High Yield Algal Biomass Production Without Concentrated CO2 Supply Under Open Pond Conditions

Abstract

Methods and systems for efficient culturing of algae in open ponds are described.

Claims

1. A method for culturing algae, the method comprising: culturing alkaliphilic algae in an open pond medium having a pH above 9.5; and incorporating into the open pond medium an inorganic carbon buffer sufficient to allow increased fixation of atmospheric CO.sub.2 into the open pond medium; wherein the open pond medium is free of any concentrated supply of CO.sub.2, and no concentrated source of CO.sub.2 is used to supply carbon into the open pond medium.

2. The method of claim 1, wherein the inorganic carbon buffer comprises either a NaHCO.sub.3/Na.sub.2CO.sub.3 mixture or a KHCO.sub.3/K.sub.2CO.sub.3 mixture.

3. The method of claim 2, wherein the NaHCO.sub.3/Na.sub.2CO.sub.3 mixture or the KHCO.sub.3/K.sub.2CO.sub.3 mixture is incorporated into the open pond medium at a concentration ranging from about 7 mM to about 1 M.

4. The method of claim 1, wherein the pH of the open pond medium is at least about 9.9.

5. The method of claim 1, further comprising incorporating glucose or other sugars or carboxylic acids into the open pond medium.

6. The method of claim 1, wherein the algae comprise a Chlorella sp., Dunaliella sp., Synechocystic sp., Cyanothece sp., Microcoleus sp., Euhalothece sp., or Spirulina sp. strain.

7. The method of claim 1, further comprising incorporating Ca and/or Mg into the open pond medium at a concentration of less than 7 mg/L.

8. The method of claim 7, wherein the Ca is incorporated into the open pond medium at a concentration of less than 1.5 mg Ca/L.

9. The method of claim 7, wherein the Mg is incorporated into the open pond medium at a concentration of less than 0.5 mg Mg/L.

10. The method of claim 1, further comprising circulating the algae within the open pond medium.

11. The method of claim 1, further comprising harvesting biomass from the cultured algae and recovering remnant media.

12. The method of claim 11, further comprising recycling the remnant media in a second open pond medium.

13. The method of claim 11, further comprising converting the harvested biomass to one or more fuels.

14. The method of claim 13, wherein the converting comprises hydrothermal liquefaction to produce biocrude having a N content of less than 4%.

15. The method of claim 1, further comprising regulating nitrogen input in the open pond medium, in a range from about 5 mg/L to about 27 mg/L, so as to modulate the biochemical composition of the microalgae.

16. The method of claim 1, wherein the open pond medium has a salinity in the range of from about 10 g/L to about 30 g/L, a pH greater than 10.0, and an alkalinity of up to about 1 M.

17. The method of claim 1, further comprising improving phycocyanin production by increasing one or more of biomass concentration, nitrogen concentration, and salinity in the open pond medium.

18. An open pond system comprising: a medium having a pH above 9.5 and exposed to solar radiation; an inorganic carbon buffer in the medium; and alkaliphilic algae in the medium; wherein the open pond system is free from any unnatural or concentrated CO.sub.2 supply.

19. The open pond system of claim 18, further comprising an organic substrate comprising glucose or other sugars or carboxylic acids in the medium.

20. The open pond system of claim 18, wherein the inorganic carbon buffer comprises either a NaHCO.sub.3/Na.sub.2CO.sub.3 mixture or a KHCO.sub.3/K.sub.2CO.sub.3 mixture present at a concentration ranging from about 7 mM to about 1 M.

21. The open pond system of claim 18, wherein the alkaliphilic algae comprises a Chlorella sp., Dunaliella sp., Synechocystic sp., Cyanothece sp., Microcoleus sp., Euhalothece sp., or Spirulina sp. strain.

22. The open pond system of claim 18, further comprising a water-moving device configured to circulate the medium within the open pond system.

23. The open pond system of claim 18, wherein the medium further comprises Ca and/or Mg at a concentration of less than 7 mg/L.

24. The open pond system of claim 18, wherein the medium further comprises one or more nutrients selected from the group consisting of: NaNO.sub.3, MgSO.sub.4, CaCl.sub.2, NaCl, ferric ammonium citrate, H.sub.3BO.sub.3, MnCl.sub.2, ZnCl.sub.2, CuCl.sub.2, Na.sub.2MoO.sub.4, CoCl.sub.2, NiCl.sub.2, V.sub.2O.sub.5, and KBr.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The patent or application file may contain one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fees.

[0020] FIGS. 1A-1B: Graphs showing pH-dependent changes in bicarbonate concentrations and mass transfer driving force (FIG. 1A), and in enhancement factor and CO.sub.2 flux (FIG. 1B).

[0021] FIG. 2: Illustration depicting the cellular DIC transport and fixation mechanisms in alkaline media.

[0022] FIGS. 3A-3C: Graphs showing cell growth, biomass productivity, rapid light curve, and nitrate utilization parameters of SLA-04 grown under different inorganic carbon conditions (Experiment A). FIG. 3A: Cell dry weight. FIG. 3B: Biomass productivity. FIG. 3C: Rapid light curve showing measurement of changes in electron transfer rate (ETR) with increasing incident photon intensity.

[0023] FIGS. 4A-4B: Graphs showing pH change during SLA-04 growth (FIG. 4A), and for Experiment A (FIG. 4B).

[0024] FIGS. 5A-5C: Graphs showing cell growth, biomass productivity, rapid light curve, and nitrate utilization parameters of SLA-04 grown under different pH and inorganic carbon conditions. FIG. 5A: Cell dry weight. FIG. 5B: Biomass productivity. FIG. 5C: Rapid light curve.

[0025] FIGS. 6A-6B: Graphs showing pH change during SLA-04 growth (FIG. 6A) and carbon balance of SLA-04 growth system (FIG. 6B).

[0026] FIG. 7: Graph showing CO.sub.2 absorption under abiotic (without algae) conditions.

[0027] FIGS. 8A-8B: Results of phototrophic SLA-04 outdoor (750 L) raceway pond cultivation (FIG. 8A), and carbon balance for cultures grown under phototrophic conditions (FIG. 8B).

[0028] FIG. 9: Results of mixotrophic cultivation of SLA-04 outdoor (750 L) raceway pond cultivation.

[0029] FIG. 10: Graph showing biomass productivity under fresh and recycling media conditions.

[0030] FIG. 11: Graph showing the effect of initial nitrate concentration on nitrogen percent of harvested biomass.

[0031] FIG. 12: SLA-04 productivities in high and low Ca/Mg media.

