Bioreactor systems and methods

Abstract

A method comprising adding photoautotrophs to a growth medium in a first bioreactor, the growth medium comprising a liquid medium, adding photons and CO2 to the first bioreactor, conducting a photoautotrophic process in the first bioreactor to produce glycolic acid, measuring, via one or more first sensors, a first operational parameter of the first bioreactor, removing the glycolic acid from the first bioreactor in response to the first operational parameter meeting a first threshold value, adding the glycolic acid and water to a second bioreactor, adding microorganisms to the second bioreactor, conducting a heterotrophic process in the second bioreactor to produce a secondary product and CO2, measuring, via one or more second sensors, a first operational parameter of the second bioreactor, and removing the secondary product from the second bioreactor in response to the first operational parameter meeting a first threshold value.

Description

TITLE: BIOREACTOR SYSTEMS AND METHODSINVENTORS: JARRETT ESHIM A, ERIK ESHIM AASSIGNEE: AIROBES, LLCCROSS REFERENCE TO RELATED APPLICATION

[0001] The application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 694,657, entitled “BIOREACTOR DEVICES, SYSTEMS, AND METHODS OF OPERATION” and filed September 13, 2024, the disclosure of which is herein incorporated by reference in its entirety.FIELD

[0002] The present disclosure generally relates to bioreactor devices, systems, and methods, and more particularly to bioreactor devices, systems, and methods for converting carbon dioxide into one or more products capable of repurposing.BACKGROUND

[0003] The subject matter discussed in the background section should not be assumed to be prior art merely as a result of its mention in the background section. Similarly, a problem mentioned in the background section or associated with the subject matter of the background section should not be assumed to have been previously recognized in the prior art. The subject matter in the background section merely represents different approaches, which in and of themselves can be inventions.

[0004] The carbon capture, removal, and reuse industry is nascent and fast growing. The ultimate aim is to reduce and / or remove carbon dioxide (CO2) (and other greenhouse gases) directly from the environment and / or abate the release at the source of generation. Such systems include but are not limited to direct air / ocean capture (DAC / DOC) technologies, bioenergy with carbon capture and storage (BECCS), capture at the source of generation (CCS), reforestation, enhanced weathering of minerals, and a controlled bioreactor format (BiCRS). The current disposition of captured CO2 is often sequestration, or the process of injecting the carbon mass deep underground. In doing so, sequestered carbon is not recycled to produce usefulDocketNo 95695.00116 1products. Consequently, there is a need for specialized biological methods that offer the ability to convert CO2 into a variety of useful outputs.SUMMARY OF THE DISLOSURE

[0005] In an exemplary embodiment, a method is disclosed, the method comprising adding photoautotrophs to a growth medium in a first bioreactor, the growth medium comprising a liquid medium, adding photons and CO2 to the first bioreactor, conducting a photoautotrophic process in the first bioreactor to produce glycolic acid, measuring, via one or more first sensors, a first operational parameter of the first bioreactor, removing the glycolic acid from the first bioreactor in response to the first operational parameter meeting a first threshold value, adding the glycolic acid and water to a second bioreactor, adding microorganisms to the second bioreactor, conducting a heterotrophic process in the second bioreactor to produce a secondary product and CO2, measuring, via one or more second sensors, a first operational parameter of the second bioreactor, and removing the secondary product from the second bioreactor in response to the first operational parameter meeting a first threshold value.

[0006] In various embodiments, the method further comprises adding a carbonic anhydrase inhibitor to the first bioreactor after the adding of photoautotrophs to the growth medium. In various embodiments, the method further comprises adding non-carbon nutrients to the second bioreactor after the adding the glycolic acid, wherein the non-carbon nutrients comprise at least one of ammonium chloride, sodium phosphate, potassium phosphate, salt, magnesium, calcium, or an antibiotic.

[0007] In various embodiments, the method further comprises conditioning the photoautotrophs before the adding the photoautotrophs to the growth medium, wherein the conditioning comprises mixing the photoautotrophic with CO2 and nutrients. In various embodiments, the growth medium comprises water. In various embodiments, the CO2 added to the first bioreactor is provided from the CO2 produced by the second bioreactor.

[0008] In various embodiments, the method further comprises purifying the glycolic acid before the adding the glycolic acid to the second bioreactor. In various embodiments, the glycolic acid is added to the second bioreactor as an unprocessed bioreactor medium. In various embodiments, the photons are added via a light source. In various embodiments, the light source is an LED-coupled fiber optic system.DocketNo 95695.00116 2

[0009] In various embodiments, the method further comprises before the adding the glycolic acid to the second bioreactor, one or more of genetically modifying the microorganism population to produce recombinant proteins, or carrying out one or more non-native enzymatic processes.

[0010] In various embodiments, the removing the secondary7product from the second bioreactor produces a barren medium, and wherein the barren medium is gasified to produce syngas. In various embodiments, the method further comprises combining the syngas with the secondary' product to produce an additional end product. In various embodiments, the secondary7product comprises at least one of hydrogen, ammonia, C1-C4 carboxylic acids, fluorescent or bioactive recombinant proteins, vitamins, lipids, antibiotics, or glycosaminoglycans.

[0011] In various embodiments, the first operational parameter is pH and the second operational parameter is cell densify. In various embodiments, the first bioreactor is a photoautotrophic bioreactor and the second bioreactor is a heterotrophic bioreactor. In various embodiments, the photoautotrophic bioreactor is an open pond system, tubular bioreactor, flat panel bioreactor, air lifted bioreactor, bag bioreactor, or a combination thereof.

[0012] In another exemplary7embodiment, a system is disclosed, the system comprising a first bioreactor comprising a growth medium, a photoautotroph population, a light source, and CO2. wherein the first bioreactor is configured to produce glycolic acid, a second bioreactor comprising carbon feedstock and a microorganism population, wherein the second bioreactor is configured to produce the secondary7product and CO2, and a first fluid connection from the first bioreactor to the second bioreactor configured to transfer the glycolic acid from the first bioreactor to the second bioreactor, wherein the system is configured to provide the glycolic acid produced by the first bioreactor as at least a portion of the carbon feedstock in the second bioreactor.

[0013] In various embodiments, the system further comprises a second fluid connection from the second bioreactor to the first bioreactor configured to transfer the CO2 produced by the second bioreactor to the first bioreactor. In various embodiments, the second bioreactor further comprises non-carbon nutrients, and wherein the noncarbon nutrients comprise at least one of ammonium chloride, sodium phosphate, potassium phosphate, salt, magnesium, calcium, and one or more antibiotics.DocketNo 95695.00116 3BRIEF DESCRPTION OF DRAWINGS

[0014] FIG. 1 illustrates a schematic diagram of a bioreactor system in accordance with various embodiments of the present disclosure.

[0015] FIG. 2 illustrates a schematic diagram of a bioreactor system in accordance with various embodiments of the present disclosure.

[0016] FIG. 3A is a graphical illustration of glycolic acid production from non-CCh preconditioned photoautotrophs in accordance with various embodiments of the present disclosure.

[0017] FIG. 3B is a graphical illustration of glycolic acid production from CO2 preconditioned photoautotrophs in accordance with various embodiments of the present disclosure.

[0018] FIG. 4 is a graphical illustration of Chlorophyll A weight in a growth medium in accordance with various embodiments of the present disclosure.

[0019] FIG. 5 illustrates a schematic diagram of a bioreactor plant system in accordance with various embodiments of the present disclosure.

[0020] FIG. 6 illustrates a bioreactor in accordance with various embodiments of the present disclosure.

[0021] FIG. 7 is a graphical illustration of the microorganism cell density of various bioreactor mediums in accordance with various embodiments of the present disclosure.

[0022] FIG. 8 illustrates a method for producing products from CO2 in accordance with various embodiments of the present disclosure.DETAILED DESCRIPTION

[0023] The following detailed description of various embodiments herein refers to the accompanying drawings, which show various embodiments by way of illustration. While these various embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosure, it should be understood that other embodiments can be realized and that changes can be made without departing from the scope of the disclosure. Thus, the detailed description herein is presented for purposes of illustration only and not of limitation. Furthermore, any reference to singular includes plural embodiments, and any reference to more than one component or step can include a singular embodiment or step. Also, any reference to attached,DocketNo 95695.00116 4fixed, connected, or the like can include permanent, removable, temporary', partial, full or any other possible attachment option. Additionally, any reference to without contact (or similar phrases) can also include reduced contact or minimal contact. It should also be understood that unless specifically stated otherwise, references to ‘"a,” “an” or “the” can include one or more than one and that reference to an item in the singular can also include the item in the plural. Further, all ranges can include upper and lower values and all ranges and ratio limits disclosed herein can be combined. The steps of any method disclosed herein can be performed any suitable order or combination.

[0024] The term “photoautotrophic” is art-recognized and refers to conditions in which the organism is capable of deriving energy needs through the production of organic compounds from sunlight and / or CO2.

[0025] The term “heterotrophic” is art-recognized and refers to conditions in which the organism derives energy needs from external sources, for example glucose.

[0026] The term “biomass” refers to material produced by growth and / or propagation of a living cell or organism, for example, algae or bacteria. Biomass can comprise cells, intracellular contents, for example cellular proteins or metabolites, and / or extracellular material.

[0027] The term “glycolic acid” refers to the chemical itself and is used interchangeably with “glycolate”, a salt or ester derivate of glycolic acid.

