Sustainable Production of Reduced Phosphorus Compounds: Mechanochemical Hydride Phosphorylation Using Condensed Phosphates as a Route to Phosphite

In pursuit of a more sustainable production of phosphorous acid (H3PO3), a versatile chemical with phosphorus in the +3 oxidation state, we herein report that condensed phosphates can be employed to phosphorylate hydride reagents under solvent-free mechanochemical conditions to furnish phosphite (HPO32–). Using potassium hydride as the hydride source, sodium trimetaphosphate (Na3P3O9), triphosphate (Na5P3O10), pyrophosphate (Na4P2O7), fluorophosphate (Na2PO3F), and polyphosphate (“(NaPO3)n”) engendered phosphite in optimized yields of 44, 58, 44, 84, and 55% based on total P content, respectively. Formation of overreduced products including hypophosphite (H2PO2–) was identified as a competing process, and mechanistic investigations revealed that hydride attack on in-situ-generated phosphorylated phosphite species is a potent pathway for overreduction. The phosphite generated from our method was easily isolated in the form of barium phosphite, a useful intermediate for production of phosphorous acid. This method circumvents the need to pass through white phosphorus (P4) as a high-energy intermediate and mitigates involvement of environmentally hazardous chemicals. A bioproduced polyphosphate was found to be a viable starting material for the production of phosphite. These results demonstrate the possibility of accessing reduced phosphorus compounds in a more sustainable manner and, more importantly, a means to close the modern phosphorus cycle.

2 Measuring the T 1 relaxation times for phosphite, phosphate, and triethyl phosphate In the fume hood, an aqueous solution (20 mL) containing triethyl phosphate (0.05 M), phosphate (0.0875 M, 1.75 mmol), and phosphite (0.0375 M, 0.75 mmol) was prepared using commercially available starting materials (OP(OEt) 3 , NaH 2 PO 4 ·H 2 O, and Na 2 HPO 3 · 5H 2 O, respectively). This solution (0.7 mL) was transferred to an NMR tube. An inversion recovery measurement was made in order to determine the T 1 of the 31 P nuclei present in the solution and the values were found to be 7.8, 4.6, and 6.9 s for triethyl phosphate, phosphite, and orthophosphate, respectively ( Figure S1). These values, roughly in the range of 4-8 s, allow for reliable integration using a scan delay (d1) of 40 s. 3 Thermal reaction of Na 3 P 3 O 9 with NaH In the glovebox, Na 3 P 3 O 9 (102 mg, 0.33 mmol) and NaH (24 mg, 1.00 mmol, 3.0 equiv) were weighed into a test tube (10 mL, 15 mm ID, borosilicate glass) and briefly mixed with a spatula. The tube was removed from the glovebox with a septum sealing the top of the tube. Nitrogen was flowed through the tube and it was placed in a heating mantle filled with aluminum shot and heated to 300 • C for 1 h to give a fused gray solid. After cooling, the material was carefully quenched with isopropyl alcohol (3 mL) then dissolved in water (5 mL) and analyzed by 31 P NMR spectroscopy ( Figure S2). A signal for phosphite was observed in the 31 P NMR spectrum, but with low conversion. Also present in the solution was phosphino-phosphate, [H 2 P-OPO 3 ] 2− , tentatively assigned based on the chemical shifts for phosphino 1 and phosphate 2 groups, and their corresponding 1 J P-H , 3 J P-H , and 2 J P-P coupling constants. 1,2 31 P NMR (162 MHz, H 2 O, δ) 19.3 (dt, 2 J P-P = 60 Hz, 3 J P-H = 13 Hz, phosphate), -178.6 (td, 1 J P-H = 195 Hz, 2 J P-P = 60 Hz, OPH 2 ).   In the glovebox, a 25 mL stainless steel milling jar was charged with the desired phosphate and hydride reagents. A 10 mm stainless steel ball bearing was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape but without the PTFE gasket (see Section 4.6). The jar was removed from the glovebox and mounted on a mixer mill. Mounting another 25 mL stainless steel milling jar on the other grinding station was required for weight balance.
The reaction mixture was first milled at 20 Hz for 2 min for pre-mixing and then milled at 30 Hz for the desired time. After the reaction, the jar was carefully opened in air, and deionized water (10-15 mL) was cautiously added to the contents. The mixture was left undisturbed for a few minutes until unreacted pyrophoric species were quenched and all water-soluble products were dissolved. Note: Residual metal hydride reacts with water to generate highly flammable hydrogen gas. Hence, the addition of water must be performed in a well-functioning fume hood and be slow at the beginning. The mixture was then transferred to a 20 mL borosilicate glass scintillation vial. Unless otherwise noted, the aqueous mixtures, which were homogeneous and strongly basic (pH 14, measured using pH strips), were carefully acidified with solid NaHCO 3 (ca. 2 equiv per hydride) to pH 12 (measured by pH strips) to improve spectral resolution between the resonances of orthophosphate and phosphite in 31 P NMR spectra. Triethyl phosphate (ca. 250 mg, measured by mass) was added as an internal standard for NMR spectroscopy, and an aliquot (0.5-1 mL) of this mixture was analyzed by 31 P NMR spectroscopy. The ratio of P-containing products to triethyl phosphate was estimated from the relative integration of the observed signals and the yields were calculated. S5

