CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/701,235 filed on July 20, 2018, the contents of which are incorporated by reference in its entirety.

STATEMENT REGARD 1NG FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under 1554511 awarded by the National Science Foundation. The government has certain rights to the invention.

BACKGROUND OF THE INVENTION

[0003] The field of the invention is related to the ability to selectively adsorb and desorb phosphate from a liquid solution under controlled conditions. This method can be used to 1) facilitate measurement of ultra-low phosphate concentrations and 2) to recover phosphate for reuse.

[0004] Phosphate (Pi) is an essential nutrient for global food supply and is a non renewable and increasingly expensive resource. (Cordell et al., 2009; Cordell and White, 2014; Mayer et al., 2016; Rittmann et al., 2011). At the same time, the presence of Pi in fresh water sources can cause severe algal blooms, leading to ecological and economic losses. (Schroder et al., 2010; USEPA, 2007). ln light of this, a system which can effectively remove Pi down to ultra-low levels (<100 pg/L) and release Pi under controlled conditions for reuse as fertilizer is essential for attaining an environmentally sustainable and economically viable solution. (Mayer et al., 2013; Rittmann et al., 2011). Additionally, improved analytical quantification techniques to accurately measure ultra-low levels of phosphate are important for monitoring its presence in aquatic matrices.

[0005] There have been numerous approaches developed and tested to try and achieve ultra-low levels of phosphate detection and removal/recovery. Prior removal methods include physical-chemical (e.g., coagulation using iron followed by sedimentation, filtration using iron-based media) as well as biological (e.g., enhanced biological phosphorus removal). To more selectively, and reversibly, recover phosphorus from heterogeneous liquids, one approach that has attracted increasing interest is ion exchange. Synthetic resins can be designed to more selectively remove phosphate. One example is LayneRT resin (a commercialized product originally developed at Lehigh University). However the current methods still lack the ability to bind ultra-low levels of phosphate or the specificity to produce a purer product (e.g. reduce non-specific binding with arsenate).

[0006] There is a need for highly sensitive and specific methods of removing and concentrating phosphate from a liquid sample, particularly when present at initially low levels, to facilitate improved recovery and quantification.

SUMMARY OF THE INVENTION

[0007] The present invention provides methods, kits and systems for concentrating and recovering phosphate from a sample. The methods, kits and systems of the present invention may facilitate measurement of ultra-low concentrations and recovery of phosphate for reuse.

[0008] ln one aspect, the present disclosure provides a method of concentrating and recovering phosphate from a sample, the method comprising: (a) exposing a liquid sample to immobilized phosphate binding protein (PBP) for a sufficient amount of time to bind phosphate to the immobilized PBP within the sample; and (b) desorbing the phosphate from the immobilized PBP by contacting the immobilized PBP with a desorption solution having a pH of 11 or greater (e.g., 11.4), preferably a pH of 12 or greater, wherein the phosphate is concentrated with the desorption solution ln some aspects, step (a) further comprises a wash step with a washing solution at a neutral pH (preferably between pH 6.8-7.5) to remove non-specific binding of other ions in the sample.

[0009] ln some aspects, the method further comprises: (c) reusing the immobilized PBP by (i) washing the immobilized PBP with a washing solution at a neutral pH between pH 6.8-7.5, and (ii) re-exposing the immobilized PBP with a second sample, (iii) desorbing the phosphate from the immobilized PBP by exposing the immobilized PBP to desorption solution of pH of 11 or higher, preferably a pH of 12 or higher, and (iv) recoving the phosphate in the desorption solution.

[0010] ln another aspect, the present disclosure provides a method of concentrating low levels of phosphate in a sample, the method comprising: (a) exposing a liquid sample to immobilized phosphate binding protein (PBP) for a sufficient amount of time to bind phosphate to the immobilized PBP within the sample; (b) desorbing the phosphate from the immobilized PBP by contacting the immobilized PBP with a desorption solution having a pH of 11 or higher, preferably a pH of 12 or greater wherein the volume of the desportion solution is at least 2 to 10 times smaller than the starting sample volume and the phosphate is concentrated to a detectable level.

[0011] ln a further aspect, the disclosure provides a kit for improved detection or quantification of low levels of phosphate in a liquid sample or concentrating phosphate from a liquid sample, the kit comprising (a) immobilized PBP; (b) a desorption solution having a pH of 11 or higher, preferably a pH of 12 or greater, and(c) instructions for adsorbing and desorbing the phosphate from the immobilized PBP.

[0012] The foregoing and other aspects and advantages of the invention will appear from the following description ln the description, reference is made to the accompanying drawings which form a part hereof, and in which there are shown, by way of illustration, preferred embodiments of the invention. Such embodiments do not necessarily represent the full scope of the invention, however, and reference is made therefore to the claims and herein for interpreting the scope of the invention.

BR1EF DESCRIPTION OF THE DRAW1NGS

[0013] F1G. 1A is a cartoon representation of the method of the current invention using the engineered systems (both recombinant bacteria expressing the phosphate binding protein [PBP] and the PBP immobilized on a bead surface, as signified by the dotted lines).

[0014] F1G. IB is an illustration depicting the approach to using bacteria cells to over express PBP on their surfaces. The PBP immobilized on the bacteria cells can then be used in adsorption applications.

[0015] F1G. 2: lllustration of the PBP bead reusability experiment. The experiment was conducted in triplicate Poly-Prep® columns with 0.25 ITILBV of PBP and control beads without PBP. Cycle 0 includes 2 steps: 1) initial Pi desorption wash and 2) neutral pH wash. Cycles 1 to 10 incude 3 steps that were repeated 10 times: 1) Pi adsorption, 2) high pH Pi desorption and 3) neutral pH wash.

[0016] F1G. 3: Pi recovery from 0.25 ITILBV PBP beads at varying pH and temperature.

The Y axis represents percent Pi desorbed out of the theoretical adsorption capacity (49 nmoles/0.25m LBV) . The X axis describes the IX buffer pH and temperature condition during the test. The error bars represent the standard deviation from triplicate analysis.

[0017] F1G. 4: Kinetics of Pi recovery from 0.25 mLBv PBP beads at varying pH. The Y axis represents percent Pi desorbed out of the theoretical adsorption capacity (49 nmoles/0.25 ITILBV) . The X axis represents exposure time in minutes. All tests were conducted in IX buffer at 25°C and the pH conditions are described in the legend. The error bars represent the standard deviation from triplicate analysis.

[0018] F1G. 5: Summary of PBP bead reusability experiment showing 10 repetitive cycles of Pi adsorption at pH 7.1 and Pi desorption at (A) pH 11.5, (B) pH 12 and (C) pH 12.5. The Y axis represents percent Pi adsorbed and desorbed out of the theoretical adsorption capacity (49 nmoles/0.25 ITILBV). ln the X axis, Cycle 0 represents the initial Pi desorption wash and Cycles 1-10 represent the subsequent 10 Pi adsorption and desorption cycles. All tests were conducted in IX buffer at 25°C. The error bars represent the standard deviation from triplicate analysis.

