h to calculate the sorption capacity of FCA beads. Initial concentration of phosphate was varied from 200 to 1200 mg/L.

(C ₀ — C _e ) x V Where q is the unit mass (mg) of P0 ₄ ^3~ -P per g of dry FCA bead, Co and C _e are the initial and equilibrium concentrations of P0 ₄ ^3~ -P in mg/L, V is the volume of P0 ₄ ^3" solution in L and m is mass of dry FCA beads in g.

Kinetic studies with dry beads: Adsorption kinetics was also examined using dry FCA beads. Dry beads were tested for kinetic studies because of their utility for their intended uses. Wet beads are not user-friendly and can pose problems stemming from their volume. Wet beads have other issues like transportability and aesthetics whereas dry beads are easy to use because they are light in weight. For dry beads, beads produced in a single batch

(0.11812 g dry alginate) were added into 50 mL phosphate solution (in multiple

polypropylene plastic tubes fitted with plastic caps (reactors)). 5 mg P0 ₄ ^3" -P/L was used for the kinetic study. The reactors and controls were then rotated at 28 rpm in a tailor-made end- over-end shaker to reduce mass transfer resistance. A set of sacrificial reactors was withdrawn at specific time intervals (0, 6, 12, 18, 24, 30, 36, 42, 48, 72 and 96 h). The phosphate concentration in the bulk solution was measured and reported as average (with standard deviations) of readings from three replicates. Ascorbic acid method (Eaton et al., 2005) was used for phosphate analysis. Blanks (no FCA beads but only P0 ₄ ^3" solution) were also run. Hach DR 5000 spectrophotometer was used for the analysis of orthophosphate in samples. The detection limit of the spectrophotometer was 19 μg P0 ₄ ^3~ -P /L.

Lower concentration phosphate removal by FCA beads: A lower concentration of phosphate ( 100 μg P0 ₄ ^3" -P/L) was tried to see the efficacy of FCA beads for phosphate removal at environmentally important concentrations. As phosphate is difficult to remove at lower concentrations, this batch study is likely to give an insight into the kinetics of removal at lower P0 ₄ ^3" -P concentrations.

Comparative ability of FCA and other alginate beads: Efficacy of FCA beads and other beads for phosphate removal were investigated using 50 mg P0 ₄ ^3" -P/L as the initial concentration. Different combinations were tried for production of different beads. Ca ²⁺ , Fe ²⁺ and Fe ³⁺ were used as crosslinking or doping ions and these ions were introduced in CaCl ₂ , FeCl ₂ and FeCl ₃ solutions. The solutions were also used for hardening purposes. Then batch studies were conducted with the different synthesized beads following the protocol as mentioned earlier.

Interference studies: Effects of competing compounds on phosphate sorption by the

FCA beads were investigated by adding common coexisting anions (chloride, bicarbonate, sulfate, or nitrate) or natural organic matter (NOM) found in surface waters. Interference studies were carried out with known concentrations of chloride (Cl ^~ , 50 to 500 mg/L), bicarbonate (HC0 ₃ ^~ , 10 to 100 mg/L), sulfate (S0 ₄ ^2~ , 50 to 1000 mg/L), nitrate (N0 ₃ ^~ , 10 to 100 mg/L as N0 ₃ ^" -N), Suwanee River NOM (10 to 50 mg/L) and humic acid (2 mg/L) using FCA beads with 5 mg/L of P0 ₄ ^3" -P solution. Humic acid was first dissolved with NaOH at pH 11. The competing ions or compounds were first mixed with the P0 ₄ ^3" solution in a 50 mL plastic tube and one batch of FCA beads was added to it. The reactors were then capped and placed in an end-over-end shaker (28 rpm) for 24 h. The batch studies were carried out at room temperature (22 ± 2 °C) and triplicate reactors were run for each study.

Pouch studies

A feasibility study was carried out to see the efficacy of FCA beads in real-life condition (e.g., eutrophic lakes). FCA beads were introduced into the P0 ₄ ^3" rich water in pouches. Two conditions were simulated, namely shaking and static. Beads produced in a single batch (0.11812 g dry alginate) were bagged into small pouches (FIG. 2) and subsequently added to 50 mL phosphate solution (50 mg P0 ₄ ^3" -P/L) in multiple

polypropylene plastic vials fitted plastic caps (reactors). Free-floating beads were also added to phosphate solution for comparison. For shaking condition, the reactors and controls were rotated end-over-end at 28 rpm in a custom-made shaker to reduce mass transfer resistance. For static water condition, the beads were introduced to the phosphate solution and the reactors were kept undisturbed for 24 h. The reactors were also withdrawn from the shaker after 24 h. The phosphate concentration in the bulk solution was measured and reported as average (with standard deviations) of readings from three replicates. Column studies

