Alkaliphilic Consortium Shifting for Production of Phycocyanins and Biochemicals

Abstract

Processes are disclosed for photosynthetic cyanobacterial production of selected proteins and biochemicals within an evolving alkaliphilic microbial consortium.

Claims

1. -32. (canceled)

33. A method of making a phycocyanin product, comprising: culturing an alkaliphilic soda lake microbial consortium under aerobic alkaline conditions in a diurnal growth cycle to establish a cyanobacterial population containing the phycocyanin within the microbial consortium in a cultured biomass in an aerobic culture comprising a growth medium; and, shifting the microbial consortium, having a proportion of cyanobacteria and a proportion of non-cyanobacterial alkaliphile microbes, to a dark-phase cycle under anaerobic alkaline conditions in an anaerobic culture to initiate auto-catabolic release of the phycocyanin intact from the cyanobacterial population as the proportion of cyanobacteria in the microbial consortium decreases and the proportion of non-cyanobacterial alkaliphile microbes increases in the consortium, to produce a biomass solids product and an aqueous phycocyanin product.

34. The method of claim 33, wherein the aerobic and/or anaerobic alkaline conditions comprise at least 0.5 M Na.sup.+, or from 0.25 M - 3 M Na.sup.+.

35. A method of making a phycocyanin product, comprising: culturing one or more members of an alkaliphilic soda lake microbial consortium under aerobic alkaline conditions in a diurnal growth cycle to establish a cyanobacterial population containing the phycocyanin within the microbial consortium in a cultured biomass in an aerobic culture comprising a growth medium; and, shifting the microbial consortium to a dark-phase cycle under anaerobic alkaline conditions in an anaerobic culture to initiate auto-catabolic release of the phycocyanin intact from the cyanobacterial population, to produce a biomass solids product and an aqueous phycocyanin product; wherein the aerobic and/or anaerobic alkaline conditions comprise at least 0.5 M Na.sup.+, or from 0.25 M - 3 M Na.sup.+.

36. The method of claim 33, wherein the aerobic and/or anaerobic alkaline conditions comprise: at least 0.5 M total carbonate alkalinity (CO.sub.3.sup.2- + HCO.sub.3.sup.-), or from about 0.25 M to about 1 M total carbonate alkalinity; and/or, a pH of at least 9, or a pH of from about 7 to about 11; and/or, one or more dissolved species that are: Na.sub.2CO.sub.3, NaHCO.sub.3, NaNO.sub.3, NH.sub.4, KH.sub.2PO.sub.4, MgSO.sub.4.7H.sub.2O, CaCl.sub.2.2H.sub.2O, NaCl, KCl, FeCl.sub.3.6H.sub.2O, H.sub.3BO.sub.3, MnCl.sub.2.4H.sub.2O, ZnCl.sub.2, CuCl.sub.2.2H.sub.2O, Na.sub.2MoO.sub.4.2H.sub.2O, CoCl.sub.2.6H.sub.2O, NiCl.sub.2.6H.sub.2O, and/or KBr; and/or, a temperature of from about 10° C. to about 30° C., or from about 4° C. to about 45° C.

37. The method of claim 35, wherein the aerobic and/or anaerobic alkaline conditions comprise: at least 0.5 M total carbonate alkalinity (CO.sub.3.sup.2- + HCO.sub.3.sup.-), or from about 0.25 M to about 1 M total carbonate alkalinity; and/or, a pH of at least 9, or a pH of from about 7 to about 11; and/or, one or more dissolved species that are: Na.sub.2CO.sub.3, NaHCO.sub.3, NaNO.sub.3, NH.sub.4, KH.sub.2PO.sub.4, MgSO.sub.4.7H.sub.2O, CaCl.sub.2.2H.sub.2O, NaCl, KCl, FeCl.sub.3.6H.sub.2O, H.sub.3BO.sub.3, MnCl.sub.2.4H.sub.2O, ZnCl.sub.2, CuCl.sub.2.2H.sub.2O, Na.sub.2MoO.sub.4.2H.sub.2O, CoCl.sub.2.6H.sub.2O, NiCl.sub.2.6H.sub.2O, and/or KBr; and/or, a temperature of from about 10° C. to about 30° C., or from about 4° C. to about 45° C.

38. The method of claim 33, wherein the aerobic alkaline conditions comprise: an oxygen partial pressure of at least 200 mbar or at least 9 mg/L; and/or, exposure to air; wherein the anaerobic alkaline conditions comprise: an oxygen partial pressure of less than 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 mbar or less than 4, 3, 2, 1, 0.5, or 0.1 mg/L; and/or, exclusion of air.

