Compositions and methods for biomass processing

Abstract

The present invention provides systems and methods for biomass processing. In particular, provided herein are coupled biomass systems that generate energy and algae from fermentation reaction products.

Description

[0001] HHAS-43824.601

[0002] COMPOSITIONS AND METHODS FOR BIOMASS PROCESSING

[0003] Cross-Reference to Related Applications

[0004] The present application claims priority to U.S. Provisional Application No. 63 / 699,356, filed September 26, 2024; U.S. Provisional Application No. 63 / 701,639, filed October 1, 2024; and U.S. Provisional Application No. 63 / 777,985, filed March 26, 2025, which are incorporated herein by reference in their entireties.

[0005] Field

[0006] The present invention provides systems and methods for biomass processing. In particular, provided herein are coupled biomass systems that generate energy and algae from fermentation reaction products.

[0007] Background

[0008] Microalgae or microphytes are microscopic algae. They are phytoplankton typically found in freshwater and marine systems, living in both the water column and sediment. They are unicellular species which exist individually, or in chains or groups. Depending on the species, their sizes can range from a few micrometers (pm) to a few hundred micrometers. Unlike higher plants, microalgae do not have roots, stems, or leaves. They are specially adapted to an environment dominated by viscous forces.

[0009] The biodiversity of microalgae is enormous and they represent an almost untapped resource. It has been estimated that about 200,000-800,000 species in many different genera exist of which about 50,000 species are described. Over 15,000 novel compounds originating from algal biomass have been chemically determined. Examples include carotenoids, fatty acids, enzymes, polymers, peptides, toxins, and sterols. Besides providing these valuable metabolites, microalgae is regarded as a potential feedstock for biofuels and has also emerged as a promising microorganism in bioremediation.

[0010] A range of microalgae species are produced in hatcheries and are used in a variety of ways for commercial purposes, including for human nutrition, as biofuel, in the aquaculture of other organisms, in the manufacture of pharmaceuticals and cosmetics, and as biofertilizer. However, the low cell density is a major bottleneck in commercial viability of many microalgae derived products, especially low cost commodities.

[0011] Additional methods for producing microalgae are needed. HHAS-43824.601

[0012] Summary of the Invention

[0013] The present invention provides systems and methods for biomass processing. In particular, provided herein are coupled biomass systems that generate energy and algae from fermentation reaction products. The systems and methods described herein improve the efficiency of microalgae and incineration plants while also aiding in waste treatment and resource recovery.

[0014] Accordingly, in some embodiments, the present invention provides a system for integrated waste processing and carbon dioxide capture, comprising: a hyperthermophilic fermentation bioreactor comprising at least one thermophilic bacteria, wherein the hyperthermophilic fermentation bioreactor is configured to ferment a biomass using the thermophilic bacteria to generate fermented solids, fermented liquids, H2, and CO2; an incineration plant or biogas plant configured to receive the fermented solids and optionally other materials and generate heat and CO2; and a mixotrophic microalgae cultivation bioreactor comprising one or more microalgae species configured to receive fermented liquids and the CO2 and generate microalgae.

[0015] In some embodiments, the system further comprises a transport component that transports fermented solids, liquids, and gas from the hyperthermophilic fermentation bioreactor to the incineration plant and / or the mixotrophic microalgae cultivation bioreactor. In some embodiments, the transport component is tubing.

[0016] The present disclosure is not limited to particular thermophilic bacteria. Examples include but are not limited to Thermotoga sp. (e.g., Thermotoga petrophila).

[0017] In some embodiments, the mixotrophic microalgae cultivation bioreactor uses acetic acid in fermented liquids as a heterotrophic carbon source for microalgae cultivation. In some embodiments, the mixotrophic microalgae cultivation bioreactor performs mixotrophic cultivation of Chlorella species (e.g., Chlorella pyrenoidosa. Chlorella vulgaris, or Chlorella sorokiniana), although other microalgae are specifically contemplated. In some embodiments, the mixotrophic microalgae cultivation bioreactor comprises a light source.

[0018] In some embodiments, the incineration plant or biogas plant produces CO2 flue gas that is transported to the mixotrophic microalgae cultivation bioreactor.