Column studies were conducted to simulate a real-world application of the FCA beads for P0 ₄ ^3" removal. Two concentrations of P0 ₄ ^3" were used in the column studies (15 and 30 mg P0 ₄ ^3" -P/L) to simulate extreme conditions. Glass columns (height 30 cm and internal diameter 1.5 cm) with an empty bed volume of 53 mL were used. Each column was filled with FCA beads (made with 1.2 grams alginate) and had a packed bed volume of 27 mL. Orthophosphate (P0 ₄ ^3~ ) solution was fed in an up-flow mode using a peristaltic pump at a flow rate of ~0.1 mL/min. Samples were collected over time from the effluent point at the top of the column (FIG. 3) and analyzed for P0 ₄ ^3~ concentration.

Desorption studies

Desorption studies with carried out with an initial phosphate concentration of 50 mg

P0 ₄ ^3" -P/L. First, sorption studies were done with FCA beads. After 24 h of sorption, the beads were separated from the solution and washed with deionized water. Afterwards, they were blotted dry with blotting paper. The beads were then put back in reactors with deionized water. Finally, the reactors were put in an end-over-end shaker and rotated at 28 rpm.

Periodically the reactors were taken out from the shaker and the solution was tested for orthophosphate.

Characterization of Alginate beads

Scanning electron microscopy along with energy dispersive spectroscopy (SEM/EDS, JEOL JSM-6300, JEOL Ltd.) was used to observe surface morphology and characterize the elemental composition of the beads. SEM analyses were performed in a wide beam current range to determine the microstructure of the dry FCA beads before (new FCA beads) and after using them for P0 ₄ ^3" removal (used FCA beads). New and used beads were dried overnight in a vacuum oven under nitrogen environment, and cross sectional samples of the dry beads were used for imaging and EDS analyses. Results and Discussion

Synthesis and characterization of alginate beads

FCA beads were synthesized successfully (FIG. 4). All the beads were approximately spherical in shape with average diameters of 3.97 ± 0.029 mm (n = 15). Average diameters of dry beads were 1.18 ± 0.034 mm (n = 15). Average number of beads produced per batch was 86.27±5.95 (n = 26) for FCA. To know the dry weights of the beads, the beads were dried overnight in a vacuum oven in nitrogen environment. Each batch of dry FCA beads weighed 0.11812 ± 0.002 g.

The size of the dry FCA beads was ~1 mm and the dry beads had a uniform hard texture (FIG. 5a and b). Nanoparticles with average size of 74.45±35.60 nm (n =97) were observed inside the fresh beads (FIG. 5e). The surface morphology of the beads changed completely once phosphate was adsorbed (fresh bead in FIG. 5c and used bead in FIG. 5d). A fragile outer layer was formed around the hard core after phosphate was adsorbed (FIG. 5d). The size of nanoparticles increased marginally after phosphate adsorption. The average size of nanoparticles was 83.65±42.83 nm (n=67) inside the used beads (FIG. 5f).

Nanoparticle size was measured using ImageJ software.

EDS analysis of fresh (FIG. 6a) and the used beads (FIG. 6b) revealed a consistent carbon weight % and similar iron weight % except for one point in the fresh beads which indicates heterogeneous distribution of iron inside the beads. Chloride (~ 30%) present in the fresh beads was not observed in the used beads. It is suspected that the nanoparticles are some form of iron but further investigations are needed to completely characterize the particles.

Batch studies

Adsorption kinetics and isotherms

Batch experiments were conducted with wet and dry FCA beads to determine the kinetic parameters for P0 ₄ ^3~ removal (Co = 5, 50 and 100 mg P0 ₄ ^3" -P/L for wet beads and Co = 5 mg P0 ₄ ^3" -P/L for dry beads). Zero, first, and second order reaction equations were fitted to determine the type of reaction and reaction rate constants (FIGS. 7, 8, 9). The second order reaction model fit better for all the concentrations with wet FCA beads and observed reaction rates were found to be 0.2979, 0.083 and 0.0181 per h for 5, 50, and 100 mg P0 ₄ ^3" - P/L, respectively (Table 2). A zero order reaction model fit well at 5 mg P0 ₄ ^3" -P/L with dry FCA beads (FIG. 12). The observed reaction rate was -0.0207 mg/L/min.