39. The method of claim 35, wherein the aerobic alkaline conditions comprise: an oxygen partial pressure of at least 200 mbar or at least 9 mg/L; and/or, exposure to air; and/or, wherein the anaerobic alkaline conditions comprise: an oxygen partial pressure of less than 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 mbar or less than 4, 3, 2, 1, 0.5, or 0.1 mg/L; and/or, exclusion of air.

40. The method of claim 33, wherein: the diurnal cycle is maintained for a light phase incubation time that is at least 1, 2, 3, 4, 5 or 6 days; and/or,the dark phase cycle is maintained for a dark phase incubation time that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 days, or from 1 - 12 days; and/or, the diurnal cycle comprises exposure of the microbial consortium to full spectrum sunlight or artificial light that covers a substantially complete visible spectrum range, optionally at a light intensity of at least 200 .Math.mol. photons/m.sup.2/s with a light:dark cycle of approximately 16:8 hr, or wherein the diurnal cycle is from about 9 to about 16 hr and the dark phase cycle is from about 8 to about 15 hr.

41. The method of claim 35, wherein: the diurnal cycle is maintained for a light phase incubation time that is at least 1, 2, 3, 4, 5 or 6 days; and/or, the dark phase cycle is maintained for a dark phase incubation time that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 days, or from 1 - 12 days; or, the diurnal cycle comprises exposure of the microbial consortium to full spectrum sunlight or artificial light that covers a substantially complete visible spectrum range, optionally at a light intensity of at least 200 .Math.mol. photons/m.sup.2/s with a light:dark cycle of approximately 16:8 hr, or wherein the diurnal cycle is from about 9 to about 16 hr and the dark phase cycle is from about 8 to about 15 hr.

42. The method of claim 33, further comprising allowing the cultured biomass to either settle or float prior to shifting the microbial consortium to the dark-phase cycle to provide a concentrated biomass for the dark-phase cycle, optionally separating the cultured biomass from the growth medium by a filtration to provide the concentrated biomass, optionally wherein the filtration comprises filtration with a filter of from about 100 to about 635 mesh; optionally, wherein the concentrated biomass has a concentration of at least 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19% in the anaerobic culture (biomass solid weight/total anaerobic culture weight) under the anaerobic alkaline conditions.

43. The method of claim 35, further comprising allowing the cultured biomass to either settle or float prior to shifting the microbial consortium to the dark-phase cycle to provide a concentrated biomass for the dark-phase cycle, optionally separating the cultured biomass from the growth medium by a filtration to provide the concentrated biomass, optionally wherein the filtration comprises filtration with a filter of from about 100 to about 635 mesh; optionally, wherein the concentrated biomass has a concentration of at least 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19% in the anaerobic culture (biomass solid weight/total anaerobic culture weight) under the anaerobic alkaline conditions.

44. The method of claim 33, wherein the microbial consortium comprises: a Phormidium, optionally comprising the Phormidium of deposit NCBI # REDN00000000.1; and/or, a Planctomycetota; and/or, an Arthrospira sp., optionally wherein the Arthrospira sp. is Arthrospira plantensis.

45. The method of claim 35, wherein the microbial consortium comprises: a Phormidium, optionally comprising the Phormidium of deposit NCBI # REDN00000000.1; and/or, a Planctomycetota; and/or, an Arthrospira sp., optionally wherein the Arthrospira sp. is Arthrospira plantensis.