[0019] F1G. 6: Pi concentration in the raw environmental waters and desorbed Pi isolution. The Pi concentration in desorbed Pi solution were 2.9 and 3.2 times higher than in the raw river and pond samples, respectively. The total Pi adsorbed and recovered was 88% and 97% of the total Pi present in the raw river and pond water, respectively.

[0020] F1G. 7: Adsorption of phosphate (Pi) and arsenate using PBP-beads exposed to varying molar ratios of arsenate to phosphate. The PBP-beads were exposed to a buffer solution (pH 7 and 25 ^Q C) containing both ions at an arsenate to phosphate molar ratio ranging from 0 to 2. Even when arsenate was present at two times the amount of phosphate, the majority of the PBP-beads’ capacity was occupied by phosphate (>95%). Bars show the average of triplicate experiments, while the error bars show ± 1 standard deviation.

[0021] F1G. 8: lnitial theoretical Pi concentration in the synthetic water samples containing ultra-low Pi concentrations ranging from 4.7 to 47.5 pg/L and the measured Pi concentration in the desorbed concentrated regenerant solution. The Pi concentrations in the desorption solution (pH 12.7) were 4.4 to 5.4 times higher than in the synthetic water samples. The total Pi adsorbed and recovered ranged from 89% to 100% of the total Pi originally present in the water samples.

[0022] F1G. 9: Percent error in Pi quantification in the synthetic water samples containing ultra-low Pi concentrations ranging from 4.7 to 47.5 pg/L and the measured Pi concentrations in the desorbed concentrated regenerant solutions. The percent error in Pi quantification in the concentrated regenerant, recovered from synthetic water samples containing ultra-low Pi concentrations was much lower compared to the initial samples. Fig

DETA1LED DESCRIPTION OF THE INVENTION

[0023] The present invention provides methods of removing, concentrating and purifying phosphate from a liquid sample, specifically ultra-low concentrations of phosphate, to facilitate downstream phosphorus quantification or reuse.

[0024] The present invention provides immobilized PBP in engineered systems (preferably either attached to a surface, e.g., a plastic bead, or expressed on the surface of bacterial cells) to recover phosphate from liquids, even in conditions in which the phosphate is found in ultra-low levels (e.g. levels below 100 pg/L, preferably below 50 pg/L, and including levels below 20 pg/L).

[0025] The present invention provides an improved process to selectively adsorb and desorb phosphorus from liquids including, but not limited to, for example, environmental waters and wastewaters. By removing the phosphorus, the environmental degradation caused by excess phosphorus in water is alleviated. The recovered phosphorus can be reused for beneficial purposes, e.g., to supplement depleting global supplies of the mineral phosphorus used to produce agricultural fertilizer.

[0026] ln some embodiments, the methods of the present invention provide selective adsorption and recovery of phosphorus that can be used for improved quantification of phosphorus in environmental water samples. Current standard methods (colorimetric or ion chromatograph) can reliably detect phosphate at >50 ug/L. Standard colorimetric methods can detect phosphate from 20-50 ug/L; however, error in measurement is more than 10% and reliability is low. lon chromatographs require an anion exchange solid phase extraction (SPE) cartridge to measure phosphorus below 50 pg/L. However, there are no known SPE cartridges available to specifically concentrate phosphorus. Phosphorus- induced eutrophication has been shown to occur at concentrations below 50 pg/L. The lack of reliable detection methods between ~0 to 50 pg/L hinders identification of issues and implementation of remedial measures that could be applied before phosphorus reaches critical levels in environmental waters. The method of the present invention provides methods to improve phosphate quantification at ultra-low levels by selectively adsorbing phosphates from water samples (<50 pg/L) and subsequently desorbing phosphorus in lower volumes (concentrated at >100 pg/L), which can be quantified using conventional methods.

[0027] The methods described herein use phosphate binding proteins (PBPs) to capture and release phosphate under controlled conditions that allow for the unexpectedly low capture and concentration of phosphate. PBP is naturally expressed in some bacteria under low phosphate conditions. Bacteria express the protein in their periplasmic space as part of a high-affinity phosphate-specific transporter system. The PBP selectively binds inorganic phosphorus (i.e., phosphate) in a deep cleft using 12 hydrogen bonds. The phosphate is then transported into the cell for use in the cell’s metabolic machinery, e.g., to build DNA or to store energy (ATP). The PBP protein may contain a tag that allows for it to be attached to the surface or inert material (e.g., bead or membrane) for use in the present invention.

[0028] Suitalbe PBP proteins are known in the art and are not limited to the examples described herein. PBP is a multispecies ABC transporter protein found in several species of proteobacteria (NCB1 Reference Sequence: WP_000867146.1) and is highly conserved between bacteria. PBP features 7 different amino acid residues that form 12 strong hydrogen bonds with Pi, as illustrated in Venkiteshwaran et al. (2018), incorporated by reference in its entirety.

[0029] Any suitable PBP that binds to phosphate can be used in the practice of this invention. Suitable PBP include, for example, wildtype and modified PBP obtained from any suitable bacterial strain, which maintain their ability to bind phosphate. Suitable PBP examples include, for example, E. coli or Shigella flexneri , among others. While the PBP protein disclosed in the Examples was derived from E. coli K-12 strain, many other PBP proteins may be used in the present invention. PBP is a highly conserved protein. According to UniProt, over 100 different microorganisms have PBP proteins with an identical amino acid sequence (100% identity) to that found in E. coli K-12 strain (SEQ ID NO: 1). These microorganisms include many other strains of E. coli, as well as several species of Shigella. Notably, among these proteins, the PBP of Shigella flexneri 301 strain is the only other PBP protein that has been manually curated and reviewed (SEQ ID NO: 7). Additionally, many microorganisms have PBP proteins that are nearly identical to that of E. coli K-12 strain (ETniProt lists over 350 with greater than 90% identity). Closely related PBP proteins include, without limitation, those of E. coli UMEA 3200-1 (SEQ ID NO: 9, 99.4% identity), Shigella boydii ATCC 9905 (SEQ ID NO: 10, 99.7% identity), and Trichuris trichiura (SEQ ID NO: 11, 98.8% identity). For example, PBP from the E. coli K- 12 strain, substrain MG1655 (NCB1 Reference Sequence: NP_418184.1; GenBank: AYG21454.1; UniProtKB - P0AG82 (SEQ 1D NO:l), including the PBP without the singal sequence (e.g., SEQ 1D NO:2, SEQ 1D NO:6) or modified E. coli PBP (e.g., SEQ 1D N0:4, SEQ 1D NO:5), or the PBP of Shingella (SEQ 1D NO:7) or sequence that have at least 80% identity to the PBP of any one of SEQ 1D NO: 1-7 can be used in the practice of the invention. Suitable PBP proteins include, for example, PBP proteins that share substantial identity to the polypeptide found in any one of SEQ ID NO: 1-7, including modified proteins that maintain the ability to bind phosphate. SEQ ID NO: 2 is PBP amino acid sequence from E. coli K-12 strain, substrain MG1655 (NCB1 Reference Sequence: NP_418184.1; GenBank: AYG21454.1; UniProtKB - P0AG82) with the signal peptide removed and replaced with start codon methionine (M,). SEQ 1D NO: 3 is gene sequence associated with the phosphate-binding protein from E. coli K- 12 strain, substrain MG1655 (GenBank: CP032667.1.). SEQ 1D N0:4 is the amino acid sequence PBP sequence PBP A197C from E. coli K-12 strain (addgene id: pET22b_PstS_l) with the signal peptide removed and replaced with methionine (M), and alanine (A) in position 197 replaced with cysteine (C). SEQ 1D NO:5 is the gene sequence or the associated PBP of SEQ 1D N0:4. SEQ 1D NO:6 is PBP without the signal sequence from in E. coli K-12 strain.