[0032] FIGS. 13A-13B: Biomass productivity for cultures grown in different N concentrations (FIG. 13A), and chlorophyll concentration for cultures grown in different N concentrations (FIG. 13B), given biomass productivity for two days' period.

[0033] FIGS. 14A-14F: Effect of media N input on SLA-04 cultures in 30 L reactors. FIG. 14A shows biomass productivity. FIG. 14B shows biomass produced/chlorophyll (g/g). FIG. 14C shows chlorophyll concentration. FIG. 14D shows nitrogen content in biomass (%). FIG. 14E shows maximum quantum yield (F.sub.V/F.sub.M). FIG. 14F shows FAME content.

[0034] FIGS. 15A-15B: Effect of media N input on SLA-04 cultures cultivated in 1100 L reactors. FIG. 15A shows the results from batch 1, and FIG. 15B shows the results from batch 2.

[0035] FIGS. 16A-16B: Biomass productivity (FIG. 16A), and maximum quantum yield of cultures grown with and without salinity conditions (FIG. 16B).

[0036] FIGS. 17A-17D: Cell dry weight (FIG. 17A), biomass productivity (FIG. 17B), chlorophyll content (FIG. 17C), and chlorophyll a/b ratio of cultures grown with salinity (0-30 g/L) (FIG. 17D).

[0037] FIGS. 18A-18E: Cell dry weight (FIG. 18A), biomass productivity (FIG. 18B), phycocyanin content (FIG. 18C), chlorophyll content (FIG. 18D), and chlorophyll a/b ratio of cultures grown with salinity (0-30 g/L) (FIG. 18E).

DETAILED DESCRIPTION OF THE INVENTION

[0038] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this invention pertains.

[0039] Provided is a method that can cultivate microalgae under high pH and alkalinity conditions at high productivity without a supply of concentrated CO.sub.2 in any form. Consequently, the method reduces production costs up to 25%. Furthermore, the method allows for open ponds to be used in geographic areas not co-located with a source of CO.sub.2. In other words, the method herein alleviates the need for an open pond to be in proximity to a flue gas source. In order to be able to achieve high growth rates using atmospheric CO.sub.2 alone, media design is key. In general, the media should have a high pH to drive atmospheric CO.sub.2 into solution at high rates, and should have a sufficient inorganic carbon “buffer” to allow rapid rates of CO.sub.2 fixation. The algae strain must also be capable of withstanding both the high pH and high inorganic carbon concentrations in the media. The high pH conditions allow the use of organic carbon (e.g., glucose or other sugars or carboxylic acids) to be used as a substrate in low-cost open ponds, without concern of a culture crash because most bacteria do not survive in the high pH conditions of the medium. Further, the method produces lower amounts of nitrogen in the algae, which is advantageous for biofuel production from the harvested biomass.

[0040] In accordance with the present disclosure, the cultivation of alkaliphilic algae under appropriately tailored media conditions can eliminate most of the obstacles encountered with mesophilic algae cultivation in open-ponds. These include (1) the need to situate open ponds close to a CO.sub.2 emission source, (2) costs associated with CO.sub.2 concentration, and (3) the energy and infrastructure costs associated with the supply of CO.sub.2 for commodity-scale biomass production. It is demonstrated herein that the high pH media conditions of alkaliphilic algae make it possible to carry out open-pond cultivation in “mixotrophic mode” without culture crash and without detrimental microbial contamination. These advantages are derived from the ability of highly alkaline solutions to efficiently absorb atmospheric CO.sub.2, and the inability of predatory microorganisms to survive under alkaline conditions. Moreover, with this method, after harvesting the microalgae, the aqueous medium which has high inorganic carbon and other nutrients can be recycled indefinitely without compromising the algal growth. In addition, the cultivation conditions reduce the nitrogen content of the biomass—an aspect that is highly advantageous for producing low nitrogen content biofuels from biomass intermediates (such as through hydrothermal liquefaction). Furthermore, cultivation of alkaliphilic microalgae under high salinity environment promotes the production of phycocyanin, a high value pigment.

[0041] Alkaliphiles are organisms that thrive at high pH values (>9.5). As such, the cultivation medium is at an initial pH ˜10 or higher, and contains high concentrations of inorganic carbon, up to 60-100 mM in the form of added NaHCO.sub.3/Na.sub.2CO.sub.3 and/or KHCO.sub.3/K.sub.2CO.sub.3. Alkaline solutions are especially effective in absorbing “atmospheric CO.sub.2” and sustaining the productivity of algae, without the need for a concentrated CO.sub.2 source and the infrastructure for CO.sub.2 distribution. Simultaneously, the liquid phase equilibrium between OH.sup.−, CO.sub.3.sup.2−, and HCO.sub.3.sup.− allows the solution to contain high concentrations of HCO.sub.3.sup.−, which is a DIC form usable by microalgae through carbonic anhydrases.

[0042] The mass transfer flux of CO.sub.2 (J.sub.CO.sub.2) from the atmosphere into highly alkaline bulk media is determined by (i) the driving force established by the concentration gradient of CO.sub.2 between the gas-liquid interface ([CO.sub.2(aq)*]) and the bulk liquid ([CO.sub.2(aq).sup.bulk]), (ii) an “enhancement factor”(E) that accounts for the enhancement of mass transfer rates due to reaction between (the acidic) CO.sub.2 and the high concentration of hydroxyl ions in the mass transfer boundary layer, and (iii) the mass transfer coefficient (k.sub.L):

J.sub.CO.sub.2=k.sub.L.Math.E.Math.([CO.sub.2(aq)*]−[CO.sub.2(aq).sup.bulk])  (1)

where k.sub.L is the physical mass transfer coefficient (m/h).