46. The method of claim 33, wherein the microbial consortium comprises: one or more of: Nodosilinea, Gloeocapsa, Phormidium, Arthrospira, Spriulina, Rhodobacteraceae, Gemmatimonadota; SG8-23, Chromatiaceae, Natronohydrobacter, Geminicoccales, Nodosilinea, Cyanobium, Wenzhouxiangella, Indibacter, Competibacteraceae, Nitriliruptoraceae, Desulfonatronum, or Thioalkalivibrionaceae; or, one or more members of the following taxa: Roseinatronobacter, Natronohydrobacter, Rhodobacteraceae, Salinarimonas, Geminicoccales, Rhodospirillales, Micavibrionales, Wenzhouxiangella, Thioalkalivibrionaceae, Ectothiorhodospira, Gammaproteobacteria, Thiohalocapsa, Competibacteraceae, Halomonas, Nitrincola, Pseudomonadales, Pseudomonas “D”, Hahellaceae, Desulfonatronum, Bdellovibrionota belonging to the family UBA2394, Myxococcota belonging to the family CA-2862545, Myxococcota belonging to the order UBA4248, Bacteroidales belonging to the family UBA7960, Bacteroidales belonging to the family UBA12077, Saprospiraceae, Chitinophagales belonging to the family UBA2359, Flavobacteriales, Schleiferia, Indibacter, Balneolaceae belonging to the genus UBA2664, Balneolaceae, Balneolales, Alkalispirochaeta, Alkalispirochaetaceae, Spirochaetales, Pirellulaceae belonging to the genus UBA6163, Pirellulaceae, Phycisphaerales of the family SM1A02, Phycisphaerales belonging to the family SM1A02, Planctomycetota belonging to the family UBA11346, Opitutaceae, Puniceicoccaceae belonging to the genus BACL24, Nitrolancea, Anaerolineae belonging to the genus GCA-2794505, Ilumatobacteraceae, Nitriliruptoraceae, Trueperaceae, Acholeplasmataceae, Izimaplasmataceae, Alkalibacterium, Bacillus “AQ”, Nodosilinea, Phormidesmiaceae, Arthrospira platensis, Phormidium (A), Nodularia, Cyanobium, Gloeocapsa, Spirulina, Gemmatimonadota belonging to the order SG8-23, and/or Nodosilinea.

47. The method of claim 35, wherein the microbial consortium comprises: one or more of: Nodosilinea, Gloeocapsa, Phormidium, Arthrospira, Spriulina, Rhodobacteraceae, Gemmatimonadota belonging to order SG8-23, Chromatiaceae, Natronohydrobacter, Geminicoccales, Nodosilinea, Cyanobium, Wenzhouxiangella, Indibacter, Competibacteraceae, Nitriliruptoraceae, Desulfonatronum, or Thioalkalivibrionaceae; or, one or more members of the following taxa: Roseinatronobacter, Natronohydrobacter, Rhodobacteraceae, Salinarimonas, Geminicoccales, Rhodospirillales, Micavibrionales, Wenzhouxiangella, Thioalkalivibrionaceae, Ectothiorhodospira, Gammaproteobacteria, Thiohalocapsa, Competibacteraceae, Halomonas, Nitrincola, Pseudomonadales, Pseudomonas “D”, Hahellaceae, Desulfonatronum, Bdellovibrionota belonging to the family UBA2394, Myxococcota belonging to the family CA-2862545, Myxococcota belonging to the order UBA4248, Bacteroidales belonging to the family UBA7960, Bacteroidales belonging to the family UBA12077, Saprospiraceae, Chitinophagales belonging to the family UBA2359, Flavobacteriales, Schleiferia, Indibacter, Balneolaceae belonging to the genus UBA2664, Balneolaceae, Balneolales, Alkalispirochaeta, Alkalispirochaetaceae, Spirochaetales, Pirellulaceae belonging to the genus UBA6163, Pirellulaceae, Phycisphaerales belonging to the family SM1A02, Phycisphaerales belonging to the family SM1A02, Planctomycetota belonging to the family UBA11346, Opitutaceae, Puniceicoccaceae belonging to the genus BACL24, Nitrolancea, Anaerolineae belonging to the genus GCA-2794505, Ilumatobacteraceae, Nitriliruptoraceae, Trueperaceae, Acholeplasmataceae, Izimaplasmataceae, Alkalibacterium, Bacillus “AQ”, Phormidesmiaceae, Arthrospira platensis, Phormidium (A), Nodularia, Cyanobium, Gloeocapsa, Spirulina, Gemmatimonadota belonging to the order SG8-23, and Nodosilinea.

48. The method of claim 33, wherein the yield of the phycocyanin product is at least 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wt% of the biomass solids product yield.

49. The method of claim 35, wherein the yield of the phycocyanin product is at least 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wt% of the biomass solids product yield.

50. The method of claim 33, wherein CO.sub.2 produced during auto-catabolic release of the phycocyanin is captured and utilized as an inorganic carbon source for culturing the alkaliphilic soda lake microbial consortium.

51. The method of claim 35, wherein CO.sub.2 produced during auto-catabolic release of the phycocyanin is captured and utilized as an inorganic carbon source for culturing the alkaliphilic soda lake microbial consortium.