[0019] In some embodiments, the hyperthermophilic fermentation bioreactor comprises one or more of a mixer, a heat exchanger, and a separator. In some embodiments, the hyperthermophilic fermentation bioreactor is a dark reactor. The present disclosure is not limited to particular fermentation temperatures. In some embodiments, the hyperthermophilic HHAS-43824.601 fermentation bioreactor is at a temperature range of from 70.0 to 105.0°C, 70.0 to 100.0°C, 70.0 to 90.0°C, 70.0 to 85.0°C, 75.0 to 100.0°C, 75.0 to 90.0°C, 75.0 to 85.0°C, 76.0 to 84.0°C, 77.0 to 83.0°C, 78.0 to 82.0°C, 75.0 to 79.0°C, or 76.0 to 79.0°C. For example, in some embodiments, the temperature is 80°C or about 80°C.

[0020] In some embodiments, the system further comprises a microalgae harvesting and processing unit.

[0021] The present disclosure is not limited to particular sources of biomass. Examples include but are not limited to, sewage, agricultural waste products, brewery grain byproducts, food waste, forestry waste, crops, grass, seaweed, plankton, algae, fish, fish waste, com, potato waste, sugar cane waste, sugar beet waste, straw, paper waste, chicken manure, fish manure, fish sludge, cow manure, hog manure, switchgrass or combinations thereof.

[0022] Further embodiments provide a process for integrated waste processing and carbon dioxide capture, comprising: fermenting a biomass a in a hyperthermophilic fermentation bioreactor comprising at least one thermophilic bacteria to generate fermented solids, fermented liquids, and CO2; transferring fermented solids and optionally additional materials to an incineration plant or biogas plant that generates heat and CO2 from the fermented solids; and transferring fermented liquids and CO2 to a mixotrophic microalgae cultivation bioreactor comprising one or more bacteria species that generates microalgae from fermented liquids and CO2. In some embodiments, the process further comprises separating microalgae from cultivation medium and recirculating the remaining liquid to the mixotrophic microalgae cultivation bioreactor. In some embodiments, the hyperthermophilic fermentation bioreactor performs fermentation in the presence of a cell density of at least one genus of a hyperthermophilic organism of greater than 109cell / ml.

[0023] Additional embodiments are described herein.

[0024] Brief Description of the Drawings

[0025] FIG. 1. Schematic depiction of systems of embodiments of the present disclosure.

[0026] FIG. 2A-B. A. Optical density of C. sorokiniana measured with a spectrophotometer at a wavelength of 680 nm; B. Cell counts using a hemocytometer. The data represent the mean ± SE from three biological replicates.

[0027] FIG. 3. Biomass of C. sorokiniana measured on day 13 in different media treatments. The data represents the mean ± SE from three biological replicates. HHAS-43824.601

[0028] Detailed Description

[0029] The present invention provides systems and methods for biomass processing. In particular, provided herein are coupled biomass systems that generate energy and microalgae from fermentation reaction products. Exemplary systems are shown in FIG. 1 and described in detail below. The systems and methods described herein improve the efficiency of microalgae and incineration plants, reduce contamination risk, provide for recycling and recirculation through the system, and maximize energy and resource recovery from biomass waste.

[0030] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

[0031] 1. Definitions

[0032] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0033] For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0034] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0035] As used herein, the term “biomass” refers to biological material which can be used as fuel, for energy production, or for industrial production. Most commonly, biomass refers to plant matter grown for use as biofuel, but it also includes plant or animal matter used for production of fibers, chemicals, or heat. Biomass may also include biodegradable wastes that HHAS-43824.601 can be used as fuel. It is usually measured by dry weight. The term biomass is useful for plants, where some internal structures may not always be considered living tissue, such as the wood (secondary xylem) of a tree. This biomass became produced from plants that convert sunlight into plant material through photosynthesis. Sources of biomass energy lead to agricultural crop residues, energy plantations, and municipal and industrial wastes. The term “biomass,” as used herein, excludes components of traditional media used to culture microorganisms, such as purified starch, peptone, yeast extract but includes waste material obtained during industrial processes developed to produce purified starch. According to the invention, biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of com cobs and com stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, cellulose and / or hemicellulose containing waste, yard waste, wood, and forestry waste. Examples of biomass include, but are not limited to, com grain, com cobs, crop residues such as com husks, com stover, com steep liquor, grasses, wheat, wheat straw, barley, barley straw, grain residue from barley degradation during brewing of beer, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from processing of grains, trees, branches, roots, leaves, wood chips, sawdust, shmbs and bushes, soybean hulls, vegetables, fruits, flowers and animal manure. In one embodiment, biomass that is useful for the invention includes biomass that has a relatively high carbohydrate value, is relatively dense, and / or is relatively easy to collect, transport, store, and / or handle.