[0043] At the interface with air, the liquid-phase concentration of CO.sub.2 ([CO.sub.2(aq)*]) is determined by the concentration of CO.sub.2 in air (assumed to be 387 ppm) and the Henry's constant for CO.sub.2 ([CO.sub.2(aq)*]=0.013 mM). In the bulk, the aqueous CO.sub.2 concentration ([CO.sub.2(aq).sup.bulk]) is determined by the simultaneous equilibria established among reactions shown in Equations 2, 3, and 4 coupled to the electro-neutrality (total alkalinity) requirement shown in Equation 5:

[00001]$\begin{array}{cc}{\mathrm{CO}}_{2.Math.\left(\mathrm{aq}\right)}^{\mathrm{bulk}}+{\mathrm{OH}}^{-}.Math.\begin{array}{c}{k}_{11}\\ \rightleftarrows \\ k_{12}\end{array}.Math.{\mathrm{HCO}}_3^{-},.Math..Math.\mathrm{with}.Math..Math..Math.K_1=\frac{k_{11}}{k_{12}}=\frac{\left[{\mathrm{HCO}}_3^{-}\right]}{\left[{\mathrm{CO}}_{2.Math.\left(\mathrm{aq}\right)}^{\mathrm{bulk}}\right].Math.{\mathrm{OH}}^{-}]}=4.5\times {10}^7.Math.\frac{L}{\mathrm{mol}}.Math..Math.\mathrm{at}.Math..Math.25.Math.°.Math..Math.C.& \left(2\right)\\ {\mathrm{HCO}}_3^{-}+{\mathrm{OH}}^{-}.Math.\begin{array}{c}{k}_{21}\\ \rightleftarrows \\ k_{22}\end{array}.Math.{\mathrm{CO}}_3^{2-}+H_2.Math.O,.Math..Math..Math.\mathrm{with},.Math.K_2=\frac{k_{21}}{k_{22}}=\frac{\left[{\mathrm{CO}}_3^{2-}\right]}{\left[{\mathrm{HCO}}_3^{-}\right].Math.{\mathrm{OH}}^{-}]}=4.9\times {10}^3.Math.\frac{L}{\mathrm{mol}}.Math..Math.\mathrm{at}.Math..Math.25.Math.°.Math..Math.C.& \left(3\right)\\ H^{+}+{\mathrm{OH}}^{-}.Math.\begin{array}{c}{k}_{31}\\ \rightleftarrows \\ k_{32}\end{array}.Math.H_2.Math.O,.Math..Math.\mathrm{with}.Math..Math..Math.K_w=\frac{k_{32}}{k_{31}}=\left[H^{+}\right]\left[{\mathrm{OH}}^{-}\right]=0.92\times {10}^{-14}.Math.\frac{{\mathrm{mol}}^2}{L^2}.Math..Math.\mathrm{at}.Math..Math.25.Math.°.Math..Math.C.& \left(4\right)\\ \mathrm{TA}=\left[{\mathrm{HCO}}_3^{-}\right]+2\left[{\mathrm{CO}}_3^{2-}\right]+\left[{\mathrm{OH}}^{-}\right]& \left(5\right)\end{array}$

where TA is the “total alkalinity” of the system, and can be measured independently via titration. The equilibrium constant (K) values are from the literature. The plots in FIG. 1A (estimated by solving Equations 2-5) show that in a high alkalinity medium and at pH 10.2, approximately 35 mM HCO.sub.3.sup.− is present along with a high CO.sub.2 mass transfer driving force.

[0044] The pH driven enhancement factor (E) can significantly increase mass transfer rates in high pH media. For high alkalinity solutions reacting with small concentrations of CO.sub.2, the concentrations of CO.sub.3.sup.2− and HCO.sub.3.sup.− can be considered essentially invariant in the mass transfer boundary layer. At these conditions, the enhancement factor can be estimated from the solution of the ordinary differential equations that describe the one-dimensional mass transport of CO.sub.2 via the reaction shown in Eq. 2. The expression for E can be given as:

[00002]$E=\begin{array}{cc}1+\frac{{{\mathrm{OH}}^{-}}_{}.Math.{{\mathrm{HCO}}_3^{-}}_{}.Math.K_1.Math.\left[{\mathrm{OH}}^{-}\right]}{{{\mathrm{CO}}_2}_{}.Math.\left(K_1.Math.{\mathrm{CO}}_{2.Math.\left(\mathrm{aq}\right)}^{*}.Math.{{\mathrm{HCO}}_3^{-}}_{}+{{\mathrm{OH}}^{-}}_{}\right)}& \left(6\right)\end{array}$

where, the subscripted 's represent diffusion coefficients of the various dissolved species. As seen from Eq (6), at a constant temperature, is unction of solution pH only (see computed values in FIG. 1B; E at pH 10.2=40).

[0045] The physical mass transfer coefficient of CO.sub.2 (k.sub.L) in open raceway ponds has been previously estimated to be 0.1 m/h. At this k.sub.L, the CO.sub.2 mass transfer flux values can be estimated as a function of pH using computed values of E and ([CO.sub.2(aq)*]−[CO.sub.2(aq).sup.bulk]) and shown in FIG. 1B. At pH 10.2, the mass transfer flux is approximately 35 mmole/m.sup.2/h, which corresponds to biomass productivity of 20 g/m.sup.2/d (assuming a biomass C-content of ˜50%).

[0046] During cultivation, HCO.sub.3.sup.− is taken up, CO.sub.2 is abstracted and fixed, resulting in a net release of OH.sup.− as shown in Eqs. 7 and 8 below:

##STR00001##

[0047] The production of OH.sup.− shifts the DIC equilibrium towards CO.sub.3.sup.2− (see Eq. 3) which, in turn, increases the driving force for CO.sub.2 dissolution. Any net increase in pH and associated decrease of HCO.sub.3.sup.− due to conversion to CO.sub.3.sup.2− can be rebalanced at night, when photosynthesis is absent (Eq. 1).

[0048] Suitable alkaliphilic algae include, but are not limited to, eukaryotic microalgae such as Chlorella sp. and Dunaliella sp., as well as cyanobacteria such as Synechocystic sp., Cyanothece sp., Microcoleus sp., Euhalothece sp., and Spirulina sp. Some non-limiting examples of alkaliphilic algae strains include Synechocystis salina, Aphanothece stagnina, Chamaesi-phon subglobosus, Rhabdoderma lineare, Synechococcus elongates, Phormidium ambiguum, Phormidium foveo-larum, Phormidium retzii, Oscillatoria splendid, Sscilla-toria limnetica, Spirulina fusiformis, and Spirulina laxissima. However, any algae that can thrive at high pH values (>9.5) and withstand high (˜60-100 mM) inorganic carbon content can be utilized.

[0049] The multi-step process of DIC transport into alkaliphilic microalgae cells and ultimate conversion to organic carbon is shown in FIG. 2. The conversion to organic carbon using light energy (i.e., photosynthesis) takes place via two parallel processes: light-dependent and light-independent reactions (FIG. 2). The light-dependent reactions occur in the thylakoid region of cells with two photosystems (PS 1 and PS II) that work in tandem to produce energy (ATP) and reducing equivalents (NADPH). When a photon strikes chlorophyll within the photosystem, the absorbed energy is either used to split H.sub.2O (at PSII) and recover electrons (e.sup.−) or is dissipated as heat/fluorescence. The recovered e are transported through an electron transport chain (through ferredoxins, Fd) and used for generation of reducing equivalents (NADPH) or for nitrate reduction and protein synthesis (see the reactions below the dotted line, FIG. 2).