[0028] The term “culturing” refers to maintaining a culture of an organism, for example, algae or bacteria, for a set period of time that is sufficient for a desired cellular process to be carried out by the microorganism. In some embodiments, the culture comprises a microorganism described herein and a liquid medium. In various embodiments, the culture comprises a carbon source, for example a carbon source dissolved in the culture medium or sourced from CO2 in the air. For example, in some embodiments, microorganisms are cultured in a bioreactor comprising a liquid medium in the presence of a carbon source and various other nutrients dissolved in the medium. Non-limiting examples of other nutrients include nitrogen source(s) like ammonium, nitrate, or urea, trace metals, such as, for example, zinc, manganese, molybdenum, copper, cobalt, magnesium, calcium, chelating agents like Ethylenediaminetetraacetic Acid (“EDTA”), and / or molecules capable of inducing a cellular response like growth factors, such as, for example, isopropyl P-D-l -thiogalactopyranoside “IPTG”).

[0029] The term “stock culture” is art-recognized and refers to a culture medium that comprises organisms not participating in the bioreactor productionDocketNo 95695.00116 5method. These organisms are grown in a separate vessel and are designed to enable semi-continuous or continuous operation of the downstream processes without waiting for sufficient biomass accumulation. In some embodiments, the stock culture can be connected to the downstream bioreactor or entirely separate. In all embodiments, the stock culture comprises a mixture of nutrients to allow for cell division and propagation of biomass.

[0030] The term "C I -C4 carboxylic acids’7refers to several carbon- comprising compounds with a carboxyl functional group. These carbon-comprising compounds have between one and four carbons covalently bound to each other. Nonlimiting examples include formic acid, acetic acid, glycolic acid, glyoxylic acid, acrylic acid, lactic acid, glyceric acid, 3-hydroxypropionic acid, pyruvic acid, butanoic acid, 2- methylpropanoic acid, 2-hydroxybutanoic acid, 3 -hydroxy butanoic acid, 4- hydroxybutanoic acid, succinic acid, fumaric acid, malic acid, tartaric acid, and oxaloacetic acid.

[0031] The term “liquid medium” refers to a liquid comprised within the bioreactor with the relevant nutrients and bioactive molecules required for the intended biological process.

[0032] The term “broth” is art-recognized and refers to the liquid medium comprising the target molecule of interest. For example, the photoautotrophic broth refers to the liquid medium comprising glycolate, the algae, and other dissolved nutrients required for the operation of the biological process.

[0033] The term “primed” refers to the culturing of microorganisms in non- traditional media to induce genetic and / or cellular changes that make them better suited to utilize nutrient comprised within the non-traditional media for cell growth and production.

[0034] The term “carbon feedstock” is art-recognized and refers to the source of carbon to be utilized by the microorganism in a bioreactor production method. For example, the carbon feedstock can be CO2 in the air or glycolate.

[0035] The term “secondary products” refers to the production of various molecules by biological means in the heterotrophic bioreactor. For example, secondary products can include hydrogen, ammonia, C1-C4 carboxylic acids, fluorescent or bioactive recombinant proteins, vitamins, lipids, antibiotics, and / or glycosaminoglycans in accordance with various embodiments.DocketNo 95695.00116 6

[0036] Disclosed herein are systems, methods, and devices for biologically converting captured CO2 into end products which can be repurposed (i.e., which can be useful in other contexts). In an exemplary embodiment, a bioreactor system that is configured around the mass balance between photosynthesis and photorespiration is disclosed.

[0037] In various embodiments, and with reference to FIG. 8, a bioreactor system can be configured to enable the biological conversion of CO2 into one or more recyclable and / or reusable products, e.g., via method 800, using photoautotrophic algae and various heterotrophic bacteria and fungi. In various embodiments, the bioreactor production method 800 can comprise a plurality of subsequent steps and / or can be modular. In various embodiments, the bioreactor system can comprise a plurality of bioreactor systems.

[0038] In various embodiments, the photoautotrophic algae species (“photoautotrophs”) can comprise Chlorella or Chlamydomonas suspended in a liquid medium comprising water to reduce operating costs and simplify downstream purification. However, the disclosure is not limited in this regard, and the photoautotrophs can comprise any of the following algae genera of Chlorella, Chlamydomonas, Cyanidioschyzon, Asterarcys, Spirogyra, Scenedesmus, Tetraselmis Pediaslrum Volvox, and Coelastrella, and / or any combination thereof. In various embodiments, the water can comprise, for example, tap, deionized, MilliQ water, and / or any water source with a resistivity of approximately 18 MQ-cm at 25°C with total organic carbon < 10 ppb, < 0.1 pg / mL of proteases, and <10 pg / mL of DNAses and RNAses.

[0039] In various embodiments, the biological production method 800 conducted in a bioreactor system can comprise an enzymatic coupling between photosynthesis (ribulose- 1,5 -biphosphate + CO2 -> 3-phosphoglyceric acid -> energy; Calvin cycle) and photorespiration (ribulose- 1,5 -biphosphate + O2 -> 2- phosphoglyceric acid + 3-phosphoglyceric acidglycolic acid). In various embodiments, the method can comprise adding carbonic anhydrase inhibitors, rendering alternative carbon sources unavailable, and thereby increasing rates of photorespiration in a low CO2 environment. Thus, all or substantially all carbon for the reaction can be (and in some cases, must be) derived from CO2.

[0040] In various embodiments, drug or genetic inhibition of glycolate dehydrogenase can prevent the enzymatic catabolism of glycolic acid, allowing forDocketNo 95695.00116 7significant accumulation in the liquid growth medium. Accordingly, the bioreactor system can be configured to track or monitor the resultant mass balance equation that arises during operation, wherein the target ratio of the mass balance equation is about 2: 1 (photosynthesis:photorespiration) which reflects the number of carbon atoms in glycolic acid and can prevent the loss or accumulation of carbon within the microorganisms. “About” as referred to herein refers to plus or minus 100%. For example, about 2: 1 would encompass a ratio of 4: 1 - 1: 1, in accordance with various embodiments.

[0041] In various embodiments, the coupling of the photoautotrophic and non-photoautotrophic steps is accomplished through utilization of the glyoxylate shunt that is present in bacteria and fungi. In this regard, glycolic acid is converted to glyoxylate by glycolate oxidase. From there, glyoxylate enters the Krebs cycle and is converted to malate in a single step via malate synthase, which then proceeds via traditional Krebs cycle enzymatic steps to produce new sugars, amino acids, etc. Benefits of doing so allow for the carbon removal process to occur independently from the production of the target output (except in the case of glycolate) which translates to a sufficiently constant rate of CO2 removal even as the target output from the non- photoautotrophic step changes.

[0042] In various embodiments, with combined reference to FIGs. 1, 2, and 8, a bioreactor production method 800 conducted in a bioreactor system (e.g.. system 100 and / or 200) can utilize CCh (e.g., environmental CO2) to produce various emission-related or high-value biomolecules. In various embodiments, emission- related molecules can comprise glycolic acid and its sodium and potassium derivatives (glycolate), methanol, (di)hydrogen, ammonia, and C1-C4 carboxylic acids. In various embodiments, high-value biomolecules include fluorescent or bioactive recombinant proteins, vitamins, retinal, retinol, folic acid, cholecalciferol, B12, tocopherol lipids including polyunsaturated fatty acids, glycosaminoglycan hyaluronic acid glycolipids, phospholipids, sphingolipids, sterols, omega-3 fatty acids, and / or antibiotics including streptomycin, tetracycline, neomycin, bacitracin, vancomycin, and violacein. In various embodiments, the production of these molecules is via a CO2 feedstock, offering advantages related to net process emissions, supply stability, and cost relative to more traditional carbon feedstocks. This approach further supports location agnostic development, as environmental CO2 is readily accessible (e.g., from ambient air).DocketNo 95695.00116

[0043] In various embodiments, the bioreactor production method 800 comprises reaching and maintaining a mass balance between photosynthesis and photorespiration pathways in photoautotrophic algae to produce glycolic acid (i.e. glycolate). In this regard, the system uses in-culture sensors to monitor the idealized mass balance ratio (e g., about 2: 1) between photosynthesis and photorespiratory events occurring at the enzyme ribulose- 1,5-biphosphate carboxylase / oxygenase (RuBisCO). Operating at this ratio allows continuous production of glycolic acid without corresponding biomass accumulation or loss. In various embodiments, ratios of O2 to CO2 in the inflowing gas can be in the range of 750: 1 to 100:1, more preferably in the range of about 450: 1 to 550: 1, more preferably about 500:1 (wherein “about'’ means plus or minus 20: 1).

[0044] Turning now to FIG. 1, and with reference to FIG. 8, a first bioreactor system 100 is illustrated. In various embodiments, at least a portion of the bioreactor production method 800 can take place in the first bioreactor system 100. In various embodiments, photoautotrophs 110 (e g., photoautotrophic algae) are added to a first, photoautotrophic bioreactor 104 (step 810). In various embodiments, a growth medium 120 is added to bioreactor 104. In various embodiments, the photoautotrophs 110 can be added to the bioreactor 104 before the addition of the grow th medium 120, or vice versa.

[0045] In various embodiments, photoautotrophs 110 can be obtained from a photoautotrophic stock 130. In various embodiments, the photoautotrophic stock 130 can be generated and / or held in a stock tank 102. The stock tank 102 can further comprise CO2 and various nutrients 101 to help feed and support the photoautotrophs in photoautotrophic stock 130 being supplied to bioreactor 104. In various embodiments, the various nutrients 101 can comprise NaN03 (Sodium Nitrate), CaC12 (Calcium Chloride), MgSO4*7H2O (Magnesium Sulfate heptahydrate), K2HPO4 (Dipotassium phosphate), KH2PO4 (Potassium phosphate), NaCl (Salt), TRIS Base, FeSO4 * 7H2O (Iron (II) Sulfate heptahydrate), Na2-EDTA * 2H2O, (Ethylenediaminetetraacetic acid disodium salt dihydrate), and / or trace metals.