[0036] As used herein, the term “bioreactor” refers to an enclosed or isolated system for containment of a microorganism and a biomass material. The “bioreactor” may preferably be configured for anaerobic growth of the microorganism.

[0037] As used herein, the term “hyperthermophilic organism” means an organism which can grow at or above 80°C. It will be understood that hyperthermophilic organisms may be used for fermentations at temperatures above or below 80°C, or above or below the optimum temperature for the culture of the hyperthermophilic organism.

[0038] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. HHAS-43824.601

[0039] 2. Biomass

[0040] The present invention contemplates the degradation of biomass with hyperthermophilic organisms. The present invention is not limited to the use of any particular biomass or organic matter. Suitable biomass and organic matter includes, but is not limited to, sewage, agricultural waste products, brewery grain by-products, food waste, organic industry waste, forestry waste, crops, grass, seaweed, plankton, algae, fish, fish waste, com potato waste, sugar cane waste, sugar beet waste, straw (e.g., wheat, rice, oat or barley straw), hay, fish sludge, fish manure, fish waste, paper mill waste, chicken manure, cow manure, hog manure, switchgrass, and marine or fresh water algas such as cyanobacteria (blue-green algae), green algae (division Chlorophyta), brown algae (Phaeophyceae, division Phaeophyta), and red algae (division Rhodophyta) and combinations thereof. In some embodiments, the biomass is harvested particularly for use in hyperthermophilic degradation processes, while in other embodiments waste or by-products materials from a pre-existing industry are utilized.

[0041] In some preferred embodiments, the biomass is lignocellulosic. In some embodiments, the biomass is pretreated with cellulases or other enzymes to digest the cellulose. In some embodiments, the biomass is pretreated by heating in the presence of a mineral acid or base catalyst to completely or partially hydrolyze hemicellulose, decrystallize cellulose, and remove lignin. This allows cellulose enzymes to access the cellulose.

[0042] 3. Hyperthermophilic organisms

[0043] The present invention contemplates the use of hyperthermophilic organisms for fermenting biomass. The present invention is not limited to the use of any particular hyperthermophilic organism. While the present disclosure is illustrated with Thermotoga petrophila. additional hyperthermophilic organisms are specifically contemplated.

[0044] In some embodiments, mixtures of hyperthermophilic organisms are utilized. In some embodiments, the hyperthermophiles are from the archaeal order Thermococcales. including but not limited to hyperthermophiles of the genera Pyrococcus, Thermococcus, and Palaeococcus. Examples of particular organisms within these genera include, but are not limited to, Pyrococcus furiosus, Thermococcus harophilus, T. aggregans, T. aegaeicus, T. litoralis, T. alcaliphilus, T. sihiricus, T. atlanticus, T. siculi, T. pacificus, T. waiotapuensis, T. zilligi, T. guaymasensis, T. fumicolans, T. gorgonarius, T. celer, T. harossii, T. HHAS-43824.601 hydrothermalis, T. acidaminovorans, T. profundus, T. stetteri, T. kodakaraenis, T. peptonophilis . In some embodiments, aerobic hyperthermophilic organisms such as Aeropyrum pernix, Sulfolobus solfataricus, Metallosphaera sedula, Sulfolobus tokadciii, Sulfolobus shibatcie, Thermoplasma acidophilum and Thermoplasma volcanium are utilized. While in other embodiments, anaerobic or facultative aerobic organisms such as Pyrobaculum calidifontis and Pyrobaculum oguniense are utilized. Other useful archaeal organisms include, but are not limited to, Sulfolobus acidocaldarius and Acidianus ambivalens. In some embodiments, the hyperthermophilic organisms are bacteria, such as Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermus ruber, Bacillus caldotenax, Geobacillus stearothermophilus, Anaerocellum thermophilus, Thermoactinomyces vulgaris, and members of the order Thermotogales, including, but not limited to Thermotoga elfeii, Thermotoga hypogea, Thermotoga maritima, Thermotoga neapolitana, Thermotoga subterranea, Thermotoga petrophila, Thermotoga thermarum, Petrotoga miotherma, Petrotoga mobilis, Thermosipho africanus, Thermosipho melanesiensis, Fervidobacterium islandicum, Fervidobacterium nodosum, Fervidobacterium pennavorans, Fervidobacterium gondwanense, Geotoga petraea, Geotoga subterranea. In some preferred embodiments, the microorganism preferably is a member of the genus Thermotoga.