[0050] In parallel, under alkaline conditions, light-independent DIC uptake occurs via carbon concentrating mechanisms (CCMs) that consist of a series of active HCO.sub.3.sup.− transporters, carbonic anhydrases, and, in some cases, conversions of C3 and C4 molecules (not depicted in FIG. 2 for simplicity). Since multiple enzymatic steps are involved, the DIC transport process can be kinetically limited by external DIC concentrations. In the presence of high DIC, the net flux through the transporters (shown as filled ovals in FIG. 2) is enhanced such that more DIC is available for carbonic anhydrases that can ultimately deliver CO.sub.2 to RuBisCO at higher rates. The half-saturation constant for carbonic anhydrases is generally in the 20 mM range, and low DIC availability severely limits the kinetics of this enzyme. In addition, the carbonic anhydrase gene expression is low under low HCO.sub.3.sup.− concentrations, which may decrease the availability of CO.sub.2 at RuBisCO. Such a decreased availability of CO.sub.2 at RuBisCO would negatively impact the rates of carbon fixation through the Calvin cycle. Simultaneously, the high cellular DIC flux drives the light-dependent reactions towards higher production of NADPH by increasing NADPH demand for use in carbon fixation. Thus, by maintaining high alkalinity, higher photosynthetic efficiencies and ultimately high rates of CO.sub.2 fixation can be achieved.

[0051] The mechanisms of inorganic carbon uptake from the atmosphere and use for photosynthesis are (as described above) innately established in natural alkaline lakes which have the highest reported aquatic photosynthetic carbon fixation rates. In addition to facilitating sustained supply of CO.sub.2 from the atmosphere (rather than flue gases), the use of high-pH and high-alkalinity media can enable the sustained cultivation of desired species due to the relatively low microbial diversity in these harsh environments. Grazer infestations are also less likely in alkaline environments. For example, Daphnia eggs lose viability when pH values exceed 10-10.5. In commercial practice, Spirulina production is successful, at least partly, due to the high pH growth conditions that enable prolonged maintenance of these cyanobacterial species in low-cost open ponds. A SLA-04 culture crash has not been observed despite several months of outdoor cultivation in high-pH and high-alkalinity media.

[0052] The adjustment of macro- and micro-nutrient concentrations results in improvements in carbohydrate and lipid productivity. One of the principal macro-nutrients important for algae cultivation is nitrogen (N). N is also a significant contributor to the net carbon footprint of algal biofuels. Low N is also very desirable for downstream conversion processes since the resulting fuels also have a low N content. Therefore, the cultivation of alkaliphilic algae on low N media was evaluated. The results showed that the high biomass productivities can be maintained at lower N in the media, and the resulting biomass also has a low N-content. An increase in pigment production (e.g., chlorophyll b) when cellular N content is high has been observed, which causes cultures to become “dark” and detrimental to light penetration. Overall, by adjusting media alkalinity and N supply, biomass with low N-content can be produced.

[0053] The requirements for the micro-nutrients Ca and Mg have also been evaluated, as the effects of these micro-nutrients are generally underappreciated in the art. Typically, these micro-nutrients are added at a concentration level of 5-7 mg/L (Bold's medium). However, under alkaline conditions their solubility in the medium is diminished. These reduced dissolved nutrient concentrations can induce “nutrient-limited stress” on the growing microalgae. It is known that N-starvation improves lipid productivity in microalgae. Therefore, whether micro-nutrient (Ca and Mg) limitations would also lead to improved biomass and lipid productivity during alkaliphilic microalgae cultivation was evaluated.

[0054] The impact of increasing medium salinity on biomass growth was also evaluated. Use of saline water (from oceans or from saline/brackish groundwater sources) improves the sustainability of microalgae cultivation by decreasing the requirements of freshwater. The results, described in the examples herein, indicate that cultivation of microalgae in high salinity media containing excess nitrogen and high biomass concentrations (i.e., conditions that limit light penetration into cultures), increased the production of phycocyanin—a high-value nutraceutical.

[0055] Recycled media can be used in the open ponds. In some embodiments, high concentrations (for example, 100 mM) of bicarbonate/carbonate salts are added to the culture media to provide high alkalinity. Hence, the ability to recycle and reuse the media is important to minimize the costs associated with replenishing these salts, and other unused nutrients. As described in the Examples herein, post-harvest media can be re-used without detrimental impact on biomass productivity.

[0056] The high pH media permits open-pond cultivation in “mixotrophic mode” without culture crash. In addition to facilitating sustained supply of CO.sub.2 from the atmosphere (rather than from flue gases), the use of high-alkalinity and high-pH media can enable sustained cultivation of desired species, since it is likely that contaminating populations will be less diverse at higher pH values. A culture crash of the alkaliphilic strain SLA-04 has not been observed in the presently described method, despite a significant number of months of outdoor cultivation in high-pH and -alkalinity media. The extreme pH and alkalinity of the medium also allows for low-cost outdoor pond mixotrophic cultivation with significantly lower chance for bacterial contamination—mesophilic (<pH 8.5) outdoor cultivation woud likely not be possible with mesophilic algae.

[0057] It is understood that an open pond utilizing the methods described herein can include any apparatuses or structures common in open pond algae systems. For example, the open ponds may include paddle wheels or other water-moving devices usable to keep the algae circulating, as well as electronic controls, pumps, pipes, sensors, and the like. Continuous mixing of algal cultures is preferred in order to prevent thermal stratification and cell sedimentation, and to maintain carbonation. In some embodiments, the open ponds are known as raceway ponds, resembling a race track. A typical open pond is about one-foot deep, from about one acre to several acres in size, where the algae is exposed to natural solar radiation which is converted into biomass. An open pond system can be constructed out of any suitable material for containing the medium, such as PVC, PE, or concrete. Further, one skilled in the art will recognize that once the algae is harvested (such as by centrifugation), any method known in the art can be utilized to convert the harvested biomass to one or more high-value downstream products such as fuels, including hydrothermal liquefaction. In some embodiments, the biomass harvested from the open ponds as described herein can be converted to biofuels with lower nitrogen content than algae from conventional open ponds.

