Chemical production in salt-containing media with halotolerant yeast

Abstract

The present disclosure provides a method for producing chemicals using halotolerant yeast. The method includes culturing halotolerant yeast cells in a medium comprising a solution of salts at a concentration of at least 0.5 times the salinity of seawater, and allowing the halotolerant yeast cells to produce one or more chemicals. The medium may include a carbon source, nitrogen source and / or phosphate source, trace elements, and additives. The halotolerant yeast cells may be selected from, e.g., Debaryomyces subglobosus, Candida taylori, Pichia anomaly Millerozyma farinosa, Blastobotrys adeninivorans, Scheffersomyces stipitis. or combinations thereof. The method enables chemical production in high-salt media, reducing freshwater usage in fermentation processes.

Description

CHEMICAL PRODUCTION IN SALT-CONTAINING MEDIAWITH HALOTOLERANT YEASTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 679.445, filed August 5, 2024, the contents of which are incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to halotolerant yeast species for chemical production, and more particularly to methods, media compositions, and halotolerant microorganisms for producing chemicals in media containing high salt concentrations.BACKGROUND

[0003] Seaweed biomass has emerged as a promising sustainable feedstock for the production of fuels, chemicals, and other valuable products. Unlike terrestrial crops, seaweed does not compete for arable land or freshwater resources, making it an attractive option for large-scale cultivation. However, the efficient utilization of seaweed biomass presents several challenges due to its unique composition and the marine environment in which it grows.

[0004] Traditional fermentation processes for converting biomass to chemicals typically rely on freshwater-based media and microorganisms adapted to low-salt conditions. When applied to seaweed-derived sugars, these processes often require energy -intensive desalination steps to remove salt from the feedstock. Additionally, the presence of salt can inhibit the growth and metabolic activity of many conventional fermentation organisms, reducing yields and productivity.

[0005] The use of halotolerant microorganisms capable of growing in high-salt environments offers a potential solution to these challenges. Halotolerant yeasts, in particular, have shown promise for their ability’ to ferment sugars in the presence of salt. However, the diversity of halotolerant yeast species and their capabilities for producing various chemicals in salt-containing media remain largely unexplored.

[0006] Another obstacle in utilizing seaweed biomass is the presence of complex polysaccharides like alginate, which are not easily fermented by many microorganisms. Developing fermentation processes that can efficiently convert these unique marinecarbohydrates into valuable products would significantly enhance the economic viability of seaweed-based biorefineries.

[0007] The scale-up of seaweed cultivation and processing also presents logistical challenges. Unlike terrestrial biomass, seaweed requires specialized harvesting and handling techniques to maintain its quality and minimize degradation. Furthermore, the seasonal variability in seaweed composition and availability can impact the consistency of feedstock supply for industrial-scale fermentation processes.

[0008] It has been appreciated that a method is needed that overcomes one or more of these problems.SUMMARY

[0009] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description.

[0010] In a first aspect, a method for producing chemicals using halotolerant yeast is provided. The method comprises culturing halotolerant yeast cells in a medium consisting of an aqueous solution of salts, a carbon source, optionally a nitrogen source, optionally a phosphate source, optionally one or more trace metals, and optionally one or more additives, the salts present in the aqueous solution at a concentration of at least 0.5 times the salinity of seawater, and allowing the halotolerant yeast cells to produce one or more chemicals.

[0011] This method enables the use of abundant and inexpensive salt water sources such as seawater or brine for fermentation processes, thereby reducing the reliance on freshwater resources and lowering operational costs for chemical production. The halotolerant nature of the yeast allows for efficient chemical production even in high-salt environments that would inhibit conventional microorganisms.

[0012] The halotolerant yeast cells may be Debaryomyces cells, Candida cells, Pichia cells, Millerozyma cells, Blastobotrys cells, Scheffer somyces cells, Wickerhamomyces cells, their synonyms or a combination thereof.

[0013] These specific genera of halotolerant yeast have been demonstrated to maintain robust grow th and metabolic activity’ in salt-containing media, ensuring reliable chemical production under saline conditions.

[0014] The halotolerant yeast cells may be Debaryomyces subglobosus cells, Candida taylori cells, Pichia anomala cells, Millerozyma farinosa cells, Blastobotrys adeninivorans cells. Scheffersomyces stipitis cells, Wickerhamomyces anomalous cells, their synonyms or a combination thereof.

[0015] These particular species have been specifically characterized for their superior performance in high-salt environments, with grow th rates and chemical yields that compare favorably to conventional yeast strains in freshwater media.

[0016] The concentration of salts (csait) may be 0.5 < Csait < 5 times a salinity of seawater.

[0017] This salt concentration range encompasses the salinity levels found in various available water sources, from diluted seawater to concentrated brine, providing flexibility in selecting appropriate w ater sources for the fermentation process.

[0018] The concentration of salts may be 1 < Csait < 5 times a salinity of seawater.

[0019] Operating at salt concentrations above seawater salinity maximizes the cost savings by enabling the use of highly saline w aste streams such as desalination brine that would otherwise require disposal.

[0020] The concentration of salts (csait) may be at least a same salinity as seawater.

[0021] Using salt concentrations at or above seawater salinity allows direct utilization of seawater without dilution, simplifying the process and maximizing resource efficiency.

[0022] The salt water may include a halide, a sulfate, a hydroxide, a bicarbonate, a fluoride, or a combination thereof.