[0045] 4. Microalgae

[0046] The present application provides for culture of microalgae. The present invention is not limited to the culture of particular species of microalgae. For example, in some embodiments, the microalgae is selected from Chlorella sp. (e.g., Chlorella pyrenoidosa, Chlorella vulgaris, or Chlorella sorokiniana), Phaeodactylum tricornutum, Nannochloropsis salina, Porphyridium cruentum, Tetraselmis chuii, Botryococcus braunii, Chlamydomonas reinhardtii, Spirulina platensis, Synechocystis sp., Dunaliella salina, and Haematococcus pluvialis.

[0047] 5. Systems and processes

[0048] In some preferred embodiments, the present invention provides coupled processes for biomass utilization and microalgae fermentation. Exemplary systems are illustrated in FIG. 1. In some embodiments, a biomass is first subjected to a hyperthermophilic fermentation with a hyperthermophilic organism in a hyperthermophilic reactor to generate fermentation liquids, fermentation solids, H2, and CO2. As shown in FIG. 1, in some HHAS-43824.601 embodiments, the hyperthermophilic reactors comprise one or more of a mixer, a heat exchanger, and a reactor.

[0049] The use of a wide variety of bioreactors, also known as biodigesters, is contemplated. Examples include, but are not limited to, floating drum digesters, fixed dome digesters, Deenbandhu digesters, bag digesters, plug flow digesters, anaerobic filters, upflow anaerobic sludge blankets, and pit storage digesters. In some embodiments, the volume of the first bioreactor is from 10 m3to 10000 m3, and most preferably from 100 m3to 10000 m3.

[0050] In some preferred embodiments, the step of fermenting a biomass with a population of at least one genus of a hyperthermophilic organism to provide a pretreated biomass is performed at a temperature range of from 70.0 to 105.0°C, 70.0 to 100.0°C, 70.0 to 90.0°C, 70.0 to 85.0°C, 75.0 to 100.0°C, 75.0 to 90.0°C, 75.0 to 85.0°C, 76.0 to 84.0°C, 77.0 to 83.0°C, 78.0 to 82.0°C, 75.0 to 79.0°C, or 76.0 to 79.0°C.

[0051] Systems of the present disclosure are illustrated in Figure 1. Referring to FIG. 1, a hyperthermophilic reactor (100) is operably linked (e.g., via tubing or other conduit, not shown)) to a microalgae bioreactor (200) and optionally to a plant (300) such as an incineration plant or a biogas plant. The present disclosure is not limited to particular types of incinerators. In some embodiments, the incinerator is commercially available. The present disclosure is not limited to particular types of biogas plants. Any suitable design may be utilized, including commercially available systems (e.g., from Hyperthermics AS, Ulsteinvik, Norway) or biodigesters as described above. In some embodiments, the incinerator or biogas plant additionally accepts or utilizes other materials in addition to fermented solids (e.g., additional source of biomass or biowaste and the like).

[0052] In some embodiments, biomass (105) is introduced into a mixer (110) in operable connection (e.g., in fluid communication) with a first heat exchanger (115) and optionally a second heat exchanger (120). The first heat exchanger (115) and / or the second heat exchanger (120) are in in turn operably connected to the hyperthermophjlic reactor (100) so that the biomass (105) may be introduced into the hyperthermophlic reactor (100) via one or both of heat exchangers (115) and (120).

[0053] In some embodiments, the systems further comprise a first separator (125). In some embodiments, fermentation products from the hyperthermophilic reactor (100), such as fermentation solids and fermentation liquids, are optionally transported to heat exchanger (115) and then to separator (125) to separate fermentation liquids from fermentation solids. In some embodiments, the fermentation liquids comprise acetic acid and / or hydrolyzed sugars (e.g., glucose, arabinose, xylose). HHAS-43824.601

[0054] In some embodiments, the fermentation liquids are then transported to the microalgae bioreactor (200) where the components of the formation liquid are utilized by microalgae for growth. In some embodiments, the microalgae reactor comprises a light source. In some embodiments, the hyperthermophilic reactor (100), separator (125), and microalgae bioreactor are operably commented (e.g., in fluid communication). In some embodiments, the systems comprise a second separator (205) downstream from and operably connected to the microalgae bioreactor (200). The separator (200) allows harvesting of microalgae (210) produced in the microalgae bioreactor (200). In some embodiments, microalgae cell residue is recirculated into the hyperthermophilic reactor. In some embodiments, the separator also provides a microalgae cell residue circulate (215) comprising components (e.g., buffers and / or carbohydrates) that can be introduced into the hyperthermophilic bioreactor (100), for example via introduction at mixer (110). In some embodiments, the systems may further comprise a CO2 post-scrubber downstream from the microalgae bioreactor (200). The postscrubber preferably removes residual CO2 and allows for recirculation of residual CO2 to the microalgae bioreactor (200).

