Systems and methods of continuous photosynthetic hydrogen production

Abstract

Disclosed herein are algae culture system and methods for continuous, photosynthetic hydrogen production. The algae culture system is a closed culture system comprising transgenic red algae and Galdieria sulphuraria. The transgenic red algae is transformed to express a hydrogenase gene from a green algae. The only substrate provided to the closed culture system is an organic carbon source. Accordingly, the methods for continuous, photosynthetic hydrogen production comprise coculturing the transgenic red algae with G. sulphuraria in a closed algae culture system; not providing the closed algae culture system with supplemental oxygen or supplemental carbon dioxide; and providing organic carbon to the closed algae culture system. In some aspects, the G. sulphuraria is from a strain that consumes oxygen without contributing to excess O2 production. In certain embodiments, the transgenic red algae is Cyanidioschyzon merolae transformed to express a hydrogenase gene from Chlamydomonas reinhardtii.

Description

Agent Reference: 11157-204WO-PCTSYSTEMS AND METHODS OF CONTINUOUS PHOTOSYNTHETIC HYDROGEN PRODUCTIONRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application 63 / 742,730, filed January 7, 2025, to Lammers, et al., titled “SYSTEMS AND METHODS OF CONTINUOUS PHOTOSYNTHETIC HYDROGEN PRODUCTION,” the entirety of the disclosure of which is hereby incorporated by this reference.SEQUENCE LISTING

[0002] In accordance with 37 C.F.R. § 1.831, the present specification makes reference to a Sequence Listing submitted electronically in the form of an XML file (entitled “204WO-PCT.xml”, created on December 11, 2025, 56,889 bytes in size). The entire contents of the Sequence Listing are herein incorporated by reference in their entirety, with the intention that, upon publication (including issuance), this incorporated Sequence Listing will be inserted in the published document immediately before the claims.TECHNICAL FIELD

[0003] This document relates genetically modified red algae systems and methods of continuously producing hydrogen via photosynthesis.BACKGROUND

[0004] Oxygenic photosynthesis and enzymatic hydrogen production are not compatible processes. This is due to the oxygen sensitivity of the hydrogenase enzymes and the release of oxygen after photosystem II water splitting. Various methods have been used to temporally separate the two activities, the most successful of which combine a growth phase followed by a nutrient deprivation step (sulfur, phosphorus, nitrogen or potassium) that inhibits photosynthesis, preventing O2 evolution (Eroglu and Melis 2016). This allows for a period of hydrogen production until cellular energy production is depleted, at which point the medium is replenished and a period of growth without hydrogen production is required.

[0005] Current methods for removing oxygen in algae to invoke hydrogen production include nutrient stress to suppress photosynthetic O2 evolution, co-culturing with heterotrophicAgent Reference: 11157-204WO-PCTmicroorganisms, inert gas purges, and the addition of oxygen scavengers (Chen, Xiang et al.2024). All of these are difficult to scale.

[0006] Accordingly improved methods for bio-hydrogen production are needed.SUMMARY

[0007] According to some embodiments, the present disclosure relates to a system for continuous, photosynthetic hydrogen production comprising an algae culture comprising transgenic red algae genetically modified to express a hydrogenase gene from a green algae, and Galdieria sulphuraria, and a closed culture system containing the algae culture.

[0008] Particular embodiments may comprise one or more of the following features. The G. sulphuraria may be from a strain that consumes oxygen without contributing to excess oxygen production. The red algae may be Cyanidioschyzon merolae. The green algae may be Chlamydomonas reinhardtii. The hydrogenase gene may be HydAl. The transgenic red algae may be genetically modified to express two copies or more of HydAl . The transgenic red algae may be transformed with CmU 02 / CmC 19 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 12) or CmU 02 / CmC 20 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 16). The G. sulphuraria may be strain 074W or strain 3377. The system may not be provided supplemental oxygen or supplemental carbon dioxide. The system may be configured to continuously produce hydrogen. The system may be configured to maintain microaerobic conditions suitable for continuous hydrogen production. The system may be configured to inhibit contamination of the algae culture. The system may be configured to operate at a pH below 4.0 and a temperature above 40 °C. The system may be configured to maintain microaerobic conditions by balancing photosynthetic O2 evolution and respiratory O2 consumption such that dissolved O2 remains below the inactivation threshold of the hydrogenase gene. The system may be configured to remove hydrogen gas from the closed culture system. The system may be configured to mitigate product inhibition and sustain production rates of hydrogen.

