Compositions and methods for synthesizing metal carbonate compounds

Abstract

Provided herein are compositions, methods and systems for precipitating metallic carbonate using cyanobacteria. The precipitated metallic carbonate can be of high purity and / or has a biogenic signature associated with the one or more cyanobacteria.

Description

WSGR Docket No. 69144-701601COMPOSITIONS AND METHODS FOR SYNTHESIZING METAL CARBONATE COMPOUNDSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 683,647, filed August 15, 2024, which application is incorporated herein by reference in its entirety.BACKGROUND

[0002] To mitigate the millions of tons of CO2 generated annually by the paper, plastics, and paints industries, companies are seeking carbon-reduced raw materials. The challenge is finding alternatives that do not alter existing processes or increase costs. These industries currently use large amounts of calcium carbonate, where current mining extraction methods are carbon-positive, adding to the detrimental effects of carbon-heavy manufacturing methods. Scalable solutions for carbon-negative carbonates are still lacking, as physicochemical methods consume excessive energy or produce chemical waste, and biological methods involve complex purification processes.SUMMARY

[0003] Disclosed herein is a metallic carbonate precipitated using one or more cyanobacteria grown in vitro. The metallic carbonate can comprise (i) a purity level of at least 90%, (ii) an impurity content of less than 200 parts per million (ppm), and / or (iii) a biogenic signature associated with the one or more cyanobacteria, wherein the biogenic signature is measurable or detectable using X-ray photoelectron spectrometer (XPS). In some embodiments, the metallic carbonate has a purity level of at least 95%. In some embodiments, the metallic carbonate has a purity level of at least 98%. In some embodiments, impurity content is associated with one or more impurities comprising arsenic (e.g., arsenate (As (V)) and arsenite (As (III)), Fe (III) hydroxides, mercury, or any combination thereof. In some embodiments, the metallic carbonate has an impurity content of less than 50 parts per million (ppm). In some embodiments, the metallic carbonate has an impurity content of less than 25 parts per million (ppm). In some embodiments, the metallic carbonate has an arsenic level of less than 100 ppm. In some embodiments, the metallic carbonate has an arsenic level of less than 30 ppm. In some embodiments, the metallic carbonate comprises a biogenic signature as measured by X-ray photoelectron spectrometer (XPS). In some embodiments, the biogenic signature comprises a wide carbon (Cl) peak with binding energy from about 280 to 294 eV as measured by XPS. InWSGR Docket No. 69144-701601 some embodiments, the biogenic signature comprises a wide carbon (Cl) peak with binding energy from about 284 to 290 eV as measured by XPS. In some embodiments, the metallic carbonate has a peak of at least 26.5° of 2-theta (29) as measured by X-ray diffraction (XRD). In some embodiments, the metallic carbonate has a peak at 27.9° 2-theta (29) as measured by X-ray diffraction (XRD). In some embodiments, the metallic carbonate is precipitated using less than 5 GJ per ton of precipitated metallic carbonate compared to another metallic carbonate that is not generated using the one or more cyanobacteria. In some embodiments, the metallic carbonate is precipitated using less than 19 GJ per ton of precipitated metallic carbonate compared to another metallic carbonate that is not generated using the one or more cyanobacteria. In some embodiments, the metallic carbonate is precipitated using less than 29 GJ per ton of energy. In some embodiments, the metallic carbonate is precipitated using less than 1 GJ per ton of energy.

[0004] Disclosed herein is metallic carbonate precipitated using one or more cyanobacteria. The metallic carbonate can be precipitated by removing at least 0.22 grams of CO2 from an atmosphere for every gram of metallic carbonate produced. In some embodiments, the one or more cyanobacteria are selected from the group consisting of a Rivulariaceae species, a Nodosilinea species, a Phormidium species, an Anabaena species, and a Synechococcus species. In some embodiments, the one or more cyanobacteria comprises at least two species of cyanobacteria. In some embodiments, the one or more cyanobacteria comprises at least three species of cyanobacteria. In some embodiments, the one or more cyanobacteria comprises Rivularia halophila. In some embodiments, the one or more cyanobacteria comprises Nodosilinea nodulosa. In some embodiments, the one or more cyanobacteria comprises a sheath that facilitates or enables the precipitation of the metallic carbonate. In some embodiments, the metallic carbonate comprises an alkaline-earth metal. In some embodiments, the metallic carbonate comprises a metal selected from Group 2 of the periodic table. In some embodiments, the metallic carbonate comprises calcium carbonate. In some embodiments, the metallic carbonate comprises at least 69% crystalline metallic carbonate. In some embodiments, the metallic carbonate comprises at least 79% crystalline metallic carbonate. In some embodiments, the metallic carbonate comprises at least 89% crystalline metallic carbonate.

[0005] Disclosed herein is a culture medium for facilitating precipitation of metallic carbonate using one or more cyanobacteria, wherein the culture medium has a salinity of at least 3.5% (w / v) NaCl. In some embodiments, the culture medium has a salinity of at least 8 % (w / v) NaCl. In some embodiments, the culture medium has a pH of at least 7.25. In someWSGR Docket No. 69144-701601 embodiments, the culture medium has a pH of at least 8.5. In some embodiments, the culture medium comprises sea water. In some embodiments, the culture medium is obtained from sea water. In some embodiments, the culture medium comprises a salt of an alkaline-earth metal. In some embodiments, the culture medium comprises a salt of a metal selected from Group 2 of the periodic table. In some embodiments, the culture medium comprises a calcium salt. In some embodiments, the culture medium comprises at least 900 mg / L calcium chloride. In some embodiments, the culture medium comprises a carbon source. In some embodiments, the carbon source comprises at least 1000 mg / L bicarbonate (HCO3-). In some embodiments, the carbon source comprises dissolved CO2. In some embodiments, the culture medium further comprises a cyanobacteria. In some embodiments, the culture medium further comprises precipitated metallic carbonate, wherein the precipitated metallic carbonate is deposited on the surface of the one or more cyanobacteria. In some embodiments, the culture medium further comprises precipitated metallic carbonate, wherein the one or more cyanobacteria comprises a sheath that facilitates or enables the precipitation of the metallic carbonate.

[0006] Disclosed herein is a system comprising the culture medium disclosed herein. The system can further comprise one or more cyanobacteria. In some embodiments, the one or more cyanobacteria are selected from the group consisting of a Rivulariaceae species, Nodosilinea species, a Phormidium species, an Anabaena species, and a Synechococcus species. In some embodiments, the one or more cyanobacteria comprises at least two species of cyanobacteria. In some embodiments, the one or more cyanobacteria comprises at least three species of cyanobacteria. In some embodiments, the one or more cyanobacteria comprises Rivularia halophila. In some embodiments, the one or more cyanobacteria comprises Nodosilinea nodulosa. In some embodiments, system comprises an open or closed system.

[0007] Disclosed herein is a method of manufacturing a metallic carbonate. The method can comprise contacting one or more cyanobacteria with a carbon source in a culture media, thereby generating a precipitated metallic carbonate, wherein the one or more cyanobacteria comprises a sheath that facilitates or enables the precipitation of the metallic carbonate, wherein the metallic carbonate has (i) a purity level of at least 90% and / or (ii) an impurity content of less than 200 ppm.

[0008] Disclosed herein is a method of forming a metallic carbonate. The method can comprise providing one or more cyanobacteria in a culture media containing high salinity; and providing a carbon source to the culture media, thereby generating a precipitated metallic carbonate, wherein the one or more cyanobacteria comprises a sheath that facilitates or enables the precipitation of the metallic carbonate. In some embodiments, the salinity of the cultureWSGR Docket No. 69144-701601 media is at least 3.5% (w / v) NaCl. In some embodiments, the salinity of the culture media is at least 8% (w / v) NaCl. The method can further comprise separating the precipitated metallic carbonate from the one or more cyanobacteria. In some embodiments, the one or more cyanobacteria comprise a sheath.INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0011] FIG. 1A shows the various biogenic processes that cyanobacteria are capable of performing, including biomineralization of CaCCL and arsenic transformation. FIG. IB is an exemplary bioreactor provided herein.

[0012] FIG. 2A shows Zeta potential values of dead cyanobacteria colonies (DC) and live cyanobacteria colonies (LC) of R halophila at different pH values (panel (a)) and Ca2+uptake in DC and LC of R. halophila (panel (b)). FIG. 2B depicts a bar plot displaying the statistics of differential precipitation and images of colonies and / or minerals from optical microscopy.

[0013] FIG. 3 depicts scanning electron micrograph (SEM) images obtained for LC and DC conditions under 5 mM, 10 mM and 15 mM of Ca2+.

[0014] FIG. 4 depicts X-ray diffraction (XRD) patterns of calcium carbonates obtained in LC, DC and without bacterial culture (WS) conditions at 10 mM and 15 mM of Ca2+.

[0015] FIG. 5 shows infrared spectra of DC and LC with 5 mM of Ca2+.

[0016] FIG. 6 depicts normalized X-ray photoelectron spectrometer (XPS) spectra. Panel (a) of FIG. 6 shows the R halophila biomass reported by Soto Rueda et al. (2023). Panel (b) of FIG. 6 shows the R. halophila biomass obtained for calcium carbonates. Solid black lines depict measured spectra; solid red lines indicate fits to measured spectra and dashed lines correspond to fits. A Shirley background is included as a solid curve at base of the peaks. Dashed vertical lines are added to guide the eye and correspond to C species identified in theWSGR Docket No. 69144-701601 analyzed samples. Values of binding energy (BE) correspond to the maximum intensity of the fitted peak are reported in Table 2 herein.

[0017] FIG. 7A depicts the XPS spectra of various arsenic (As) species in calcium carbonate precipitated by R. halophila. Both As(III)-S and As(V)-0 were identified in the XPS spectra. The cyanobacteria had been cultured in media supplemented with 300 pg / L As(V) (left panel) or 300 pg / L As(III) (right panel). FIG. 7B shows the percentage of As(V) removal by cyanobacteria under various conditions. Panels A, B, and C of FIG. 7B present the results of As(V) removal at 5 mM Ca2+, 10 mM Ca2+, and 15 mM Ca2+, respectively. FIG. 7C shows the percentage of As(III) removal by cyanobacteria under various conditions. Panels A, B, and C of FIG. 7C present the results of As(III) removal at 5 mM Ca2+, 10 mM Ca2+, and 15 mM Ca2+, respectively. FIG. 7D depicts a summary of the multivariate analysis of the As removal data, which takes into account the concentrations of Ca2+and As (III and V) in the precipitation experiments, both with live R. halophila (LC; left panel) and without R. halophila (WC; right panel).

[0018] FIG. 8 shows a graphic experimental design. Panel (a) of FIG. 8 shows the bioreactor used in the experiment. Panel (b) of FIG. 8 shows Macrocolonies of R. halophila. Panel (c) of FIG. 8 depicts an optical microscopy image of cyanobacteria with CaCCL.

[0019] FIG. 9A depicts images obtained using Polarized Light Microscopy (PLM) at different Ca2+concentrations, (i), (iii) and (v) correspond to live cyanobacteria (LC); (ii), (iv) and (vi) correspond to dead cyanobacteria (DC). FIG. 9B depicts images obtained using petrographic microscope showing the presence of nucleated calcium carbonate crystals in the sheath (a) and (b) and other more incipient ones within the trichrome of cyanobacteria (c) and (d).

[0020] FIG. 10 panels a) and b) depict confocal laser scanning microscopy (CLSM) images from medium containing 15 mM of Ca2+and LC or DC. FIG. 10 panels c) and d) show transversals cuts showing a strong signal of calcein staining (green) in close relation with the cell surface as well as the sheath.

[0021] FIGS. 11A-11C depict Scanning Electron Microscopy - Energy Dispersive X-ray Spectroscopy (SEM-EDS) images of calcite crystals formed at different Ca2+concentrations. Panels of FIG. 11A depict images of (a) dead cyanobacteria (DC) under 5mM Ca2+(b) DC under lOmM Ca2+and (c) DC under 15mM Ca2+. Panels of FIG. 11B depict images (d) corresponding to living cyanobacteria (LC) at 5mM Ca2+(e) LC under lOmM Ca2+and (f) LC under 15mM Ca2+. Samples were coated with gold (Au) and palladium (Pd). FIG. 11C depicts minerals obtained from a control without cyanobacteria (WC) under various conditions,WSGR Docket No. 69144-701601 including (a) in media with 10 mM Ca2+and (b) 15 mM Ca2+. There are no photos at 5 mM Ca2+since no precipitation was observed at this concentration in the media without R. halophila.

[0022] FIGS. 12A-12B show the changes in pH in culture medium comprising cyanobacteria Rivularia halophila. FIG. 12A shows exemplary data illustrating pH changes at multiple time points over a 45-day period in either a control culture medium lacking bacteria or a culture medium comprising Rivularia halophila. FIG. 12B depicts a table outlining exemplary pH values at multiple time points over a 45-day period in either a control culture medium lacking bacteria or a culture medium comprising Rivularia halophila.

[0023] FIGS. 13A-13B depict crystals outside of the cyanobacterial sheath (cellular structure). FIG. 13A depicts images of CaCCh crystals precipitated at a bottom of a bioreactor in experiments with cyanobacteria. FIG. 13B depicts images of CaCCh crystals associated with cyanobacterial filaments and extracellular polymeric substance (EPS).

[0024] FIG. 14A depicts the CaCCh crystals associated with cyanobacteria. FIG. 14B depicts the CaCCh crystals precipitated at the bottom of the bioreactor in experiments with cyanobacteria.

[0025] FIG. 15 are images of R. halophila macrocolonies used in the CaCCh precipitation process.

[0026] FIG. 16 depicts the XRD patterns of the precipitated metallic carbonate, including XRD patterns of the precipitated metallic carbonate at the bottom of the bioreactor (BBLC).

[0027] FIG. 17 depicts the precipitated calcium carbonate, both associated with the cyanobacterial EPS and at the bottom of the bioreactor.

[0028] FIG. 18A shows composite CLSM images of a resin-embedded black pustular microbialite, stained with calcein. FIG. 18B is an optical microscopy image of a Rivularia filament (Riv.) FIG. 18C is an optical microscopy image of a Rivularia with CaCCE crystals precipitated on the sheath.

[0029] FIG. 19A depicts a table of major chemical components of seawater. FIG. 19B depicts a table showing an exemplary artificial seawater composition.

[0030] FIGs. 20A-20D are scanning electron microscopy image obtained for the cyanobacteria R. halophila and N. nodulosa. FIG. 20A shows the filaments of R. halophila connected by EPS; the white asterisks indicate this interaction. FIG. 20B shows the sheath covering the cyanobacteria R. halophila. FIG. 20C shows the cyanobacteria of the genus N. nodulosa; the white point indicates a well-defined cyanobacterium. FIG. 20D shows the filaments of N. nodulosa.WSGR Docket No. 69144-701601

[0031] FIG. 21 shows the pH species distribution diagram of H2CO3.

[0032] FIGS. 22A-22B demonstrate the calibration curves for CO2 and Ca2+measurements respectively. FIG. 22A depicts the calibration curves for CO2 measurements. FIG. 22B depicts calibration curves for Ca2+measurements.

[0033] FIG. 23 depicts the polarized light optical microscopy images of CaCCh precipitated on R. halophila filaments. FIGs. 24A-24D show scanning electron microscopy (SEM) images illustrating two fields of view of the precipitate obtained from: the control (FIG. 24A and FIG. 24B); the experiments in Example 5 using pure CO2 gas with R. halophila (FIG. 24C and FIG. 24D).

[0034] FIGs. 25A-25C depicts the XRD patterns of the mineral samples precipitated in the experiment using a culture medium with pure CO2 bubbling. FIG. 25A shows the X-ray diffraction patterns (XRD) of the control. FIG. 25B shows the XRD patterns of the precipitate at the bottom of the flask from the experiment with cyanobacteria. FIG. 25C shows the XRD patterns of the precipitate on the cyanobacteria.

[0035] FIG. 26 shows the pH changes before and after CO2 additions to the culture medium.

[0036] FIG. 27A-27C show the pH (FIG. 27A), calcium concentration in the medium (FIG. 27B), and dissolved inorganic carbon (DIC) (FIG. 27C) in treatments with R halophila and N nodulosa. One-way ANOVA analysis, bars indicate standard error, and letters represent significant differences between treatments (p < 0.05).

[0037] FIGs. 28A-28D depicts the images acquired using a petrographic optical microscope with polarized light (Leica C2000 optical microscope). FIG. 28A and FIG. 28B show R. halophila, and FIG. 28C and FIG. 28D show N nodulosa.

[0038] FIG. 29A and FIG. 29B depicts the scanning electron microscope (SEM) images of R. halophila and N. nodulosa, respectively. FIG. 29A depicts SEM images of R halophila. FIG. 29B depicts an SEM image of N nodulosa.

[0039] FIGs. 30A-30C depicts the X-ray diffraction patterns (XRD) for the Control, R halophila, and TV. nodulosa respectively. FIG. 30A shows XRD for control samples. FIG. 30B shows XRD for R. halophila. FIG. 30C shows XRD for N. nodulosa.

[0040] FIGs. 31A-31F are the C Is spectra obtained for the experiments using a mix of CCE / air in a ratio of 3:97 as a carbon source, along with the corresponding control media and reference materials. Solid black lines depict measured spectra; solid grey lines indicate fits to measured spectra and dashed lines correspond to fits obtained with parameters indicated in Table 13. A Shirley background is included as a solid curve at the base of the peaks. The shaded area under the curve is assigned to carbonate contribution. FIG. 31A shows referenceWSGR Docket No. 69144-701601C is spectra in the control condition. FIG. 31B shows reference C is spectra obtained with R. halophila. FIG. 31C shows reference C is spectra obtained with TV. nodulosa. FIG. 31D shows Cis spectra obtained experimentally using control media. FIG. 31E shows Cis spectra obtained experimentally using a mix of CCE / air in a ratio of 3:97 with R. halophila. FIG. 31F shows Cis spectra obtained experimentally using a mix of CCE / air in a ratio of 3:97 with N nodulosa.

[0041] FIGs. 32A-32C show the pH (FIG. 32A), calcium concentration in the medium (FIG. 32B), and DIC (FIG. 32C) in treatments with R. halophila and N nodulosa. One-way ANOVA analysis, bars indicate standard error, and letters represent significant differences between treatments (p < 0.05).

[0042] FIGs. 33A-33D are polarized light optical microscopy images of CaCOs precipitated on R. halophila (FIG. 33A and FIG. 33B) and N. nodulosa (FIG. 33C and FIG. 33D). FIG. 33A shows a polarized light optical microscopy image of CaCCE precipitated on R halophila, where the images was taken with parallel Nicols. FIG. 33B shows a polarized light optical microscopy image of CaCOs precipitated on R. halophila, where the images was taken with crossed Nicols. FIG. 33C shows a polarized light optical microscopy image of CaCCE precipitated on TV. nodulosa, where the images was taken with parallel Nicols. FIG. 33D shows a polarized light optical microscopy image of CaCCE precipitated on TV. nodulosa, where the images was taken with crossed Nicols. FIG. 34A-34C are scanning electron microscopy and elemental analysis (SEM-EDS) images of the precipitate obtained from: FIG. 34A control, FIG. 34 BA’, halophila and FIG. 34CTV. nodulosa.

[0043] FIGs. 35A-35C are X-ray diffraction patterns (XRD). FIG. 35A shows XRD for the control without cyanobacteria, FIG. 35B shows XRD for R. halophila, FIG. 35C shows XRD for TV. nodulosa.

[0044] FIG. 36 depicts survival percentage of R. halophila and TV. nodulosa to increasing produced water (PW) concentrations. Data are presented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test was used to assess statistical significance, with p < 0.05 considered significant. Since the 100:0 ratio and the negative control yielded values close to zero and are not visible in the graph, they are denoted as (E) and (F), respectively. The positive control was significantly different from all treatment groups (p < 0.05), except for the 25:75 ratio. For clarity, these pairwise comparisons are not shown in the figure.

[0045] FIG. 37 depicts survival percentage of R. halophila and TV. nodulosa at increasing PW concentrations. The graphed values correspond to the average across replicates, while the bars represent the standard error.FIGs. 38A-38F depicts microscopy images of R. halophilaWSGR Docket No. 69144-701601 under different PW-C200 concentrations. FIG. 38A shows a positive control; FIG. 38B shows 25:75 PW-C200; FIG. 38C shows 50:50 PW-C200; FIG. 38D shows 75:25 PW-C200; FIG. 38E shows 100:0 PW-C200; and FIG. 38F shows a negative control.

[0046] FIGs. 39A-39F are the microscopy images of N. nodulosa under different PW-C200 concentrations. FIG. 39A shows a positive control; FIG. 39B shows 25:75 PW-C200; FIG. 39C shows 50:50 PW-C200; FIG. 39D shows 75:25 PW-C200; FIG. 39E shows 100:0 PW- C200; and FIG. 39F shows a negative control.

[0047] FIG. 40A and FIG. 40B show the pH variation over time for PW-C200-800 (FIG. 40A) and PW-C200-1600 (FIG. 40B). The top lines correspond to the experiments with R. halophila and the bottom lines are the control media.

[0048] FIGs. 41A-41B depict the Two-way ANOVA statistical analysis of the pH (FIG. 41A) and Ca concentration (FIG. 41B) in PW-C200 with R. halophila (Rl) compared to the control (PW-C200 with 800 and 1600 ppm of NaHCCh). Tukey analysis identified two groups: a and b. Bars indicate standard error and (*) represent a significant difference between R. halophila and the control (p < 0.05).

[0049] FIGs. 42A-42D depict images of R. halophila acquired with a petrographic optical microscope using polarized light. FIG. 42A shows an image of R. halophila acquired with a petrographic optical microscope using polarized light of pre-equilibration treatment with 800 ppm NaHCCE, where the images was taken with parallel Nicols. FIG. 42B shows an image of R. halophila acquired with a petrographic optical microscope using polarized light of preequilibration treatment with 800 ppm NaHCCE, where the images was taken with crossed Nicols. FIG. 42C shows an image of R. halophila acquired with a petrographic optical microscope using polarized light of pre-equilibration treatment with 1600 ppm NaHCCE, where the images was taken with parallel Nicols. FIG. 42D shows an image of R. halophila acquired with a petrographic optical microscope using polarized light of pre-equilibration treatment with 1600 ppm NaHCCE, where the images was taken with crossed Nicols. FIG. 43A-43D show XRD patterns of the precipitates collected after the experiments. FIG. 43A shows the control and FIG. 43B shows the precipitate in the experiment with cyanobacteria at 800 ppm NaHCCh. FIG. 43C shows the control and FIG. 43D shows the precipitate in the experiment with cyanobacteria at 1600 ppm NaHCCh. Calcite was referred to C.

[0050] FIGs. 44A-C. SEM images and EDS analysis of the precipitates associated with cyanobacteria and the control in the experiments with 800 and 1600 ppm of NaHCCE. FIG. 44A Experiment with 800 ppm, FIG. 44B 1600 ppm, and FIG. 44C control at 1600 ppm (precipitates without cyanobacteria).WSGR Docket No. 69144-701601

[0051] FIGs. 45A-45D depict the Ca 2p spectra obtained for PW-C200-800 and PW-C200- 1600 along with the corresponding control media. FIG. 45A depicts Ca 2p spectra obtained for PW-C200-800 with control media. FIG. 45B depicts Ca 2p spectra obtained forPW-C200-800 with R. halophila. FIG. 45C depicts Ca 2p spectra obtained for PW-C200-1600 with control media. FIG. 45B depicts Ca 2p spectra obtained for PW-C200-1600 with N. nodulosa.

[0052] FIGS. 46A-46F depict the C Is spectra obtained for PW-C200-800 and PW-C200- 1600 along with the corresponding control media and reference materials. FIG. 46A shows reference C is spectra in the control condition. FIG. 46B shows reference C is spectra obtained with R. halophila. FIG. 46C shows Cis spectra obtained for PW-C200-800 without R halophila. FIG. 46D shows Cis spectra obtained for PW-C200-800 with R. halophila. FIG. 46E shows Cis spectra obtained for PW-C200-1600 without R. halophila. FIG. 46F shows Cis spectra obtained for PW-C200-1600 with R halophila.

[0053] FIGs. 47A-47Bdepict the calibration curve for CO2(FIG. 47A) and Ca2+(FIG. 47B) measurements in Example 7.

[0054] FIG. 48 shows the percentage growth of R. halophila and N. nodulosa cultures. The columns represent the average of the replicates and the bar the standard error.

[0055] FIGs. 49A-49C depict the pH (FIG. 49A), DIC (FIG. 49B), and [Ca2+] (FIG. 49C) of the media with cyanobacteria compared with initial values (NSWi and ASWi) and chemical controls without cyanobacteria. The columns represent the average of the replicates, and the bar represents the standard error.

[0056] FIGs. 50A-50H show the images taken with the petrographic microscope with polarized light using parallel nicols (left) and crossed nicols (right): R. halophila W^i (FIGs. 50A-50B), R. halophila ASW (FIGs. 50C-50D), N. nodulosa NSW (FIGs. 50E-50F), N. nodulosa ASW (FIGs. 50G-50H). FIG. 50A shows an image taken with the petrographic microscope with polarized light using parallel nicols of R. halophila NSW. FIG. 50B shows an image taken with the petrographic microscope with polarized light using crossed nicols of R. halophila NSW. FIG. 50C shows an image taken with the petrographic microscope with polarized light using parallel nicols of R. halophila ASW. FIG. 50D shows an image taken with the petrographic microscope with polarized light using crossed nicols of R. halophila ASW. FIG. 50E shows an image taken with the petrographic microscope with polarized light using parallel nicols of N. nodulosa NSW. FIG. 50F shows an image taken with the petrographic microscope with polarized light using crossed nicols of N. nodulosa NSW. FIG. 50G shows an image taken with the petrographic microscope with polarized light using parallelWSGR Docket No. 69144-701601 nicols of N. nodulosa ASW. FIG. 50H shows an image taken with the petrographic microscope with polarized light using crossed nicols of N nodulosa ASW.

[0057] FIGs. 51A-51F are the images obtained with the scanning electron microscope (SEM) of the precipitate recovered from: R. halophila NSW (FIGs. 51A-51B), R. halophila ASW (FIGs. 51C-51D) and A. nodulosa S cultures (FIGs. 51E-51F).

[0058] FIGs. 52A-52F depict X-ray diffraction patterns of the precipitate recovered from: FIG. 52A) NSW medium control, FIG. 52B) R halophila in NSW medium, FIG. 52C) N. Nodulosa in NSW medium, FIG. 52D) ASW medium control, FIG. 52E) R. halophila in ASW medium, FIG. 52F) N Nodulosa in ASW medium.

[0059] FIGs. 53A-53C depict precipitate mass (FIG. 53A), productivity (FIG. 53B), and percentage yield (FIG. 53C) obtained from media containing cyanobacteria and chemical controls without cyanobacteria. The columns represent the average of the replicates, and the bar represents the standard error.

[0060] FIGs. 54A-54D depict effects of phosphorus and nitrogen variation on Rivularia halophila growth. Cyanobacteria were exposed to different phosphorus (FIGs. 54A-54B) and nitrogen concentrations (FIGs. 54C-54D) for 5 days. FIG. 54A shows the relative growth of R. halophila following administration of 0.03, 0.05, or 0.1 g / L of phosphorus. FIG. 54B shows the fold change in relative growth of R. halophila following administration of 0.03, 0.05, or 0.1 g / L of phosphorus. FIG. 54C shows the relative growth of R. halophila following administration of 0.03, 0.05, or 0.1 g / L of nitrogen. FIG. 54D shows the fold change in relative growth of R halophila following administration of 0.03, 0.05, or 0.1 g / L of nitrogen.

[0061] FIGs. 55A-55D depict effects of phosphorus and nitrogen variation on Nodosilinea nodulosa growth. Cyanobacteria were exposed to different phosphorus (FIGs. 55A-55B) and nitrogen concentrations (FIGs. 55C-55D) for 5 days. FIG. 55A shows the relative growth of N. nodulosa following administration of 0.03, 0.05, or 0.1 g / L of phosphorus. FIG. 55B shows the fold change in relative growth of N. nodulosa following administration of 0.03, 0.05, or 0.1 g / L of phosphorus. FIG. 55C shows the relative growth of N nodulosa following administration of 0.5, 0.75, or 3 g / L of nitrogen. FIG. 55D shows the fold change in relative growth of N. nodulosa following administration of 0.5, 0.75, or 3 g / L of nitrogen.

[0062] FIGs. 56A-56H. Petrographic microscope images of R. halophila and N. nodulosa after 5 days of exposure to different phosphorus concentrations. FIG. 56A shows a petrographic microscope image of R. halophila after 5 days of exposure to 0 g / L phosphorus. FIG. 56B shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0 g / L phosphorus. FIG. 56C shows a petrographic microscope image of R. halophila after 5WSGR Docket No. 69144-701601 days of exposure to 0.03 g / L phosphorus. FIG. 56D shows a petrographic microscope image of TV. nodulosa after 5 days of exposure to 0.03 g / L phosphorus. FIG. 56E shows a petrographic microscope image of R. halophila after 5 days of exposure to 0.05 g / L phosphorus. FIG. 56F shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0.05 g / L phosphorus. FIG. 56G shows a petrographic microscope image of R. halophila after 5 days of exposure to 0.1 g / L phosphorus. FIG. 56H shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0.1 g / L phosphorus.

[0063] FIGs. 57A-57H depict petrographic microscope images of R. halophila and N. nodulosa after 5 days of exposure to different nitrogen concentrations. FIG. 57A shows a petrographic microscope image of R. halophila after 5 days of exposure to 0 g / L nitrogen. FIG. 57B shows a petrographic microscope image of N. nodulosa after 5 days of exposure to 0 g / L nitrogen. FIG. 57C shows a petrographic microscope image of R. halophila after 5 days of exposure to 0.5 g / L nitrogen. FIG. 57D shows a petrographic microscope image of TV nodulosa after 5 days of exposure to 0.5 g / L nitrogen. FIG. 57E shows a petrographic microscope image of R. halophila after 5 days of exposure to 0.75 g / L nitrogen. FIG. 57F shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0.75 g / L nitrogen. FIG. 57G shows a petrographic microscope image of R. halophila after 5 days of exposure to 3 g / L nitrogen. FIG. 57H shows a petrographic microscope image of N nodulosa after 5 days of exposure to 3 g / L nitrogen.

[0064] FIG. 58A-58F depict the effect of nitrogen and phosphorus concentration on titer, productivity, and substrate yield (%SY) of R. halophila and N nodulosa. FIG. 58A depicts the effect of phosphorus concentration on titer of R. halophila and TV nodulosa. FIG. 58B depicts the effect of phosphorus concentration on productivity of R. halophila and TV nodulosa. FIG. 58C depicts the effect of phosphorus concentration on %SY of R. halophila and N. nodulosa. FIG. 58D depicts the effect of nitrogen concentration on titer of R. halophila and N. nodulosa. FIG. 58E depicts the effect of nitrogen concentration on productivity of R. halophila and N. nodulosa. FIG. 58F depicts the effect of nitrogen concentration on %SY of R. halophila and N. nodulosa.

[0065] FIGs. 59A-59B depict the effect of agricultural- and laboratory-grade (NFL^zHPCL as nitrogen and phosphorus sources on the growth of Rivularia halophila and Nodosilinea nodulosa. Cyanobacteria were exposed to the two fertilizer grades for 7 days. Growth was assessed by measuring wet biomass and is expressed as relative growth. FIG. 59A: R. halophila, FIG. 59B: N. nodulosa.WSGR Docket No. 69144-701601

[0066] FIGs. 60A-60F depict petrographic microscope images of R. halophila and N nodulosa after 7 days of exposure to (NH^HPC FIG. 60A depicts a petrographic microscope image of R. halophila after 7 days of exposure to a control solution. FIG. 60B depicts a petrographic microscope image of N nodulosa after 7 days of exposure to a control solution. FIG. 60C depicts a petrographic microscope image of R. halophila after 7 days of exposure to agri cultural -grade (NH^HPC FIG. 60D depicts a petrographic microscope image of N nodulosa after 7 days of exposure to agri cultural -grade (NHQJFPCU. FIG. 60E depicts a petrographic microscope image of R halophila after 7 days of exposure to laboratory-grade (NH^HPC FIG. 60F depicts a petrographic microscope image of N nodulosa after 7 days of exposure to laboratory-grade (NH hHPC

[0067] FIGs. 61A-61B depict the effect of FeSCL and FeCh fertilizers as iron sources on the growth of Rivularia halophila (FIG. 61A) and Nodosilinea nodulosa (FIG. 61B). Cyanobacteria were exposed to both iron sources for 7 days. Growth was assessed by measuring wet biomass and is expressed as relative growth.

[0068] FIGs. 62A-62F depict petrographic microscope images of R. halophila and N nodulosa after 7 days of exposure to different iron sources. FIG. 62A depicts a petrographic microscope image of R. halophila after 7 days of exposure to a control solution. FIG. 62B depicts a petrographic microscope image of N nodulosa after 7 days of exposure to a control solution. FIG. 62C depicts a petrographic microscope image of R. halophila after 7 days of exposure to FeSCU. FIG. 62D depicts a petrographic microscope image of N nodulosa after 7 days of exposure to FeSO44. FIG. 62E depicts a petrographic microscope image off?, halophila after 7 days of exposure to FeCh. FIG. 62F depicts a petrographic microscope image of N nodulosa after 7 days of exposure to FeCh.

[0069] FIGs. 63A-63B depict calibration curves for CO2 (FIG. 63A) and Ca measurements (FIG. 63B) in Example 9.

[0070] FIGs. 64A-64C depict pH (FIG. 64A), dissolved carbon dioxide (DIC) (FIG. 64B), and calcium concentration in the medium (FIG. 64C) for the co-culture experiments using NaHCCh as a carbon source; controls and the initial culture medium (ICM). One-way ANOVA analysis, bars indicate standard error, and letters represent significant differences between treatments (p < 0.05).

[0071] FIGs. 65A-65D depict images acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope). FIG. 65A shows an image acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope) with 80:20 co-culture, where the images was taken with parallel Nicols. FIG. 65BWSGR Docket No. 69144-701601 shows an image acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope) with 80:20 co-culture, where the images was taken with crossed Nicols. FIG. 65C shows an image acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope) with 95:5 co-culture, where the images was taken with parallel Nicols. FIG. 65D shows an image acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope) with 95:5 co-culture, where the images was taken with crossed Nicols.

[0072] FIGs. 66A-66F depict scanning electron microscopy images. FIGs. 66A-66B show two different fields of view of the control culture medium without cyanobacteria. FIGs. 66C- 66D show two different fields of view of CaCCL precipitates obtained from the 80:20 coculture; FIGs. 66E-66F show two different fields of view of CaCCh precipitates obtained from the 95:5 co-culture.

[0073] FIGs. 67A-67C depict XRD patterns of the precipitates collected after the experiments. FIG. 67A shows the control, FIG. 67B shows the 80:20 co-culture, and FIG. 67C shows the 95:5 co-culture. Calcite was referred to C.

[0074] FIGs. 68A-68F depict C is spectra obtained for the co-culture experiments using NaHCCL as a carbon source, along with the corresponding control media and reference materials. Solid black lines depict measured spectra; solid red lines indicate fits to measured spectra and dashed lines correspond to fits obtained with parameters indicated in Table 38. FIG. 68A shows reference C is spectra in the control condition. FIG. 68B shows reference C 1 s spectra obtained with R. halophila. FIG. 68C shows reference C is spectra obtained with N nodulosa. FIG. 68D shows Cis spectra obtained via co-culture experiments using NaHCCh as a carbon source in the control condition without cyanobacteria. FIG. 68E shows Cis spectra obtained via co-culture experiments using NaHCCh as a carbon source with an 80:20 co-culture of R. halophila : N. nodulosa. FIG. 68F shows Cis spectra obtained via co-culture experiments using NaHCCh as a carbon source with a 95:5 co-culture of R. halophila : N. nodulosa .FIGs. 69A-69B depict the productivity (FIG. 69A) and percentage yield (%SY) (FIG. 69B). Oneway ANOVA analysis, bars indicate standard error, and letters represent significant differences between treatments (p < 0.05).