[0023] These salt types are commonly found in natural seawater and industrial brine streams, making the method compatible with readily available saline water sources.

[0024] The halide may be NaCl, NaBr, MgCh, MnCh, KC1, or a combination thereof, the sulfate may include MgSCh. MnSO-i. (NTU^SCh, or a combination thereof, the hydroxide may include KOH, or a combination thereof.

[0025] These specific salt compositions mirror the ionic composition of natural seawater and synthetic seawater formulations, ensuring optimal compatibility with marine-derived water sources.

[0026] The carbon source may be glucose, galactose, cellobiose, xylose, mannitol, sucrose, lactose, glycerol, alginate, or a combination thereof.

[0027] This diverse range of carbon sources enables the utilization of various feedstocks, including marine biomass components such as seaweed-derived sugars, further enhancing the sustainability of the process.

[0028] The alginate may be present in a total amount of 0.5% - 2% w / v of the medium.

[0029] This alginate concentration range provides sufficient carbon source for yeast metabolism while maintaining cost-effectiveness and avoiding substrate inhibition effects.

[0030] The medium may consist of the aqueous solution of salts, the carbon source, the nitrogen source / phosphate source, optionally the one or more trace metals, and optionally the one or more additives.

[0031] Including a nitrogen source ensures adequate nutrient availability for yeast growth and metabolic activity, supporting efficient chemical production in the saline environment.

[0032] The nitrogen source may be selected from the group comprising ammonium sulfate, glutamate, amino acids, and urea.

[0033] These nitrogen sources are readily available and provide essential nutrients for yeast metabolism, supporting robust growth and chemical production in salt-containing media.

[0034] Similar to nitrogen source, a phosphate source is often added to ensure adequate nutrient availability and simultaneously offer some buffering capacities. The phosphate source may be selected from potassium dibasic phosphate, potassium monobasic phosphate, potassium phosphate, phosphoric acid. Additional phosphate sources include sodium phosphate salts.

[0035] The medium may consist of the aqueous solution of salts, the carbon source, the nitrogen source / phosphate source, the one or more trace metals, and optionally the one or more additives.

[0036] The inclusion of trace metals provides essential micronutrients required for enzymatic functions and metabolic pathways, ensuring optimal yeast performance in the challenging high-salt environment.

[0037] The one or more trace elements may be zinc, cadmium, bromine, gold, copper, iron, aluminum, silicon, lithium, or a combination thereof.

[0038] These trace elements support various enzymatic processes and cellular functions, maintaining yeast viability and metabolic efficiency under saline stress conditions.

[0039] The medium may consist of the aqueous solution of salts, the carbon source, the nitrogen source / phosphate source, the one or more trace metals, and the one or more additives.

[0040] The inclusion of additives provides additional nutrients and growth factors that enhance yeast performance and chemical production yields in salt-containing media.

[0041] The one or more additives may be vitamin B2, vitamin Bl 2, inositol, yeast extract, peptone, tryptone, or a combination thereof.

[0042] These additives supply essential vitamins, growth factors, and complex nutrients that support yeast metabolism and improve chemical production efficiency in challenging saline conditions.

[0043] Culturing the halotolerant yeast cells may comprise incubating the cells at a temperature between 10°C and 45°C.

[0044] This temperature range accommodates the optimal growth conditions for various halotolerant yeast species while providing operational flexibility for different industrial settings and climatic conditions.

[0045] Culturing the halotolerant yeast cells may comprise maintaining a pH between 2 and 9.

[0046] This broad pH range allows for process optimization based on specific yeast strains and desired chemical products, while accommodating the natural pH variations that may occur in different saline water sources, and unbuffered saline water sources in particular.

[0047] The one or more chemicals produced may comprise ethanol, glycerol, acetate, xylitol, acetoin, or a combination thereof.

[0048] These chemicals represent valuable industrial products including biofuels, chemical intermediates, and specialty chemicals, demonstrating the commercial viability of the salt-tolerant fermentation process.

[0049] The yield of the one or more chemicals produced, on a g / g basis, may be equal to or greater than the yield of the one or more chemicals in the same system but with fresh water instead of the aqueous solution of salts.

[0050] Achieving yields comparable to or exceeding those obtained with freshwater demonstrates that the salt-tolerant approach does not compromise production efficiency while providing the additional benefits of reduced freshwater consumption and low er operational costs.BRIEF DESCRIPTION OF FIGURES

[0051] Embodiments of the invention will be described, by way of example, with reference to the following drawings, in which:

[0052] Figure 1 illustrates a flowchart for producing chemicals using halotolerant yeast cells, according to aspects of the present disclosure.

[0053] Figure 2 illustrates a flowchart for a method of chemical production using halotolerant yeast species, according to an embodiment.

[0054] Figures 3A and 3B are graphs showing ethanol production (3 A) and yields (3B) after 48 hour fermentation in a glucose media with salinities of Ox ,lx. 3x. and 5x the salinity of seaw ater.

[0055] Figure 4 is a graph showing glycerol production after 48 hour fermentation in glucose media.

[0056] Figures 5A and 5B are graphs showing ethanol (5A) and glycerol (5B) production after 48 hour fermentation in xylose media.

[0057] Figure 6 is a graph showing ethanol production after 48 hour fermentation in cellobiose media

[0058] Figures 7A, 7B, and 7C are graphs showing growth rates in glucose media (7A), xylose media (7B), and cellobiose media (7C). A and 6B are graphs showing ethanol (6 A) and glycerol (6B) production after 48 fermentation in xylose media.