Table 2. Reaction rate constants calculated based on the obtained results

Equilibrium

Co C _e time Zero Order First Order Second Order mg/L H Kobs* R ² Kobs* * R ² K ^" , * * *

i^-obs R ²

5 0.085 96 -0.004 0.176 -0.02 0.295 0.298 0.471

50 5.407 96 -0.007 0.615 -0.021 0.819 0.083 0.909

100 33.807 96 -0.005 0.682 -0.009 0.836 0.0181 0.935 g/L/min; ** per min; *** L/mg/min

Rapid phosphate removal by wet FCA beads was observed for the lower

concentrations (FIG. 10). About 96% of phosphate was removed within 6 h at the lowest loading of phosphate (5 mg P0 ₄ ^3~ -P/L) and negligible removal was observed beyond that point. At 50 mg P0 ₄ ^3" -P/L, -41% of phosphate was removed within 6 h. The percentage of removal increased to -66% and 89% after 24 h and 96 h, respectively. At the highest loading of phosphate, removal of phosphate was -27% after 6 h and the removal percentage was -67% after 96 h.

From the kinetic study done with dry beads, phosphate was found to be removed steadily and gradually at 5 mg P0 ₄ ^3" -P/L (FIG. 11). Around 28% phosphate was removed after 6 h. Removal percentage was -42%, -56% and -62% after 12 h, 18 h and 24 h, respectively. After 42 h, -94% was removed by wet beads and phosphate concentration went beyond the detection limit of spectrophotometer after 48 h. The kinetic data was found to fit the zero order reaction (FIG. 12).

Langmuir isotherm was found to most closely fit with experimental data (FIG. 13). Experimental and Langmuir maximum adsorption capacity were found to be 91.74 and 147.06 mg/g of dry FCA beads, respectively. Others (Chitrakar et al, 2006; Ogata et al, 2011) have reported that Freundlich describes sorption behavior better when dual sorbents are present. Freundlich isotherm model has been used to describe P0 ₄ ^3" adsorption behavior onto sulfate-coated zeolite, hydrotalcite, and activated alumina while the adsorption behaviors of the same materials without coating were described better by Langmuir isotherm model (Choi et al, 2012). Lower concentration phosphate removal by FCA beads

Phosphate removal by FCA beads was also investigated for 100 μg P0 ₄ ^3~ -P/L (FIG. 14). About 64% of phosphate was removed within 6 h. After 24 h, removal percentage was -71% and after 48 h the removal percentage remained almost the same until 96 h.

Comparative ability of FCA and other alginate beads

From the comparative study it was found that ability of different kind of sodium alginate beads differed depending on how FeCl ₂ , FeCl ₃ and CaCl ₂ are used in different combinations (FIG. 15). It was seen that when FeCl ₃ is used for crosslinking and FeCl ₂ for hardening, the efficacy of alginate beads increased markedly. Even the beads performed better than the FCA beads used here in this research for batch studies.

What is the removal mechanism - cross-linking or hardening?

Cross-linking ions and hardening ions were added in different combinations to synthesize alginate beads. The beads performed differently depending on how cross-linking ions were added. CaCl ₂ ions were not found to be a good hardener. When CaCl ₂ was used as hardening solution with CaCl ₂ as the cross-linking ions, the alginate beads removed only ~10%> phosphate from the spiked solution (FIG. 15). CaCl ₂ worked very well as cross-linking ions with FeCl ₂ and FeCl ₃ as hardening solutions. It is evidently clear that hardening solution is playing the main role in the phosphate removal process by alginate beads. FeCl ₃ worked better than the FeCl ₂ as cross-linking ions with FeCl ₂ as the hardening solution. From this study, it is obvious that FeCl ₂ is the best for the hardening purpose. There is not much difference between CaCl ₂ and FeCl ₂ when they are used as cross-linking ions with FeCl ₂ as the hardener. FeCl ₃ really stood out among the cross-linkers; FeCl ₂ -hardened alginate beads performed best when FeCl ₃ was used as the cross-linker.

Interference studies

Effect of the presence of CI ^" , HC0 ₃ ^" , S0 ₄ ^2" , N0 ₃ , NOM and humic acid on P0 ₄ ^3" (C ₀