52. The method of claim 33, wherein a residual solids fraction is collected after auto-catabolic release of the phycocyanin and at least a portion of the residual solids fraction is re-cycled back to the step of culturing the alkaliphilic soda lake microbial consortium; optionally wherein at least a portion of the residual solids fraction is directed to a microbial process to produce methane.

53. The method of claim 35, wherein a residual solids fraction is collected after auto-catabolic release of the phycocyanin and at least a portion of the residual solids fraction is re-cycled back to the step of culturing the alkaliphilic soda lake microbial consortium; optionally, wherein at least a portion of the residual solids fraction is directed to a microbial process to produce methane.

54. The method of claim 33, wherein organic acids produced during the auto-catabolic release of the phycocyanin are separated from the phycocyanin product; optionally, wherein the organic acids separated from the phycocyanin product are collected to provide collected organic acids; optionally, wherein the collected organic acids are directed for use in culturing the alkaliphilic soda lake microbial consortium mixotrophically; optionally, wherein the organic acids are separated from the phycocyanin product by an organic acid filtration; optionally, wherein the organic acid filtration comprises filtration with a molecular weight cut-off filter, optionally a molecular weight cut-off filter in a range of 10-30 kDa.

55. The method of claim 35, wherein organic acids produced during the auto-catabolic release of the phycocyanin are separated from the phycocyanin product; optionally, wherein the organic acids separated from the phycocyanin product are collected to provide collected organic acids; optionally, wherein the collected organic acids are directed for use in culturing the alkaliphilic soda lake microbial consortium mixotrophically; optionally, wherein the organic acids are separated from the phycocyanin product by an organic acid filtration; optionally, wherein the organic acid filtration comprises filtration with a molecular weight cut-off filter, optionally a molecular weight cut-off filter in a range of 10-30 kDa.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1: is a schematic process flow diagram illustrating an example of a bio-conversion process, used to produce both phycocyanin and organic acids from a cyanobacterial consortium. The block flow diagram shows the process for extraction of phycocyanin and production of organic acids from cyanobacterial consortium. Carbon dioxide and digested solids obtained after bio-conversion process will be re-cycled back to cultivation process. Recovered organic acids may be used for either production of other chemicals or natural gas (via anaerobic digestion).

[0022] FIG. 2 is a schematic process flow diagram illustrating an example of a bio-conversion process used to produce both phycocyanin and organic acids from a cyanobacterial consortium. The block flow diagram shows the process for extraction of phycocyanin and production of organic acids from cyanobacterial consortium. Carbon dioxide obtained after bio-conversion process will be re-cycled back to cultivation process Recovered digested solids and organic acids may be used for the production of natural gas (via anaerobic digestion).

[0023] FIG. 3 is a schematic process flow diagram illustrating an example of a bio-conversion process used to produce both phycocyanin and organic acids from a cyanobacterial consortium. The block flow diagram shows the process for extraction of phycocyanin and production of organic acids from cyanobacterial consortium. Recovered carbon dioxide, digested solids and organic acids will be recycled back to cultivation process.

[0024] FIG. 4 (a), (b) and (c) are microscopic images of a cyanobacterial consortium and (d) and (e) shows microscopic images of Arthrospira plantensis during the dark-phase incubation period.

[0025] FIG. 5 is a graph illustrating a decrease in weight of the biomass during the dark-phase bio-conversion process.

[0026] FIG. 6 includes a photograph showing the change in color of supernatant during the dark-phase incubation period; and, b) spectral data showing increase in the peak area at 620 nm and c) spectral data normalized to Day 0.

[0027] FIG. 7 includes graphs showing (a) Increase in the mass of phycocyanin and (b) purity of phycocyanin during a dark-phase bio-conversion process of biomass obtained from a 20 L bioreactor. The errors bars are obtained mean values of two replicates.

[0028] FIG. 8 includes two graphs illustrating an increase in both phycocyanin weight percentage (a) and purity (b) with increase in biomass concentration. The errors bars are obtained mean values of two replicates.

[0029] FIG. 9 includes graphs showing the increase in the organic acid concentration and (b) CO.sub.2 release during the dark-phase bio-conversion of cyanobacterial consortium.

[0030] FIG. 10 is a photographic image showing the change in color of supernatant during the incubation period under varied Na.sup.+ concentrations. The supernatant obtained from trial 1 (Na+ = 1 M) experiments has intense blue color compared to trial 2 (Na+ = 0.5 M) and trial 3 (Na+ = 0.25 M) experiments.