[0075] FIG. 70 depicts changes in pH before and after adding CO2 to the culture medium.

[0076] FIGs. 71A-71C depict the pH (FIG. 71A), DIC (FIG. 71B), and calcium concentration in the medium (FIG. 71C) in the co-culture treatments compared to the controls and the initial culture medium (ICM). One-way ANOVA analysis, bars indicate standard error, and letters represent significant differences between treatments (p < 0.05).WSGR Docket No. 69144-701601

[0077] FIGs. 72A-72D depict images acquired using a petrographic optical microscope with polarized light (Leica C2000 optical microscope). FIGs. 72A-72B. 80:20 co-culture, FIGs. 72C-72D. 95:5 co-culture experiments with CO2 bubbling. FIG. 72A shows an image acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope) with 80:20 co-culture, where the images was taken with parallel Nicols. FIG. 72B shows an image acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope) with 80:20 co-culture, where the images was taken with crossed Nicols. FIG. 72C shows an image acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope) with 95:5 co-culture, where the images was taken with parallel Nicols. FIG. 72D shows an image acquired using a petrographic optical microscope using polarized light (Leica C2000 optical microscope) with 95:5 co-culture, where the images was taken with crossed Nicols.

[0078] FIGs. 73A-73D depict the scanning electron microscopy (SEM) images. FIGs. 73A- 73B show two fields of view of CaCCL precipitates obtained from the 80:20 co-culture, FIGs. 73C-73D show two fields of view of CaCCL precipitates obtained from the 95:5 co-culture.

[0079] FIGs. 74A-74C depict the X-ray diffraction (XRD) patterns in Control without cyanobacteria (FIG. 74A), precipitates obtained from the 80:20 co-culture (FIG. 74B), (b) precipitates from the 95:5 co-culture (FIG. 74C).

[0080] FIGs. 75A-75B depict the productivity (FIG. 75A) and percentage yield (%SY) (FIG. 75B) One-way ANOVA analysis, bars indicate standard error, and letters represent significant differences between treatments (p < 0.05).DETAILED DESCRIPTION

[0081] The methods, compositions, and systems provided here relate to biomineralization, such as the precipitation of metallic carbonates (e.g., CaCOs) using one or more cyanobacteria (e.g., Rivularia halophila; Nodosilinea nodulosa). The approach offers distinct advantages over other methods of manufacturing metallic carbonates. Additionally, the cyanobacteria used (e.g., R. halophila) demonstrate clear benefits compared to other cyanobacterial strains. In some aspects, provided herein are improved methods, compositions and systems for precipitating metallic carbonates using cyanobacteria, including media for culturing cyanobacteria, culturing environment or conditions and bioreactors. The methods, composition and systems described herein can enable improvements in yield, scalability, and / or purity of the precipitated metallic carbonates, reduction in carbon footprint or environment impact of the processes, utilizing industrial waste, or reduction in cost.WSGR Docket No. 69144-701601Advantages of precipitating metallic carbonate using cyanobacteria

[0082] The methods, compositions, and systems herein relate to manufacturing of precipitated metallic carbonates (e.g., CaCCE) using one or more cyanobacteria (e.g., Rivularia halophila; Nodosilinea nodiilosct). and provide several advantages. For example, the precipitated metallic carbonate produced using cyanobacteria can have high purity or contain low levels of impurities. The high purity levels of the metallic carbonate produced by the methods described herein make it suitable for various industrial applications, including those requiring stringent quality standards, such as in pharmaceuticals, electronics, and high- performance materials. The ability to achieve such high purity levels with cyanobacteria-driven biomineralization underscores the efficiency and effectiveness of the process. In some embodiments, the precipitated metallic carbonate has a purity level of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the highly pure precipitated metallic carbonate can be extracted from the bottom of cyanobacteria cultures and requires no further purification. Purity of the precipitated metallic carbonates (e.g., CaCCE) can be assessed through one or more analytical techniques. In some cases, the purity (or the impurity) of the precipitated metallic carbonates can be measured and / or calculated by Energy- Dispersive X-ray Spectroscopy (EDS), which can be coupled with a Scanning Electron Microscope (SEM). EDS can provide an elemental analysis of the sample's surface, allowing for the detection of elements in CaCCE and elements in the impurities (e.g., Mg, Mn, Fe). The results from EDS can indicate the relative weight percentages of the elements, thereby providing an estimate of purity and identifying the presence and proportion of the impurities. Examples of methods for calculating the purity of the precipitated metallic carbonates is provided in Examples 5-9. In some cases, purity or impurity can be measured and / or calculated by X-ray Photoelectron Spectroscopy (XPS), X-ray Diffraction (XRD), SEM-EDS, or any combination thereof. In some cases, purity or impurity can be measured and / or calculated by XPS.

[0083] In some embodiments, the precipitated metallic carbonate comprises amorphous metallic carbonate. In some embodiments, the precipitated metallic carbonate comprises crystalline metallic carbonate. In some embodiments, the crystalline metallic carbonate produced by the methods and systems herein settles at the bottom of a bioreactor and does not require further purification, which further reduces cost of production.

[0084] In some embodiments, the precipitated metallic carbonate provided herein has a low impurity level, with less than 100 parts per million (ppm), less than 90 ppm, less than 80 ppm,WSGR Docket No. 69144-701601 less than 70 ppm, less than 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm, or less than 5 ppm. In some embodiments, the impurity comprises one or more members selected from the group consisting of arsenic (e.g., arsenate (As (V)), arsenite (As (III))), iron (Fe (III)) hydroxides, silica, alumina, magnesium oxide, and mercury. In some embodiments, the impurity comprises arsenic (e.g., arsenate (As (V)), arsenite (As (III))), iron (Fe (III)) hydroxides, silica, alumina, magnesium oxide, or mercury, or any combination thereof.

[0085] In some embodiments, the metallic carbonate precipitated using cyanobacteria has a biogenic signature. This biogenic signature can be measured by various analytical methods, including X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Fourier- transform infrared spectroscopy (FTIR), Raman spectroscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS).

[0086] In some cases, the biogenic signature of the precipitated metallic carbonate described herein can be measured by X-ray Photoelectron Spectroscopy (XPS). The spectra generated by XPS can include one or more carbon peaks at a specific binding energy (BE). These carbon peaks can appear as a single sharp peak or as a wider peak composed of two or more overlapping peaks. The carbon peaks can originate from a variety of bonds selected from the group consisting of: C-C, C-H, C-O, C=O, C-SH, C-OH, C-N, C=N, O-C-O, and HO-C=O, wherein the dash represents a covalent bond. In some embodiments, the biogenic signature of the precipitated metallic carbonate described herein can comprise one or more overlapping carbon peaks measured by XPS. In some embodiments, the biogenic signature comprises a wide carbon (Cl) peak with a binding energy of from about 284.6 to 291.5 eV. In some embodiments, the biogenic signature comprises a wide carbon (Cl) peak with a binding energy of from about 283 to 288.8 eV, about 283 to 289.0 eV, about 283 to 289.2 eV, about 283 to 289.4 eV, about 283 to 289.6 eV, about 283 to 289.8 eV, about 283 to 290.0 eV, about 283 to290.2 eV, about 283 to 290.4 eV, about 283 to 290.6 eV, about 283 to 290.8 eV, about 283 to291.0 eV, about 283 to 291.2 eV, about 283 to 291.4 eV, about 283 to 291.6 eV, about 283 to291.8 eV, about 283 to 292.0 eV, about 283 to 292.2 eV, about 283 to 292.4 eV, about 283 to292.6 eV, about 283 to 292.8 eV, about 283 to 293.0 eV.

[0087] In some cases, the precipitated metallic carbonate described herein can be identified by X-ray diffraction (XRD). In some embodiments, the precipitated metallic carbonate provided herein has a peak of at least 29.3° of 2-theta (29) as measured by XRD. In some embodiments, the precipitated metallic carbonate provided herein has a peak of at least 29°, at least 29.1°, at least 29.2°, at least 29.3°, at least 29.4°, at least 29.5°, at least 29.6°, at leastWSGR Docket No. 69144-79169129.7°, at least 29.8°, at least 29.9°, or at least 30° of 2-theta (29) as measured by XRD. In some embodiments, the precipitated metallic carbonate provided herein has a peak of at most 50° of 2-theta (29) as measured by XRD. In some embodiments, the precipitated metallic carbonate provided herein has a peak of at most 39°, at most 31°, at most 32°, at most 33°, at most 34°, at most 35°, at most 36°, at most 37°, at most 38°, at most 39°, at most 49°, at most 41°, at most 42°, at most 43°, at most 44°, at most 45°, at most 46°, at most 47°, at most 48°, at most 49°, or at most 59° of 2-theta (29) as measured by XRD. In some embodiments, the precipitated metallic carbonate provided herein has a peak or a broad band of about 29.3° to 29.5° (CaCO3 with impurities lower than 19 %), 29.3° to 29.9° (CaCO3 with impurities higher than 19%) and 39° to 59° of 2-theta (29) (ACC, amorphous calcium carbonate) as measured by XRD. In some cases, the purity of the precipitated metallic carbonate can be calculated or inferred from the position of its XRD peak(s). For example, the precipitated metallic carbonate (e.g., CaCCh) provided herein can have a prominent peak at about 29.3° to 29.9° 2-theta (29), which is consistent with the primary peak for crystalline CaCCh. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.2° of 29.3° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.3° of 29.3° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.4° of 29.3° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.5° of 29.3° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.2° of 29.4° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.3° of 29.4° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.4° of 29.4° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.5° of 29.4° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.2° of 29.5° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.3° of 29.5° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.4° of 29.5° 29 as measured by XRD. In some embodiments, the peak of the precipitated metallic carbonate is within + / - 9.5° of 29.5° 29 as measured by XRD.

[0088] The compositions, methods and systems provided herein are carbon-negative because the metallic carbonate (e.g., CaCCh) is produced through the photosynthetic activity of cyanobacteria, which absorb carbon dioxide from the atmosphere as dissolved CO2 or bicarbonate. This carbon-negative, chemi cal -grade metallic carbonate can be utilized acrossWSGR Docket No. 69144-701601 various industries as an additive or filler in plastics, paper, and paints. Additionally, it has the potential to replace carbon-positive calcium carbonate in existing applications, including in the manufacture of bio-limestone. In some embodiments, the compositions, methods, and systems can reduce the carbon footprint of metallic carbonate synthesis by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% as compared to carbon-positive manufacturing methods. An example of calculating this reduced carbon footprint can be found in Example 3.

[0089] In some embodiments, metallic carbonate precipitation using cyanobacteria can remove at least 500 grams of carbon from an atmosphere (e.g., CO2 or CH4) for every kilogram of precipitated metallic carbonate produced. In some embodiments, the metallic carbonate precipitation using cyanobacteria can remove at least 100 grams, at least 200 grams, at least 300 grams, at least 400 grams, at least 500 grams, at least 600 grams, at least 700 grams, at least 800 grams, at least 900 grams, at least 1000 grams, at least 1500 grams, or at least 2000 grams of atmospheric carbon for every kilogram of precipitated metallic carbonate produced. In some embodiments, the carbon footprint for precipitating one kilogram of metallic carbonate is at least negative 50 grams, at least negative 100 grams, at least negative 150 grams, at least negative 200 grams, at least negative 250 grams, at least negative 300 grams, at least negative 350 grams, at least negative 400 grams, at least negative 450 grams, or at least negative 500 grams CO2 equivalent.

[0090] In some embodiments, CO2 can be injected into the composition or system comprising the cyanobacteria for metallic carbonate precipitation. In some embodiments, the compositions, methods and systems do not or need not rely on exogenous carbon source (e.g., HCO3 ) from outside the system. In some embodiments, direct injection of CO2 into the cyanobacteria culture (e.g., tank, bioreactor comprising the culture) can drastically improve growth rates of the cyanobacteria and calcium carbonate precipitation. This process not only boosts the cyanobacteria's metabolic activity but also elevates the concentration of bicarbonate ions in the solution. As CO2 levels increase, natural bicarbonate formation occurs, negating the need for external bicarbonate supplementation. As photosynthetic microorganisms, cyanobacteria use CO2 as a primary carbon source during photosynthesis. Elevated CO2 levels lead to increased photosynthetic efficiency and greater cyanobacteria biomass production. In some embodiments, the increased metabolic activity of the cyanobacteria also enhances the pH level in the medium, favoring the precipitation of calcium carbonate. Methods of injecting CO2 into the cyanobacteria culture media is provided in Example 4 and Example 5.WSGR Docket No. 69144-701601

[0091] The compositions, methods and systems provided herein rely on using cyanobacteria (e.g., Rivularia halophila; Nodosilinea nodulosd) and thus consume less energy compared to other compositions, methods and systems that do not rely on using cyanobacteria or using a different strain of the cyanobacteria (e.g., not Rivularia halophila; Nodosilinea nodulosa). In some embodiments, precipitating metallic carbonate using cyanobacteira uses less energy than physiochemical methods of manufacturing metallic carbonates. In some embodiments, precipitating metallic carbonate using cyanobacteria uses less energy than manufacturing biolimestone. In some embodiments, precipitating metallic carbonate using cyanobacteria uses less than about 20 gigajoule (GJ) per ton of product. In some embodiments, precipitating metallic carbonate using cyanobacteria uses less than 20 GJ, less than 19.5 GJ, less than 19.0 GJ, less than 18.5 GJ, less than 18.0 GJ, less than 17.5 GJ, less than 17.0 GJ, less than 16.5 GJ, less than 16.0 GJ, less than 15.5 GJ, less than 15.0 GJ, less than 14.5 GJ, less than 14.0 GJ, less than 13.5 GJ, less than 13.0 GJ, less than 12.5 GJ, less than 12.0 GJ, less than 11.5 GJ, less than 11.0 GJ, less than 10.5 GJ, less than 10.0 GJ, less than 9.5 GJ, less than 9.0 GJ, less than 8.5 GJ, less than 8.0 GJ, less than 7.5 GJ, less than 7.0 GJ, less than 6.5 GJ, less than 6.0 GJ, less than 5.5 GJ, less than 5.0 GJ, less than 4.5 GJ, less than 4.0 GJ, less than 3.5 GJ, less than 3.0 GJ, less than 2.5 GJ, less than 2.0 GJ, less than 1.5 GJ, less than 1.0 GJ, or less than 0.5 GJ per ton of product. In some embodiments, the closed system provided herein uses more energy than the open system herein. In some embodiments, the open system uses less than 5.0 GJ, less than 4.5 GJ, less than 4.0 GJ, less than 3.5 GJ, less than 3.0 GJ, less than 2.5 GJ, less than 2.0 GJ, less than 1.5 GJ, less than 1.0 GJ, or less than 0.5 GJ per ton of product.

[0092] In some embodiments, the improved compositions, methods and systems further comprise having co-cultures of cyanobacteria to improve efficacy. Various strains of cyanobacteria can be selected for their ability to excrete specific nutrients or metabolites that are beneficial for the growth of Rivularia halophila. This can create a synergistic relationship, further enhancing the productivity of the methods and systems disclosed herein. In some cases, co-culturing can increase the yield, the purity, and / or the crystallinity of the precipitated metallic carbonates. See Example 9. In some cases, co-culturing can increase calcite yield by increasing the pH of the culture medium. Furthermore, co-cultures can be adjusted and optimized based on different growth parameters such as light intensity, temperature, and nutrient concentrations to maximize the proliferation of cyanobacteria, such as Rivularia halophila, and the production via precipitation of metallic carbonate. By creating a self- sustaining system, this not only minimizes the need for external inputs, making it more cost- effective, but also potentially increases the cyanobacteria biomass production of RivulariaWSGR Docket No. 69144-701601 halophila., thereby enhancing its commercial viability. Methods of using cyanobacteria cocultures are provided in Example 4 and Example 9.

[0093] In some embodiments, Rivularia halophila can be co-cultured with another Rivularia species. In some embodiments, Rivularia halophila can be co-cultured with other cyanobacteria species (e.g., a Nodosilinea species). In some embodiments, Nodosilinea nodulosa can be co-cultured with another Nodosilinea species. In some embodiments, Nodosilinea nodulosa can be co-cultured with other cyanobacteria species (e.g., a Rivularia species). Co-cultures of cyanobacteria can be done at various ratios of cyanobacteria species. In some embodiments, the co-culture can comprise a first species of cyanobacteria and a second species of cyanobacteria, with a ratio of the first species of cyanobacteria to the second species of cyanobacteria of about 50:50, about 60:40, about 70:30, about 80:20, about 85: 15, about 90: 10, about 95:5, or about 98:2. In some embodiments, the co-culture can comprise a ratio of Rivularia halophila to Nodosilinea nodulosa of about 50:50, about 60:40, about 70:30, about 80:20, about 85: 15, about 90: 10, about 95:5, or about 98:2. In some embodiments, the coculture can comprise a ratio of Rivularia halophila to Nodosilinea nodulosa of about 80:20.

[0094] In some embodiments, the compositions, methods and systems provided herein can utilize industrial by-product which minimizes waste and exogenous nutrient input. In some embodiments, the industrial by-product comprises agricultural by-product, wastewater, or industrial effluents. In some embodiments, the approaches provided herein can enable costeffectiveness by reusing brine waste, mitigating its environmental impact, contributing to a circular economy, and addressing water scarcity by repurposing water typically considered waste. In some embodiments, the industrial by-product comprises brine. In some embodiments, the brine comprises lithium brine. Lithium brine is a by-product of the mining industry. In some embodiments, the brine comprises sodium chloride brine. In some embodiments, the brine comprises calcium and bicarbonate-rich brines. Using such brines as a growth medium for cyanobacteria boosts cyanobacteria biomass production and metallic carbonate precipitation due to the nutrient-dense environment. Methods of using brine as cyanobacteria culture media are provided in Example 4 and Example 6.

[0095] In some embodiments, cyanobacteria are cultured in a high salinity solution. In some instances, the high salinity solution comprises sea water. Seawater, being nutrient-rich and freely available, can effectively enhance the cyanobacteria biomass production and calcium carbonate precipitation when used as a cyanobacteria growth medium. In some embodiments, the inherent salinity of seawater optimizes cyanobacterial growth and reduces the need for extra nutrients. Methods of using sea water as cyanobacteria culture media are provided in ExampleWSGR Docket No. 69144-7016014 and Example 7. In some embodiments, the compositions, methods and systems herein can utilize any solution with high salinity.

[0096] The present disclosure also provides improved methods for separating the precipitated metallic carbonate from the cyanobacteria and culture media. In some embodiments, the precipitated metallic carbonate can be separated from the cyanobacteria biomass chemically (e.g., hydrogen peroxide). In some embodiments, the precipitated metallic carbonate can be separated from the cyanobacteria biomass physically. In some embodiments, the precipitated metallic carbonate can be separated from the cyanobacteria biomass by centrifugation, flocculation or membrane filtration. Methods of separating the precipitated metallic carbonate from the cyanobacteria are provided in Example 4. In some embodiments, the precipitated metallic carbonate of the compositions, methods and systems can be separated from the cyanobacteria biomass without further purification.

[0097] In some embodiments, the compositions, methods and systems relate to high growth rate of the cyanobacteria provided herein, thereby generating a large amount of cyanobacteria biomass rapidly. In some embodiments, the high growth rate of the cyanobacteria leads to high production rate and yield of the precipitated metallic carbonate.Cyanobacteria

[0098] Biomineralization is a process performed by both eukaryotic and prokaryotic organisms to create a diverse range of mineralized structures and nanostructures commonly found in nature. Microorganisms may influence the formation of minerals such as oxides, halides, silicates, among others, including calcium phosphate ((Cas PCU)?) and calcium carbonate (CaCCh). The morphology and elemental composition of crystals resulting from biomineralization processes may differ from those of inorganic mineralization. Microbial metabolism, such as photosynthesis, ureolysis, denitrification and sulfate reduction are known to promote CaCCE precipitation by locally increasing pH, leading to alkalinization, while other metabolic processes, such as aerobic heterotrophy or fermentation processes, promote carbonate dissolution through acidification. In addition, exopolymeric substances (for example, extracellular polymeric substance (EPS)), which consist of a mixture of carbohydrates, proteins, and nucleic acids excreted in varying amounts, are also known to influence CaCCh formation both positively and negatively. This influence depends on their capacity to bind divalent cations (such as Ca2+or Mg2+) and to make them either available or unavailable for precipitation. This intricate process has had a significant impact on the evolution of Earth's surface by influencing the cycling of biogeochemical elements and providing signals in the geological record. Cyanobacteria, in particular, are recognized as primary contributors to theWSGR Docket No. 69144-701601 production of CaCCh rocks throughout Earth's history. Ancient cyanobacterial stromatolites and modern microbialites can be found in both marine and terrestrial environments.

[0099] The mechanism by which photosynthesis induce CaCCh precipitation involves an HCOs' / OH- exchange process across the cell membrane. Atmospheric CO2 is converted to bicarbonate ion (HCO3 ) in the external aqueous solution, then HCO3 - is transported into cells and spontaneous dehydration of HCOf occurs catalyzed by carbonic anhydrase. This process exchanges OH' thereby increasing the pH in the microenvironment around cells, resulting in the formation of insoluble CaCOs. Numerous evidence demonstrates the potential of cyanobacterial surfaces to act as a template for mineral nucleation. Additionally, the proteinaceous surface layer (S-layer) attached to the outer membrane of the cells can also play a significant role in this process. However, only certain genera are capable of calcification, and none of them are obligate calcifiers. In this regard, various cyanobacterial strains have been investigated as promising methods for reducing CO2 emissions by facilitating its conversion and storage into carbonate minerals such as calcite, aragonite, and dolomite.

[0100] Provided herein are compositions, methods and systems for precipitating metallic carbonate using one or more cyanobacteria. In some embodiments, the cyanobacteria comprise a benthic extremophile. In some embodiments, the cyanobacteria comprise a Rivularia species (or Rivulariaceae species In some embodiments, the cyanobacteria comprise a Rivularia halophila (R halophila). In some embodiments, the cyanobacteria comprise a cyanobacteria species isolated from Laguna Negra. In some embodiments, the cyanobacteria comprise a Nodosilinea species. In some embodiments, the cyanobacteria comprise a Nodosilinea nodulosa. Previously, in the U.S. Provisional Application No. 63 / 683,647, Nodosilinea nodulosa, a species of cyanobacteria used in the co-cultivation experiments in Example 4, had been mis-identified as a Phormidium species.

[0101] In some embodiments, the one or more cyanobacteria comprises at least two, at least three, at least four, at least five or at least six cyanobacteria. In some embodiments, the one or more cyanobacteria are co-cultured with cyanobacteria. In some embodiments, the one or more cyanobacteria are co-cultured with non-cyanobacteria microorganisms. In some embodiments, the one or more cyanobacteria comprises at least two cyanobacteria. In some embodiments, the least two cyanobacteria comprise a. Rivularia sp. and a Nodosilinea sp.

[0102] In some embodiments, the one or more cyanobacteria comprises an isolated cyanobacteria species. In some embodiments, the isolated cyanobacteria behave differently in controlled conditions compared to their natural environment. In some embodiments, theWSGR Docket No. 69144-701601 isolated cyanobacteria (e.g., R. halophila; Nodosilinea nodulosa) show unique characteristics to precipitate high purity metallic carbonate (e.g., calcium carbonate) compared to other microorganisms which can only produce a low purity product suitable for use in construction. In some embodiments, the one or more cyanobacteria are capable of precipitating crystalline metallic carbonate. In some embodiments, the one or more cyanobacteria are capable of precipitating amorphous metallic carbonate.

[0103] In some embodiments, the one or more cyanobacteria comprises a member selected from the group consisting of: w Anabaena species (sp.), Nostoc sp., Spirulina (Arthrospira) sp., a Microcystis sp., a Synechococcus sp., a Prochlorococcus sp., an Oscillatoria sp., a Gloeocapsa sp., a Lyngbya sp., a Rivularia sp., a Nodosilinea sp., a Fischer ella sp., a Cyanothece sp., a Trichodesmium sp, a Gloeobacter sp., a Chroococcidiopsis sp., a Phormidium sp., a Aphanizomenon sp., and a Planktothrix sp. In some embodiments, the one or more cyanobacteria comprises Rivularia halophila and one or more cyanobacteria species commonly used for biofuel production.

[0104] The cyanobacteria provided here (e.g., Rivularia or Nodosilinea species) can comprise organic functional groups on the surface (e.g., extracellular polymeric substances (EPS)) of the organisms, resulting in a net negative surface charge that facilitates the accumulation of Ca2+on the surface of the cyanobacteria. In some cases, the cyanobacteria comprise an extracellular sheath on the surface of the cell (e.g., Rivularia halophilia). In some cases, the cyanobacteria do not comprise an extracellular sheath on the surface of the cell (e.g., Nodosilinea nodulosa). The cyanobacteria also can comprise filaments. In some embodiments, the cyanobacteria generate favorable conditions for precipitation of the metallic carbonate provided herein (e.g., CaCCh). In some embodiments, the cyanobacteria provide nucleation points on its surface further enhancing the precipitation of metallic carbonate. In some embodiments, the nucleation points comprise sheath of the cyanobacteria. In some embodiments, the nucleation points comprise filaments of the cyanobacteria.

[0105] In some embodiments, the preferred cyanobacteria comprise Rivulariaceae species. In some embodiments, the preferred cyanobacteria comprises Rivularia halophila. Table 1 lists the advantages of Rivularia halophila compared to other microorganisms.WSGR Docket No. 69144-701601Precipitated Metallic Carbonate

[0106] The precipitated metallic carbonate provided herein is produced by cyanobacteria through a process known as biologically-induced mineralization or biomineralization. In some embodiments, “precipitation” or “precipitating” refers to the process by which metallic carbonates are formed and solidified from dissolved ions in the surrounding environment (e.g.,WSGR Docket No. 69144-701601 culturing medium, bioreactor, system). In some embodiments, precipitation occurs when cyanobacteria induce the formation of calcium carbonate (CaCCri) by altering the local chemistry (e.g., increase pH; remove CO2). In some embodiments, the precipitated metallic carbonate can be deposited on and around the outside of the cyanobacteria. In some embodiments, the precipitated metallic carbonate can be deposited on and around the sheath of the cyanobacteria that comprise a sheath outside the cell. In some embodiments, the precipitated metallic carbonate can be deposited on and around the filament of the cyanobacteria. In some embodiments, precipitation of the precipitated metallic carbonate can be facilitated by the cyanobacteria Extracellular Polymeric Substances (EPS), which is a gellike matrix comprising a mixture of polysaccharides among other marcromolecules. The precipitated metallic carbonate can be collected without lysing the cells.

[0107] In some embodiments, the metallic carbonate comprises an alkali metal, an alkaline earth metal, a transitional metal, or any combination thereof. In some embodiments, the metallic carbonate comprises a metal selected from Group 1 to Group 12 of the periodic table. In some embodiments, the metallic carbonate comprises a metal, wherein the metal comprises Calcium (Ca), Magnesium (Mg), Iron (Fe), Zinc (Zn), Copper (Cu), Aluminum (Al), Lead (Pb), Barium (Ba), Silver (Ag), Lithium (Li) or Strontium (Sr). In some embodiments, the metallic carbonate comprises sodium carbonate (Na2COs), potassium carbonate (K2CO3), lithium carbonate (Li2CO3), calcium carbonate (CaCCri), magnesium carbonate (MgCCri), barium carbonate (BaCCL), strontium carbonate (SrCCri), iron(II) carbonate (FeCCri), copper(II) carbonate (CuCCri), zinc carbonate (ZnCCri), lead carbonate (PbCCri), aluminum carbonate (A12(CO3)3), tin(II) carbonate (SnCCri) and manganese carbonate (MnCCri). In some embodiments, the metallic carbonate comprises an alkaline-earth metal. In some embodiments, the metallic carbonate comprises a metal selected from Group 2 of the periodic table. In some embodiments, the metallic carbonate comprises calcium carbonate (CaCCh). In some embodiments, the metallic carbonate can be soluble in water but precipitate in the culture medium provided herein.

[0108] In some embodiments, the metallic carbonate comprises crystalline metallic carbonate. In some embodiments, the metallic carbonate comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to 100% crystalline metallic carbonate. In some embodiments, the precipitated metallic carbonate comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to 100% crystalline metallic carbonate. In some embodiments, the metallic carbonate comprisesWSGR Docket No. 69144-701601 amorphous metallic carbonate. In some embodiments, the metallic carbonate comprises at most 10%, at most 20%, at most 30%, at most 40%, or at most 50% amorphous metallic carbonate.Culture Medium

[0109] Another aspect of the disclosure relates to a culture medium for maintaining or growing a cyanobacteria cell culture. In some embodiments, the culture medium can facilitate precipitation of metallic carbonate using one or more cyanobacteria.

[0110] In some embodiments, the culture medium has an alkaline pH (pH>7). In some embodiments, the culture medium has a pH of at least 7.25, at least 7.5, at least 7.75, at least 8, at least 8.25, at least 8.5, at least 8.75, at least 9, at least 9.25, at least 9.5, at least 9.75, at least 10, at least 10.25, at least 10.5, at least 10.75, or at least 11. In some embodiments, the culture medium has a pH of at least 7.25. In some embodiments, the culture medium has a pH of at least 8. In some embodiments, the culture medium comprises a buffer. In some embodiments, the buffer comprises a carbonate, a bicarbonate, a phosphate, or a sulfate.[OHl] In some embodiments, the culture medium has high concentrations of at least one salt (e.g., high salinity). The salinity of a solution can refer to the concentration of the at least one salt provided herein. In some embodiments, the concentration can comprise percentage concentration, wherein the percentage concentration can comprise weight to volume percentage (w / v) or weight to weight percentage (w / v). In some embodiments, the weight to volume percentage (w / v) can be calculated as (grams of solute per liter of solution) / (1000 ml of solution) x 100. For example, normal saline comprises 9 grams of NaCl per liter solution, which is 0.9% (w / v). In some embodiments, the culture medium comprises a hypertonic saline (or hypersaline) with a concentration of at least 1% (w / v), at least 1.5% (w / v), at least 2% (w / v), at least 2.5% (w / v), at least 3% (w / v), at least 3.5% (w / v), at least 4% (w / v), at least 4.5% (w / v), at least 5% (w / v), at least 5.5% (w / v), at least 6% (w / v), at least 6.5% (w / v), at least 7% (w / v), at least 7.5% (w / v), at least 8% (w / v), at least 8.5% (w / v), at least 9% (w / v), at least 9.5% (w / v), at least 10% (w / v), at least 10.5% (w / v), at least 11% (w / v), at least 11.5% (w / v), at least 12% (w / v), at least 12.5% (w / v), at least 13% (w / v), at least 13.5% (w / v), at least 14% (w / v), at least 14.5% (w / v), at least 15% (w / v), at least 15.5% (w / v), at least 16% (w / v), at least 16.5% (w / v), at least 17% (w / v), at least 17.5% (w / v), at least 18% (w / v), at least 18.5% (w / v), at least 19% (w / v), at least 20% (w / v), at least 25% (w / v), at least 30% (w / v), at least 35% (w / v), at least 40% (w / v), at least 45% (w / v), or at least 50% (w / v) of the at least one salt. Example compositions of high salinity culture media comprising various salts are provided in Example 4, Example 6, and Example 7.WSGR Docket No. 69144-701601

[0112] In some embodiments, the at least one salt comprises a calcium, a potassium, a magnesium, a sodium, a lithium or iron. In some embodiments, the at least one salt comprises sodium chloride (NaCl), potassium chloride (KC1), calcium carbonate (CaCCh), magnesium sulfate (MgSCh), sodium bicarbonate (NaHCCh), potassium nitrate (KNCh), calcium sulfate (CaSO4), sodium carbonate (Na2COs), ammonium nitrate (NH4NO3), copper sulfate (CuSO4), aluminum sulfate (Ah(SO4)3), calcium chloride (CaCh), magnesium chloride (MgCh), sodium sulfate (Na2SO4), lithium carbonate (U2CO3), or iron(III) chloride (FeCh).

[0113] In some embodiments, the at least one salt comprises a sodium salt. In some embodiments, the sodium salt comprises sodium chloride (NaCl), sodium bicarbonate (NaHCOs), sodium carbonate (Na2COs), sodium sulfate (Na2SC>4), sodium nitrate (NaNOs), sodium nitrite (NaNCh), sodium hydroxide (NaOH), sodium phosphate (NasPCh), sodium citrate (NasCkHsO?), sodium chlorite (NaCICh).

[0114] In some cases, sodium bicarbonate can be added to the culture medium. In some embodiments, sodium bicarbonate is added to the culture medium at at least 600 ppm, at least 700 ppm, at least 800 ppm, at least 900 ppm, at least 1000 ppm, at least 1100 ppm, at least 1200 ppm, at least 1300 ppm, at least 1400 ppm, at least 1500 ppm, at least 1600 ppm, at least 1700 ppm, at least 1800 ppm, at least 1900 ppm, or at least 2000 ppm. The addition of sodium bicarbonate can increase the pH of the culture medium, increase biomass of the cyanobacteria in the culture medium, and / or increase the yield of precipitated calcite from the cyanobacteria. In some cases, the addition of sodium bicarbonate can increase the yield of precipitated calcite from the cyanobacteria in the culture medium by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, or at least 15-fold. See Examples 5-6

[0115] In some cases, a nitrogen source can be added to the culture medium. In some cases, the nitrogen source comprises nitrates, ammonium, or urea. In some cases, the addition of the nitrogen source into the culture medium can increase the growth of the cyanobacteria in the culture medium, thereby increasing yield of the precipitated calcite. See Example 8.

[0116] In some cases, an iron source can be added to the culture medium. In some cases, the iron source comprises Iron(II) (e.g., FeSCN) or iron(III) (e.g., FeCh). In some cases, the addition of the iron source into the culture medium can increase the growth of the cyanobacteria in the culture medium, thereby increasing yield of the precipitated calcite. See Example 8.

[0117] In some embodiments, the culture medium comprises at least 1% (w / v), at least 1.5% (w / v), at least 2% (w / v), at least 2.5% (w / v), at least 3% (w / v), at least 3.5% (w / v), at least 4%WSGR Docket No. 69144-701601(w / v), at least 4.5% (w / v), at least 5% (w / v), at least 5.5% (w / v), at least 6% (w / v), at least 6.5% (w / v), at least 7% (w / v), at least 7.5% (w / v), at least 8% (w / v), at least 8.5% (w / v), at least 9% (w / v), at least 9.5% (w / v), at least 10% (w / v), at least 11% (w / v), at least 12% (w / v), at least 13% (w / v), at least 14% (w / v), at least 15% (w / v), at least 16% (w / v), at least 17% (w / v), at least 18% (w / v), at least 19% (w / v), at least 20% (w / v), at least 25% (w / v), at least 30% (w / v), at least 35% (w / v), at least 40% (w / v), at least 45% (w / v), or at least 50% (w / v) of a NaCl.