= 5 mg P0 ₄ ^3" -P/L) removal efficiency of FCA beads was examined. Little or no interference in the removal of P0 ₄ ^3" was observed because of the presence of these ions (FIG. 16). The ions used in this interference study are usually present in wastewater, surface water, and groundwater. Lee et al. (Desalination and Water Treatment 44:229-236 (2012)) reported a 78% reduction in P0 ₄ ^3~ removal by slag microspheres in the presence of HCCV.The addition of S0 ₄ ^2" was reported to decrease the P0 ₄ ^3" removal efficiency by -60% in a polymer-based nanosized hydrated ferric oxides system (Pan et al. 2009), and the efficiency reduction was 24.5 % when layered double hydroxides were used (Das et al, 2006, Appl Clay Sci 32:252- 260). S0 ₄ ^2" and Cr were found have a negative impact on P0 ₄ ^3" removal from lake water using high gradient magnetic separation (de Vicente et al. 2011). In the presence of N0 ₃ ^" , P0 ₄ ^3" removal decreased by 29.2% while using layered double hydroxides (Das et al, 2006, Appl Clay Sci 32:252-260) and by 6.27% while using NZVI (Almeelbi and Bezbaruah, 2012). NOMs are present in surface waters, and known to interfere with P0 ₄ ^3" removal in adsorption processes (Guan et al. 2006, Vicente et al, 2008, Environ Sci Technol 42:6650- 6655). However, no effect of NOM on P0 ₄ ^3" removal was observed in this study. Similar findings were reported earlier with bare NZVI (Almeelbi and Bezbaruah, 2012). The lack of interference by the dominant ions and NOM makes an FCA bead system a potential candidate for field application for P0 ₄ ^3" removal.

Pouch studies

From the "Pouch studies" conducted, the bagged beads in pouches were found to work almost as well as the free loose beads in shaking condition. In 24 h, bagged beads removed -58%) phosphate from the solution whereas loose beads removed ~60%> phosphate (FIG. 17(a)). However, in static condition there has been a significant decrease in phosphate removal by FCA beads. Bagged FCA beads removed ~15% phosphate from the phosphate- spiked solution whereas the loose beads removed ~17% phosphate from the solution (FIG. 17(b)). Of course, in lake condition, there will be a lot of turbulence that will reduce the mass transfer resistance enabling the FCA beads perform better.

Effect of pH

The effect of pH on phosphate removal (C ₀ = 5 mg P0 ₄ ^3" -P /L) by FCA was investigated at pH of 4, 7, 8 and 9 (FIG. 18). Changing the pH did not affect removal of

P0 ₄ ^3" by FCA beads; approximately 96%, 96%, 95% and 96% removal was achieved at pH 4, 7, 8 and 9 values, respectively. That pH did not affect the P0 ₄ ^3" removal efficiency of FCA has important practical implications. The pH in eutrophic lakes ranges from 7.5 to 8.5

(Michaud JP (1991) A citizen's guide to understanding and monitoring lakes and streams. Publ. # 94-149. Washington State Department of Ecology, Publications Office, Olympia, WA, USA 360-407-7472) and FCA can possibly be used for phosphate removal in eutrophic lakes. Column studies

Breakthrough behavior in FCA bead columns was studied with 15 and 30 mg P0 ₄ ^3~ - P/L and a flow rate of ~0.1 mL/min. (FIG. 19). For the higher concentration (30 mg P0 ₄ ^3" -P /L), the breakthrough (C _e = 0.05 Co) was achieved after 2 bed volumes when removal dramatically decreased from 99 to 57%. In the case of the lower concentration (15 mg P0 ₄ ^3" - P/L) removal decreased gradually in the first 3 bed volumes (only -5% decrease). The adsorption capacity increased from 0.97 to 1.81 mg/g of dry beads when initial P0 ₄ ^3" -P concentration was increased from 15 to 30 mg P0 ₄ ^3" -P/L which is much lower than the adsorption capacity obtained in batch study (14.77 mg/g of dry FCA beads).

Desorption studies

Phosphate desorption studies were carried out to see the desorbability of phosphate by

FCA beads. Sorbed phosphate was found to desorb slowly and steadily at a rate that is deemed to be sufficient to meet the demand of growing plants (FIG. 20). This desorption has significant implications because these beads hold potential as slow-releasing fertilizers. Moreover, these FCA beads also contains iron which is also a plant nutrient element. So these FCA beads can serve dual purpose with respect to plant nutrient element.

Conclusions

Ferrous iron cross-linked alginate (FCA) beads were successfully synthesized and utilized for phosphate removal. 94% removal of aqueous phosphate was achieved after 6 h. Further, there was no interference by CI ^" , HCO ₃ ^" , S0 ₄ ^2" , NO ₃ ^" , NOM and humic acid in phosphate removal with FCA beads. Langmuir isotherm best described the phosphate sorption behavior of FCA beads. It is inferred that presence of iron in alginate beads increased the phosphate removal capacity of the beads. And hardening by FeCl ₂ was found to be the dominant mechanism for aqueous phosphate removal. From the "pouch studies", FCA beads seem to hold a lot of promise for the reclamation of eutrophic lakes. And then they can be applied in agricultural soils for crop growth. Therefore, these FCA beads can potentially solve two global problems of gargantuan importance. References

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Example 2. Compositional Analysis of Iron-Functionalized Alginate Beads

Compositional analysis was performed on dry FFA beads before and after sorption of phosphate. FIGS. 21 and 22 show SEM/EDS images of cross sections of dry FFA beads. Beads were dried using a vacuum oven under an N ₂ environment. FIG. 21 shows SEM/EDS images of cross sections of new dry FFA beads (before sorbing phosphate). FIG. 22 shows SEM/EDS images of cross sections of spent dry FFA beads (after sorbing phosphate).