[0046] In various embodiments, conditioning the photoautotrophs 110 in a high CO2 environment (> 2%) in stock tank 102 prior to adding them to the bioreactor 104 can increase rates of growth and glycolate production inside bioreactor 104, as illustrated in FIGs. 3A and 3B. In FIG. 3A, glycolate was produced from photoautotrophs that were not conditioned in a high CO2 environment, while FIG. 3BDocketNo 95695.00116 9shows glycolate production from photoautotrophs that were conditioned in a high CO2 environment. As can be seen, conditioning the photoautotrophs in a high CO2 environment before introducing them to the bioreactor 104 results in higher rates of glycolic acid (i.e. glycolate) production in bioreactor 104.

[0047] In various embodiments, the photoautotrophs 110 can be processed via centrifugation and resuspension in a medium after removal from the photoautotrophic stock 130 before being added to the photoautotrophic bioreactor 104. In various embodiments, the concentration of photoautotrophs 110 can be measured by the amount of Chlorophyll A present. In various embodiments, the concentration of photoautotrophs 110 added to the photoautotrophic bioreactor 104 is in the range of 1 mg / L to 20 mg / L Chlorophyll A.

[0048] In various embodiments, the growth medium 120 added to bioreactor 104 can comprise a liquid medium (e.g. a sterile liquid medium). In various embodiments, the liquid medium can comprise water, such as, for example, deionized water, MilliQ water, and / or any water source with a resistivity of approximately 18 MQ-cm at 25°C with total organic carbon < 10 ppb. < 0. 1 pg / mL of proteases, and <10 pg / mL of DNases and RNAses. In various embodiments, the liquid medium can further comprise trace levels (<1% by weight) of nutrients including phosphates, metals (e.g., Zinc, Iron, Copper, Magnesium, Manganese, Cobalt, and / or Molybdenum), sodium or ammonium nitrate, and / or urea.

[0049] In various embodiments, and with continued reference to FIG. 8, various additives 111, such as, for example, photons from light, air, CO2, and / or sodium hydroxide can be added to the bioreactor 104 (step 820). In various embodiments, in response to the addition of photoautotrophs 110 (step 810) and various additives (step 820) to the photoautotrophic bioreactor 104, a photoautotrophic process is carried out in the bioreactor 104 (step 830). The photoautotrophic process can produce glycolic acid.

[0050] In various embodiments, the bioreactor 104 can comprise a gas pump for introduction of air through a sparging / air stone apparatus at or near the bottom of an internal chamber of the bioreactor 104, with an optional mixing component achieved by electromagnetic movement of an in-culture stir bar. In various embodiments, bioreactor 104 can be configured for continuous pumping or mixing of air into the liquid mixture to enhance the efficiency and / or continuance of the photoautotrophic process. In various embodiments, bioreactor 104 can comprise anDocketNo 95695.00116 10open pond system, tubular bioreactor, flat panel bioreactor, air lifted bioreactor, bag bioreactor, or a combination thereof. In various embodiments, bioreactor 104 can comprise an enclosed tank bioreactor.

[0051] In various embodiments, the photoautotrophic process can be carried out in bioreactor 104 due to a two-stage photoautotrophic process system. In various embodiments, the two-stage photoautotrophic process system can comprise (i) continuous recycling of headspace gas using a positive displacement pump, and / or (ii) venting of the headspace and dissolved gases using a vacuum pump. In various embodiments, an inlet gas can be injected into bioreactor 104 via the gas pump. The inlet gas can comprise a blend / mixture of compressed / pressured CO2 and / or high purity nitrogen (N2), ranging anywhere from 5-100% CO2 with the balance being nitrogen. Because O2 is released during photosynthesis, the bioreactor 104 can be configured without an external oxygen source. During the first stage (i.e., recycling), internal sensors in bioreactor 104 (e.g., in the gaseous headspace and / or in the grow th medium 120) can monitor the concentration and / or amount of CO2 and O2 over time to determine when the target ratio (e.g., 2: 1 CChiCh) is met (e.g., when a calculated — or measured — ratio is within a threshold range). As gases have unique partial pressures, the sensors can convert the CO2 and O2 concentrations to weight (moles) (e.g., using the ideal gas law) and apply the desired target ratio to the converted concentrations.

[0052] In various embodiments, the two-stage photoautotrophic process system can be configured for continuous recycling (e.g., through the sparge / air stone at the bottom of bioreactor 104). This continuous recycling can speed up the overall carbon removal rate. Once the target mass balance ratio (e.g., 2:1 CO2:O2) is met, or is within a target mass balance ratio range (e.g.. between 2.5: 1 and 1.5: 1), the recycling stage can cease, and the venting stage can begin. The venting stage can involve continuous removal of the headspace gas (e.g., via opening a vent line) with the optional injection of high purity nitrogen (w ithout CO2) to enhance the flushing of O2 from the reactor. When a sufficiently low O2 concentration and / or amount is met (e.g. less than 1-5% O2 remaining in bioreactor 104 ), the venting line can be closed, a new injection mixture of CO2 and N2 can be introduced into the bioreactor 104 (e g., via the recycling line being opened), and a recycling stage can begin again. The process can be repeated until a minimum amount of CO2 is treated (converted to O2) and / or a minimum amount of glycolic acid is produced during a process, a maximum time threshold is exceeded, and / or a desired amount of glycolic acid is produced.DocketNo 95695.00116 11

[0053] In various embodiments, the body of bioreactor 104 is sufficiently optically transparent to allow introduction of light, such as, for example, ambient light from external sources (e.g. the Sun). In various embodiments, the bioreactor 104 can comprise a light mechanism that can be configured to generate light, for example, by incandescent and / or light emitting diode (“LED”) lights to introduce photons into bioreactor 104. In various examples, light can be generated and provided by a plurality of independent systems providing broad spectrum light (e.g., warm light temperature and cool light temperature).

[0054] In various embodiments, the light mechanism can circumvent traditional issues surrounding algal self-shading, such as in embodiments wherein the light mechanism is at least partially placed within the medium inside bioreactor 104. In this regard, productivity of the bioreactor 104 can be improved by enhancing photosynthesis while photorespiration rates can be optimized through the modulation of LED intensity and wavelength.

[0055] In various embodiments, bioreactor 104 can comprise a plurality of internal sensors (e.g., 416 in FIG. 6) disposed within the bioreactor. In this regard, the plurality of sensors can provide measurements to a controller (e.g., 418 in FIG. 6) to facilitate monitoring the system and achieving a target mass balance rate of about 2: 1 (photosynthesis: photorespiration), as estimated by monitoring dissolved O2 and dissolved CO2. In various embodiments, each sensor in the plurality of sensors can be selected and configured to provide measurements to a controller for monitoring the growth environment. For example, the plurality7of sensors can be configured to monitor the pH, salinity, dissolved gas concentration, temperature, and any other operational parameter beneficial to monitor bioreactor 104. In various embodiments, in response to a parameter falling outside of a desired range, a control system can be configured to generate an alert, stop the current process, and / or begin recovery of glycolate, biomass, and / or liquid medium, and / or to perform an action to correct the operational parameter that has fallen outside of the desired range.

[0056] For example, in various embodiments, a nutrient addition port(s) (e.g. 412 in FIG. 6) can be positioned within the lid of bioreactor 104 and, in response to an operational parameter falling outside of a desired range (e.g., as determined by the controller based on data received from various sensors), nutrients and / or other additives can be added through the nutrient addition port to adjust the respective parameter.DocketNo 95695.00116 12

[0057] In various embodiments, and contrary' to ty pical approaches, the amount (e.g.. concentration / density) of the photoautotrophs 110 can be maintained at a constant level or relatively constant level throughout the first bioreactor system 100. Stated another way, bioreactor 104 can be configured to maintain the amount of the photoautotrophs 110 allowing for production of glycolic acid 140 that is unrelated to the generation of biomass. This effect is demonstrated in FIG. 4. which shows that after production of glycolic acid for two weeks, the amount by weight of Chlorophyll A in a growth medium decreased. Because the amount of Chlorophyll A produced is correlated with biomass, a decrease in Chlorophyll A generally indicates that there is not an increase in biomass. Therefore, the decrease in Chlorophyll A indirectly shows constant and / or decreasing cell density and not the accumulation of biomass.

[0058] Advantageously, this approach yields high concentrations of glycolic acid 140 and offers enhanced traceability' (i.e. the tracking of how much CO2 is captured / removed from the environment by bioreactor system 100) of CCh-sourced carbon. In various embodiments, when the growth medium 120 is water, this photoautotrophic process is further encouraged by the absence of nutrients in the growth medium 120, as the growth medium 120 is being utilized within bioreactor 104 to produce glycolic acid 140 from CO2, and not to grow' additional grow th medium 120. In various embodiments, in the absence of nutrients, no part of the growth medium 120 is used in a process for feeding and multiplying the growth medium 120, and all reaction energy is put towards the production of glycolic acid 140 from CO2. Under long durations of operation (i.e., greater than 1 w'eek), some loss of biomass can be inevitable which can be compensated for with the selective addition of nutrients through the nutrient addition ports.

[0059] In various embodiments, the photoautotrophic process can be further enhanced by the addition of glycolate dehydrogenase / oxidase inhibitors such as aminooxyacetic acid or 6-ethoxy-2-benzothiazolesulfonamide. In various embodiments, the glycolate dehydrogenase / oxidase inhibitors inhibit enzymatic activity of glycolate dehydrogenase / oxidase.

[0060] In various embodiments, the traceability of C 02-based carbon and efficiency of CO2 conversion in the photoautotrophic bioreactor 104 can be enhanced through the addition of carbonic anhydrase inhibitors such as acetazolamide and ethoxzolamide. as such inhibitors remove various variables that influence the rate of photosynthesis and photorespiration, thus allowing for better control over the rate byDocketNo 95695.00116 13limiting the influential variables to those controlled within system 100. In various embodiments, wherein inhibitors are added to bioreactor 104, sodium bicarbonate is not converted to CO2, and the localized availability of CO2 near RuBisCO remains low and proportional to the dissolved CO2 concentration in the medium, allowing for a more accurate estimation of the ratio of photosynthesis to photorespiration.

[0061] In various embodiments, the efficiency of glycolate production and excretion by the photoautotrophic process can be enhanced by genetically modifying phosphoglycolate phosphatase, RuBisCO, bile acid sodium symporter, and / or plastidal glycolate / glycerate translocator 1 in the photoautotrophic algae. Genetic modification can additionally be combined with the addition of specialized nutrients like aspartate to further enhance the rate of glycolate production and excretion.

[0062] In various embodiments, first bioreactor system 100 can be configured to produce glycolic acid (i.e. glycolate) 140 in the photoautotrophic bioreactor 104. In various embodiments, first bioreactor system 100 can be operated for a certain period of time optimized to produce glycolic acid 140. In various embodiments, glycolic acid 140 production is measured by monitoring pH. with a change in pH indicating production has completed. In various embodiments, first bioreactor system 100 is operated until the pH stops decreasing and / or begins to increase. In various embodiments, first bioreactor system 100 can be operated for at least 7 days.

[0063] In various embodiments, and with continued reference to FIGs. 1 and 8, the glycolic acid 140 is recovered from the bioreactor 104 (step 840 from FIG. 8) and / or purified in processing system 150 via any known process sufficient to separate one substance from another, such as, for example, one or more rounds of filtration, centrifugation, evaporative concentration, one or more rounds of chromatographic separation (e.g. size exclusion, ion exchange, activated carbon, etc.), and / or recrystallization.

[0064] In various embodiments, the processing system 150 can be configured to operate the process of: (1) separation of the photoautotrophs 110 from the growth medium 120 via flocculation (by addition of aluminum sulfate) or centrifugation; (2) concentrating the liquid medium via evaporation or similar method; (3) passing of liquid supernatant through a filter (e g., with <1 micron pore size); (4) packing of a chromatography column with size exclusion resin (e.g., Sephadex G-10); (5) introduction of the filtered bioreactor contents into the packed chromatographyDocketNo 95695.00116 14column; (6) an eluate (i.e., a solution obtained by elution that is output from the column) is disposed into a large collection container until a time threshold is met (fractionation); (7) after a time threshold is met, the eluting glycolate can be collected; (8) a second chromatography column is packed with anion exchange resin (e.g., Amberlite IRC120); (9) introduction of the partially purified glycolic acid solution into the second packed chromatography column; (10) recovering the eluate fraction comprising neutral glycolic acid by monitoring pH of the eluate; (11) further concentration of the glycolic acid solution can then be achieved using heat or reduced pressure (vacuum) or a combination thereof; (12) the concentrated glycolic acid solution can then be crystallized or distilled to achieve high purity; (13) a small quantity can be retained for quality control analysis by LC-MS (Liquid Chromatography coupled with one or more Mass Spectrometers); (14) remaining glycolic acid 140 can then be packaged for storage, sale, or immediate introduction into a second, heterotrophic bioreactor 204; and / or (15) the chromatography column material is regenerated and can be replaced at regular time intervals determined through experimentation. In various embodiments the previous steps can occur in any suitable order or combination.

[0065] In various embodiments, the chromatography column can be configured to be gravity' fed. However, the present disclosure is not limited in this regard. For example, the chromatography column can be pressurized and would still be within the scope of this disclosure. Similarly, custom centrifugation columns packed with the same material can be used to speed processing depending on batch size.

[0066] In alternative embodiments, and as shown in FIG. 2, the glycolic acid 140 can be removed from the bioreactor 104 as an unprocessed bioreactor product / medium. wherein the bioreactor product comprises the algae 110 and the produced glycolic acid 140. In various embodiments, the unprocessed bioreactor product can undergo a process to disrupt the algae 110 membrane and release the cellular contents into the liquid to create a carbon feedstock 160, such as, by way of nonlimiting example, electroporation, mechanical homogenization, and / or sonication. In addition to disrupting the algae 110 membrane, the unprocessed bioreactor product can be purified as detailed above.

[0067] In various embodiments, the medium remaining in bioreactor 104 after the glycolic acid 140 is removed can be reintroduced to a new photoauto trophic bioreactor (not shown) comprising fresh growth medium 120 to begin a new photoautotrophic process. The fresh growth medium 120 can be recycled in part or inDocketNo 95695.00116 15whole from prior photoautotrophic bioreactor processes. In various embodiments, the new photoautotrophic bioreactor can comprise a lesser concentration of glycolate dehydrogenase / oxidase inhibitors. In various embodiments, the concentration of glycolate dehydrogenase / oxidase inhibitors can be less than 50% of the original concentration of glycolate dehydrogenase / oxidase inhibitors, preferably in the range of 10%-25% of the original concentration, more preferably 0%.

[0068] Processing and reusing the residual medium from bioreactor 104 in additional photoautotrophic processes minimizes cultivation and operating costs, as well as improves process circularity, thus reducing overall emissions from the process.

[0069] In various embodiments, and with continued reference to FIGs. 2 and 8, the carbon feedstock 160, which comprises glycolic acid 140. is used in a second bioreactor system 200. In various embodiments, at least a portion of the bioreactor production method 800 disclosed herein can take place in the second bioreactor system 200. In various embodiments, second bioreactor system 200 can utilize the carbon feedstock 160 to produce secondary products 210.

[0070] In various embodiments, the carbon feedstock 160 can comprise the glycolic acid 140 and / or unprocessed bioreactor product from the first bioreactor system 100. In various embodiments, the carbon feedstock 160 can comprise the glycolic acid 140 in combination with other carbon feedstocks sources, such as, for example, algal biomass, glucose, xylose, and / or agricultural waste. In such embodiments, the other carbon feedstocks are preferably less than 50% of the carbon feedstock 160. In such embodiments, at least 75% of the carbon originating from glycolic acid is metabolized as an energy source to aid production of the target molecule(s).

[0071] In various embodiments, glycolic acid 140, either alone or as a portion of carbon feedstock 160, is fed into a second, heterotrophic bioreactor 204 (step 850). In various embodiments, wherein carbon feedstock 160 comprises other carbon feedstock sources in addition to the glycolic acid 140 produced in bioreactor 104, the other carbon feedstock sources can be used as a supplementary nutrient source, thereby enhancing process circularity and reducing waste burden.

[0072] In various embodiments, microorganisms 220 can be added to the heterotrophic bioreactor 204 (step 860). In various embodiments, the heterotrophic microorganisms 220 can comprise Escherichia coli, Saccharomyces serevisiae, Anaerobiospirillum succiniciproducens , Chromobacterium violaceum, methanotrophsDocketNo 95695.00116 16of the genera Methylocella and Methylococcus , fungi of the genus Aspergillus, and / or bacteria of the genera Azotobacter.Anabaena. Bacillus, Clostridium, Corynebacterium, Citrobacter, Cyanothece, Enterobacter, Klebsiella, Lactobacillus, Pseudomonas, Staphylococcus, Streptomyces, and / or Trichodesmium The heterotrophic microorganisms 220 can comprise and / or be combined with acidophilic or acid-tolerant characteristics.

[0073] In various embodiments, the microorganisms 220 can be genetically modified to produce recombinant proteins and / or carry out one or more non-native enzymatic processes. This modification can allow for the programmable production of the target molecule (e.g. secondary products) or protein with carbon originally sourced from CO2. This methodology differs from common standard approaches which do not perform genetic modification on the microorganisms 220, but instead modify the photoautotrophic algae used in stock culture 120. However, genetically modifying the microorganisms 220 can enhance the efficiency of the CO2 removal process and optimize chemical reactions which take place in bioreactor 204, as the modified microorganisms are better suited to encourage the reactions, making genetic modification of microorganisms a more robust and repeatable process than that of modifying algae. Furthermore, the simplicity of modifying microorganisms (i.e. bacteria) lowers technical barriers, reduces costs, and simplifies upstream (formulation of the culture medium) and downstream (purification) steps. For example, algae grow slower than microorganisms, and downstream purification processes have not been optimized to the same extent as those for purifying microorganisms. Thus, the production of recombinant proteins and speed of non-native enzymatic processes is increased with the use of modified microorganisms when compared to the use of modified algae.

[0074] In various embodiments, the heterotrophic microorganisms 220 are pre-conditioned (“primed'’) for glycolate metabolism through iterative rounds of selective pressure in stock tank 202. In various embodiments, the priming of the microorganisms 220 compnses subjecting the microorganisms 220 to repeated growth in a liquid medium comprising glycolate as the sole carbon source, in turn, causing genetic mutations over many generations that improve its growth capabilities in the medium. These genetic mutations can occur in as few as 1 sequential culture to as many as 50 or more. The resulting mutant strain is then isolated, cryogenically preserved, and revived as needed for the generation of the heterotrophic stock 230 as shown in variousDocketNo 95695.00116 17embodiments. In this regard, the glycolate medium acts as a natural combatant to contamination from other microorganisms that grow less favorably on this carbon source (i.e., other microorganism cannot reproduce or grow as rapidly, or at all, relative to the primed heterotrophic microorganisms). In various embodiments, primed heterotrophic organisms are mixed with unprimed organisms from the same genus and species or a different genus and species to induce competition within the bioreactor.

[0075] In various embodiments, in response to the addition of glycolic acid 140 and microorganisms 220 to the bioreactor 204, a heterotrophic process can be carried out in the bioreactor 204 (step 870). In various embodiments, the heterotrophic process can produce the secondary products 210. which are then recovered from the heterotrophic bioreactor 204 (step 880).

[0076] In various embodiments, various additives 201 can be added to the heterotrophic bioreactor 204 to enhance the heterotrophic process, such as, for example, nutrients and / or antibiotics. In various embodiments, the nutrients 201 can be noncarbon nutrients, such as. for example, disodium phosphate, monopotassium phosphate, sodium chloride, ammonium chloride, magnesium sulfate, and / or calcium chloride. In various embodiments, the various nutrients 201 are premixed before being added into bioreactor 204. In various embodiments, the dosing of non-carbon nutrients 201, microorganisms 220 and / or carbon feedstock 160 can be continuous, pulsed, or fixed. In various embodiments, finding particular use with a modular bioreactor system, second bioreactor system 200 can be aerobic or anaerobic.

[0077] In various embodiments, bioreactor 204 can comprise one or more input / output valves to enable the addition of water, nutrients, microorganisms 220 as well as support sterilization of the bioreactor 204 and post-operational processing. In various embodiments, bioreactor 204 can comprise a water jacket to maintain temperature and a mixing element. In various embodiments, bioreactor 204 can comprise a plurality7of sensors integrated into the inner chamber of bioreactor 204. In various embodiments, the plurality of sensors can monitor relevant parameters such as, for example, pH. temperature, salinity, and dissolved oxygen.

[0078] In various embodiments, CO2 produced in the second bioreactor system 200 can be reused throughout the bioreactor production method 800, such as by reintroduction to the photoautotrophic stock 130 and / or bioreactor 104. In such embodiments, some or all of the CO2 requirements for the bioreactor production method 800 can be recovered from the second bioreactor system 200. In other embodiments, atDocketNo 95695.00116 18least a portion of the CO2 required by the bioreactor production method 800 can come from a direct air capture (DAC) and / or flue gas from a carbon capture and storage (CCS) facility (e.g. 302 from FIG. 5). In various embodiments, the bioreactor production method 800 can be supplemented with alternative carbon sources (e.g. urea) in addition to CO2.

[0079] In various embodiments, the secondary’ products 210 can comprise hydrogen, ammonia. C1-C4 carboxylic acids, fluorescent or bioactive recombinant proteins, vitamins, lipids, antibiotics, and / or glycosaminoglycans in accordance with various embodiments. In various embodiments, the secondary products 210 can be used in a variety of industries. For example, some, such as hydrogen and ammonia, are high usage chemicals while others can be used to synthesize complex chemicals, such as Cl- C4 carboxylic acids. Others, such as recombinant proteins and antibiotics, can find use in biotechnology7research and development, pharmaceuticals, cosmetics, agriculture, and enzyme-based chemical synthesis.

[0080] In various embodiments, the barren medium remaining in bioreactor 204 after removal of the secondary products 210 can be reacted with gasified algae waste to produce an unrefined syngas, which in turn can be refined and mixed with the secondary7products to product additional end products. For example, C1-C4 carboxylic acids can be converted into other chemicals, finding particular use in the production of plastic precursors.

[0081] In various embodiments, the second bioreactor system 200 can be operated for a certain period of time optimized to effectively produce the secondary products 210 in the heterotrophic process. In various embodiments, the production of secondary products 210 is monitored by cell density of the microorganisms 220, with a target optical density (monitored using 600 nm wavelength) designating that production has completed. To state it another way, the growth of the microorganisms 220 will reach a density plateau (which can be measured using a 600 nm wavelength) and begin to decrease, after which further culturing of the microorganisms 220 will not result in additional increases in production of the secondary products 210. This concept is illustrated in FIG. 7, wherein each trial comprising microorganisms shows a plateau in cell density7after a period of time (generally about 1-2 days), with the exception of “Algae Only (No Microorganisms)'’, which contained no microorganisms 220. This trial was conducted to demonstrate that the cell density growth seen is the cell density of the microorganisms 220 and no other component in second bioreactor system 200.DocketNo 95695.00116 19

[0082] In various embodiments, second bioreactor system 200 is operated until a lag phase (i.e. cell density’ plateau) is reached and / or a time threshold is met. In various embodiments, second bioreactor system 200 can be operated for at least 12 hours, preferably in the range of 24 hours to 48 hours. In various embodiments, once the second bioreactor system 200 has completed the heterotrophic process, the contents of bioreactor 204 can be removed for processing and extraction of the secondary products 210 and / or additional end products.

[0083] In various embodiments, the processing and extraction of the secondary’ products 210 and / or additional end products can comprise centrifugation, with optional lysing of the membrane depending on excretion characteristics of the specific product, filtration, pH adjustment, one or more rounds of chromatography, tangential flow filtration, distillation, lyophilization I freeze-drying, and / or gas- and / or temperature-controlled storage. In various embodiments, specific steps are subject to variation as required by the product or molecule to maintain stability and achieve high purity’.

[0084] In various embodiments, the bioreactor production method 800 can comprise a co-culture of photoautotrophic and non-photoautotrophic species (i.e., both within one bioreactor) to achieve production of the target biomolecule in a single bioreactor. In various embodiments, the photoautotrophic species can be encapsulated in alginate or hydrogel to promote co-culture conditions with the microorganisms 220. In such embodiments, where one bioreactor comprises both a photoautotrophic and heterotrophic bioreactor, the bioreactor can be aerated to support the function of the photoautotrophs 110. In such embodiments, microorganisms 220 can comprise aerobic bacteria or fungi. In various embodiments, microorganisms 220 can comprise acid tolerant bacteria or fungi.

[0085] In various embodiments, bioreactor 104 and / or 204 can be sterilized prior to initiation of the bioreactor production method 800. In such embodiments, a solution of solution of >70% ethanol or isopropyl alcohol can be utilized for sterilization. However, the present disclosure is not limited in this regard and various forms of sterilization could be utilized, such as sterilization via heating, via UV-C light, steam, ethylene oxide, hydrogen peroxide or any other sterilization method sufficient to sterilize bioreactor 104 and / or 204.

[0086] In various embodiments, once the bioreactor production method 800 has concluded, the contents of bioreactor 104 and / or 204 can be emptied into aDocketNo 95695.00116 20temporary storage container or directly processed. In various embodiments, a small quantity of the contents can be retained during operation and at the conclusion of operation for quality control analysis, quantification of Chlorophyll A, gene expression, and / or nutrient assays. In various embodiments, after bioreactor 104 and / or 204 are emptied, they can be sterilized as described above.

[0087] Turning now to FIG. 7, and wi th reference to FIG. 8. the efficacy of the bioreactor production method 800 can be seen. As graphically demonstrated in FIG. 7, a series of trials were conducted to determine efficacy of the production of secondary products using within a bioreactor using various sources of glycolic acid. In various trials, the source of glycolic acid was either synthetic glycolic acid (‘‘Synthetic’' in FIG. 7), glycolic acid produced from photoautotrophic process 830 (“Algae” in FIG. 7), or a combination thereof (“Synthetic + Algae” in FIG. 7. As described above, cell density of the microorganisms 220 indicates production of the secondary products, therefore, the higher the cell density, the greater the production of secondary' products.

[0088] As can be seen, the glycolic acid produced from photoautotrophic process 830 performed as well as. if not better, than the synthetic glycolic acid in the production of secondary products. In fact, the trials using glycolic acid produced from photoautotrophic process 830 reached their production plateau at faster rate than that of just synthetic glycolic acid. This demonstrates that glycolic acid produced from photoautotrophic process 830 can be used alone or to supplement synthetic glycolic acid without any detriment to the production of secondary products.

[0089] While not being bound to any particular theory, the glycolic acid produced from photoautotrophic process 830 may perform well in the production of secondary products as other energy rich compounds, such as, for example, lipids, may be produced by the algae used in photoautotrophic process 830, which microorganisms 220 can use as an additional carbon source, speeding up the growth of the microorganisms 220.

[0090] Turning now to FIG. 5, and with continued reference to FIG. 8, a bioreactor plant system 300 is shown. In various embodiments, bioreactor plant system 300 can be utilized in the performance of the bioreactor production method 800 disclosed herein. In various embodiments, the bioreactor plant system 300 can generate carbon credits associated with emission offsets, high purity (> 90%) glycolic acid, and secondary products like hydrogen, ammonia. C1-C4 carboxylic acids, fluorescent or bioactive recombinant proteins, vitamins, lipids, antibiotics and glycosaminoglycans.DocketNo 95695.00116 21

[0091] In various embodiments, bioreactor plant system 300 can comprise a plurality of bioreactors, such as bioreactor 104 and bioreactor 204. In various embodiments, each bioreactor in the plurality of bioreactors can be connected in sequential or modular configuration. In embodiments having a modular configuration, one bioreactor in the plurality of bioreactors can be spatially separated within plant system 300 from another bioreactor in the plurality of bioreactors, so as to enable various processes taking place within the plurality of bioreactors to be separate and independent.

[0092] In various embodiments, bioreactor plant system 300 can comprise an indoor bioreactor facility or a facility with indoor and outdoor operations. In various embodiments, the bioreactor plant system 300 can be a fully or partially autonomous system.

[0093] In various embodiments, bioreactor plant system 300 can further comprise a stock tank, such as stock tank 102, configured to operate as a conditioning module and condition and hold a stock culture, such as stock culture 130, comprising photoautotrophs 110. In various embodiments, stock tank 102 can support continuous operation of bioreactor 104, as bioreactor 104 will not need to cease operation to condition and grow photoautotrophs 110. In various embodiments, stock tank 102 can be configured to enhance glycolic acid 140 production from the photoautotrophs 110 in bioreactor 104 and accelerate growth through high concentration CO2 conditioning of the photoautotrophs 1 10 to enable more frequent operation of the bioreactor 104.

[0094] In various embodiments, stock tank 102 can be a standalone unit remote from bioreactor 104, requiring manual transport of the photoautotrophs 110 to bioreactor 104. In other embodiments, stock tank 102 comprises one or more pipes fluidly coupled to bioreactor 104 and configured to feed the photoautotrophs 110 into the bioreactor 104 directly from stock tank 102. In various embodiments, stock tank 102 can be configured to regulate temperature of and maintain a desired concentration of dissolved CO2 in the stock culture.

[0095] In various embodiments, stock tank 102 can be fabricated from any material sufficient to condition and hold a stock culture such as, for example, metal, glass, a composite material, polymeric-based material, or any combination thereof. In various embodiments, stock tank 102 can comprise a mixing device, such as a mechanical device and / or gas pumped through a sparging apparatus. In various embodiments, stock tank 102 can comprise a plurality of sensors to monitor the internalDocketNo 95695.00116 22environment within stock tank 102. In various embodiments, stock tank 102 can comprise a lighting source, such as, for example, LEDs, light bulbs, or natural sunlight can provide a desired light intensity. In various embodiments, the desired light intensity can mimic the light intensity within bioreactor 104.

[0096] In various embodiments, the stock culture in stock tank 102 can be transferred, in part or in whole, to bioreactor 104. In various embodiments, the photoautotrophs 110 in the stock culture can be removed from the stock culture and transferred independently to bioreactor 104, while the stock culture, now devoid of photoautotrophs 110, remains in stock tank 102. While shown as only one stock tank 102, bioreactor plant system 300 can comprise a plurality of stock tanks.

[0097] In various embodiments, bioreactor plant system 300 can further comprise a DAC or CCS facility 302 configured to provide at least a portion of the CO2 used in the bioreactor production method 800. Similarly, an industrial atmospheric nitrogen concentrator 304 can be attached to the facility to provide a continuous and / or endless source of nitrogen (e.g., high-purity nitrogen). In various embodiments, the bioreactor plant system 300 can generate sellable carbon credits and high purity- (> 97%) glycolic acid, in accordance with various embodiments.

[0098] In various embodiments, the bioreactor plant system 300 can provide synergy with DAC. For example, the bioreactor plant system 300 can eliminate or lessen the need for expanding underground infrastructure for long term storage of captured CO2. In this regard, the bioreactor plant system 300, can be scaled to meet the daily recovery of a specific DAC site, which can at least partially eliminate the need for underground infrastructure. Stated another way, the bioreactor plant system 300, can be retrofitted into existing DAC sites, in accordance with various embodiments

[0099] In various embodiments, bioreactor 104 and / or bioreactor 204 can be comprised of any material suitable to perform the bioreactor production method 800, such as, for example, stainless steel, plastic, and / or other corrosion-resistant alloys, and can have any suitable shape or geometry. For example, with reference to FIG. 6, bioreactor 400 (an example of bioreactor 104 and / or 204) can comprise a cylindrical body 402. Bioreactor 400 can include a bottom surface 404 that tapers towards the ground. In various embodiments, bottom surface 404 can comprise one or more apertures 406 configured to couple to one or more pipes 408. In various embodiments, the one or more apertures 406 can serve as an inlet for gas and / or liquid medium into the body 402. In various embodiments, inlet apertures for fluid addition into the bodyDocketNo 95695.00116 23402 can be disposed on any other suitable portion of bioreactor 400, such as sidewalls or the lid.

[0100] In various embodiments, such as when a gas medium is fed into the body 402 via the one or more pipes 408, the gas can pass through a sparging devices 420 to facilitate the dissolving of the gas into a liquid medium already contained in the body 402. In various embodiments, the sparging device 420 can comprise a plurality of sparging devices.

[0101] In various embodiments, the body 402 can be raised off the ground to allow for access to the one or more apertures 406 as well as utilize potential energy to induce natural liquid flow in place of or in combination with commercial pumps. In various embodiments, the body 402 can be raised off the ground by any suitable support structure, such as, for example, a plurality of legs attached to the body 402 or a raised platform.

[0102] In various embodiments, such as in an open-pond and / or plastic bag configuration, bioreactor 104 and / or 204 can be open to the environment at the top surface (i.e. lidless).

[0103] In other embodiments, bioreactor 104 and / or 204 can comprise a plurality of coupling devices configured to secure a lid 410 to the body 402. In such embodiments, lid 410 can be configured to maintain the desired reactions inside bioreactor 400. In various embodiments, lid 410 can comprise any suitable material, such as, for example, stainless-steel and / or any alloy with corrosion resistant properties. In various embodiments, lid 410 can coupled directly to body 402 and provide a continuous, airtight seal during bioreactor operation to prevent gas exchange with the surrounding environment. In various embodiments, lid 410 can be removably coupled to body 402.

[0104] In various embodiments, lid 410 can comprise one or more fluid ports 412, configured to provide an access for introducing various materials into the body 402 such as, for example, one or more sensor probes 418 (e.g., suspended above the liquid medium and optionally within the liquid medium in the body 402), nutrients, a pH balancing solution, pressurized gas, and / or steam for sterilization. In various embodiments, the one or more fluid ports 412 can comprise a threaded aperture, piloted holes, and / or any other suitable opening structure.

[0105] In various embodiments, the one or more fluid ports 412 can be coupled to a pressure relief valve (not shown) configured to provide pressure relief to DocketNo 95695.00116 24bioreactor 104 and / or 204 in response to a threshold pressure value being exceeded. In various embodiments, the threshold pressure value can be in the range of 1 atm to 10 atm.

[0106] In various embodiments, bioreactor 400 can comprise a light mechanism 414 that can be configured to generate light by a plurality7of independent light emitting diode (“LED”) systems providing broad spectrum light (e.g., warm light temperature and cool light temperature). In various embodiments, light mechanism 141 can be fully or partially submerged within the medium contained in body 402. In various embodiments, light mechanisms 141 can be positioned above the surface of medium contained in body 402.

[0107] In various embodiments, the light mechanism 414 is an LED- coupled fiber optic system. In various embodiments, the light mechanism can emit broad-spectrum white light or use a wavelength specific LED ranging between 400nm and 700nm. Alternatively, chromatic filters can be introduced into the light mechanism 414 to permit specific wavelengths into the bioreactor body. In various embodiments, light intensity can be further modulated through the pulsing of light through the light mechanism 414 allowing for additional control of heat / temperature within the bioreactor. Additionally, wavelength can be modulated by use of spectral filters and / or wavelength specific LEDs, which typically emit light in a narrow range of wavelengths. Finally, in embodiments wherein the light mechanism 414 is an LED-coupled fiber optic system, the various length fibers allow for the placement of such fibers at various depths within the body 402 to ensure the diffusion of light in the liquid medium is at least substantially consistent throughout the medium contained in the body 402.

[0108] In various embodiments, the light mechanism 414 can comprise a plurality of LED systems without a fiber optic system. In such embodiments, the plurality of LED systems can be coupled directly to the lid 410 and / or coupled to an adapter which is in turn coupled to the lid 410, such that the adapter forms a continuous body with the plurality of LED systems and the lid 410.

[0109] In various embodiments, bioreactor 400 can comprise a plurality of sensor probes 416. In various embodiments, at least one sensor probe in the plurality of sensor probes 416 is a gas sensor probe. In various embodiments, the gas sensor probe can be a custom fabricated cylindrical probe that is constructed using off-the-shelf sensors. However, the present disclosure is not limited in this regard. For example, the gas sensor probe can be upgraded to industrial grade sensors. In various embodiments,DocketNo 95695.00116 25the gas sensor probe can comprise a housing and can be configured to measure at least one of temperature, pressure, O2 concentration, CO2 concentration, and / or light intensity. In various embodiments, the gas sensor probe can comprise a temperature sensor, a pressure sensor, an O2 concentration sensor, a CO2 concentration sensor, and / or a light intensity sensor. Although described as comprising separate and distinct sensors within the gas sensor probe, the present disclosure is not limited in this regard. For example, some of the sensors can be combined measurement sensors and would still be within the scope of this disclosure.

[0110] In various embodiments, at least one of the sensor probes in the plurality of sensor probes 416 can comprise a liquid probe. In various embodiments, the liquid probe can be configured to measure temperature, dissolved oxygen, dissolved CO2, cell density, pH, and / or salinity / total dissolved solids. In various embodiments, the plurality of sensor probes 416 can be coupled to lid 410 and / or body 402 and be accessed through a probe inlet (not shown).

[0111] In various embodiments, controller 418 configured to enable automated regulation of various parameters can be placed on or near lid 410. In various embodiments, controller 418 can regulate light intensity, pH, gas composition, and addition of nutrients or reagents based on readings recorded from the one or more sensor probes 416. In various embodiments, controller 418 can comprise one or more processors and one or more memories and can be configured to receive readings from the one or more sensor probes 416. In various embodiments, controller 418 can temporarily store the measurements, transmit data to cloud-based storage, perform analysis based on the measurements, and / or make quality control determinations based on the measurements, in accordance with various embodiments.

[0112] In various embodiments, the housing of the gas sensor probe can be manufactured using stainless steel or any similar corrosive resistant alloy. In various embodiments, the housing can be printed through additive manufacturing using corrosive resistant polymeric materials, however, the disclosure is not limited in this regard.

[0113] The Examples set forth herein are illustrative of exemplary embodiments of the present invention. The process, conditions and parameters reflected therein are intended to exemplify various aspects of the invention and are not intended to limit the scope of the claimed invention.