[0031] FIG. 11 is a stacked bar plot showing the community composition of the processed biomass throughout the course of a 12 day process. Bacterial species that contributed to at least 5% of the relative abundance in at least one sample during the time series experiment are included. Relative abundance is based on proportion of community DNA. Table 1: Shows the taxonomy of the species.

TABLE-US-00002 Taxonomy Code Taxonomy A1 Proteobacteria: Rhodobacteraceae (f) A3 Proteobacteria: Rhodobacteraceae (f) A4 Proteobacteria: Salinarimonas (g) C1 Cyanobacteria: Phormidium (g) F1 Firmicutes: Alkalibacterium (g) G1 Proteobacteria: Wenzhouxiangella (g) G6 Proteobacteria: Alkalimonas (g) P1 Planctomycetota: SM1A02 (f) V1 Verrucomicrobiota: UBA6053 (f) V2 Verrucomicrobiota: UBA6053 (f) V3 Verrucomicrobiota: Opitutales (o)

[0032] FIG. 12 includes two photographs showing phycocyanin passively released during a dark-phase incubation of Arthrospira Plantensis.

DETAILED DESCRIPTION

[0033] In the context of the present disclosure, various technical terms are used in accordance with definitions that are commonly understood in the art, as follows. “Total carbonate alkalinity” is calculated as the sum of the concentrations of bicarbonates and carbonates. Alkaliphilic microorganisms are defined as organisms which exhibit optimum growth in an alkaline pH environment, particularly in excess of pH 8, and generally in the range between pH 9 and 10. Alkaliphiles may also be found living in environments having a pH as high as 12. Obligate alkaliphiles are incapable of growth at neutral pH. Halophilic bacteria are microorganisms that grow optimally in the presence of salt (sodium chloride), for example microorganisms having a minimum requirement in excess of the concentration found in sea water (ca. 0.5 M or 3%). The term “haloalkaliphile” may be used to describe bacteria that are both halophilic and alkaliphilic (see for example U.S. Pat. Nos. 6,420,147 and 6,291,229). The “soda lake” environments, which host many haloalkaliphiles, are characterized by relatively high total carbonate alkalinities, brackish or saline salt concentrations, and alkaline pH.

[0034] In select embodiments, processes are provided that make use of a culture of an organism found in soda lake environmens, such as Spirulina. In this context, we note that Arthrospira platensis is the current genus/species name for the African cyanobacteria commonly used as a human food product. This organism was previously referred to as Spirulina platensis, but is now generally recognized as an Arthrospira species, distinct from the Spirulina genus. In common usage, in keeping with existing custom in the fields of foods and dietary supplements, many formulations designated commercially as Spirulina are actually Arthrospira platensis.

[0035] Methods are provided that facilitate extraction and purification of phycocyanin from wet biomass, and in addition result in production of organic acids that may for example be converted into high value chemicals or natural gas. Referring now to FIG. 1, a method is provided for a bio-conversion process to both extract phycocyanin and produce organic acids from cyanobacterial consortium. The block flow diagram in FIG. 1 shows a process wherein cyanobacterial consortium is first cultivated under high pH and high alkalinity growth conditions. The high pH and high alkalinity growth conditions facilitate efficient capture of carbon dioxide either from air or flue gas. The biomass produced is then concentrated via settling process. The spent medium obtained after this step is recycled back to cultivation system. The concentrated biomass obtained is subjected to a dark-phase anaerobic bio-conversion process. During this process, biomass is for example incubated at room temperature (20° C.) under dark and anoxic conditions. This process is carried out so as to lead to cyanobacterial cell disruption and the concomitant release of phycocyanin. In addition, this process would results in conversion of carbohydrates (storage compound in cyanobacteria) into organic acids. Following the bio-conversion process, the extract containing phycocyanin and organic acids may be separated from digested biomass. In selected embodiments, residual biomass (rich in nitrogen and phosphorous) and carbon dioxide, a bi-product, produced during the bio-conversion process may be recycled back to cultivation system. The extract is then subjected to a second separation step wherein phycocyanin is separated from organic acids. While phycocyanin can further be purified, organic acids are either converted into high value chemicals or used to produce natural gas via anaerobic digestion.

[0036] Referring now to FIG. 2, a method is provided for a bio-conversion process to both extract phycocyanin and produce organic acids from the cyanobacterial consortium. In the FIG. 2 embodiment, the digested solids (rich in nitrogen and phosphorous) obtained after the bio-conversion process will be supplied to anaerobic digestion to enhance the natural gas production.