[0055] In some embodiments, fermentation solids are transported to the plant (300), which may preferably be an incinerator or biogas plant, to generate energy and CO2 (e.g., from flue gas) for use in microalgae fermentation. In some embodiments, the plant (300) comprises one or more flues (305). In some embodiments, CO2 from the plant 300, for example flue gas from flue (305) is introduced to the microalgae bioreactor (200) where it is utilized by the microalgae. In some embodiments, the plant (300) is operably connected to microalgae bioreactor (200), for example, by pipes or tubing. In some embodiments, the flue gas is directly introduced into microalgae bioreactor (200), optionally via a cooler and drier (310). In some embodiments, the system comprises one or more scrubbers between the plant (300) and the microalgae bioreactor (200) so that scrubbed CO2 may be introduced into the microalgae bioreactor (200).

[0056] Example 1

[0057] This example provides data demonstrating that supplementing algae growth media with fermentation fluid (FF) from a hyperthermophilic fermentation enhances algal growth under mixotrophic conditions.

[0058] Materials and Methods HHAS-43824.601

[0059] Residual fermentation fluid. Two fermentation fluids (FFs) were supplied by the biogas production company Hyperthermics. The first fermentation fluid (FF1) was derived from 5% barley straw, centrifuged, and filtered through a 0.22 pm membrane. It had a pH of 7.8, a conductivity of 10.01 mScm'1(18 PSU), an acetic acid concentration of 22.7 mM, and an ammonium chloride (NH4CI) concentration of less than 0.5 g / L. The second fermentation fluid (FF2) originated from 75% vinasse. It had a pH of 7.5, a conductivity of 34.81 mS / cm (62 PSU), and contained acetic acid at a concentration of 44. 1 mM. Both fermentation fluids may contain glucose, arabinose, xylose, proteins, and carbonic acid.

[0060] Pre-trial. A pre-trial was conducted to determine the appropriate concentrations of fermentation fluid for the experiment. C. sorokinianci was cultivated in BG- 11 media supplemented with 10%, 20%, 40%, and 60% of both FFs. Cultures were grown in culture tubes and placed on a shaker under continuous light (24 hours) at a temperature of 19-20°C. Visual observations were made to assess the concentrations that supported the growth of C. sorokinianci. Two of the best preforming concentrations of were selected from each FF to be used in the trial.

[0061] Inoculum Preparation. Seed cultures were prepared by inoculating 10 m of C. sorokiniana into a 250 m Erlenmeyer flask containing 90 mb of BG-11 media. Seed cultures were incubated photoautotrophically at a room temperature of 19-21 °C with a photoperiod of an 18:6 light-dark cycle.

[0062] Experimental set up. BG-11 media supplemented with FF was sterilized by autoclaving. Experimental cultures were prepared by inoculating 5 mL of C. sorokiniana into 45 mL of media in a 250 mL single-use culture flask (Fig. 1A.). Cultures were incubated photoautotrophically at a room temperature of 19-21 °C with a photoperiod of an 18:6 lightdark cycle.

[0063] FF was added as a carbon source at four different concentrations: 40% FF1, 60% FF1, 10% FF2, and 20% FF2, based on the results from the pre-trial. Each concentration was tested in triplicate, and a control group without FFs (BG-11 only) was included. Cultures were shaken by hand when sampling, once a day, and growth rates were measured at the same time each day. Sampling was conducted under a laminar flow hood using aseptic techniques to ensure sterility.

[0064] Growth rates and biomass. Growth rates were monitored using optical density at 680 nm with a spectrophotometer. Media with different concentrations of FF were used as blanks before measuring the samples. Cultures were diluted prior to measuring with spectrophotometer when they reached an optical density higher than 1.2. Cells were visually HHAS-43824.601 observed (Fig. IB.) and counted using a haemocytometer on day 2, 5 and 7. The cultures were maintained until the end of the exponential growth phase, where optical density plateaued.