[0009] According to some embodiments, the present disclosure relates to a method for continuous, photosynthetic hydrogen production comprising providing the algae culture system described above, supplying organic carbon and light to the algae culture system, and maintaining the algae culture system without supplemental oxygen or supplemental carbon dioxide.

[0010] According to some embodiments, the present disclosure relates to a method for continuous, photosynthetic hydrogen production comprising coculturing transgenic red algaeAgent Reference: 11157-204WO-PCTand Galdieria sulphuraria in a closed algae culture system, wherein the transgenic red algae is genetically modified to express a hydrogenase gene from a green algae, and the G. sulphuraria strain consumes oxygen without contributing to excess oxygen production, maintaining the closed algae culture system without supplemental oxygen or supplemental carbon dioxide, and providing organic carbon and light to the closed algae culture system.

[0011] Particular embodiments may comprise one or more of the following features. The method may further comprise purging the closed algae culture system with nitrogen. The method may further comprise removing hydrogen product from the closed algae culture system. In some aspects, this avoids product inhibition. The method may further comprise maintaining culture pH below 4.0 and temperature above 40 °C. The red algae may be Cyanidioschyzon merolae. The green algae may be Chlamydomonas reinhardtii. The hydrogenase gene may be HydAl. The transgenic red algae may be genetically modified to express more than one copy of HydAl. The transgenic red algae may be transformed with CmU 02 / CmC 19 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 12) or CmU 02 / CmC 20 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 16). The G. sulphuraria may be strain 074W or strain 3377.

[0012] According to some embodiments, the present disclosure relates to transgenic Cyanidioschyzon merolae genetically modified to express HydAl gene from Chlamydomonas reinhardtii.

[0013] Particular embodiments may comprise one or more of the following features. The transgenic C. merolae may be transformed with CmU 02 / CmC 19 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 12) or CmU 02 / CmC 20 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 16).

[0014] According to some embodiments, the present disclosure relates to use of a system comprising transgenic red algae expressing a hydrogenase gene from a green algae for the continuous production of hydrogen gas.

[0015] Particular embodiments may comprise one or more of the following features. The system may be operated under microaerobic conditions. The microaerobic conditions may be maintained by respiratory oxygen consumption of an organic carbon source. The red algae may be Cyanidioschyzon merolae. The green algae may be Chlamydomonas reinhardtii. The hydrogenase gene may be HydAl. The transgenic red algae may be genetically modified to express two or more copies of HydAl . The transgenic red algae may be transformed with CmU 02 / CmC 19 or CmU 02 / CmC 20. The system may further comprise Galdieria sulphuraria. The G. sulphuraria may be strain 074W or strain 3377.Agent Reference: 11157-204WO-PCT

[0016] The foregoing and other aspects, features, and advantages will be apparent from the DESCRIPTION and DRAWINGS, and from the CLAIMS.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] Implementations will hereinafter be described in conjunction with the appended DRAWINGS, where like designations denote like elements.

[0019] FIG. 1A, in accordance with certain embodiments, illustrates the plasmid design and transgene integration. The synthetic plasmids were designed in silico and constructed de novo after codon optimizing each gene for nuclear gene expression in C. merolae (reference Seger et al. 2023). The CmU 02 construct (nucleic acid sequence set forth in SEQ ID NO: 18) contains the C. renhardtii HydAl gene (corresponding amino acid sequence set forth in SEQ ID NO: 6) and the wild-type URA 5.3 gene (corresponding amino acid sequence set forth in SEQ ID NO: 17) to complement the URA deficiency of the T1 strain using left and right homology arms (HR-L and -R) directing integration into the URA 5.3 region. Subsequently, the CmC 19 (HydG + HydEF) or CmC 20 (HydAl, HydG, HydEF) transgenes were integrated into the 184C-185C neutral locus on C. merolae chromosome 4 with selection for chloramphenicol resistance. The CmC 19 strain has a single copy of HydAl while the CmC20 strain has two. PCPCC - phycocyanin-associated rod linker protein promoter, StrepII - C-terminal peptide tag with stop codon, CMS243C - chloroplast targeting peptide, 3xHA -peptide tag with stop codon, CMO250C, chloroplast targeting peptide, pAPCC - promoter from allophycocyanin-associated rod linker protein gene, CTP CMH166C - DNA Gyrase B chloroplast targeting peptide, tNOS - nopaline synthase terminator, tUbq - C. merolae ubiquitin terminator, tp-tub - C. merolae P-tubulin terminator CMN263C.