[0037] Referring now to FIG. 3, a method is provided for a bio-conversion process to both extract phycocyanin and produce organic acids from the cyanobacterial consortium. In the FIG. 3 embodiment, carbon dioxide, digested solids (rich in nitrogen and phosphorous) and organic acids obtained after the bio-conversion process will be recycled back to cultivation system. In this way, organic carbon (provided in the form of organic acids) and organic nitrogen and phosphorous from digested solids may be used to enhance biomass yields.

EXAMPLES

Example 1: Digestion of Biomass During Bio-Conversion Process

[0038] Cyanobacterial consortium collected from Soda Lakes located on the Cariboo Plateau, British Columbia, Canada was used in these examples. Culture incubations were performed for 4 days at room temperature (20° C.) in 25 L glass carboys with a working volume of 20 L. The glass carboys were placed on shaker incubator and mixed at a speed of 300 rpm. Full spectrum led lights (Model T5H0; 6400K, Sunblaster Holdings ULC, Langley, BC, Canada) were used and a light intensity of 200 .Math.mol. photons/m.sup.2/s with a light:dark cycle of 16:8 hr was maintained. To simulate the high pH and alkalinity conditions of soda lakes, a synthetic medium is used in these experiments. The high pH and high alkalinity growth medium contained the following: Na.sub.2CO.sub.3 (210.98 mM), NaHCO.sub.3 (77.85 mM), NaNO.sub.3 (3.06 mM), NH.sub.4 (0.92 mM), KH.sub.2PO.sub.4, (1.44 mM), MgSO.sub.4.7H.sub.2O (1 mM), CaCl.sub.2.2H.sub.2O (0.17 mM), NaCl (0.43 mM), KCI (6.04 mM) FeCl.sub.3.6H.sub.2O (0.04 mM) and 300 uL of trace metal solution. The trace metal solution comprised -H.sub.3BO.sub.3 (9.7 mM), MnCl.sub.2.4H.sub.2O (1.26 mM), ZnCl.sub.2 (0.15 mM), CuCl.sub.2.2H.sub.2O (0.11 mM), Na.sub.2MoO.sub.4.2H.sub.2O (0.07 mM), CoCl.sub.2.6H.sub.2O (0.06 mM), NiCl.sub.2.6H2O (0.04 mM), KBr (0.08 mM). The biomass obtained after 4 days of incubation is subjected to “settling” process in order to concentrate the biomass.

[0039] To illustrate the effect of bio-conversion process on cyanobacterial consortium, 16 sacrificial samples containing 2 grams of concentrated biomass (20% (w/w)) were incubated at room temperature (20° C.) under dark and anoxic conditions. The process was carried out for 12 days and two sacrificial samples were removed on every alternate day and analyzed for cell integrity and biomass dry weight. In another set of experiments, a commercially available algae, Arthrospira Plantensis, was purchased and incubated in dark and anoxic conditions as a control.

[0040] FIGS. 4 a, b, and c shows the microscopic images of sacrificial samples obtained on days 0, 6 and 8 respectively. It can be seen that cyanobacterial cells are intact on day 0. However, as the incubation time progressed (i.e. on days 6 and 8), the cell integrity was lost (see FIGS. 4)b and c. This illustrates that the cyanobacterial consortium was digested during the bio-conversion process. Consistent with this, the dry weights of sacrificial samples decreased over the incubation time (See FIG. 5). In contrast, microscopic images of the control experiments reveal that the algae cells were still intact even after 8 days of incubation (see FIGS. 4) d and e.

Example 2: Effect of Incubation Period on Extraction and Purification of Phycocyanin During Bio-Conversion Process.

[0041] For phycocyanin recovery and analysis, a 0.1 M phosphate buffer is used. 5 mL of 0.1 M phosphate buffer was added to the previously obtained sacrificial samples and vortexed for 10 min. The slurry was then centrifuged at 4000 rpm for 5 min to recover the extracted phycocyanin. As illustrated in grayscale in FIG. 6a , the color of the supernatant gradually changed from pale yellow to dark blue. This confirms that dark-phase incubation of concentrated biomass under anoxic conditions not only resulted in digestion of biomass but also resulted in phycocyanin release. Further evidence of this is available from spectroscopic data, based on the fact that for phycocyanin, a maximum absorbance is observed at a wavelength of 620 nm. When absorption spectra of phycocyanin was measured on a UV-Vis Spectrophotometer over a spectral range of 300 - 700, it was evident that the spectral absorbance is significantly high at a wavelength of 620 (see FIGS. 6 b and c).