[0114] Example 1DocketNo 95695.00116 26

[0115] In an exemplary embodiment, a bioreactor is disclosed. In various embodiments, the bioreactor comprises a body comprising a chamber and a gas inlet, the chamber configured to receive a growth medium therein; one or more mixing devices, each of the one or more mixing devices extending at least partially into the chamber of the body; a sparging device disposed within the body; a lid removably coupled to the body, wherein the lid comprises two or more openings; one or more light sources disposed through a first of the two or more openings in the lid; one or more sensors threaded through a second of the two or more openings in the lid, the one or more sensors configured to measure one or more operational parameters within the body during operation of the bioreactor; one or more venting pipes coupled to the body; one or more recycling pipes coupled to the body; a venting stage configuration that comprises the one or more venting pipes in fluid communication with the chamber and the one or more recycling pipes fluidly isolated from the chamber; and a recycling stage configuration that comprises the one or more recycling pipes in fluid communication with the chamber and the one or more venting pipes fluidly isolated from the chamber.

[0116] In various embodiments, the bioreactor further comprises a gas supply system, the gas supply system comprising a gas pipe extending from a gas supply to the gas inlet, wherein the gas supply system is configured to inject a gas mixture into the chamber of the body through the gas pipe and the gas inlet, and the gas supply system is configured to pass the gas mixture through the sparging device before entering the growth medium. In various embodiments, the gas pipe is coupled to a mixing chamber, wherein the mixing chamber is configured to receive two or more gases from the gas supply and combine them to produce the gas mixture.

[0117] In various embodiments, the bioreactor further comprises a controller in electronic communication with the one or more sensors, wherein the controller is configured to monitor a mass balance ratio of the growth medium during operation in the recycling stage configuration; determine, based on a first set of data received from the one or more sensors, the mass balance ratio is about 2: 1 between photosynthesis and photorespiration, and responsive to determining the mass balance ratio is about 2: 1 between photosynthesis and photorespiration, transition from the recycling stage configuration to the venting stage configuration.

[0118] In various embodiments, the controller is further configured to monitor an O2 concentration during operation in the venting stage configuration, determine, based on a second set of data received from the one or more sensors, thatDocketNo 95695.00116 27the O2 concentration has fallen below a threshold level; and responsive to determining that the O2 concentration has fallen below the threshold level, transition from the venting stage configuration back to the recycling stage configuration.

[0119] Example 2

[0120] In an exemplary embodiment, a method for operating a bioreactor is disclosed. In various embodiments, the method comprises pumping, via a solution pipe, a growth medium into a body of the bioreactor, coupling a lid comprising one or more openings to a top surface of the body, introducing, via a light source, photons into the growth medium, injecting, via a gas pipe, a first gas into the growth medium, wherein the gas pipe is configured to inject the first gas through a sparging device within the body at a bottom surface of the body, venting, via a venting pipe, a dissolved gas from the body, measuring, via one or more sensors, a first operational parameter of the bioreactor, swapping, in response to the first operational parameter meeting a first threshold value, from the first gas to a second gas, switching, via a regulating device located on the lid, from the venting pipe to a recycling pipe, recycling, via the recycling pipe, the second gas within the body, swapping, in response to a second operational parameter meeting a second threshold value, from the second gas to the first gas, switching, via the regulating device located on the lid, from the recycling pipe to the venting pipe, repeating a cycle of venting and recycling until a third operational parameter reaches a third threshold value, and producing, via the cycle of venting and recycling, a byproduct.

[0121] In various embodiments, the growth medium comprises a carrier liquid, a microorganism population, and a nutrient source. In various embodiments, the microorganism population comprises at least one of a photoautotroph, green algae, red algae, and diatom. In various embodiments, the microorganism population consists essentially of diatoms. In various embodiments, the microorganism population is conditioned under a CO2 environment for a set duration of time before being combined into the growth medium.

[0122] In various embodiments, the first operational parameter is an O2 concentration in the bioreactor, the first threshold value is between 0.1% and 50% of O2, and the swapping from the first gas to the second gas is performed in response to the first operational parameter falling below the first threshold value. In various embodiments, the first threshold value is between 15% and 25 of O2.DocketNo 95695.00116 28

[0123] In various embodiments, the second operational parameter is a mass balance ratio, the second threshold value is 2: 1 between photosynthesis and photorespiration, and the swapping from the second gas to the first gas is performed in response to the second operational parameter falling within a threshold range that includes the second threshold value. In various embodiments, the first gas is N2 and the second gas comprises a mixture of CO2 and N2. In various embodiments, the third operational parameter is at least one of a byproduct concentration, a bioreactor weight, an amount of CO2 inj ected into the body, and a time elapsed.

[0124] In various embodiments, the byproduct is glycolic acid, a sodium derivative thereof, or a potassium derivative thereof. In various embodiments, the method further comprises collecting, via a collection pipe, the byproduct, and purifying, via a purification process, the byproduct.

[0125] Example 3

[0126] In an exemplar}' embodiment, a bioreactor sy stem is disclosed. In various embodiments, the bioreactor system comprises a plurality of bioreactors, each of the plurality of bioreactors comprising: a body comprising a chamber and a gas inlet, the chamber configured to receive a growth medium therein, one or more magnetic field generators located outside of and adjacent to the body configured to rotate one or more stir bars located within the body. a sparging device located within the body, a lid coupled to the body, wherein the lid comprises two or more openings, one or more light sources disposed within the body, one or more sensors threaded through a first of the two or more openings in the lid, the one or more sensors configured to measure one or more operational parameters within the body during operation, one or more venting pipes coupled to the body, one or more recycling pipes coupled to the body, a venting stage configuration that comprises the one or more venting pipes in fluid communication with the chamber and the one or more recycling pipes fluidly isolated from the chamber, and a recycling stage configuration that comprises the one or more recycling pipes in fluid communication with the chamber and the one or more venting pipes fluidly isolated from the chamber. one or more transfer pipes configured to move one or more materials into or out of the plurality of bioreactors, wherein one or more pumps are coupled to the one or more transfer pipes, one or more holding tanks configured to couple to the one or more transfer pipes and store the one or more materials, and an industrial scale, wherein at least one of the plurality’ of bioreactors is disposed on the industrial scale.DocketNo 95695.00116 29

[0127] In various embodiments, each of the plurality of bioreactors is an independently contained system. In various embodiments, at least two of the plurality of bioreactors are apart of the same system, and wherein the at least two of the plurality of bioreactors are configured to share at least one of the one or more transfer pipes and the one or more holding tanks. In various embodiments, at least one bioreactor in the plurality of bioreactors further comprises a raised platform located underneath the industrial scale and configured to suspend the at least one of the plurality of bioreactors off a ground surface. In various embodiments, the grow th medium comprises a carrier liquid, a microorganism population, and a nutrient source. In various embodiments, the microorganism population comprises at least one of a photoautotroph, green algae, red algae, and diatom. In various embodiments, the microorganism population consists essentially of diatoms.

[0128] In various embodiments, the bioreactor system further comprises one or more controllers operably coupled to each of the plurality of bioreactors, wherein the one or more controllers is configured to: monitor a mass balance ratio of the growth medium during operation in the recycling stage configuration for at least one of the plurality of bioreactors, determine, based on a first set of data received from the one or more sensors, the mass balance ratio is about 2:1 between photosynthesis and photorespiration for the at least one of the plurality of bioreactors, and responsive to determining the mass balance ratio is about 2: 1 between photosynthesis and photorespiration, transition from the recycling stage configuration to the venting stage configuration for the at least one of the plurality of bioreactors.

[0129] In various embodiments, at least one transfer pipe in the one or more transfer pipes is a gas pipe configured move a gas mixture from a mixing chamber to the body of each of the plurality of bioreactors. In various embodiments, the gas mixture passes through the sparging device before entering the growth medium. In various embodiments, the gas pipe comprises a sterile filter configured to remove contaminants from the gas mixture.

[0130] In various embodiments, the lid forms a continuous seal with the body. In various embodiments, the bioreactor system further comprises one or more sterilization pipes coupled to configured to inject sterilization fluid into the body. In various embodiments, the gas mixture comprises CO2 and optionally at least one of O2 and N2.

[0131] Example 4DocketNo 95695.00116 30

[0132] In an exemplary embodiment, a control system (i.e. controller) for a bioreactor is disclosed. In various embodiments, the control system comprises a first set of sensors configured to measure one or more operational parameters within a body of the bioreactor, and a control module in electronic communication with the first set of sensors, the control module configured to: receive, from the first set of sensors, a plurality of sensor measurements, calculate a mass balance ratio of a growth medium disposed in the body of the bioreactor; compare the mass balance ratio of the growth medium to a desired mass balance ratio, responsive to the mass balance ratio matching the desired mass balance ratio within a threshold tolerance, transition the bioreactor from a recycling stage mode of operation to a venting stage mode of operation, monitor one or more gas concentrations during operation in the venting stage mode of operation, and responsive to determining the one or more gas concentrations exceeds or falls below a first threshold value, transitioning from the venting stage mode of operation back to the recycling stage mode of operation.

[0133] In various embodiments, the control module is further configured to alternate between the venting stage mode of operation and the recycling stage mode of operation, and responsive to the alternating between the venting stage mode of operation and the recycling stage mode of operation, produce a byproduct. In various embodiments, the byproduct is glycolic acid, a sodium derivative thereof, or potassium derivative thereof. In various embodiments, the growth medium comprises a carrier liquid, a microorganism population, and a nutrient source. In various embodiments, the desired mass balance ratio is 2: 1 between photosynthesis and photorespiration. In various embodiments, the one or more gas concentrations comprise at least one of an O2 concentration or a CO2 concentration.

[0134] In various embodiments, the control system further comprises a second set of sensors that are configured to be positioned external to the body of the bioreactor and configured to measure one or more external operational parameters outside of the body, wherein each of the second set of sensors is in electronic communication with the control module.