Optimization of hydride source
Mechanochemical reactions of Na 3 P 3 O 9 with various commercial hydrides were carried out as described in Section 4.1. Results are summarized in Table S1. Typical spectra are shown in Section 4.8.   (n+2)− , bracketed value of yield is based on (n − 1) reactive P units (two for triphosphate, one for pyrophosphate). g Graham's salt (300 mg) was used. The phosphorus content of Graham's salt was determined by 31 P NMR spectroscopy ( Figure  S10).

Optimization of reaction conditions
Mechanochemical reactions of various phosphate sources and KH were carried out as described in Section 4.1. Results are summarized in Table S3. Typical spectra are shown in Section 4.8.  Method I : Two parallel runs each comprising Na 4 P 2 O 7 (532 mg, 2.0 mmol) and KH (80 mg, 2.0 mmol, H:P = 1:2, H:(reactive P) = 1:1) were set up and carried out as described in Section 4.1 for 60 minutes. The obtained aqueous solutions were combined. Hydrochloric acid (37 wt.%, 2 mL) was added and the solution was heated at 95 • C for 1 h to hydrolyze pyrophosphoric acid. After cooling to room temperature, potassium hydroxide was added until the solution reached a pH of 6 to 8 measured using pH strips. Magnesium chloride (500 mg, 1.3 equiv) and ammonium hydroxide (28 wt.%, 1 mL, 3.5 equiv) were added, resulting in rapid formation of a white precipitate, presumably struvite (NH 4 MgPO 4 · 6 H 2 O). The slurry was stirred for 5 min and filtered through filter paper. Removal of all phosphates was confirmed by analyzing an aliquot of the filtrate by 31 P NMR spectroscopy. To the filtrate was added barium acetate (1.0 g, 1.0 equiv), and the formed barium phosphite (BaHPO 3 ·H 2 O) 3 was collected on a frit (15 mL, medium porosity), transferred to a vial and dried at 120 • C until constant mass was achieved, providing BaHPO 3 ·H 2 O (630 mg, 2.68 mmol, 67% yield based on KH as the limiting reagent).
Method II : Two parallel runs each comprising Na 4 P 2 O 7 (399 mg, 1.5 mmol) and KH (60 mg, 1.5 mmol, H:P = 1:2, H:(reactive P) = 1:1) were set up and carried out as described in Section 4.1 for 60 minutes. The obtained aqueous solutions were combined. Magnesium chloride (371 mg, 1.3 equiv) and ammonium hydroxide (28 wt.%, 1 mL, 4.5 equiv) were added, resulting in rapid formation of a white precipitate, presumably struvite and magnesium pyrophosphate (Mg 2 P 2 O 7 ). The slurry was stirred for 5 min and filtered through filter paper. Removal of all phosphates was confirmed by analysing an aliquot of the filtrate by 31 P NMR spectroscopy. To the filtrate was added barium acetate (0.8 g, 1.0 equiv), and the formed barium phosphite (BaHPO 3 ·H 2 O) was collected on a frit (15 mL, medium porosity), transferred to a vial and dried at 120 • C until constant mass was achieved, providing