[0059] Figure 8A is a graph showing ethanol production after 72 hour fermentation in minimal glucose media.

[0060] Figures 8B and 8C are graphs showing ethanol production after 60 hour fermentation in minimal xylose media (8B) and minimal cellobiose media (8C).

[0061] Figure 9 is a graph showing the amount of alginate degradation after 24hr fermentation in an alginate + glucose media.

[0062] Figure 10A and 10B are graphs showing growth rates in minimal glucose media (10A) and minimal glucose + alginate media (10B).

[0063] Figure 11 A, 11B, and 11C are tables showing ODeoo results of growing various species grown in salt water with various carbon sources.

[0064] Common reference numerals are used throughout the figures to indicate similar features.DETAILED DESCRIPTION

[0065] The present disclosure relates to methods for producing chemicals using halotolerant yeast species in media compositions containing high concentrations of salt. Halotolerant yeast are capable of growing and producing chemicals in environments with salinity7levels similar to or higher than seawater. This approach enables the use of non-potable water sources, such as seawater or industrial effluents, for fermentation processes.

[0066] Utilizing salt-containing media for chemical production offers several advantages in terms of sustainability' and resource utilization. By reducing reliance on freshwater resources, these methods can help conserve potable water for other essential uses. Additionally, the ability' to use seawater or industrial effluents as a water source may reduce costs associated with water purification or synthetic media preparation.

[0067] The disclosed methods involve culturing halotolerant yeast species in media containing high salt concentrations to produce various chemicals of interest. These yeast species are capable of maintaining metabolic activity7and chemical production in saline conditions that would be inhibitory to many conventional production organisms. The saltcontaining media may be derived from natural sources like seawater or from industrial processes that generate high-salinity effluents.

[0068] The method utilizes halotolerant yeast species capable of growing and producing chemicals in high-salt environments. Examples of suitable halotolerant yeast species include Debaryomyces subglobosus. Candida taylori. Pichia anomala (also known as Wickerhamomyces a no mains). Millerozyma farinosa, Blastobotrys adeninivorans , andScheffersomyces stipitis. These yeast species demonstrate the ability to tolerate salt concentrations ranging from 0.5 to 5 times the salinity of seawater, and growth and production are especially surprising at elevated salinities.

[0069] The yeast may be from the family Debaryomycetaceae. such as from the genus Scheffersomyces (e.g. Scheffersomyces stipitis, Scheffersomyces shehatae) or genus Milleroz ma (e.g. Millerozyma farinosa, Millerozyma miso'). Other suitable yeast species include Issatchenkia orientalis and Kluyveromyces marxianus. which have demonstrated halotolerance and chemical production capabilities in high-salt media.

[0070] FIG. 1 illustrates a method for producing chemicals using halotolerant yeast cells. The method includes a step 102 of providing halotolerant yeast cells. These yeast cells may be selected based on their ability to grow and produce desired chemicals in salt-containing media.

[0071] FIG. 2 depicts a more detailed method 200 for chemical production using halotolerant yeast species. The method 200 begins with a step 102 of selecting halotolerant yeast species. This selection may be based on the specific chemical production goals and the salt concentration of the medium to be used.

[0072] The halotolerant yeast species used in the method may include novel yeast species capable of utilizing sugars derived from Sargassum, a genus of seaweed. These novel species may be identified through screening processes that evaluate grow th and chemical production on media containing Sargassum-derived sugars and elevated salt concentrations.

[0073] The selected halotolerant yeast species are cultured in salt-containing media as shown in step 106 of both FIG. 1 and FIG. 2. The salt concentration of the media may range from 0.5 to 5 times the salinity of seawater, allowing for the use of various non-potable w ater sources in the fermentation process. The medium may comprise or consist of an aqueous solution of salts, a carbon source, optionally a nitrogen source, optionally a phosphate source, optionally one or more trace metals, and optionally one or more additives.

[0074] The salt-containing media used for culturing halotolerant yeast may include a solution of salts at concentrations ranging from 0.5 to 5 times the salinity of seaw ater. As shown in step 104 of FIG. 1 and FIG. 2, an aqueous medium is prepared with an appropriate salt concentration. The salts in the medium may include halides, sulfates, bicarbonates, fluondes. or combinations thereof. Examples of halides include metal halides such as NaCl, NaBr, MgCh, MnCh, and KC1. Sulfates may include MgSCh, MnSCb, and (NF hSCh.

[0075] The aqueous solution may contain halides, sulfates, hydroxides, bicarbonates, fluorides, or combinations thereof. For example, the hydroxide may be KOH. The bicarbonatemay be the bicarbonate ion (HCCh ), which may be at a concentration of 100-150 mg / L. The fluoride may be present as a fluoride ion (F ), and may be at a concentration of 0.8- 1.5 mg / L.

[0076] The medium also includes a carbon source to support yeast growth and chemical production. Carbon sources may be selected from sugars such as glucose, galactose, cellobiose, sucrose, lactose, and xylose. The carbon source may a sugar alcohol, such as mannitol. The carbon source may be glycerol. Additionally, alginate, a polysaccharide found in seaweed, may serve as a carbon source. In some examples. CO2 or formate may be used as alternative carbon sources.

[0077] A nitrogen source may be incorporated into the medium to provide essential nutrients for yeast metabolism. Nitrogen sources may be selected from compounds such as ammonium sulfate, glutamate, amino acids, and urea. These nitrogen-containing compounds support protein synthesis and cellular growth of the halotolerant yeast.