[0065] The specific growth rate (p, day-1) was calculated using the equation (1), where N1 and N2 were the optical density of cells at the time period (tl) and (t2).

[0066] Biomass was determined by measuring dry weight on day 13. A 5 mb sample of culture was filtered through a GFF filter, rinsed with PBS buffer, and dried in an oven (Fig. 1C.).

[0067] Statistical analysis. One-way ANOVA was performed on biomass result. Shapiro- wilk normality test was conducted to ensure the distribution of normality was met prior to conducting one-way ANOVA.

[0068] Results and Discussion

[0069] The results of this example demonstrate that supplementing BG-11 media with FF significantly enhances the growth parameters of C. sorokinianci under mixotrophic conditions compared to photoautotrophic growth in standard BG-11 media. The optical density, specific growth rates, cell counts, and biomass concentration were all higher in cultures supplemented with FF, indicating that the addition of FF provides a more favorable environment for microalgal growth.

[0070] Enhanced growth parameters in mixotrophic mode. The optical density of C. sorokiniana was higher in all FF-supplemented treatments compared to the control (Fig. 2A.), with the highest optical density (4. 1 ± 0.31) observed on day 13 in 10% FF2. This suggests that the mixotrophic mode of nutrition, which combines photosynthesis and organic carbon utilization, is more efficient for C. sorokinicma growth than photoautotrophy alone. The specific growth rates further support this observation, with the highest rates occurring between days 2 and 5 in 20% FF2 (1.35 day1± 0.058), followed by 10% FF1 (0.84 day1± 0.016) and 40% FF1 (0.72 day1± 0.007). These rates are substantially higher than the control (0.47 day '). indicating that FF supplementation accelerates growth during the exponential phase.

[0071] Contrary to optical density results, the higher cell counts were observed in cultures supplemented both types of FF, with 59.4 M cells / ml ± 1.79 when grown in 40% FF1 and 55.3 M cells / ml ± 3.93 when grown in 10% FF2 on day 7 (Fig. 2B.). These values were 20- 30% higher than the control, highlighting the potential of both types of FFs to enhance cell division and growth. HHAS-43824.601

[0072] Increased biomass production with fermentation fluid supplemented media. In line with the cell counts and optical density result, the dry biomass was higher in FF- supplemented media compared to the control, with the highest biomass productivity values of 0.160 g l / day’1± 0.001 and 0.159 g L’May1± 0.002 achieved in 10% FF2 and 20% FF2 respectively. The biomass productivity value of 40% FF1 was 0.152 g L' ay'1± 0.006, which was comparable to the biomass productivity values from FF2. These results represent a 65% higher biomass productivity over the control 0.096 g L' ay'1± 0.002 (Fig. 3), underscoring the potential of both FFs to boost biomass yield. The ANOVA results (F- statistic = 60.46, p-value < 0.001) confirm that the differences in biomass among the treatments are statistically significant, further validating the efficacy of FF supplementation.

[0073] Implications of Fermentation Fluid Supplementation. The higher growth rates and biomass productivity of C. sorokiniana in FF-supplemented media is likely attributed to the presence of organic carbon sources, such as barley extract, vinasse, acetic acid, and nitrogen sources such as ammonium chloride in the FF. Although barley extract and vinasse have not been reported as a carbon source for mixotrophic cultivation of Chlorella sp., acetic acid has been successfully used to cultivate Chlorella sp. in mixotrophic and heterotrophic conditions (Kumar et al., 2014; Li et al., 2022). The presence of ammonium in cultivation media can be detrimental to some microalgal species but beneficial to others that can utilize it in low concentrations (Metin & Altmbas. 2024). However, Chlorella sp. is highly resistant to ammonium and can utilize it as a nitrogen source (Ahmed et al., 2017; Tam & Wong, 1996). Despite the utilization of some nutrients during biogas production, the remaining nutrients in the FF were sufficient to enhance the growth and biomass production of C. Sorokiniana.

[0074] Chlorella sp. has been cultivated in a variety of residual waste, including aquaculture wastewater (Lugo et al., 2020), dairy wastewater (Hamidian & Zamani, 2022), and molasses (Qurat-ul-Ain et al., 2022). Our results suggest that FF2 is a promising addition for supplementing the growth of C. sorokiniana for mixotrophic growth. This is supported by the fact that the biomass productivity of C. sorokiniana cultivated in 10% and 20% FF2 was 25% higher than in other reported lab-scale experiments where C. sorokiniana was grown in BG- 11 media supplemented with 0.2% (w / v) sugarcane molasses (Qurat-ul-Ain et al., 2022). Additionally, the dry biomass yield from 10% and 20% FF2 outperformed that from dairy wastewater by 36% (Hamidian & Zamani, 2022).