[0118] In some embodiments, the at least one salt comprises a calcium salt. In some embodiments, the calcium salt comprises calcium chloride (CaCh), calcium carbonate (CaCCh), calcium sulfate (CaSO4), calcium nitrate (Ca(NOs)2), calcium phosphate (Cas PO^), calcium hydroxide (Ca(OH)2), calcium acetate (Ca^HsCh^), calcium citrate (Cas^eHsO?^), calcium gluconate (CnILoCaOio), and calcium fluoride (CaF2). In some embodiments, the culture medium contains a high amount of CaCh. In some embodiments, the culture medium comprises at least 5% (w / v), at least 5.5% (w / v), at least 6% (w / v), at least 6.5% (w / v), at least 7% (w / v), at least 7.5% (w / v), at least 8% (w / v), at least 8.5% (w / v), at least 9% (w / v), at least 9.5% (w / v), at least 10% (w / v), at least 10.5% (w / v), at least 11% (w / v), at least 11.5% (w / v), at least 12% (w / v), at least 12.5% (w / v), at least 13% (w / v), at least 13.5% (w / v), at least 14% (w / v), at least 14.5% (w / v), at least 15% (w / v), at least 15.5% (w / v), at least 16% (w / v), at least 16.5% (w / v), at least 17% (w / v), at least 17.5% (w / v), at least 18% (w / v), at least 18.5% (w / v), at least 19% (w / v), at least 20% (w / v), at least 25% (w / v), at least 30% (w / v), at least 35% (w / v), at least 40% (w / v), at least 45% (w / v), or at least 50% (w / v) CaCh.

[0119] In some embodiments, the at least one salt comprises a lithium salt. In some embodiments, the lithium salt comprises lithium carbonate (L COs), lithium hydroxide (LiOH), lithium chloride (LiCl), lithium bromide (LiBr), lithium fluoride (LiF), lithium phosphate (LisPO^, lithium sulfate (Li2SO4), lithium nitrate (LiNCh), lithium acetate (LiC2H3O2), and lithium tetraborate (L EhO?). In some embodiments, the culture medium contains a high amount of Li2CO3 or LiOH. In some embodiments, the culture medium comprises at least 5% (w / v), at least 5.5% (w / v), at least 6% (w / v), at least 6.5% (w / v), at least 7% (w / v), at least 7.5% (w / v), at least 8% (w / v), at least 8.5% (w / v), at least 9% (w / v), at least 9.5% (w / v), at least 10% (w / v), at least 10.5% (w / v), at least 11% (w / v), at least 11.5% (w / v), at least 12% (w / v), at least 12.5% (w / v), at least 13% (w / v), at least 13.5% (w / v), at least 14% (w / v), at least 14.5% (w / v), at least 15% (w / v), at least 15.5% (w / v), at least 16% (w / v), at least 16.5% (w / v), at least 17% (w / v), at least 17.5% (w / v), at least 18% (w / v), at least 18.5% (w / v),WSGR Docket No. 69144-701601 at least 19% (w / v), at least 20% (w / v), at least 25% (w / v), at least 30% (w / v), at least 35% (w / v), at least 40% (w / v), at least 45% (w / v), or at least 50% (w / v) Li2COs or LiOH.

[0120] In some embodiments, the at least one salt comprises a bicarbonate, chloride, sulfate, nitrate, carbonate, phosphate, acetate, hydroxide, fluoride, bromide or iodide. In some embodiments, the at least one salt comprises a bicarbonate (HCO3 ). In some embodiments, the culture medium comprises a high level of bicarbonate. In some embodiments, the culture medium comprises at least 5% (w / v), at least 5.5% (w / v), at least 6% (w / v), at least 6.5% (w / v), at least 7% (w / v), at least 7.5% (w / v), at least 8% (w / v), at least 8.5% (w / v), at least 9% (w / v), at least 9.5% (w / v), at least 10% (w / v), at least 10.5% (w / v), at least 11% (w / v), at least 11.5% (w / v), at least 12% (w / v), at least 12.5% (w / v), at least 13% (w / v), at least 13.5% (w / v), at least 14% (w / v), at least 14.5% (w / v), at least 15% (w / v), at least 15.5% (w / v), at least 16% (w / v), at least 16.5% (w / v), at least 17% (w / v), at least 17.5% (w / v), at least 18% (w / v), at least 18.5% (w / v), at least 19% (w / v), at least 20% (w / v), at least 25% (w / v), at least 30% (w / v), at least 35% (w / v), at least 40% (w / v), at least 45% (w / v), or at least 50% (w / v) HCOf.

[0121] In some embodiments, the culture medium can comprise brines obtained from industrial waste. For example, the brines provided herein can comprise one or more desalination brines, which are highly concentrated saltwater byproducts generated during the desalination process. In some embodiments, the brines can comprise seawater. In some instances, the brines can comprise wastewater from the lithium battery industry. In some cases, the brines can comprise produced water (PW), which is a high salinity byproduct from oil and gas industry. In some cases, the PW can comprise high levels of dissolved Ca2+(e.g., about 26.000 ppm) and / or bicarbonate (e.g., about 200 ppm). In some cases, the PW can further comprise Na+, Cl’, Mg2+, Fe3+, sulfate. Example compositions of seawater and various brines are provided in Example 4 and Examples 6-7.

[0122] In some embodiments, the culture medium provided herein comprises components for growth and propagation of the cyanobacteria provided herein. In some embodiments, the culture medium can comprise macronutrients and / or micronutrients. In some embodiments, the macronutrients comprise a nitrogen source, a carbon source, a phosphorus source, a potassium source, a calcium source, or a magnesium source. In some embodiments, the micronutrients comprise iron, manganese, zinc, copper, boron, various vitamins, various growth factors, or trace elements. In some embodiments, various components of the culture medium provided herein can be optimized to increase the yield, productivity and titer of the cyanobacteria. In some embodiments, the culture medium comprises from 10 mM to 70 mM EEBCh, from 5 mM to 15 mM MnCE, from 0.5 mM to 1 mM ZnSO4, from 1 mM to 2.5 mM Na2MoO4, from 0.1WSGR Docket No. 69144-701601 mM to 0.5 mM CuSO4, or from 0.1 mM to 0.3 mM Co(NOs)2. Examples of various culture media compositions are provided in Example 4 and Examples 5-9 In some embodiments, the culture medium provided herein has an improved productivity, titer, and yield of cyanobacteria compared to other culture media. In some embodiments, the culture medium provided herein has an improved yield of precipitated metallic carbonate compared to other culture media.Bioreactor

[0123] Another aspect of the present disclosure relates to a bioreactor for precipitating metallic carbonate using one or more cyanobacteria. In some embodiments, the bioreactor comprises the composition or the culture medium provided herein. In some embodiments, the bioreactor further comprises one or more cyanobacteria.

[0124] The bioreactor provided herein can comprise a component selected from a group consisting of: a reaction vessel, an agitator for mixing contents, a sparger to introduce gases, one or more sensors to monitor parameters (e.g., temperature, pH), a control system (e.g., regulator), a heating / cooling jacket, inlet / outlet ports for adding or removing materials, one or more pumps, a sterilization system, and a sampling port for extracting samples. In some embodiments, the bioreactor comprises one or more sensors to monitor a temperature, pH, dissolved inorganic carbon (DIC), or calcium concentration of the cyanobacteria culture media used in the methods or systems provided herein. FIG. IB and FIG. 8 (top panel) depict an exemplary bioreactor provided herein. In some embodiments, the bioreactor has a collection plate at the bottom of the bioreactor for collecting the precipitated metallic carbonate.

[0125] In some embodiments, the bioreactor has the capacity to house at least 500 ml, at least 1 liter, at least 1.5 liters, at least 2 liters, at least 2.5 liters, at least 3 liters, at least 3.5 liters, at least 4 liters, at least 4.5 liters, at least 5 liters, at least 5.5 liters, at least 6 liters, at least 6.5 liters, at least 7 liters, at least 7.5 liters, at least 8 liters, at least 8.5 liters, at least 9 liters, at least 9.5 liters, or at least 10 liters of the culture media provided herein.

[0126] In some embodiments, the bioreactor provided herein can comprise a closed bioreactor (e.g., close bioreactor). In some embodiments, the bioreactor herein comprises an open bioreactor (e.g., continuous bioreactor). In some embodiments, the open bioreactor can comprise an open culture tank or a pond. In some embodiments, the pond comprises a raceway pond, a shallow pond, an evaporation pond, or a photobioreactor pond.System

[0127] Another aspect of the present disclosure relates to a system for precipitating metallic carbonate using one or more cyanobacteria. In some embodiments, the system comprises a closed system. In some embodiments, the closed system comprises a bioreactor, an incubator,WSGR Docket No. 69144-701601 or a culturing tank. In some embodiments, the system comprises an open system. In some embodiments, the open system comprises a bioreactor, an incubator, a culturing tank, or a pond for cyanobacteria cultivation. In some embodiments, the pond comprises a raceway pond, a shallow pond, an evaporation pond, or a photobioreactor pond. In some embodiments, the open system provided herein exchanges matter and / or energy with its environment during operation. In some embodiments, the open system allows the cyanobacteria to utilize resources outside the system, thereby reducing the need for external nutrient supplementation. In some embodiments, the closed system provided herein does not exchange matter with its environment while operating. In some embodiments, the closed system enables an in vitro growth environment that minimizes or avoids exposing the cyanobacteria herein to potential contamination.

[0128] In some embodiments, the system further comprises components involved in supporting growth, maintenance, and / or experimentation of cells (e.g., cyanobacteria). In some embodiments, the system further comprises cell culture media, incubators, flasks, plates, pipettes, dispensers, stirring and agitating systems, sensors, filtration systems, pumps, sterilization equipment, cryopreservation equipment, imaging and monitoring devices, control systems, data logging / analysis systems, or any combination thereof.

[0129] In some embodiments, the system has the capacity to house at least 500 ml, at least 1 liter, at least 1.5 liters, at least 2 liters, at least 2.5 liters, at least 3 liters, at least 3.5 liters, at least 4 liters, at least 4.5 liters, at least 5 liters, at least 5.5 liters, at least 6 liters, at least 6.5 liters, at least 7 liters, at least 7.5 liters, at least 8 liters, at least 8.5 liters, at least 9 liters, at least 9.5 liters, or at least 10 liters of the culture media provided herein. In some embodiments, the open system provided herein has the capacity for at least 1 gallon, at least 5 gallons, at least 10 gallons, at least 15 gallons, at least 20 gallons, at least 25 gallons, or at least 30 gallons of the culture media provided herein. In some embodiments, the open system provided herein has the capacity for at least 1 cubic kilometer (km3), at least 5 km3, at least 10 km3, at least 15 km3, at least 20 km3, at least 25 km3, or at least 30 km3of the culture media provided herein.Methods

[0130] Another aspect of the present disclosure relates to methods for precipitating metallic carbonate using one or more cyanobacteria. The methods are described in various sections herein, for example in the EXAMPLES. In some embodiments, the methods comprise using the one or more cyanobacteria that secretes exopolymeric substances (for example, extracellular polymeric substance (EPS)). In some embodiments, the methods comprise using the one or more cyanobacteria that produce the proteinaceous surface layer (S-layer) on outerWSGR Docket No. 69144-701601 membrane. In some embodiments, the methods comprise precipitating metallic carbonate onto the EPS or S-layer.

[0131] The methods herein relate to manufacturing of precipitated metallic carbonates (e.g., CaCCh) using one or more cyanobacteria (e.g., Rivularia halophild), and provide several advantages. For example, the methods herein are carbon-negative and yield precipitated metallic carbonate of high purity and has zero to minimum impurities.

[0132] In some embodiments, the methods comprise using the culture media, bioreactor or system provided herein to grow the cyanobacteria herein, thereby generating the precipitated metallic carbonate. In some embodiments, the methods comprise injecting CO2 into the culture media, bioreactor or system provided herein to grow the cyanobacteria herein, thereby generating the precipitated metallic carbonate. In some embodiments, the methods are carbonnegative. In some embodiments, the methods use less energy than existing methods of manufacturing metallic carbonate. In some embodiments, the methods reduce industrial waste.

[0133] In some embodiments, the methods provided herein can remove atmospheric CO2. In some embodiments, the methods can remove at least 0.22 grams of CO2 from an atmosphere for every gram of precipitated metallic carbonate produced. In some embodiments, the methods can remove at least 0.22 g, at least 0.25 g, at least 0.30 g, at least 0.35 g, at least 0.40 g, or at least 0.45 g of CO2 from an atmosphere for every gram of precipitated metallic carbonate produced. In some embodiments, the methods can remove up to 0.44 grams of CO2 from an atmosphere for every gram of precipitated metallic carbonate produced.

[0134] In some embodiments, the methods herein further comprise analyzing the biogenic signature of the precipitated metallic carbonate using cyanobacteria. In some embodiments, the methods comprise performing X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, scanning electron microscopy (SEM), or energy-dispersive X-ray spectroscopy (EDS), thereby analyzing the precipitated metallic carbonate herein.

[0135] In some embodiments, the methods provided herein further comprises separating the precipitated metallic carbonate from the cyanobacteria and / or the culture media. In some embodiments, methods can comprise separating the precipitated metallic carbonate from the cyanobacteria and / or culture media chemically (e.g., hydrogen peroxide). In some embodiments, methods can comprise separating the precipitated metallic carbonate from the cyanobacteria and / or culture media physically. In some embodiments, separating physically comprises separating by centrifugation, flocculation or membrane filtration. Methods of separating the precipitated metallic carbonate from the cyanobacteria are provided in ExampleWSGR Docket No. 69144-7016014. In some embodiments, the method comprises further purification of the precipitated metallic carbonate after separating the precipitated metallic carbonate from the cyanobacteria.EXAMPLES

[0136] The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.Example 1: CaCCh biomineralization by the benthic-extremophile cyanobacteria

[0137] 1. Introduction

[0138] Laguna Negra (LN, Puna region, Catamarca, Argentina) is a shallow hypersaline lake with high evaporation rates, elevated dissolved inorganic carbon concentrations, and enrichment in ions such as Ca2+, Na+, and CP. This results in mineral precipitation within an extensive microbial mat system. Rivularia halophila PUNA-NP3 (R halophila) is a cyanobacteria isolated from LN from a black mucilaginous microbial mat in close relation with CaCCh crust and microbialites. Hence, in this study, the capacity of R. halophila to precipitate CaCCri was evaluated. To achieve this, cell surface properties of both living and dead R halophila cyanobacteria were analyzed to determine their ability to bind Ca2+ions. The biogenic and abiotic CaCCri minerals obtained were analyzed using electron scanning microscopy (SEM), confocal laser scanning microscopy (CLSM), X-ray diffraction (XRD), and p -Fourier transform infrared spectroscopy (p-FTIR). Additionally, X-ray photoelectron spectroscopy (XPS) was employed to identify characteristic signals of biogenic carbon in the calcium carbonate formed. The findings highlight the ability of the extremophile cyanobacterium R. halophila to induce CaCCri precipitation through photosynthesis, demonstrating significant potential for use as a new material in processes aimed at reducing carbon footprints.

[0139] Additionally, the calcium carbonate precipitated from cyanobacterial has high purity. The cyanobacterium R. halophila are naturally exposed to Arsenic (As) and has the ability to tolerate, accumulate and biotransform this metalloid. Thus the amount of contaminants in the calcium carbonate precipitated from R. halophila, such as arsenic, can be very low. FIG. 1A depicts the various biogenic processes that cyanobacteria can perform, including biomineralization of CaCCh, redox reactions, and arsenic transformation. R. halophila generates favorable conditions for the precipitation of CaCCh, such as an increase in the pH due to photosynthetic activity, but it also favors the nucleation of CaCCh on its surface, sinceWSGR Docket No. 69144-701601 cyanobacteria have organic functional groups that give them a net negative surface charge that facilitates the accumulation of Ca2+on its surface.

[0140] 2. Materials and methods

[0141] 2.1. Cyanobacteria cultivation and growth

[0142] Non-axenic cultures of the cyanobacteria R. halophila were used in this study. R. halophila has been previously described in the work of Shalygin (2018) and culture specifications were also described in the previous study of Soto (2023). Briefly, cyanobacterial cultures were grown in BG11 medium with 3.6 g L'1of NaCl (pH 8) (BG11 -saline), under 12: 12 hours light: dark regimen (30 pEm'1) at 25 °C. Cyanobacterial cultures used for the experiments were mature, healthy at the end of the exponential phase reaching the stationary phase and cyanobacteria biomass concentration was determined by wet weight (mg / L) estimation (Soto Rueda et al., 2023). Cultures were harvested and separated on live colonies in BGl l-saline (LC) and inactivated cyanobacterial colonies or dead cells (DC). DC were obtained by exposure to high temperature and pressure treatment (120 °C and 1.5 bar). This method is widely used for inactivating cells, keeping the surfaces physically and chemically intact. All the experiments were performed by triplicates and incubated with the corresponding solutions for 48 h at 25°C.

[0143] 2.2. Zeta potential measurements of R. halophila and calcium uptake

[0144] The potential of Ca2+uptake in the surface / biomass of R halophila, along with the net surface charge (i.e.: zeta potential, Q of the cyanobacteria was evaluated. First, the zeta potential measurements were conducted with an initial cyanobacterial biomass of 10 mg / L of LC and DC in BGl l-saline solution, in a variable pH range (3-12) and with a stable ionic strength (0.125) using a Delsa™Nano Zeta (Beckman Coulter). On the other hand, calcium (Ca2+) adsorption / incorporation experiments were carried out on LC and DC (50 mg / L), at constant pH (~8) in modified BG11 -saline, under five different concentrations (2, 5, 10, 15 and 20 mM) of CaCh, as Ca2+source. After 48 hours, LC and DC colonies were centrifuged (at 3000 ref) and the corresponding supernatant was separated for Ca2+measurement with a flame atomic absorption spectroscopy (Perkin Elmer AAnalyst 400). Additional blank treatments were conducted with the same initial Ca2+concentration in modified BG11 -saline, but without the addition of live or dead cyanobacterial biomass (WC; abiotic control).

[0145] 2.3. Calcium carbonate precipitation

[0146] CaCCL experiments were conducted with ~1.7g (fresh weight) of LC and DC cyanobacteria biomass of R halophila, using a 1 L biological bioreactor (Winpact EVO) in modified BGl l-saline medium for 48 h. The pH was set around ~8.9. Three differentWSGR Docket No. 69144-791691 concentrations of CaCh, as a source of Ca2+(5, 10 and 15 mM), were tested with 10 mM NaHCCh (HCCU source). WC was used as abiotic control. All solutions were checked and performed using the MINTEQ program (Allison et al., 1991) (See Table 4.)

[0147] 2.4. Mineral analysis

[0148] 2.4.1. Optical and electron scanning microscopy (SEM). Minerals shaped under different experimental conditions (e.g., different Ca2+concentrations) were isolated following the methodology described in FIG. 8. A first visualization of CaCOs minerals associated with R. halophila filaments was performed using a Leica DM 4500 P LED polarized light microscope. In addition, minerals associated with LC, DC and WC were also analyzed using a ZEISS Sigma 300 scanning electron microscope, equipped with a field emission gun and operating at 2 kV accelerating voltage, at the LAMARX laboratory (Universidad Nacional de Cordoba, Argentina). The samples were metallized with gold / palladium in a 70 / 30 ratio respectively. The SEM was equipped with an EDXS spectrometer (X flash Quad detector, Brucker) (LAMARX, UNC, Argentina) in order to perform elemental semi-quantification. Images were collected using secondary electron detectors.

[0149] 2.4.2. X-ray diffraction (XRD) and p -Fourier transform infrared (p-FTIR). XRD was used to identify the polymorph of CaCCL formed at 10 and 15 mM Ca2+concentration. Powder XRD (PXRD) patterns of the carbonate precipitates were collected from 10-60 29, using a PANalytical X’Pert PRO diffractometer with Cu-Ka-radiation at INFIQC-CONICET, Universidad Nacional de Cordoba, Argentina. The normal-focus Cu X-ray tube was operated at 40 kV and 40 mA and using Cu-Ka radiation (step size: 0.026 29, 34.425 s count time per step). The mineralogical identification was performed using the X'Pert HighScore software package by PANalytical. For 5mM of Ca2+concentration, p-FTIR was used for mineralogical analysis, since it was not possible to obtain abundant quantities of minerals for XRD analysis. For this, a Thermo Scientific iN10 MX microscope (Ever-Glo™ conventional infrared source) was used (IPQA-CONICET, Universidad Nacional de Cordoba, Argentina). The cooled mercury cadmium telluride detector (MCT-LN2) facilitated the analysis of particles with a precision down to 5 to 19 pm in size. Spectra were taken by reflection on a silver mirror sample holder, where a volume of 5 pL per sample was placed. Subsequently, the sample was allowed to dry at room temperature and then the analysis of the minerals associated with cyanobacteria were carried out in a range of 499 to 4999 cm’1. Background spectra were acquired between each spectrum under the same analytical conditions. Data was processed using OMNIC™ software (Thermo Fisher Scientific).WSGR Docket No. 69144-701601

[0150] 2.4.3. X-ray photoelectron spectroscopy. X-ray photoelectron spectroscopy (XPS) analysis was used to identify the presence of C signal related to R. halophila. The analyses were performed using an X-ray photoelectron spectrometer (K-Alpha+ XPS spectrometer, Thermo Fisher Scientific) at the LAMARX facility (Universidad Nacional de Cordoba, Argentina). XPS data were collected using an electron flood gun during spectral acquisition and three spectra of the same element, with no evidence of beam damage, were collected. The spectrum of the C Is was monitored from the beginning to the end of the measurements to determine the extent of the charging effect. The binding energies (BE) of the photoelectrons were referenced to the C Is contaminant peak at 284.8 eV (Biesinger, 2022). The C is spectra were fitted using the software of XPSPeak 4.1 (http: / / www.uksaf.Org / software.html#l) with peaks of Gaussian-Lorentzian mixed function using Shirley background correction.

[0151] 2.5. Statistical analysis. All data were representative of at least three independent experimental samples and values were given as Mean ± Standard Error of the Mean. Statistical analysis was carried out using a two-way or one-way ANOVA test, followed by post-Aoc Duncan's analysis to enable specific group comparison. The level of significance was set at a=0.05 for all tests. All analyses were performed using Statistic 7 (StatSoft, Inc.; OK, USA) and R package (Core, version 3.6.3).

[0152] 3. Results

[0153] 3.1. Net surface charge of Rivularia halophila and Ca2+uptake.

[0154] The surface of bacteria is a very reactive interface due to the presence of carboxyl, hydroxyl, phosphoryl and amine groups. These surface functional groups deprotonate with increasing pH and thus microorganisms have a net negative surface charge. However, for a number of bacteria (including cyanobacteria), the cell wall is not the outermost surface. Microorganisms can generate an extracellular network of polysaccharides and proteins (e.g., exopolymers) which acts as reactive sites capable of protecting the cell proper against detrimental metal uptake or biomineralization. In particular, it was reported that cyanobacteria also have an outermost surface layer, called S-layer. Chemical analysis revealed that they are protein or glycoprotein subunits forming a monomer arrangement that provides nucleation sites for mineral growth. Mineral formation occurs when calcium ions bind to negatively charged sites of the S-layer proteins.

[0155] FIG. 2A (left panel) shows the zeta potential measurements of cyanobacterial suspensions of LC and DC at different pH values. Both groups behave similarly. For the entire pH range evaluated, the C, was always negative indicating that the net surface charge was negative. Even though both experimental groups displayed a similar C, potential pattern, aWSGR Docket No. 69144-701601 significant difference was observed between LC and DC (F(8, 36)=2.7, p<0.05; Figure la) at pH 6 and 9. Between these pH, the net surface charge was negative and almost constant; however, for pH <5 the C, slightly decreased to less negative values, probably due to the protonation of the surface groups (eg.: carboxyl, amine groups). Interestingly, LC behaves differently at alkaline pH. The C, is even less negative since pH 10 (-23 to -12 mV) in comparison with DC. These changes in the potential values at alkaline pH may be a consequence of the bacteria metabolic. Martinez (2008) found a similar decrease in the potential values for suspensions of active cyanobacteria (Planktothrix sp. and Synechococcus sp.) which was explained as a movement of H+ across the membrane to the outside of membrane during metabolism that explain the decrease in the negative surface charge (Urritia- Mera et al., 1992).

[0156] Likewise, experiments on Ca2+uptake showed that cyanobacteria can accumulate and / or adsorb calcium in both LC and DC (FIG. 2A, right panel). Although the process seems to be far from equilibrium, the analysis showed that the cyanobacterial can retain Ca2+in a similar way for LC and DC. Although it is expected that Ca2+adsorb onto the negatively charged sites of R. halophila, the incorporation in the biomass cannot be completely ruled out. In fact, the ANOVA did not show significant differences between LC and DC. However, a tendency was detected for LC to a more uptake capacity (p=0.11), particularly at 5 mM of Ca2+, as a significant difference was detected by Duncan post-hoc test (p < 0.05). These results demonstrated the ability of R. halophila to uptake cations that may potentially act as nucleation sites for calcium carbonate precipitation.

[0157] 3.2. CaCCL mineral precipitation

[0158] Calcium carbonate precipitation was evidenced by microscopic examination in all of the Ca+2doses tested. The presence of CaCCL associated to the cyanobacterial filaments was observed in LC and DC, with a clear tendency of a higher abundance of minerals in LC compared to DC [F(l, 22)=17.546; p<0.01)] and at higher calcium doses [F(2, 22)=21.025; p<0.01)]. A significant difference between LC and DC was detected under 15 mM of Ca+2(p< 0.05) (FIG. 2B). Microscopy observation showed that the minerals formed under 5 mM of Ca+2exhibit a granular texture with no clear shape, as it can be observed at the highest doses in DC experiments (see FIG. 9A, (vi)). Besides, treatment with 5 mM of Ca+2did not show mineral formation without the presence of cyanobacteria (WC). Interestingly, incipient CaCCL minerals were detected using optical polarized microscopy (petrographic microscope), within the trichomes of the cyanobacterial filaments (see FIG. 9B). Moreover, CLSM confirmed the accumulation of calcium signals (potential nucleation points) within the cells surface of theWSGR Docket No. 69144-701601 cyanobacteria, which is closely related to the formation of emerging minerals and the concomitant growth of these minerals (FIG. 10).

[0159] 3.3. Identification and characterization of calcium carbonate minerals.

[0160] The SEM-EDS images of calcite formed in LC, DC and WC under different Ca+2doses are shown in FIG. 3 and FIGS. 11A-11B. Although calcium carbonate precipitation was identified in all the treatments, the shape and size of the CaCCE crystals were different depending on whether they were associated with the cyanobacterial filaments or located at the bottom of the bioreactor glass. WC treatment produced typical euhedral mostly rhombohedral crystals of around 17 to 36 pm long, frequently associated into clusters in all the Ca+2doses tested, except for 5mM of Ca+2(FIG. 11 A, top). On the other hand, in the presence of cyanobacteria, more spherical, rounded-shape amorphous carbonate minerals were observed (FIG. 3). In addition, a clear difference between minerals formed under LC and DC conditions was also observed. Under 5 mM of Ca+2, LC showed micritic texture and subhedral porous crystals with rhombohedral shapes, strictly associated with the cyanobacterial filaments. Ovalshape clusters of these porous rhombohedral structures were observed within the filaments (FIG. 3, panels (a) and (b)). On the contrary, DC showed a thin layer of CaCOs covering the sheaths of the cyanobacterial filaments, mixed with organic matter (EPS). No clear crystal grade was observed, indeed resembling a crustification (FIG. 3, panels (c) and (d)), formed by small spherical anhedral nanocrystals. The presence of EPS associated with the CaCOs crust was confirmed by the EDS analysis which demonstrated C content at the surface of these CaCOs crust (FIG. 11B, top). Minerals obtained from a control without cyanobacteria (WC) under conditions including in media with 10 mM Ca2+(panel a) and 15 mM Ca2+(panel b) are depicted in FIG. 11C.

[0161] Minerals formed under 10 mM of Ca+2with LC showed rhombohedral rounded- shapes. Aggregates of porous minerals were observed within and along the cyanobacterial filaments (FIG. 3, panels (e) and (f)). DC showed a mix between euhedral and subhedral crystals with squared shapes. The dispositions were dispersed alone beside the filaments, with a mineral size lower than those corresponding to LC and for the same dose (FIG. 3, panels (g) and (h)).

[0162] Finally, treatments under 15 mM of Ca2+showed conspicuous quantities of mineral clusters along and within the cyanobacterial filaments. Under LC condition, subhedral crystals with rhombohedral shapes were observed. Besides, euhedral crystals with squared shape, resembling minerals formed by chemical precipitation, were also observed (FIG. 3, panels (i)WSGR Docket No. 69144-701601 and (j)). Conversely, under DC condition, the majority of the minerals formed were euhedral crystals with a low porosity (FIG. 3, panels (k) and (1)).

[0163] 3.4. XRD and p-FTIR results

[0164] The XRD patterns of the precipitates formed in LC and DC conditions, at lOmM and 15 mM of Ca2+, identified the presence of calcite (29= 29.35). Interestingly, the slightly raised baseline suggests the concomitant presence of amorphous phases, likely amorphous calcium carbonate (ACC) (FIG. 4). Furthermore, minerals produced in LC and WC conditions disclosed more crystallinity than those obtained from DC, as it was revealed by the stronger intensity of the main peak of calcite. Calcite formed in culture media with LC and DC has lower crystallinity than calcite formed under WC conditions.

[0165] The mineralogical composition under 5 mM of Ca2+was conducted by p-FTIR due to the low quantity of mineral material recovered after the experiment. The p-FTIR spectra acquired were compared with the standard spectrum of synthetic calcite, taking into account the bands reported for CaCCL polymorphs. Carbonates show vibrational absorption bands due to the planar CCL2' ion. There are four vibrational modes in the free carbonate ion; however, the vibrational absorptions vary with the mineral structure and the size of the cation and the different polymorphs result in different spectral features. Thus, the symmetric stretch band (vl) occurs between 1040 to 1105 cm'1; the out-of-plane bend (v2) from 810 to 906 cm'1; the asymmetric stretch (v3) from 1275 to 1590 cm'1and the in-plane bend (v4) from 670 to 756 cm'1. Vagenas (2003) examined the region between 700 to 900 cm'1to quantify and differentiate the three polymorphs of carbonate. The selected absorption bands chosen were the 713 cm'1for calcite, the 700 and 713 cm'1for aragonite, and the 745 cm'1for vaterite. In this work, the same region was analyzed, as depicted in FIG. 5. For LC and DC samples, the presence of calcium carbonate is identified. Additionally, a ternary mixture of cal cite / aragonite and vaterite can be inferred.

[0166] Table 2 Wavenumber (in cm'1) corresponding to the internal modes of carbonate in calcite, aragonite, vaterite and amorphous calcium carbonate (ACC) reported in literature in comparison with LC and DC samples at 5 mM of Ca2+.aSato and Mantsuda (1969);bAndersen and Brecevic (1991);cVagenas et. al (2003).WSGR Docket No. 69144-701601

[0167] 3.5. XPS results

[0168] XPS analysis was performed to elucidate signals of biogenicity in the carbonate mineral formed. This analysis is being conducted for the first time on biogenic carbonate samples. FIG. 6 displays the results obtained in comparison with the results obtained in R. halophila biomass (Soto Rueda et al., 2023). Panel (a) of FIG. 6 shows the R. halophila biomass reported by Soto Rueda et al. (2023). Panel (b) of FIG. 6 shows the R. halophila biomass obtained for calcium carbonates. Solid black lines depict measured spectra; solid red lines indicate fits to measured spectra and dashed lines correspond to fits. A Shirley background is included as a solid curve at base of the peaks. Dashed vertical lines are added to guide the eye and correspond to C species identified in the analyzed samples. Values of BE correspond to the maximum intensity of the fitted peak are reported in Table 3. The deconvolution of the C is signal yields four peaks (Cl, C2, C3, and C4) with binding energies ranging from 284.8 to 289.2 eV (Table 3), resembling a pattern identified in the R. halophila biomass. The similarity of both spectra confirms the biogenicity of the mineral, in line with the results obtained from the SEM-EDS analysis. Interestingly, the relative proportion obtained of C2, C3 and C4 components increases in the calcium carbonate compared with R Rivularia cbiomass.

[0169] Table 3 XPS Cis binding energy (eV) assignments and ratios area (%) of C species for calcium carbonate samples. (*) reported by Soto Rueda et al. (2023).WSGR Docket No. 69144-701601

[0170] 3.6. Arsenic removal results

[0171] In some cases, CaCCh was precipitated using R. halophila cultured in medium with arsenic. To determine the removal of As, different concentrations of this metalloid were used: 25, 70, 150, and 300 pg L'1As(V) and As(III). The As removed from the medium was quantified by inductively coupled plasma-optical emission spectrometry (ICP-OES). The percentage of As removal after carbonates precipitation showed a significant difference in the presence of R. halophila (LC) compared with abiotic precipitation (WC) [F(l, 132)=6.817; p<0.05)] in all the Ca+2doses tested (FIGS. 7B-7D). In this regard, it was observed that under LC condition, As (V) was the species with more percentage of removal under 5mM, lOmM and 15 mM of Ca+2doses [F=(2, 66)=3.925; p<0.05]. In addition, 10 mM and 15 mM of Ca+2uptake a more significant percentage of As(V) than 5 mM of Ca+2, reaching almost 90% of the As removal. R. halophi Za-induced carbonate precipitation demonstrated a higher potential to remove As relative to purely chemical precipitation processes (WC). The presence of R halophila in the carbonate precipitation processes demonstrated that the biological process has a higher potential to remove As from the culture medium compared to the chemical process. This result may be due to the accumulation of this metalloid in both the biomass and the bioprecipitated minerals.

[0172] XPS was also performed here in order to identify the presence and the As-solid speciation in the calcium carbonate. FIG. 7A displays the XPS spectra of the carbonate samples in presence of 300 pg / L of As(III) and As(V) in the medium. In both cases, the As 3d signal is represented by two peaks at ~43 eV and 46-47 eV. Based on reference values (Bia et al., 2015; Soto et al., 2022), peaks at ~ 43 eV correspond to As(III)-S species, while peaks fitted at about 46-47 eV correspond to As(V)-0 species. The peak at ~44 eV could not be assigned to the As(III)-0 signal, as it is attributed to the Ca 3s signal of the carbonate mineral. The identification of the As(III)-S signal is important because it serves as another evidence of theWSGR Docket No. 69144-701601 biogenicity of the mineral. Soto et al. (2022) reported that R halophila is capable of bioreduce As(V) to As(III). The As(III) species then interacts with the sulfhydryl groups (-SH) of proteins, heightened the toxicity and ROS response levels. The discernible As(III)-S signal found in the formed mineral unequivocally indicates that R. halophila first reduces the As(V) species to As(III), and then incorporates within the carbonate mineral structure.

[0173] 4. Discussion

[0174] This study focuses on the significant potential of the photosynthetic cyanobacterium Rivularia halophila for inducing CaCOs biomineralization. This cyanobacterium fosters favorable conditions for carbonate precipitation by increasing the pH in the microenvironment near the cell surface through photosynthetic activity. Furthermore, living cyanobacteria enhance CaCCh nucleation in their extracellular structures due to their negatively charged surfaces, which facilitates the accumulation of Ca2+ions. Biotic experiments conducted in saturated solutions resulted in various types of crystals, suggesting that CaCCh precipitation in these systems may be driven by different mechanisms. Specifically, precipitation in the absence of cells resulted in rhombohedral calcite crystals, typical of abiotic conditions, while cell- induced precipitation produced more irregular shapes, including anhedral and subhedral crystals, granular textures, and porous structures on the nanometer scale. In all cases, the primary mineral was calcite, followed by vaterite and amorphous calcium carbonate (ACC) associated with R. halophila. Notably, calcite minerals exhibited similar C signals by XPS, reported previously for R. halophila, which strongly suggests a biogenic imprint.