[0078] A phosphate source may be added to ensure adequate nutrient availability and simultaneously offer some buffering capacities. The phosphate source may be selected from potassium dibasic phosphate, potassium monobasic phosphate, potassium phosphate, phosphoric acid. Additional phosphate sources include sodium phosphate salts.

[0079] In some implementations, only as nitrogen source is utilized. In some implementations, only as phosphate source is utilized. In preferred implementations, a nitrogen source and a phosphate source are utilized.

[0080] Trace elements are included in the medium to facilitate various enzymatic reactions and cellular processes. Examples of trace elements that may be added include zinc, cadmium, bromine, gold, copper, iron, aluminum, silicon, and lithium. These elements, while required in small quantities, play important roles in yeast physiology and chemical production.

[0081] The medium may further comprise one or more additives to enhance yeast growth and chemical production. Additives may be selected from compounds such as vitamin B2, vitamin Bl 2, inositol, yeast extract, peptone, tryptone, and alginate. These additives provide additional nutrients, growth factors, or serve as stabilizing agents in the high-salt environment.

[0082] The composition of the salt-containing media is designed to support the growth and metabolic activities of halotolerant yeast species. As illustrated in step 106 of FIG. I and FIG. 2, the yeast cells are cultured in this specially formulated medium. The combination of salts, carbon sources, nitrogen sources, phosphate sources, trace elements, and additives creates an environment that allows the halotolerant yeast to thrive and produce desired chemicals even under high-salinity conditions.

[0083] The method 200 includes a step 202 of setting temperature and pH parameters for culturing the halotol erant yeast cells. The temperature for culturing may be set between 10°C and 45°C. In some examples, the temperature range may be 15°C to 40°C, 20°C to 35°C, or 25°C to 30°C. The pH for culturing may be maintained between 2 and 9. In certain examples, the pH range may be 3 to 8, 4 to 7, or 5 to 6. One or more buffers may be utilized as additives to gain control over the media pH.

[0084] The temperature and pH conditions affect the growth rate and metabolic activity of the halotolerant yeast cells in high-salt environments. Higher temperatures within the specified range may increase the grow th rate and chemical production of the yeast cells, while lower temperatures may slow- down cellular processes. The pH level influences enzyme activity and nutrient uptake by the yeast cells.

[0085] As shown in step 106 of FIG. 1 and FIG. 2, the halotolerant yeast cells are cultured in the salt-containing medium under the specified temperature and pH conditions. The incubation time for culturing may vary depending on the specific yeast species, desired chemical production, and salt concentration of the medium. Incubation times may range from 24 hours to 72 hours, with some examples requinng 36 to 60 hours or 48 to 56 hours.

[0086] During the culturing period, the halotolerant yeast cells adapt to the high-salt environment and begin producing chemicals of interest. The step 108 of FIG. 1 and the step 204 of FIG. 2 involve allowing the yeast cells to produce chemicals such as ethanol, glycerol, acetate, xylitol, or acetoin. The production of these chemicals may be monitored throughout the culturing period to determine optimal harvest times. As will be understood, the yeast cells being fermented will necessarily need to, collectively or individually, have all of the necessary biosynthetic pathways to generate these chemicals.

[0087] The culturing conditions may be adjusted based on the specific halotolerant yeast species being used and the desired chemical output. For example, Debaryomyces subglobosus may have different optimal temperature and pH ranges compared to Pichia anomala for maximum ethanol production in high-salt media. Monitoring and adjusting these parameters throughout the culturing process may help optimize chemical yields and production efficiency.

[0088] The halotolerant yeast cells produce various chemicals during the culturing process. As shown in step 108 of FIG. 1 and step 204 of FIG. 2, the method allows the yeast cells to produce chemicals such as ethanol, glycerol, acetate, and acetoin. The production of these chemicals occurs as the yeast metabolize the carbon sources present in the salt-containing medium.

[0089] Ethanol is a primary' product of yeast fermentation. The halotolerant yeast cells convert sugars like glucose into ethanol through the glycolysis pathway and subsequent fermentation. The ethanol production may be influenced by factors such as the specific yeast strain, salt concentration, temperature, and availability of nutrients.

[0090] Glycerol is another metabolic product generated by the yeast cells. Glycerol production often increases in high-salt environments as the yeast use it as an osmoprotectant. The synthesis of glycerol helps the halotolerant yeast maintain cellular osmotic balance in the presence of high salt concentrations.

[0091] Acetate production occurs as a byproduct of yeast metabolism. The formation of acetate may result from the oxidation of acetaldehyde, an intermediate in ethanol production. The accumulation of acetate in the medium can affect the pH and potentially influence yeast growth and chemical production.

[0092] Acetoin is a flavor compound produced by some yeast species. The production of acetoin may be influenced by the carbon source, oxygen availability, and specific metabolic pathways of the halotolerant yeast strain used in the method.

[0093] In addition to these chemicals, the method may produce citramalate. Citramalate is a precursor for methacrylate production and may be synthesized by certain engineered halotolerant yeast strains. The production of citramalate depends on the specific metabolic pathways present in the yeast and may require genetic modifications to enhance yields.

[0094] The method may also produce isobutanol, a potential biofuel precursor. Isobutanol production by halotolerant yeast involves the conversion of pyruvate, an intermediate in glucose metabolism, through a series of enzymatic reactions. The production of isobutanol may require metabolic engineering of the yeast strains to optimize the biosynthetic pathways.