[0135] In various embodiments, the control module is further configured to monitor, based on at least one sensor measurement from the first set of sensors, a third operational parameter, and in response to the third operational parameter reaching or exceeding a threshold value, stop operation of the bioreactor.DocketNo 95695.00116 31

[0136] In various embodiments, the recycling stage mode of operation comprises injecting, via a gas pipe, a first gas through a sparging device into the growth medium to produce a modified first gas, wherein the sparging device is located at a bottom surface within the body, pumping, via a recycling pipe, the modified first gas out of the body and into the gas pipe, and reinjecting, via the gas pipe, the modified first gas through the sparging device into the growth medium. In various embodiments, the venting stage mode of operation comprises injecting, via the gas pipe, a second gas through the sparging device into the growth medium to produce a modified second gas, venting, via a venting pipe, the modified second gas out of the body, wherein the venting pipe comprises an activated carbon trap configured to remove contaminants from the modified second gas and produce a decontaminated gas, and releasing, via the venting pipe, the decontaminated gas into an external atmosphere.

[0137] In various embodiments, the first gas comprises CO2 and optionally at least one of O2 and N2. In various embodiments, the control module is further configured to alternate between the venting stage mode of operation and the recycling stage mode of operation until a third operational parameter reaches or exceeds a second threshold value, and ceasing operation of the bioreactor in response to the third operational parameter reaching or exceeding the second threshold value. In various embodiments, the third operational parameter is at least one of byproduct concentration, a bioreactor weight, an amount of CO2 injected into the body, and time elapsed.

[0138] Example 5

[0139] In an exemplary embodiment, a condition module is disclosed. In various embodiments, the conditioning module comprises a body comprising an enclosed chamber, an intake pipe configured to transfer a portion of a microorganism population from a growth vessel into the enclosed chamber, one or more sensors configured to measure one or more characteristics of the enclosed chamber, a discharge pipe configured to transfer a conditioned microorganism population that is formed in the enclosed chamber out of the enclosed chamber, and a control module in electronic communication with the one or more sensors, the control module configured to control at least one of temperature and CO2 concentration within the enclosed chamber to produce the conditioned microorganism population, and in response to a gas concentration reaching, exceeding, or falling below a threshold value, transferring the conditioned microorganism population out of the enclosed chamber.DocketNo 95695.00116 32

[0140] In various embodiments, the conditioning module further comprises a mixing device extending at least partially within the enclosed chamber. In various embodiments, the conditioning module further comprises an electromagnet located proximate a bottom side of the enclosed chamber configured to generate a magnetic field and rotate the mixing device, and wherein the mixing device is a stir bar. In various embodiments, one or more light sources configured to direct photons into the enclosed chamber, the one or more light sources are in electronic communication with the control module. In various embodiments, the one or more light sources are an LED system. In various embodiments, the one or more light sources produce a wavelength in a range of about 550 nm to about 650 nm. In various embodiments, the conditioning module further comprises a photoreceiver configured to receive the photons, wherein the photoreceiver is in electronic communication with the control module.

[0141] In various embodiments, the discharge pipe is coupled at one end to the enclosed chamber and coupled to a bioreactor at a second end. In various embodiments, the microorganism population comprises at least one of a photoautotroph, green algae, red algae, and diatom. In various embodiments, the conditioning module further comprises a sampling port configured to act as a barrier at an interface between the intake pipe and the growth vessel, wherein at periodic intervals, the sampling port opens to allow the portion of the microorganism population to flow through the intake pipe into the enclosed chamber.

[0142] In various embodiments, the one or more light sources and the photoreceiver are configured to measure one or more traits of the portion of the microorganism population. In various embodiments, the one or more traits comprises concentration per liter of the portion of the microorganism population. In various embodiments, the one or more characteristics comprises at least one of pH, salinity, CO2 concentration, and temperature.

[0143] Benefits, other advantages, and solutions to problems have been described herein regarding specific embodiments. Furthermore, the connecting lines shown in the various figures contained herein are intended to represent exemplary functional relationships and / or physical couplings between the various elements. It should be noted that many alternative or additional functional relationships or physical connections can be present in a practical system. However, the benefits, advantages, solutions to problems, and any elements that can cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as critical,DocketNo 95695.00116 33required, or essential features or elements of the disclosure. The scope of the disclosure is accordingly to be limited by nothing other than the appended claims, in which reference to an element in the singular is not intended to mean “one and only one” unless explicitly so stated, but rather “one or more.” Moreover, where a phrase similar to “at least one of A, B, or C” is used in the claims, it is intended that the phrase be interpreted to mean that A alone can be present in an embodiment, B alone can be present in an embodiment, C alone can be present in an embodiment, or that any combination of the elements A, B and C can be present in a single embodiment; for example, A and B, A and C, B and C, or A and B and C. Different cross-hatching is used throughout the figures to denote different parts but not necessarily to denote the same or different materials.

[0144] Systems, methods, and apparatus are provided herein. In the detailed description herein, references to “one embodiment,” “an embodiment,” “various embodiments,” etc., indicate that the embodiment described can include a particular feature, structure, or characteristic, but every embodiment can not necessarily include the feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether explicitly described. After reading the description, it will be apparent to one skilled in the relevant art(s) how to implement the disclosure in alternative embodiments.

[0145] Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed under the provisions of 35 U.S.C. 112(f) unless the element is expressly recited using the phrase “means for.” As used herein, the terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0146] Finally, any of the above-described concepts can be used alone or in combination with any or all the other above-described concepts. Although various embodiments have been disclosed and described, one of ordinary skill in this art wouldDocketNo 95695.00116 34recognize that certain modifications would come within the scope of this disclosure. Accordingly, the description is not intended to be exhaustive or to limit the principles described or illustrated herein to any precise form. Many modifications and variations are possible considering the above teaching.DocketNo 95695.00116 35

Claims

CLAIMSWhat is claimed is:

1. A method, comprising: adding photoautotrophs to a growth medium in a first bioreactor, the growth medium comprising a liquid medium; adding photons and CO2 to the first bioreactor; conducting a photoautotrophic process in the first bioreactor to produce glycolic acid; measuring, via one or more first sensors, a first operational parameter of the first bioreactor; removing the glycolic acid from the first bioreactor in response to the first operational parameter meeting a first threshold value; adding the glycolic acid and water to a second bioreactor; adding microorganisms to the second bioreactor; conducting a heterotrophic process in the second bioreactor to produce a secondary product and CO2; measuring, via one or more second sensors, a first operational parameter of the second bioreactor; and removing the secondary product from the second bioreactor in response to the first operational parameter meeting a first threshold value.

2. The method of claim 1, further comprising adding a carbonic anhydrase inhibitor to the first bioreactor after the adding of photoautotrophs to the growth medium.

3. The method of claim 1, further comprising adding non-carbon nutrients to the second bioreactor after the adding the glycolic acid, wherein the non-carbon nutrients comprise at least one of ammonium chloride, sodium phosphate, potassium phosphate, salt, magnesium, calcium, or an antibiotic.

4. The method of claim 1, further comprising conditioning the photoautotrophs before the adding the photoautotrophs to the growth medium, wherein the conditioning comprises mixing the photoautotrophic with CO2 and nutrients.DocketNo 95695.00116 365. The method of claim 1. wherein the grow th medium comprises water.

6. The method of claim 1 , wherein the CO2 added to the first bioreactor is provided from the CO2 produced by the second bioreactor.

7. The method of claim 1, further comprising purifying the glycolic acid before the adding the glycolic acid to the second bioreactor.

8. The method of claim 1, wherein the glycolic acid is added to the second bioreactor as an unprocessed bioreactor medium.

9. The method of claim 1 , wherein the photons are added via a light source.

10. The method of claim 9, wherein the light source is an LED-coupled fiber optic system.1 1. The method of claim 1, further comprising, before the adding the glycolic acid to the second bioreactor, one or more of: genetically modifying the microorganism population to produce recombinant proteins; or carrying out one or more non-native enzymatic processes.

12. The method of claim 1, wherein the removing the secondary product from the second bioreactor produces a barren medium, and wherein the barren medium is gasified to produce syngas.

13. The method of claim 12, further comprising combining the syngas with the secondary product to produce an additional end product.

14. The method of claim 1, wherein the secondary product comprises at least one of hydrogen, ammonia, C1-C4 carboxylic acids, fluorescent or bioactive recombinant proteins, vitamins, lipids, antibiotics, or glycosaminoglycans.DocketNo 95695.00116 3715. The method of claim 1, wherein the first operational parameter is pH and the second operational parameter is cell density.

16. The method of claim 1, wherein the first bioreactor is a photoautotrophic bioreactor and the second bioreactor is a heterotrophic bioreactor.

17. The method of claim 16, wherein the photoautotrophic bioreactor is an open pond system, tubular bioreactor, flat panel bioreactor, air lifted bioreactor, bag bioreactor, or a combination thereof.

18. A system, comprising: a first bioreactor comprising a growth medium, a photoautotroph population, a light source, and CO2, wherein the first bioreactor is configured to produce glycolic acid; a second bioreactor comprising carbon feedstock and a microorganism population, wherein the second bioreactor is configured to produce the secondary product and CO2; and a first fluid connection from the first bioreactor to the second bioreactor configured to transfer the glycolic acid from the first bioreactor to the second bioreactor, wherein the system is configured to provide the glycolic acid produced by the first bioreactor as at least a portion of the carbon feedstock in the second bioreactor.

19. The system of claim 18, further comprising a second fluid connection from the second bioreactor to the first bioreactor configured to transfer the CO2 produced by the second bioreactor to the first bioreactor.

20. The system of claim 18, wherein the second bioreactor further comprises noncarbon nutrients, and wherein the non-carbon nutrients comprise at least one of ammonium chloride, sodium phosphate, potassium phosphate, salt, magnesium, calcium, and one or more antibiotics.DocketNo 95695.00116 38

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