EXAMPLES

Example 1: Effect of HCO.SUB.3..SUP.− .Content on SLA-04

[0058] Biomass Growth and Productivity

[0059] The Chlorella sp. strain SLA-04 (henceforth referred to as SLA-04) was isolated from Soap Lake in the State of Washington (USA). Cultures were grown in a medium that comprises the nutrients: NaNO.sub.3 (1.05 mM), KH.sub.2PO.sub.4 (0.3 mM), MgSO.sub.4.7H.sub.2O (0.3 mM), CaCl.sub.2.2H.sub.2O (0.17 mM), NaCl (0.42 mM), ferric ammonium citrate (10 mg/L)), and 1 mL 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.2.6H.sub.2O (0.04 mM), V.sub.2O.sub.5(0.01 mM), and KBr (0.08 mM). For experiments that were started in a mildly alkaline pH medium (8.7 and 8.2), NaHCO.sub.3.sup.− was added as an inorganic carbon source at HCO.sub.3.sup.− concentrations in the range of 7-40 mM. For pH-controlled cultures, pH was controlled by periodic CO.sub.2 addition through a solenoid-regulated control system that maintained the pH at an approximate value of 8.7 (Neptune Systems Apex, NC, USA). (Experiment A.) For experiments that were started under significantly higher alkaline pH conditions (pH 10), equal molar concentrations of NaHCO.sub.3.sup.− and Na.sub.2CO.sub.3 were added to achieve final HCO.sub.3.sup.− concentrations of 4.5-30 mM. (Experiment B.)

[0060] Open raceway ponds with dimensions of 2′×1′×1′ (L×W×D) were constructed and used in these experiments. These ponds were equipped with a real-time temperature and pH monitoring and data logging system (Neptune Systems Apex, NC, USA). The ponds were placed in a heated greenhouse. Tap water available at the greenhouse facility was first filtered through a 10 μm filter (to remove sediments) and then used for medium preparation. The working volume of the culture was kept at 5″ for Experiment A and 6″ for Experiment B.

[0061] During Experiment A, biomass concentrations (measured as cell dry weight (CDW) and productivity of cultures grown in media with varying HCO.sub.3.sup.− concentrations and without pH control were assessed and compared with pH-controlled controls (FIGS. 3A-3B). The results show that biomass concentrations as well as biomass productivity were greater when HCO.sub.3.sup.− concentrations in the media were higher. Maximum biomass productivity was observed to be 24.2±1.0 g CDW/m.sup.2/day for cultures that initially contained 40 mM HCO.sub.3.sup.−. These productivity values were similar (23.4±0.28 g CDW/m.sup.2/day) to the productivities measured in pH-controlled cultures containing 20 mM HCO.sub.3.sup.− (FIG. 3A).

[0062] Because of CCMs, microalgae can accumulate HCO.sub.3.sup.− in cytosol and subsequently deliver high CO.sub.2 concentrations around the ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) enzyme, and thus increase the rates of photosynthetic carbon fixation (FIG. 2). However, the HCO.sub.3.sup.− transfer rate into cells is an important factor to maintain high cytosolic HCO.sub.3.sup.− concentration as well as photosynthetic rate. Higher media concentrations of HCO.sub.3.sup.− lead to improved mass transfer rates and cellular uptake.

[0063] To assess the impact of HCO.sub.3.sup.− on photosynthetic efficiency (i.e., the efficient use of incident photons), “rapid light curve” measurements were made. As shown in FIG. 2, only a portion of the electrons generated from the incident light are utilized towards photosynthesis while the rest are dissipated. The electrons participating in photosynthesis serve to electrochemically reduce cellular DIC to carbohydrates via NADPH mediated reactions at RuBisCO (FIG. 2). The flow of electrons towards photosynthesis is referred to as the “electron transfer rate” (ETR). Higher values of ETR were observed in cultures containing higher concentrations of bicarbonate in the media (FIG. 3C). Cultures fed with high HCO.sub.3.sup.− content (40 mM) showed a maximum ETR value (ETR.sub.max) of 32 μmol/m.sup.2s, while cultures fed with 7 mM HCO.sub.3.sup.− showed much lower ETR.sub.max values of 13 μmol/m.sup.2s (FIG. 3C). Overall, the higher alkalinity medium allowed better photon utilization.

[0064] pH Change

[0065] An increase in pH was observed during algal growth at day time (light cycle) due to uptake of bicarbonate and release of hydroxyl ions (Eq 7). pH decreased at night due to CO.sub.2 release from microalgae respiration (FIG. 4A).

Example 2: Biomass Growth and Productivity in High-pH Media with Varying Levels of Alkalinity

[0066] Biomass Growth, Productivity

[0067] Experiment B was performed to assess biomass productivity at high pH. Since Experiment A (FIGS. 3-4) showed improvement in productivity with higher alkalinity, a similar approach was used in Experiment B and media alkalinity was varied. Experiment B was performed in media initially at pH 10. The alkalinity of the media was set such that initial bicarbonate concentrations were 7, 20, and 30 mM. An experiment at an initial pH of 8.2 and initial HCO.sub.3.sup.− concentration of 30 mM served as a control. FIGS. 5A-5C depict the growth, productivity, and ETR of SLA-04 cultures during Experiment B. As observed in Experiment A (Example 1), biomass growth, productivity, and ETR increased when HCO.sub.3.sup.− concentration in medium increased. The results revealed that with the same HCO.sub.3.sup.− availability (30 mM), cultures started with higher pH (9.9) showed higher growth as well as maximum productivity (34.7±1.5 g CDW/m.sup.2/day) than cultures started with low pH (8.2) (FIGS. 5B, 5C). Table 1 (FIG. 13) shows that when compared to low HCO.sub.3.sup.− conditions, the parameters related to efficient energy capture towards carbon fixation were higher, and the parameters related to energy dissipation were lower under high HCO.sub.3.sup.− conditions.

TABLE-US-00001 TABLE 1 Rapid light curve parameters for cultures grown under high and low HCO.sub.3.sup.− concentrations. High Low Param- HCO.sub.3.sup.− HCO.sub.3.sup.− Description eter (30 mM) (7 mM) Energy Effective PS II quantum yield Y (II) 0.361 0.209 capture Maximum electron transfer ETR.sub.max 17.9 9.4 towards rate (μmol/m.sup.2s) carbon Photosynthetic efficiency α 0.167 0.098 fixation (el./ph.) Light saturation I.sub.k 126 96 (μmol/m.sup.2s) Energy Quantum yield of regu- Y (NPQ) 0.066 0.094 dissi- lated energy dissipation pation Quantum yield of non regu- Y (NO) 0.573 0.697 lated energy dissipation

[0068] Nitrate utilization efficiency (g biomass/g nitrate utilized) was observed to be higher in cultures with high HCO.sub.3.sup.− availability (FIG. 5D), resulting in low nitrogen content in the biomass. This is important to produce low nitrogen content biofuels from biomass intermediates through hydrothermal liquefaction (HTL). Moreover, it reduces the cost and greenhouse gas emissions associated with industrial nitrate production.