[0095] The production of these chemicals by the halotolerant yeast cells is influenced by various factors. The type and concentration of carbon sources in the medium affect the metabolic pathways utilized by the yeast. For example, glucose may be preferentially fermented to ethanol, while other sugars like xylose may lead to different product distributions.

[0096] The salt concentration in the medium impacts the metabolic activities of the yeast cells. Higher salt concentrations may shift metabolism towards the production of osmoprotectants like glycerol, potentially affecting the yields of other chemicals.

[0097] Temperature and pH conditions, as set in step 202 of FIG. 2, influence enzyme activities and metabolic rates, thereby affecting the production of various chemicals. Optimal temperature and pH ranges may vary for different chemical products and yeast strains.

[0098] Oxygen availability in the culture medium affects the balance between fermentative and respiratory’ metabolism in the yeast cells. This balance can influence the distribution of metabolic products, with anaerobic conditions generally favoring ethanol production.

[0099] The specific halotolerant yeast strain used in the method significantly impacts the types and quantities of chemicals produced. Different yeast species and strains may have varying metabolic capabilities and tolerances to high-salt conditions, resulting in distinct chemical production profiles.

[0100] The method allows for the accumulation of these chemicals in the culture medium as the yeast cells grow and metabolize the available nutrients. The production of chemicals may continue throughout the culturing period, with yields and product ratios potentially changing over time as nutrients are consumed and metabolic byproducts accumulate.

[0101] The collection and analysis of chemicals produced by the halotolerant yeast cells are important steps in the method for chemical production. As shown in step 110 of FIG. 1 and FIG. 2, the produced chemicals are collected from the culture medium.

[0102] Collection of the produced chemicals may involve separating the yeast cells from the liquid medium. A step of centrifugation may be used to pellet the yeast cells, allowing the supernatant containing the dissolved chemicals to be collected. Alternatively, a step of filtration may be employed to remove the yeast cells and collect the cell-free medium containing the produced chemicals.

[0103] Following the separation of yeast cells, the collected liquid may undergo further processing steps to isolate and purify specific chemicals of interest. A step of distillation may be used to separate volatile compounds such as ethanol from the aqueous medium. For less volatile compounds like glycerol or organic acids, a step of liquid-liquid extraction using organic solvents may be employed to isolate the target chemicals.

[0104] The method may include a step of concentrating the collected chemicals through evaporation or membrane filtration techniques. This concentration step can increase the purity of the final product and reduce downstream processing requirements.

[0105] Analysis of the produced chemicals typically involves quantitative and qualitative measurements. High-performance liquid chromatography (HPLC) may be used to separate and quantify the various chemical components in the collected samples. The HPLC analysis may utilize an ion-exchange column, such as an Aminex HPX-87H column, to separate the chemical species based on their ionic properties.

[0106] The method 200 may include a step of sample preparation for HPLC analysis. This step may involve diluting the samples to an appropriate concentration range and filtering to remove any particulates that could interfere with the chromatographic separation.

[0107] The HPLC analysis conditions may include using a mobile phase of dilute sulfuric acid (e.g., 5 mM H2SO4) with a flow rate of 0.6 mL / min. The column temperature may be maintained at 55°C, and the analysis may run for approximately 30 minutes per sample.

[0108] Detection of the separated chemical species may be accomplished using a refractive index detector or a diode array detector, depending on the specific chemicals being analyzed. The method may include a step of calibrating the HPLC system using standard solutions of known concentrations for each chemical of interest.

[0109] The concentrations of produced chemicals may be calculated based on the peak areas obtained from the HPLC chromatograms. The method may include a step of data analysis using specialized software to integrate peak areas and calculate concentrations based on the calibration curves.

[0110] In addition to HPLC analysis, the method may include other analytical techniques for characterizing the produced chemicals. Gas chromatography (GC) may be used for analyzing volatile compounds like ethanol or acetoin. Mass spectrometry (MS) may be coupled with HPLC or GC to provide additional structural information and improve the specificity of chemical identification.[01111 The method may include a step of calculating yields and productivities for the various chemicals produced. These calculations take into account the initial substrate concentrations, fermentation time, and final product concentrations to assess the efficiency of the chemical production process.

[0112] The collected and analyzed data may be used to optimize the fermentation conditions, select the most productive yeast strains, and evaluate the overall performance of the chemical production process using halotolerant yeast in salt-containing media.

[0113] The method 200 integrates various process elements to achieve efficient chemical production using halotolerant yeast in high-salt environments. FIG. 2 illustrates the overall process flow, incorporating steps for yeast selection, media preparation, culturing conditions, and chemical production.

[0114] The method 200 begins with the step 102 of selecting halotolerant yeast species. The selection process considers the yeast's ability to grow and produce desired chemicals in salt-containing media. The method 200 may use genetic engineering techniques to modify the selected yeast species, enhancing chemical production capabilities in high-salt environments.For example, the yeast may be engineered with improved metabolic pathways for ethanol or isobutanol production.

[0115] Following yeast selection, the method 200 proceeds to the step 104 of preparing salt-containing media. The media composition is tailored to support the grow th and metabolic activities of the selected halotolerant yeast species. The salt concentration, carbon sources, nitrogen I phosphate sources, and trace elements are balanced to create an optimal environment for chemical production.