[0175] Calcification in cyanobacteria is highly dependent on conditions that favor this process and occurs predominantly in waters supersaturated with respect to CaCCh. However, only certain cyanobacterial genera have the ability to calcify, and none are obligate calcifiers. Various mechanisms for carbonate biomineralization involving cyanobacterial cellular metabolism and surface composition have been proposed. Cyanobacterial EPS are composed of diverse organic acids and sugar monomers, and their external cellular structures contain functional groups such as carboxyl, phosphoryl, amine, and hydroxyl groups. These groups significantly influence the electrostatic properties of the cell surface, promoting the accumulation of ions like Ca2+.

[0176] R. halophila exhibits surface properties similar to those previously proposed for other cyanobacteria (i.e., negative surface charge) that help explain the precipitation of CaCCh near the cell surface. The entire process involves several stages: initially, Ca2+ions in the culture media interact electrostatically with the negatively charged surface groups (and cell exopolysaccharides, which may serve as nucleation sites for Ca2+accumulation). Once bound,WSGR Docket No. 69144-701601Ca2+attracts carbonate ions, a process facilitated by the cyanobacterial metabolism that increases the microenvironmental pH. Indeed, experiments with LC showed an increase in the negative zeta potential, compared to DC at pH 9, indicating the role of photosynthesis in pH elevation and nucleation site formation. This hypothesis was further supported by the observation of mineral clusters at 5 mM of Ca2+in LC, while in DC, ACC coated cyanobacterial filaments and more closely resembled crust formation than nucleation points. Here, EPS might act as simple adsorption templates. Similar results were reported for Synechococcus leopoliensis cells, where negative net surface charges decreased due to inhomogeneous specific adsorption of Ca2+, leading to CaCCL nucleation. Although some Ca2+may be incorporated into cells to meet metabolic needs, cytosolic Ca2+concentrations are tightly regulated by Ca-binding proteins, pumps, and transporters to maintain low intracellular levels. The lack of significant differences in Ca2+adsorption between LC and DC suggests that R. halophila does not internalize Ca2+. Instead, cell surface functional groups exceed saturation in the microenvironment by binding Ca2+and interacting with ambient bicarbonate-carbonate ions.

[0177] An additional biomineralization mechanism, proposed by Douglas and Beveridge (1998), involves the continuous shedding of mineralized S-layers by cyanobacteria, which are rapidly replaced by new material. Although this study cannot quantify this mechanism's contribution to R. halophila biocalcification, the presence of precipitated CaCCL adjacent to and at a micron-scale distance from cell surfaces suggests the plausibility of an exopolysaccharide shedding pathway. Recent studies with other anaerobic and aerobic heterotrophic bacteria have also shown that dead cyanobacteria are coated with calcium carbonate more quickly and extensively than live cells. This observation, alongside the well- documented S-layer shedding, may be linked to the metabolic maintenance of positive surface potential and the protection of live cyanobacterial cells from incrustation.

[0178] SEM-EDXS analysis revealed that most cells were associated with calcium, supporting the hypothesis that calcium carbonate nucleation begins in microenvironments with high ion saturation due to photosynthesis. SEM, CLSM, and polarized microscopy images showed that precipitates formed extracellularly, grew, and aggregated. These results align with studies by Bundeleva (2013) and Zhu (2015) on Gloeocapsa sp. and Synechococcus sp., although in these studies, cells were embedded in minerals over time. The presence of diverse crystal morphologies underscores the role of organic components on cell surfaces in CaCCL biomineralization. XRD analysis showed a shift in the calcite (104) peak (29 = 29.39), absent in calcite samples precipitated without cyanobacteria, a phenomenon previously observed in cyanobacterial-influenced calcite. Additionally, XPS spectra revealed a similar pattern of CWSGR Docket No. 69144-701601 signals described previously for R. halophila (Soto Rueda et al., 2023). The deconvolution process identified typical signs of biogenicity (eg.: the presence of C2, C3 and C4 signals in FIG. 6). In addition, the relative proportion of C2, C3 and C4 increases compared with R. halophila biomass and may indicate a participation of some of these functional groups in the interaction of Ca2+ions with the cell surface, and a further nucleation of CaCCh. All the evidence strongly suggests an active participation off?, halophila in the precipitation of CaCCE.

[0179] Additional Materials and Methods

[0180] 1.1 Confocal laser scanning microscopy (CLSM). R. halophila colonies were fixed with paraformaldehyde 4% P / V at 4°C for 2 h and then washed with phosphate-buffered saline (PBS) solution, pH ~ 7.4. After, 0.1 mg / ml of Calcein (2,4-bis-[N,N'-di(carbomethyl)- aminomethyl]-fluorescein) was added in each treatment condition, at 4 °C for 48 h. Calcein produces a stable fluorescent complex in the presence of calcium ions and fluoresces (in the green region of visible light). After staining, samples were examined at the CEMECO (Universidad Nacional de Cordoba, Argentina) using a FluoViewTM F VI 000 confocal laser scanning microscope (Olympus), with a spectral resolution of 2 nm and a spatial resolution of 0.2 pm. The FluoViewTM 10 FV1000 was equipped with a 405 nm laser diode and multi-line argon (458 nm, 488 nm, and 515 nm), helium-neon-green (543 nm) and helium-neon-red (633 nm) lasers. Fluorescence image were obtained with concomitant excitation at wavelengths of 405 nm, 488 nm and 543 nm, by collecting the emitted fluorescence between 425-475 nm, 500- 530 nm, and 560-660 nm, respectively. For CSLM image acquisitions an oil immersion objective, UPLSAPO 60X0182 (Olympus; 60x magnification, N. A.= 1.35) was used. 3D images were acquired, visualized, and processed using the F10-ASW FLUOVIEW software.

[0181] Table 4. MINTEQ results.aSolution at pH 8.3; SI: Saturation index = log (IAP / K)WSGR Docket No. 69144-701601Example 2: Analysis of the precipitated metallic carbonate generated by cyanobacteria Part 1: Further analysis of CaCCh precipitated at the bottom of the bioreaction using R. halophila

[0182] 1. pH of the media: The Rivularia halophila species maintains a more alkaline environment in the culture medium, which favors the precipitation of CaCCf. The control sample of the medium without cyanobacteria precipitates due to the presence of bicarbonate, but when it decreases, the pH also drops and there is no other agent to maintain the pH. The technology herein, which comprises working with these cyanobacteria, generates favorable conditions for carbonate precipitation and, by having nucleation site such as extracellular polymeric substances (EPS) and structures such as the S-layer, also facilitates the formation of carbonate minerals. The results of various experiments carried out demonstrated greater precipitation in the presence of live cyanobacteria (LC) than in the controls (dead cyanobacteria (DC) and the medium without cyanobacteria (WC)). FIGS. 12A-12B show the changes in pH in culture medium comprising cyanobacteria Rivularia halophila. pH changes at multiple time points over a 45-day period in either a control culture medium lacking bacteria or a culture medium comprising Rivularia halophila are depicted in FIG. 12A. A table outlining exemplary pH values at multiple time points over a 45-day period in either a control culture medium lacking bacteria or a culture medium comprising Rivularia halophila is depicted in FIG. 12B.

[0183] 2. Microscopy data: FIGS. 13A-13B depict crystals outside the cyanobacterial sheath (cellular structure). FIG. 13A depicts images of CaCCh crystals precipitated at the bottom of the bioreactor in experiments with cyanobacteria. FIG. 13B depicts images CaCCh crystals associated with cyanobacterial filaments and EPS.

[0184] 3. Biogenic and bioinduced crystals do not need pulverization or post treatment to reduce size and brake structure like coccolithophores. The calcium carbonate crystals obtained by the bioinduced precipitation process by cyanobacteria do not require a grinding process since their size is already micrometric. FIG. 14A shows sizes of crystals associated with cyanobacteria. FIG. 14B shows sizes of crystals precipitated at the bottom of the bioreactor in experiments with cyanobacteria.

[0185] 4. Images of microcolonies formation proved that easy dewatering was achieved. Dewatering represents a significant challenge in the production and utilization of microalgae, contributing to 13% to 30% of the overall product cost. The use of microorganisms capable of forming macrocolonies ranging from 1 to 5 mm in size offers a potential solution, as these larger aggregates can be easily separated from the culture medium using simple filtration methods. In contrast, most competitors utilize organisms that do not naturally form suchWSGR Docket No. 69144-701601 structures, necessitating high-cost technological interventions to separate their micrometricsized cells. FIG. 15 shows images of R. halophila macrocolonies used in the CaCCh precipitation process.

[0186] 5. Search for weight / quantity of bioinduced vs control vs biogenic. FIG. 16 panel A) depicts the XRD patterns of the samples of the minerals precipitated near the cyanobacteria (LC) and at the bottom of the bioreactor (BBLC) formed in BG11- saline culture medium at two different Ca2+concentrations (10 mM and 15 mM). The red dashed lines indicate the main calcite peak. FIG. 16 panels B) and C) depict XRD patterns showing a detailed view of the main calcite (104) peak (29 = 29.39). B) Calcite precipitated on the cyanobacteria (LC) and C) calcite from the bottom of the bioreactor (BBLC). Calcium carbonate crystals precipitated in experiments with R. halophila in BG11- saline culture medium with 10 mM calcium. FIG. 17 shows large minerals associated with the sheath (EPS) of the cyanobacteria that can be separated by some physical process. In turn, the presence of precipitated minerals at the bottom of the bioreactor was also observed in the same experiment. The total amount of precipitated calcium carbonate was higher in the experiments with cyanobacteria than in the control medium without cyanobacteria.

[0187] 6. Comparing morphology of R. halofila in microbial mat vs isolated in the lab. FIG. 18A shows composite CLSM images of a resin-embedded black pustular microbialite, stained with calcein. Panel (A) of FIG. 18A: Mineral aggregates (Min. and white arrow) are observed in blue. Their surfaces are partially stained with calcein, hence indicating the presence of free Ca2+ion. Inside and surrounding the mineral aggregate, diatom frustules (x) and living diatoms (Diat.) are visible.

[0188] Photosynthetic pigments. Some Rivularia filaments (Riv.) seem to be separated from the mineralized aggregates, while other filaments are close but not entombed in the aggregate and occur with diatoms. Calcein stained the space inside the filaments, i.e., between the sheath and the cells. In FIG. 18A Panel (B) yellow / light green dots are observed in one of the Rivularia filament (*). Calcein also strongly stained the contours of the pigmented cl cocci- shaped cell colonies that were found closely associated with the mineral aggregates (white arrow). FIG. 18B is an optical microscopy image of a Rivularia filament (Riv.) with which numerous microorganisms are associated, hence suggesting the presence of a consortium (Cons.) (dotted circle). A large mineral aggregate is also visible near the filament (red arrow). Sulfur filaments (SF.), diatoms (Diat.) and mineral grains (Min.) can also be observed around the filament. FIG. 18C is an optical microscopy image of a Rivularia with CaCCL crystals over the sheath and no other microorganism associated.WSGR Docket No. 69144-701601Part 2: Analyzing the Biogenic Signature and Purity of Metallic Carbonate

[0189] Provided herein relates to metallic carbonate precipitated by cyanobacteria, featuring a high purity level, low arsenic content, and a biogenic signature. It is recognized that not all crystals formed by the methods provided herein will possess a biogenic signature detectable by X-ray photoelectron spectroscopy (XPS). Specifically, crystals formed due to excess calcium ions or localized pH changes may lack this signature, whereas those precipitated directly on the cyanobacterial surface are more likely to exhibit it. To accurately quantify the proportion of biogenically influenced crystals, a detailed experimental approach is provided herein, including an exemplary experimental setup designed to separate and analyze the different types of calcium carbonate precipitates, with a focus on identifying the biogenic signature.

[0190] Experimental Setup for Separation and Analysis of Metallic Carbonate Precipitates

[0191] Objective: To separate and characterize the different types of calcium carbonate precipitated by Rivularia halophila, with a focus on identifying and quantifying the crystals that exhibit a biogenic signature as detected by X-ray photoelectron spectroscopy (XPS).

[0192] Materials and Equipment

[0193] Cyanobacterial cultures: Pure culture of Rivularia halophila.

[0194] Growth medium: Baseline growth medium supplemented with calcium and bicarbonate.

[0195] Calcium source: CaCh solution, various concentrations (5, 10, 15 mM).

[0196] Bicarbonate source: NaHCCE solution, 10 mM.

[0197] Arsenic source: Trace amounts of arsenic-containing compound (optional, if applicable).

[0198] pH meter: For monitoring and adjusting pH.

[0199] Incubator: With controlled temperature and light conditions.

[0200] Bioreactors: Small-scale bioreactors or flasks with airtight seals.

[0201] Centrifuge: For separating precipitated calcium carbonate.

[0202] Filtration setup: To collect calcium carbonate precipitates.

[0203] Microscope: For observing cyanobacterial cells and calcium carbonate crystals.

[0204] X-ray photoelectron spectroscopy (XPS): For detecting biogenic signatures.

[0205] X-ray diffraction (XRD): For phase identification of calcium carbonate crystals.

[0206] Scanning electron microscopy (SEM): For morphological analysis of crystals.

[0207] Electron dispersive X-ray spectroscopy (EDS): For elemental analysis and arsenic content.

[0208] Experimental DesignWSGR Docket No. 69144-701601

[0209] Step 1 : Cultivation and Precipitation

[0210] Prepare the growth medium by dissolving CaCh and NaHCCh in distilled water. Adjust the pH to approximately 8.5 using dilute NaOH or HC1 solution.

[0211] Inoculate the medium with a standardized concentration of Rivularia halophila (typically 1 g / L).

[0212] Incubate the cultures under controlled light and temperature conditions (e.g., 25- 30°C, 12: 12 hours light: dark cycle).

[0213] Monitor pH regularly to ensure it remains stable around the desired level, promoting calcium carbonate precipitation.

[0214] Step 2: Separation of Precipitated Carbonates

[0215] After a set incubation period (e.g., 48-72 hours), collect samples from each bioreactor.

[0216] Separate the precipitated calcium carbonate by centrifuging the culture at 3000 ref for 10 minutes.

[0217] Filter the supernatant to collect remaining calcium carbonate precipitates.

[0218] Sort the collected precipitates into distinct fractions based on their morphology and attachment to cyanobacterial cells:

[0219] Fraction A: Crystals visibly attached to cyanobacterial filaments.

[0220] Fraction B: Crystals found in the bulk medium, not attached to cells.

[0221] Fraction C: Crystals associated with EPS or other organic material.

[0222] Step 3: Characterization of Carbonate Fractions

[0223] Analyze each fraction using XRD to determine the phase (e.g., calcite, aragonite, vaterite) and assess purity levels.

[0224] Perform SEM and EDS analysis on each fraction to observe crystal morphology and determine elemental composition, including arsenic content.

[0225] Use of XPS to detect biogenic signatures in the crystals. Focus on identifying Cis peaks and other relevant signals that indicate the presence of organic material or cyanobacterial influence and / or focus on identifying Cis, Ca2p peaks and other relevant signals that indicate the presence of organic material or cyanobacterial influence.

[0226] Step 4: Data Analysis and Correlation

[0227] Quantify the proportion of crystals in each fraction that exhibit a biogenic signature.

[0228] Correlate the presence of biogenic signatures with the purity levels and arsenic content of the crystals.

[0229] Determine the relationship between the mode of precipitation (nucleation on cyanobacteria vs. abiotic precipitation) and the presence of biogenic signatures.WSGR Docket No. 69144-701601

[0230] Expected Outcomes

[0231] This experimental setup is expected to identify and quantify the calcium carbonate crystals that possess a biogenic signature, providing empirical support for the patent claim. The results will also clarify the proportion of biogenically influenced crystals in the overall precipitate, helping to refine the understanding of the process and its implications for purity and arsenic content.Example 3: Reduced carbon footprint from precipitating CaCO3using Cyanobacteria

[0232] 1. Carbon Dioxide Fixation Quantification.

[0233] Stoichiometric calculations suggest that for each gram of metallic carbonate produced, at least 0.44 grams of CO2 should be fixed, assuming CO2 is the sole carbon source for photosynthesis and that HCO3‘ is incorporated into the crystals. To confirm these estimates, a Dissolved Inorganic Carbon (DIC) analysis can be performed. This experiment would provide a direct measurement of the carbon content in the precipitated metallic carbonate, offering empirical support for the carbon sequestration potential of the process. The following experimental setup outlines the procedure for performing DIC analysis.

[0234] Experimental Setup: Dissolved Inorganic Carbon (DIC) Analysis

[0235] Objective: To quantify the amount of carbon fixed during the precipitation of calcium carbonate by Rivularia halophila using Dissolved Inorganic Carbon (DIC) analysis.

[0236] Materials and Equipment

[0237] Cyanobacterial cultures: Pure culture of Rivularia halophila.

[0238] Growth medium: Baseline growth medium supplemented with calcium and bicarbonate.

[0239] Calcium source: CaCh solution, various concentrations (5, 10, 15 mM).

[0240] Bicarbonate source: NaHCO3solution, 10 mM and / or CO2 injection that would result in HCO3‘ formation.

[0241] pH meter: For monitoring and adjusting pH.

[0242] Incubator: With controlled temperature and light conditions.

[0243] Bioreactors: Small-scale bioreactors or flasks with airtight seals.

[0244] DIC analyzer: The DIC will be measured by assessing alkalinity and pH values (Beeler et al., 2020. Controls of extreme isotopic enrichment in modern microbialites and associated abiogenic carbonates. Geochimica et Cosmochimica Acta). Additionally, an approximation will be made by acidifying the samples, which causes CO32- and HCO3‘ to deprotonate and convert into CO2, which is then measured. DIC will be calculated as the sum of CO2, CO32-, and HCO3’. DIC= CO2+CO3-2 and HCO3’.WSGR Docket No. 69144-701601

[0245] Centrifuge: For separating precipitated calcium carbonate.

[0246] Filtration setup: To collect calcium carbonate precipitates.

[0247] Analytical balance: For weighing precipitates and final biomass.

[0248] Microscope: For observing cyanobacterial cells and calcium carbonate crystals.

[0249] Experimental Design

[0250] Preparation of Growth Media: Growth medium preparation by dissolving appropriate amounts of CaCh and NaHCCh in distilled water. Adjust the pH to approximately 8.5 using a dilute NaOH or HC1 solution.

[0251] Medium inoculation with a standardized concentration of Rivularia halophila (typically 1 g / L).

[0252] Inoculation and Incubation: Bioreactor Inoculation with the prepared culture medium containing R. halophila. Cultures Incubation under controlled light and temperature conditions (e.g., 25-30°C, 12: 12 hours light: dark cycle). pH Monitoring regularly to ensure it remains stable around the desired level.

[0253] Calcium Carbonate Precipitation: After a set incubation period (e.g., 48 hours), collect samples from each bioreactor. Calcium Carbonate precipitated separation by centrifuging the culture at 3000 ref for 10 minutes. Supernatant Filtration to collect any remaining calcium carbonate precipitates.

[0254] DIC Analysis: Transfer a known volume of the culture supernatant (without precipitate) to the DIC analyzer. Measure the DIC content using the analyzer. This will provide a direct measurement of the inorganic carbon in the solution, including CO2, HCCh', and CO32-. Calculate the amount of carbon fixed in the precipitated calcium carbonate by comparing the initial and final DIC values.

[0255] Data Analysis

[0256] Compare the DIC values before and after the experiment to determine the amount of carbon removed from the solution and fixed in the calcium carbonate. Correlate the amount of carbon fixed with the mass of the precipitated calcium carbonate to establish the relationship between carbon removal and carbonate production.

[0257] Expected Outcomes

[0258] The DIC analysis will provide quantitative data on the amount of CO2 fixed during the precipitation of calcium carbonate by Rivularia halophila, and that a specific amount of carbon is removed from the atmosphere for every gram of metallic carbonate produced.

[0259] 2. CO2 Uptake and Assimilation Rate MeasurementWSGR Docket No. 69144-701601

[0260] Obj ective: To measure the rate at which CO2 is taken up by the culture and how much of it is converted into biomass versus CaCO3.

[0261] Method:

[0262] Use a closed-loop gas exchange system to monitor CO2 concentrations in the culture environment over time. Measure the decrease in CO2 concentration in the headspace and the corresponding increase in biomass and CaCO3. Calculate the CO2 assimilation rate by analyzing the rate of CO2 depletion.

[0263] Expected Outcome: Determine the rate of CO2 consumption and partitioning into biomass and CaCO3.

[0264] 3. Respiration and Photosynthesis Rate Analysis (Oxygen Evolution)

[0265] Objective: To correlate the rate of photosynthesis (oxygen evolution) with CO2 uptake and CaCO3 formation.

[0266] Method: Measure the rate of oxygen production in the culture using an oxygen electrode under controlled light conditions. Correlate oxygen production rates with CO2 uptake rates measured by a CO2 sensor. Compare these rates with the accumulation of biomass and CaCO3 to infer the proportion of CO2 used for each process.

[0267] Expected Outcome: Provide insights into the efficiency of CO2 fixation into biomass and CaCO3 during photosynthesis.

[0268] 4. pH Drift Experiments

[0269] Objective: To estimate CO2 fixation by observing changes in pH during photosynthesis-driven CaCO3 precipitation.

[0270] Method: Monitor the pH of the culture medium over time as CO2 is fixed into biomass and CaCO3. Use pH drift curves to estimate the amount of CO2 being fixed. Supplement with DIC analysis to confirm the amount of carbon fixed.

[0271] Expected Outcome: Determine the correlation between pH changes and CO2 fixation, supporting estimates of carbon partitioning.

[0272] 5. Biomass and CaCO3 Yield Analysis

[0273] Objective: To quantify the relative amounts of CO2 incorporated into biomass versus CaCO3 by comparing yields.

[0274] Method: Grow cyanobacteria under controlled conditions with known initial CO2 levels.

[0275] Harvest and separate the biomass and CaCO3 at regular intervals.

[0276] Measure the dry weight of both the biomass and CaCO3 precipitates.

[0277] Calculate the carbon content in each fraction and relate it to the initial CO2 levels.WSGR Docket No. 69144-701601

[0278] Expected Outcome: Provide a clear ratio of carbon distribution between biomass growth and CaCO3formation.

[0279] 6. Continuous Monitoring in a Carbon Flux Chamber

[0280] Objective: To continuously monitor CO2 flux in and out of the system, providing realtime data on CO2 consumption.

[0281] Method: Place the culture in a sealed carbon flux chamber with CO2 sensors at both the inlet and outlet. Continuously measure CO2 levels entering and exiting the chamber.

[0282] Monitor changes in biomass and CaCO3over time to correlate with CO2 flux.

[0283] Expected Outcome: Real-time data on CO2 uptake, with direct correlation to biomass growth and CaCO3 precipitation.Example 4: Improvements and benefits of the compositions, methods and systems provided herein

[0284] A summary of improved methods and compositions from the inventions disclosed herein are described in this section:

[0285] 1. Reactive cell surface: Simplifies purification of the precipitated calcium carbonate using cyanobacteria.

[0286] 2. CO2 injection, which can increase productivity and titer. Avoids using exogenous HCO3-.

[0287] 3. Industrial brines as culture media.

[0288] 4. Seawater as culture media.

[0289] 5. Cost effective culture media.

[0290] 6. Optimized culture media.

[0291] 7. Open system.

[0292] 8. Co-culture with other microorganisms to improve productivity / titer / yield.

[0293] 9. Efficient separation methods.1. Reactive cell surface: simplifies purification

[0294] The precipitation induced by the cyanobacterium R. halophila is generated extracellularly, in structures such as the sheath (formed by mucilaginous substances known as EPS) or even some can form in the S layer. The calcium carbonate (CaCO3) crystals that remain attached to these external structures can be separated by physical methods (centrifugation, vibration or sonication, etc.) and subsequently purified by washing with distilled water (or drinking water). In addition, the potential of this cyanobacterium to shed the sheath or the S- layer will be tested. Some filamentous cyanobacteria use this process as a strategy to avoidWSGR Docket No. 69144-701601 being buried by carbonates. This can release the CaCCh with the organic matter of the sheath or the S-layer that can be eliminated by washing.

[0295] Experimental Setup

[0296] Materials and Equipment: Cultures of cyanobacteria (R halophila , Calcium (CaCh) solution (varying concentrations); Control group (dead cells of R. halophila} Growth media (BG11 -Saline)

[0297] Microscopy equipment: Transmission electron microscope (TEM), Scanning electron micro-scope-energy dispersive X-ray spectroscopy (SEM-EDS) and fluorescence microscopy.

[0298] Experimental Design

[0299] Preparation of the medium and cyanobacteria: The cyanobacteria inoculum will be prepared in saline BG11 culture medium. Dead cyano-bacteria inoculum (inactivated with autoclave) will be evaluated as a control. The experiments will be carried out by triplicate. Different calcium concentrations will be tested (e.g. 0.74, 0.96 and 1.5 g / L).

[0300] Precipitation of CaCCh with the cyanobacterium R. halophilc . The carbonate precipitation process will be monitored in R halophila cultures and samples will be taken periodically (e.g., every 48 hours, 72 hours and 1 week) for study by microscopic analysis (SEM- EDS, TEM and fluorescence microscopy). In addition, light and temperature conditions will be kept constant in all groups and pH changes will be monitored during the process.

[0301] Analysis:

[0302] Use microscopy (SEM-EDS and TEM microscopy) to visualize the sheath and S- layer before and after exposure to the calcium carbonate precipitation process. The percentage of free cyanobacteria in the medium versus those buried by CaCCh precipitation will be determined by processing images acquired by fluorescence microscopy at different times for the Ca concentrations evaluated. Use biochemical assays to quantify any dissolved or released components (such as polysaccharides) in the supernatant.

[0303] Data analysis:

[0304] Graphical representation of cyanobacteria growth (g / 1) as a function of calcium concentrations and time. ANOVA analysis to compare treatments to determine the conditions that have a significant effect on the detachment of external structures of cyanobacteria.

[0305] Expected Outcomes

[0306] Determination of the capacity of R. halophila to shed its external structures in order not to become trapped in the precipitated calcium carbonates. Identification of the times andWSGR Docket No. 69144-701601 under what conditions this process occurs: Important data to calculate the possible impact of this process on the performance and costs of the CaCCh precipitation process.2. CO2 injection: Increases productivity and titer. Avoids using exogenous II CO.;

[0307] Enhanced Growth and Precipitation Through CO2 Injection: Injecting CO2 directly into the tank inhabited by cyanobacteria is anticipated to significantly enhance their growth rates and the precipitation of calcium carbonate (CaCCh). This process not only promotes the metabolic activity of the cyanobacteria but also increases the concentration of bicarbonate ions (HCO3 ) in the solution (at pH > 7). As CO2 levels rise, bicarbonate formation will naturally occur, eliminating the need for external supplementation of HCCh'. This integrated approach can lead to efficient cyanobacteria biomass production and carbonate mineralization, aligning with sustainable practices in carbon management.

[0308] On the other hand, cyanobacteria, as photosynthetic microorganisms, utilize carbon dioxide (CO2) as a primary carbon source during the process of photosynthesis. When CO2 levels are elevated, several beneficial effects can be observed like increased photosynthetic efficiency as higher concentrations of CO2 can enhance the photosynthetic rate of cyanobacteria, leading to greater biomass production. In addition, the metabolic activity of cyanobacteria increases the pH in the medium, which positively affects the CaCCh precipitation process.

[0309] Experimental Setup

[0310] Materials and Equipment: Cyanobacterial cultures R. halophila. Growth medium: BG11 -saline medium as a base condition or desalination brines or lithium brines or salt mining brines or seawater as scalable conditions. Calcium source: Typically, calcium chloride (CaCh) or alternatively another soluble calcium salt. CO2 supply system: CCh gas cylinder with a flow meter and diffuser. pH meter: For monitoring and adjusting the pH of the medium. Electrodes: For monitoring CO2 and Calcium concentration. Incubator: With controlled temperature and light conditions. Bioreactors: Flasks and / or small-scale bioreactors and / or small-scale raceway ponds (SSRP) with CO2 injection ports and airtight seals if applicable. Centrifuge: For harvesting cyanobacterial cells. Filtration setup: To separate precipitated calcium carbonate. Microscope: To observe cyanobacterial cells and calcium carbonate crystals.

[0311] Analytical techniques for crystal studying: Use SEM-EDS, X-ray diffraction (XRD), p -Fourier transform infrared (p-FTIR), and X-ray photoelectron spectroscopy (XPS) to analyze and characterize the calcium carbonate crystals.

[0312] Experimental DesignWSGR Docket No. 69144-701601

[0313] Preparation of Growth Media: Prepare BGl l-saline medium with a fixed concentration of calcium (e.g., 400 or 900 mg / L Ca2+). Adjust the initial pH to a suitable range for cyanobacterial growth (typically around 7.5-8.5).

[0314] Inoculation and Incubation: Inoculate each bioreactor with a standardized concentration of cyanobacterial cells ranging between 0,1 g / 1 to 1,7 g / 1. Incubate under controlled light and temperature conditions, typically around 25-30°C with a light / dark cycle.

[0315] CO2 Injection: Set up multiple bioreactors / flasks / SSRP with different CO2 injection rates (e.g., 0, 0.5, 1, 2, and 4 mL / min). Inject CO2 continuously or at set intervals to maintain a stable concentration in the bioreactor. Monitor the pH of the medium, as CO2 injection will decrease the pH; adjust if necessary to maintain optimal conditions for cyanobacterial growth.

[0316] Growth Monitoring: Due to the nature of macrocolonies and crystals, present growth can be monitored by OD. Experiments must be stopped, and cyanobacteria biomass will be weighted. Take samples at regular intervals (e.g., every 24 hours) for calcium carbonate precipitation analysis.

[0317] Calcium Carbonate Precipitation Analysis: Extract calcium carbonate precipitates by peroxide treatment or less harsh methods. Wash the precipitates with distilled water to remove any medium residues. Dry the precipitates at a constant temperature (e.g., 60°C) until a constant weight is achieved. Weigh the dried precipitates using an analytical balance.

[0318] Microscopic Examination: Examine samples under a microscope to observe the morphology of cyanobacteria and calcium carbonate crystals. Take images for documentation and further analysis.

[0319] Analytical Techniques for Crystal Studying: Use SEM-EDS and X-ray diffraction (XRD) to analyze and characterize the calcium carbonate crystals.

[0320] Data Analysis: Plot cyanobacterial growth (g / 1) against time for each CO2 injection rate. Plot the weight of calcium carbonate precipitates against cyanobacterial growth for each CO2 injection rate. Determine the optimal CO2 injection rate by comparing the maximum cyanobacterial growth with the highest calcium carbonate precipitation.

[0321] Experimental Variables and Controls

[0322] Independent Variable: CO2 injection rate.

[0323] Dependent Variables: Cyanobacterial growth and the amount of calcium carbonate precipitate.

[0324] Control Variables: Initial pH, temperature, light intensity, initial cyanobacterial concentration, calcium concentration in the medium.

[0325] Expected OutcomesWSGR Docket No. 69144-701601

[0326] Identification of the optimal CO2 injection rate that maximizes both cyanobacterial growth and calcium carbonate precipitation. Understanding the relationship between CO2 concentration, pH changes, cyanobacterial growth, and calcium carbonate precipitation.3. Brines as culture media

[0327] Brines are hypersaline concentrates, which generally contain high concentrations of calcium together with other elements that form complex mixtures. Brines can be a by-product of diverse human activities such as water desalination or naturally occurring lithium baring brines. Traditionally, the most common brine were sodium chloride brines, however, in recent years, the surge in lithium extraction has significantly increased the mining or extraction of lithium containing brines.

[0328] Using brines rich in calcium and bicarbonate as a growth medium for cyanobacteria offers several advantages. First, the nutrient-rich environment can enhance cyanobacteria biomass production and calcium carbonate precipitation, promoting efficient product formation. This approach also provides a cost-effective solution by repurposing brine waste, contributing to a circular economy. Treating these brines mitigates the environmental impact of their disposal into oceans or other water bodies, which can harm ecosystems. Additionally, this method aligns with industrial practices, making it scalable for larger operations and addressing water scarcity by utilizing water typically deemed waste.

[0329] Desalination brines are produced when sea water is desalinated to make fresh water. By 2018, 95 million m3 / day were produced annually and are expected to increase significantly in the next decade. For every liter of fresh water produced, 1,48 liters of brines are produced. This vast majority of this hypersaline concentrates are discharged back to the ocean posing an environmental risk.

[0330] Lithium extraction from brines involves the processing of hypersaline solutions to isolate lithium, a component for batteries and other technologies. By 2023, the global production of lithium brine reached millions of cubic meters annually, with projections showing significant increases in the coming decade. For every ton of lithium extracted, approximately 500,000 liters of brine are processed.

[0331] Previously, R. halophila was cultured in BGl l-saline medium (0.36% NaCl), a synthetic medium designed for the growth of cyanobacteria. For the precipitation of calcium carbonate, calcium (CaCL) and bicarbonate (NaHCCh) were added exogenously. This culture medium is very expensive and difficult to scale up to industrial volumes. As growing the cyanobacteria in desalination brines or desalination brines plus nutrients that might be lacking poses an effective alternative as it has currently 0 value.WSGR Docket No. 69144-701601

[0332] Table 5. Comparison between BG11 -saline culture medium and brine, composition and costs.

[0333] Table 6. An exemplary composition of 7 lithium mine brine samples.WSGR Docket No. 69144-701601

[0334] Experimental Setup

[0335] Objective: To assess R. halophila growth and precipitation in different brine compositions and test different co-nutrients to help tolerate the brine composition and enhance calcium carbonate precipitation.

[0336] Materials and Equipment

[0337] Cyanobacterial cultures: Rivularia halophila.

[0338] Growth medium: Desalination and / or lithium mining brines and / or hypersaline concentrates, supplemented with essential nutrients as needed (e.g., nitrogen, phosphorus, trace minerals).

[0339] Calcium source: Intrinsic calcium present in desalination brines; additional calcium chloride (CaCL) if required.

[0340] Bicarbonate source: Intrinsic bicarbonate present in desalination brines; additional sodium bicarbonate (NaHCCh) if required.

[0341] pH meter: For monitoring and adjusting the pH of the medium.

[0342] Electrodes: For monitoring calcium concentration.

[0343] Incubator: With controlled temperature and light conditions.

[0344] Bioreactors: Flasks and / or small-scale bioreactors and / or small-scale raceway ponds (SSRP) with CO2 injection and / or airtight seals if applicable.

[0345] Centrifuge: For harvesting cyanobacterial cells.

[0346] Filtration setup: To separate precipitated calcium carbonate.

[0347] Analytical balance: For weighing precipitates and final cyanobacteria biomass.

[0348] Microscope: To observe cyanobacterial cells and calcium carbonate crystals.

[0349] Analytical techniques for crystal studying: Use SEM-EDS, confocal laser and X-ray diffraction (XRD) to analyze and characterize the calcium carbonate crystals.

[0350] Experimental Design

[0351] Preparation of Growth Media: Obtain desalination brine or hypersaline concentrate. Brine composition ranges provided in Table 5. Supplement the brine with any lacking essential nutrients, such as nitrogen (e.g., sodium nitrate between 0- 1,4 g / L), phosphorus (e.g., potassium phosphate, typically between 0.03 - 0.04 g / L or lower than 0,03), and trace metals. Adjust the initial pH to a suitable range for / ?, halophila growth (typically around 7.5-8.5).WSGR Docket No. 69144-701601

[0352] Inoculation and Incubation: Inoculate each bioreactor with a standardized concentration of cyanobacterial cells ranging between 0,1 g / 1 to 1,7 g / 1. Incubate under controlled light and temperature conditions, typically around 25-30°C with a light / dark cycle.