Results for fresh FFA beads (before sorbing phosphate)

Weight %

Results for used FFA beads (after sorbing phosphate)

Weight %

C-K O K P-K K-K Ca-K Fe-L

125560 USED ALGINATE DRYXSECT(2) _ptl 25.27 27.16 6.28 3.41 37.88

125560 USED ALGINATE DRYXSECT(2) _pt2 22.21 26.94 6.38 3.38 0.55 40.54

125560 USED ALGINATE DRYXSECT(2) _pt3 24.00 25.99 6.61 3.97 39.43

Example 3. Crosslinked Alginates

The use of a number of biopolymers like alginate, poly(methyl methacrylate) (PMMA), chitosan, and carrageenan have been explored for environmental remediation applications (Bezbaruah et al., 2009; Bezbaruah et al., 2011).

Alginate is a biopolymer derived from seaweeds, and it has been used for entrapment for microbial cells and NZVI (Bezbaruah et al., 2009, Kim et al., 2010). The entrapped NZVI was found to work very efficiently for contaminants like trichloroethylene (TCE). The entrapment process does not require sophisticated instrumentation and can be performed at ambient temperature and pressure. Ca-alginate is non-toxic, biodegradable, and sparsely soluble in water making it an ideal polymer for use in environmental applications (Bezbaruah et al., 2009a). The porous nature of Ca-alginate allows solutes to diffuse and come in contact with the entrapped NZVI (Bezbaruah et al., 2009).

Ionic cross-linking refers to the ion exchange process between the monovalent ion on the water soluble alginate (e.g., sodium or potassium ions) and the multivalent ion (e.g., Ca ²⁺ ) to give a sol/gel transition (Draget et al, 1998). The characteristic chelate-type ion- binding properties of alginates can be explained by 'egg-box' model in which electronegative cavities are formed by polyguluronic chains in alginate to host divalent cations (Grant et al., 1973; Morris et al, 1978). In this model, guluronate sequences are responsible for creating cavities where the multivalent ions coordinate along the alginate chains (Mehrotra, 1983). The coordination of metal-carboxylate can occur in different ways: (a) an ionic or uncoordinated form, (b) unidentate coordination, (c) bidentate chelating coordination, and (d) bidentate bridging coordination (Papageorgiou et al, 2010).

a b c d Types of metal-carboxylate coordination (After Papageorgiou et al., 2010). (a) an ionic or uncoordinated form, (b) unidentate coordination, (c) bidentate chelating coordination, and (d) bidentate bridging coordination.

Sodium alginate is the salt of alginic acid which consists of two uronic acids, b-D- mannuronic acid and a-L-guluronic acid. Ca ²⁺ is typically used to replace Na ⁺ in alginate to produce Ca-alginate. This stable gels is formed as Ca ²⁺ interact ionically with blocks of uronic acid residues to form a three-dimensional network that is usually described by the 'egg-box' model (Papageorgiou et al, 2010). Other di-valent ions such as Fe ⁺² can also be used to cross-link with alginate. Fe ² has been cross-linked with alginate and used in the biomedical research (Machida-Sano et al., 2009).

Formation and chemical structure of Fe (II) alginate coordination polymer:

Based on the molar ratio of alginate to Fe (II) of 1 : 2 (from the conductivity study performed), the above structure can be predicted where the iron ion coordinates with carboxyl group on the L-guluronic acid (G units). Other forms of hydrogen bonds between the iron ion and other hydroxyl groups might take place as well.

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Example 4. Bare NZVI and Iron Cross-Linked Alginate Beads: Applications for Phosphate

Removal From Actual Wastewaters Applications of nanoscale zero-valent iron (NZVI) and iron cross-linked alginate

(FCA) beads were explored in this study for phosphate removal from actual wastewaters. Wastewater treatment plant effluent (WTPE) and animal feedlot effluent/runoff (AFLE) samples were used in the phosphate removal studies. While FCA beads removed 97% of the P0 ₄ ^3~ in 2 h from WTPE, NZVI removed 84%. However, the difference was not statistically significant. Fast removal rate was observed with FCA used to remove phosphate from AFLE (-77% removal at the end of 15 min). The FCA beads continued to remove phosphate faster than NZVI until ~ 60 min. Results have indicated that FCA beads were more efficient (85%) as compared to NZVI particles (57%) in the first hour. The overall P0 ₄ ^3" removal by FCA beads reduced from 85% in 1 h to 75% at 24 h. This removal rate has possible application in the field with high flow rate systems.

Introduction

Excessive discharge of phosphorus (P) in surface water causes deterioration of water quality. Nutrient (P) richness in surface water bodies results in eutrophication of the water bodies. Eutrophication has significant economic impacts on local communities.

Two of the major sources of phosphate in surface water are wastewater effluent (point- source) and animal feedlot runoff (nonpoint-source). The estimated contributions of P sources to municipal wastewater from human wastes, laundry detergents, and other cleaners are 0.6, 0.3, and 0.1 kg P/capita/year, respectively (Sengupta et al, 2011, Wat Res 45:3318- 3330). Municipal wastewater contains adequate amount (5 -15 mg/L) of P (Blackall et al,

2002). Even though the contribution of laundry detergents in increasing P in wastewater successfully reduced nowadays, P concentration in WTPE would reduce by only to 4-5 mg/L P (USGS, 1999). This effluent with high concentration of P finds its way to lakes and surface waters. Various studies have indicated that concentrations of P above 0.02 mg/L accelerate eutrophication of water bodies (Sharpley et al, 2003; Seviour et al, 2003, FEMS Microbiol Rev 27:99-127).

Materials and Methods

Chemicals

Iron (II) chloride tetrahydrate (FeC ^F^O, reagent grade, J.T. Baker), calcium chloride (CaCl ₂ , ACS grade, BDH), monopotassium phosphate (KH ₂ P0 ₄ , 99% pure, EMD), sodium alginate (production grade, Pfaltz & Bauer), potassium nitrate (K 0 ₃ , 99%, Alfa Aesar), sodium hydroxide (NaOH, ACS Grade, BDH), sodium borohydride (NaBH ₄ , 98%, Aldrich), methanol (production grade, BDH) were used as received unless and otherwise specified. NZVI synthesis

NZVI was prepared as described by (Almeelbi, 2012). Briefly, FeCl ₃ solution was dropped into sodium borohydride solution and stirred for 30 min. The black resultant black precipitate (NZVI) was separated, washed by methanol and water using a centrifuge. The washed (NZVI) particles were dried using a vacuum oven under N ₂ environment overnight and then ground using a mortar and pestle. The fine black powder was stored in a 20 mL vial for later use. Particles were not stored more than two weeks. The detailed method of NZVI has been reported by Almeelbi and Bezbaruah (2012).

Beads synthesis

Alginate solution (5 mL of 2% w/v) was dropped into FeCl ₂ solution (35 mL of 2% w/v) in a 50 mL polypropylene plastic vial using a pump with very small tube track to reduce the loss of alginate. Moreover, the first batch was sacrificed to ensure eliminate any effect of alginate volume reduction due to alginate that might have remained within the tubings.

Beads were kept in the FeCl ₂ solution for at least 6 h with vial was capped. Samples collection and storage

Municipal Wastewater Treatment Plant (WTPE) Effluent: Samples were obtained from the City of Moorhead Wastewater Treatment Plant (Moorhead, MN, USA). Moorhead follows a pure oxygen activated sludge treatment scheme. The secondary treated wastewater is subjected to tertiary treatment that involved nitrogen removal and additional polishing for organics and suspended solids. Tertiary treated wastewater samples from the effluent sampling point in outlet leading to the Red River outfall were collected in plastic containers (~8L). The WTPE was filtered through a 1.2 μιη pore-size Whatman glass microfiber filter (GF/C) before use in the experiments or stored in the refrigerator at 4°C for later use. Stored samples were used within a month.

Animal Feedlot Effluent (AFE): Samples were collected from a privately owned cattle feedlot at Sargent County, North Dakota, USA. Unfiltered samples were used immediately or stored in a plastic container (~8L) in the refrigerator at 4°C for later use. Stored samples were used within a month.

Batch studies

WTPE and AFLE samples were used in P0 ₄ ^3~ removal studies with NZVI and FCA beads as the sorbents. One batch of FCA beads (0.121g dry weight) or 0.02 g NZVI were added to 50 mL of wastewater in multiple polypropylene plastic vials fitted plastic caps (reactors). The reactors were rotated end-over-end at 28 rpm in a custom-made shaker to reduce mass transfer resistance. A set of sacrificial reactors was withdrawn at specific time interval. The phosphate concentration in the bulk solution was measured and reported as average (with standard deviations) of readings from three replicate studies. Phosphate analysis

Ascorbic acid method (Eaton et al, 2005) was used for phosphate analysis. This method depends on the direct reaction of orthophosphate with molybdate anions to form a yellow-colored phosphomolybdate complex. Ascorbic acid reduces phosphomolybdic to form molybdenum blue species that has a broad absorbance range in between 700 nm to 900 nm. The color was measured in a UV-vis spectrophotometer (HACH, DR 5000) at wavelength of 880 nm. A five-point calibration was done routinely. Quality control

All experiments were done in triplicates during this research and the average values are reported along with the standard deviations. Blanks with only wastewater/runoff (without NZVI/FCA beads) were run along with the NZVI and FCA bead experiments. The analytical instruments and tools were calibrated before the day's measurements. One-way ANOVA tests were performed to compare the variance between data sets as needed. Minitab 16 software (Minitab, USA) was used for all statistical analyses. Results and Discussion

Beads characterization

Beads were approximately spherical in shape with average diameters of 3.09±0.16 mm and each batch of dry FCA beads weighted 0.121±0.002 g. SEM analysis of the beads were done after drying the beads for 24 h in a vacuum oven under nitrogen environment. Iron nanoparticles was observed inside the dried the beads (FIG. 23), and the nanoparticles had an average size of 74.45±35.60 nm (n = 97).

NZVI characterization

Almeelbi and Bezbaruah (2012) have used TEM to determine the size of NZVI and reported the particle size as 16.24±4.05 nm (n = 109).

Phosphate removal from WTPE

In batch studies conducted using NZVI and FCA beads for P0 ₄ ^3~ removal from WTPE, FCA beads removed 97% of the P0 ₄ ^3" in 2 h while NZVI removed only 84% (FIG. 24). NZVI was faster in removing P0 ₄ ^3" as compared FCA in the first 15 min, and removed 80% P0 ₄ ^3" while FCA beads removed only 63%. NZVI continued to perform better till ~30 min beyond which FCA removed P0 ₄ ^3" at better rate than NZVI. However, ANOVA analysis indicates that there is no significant difference between the P0 ₄ ^3" removal efficiencies by NZVI and FCA beads after 2 h (p = 0.629). The finding is important from field application perspective. While it may be difficult to use and then recover NZVI particles (average diameter ~16 nm) in wastewater treatment plant or similar set-ups, the FCA beads which are much larger (average diameter ~3 mm) will be easily recoverable. Further, there are still a number of unknowns about the toxicity of NZVL Saleh et al., (2008) found that coated NZVl can remain mobile in aqueous media even after 8 months of application and may be toxic to humans. There are also other reports on toxicity of NZVl (Keller, 2012; Li, 2010; Phenrat, 2009; Xiu, 2010) that call for caution in wide scale application of the bare or unmodified nanoparticles.

Phosphate removal from AFLE

Batch study results have indicated that FCA beads were more efficient (85%) as compared to NZVl particles (57%) in the first hour (FIG. 25) of reaction in removing P0 ₄ ^3~ from animal feedlot effluent (AFLE). Statistical analysis indicate that the results from these two sets of experiments are significantly different (one-way ANOVA, p = 0.00). Data points could not be collected exactly at 2 h for all the samples due to management issues and, therefore, have not been compared.

The batch studies with the AFLE were continued till 24 h (FIG. 26) and it was observed that the overall P0 ₄ ^3" removal by FCA beads reduced from 85% in 1 h to 75% at 24 h. There is no immediate explanation for this behavior of the beads till further research is conducted. However, a possible reason may have to do with the presence of orthophosphate in the particulate form. AFLE was used as received (without any filtration) for P0 ₄ ^3" removal using NZVl and FCA beads. A layer of visible black particles was observed on the beads at the end of the reaction which may be the particulate P0 ₄ ^3" and they might have contributed to the increase in P0 ₄ ^3" concentration. Further studies may be needed to understand this behavior of the beads. It is, however, clear that FCA beads can be used to remove phosphate from AFLE. P0 ₄ ^3" removal was -77% at the end of 15 min (Table 3). The short contact time needed for P0 ₄ ^3" removal is expected to have major ramifications as FCA beads can possibly be used in high flow system (e.g., pumped system). Table 3: P0 ₄ ^3~ removal from AFLE using NZVI and FCA beads

Time, h % P0 ₄ ³ Removal

FCA NZVI

0.25 76.85 35.23

0.50 84.07 50.39

1.00 85.27 57.22

2.75 85.66 72.91

4.00 83.80 -

6.00 81.05 -

8.00 81.67 -

12.00 79.50 -

18.00 83.03 -

24.00 75.21 94.06

Data at 4, 6, 8, 12, and 18 h were not collected for NZVI studies Conclusions

NZVI and FCA beads successfully removed P0 ₄ ^3" from both municipal wastewater (WTPE) and animal feedlot effluent (AFLE). The fact that FCA beads could remove 63% and 77% P0 ₄ ^3" from WTPE and AFLE, respectively, within the first 15 min provides a huge advantage for their application in high flow systems. NZVI particles were also effective in removing P0 ₄ ^3" from waters. However, FCA beads performed better with AFLE. More experiments need to be conducted to determine the possibility of P0 ₄ ^3" recovery from FCA beads.

References

Almeelbi T, Bezbaruah AN (2012) Aqueous phosphate removal using nanoscale zero-valent iron. Journal of Nanoparticle Research, 14(7), 1-14

Saleh N, KimHJ, Phenrat T, Matyjaszewski K. Tilton R D. Lowry GV. (2008) Ionic strength and composition affect the mobility of surface-modified Fe° nanoparticles in water- saturated sand columns. Environ. Sci. Technol, 42, 3349-3355. Li ZQ, Greden K, Alvarez PJJ, Gregory KB, Lowry GV (2010) Adsorbed Polymer and NOM Limits Adhesion and Toxicity of Nano Scale Zerovalent Iron to E. coli. Environ Sci Technol 44:3462-3467. doi: 10.1021/es9031198

Phenrat T, Long TC, Lowry GV, Veronesi B (2009) Partial oxidation ("aging") and surface modification decrease the toxicity of nanosized zerovalent iron. Environ. Sci.

Technol, 43: (1), 195-200.

Xiu ZM, Gregory KB, Lowry GV, Alvarez PJ (2010). Effect of Bare and Coated Nanoscale Zerovalent Iron on tceA and vcrA Gene Expression in Dehalococcoides spp. Environmental science & technology, 44(19), 7647-7651.

Keller AA, Garner K, Miller RJ, Lenihan HS (2012) Toxicity of Nano-Zero Valent

Iron to Freshwater and Marine Organisms. PLoS ONE 7(8) e43983

Blackall LL, Crocetti G, Saunders AM, Bond PL (2002) A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology 81(1-4):681-691.

USGS (1999) Phosphorus in a Ground- Water Contaminant Plume Discharging to Ashumet Pond, Cape Cod, Massachusetts, Northborough, MA.

Sharpley AN, Daniel T, Sims T, Lemunyon J, Stevens R, Parry R (2003)Agricultural Phosphorus and Eutrophication (second ed.) United States Department of Agriculture, Agricultural Research Service

Seviour RJ, Mcllroy S (2008) The microbiology of phosphorus removal in activated sludge processes - the current state of play. Journal of Microbiology 46(2): 115.

Example 5. Dry Bead Sorption Results

Beads (FeCl ₂ crosslinked/FeCl ₂ hardened) were tested to ascertain how much phosphate they could sorb once they had been dried out. Different concentrations of aqueous phosphate were tested. At an initial phosphate concentration of 5 mg/L, after 24 hours approximately 4.31 mg/L of phosphate had been sorbed (83.4%). At an initial phosphate concentration of 50 mg/L, after 24 hours approximately 19.7 mg/L of phosphate had been sorbed (38.8%). At an initial phosphate concentration of 100 mg/L, after 24 hours approximately 37.3 mg/L of phosphate had been sorbed (33.3%). At an initial phosphate concentration of 200 mg/L, after 24 hours approximately 46.8 mg/L had been sorbed (22.4%). At an initial concentration of 500 mg/L, after 24 hours approximately 82.8 mg/L of phosphate had been sorbed (15.6%). FIG. 27 shows an isotherm constructed from the phosphate sorption data, indicating that maximum capacity of the beads has likely not been reached. It is expected that capacity will exceed 100 mg/L, and that results will be similar for FeCl ₃ crosslinked/FeCb hardened beads as well as other embodiments of the beads described herein.

The complete disclosures of all patents, patent applications including provisional patent applications, publications including patent publications and nonpatent publications, and electronically available material (e.g., GenBank amino acid and nucleotide sequence submissions) cited herein are incorporated by reference. The foregoing detailed description and examples have been provided for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described; many variations will be apparent to one skilled in the art and are intended to be included within the invention defined by the claims.