[0069] pH Change, and Atmospheric CO.sub.2 Capture

[0070] Change in pH during algal growth is illustrated in FIG. 6A. Cultures fed with high HCO.sub.3.sup.− content showed higher buffering capacity than cultures fed with low HCO.sub.3.sup.− content. Detailed OH.sup.− increase and controlled mechanisms are shown in Table 2.

TABLE-US-00002 TABLE 2 OH− ion balance of SLA-04 grown under different pH and inorganic carbon conditions. OH.sup.− Control mechanisms Increase HCO.sub.3.sup.− Atm. CO.sub.2 Inorganic in OH.sup.− Atm. CO.sub.2 to CO.sub.3.sup.2− absorption/ carbon conc. absorption conversion Δ OH.sup.− HCO.sub.3.sup.− to CO.sub.3.sup.2− (mM) (mM) (mM) (mM) (mM) conversion 7 6.74 5.26 0.56 0.91 9.33 40 12.39 6.06 6.10 0.23 1.00 60 13.44 7.62 5.70 0.12 1.34 30 pH 8.2 11.35 2.46 8.77 0.12 0.28 Note: Increase in OH− concentration generated was calculated from sum of increase in TOC and NO.sub.3.sup.− and phosphate ut ilization data. Increase in CO.sub.3.sup.2− indicates HCO.sub.3.sup.− to CO.sub.3.sup.2− conversion. Atm. CO.sub.2 absorption was calculated by: Atm. CO.sub.2 absorption = OH.sup.− generation − HCO.sub.3.sup.− to CO.sub.3.sup.2− conversion + ΔOH.sup.−

[0071] The data indicate that with the same initial inorganic carbon availability (60 mM), cultures started with pH 9.9 showed higher (1.34) atm. CO.sub.2 absorption/HCO.sub.3.sup.− to CO.sub.3.sup.2− conversion than cultures started with pH 8.2 (0.28). These results indicate that under high pH algal growth conditions, atmospheric CO.sub.2 absorption dominates over HCO.sub.3.sup.− to CO.sub.3.sup.− conversion and results in low inorganic carbon drain. Carbon content of dried biomass was observed to be in the range of 44-47%. FIG. 5B depicts the carbon balance data for all cultures. Maximum ΔTOC (12.3±0.7 g C/m.sup.2) was observed in cultures fed with high inorganic carbon content (60 mM). For these cultures, the inorganic carbon drain ΔIC was observed to be 4.75±0.13 g C/m.sup.2, revealing that the remaining 61% (7.55±0.61 g C/m.sup.2) of biomass carbon was derived from atmospheric CO.sub.2 absorption (FIG. 6B). The Example 1 and 2 results demonstrate that increasing initial pH from 8.7 to 9.9 results in an increase in atmospheric CO.sub.2 fixation in biomass from 37% to 61%.

Example 3: Phototrophic and Mixotrophic Cultivation of Microalgae Under High pH and Alkalinity

[0072] Sustainability of microalgal cultivation under phototrophic and mixotrophic conditions was studied in 1100 L ponds with efficient mixing by paddle wheel (Commercial algae Professionals, NC, USA) with a working volume of 750 L and a depth of 7″ in outdoor raceway ponds under high pH (˜10) and high inorganic carbon (˜100 mM) conditions without CO.sub.2 supplementation. Under the phototrophic conditions, cell dry weight, and biomass and lipid productivities, were determined to be 23 g/m.sup.2/day and 2 g/m.sup.2/day, respectively (FIG. 8A). The results indicate that ˜95% of the biomass carbon was derived from atmospheric CO.sub.2, rather than from the added inorganic carbon (FIG. 8B). This shows that when compared to Example 2, the results demonstrate that efficient mixing of the culture medium improved atmospheric CO.sub.2 derived biomass carbon from 61% to 95%. Under mixotrophic cultivation with glucose, SLA-04 cultures grew without any measurable signs of contamination and showed significantly higher biomass productivity (57 g/m.sup.2/day) due to the availability of additional organic carbon and associated reducing equivalents (FIG. 9). Lipid productivities were also higher under mixotrophic conditions (7 g/m.sup.2/day) relative to phototrophic SLA-04 cultures (2 g/m.sup.2/day) (FIG. 9).

Example 4: Remnant Media Nutrients Recycling

[0073] After growth, remnant media was recovered by harvesting the algal biomass through centrifugation. Then, the effect of remnant media (which contain high amount of inorganic carbon (˜60 mM)) on biomass growth was evaluated by adding the used portion of nutrients only. The remnant media was recycled 8 times without any deleterious effects on algal biomass growth. FIG. 10 depicts a comparison between algal biomass growth pattern using remnant media and fresh media.

Example 5: Nitrogen Utilization by SLA-04 and the Effect of N Input on SLA-04 Biochemical Composition

[0074] Nitrogen is a macronutrient and N content in biomass can determine the end-use of microalgae. For instance, high N-content (i.e., high protein) is desirable for microalgae use as food/feed ingredient. However, for biofuel production low N in biomass is desirable since presence of N in fuel is detrimental to fuel quality. Conventional cultivation methods use high concentration of N in the medium, which leads to production of biomass with high N content. The concentration of these nitrogenous compounds in the biomass can be decreased by growing microalgae under nitrogen limitation conditions. But severe nitrogen limitation can also impair growth. It was demonstrated that by maintaining an optimal concentration of N in the media, the N-content of biomass can be decreased without significant detrimental impact on biomass productivity.

[0075] The results from 450 mL e-PBR experiments (FIG. 11) show that the high biomass productivities can be maintained at lower N in the media. The resulting biomass also has a low N-content (FIG. 11).

[0076] Additional indoor experiments were performed with SLA-04 cultures grown in 3 L reactors. Cultures were grown in a medium that comprised the nitrogen concentration in the range of 5-15 mg/L using NaNO.sub.3 as a nitrogen source. NaHCO.sub.3.sup.− and Na.sub.2CO.sub.3 were added in a molar ratio of 2:3 to get a final HCO.sub.3.sup.− concentration (30 mM) and initial pH 10.1. Cultures adapted to high media N input (27 mg/L) with initial nitrogen content in biomass about 7% was used as an inoculum. The reactors were placed on a stir plate and illuminated by a bank of 4 Ecolux Starcoat 54 W fluorescent tubes (GE Lighting, Cleveland, Ohio) on each side. Light cycle was maintained at a PAR intensity ˜400 μmol/m.sup.2/s on each side for 10 h.