[0353] Brine Composition: Calcium and Bicarbonate Variations. Set up multiple bioreactors / flasks / SSRP with different brine compositions by varying the concentration of intrinsic calcium and bicarbonate and adding different co-nutrients.

[0354] Possible Variations:

[0355] Base Composition for Reference: Calcium (Ca2+): 960 mg / L; Bicarbonate (HCO3 ): 1,829 mg / L;

[0356] Brine A: High Calcium, Low Bicarbonate; Calcium (Ca2+): 1,200 mg / L; Bicarbonate (HCO3): 1,000 mg / L;

[0357] Brine B: Low Calcium, High Bicarbonate; Calcium (Ca2+): 600 mg / L; Bicarbonate (HCO3 ): 2,200 mg / L;

[0358] Brine C: Balanced Calcium and Bicarbonate; Calcium (Ca2+): 960 mg / L (close to the base composition, optimized for balanced growth); Bicarbonate (HCO3 ): 1,500 mg / L (adjusted for a balance between the base and optimal growth conditions)

[0359] Brine D: High Calcium, High Bicarbonate; Calcium (Ca2+): 1,200 mg / L; Bicarbonate (HCO3 ): 2,200 mg / L

[0360] Brine E: Low Calcium, Low Bicarbonate; Calcium (Ca2+): 600 mg / L; Bicarbonate (HCO3 ): 1,000 mg / L

[0361] Brine Composition Variations: Other Component Variations.

[0362] Set up multiple bioreactors / flasks / SSRP with different brine compositions by varying the concentration of intrinsic calcium and bicarbonate and adding different co-nutrients.

[0363] Base Composition: Calcium (Ca2+): 960 mg / L; Bicarbonate (HCO3 ): 1,829 mg / L

[0364] Variation A (High Potassium, Low Magnesium): Potassium (K): 1,200 mg / L; Magnesium (Mg): 1,500 mg / L; Strontium (Sr): 14.5 mg / L; Iron (Fe): 1.8 mg / L; Sodium (Na): 25,237 mg / L; Bromine (Br): 8 mg / L; Chloride (C1‘): 41,829 mg / L; Nitrate (NO3-): 0 mg / L; Phosphate (PO42-): 40 mg / L; Sulfate (SO42-): 6.05 mg / L

[0365] Variation B (Low Potassium, High Magnesium): Potassium (K): 600 mg / L; Magnesium (Mg): 3,000 mg / L; Strontium (Sr): 14.5 mg / L; Iron (Fe): 1.8 mg / L; Sodium (Na): 25,237 mg / L; Bromine (Br): 8 mg / L; Chloride (CF): 41,829 mg / L; Nitrate (NO3-): 1400 mg / L; Phosphate (PO42-): 30 mg / L; Sulfate (SO42-): 6.05 mg / L

[0366] Variation C (Balanced Potassium and Magnesium):; Potassium (K): 900 mg / L; Magnesium (Mg): 2,867 mg / L; Strontium (Sr): 14.5 mg / L; Iron (Fe): 1.8 mg / L; Sodium (Na):WSGR Docket No. 69144-70160125,237 mg / L; Bromine (Br): 8 mg / L; Chloride (Cl’): 41,829 mg / L; Nitrate (NO3-): 700 mg / L; Phosphate (PO42-): 30 mg / L; Sulfate (SO42-): 6.05 mg / L

[0367] Variation D (High Sodium, Low Potassium): Potassium (K): 500 mg / L; Magnesium (Mg): 2,867 mg / L; Strontium (Sr): 14.5 mg / L; Iron (Fe): 1.8 mg / L; Sodium (Na): 30,000 mg / L; Bromine (Br): 8 mg / L; Chloride (C1‘): 45,000 mg / L; Nitrate (NO3-): 100 mg / L; Phosphate (PO42-): 30 mg / L; Sulfate (SO42-): 6.05 mg / L

[0368] Variation E (Low Sodium, High Potassium):; Potassium (K): 1,200 mg / L; Magnesium (Mg): 2,867 mg / L; Strontium (Sr): 14.5 mg / L; Iron (Fe): 1.8 mg / L; Sodium (Na): 20,000 mg / L; Bromine (Br): 8 mg / L; Chloride (O’): 35,000 mg / L; Nitrate (NO3-): 50 mg / L; Phosphate (PO42-): 30 mg / L; Sulfate (SO42-): 6.05 mg / L

[0369] Brine E: Low calcium, low bicarbonate.

[0370] Growth Monitoring:

[0371] Monitor cyanobacterial growth by dry weight. Take samples at regular intervals (e.g., every 24 hours) for calcium carbonate precipitation analysis.

[0372] Calcium Carbonate Precipitation Analysis: Extract calcium carbonate precipitates by peroxide treatment or less harsh methods. Wash the precipitates with distilled water to remove any medium residues. Dry the precipitates at a constant temperature (e.g., 60°C) until a constant weight is achieved. Weigh the dried precipitates using an analytical balance.

[0373] Microscopic Examination: Examine samples under a microscope to observe the morphology of cyanobacteria and calcium carbonate crystals. Take images for documentation and further analysis.

[0374] Analytical Techniques for Crystal Studying: Use SEM-EDS, X-ray diffraction (XRD), p-Fourier transform infrared (p-FTIR), and X-ray photoelectron spectroscopy (XPS) to analyze and characterize the calcium carbonate crystals.

[0375] Data Analysis: Plot cyanobacterial growth (g / L) against time for each brine composition. Plot the weight of calcium carbonate precipitates against cyanobacterial growth for each brine composition. Determine the optimal brine composition by comparing the maximum cyanobacterial growth with the highest calcium carbonate precipitation.

[0376] Experimental Variables and Controls

[0377] Independent Variable: Brine composition and co-nutrient supplementation.

[0378] Dependent Variables: Cyanobacterial growth and the amount of calcium carbonate precipitate.

[0379] Control Variables: Initial pH, temperature, light intensity, initial cyanobacterial concentration.WSGR Docket No. 69144-701601

[0380] Expected Outcomes

[0381] Identification of the optimal brine composition and co-nutrient combination that maximizes both cyanobacterial growth and calcium carbonate precipitation.

[0382] Understanding the relationship between brine composition, nutrient supplementation, cyanobacterial growth, and calcium carbonate precipitation.

[0383] Evaluation of the effectiveness of using desalination brines as a growth medium for cyanobacteria.4. Seawater as culture media

[0384] Utilizing seawater as a growth medium for cyanobacteria presents numerous advantages, particularly given the high costs associated with current media. Seawater is inherently nutrient-rich, providing essential minerals and trace elements that can enhance cyanobacteria biomass production and calcium carbonate precipitation. This approach is also cost-effective, as seawater is abundant and freely available, significantly reducing cultivation expenses. Additionally, growing cyanobacteria in seawater aligns with sustainable practices by promoting the utilization of natural resources while potentially mitigating the environmental impact of saline waste disposal. The natural salinity of seawater can also create an optimal environment for cyanobacterial growth, decreasing the need for supplemental nutrients. Overall, employing seawater as a culture medium not only supports efficient cyanobacterial cultivation but also contributes to more sustainable and economically viable production methods. FIG. 19A is a table of major chemical components of seawater. FIG. 19B is a table showing an exemplary artificial seawater composition.

[0385] Experimental Design for calcium carbonate precipitation with R. halophila in seawater

[0386] Objective: To investigate the effect of nutrient supplementation on the growth of R. halophila and its capacity for CaCOs precipitation in seawater culture media.

[0387] Experimental Setup

[0388] Materials and Equipment

[0389] Biological Materials: Rivularia halophila culture

[0390] Culture Media: Trace elements (e.g., Fe, Zn, Mn) as needed. Nutrients for supplementation (e.g., nitrogen sources like NaNO3, phosphorus sources like KH2PO4

[0391] Chemicals for Precipitation Evaluation: Calcium chloride (CaC12) for calcium ion supplementation. Sodium bicarbonate (NaHCO3) to provide bicarbonate ions.WSGR Docket No. 69144-701601

[0392] In artificial sea water it is necessary to supplement both Ca2+(between 300 - 900 mg / L) and HCCh'10 mM). In natural seawater it will also be evaluated whether any type of supplementation of these compounds could aid the precipitation process.

[0393] Laboratory Equipment: Erlenmeyer flasks (Probably IL); BOD incubator or culture chamber with controlled temperature and light; pH meter; Spectrophotometer (for measuring chlorophyll-a concentration); Microscope:polarized light microscope (PLM); SEM-EDS.

[0394] Analytical Tools: Ion chromatography, colorimetric methods for determining calcium and carbonate concentrations or determination with specific electrodes; Standard laboratory equipment for preparing and sterilizing media.

[0395] Methods

[0396] Preparation of Culture Media: Prepare natural seawater by filtering or sterilizing the collected seawater. Artificial seawater (ASW) media are prepared according to the components described in the table depicted in FIG. 19B.

[0397] Experimental Design

[0398] Experimental Groups:

[0399] Group 1 :

[0400] Control Group: Seawater without nutrient supplementation.

[0401] Nutrient Treatment Groups: Seawater supplemented with different concentrations of: Nitrogen (NaNO3) and Phosphorus (KH2PO4)

[0402] Group 2:

[0403] Control Group: ASW without nutrient supplementation.

[0404] Nutrient Treatment Groups: ASW supplemented with different concentrations of: Nitrogen (NaNO3) and Phosphorus (KH2PO4),

[0405] Experimental Setup:

[0406] Prepare 5 different treatment conditions (1 control and 4 nutrient treatments) with 3 replicates each.

[0407] Option 1 : Nutrient-Rich Medium has NaNO3 (Sodium Nitrate): 2.5 g / L; KH2PO4 (Potassium Dihydrogen Phosphate): 0.1 g / L

[0408] Option 2: Balanced Medium has NaNO3 : 1.5 g / L; KH2PO4: 0.05 g / L

[0409] Option 3: High-Phosphorus Medium has NaNO3: 2.0 g / L; KH2PO4: 0.5 g / L

[0410] Option 4: Minimal Medium hasNaNO3: 1.0 g / L; KH2PO4: 0.01 g / L; Trace Elements (e.g., Fe-EDTA): 0.01 g / L (Iron source, essential trace elements can be included)WSGR Docket No. 69144-701601

[0411] Option 5 : Seawater and ASW without nutrient supplementation (Control).

[0412] The experiments will be carried out in a volume of 500 mL of natural seawater or ASW. These will be incubated under controlled conditions (constant light: light: dark cycle 12: 12 h, temperature: 25°C).

[0413] Inoculation: Inoculate each flask with a standard volume of R. halophila biomass ( < 1.7 g / L). Allow cultures to grow for a specified period (2-15 days).

[0414] Monitoring Growth and Precipitation

[0415] Measure the growth of R. halophila by: Sampling every few days to measure chlorophyll-a concentration using a spectrophotometer. Counting cells using a hemocytometer or flow cytometry. Monitor the pH daily using a pH meter, as it influences CaCCE precipitation.

[0416] To assess CaCCE precipitation: Collect samples at the end of the experiment (biomass, medium and CaCO3 precipitated at the bottom of the Erlernmeyer flask). Measure the remaining calcium and bicarbonate concentrations in solution (using electrodes, chromatography or spectrophotometry). Visually under a microscope for the precipitates formed in these experiments with and without R. halophila (in seawater and in ASW). SEM- EDS and XRD are used to analyze and characterize calcium carbonate crystals.

[0417] Data Analysis

[0418] Growth rate is determined based on chlorophyll-a measurements and cyanobacteria biomass weight (cell counts can optionally be used). Total calcium carbonate precipitation is determined indirectly by evaluating the differences in calcium concentrations before and after the experiment. It is also quantified by the weight of CaCCE at the end of each experiment. Statistical analysis (ANOVA) is performed to compare growth rates and precipitation between all treatment groups. Corresponding statistical analyses will be performed to determine which system has the best performance in carbonate precipitation.

[0419] The results are important to understand the processes of biogenic CaCCE precipitation in a potentially economic system such as seawater and what nutrient conditions can facilitate an optimal process of both cyanobacterial growth and carbonate precipitation.5. Cost effective culture media

[0420] Exploring more economical alternatives to the current growth media for cyanobacteria can significantly enhance the economic viability of large-scale cultivation. Utilizing low-cost resources, such as agricultural by-products, wastewater, or industrial effluents, can provide essential nutrients while reducing overall operational expenses. These alternatives can be rich in organic compounds and minerals, promoting healthy cyanobacterialWSGR Docket No. 69144-701601 growth and calcium carbonate precipitation. Additionally, leveraging economical media can decrease dependency on commercially available formulations, making cultivation more accessible to various industries. This approach also aligns with sustainability goals by recycling waste materials and minimizing environmental impact. Overall, finding and utilizing cost- effective growth media options can optimize cyanobacterial production while supporting sustainable practices.6. Optimized culture media

[0421] To address the current challenges of low productivity, titer, and yield in cyanobacterial cultivation, optimizing culture media is essential. By systematically assessing and refining the composition of growth media, the optimal nutrient ratios were identified and concentrations that support enhanced metabolic activity and cyanobacteria biomass accumulation. This optimization may involve experimenting with various nitrogen phosphorus, and micronutrients sources, as well as exploring the inclusion of micronutrients and growth factors that can stimulate cyanobacterial performance. Improved culture media can lead to higher cyanobacteria biomass productivity and titer, ultimately increasing yield and making the cultivation process more economically viable.

[0422] Experimental Setup:

[0423] Objective: To optimize the culture media for cyanobacterial cultivation to improve productivity, titer, and yield by systematically assessing and refining the composition of growth media.

[0424] Materials and Equipment

[0425] Cyanobacterial cultures: Pure culture of Rivularia halophila.

[0426] Growth medium: Baseline BG11-Saline and brine / hypersaline concentrate.

[0427] Nutrient sources: Various sources of nitrogen, phosphorus, and micronutrients. Micronutrients and growth factors: Essential trace metals, vitamins, and other growthpromoting substances.

[0428] pH meter: For monitoring and adjusting pH.

[0429] Incubator: With controlled temperature and light conditions.

[0430] Bioreactors: Flasks and / or bioreactors and / or small-scale raceway ponds (SSRP) with CO2 injection and / or airtight seals if applicable.

[0431] Centrifuge: For harvesting cyanobacterial cells.

[0432] Filtration setup: To separate biomass and precipitates.

[0433] Analytical balance: For weighing biomass and precipitates.

[0434] Microscope: To observe cyanobacterial cells.WSGR Docket No. 69144-701601

[0435] Analytical techniques for cyanobacteria biomass analysis: Dry weight measurement, chlorophyll content, and biochemical assays.

[0436] Experimental Design

[0437] Preparation of Baseline Growth Media: Obtain desalination brine or hypersaline concentrate. Prepare a baseline growth medium with the following composition:

[0438] Nitrogen (N): Sodium nitrate (NaNO3)

[0439] Phosphorus (P): Potassium phosphate (K2HPO4)

[0440] Micronutrients: Trace metals and other essential nutrients.

[0441] Inoculation and Incubation: Inoculate each bioreactor with a standardized concentration of cyanobacterial cells (0,1 g / 1 to 1,7 g / 1 ). Incubate under controlled light and temperature conditions, typically around 25-30°C with a light / dark cycle.

[0442] Nutrient Optimization Experiments

[0443] Nitrogen Source Variations: Sodium nitrate (NaNO3); Ammonium chloride (NH4C1); Urea (CO(NH2)2); Organic nitrogen sources (e.g., yeast extract)

[0444] Prepare media with different concentrations of each nitrogen source (e.g., 0.5, 1.0, 1.5, 2.0 g / L).

[0445] Phosphorus Source Variations: Potassium phosphate (K2HPO4); Sodium phosphate (NaH2PO4); Organic phosphorus sources (e.g., phytate)

[0446] Prepare media with different concentrations of each phosphorus source (e.g., 0.03, 0.05, 0.1, 0.2 g / L).

[0447] Micronutrient Variations: Trace metals (e.g., Fe, Mg, Zn, Cu, Mn); Vitamins (e.g., B12, biotin); Other growth factors (e.g., amino acids, growth hormones)

[0448] Prepare media with different combinations and concentrations of micronutrients.

[0449] Growth Monitoring and Analysis

[0450] Monitor cyanobacterial growth by measuring dry weight and chlorophyll content. Take samples at regular intervals (e.g., every 24 hours) for biomass and biochemical analysis. Measure the titer of desired metabolites (e.g., lipids, carbohydrates, proteins) using biochemical assays.

[0451] Data Collection and Analysis

[0452] Plot cyanobacterial growth (g / L) against time for each variation in nutrient source and concentration. Analyze the titer of desired metabolites for each nutrient variation. Determine the optimal nutrient composition by comparing the highest cyanobacteria biomass productivity, titer, and yield.

[0453] Microscopic ExaminationWSGR Docket No. 69144-701601

[0454] Examine samples under a microscope to observe the morphology and health of cyanobacterial cells. Take images for documentation and further analysis.

[0455] Experimental Variables and Controls

[0456] Independent Variables: Source and concentration of nitrogen, phosphorus, and micronutrients.

[0457] Dependent Variables: Cyanobacterial growth, cyanobacteria biomass productivity, titer of desired metabolites, and overall yield.

[0458] Control Variables: Initial pH, temperature, light intensity, initial cyanobacterial concentration.

[0459] Expected Outcomes

[0460] Identification of the optimal nutrient sources and concentrations that maximize cyanobacterial growth, productivity, and titer. Improved understanding of the specific nutrient requirements for Rivularia halophila. Enhanced cyanobacteria biomass yield and economic viability of the cultivation process.

[0461] Additional Notes:

[0462] Replicates: Conduct each experiment in triplicate to ensure statistical validity. Standardization: Ensure all other conditions (e.g., pH, light intensity) are kept constant across all experiments. Data Analysis: Use statistical software to analyze the data and identify significant differences between treatments.7. Co-culture with other microorganisms to improve productivity / titer / yield

[0463] Implementing co-culture with other microorganisms can significantly enhance productivity, titer, and yield in cyanobacterial cultivation. By cultivating R. halofila alongside complementary species of cyanobacteria, a synergistic environment can be created that promotes nutrient exchange and enhances metabolic activity. For instance, heterotrophic bacteria can help by decomposing organic matter, releasing nutrients that cyanobacteria can readily utilize, while also providing growth factors that stimulate their performance. Additionally, certain microorganisms may contribute to improved carbon fixation or facilitate the removal of inhibitory byproducts, leading to healthier cultures. This integrated approach can optimize resource utilization, increase cyanobacteria biomass production, and ultimately enhance the overall efficiency of the cultivation process, making it a promising strategy for scaling up cyanobacterial production.

[0464] Given that calcium carbonate precipitation is a multifactorial process, a potential approach is to leverage the varying capabilities of different cyanobacteria species. For instance, one species may excel in generating extracellular nucleation points, while anotherWSGR Docket No. 69144-701601 complementary species can induce a localized pH increase, thereby facilitating enhanced precipitation.

[0465] Experimental Setup: Co-Cultivation of R. halophila with Other Cyanobacteria

[0466] Objective: To enhance productivity, titer, and yield in cyanobacterial cultivation by co-cultivating Rivularia halophila with complementary cyanobacteria species, leveraging their synergistic interactions to promote nutrient exchange, metabolic activity, and calcium carbonate precipitation.

[0467] Materials and Equipment

[0468] Cyanobacterial cultures: Rivularia halophila;

[0469] Complementary cyanobacteria species: Phormidium, Anabaena sp., Synechococcus sp., etc.

[0470] Growth medium: Desalination brine and / or hypersaline concentrate, supplemented with essential nutrients.

[0471] Nutrient sources: Various sources of nitrogen, phosphorus, and micronutrients.

[0472] pH meter: For monitoring and adjusting pH.

[0473] Incubator: With controlled temperature and light conditions.

[0474] Bioreactors: Flasks and / or bioreactors and / or small-scale raceway ponds (SSRP) with CO2 injection and / or airtight seals if applicable.

[0475] Centrifuge: For harvesting cyanobacterial cells.

[0476] Filtration setup: To separate biomass and precipitates.

[0477] Analytical balance: For weighing biomass and precipitates.

[0478] Microscope: To observe cyanobacterial cells and calcium carbonate crystals.

[0479] Analytical techniques for cyanobacteria biomass analysis: Dry weight measurement, chlorophyll content, and biochemical assays.

[0480] Experimental Design

[0481] Preparation of Baseline Growth Media and obtain desalination brine or hypersaline concentrate. Prepare a baseline growth medium with the following composition: Nitrogen (N): Sodium nitrate (NaNO3); Phosphorus (P): Potassium phosphate (K2HPO4); Micronutrients: Trace metals and other essential nutrients.

[0482] Inoculation and Incubation: Inoculate each bioreactor with a standardized concentration of cyanobacterial cells (0,1 g / 1 to 1,7 g / 1 ).

[0483] Introduce complementary species of cyanobacteria into selected experimental groups: Phormidium, Anabaena p., Synechococcus sp., or Others strains.WSGR Docket No. 69144-701601

[0484] Incubate under controlled light and temperature conditions, typically around 25-30°C with a light / dark cycle.

[0485] Co-Cultivation Experiments

[0486] Experiment 1 : Co-Cultivation with Phormidium

[0487] Rivularia halophila + Phormidium: Vary the ratio of R. halophila to Phormidium. (e.g., 1 : 1, 1 :2, 2: 1)

[0488] Experiment 2: Co-Cultivation with Anabaena sp.

[0489] Rivularia halophila + Anabaena sp.: Vary the ratio of R. halophila to Anabaena sp. (e.g., 1 : 1, 1 :2, 2: 1)

[0490] Experiment 3: Co-Cultivation with Synechococcus sp.

[0491] Rivularia halophila + Synechococcus sp.: Vary the ratio of R. halophila to Synechococcus sp. (e.g., 1 : 1, 1 :2, 2: 1)

[0492] Experiment 4: Tri-Culture

[0493] Rivularia halophila + Phormidium + Anabaena sp. or Rivularia halophila + Phormidium + Synechococcus sp.: Vary the ratio of the three species (e.g., 1 : 1 : 1, 1 :2: 1, 2: 1 : 1)

[0494] Growth Monitoring and Analysis

[0495] Monitor cyanobacterial growth by measuring dry weight and chlorophyll content. Take samples at regular intervals (e.g., every 24 hours) for cyanobacteria biomass and biochemical analysis.

[0496] Measure the titer of desired metabolites (e.g., lipids, carbohydrates, proteins) using biochemical assays. Measure calcium carbonate precipitation by filtering, washing, drying, and weighing the precipitates.

[0497] Microscopic Examination: Examine samples under a microscope to observe the morphology and health of cyanobacterial cells and calcium carbonate crystals. Take images for documentation and further analysis.

[0498] Analytical Techniques for Crystal Studying: Use SEM-EDS and XRD to analyze and characterize the calcium carbonate crystals.

[0499] Data Collection and Analysis: Plot cyanobacterial growth (g / L) against time for each co-cultivation experiment. Analyze the titer of desired metabolites for each co-cultivation experiment. Measure calcium carbonate precipitation for each co-cultivation experiment. Determine the impact of each co-cultivation strategy by comparing the highest cyanobacteria biomass productivity, titer, and yield.

[0500] Experimental Variables and Controls

[0501] Independent Variables: Type and ratio of co-cultivated cyanobacteria species.WSGR Docket No. 69144-701601

[0502] Dependent Variables: Cyanobacterial growth, cyanobacteria biomass productivity, titer of desired metabolites, and overall yield.

[0503] Control Variables: Initial pH, temperature, light intensity, initial cyanobacterial concentration.

[0504] Expected Outcomes

[0505] Identification of optimal co-cultivation strategies that maximize cyanobacterial growth, productivity, and titer. Enhanced understanding of the synergistic interactions between R. halophila and complementary cyanobacteria species. Increased cyanobacteria biomass yield and economic viability of the cultivation process.

[0506] Additional Notes

[0507] Replicates: Conduct each experiment in triplicate to ensure statistical validity.

[0508] Standardization: Ensure all other conditions (e.g., pH, light intensity) are kept constant across all experiments.

[0509] Data Analysis: Use statistical software to analyze the data and identify significant differences between treatments.

[0510] Promoters of nitrogen fixation: Increased nitrogen fixation enhances the availability of ammonium, which is crucial for the synthesis of vital compounds that promote the growth of cyanobacteria. This growth can result in greater cyanobacteria biomass and heightened photosynthetic activity. The subsequent rise in metabolic processes significantly impacts calcium carbonate (CaCCE) biomineralization. Thus, investigating the effects of nitrogen fixation in R. halophila under various culture conditions is essential for optimizing the carbonate precipitation process.8. Trace metals alternatives

[0511] In order to reduce the costs of trace metals used in the growth of cyanobacteria, optimizations of the concentrations which can facilitate optimal growth or some more economical alternative sources of these trace metals can be implemented.

[0512] Optimization of Metal Concentrations

[0513] Conduct Dose-Response Experiments: Test lower concentrations of each metal to establish the minimum effective concentrations needed for optimal cyanobacterial growth.

[0514] Phased Reductions: Gradually reduce concentrations of each trace metal, monitoring growth and physiological responses, to identify thresholds for essential versus non-essential metals.

[0515] Alternative Sources for MetalsWSGR Docket No. 69144-701601

[0516] Bio-Based Sources: Explore using agricultural by-products or other organic waste as potential sources of trace metals. For example, using compost extracts known to contain trace metals can reduce costs.

[0517] Utilizing Economically Advantageous Compound Forms: Instead of high-purity reagents, consider less expensive or bulk forms of the required trace metals, as they may contain sufficient purity for the intended use.

[0518] Recycling and Recovery of Metals

[0519] Metal Recovery Systems: Develop methods to recover trace metals from spent media or cyanobacteria biomass, allowing reuse in new media formulations.

[0520] A. Minimal Media Approaches

[0521] Defined Minimal Media: Develop and test minimal media formulations that only include essential nutrients for cyanobacteria to thrive, while omitting any trace metals deemed non-essential based on experimental results.

[0522] B. Tailored Metal Supplementation

[0523] Metals on Demand: Instead of supplementing the full range of metals at set concentrations, implement a system where metals are added based on real-time growth monitoring (e.g., using sensors to detect deficiencies).

[0524] Experimental Setup: Testing Individual Trace Metal Reductions

[0525] Objective: To determine the effect of reducing individual trace metals on the growth of R. halophila and calcium carbonate precipitation.

[0526] Materials and Equipment

[0527] Cyanobacterial cultures: Pure culture of Rivularia halophila.

[0528] Growth medium: BG11 Saline, desalination brine, hypersaline concentrate, seawater, supplemented with essential nutrients (e.g., nitrogen, phosphorus, trace metals).

[0529] Trace metals: H3BO3; MnCl24H2O; ZnSO47H2O; Na2MoO42H2O; CuSO45H2O; CO(NO3)26H2O

[0530] Standard lab equipment as listed in the previous setup.

[0531] Experimental Design Preparation of Growth Media

[0532] Obtain desalination brine or hypersaline concentrate. Supplement with essential nutrients as needed.

[0533] Prepare a control growth medium with full concentration of trace metals:

[0534] H3BO3: 46 mM

[0535] MnC12 4H20 : 9 mM

[0536] ZnSO4 7H2O: 0.77 mMWSGR Docket No. 69144-701601

[0537] Na2MoO4 2H2O: 1.6 mM

[0538] CuSO4 5H2O: 0.3 mM

[0539] Co(NO3)2 6H2O: 0.17 mM

[0540] Inoculate each bioreactor with a standardized concentration of cyanobacterial cells (0,1 g / 1 to 1,7 g / 1 ). Incubate under controlled light and temperature conditions, typically around 25-30°C with a light / dark cycle.

[0541] Individual Trace Metal Reduction Experiments

[0542] Reduce the concentration of each trace metal individually while keeping the others at full concentration. The reductions can be: Full concentration (control); Half concentration; Quarter concentration; Minimal concentration; Close to zero concentration.

[0543] Exemplary Experiment 1 : Reducing H3BO3

[0544] Full concentration: 46 mM

[0545] Half concentration: 23 mM

[0546] Quarter concentration: 11.5 mM

[0547] Minimal concentration: 5 mM

[0548] Close to zero concentration: 0 mM

[0549] Exemplary Experiment 2: Reducing MnC12 4H2O

[0550] Full concentration: 9 mM

[0551] Half concentration: 4.5 mM

[0552] Quarter concentration: 2.25 mM

[0553] Minimal concentration: 1 mM

[0554] Close to zero concentration: 0 mM

[0555] Exemplary Experiment 3 : Reducing ZnSO4 • 7H2O

[0556] Full concentration: 0.77 mM

[0557] Half concentration: 0.385 mM

[0558] Quarter concentration: 0.1925 mM

[0559] Minimal concentration: 0.1 mM

[0560] Close to zero concentration: 0 mM

[0561] Exemplary Experiment 4: Reducing Na2MoO4 2H2O

[0562] Full concentration: 1.6 mM

[0563] Half concentration: 0.8 mM

[0564] Quarter concentration: 0.4 mM

[0565] Minimal concentration: 0.2 mM

[0566] Close to zero concentration: 0 mMWSGR Docket No. 69144-701601

[0567] Exemplary Experiment 5: Reducing CuSO4 5H2O

[0568] Full concentration: 0.3 mM

[0569] Half concentration: 0.15 mM

[0570] Quarter concentration: 0.075 mM

[0571] Minimal concentration: 0.05 mM

[0572] Close to zero concentration: 0 mM

[0573] Exemplary Experiment 6: Reducing Co(NO3)2 6H2O

[0574] Full concentration: 0.17 mM

[0575] Half concentration: 0.085 mM

[0576] Quarter concentration: 0.0425 mM

[0577] Minimal concentration: 0.02 mM

[0578] Close to zero concentration: 0 mM

[0579] Growth Monitoring and Calcium Carbonate Precipitation Analysis

[0580] Monitor cyanobacterial growth by dry weight. Take samples at regular intervals (e.g., every 24 hours) for calcium carbonate precipitation analysis.

[0581] Extract calcium carbonate precipitates by peroxide treatment or less harsh methods. Wash the precipitates with distilled water to remove any medium residues. Dry the precipitates at a constant temperature (e.g., 60°C) until a constant weight is achieved. Weigh the dried precipitates using an analytical balance.

[0582] Microscopic Examination

[0583] Examine samples under a microscope to observe the morphology of cyanobacteria and calcium carbonate crystals. Take images for documentation and further analysis.

[0584] Analytical Techniques for Crystal Studying

[0585] Use SEM-EDS, XRD, and XPS to analyze and characterize the calcium carbonate crystals.

[0586] Data Analysis

[0587] Plot cyanobacterial growth (g / L) against time for each trace metal reduction experiment. Plot the weight of calcium carbonate precipitates against cyanobacterial growth for each trace metal reduction experiment. Determine the impact of each trace metal by comparing the maximum cyanobacterial growth with the highest calcium carbonate precipitation.

[0588] Expected Outcomes

[0589] Identification of the concentrations of each trace metal for optimal cyanobacterial growth and calcium carbonate precipitation.WSGR Docket No. 69144-701601

[0590] Understanding the specific role of each trace metal in cyanobacterial metabolism and biomineralization.

[0591] Evaluation of potential cost savings by reducing trace metal concentrations without compromising growth and product yield.

[0592] Experimental Setup: Testing Alternative Sources for Trace Metals

[0593] Objective: To assess R. halophila growth and calcium carbonate precipitation using alternative sources of trace metals from bio-based sources and economically advantageous compound forms.

[0594] Materials and Equipment

[0595] Cyanobacterial cultures: Rivularia halophila. Growth medium: BG11 Saline, desalination brine, hypersaline concentrate, sea water, supplemented with essential nutrients (e.g., nitrogen, phosphorus, trace metals).

[0596] Alternative trace metal sources:

[0597] Agricultural by-products.

[0598] Bulk forms of trace metals

[0599] Standard lab equipment as listed in the previous setups.

[0600] Experimental Design

[0601] Preparation of Growth Media: Obtain desalination brine or hypersaline concentrate. Supplement with essential nutrients as needed. Prepare a control growth medium with standard trace metal concentrations: H3BO3: 46 mM; MnC12 4H2O: 9 mM; ZnSO4 7H2O: 0.77 mM; Na2MoO4 2H2O: 1.6 mM; CuSO4 5H2O: 0.3 mM; Co(NO3)2 6H2O: 0.17 mM

[0602] Inoculate each bioreactor with a standardized concentration of cyanobacterial cells (0, 1 g / 1 to 1 ,7 g / L). Incubate under controlled light and temperature conditions, typically around 25-30°C with a light / dark cycle.

[0603] Alternative Source Experiments

[0604] Bio-Based Sources: Use agricultural by-products known to contain trace metals.

[0605] Example sources include:

[0606] Compost extract: Obtain a liquid extract from compost rich in organic matter and trace metals.

[0607] Economically Advantageous Compound Forms: Utilize bulk or less expensive forms of the trace metals:

[0608] Iron: Use ferrous sulfate (FeSO4) instead of iron nitrate (Fe(NO3)3).

[0609] Magnesium: Use magnesium oxide (MgO) instead of magnesium chloride (MgC12).

[0610] Potassium: Use potassium chloride (KC1) instead of potassium nitrate (KNO3).WSGR Docket No. 69144-701601

[0611] Examples of Alternative Source Variations:

[0612] Experiment 1 : Compost Extract. Prepare a series of growth media with varying concentrations of compost extract. Compare cyanobacterial growth and CaCCE precipitation to the control.

[0613] Experiment 3: Bulk Iron (FeSO4). Replace Fe(NO3)3 with FeSO4 in the growth medium. Prepare a series of media with varying FeSO4 concentrations.

[0614] Experiment 4: Bulk Magnesium (MgO). Replace MgC12 with MgO in the growth medium. Prepare a series of media with varying MgO concentrations.

[0615] Experiment 5: Bulk Potassium (KC1). Replace KN03 with KC1 in the growth medium. Prepare a series of media with varying KC1 concentrations.

[0616] Growth Monitoring: Monitor cyanobacterial growth by dry weight. Take samples at regular intervals (e.g., every 24 hours) for calcium carbonate precipitation analysis.

[0617] Calcium Carbonate Precipitation Analysis: Extract calcium carbonate precipitates by peroxide treatment or less harsh methods. Wash the precipitates with distilled water to remove any medium residues. Dry the precipitates at a constant temperature (e.g., 60°C) until a constant weight is achieved. Weigh the dried precipitates using an analytical balance.

[0618] Microscopic Examination: Examine samples under a microscope to observe the morphology of cyanobacteria and calcium carbonate crystals. Take images for documentation and further analysis.

[0619] Analytical Techniques for Crystal Studying: Use SEM-EDS and XRD to analyze and characterize the calcium carbonate crystals.

[0620] Data Analysis: Plot cyanobacterial growth (g / L) against time for each alternative source experiment. Plot the weight of calcium carbonate precipitates against cyanobacterial growth for each alternative source experiment. Determine the effectiveness of each alternative source by comparing the maximum cyanobacterial growth with the highest calcium carbonate precipitation.

[0621] Experimental Variables and Controls

[0622] Independent Variable: Source and concentration of trace metals.

[0623] Dependent Variables: Cyanobacterial growth and the amount of calcium carbonate precipitate.

[0624] Control Variables: Initial pH, temperature, light intensity, initial cyanobacterial concentration.

[0625] Expected OutcomesWSGR Docket No. 69144-701601

[0626] Identification of cost-effective alternative sources of trace metals that maximize cyanobacterial growth and calcium carbonate precipitation.

[0627] Understanding the potential of bio-based sources and economically advantageous compound forms for trace metal supplementation.

[0628] Evaluation of the environmental and economic benefits of using alternative sources of trace metals in cyanobacterial cultivation.9. Open System.

[0629] Utilizing small scale open systems resembling raceway ponds for cyanobacterial cultivation not only enhances scalability and efficiency and allows a more accurate but also acts as a proven method. This setup promotes better light penetration, improved gas exchange, and natural mixing, effectively de-risking the process and demonstrating its feasibility for optimized growth and calcium carbonate precipitation.

[0630] Objective: To evaluate the growth of cyanobacteria in open pond systems at laboratory scale and quantify calcium carbonate precipitation as a function of various environmental factors.

[0631] Materials and equipment

[0632] Raceway pond setup: Small-scale raceway ponds (e.g., dimensions 1 m x 0.5 m x 0.5 m); Water circulation system (mixing paddles); Light sources (LED or sunlight exposure through transparent covers)

[0633] Culture conditions:

[0634] Culture medium: BG-l l-saline or any previously evaluated medium that has better results in the CaCCL precipitation process.

[0635] Nutrient solutions: nitrogen and phosphorus sources that presented the best growth results for cyanobacteria and that influence the precipitation of CaCCh.

[0636] Microscopic examination: Analysis of cyanobacteria and mineral morphology by microscopy (optical and SEM).

[0637] Analytical techniques: SEM-EDS, XRD, p-FTIR and XPS are used to analyze and characterize calcium carbonate crystals.

[0638] Experimental Design

[0639] Establish basic conditions for the ponds (laboratory scale), including appropriate media (BG11 -saline, seawater or others previously evaluated) and strains of cyanobacteria (R. halophila. Phormidium and others) and light conditions.

[0640] Treatment groups: Create multiple experimental conditions to test the effects of various factors on cyanobacteria growth and calcium carbonate precipitation:WSGR Docket No. 69144-701601

[0641] Option 1 : BG11 saline medium at the three best calcium conditions previously evaluated (concentrations between 400 - 1000 mg / L)

[0642] Option 2: Seawater under the best nutrient and calcium conditions for carbonate precipitation.

[0643] Option 3. Another culture medium that provides good performance in cyanobacteria growth and CaCOs precipitation

[0644] Monitoring Parameters: Biomass concentration (using dry weight); pH measurements (daily); Daily light / dark cycles (to mimic natural conditions); Temperature measurements (to maintain optimal growth); Calcium carbonate precipitation (measure larval precipitate on specific intervals using filtration or colorimetric assays)

[0645] Data collection time points: Collect data at regular intervals (e.g. every 2-3 days) for cyanobacteria biomass, pH and calcium concentration over a set experimental period.

[0646] Statistical analysis: Analyze the effect of different treatments on cyanobacterial growth and calcium carbonate precipitation using ANOVA or other suitable statistical tests. Evaluate correlations between growth rates and levels of carbonate precipitation.

[0647] These experiments allow for evaluation of cyanobacteria growth under different conditions and their ability to induce calcium carbonate precipitation in open systems such as ponds. The results may indicate the most important data for larger scale applications.10. Efficient separation methods

[0648] To enhance the scalability and economic viability of the cyanobacterial cultivation process, it is essential to prioritize the effective recovery of products such as calcium carbonate and cyanobacteria biomass. Previous methods for separation, like harsh agents such as hydrogen peroxide, can compromise cell integrity and make the process less economically feasible. Instead, implementing gentler separation techniques — such as centrifugation, flocculation, or membrane filtration — can effectively isolate these valuable components while maintaining their quality. By optimizing these recovery methods, the overall efficiency of the production process is improved, making it more sustainable and cost-effective, ultimately supporting the large-scale deployment of cyanobacterial cultivation for producing calcium carbonate and biomass.

[0649] Methodologies for Separation of CaCCh Crystals

[0650] Objective: To separate bioprecipitated CaCOs from cyanobacterial biomass.

[0651] 1. Centrifugation Procedure:

[0652] Collection: Collect the cyanobacterial culture containing the adhered CaCOs by sedimentation or initial low-speed centrifugation (e.g., 1000-2000 rpm for 5-10 minutes).WSGR Docket No. 69144-701601

[0653] Resuspension: Resuspend the sedimented biomass in a suitable buffer or distilled water.

[0654] High-speed centrifugation: Perform high-speed centrifugation (e.g., 4000-6000 rpm for 10-15 minutes) to separate the heavier particles (CaCCh and cyanobacterial structures).

[0655] Collection: Carefully decant the supernatant and collect the sediment, which contains both the cyanobacteria biomass and the precipitated crystals.

[0656] 2. Sieving Procedure:

[0657] Prepare sieves: use a series of sieves or filter meshes of different pore sizes (e.g., 50 pm, 100 pm, 200 pm) to allow passage of smaller particles and retain macrocolonies and let CaCCh crystals pass through.

[0658] Sieving process: pour the resuspended sample onto the sieve. Wash gently with distilled water or buffer solution to help dislodge and separate remaining crystals.

[0659] Collect fractions: separate the fraction with biomass (retained on the sieve) from that with CaCCh (which can pass through these sieves).

[0660] 3. Flocculation Procedure:

[0661] Flocculant Selection: Identify appropriate flocculants based on system compatibility and application goals. Flocculant Addition: Introduce flocculants into the cyanobacterial culture medium at low concentrations.

[0662] Mixing: Mix the solution to allow even distribution of flocculants.

[0663] Settling Time: Allow the solution to sit undisturbed for a predetermined time (e.g. 30 minutes to 1 hour) to allow floc formation.

[0664] Separation: Decant or centrifuge the aggregated flocs, separately collecting both cyanobacterial biomass and CaCCh.Example 5: CO2 injection: increases productivity and titer. Avoids using exogenous HCO3

[0665] Injecting CO2 directly into the tank inhabited by cyanobacteria is anticipated to significantly enhance their growth rates and the precipitation of calcium carbonate (CaCCh). This process not only promotes the metabolic activity of the cyanobacteria but also increases the concentration of bicarbonate ions (HCO3 ) in the solution (at pH > 7). As CO2 levels rise, bicarbonate formation will naturally occur, eliminating the need for external supplementation of HCCh'. This integrated approach can lead to efficient biomass production and carbonate mineralization, aligning with sustainable practices in carbon management. On the other hand, cyanobacteria, as photosynthetic microorganisms, utilize carbon dioxide (CO2) as a primary carbon source during the process of photosynthesis. When CO2 levels are elevated, severalWSGR Docket No. 69144-701601 beneficial effects can be observed, like increased photosynthetic efficiency, as higher concentrations of CO2 can enhance the photosynthetic rate of cyanobacteria, leading to greater biomass production. In addition, the metabolic activity of cyanobacteria increases the pH in the medium, which positively affects the CaCCh precipitation process.

[0666] Objective

[0667] Evaluate different sources of dissolved inorganic carbon (DIC) in the culture medium and their effect on the growth of R. halophila and N. nodulosa. as well as their impact on the CaCOs precipitation process.

[0668] 1.1. Experimental setup: materials and methods

[0669] A) Cyanobacterial cultures: R. halophila and N. nodulosa

[0670] FIGs. 20A-20D show scanning electron microscopy images obtained for the cyanobacteria R. halophila and N. nodulosa. Cyanobacteria of the genus R. halophila are formed by filaments connected to EPS (FIG. 20A), as well as a sheath that covers the entire cyanobacterium (FIG. 20B), while the genus N nodulosa lacks a sheath and EPS aggregates (FIGs. 20C-20D). An exemplary cyanobacteria of the genus N nodulosa is depicted in FIG. 20C. An exemplary image of the filaments of A. nodulosa is depicted in FIG. 20D.

[0671] Growth medium: BGl l-saline medium supplemented with essential nutrients (nitrogen, phosphorus, and micronutrients).

[0672] Calcium source: calcium chloride (CaCh).

[0673] Carbon supplements in the system: NaHCCE, CO2 (100%) and a mix of CCh / air in a ratio of 3:97%.

[0674] pH meter: for monitoring and adjusting the pH of the medium.

[0675] Electrodes: for monitoring DIC and calcium concentration.

[0676] Incubator: with controlled temperature and light conditions.

[0677] Bioreactors: flasks and / or small-scale bioreactors with CO2 injection ports.

[0678] Centrifuge: for harvesting cyanobacterial cells.

[0679] Filtration setup: to separate precipitated calcium carbonate.

[0680] Microscope: petrographic optical microscope under polarized light to observe cyanobacterial cells and calcium carbonate crystals.

[0681] Analytical techniques for crystal studying: use scanning electron microscopy (SEM) coupled with EDS and X-ray diffraction (XRD) to analyze and characterize the precipitated calcium carbonate crystals and XPS for the identification of the biogenic signal.

[0682] 1.2. Experimental designWSGR Docket No. 69144-701601

[0683] 1.2. a) Preparation of growth media: BGl l-saline supplemented with 3.6 g L'1of NaCl. For Ca2+supplementation, a 2 M CaCh stock solution was prepared and sterilized at 121°C and 0.13 MPa. The initial calcium (Ca2+) concentration (for all the conditions evaluated) was maintained within the range of 600 to 800 mg / L in the culture medium.

[0684] Table 7. Composition of BG11 -saline medium. Culture media also contains 3.6 g L’1of NaCl and 600 mg / L of Ca. A volume of 1 mL from the trace metals stock solution was added to the BG-11 medium.

[0685] 1.2.b) Inoculation, incubation, and carbon sources added: In order to compare the final productivity and discard the use of exogenous HCCL' (NaHCCL) as a source of C, two types of C sources were added to the culture medium:

[0686] l.l.b.l) CO2 gas:

[0687] 1.2.b.l.a) Pure CO2gas (100%)

[0688] For this experiment, the pH of the BGl l-saline culture medium was initially set to 11.85 with the addition of IM NaOH, followed by the subsequent addition of CO2 by bubbling the gas directly into the medium. According to FIG. 21, to achieve a concentration of HCCL' higher than 97%, the pH should be near 8. Thus, the prior step of raising the pH is essential, as the introduction of CO2 decreases the pH.

[0689] The inoculation was performed using 100 mL of the aforementioned medium with 0.17 g of biomass to achieve a ratio of 1.7 g of biomass per liter of medium. The experiments were performed in triplicate. A control (culture medium without cyanobacteria) was also prepared. All media were incubated at a temperature of 21 ± 2°C and under 12-h: 12-h lightdark cycles, with light intensity maintained at 110 pmol / m2 / s. The test time of the precipitation process with cyanobacteria was 48 hours. At the beginning and end of experiments, the pH, DIC, and dissolved Ca concentration were measured.

[0690] 2.2.b.l.b) A mix of CCL / air in a ratio of 3:97%: To provide a source of carbon, CO2 was added by bubbling the gas mixture into the BGl l-saline culture medium. Similar to theWSGR Docket No. 69144-701601 previous experiment, the initial pH of the culture medium was previously adjusted to 11.75 by adding 1 M NaOH, followed by the subsequent addition of CO2. The pH of the culture medium after bubbling dropped to 7.93 and the concentration of CO2 was 393 ppm. A fed-batch process was carried out for 7 days; every 24 hours, 265 ppm of CO2 was added to the culture medium.

[0691] Inoculation was carried out following the same biomass / culture medium ratio mentioned above (1.7 g / L). The experiments were performed in triplicate. As a control, the culture medium without inoculum was evaluated. All media were incubated at a temperature of 21 ± 2 °C and under 12 h: 12 h light-dark cycles, with a light intensity maintained at 100 pmol / m2 / s. pH changes were monitored before and after each CO2 addition. On the other hand, DIC and dissolved Ca concentration were measured at the beginning and end of the process.

[0692] 2.2.b.2) Solid NaHCOs: In this experiment, the carbon source added was NaHCCE incorporated into the culture medium in a concentration of -840 ppm. The pH was adjusted to 8 by adding HC1 0.1 M. Experiments were performed in triplicate and the biomass / culture medium ratio used was the same as described for previous processes (1.7 g / L). All media were incubated at a temperature of 21 ± 2 °C under 12h: 12h light-darkness cycles, with a light intensity maintained at 100 pmol / m2 / s. The test time of the precipitation process with cyanobacteria was 7 days. pH, DIC, and Ca2+concentration were measured at the beginning and end of experiments.

[0693] 2.2. c) Quantification of dissolved inorganic carbon (DIC) and Calcium (Ca2+)

[0694] DIC was measured as CO2. In analytical procedures, carbon dioxide buffer solutions were added to samples and standards before measurement. After the addition of the buffer solution, all samples and standards should fall within the pH 4.8 to 5.2 range so that all dissolved bicarbonate and carbonate species in solution are converted to H2CO3 / CO2 and so that possible interferences are minimized. For quantification, the Orion™ 9502BNWP carbon dioxide selective electrode was used. The calibration curve was created following the manufacturer's instructions (Thermo Scientific). The curve showed an R2of 0.999 (FIG. 22A). Dissolved CO2 concentration measurements were conducted in the BGl l-saline medium before and after 0.2 pm filtration (medium sterilization). Calcium was measured as Ca2+. For quantification, the 9729BNWP Calcium Combination Electrode was used. The calibration curve was created following the manufacturer instructions (Thermo Scientific). The curve showed an R2of 0.9995 (FIG. 22B). Dissolved Ca2+concentration measurements were made in the BG11 -saline medium before and after 0.2 pm filtration (medium sterilization).

[0695] 2.2.d) Calcium carbonate precipitation analysis: The CaCCE precipitate was extracted using H2O2 and washed with distilled water to remove any medium residues. Then,WSGR Docket No. 69144-701601 the residue was dried at 60°C until a constant weight was achieved. Finally, the precipitate was weighed using an analytical balance and analyzed by polarized microscopy, XRD, SEM-EDS, and XPS.

[0696] 2.2. e) Statistical Analysis

[0697] One-way ANOVA followed by Tukey's multiple comparisons test was used to assess statistical significance, with p< 0.05 considered significant. This analysis was performed on data for pH, Ca and DIC concentrations, productivity, and yield percentage.

[0698] 2.3. Results

[0699] 2.3.1. Experiments using pure CO2 gas and monocultures of R. halophila

[0700] 2.3.1.a). Changes in pH, Ca2+concentration and DIC

[0701] Table 8 shows the initial and final Ca2+concentrations of the experiments with their corresponding replicates. Several replicates of the experiments with cyanobacteria were carried out, as well as the controls. After 48 h of incubation, a decrease in Ca2+concentration was determined (51.0 ± 1.3%) due to the precipitation process biologically induced by cyanobacteria. The decrease was higher than that of the control (18 ± 3%).WSGR Docket No. 69144-701601

[0702] Additionally, R. halophila slightly raised the pH of the culture medium after 48 hours of incubation, increasing from 8 (initial) to 8.4. The pH of the control exhibited no significant changes, consistently remaining at 8 (Table 9).

[0703] Regarding DIC, the content decreased by 60% after 48 hours in experiments with cyanobacteria compared to 28% in the control (see Table 9). This higher reduction in dissolved CO2 in the medium is attributed to the precipitation process as well as the metabolic activity of the cyanobacteria.

[0704] 2.3. l.b). Mineral identification and characterization: images with petrographic microscope, SEM-EDS, and XRD results.

[0705] The images obtained with polarized microscopy show crystals of CaCCE around the R. halophila cyanobacteria filaments (FIG. 23. In order to study the morphology of the precipitate powders, SEM images were collected (FIGs. 24A-24D). SEM images of the precipitate obtained from control samples are depicted in FIG. 24A and FIG. 24B, while SEM images of the precipitate obtained from samples comprising A. halophila are depicted in FIG. 24C and FIG. 24D. The carbonate powder from R. halophila appeared as a mixture of big particles (around 5 pm) and small ones of less than 1 pm size. The big particles show typical crystalline shapes in which rhombohedral faces are visible. The small grains are aggregates in which the crystalline faces are not clearly defined (FIG. 24A and FIG. 24C). The EDS results identified a chemical composition dominated by 97% CaCCE (C, O, and Ca) with other elements in smaller proportions, such as phosphorus (P) and iron (Fe) (Table 10), which are common elements present in the culture medium. Table 10. Elemental composition of CaCCE precipitates according to EDS analysis in the treatments. SD: Standard deviation.

[0706] The analyses of the XRD diffraction patterns showed that the control (FIG. 25A) and the precipitates at the bottoms of the flasks (FIG. 25B) are free of calcite, while the powders precipitated on the R. halophila contain calcite as the unique mineral phase (FIG. 25C). TheWSGR Docket No. 69144-701601 shapes of the XRD diffraction patterns for the control and bottom sample identified a high content of amorphous phases.

[0707] 2.3. l.c). CaCOs productivity.

[0708] The average CaCO3productivity using R. halophila and CO2 (100%) as a source of C increased fivefold compared to the control (Table 11). The results obtained clearly demonstrate greater efficiency in CaCCE precipitation by the R. halophila culture compared to the control. A significant increase is observed in the total amount of CaCCE precipitated, the titer (CaCCE concentration), productivity, and substrate yield in the treatment with R. halophila. In contrast, the low yield in the control treatment indicates limited CaCCE precipitation in the absence of cyanobacteria, suggesting that the presence of R halophila is essential for biomineralization under the experimental conditions evaluated.

[0709] Table 11. pH, titer, productivity, and substrate yield (SY).

[0710] 2.3.2. Experiments using a mix of CCE / air in a ratio of 3 :97% and monocultures of R halophila and N. nodulosa.

[0711] 2.3.2. a). Changes in pH, Ca2+concentration and DIC

[0712] FIG. 26 shows the changes in the pH obtained after each addition of CO2 (265 ppm) during 7 days. The pH of the control decreases progressively over time, reaching the lowest values at the end of the experiment (pH 5). The experiments with R halophila and N. nodulosa show very different behavior. The CO2 additions cause a slight decrease in pH, which is compensated by a subsequent increase resulting from the cyanobacteria metabolism. This increase is higher for N. nodulosa. During day 5, the increase in pH was the highest for both cyanobacteria. The decrease in DIC is accompanied by a decrease in Ca2+concentration, associated with the precipitation of CaCCE (Table 12).

[0713] Table 12. Calcium concentration [Ca], DIC (dissolved carbon dioxide).WSGR Docket No. 69144-701601

[0714] The increase in pH (FIG. 27A) and decrease in CO2 (FIG. 27C) were compared between the treatments and control, resulting in statistical differences (). Regarding Ca concentration (FIG. 27B), the control showed the highest value, showing significant differences when compared to N. nodulosa, but not with R. halophila.

[0715] 2.4.2.b). Mineral identification and characterization: images with petrographic microscope, SEM-EDS, and XRD results.

[0716] Images obtained with a polarized light microscope reveal crystals of minerals around both cyanobacteria (FIGs. 28A-28D). R. halophila appears to promote more abundant precipitation compared to N nodulosa, as evidenced by the higher birefringence observed in the crossed prism images. FIG. 28A and FIG. 28B show R. halophila, and FIG. 28C and FIG. 28D show M nodulosa. FIG. 28A and FIG. 28B were taken with parallel nicols and FIG. 28C and FIG. 28D with crossed nicols to observe birefringence. The arrows indicate the minerals precipitated by the cyanobacteria.

[0717] The SEM images of the powders obtained for both cyanobacteria show aggregates in which the crystalline faces are not clearly defined (FIG. 29A and FIG. 29B). The typical rhombohedral calcite was not identified. However, XRD results identified the main peak related to calcite in the precipitates obtained with both R. halophila (FIG. 30B) and N nodulosa (FIG. 30C). The crystallinity of precipitated calcite from R halophila was higher than N. nodulosa (FIGs. 30A-30C). Interestingly, the XRD of the control indicated that the obtained precipitate is amorphous, as no defined peak could be detected (FIG. 30A).

[0718] 2.3.2.c). Identification of the biogenic signal on the CaCCh precipitate: XPS results

[0719] The identification of functional groups of the extracellular polymeric substances (EPS) in the Ca-carbonates is a fingerprint of the biogenic origin of the minerals. The biogenic signal was identified after the deconvolution of the XPS C Is peaks. FIG. 31 shows the photoemission spectra of C Is, measured for Ca-carbonates precipitated for the experiments using a mix of CCh / air in a ratio of 3:97 with R. halophila, along with the corresponding precipitate in the control media. Specifically, FIG. 31 A shows reference C is spectra in the control condition. FIG. 31B shows reference C is spectra obtained with R halophila. FIG. 31C shows reference C is spectra obtained with N nodulosa. FIG. 31D shows Cis spectra obtained experimentally using control media. FIG. 31E shows Cis spectra obtainedWSGR Docket No. 69144-701601 experimentally using a mix of CO2 / air in a ratio of 3:97 with R. halophila. FIG. 31F shows Cis spectra obtained experimentally using a mix of CO2 / air in a ratio of 3:97 with TV. nodulosa. XPS parameters of core level spectra are given in Table 13. Table 13 shows the Parameters of high-resolution XPS C is spectra for experiments using a mix of CO2 / air as a carbon source, along with the corresponding control media and reference materials. The full width at half maximum (FWHM, eV) and binding energies (BE, eV) were allowed to vary during the fitting.

[0720] The deconvolution of the C Is signal generates four or five peaks referred to as Cl,C2, C3, C4, and C5, with binding energies between 284.8 and 290 eV. The first peak at 284.8 eV (i.e., Cl) was taken as an internal reference and was used to correct other spectra; it corresponds to aliphatic C atoms (C-C and C-H groups). The last peak at -290 eV (i.e., C5) is attributed to C-0 in carbonate samples (Gopinath et al., 2002; Bia et al., 2021).

[0721] The Cl, C2, and C3 are common contributions attributed to carbon contamination. These peaks are detectable in most of the samples that have been exposed to the atmosphere, and there was the main signal obtained in the Ca-carbonates precipitated in both control media. However, in the Ca-carbonate precipitated with R. halophila and N. nodulosa, the relative proportion of the C is peaks changed. Thus, the C2, C3, and C4 components increase (Table 13 and FIG. 31E and FIG. 31F). The larger peak areas of C2, C3, and C4 may suggest the formation of alcohol, primary amide or amine, aldehyde, ester, and sulfhydryl functionalWSGR Docket No. 69144-701601 groups, presumably due to polysaccharide-like components of EPS incorporated or mixed with the Ca-carb onate.

[0722] FIGs. 31A-31F. C Is spectra were obtained for the experiments using a mix of CCh / air in a ratio of 3:97 as a carbon source, along with the corresponding control media and reference materials. Solid black lines depict measured spectra; solid red lines indicate fits to measured spectra and dashed lines correspond to fits obtained with parameters indicated in Table 13. A Shirley background is included as a solid curve at the base of the peaks. The shaded area under the curve is assigned to carbonate contribution. Synthetic calcite was obtained from Bia et al. (2025).

[0723] 2.3.2.d). CaCOs productivity

[0724] Table 14 shows the results obtained for both cyanobacteria. R. halophila has a higher yield compared to N nodulosa, while the control almost does not produce precipitation.

[0725] 2.3.3. Experiments using NaHCCE with monocultures of R. halophila and N nodulosa

[0726] 2.3.3. a). Changes in pH, Ca2+concentration and DIC

[0727] The results presented in Table 15 show the impact of the precipitation on the dynamics of DIC, calcium concentration, and pH. The control shows a decrease in DIC levels compared to the initial condition, a slight decrease in Ca concentration (Ca initial: 806 ppm) and a pH increase, related to the equilibrium of bicarbonate in solution. In contrast, the presence of R. halophila and N. nodulosa leads to a drastic reduction in DIC (FIG. 32C) and Ca (FIG. 32B), along with an increase in pH (FIGs 32A and Table 15). Both cyanobacteria consume bicarbonate as a C source and also use it to precipitate CaCCh precipitation. The increase of pH, due to metabolism, is more pronounced with N. nodulosa.

[0728] Table 15. Values of DIC, [Ca], initial and final pHWSGR Docket No. 69144-701601

[0729] 2.3.3.b). Mineral identification and characterization: images with petrographic microscope, SEM-EDS, and XRD results. Images obtained with a polarized light microscope reveal the presence of many crystals of minerals around both cyanobacteria (FIGs. 33A-33D). The crystal morphology is processed by SEM-EDS. FIG. 33A shows a polarized light optical microscopy image of CaCCE precipitated on R. halophila, where the images was taken with parallel Nicols. FIG. 33B shows a polarized light optical microscopy image of CaCCE precipitated on R. halophila, where the images was taken with crossed Nicols. FIG. 33C shows a polarized light optical microscopy image of CaCCE precipitated on N. nodulosa, where the images was taken with parallel Nicols. FIG. 33D shows a polarized light optical microscopy image of CaCCE precipitated on N nodulosa, where the images was taken with crossed Nicols. R. halophila and N. nodulosa produce well-defined rhombohedral crystals accompanied by amorphous carbonate material (FIG. 34B and FIG. 34C), while the control produces similar crystals, but with higher porosity (FIG. 34A). XRD results identify the presence of calcite in all samples, all with high crystallinity (FIG. 35A-C). FIG. 35A shows XRD for the control without cyanobacteria, FIG. 35B shows XRD for R. halophila, FIG. 35C shows XRD for N nodulosa. This is an important difference from previous experiments with CCE as a carbon source (exp 3.2), where the control did not generate a crystal precipitate due to the final pH reached in that case, compared to the final pH obtained here.

[0730] 2.3.3.c). CaCCE productivity

[0731] R halophila and N. nodulosa cultures notably enhanced the total amount of CaCCE produced, as well as the titer, productivity, and substrate yield. R. halophila exhibited a greater overall yield, surpassing that of N nodulosa (Table 15).

[0732] Table 15. CaCCE production data in culture media containing R. halophila and N nodulosaWSGR Docket No. 69144-701601

[0733] 2.4. Discussion

[0734] The obtained results demonstrate the significant influence of R halophila and N nodulosa on CaCOs biomineralization. The increase in total production, titer, productivity, and substrate yield compared to the abiotic control confirms this potential. These findings are consistent with previous studies that have reported the capacity of certain microorganisms, such as cyanobacteria (Bundeleva et al., 2014; Ramasamy et al., 2015; Mlweski et al., 2018), to induce carbonate precipitation through photosynthesis, as they generate an elevation in local pH, favoring CaCCf supersaturation (De Muynck et al., 2010) and providing nucleation sites for mineral formation.

[0735] The difference in precipitation efficiency observed between R. halophila and N. nodulosa could be attributed to various factors, including differences in photosynthetic activity, cell morphology, extracellular matrix composition, and the ability to modulate the chemical microenvironment. N. nodulosa exhibits higher metabolic activity than R halophila and can induce a higher pH increase.

[0736] X-ray diffraction (XRD) analysis (FIGs. 29A-29B) confirmed the presence of calcite as the main crystalline phase in the precipitates obtained with both R. halophila and N nodulosa, suggesting a similar biomineralization pathway in both species (Garcia-Ruiz et al., 2003). The difference in the crystallinity obtained is perhaps related to the type of C source used, the final pH, and the way as the cyanobacteria nucleate the calcium carbonate. The presence of a sheath and the capacity of R. halophila to produce extracellular polymeric substances (EPS) may facilitate nucleation, whereas N nodulosa, lacking a sheath, is limited in the EPS production.

[0737] 2.5. Conclusions

[0738] The addition of NaHCCh to the cyanobacterial culture medium was demonstrated to induce significant alterations in key parameters influencing CaCCh precipitation, including pH, Ca2+concentration, and dissolved inorganic carbon (DIC), in comparison to experiments involving CO2 supplementation.

[0739] Experiments with NaHCCE as a carbon source improve the crystallinity of the precipitates

[0740] The extent of CaCCh precipitation was significantly enhanced in the presence of cyanobacteria when utilizing both CO2 and NaHCCh as carbon substrates, relative to the uninoculated control.WSGR Docket No. 69144-701601

[0741] CaCOs productivity and biomass yield were consistently enhanced in the presence of cyanobacterial strains R halophila and N. nodulosa compared to the uninoculated control medium. These increases were corroborated across experimental conditions employing both CO2 and NaHCCh as inorganic carbon sources.

[0742] Productivity values increased significantly with the addition of NaHCCh. For the R. halophila strain, productivity was ten times higher compared to using CO2 alone, while for N nodulosa, it was three times higher.

[0743] The identification of organic compounds in the CaCCh could be used as a spectroscopic indicator to predict a possible biogenic origin of the precipitates. Additionally, high proportions of C-N and C-S bonds in the CaCCh could be a valuable proxy of the presence of cyanobacteria in the nearby region where precipitation occurs.

[0744] This study demonstrates the potential of the cyanobacteria R. halophila and N nodulosa for CaCCh biomineralization, presenting a sustainable alternative to conventional chemical methods. The presence of these cyanobacteria was shown to enhance CO2 assimilation and CaCCh precipitation, exhibiting higher productivity, crystallinity, and purity compared to the control. Optimization of cultivation conditions and elucidation of the underlying mechanisms of cyanobacteria-mediated biomineralization are essential for advancing biotechnological applications in areas such as carbon capture and eco-friendly material synthesis.Example 6. Testing cost-effective alternative media (Brines and other cheaper media)

[0745] Brines are hypersaline concentrates, typically comprising sodium chloride (NaCl) or calcium chloride (CaCh) along with various other compounds forming complex mixtures. Brines can be a by-product of diverse human activities such as water desalination or naturally occurring lithium (Li)-bearing brines. Traditionally, the most common brines used as a growth media for cyanobacteria were NaCl brines; however, in recent years, brines rich in calcium (Ca) and bicarbonate (HCO3 ) have offered several advantages. The nutrient-rich environment can enhance biomass production and calcium carbonate (CaCCh) precipitation, promoting efficient product formation. This approach also provides a cost-effective solution by repurposing brine waste, contributing to a circular economy. Treating these brines mitigates the environmental impact of their disposal into oceans or other water reservoirs, which can harm ecosystems. Additionally, this method aligns with industrial practices, making it scalable for larger operations and addressing water scarcity by utilizing water typically deemed waste. Previously, R halophila was cultured in BG-11 saline medium (0.36% NaCl), a synthetic medium designed for the growth of cyanobacteria. For the precipitation of calcium carbonate,WSGR Docket No. 69144-701601 calcium (added as CaCh) and bicarbonate (added as NaHCCh) were used as analytical-grade reagents without further purification. This culture media is very expensive and difficult to scale up to industrial volumes.

[0746] Exploring more economical alternatives to the current growth media for cyanobacteria can significantly enhance the economic viability of large-scale cultivation. Utilizing low-cost resources, such as agricultural by-products, wastewater, or industrial effluents, can provide essential nutrients while reducing overall operational expenses. These alternatives can be rich in organic compounds and nutrients, promoting healthy cyanobacterial growth and calcium carbonate precipitation. Additionally, leveraging economical media can decrease dependency on commercially available formulations, making cultivation more accessible to various industries. This approach also aligns with sustainability goals by recycling waste materials and minimizing environmental impact. Overall, finding and utilizing cost- effective growth media options can optimize cyanobacterial production while supporting sustainable practices.

[0747] The experiments in this example were carried outusing produced water (PW) from a Patagonian oil and gas industry (Argentina). The PW is high in saline concentration (i.e., Na+, Cl’, Mg2+, Fe3+, sulfate) and also contains high levels of dissolved Ca2+(-26.000 ppm) and moderate levels of bicarbonate (-200 ppm). The use of PW as a culture media may explain both experimental outcomes.

[0748] Objectives: I) Evaluate the viability and growth of R. halophila and N. nodulosa in produced water. II) Evaluate the precipitation of CaCOs in produced water.

[0749] I. Growth and viability of R. halophila and N. nodulosa cells in produced water

[0750] 1.1. Experimental setup: materials and methods

[0751] a. Cyanobacterial cultures: Rivularia halophila and Nodosilinea nodulosa.

[0752] b. Growth media: produced water from a Patagonian oil and gas industry. The major chemical composition is summarized in Table 16.

[0753] The PW samples were collected by the company and kept in the cold room at a temperature of 4 °C. The PW has high turbidity, so before conducting the cell viability and precipitation experiments, a clarifier was used to reduce the amount of dissolved and suspended solids in wastewater. In this way, clarifiers ensure water with lower turbidity, allowing better light transmission, which is optimal for conducting viability experiments of cyanobacteria and Ca-carbonate precipitation. The experiment consisted of dissolving the clarifier (i.e., aluminum sulfate, commercial grade) in PW with a final concentration of 200 mg / L. After the addition ofWSGR Docket No. 69144-701601 the clarifier, the pH of the PW decreases to 4.3 (see Table 16). This culture media was referred to as PW-C200.

[0754] Table 16. Chemical composition and physicochemical parameters of produced water. Fluoride, nitrite, nitrate, and phosphate were below detection limits. Ca and HCO3' contents were determined using a specific combination electrode (ISE). The ionic strength (I) was calculated using Visual MINTEQ 3.1 software (Allison et al., 1991). n.d. = not determined

[0755] c. Calcium source: natural content of calcium in PW-C200 (see Table 16).

[0756] d. Bicarbonate source: natural content of HCO3- in PW-C200 (see Table 16).

[0757] e. pH meter: for monitoring and adjusting the pH of the medium.

[0758] f. Calcium and CO2 selective electrodes: for monitoring dissolved calcium and DIC concentration of the medium.

[0759] g. Incubator: with controlled temperature and light conditions.

[0760] h. Bioreactors: flasks and / or small-scale bioreactors.

[0761] i. Centrifuge: for harvesting cyanobacterial cells.

[0762] j. Analytical balance: for weighing precipitates and final biomass.

[0763] k. Microscope: petrographic optical microscope under polarized light to observe cyanobacterial cells.

[0764] 1.2. Experimental design:

[0765] 1.2. a. Preparation of growth media

[0766] - Positive control'. BG-11 saline medium (modified from Phoenix et al., 2002) supplemented with 8.0 g L'1of NaCl. The composition is listed in Table 17.

[0767] Table 17. Composition of BG-11 saline medium. A volume of 1 mL from the trace metals stock solution was added to the BG-11 medium.WSGR Docket No. 69144-701601

[0768] Negative control'. PW-C200 without the addition of macro-, micronutrients, and trace metals.

[0769] - Culture media'. PW-C200 diluted using RO water.

[0770] Culture media were prepared from PW-C200 at different dilutions with the addition of macronutrients (NaNO3, K2HPO4, and NaHCO3), micronutrients (ferric ammonium citrate and Na2EDTA), and trace metals. In addition, KC1 was added to adjust the Na / K ratio to a value of 25.

[0771] PW-C200 was diluted with RO water in the following proportions: 25:75, 50:50, and 75:25 for PW-C200:RO water, respectively. Also, an experiment with 100% ofPW-C200 was tested. Two control conditions were included: a positive control using BG11 medium as the standard culture condition, and a non-supplemented 100% PW-C200 condition, used as a negative control, to assess cyanobacterial performance in nutrient-deprived produced water. The PW-C200, enriched with nutrients for cyanobacteria growth, was adjusted to a pH of 6.8 by adding 0.1 M NaOH while monitoring the pH using an electrode and a magnetic stirrer to homogenize the medium. The pH was set so that it would not be too acidic for the survival of cyanobacteria nor too basic to induce spontaneous precipitation of CaCO3.

[0772] I.2.b Inoculation, incubation, growth, pH, and Ca monitoring

[0773] Each bioreactor was inoculated with 10 mg of biomass in 10 mL of culture media (biomass / volume ratio of 1 g / L) in duplicate (n=2) for each condition. The incubation was under controlled light and temperature conditions (12 hours of light at 21°C and 12 hours of darkness at 19°C, 7 days). The cyanobacterial growth was monitored by wet weight. The Ca concentration and pH were also monitored during experiments. After the experiments, the samples were examined under a microscope to observe the morphology of cyanobacteria. To compare growth rates between all treatment groups, the data was statistically analyzed using ANOVA.

[0774] I .3. Results and discussion

[0775] 1.3.1 Effect of produced water dilution on cyanobacteria growth and viability

[0776] Table 18 and FIG. 36 show the growth results of R. halophila and N. nodulosa in PW-C200 at different dilutions with RO water.WSGR Docket No. 69144-701601

[0777] In R. halophila, the results of the two-way ANOVA did not show statistically significant differences between the treatments (FIG. 36). This suggests that the strain exhibits similar levels of tolerance across the different PW-C200 concentrations, with a survival range between 60% and 75% (FIG. 37). Regarding control conditions, R. halophila successfully grew in BG11 but not in 100% PW-C200. The addition of nutrients to PW-C200 did not significantly improve biomass growth, as no differences were found between the negative control (PW-C200 without supplements) and PW-C200 with added supplements. However, according to microscopic observations, differences in morphology were identified between the two treatments (FIGs. 38A-F), with more affected morphology observed in those without nutrient addition. These findings suggest that nutrient supplementation or treatments prior to using PW should be optimized to improve conditions for strain growth, since PW-C200 is a chemically complex system very different from the BG11 medium to which the cyanobacteria are adapted. Cyanobacteria are versatile organisms capable of adapting to diverse ecosystems and harsh conditions, equipped with a range of physiological and molecular mechanisms that facilitate their survival and proliferation under stress. Therefore, a gradual adaptation process is being carried out using increasing concentrations of PW-C200. This will give the cyanobacteria time to make genetic, metabolic, or morphological adjustments to thrive in this new environment.

[0778] As for TV. nodulosa (FIG. 36), from 75% PW-C200 and above, there was a significant decrease in the survival rate, with values close to 0%, with no statistically significant differences among them. In the case of undiluted PW-C200 and the negative control with PW- C200, no viable cells were evident. This indicates that PW-C200 is particularly aggressive for this species, probably due to its morphological differences. At concentrations of 50% PW-C200 and below, although cell death also occurs, the survival rate is significantly higher than in the less diluted conditions, with the difference being statistically significant. The same pattern is observed in R halophila, but to a lesser extent (FIG. 37). This indicates that the toxic effects of PW-C200 decrease with dilution, resulting in a lower percentage of cell death (higherWSGR Docket No. 69144-701601 survival rate). These findings support the proposal for gradual adaptation strategies, in which cyanobacteria are exposed to progressively increasing concentrations of produced water to facilitate genetic, metabolic, and morphological adaptation.

[0779] Microscopic observations revealed a noticeable thinning of the sheath and trichomes in R. halophila (FIGs. 38A-38F). FIG. 38A shows a positive control; FIG. 38B shows 25:75 PW-C200; FIG. 38C shows 50:50 PW-C200; FIG. 38D shows 75:25 PW-C200; FIG. 38E shows 100:0 PW-C200; and FIG. 38F shows a negative control. While green pigmentation persisted, partially transparent or white filaments were evident at the 50:50, 75:25, and 100:0 PW-C200 ratios (FIG. 38C, FIG. 38D, and FIG. 38E) and in the negative control, a marked loss of pigmentation was observed (FIG. 38F). In contrast, N nodulosa exhibited severe morphological alterations (FIGs. 39A-39F. FIG. 39A shows a positive control; FIG. 39B shows 25:75 PW-C200; FIG. 39C shows 50:50 PW-C200; FIG. 39D shows 75:25 PW-C200; FIG. 39E shows 100:0 PW-C200; and FIG. 39F shows a negative control. N. nodulosa appeared almost entirely degraded from the 75:25 dilutions onward (FIG. 39D and FIG. 39E). At 50:50 and 25:75 ratios, some pigmentation remains detectable, though it appears yellowish (FIGs. 39B and FIG. 39C) These observations are consistent with the survival rate, which approached 0% at the 75:25 ratios and higher, while higher survival rates — around 50 - 75% — were observed at the 25:75 and 50:50 dilutions. Additionally, microscopic analysis revealed that PW-C200 dilutions improve cyanobacterial viability indicators, such as the presence of chlorophyll, a lower number of dead filaments, and reduced contamination by other organisms FIGs. 38A-38F and FIGs. 39A-39F). Although no increase in biomass was observed, cell viability improved compared to the unsupplemented PW-C200 negative control (FIGs. 38E and FIG. 38F)) This effect is particularly evident in R. halophila, which showed greater resistance to PW-C200.

[0780] 1.3.2 pH changes in the cultures with different PW-C200 dilutions

[0781] Regarding pH (Table 19), all conditions showed an increase in final pH compared to the initial value. However, in all treatment conditions involving PW-C200 dilutions — except for N. nodulosa at the 25:75 ratio — the final pH was equal to or slightly lower than that of the abiotic control. This suggests that the pH increase in PW-C200 treatments is mainly due to abiotic factors, such as chemical equilibration within the medium, as it is similar to the abiotic control and accompanied by reduced biomass. In contrast, in the positive control with BG11, the final pH was higher than that of the abiotic control for both cyanobacterial strains, which can be attributed not only to chemical processes but also to the metabolic activity of the cyanobacteria.WSGR Docket No. 69144-701601

[0782] I .4. Conclusions

[0783] a) The addition of macronutrients, micronutrients, and trace metals does not significantly increase the growth of R halophila and N. nodulosa in concentrated PW-C200. However, it does improve morphological stability in both strains, especially in R. halophila.

[0784] b) Although PW-C200 dilutions affected the growth of R. halophila, no statistically significant differences were observed between the different dilutions, suggesting similar tolerance across all concentrations, including undiluted PW-C200. After 7 days, survival rates ranged from 50% to 75%.

[0785] c) R halophila is the most suitable cyanobacterium for a precipitation assay, as it showed a higher survival rate than N nodulosa. Since no significant differences in survival rate were observed among the different dilutions of PW-C200, using the undiluted condition is preferable for precipitation assays, as it retains a higher calcium concentration.

[0786] II. Precipitation of CaCCh by R. halophila in PW-C200

[0787] II.1 Experimental setup: materials and methods

[0788] a. Cyanobacterial cultures: Rivularia halophila and Nodosilinea nodulosa.

[0789] b. Growth media: PW-C200.

[0790] c. Calcium source: natural calcium content in PW-C200.

[0791] d. Bicarbonate source: NaHCCh was added for precipitation experiments in order to obtain a pre-equilibrium condition for CaCCh precipitation. The concentration of NaHCCh was 800 and 1600 ppm.

[0792] e. pH meter: for monitoring and adjusting the pH of the culture media.

[0793] f. Calcium and CO2 selective electrodes: for monitoring dissolved Ca and DIC concentration of the medium.

[0794] g. Incubator: with controlled temperature and light conditions.

[0795] h. Bioreactors: flasks and / or small-scale bioreactors.WSGR Docket No. 69144-701601

[0796] i. Centrifuge: for harvesting cyanobacterial cells.

[0797] j. Filtration setup: to separate precipitated calcium carbonate.

[0798] k. Analytical balance: for weighing precipitates and final biomass.

[0799] 1. Microscope: petrographic optical microscope under polarized light to observe cyanobacterial cells and calcium carbonate crystals.

[0800] m. Analytical techniques for chemical and mineralogical characterization: use electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy microanalysis (EDXA) and X-ray diffraction (XRD) to analyze and characterize the precipitated calcium carbonate crystals and X-ray photoelectron spectroscopy (XPS) for the identification of the biogenic signal.

[0801] II.2. Experimental design:

[0802] II.2.a Preparation of growth media

[0803] Two growth media were used: PW-C200-800 and PW-C200-1600. These culture media contain PW-C200 with 800 and 1600 ppm of NaHCCh, and the pH was set at 6.5. The growth media were prepared as follows:

[0804] 1. The initial pH of PW-C200 (pH 4.3) was adjusted to 7.5 with 0.1 M NaOH.

[0805] 2. The PW-C200 with pH 7.5 was divided into two 150 mL portions and then 800 and 1600 ppm of NaHCCE were added.

[0806] 3. The mixtures were left in pre-equilibration for 24 hours to chemically precipitate the CaCCE.

[0807] 4. After this time, the pH was measured again and adjusted to 6.5.

[0808] 5. Finally, the supernatant was filtered and a portion was separated for use in the subsequent biogenic precipitation experiments.

[0809] - Control- PW-C200-800 and PW-C200- 1600 without R. halophila.

[0810] II.2.b Inoculation, incubation, and pH-Ca monitoring

[0811] The R. halophila strain was inoculated in PW-C200-800 and PW-C200-1600 in duplicate (n=2) for each condition. An amount of 1.7 g / L of biomass per litre of medium was used. The incubation was performed under control of light intensity (average daily light intensity 57.1 pmol / m2 / s). Samples were kept at room temperature for 7 days. The calcium concentration and changes in pH were monitored daily.

[0812] II.2.C Calcium carbonate precipitation analysis:

[0813] The CaCOs precipitate was extracted using H2O2 and washed with distilled water to remove any medium residues. Then, the solids were dried at 60°C until a constant weight wasWSGR Docket No. 69144-701601 achieved. Finally, the precipitate was weighed using an analytical balance and analyzed by polarized microscopy, XRD, SEM-EDS, and XPS.

[0814] II.3. Results and discussion

[0815] II.3.1 Changes in dissolved Ca concentrations and pH

[0816] FIG. 40A and FIG. 40B show the changes in pH over time. The experiments with R. halophila showed a greater increase in pH than the control during the first 120 hours, reaching pH values between 7.2 and 7.4 with PW-C200-800 (FIG. 40A) and PW-C200-1600 (FIG. 40B), respectively). In the PW-C200-800 experiments, no significant differences were observed in the pH compared with the control (FIG. 41A). However, in the PW-C200-1600, significant differences were identified between the cyanobacteria treatments and the control media, with a greater pH increase in the presence of microorganisms (FIG. 41A).

[0817] Regarding the dissolved Ca concentration that remained after precipitation, there were no significant differences between R. halophila experiments (617 ppm / day) and the control (590 ppm / day) in the PW-C200-800 experiments (FIG. 41B). However, under the 1600 ppm condition, a significant difference was found between the cyanobacteria treatments (768 ppm / day) and the control (509 ppm / day). This may be due to an excess of free Ca2+remaining in solution that was not incorporated into the precipitate, because part of the inorganic carbon was used by the cyanobacteria for their metabolism and not to precipitate Ca-carbonate.

[0818] II.3.2 Mineral identification and characterization: images with petrographic microscope, XRD, and SEM-EDS results

[0819] FIGs. 42A-42D include images, obtained with a petrographic optical microscope under polarized light, of the cyanobacteria samples from the experiments with 800 and 1600 ppm of NaHCCf. FIG. 42A shows an image of R. halophila acquired with a petrographic optical microscope using polarized light of pre-equilibration treatment with 800 ppm NaHCCE, where the images was taken with parallel Nicols. FIG. 42B shows an image of R. halophila acquired with a petrographic optical microscope using polarized light of pre-equilibration treatment with 800 ppm NaHCCE, where the images was taken with crossed Nicols. FIG. 42C shows an image of R. halophila acquired with a petrographic optical microscope using polarized light of pre-equilibration treatment with 1600 ppm NaHCCE, where the images was taken with parallel Nicols. FIG. 42D shows an image of R. halophila acquired with a petrographic optical microscope using polarized light of pre-equilibration treatment with 1600 ppm NaHCCE, where the images was taken with crossed Nicols. The CaCCE crystals are visible both under parallel nicols (FIGs. 42A and 42C) and crossed at 90° (FIGs. 42B and 42D). AWSGR Docket No. 69144-701601 greater presence of CaCCh crystals is observed in the R. halophila samples from the 1600 ppm experiment (FIGs. 42C and 42D) compared to the 800-ppm treatment (FIGs. 42A and 42B).

[0820] FIGs. 43A-43D show the XRD patterns of the samples treated with 800 ppm and 1600 ppm of NaHCCh. FIG. 43A shows the control and FIG. 43B shows the precipitate in the experiment with cyanobacteria at 800 ppm NaHCCE. FIG. 43C shows the control and FIG. 43D shows the precipitate in the experiment with cyanobacteria at 1600 ppm NaHCCE. All samples display broad bands between 20 and 60 29, similar to the band positions of ACC (amorphous calcium carbonate) as reported by Faatz et al. (2005) and Wang et al. (2015), indicating that the mineralized product is completely amorphous, with no crystallized phases. On the other hand, at 1600 ppm, the presence of crystalline minerals can be identified (calcite peak at 29 = 29.39). Despite the increased crystallinity, the mineral obtained remains more amorphous compared to the chemical control. This may be attributed to the ongoing reduction of bicarbonate in the medium, resulting not only from the precipitation process but also from its consumption by the cyanobacteria.

[0821] The SEM-EDS images show the morphology and chemical composition of the minerals precipitated with the cyanobacteria. Amorphous CaCCh structures were observed at the 800 ppm NaHCCh concentration (FIG. 44A), which is consistent with the XRD patterns identifying an amorphous material. At the 1600 ppm NaHCCE concentration, columnar flowerlike aggregates typical of aragonite were observed. However, this polymorph was not detected in the XRD analysis, where calcite was identified as the predominant crystalline phase (FIG. 44B) On the other hand, the control also showed both crystalline and amorphous structures, with a composition primarily of C, O, and Ca, along with other elements in smaller proportions (FIG. 44C)

[0822] II.3.3 Identification of the biogenic signal on the CaCOs precipitated: XPS results

[0823] After precipitation, polysaccharide-like components of EPS could be incorporated or mixed with the Ca-carbonate. Thus, the identification of functional groups of EPS in the Ca- carbonates is a fingerprint of the biogenic origin of the minerals. The biogenic signal was identified after the deconvolution of the XPS C is peaks.

[0824] FIGs. 45A-45D and FIGs. 46A-46F show the photoemission spectra of Ca 2p and C Is, respectively, measured for Ca-carbonates precipitated at PW-C200-800 and PW-C200- 1600 along with the corresponding precipitate in the control media. FIG. 45A depicts Ca 2p spectra obtained for PW-C200-800 with control media. FIG. 45B depicts Ca 2p spectra obtained for PW-C200-800 with R. halophila. FIG. 45C depicts Ca 2p spectra obtained for PW-C200-1600 with control media. FIG. 45B depicts Ca 2p spectra obtained for PW-C200-WSGR Docket No. 69144-7016011600 with N. nodulosa. FIG. 46A shows reference C is spectra in the control condition. FIG. 46B shows reference C is spectra obtained with R. halophila. FIG. 46C shows Cis spectra obtained for PW-C200-800 without R. halophila. FIG. 46D shows Cis spectra obtained for PW-C200-800 with R halophila. FIG. 46E shows Cis spectra obtained for PW-C200-1600 without R. halophila. FIG. 46F shows Cis spectra obtained for PW-C200-1600 with R. halophila. XPS parameters of core level spectra are given in Table 20.

[0825] According to literature, the Ca 2p peak for Ca-carbonate polymorphs occurs in a narrow range of binding energy values and cannot be well differentiated between them (Gopinath et al., 2002). Bands assigned to natural Mg-calcite and aragonite occur at 347.3 and 347.5 eV, respectively (Bia et al., 2021), and it is in good agreement with the studied samples.

[0826] Table 20. Parameters of high-resolution XPS C is spectra for PW-C200-800 and PW- C200-1600 along with the corresponding control media and reference materials. The full width at half maximum (FWHM, eV) and binding energies (BE, eV) were allowed to vary during the fitting.

[0827] FIGs. 45A-45D. Ca 2p spectra obtained for PW-C200-800 and PW-C200-1600 along with the corresponding control media. Solid black lines depict measured spectra; solid red lines indicate fits to measured spectra and dashed lines correspond to fits obtained with parameters indicated in Table 20. A Shirley background is included as a solid curve at the base of the peaks. The Ca 2p spectrum displays the spin-orbit doublet of 2p3 / 2 and 2pl / 2 of 3.5 eV.WSGR Docket No. 69144-701601

[0828] The deconvolution of the C Is signal generates four or five peaks referred to as Cl, C2, C3, C4, and C5, with binding energies between 284.8 and 290 eV. The first peak at 284.8 eV (i.e., Cl) was taken as an internal reference and was used to correct other spectra; it corresponds to aliphatic C atoms (C-C and C-H groups). The last peak at -290 eV (i.e., C5) is attributed to C-0 in carbonate samples (Gopinath et al., 2002; Bia et al., 2021).

[0829] The Cl, C2, and C3 are common contributions attributed to carbon contamination. These peaks are detectable in most of the samples that have been exposed to the atmosphere, and there was the main signal obtained in the Ca-carbonates precipitated in both control media. However, in the Ca-carbonate precipitated with R. halophila, the relative proportion of the C Is peaks changed. Thus, the C2, C3, and C4 components increase (Table 20 and FIGs. 46A- 46F). The larger peak areas of C2, C3, and C4 may suggest the formation of alcohol, primary amide or amine, aldehyde, ester, and sulfhydryl functional groups, presumably due to polysaccharide-like components of EPS incorporated or mixed with the Ca-carbonate.

[0830] FIGs. 46A-46F. C Is spectra obtained for PW-C200-800 and PW-C200-1600 along with the corresponding control media and reference materials. Solid black lines depict measured spectra; solid red lines indicate fits to measured spectra and dashed lines correspond to fits obtained with parameters indicated in Table 20. A Shirley background is included as a solid curve at the base of the peaks. The shaded area under the curve is assigned to carbonate contribution. Synthetic calcite was obtained from Bia et al. (2025).

[0831] II.3.4 CaCCh productivity

[0832] The average CaCO3productivity using R. halophila is 0.166 and 0.367 mg CaCCh / L per h, using 800 and 1600 ppm of NaHCCh, respectively (see Table 21). There is an increase in the CaCO3mass obtained of 0.055 and 0.23 mg for the 800 ppm and 1600 conditions, respectively.

[0833] Table 21. Data from the CaCO3precipitation (mg / L) experiment in production water (PW-C200).WSGR Docket No. 69144-701601

[0834] II .4. Conclusions

[0835] The pre-equilibration condition with 1600 ppm of NaHCCh showed significant differences compared to the control, both in the reduction of Ca2+concentration and in the increase of the medium’s pH.

[0836] The percentage reduction of Ca2+in the PW-C200 sample was 0.102%, compared to 0.035% in the control. These results indicated that this method can be used to reduce the salinity of an industrial wastewater, like PW.

[0837] The average CaCCh productivity under the 1600 ppm NaHCCh condition using R. halophila was 0.352 mg CaCCh / L h.

[0838] XRD and SEM analyses show that the precipitates formed under the 1600 ppm NaHCCh condition exhibit greater crystallinity than those formed at 800 ppm.

[0839] The identification of surface organic compounds in the CaCCh could be used as a spectroscopic indicator to predict a possible biogenic origin of the precipitates. Additionally, high proportions of C-N and C-S bonds on the surface of CaCCh could be a valuable proxy of the presence of cyanobacteria in the nearby region where precipitation occurs.Example 7. Seawater as culture media

[0840] Utilizing seawater as a growth medium for cyanobacteria presents numerous advantages, particularly given the high costs associated with current media. Seawater is inherently nutrient-rich, providing essential minerals and trace elements that can enhance biomass production and calcium carbonate precipitation. This approach is also cost-effective, as seawater is abundant and freely available, significantly reducing cultivation expenses. Additionally, growing cyanobacteria in seawater aligns with sustainable practices by promoting the utilization of natural resources while potentially mitigating the environmental impact of saline waste disposal. The natural salinity of seawater can also create an optimal environment for cyanobacterial growth, decreasing the need for supplemental nutrients. Overall, employing seawater as a culture medium not only supports efficient cyanobacterial cultivation but also contributes to more sustainable and economically viable production methods.

[0841] ObjectiveWSGR Docket No. 69144-701601

[0842] To investigate the growth of R. halophila and N nodulosa and their CaCCh precipitation capacity in natural and artificial seawater culture media.

[0843] 1. Experimental setup: materials and methods a) Cyanobacterial cultures: Rivularia halophila and Nodosilinea nodulosa. b) Growth culture medium: c) b.1) Seawater as culture media: a natural seawater (NSW) medium was prepared using seawater obtained from the Sauce Grande area, Buenos Aires Province (Argentina).

[0844] b2) Artificial seawater as culture media: artificial seawater (ASW) was prepared based on the concentrations of ions present in seawater. d) Autoclave: To sterilize the stock solutions, vials, and pipette tips. The usual conditions of temperature and pressure used for this are 121°C and 0.13 MPa for 30 minutes. e) Laminar air flow cabin: To prepare the culture medium and inoculate the cultures in sterile conditions. f) pH meter: for monitoring and adjusting the pH of the medium. g) Calcium and CO2 electrodes: for monitoring calcium concentration and DIC concentration of the medium. h) Incubator: with controlled temperature and light conditions. i) Bioreactors: vials and / or small-scale bioreactors. j) Centrifuge: for harvesting cyanobacterial cells. k) Analytical balance: for weighing precipitates and final biomass. l) Microscope: petrographic optical microscope under polarized light to observe cyanobacterial cells and calcium carbonate crystals. m) Analytical techniques for crystal studying: use electron microscopy (SEM) coupled with EDS and X-ray diffraction (XRD) to analyze and characterize the precipitated calcium carbonate crystals.

[0845] 2. Experimental design.

[0846] 2.1) Preparation of growth media

[0847] 2.1a) Natural seawater as culture media: an NSW medium was prepared using seawater to which macro- and micronutrients were added to optimize cyanobacteria growth (Table 22). Visual MINTEQ software was used to determine ideal pH, macronutrient, and bicarbonate values to prevent the formation of unwanted precipitates.

[0848] The seawater was filtered twice using paper filters to remove sediment and sterilized using 0.2 pm Sartorius filters, after which nutrients were added. Then pH, dissolved inorganic carbon (DIC), and free [Ca2+] were immediately measured using ThermoFisher ScientificWSGR Docket No. 69144-701601Orion™ electrodes. The calibration curves for CO2 and Ca2+measurements are depicted inFIG. 47A and FIG. 47B, respectively.

[0849] 2.1b) Artificial seawater as culture media: precipitation in the NSW environment was compared with the same process performed in ASW, which was prepared based on the ion concentrations present in seawater (Tables 23-and 25).WSGR Docket No. 69144-701601

[0850] 2.1c). Preparation of stock solutions: in both cases, the stock solutions were prepared and autoclaved separately, then mixed during medium preparation to prevent precipitation. Sodium bicarbonate was added after measuring the DIC in the natural seawater at the time of the experiment, and the pH was adjusted to similar values (7.86) using hydrochloric acid and sodium hydroxide.

[0851] 2.2) Calcium and bicarbonate sources: Intrinsic calcium and bicarbonate present in seawater (434.8 ppm Ca2+and 364.2 ppm HCO3 ) were supplemented to enhance the precipitation process. [Ca2+] was increased to match the magnesium concentration naturally present in seawater since lower Ca / Mg ratios interfere in the precipitation of calcium carbonate. In artificial seawater it is necessary to add both Ca2+(between 5 - 25 mM) and HCCh' ( 10 mM) in order to equal the concentration, present in the natural seawater medium.

[0852] 2.2) Quantification of dissolved inorganic carbon (DIC) and Calcium (Ca2+)

[0853] DIC was measured as CO2. In analytical procedures, carbon dioxide buffer solutions were added to samples and standards before measurement. After the addition of the buffer solution, all samples and standards should fall within the pH 4.8 to 5.2 range so that all dissolved bicarbonate and carbonate species in solution are converted to H2CO3 / CO2 and so that possible interferences are minimized. For quantification, the Orion™ 9502BNWP carbon dioxide selective electrode was used. The calibration curve was created following the manufacturer's instructions (Thermo Scientific). The curve showed an R-squared value of 0.999 (FIG. 22A). Dissolved CO2 concentration measurements were made in the BG11-saline medium before and after 0.2 pm filtration (medium sterilization).

[0854] Calcium was measured as Ca2+. For quantification, the 9729BNWP Calcium Combination Electrode was used. The calibration curve was created following the manufacturer's instructions (Thermo Scientific). The curve showed an R-squared value of 0.9995 (FIG. 22A). Dissolved Ca2+concentration measurements were made in the BG11- saline medium before and after 0.2 pm filtration (medium sterilization).

[0855] 2.3) Inoculation, incubation, and data collection:

[0856] Cultures of R. halophila and N. nodulosa were grown on NSW and ASW media, along with a chemical control consisting of media without cyanobacteria, to compare the precipitate formed in the presence and absence of cyanobacteria (FIG. 48). Approximately 35WSGR Docket No. 69144-701601 mg of biomass of each strain were inoculated into 30 mL glass vials using 20 mL of medium, maintaining a biomass / volume ratio of 1.7 g / L. Cultures were grown in a germinator at a constant temperature of 21 °C for 7 days under artificial light in a 12 h light / 12 h dark photoperiod. Luminosity was measured using a Phantom PhotoBio PAR meter to avoid light conditions that were too intense, which could cause photoinhibition of growth or be too low to allow photosynthesis, maintaining cultures within a range of 50-80 pmol.m2 / s.

[0857] Each condition was evaluated in triplicate, and the experiment was finished 7 days after it began. Once the experiment was completed, DIC (FIG. 49B), [Ca2+] (FIG. 49C), pH (FIG. 49A), and final biomass were quantified. Samples were also taken to analyze cell morphology and the formation of crystals by petrographic microscopy. The biomass was then treated with 30% hydrogen peroxide to remove it, and the remaining mineral precipitate was weighed. This precipitate was subsequently analyzed by XRD and SEM to evaluate composition and crystallinity. Finally, the productivity and yield of the calcium carbonate precipitation process with and without cyanobacteria were evaluated. The results obtained in ASW and NSW media were compared using a one-way ANOVA test.

[0858] 3. Results and discussion

[0859] 3.1) Growth and viability of R halophila and N. nodulosa in NSW and ASW

[0860] Both R. halophila and N. nodulosa significantly increased their biomass in NSW and ASW media. No statistically significant differences were observed in either medium (p- value >0.05). This increase in biomass indicates that seawater supplemented with nutrients (NaNCh, K2HPO4, ammonium citrate and iron, trace metals, and vitamin B12) is a viable culture medium for the growth of R. halophila and N nodulosa (Mouga et al., 2024; Lu et al., 2019; Ruter et al., 1987).WSGR Docket No. 69144-701601

[0861] 3.2) Changes in pH, DIC, and [Ca2+]

[0862] R. halophila cultures increased the pH of the culture media due to the cyanobacterial metabolism (Zerveas et al., 2021), favoring the precipitation of calcium carbonate. The increase was much lower for N. nodulosa but still evident in ASW medium. In line with precipitation, a significant decrease in [Ca2+] levels was observed compared to the initial values.

[0863] The DIC concentration also decreased significantly in the media with cyanobacteria but not in the chemical controls. Part of this decrease is due to the fact that cyanobacteria favor the selective precipitation of calcium carbonate. This is why, in the controls, the [Ca2+] concentration decreased but not the DIC concentration. No significant differences were observed in the [Ca2+] and DIC levels after the experiment between the NSW and ASW media (Dittrich et al, 2010; Jansson et al., 2010). The DIC decrease is also due to the metabolism of the cyanobacteria, which captures and internalizes soluble carbon for carbohydrate biosynthesis, incorporating it into their biomass (Bhai et al., 2025; Zhang et al., 2023).

[0864] Table 27. pH, DIC, and [Ca2+] quantified in the media with cyanobacteria, the chemical controls without cyanobacteria, and the medium at the beginning of the experiment (NSWi and ASWi).WSGR Docket No. 69144-701601

[0865] 3.3 Mineral identification and characterization: images with petrographic microscope, SEM-EDS, and XRD results

[0866] FIGs. 50A-50H are petrographic images obtained of N. nodulosa cultures, both in NSW and ASW medium, where a large number of crystals of varying size and shape were observed. FIG. 50A shows an image taken with the petrographic microscope with polarized light using parallel nicols of R. halophila NSW. FIG. 50B shows an image taken with the petrographic microscope with polarized light using crossed nicols of R. halophila NSW. FIG. 50C shows an image taken with the petrographic microscope with polarized light using parallel nicols of R. halophila ASW. FIG. 50D shows an image taken with the petrographic microscope with polarized light using crossed nicols of R. halophila ASW. FIG. 50E shows an image taken with the petrographic microscope with polarized light using parallel nicols of N nodulosa NSW. FIG. 50F shows an image taken with the petrographic microscope with polarized light using crossed nicols of N nodulosa NSW. FIG. 50G shows an image taken with the petrographic microscope with polarized light using parallel nicols of N nodulosa ASW. FIG. 50H shows an image taken with the petrographic microscope with polarized light using crossed nicols of N. nodulosa ASW. Many of the crystals formed around N. nodulosa cultures in NSW medium exhibit rosette-like shaped aggregates that could indicate an early stage of aWSGR Docket No. 69144-701601 crystallization process, perhaps due to the presence of interferences in the NSW that reduce the crystallization process. On the contrary, in the ASW medium, well-defined calcite crystals can be observed (FIG. 50D).

[0867] The presence of calcium carbonate crystal was scarce for R. halophila cultures, although in the ASW medium, the precipitation was more abundant, and also the presence of rosette-like shaped aggregates of early crystals could be observed. However, in the NSW medium, crystalline phases were not clearly defined, and the calcium carbonate is presented as amorphous aggregates.

[0868] It is important to mention that the cell morphology was not adversely affected by growth in either medium. No apparent decrease in chlorophyll levels or abnormally shaped trichomes was observed. No dead cells or broken sheaths that could indicate cell lysis were found. There was good cohesion between colonies, suggesting that extracellular polymeric substances (EPS, in the case of R. halophila) and biofilm formation (in the case of N. nodulosa) were not altered. This reaffirms the viability of nutrient-supplemented seawater as a culture medium for R. halophila and N. nodulosa.

[0869] Similar to those obtained using a petrographic microscope, the images obtained with SEM of R halophila in NSW medium reveal amorphous calcium carbonate aggregates, which are more crystalline structures in ASW. According to the chemical composition (Table 28), some biomass is still present, as is indicated by the low calcium-to-carbon ratio determined in the EDS analysis.

[0870] N nodulosa cultures in NSW medium produce elongated crystals, exhibiting tabular structures (FIG. 51E). Although this morphology could respond to the presence of some impurities in the medium, the purity of the calcium carbonate (C, O, and Ca) obtained for N nodulosa in NSW (94.7%, see Table 28) was the highest. This aligns with the high crystallinity observed in the XRD results under these similar conditions (see FIGs. 51A-51F). FIGs. 51A- 51F depict the images obtained with the scanning electron microscope (SEM) of the precipitate recovered from: R halophila NSW (FIGs. 51A-51B), R. halophila ASW (FIGs. 51C-51D) and A. nodulosa NSW cultures (FIGs. 51E-51F).

[0871] The presence of other elements (i.e., P, Cl, Na, Fe, and Mg; see Table 28) remaining in the NSW with R. halophila may act as impurities that prevent the formation of well-defined crystals.WSGR Docket No. 69144-701601

[0872] The XRD patterns confirm the presence of calcite crystals in the precipitate formed by N. nodulosa, but not for R. halophila (FIGs. 52A-52F). FIGs. 52A-52F depict X-ray diffraction patterns of the precipitate recovered from: FIG. 52A) NSW medium control, FIG. 52B) R. halophila in NSW medium, FIG. 52C) N. Nodulosa in NSW medium, FIG. 52D) ASW medium control, FIG. 52E) R halophila in ASW medium, FIG. 52F) N Nodulosa in ASW medium. In fact, better crystallinity was obtained for TV. nodulosa in ASW, while a rather amorphous calcium carbonate (ACC) was developed for R halophila in the same medium. In the control patterns, halite (sodium chloride) was identified.

[0873] 3.4 CaCCh productivity

[0874] The amount of precipitation produced in the chemical controls was very low for both ASW and NSW media. According to XRD, the main precipitate produced was halite. R. halophila cultures also did not show a significant increase in precipitate formation. In contrast, N nodulosa cultures were able to produce much more precipitation. Therefore, the productivity and yield of the precipitation process are greatly enhanced in culture media with the presenceWSGR Docket No. 69144-701601 of cyanobacteria compared to the chemical control. This improvement is more pronounced for N. nodulosa cultures than for R. halophila cultures. FIGs. 53A-53C. Precipitate mass (FIG. 53A), productivity (FIG. 53B), and percentage yield (FIG. 53C) obtained from media containing cyanobacteria and chemical controls without cyanobacteria. The columns represent the average of the replicates, and the bar represents the standard error.WSGR Docket No. 69144-701601

[0875] 4. Conclusion

[0876] 1) Nutrient-supplemented seawater proved to be an optimal culture medium for the growth of the cyanobacteria strains R. halophila and N. nodulosa. promoting both cell growth and viability.

[0877] 2) The precipitation of CaCCh in seawater with the addition of [Ca2+] and NaHCCh is an effective bioprocess where cyanobacteria favor the selective precipitation of [Ca2+] and HCCh' over other ions present in the medium.

[0878] 3) Well-defined calcite crystals were obtained only with N. nodulosa in NSW and ASW, while ACC was produced by R. halophila.

[0879] 4) A marked decrease in dissolved carbon levels in the medium was determined, demonstrating the efficiency of cyanobacteria in carbon uptake. This suggests that microbial precipitation of CaCCh by cyanobacteria is a carbon-negative process. It should be emphasized that obtaining more crystalline precipitates in the artificial seawater medium opens the possibility of optimizing the calcium carbonate precipitation bioprocess in natural seawater to obtain a higher-quality product with improved productivity. This is primarily due to the fact that the ASW medium is a more controlled medium with well-defined concentrations, unlike the NSW medium, whose composition is not known with absolute precision, resulting in a lower presence of interfering agents that could affect the calcium carbonate precipitation reaction.Example 8. Optimized culture media

[0880] To address the current challenges of low productivity, titer, and yield in cyanobacterial cultivation, optimizing culture media is essential. By systematically assessing and refining the composition of growth media, we can identify the optimal nutrient ratios and concentrations that support enhanced metabolic activity and biomass accumulation. This optimization may involve experimenting with various nitrogen, phosphorus, and micronutrient sources, as well as exploring the inclusion of micronutrients and growth factors that can stimulate cyanobacterial performance. Improved culture media can lead to higher biomass productivity and titer, ultimately increasing yield and making the cultivation process more economically viable.

[0881] Objective

[0882] To optimize the culture media for cyanobacterial cultivation to improve productivity, titer, and yield by systematically assessing and refining the composition of growth media.

[0883] 6.1. Experimental setup: materials and methods a) Cyanobacterial cultures: pure culture of A. halophila and A. nodulosaWSGR Docket No. 69144-701601 b) Growth medium: baseline BG11-saline media. c) Nutrient sources: nitrogen and phosphorus d) Micronutrients and growth factors: essential trace metals, vitamins, and other growthpromoting substances. e) pH meter: for monitoring and adjusting pH. f) Incubator: with controlled temperature and light conditions. g) Bioreactors: flasks and / or bioreactors h) Centrifuge: for harvesting cyanobacterial cells. i) Analytical balance: for weighing biomass and precipitates. j) Microscope: to observe cyanobacterial cells and calcium carbonate crystals.

[0884] 6.2. Experimental design

[0885] 6.2. a. Preparation of growth media

[0886] BGl l-saline medium, supplemented with 3.6 gL1of NaCl, was used as the basal culture medium for all experiments.

[0887] 6.2.a.l Growth medium with variation in the N:P ratio

[0888] All culture media were based on standard BG11 composition, maintaining constant concentrations of all components except for nitrogen (NaNCh) and phosphorus (K2HPO4), which were selectively varied. Two experimental sets were designed and three conditions were studied for each macronutrient (Table 31):

[0889] Nitrogen-fixed, phosphorus-variable: NaNCh was maintained at 17.6 mM, while K2HPO4 was adjusted to 0.165 mM, 0.275 mM and 0.55 mM.

[0890] Phosphorus-fixed, nitrogen-variable: K2HPO4 was kept constant at 0.23 mM, while NaNCh was varied to 5.87 mM, 8.8 mM and 35.2 mM.

[0891] All other components, including trace metals, vitamins, antifungal agents, and chelators, remained unchanged across conditions. On the other hand, the BG11 medium without any modifications was used as the control condition.WSGR Docket No. 69144-701601

[0892] The data refers to the final concentrations and ratio. Sodium nitrate (NaNOs) solution stock: 1,76M; Dipotassium phosphate (K2HPO4) solution stock: 22mM. *BGl l-saline N:P radio is 76.52. **It represents how many times higher or lower the phosphorus or nitrogen concentration is in the treatment relative to the control.

[0893] 6.2. a.2 Growth medium using commercial fertilizers and laboratory-grade fertilizer

[0894] Two culture media were prepared by replacing the nitrogen and phosphorus sources. In both conditions, sodium nitrate (NaNCh) and potassium phosphate (K2HPO4) were omitted and substituted with ammonium phosphate ((NIT^HPC ) at an equivalent molar concentration (17.6 mM) to BG11. Condition 1 used fertilizer-grade (NIT^HPCX while condition 2 used a laboratory-grade version. All other components — such asNa2COs, MgSO4 7ILO, CaCb 2EL>0, ferric ammonium citrate, EDTA, trace metals, and vitamins — remained unchanged.

[0895] 6.2. a.3 Micronutrient variation

[0896] Iron(II) sulfate (FeSO4.7 H2O) and iron(III) chloride (FeCh) were tested as alternative iron sources. Hereafter, iron(II) sulfate is referred to simply as FeSCk Their concentrations were adjusted to match the iron level present in standard BG11 medium (0.021 mM). For this purpose, 21 mM stock solutions of each compound were prepared.

[0897] 6.2.b. Inoculation, incubation, and growth monitoring

[0898] Each bioreactor was inoculated with a biomass / volume ratio of 1 g / L in triplicate for each condition. The incubation was under controlled light and temperature conditions (12 hours of light at 21°C and 12 hours of darkness at 19°C, 7 days). The cyanobacterial growth was monitored by wet weight. The pH was measured at the beginning and at the end of the experiment, after which the samples were examined under a microscope to assess cyanobacterial morphology.

[0899] 6.2. c. Statistical analysis

[0900] Final wet biomass was measured, and the results are presented as: A) relative growth and B) fold change in relative growth. The former was calculated as the ratio between final and initial fresh biomass, while the latter corresponds to the normalization of each treatment's relative growth against the control. For relative growth, a value of 1 indicates no net growth; values >1 indicate biomass increase; values <1 indicate biomass loss. On the other hand, a fold change of 1 indicates growth equal to the control; values >1 indicate growth higher than the control; values <1 indicate growth lower than the control. Data are presented as mean ± SEM. One-way ANOVA followed by Tukey ’ s multiple comparisons test was used to assess statistical significance, with p < 0.05 considered significant.WSGR Docket No. 69144-701601

[0901] 6.3. Results and discussion

[0902] 6.3.1 Effect of using different N:P ratios in the culture medium on cyanobacteria growth

[0903] To evaluate the impact of phosphorus and nitrogen availability on Rivularia halophila and Nodosilinea nodulosa growth, cultures were exposed to different concentrations of sodium phosphate and sodium nitrate in BG11 medium and compared to the standard formulation.

[0904] For phosphorus, three concentrations of sodium phosphate (0.03, 0.05, and 0.1 g / L) were tested alongside the control (FIGs. 54A-54B). FIG. 54A shows the relative growth of R. halophila following administration of 0,03, 0,05, or 0, 1 g / L of phosphorus. FIG. 54B shows the fold change in relative growth of R. halophila following administration of 0,03, 0,05, or 0,1 g / L of phosphorus. The control condition (BG11) yielded the highest average relative growth (1.64), while lower values were observed in all modified treatments (ranging from 1.11 to 1.46). Fold-change analysis revealed that none of the tested phosphorus concentrations enhanced growth compared to the control. The highest phosphate condition (0.1 g / L) resulted in a fold change of -0.84, indicating slightly reduced growth, whereas the lowest phosphate condition (0.03 g / L) showed the most pronounced decrease (fold change -0.64). These results suggest R. halophila grows optimally under standard phosphorus conditions, with reductions leading to moderate biomass declines.

[0905] Regarding nitrogen, cultures were grown with three NaNCh concentrations (0.5, 0.75, and 3.0 g / L) compared to the standard 1.5 g / L NaNCh control (FIGs. 54C-54D). FIG. 54C shows the relative growth of R. halophila following administration of 0,03, 0,05, or 0,1 g / L of nitrogen. FIG. 54D shows the fold change in relative growth of R. halophila following administration of 0,03, 0,05, or 0, 1 g / L of nitrogen. The control medium supported robust growth (average relative growth = 1.64-1.81). Low nitrogen availability (0.5 g / L) led to reduced biomass accumulation (relative growth - 1.24-1.56; fold change - 0.72-0.90). Conversely, high nitrogen concentration (3.0 g / L) slightly enhanced growth (relative growth - 1.78-2.07; fold change - 1.03-1.20), indicating that / ?, halophil tolerates and may even benefit from elevated nitrogen levels. Intermediate concentrations, such as 0.75 g / L, produced growth values similar to the control (relative growth - 1.57-1.98; fold change - 0.91-1.14). However, these differences were moderate, with no strong inhibition or stimulation observed across the tested range. Therefore, using 0.75 g / L NaNCh could be a viable strategy to grow cyanobacteria without compromising biomass production, potentially allowing a 50% reduction in sodium nitrate usage.WSGR Docket No. 69144-701601

[0906] As for Nodosilinea nodulosa, no significant differences were observed among the different sodium phosphate (FIGs. 55A-55B) and sodium nitrate (FIGs. 55C-55D) concentrations tested, with growth levels similar to the control. FIG. 55A shows the relative growth of N. nodulosa following administration of 0,03, 0,05, or 0,1 g / L of phosphorus. FIG. 55B shows the fold change in relative growth of N. nodulosa following administration of 0,03, 0.05, or 0, 1 g / L of phosphorus. FIG. 55C shows the relative growth of N. nodulosa following administration of 0,5, 0.75, or 3 g / L of nitrogen. FIG. 55D shows the fold change in relative growth of N. nodulosa following administration of 0,5, 0,75, or 3 g / L of nitrogen. For phosphorus, the fold-change values were very close to 1 across the three concentrations used (0.03 g / L: 0.93-1.19; 0.05 g / L: 0.96-1.11; 0.1 g / L: 0.84-1.01), indicating growth comparable to the control (FIG. 55A and FIG. 55B). Although the 0.1 g / L phosphorus concentration showed a slightly wider range of lower values, no statistically significant differences were found. This suggests that none of the phosphorus concentrations tested negatively affected or reduced biomass production in this cyanobacterium. Therefore, using the lowest concentration of 0.03 g / L is feasible, which could reduce sodium phosphate concentration by approximately 25%.

[0907] On the other hand, as for nitrogen, a similar situation was observed. No significant differences were found among the tested concentrations, as values were close to the control (fold-change: 0.5 g / L: 0.97-0.99; 0.75 g / L: 0.87-0.98; 3 g / L: 0.88-1.15) (FIG. 55C and FIG. 55D). Therefore, the sodium nitrate concentration could be reduced up to threefold, using 0.5 g / L, without compromising the growth or biomass production of the cyanobacterium.

[0908] Microscopic images revealed no apparent morphological alterations for either of the cyanobacteria species, indicating that the experimental conditions did not adversely affect the structural integrity of the organisms (FIGs. 56A-56Hand FIGs. 57A-57H). FIG. 56A shows a petrographic microscope image of R. halophila after 5 days of exposure to 0 g / L phosphorus. FIG. 56B shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0 g / L phosphorus. FIG. 56C shows a petrographic microscope image of R. halophila after 5 days of exposure to 0.03 g / L phosphorus. FIG. 56D shows a petrographic microscope image of A. nodulosa after 5 days of exposure to 0.03 g / L phosphorus. FIG. 56E shows a petrographic microscope image of R. halophila after 5 days of exposure to 0.05 g / L phosphorus. FIG. 56F shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0.05 g / L phosphorus. FIG. 56G shows a petrographic microscope image of R. halophila after 5 days of exposure to 0.1 g / L phosphorus. FIG. 56H shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0.1 g / L phosphorus. FIGs. 57A-57H depict petrographicWSGR Docket No. 69144-701601 microscope images of R. halophila and N. nodulosa after 5 days of exposure to different nitrogen concentrations. FIG. 57A shows a petrographic microscope image of R. halophila after 5 days of exposure to 0 g / L nitrogen. FIG. 57B shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0 g / L nitrogen. FIG. 57C shows a petrographic microscope image of R halophila after 5 days of exposure to 0.5 g / L nitrogen. FIG. 57D shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0.5 g / L nitrogen. FIG. 57E shows a petrographic microscope image of R. halophila after 5 days of exposure to 0.75 g / L nitrogen. FIG. 57F shows a petrographic microscope image of N nodulosa after 5 days of exposure to 0.75 g / L nitrogen. FIG. 57G shows a petrographic microscope image of R. halophila after 5 days of exposure to 3 g / L nitrogen. FIG. 57H shows a petrographic microscope image of N nodulosa after 5 days of exposure to 3 g / L nitrogen.Long, flexible trichomes were observed, showing no signs of fragmentation, deformation, or changes in pigmentation, which aligns with the growth measurements, as all strains grew under the tested conditions. This suggests a normal morphological and physiological response to the applied treatments.

[0909] 6.3.1.1 Biomass productivity

[0910] The results presented here, together with previous data, provide insight into the effect of phosphorus and nitrogen availability on biomass production in both cyanobacteria. Regarding phosphorus availability (FIGs. 58A-58F and Tables 32A-32B), all tested concentrations led to reduced productivity in R halophila compared to the control (titer: control = 1.74 g / L; 0.03 g / L = 1.22 g / L; 0.05 g / L = 1.37 g / L; 0.1 g / L = 1.36 g / L). FIG. 58A depicts the effect of phosphorus concentration on titer of R halophila and N. nodulosa. FIG. 58B depicts the effect of phosphorus concentration on productivity of R halophila and N nodulosa. FIG. 58C depicts the effect of phosphorus concentration on %SY of R. halophila and N. nodulosa. FIG. 58D depicts the effect of nitrogen concentration on titer of R. halophila and N. nodulosa. FIG. 58E depicts the effect of nitrogen concentration on productivity of R. halophila and N. nodulosa. FIG. 58F depicts the effect of nitrogen concentration on %SY of R. halophila and N. nodulosa. The corresponding %SY values also showed a marked decline, from 10.34% in the control to just 1.83% at 0.1 g / L. These results suggest that neither low nor high phosphorus levels are favorable for this species, possibly due to metabolic imbalances under nutrient-limiting or excess conditions. In contrast, N. nodulosa showed its highest productivity and titer at 0.03 g / L phosphorus (titer = 1.21 g / L, productivity = 0.00015 g / L / day), slightly outperforming the control (titer = 1.09 g / L, productivity = 0.00009 g / L / day). Additionally, %SY more than doubled at 0.03 g / L (3.38%) compared to the control (1.43%),WSGR Docket No. 69144-701601 indicating that reduced phosphorus supply may improve metabolic efficiency in this species without compromising biomass yield.

[0911] For nitrogen levels (Table 32B), R. halophila exhibited a clear positive response to moderate nitrogen concentrations, with 0.75 g / L yielding the highest values for titer (1.88 g / L), productivity (0.00069 g / L / day), and %SY (0.667%). These results suggest that R. halophila benefits from nitrogen levels around or below the standard BG11 concentration (1.5 g / L). However, increasing nitrogen to 3 g / L led to a decrease in %SY (0.188%), suggesting that excessive nitrogen may reduce metabolic efficiency and lead to diminishing returns. In contrast, N. nodulosa displayed relatively stable titers across all nitrogen treatments (ranging from 1.03 g / L to 1.16 g / L), with only minor changes in productivity and %SY. The best result was obtained at 3 g / L (titer = 1.16 g / L, productivity = 0.00010 g / L / day, %SY = 0.025%), slightly exceeding the control (titer = 1.09 g / L, productivity = 0.00009 g / L / day, %SY = 0.041%), but the differences were not substantial. These observations suggest that N nodulosa is less sensitive to nitrogen variation and that 0.5 g / L sodium nitrate may be sufficient to support biomass production while potentially reducing cultivation costs.

[0912] Table 32A and 32B. Yield data is expressed as a function of phosphorus variation (A) and nitrogen variation (B) in optimized culture media.

[0913] 6.3.2 Effect of using commercial fertilizer and lab oratory -grade fertilizer on cyanobacteria growthWSGR Docket No. 69144-701601

[0914] Ammonium hydrogen phosphate ((NFL^HPCN) was tested as a combined nitrogen and phosphorus source, with its concentration adjusted to match the nitrogen level of standard BG11. Two grades were evaluated: a laboratory -grade reagent and an agri cultural -grade fertilizer commonly used in farming. The results showed that neither source was a suitable replacement for nitrogen and phosphorus, as growth was minimal or even resulted in biomass loss, both being lower than the control for both cyanobacteria species. For Rivularia halophila (FIG. 59A), relative growth values ranged from 0.94 to 1.00 for the agricultural-grade, and 1.16 to 1.20 for the laboratory-grade. In Nodosilinea nodulosa (FIG. 59B), values ranged from 0.70 to 0.86 for the agri cultural -grade, and 0.79 to 0.96 for the laboratory-grade. The negative effect was more pronounced in N. nodulosa, and in particular with the agricultural-grade fertilizer, likely due to the presence of impurities or additional compounds commonly found in this type of fertilizer, which may negatively affect cyanobacterial physiology. Overall, these results indicate that using ammonium hydrogen phosphate in either grade is not recommended as a nitrogen and phosphorus source for these cyanobacteria.

[0915] Additionally, optical microscopy images revealed morphological alterations in both cyanobacteria (FIGs. 60A-60F), most notably a reduction in pigmentation. FIG. 60A depicts a petrographic microscope image of R. halophila after 7 days of exposure to a control solution. FIG. 60B depicts a petrographic microscope image of N nodulosa after 7 days of exposure to a control solution. FIG. 60C depicts a petrographic microscope image of R halophila after 7 days of exposure to agri cultural -grade (NFUjjHPC FIG. 60D depicts a petrographic microscope image of N nodulosa after 7 days of exposure to agri cultural -grade (NHThHPC FIG. 60E depicts a petrographic microscope image of R halophila after 7 days of exposure to laboratory-grade (NHThHPC FIG. 60F depicts a petrographic microscope image of N nodulosa after 7 days of exposure to lab oratory -grade (NHThHPC In some cases, yellow or transparent filaments were observed, along with thinning of the trichomes, particularly in Rivularia halophila (FIGs. 60C-60E). These results indicate that using (NH4)2HPO4 as a source of nitrogen and phosphorus affects not only biomass production but also overall cellular metabolism, ultimately influencing biomass yield. This highlights that the origin and quality of the nutrient source can be sufficient to impact R. halophila or N. nodulosa growth.

[0916] 6.3.2.1 Biomass productivity

[0917] Table 33 shows the efficiency of biomass production for each (NH4)2HPO4 type tested. In both cyanobacteria, all evaluated parameters were higher in the control than in the treatments, regardless of the grade of (NH4)2HPO4 used. These findings are consistent with previous results. The difference between the control and the treatments was more pronouncedWSGR Docket No. 69144-701601 in R. halophila than in N. nodulosa, which may indicate that the nitrogen and phosphorus source has a stronger effect on the former. The control condition reached a titer of 1 .46 g / L, surpassing the values obtained with agricultural-grade (1 .00 g / L) and laboratory-grade (1.18 g / L) (NH4)2HPO4. A similar pattern was observed for productivity, with the control showing a value of 0.004 g / L-h, while the agricultural- grade and laboratory-grade treatments showed even lower or negative values (-0.0003 and 0.0015 g / L-h, respectively). In contrast, in N. nodulosa, although the differences were less pronounced, the control still showed higher titer and productivity values (1.00 g / L and 0.0003 g / L h, respectively).

[0918] Regarding yield, the same pattern was observed. The highest values were recorded in the control (0.019 g biomass / g substrate for A. halophila and 0.002 g biomass / g substrate for N nodulosa), followed by the (NH4)2HPO4laboratory-grade treatment (0.004 and -0.003 g biomass / g substrate, respectively), and lastly the agricultural-grade (NH4)2HPO4(-0.001 and -0.005 g biomass / g substrate, respectively), with some values even reaching negative yields.

[0919] * Yield calculations were performed based on the amount of nitrogen added to the culture medium.

[0920] 6.3.3 Effect of using other sources of iron on cyanobacteria growth: micronutrients variations

[0921] Iron(II) sulfate (FeSCk? H2O) and iron(III) chloride (FeCL) were tested as alternative iron sources, with their concentrations adjusted to match the iron level in standard BG11 medium. The aim was to evaluate whether they could serve as suitable replacements for ferric ammonium citrate, which is commonly used in BG11. In Rivularia halophila, the results indicated that neither iron source led to biomass loss or growth inhibition, suggesting that both could serve as viable alternatives to ferric ammonium citrate (FIG. 61A). Although the differences between treatments and the control were not statistically significant, a slight trendWSGR Docket No. 69144-701601 toward enhanced growth was observed with FeSO4, and even more so with FeCF. Relative growth values ranged from 1.40 to 1.55 for FeSO4 and from 1.41 to 1.60 for FeCL, compared to 1.32 to 1.39 in the control, indicating a modest yet consistent increase in biomass accumulation. In the case of Nodosilinea nodulosa (FIG. 61B), replacing ferric ammonium citrate with FeSO4 or FeCh resulted in minimal or no growth. Relative growth values ranged from 1.04 to 1.69 for FeSO4, 0.92 to 1.14 for FeCh, and 1.07 to 1.19 for the control.

[0922] FIG. 62A depicts a petrographic microscope image of R. halophila after 7 days of exposure to a control solution. FIG. 62B depicts a petrographic microscope image of N nodulosa after 7 days of exposure to a control solution. FIG. 62C depicts a petrographic microscope image of R. halophila after 7 days of exposure to FeSC . FIG. 62D depicts a petrographic microscope image of N nodulosa after 7 days of exposure to FeSO44. FIG. 62E depicts a petrographic microscope image of R halophila after 7 days of exposure to FeCh. FIG. 62F depicts a petrographic microscope image of N nodulosa after 7 days of exposure to FeCh. Images of R. halophila obtained with microscopy showed no signs of depigmentation or trichome thinning, indicating that the treatments did not cause noticeable morphological alterations (FIG. 62A, FIG. 62C, and FIG. 62E). Similarly, in N nodulosa, a greenish pigmentation is observed across both treatments and the control, with no apparent morphological alterations (FIG. 62B, FIG. 62D, and FIG. 62F). These observations are consistent with the growth results obtained.

[0923] 6.3.3.1 Biomass productivity

[0924] Biomass production parameters for Rivularia halophila and Nodosilinea nodulosa under different iron sources are shown in Table 34. In line with previous results, all measured parameters — titer, productivity, yield, and %SY — showed improved growth in R. halophila under both FeSO4 and FeCL treatments compared to the control. The FeSCb treatment yielded the highest titer (1.81 g / L), productivity (0.0046 g / L h), and %SY (653.85), followed by FeCh (1.59 g / L; 0.0034 g / L h; 490.60 %SY), whereas the control condition showed the lowest values (1.43 g / L; 0.0023 g / L h; 281.27 %SY). Although the final pH of the culture medium was not as high as in the control condition, this may indicate that the treatment affects cyanobacterial metabolism differently than ferric ammonium citrate. Changes in pH depend on differences in photosynthetic activity, carbon uptake mechanisms, or proton / anion exchange rates. Medium pH changes should not be interpreted exclusively as indicators of biomass accumulation, given that pH and biomass are not always directly correlated. Rather, they may represent distinct metabolic traits of each species (Becker, 1994; Gomez-Luna et al., 2021; Raven, 1988; Sanchez-Bayo et al., 2020). Similarly, N. nodulosa exhibited enhanced growth with FeSCLWSGR Docket No. 69144-701601(1.30 g / L; 0.0017 g / L h; 246.44 %SY) compared to the control (1.07 g / L; 0.0009 g / L h; 105.47 %SY). In contrast, FeCk resulted in lower values for all parameters (0.96 g / L; -0.0004 g / L h; -58.12 %SY). Unlike R. halophila. the treatment with FeSCh in N. nodulosa reached pH values as high as those observed in the control (10.10 and 10.38, respectively). Although the FeCk treatment resulted in the lowest biomass values, the final pH remained close to 10, indicating active cyanobacterial metabolism. The difference in pH increase compared to R. halophila may be attributed to the fact that N. nodulosa possesses an intrinsically more alkalinizing metabolism. Members of the genus Nodosilinea are well-known for frequently dominating natural and engineered alkaline environments, such as soda lakes with pH values ranging from 10 to 11.4 (Yi et al., 2024).

[0925] 6.4 Conclusions

[0926] 1) Rivularia halophila grows best under standard phosphorus and nitrogen concentrations in BG11. Reducing phosphorus leads to moderate biomass decreases, while increasing nitrogen slightly enhances growth. Using an intermediate nitrogen concentration (0.75 g / L) maintains growth comparable to the control, suggesting a potential 50% reduction in sodium nitrate usage without compromising biomass production. Nodosilinea nodulosa growth is not significantly affected by reduced phosphorus and nitrogen levels. Phosphorus can be decreased by approximately 25%, and nitrogen reduced up to threefold (to 0.5 g / L) without impacting biomass or morphology, offering opportunities for cost savings in culture media preparation.

[0927] 2) Testing ammonium hydrogen phosphate ((NH^HPCL) as a combined nitrogen and phosphorus source, using both laboratory-grade and agri cultural -grade fertilizers, demonstrated poor suitability for replacing standard BG11 nutrients. Both sources resulted in minimal growth or biomass loss compared to the control, with a stronger negative effect observed in Nodosilinea nodulosa and with the agri cultural -grade fertilizer. Additionally, morphological changes such as reduced pigmentation and thinning trichomes were observed,WSGR Docket No. 69144-701601 indicating physiological stress. Therefore, the use of ammonium hydrogen phosphate is not recommended as a cost-saving alternative for these cyanobacteria due to its negative impact on growth and cell integrity.

[0928] As a future perspective, urea (CO(NH2)2) could be evaluated as an alternative nitrogen source, given its widespread use and affordability, to potentially improve growth performance while reducing costs.

[0929] 1) Both iron(II) sulfate (FeSCL) and iron(III) chloride (FeCh) can effectively replace ferric ammonium citrate as iron sources in BG11 without inhibiting Rivularia halophila growth, with FeSO4 proving to be the better option for both R. halophila and Nodosilinea nodulosa. These alternatives offer cost-effective options to improve or maintain production. FeSCb notably increased biomass titer, productivity, and yield compared to FeCh, which may be attributed to the higher bioavailability or lower toxicity of ferrous iron (Fe2+) compared to ferric iron (Fe3+) (Morel et al., 2008; Shaked & Lis, 2012; Qiu et al., 2022). Furthermore, replacing ferric ammonium citrate with FeSCL could enhance economic viability for large-scale cultivation. Future studies could explore iron oxides or hydroxides (Fe2Os, Fe(OH)s) and agricultural micronized iron oxide powder (FesCL) as potentially affordable and sustainable iron sources for cyanobacterial cultivation.Example 9. Co-culture with other microorganisms to further improve productivity, titer and yield

[0930] Implementing co-culture with other microorganisms can significantly enhance productivity, titer, and yield in cyanobacterial cultivation. By cultivating R halophila alongside complementary species of cyanobacteria, we can create a synergistic environment that promotes nutrient exchange and enhances metabolic activity. For instance, heterotrophic bacteria can help by decomposing organic matter, releasing nutrients that cyanobacteria can readily utilize, while also providing growth factors that stimulate their performance. Additionally, certain microorganisms may contribute to improved carbon fixation or facilitate the removal of inhibitory byproducts, leading to healthier cultures. This integrated approach can optimize resource utilization, increase biomass production, and ultimately enhance the overall efficiency of the cultivation process, making it a promising strategy for scaling up cyanobacterial production.

[0931] The precipitation of calcium carbonate depends on several biological and chemical factors. Therefore, one possible approach is to conduct experiments with different species of cyanobacteria that could promote the CaCCL precipitation process. For instance, one species could be particularly effective at generating extracellular nucleation sites, while anotherWSGR Docket No. 69144-701601 complementary species could induce a localized pH increase, thereby facilitating enhanced precipitation.

[0932] Objective

[0933] To enhance productivity, titer, and yield in cyanobacterial cultivation by cocultivating Rivularia halophila with Nodosilinea nodulosa cyanobacteria, leveraging their synergistic interactions to promote metabolic activity and calcium carbonate precipitation.

[0934] 7.1) Experimental setup: materials and methods a) Cyanobacterial cultures: Rivularia halophila and Nodosilinea nodulosa. b) Growth media: BG-11 saline medium supplemented with essential nutrients (nitrogen, phosphorus, and micronutrients). c) Calcium source: calcium chloride (CaCh). d) Carbon source: NaHCCh and CO2 were used for precipitation experiments. The concentration of NaHCCh was 800 ppm, while for CO2, a gas supply system was used, mixing CO2 with air (3 / 97%) from a gas cylinder with a flow meter and diffuser. In addition, 100% CO2 was also tested. e) pH meter: for monitoring and adjusting the pH of the culture medium. f) Calcium and CO2 selective electrodes: for monitoring dissolved calcium and DIC concentration of the medium. g) Incubator: with controlled temperature and light conditions. h) Bioreactors: flasks and / or small-scale bioreactors with CO2 injection ports. i) Centrifuge: for harvesting cyanobacterial cells. j) Filtration setup: to separate precipitated calcium carbonate. k) Analytical balance: for weighing precipitates and final biomass l) Microscope: petrographic optical microscope under polarized light to observe cyanobacterial cells and calcium carbonate crystals. m) Analytical techniques for chemical and mineralogical characterization: use electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy microanalysis (EDXA) and X-ray diffraction (XRD) to analyze and characterize the precipitated calcium carbonate crystals and X-ray photoelectron spectroscopy (XPS) for the identification of the biogenic signal.

[0935] 7.2) Experimental design:

[0936] 7.2a) Preparation of growth media

[0937] BGl l-saline media (modified from Phoenix et al., 2002): the media was supplemented with 3.6 g L'1of NaCl. For Ca supplementation, a 2 M CaCh stock solution wasWSGR Docket No. 69144-701601 prepared and sterilized at 121°C and 0.13 MPa. The Ca concentration in the medium was between 600 and 800 mg / L. Table 35 shows the composition of the culture medium without dissolved Ca and HCCh'. Table 35. Composition of BG11-saline medium. Culture media also contains 3.6 g L'1of NaCl. A volume of 1 mL from the trace metals stock solution was added to the BG-11 medium.

[0938] 7.2b) Inoculation, incubation, and carbon sources added

[0939] To assess the efficiency of different carbon sources, 3% CO2 in air and solid NaHCCh were utilized as carbon sources. The growth of cyanobacteria and the precipitation of CaCO3were evaluated. In all the experiments, the cyanobacteria were inoculated in two proportions: 80:20 and 95:5 (R. halophila.N. nodulosa). The biomass-to-culture medium ratio was maintained at 1.7 g / L.

[0940] 7.2bl) Solid NaHCCh: the NaHCCh concentration solution was 10 mM (800 ppm NaHCCh) and was prepared from a sterile solution, adjusted to pH 8 by a...

Claims

1. WSGR Docket No. 69144-701601CLAIMSWHAT IS CLAIMED IS:

1. A metallic carbonate precipitated using one or more cyanobacteria grown in vitro, wherein the metallic carbonate comprises (i) a purity level of at least 90%, (ii) an impurity content of less than 200 parts per million (ppm), and / or (iii) a biogenic signature associated with the one or more cyanobacteria, wherein the biogenic signature is measurable or detectable using X-ray photoelectron spectrometer (XPS).

2. The metallic carbonate of claim 1, wherein the metallic carbonate has a purity level of at least 95%.

3. The metallic carbonate of claim 1 or 2, wherein the metallic carbonate has a purity level of at least 98%.

4. The metallic carbonate of any one of the preceding claims, wherein impurity content is associated with one or more impurities comprising arsenic (e.g., arsenate (As (V)) and arsenite (As (III)), Fe (III) hydroxides, mercury, or any combination thereof.

5. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate has an impurity content of less than 50 parts per million (ppm).

6. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate has an impurity content of less than 25 parts per million (ppm).

7. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate has an arsenic level of less than 100 ppm.

8. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate has an arsenic level of less than 30 ppm.

9. A metallic carbonate precipitated using one or more cyanobacteria, wherein the metallic carbonate comprises a biogenic signature as measured by X-ray photoelectron spectrometer (XPS).

10. The metallic carbonate of claim 9, wherein the biogenic signature comprises a wide carbon (Cl) peak with binding energy from about 280 to 294 eV as measured by XPS.

11. The metallic carbonate of claim 9 or 10, wherein the biogenic signature comprises a wide carbon (Cl) peak with binding energy from about 284 to 290 eV as measured by XPS.

12. The metallic carbonate of any one of claims 9-11, wherein the metallic carbonate has a peak of at least 26.5° of 2-theta (29) as measured by X-ray diffraction (XRD).

13. The metallic carbonate of any one of claims 9-11, wherein the metallic carbonate has a peak at 27.9° 2-theta (29) as measured by X-ray diffraction (XRD).WSGR Docket No. 69144-70160114. A metallic carbonate precipitated using one or more cyanobacteria, wherein the metallic carbonate is precipitated using less than 5 GJ per ton of precipitated metallic carbonate compared to another metallic carbonate that is not generated using the one or more cyanobacteria.

15. The metallic carbonate of claim 14, wherein the metallic carbonate is precipitated using less than 10 GJ per ton of precipitated metallic carbonate compared to another metallic carbonate that is not generated using the one or more cyanobacteria.

16. The metallic carbonate of claim 13 or 14, wherein the metallic carbonate is precipitated using less than 20 GJ per ton of energy.

17. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate is precipitated using less than 10 GJ per ton of energy.

18. A metallic carbonate precipitated using one or more cyanobacteria, wherein the metallic carbonate is precipitated by removing at least 0.22 grams of CO2 from an atmosphere for every gram of metallic carbonate produced.

19. The metallic carbonate of any one of the preceding claims, wherein the one or more cyanobacteria are selected from the group consisting of a Rivulariaceae species, a Nodosilinea species, a Phormidium species, an Anabaena species, and a Synechococcus species.

20. The metallic carbonate of any one of the preceding claims, wherein the one or more cyanobacteria comprises at least two species of cyanobacteria.

21. The metallic carbonate of any one of the preceding claims, wherein the one or more cyanobacteria comprises at least three species of cyanobacteria.

22. The metallic carbonate of any one of the preceding claims, wherein the one or more cyanobacteria comprises Rivularia halophila.

23. The metallic carbonate of any one of the preceding claims, wherein the one or more cyanobacteria comprises Nodosilinea nodulosa.

24. The metallic carbonate of any one of the preceding claims, wherein the one or more cyanobacteria comprises a sheath that facilitates or enables the precipitation of the metallic carbonate.

25. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate comprises an alkaline-earth metal.

26. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate comprises a metal selected from Group 2 of the periodic table.WSGR Docket No. 69144-70160127. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate comprises calcium carbonate.

28. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate comprises at least 60% crystalline metallic carbonate.

29. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate comprises at least 70% crystalline metallic carbonate.

30. The metallic carbonate of any one of the preceding claims, wherein the metallic carbonate comprises at least 80% crystalline metallic carbonate.

31. A culture medium for facilitating precipitation of metallic carbonate using one or more cyanobacteria, wherein the culture medium has a salinity of at least 3.5% (w / v) NaCl.

32. The culture medium of claim 30, wherein the culture medium has a salinity of at least 8 % (w / v) NaCl.

33. The culture medium of claim 30 or 31, wherein the culture medium has a pH of at least 7.25.

34. The culture medium of any one of the preceding claims, wherein the culture medium has a pH of at least 8.5.

35. The culture medium of any one of the preceding claims, wherein the culture medium comprises sea water.

36. The culture medium of any one of the preceding claims, wherein the culture medium is obtained from sea water.

37. The culture medium of any one of the preceding claims, wherein the culture medium comprises a salt of an alkaline-earth metal.

38. The culture medium of any one of the preceding claims, wherein the culture medium comprises a salt of a metal selected from Group 2 of the periodic table.

39. The culture medium of any one of the preceding claims, wherein the culture medium comprises a calcium salt.

40. The culture medium of any one of the preceding claims, wherein the culture medium comprises at least 900 mg / L calcium chloride.

41. The culture medium of any one of the preceding claims, wherein the culture medium comprises a carbon source.

42. The culture medium of any one of the preceding claims, wherein the carbon source comprises at least 1000 mg / L bicarbonate (HCO3 ).

43. The culture medium of any one of the preceding claims, wherein the carbon source comprises dissolved CO2.WSGR Docket No. 69144-70160144. The culture medium of any one of the preceding claims, further comprising a cyanobacteria.

45. The culture medium of any one of the preceding claims, further comprising precipitated metallic carbonate, wherein the precipitated metallic carbonate is deposited on the surface of the one or more cyanobacteria.

46. The culture medium of any one of the preceding claims, further comprising precipitated metallic carbonate, wherein the one or more cyanobacteria comprises a sheath that facilitates or enables the precipitation of the metallic carbonate.

47. A system comprising the culture medium of claims 30-45, wherein the system further comprises one or more cyanobacteria.

48. The system of claim 46, wherein the one or more cyanobacteria are selected from the group consisting of a Rivulariaceae species, a Nodosilinea species, a Phormidium species, w\ Anabaena species, and a Synechococcus species.

49. The system of claim 47, wherein the one or more cyanobacteria comprises at least two species of cyanobacteria.

50. The system of any one of the preceding claims, wherein the one or more cyanobacteria comprises at least three species of cyanobacteria.

51. The system of any one of the preceding claims, wherein the one or more cyanobacteria comprises Rivularia halophila.

52. The system of any one of the preceding claims, wherein the one or more cyanobacteria comprises Nodosilinea nodulosa.

53. The system of any one of the preceding claims, wherein the system comprises an open or closed system.

54. A method of manufacturing a metallic carbonate, comprising: contacting one or more cyanobacteria with a carbon source in a culture media, thereby generating a precipitated metallic carbonate, wherein the one or more cyanobacteria comprises a sheath that facilitates or enables the precipitation of the metallic carbonate, wherein the metallic carbonate has (i) a purity level of at least 90% or (ii) an impurity content of less than200 ppm.

55. A method of forming a metallic carbonate, comprising: a) providing one or more cyanobacteria in a culture media containing high salinity; and b) providing a carbon source to the culture media, thereby generating a precipitated metallic carbonate.WSGR Docket No. 69144-70160156. The method of claim 53 or 54, wherein the salinity of the culture media is at least 3.5% (w / v) NaCl.

57. The method of claim 55, wherein the salinity of the culture media is at least 8% (w / v) NaCl.

58. The method of any one of the preceding claims, further comprising separating the precipitated metallic carbonate from the one or more cyanobacteria.

59. The method of any one of the preceding claims, wherein the one or more cyanobacteria comprise a sheath.

Images