[0042] The mass and purity of phycocyanin released during the dark-phase bio-conversion process was also quantified. The absorption spectra of phycocyanin was measured on a UV-Vis Spectrophotometer at wavelengths 280 and 620. Mass of phycocyanin in the supernatant was estimated from a calibration curve generated by applying the same protocol as the samples to pure phycocyanin of known concentrations (obtained from Sigma Aldrich). And using the ratio of A.sub.620 to A.sub.280 purity of phycocyanin was determined.

[0043] The results indicate that a maximum phycocyanin content of ~35 mg (~10% (w/w), see FIG. 7) awas obtained over a period of 6 to 8 days. Moreover, both the mass and purity increased up till day 8 after which they started to decline (see FIGS. 7) a and b. In contrast, when similar analysis were carried out for control experiments, no phycocyanin release was observed.

Example 3: Effect of Biomass Concentration on Phycocyanin Extraction and Its Purity.

[0044] To illustrate the effect of biomass concentration on phycocyanin extraction and purity, four trial-scale processes were conducted with four different concentrations. Trial 1 with 19% (w/w) solids, trial 2 with 10% (w/w) solids, trial 3 with 6.4% (w/w) solids and trial 4 with 0.9% (w/w) solids. In each trial, 16 sacrificial samples were incubated under dark and anoxic conditions. In these trials, the volume of concentrated biomass is varied accordingly to maintain the same amount of biomass across all trials. The trails were carried out for 8 days and two sacrificial samples were removed from each trial on every alternate day and analyzed for phycocyanin content and purity. It was observed that for trial 1 and trial 2, phycocyanin content of 8.4% (w/w) was obtained (See FIG. 8) a. It was also observed that the highest purity was achieved when the biomass concentrations were 19% (trial 1) and 10% (trial 2) (see FIG. 8) b. In contrast, in trial 3 and trial 4, where the biomass concentrations were low, both phycocyanin content and purity were relatively low.

Example 4: Effect of Biomass Concentration on Organic Acid Production and Carbon Dioxide Release During Bio-Conversion Process

[0045] For organic acid recovery and analysis, a 0.1 M phosphate buffer is used. 5 mL of 0.1 M phosphate buffer was added to the previously obtained sacrificial samples from all trials. The digested biomass in the sacrificial samples along with the phosphate buffer were vortexed for 10 min. The slurry was then centrifuged at 4000 rpm for 5 min to recover the produced organic acids. The recovered organic acids were first filtered through a 0.22 .Math.m membrane filter and then analyzed on HPLC equipped with Aminex HPX-87H column and a UV detector. The results show that the organic acid production for trial 2 (10% (w/w) solids) and trial 3 (6.4 % (w/w) solids) were significantly higher when compared to trial 1 experiments (see FIG. 9) a. Additionally, the head space gas was also analyzed using gas chromatography equipped with TCD and FID detectors. It was observed that there is no measurable carbon dioxide production in trial 3 and trial 4. In contrast, there was significant production of carbon dioxide in both trial 1 and trial 2 (~20 Mole %, see FIG. 9) b.

Example 5: Effect of Na.SUP.+ Concentration on Phycocyanin Release During the Bio-Conversion Process.

[0046] This example illustrates the effect of Na.sup.+ concentrations in the growth medium on lysis of cyanobacterial cells during the dark-phase bio-conversion process. Three trials were conducted with varying Na.sup.+ concentrations. Trial 1 with a Na.sup.+ concentration of 1 M, trial 2 with a Na.sup.+ concentration of 0.5 M, and trial 3 with 0.25 M Na.sup.+ concentration. The trails were carried out for 10 days. Visual observations indicates that the phycocyanin release in trial 1 (see day 4 and day 5 samples of FIG. 10) was much faster compared to trail 2 and trial 3. This illustrates that the rate at which cyanobacterial cells are lysed was significantly higher under high Na.sup.+ concentration (trial 1) compared to low Na.sup.+ concentrations.

Example 6: Metagenomic Analysis

[0047] 16 sacrificial samples containing 2 grams of concentrated biomass (20% (w/w)) were incubated at room temperature (20° C.) under dark and anoxic conditions. The trial was carried out for 12 days and two sacrificial samples were removed on every alternate day to carry out a metagenomic analysis.

Day 0 - Day4

[0048] Species C1 (Phormidium) remained the dominant community member (>60% relative abundance) throughout Day 2 and Day 4. During Day 2 and Day 4, species P1 and V3 remained the second and third most abundant species respectively (FIG. 11).

Day 6 - Day 12

[0049] On Day 6, coinciding with the noticeable release in phycocyanin, there was a dramatic decrease of the Phormidium C1 population from 65% (Day 4) relative abundance to 10% relative abundance (Day 6, FIG. 11). The species P1 (Planctomycetota: SM1A02) increased from 8.5% (Day 4) to 21% (Day 6) relative abundance and became the most abundant species in the biomass with respect to DNA concentration. Species G1 (Proteobacteria: Wenzhouxiangella) increased in abundance to 14% on Day 6. Species F1 (Firmicutes: Alkalibacterium), A3 (Proteobacteria: Rhodobacteraceae), A4 (Proteobacteria: Salinarimonas), and A1 (Proteobacteria: Rhodobacteraceae), all increased on Day 6 to 5% relative abundance or greater.

[0050] From Day 8 to Day 12, C1 (Cyanobacteria: Phormidium) continued to decrease to less than 0.2% of the community DNA. Species P1 (Planctomycetota:SM1A02) continued to increase to 36% of the community relative abundance, and species A3 (Proteobacteria: Rhodobacteraceae), F1 (Firmicutes: Alkalibacterium), A4 (Proteobacteria: Salinarimonas), and A1 (Proteobacteria: Rhodobacteraceae) each persisted at between 5-10% of community relative abundance. Species G1 (Proteobacteria: Wenzhouxiangella), abundant on Day 6 (14%), decreased to less than 3.5% by Day 12.

Example 7: Effect of Na.SUP.+ Concentration on Phycocyanin Release During the Bio-Conversion Process of Commercially Available Algae, Arthrospira Plantensis

[0051] This example illustrates the effect of Na+ concentration on lysis of Arthrospira Plantensis cells during a dark-phase bio-conversion process. In brief, 12 g of NaCl was added to 230 g of Arthrospira Plantensis paste (~ 15% (w/w) dry weight) to bring overall concentration of Na.sup.+ ions of the algae paste to 1 M. Following the NaCl addition, the algae paste was incubated in dark for 15 days. Visual observations clearly indicate that phycocyanin was passively released (FIG. 12). On the contrary, when the same algae were incubated in dark without any addition of Na.sup.+ ions, phycocyanin was not released and the cells were intact even after 8 days (FIGS. 4) e & f. This illustrates that high Na.sup.+ concentrations during a dark incubation facilitates cell lysis and passive release of phycocyanin.

INCORPORATED REFERENCES

[0052] Boros E, Kolpakova M (2018) “A review of the defining chemical properties of soda lakes and pans: An assessment on a large geographic scale of Eurasian inland saline surface waters.” PLoS ONE 13(8): e0202205.

[0053] Foulds and Carr (1977) “A Proteolytic Enzyme Degrading Phycocyanin in the Cyanobacterium Anabaena Cylindrica” FEMS Microbiology Letters 2: 117-119.

[0054] Kuddus M, Singh P, Thomas G, Al-Hazimi A (2013). “Recent developments in production and biotechnological applications of C-phycocyanin”. BioMed Research International. 2013.

[0055] Pagels, Guedes, Amaro, Kijjoa, Vasconcelos (2019) “Phycobiliproteins from cyanobacteria: Chemistry and biotechnological applications.” .Biotechnol Adv. 2019 May - Jun;37(3):422-443. Epub 2019 Feb 21.

[0056] Sorokin, Gijs Kuenen (2005) “Chemolithotrophic haloalkaliphiles from soda lakes” FEMS Microbiology Ecology, Volume 52, Issue 3, May 2005, Pages 287-295.

[0057] Zorz, Sharp, Kleiner, Gordon, Pon, Dong & Strous (2019) “A shared core microbiome in soda lakes separated by large distances” Nature Communications 10:4230.

[0058] Citation of references herein is not an admission that such references are prior art to the present invention. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification, and all documents cited in such documents and publications, are hereby incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

[0059] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Terms such as “exemplary” or “exemplified” are used herein to mean “serving as an example, instance, or illustration.” Any implementation described herein as “exemplary” or “exemplified” is accordingly not to be construed as necessarily preferred or advantageous over other implementations, all such implementations being independent embodiments. Unless otherwise stated, numeric ranges are inclusive of the numbers defining the range, and numbers are necessarily approximations to the given decimal. The word “comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing.