Thin-film photomicrobial bioreactor systems for the production of secreted chemicals

Abstract

Provided herein are photobioreactor systems that facilitate the culture of thin films of phototrophic microorganisms to produce extracellular chemicals using sunlight and CO₂. These systems greatly reduce water and energy needs compared to traditional (e.g., suspension-based) methods employed in bioreactors. By immobilizing the microorganisms in a thin layer, these systems optimize light exposure and enable higher cell density and easier product separation, making the process more sustainable and cost-effective. These systems can be used to culture a variety of phototrophic microorganisms, including bacteria, cyanobacteria, algae, and fungi. These systems and methods can be used to produce a wide variety of important chemical products, including fuels, pharmaceuticals, and industrial chemicals including ammonia.

Description

[0001] Thin-Film Photomicrobial Bioreactor Systems for the Production of Secreted Chemicals

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant number 1828902 awarded by the National Science Foundation. The government has certain rights in the invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims benefit of priority of U.S. Provisional Application No. 63 / 678,263, filed August 1, 2024, which is hereby incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Microbes have long been utilized for the production of industrially and pharmaceutically relevant chemicals, including amino acids, organic acids, alcohols, antibiotics, and biopolymers. These products are commonly synthesized by microorganisms such as bacteria, yeasts, or filamentous fungi, either naturally or through metabolic engineering. In many cases, the target compound is secreted from the microbial cells into the extracellular medium, from which it can be recovered and purified.

[0008] While microbial biosynthesis offers significant advantages in terms of sustainability, renewability, and product specificity, there remain several limitations that hinder commercial efficiency. These include challenges in maintaining high cell viability and productivity over time, controlling contamination, managing toxic byproducts, and optimizing conditions to promote secretion rather than intracellular accumulation. Moreover, conventional bioreactor systems often lack the flexibility or control needed to accommodate dynamic metabolic shifts or cell population heterogeneity during long-term cultivation.

[0009] Various reactor configurations, such as batch, fed-batch, and continuous systems, have been developed to enhance productivity. However, each has trade-offs related to throughput, product yield, ease of scale-up, and process control. In particular, processes aimed at maximizing extracellular secretion benefit from reactor designs that facilitate nutrient delivery, waste removal, and product separation without disrupting cell viability or culture integrity.

[0010] Accordingly, there is a continuing need for improved methods and bioreactor systems that enhance the efficiency and scalability of microbial production processes, particularly for secreted target compounds. The present invention addresses these and other needs by providing systems and methods for culturing microorganisms that improve product yields, maintain culture stability, reduce cost, and simplify downstream processing.

[0011] SUMMARY

[0012] This application relates generally to methods and systems for culturing microorganisms. More specifically, the application relates to improved processes and systems for cultivating microorganisms that produce and secrete desirable chemicals.

[0013] For example, provided herein are photobioreactor systems that facilitate the culture of thin films of phototrophic microorganisms to produce extracellular chemicals using sunlight and CO2. These systems greatly reduce water and energy needs compared to traditional (e.g., suspension-based) methods employed in bioreactors. By immobilizing the microorganisms in a thin layer, these systems optimize light exposure and enable higher cell density and easier product separation, making the process more sustainable and cost-effective. These systems can be used to culture a variety of phototrophic microorganisms, including bacteria, cyanobacteria, algae, and fungi. These systems and methods can be used to produce a wide variety of important chemical products, including fuels, pharmaceuticals, and industrial chemicals including ammonia.

[0014] In some embodiments, the photobioreactor system for producing a chemical product via bioconversion can comprise a housing; one or more thin films disposed within the housing, wherein each of the one or more thin films comprises a phototrophic microorganism dispersed within a transparent matrix material; a gas inlet configured to direct a flow of gas into the housing; and a gas outlet configured to remove gas from the housing.

[0015] Also described herein are methods for producing a chemical product via bioconversion that comprise forming a thin film comprising a phototrophic microorganism dispersed within a transparent matrix material; contacting the thin film with water and nutrients within a photobioreactor; exposing the thin film to light under conditions effective to support bioconversion and secretion of the chemical product by the phototrophic microorganism; and isolating the chemical product secreted by the phototrophic microorganism.

[0016] Unlike traditional suspension cultures, these systems and methods can require less water and energy because the microorganisms are held in place, eliminating the need for intensive mixing and separation steps. These systems and methods can support the production of both volatile and non-volatile extracellular compounds. Volatile products can easily be isolated in the gas phase, while non-volatile products are harvested without significant energy input. By reducing nutrient requirements after initial cell growth, these systems and methods can also minimize ongoing nutrient demands, making them highly efficient. These systems and methods can also mitigate contamination risks, allowing continuous operation and improving product yield while reducing the need for constant resource input.

[0017] DESCRIPTION OF DRAWINGS

[0018] Figure 1. Schematic illustration of thin-film cultivation in a photomicrobial bioreactor described herein. The top left image illustrates standard methods used for planktonic cultivation in aqueous suspension. In these standard methods, cell concentrations are relatively low and water use relatively high. The top right image illustrates methods for the thin-film cultivation in a photomicrobial bioreactor. Thin-film cultivation results in higher cell concentrations and lower water use. As shown in the bottom image, thin-film cultivation of cells that secrete chemical products allows for efficient continuous cultivation with simple separation of product from cells.

[0019] Figure 2A. Photographs showing the growth of an example microorganism (a cyanobacterium) in a thin film of a hydrogel (calcium alginate) at day 1 and day 10 of cultivation.

[0020] Figure 2B. Photographs showing the growth of an example microorganism (a cyanobacterium) in a thin film of a hydrogel (F127-DMA) at day 1 and day 10 of cultivation.

[0021] Figure 3. Plot showing the amount of chlorophyll a (a proxy for biomass) extracted thin film hydrogel (F127-DMA and calcium alginate) containing an example microorganism (a cyanobacterium) over the course of a 3 -week cultivation in a bioreactor.

[0022] Figures 4A-4B. Comparison of thin-film cultivation in two hydrogels (calcium alginate and F127-DMA) to standard planktonic cultivation. Figure 4 A is a plot showing chlorophyll a concentration (a proxy for biomass), illustrating much higher cell concentration in thin-film systems. Figure 4B is a plot showing the productivity of the secreted product trans-cinnamic acid (tCA) on a per-cell basis, indicating that thin-film cells can produce as well or better than planktonic cells. Note that the combination of these plots shows that volumetric productivity (mg tCA / mL day) is 4-6 fold higher in thin-film cultivations. Asterisks denote significant differences at p < 0.05.

[0023] Figure 5A. Structure of trans-cinnamic acid.

[0024] Figure SB. Plot comparing the production of a secreted product trans-cinnamic acid (tCA) obtained by cultivating Synechocystis (engineered to secrete trans-cinnamic acid) in thin films of calcium alginate and F127-DMA. F127-DMA encapsulated cells had a higher productivity than the calcium alginate encapsulated cells.

[0025] Figures 6A-6B. Photographs showing a vertical cross-section (lOx magnification) of cyanobacteria in a thin hydrogel film after 4 days of cultivation (Figure 6A) and 14 days of cultivation (Figure 6B). Light exposure was on the right of the images. Gradients of cell concentration within encapsulated cultures are indicative of carbon and light limitations.

[0026] Figure 7. Photograph showing a 30-micron horizontal cross-section of an example thin film after 14 days of cultivation.

[0027] Figure 8. Chlorophyll content and per-chlorophyll production of tCA from planktonic, F127-DMA immobilized, and calcium alginate immobilized Synechocystis pDFlb-NT. Chlorophyll contents for immobilized cultures are those contained within the hydrogel. Error bars are representative of standard error. Three samples were taken for each group at each time point.

[0028] Figure 9. Maximum chlorophyll concentrations from an 18-day cultivation of planktonic, F127-DMA immobilized, and calcium alginate immobilized cultures of Synechocystis pDFlb-NT. Error bars are representative of standard error.

[0029] Figure 10A. Representative confocal fluorescent microscopy images (excitation at 561 nm) of Synechocystis WT immobilized within F127-DMA 1 day and 3 days after gel casting.

[0030] Figure 10B. Representative confocal fluorescent microscopy images (excitation at 561 nm) of Synechocystis / I tics A immobilized within F127-DMA 1 day and 3 days after gel casting.

[0031] Figure 10C. Colony concentration of Synechocystis WT and tacsA immobilized within F127-DMA 1 day and 3 days after gel casting. Error bars represent standard error. Three replicates were imaged in 6 different locations each ( 18 images total) for each group. Figure 10D. Average colony volume of Synechocystis WT and AacsA immobilized within F127-DMA 1 day and 3 days after gel casting. Error bars represent standard error. Three replicates were imaged in 6 different locations each (18 images total) for each group.

[0032] Figure 11. Chlorophyll content extracted from ~1 mm thick hydrogels containing WT and AacsA Synechocystis 1 day and 4 days after casting. Error bars represent standard error. Three samples were taken for each group at each time point.

[0033] Figure 12. Non-photochemical quenching (NPQ) measured from WT and pDFlb-NT Synechocystis grown planktonically and immobilized within F127-DMA. Approximately I pg chlorophyll a was used for each sample. Error bars represent standard error. 5 samples were assessed for each group.

[0034] Figures 13A-13B. 77K fluorescence assessments of immobilized and planktonic WT and pDFlb-NT Synechocystis. Figure 13A shows recorded fluorescent spectra from each sample group after excitation with an LED with a peak intensity at 595nm. Spectra are averages from 3 biological replicates, with 100 individual spectra taken for each replicate over a 10 second interval. Figure 13 shows comparisons of peak intensities corresponding to phycocyanin (646nm), PSII (685nm and 695nm), and PSI (726nm) fluorescent emissions. Error bars represent standard error.

[0035] Figure 14. Absorbance spectra from cultures of Fremyella and Synechocystis with an OD750 of 1 normalized such that 0750 is equal to zero and the maximum absorbance is equal to 1.

[0036] Figure 15. Absorbance spectra of F127-DMA immobilized cultures of Synechocystis and Synechocystis stacked on top of Fremyella. Individual layers are 200 um thick, such that each stacked culture is 400 um. Individual layers of Synechocystis and Fremyella cast with equivalent initial cell concentrations were grown for 7 days prior to stacking.

[0037] Figure 16. Normalized absorbance spectra of F127-DMA immobilized cultures of Synechocystis and Synechocystis stacked on top of Fremyella 5 days after stacking. Normalized such that OD750 is equal to zero and OD400 is equal to one.

[0038] Figure 17. Schematic illustration of an example photobioreactor system described herein.

[0039] DETAILED DESCRIPTION

[0040] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0041] “Biocompatible” and “biologically compatible”, as used herein, generally refer to materials that are, along with any metabolites or degradation products thereof, generally nontoxic to the phototrophic microbial cells present in the thin film, and do not cause any significant adverse effects to the phototrophic microbial cells present in the thin film.

[0042] As used herein, the term “insoluble” refers to a material or combination of materials that is not soluble in water at 37 °C. As used herein, the term “non-degradable” refers to a material or combination of materials which does not lose more than approximately 15% of its weight following immersion in water at 37 °C for 10 days.

[0043] “Bioconversion,” as used herein, generally refers to a process wherein a microorganism produces a chemical product of interest from a different chemical species, such a nutrient provided to the microorganism in culture. Bioconversion can occur via a biosynthetic pathway or metabolic pathway present within the microorganism. A "biosynthetic pathway" or "metabolic pathway" refers to a set of anabolic or catabolic biochemical reactions for converting (transmuting) one chemical species into another. For example, a hydrocarbon biosynthetic pathway refers to the set of biochemical reactions that convert inputs and / or metabolites to hydrocarbon product-like intermediates and then to hydrocarbons or hydrocarbon products. Anabolic pathways involve constructing a larger molecule from smaller molecules, a process requiring energy. Catabolic pathways involve breaking down of larger molecules, often releasing energy. Such biosynthetic pathways and metabolic pathways may be naturally occurring within the microorganism. Alternatively, the microorganisms can be engineered to exhibit desired biosynthetic pathways or metabolic pathways that can generate a chemical product of interest.

[0044] "Phototrophs" or "photoautotrophs" are organisms that carry out photosynthesis such as eukaryotic plants, algae, protists and prokaryotic cyanobacteria, green- sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria. Phototrophs include natural and engineered organisms that carry out photosynthesis.

[0045] As used herein, "organisms" encompasses autotrophs, phototrophs, heterotrophs, engineered light capturing organisms and at the cellular level, e.g., unicellular and multicellular.

[0046] As used herein, "light" generally refers to sunlight but can be solar or from artificial sources including incandescent lights, LEDs, fiber optics directing / transmitting light from a natural or artificial light source, or metal halide, neon, halogen and fluorescent lights. The light can include light of a wavelength that is photosynthetically active in the phototrophic microorganism, meaning light that can be utilized by the microorganism to grow and / or produce chemical products of interest.

[0047] Ranges of values defined herein include all values within the range as well as all subranges within the range. For example, if the range is defined as an integer from 0 to 10, the range encompasses all integers within the range and any and all subranges within the range, e.g., 1-10, 1-6, 2-8, 3-7, 3-9, etc.

[0048] Photobioreactor Systems and Methods of Generating Chemical Products

[0049] Described herein are photobioreactor systems for producing chemical products via bioconversion. Referring now to Figure 17, in some embodiments, these photobioreactor systems (100) can comprise a housing (102); one or more thin films (104) disposed within the housing, wherein each of the one or more thin films comprises a phototrophic microorganism dispersed within a transparent matrix material; a gas inlet (106) configured to direct a flow of gas into the housing; and a gas outlet (108) configured to remove gas from the housing. In some embodiments, the system can further comprise a liquid inlet (110) configured to direct an aqueous fluid (114) into the housing so as to contact the one or more thin films; and a liquid outlet (112) configured to remove the aqueous fluid from the housing.

[0050] In some embodiments, the housing can comprise a transparent region (115) positioned to allow incident light (116, for example, sunlight) to reach the one or more thin films disposed within the housing. In some examples, the transparent region can comprise a window formed from a material transparent to incident light present within a portion of an otherwise non-transparent housing. In other examples, the entire housing can be formed from a material that is not transparent to light. In these embodiments, an artificial light source can be present within the housing to supply a light source to the culture. In some embodiments, the one or more thin films can be disposed on a scaffold (105). The scaffold can provided added mechanical integrity to a thin film. Depending on the orientation of the thin film and scaffold with respect to the light source, the scaffold may or may not be transparent. In some examples, the scaffold can comprise a polymer membrane, a carbon resin sheet, metal sheet, or a glass sheet.

[0051] The photobioreactor system as a whole, as well as the components of the system including the housing, the thin film, and the scaffold (when present), can be of different shapes (e.g., elongated semi-circle shaped, flat, etc.) and sizes. Typically, however, they are substantially flat (planar). This can be advantageous, for example, for facilitating impingement of light on the thin films as well as positioning of the photobioreactor systems on flat / planar surfaces, such as flat ground, a roof, or a body of water, for example, a lake. In certain embodiments, the photobioreactor system as a whole is formed in the general shape of a panel.

[0052] The photobioreactor system as a whole, as well as the components of the system including the housing, the thin film, and the scaffold (when present), can be of any suitable size. The size can be influenced by material and manufacturing choices. A further consideration is transportability of photobioreactor system as a whole, as well as the components of the system.

[0053] In some embodiments, a plurality of thin films described herein are disposed within the housing. In some embodiments, each of the plurality of thin films comprises a different phototrophic microorganism.

[0054] Also described herein are methods for producing a chemical product via bioconversion, for example, using the photobioreactor systems described herein. For example, described herein are methods that comprise forming a thin film comprising a phototrophic microorganism dispersed within a transparent matrix material; contacting the thin film with water and nutrients within a photobioreactor; exposing the thin film to light under conditions effective to support bioconversion and secretion of the chemical product by the phototrophic microorganism; and isolating the chemical product secreted by the phototrophic microorganism. The conditions effective to support bioconversion and secretion of the chemical product can include, for example, a certain intensity and / or duration or light, a certain temperature, a certain period of time, the presence of certain nutrients, or a combination thereof. One of ordinary skill in the art can readily ascertain the conditions effective to support bioconversion and secretion of a particular chemical product from a particular microorganism.

[0055] The methods described herein can be used to produce a variety of chemical products. Examples of chemical products that can be produced via bioconversion using the methods described herein include hydrogen, ammonia, alcohols such as ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, ethyl esters, wax esters; hydrocarbons and alkanes such as ethane, propane, octane; polymers such as terephthalate, 1,3-propanediol, 1 ,4-butanediol, polyols, polyhydroxyalkanoates (PHAs) such as poly-beta-hydroxybutyrate (PHB), acrylate, adipic acid, 8-caprolactone, isoprene, caprolactam, rubber; commodity chemicals such as lactate, docosahexaenoic acid (DHA), 3-hydroxypropionate, y- valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate, 1 ,3 -butadiene, ethylene, propylene, succinate, citrate, citric acid, glutamate, malate, 3-hydroxypropionic acid (HP A), lactic acid, THF, gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid, levulinic acid, acrylic acid, malonic acid; specialty chemicals such as carotenoids, isoprenoids, itaconic acid; pharmaceuticals and pharmaceutical intermediates such as 7- aminodeacetoxycephalosporanic acid (7-ADCA) / cephalosporin, erythromycin, polyketides, statins, paclitaxel, docetaxel, terpenes, peptides, steroids, omega fatty acids and other such suitable products of interest. Such products are useful in the context of biofuels, industrial and specialty chemicals, as intermediates used to make additional products, such as nutritional supplements, nutraceuticals, polymers, paraffin replacements, personal care products and pharmaceuticals.

[0056] In some embodiments, the chemical product comprises a gas, such as ammonia or hydrogen gas.

[0057] In some embodiments, the chemical product comprises an alcohol (e.g., ethanol, methanol, isopropanol, butanol, glycerol, or a combination thereof), an amino acid (e.g., glutamic acid, lysine), an organic acid (e.g., lactic acid, acetic acid, butyric acid, succinic acid, propionic acid, or a combination thereof), an aldehyde (e.g., isobutyraldehyde), an alkane or alkene (e.g., isoprene, ethane, propane, butane), a carbohydrate, a biopolymer (e.g., a polysaccharide, a poly hydroxy alkanoate), a lipid, an enzyme, a peptide, a vitamin, a pharmaceutical agent (e.g., an antibiotic agent, an anticancer agent), or a combination thereof.

[0058] In some embodiments, contacting the thin film with water and nutrients comprises contacting the thin film with an aqueous fluid. In some cases, this can comprise flowing an aqueous fluid over the thin film. In other cases, the thin film can be contacted with an aqueous fluid by spraying droplets of water over the thin film, or by introducing a nebulized / aerosolized aqueous fluid into the photobioreactor such that the nebulized / aerosolized droplets contact the thin film. The aqueous fluid comprises one or more nutrients, such as a carbon source (e.g., bicarbonate or a carbohydrate), a nitrogen source (e.g., a nitrate salt, an ammonium salt, or urea), a phosphorus source (e.g., a phosphate salt), a potassium source (e.g., a potassium salt such as potassium chloride or potassium nitrate), a magnesium source (e.g., a magnesium salt such as magnesium sulfate), a calcium source (e.g., a calcium salt such as calcium chloride), a sulfur source (e.g., a sulfate salt), or a combination thereof, dissolved or dispersed in water.

[0059] In some embodiments, the chemical product is dissolved or dispersed in the aqueous fluid following secretion of the chemical product by the phototrophic microorganism. In these embodiments, isolating the chemical product secreted by the phototrophic microorganism can comprise removing the aqueous fluid from the photobioreactor and separating the chemical product from the aqueous fluid. This separation can be performed via a variety of suitable methods known in the art, such as adsorption (e.g., using a stationary phase such as an ion exchange resin), membrane separation, and / or chromatography.

[0060] In some embodiments, contacting the thin film with water and nutrients comprises flowing a gas into the photobioreactor. The gas can comprise, for example, carbon dioxide, nitrogen, oxygen, air, water vapor, gaseous and / or aerosolized nutrients, or a combination thereof.

[0061] In some embodiments, the chemical product is a gas or volatile product secreted by the phototrophic microorganism. In these embodiments, isolating the chemical product secreted by the phototrophic microorganism can comprise removing the gas from the photobioreactor and separating the chemical product from the gas. This separation can be performed via a variety of suitable methods known in the art, such as membrane separation.

[0062] Exposing the thin film to light can comprise exposing the thin film to sunlight, irradiating the thin film with an artificial light source, or a combination thereof.

[0063] Thin Films

[0064] The methods and systems described herein include one or more thin films that comprise a phototrophic microorganism dispersed within a transparent matrix material.

[0065] In the thin films described herein, the transparent matrix material can comprise a variety of suitable materials that can encapsulate and / or entrap a population of microorganisms during culture in the photobioreactor. In preferred embodiments, the transparent matrix material can be insoluble and non-degradable, such that it can immobilize the microorganisms for a length of time effective to generate the chemical products in the presence of an aqueous fluid. In preferred embodiments, the transparent matrix material can also be biocompatible.

[0066] Examples of suitable transparent matrix materials can include for example, hydrophilic and biocompatible grades of the following materials and their derivatives: poly( vinyl alcohol); ethylene vinyl alcohol co-polymers (typically non-biodegradable materials which degree of hydrophilicity depends on distribution of ethylene (hydrophobic) and vinyl alcohol (hydrophilic) groups); co-polymers of polyvinyl alcohol and ethylene vinyl alcohol; polyacrylate compositions; polyurethane compositions; poly(ethylene glycol) (PEG), otherwise known as poly(oxyethylene) (POE) and poly(ethylene oxide) (PEO), and its derivatives including but not limited to polyethylene glycol methacrylate (PEGMA), polyethylene glycol dimethacrylate (PEGDMA) and polyethylene glycol diacrylate (PEGDA); cellulose and its derivatives including but not limited to methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose; hydrophilic alginic acid, also known as algin or alginate and its derivatives; nitrogencontaining materials such as polyacrylamide (without acrylamide toxic residuals), polyvinylpyrrolidone, polyvinylamine, and polyethyleneimine; electrically charged materials such as poly(lactic acid) also known as polylactide (PLA) in various forms (e.g. poly-L- lactide (PLLA) and its derivatives, poly-D-lactide (PDLA) and its derivatives, poly(L-lactide- co-D,L-lactide) (PLDLLA) and its derivatives), poly(glycolic acid) (PGA) also known as polyglycolide, co-polymers of lactic acid and glycolic acid poly(lactic-co-glycolic acid) (PLGA), co-polymers of PLA and / or PGA with PEG; polymethacrylic acid; poly(hydroxyethyl methacrylate) poly-HEMA, among other absorbent, hydrophilic and biocompatible materials known in the art. Other suitable materials may be derived from a mixture of water-soluble polymers. For example, a mixture of an anionic water-soluble polymer and a cationic water-soluble polymer can produce a gel-like insoluble material as a result of neutralization. A mixture of an uncharged water-soluble polymer and an anionic water-soluble polymer may also be neutralized with a mixture of an uncharged water-soluble polymer and a cationic water-soluble polymer.

[0067] In some embodiments, the transparent matrix material comprises a polymer matrix, such as a hydrogel matrix. In certain embodiments, the transparent matrix material comprises a polysaccharide (e.g., alginate, chitosan, agarose, carrageenan, cellulose, hyaluronic acid, or a combination thereof), a protein (e.g., collagen, gelatin, or a combination thereof), polyvinyl alcohol, polyvinyl acetate, a polyalkylene oxide (e.g., a crosslinked polyethylene glycol), a polyacrylamide, a polyester (e.g., polylactic acid, polyglycolic acid, polycaprolactone), and combinations thereof.

[0068] In some embodiments, the thin film has a thickness of less than 10 mm (e.g., less than 9 mm, less than 8 mm, less than 7 mm, less than 6 mm, less than 5 mm, less than 4 mm, less than 3 mm, less than 2 mm, or less than 1 mm), such as a thickness of from 10 microns to 10 mm (e.g., from 10 microns to 9 mm, from 10 microns to 8 mm, from 10 microns to 7 mm, from 10 microns to 6 mm, from 10 microns to 5 mm, from 10 microns to 4 mm, from 10 microns to 3 mm, from 10 microns to 2 mm, from 10 microns to 1 mm, from 10 microns to 500 microns, from 10 microns to 250 microns, from 10 microns to 100 microns, from 25 microns to 10 mm, from 100 microns to 9 mm, from 100 microns to 8 mm, from 100 microns to 7 mm, from 100 microns to 6 mm, from 100 microns to 5 mm, from 100 microns to 4 mm, from 100 microns to 3 mm, from 100 microns to 2 mm, from 100 microns to 1 mm, from 25 microns to 500 microns, from 25 microns to 250 microns, from 25 microns to 100 microns, from 50 microns to 10 mm, from 50 microns to 9 mm, from 50 microns to 8 mm, from 50 microns to 7 mm, from 50 microns to 6 mm, from 50 microns to 5 mm, from 50 microns to 4 mm, from 50 microns to 3 mm, from 50 microns to 2 mm, from 50 microns to 1 mm, from 50 microns to 500 microns, from 50 microns to 250 microns, from 50 microns to 100 microns, from 100 microns to 10 mm, from 100 microns to 9 mm, from 100 microns to 8 mm, from 100 microns to 7 mm, from 100 microns to 6 mm, from 100 microns to 5 mm, from 100 microns to 4 mm, from 100 microns to 3 mm, from 100 microns to 2 mm, from 100 microns to 1 mm, from 100 microns to 500 microns, from 100 microns to 250 microns, from 250 microns to 10 mm, from 250 microns to 9 mm, from 250 microns to 8 mm, from 250 microns to 7 mm, from 250 microns to 6 mm, from 250 microns to 5 mm, from 250 microns to 4 mm, from 250 microns to 3 mm, from 250 microns to 2 mm, from 250 microns to 1 mm, from 250 microns to 500 microns, from 500 microns to 10 mm, from 500 microns to 9 mm, from 500 microns to 8 mm, from 500 microns to 7 mm, from 500 microns to 6 mm, from 500 microns to 5 mm, from 500 microns to 4 mm, from 500 microns to 3 mm, from 500 microns to 2 mm, or from 500 microns to 1 mm).

[0069] In some embodiments, the thin film has an area of at least 100 cm2, such as an area of from 100 cm2to 5 m2, such as from 100 cm2to 3 m2. The phototrophic microorganisms present in the thin films described herein can include, but are not limited to, naturally photosynthetic microorganisms, such as plants, algae, and / or cyanobacteria, or engineered photosynthetic microorganisms, such as artificially photosynthetic bacteria. Example microorganisms that are either naturally photosynthetic or can be engineered to be photosynthetic include, but are not limited to, bacteria, fungi, archaea, protists, microscopic plants, such as a green algae, and animals such as plankton, planarian, and amoeba. Examples of phototrophic microorganisms can include, but are not limited to, Spirulina maximum, Spirulina platensis, Dunaliella salina, Botrycoccus braunii, Chlorella vulgaris, Chlorella pyrenoiclosa, Serenastrum capricomutum, Scenedesmus auadricauda, Porphyridium cruentum, Scenedesmus acutus, Dunaliella sp., Scenedesmus obliqttus, Anabaenopsis sp., Aulosira sp., Cylindrospermum sp., Synechococcussp., Synechocystis sp., Tolypothrix sp., or a combination thereof.

[0070] In some embodiments, the phototrophic microorganism is engineered to produce and / or increase production of the chemical product, resist the toxicity of the matrix material (e.g., resist the toxicity of a crosslinker used to form the matrix material), increase secretion of the chemical product, or any combination thereof.

[0071] In some embodiments, the thin film comprises two or more different phototrophic microorganisms dispersed within the transparent matrix material. The two or more different phototrophic microorganisms can be, for example, phototrophic microorganisms that produce different chemical products, phototrophic microorganisms that consume different nutrients, phototrophic microorganisms that possess different photosynthetic characteristics, or any combination thereof. The two or more different phototrophic microorganisms can be mixed together or separately patterned within different regions of the thin film. In certain embodiments, the two or more different phototrophic microorganisms can be disposed in different layers (e.g., sandwiched one above the other) within the thin film.

[0072] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0073] EXAMPLES

[0074] Example 1. Bioreactor System for the Production of Secreted Chemicals.

[0075] Overview

[0076] Phototrophic microorganisms (primarily cyanobacteria and microalgae) have the potential to produce a wide range of fuels and other chemicals from sunlight and carbon dioxide. However, current methods for this production require substantial water, nutrient, and energy inputs. These methods generally involve growing cyanobacteria and microalgae in suspension cultures, typically reaching cell concentrations of only 1% by volume. Low cell concentrations correlate to high water requirements per cell biomass, and, in turn, significant amounts of energy required to mix cultures. High energy use is additionally required in separations, with a need to isolate small cells (on the order of single microns in diameter) from surrounding liquid. Lastly, this phototrophic production requires nitrogen, phosphorous, and additional nutrients, which leads to resource competition particularly with food production.

[0077] In this example, we describe improved systems and methods for phototrophic chemical production. These systems and methods can include (1) a thin film of natural or engineered photoautotrophic microorganisms; (2) maintaining the photomicrobial film in a moist state; (3) supplying nutrients to the photomicrobial film; and (4) removing extracellular products for downstream processing.

[0078] In this system, the phototrophic microorganisms are immobilized in a thin film, either by natural or engineered biofilm processes or by entrapment in a hydrogel or polymeric matrix. Examples of suitable matrix materials that were initially investigated include but are not limited to F127-dimethacrylate, polyethylene glycol diacrylate, and calcium alginate; however, other materials can be used. The cell concentration within the photomicrobial film is high, resulting in high rates of areal metabolism. In a sense, the photomicrobial film is a solar cell, albeit for chemical synthesis rather than generation of electrical current.

[0079] The photoautotrophic microorganisms can be any suitable microorganism that is selected or engineered to produce and secrete a chemical product. These molecules could be highly volatile such as isobutyraldehyde, isoprene, and ethane. High volatility would allow facile partitioning of the product into the gaseous phase of the reactor (which will supply carbon dioxide and potentially nitrogen and phosphorous for cell growth and maintenance). This partitioning into the gas phase prevents inhibitory effects of product accumulation and prevents consumption of the product by microorganisms that may be present in the aqueous phase. Due to the simple chemical composition of the gas phase, isolation of these products could be performed with a simple membrane device.

[0080] The photoautotrophic microorganisms could also produce and secrete small molecules with low volatility. Harvesting of the secreted product from the thin-film bioreactor is simpler in comparison to suspension cultures. In suspension cultures, the necessary isolation of cells from the bulk liquid phase can be highly energy or time intensive. Due to the inherent separation of cells from the bulk liquid in the thin-film bioreactor, this isolation is not required. The immobilization of cells in the thin-film bioreactor also allows for continuous flow of both liquid and gas through the bioreactor. Similar to volatile products, this continuous flow will prevent significant buildup of a product, avoiding inhibitory effects.

[0081] Most photoautotrophic microorganisms must be provided with moisture to remain metabolically active. Moisture could be applied in several ways, including (but not limited to) the following: (a) by diffusing water from the side of the photomicrobial film not exposed to light, (b) by maintenance of very high (near 100%) relative humidity levels in the gas phase over the photomicrobial film, (c) by the flow of a thin stream of water over the photomicrobial film, and / or (d) by periodic spraying of the photomicrobial film with water.

[0082] Photoautotrophic microorganisms require nutrients, especially nitrogen and phosphorous, for growth. In suspension cultures, large quantities of nutrients are continuously required for substantial growth of organisms as high cell densities are required for economically viable production of intra- or extra-cellular products. In a photomicrobial film, substantial nutrient application is typically only required in the initial growth phase to achieve a high concentration of cells within the film. After a high cell density has been achieved, nutrient application can be drastically reduced to minimize further growth while not inhibiting product formation. This separation of a growth phase and production phase is possible as the thin-film bioreactor can be operated continuously without washout of cells.

[0083] An additional advantage of this photomicrobial film bioreactor system is its resistance to contaminating organisms. It is increasingly recognized that suspension cultivation of algae and cyanobacteria are challenged by the presence of bacteria, fungi, and protists. These contaminating organisms are often benign, sometimes beneficial, but also sometimes harmful to phototrophic organisms. A photomicrobial film is less susceptible to the effects of contamination as high concentrations of the photoautotrophic microorganisms are present from the start of cultivation, decreasing the likelihood of being outcompeted for resources. Additionally, immobilized cells may be surrounded by a protective synthetic or natural polymeric matrix, preventing invasion by contaminating organisms and resulting in the eventual washout of contaminants from the reactor while the photoautotrophic microorganisms persist. Finally, while most products likely to be produced with this photobioreactor system are hydrocarbons, the technology could also be used to produce ammonia, replacing the energy-intensive Haber-Bosch process. Certain cyanobacteria fix atmospheric nitrogen and can be manipulated (genetically or by environmental means) to secrete the reduced nitrogen in the form of ammonia.

[0084] These bioreactor systems allow for low water, nutrient, and energy use in the photomicrobial production of chemical intermediates for the synthesis of fuels or other chemicals. Photomicrobial production of these chemicals can result in a lower greenhouse gas footprint than traditional, fossil fuel-based processes used for their production. However, current methods for cultivation of photomicrobial organisms are not economically competitive, in part due to high resource requirements. This system will substantially lower resource requirements for photomicrobial cultivation, in turn lowering the cost of chemical production.

[0085] Initial Proof of Principle Studies

[0086] Initial studies were performed using cyanobacteria as a representative microorganism. Cyanobacteria are single-cell, photosynthetic organisms. Cyanobacteria have been engineered to produce a variety of desirable chemical products via bioconversion, including numerous chemicals normally sourced from fossil fuels. This bioproduction has the potential to be more sustainable than fossil fuel sourcing. However, commonly used planktonic cultivation results in high water, nutrient, and energy use which limits sustainability and economic viability.

[0087] Figure 1 schematically illustrates the thin- film cultivation of cyanobacteria in a photomicrobial photobioreactor. As shown in Figure 1 , thin- film cultivation of cells that secrete chemical products allows for efficient continuous cultivation with simple separation of product from cells.

[0088] In initial studies, thin films were formed by dispersing Synechocystis engineered to secrete trans-cinnamic acid (tCA) in two different polymeric matrix materials: a calcium alginate hydrogel and a F127-DMA hydrogel. Figures 2A-2B show photographs of these example thin films at day 1 and day 10 of cultivation. Figure 3 is a plot showing the amount of chlorophyll a (a proxy for biomass) extracted thin film hydrogel (F127-DMA and calcium alginate) over the course of a 3 -week cultivation in a bioreactor.

[0089] Thin-film cultivation in these two hydrogels (calcium alginate and F127-DMA) was also compared to standard planktonic cultivation. Figure 4A is a plot showing chlorophyll a concentration (a proxy for biomass) present in the thin film and suspension cultures following 10 days of cultivation. As shown in Figure 4 A, both thin film systems exhibit higher volumetric cell concentrations than similar planktonic (suspension) cultures. Figure 4B is a plot showing the productivity of the secreted product trans -cinnamic acid (tCA) on a per-cell basis, indicating that thin-film cells can produce as well or better than planktonic cells. Note that the combination of these plots shows that volumetric productivity (mg tCA / mL day) is 4-6 fold higher in thin-film cultivations than similar suspension cultures.

[0090] Figure 5B compares the production of a secreted product (trans -cinnamic acid (tCA)) obtained by cultivating Synechocystis (engineered to secrete trans-cinnamic acid) in thin films of calcium alginate and F127-DMA. F127-DMA encapsulated cells had a higher productivity than the calcium alginate encapsulated cells.

[0091] Vertical and horizontal cross-sections of thin films (Figures 6A-6B and 7) reveal gradients of cell concentration formed following cultivation, indicative of carbon and light limitations on the growing microbial cells. If desired, assessments of spatially dependent characteristics can be made through RNA extraction from thin-slices of encapsulated cultures.

[0092] Further Studies

[0093] Growth and bioproduction: Planktonic cultures of Synechocystsis pDFlb-NT along with those immobilized within F127-DMA and calcium alginate were grown in BG1 1 under 24-hour light for 18 days. Chlorophyll a and trans-cinnamic acid concentrations were monitored every three days of the cultivation (Figure 8). Chlorophyll a concetration serves as an estimate of biomass.

[0094] Planktonic cultures accumulated the most chlorophyll, with a maximum average content of 80.0 pg. Chlorophyll content seemed to stabilize over the last 6 days of cultivation. Growth was observed from cultures immobilized within F127-DMA for the first 9 days of cultivation, after which chlorophyll content plateaud, just over 40 pg. At this point, the hydrogel may have been saturated with cells, preventing further growth within it. Calcium alginate immobilized cultures showed slower growth than either planktonic or F127-DMA immobilized cells, achieving a maximum chlorophyll content of 37 pg after 12 days of cultivation. The last 6 days of cultivation chlorophyll content rapidly dropped, as gels were observed to degrade, releasing chlorophyll into the surrounding media.

[0095] Although total chlorophyll achieved in the planktonic system exceeded that of the F127-DMA or calcium algiante immobilized cultures, the volumetric concetration of chlorophyll was highest when cells are immobilized (Figure 9). Two mL of medium was present within each culture; however, in planktonic cultures, cells are distributed throughout the entirety of the medium. In immobilzed systems, cells are isolated within the confines of the hydrogel, occupying 375 uL. Volumetric cell concentration, therefore, was higher in the immobilized systems for much of the cultivation than the planktonic cultures.

[0096] For all but the first and last samples, per chlorophyll productivity of tCA from the F127-DMA immobilized cultures exceeded that of the planktonic cells and calcium alginate immobilized cells. Both the lower chlorophyll accumulation and productivity of the calcium alginate cultures may be due to lower carbon availability. As calcium alginate cultures received calcium chloride in the medium, precipitates, presumed to be of calcium carbonate, formed. These precipitates remove carbon from the aqueous environment, potentially limiting the growth and trans -cinnamic acid produced in these cultures.

[0097] Effects of photopolymerization: The initial chlorophyll concentration in F127-DMA cultures was significantly lower than that within planktonic and calcium alginate cultures, despite all three receiving the same cell concentration inoculum. This may be due to the effects of photopolymerization. We hypothesized that the presence of acrylate as a functional group of F127-DMA was likely contributing to cell death during casting, due to the high toxicity of acrylate for Synechocystis. Following casting, acrylate groups are cross-linked to one another and therefore less likely to be inhibitory.

[0098] To subvert the effects of toxicity, a Synechocystis mutant was generated in which the gene coding for an acetyl coenzyme A ligase (acsA, sll0542) was replaced with a kanamycin resistance cassette. Begemann et al. previously showed that knocking out a homolog of this gene in Synechococcus sp. PCC 7002 increased acrylate tolerance of the organism while also improving tolerance to other organic acids1. Knocking out this gene in Synechocystis was similarly shown to greatly increase tolerance to acrylate. Following generation of the knockout strain, both WT and zlascA Synechocystis were immobilized within 200pm thick hydrogels of F127-DMA. Twenty-four hours and 72 hours after casting, the hydrogels were imaged with confocal microscopy, using autofluorescence of chlorophyll to detect cells. Average colony volume, colony concentration, and colony locations were calculated (Figures 10A-10D).

[0099] The WT cultures had a significant drop in colony concentration between day 1 and day 3 (65%), suggesting nearly two thirds of the cast cells died in this time. Average colony volume, however, increased by 120%, suggesting those cells which survive the initial effects of photopolymerization remain viable. The AascA cultures had a lower drop in colony concentration between days 1 and 3 (24%) and a substantially greater increase in colony volume (1340%) when compared to WT. While some of the increase in colony volume may be attributed to merging colonies, the degree of the increase (in combination with the lower decrease in colony concentration) clearly shows an improved tolerance to the conditions of photopolymerization by the AascA compared to WT.

[0100] When grown at higher cell concentrations and in thicker hydrogels, the benefits of the AascA strain are less but still apparent. Chlorophyll concentrations one day after casting are surprisingly lower than WT (Figure 11), suggesting that in these systems differences in initial chlorophyll concentrations may be more attributed to cells washing out during and immediately after the photopolymerization process rather than cell death due to hydrogel toxicity. During the first 4 days of cultivation, however, the accumulation of chlorophyll from AascA cultures exceeds that of WT, suggesting the improved tolerance to acrylate may drive increased growth rates in early cultivation (5.65 pg chl / day vs. 3.18 pg chl / day, p < 0.05).

[0101] Photophysiological effects of immobilization: The higher per chlorophyll productivity observed in the F127-DMA immobilized cultures could be driven by mechanical forces on cells. Frictional force could inhibit growth, requiring dissipation of energy through non-biomass accumulating pathways. Native pathways for this dissipation in Synechocystis are non-photochemical quenching (NPQ) and fluorescent emissions, which can be estimated through PAM fluorometry and 77K fluorometry respectively. The introduction of a secreted product pathway, such as trans-cinnamic acid secretion exhibited by the pDFlb-NT transformed Synechocystis, presents a separate option for non-intracellular biomass accumulating energy dissipation. To probe if the higher observed productivity is a response to the physical constraints of immobilization, NPQ and fluorescent dissipation were assessed in both WT and pDFlb-NT transformed cultures immobilized within F127-DMA and grown planktonically (Figure 12).

[0102] NPQ is significantly lower in the immobilized pDFlb-NT transformed cultures than all other experimental groups (p < 0.05). This suggests that the secreted product pathway may present an alternative option for energy dissipation from NPQ which Synechocystis makes use of when mechanically stressed. No statistically significant difference in NPQ was observed between immobilized WT, planktonic WT, or planktonic pDFlb-NT. Therefore, the presence of the secreted product pathway alone does not drive re-direction of energy dissipation through NPQ, but rather mechanical stress may be required for this response. Further, with no difference between WT immobilized and WT planktonic NPQ, mechanical stress may not result in energy dissipation through NPQ, but rather via other pathways. This result is consistent with findings from Moore et al. who showed no change in NPQ between Synechococcus elongatus cells under different degrees of mechanical stress but rather saw increased dissociation of phycobilisomes (PBSs) from PSII2.

[0103] 77K fluorometry was used to probe this PBS dissociation in planktonic and F127- DMA immobilized cultures of WT and pDFlb-NT Synechocystis. Cells submerged in liquid nitrogen were excited with an LED with a peak wavelength of 590 nm (targeting PBSs) and resulting fluorescent emissions were recorded (Figures 13A-13B). Fluorescent emission peaks at wavelengths of approximately 650 nm represent PBS rods completely dissociated from allophycocyanin cores, 665 nm represent whole PBS (rod and core) dissociated from PSII, and 695 nm represent emissions at PSII34. Relative peak intensities can therefore be representative of the degree of dissociation of phycobilisome rods and cores from PSII.

[0104] PC:PSII is lower in planktonic cells relative to immobilized cells for both WT and pDFlb-NT, albeit this trend is only statistically significant for WT (p = 0.028 and p = 0.072 respectively). This suggests that a greater degree of PBS dissociation may be occurring in immobilized cells relative to planktonic, consistent with results of Moore et al. The ratio of these peaks between immobilized pDFlb-NT and immobilized WT was also significantly different (p = 0.032), with transformed cells showing lower PBS dissociation.

[0105] Taking advantage of spatial light heterogeneity:

[0106] Changing spatial and temporal light spectra within an immobilized culture of Synechocystis is a result of chromophores contained within light-harvesting complexes of this organism tending to absorb red, orange, and violet light to a greater extent than green and blue light5,6(Figure 14). Therefore, as cell concentration increases, the proportion of green and blue light passing through a system relative to red, orange, and violet increases. In an immobilized culture, this phenomenon will alter the spectra of light reaching cells at the top of a matrix (a normal solar spectra) as compared to those further down (a spectra dominated by blue, green, and far-red light).

[0107] Cells can be immobilized in individual, thin layers of hydrogel (200 um in height). Therefore, a layered system could be constructed in which cells within each 200 um segment are engineered or selected such that they are tolerant to the light conditions they will experience. An approach for improving light-use efficiency from immobilized bioproduction systems is to engineer a cyanobacteria which naturally absorbs blue and green light to produce tCA. Heterologous expression of proteins through plasmid transformations has been shown with several of these organisms14-16. Fremyella diplosiphon (hereafter referred to as Fremyella) can be transformed, has the capability of producing substantial amounts of phycoerythrin (over 80% of its total phycobiliprotein content when grown under greenlight17), and grows optimally under similar conditions to Synechocystis18. As such, it is an ideal candidate for co-culturing in a stacked hydrogel with Synechocystis to improve light-use efficiency.

[0108] We cast 200-um thick F127-DMA immobilized cultures of Fremyella and Synechocystis with an initial OD750 of approximately 3 and grew them under equivalent intensities of green and white light respectively for 7 days. After 7 days, individual layers were stacked to form 400 um thick immobilized cultures of either just Synechocystis or Synechocystis on top of Fremyella. These stacked cultures were grown for 5 days under white light. Absorbance measurements across the visible spectrum were taken on day one and day five (Figure 15). In both instances, total absorbance was higher in the multiculture as compared to the monoculture.

[0109] Summary of Further Studies: In these further studies, we found an approximately 3- fold increase in cell concentration when comparing planktonic Synechocystis cultures to those immobilized within F127-DMA after an 18-day cultivation.

[0110] Further, the average specific productivity of trans-cinnamic acid from F127-DMA immobilized cultures was found to be 35.9% higher than planktonic cultures over an 18-day cultivation. This increased productivity may be due to shifts in energy dissipation strategies by cells when they’re immobilized, as evidenced by measurements of PBS dissociation and NPQ. Lower NPQ and PBS dissociation in cultures producing trans-cinnamic acid compared to wild-type suggests that the presence of a metabolic sink (trans-cinnamic acid) shifts cellular energy allocations when stressed by immobilization within F127-DMA.

[0111] Studies performed using a genetically engineered strain of Synechocystis with a higher tolerance to acrylate grows more than wild-type within the first several days after immobilization within F127-DMA (78% higher growth rate in 1-mm thick gels with a relatively high initial cell concentration).

[0112] Finally, stacked cultures of F127-DMA immobilized Synechocsylis on top of Fremyella diplosiphon have higher and more uniform light absorption across the visible spectrum than monocultures of immobilized Synechocystis. This shows the potential for improved productivity from multicultures as compared to monocultures. References

[0113] 1. Begemann, M. B. et al. An Organic Acid Based Counter Selection System for Cyanobacteria. PLoS One 8, (2013).

[0114] 2. Moore, K. A. et al. Mechanical regulation of photosynthesis in cyanobacteria. Nat Microbiol 5, 757-767 (2020).

[0115] 3. Fuente, D., Lazar, D., Oliver-Villanueva, J. V. & Urchueguia, J. F. Reconstruction of the absorption spectrum of Synechocystis sp. PCC 6803 optical mutants from the in vivo signature of individual pigments. Photosynth Res 147, 75-90 (2021).

[0116] 4. Remelli, W. & Santabarbara, S. Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: Influence on the estimation of Photosystem II maximal quantum efficiency. Biochim Biophys Acta Bioenerg 1859, 1207-1222 (2018).

[0117] 5. Dartnell, L. R. et al. Degradation of cyanobacterial biosignatures by ionizing radiation. Astrobiology 11, 997-1016 (2011).

[0118] 6. Hotos, G. N. & Bekiari, V. Absorption Spectra as Predictors of Algal Biomass and Pigment Content of the Cultured Microalgae Amphidinium carterae, Isochrysis galbana, Nephroselmis sp., and Anabaena sp. International Journal of Plant Biology 14, 879-895 (2023).

[0119] 7. Cano, M. et al. Glycogen Synthesis and Metabolite Overflow Contribute to Energy Balancing in Cyanobacteria. Cell Rep 23, 667-672 (2018).

[0120] 8. Jahn, M. et al. Growth of Cyanobacteria Is Constrained by the Abundance of Light and Carbon Assimilation Proteins. Cell Rep 25, 478-486.e8 (2018).

[0121] 9. Sanfilippo, J. E., Garczarek, L., Partensky, F. & Kehoe, D. M. Chromatic Acclimation in Cyanobacteria: A Diverse and Widespread Process for Optimizing Photosynthesis. 44, 2 (2024).

[0122] 10. Calzadilla, P. I. & Kirilovsky, D. Revisiting cyanobacterial state transitions. Photochemical and Photobiological Sciences vol. 19 585-603 Preprint at https: / / doi.org / ! 0.1039 / c9pp00451 c (2020).

[0123] 11. Grebert, T. et al. Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria. doi: 10.1073 / pnas.2019715118 / - / DC Supplemental. 12. Hirose, Y., Shimada, T., Narikawa, R., Katayama, M. & Ikeuchi, M.

[0124] Cyanobacteriochrome CcaS Is the Green Light Receptor That Induces the Expression of Phycobilisome Linker Protein. www.pnas.orgcgidoil0.1073pnas.0801826105 (2008).

[0125] 13. Hirose, Y. et al. Diverse Chromatic Acclimation Processes Regulating Phycoerythrocyanin and Rod-Shaped Phycobilisome in Cyanobacteria. Mol Plant 12, 715— 725 (2019).

[0126] 14. Brahamsha, B. A Genetic Manipulation System for Oceanic Cyanobacteria of the Genus Synechococcus. APPLIED AND ENVIRONMENTAL MICROBIOLOGY vol. 62 (1996).

[0127] 15. Gichuki, S. M. et al. Augmentation of the Photoreactivation Gene in Fremyella diplosiphon Confers UV-B Tolerance. ACS Omega 7, 35092-35101 (2022).

[0128] 16. Tabatabai, B. et al. Overexpression of hlyB and mdh genes confers halotolerance in Fremyella diplosiphon, a freshwater cyanobacterium. Enzyme Microb Technol 103, 12-17 (2017).

[0129] 17. Stowe, W. C., Brodie-Kommit, J. & Stowe-Evans, E. Characterization of complementary chromatic adaptation in gloeotrichia UTEX 583 and identification of a transposon-like insertion in the cpeBA operon. Plant Cell Physiol 52, 553-562 (2011).

[0130] 18. Tan, H. T. et al. A Review on a Hidden Gem: Phycoerythrin from Blue-Green Algae. Marine Drugs vol. 21 Preprint at https: / / doi.org / 10.3390 / md21010028 (2023).

[0131] 19. Wyatt, L. D., Gichuki, S., Yalcin, Y. S. & Sitther, V. Impact of Ascorbic Acid on Zero-Valent Iron Nanoparticle and UV-B Mediated Stress in the Cyanobacterium, Fremyella diplosiphon. Microorganisms 11, (2023).

[0132] 20. Agostoni, M. et al. Competition-based phenotyping reveals a fitness cost for maintaining phycobilisomes under fluctuating light in the cyanobacterium Fremyella diplosiphon. Algal Res 15, 110-119 (2016).

[0133] The compositions, articles, and methods of the appended claims are not limited in scope by the specific compositions, articles, and methods described herein, which are intended as illustrations of a few aspects of the claims. Any compositions, articles, and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions, articles, and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions, articles, and method steps disclosed herein are specifically described, other combinations of the compositions, systems, kits, and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

[0134] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

[0135] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

WHAT IS CLAIMED IS:1 . A method for producing a chemical product via hi ocon version, the method comprising: forming a thin film comprising a phototrophic microorganism dispersed within a transparent matrix material; contacting the thin film with water and nutrients within a photobioreactor; exposing the thin film to light under conditions effective to support bioconversion and secretion of the chemical product by the phototrophic microorganism; and isolating the chemical product secreted by the phototrophic microorganism.

2. The method of claim 1, wherein the chemical product comprises a gas, such as ammonia or hydrogen gas.

3. The method of claim 1, wherein the chemical product comprises an alcohol (e.g., ethanol, methanol, isopropanol, butanol, glycerol, or a combination thereof), an amino acid (e.g., glutamic acid, lysine), an organic acid (e.g., lactic acid, acetic acid, butyric acid, succinic acid, propionic acid, or a combination thereof), an aldehyde (e.g., isobutyraldehyde), an alkane or alkene (e.g., isoprene, ethane, propane, butane), a carbohydrate, a biopolymer (e.g., a polysaccharide, a polyhydroxyalkanoate), a lipid, an enzyme, a peptide, a vitamin, a pharmaceutical agent (e.g., an antibiotic agent, an anticancer agent), or a combination thereof.

4. The method of any one of claims 1-3, wherein contacting the thin film with water and nutrients comprises contacting the thin film with an aqueous fluid, such as flowing an aqueous fluid over the thin film.

5. The method of claim 4, wherein the aqueous fluid comprises one or more nutrients, such as a carbohydrate, a nitrogen source (e.g., a nitrate salt, an ammonium salt, or urea), a phosphorus source (e.g., a phosphate salt), a potassium source (e.g., a potassium salt such as potassium chloride or potassium nitrate), a magnesium source (e.g., a magnesium salt such as magnesium sulfate), a calcium source (e.g., a calcium salt such as calcium chloride), a sulfur source (e.g., a sulfate salt), or a combination thereof, dissolved or dispersed in water.

6. The method of any one of claims 4-5, wherein the chemical product is dissolved or dispersed in the aqueous fluid following secretion of the chemical product by the phototrophic microorganism; and wherein isolating the chemical product secreted by the phototrophic microorganism comprises removing the aqueous fluid from the photobioreactor and separating the chemical product from the aqueous fluid.

7. The method of any one of claims 1-6, wherein contacting the thin film with water and nutrients comprises flowing a gas into the photobioreactor.

8. The method of claim 7, wherein the gas comprises carbon dioxide, nitrogen, oxygen, air, water vapor, gaseous and / or aerosolized nutrients, or a combination thereof.

9. The method of any one of claims 7-8, wherein the chemical product is a gas or volatile product secreted by the phototrophic microorganism; and wherein isolating the chemical product secreted by the phototrophic microorganism comprises removing the gas from the photobioreactor and separating the chemical product from the gas.

10. The method of any one of claims 1-3, wherein the transparent matrix material comprises a polymer matrix, such as a hydrogel matrix.1 1. The method of any one of claims 1-10, wherein the transparent matrix material comprises a polysaccharide (e.g., alginate, chitosan, agarose, carrageenan, cellulose, hyaluronic acid, or a combination thereof), a protein (e.g., collagen, gelatin, or a combination thereof), polyvinyl alcohol, polyvinyl acetate, a polyalkylene oxide (e.g., a crosslinked polyethylene glycol), a polyacrylamide, a polyester (e.g., poly lactic acid, polyglycolic acid, polycaprolactone), and combinations thereof.

12. The method of any one of claims 1-11, wherein the transparent matrix material is non- biodegradable.

13. The method of any one of claims 1-12, wherein the thin film has a thickness of less than 10 mm (e.g., less than 9 mm, less than 8 mm, less than 7 mm, less than 6 mm, less than 5 mm, less than 4 mm, less than 3 mm, less than 2 mm, or less than 1 mm), such as a thickness of from 10 microns to 10 mm (e.g., from 10 microns to 9 mm, from 10 microns to 8 mm, from 10 microns to 7 mm, from 10 microns to 6 mm, from 10 microns to 5 mm, from 10 microns to 4 mm, from 10 microns to 3 mm, from 10 microns to 2 mm, from 10 microns to 1 mm, from 10 microns to 500 microns, from 10 microns to 250 microns, from 10 microns to 100 microns, from 25 microns to 10 mm, from 100 microns to 9 mm, from 100 microns to 8 mm, from 100 microns to 7 mm, from 100 microns to 6 mm, from 100 microns to 5 mm, from 100 microns to 4 mm, from 100 microns to 3 mm, from 100 microns to 2 mm, from 100 microns to 1 mm, from 25 microns to 500 microns, from 25 microns to 250 microns, from 25 microns to 100 microns, from 50 microns to 10 mm, from 50 microns to 9 mm, from 50 microns to 8 mm, from 50 microns to 7 mm, from 50 microns to 6 mm, from 50 microns to 5 mm, from 50 microns to 4 mm, from 50 microns to 3 mm, from 50 microns to 2 mm, from 50 microns to 1 mm, from 50 microns to 500 microns, from 50 microns to 250 microns, from 50 microns to 100 microns, from 100 microns to 10 mm, from 100 microns to 9 mm, from 100 microns to 8 mm, from 100 microns to 7 mm, from 100 microns to 6 mm, from 100 microns to 5 mm, from 100 microns to 4 mm, from 100 microns to 3 mm, from 100 microns to 2 mm, from 100 microns to 1 mm, from 100 microns to 500 microns, from 100 microns to 250 microns, from 250 microns to 10 mm, from 250 microns to 9 mm, from 250 microns to 8 mm, from 250 microns to 7 mm, from 250 microns to 6 mm, from 250 microns to 5 mm, from 250 microns to 4 mm, from 250 microns to 3 mm, from 250 microns to 2 mm, from 250 microns to 1 mm, from 250 microns to 500 microns, from 500 microns to 10 mm, from 500 microns to 9 mm, from 500 microns to 8 mm, from 500 microns to 7 mm, from 500 microns to 6 mm, from 500 microns to 5 mm, from 500 microns to 4 mm, from 500 microns to 3 mm, from 500 microns to 2 mm, or from 500 microns to 1 mm).

14. The method of any one of claims 1-13, wherein the thin film has an area of at least 100 cm2, such as an area of from 100 cm2to 5 m2, such as from 100 cm2to 3 m2.

15. The method of any one of claims 1-14, wherein a plurality of the thin films are disposed within the photobioreactors.

16. The method of claim 15, wherein each of the plurality of thin films comprises a different phototrophic microorganism.

17. The method of any one of claims 1-16, wherein the thin film is disposed on a scaffold, such as a polymer membrane, a carbon resin sheet, metal sheet, or a glass sheet.

18. The method of claim 17, wherein the scaffold is substantially planar.

19. The method of any one of claims 17-18, wherein the scaffold is formed from a nontransparent material.

20. The method of any one of claims 1-19, wherein the phototrophic microorganism is engineered to produce and / or increase production of the chemical product, resist the toxicity of the matrix material (e.g., resist the toxicity of a crosslinker used to form the matrix material), increase secretion of the chemical product, or any combination thereof.

21. The method of any one of claims 1-20, wherein the phototrophic microorganism comprises a bacterium, a cyanobacterium, an alga, a fungus, or a combination thereof.

22. The method of any one of claims 1-21 , wherein the thin film comprises two or more different phototrophic microorganisms dispersed within the transparent matrix material.

23. The method of claim 22, wherein the two or more different phototrophic microorganisms are disposed in different layers within the thin film.

24. The method of any one of claims 22-23, wherein the two or more different phototrophic microorganisms possess different photosynthetic characteristics.

25. The method of any one of claims 1-24, wherein exposing the thin film to light comprises exposing the thin film to sunlight, irradiating the thin film with an artificial light source, or a combination thereof.

26. A photobioreactor system for producing a chemical product via bioconversion, the system comprising a housing; one or more thin films disposed within the housing, wherein each of the one or more thin films comprises a phototrophic microorganism dispersed within a transparent matrix material; a gas inlet configured to direct a flow of gas into the housing; and a gas outlet configured to remove gas from the housing.

27. The photobioreactor system of claim 26, further comprising a liquid inlet configured to direct an aqueous fluid into the housing so as to contact the one or more thin films; and a liquid outlet configured to remove the aqueous fluid from the housing.

28. The photobioreactor system of any of claims 26-27, wherein the housing comprises a transparent region positioned to allow incident sunlight to reach the one or more thin films disposed within the housing.

29. The photobioreactor system of any of claims 26-28, wherein the one or more thin films are disposed on a scaffold.

30. The photobioreactor system of claim 29, wherein the scaffold is planar.

31. The photobioreactor system of any one of claims 26-30, wherein the transparent matrix material comprises a polymer matrix, such as a hydrogel matrix.

32. The photobioreactor system of any one of claims 26-31, wherein the transparent matrix material comprises a polysaccharide (e.g., alginate, chitosan, agarose, carrageenan, cellulose, hyaluronic acid, or a combination thereof), a protein (e.g., collagen, gelatin, or a combination thereof), polyvinyl alcohol, polyvinyl acetate, a polyalkylene oxide (e.g., a crosslinked polyethylene glycol), a polyacrylamide, a polyester (e.g., polylactic acid, polyglycolic acid, polycaprolactone), and combinations thereof.

33. The photobioreactor system of any one of claims 26-32, wherein the transparent matrix material is non- biodegradable.

34. The photobioreactor system of any one of claims 26-33, wherein the thin film has a thickness of less than 10 mm (e.g., less than 9 mm, less than 8 mm, less than 7 mm, less than 6 mm, less than 5 mm, less than 4 mm, less than 3 mm, less than 2 mm, or less than 1 mm), such as a thickness of from 10 microns to 10 mm (e.g., from 10 microns to 9 mm, from 10 microns to 8 mm, from 10 microns to 7 mm, from 10 microns to 6 mm, from 10 microns to 5 mm, from 10 microns to 4 mm, from 10 microns to 3 mm, from 10 microns to 2 mm, from 10 microns to 1 mm, from 10 microns to 500 microns, from 10 microns to 250 microns, from 10 microns to 100 microns, from 25 microns to 10 mm, from 100 microns to 9 mm, from 100 microns to 8 mm, from 100 microns to 7 mm, from 100 microns to 6 mm, from 100 microns to 5 mm, from 100 microns to 4 mm, from 100 microns to 3 mm, from 100 microns to 2 mm, from 100 microns to 1 mm, from 25 microns to 500 microns, from 25 microns to 250 microns, from 25 microns to 100 microns, from 50 microns to 10 mm, from 50 microns to 9 mm, from 50 microns to 8 mm, from 50 microns to 7 mm, from 50 microns to 6 mm, from 50 microns to 5 mm, from 50 microns to 4 mm, from 50 microns to 3 mm, from 50 microns to 2 mm, from 50 microns to 1 mm, from 50 microns to 500 microns, from 50 microns to 250 microns, from 50 microns to 100 microns, from 100 microns to 10 mm, from 100 microns to 9 mm, from 100 microns to 8 mm, from 100 microns to 7 mm, from 100 microns to 6 mm, from 100 microns to 5 mm, from 100 microns to 4 mm, from 100 microns to 3 mm, from 100 microns to 2 mm, from 100 microns to 1 mm, from 100 microns to 500 microns, from 100 microns to 250 microns, from 250 microns to 10 mm, from 250 microns to 9 mm, from 250 microns to 8 mm, from 250 microns to 7 mm, from 250 microns to 6 mm, from 250 microns to 5 mm, from 250 microns to 4 mm, from 250 microns to 3 mm, from 250 microns to 2 mm, from 250 microns to 1 mm, from 250 microns to 500 microns, from 500 microns to 10 mm, from 500 microns to 9 mm, from 500 microns to 8 mm, from 500 microns to 7 mm, from 500 microns to 6 mm, from 500 microns to 5 mm, from 500 microns to 4 mm, from 500 microns to 3 mm, from 500 microns to 2 mm, or from 500 microns to 1 mm).

35. The photobioreactor system of any one of claims 26-34, wherein the thin film has an area of at least 100 cm2, such as an area of from 100 cm2to 5 m2, such as from 100 cm2to 3

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