[0030] In some embodiments, the PBP have at least 75% sequence identity to any one of

SEQ ID NO: 1-7, alternatively at least 80% sequence identity, alternatively at least 85% sequence identity, alternatively at least 90% sequence identity, alternatively at least 95% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 99% sequence identity, alternatively at least 100% sequence identity to any one of SEQ ID NO: 1-7. The term "substantial identity" of protein sequences means that a protein comprises a sequence that has at least 75% sequence identity to the polypeptide of interest described herein. Alternatively, percent identity can be any integer from 75% to 100%. More preferred embodiments include at least: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs known in the art; for example BLAST using standard parameters. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0031] Additionally, the PBP protein or modified protein may further comprise a tag that allows for ease of mass producing the PBP protein for use in the methods described herein. Suitable tags are known in the art and include but are not limited to, for example, epitope tags which are known in the art and include, but are not limited to, 6-Histidine (His, HHHHHH), cMyc (EQKLISEEDL), FLAG (DYKDDDDK), V5-tag (GKPIPNPLLGLD ST), HA- tag (YPYDVPDYA), NE-tag (TKENPRSNQEESYDDNES), S-tag (KETAAAKFERQHMDS), Ty tag (EVHTNQDPLD), among others. Epitope tags are commonly used as a purification tag. A purification tag is an agent that allows isolation of the polypeptide from other non-specific proteins.

[0032] The wildtype E. coli K-12 PBP has 346 amino acid bases (SEQ 1D NO:l). The complete genome sequence of E. coli K-12 strain, substrain MG1655 can be found using the reference 1D GenBank: CP032667.1 where the protein gene sequence is located between base pairs 4611542 to 4612582. The length of the PBP gene is 1041 nucleotides. Expressed wild type PBP amino acid sequence from the E. coli K-12 strain, substrain MG1655 (NCB1 Reference Sequence: NP_418184.1; GenBank: AYG21454.1; UniProtKB - P0AG82) is found in SEQ 1D NO:l (346 amino acids with a 22 aa signal peptide at the beginning). Suitable, for producing large quantities Other PBP sequences This sequence is similar to the reference sequence for multispecies PBP ABC transporter in the database (NCB1 Reference Sequence: WP_000867146.1).

[0033] ln the newly developed system, PBP is used to selectively capture phosphate as a reversible ligand such that the phosphate can be released back into solution in a controlled fashion at a high pH (e.g. a pH of about 12 or greater). The phosphate is released back into solution in a concentrated and pure form.

[0034] The high specificity of the use of PBP allows for the recovered and concentrated phosphate isolated by the current invention to be of high purity, e.g. at least 50% phosphate, more preferably at least 75% phosphate, alternatively at least 80% phosphate, alternatively at least 85% phosphate, alternatively at least 90% phosphate, alternatively at least 95% phosphate ln a preferred embodiment, the purity is at least about 90%.

[0035] ln one embodiment, the present invention provides a method of concentrating and recovering phosphate from a sample, the method comprising: (a) exposing a liquid sample to immobilized phosphate binding protein (PBP) for a sufficient amount of time to bind phosphate to the immobilized PBP within the sample; and an optional wash step with a washing solution at a neutral pH (preferably between pH 6.8-7.5) to remove non-specific binding of other ions in the water, and (b) desorbing the phosphate from the immobilized PBP by contacting the immobilized PBP with a desorption solution having a pH of 11 or greater, preferably a pH of 11.4 or greater, more preferably having a pH of 12 or greater, wherein the phosphate is concentrated with the desorption solution. The volume of the desorption solution can be 2 to 10 times smaller than the raw liquid sample ln some embodiments, the desorption solution containing the phosphate is altered to a neutral pH by the addition of an acid (for example, but not limited to, HC1, citric acid, etc.) . Once neutralized, the desorption solution containing the phosphate may be used for quantification of phosphate or recovered for reuse applications, e.g., as struvite. Suitable desorption solutions are able to be determined by one skilled in the art, and include any solution able to have a pH of 11 or greater, preferably a pH of 11.4 or greater, more preferably having a pH of 12 or greater (e.g., pH of 12.5). Suitable desorption solutions include any buffer with a pH greater than 11, preferably greater than 12, including, but not limited to, water, Tris buffer, among others. For example, as described in the Examples below, some examples of suitable desorption solutions include deionized water, Milli-Q water, lOmM Tris + 1 mM MgCk buffer at a pH greater than 11, preferably greater than 12, for example, a pH of 12.5. As disclosed in the Examples, the higher the pH above 11, the more effective the desorption is. For example, a desorption solution of a pH of 11.4 allows for desorption of about 60% of the phosphate, while a desorption solution of pH of 12 allows for desorption of over 80%, preferably over 90% and up to 100% of the desportion of the phosphate from the PBP as demonstrated in the examples. As denoted in the examples, a desorption solution at a pH of 12.5 was able to desorb 100% of the phosphate. Again, suitable desorption solutions with a pH greated than 11, preferably greater than 12 can be determined by one skilled in the art and are not limited by the Examples and embodiments provided herein.

[0036] ln some embodiments, the method further comprises (c) quantifying the phosphate within the desorption solution of step (b). Suitable methods of detection or quantification are known in the art and include, but are not limited to, for example, detected by colorimetric methods or ion chromatography.

[0037] The term "immobilized PBP" as referred to herein refers to PBP attached or complexed to a surface or inert material, for example, a bead, membrane or bacteria ln some methods, the PBP is expressed on the surface of bacteria.