[0116] The method 200 includes a step 202 of seting temperature and pH parameters for culturing. These parameters are adjusted based on the specific requirements of the selected yeast species and the desired chemical products. The temperature and pH conditions influence the growth rate, metabolic activity, and chemical production efficiency of the halotolerant yeast.

[0117] The step 106 of culturing the halotolerant yeast cells integrates the selected yeast species, prepared media, and set culturing conditions. During this step, the yeast cells adapt to the high-salt environment and begin producing chemicals of interest. The culturing process may involve feedback mechanisms to monitor and adjust conditions for optimal chemical production.

[0118] The method 200 incorporates a step 204 of allow ing chemical production by the halotolerant yeast cells. This step involves the metabolic conversion of carbon sources into desired chemicals such as ethanol, glycerol, acetate, xylitol, or acetoin. The production of these chemicals is influenced by the integrated elements of yeast metabolism, media composition, and culturing conditions.

[0119] The method 200 may include a step 300 of optimizing the chemical production process. This step involves a series of sub-steps and decisions to enhance the efficiency and yield of chemical production. A decision 302 may be made to determine if the current process parameters are optimal for chemical production. Based on this decision, the method 200 may proceed to a step 304 of adjusting media composition, a step 306 of modifying culturing conditions, or a step 308 of altering yeast strain selection.

[0120] The method 200 may use directed evolution techniques in a step 310 to increase the halotolerance of the yeast strains. This process involves subjecting the yeast to progressively higher salt concentrations over multiple generations, selecting for individuals with improved salt tolerance. The evolved yeast strains may demonstrate enhanced growth and chemical production capabilities in high-salt environments.

[0121] A step 312 may involve modifying the yeast with assimilation pathways for Sargassum sugars. This genetic modification enables the yeast to utilize sugars derived from Sargassum seaweed as a carbon source, expanding the range of potential feedstocks for chemical production in salt-containing media.

[0122] The method 200 may include a step 314 of analyzing chemical production data and implementing process improvements. This step integrates information from various stages of the process to identify opportunities for optimization. Feedback from chemical analysis may inform adjustments to media composition, culturing conditions, or yeast strain selection in subsequent production cycles.

[0123] The integration of these process elements in the method 200 creates a comprehensive approach for chemical production using halotolerant yeast in high-salt environments. The combination of yeast selection, media optimization, controlled culturing conditions, and continuous process improvement enables efficient and sustainable production of valuable chemicals using non-potable water sources.

[0124] Example 1 - Products of various yeast strains when grown in glucose at different salt concentrations.

[0125] Several strains, including species of Saccharomyces cerevisiae, Debaryomyces subglobosus , Candida taylori, Pichia anomala (also known as Wickerhamomyces anomalus) Millerozyma far ino a. Blaslobotrys adenimvorans, and Scheffersomyces stipitis, were fermented, using synthetic sea ater media containing peptone (2% w / v). yeast extract (1% w / v), 1 -tryptophan (0.015% w / v), and three different carbon sources: glucose, xylose and cellobiose at 2% w / v. The formulation of artificial seawater contained NaCl (464mM), MgCl2-6H2O (12mM), MgSO4-7H2O (14mM), KC1 (lOmM), and NaBr (0.83mM). The media was adjusted to result in four variants: one that included no added salt (OS), and three variants that had salt concentrations equivalent to 1 (I S), 3 (3S), and 5 (5S) times the concentration of seawater.

[0126] The products of the fermentation were analyzed at various times (e.g., after 24 or 48 hours) by high performance liquid chromatography using an Agilent 1260 Infinity instrument, using an Aminex HPX-87H ion exchange column from Bio-Rad. Samples were eluted with 0.6 mL / min of 5 mM FESCh at 55 °C for 30 minutes. The summary of the results are shown in Table 1, below;

[0128] Example 2 - Ethanol yields

[0129] For the same species in Example 1, the strains were fermented in various salt concentrations (OS, IS, 3S, and 5S), using Glucose as a carbon source. Ethanol yields were measured at 48 hours. The results are summarized in Table 2, below.

[0130] Table 2

[0131] Example 3 - Screening of yeasts growing in salt water.

[0132] 47 yeast species were fermented with various carbon sources for growing in salt water. Specifically, glucose (at IS, 2S, and 5S salt concentrations), and Xylose, Cellobiose,1% Alginate, 2% Glucose and 1% Alginate Mannitol, Sucrose, Lactose, Galactose, and Glycerol, each at I S salt concentrations, and the ODeoo of each was measured after 24 hours. All of the yeast species grew in salt water with at least one of the carbon sources 42 of the 47 yeast species performed well (e.g.. had ODeoo > 2) in salt water with at least one of the carbon sources. The top 14 best performers are shown in Table 2. below. More results can be seen in FIGS. 11A-11C.

[0133] Table 3

[0134] Example 3 -Chemical Production in Glucose Media

[0135] A comparison was performed, using S. cerevisiae as a control, for production of ethanol and ethanol yield in glucose media for various halotolerant species. In this example, two strains of D. hansenii, and one strain each of D. subglobosus, M. farinose., B. adeninivorans, C. taylori, P. anomala, and S. stipitis were tested. A synthetic seawater was used, the synthetic seawater having - at a 1% salinity’ level - a general composition of water combined with 3.4420 w / w% salts (predominantly NaCl, but also inclusive of -0.25 w / w% MgCh, -0.33 w / w% MgSCh, -0.1 1 w / w% CaCk, and less than 0.1 w / w% each of KC1, NaHCCh, and NaBr). The amount of salts were adjusted from Ox - 5x amounts as desired to provide a target salinity.