[0075] The results also suggest that the concentration and type of FF play a critical role in optimizing growth, with 10% and 20% FF2 emerging as the most effective formulation. However, it would be premature to conclude that vinasse present in FF2 is a superior carbon HHAS-43824.601 source compared to barley extract, which is present in FF1, given the variations in concentrations of barley, vinasse, and acetic acid present in FF. This is because excessive carbon can reduce growth rates of Chlorella, which may explain our results where C. sorokiniana displayed a decrease in biomass production and lower growth rates when growing in 40% FF1 compared to 60% FF1. Furthermore, the types and concentrations of carbon sources have been shown to influence the protein and lipid composition of Chlorella sp. (Cho, 2011; Zhang et al., 2014). Therefore, the type of fermentation waste selected should be tailored to achieve the desired composition of C. sorokiniana.

[0076] In conclusion, the supplementation of BG- 11 media with fermentation fluids significantly enhances the growth parameters of C. sorokiniana under mixotrophic conditions. The highest optical density, specific growth rates, and biomass concentration were observed in cultures supplemented with 10% FF2 and 20% FF2, whereas the highest cell counts were observed in 10% FF2 and 40% FF1, demonstrating its potential as a sustainable, cost-effective strategy for improving microalgal cultivation.

[0077] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0078] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

HHAS-43824.601ClaimsWhat is claimed is:

1. A system for integrated waste processing and carbon dioxide capture, comprising: a hyperthermophilic fermentation bioreactor comprising at least one thermophilic bacteria, wherein said hyperthermophilic fermentation bioreactor is configured to ferment a biomass using said hyperthermophilic bacteria to generate fermented solids, fermented liquids, and CO2; a plant that produces CO2; and a mixotrophic microalgae cultivation bioreactor comprising one or more microalgae species configured to receive said fermented liquids from said hyperthermophilic bioreactor and said CO2 from said plant to grow microalgae.

2. The system of claim 1, wherein said plant is an incineration plant or biogas plant configured to receive said fermented solids and generate heat and / or CO2, wherein is said CO2 is utilized in said mixotrophic microalgae cultivation bioreactor.

3. The system of claims 1 or 2, further comprising a transport component that transports fermented solids and liquids from said hyperthermophilic fermentation bioreactor to said incineration reactor and / or said mixotrophic microalgae cultivation bioreactor.

4. The system of claim 3, wherein said transport component is tubing.

5. The system of any one of the preceding claims, wherein said thermophilic bacteria is a Thermotoga strain.

6. The system of any one of the preceding claims, wherein said hyperthermophilic fermentation bioreactor operates in the dark.

7. The system of any one of the preceding claims, wherein said fermented liquids comprise acetic acid and hydrolyzed sugars and said mixotrophic microalgae cultivation bioreactor uses said acetic acid and / or hydrolyzed sugars as a heterotrophic carbon source for microalgae cultivation.HHAS-43824.6018. The system of any one of the preceding claims, wherein said mixotrophic microalgae cultivation bioreactor performs mixotrophic cultivation of Chlorella species.

9. The system of claim 7, wherein said Chlorella species is selected from the group consisting of Chlorella pyrenoidosa. Chlorella vulgaris, and Chlorella sorokiniana.

10. The system of any one of the preceding claims, wherein said plant produces CO2 flue gas that is transported to said mixotrophic microalgae cultivation bioreactor.

11. The system of any one of the preceding claims, wherein said mixotrophic microalgae cultivation bioreactor comprises a light source.

12. The system of any one of the preceding claims, wherein said hyperthermophilic fermentation bioreactor comprises one or more of a mixer, a heat exchanger, and a separator.

13. The system of any one of the preceding claims, wherein the biomass is selected from the group consisting of sewage, agricultural waste products, brewery grain byproducts, food waste, forestry waste, crops, grass, seaweed, plankton, algae, fish, fish waste, com, potato waste, sugar cane waste, sugar beet waste, straw, paper waste, chicken manure, fish manure, fish sludge, cow manure, hog manure, switchgrass and combinations thereof.