[0038] ln one embodiment, the PBP is immobilized on bacteria. By "immobilized on bacteria" as used herein, the PBP is expressed on the surface of the bacterial cell. Suitable methods of expressing a protein on the surface of a cell are known in the art, as described in, for example, Chen and Georgiou, 2002 , Georgiou et al., 1997; Li et al., 2004, the contents of which are incorporated by reference. Any suitable method to surface display the PBP on the surface of the bacteria are contemplated, including, but not limited to, for example, cell surface display of heterologous PBP on the cell’s outer membrane using the N-terminal domain of the ice nucleation protein as its anchoring motif as described in the Examples below. Briefly, the bacteria are transformed using the recombinant plasmid containing the relevant ice nucleation protein and PBP into E. coli cells using standard procedures.

[0039] A benefit of the present methods and systems is the ability to re-use the immobilized PBP for additional adsorption/desorption cycles ln some embodiments, the method further comprises: d) reusing the immobilized PBP by (i) washing the immobilized PBP with a washing solution at a neutral pH (preferably between pH 6.8-7.5), (ii) re-exposing the immobilized PBP with a second sample, (iii) desorbing the phosphate from the immobilized PBP by exposing the immobilized PBP to desorption solution of pH of 12 or higher, and (iv) removing the phosphate in the desorption solution.

[0040] ln some embodiments, the immobilized PBP can be used for at least 5 adsorption/desorption cycles, alternatively at least 7 adsorption/desorption cycles, alternatively at least 10 adsorption/desorption cycles. [0041] The methods described herein use a desorption solution with a pH of 11 or greater, preferably a pH of 11.4 or greater, more preferably having a pH of 12 or greater, for example, a pH of 12.5.

[0042] The methods described herein are able to facilitate quantification of concentrations of phosphate from about 0-50 pg/L in the original sample ln some embodiments, the concentration of the phosphate in the sample is from about 0-20 pg/L.

[0043] The liquid samples are any suitable liquid sample in which phosphate can be detected. For example, the liquid sample may be an environmental water sample, wastewater, or liquid laboratory sample. For example, suitable environmental water samples include, but are not limited to, river water, lake water, pond water, stream water or other water found in the environment.

[0044] Suitable wastewater samples include domestic wastewater or agricultural wastewater.

[0045] The present invention can further comprise a method of concentrating low levels of phosphate in a sample, the method comprising: (a) exposing a liquid sample to immobilized phosphate binding protein (PBP) for a sufficient amount of time to bind phosphate to the immobilized PBP within the sample; (b) desorbing the phosphate from the immobilized PBP by contacting the immobilized PBP with a desorption solution having a pH of 12 or greater, wherein the amount of the desorption solution is at least 2 times less than the starting volume of the sample ln some embodiments, the amount of the desorption solution is at least 10 times less than the starting volume of the sample ln some embodiments, the method further comprises (c) quantifying the phosphate within the desorption solution. This quantification may allow for the ability to determine the amount of phosphate in the original sample.

[0046] ln some embodiments, the step (b) further comprises neutralizing the desorption solution containing the phosphate to a neutral pH with an acid. Suitably, a neutralized pH is from about 6-8, preferably about 6.8-7.5.

[0047] The methods described herein are able to desorb the phosphate from the immobilized PBP in a volume of desorption solution sufficient to concentrate the phosphate to a detectable level of more than 100 pg/L. [0048] The present invention also provides kits for performing the methods described herein. For example, in one embodiment, the present disclosure provides a kit for quantifying ultra low levels of phosphate (e.g. levels below 100 pg/L, preferably less than 50 pg/L, or less than 20 pg/L) of phosphate in a liquid sample or concentrating phosphate from a liquid sample; the kit comprising (a) immobilized PBP, (b) a desorption solution having a pH of 11 or greater, preferably 11.4 or greater, most preferably pH of 12 or greater (e.g. pHof 12.5), (c) instructions for adsorbing and desorbing the phosphate from the immobilized PBP. ln some embodiments, the immobilized PBP is PBP bound to a bead or membrane ln other embodiments, the immobilized PBP is PBP bound to a bacteria.

[0049] The present methods facilitate recovery of concentrated phosphate for more accurate measurements or as a phosphorus-rich product which can subsequently be used as agricultural fertilizer.

[0050] ln one example, an engineered PBP (e.g., the high-affinity PstS protein, e.g., SEQ 1D NO:5) system has been made comprising the PBP immobilized on a bead to ensure efficient separation from the liquid.

[0051] ln one embodiment, the system for performing the methods of the present invention comprises purified PBP immobilized on inert surfaces or on the surface of bacteria cells. Suitable inert surface include glass, silicon, plastic and agarose-based material shaped as beads or flat membranes which can be charged with a functional group that covalently binds to the proteins. These functional groups can include, e.g., Aldehyde, Glutaraldehyde, N-hydroxysuccinimide (NHS), Carbodiimide, Epoxides, lsothiocyanate, Azlactone, p- nitrophenyl. PBP can also be immobilized on bacteria by genetically modifying the bacteria with a plasmid that promotes expression of the PBP on the bacterial cell surface. Suitable PBP proteins for bacterial expression are known in the art.

[0052] Suitable methods of producing PBP are known in the art. For example, a recombinant bacteria cell modified to include a plasmid harboring the PBP gene may be used to express and purify the PBP. The bacterial cells are induced to over-express PBP proteins via either low phosphate conditions or 1PTG (isopropyl b-D-l-thiogalactopyranoside). The PBP proteins are extracted from the cells and purified.

[0053] The purified PBP are subsequently immobilized on an inert surface, e.g., Sepharose beads. The water sample is contacted with the immobilized proteins and allowed to bind with phosphate for 5 to 10 minutes or until the beads are saturated and can no longer remove additional phosphate. Subsequently, the phosphate is released by controlling the desorption solution and desorption parameters. The desorption solution is preferably at a pH greater than 12 (e.g., pH of 12, 12.5, 13, etc or any pH range in between) lt is this high pH solution that allows for the desorption and concentration of phosphate ln one embodiment, the immobilized PBP may be operated in a flow-through column mode for phosphate removal and recovery in scaled-up applications.

[0054] ln yet another embodiment, the system comprises E. coli bacteria modified to express the PBP protein on the cell surface using the ice nucleation protein as an anchor (See, e.g., SEQ 1D NO:8, providing the gen sequence of the ice nucleation protein (anchor protein) and heterologous protein (PBP), which is known in the art (e.g. Chen and Georgiou (2002), among others). The bacteria is used in a similar fashion to the bead system, in that sample will be passed over the cells until saturation, followed by a regeneration period where adjustments to desorption parameters (e.g. pH of greater than 11, preferably pH of greater than 12 (e.g., 12.5)) will be used for controlled release of the concentrated phosphate.