[0136] The various strains were fermented in glucose media and OS, 1 S. 3S. or 5S salinities for 48 hours, and their chemical production was tested.

[0137] As seen in FIGS. 3A and 3B, many strains of halotolerant yeasts produce ethanol, and have ethanol yields, comparable to S', cerevisiae in fresh water (OS), even when those HT yeasts are at relatively high salinities (e.g., 3S, 5S). This is surprising and unexpected. Indeed, it is extremely surprising that in some cases, high salinities resulted in even greater yields than at lower salinities.

[0138] As seen in FIG. 4, the general increase in glycerol production as salinity’ increased across HT tolerant yeast strains was also surprising and unexpected. Indeed, for P. anomala and one of the D. hansenii strains the glycerol titers continued to increase as salinity increased from OS to 3S and still further on to 5S salinities. The M. farinose strain also showed large increases in g / L of glycerol when increasing from OS to 3S salinities, but had a sharp drop off above 3S.

[0139] Example 4 -Chemical Production in Xylose and Cellobiose Media

[0140] A comparison was performed, using S. cerevisiae as a control, for production of various chemicals in different media for various halotolerant species. The same strains as Example 3 were used, along with the same synthetic seawater.

[0141] The various strains were first fermented in xylose media and OS. IS, 3S, or 5S salinities for 48 hours, and their chemical production was tested.

[0142] As seen in FIG. 5A, some strains of halotolerant yeasts naturally ferment xylose, producing ethanol at levels comparable to S. cerevisiae' s fermentation of glucose.

[0143] In FIG. 5B, similar to what was seen in FIG. 4, glycerol production in certain HT yeast strains was surprisingly seen to increase with increasing media salinity.

[0144] The various strains were then fermented in cellobiose media and OS, IS, 3S, or 5S salinities for 48 hours, and their chemical production was tested.

[0145] As seen in FIG. 6, some strains of halotolerant yeasts naturally ferment cellobiose, producing ethanol at levels comparable to S. cerevisiae"s fermentation of glucose.

[0146] Example 5 - Growth Rates in various media

[0147] A comparison was performed, using S. cerevisiae as a control, for grow th rates of various halotolerant species in different media types, at varying levels of salinity. The same strains as Example 3 were used, along with the same synthetic seawater.

[0148] As seen in FIGS. 7A-7C, FIT yeasts sustain growth rates in glucose, xylose, or cellobiose media, despite increasing salinity.

[0149] Example 6 - production in minimal media

[0150] A comparison was performed, using S. cerevisiae as a control, for ethanol production of various halotolerant species in different minimal media types, at varying levels of salinity. The same strains as Example 3 were used, along with the same synthetic seawater.

[0151] As seen in FIG. 8A, after fermenting in a minimal glucose media for 72 hours, surprisingly, M. farinose, one of the D. hansenii strains, and especially P. anomala. produced ethanol comparable to S. cerevisiae, even at high salinity and a minimal media.

[0152] As seen in FIG. 8B, some HT yeast naturally ferment xylose in minimal media. After fermenting in a minimal xylose media for 60 hours, at least some HT yeast produced ethanol comparable to 5. cerevisiae" s fermentation of glucose.

[0153] As seen in FIG. 8C, some HT yeast naturally ferment cellobiose in minimal media. After fermenting in a minimal cellobiose media for 60 hours, at least some HT yeast produced ethanol comparable to S. cerevisiae" s fermentation of glucose.

[0154] Example 7 - Alginate Degradation and Growth Rates in Minimal Media

[0155] A test was performed, using S. cerevisiae as a control, for alginate degradation after fermentation for various species. As seen in FIG. 9, after 24 hours of fermentation in an alginate (1%) + glucose (2%) minimal media, it can be seen that some HT yeast naturally degrade alginate in minimal media.

[0156] Growth rates in minimal media was tested. As seen in FIG. 10A, HT yeasts sustain growth rates despite increasing media salinity in minimal glucose media. Similar results are seen in minimal xylose media, and minimal cellobiose media.

[0157] As seen in FIG. 10B, which compares 2% glucose minimal media to 2% glucose + 1% alginate minimal media, the addition of alginate increases some HT growth rates.Interestingly, the greatest beneficiaries of the alginate addition do not correspond to the strains having the greatest amount of alginate degradation (see FIG. 9).

[0158] Features of any of the examples or embodiments outlined above may be combined to create additional examples or embodiments without losing the intended effect. It should be understood that the description of an embodiment or example provided above is by way of example only, and various modifications could be made by one skilled in the art. Furthermore, one skilled in the art will recognize that numerous further modifications and combinations of various aspects are possible. Accordingly, the described aspects are intended to encompass all such alterations, modifications, and variations that fall within the scope of the appended claims.

Claims

CLAIMS1 . A method for producing chemicals using halotolerant yeast, the method comprising: culturing halotolerant yeast cells in a medium consisting of an aqueous solution of salts, a carbon source, optionally a nitrogen source, optionally a phosphate source, optionally one or more trace metals, and optionally one or more additives, the salts present in the aqueous solution at a concentration of at least 0.5 times the salinity of seawater; and allowing the halotolerant yeast cells to produce one or more chemicals.

2. The method of claim 1, wherein the halotolerant yeast cells are Debaryomyces cells. Candida cells. Pichia cells, Millerozyma cells, Blastobotrys cells, Scheffer somyces cells, or a combination thereof.

3. The method of claim 2, wherein the halotolerant yeast cells are Debaryomyces subglobosus cells, Candida taylori cells, Pichia anomala cells, Millerozyma farinosa cells, Blastobotrys adeninivorans cells, Scheffersomyces stipitis cells, or a combination thereof.

4. The method of any one of claims 1 to 3, wherein the concentration of salts (csait) is 0.5 < Csait < 5 times a salinity of seawater.

5. The method of claim 4, wherein 1 < Csait < 5 times a salinity of seawater.

6. The method of any one of claims 1 to 3, wherein the concentration of salts (csait) is at least a same salinity as seawater.

7. The method of any one of claims 1 to 6, wherein the aqueous solution of salts includes a halide, a sulfate, a hy droxide, a bicarbonate, a fluoride, or a combination thereof.

8. The method of claim 7, wherein the halide is NaCl, NaBr, MgCh, MnCh, KC1, or a combination thereof, wherein cells the sulfate includes MgSO4, MnSO-i. (NH4)2SO4, or a combination thereof, wherein the hydroxide includes KOH, or a combination thereof.

9. The method of any one of claims 1 to 8. wherein the carbon source is glucose, galactose, cellobiose, xylose, alginate, mannitol, sucrose, lactose, glycerol, or a combination thereof.

10. The method of claim 9, wherein the alginate is present in a total amount of 0.5% - 2% w / v of the medium.1 1. The method of any one of claims 1 to 10, wherein the medium consists of the aqueous solution of salts, the carbon source, the nitrogen source and / or the phosphate source, optionally the one or more trace metals, and optionally the one or more additives.

12. The method of claim 11, wherein the nitrogen source is selected from the group comprising ammonium sulfate, glutamate, amino acids, and urea.

13. The method of any one of claims 1 to 12, wherein the medium consists of the aqueous solution of salts, the carbon source, the nitrogen source and / or the phosphate source, the one or more trace metals, and optionally the one or more additives.

14. The method of claim 13, wherein the one or more trace elements are zinc, cadmium, bromine, gold, copper, iron, aluminum, silicon, lithium, or a combination thereof.

15. The method of any one of claims 1 to 14, wherein the medium consists of the aqueous solution of salts, the carbon source, the nitrogen source and / or the phosphate source, the one or more trace metals, and the one or more additives.

16. The method of claim 15, wherein the one or more additives are vitamin B2, vitamin Bl 2, inositol, yeast extract, peptone, tryptone, or a combination thereof.

17. The method of any one of claims 1 to 16, wherein culturing the halotol erant yeast cells comprises incubating the cells at a temperature between 10°C and 45°C.

18. The method of any one of claims 1 to 17, wherein culturing the halotol erant yeast cells comprises maintaining a pH between 2 and 9.

19. The method of any one of claims 1 to 18, wherein the one or more chemicals produced comprise ethanol, glycerol, acetate, xylitol, acetoin, or a combination thereof.

20. The method of claim 19, wherein the yield of the one or more chemicals produced, on a g / g basis, are equal to or greater than the yield of the one or more chemicals in the same system but with fresh water instead of the aqueous solution of salts.

21. The method of claim 1, wherein the aqueous solution of salts includes a halide, a sulfate, a hydroxide, a bicarbonate, a fluoride, or a combination thereof.

22. The method of claim 21. wherein the halide is NaCl, NaBr, MgCb, MnCh, KC1, or a combination thereof, wherein cells the sulfate includes MgSO-i. MnSCh, (NH4)2SO4, or a combination thereof, wherein the hydroxide includes KOH, or a combination thereof.

23. The method of claim 1, wherein the carbon source is glucose, galactose, cellobiose, xylose, alginate, or a combination thereof.

24. The method of claim 23, wherein the alginate is present in a total amount of 0.5% - 2% w / v of the medium.

25. The method of claim 1, wherein the medium consists of the aqueous solution of salts, the carbon source, the nitrogen source and / or the phosphate source, optionally the one or more trace metals, and optionally the one or more additives.

26. The method of claim 25, wherein the nitrogen source is selected from the group comprising ammonium sulfate, glutamate, amino acids, and urea.

27. The method of claim 1, wherein the medium consists of the aqueous solution of salts, the carbon source, the nitrogen source and / or the phosphate source, the one or more trace metals, and optionally the one or more additives.

28. The method of claim 27, wherein the one or more trace elements are zinc, cadmium, bromine, gold, copper, iron, aluminum, silicon, lithium, or a combination thereof.

29. The method of claim 1, wherein the medium consists of the aqueous solution of salts, the carbon source, the nitrogen source and / or the phosphate source, the one or more trace metals, and the one or more additives.

30. The method of claim 29, wherein the one or more additives are vitamin B2, vitamin Bl 2, inositol, yeast extract, peptone, tryptone, or a combination thereof.

31. The method of claim 1 , wherein culturing the halotol erant yeast cells comprises incubating the cells at a temperature between 10°C and 45°C.

32. The method of claim 1, wherein culturing the halotolerant yeast cells comprises maintaining a pH between 2 and 9.

33. The method of claim 1, wherein the one or more chemicals produced comprise ethanol, glycerol, acetate, xylitol, acetoin, or a combination thereof.

34. The method of claim 33, wherein the yield of the one or more chemicals produced, on a g / g basis, are equal to or greater than the yield of the one or more chemicals in the same system but with fresh water instead of the aqueous solution of salts.

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