14. The system of any one of the preceding claims, wherein the hyperthermophilic fermentation bioreactor is at a temperature range of from 70.0 to 105.0°C, 70.0 to 100.0°C, 70.0 to 90.0°C, 70.0 to 85.0°C, 75.0 to 100.0°C, 75.0 to 90.0°C, 75.0 to 85.0°C, 76.0 to 84.0°C, 77.0 to 83.0°C, 78.0 to 82.0°C, 75.0 to 79.0°C, or 76.0 to 79.0°C.

15. The system of any one of the preceding claims, wherein the system further comprises a microalgae harvesting and processing unit.

16. The system of any one of the preceding claims, wherein said incineration plant or biogas plant is further configured to accept additional materials.HHAS-43824.60117. A process for integrated waste processing and carbon dioxide capture, comprising: fermenting a biomass in a hyperthermophilic fermentation bioreactor comprising at least one hyperthermophilic bacteria to generate fermented solids, fermented liquids, and CO2; producing CO2 at a plant; and transferring said fermented liquids and said CO2 from said plant and / or said hyperthermophilic fermentation bioreactor to a mixotrophic microalgae cultivation bioreactor comprising one or more microalgae species.

18. The process of claim 17, wherein said step (b) further comprises transferring said fermented solids to an incineration plant or biogas plant that generates heat and CO2 from said fermented solids.

19. The process of claims 17 or 18, further comprising separating microalgae from cultivation medium and recirculating the remaining liquid to said mixotrophic microalgae cultivation bioreactor.

20. The process of any one of the preceding claims, further comprising a transport component that transports fermented solids and liquids from said hyperthermophilic fermentation bioreactor to said incineration reactor and / or said mixotrophic microalgae cultivation bioreactor.

21. The process of claim 20, wherein said transport component is tubing.

22. The process of any one of the preceding claims, wherein said hyperthermophilic fermentation bioreactor comprises one or more of a mixer, a heat exchanger, and a separator.

23. The process of any one of the preceding claims, wherein said thermophilic bacteria is Thermotoga petrophila.HHAS-43824.60124. The process of any one of the preceding claims, wherein said mixotrophic microalgae cultivation bioreactor uses acetic acid in said fermented liquids as a heterotrophic carbon source for microalgae cultivation.

25. The process of any one of the preceding claims, wherein said mixotrophic microalgae cultivation bioreactor performs mixotrophic cultivation of Chlorella species.

26. The process of claim 25, wherein said Chlorella species is selected from the group consisting of Chlorella pyrenoidosa. Chlorella vulgaris, and Chlorella sorokiniana.

27. The process of any one of the preceding claims, wherein said incineration plant or biogas plant produces CO2 flue gas that is transported to said mixotrophic microalgae cultivation bioreactor.

28. The process of any one of the preceding claims, wherein said mixotrophic microalgae cultivation bioreactor comprises a light source.

29. The process of any one of the preceding claims, wherein the biomass is selected from the group consisting of sewage, agricultural waste products, brewery grain byproducts, food waste, forestry waste, crops, grass, seaweed, plankton, algae, fish, fish waste, com, potato waste, sugar cane waste, sugar beet waste, straw, paper waste, chicken manure, fish manure, fish sludge, cow manure, hog manure, switchgrass and combinations thereof.

30. The process of any one of the preceding claims, wherein the hyperthermophilic fermentation bioreactor is at a temperature range of from 70.0 to 105.0°C, 70.0 to 100.0°C, 70.0 to 90.0°C, 70.0 to 85.0°C, 75.0 to 100.0°C, 75.0 to 90.0°C, 75.0 to 85.0°C, 76.0 to 84.0°C, 77.0 to 83.0°C, 78.0 to 82.0°C, 75.0 to 79.0°C, or 76.0 to 79.0°C.

31. The process of claim 28, wherein hyperthermophilic fermentation bioreactor is at a temperature of 80 C.

32. The process of any one of the preceding claims, wherein the hyperthermophilic fermentation bioreactor performs fermentation in the presence of a cell density of the at least one genus of a hyperthermophilic organism of greater than 107cell / ml.HHAS-43824.60133. The process of any one of the preceding claims, further comprising harvesting and processing said microalgae using a microalgae harvesting and processing unit.

34. The process of any one of the preceding claims, wherein said hyperthermophilic fermentation bioreactor operates in the dark.

35. The process of any one of the preceding claims, wherein said incineration plant or biogas plant further accepts additional materials.

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