Comparison of experiments run with and without gaskets
In the glovebox, two stainless steel milling jars were individually charged with Na 4 P 2 O 7 (399 mg, 1.50 mmol), KH (60 mg, 1.50 mmol, H:P = 1:2, H:(reactive P) = 1:1), and a stainless steel ball. One of the jars was sealed with a PTFE gasket and electrical tape ( Figure S3(a,c)), and the other jar with PTFE thread sealant tape and electrical tape ( Figure S3(b,c)). Both jars were removed from the glovebox and mounted on a mixer mill. The reaction mixtures were milled at 30 Hz for 60 min. Each reaction mixture was completely extracted with water (10 mL), and the aqueous extract was transferred to a scintillation vial. The aqueous solution obtained from the jar sealed with a PTFE gasket was apparently more colored than that from the jar sealed with PTFE thread sealant tape ( Figure S3(d)). 31 P NMR analysis of the solutions showed that when the milling jar was sealed with a PTFE gasket, the run produced less phosphite (30% yield by NMR spectroscopy) than the run carried out with PTFE thread sealant tape (34% yield by NMR spectroscopy). Deformation of the PTFE gasket was also observed after the reaction. These observations indicated mechanical (or mechanochemical) erosion of PTFE, which may deteriorate in the presence of strong reducing agents and thus interfere with the reduction of phosphates. Therefore, we proceeded with a sealing protocol using PTFE thread sealant tape instead of PTFE gaskets, as described in Section 4.1.

Notes on yield calculation and reactive phosphorus
Unless otherwise noted, we define all reported percentage yields in this work based on total P content from the phosphate source. Such definition allows a broader comparison of yields among the structurally diverse phosphate sources yet, unfortunately, fails to accurately report the reactivity of linear phosphates, which will furnish orthophosphate which is unreactive under typical ball milling conditions as byproduct. Taking this experimental observation into account, we introduce the term of "reactive phosphorus (P)" to describe the quantity of PO 3 − units that are available for hydride phosphorylation under the reaction conditions. Cyclic phosphates, fluorophosphate, and linear polyphosphate with an infinite chain length all exhibit an ideal O:P atom ratio of three. Therefore, all of their phosphorus atoms are reactive by definition, and a new definition of yield based on reactive P is unnecessary. In contrast, linear phosphates P n O 3n+1 (n+2)− (n = 1, orthophosphate; n = 2, pyrophosphate; n = 3, triphosphate; etc.) only contain (n − 1) units of reactive P. For example, no P of orthophosphate is reactive, while 50% P of pyrophosphate and 67% P of triphosphate are reactive. Yields based on reactive P for well-defined linear phosphates (bracketed values in Tables S2 and S3) will provide more intuitive insights to the reaction outcome.   Figure S10: 31 P NMR spectrum (162 MHz, H 2 O) of a solution comprising (NaPO 3 ) n (commercial Graham's salt, 289 mg) and OP(OEt) 3 (286 mg). Inset: expansion of the region of internal phosphates. a. Orthophosphate; b. OP(OEt) 3 , internal standard; c. terminal Punits of linear phosphates; d. bridging P-units of linear phosphates; e. trimetaphosphate; f. other cyclic phosphates. The total P content of the commercial Graham's salt is 9.4 mmol/g (29.3 wt.%) as quantified by NMR spectroscopy. The P compounds in aqueous solution include linear polyphosphates (92% P, averaged chain length = 12.7), trimetaphosphate (4% P), other cyclic phosphates (4% P), and orthophosphate (0.3% P).      Figure S15: 31 P NMR spectrum (162 MHz, H 2 O) of the reaction mixture of (NaPO 3 ) n (Graham's salt, 300 mg, 2.84 mmol P) with KH (4.24 mmol) after 90 min (Table S3,  5 Mechanistic studies 5.1 Deuterium oxide quenching experiment Na 4 P 2 O 7 (399 mg, 1.5 mmol) and KH (61 mg, 1.5 mmol, H:P = 1:2, H:(reactive P) = 1:1) were mechanically mixed for 60 min as described in Section 4.1. Deuterium oxide (99.8% D, 10 mL) instead of H 2 O or NaHCO 3 was added to extract the water-soluble products. OP(OEt) 3 (219 mg) was added as an NMR internal standard for NMR spectroscopy. An aliquot (0.5 mL) was analyzed by 31 P NMR spectroscopy within 2 hours after sample preparation; no deuterio-phosphite (DPO 3 2− ) was detected from the 31 P and 31 P{ 1 H} spectra. These results confirmed that the hydrogen atom in the phosphite product originated from KH instead of proton sources added during the workup. The lack of H/D exchange of phosphite in alkaline aqueous solution has been well documented. 4,5 Figure S16: In the glovebox, a stainless steel milling jar was charged with Na 5 P 3 O 10 (368 mg, 1.00 mmol). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reactant was milled at 30 Hz for 30 min to yield a white powdery residue. The residue was completely extracted with water (10 mL), and the mixture was transferred to a scintillation vial. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S17), and the sum of integrals was set as 3.00 for the convenience of quantification. NMR integrals indicated that 51% of triphosphate was consumed to yield pyrophosphate (0.34 mmol), tetraphosphate (0.19 mmol), and a small amount of longer polyphosphates (0.02 mmol). These results suggest that triphosphate can phosphorylate linear phosphates to increase chain length and yield pyrophosphate. Pyrophosphate is essentially inactive under these conditions, as evidenced by the observation that orthophosphate was not significantly formed. In the glovebox, a stainless steel milling jar was charged with Na 3 P 3 O 9 (306 mg, 1.00 mmol). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reactant was milled at 30 Hz for 90 min to yield a white powdery residue. The residue was completely extracted with water (10 mL), and the mixture was transferred to a scintillation vial. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S18), and the sum of integrals was set as 3.00 for the convenience of quantification. 31 P NMR analysis revealed that 93% of trimetaphosphate was converted to a variety of cyclic phosphates and at least one linear phosphate species. The 31 P resonance of unreacted trimetaphosphate was identified by adding untreated Na 3 P 3 O 9 to the NMR sample followed comparing 31 P NMR spectra before and after the addition ( Figure S19). The presence of a linear phosphate species was further confirmed by observation of 31 P-31 P COSY correlation ( Figure S20).

Na 2 PO 3 F
In the glovebox, a stainless steel milling jar was charged with Na 2 PO 3 F (400 mg, 2.78 mmol). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reactant was milled at 30 Hz for 90 min to yield a white powdery residue. The residue was completely extracted with water (10 mL), and the mixture was transferred to a scintillation vial. An aliquot of this mixture was analyzed by 31 P and 19 F NMR spectroscopy ( Figures S21 and S22). NMR analysis revealed that 18% of fluorophosphate was converted to a variety of new species, among which fluoropyrophosphate (4.4% P) was identified. 31 P NMR (162 MHz, H 2 O, δ) -6.4 (d, 2 J P-P = 21 Hz), -16.6 (dd, 1 J P-F = 921 Hz, 2 J P-P = 21 Hz). 19  In the glovebox, a stainless steel milling jar was charged with Na 4 P 2 O 7 (266 mg, 1.00 mmol) and Na 2 HPO 3 (126 mg, 1.00 mmol, 1.00 equiv). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reaction mixture was milled at 30 Hz for 30 min to yield a white powdery residue. The residue was completely extracted with water (10 mL), and the mixture was transferred to a scintillation vial. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S23), and the sum of integrals was set as 3.00 for the convenience of quantification. NMR integrals indicated that only 2% phosphite was converted to isohypophosphate, which was the only P(III)-containing product. In the glovebox, a stainless steel milling jar was charged with Na 5 P 3 O 10 (368 mg, 1.00 mmol) and Na 2 HPO 3 (126 mg, 1.00 mmol, 1.00 equiv). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reaction mixture was milled at 30 Hz for 30 min to yield a white powdery residue. The residue was completely extracted with water (10 mL), and the mixture was transferred to a scintillation vial. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S24), and the sum of integrals was set as 4.00 for the convenience of quantification. 31 P NMR analysis showed that phosphite was phosphorylated to yield isohypophosphate (0.23 mmol) and trimer [HPO 2 -OPO 2 -OPO 3 ] 4− (0.06 mmol). Furthermore, a small amount of pyrophosphite (0.01 mmol) also formed. Extending the reaction time to 90 min increased the conversion of phosphite from 30 to 54% ( Figure S25).  In the glovebox, a stainless steel milling jar was charged with Na 3 P 3 O 9 (306 mg, 1.00 mmol) and Na 2 HPO 3 (126 mg, 1.00 mmol, 1.00 equiv). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reaction mixture was milled at 30 Hz for 30 min to yield a white powdery residue. The residue was completely extracted with water (10 mL), and the mixture was transferred to a scintillation vial. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S27), and the sum of integrals was set as 4.00 for the convenience of quantification. 31 P NMR analysis showed that 72% of phosphite was converted to a variety of phosphorylated phosphites with different chain lengths and pyrophosphite. Extending the reaction time to 90 min led to essentially complete conversion of phosphite ( Figure S28).

Na 2 PO 3 F and Na 2 HPO 3
In the glovebox, a stainless steel milling jar was charged with Na 2 PO 3 F (144 mg, 1.00 mmol) and Na 2 HPO 3 (126 mg, 1.00 mmol, 1.00 equiv). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reaction mixture was milled at 30 Hz for 30 min to yield a white powdery residue. The residue was completely extracted with water (5 mL), and the mixture was transferred to a scintillation vial. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S30), and the sum of integrals was set as 2.00 for the convenience of quantification. 31 P NMR analysis showed that 28% of phosphite was converted to phosphorylated phosphite species (0.18 mmol) and pyrophosphite (0.04 mmol).

Graham's salt and Na 2 HPO 3
In the glovebox, a stainless steel milling jar was charged with Graham's salt (108 mg, 1.00 mmol P) and Na 2 HPO 3 (126 mg, 1.00 mmol, 1.00 equiv). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reaction mixture was milled at 30 Hz for 30 min to yield a white powdery residue. The residue was completely extracted with water (5 mL), and the mixture was transferred to a scintillation vial. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S31), and the sum of integrals was set as 2.00 for the convenience of quantification. 31 P NMR analysis showed that 62% of phosphite was converted to phosphorylated phosphite species (0.40 mmol) and pyrophosphite (0.10 mmol). In contrast, only 31% of phosphite was converted using sodium triphosphate (Na 5 P 3 O 10 ) under similar conditions (Section 5.3.2).  were mechanically mixed for 30 min as described in Section 4.1. To the obtained aqueous mixture was added OP(OEt) 3 (203 mg) as an internal standard for NMR spectroscopy. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S32). 31 P NMR analysis showed that phosphite and pyrophosphate were the major products, while small amounts of hypophosphite, isohypophosphate, and tetraphosphate were detected.  5.5 Reaction of Na 3 P 3 O 9 with substoichiometric amount of KH Na 3 P 3 O 9 (306 mg, 1.0 mmol) and KH (40 mg, 1.0 mmol, H:P = 1:3) were mechanically mixed for 30 min as described in Section 4.1. To the obtained aqueous mixture was added OP(OEt) 3 (203 mg) as an internal standard for NMR spectroscopy. An aliquot of this mixture was analyzed by 31 P NMR spectroscopy ( Figure S33). The analysis shows that although linear polyphosphates were the major products, several phosphorylated phosphites with different chain lengths were the major reduction products, accompanied by minor reduction products including hypophosphite, phosphite, and pyrophosphite.    6 Accumulation and production of polyphosphate (bio-polyP) using Saccharomyces cerevisiae

General considerations
The protocols of yeast growth, phosphate accumulation, and isolation of bio-polyP from baker's yeast were adapted from published work by Blank and co-workers. 7,8 Red Star's fresh cake yeast was purchased from Amazon. The yeast was shipped in a Styrofoam box with cold packs and then transferred to a -20 • C freezer for long-term storage due to the perishable nature of this material. A stick of yeast (57 g, Figure S34) was thawed in a food refrigerator (at 7 • C) overnight for the experiments. Once thawed, the yeast can be stored at 7 • C over an estimated period of time of two months. Although one must be wary of potential reproducibility issues raised from degradation of yeast over storage, we were able to conduct this experiment with uncompromised yield and properties of polyphosphate using yeast stored at 7 • C for up to 30 days.
Chemicals for preparation of yeast growth media were obtained from Sigma-Millipore and used as received. Deionized water was purified through a Millipore Milli-Q Academic Water Purification System before use.  . After dilution to 750 mL, all other components were dissolved under pH control (pH 6.5 with HCl and NaOH). The volume was filled up to 1 L, the solution was filter sterilized and stored at 4 • C.

Phosphate-feeding medium
Phosphate feeding medium component A comprised glucose (45 g/L, 278 mM) and KH 2 PO 4 (8.17 g/L, 66.6 mM). The pH was adjusted to 6.4 using HCl (aq) (1 M) or NaOH (aq) (1 M). The solution was filter sterilized and stored at 4 • C. Phosphate feeding medium component B comprised MgCl 2 · 6H 2 O (40.7 g/L, 200 mM) and was filter sterilized and stored at 4 • C. Components A and B were mixed in a volume ratio of 9:1 immediately before inoculation to avoid possible formation of magnesium phosphate precipitate. Upon mixing, the pH of the medium will change to 6.0.

Growth of phosphate-starved yeast
Each of four 4 L baffled Erlenmeyer flasks (autoclaved) was charged with phosphate starvation medium (1 L) and capped with aluminum foil. The flasks were equilibrated at 30 • C. The medium was inoculated with fresh baker's yeast (10.0 g in total, corresponding to 2.5 g/L medium) and then incubated aerobically in a rotary shaker for 6 h at 200 rpm at 30 • C ( Figure S35(a)). Yeast cells were harvested by centrifugation (5000g, 10 min) and washed once with autoclaved Milli-Q water. The cell mass was streaked out on a few layers of filter paper (Whatman), air-dried for 5 min, and stored in a tared centrifuge tube in a 4 • C room overnight (17 h from the end of incubation to the beginning of phosphate accumulation, Figure S35(b)). The cell mass was collected (20.33 g).

Phosphate accumulation
Phosphate feeding medium component A (900 mL) and component B (100 mL) were freshly mixed. A 4 L baffled Erlenmeyer flasks (autoclaved) was charged with the mixed medium (660 mL) and capped with aluminum foil. The flask was equilibrated at 30 • C. The medium was inoculated with phosphate-starved yeast (20.0 g, corresponding to 30 g/L medium) and then incubated aerobically in a rotary shaker for 2.5 h at 200 rpm at 30 • C ( Figure  S35(c)). Yeast cells were harvested by centrifugation (5000g, 10 min) and washed twice with autoclaved Milli-Q water. The cell mass was streaked out on a few layers of filter paper, air-dried for 5 min, and stored in tared centrifuge tubes ( Figure S35(d)) in a -20 • C freezer overnight. The cell mass was collected (27.18 g).

Isolation
The frozen yeast mass was thawed in a 22 • C water bath for 15 min. The melt lump of yeast was transferred to a 200 mL glass Erlenmeyer flask equipped with a stir bar. Milli-Q water (135 mL) was added to facilitate the transfer. The flask was placed in a 70 • C water bath, and the mixture was vigorously stirred. (A thermometer was placed in the mixture to monitor the temperature change; it took ca. 8 min to reach 65 • C.) The mixture was stirred for 10 min at 65-75 • C and then cooled in an ice bath for >5 min. The cooled mixture was transferred to a 500 mL centrifuge bottle and centrifuged (10000g for 10 min, Figure  S35(e)). The precipitate was discarded.
The clear liquid (157 mL) was transferred to a second centrifuge bottle, and HCl (aq) (2.5 M, 3.14 mL) was added using a pipette. The mixture was shaken to mix, leading to a white flocculent precipitate (presumably protein and nucleic acid). The mixture was quickly subjected to centrifugation (10000g, 15 min, Figure S35(f)), and the precipitate was discarded. The clear liquid was transferred to a third centrifuge bottle, and NaOH (aq) (2.5 M, 3.14 mL) was added using a pipette. By reference to a pH electrode (Thermo Scientific Orion 9107BNMD pH Probe), NaOH (aq) (2.5 M, 0.50 mL) was added to adjust the pH to 7.10. NaCl (aq) (5 M, 1.71 mL) was added to adjust the concentration of Na + to ca. 100 mM. To the centrifuge bottle was added ethanol (200 proof, 165 mL), leading to rapid formation of a white flocculent precipitate. The tube was allowed to settle for 45 min and then centrifuged (10000g, 10 min, Figure S35(g)) to yield a white pellet, and the clear liquid was discarded. The pellet was gently washed several times with a total of 80 mL of ethanol:water (50:50 by volume) and then dried under vacuum overnight to yield a white pellet of polyphosphate (2.00 g, Figure S35(h)). The solid was crushed and dried under vacuum over 13 days. A white solid (1.95 g) remained.

Analyses
The bio-polyP solid (75.1 mg) was suspended in water (ca. 4 mL), and the mixture was vigorously stirred at 22 • C for 2 h until the bio-polyP was fully dissolved. Triethyl phosphate (49.7 mg, 0.273 mmol) was added as an internal standard for NMR spectroscopy. The resulting mixture was analyzed by 31 P NMR spectroscopy ( Figure S36). The P content of polyphosphate was calculated to be 23.0 wt.% based on integration of the corresponding resonances versus the internal standard resonance. Approximately 36 mol% of the phosphate feeding was recovered in the form of polyP. The averaged chain length of polyP n was calculated to be n = 37.0 according to Eq. 1, where PP1, PP2-PP3, and PP4 represent integration of the resonance for the terminal, the penultimate, and the core P-groups of polyP, respectively, and x equals 1, 2, or 3 depending on which signal is best resolved.
Averaged chain length n = 2 PP1+PP2+PP3+PP4 PPx (1) The pH value of a 1 wt.% aqueous solution of bio-polyP was measured at 22 • C to be 6.9, in good agreement with the pH neutral media from which bio-polyP was isolated. At neutral pH, each polyP chain is protonated with one proton on average. To determine cationic compositions (x) in the formula of polyP, the ratios among each two of Na + (after subtraction of the contribution from NaCl according to the content of soluble Cl − measured by titration), Mg 2+ , K + , and Ca 2+ were first calculated based on their contents measured by ICP-OES. Then, individual cationic composition was calculated based on charge balance (Eq. 2): The purity of the polyP (containing metal cations) was calculated based on the formula and the P content of polyP measured by NMR spectroscopy. Other phosphorus species detected by NMR included orthophosphate and trimetaphosphate. The content of either species was quantified based on 31 P NMR integration. The presence of organic impurities S56 (presumably nucleic acid and proteins) and water as impurities in the polyP was confirmed through combustion analysis (C,H,N). NaCl used in the isolation protocol contaminated the polyP material, as evidenced by a notable Cl − content measured by titration.   These assignments are adopted according to work by Christ et al. 9 PP1, PP2-PP3, and PP4 represent the terminal, the penultimate, and the core P-groups of polyP, respectively.

Ball milling bio-polyP
In the glovebox, a stainless steel milling jar was charged with bio-polyP containing 23.0 wt.% P (100 mg, 0.74 mmol P). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reaction mixture was first milled at 20 Hz for 2 min and then at 30 Hz for 90 min. After the reaction, the jar was carefully opened in air to reveal a brown powdery residue. The solid residue was extracted with water (10 mL) to yield a brown translucent mixture, which was filtered through a glass microfiber filter to yield a pale yellow clear solution and a dark brown tar. Triethyl phosphate (103 mg, 0.565 mmol, measured by mass) was added as an internal standard for NMR spectroscopy, and 31 P NMR quantification revealed formation of orthophosphate, pyrophosphate, and linear phosphates with 3-5 repeating units with >99% overall phosphorus recovery ( Figure S38).

Mechanochemical reaction of bio-polyP with KH
Two parallel runs were set up in the glovebox according to the procedure as follows. A stainless steel milling jar was charged with bio-polyP containing 23.0 wt.% P (200 mg, 1.49 mmol P) and KH (121 mg, 3.00 mmol, H:P = 2.0:1). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reaction mixture was first milled at 20 Hz for 2 min for pre-mixing and then milled at 30 Hz for 90 min. The jar was carefully opened in air to reveal a pale brown powdery residue.
Aqueous workup procedure #1: Deionized water (10 mL) was added to the contents to yield a pale yellow suspension. Note: Residual metal hydride reacts with water to generate highly flammable hydrogen gas, so the addition of water must be performed in a wellfunctioned fume hood and be slow at the beginning. HCl (aq) (1 M) was added under vigorous stirring until pH 7 to yield an off-white translucent mixture. Triethyl phosphate (286 mg, 1.57 mmol, measured by mass) was added as an internal standard for NMR spectroscopy, and 31 P NMR quantification revealed 95% overall phosphorus recovery with 28% phosphite and 67% orthophosphate ( Figure S39).
Aqueous workup procedure #2: Deionized water (10 mL) was added to the contents to yield a pale yellow suspension. Note: Residual metal hydride reacts with water to generate highly flammable hydrogen gas, so the addition of water must be performed in a wellfunctioned fume hood and be slow at the beginning. NaHCO 3 (250 mg, 3.0 mmol) was added. The mixture was stirred for 5 min and filtered through a glass microfiber filter to yield a pale yellow clear filtrate and a pale brown solid. Triethyl phosphate (224 mg, 1.23 mmol, measured by mass) was added to the filtrate as an internal standard for NMR spectroscopy, and 31 P NMR quantification revealed 83% phosphorus recovery with 30% phosphite, 52% orthophosphate, and 1.6% [H 2 P-OPO 3 ] 2− ( Figure S40). The brown solid was washed off with HCl (aq) (1 M, 1 mL), and 31 P NMR showed orthophosphate as the only phosphoruscontaining species.

Vacuum calcination of bio-polyP
A round bottom flask was charged with "pristine" bio-polyP containing 23.0 wt.% P (647 mg) and then equipped with a Schlenk hose adapter. The flask was then evacuated on a Schlenk line. The solid was calcined for 30 min under dynamic vacuum (ca. 200 mTorr) at ca. 500 • C in a heating mantle filled with aluminum shot and turned dark brown ( Figure S37). The solid was cooled to ambient temperature and transferred into a scintillation vial in the glovebox. 534 mg (82%) was recovered. ICP-OES measurements revealed the elemental contents as follows: Na, 6.88%; Mg, 4.28%; K, 13.12%. These results show that loss of water and pyrolysis of organic impurities increased the content of "polyphosphate" by a factor of 1.10. Therefore, the calcined bio-polyP contained 25.4 wt.% P.
Quantification of P content in the calcined bio-polyP by 31 P NMR was unsuccessful as the phosphate component could not be readily dissolved in water at ambient temperature. Nonetheless, 31 P NMR analysis suggested that the structure of polyphosphate persisted during the calcination ( Figure S41).

Reaction of calcined bio-polyP and KH
A stainless steel milling jar was charged with calcined bio-polyP containing 25.4 wt.% P (200 mg, 1.64 mmol P) and KH (121 mg, 3.00 mmol, H:P = 1.8:1). A stainless steel ball was added, and the jar was sealed with the aid of PTFE thread sealant tape and electrical tape. The jar was removed from the glovebox and mounted on a mixer mill. The reaction mixture was first milled at 20 Hz for 2 min for pre-mixing and then milled at 30 Hz for 90 min. The jar was carefully opened in air to reveal a dark brown powdery residue. Deionized water (10 mL) was added to the contents to yield a suspension with brown foam on top. Note: Residual metal hydride reacts with water to generate highly flammable hydrogen gas, so the addition of water must be performed in a well-functioned fume hood and be slow at the beginning. HCl (aq) (1 M) was added under vigorous stirring until pH 5 to yield a brown opaque mixture. Triethyl phosphate (216 mg, 1.19 mmol, measured by mass) was added as an internal standard for NMR spectroscopy, and then the mixture was filtered through a glass microfiber filter to yield a pale brown filtrate and a dark brown tar. 31 P NMR quantification revealed 87% overall phosphorus recovery with 42% phosphite and 44% orthophosphate ( Figure S42).