[0055] The PBP-based phosphate recovery systems, methods and compositions of the present invention offer several advantages over existing technology (e.g., ion exchangers) including, but not limited to, for example, (1) ability to achieve ultra-low phosphate concentrations (e.g. levels below 100 pg/L, preferably less than 50 pg/L, or less than 20 pg/L), even when starting at low levels (which surpass the ability of currently available ion exchangers), (2) operation in both wastewater matrices (e.g. where enhanced biological phosphorus removal is often employed) as well as in more dilute organic-poor environmental waters where ultra-low phosphorus regulations or guidelines may be implemented, and (3) enhanced selectivity for phospate (PBP is extraordinarily selective for phosphate, with only one other ion -arsenate- known to attach at a much lower affinity). The ability to get more selective, reversible recovery of phosphate leads to separation of a more pure phosphate-rich product. This in turn leads to higher value reuse applications. For example, in agriculture, the presence of heavy metals, pharmaceuticals, or other contaminants (which may be co-concentrated/ released during ion exchange) in addition to the recovered phosphorus causes a problem for reuse. [0056] The PBP systems and methods described herein provide improved separation and concentration to facilitate detection and reuse of low concentrations of phosphate in a range of water matrices, including environmental waters.

[0057] The PBP systems are believed to be able to capture phosphorus even more selectively compared to resins, which facilitates recovery of a more pure (thus, more valuable) phosphorus fertilizer product. Additionally, the proteins appear to be capable of removing phosphate even when it is present at initially low levels, providing improved performance over ion exchange resins in these conditions.

[0058] lmmobilized PBP has advantages for removing phosphate from a variety of liquid matrices. Suitable liquid matrices in which the systems and methods can be used include, but are not limited to, for example, wastewaters including domestic, agricultural, and others, low-phosphorus environmental waters, drinking water, laboratory samples, and any other water sources in which phosphate is to be detected and/or removed. For example, in one embodiment, the methods described herein can be used in analytical labwork to improve phosphate quantification.

[0059] ln addition to removing the phosphate from water matrices, the recovered product from use of the system and methods described herein is able to be used in phosphorus markets, for example, but not limited to, the production of agricultural fertilizer.

USES

[0060] lmmobilized PBP proteins can be used in applications targeting selective capture and controlled release of phosphate in aqueous solution ln one example, the methods, kits and compositions of the present invention can be used for analytical phosphate measurements. The immobilized PBP protein can be used as a solid-phase extraction column in order to concentrate phosphate samples and, in some embodiments, undergo subsequent analysis (e.g., via the standard ascorbic acid method, ion chromatography, etc.) to quantify its presence. The concentration step enables quantification of phosphate when it is initially present in low concentrations precluding reliable quantification.

[0061] ln another example, the present methods, compositions and kits can be used for water and wastewater treatment for removal and/or recovery of phosphate. The immobilized PBP protein (e.g., PstS) can be used as a granular adsorbent media to selectively remove phosphate from complex aqueous matrices in order to meet water quality goals. Moreover, the removed phosphate can be released using high pH (e.g. pH 12) regenerant solution to regenerate adsorption capacity and facilitate recovery of concentrated phosphate, which can then be reused, e.g., in agricultural fertilizer applications.

[0062] A conceptual diagram showing the engineered systems (both recombinant bacteria expressing the protein and the PBP immobilized on a bead surface, as signified by the dotted lines) is presented in F1G. 1A. F1G. IB shows an additional illustration showing phosphate recovery using engineered bacteria cells over-expressing PBP on the cell surface.

[0063] The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.

[0064] The invention will be more fully understood upon consideration of the following non-limiting examples.

EXAMPLES

[0065] Example 1 Phosphate removal and recovery using immobilized phosphate binding proteins

[0066] PstS is a ubiquitous, selective high affinity PBP. (Brune et al., 1998, 1994; Solscheid et al., 2015). PstS has been tested as a Pi removal recovery system previously. (Choi et al., 2013; Kuroda et al., 2000; Yang et al., 2016) While the Pi adsorption capabilities of PstS are well known, conditions that can provide controlled release of Pi from PBP are not fully understood or characterized. Previous Pi recovery studies did not show significant recovery potential using a wild type PstS. (Kuroda et al., 2000; Yang et al., 2016). Further, it was not known if the protein can be reused for multiple removal and recovery cycles. Reusability of the protein is crucial for making this system a viable alternative for Pi removal and recovery.

[0067] The present Example demonstrates a comparison to wild type periplasmic PBP, extracellular PBP immobilized on an inert surface is more conducive to both removal and controlled recovery through specific regulation of environmental factors such as pH and temperature. [0068] Materials and Methods

[0069] Expression and Purification of PBP

[0070] The PstS PBP used in this study was a single-cysteine mutant variant (A197C) of the mature E. coli PBP, developed by Solscheid et al. (2015) for use as a phosphate biosensor. A pET22b plasmid harboring the pstS gene (A197C) was procured as DH5alpha E. coli bacteria stab culture (plasmid # 78198, Addgene, Cambridge, MA, USA). Following the depositor’s protocol, the culture was initially streaked onto a Lysogeny broth (LB) agar plate containing 100 pg/mL ampicillin and incubated overnight at 37°C. A single colony was isolated and transferred into 5 mL LB solution augmented with 100 pg/mL ampicillin and grown overnight at 37°C with vigorous shaking (200 rpm) to provide aeration. The overnight culture was centrifuged at 4000 rpm for 10 minutes and the plasmid from the resulting cell pellet was extracted using a QlAprep Spin Miniprep Kit (Qiagen®, Germantown, MD, USA). The extracted plasmid was introduced into BL21(DE3) E. coli competent cells, which were subsequently cultured for protein expression and purification using the procedure described by Solscheid et al. (2015).

[0071] A 5 mL overnight culture of the transformed BL21(DE3) cells was grown in LB medium containing 100 pg/mL ampicillin at 37°C. This culture was diluted by transferring 2 mL of the overnight culture into 1 L fresh LB growth media. The baffled glass flasks were incubated at 37°C with vigorous shaking and the culture was allowed to grow to an OD 600 of approximately 0.8 before inducing protein expression using 500 pM 1PTG. After 4-hour induction, cells were centrifuged for 15 min at 4000G and 4°C.

[0072] To purify the cells, the pellets were re-suspended in 100 mL 10 mM Tris-HCl, 1 mM MgCk pH 8.0 buffer solution and sonicated 4 times for 30 s at 200 W with a 5 s on/off pulse cycle. The lysate was decanted following centrifugation at 6000G for 45 minutes. The lysate was passed through a 100 ITILBV (mLBv= settled bead volume) QSepharose column (GE Healthcare Bio-Sciences, Pittsburg, PA, USA), previously equilibrated with 10 mM Tris-HCl, 1 mM MgCk pH 8.0 buffer. The protein was eluted in a 100 mL gradient of 0-200 mM NaCl in 10 mM Tris-HCl, 1 mM MgCk pH 8.0 buffer. The presence of the protein was verified in the eluted fractions using SDS-PAGE, followed by pooling of the fractions to yield 30 mL purified PBP. The concentration of the purified PBP was quantified as 221±0.6 pM (average ±standard deviation) using a Quick Start™ Bradford Protein Assay (Bio-Rad Laboratories lnc., Hercules, CA, USA) .

[0073] Immobilization of PBP

[0074] The purified PstS PBP was immobilized on NHS-activated Sepharose 4 Fast Flow beads in accordance with the manufacturer’s instructions (GE Healthcare Bio- Sciences). A Spectra/Por 2 Dialysis Membrane (MWCO 12-14 kDa, Spectrum Laboratories, lnc., Rancho Dominguez, CA, USA) was used to dialyze the PBP. Dialysis was conducted for 16 hours at 4°C and included 6 exchanges of 0.2 M NaHCCh, 0.5 M NaCl pH 8.3 buffer. The Bradford Protein Assay was used to quantify the concentration of the dialyzed PBP as 202±2 mM.

[0075] Fresh NHS beads (GE Healthcare Bio-Sciences, stored in 100% isopropanol) were transferred into a 100 mL Econo-Column® (Bio-Rad Laboratories lnc.) and washed with 10 bed volumes 4°C 1 mM HC1 solution. For the coupling reaction, 20 mL of dialyzed PBP solution was loaded into the Econo-Column ^® containing 20 mL of washed NHS beads. The Econo-Column ^® was mixed at 30 rpm on an end over end rotator for 16 hours at 4°C to promote coupling. The flowthrough was collected and analyzed for PBP concentration using the Bradford assay. Of the initial PBP loaded onto the column (202±2 mM), 98±0.6% was immobilized onto the NHS beads, providing a coupling density of 197±0.2 nmoles PBP/mLBv NHS beads. Knowing that 1 mole of PBP can adsorb 1 mole of Pi (Brune et al., 1998, 1994; Solscheid et al., 2015), the theoretical Pi adsorption capacity of the PBP beads was 197±0.2 nmoles/mLBv. ln accordance with manufacturer instructions, the PBP beads were washed with IBV of 0.1 M Tris-HCl buffer pH 8.5 followed by IBV 0.1 M acetate, 0.5 M NaCl buffer pH 4.5. This cycle was repeated 3 times followed by five washes with IBV 10 mM Tris-HCl, 1 mM MgCk pH 7.0 buffer.

[0076] To remove any Pi that already adsorbed on the PBP during the expression, purification and immobilization process, the PBP beads were mopped using 0.1 unit/mL purine nucleoside phosphoiylase (PNPase) and 300 mM 7-methylguanosine (7-MEG) (Brune et al., 1998, 1994). To facilitate mixing, 20 mL of 10 mM Tris-HCl, 1 mM MgCk pH 7.0 buffer was added to 20 ITILBV PBP beads. Next, 0.1 unit/mL PNPase enzyme and 300 mM 7-MEG was added to the 40 mL PBP bead solution (50% suspension). The mopping reaction was carried out overnight at 4°C at 30 rpm using a rotary shaker. After 16 hours, the PBP beads were washed with 5 x IBV 10 mM Tris-HCl, 1 mM MgCh pH 7.0 buffer to remove the Pi-mop. This concluded the PBP immobilization procedure and these beads, which from hereon will be referred to as PBP beads, were either used immediately or stored at 4°C for up to 48 hours prior to use.

[0077] A control set of beads was prepared using 20 ITILBV of fresh NHS beads following the same procedure used for the PBP bead, except without the addition of PBP.

[0078] Recovery of Pi from immobilized PBP as a function of temperature and pH

[0079] Triplicate Pi recovery experiments were conducted in batch tests in 2 mL centrifuge tubes containing 0.25 ITILBV PBP beads ln all tests, excess Pi was initially added, 60 mM versus the maximum theoretical capacity of the PBP beads of 197±0.2 nmoles/mLBv (or 49 nmoles/0.25 ITILBV), to ensure maximum adsorption. Tubes were gently mixed, and then the beads were allowed to settle for 10 min. A 1 mL aliquot was analyzed for Pi using the standard ascorbic acid method (APHA, 2012). The beads were washed 3 times using 1 mL IX buffer pH 7 to remove unbound Pi. Each tube was then loaded with 1 mL of IX buffer (10 mM Tris-HCl, 1 mM MgCh) solution ln separate experiments, the influence of temperature was evaluated by adjusting the buffer temperature to 25°C, 35°C, or 45°C (pH 7). Additional tests were performed to evaluate the influence of pH by adjusting 25°C buffer to final values of 4.7, 6.5, 7.1, 8.5, 9.2, 11.4, 12.5 using 1 M HC1 or NaOH. The pH in the tubes was measured using a micro pH probe (Orion™ 9810BN, Thermo Scientific™, Waltham, MA, USA). The beads were gently mixed, allowed to settle for 10 min before 1 mL aliquot was collected and analyzed for Pi. For each condition tested, controls were tested in parallel using control beads in place of PBP beads.

[0080] Kinetics of Pi recovery from immobilized PBP

[0081] The extent of Pi release from PBP beads was assessed as a function of time for 4 different pH conditions. Triplicate experiments were conducted for both PBP beads and control beads. An initial Pi adsorption cycle was performed by adding 1 mL of 60 mM Pi solution in IX buffer at pH 7 and 25°C to the beads. The supernatant was collected and analyzed for Pi concentration. The beads were then washed 3 times with 1 mL IX buffer pH7 to remove the Pi solution. Next, the beads were washed with 1.5 mL of IX buffer, yielding final pH values in the tubes of 7.06, 10.82, 11.88 and 12.5. Aliquots of 0.2 mL were collected for Pi analysis after 5, 10, 20, 30, 40 and 50 minutes of reaction. [0082] Reusability of immobilized PBP

[0083] The ability of the PBP beads to adsorb and desorb Pi over 10 cycles of high and neutral pH (promoting desorption and adsorption, respectively) was investigated. Tests were conducted in 10 mL disposable Poly-Prep ^® columns with 0.25 ITILBV of beads. Although operated in batch mode, the columns were more convenient than centrifuge tubes for processing multiple cycles. Based on results of previous pH tests, 3 different high pH conditions, 11.5, 12 and 12.5, were tested in independent triplicate experiments using PBP beads or control beads. F1G. 2 provides the illustration of the experimental procedure used. The first step in these experiments was to release any Pi previously adsorbed on the PBP beads by washing them for 10 min with 1.75 mL of IX buffer at 25°C at pH 11.5, 12 or 12.5. lmmediately after this, 1.75 mL IX buffer at pH 7.1 and 25°C was added to adjust the beads to near neutral pH for the subsequent Pi adsorption cycle.

[0084] Following the initial Pi release step, 10 cycles of Pi adsorption/desorption were performed. The first step in each cycle consisted of 10 min Pi adsorption using 1 mL IX buffer at pH 7 and 25°C containing 60 mM of Pi ln the second step, Pi was desorbed using 1.75 mL of IX buffer at 25°C and pH 11.5, 12 or 12.5 for 10 minutes ln the third step, 1.75 mL IX buffer at pH 7.1 and 25°C was added for 10 minutes to wash away any remaining high pH buffer.

[0085] Data analysis

[0086] All PBP bead Pi concentration data was normalized to the corresponding control bead tested. The normalized data was also compared to the theoretical maximum Pi adsorption capacity of the PBP beads, to obtain percent Pi adsorbed and desorbed data. Statistical differences in Pi concentrations between different conditions were assessed using one-way ANOVA conducted on Excel 2010 (Version 14.3.2 e Microsoft, USA) with an added statistical software package XLStat Pro 2014 (Addinsoft, USA).

[0087] Results and Discussion

[0088] Pi removal from immobilized PBP

[0089] 0.25 mL of PBP beads should adsorb 49 nmoles of Pi. However, the PBP beads only adsorbed 11.8±4 nmoles Pi (n = 39) or 24±8% of the theoretical capacity. One possible reason for the low Pi adsorption could be that the PBP active sites are already bound to Pi ions. Previous studies have successfully applied PNPase (0.1 unit/mL) and 7-MEG (300 mM) to mop PBP (Brune et al., 1998, 1994). However, there are no reported studies which have applied PNPase and 7-MEG to mop immobilized PBP. Subsequent recovery experiments with PBP beads confirmed that the observed low adsorption capacity was due to insufficient removal of contaminant Pi ions.

[0090] Recovery of Pi as a function of temperature and pH

[0091] The temperature range tested in this study did not show any influence on Pi recovery (p value <0.05, n = 3). The Pi released from the PBP beads after 10 minute exposure to 25°C, 35°C and 45°C was 1.6±5.1 nmoles, 3.9±1.3 nmoles and 4.5±3.5 nmoles, respectively. On the other hand, pH was observed to have a strong influence on Pi desorption. Exposure to a pH of 4.7, 6.5, 7.1, 8.5, 9.2, 11.4 and 12.5 released 7.9±2 nmoles Pi, 1.9±2.4 nmoles Pi, 1.6±5.1 nmoles Pi, 6.7±4.9 nmoles Pi, 14.7±4.3 nmoles Pi, 30.2±3.9 nmoles Pi and 42.2±5.5 nmoles Pi, respectively.

[0092] lt is important to note that Pi released at pH 11.4 (30.2±3.9 nmoles ) and 12.5 (42.2±5.5 nmoles ) was significantly higher (p value <0.05, n=3) than the amount that was adsorbed by the PBP beads (11.8±4 nmoles). This confirms that Pi ions were previously bound to the PBP, which resulted in decreased Pi adsorption capacity.

[0093] For the purpose of Pi recovery, pH >12 was observed to be the ideal condition for inducing Pi release. Exposing the PBP beads to pH 11.4 and 12.5 released 62% and 86%, respectively, of the theoretical Pi adsorption capacity (49 nmoles/0.25 ITILBV) (F1G. 3).

[0094] Kinetics of Pi recovery from immobilized PBP

[0095] The exposure time did not show any influence on Pi desorption, in all pH conditions tested (F1G. 4). The Pi released after 5 and 50 minutes exposure were statistically the same (p value <0.05, n = 3).

[0096] However, pH did have a significant influence on Pi desorption. The average percent Pi (or nmoles Pi) desorbed within 5 minutes of exposure to pH 7.06, 10.82, 11.88 and 12.5 was 2±9.6% (1±4.8 nmoles), 35±4.4% (17±2.1 nmoles), 79±13% (39±6 nmoles), and 97±9.4% (48±4.6 nmoles), respectively.

[0097] This indicates that high pH condition, not exposure time, is the important factor for inducing Pi release lt is possible that the Pi release occurs instantaneously after exposure, which can be an advantageous feature for a Pi removal and recovery system and should be further investigated. [0098] Reusability of immobilized PBP

[0099] For the immobilized PBP to be a viable Pi removal and recovery system, it is important that PBP is reusable after being exposed repeatedly to the conditions that induce Pi release.

[00100] From the initial Pi desorption wash (Cycle 0), the average percent Pi (or nmoles Pi) desorbed at pH 11.5, 12 and 12.5 was 46±0.6% (or 23±0.3 nmoles), 69±1.2% (or 34±0.6 nmoles) and 77±1.2% (or 38±0.6 nmoles), respectively (F1G. 5).

[00101] F1G. 5 shows the PBP bead’s Pi adsorption and desorption capacity after 10 repetitive cycles of exposure to pH 11.5, 12 and 12.5. No decreasing trend in PBP bead’s Pi adsorption or desorption capacity can be observed.

[00102] Between Cycle 1 and Cycle 10, the average percent Pi (or nmoles Pi) adsorbed by PBP beads exposed to pH 11.5, 12 and 12.5 was 63±8.8% (31±4.3 nmoles), 65±6.2% (32±3 nmoles) and 83±5% (41±2.5 nmoles), respectively (F1G. 5).

[00103] The average percent Pi (or nmoles Pi) desorbed between Cycle 1 and Cycle 10 by pH 11.5, 12 and 12.5 PBP beads were 71±6.8% (35±3.3 nmoles), 71±5% (35±2.5 nmoles) and 89±4.1% (44±2 nmoles), respectively.

[00104] The average Pi adsorbed and desorbed over 10 cycles were statistically similar (p value < 0.05, n=30) for all pH conditions, i.e., all Pi adsorbed by PBP beads can be recovered.

[00105] The PBP beads successfully demonstrated that they can remove and recover Pi for at least 10 cycles, with the highest average Pi adsorption/desorption condition being pH 12.5, followed by pH 12 and pH 11.5 at room temperature.

[00106] Example 2: Concentration of phosphate from environmental water samples

[00107] The PBP-beads were tested for Pi adsorption from environmental water samples, with subsequent Pi release in a concentrated solution.

[00108] Two different environmental water samples (river and pond) with 100 pg/L Pi were tested. The results show that the PBP-beads can successfully adsorb and desorb Pi from environmental water samples, with Pi concentrations as low as 100 pg/L.

[00109] The average P recovered from PBP-beads was 88 and 97% of the total Pi present in the river and pond water, respectively (F1G. 6). [00110] The P concentration in the desorbed solution was 2.9 to 3.2 times higher than in the raw water samples.

[00111] The results demonstrate that PBP-beads can adsorb and release Pi in a concentrated solution from environmental water samples.

[00112] Phosphate binding protein (PBP) source

[00113] The wildtype PstS gene is sourced from E. coli K12 bacteria. The wildtype PstS gene can be modified to remove its signal peptide sequence or to include an affinity tag sequence. The affinity tag can be a histidine chain (His-tag), glutathione S-transferase (GST- tag) or chitin binding domain (CBD-tag). The tag sequence can be inserted at either the N or C terminus of the PstS gene sequence.

[00114] The PstS gene is cloned in an appropriate expression vector (e.g., the pET2 lb+ plasmid) and transformed into an appropriate expression host cell (e.g., BL21 DE3 E. coli cells).

[00115] Example 3: Competition between arsenate and phosphate

[00116] ln this example, experimental results indicated that the PBP-beads preferentially adsorb phosphate over arsenate (anticipated to be the main competitor).

[00117] PBP proteins were immobilized onto NHS activated sepharose beads in accordance with the manufacturer’s protocols (GE life sciences, Marlborough, MA, USA). More than 98% of the PBP protein added was successfully immobilized onto the NHS activated beads.

[00118] Results: Affinity of PBP during simultaneous additions of arsenate and phosphate. The objective of this experiment was to test the affinity of PBP-beads for arsenate or Pi when the ions were added simultaneously at variable molar ratios. The PBP-beads were exposed to buffer (pH 7 and 25 ^Q C) solution containing molar ratios of arsenate to Pi ranging from 0 to 2 (moles of arsenate/moles of Pi). All Pi and arsenate adsorption/desorption results were normalized to percent of the PBP-beads’ theoretical capacity.

[00119] The results show that PBP-beads have a higher affinity for Pi relative to arsenate lncreasing the arsenate to Pi ratio did not significantly reduce Pi adsorption to PBP- beads. Even at an arsenate to Pi ratio of 2, >95% of the PBP-beads’ adsorption capacity was occupied by Pi (F1G. 7). [00120] Example 4: Concentration of ultra-low phosphate from water samples

[00121] The PBP-beads were tested for Pi adsorption from synthetic laboratory water samples containing ultra-low Pi concentrations, with subsequent Pi release in a concentrated solution. This approach concentrates the phosphate such that it can either be recovered as a value-added product (e.g., nutrient-rich fertilizer product) or for further quantification using known laboratory techniques.

[00122] Four different synthetic laboratory water samples with low Pi concentrations - 47.5 pg/L, 19 pg/L, 9.5 pg/L and 4.7 pg/L - were tested. The results showed that the PBP- beads can successfully adsorb and desorb Pi from water samples with ultra-low Pi concentration ranging from 4.7 - 47.5 pg/L (F1G. 8). The average Pi recovered from the PBP- beads ranged from 89% to 100% of the total Pi originally present in the water samples tested. The Pi concentration in the desorbed solution was 4.4 to 5.4 times higher than in the synthetic water samples.

[00123] P i was quantified in the synthetic water samples and in the concentrated regenerant using a conventional colorimetric technique (Standard Method 4500-P E, the Ascorbic Acid method, APHA, 2012, (American Public Health Association (APHA); American Waterworks Association (AWWA); Water Environment Federation (WEF). 2012. Standard Methods for the Examination of Water and Wastewater. Standard Methods. New York, NY, USA.: McGraw-Hill Companies, lnc. https://doi.org/lSBN 9780875532356.).

For each sample, the difference between the known mass Pi (based on initial spiked amounts) per volume solution was calculated. The results are presented in F1G. 9 as“percent error in Pi quantification”. As shown, higher percent errors in Pi quantification were observed in the synthetic water samples containing ultra-low Pi concentration: 24% error for the 19 pg/L sample, 77% error for the 9.5 pg/L sample, and 153% error for the 4.7 pg/L sample. Alternately, much lower Pi quantification errors were observed in the concentrated regenerant recovered from synthetic water samples containing ultra-low Pi concentrations: 13% error for the concentrated 19 pg/L sample, 10% for the concentrated 9.5 pg/L sample, and 11% error for the concentrated 4.7 pg/L sample. [00124] Example 5: Suitable methods for use in the present invention

[00125] PBP production

[00126] The transformed E. coli expression cells are grown to an optical density (OD) of 0.8 in Luria Broth at 37°C and induced with 1 mM lsopropyl b-D-l-thiogalactopyranoside (IPTG). The cells are harvested by centrifugation at 4000 x g for 15 min and 4°C. The PstS protein is then extracted from the cells by lysis by methods known in the art. For example, suitable lysis procedures include enzymatic digestion (lysozyme treatment), freeze thaw, ultrasonic treatment, and the like.

[00127] PBP purification

[00128] The untagged PstS can be purified using a Q-Sepharose bead column following established protocols (GE Healthcare Bio-Sciences, Pittsburg, PA, USA). The PstS tagged with a His-tag, GST-tag, or CBD-tag is purified using a sepharose bead incorporated with ND-NTA, glutathione, or chitin, respectively, following established protocols (GE Healthcare Bio- Sciences, Pittsburg, PA, USA). Use of a tagged Psts protein has the advantage of providing a higher purity product after the purification step.

[00129] PBP immobilization

[00130] Several products are commercially available for immobilizing proteins onto inert beads. The untagged and His-tagged PstS can be immobilized onto NHS-activated sepharose beads following established protocols (GE Healthcare Bio-Sciences, Pittsburg, PA, USA). PstS tagged with a GST-tag or CBD-tag can be purified using glutathione or chitin sepharose beads, respectively. The advantage of GST-tagged or CBD-tagged PstS is that protein purification and immobilization can be performed in a single step.

[00131] Phosphate Desorption

[00132] Phosphate adsorbed on the immobilized PstS can be desorbed by exposing the

PstS to a high pH condition. Desorption solutions used herein should have a pH greater than 11, preferably a pH of greater than 11.4, for example, but not limited to, e.g., pH 11.4, pH 12, pH 12.5 or greater, among others. For maximum desorption, the pH should be adjusted to >12. Following the high pH desorption step, the PstS is washed with a neutral pH solution and maintained at pH 7 in preparation for subsequent adsorption/desorption cycles.

[00133] After initial desorption to remove phosphate, the immobilized PstS is ready for phosphate adsorption. Filtered water samples can be passed through these beads for phosphate adsorption. Subsequently, phosphate can be desorbed in a concentrated form by passing a high pH solution (pH 12 to 13) through the beads. The pH of the desorbed phosphate solution can be neutralized using an acid (e.g., HC1) and used for subsequent processing.

[00134] The immobilized PstS beads have demonstrated phosphate adsorption at levels greater than 85% the maximum theoretical phosphate capacity (based on 1:1 phosphate:PstS binding), with 100% phosphate desorption at pH 12 or higher, for at least 10 sequential cycles.

A sequence listing in text format is concurrently submitted and is incorporated into this specification in its entirety.