[0077] The results (3 L reactors) show that the high biomass productivities can be maintained even at N content 5 mg/L in the media (FIG. 13A) because the low N availability avoids excess production of nitrogen storage compounds (e.g., for pigments). Also, an increase in chlorophyll pigment production (FIG. 13B) was observed when medium nitrate content was high, which causes cultures to become “dark” and detrimental to light penetration. The results indicate all the cultures were photosynthetically active, however there is no significant difference in photosynthesis efficiency (F.sub.v/F.sub.m) and maximum electron transfer rate (ETR.sub.max) when N availability for the culture is decreased (Table 3).

TABLE-US-00003 TABLE 3 Rapid light curve parameters for cultures grown in 3 L reactors and under different NO.sub.3 concentrations (time = 2 days). Media N ETR.sub.max PSII input (mg/L) Fv/Fm (μmol/m.sup.2s) 5 0.649 17.6 10 0.685 17.7 15 0.711 19.1 Note: Fv/Fm: maximal PS II quantum yield; ETR.sub.max PSII: Maximum electron transfer rate (μmol/m.sup.2s).

Example 6: Nitrogen Utilization by SLA-04 and the Effect of N Input on SLA-04 Biochemical Composition—Outdoor Experiments at 30 L Scale

[0078] The outdoor experiment was conducted as a follow-up experiment to the indoor experiment with the same media conditions to examine the application of low-N, high-productivity cultivation in open ponds. Initial media N input was adjusted to a range of 5-27 mg/L using sodium nitrate as a nitrogen source. In contrast to the indoor experiments, cultures were first adapted to experimental nitrogen conditions for ten batches to get constant N content in biomass relative to the media N input. Then the experiments were conducted in open raceway ponds (30 L) with working volume of 20 L and performed in sequential batches, with each batch lasting for a duration of two days.

[0079] FIG. 14A depicts biomass productivity of SLA-04 on media N input conditions. The results show when the media N input increased, there was an increase in biomass productivity (FIG. 14A). Since chlorophyll is the nitrogenous compound among others, as media N concentrations increases, there is an increase in chlorophyll content (FIG. 14C). Hence, when considering chlorophyll concentration (FIG. 14C), the biomass productivity of SLA-04 per chlorophyll content was observed to decrease along with an increase in media N input (FIG. 14B). Without wishing to be bound by theory, it is believed this is due to the light limitation to chlorophyll present in cells being present at greater pond depths that results in a decrease in biomass productivity relative to chlorophyll content. As the N input increases, there is an abundant availability of nitrogen source and cultures tend to accumulation nitrogenous compounds (e.g., chlorophyll and related proteins). This results in increased nitrogen content in biomass (range: 3.5-7.2%) (FIG. 14D). F.sub.v/F.sub.m factor, which represents PSII maximum quantum yield at dark adapted state for cultures, is shown in FIG. 14B. Among other nutrients, nitrogen stress for culture (that damages PSII) decreases F.sub.v/F.sub.m. The results show F.sub.v/F.sub.m is increased in increase in order with media N content (5-27 mg/L). Even though F.sub.v/F.sub.m of cultures fed with low N (5-8 mg/L) had low F.sub.v/F.sub.m, it did not show a significant effect on biomass growth and productivity (FIG. 14A). Nitrogen stress condition in microalgae can inhibit further cell reproduction and induce lipid synthesis as an energy storage process. Fatty acid methyl esters (FAME) data indicate that the cultures fed at N input 5 mg/L had four folds higher FAME content (16%) than cultures fed at N input 27 mg/L (4.1%) (FIG. 14F). Low F.sub.v/F.sub.m is the indicator of stress condition for cultures fed with low (5 mg/L) N input prone to induce lipid biosynthesis and thereby shown increased FAME content. Table 4 depicts the biochemical composition of SLA-04 fed at different N input. The results confirm one can modulate the biochemical composition of the microalgae by regulating media N input.

TABLE-US-00004 TABLE 4 Biochemical composition of SLA-04 grown under different N input environment Carbohydrate Ash Protein Fame Moisture Sample Name (%) (%) (%) (%) (%) Total 5 mg/L N-Batch1 33.15 6.47 22.724 17.34 6.8 86.48 8 mg/L N-Batch1 30.49 6.70 35.88 6.20 7.58 86.85 11 mg/L N-Batch1 15.00 6.30 42.458 7.00 7.93 78.69 27 mg/L N-Batch1 13.88 8.50 43.056 4.00 8.03 77.47 5 mg/L N-Batch3 36.31 5.75 22.724 16.56 6.39 87.73 8 mg/L N-Batch3 27.91 5.20 33.488 6.00 7.73 80.33 11 mg/L N-Batch3 13.40 6.50 43.056 4.00 8.2 75.15

Example 7: Nitrogen Utilization by SLA-04 and Effect of N Input on SLA-04 Biochemical Composition—Outdoor Experiments at 1100 L Scale

[0080] Based on the above experiment, it is important to start cultures with the same chlorophyll concentration to evaluate the effect of media N input on biomass production. The initial chlorophyll concentration was adjusted to a similar concentration by appropriate dilution of inoculum for all N input culture conditions. The experiments were conducted in big raceway ponds (1100 L) with working volume of 500 L and at ˜5 inches' depth. Initial media N input was adjusted to a range of 5-15 mg/L using sodium nitrate as a nitrogen source.

[0081] Cultures fed with N concentration 5 mg/L showed higher biomass productivity than cultures fed with N concentration 10 and 15 mg/L (FIGS. 15A-15B). The results indicate the biomass productivity increases as the N input decreases in media because of two factors: i) high cell concentration at N limitation conditions provide more cellular machinery for carbon fixation, and ii) as the growth proceeds, the chlorophyll content of cultures supplemented with high N availability increases, prone to light limitation to deeper layers when compared with cultures supplemented with low N input.

[0082] The presence of high media alkalinity also decreases the N uptake by SLA-04 (FIG. 5D). Overall, these results show that by adjusting media alkalinity and N supply, biomass with low N-content (˜3%) can be produced. Cultures grown outdoors (FIGS. 8A, 9) also had an N-content of ˜3%. Most other microalgae have an N content of 4-8%.

Example 8: Optimization of Micro-Nutrient Utilization by SLA-04

[0083] Effect of Ca and Mg on Growth of SLA-04

[0084] It was observed that biomass and lipid productivities can be improved (up to 33%) through use of low concentrations of Ca and Mg (<1.5 mg-Ca/L and <0.5 mg-Mg/L) (FIG. 12). Most microalgae growth media recipes have much higher Ca and Mg. For example, Ca and Mg concentrations in Bold's medium are nearly 7 mg/L. This finding—that under alkaline conditions, it is possible to achieve higher biomass productivities and lipid accumulation at significantly lower levels of Ca and Mg compared to traditional Bold's medium—has very favorable economic implications in large-scale outdoor algae cultivations.