[0020] FIG. IB, in accordance with certain embodiments, depicts the polymerase chain reaction confirmation of plasmid integration at the uracil or 184-195C neutral locus, presence of transgenes, and unialgal status (RuBisCO Haelll digestion).

[0021] FIGs. 2A and 2B, in accordance with certain embodiments, respectively illustrate hydrogen and oxygen production in a closed co-culture of C. merolae clones transformed with CmU 02 / CmC 20 (U20_15 clone) or with CmU 02 / CmC 19 (U19 1 and U19 14 clones) with G. sulphuraria 074W. Hydrogen and oxygen values are normalized to the culture volume. The U20 15 clone contains two HydAl genes compared to one in the U19 clones.Agent Reference: 11157-204WO-PCT

[0022] FIG. 3A illustrates the impact of G. sulphuraria partner strain on H2 and O2 levels in co-cultures with C. merolae clone U20 15. Gas levels normalize to culture volume.

[0023] FIG. 3B, in accordance with certain embodiments, illustrates the photosynthetic pigment phenotype of G. sulphuraria strains in the presence of glucose, showing that pigment retention results in high oxygen production that inhibits hydrogenase activity.

[0024] FIGs. 4A-4C, in accordance with certain embodiments, illustrate that HydAl enzyme activity is dependent on photosynthesis and is sensitive to product inhibition. FIG. 4A shows the hydrogen production in closed co-cultures of C. merolae clone U_20 with G. sulphuraria 074W (short-dash line), after N2 purges at 47 and 71.5 hours to remove H2 (long-dash line), and after addition of 20 pM DCMU (solid line) at the same times as the N2 purges.

[0025] FIG. 5 depicts a schematic for the conversion of sugars into algae biomass, which can be the source of hydrogen production and other products.DETAILED DESCRIPTION

[0026] Detailed aspects and applications of the disclosure are described below in the following drawings and detailed description of the technology. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

[0027] In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the disclosure. It will be understood, however, by those skilled in the relevant arts, that embodiments of the technology disclosed herein may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices and technologies to which the disclosed technologies may be applied. The full scope of the technology disclosed herein is not limited to the examples that are described below.

[0028] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.

[0029] The word “exemplary,” “example,” or various forms thereof are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” or as an “example” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Furthermore, examples are provided solely for purposes of clarity and understanding and are not meant to limit or restrict the disclosed subject matter or relevant portions of this disclosure in any manner. It is to be appreciated that a myriad of additional orAgent Reference: 11157-204WO-PCTalternate examples of varying scope could have been presented but have been omitted for purposes of brevity.

[0030] When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0031] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and are not intended to (and do not) exclude other components.

[0032] As required, detailed embodiments of the present disclosure are included herein. It is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limits, but merely as a basis for teaching one skilled in the art to employ the present invention. The specific examples below will enable the disclosure to be better understood. However, they are given merely by way of guidance and do not imply any limitation.

[0033] The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific materials, devices, methods, applications, conditions, or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed inventions. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0034] Disclosed herein is a process that produces hydrogen by using photosynthesis that does not rely on limiting photosynthetic oxygen production. Instead, a composition of organisms that produce and share oxygen and carbon dioxide in enclosed, lighted reactor systems that admit no air, can simultaneously exhibit oxygenic photosynthesis and enzymatic hydrogen production. The continuous hydrogen production process disclosed herein relies on respiratory oxygen consumption of organic substrates to achieve microaerobic conditions in the algal culture. In the closed culture system with light but not supplemental air, the rate of respirationAgent Reference: 11157-204WO-PCTis faster than the rate of photosynthesis leading to microaerobic conditions suitable for enzymatic hydrogen biosynthesis. In some aspects, the total oxygen production rate is matched to total oxygen consumption rate in reactors that admit minimal atmospheric gas. When those rates are near equal, the culture maintains microaerobic conditions that allow hydrogenase to function.

[0035] The composition of organisms for continuous photosynthetic hydrogen production comprises a transgenic red algae and Galdieria sulphuraria. A unique aspect of disclosed system and methods is the use of two compatible acidophiles and conditions providing resistance to contamination by non-compatible invading organisms: growth at low pH and elevated temperatures. Accordingly, a system for continuous, photosynthetic hydrogen production is disclosed. The system comprises an algae culture comprising the transgenic red algae and G. sulphuraria in a closed culture system, for example, as shown in FIG. 5. The disclosed system is the only current system with potential for continuous algal H2 production at large scales as a source of bio-hydrogen.

[0036] The transgenic red algae is genetically modified to express a hydrogenase from a hydrogen-producing microorganism. In some aspects, the transgenic red algae is a mixotrophic Cyanidiophyceae algal species genetically modified to express a hydrogenase from a hydrogenproducing microorganism, for example a green algae. The transgenic red algae may be selected from any known red algae species that can be genetically modified, for example, Cyanidioschyzon merolae or Galdieria partida, or other Galdieria species. In some embodiments, the transgenic red algae is transformed to express a hydrogenase gene from Chlamydomonas reinhardtii. for example its HydAl gene. In certain embodiments, the transgenic red algae is C. merolae genetically modified to express the HydAl gene from C. reinhardtii. In some aspects, the amino acid sequence of the HydAl expressed by the transgenic red algae has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity as the sequence set forth in SEQ ID NO. 6. In certain embodiments, the transgenic red algae is genetically modified to express two or more copies of the HydAl gene from C. reinhardtii. In particular embodiments, the transgenic red algae is transformed with the CmU 02 plasmid in combination with the CmC 19 plasmid or the CmC 20. The plasmid sequence of CmU 02 is set forth in SEQ ID NO: 18. The plasmid sequence of CmC 19 is set forth in SEQ ID NO: 12. The plasmid sequence of CmC 20 is set forth in SEQ ID NO: 16. In certain embodiments, the transgenic red algae is C. merolae.

[0037] In some aspects, the G. sulphuraria is from a strain that consumes oxygen without contributing to excess O2 production. This may happen as a result of loss of pigmentation inAgent Reference: 11157-204WO-PCTpresence of organic carbon source. Accordingly, in some embodiments, the G. sulphuraria is strain 074W, strain 107.19, strain 3377, or strain 21.92 (see, for example, FIG. 3B). In other aspects, the G. sulphuraria is from a strain that has been engineered to prevent photosystem II activity.

[0038] In some embodiments, the system is not provided supplemental oxygen and / or supplemental carbon dioxide, and the algae instead relies on sources of oxygen and / or carbon dioxide that are internal to the system for growth. In certain embodiments, the disclosed system is configured to continuously produce hydrogen. In some embodiments, the system is configured to maintain conditions that prevent inhibition of hydrogenase activity and allow sustained enzymatic hydrogen evolution. Continuous production may be achieved by operating the system under microaerobic conditions, wherein the rate of respiratory oxygen consumption exceeds the rate of photosynthetic oxygen evolution. This balance ensures that dissolved oxygen remains below the inactivation threshold of the hydrogenase enzyme, thereby enabling uninterrupted hydrogen generation. The system may further be configured to remove hydrogen gas from the closed culture vessel, which helps mitigate product inhibition and sustain production rates over extended periods. In some aspects, hydrogen removal is accomplished by purging the system with an inert gas such as nitrogen or by active withdrawal through a gas outlet connected to a purification unit.

[0039] The system may also be configured to inhibit contamination of the algae culture by employing growth conditions that are selective for the disclosed acidophilic organisms. In particular embodiments, the system operates at a pH below 4.0 and a temperature above 40 °C, such as between 40 °C and 56 °C. These conditions are inhospitable to most contaminating microorganisms and provide a robust environment for large-scale hydrogen production. By combining low pH and elevated temperature with controlled gas exchange and organic carbon feeding, the system maintains microaerobic conditions suitable for continuous hydrogen production while reducing contamination risk. These operational parameters enable a scalable and efficient process for biohydrogen generation.

[0040] Methods for continuous, photosynthetic hydrogen production are also disclosed. In one implementation, the method comprises providing light to the algae culture system described above while not providing supplemental oxygen. In some aspects, an organic carbon source is provided to the algae culture system. The fed organic carbon is oxidized to CO2 in mitochondria at the expense of O2 produced by photosynthesis in the chloroplast of the algae. The produced metabolic gases are consumed as fast as they are produced leading to a very low concentration of oxygen. As shown in the examples, the co-cultures of transgenic red algae and a G.Agent Reference: 11157-204WO-PCTsulphuraria strain that lose photopigments in the presence of reduced carbon sources generated hydrogen continuously with removal of the H2 product. The organic carbon can come from primary municipal wastewater, as G. sulphuraria is able to consume this organic carbon to mediate its respiration. The wastewater is also a source of nitrogen and phosphate, which are needed for algae culture growth.

[0041] In another implementation, the method for continuous, photosynthetic hydrogen production comprises coculturing a transgenic red algae with G. sulphuraria in a closed algae culture system; not providing the closed algae culture system with supplemental oxygen; and providing organic carbon and light to the closed algae culture system. The transgenic red algae is genetically modified to express a hydrogenase from a hydrogen-producing microorganism, for example a green algae. The G. sulphuraria is from a strain that consumes oxygen without contributing to excess O2 production, for example, strain 074W or strain 3377. In some aspects, the red algae is C. merolae. In some aspects, the green algae is C. reinhardtii. Thus in some embodiments, the hydrogenase is HydAl. In some aspects, the amino acid sequence of the HydAl expressed by the transgenic red algae has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity as the sequence set forth in SEQ ID NO. 6. In some embodiments, the transgenic red algae is genetically modified to express two or more copies of the HydAl gene. In particular embodiments, the transgenic red algae is genetically modified to express two copies of the HydAl gene. For example, the transgenic red algae is transformed with CmU 02 / CmC 19 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 12) or CmU 02 / CmC 20 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 16). In some aspects, the organic carbon is provided to the algae closed algae culture system using primary municipal wastewater.

[0042] In some embodiments, the method further comprises purging the closed algae culture system with an inert gas, such as nitrogen, to remove accumulated hydrogen from the system. In some aspects, this step mitigates product inhibition of hydrogenase activity and restores maximum hydrogen production rates. In certain implementations, hydrogen removal may also be accomplished by active withdrawal through a gas outlet connected to a purification unit. These operations help sustain continuous hydrogen generation over extended periods without interruption.

[0043] The disclosed methods may also comprise maintaining selective growth conditions that inhibit contamination and favor the acidophilic organisms in the co-culture. In certain implementations of the methods for continuous, photosynthetic hydrogen production, the algae culture system has low pH conditions with high temperature. For example, the pH of the cultureAgent Reference: 11157-204WO-PCTis less than 4.0, such as around 2.0. The temperature of the algae culture system is over 40 °C. In some aspects, the temperature of the algae culture system is not over 56 °C. Thus, in particular implementations, the temperature of the algae culture system is about 42 °C. These conditions, combined with controlled gas exchange and organic carbon feeding, enable microaerobic conditions suitable for continuous hydrogen production while reducing contamination risk.

[0044] In certain embodiments, the present disclosure is related to a transgenic Cyanidioschyzon merolae genetically modified to express the HydAl hydrogenase gene from Chlamydomonas reinhardtii. In some aspects, the transgenic C. merolae is transformed to express at least one copy of the hydrogenase gene. In certain embodiments, the transgenic C. merolae is transformed using homologous recombination with constructs CmU 02 / CmC 19 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 12) or CmU 02 / CmC 20 (plasmid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 16), which enable expression of one or two copies of the HydAl gene. These genetic modifications confer the ability to catalyze hydrogen evolution under microaerobic conditions, a capability not present in wildtype red algae. In particular embodiments, the HydAl gene is operably linked to promoters and targeting sequences that direct the enzyme to photosynthetic compartments, thereby facilitating efficient hydrogen production.

[0045] Also disclosed are uses of the system described above for the continuous production of hydrogen gas. In some implementations, the system comprises transgenic red algae expressing a hydrogenase gene from a green algae and is operated under microaerobic conditions maintained by respiratory oxygen consumption of an organic carbon source, as described above.Examples

[0046] The present disclosure is further illustrated by the following examples that should not be construed as limiting.Example 1. Gene design, synthesis and transfer to C. merolae by homologous recombination

[0047] CmU 02 was integrated with selection for reversion to uracil prototrophy in the T1 line of C. merolae (Taki, Sone et al. 2015). CmC 19 and CmC 20 were introduced into the 184C- 185C neutral site as described by Seger et al., 2023. The CmU 02 / CmC 19 combination provides one copy of the HydAl hydrogenase gene, while the CmU 02 / CmC 20 combination provides two copies. FIG. 1 A shows the genes, promotors, terminators, chloroplast targetingAgent Reference: 11157-204WO-PCTpeptides and transcription terminators. FIG. IB provides PCR-based confirmation for homologous recombination at the proper locations.Example 2. Eh production rates: two copies ofHydAl are better than one.

[0048] Co-cultures of C. merolae transformed with CmU 02 / CmC 19 or CmU 02 / CmC 20 and G. sulphuraria 074W were grown in gas-tight test tubes with 50 mM glucose in MA2 medium (8 mL) as a carbon source, at pH 2 and 42 °C. Hydrogen accumulation in the head space (8 mL) was measured via gas chromatography. H2 and O2 production levels are shown in FIGs. 2A and 2B. Co-cultures with wild type C. merolae are shown as negative controls. H2 is shown as pmoles per L of head space. Two copies of the HydAl gene present in the CmU 02 / CmC 20 construct provide a dramatic increase in product. The HydEF and HydG gene products are required for assembly / repair of the HydAl enzyme.

[0049] FIG. 3A demonstrates that the rates of oxygen production via photosynthesis and oxygen consumption via respiration of organic carbon sources can be equalized by choosing the appropriate Galdieria strain as the partner in a co-culture system. Strain 074W loses photopigments in the presence of sugars, which inhibits photosynthesis and oxygen evolution (FIG. 3B) At the same time the 074W strain consumes the oxygen produced by C. merolae via respiration and provides carbon dioxide to C. merolae (FIG. 3A). This metabolic gas exchange creates an “enforced mutualism” in which neither strain is able to grow without the other in a gas-restricted culture system. The choice of the Galdieria strains depends on the photosynthetic rate of Cyanidioschyzon strain. Based on conditions used in FIG. 3A, G. sulphuraria 074W provides a better match that G. sulphuraria 5587.1.Example 3. Hydrogen production rates are reversibly inhibited by product and dependent on photosynthesis.

[0050] The same experimental set up used for FIG. 1 was employed with the CmU 02 / CmC 20 + GsO74W co-culture for a 96-h time course. One set of cultures (FIG.4A) was purged with N2 at 47 and 71.5 hours to remove H2 product (long-dash line), one was not purged with N2 (short-dash line) or purged with N2 with the photosynthesis inhibitor, DCMU (solid line). The maximum production rate was recovered as H2 was removed while that was blocked by the photosystem II inhibitor, DCMU. The rise in oxygen (FIG. 4B) at 72 hours is due to the loss of glucose (FIG. 4C).Agent Reference: 11157-204WO-PCTREFERENCES CITED AND INCORPORATED BY REFERENCE• Chen, et al., (2024). “Enhancing strategies of photosynthetic hydrogen production from microalgae: Differences in hydrogen production between prokaryotic and eukaryotic algae.” Bioresource Technology 406.• Eroglu and Melis (2016). “Microalgal hydrogen production research.” Int. J of Hydrogen Energy 41: 12772-12798.• Henkanatte-Gedera, et al., (2017). “Removal of dissolved organic carbon and nutrients from urban wastewaters by Galdieria sulphuraric. Laboratory to field scale demonstration.” Algal Research 24: 450-456.• Seger, et al., (2023). “Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D .” Metabolic Engineering Communications 17.• Taki, et al., (2015). “Construction of a URA5.3 deletion strain of the unicellular red alga Cyanidioschyzon merolae: A backgroundless host strain for transformation experiments.” J. Gen. Appl. Microbiol 61: 211-214.

Claims

Agent Reference: 11157-204WO-PCTCLAIMSWe claim:

1. A system for continuous, photosynthetic hydrogen production comprising:an algae culture comprising:transgenic red algae genetically modified to express a hydrogenase from a green algae; andGaldieria sulphuraria anda closed culture system containing the algae culture.

2. The system of claim 1, wherein the G. sulphuraria is from a strain that consumes oxygen without contributing to excess oxygen production.

3. The system of claim 2, wherein the G. sulphuraria is strain 074W or strain 3377.

4. The system of any one of claims 1-3, wherein the red algae is Cyanidioschyzon merolae.

5. The system of claim 4, wherein the green algae is Chlamydomonas reinhardtii.

6. The system of claim 5, wherein the transgenic red algae is genetically modified to express two copies of a HydAl gene.

7. The system of claim 5, wherein the hydrogenase is HydAl .

8. The system of any one of claims 1-3, wherein the green algae is Chlamydomonas reinhardtii.

9. The system of claim 8, wherein the hydrogenase is HydAl .

10. The system of claim 8, wherein the transgenic red algae is genetically modified to express two copies of a HydAl gene.

11. The system of claim 8, wherein the transgenic red algae is transformed with a plasmid having the sequence set forth in SEQ ID NO: 18 in combination with a plasmid having the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 16.

12. The system of any one of claims 1-2, wherein the transgenic red algae is transformed with a plasmid having the sequence set forth in SEQ ID NO: 18 in combination with a plasmid having the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 16.

13. The system of any one of claims 1-12, wherein the system is not provided supplemental oxygen or supplemental carbon dioxide.

14. The system of any one of claims 1-13, wherein the system is configured to continuously produce hydrogen.

15. The system of any one of claims 1-13, wherein the system is configured to maintain microaerobic conditions suitable for continuous hydrogen production.Agent Reference: 11157-204WO-PCT16. The system of any one of claims 1-15, wherein the system is configured to inhibit contamination of the algae culture.

17. The system of any one of claims 1-16, wherein the system is configured to operate at a pH below 4.0 and a temperature above 40 °C.

18. The system of any one of claims 1-17, wherein the system is configured to maintain microaerobic conditions by balancing photosynthetic O2 evolution and respiratory O2 consumption such that dissolved O2 remains below the inactivation threshold of the hydrogenase gene.

19. The system of any one of claims 1-18, wherein the system is configured to remove hydrogen gas from the closed culture system.

20. The system of any one of claims 1-19, wherein the system is configured to mitigate product inhibition and sustain production rates of hydrogen.

21. A method for continuous, photosynthetic hydrogen production comprising:providing the algae culture system of any one of claims 1-20;supplying organic carbon and light to the algae culture system; andmaintaining the algae culture system without supplemental oxygen or supplemental carbon dioxide.

22. A method for continuous, photosynthetic hydrogen production comprising:coculturing transgenic red algae and Galdieria sulphuraria in a closed algae culture system, wherein:the transgenic red algae is genetically modified to express a hydrogenase from a green algae; andthe G. sulphuraria strain consumes oxygen without contributing to excess oxygen production;maintaining the closed algae culture system without supplemental oxygen or supplemental carbon dioxide; andproviding organic carbon and light to the closed algae culture system.

23. The method of claim 22, further comprising purging the closed algae culture system with nitrogen.

24. The method of claim 22 or 23, further comprising removing hydrogen product from the closed algae culture system.

25. The method of any one of claims 22-24, further comprising maintaining culture pH below 4.0 and temperature above 40 °C.

26. The method of claim 22 or 23, wherein the red algae is Cyanidioschyzon merolae.Agent Reference: 11157-204WO-PCT27. The method of claim 26, wherein the green algae is Chlamydomonas reinhardtii.

28. The method of claim 27, wherein the hydrogenase is HydAl .

29. The method of claim 27, wherein the transgenic red algae is genetically modified to express two copies of a HydAl gene.

30. The method of claim 26, wherein the transgenic red algae is transformed with a plasmid having the sequence set forth in SEQ ID NO: 18 in combination with a plasmid having the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 16.

31. The method of claim 30, wherein the G. sulphuraria is strain 074W or strain 3377.

32. The method of any one of claims 22 or 23, wherein the G. sulphuraria is strain 074W or strain 3377.

33. Transgenic Cyanidioschyzon merolae genetically modified to express HydAl gene from Chlamydomonas reinhardtii.

34. The transgenic C. merolae of claim 33, wherein the C. merolae is transformed with a plasmid having the sequence set forth in SEQ ID NO: 18 in combination with a plasmid having the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 16.

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