[0085] Effect of Salinity (NaCl) on Growth of SIA-04

[0086] Lowering fresh water requirements is important for sustainable microalgae cultivation. Saltwater is a more sustainable water source than fresh water. For instance, seawater is inexpensively accessible in coastal areas of the southeast US (e.g. Florida and other Gulf states) and brackish water is abundant in southwest US (e.g. Arizona, New Mexico, Texas). These locations also have the most appropriate weather for microalgae cultivation. Str. SLA-04 can thrive in high salinity media as it was isolated from saline-alkaline lake (Soap Lake, State of Washington).

[0087] Growth of the isolated strain C. sorokiniana str. SLA-04 was examined in a BG-11 medium with nitrate content 40 mg/L and similar salinity to seawater (30 g/L). An appropriate proportion (2:3) of NaHCO.sub.3.sup.− and Na.sub.2CO.sub.3.sup.2− were added as an inorganic carbon source to get a final HCO.sub.3.sup.− concentration (30 mM) and initial pH 10.1. The experiment was performed in 3 L reactors under 800 μmoles/m.sup.2/s light illumination and light-dark cycles of 10 h/14 h.

[0088] Interestingly, the results show improvement of biomass productivity of SLA-04 with medium containing high salinity (FIG. 16A). Such improvement can be assigned to three factors: 1) increased availability of sodium ions, which are required for pH homeostasis of alkaliphilic C. sorokiniana str. SLA-04; 2) the Henry constant for dissolution of CO.sub.2 in water increases by increase in salinity, which improves carbon capturing and inorganic carbon availability for salty cultures; and 3) there is a lower pigmentation of microalgae cells grown in saltwater than fresh water, which therefore increase light availability. F.sub.v/F.sub.m factor, which represents PSII maximum quantum yield at dark adapted state for indoor cultures, is shown in FIG. 16B. Any environmental or nutritional stress for culture (that damages PSII) decreases F.sub.v/F.sub.m. The results show there is no significant difference in F.sub.v/F.sub.m between cultures grown with salinity and cultures grown without salinity.

[0089] The outdoor experiment was conducted with the same media conditions as the indoor experiment to examine the application of this method for more cost-efficient and manageable open ponds. To evaluate the effect of salt concentration, two different salt concentrations (18 g/L and 30 g/L) were used. The experiments were conducted in open raceway ponds (30 L) with working volume of 18 L, and were performed in sequential batches that each lasted 2 days.

[0090] FIG. 17A and FIG. 17B illustrate the growth pattern and biomass productivity of SLA-04. The results indicate biomass productivity is increased on the order of salt concentration increases in the medium (FIG. 17A). Chlorophyll a is the pigment that interacts directly in the light requiring reactions of photosynthesis, whereas chlorophyll b is an accessory pigment and acts indirectly in photosynthesis by transferring light it absorbs to chlorophyll a. Excessive accumulation of chlorophyll b limits the light penetration in to deeper layers. Low light availability further decrease photosynthetic activity and increase respiration rates, and is thereby attributed to low productivity. The lower chlorophyll concentration and higher chlorophyll a/b ratio of the cultures is the indication of efficient light penetration and photosynthetic efficiency for the cultures with high salinity (FIGS. 17C-17D). The Henry constant for dissolution of CO.sub.2 in water increases by the increase in salinity, which results in higher atmospheric carbon capture under salinity conditions (Table 5). Overall, these results are in accordance with indoor experiments and indicate that saltwater can be used as an inexpensive water source to enhance the biomass productivity and carbon capturing.

TABLE-US-00005 TABLE 5 Carbon balance of SLA-04 grown under different salinity conditions Assimilated organic Inorganic carbon Atmospheric carbon carbon (mM) depletion (mM) captured (mM) 0 g/L 18 g/L 30 g/L 0 g/L 18 g/L 30 g/L 0 g/L 18 g/L 30 g/L Batch 1 5.3 6.7 7.4 0.3 0.5 0.4 5 6.1 7 Batch 2 4.5 4.3 6.1 0.6 0.1 0.3 3.9 4.2 5.8

[0091] Effect of Salinity on Phycocyanin Production

[0092] Phycocyanin is a light-harvesting pigment and nitrogen-storing protein found in the prokaryotic cyanobacteria species, as well as in eukaryotic microalgae. Phycocyanin is widely used in pharmaceuticals and blue pigments. It is used as a natural dye for foods and cosmetics. Chlorella sorokiniana is one of the highest natural sources of phycocyanin and chlorophyll. Hence, the strain C. sorokiniana str. SLA-04 has the ability to produce phycocyanin. Environmental stresses such as light intensity, culture concentration, salinity, pH, and nitrogen availability can influence phycocyanin production in microalgae. In this example, the effect of salt concentration, inoculum concentration, and nitrogen content on phycocyanin production of C. sorokiniana str. SLA-04 was evaluated.

[0093] Culture conditions: since phycocyanin is the nitrogen storage compound, when compared to the above-described outdoor experiment, the medium nitrate concentration was increased from 40 mg/L to 150 mg/L to provide nitrogen abundant environment. Also, inoculum concentration was increased from 0.32 to 0.75 g/L.

[0094] FIGS. 18A-8B depict growth and average biomass productivity of the SLA-04 under the culture conditions. In contrast to the above-described experiments, the results show there is no significant difference in biomass growth and productivity between cultures grown with and without salinity. Without wishing to be bound by theory, it is believed this is due to the high pigmentation noticed in saline cultures (FIGS. 18C-18D). A comparative analysis between batch 1 and batch 2 cultures indicated that cultures started with low inoculum concentration showed higher biomass productivity. This is attributed to better light penetration.

[0095] FIGS. 18C-18D show pigmentation (phycocyanin and chlorophyll) of SLA-04 under culture conditions. The results show salt stress increased the phycocyanin content in biomass. As the salt concentration increases, phycocyanin content in biomass was observed to be increased in order (FIG. 18C). Like the above-described experiments, the results revealed a decrease in total chlorophyll content and an increase in chlorophyll a/b ratio with respect to salt concentration in the medium (FIGS. 18D-18E).

[0096] Certain embodiments of the methods and systems disclosed herein are defined in the above examples. It should be understood that these examples, while indicating particular embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the compositions and methods described herein to various usages and conditions. Various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof.