Method for producing alcohol

Abstract

The purpose of the present disclosure is to provide a method for efficiently producing an alcohol from a dry large alga as a raw material. A method according to the present disclosure includes (a) performing alcohol fermentation in a culture medium containing a saccharified product of a dry large alga by using an alcohol-fermenting microorganism, wherein the water content of the dry large alga is 20 wt% or less. In one embodiment, the method may further include, as a step performed before step (b) or simultaneously with step (b), (a) performing a saccharification treatment of the dry large alga by using at least one saccharification enzyme to obtain the saccharified product.　

Description

Methods for producing alcohol

[0001] The present invention relates to a method for producing alcohol. This application claims priority based on Japanese Patent Application No. 2024-226292, filed with the Japan Patent Office on December 23, 2024, the contents of which are incorporated herein by reference for all purposes and constitute part of this specification.

[0002] In recent years, against the backdrop of global population growth, depletion of fossil fuel resources, and global warming, the development of material production technologies utilizing microbial reactions that can enable sustainable material production as an alternative to petrochemical technology has been actively pursued. While such microbial material production technologies mainly utilize terrestrial biomass resources as raw materials, the use of marine biomass, which is abundant on Earth, has recently been attracting attention.

[0003] For example, Patent Document 1 discloses a method for producing ethanol from seaweed by culturing microorganisms capable of producing ethanol from sugars and microorganisms capable of producing ethanol from sugar alcohols in a culture medium using a saccharified solution obtained by treating seaweed with alginate lyase and cellulase as a culture medium, and carrying out a fermentation reaction. Specifically, Patent Document 1 describes the actual production of ethanol by liquefying and saccharifying kelp flakes using alginate lyase and cellulase to prepare a kelp saccharified solution, and then using this saccharified solution as a substrate and carrying out alcohol fermentation using budding yeast and mannitol-assimilating microorganism AW strain isolated by the inventor.

[0004] Furthermore, Patent Document 2 discloses a method for producing ethanol using mannitol as a raw material by utilizing yeast that has the ability to assimilate mannitol and can produce ethanol from mannitol. Specifically, Patent Document 2 describes the isolation of various ethanol-producing yeasts that have the ability to assimilate mannitol and the production of ethanol by culturing these yeasts in a mannitol synthesis medium, but it does not describe the actual production of ethanol using marine biomass resources. Patent Document 2 suggests that ethanol can be produced using the above-mentioned yeast with macroalgae containing alginic acid as a component as a raw material.

[0005] Furthermore, Non-Patent Literature 1 describes the creation of a recombinant Saccharomyces cerevisiae strain conferring alginate assimilation by introducing heterologous genes encoding endo-type alginate lyase, which decomposes alginate into oligosaccharides, exo-type alginate lyase, which further decomposes the above oligosaccharides into monosaccharides, and predetermined metabolic enzymes. It also describes the breeding of a strain (AM1 strain) that further acquired mannitol assimilation in addition to alginate assimilation, and the production of ethanol by culturing AM1 strain in an artificial medium containing alginate and mannitol. Although Non-Patent Literature 1 does not describe the actual production of ethanol using marine biomass resources such as algae, it suggests that ethanol can be produced from brown algae using the above-mentioned alginate / mannitol assimilation yeast, AM1 strain.

[0006] Furthermore, Non-Patent Document 2 describes how a recombinant Saccharomyces cerevisiae strain capable of alginate monomer / mannitol utilization was obtained by introducing various heterologous genes encoding alginate monomer (4-deoxy-L-erythro-5-hexoceuroseuronic acid) transporters, etc. Although it does not describe the actual production of ethanol using brown algae, it suggests the flow of a biorefinery process for ethanol using brown algae.

[0007] Furthermore, Non-Patent Literature 3 describes the creation of recombinant Escherichia coli strains BAL1366 and BAL1611, which were produced by introducing genes encoding alginate lyase derived from Vibrio splendidas to confer alginate assimilation ability, and by introducing genes encoding pyruvate decarboxylase (Pdc) and alcohol dehydrogenase B (AdhB) derived from Zymomonas movilis to construct a heterologous ethanol production pathway. Non-Patent Literature 3 further describes the production of ethanol using the above strains, with ethanol produced using a saccharified liquid obtained by saccharifying kelp (Saccharina japonica) with a saccharifying enzyme as the raw material.

[0008] Japanese Patent Publication No. 2011-244789 Japanese Patent Publication No. 2013-51914

[0009] Apple Microbiol Biotechnol. 2016 Feb;100(4) pp1723-1732, NATURE, VOL. 505, 239 (2014), and SUPPLEMENTARY INFORMATION SCIENCE, VOL. 335, 20, pp308-313 (2012), and the Supporting Online Material section of the same document.

[0010] This disclosure aims to provide a method for more efficiently producing alcohol from dried macroalgae as a raw material.

[0011] According to aspects of the present invention, the following is provided:

[0012] [1] (b) A method for producing alcohol, comprising carrying out alcohol fermentation using alcohol-fermenting microorganisms in a culture medium containing saccharified products of dried macroalgae, wherein the moisture content of the dried macroalgae is 20 wt% or less.

[0013] [2] The saccharified product is obtained by performing a saccharification treatment on the dried macroalgae using at least one saccharifying enzyme, according to the method of [1].

[0014] [3] The method according to [1] or [2], further comprising: (a) performing a saccharification treatment on the dried macroalgae using at least one saccharifying enzyme to obtain the saccharified product, as a step performed before or simultaneously with step (b).

[0015] [4] The method according to [3], wherein a solution containing the above-mentioned dried macroalgae and saccharifying microorganisms is prepared and step (a) is performed.

[0016] [5] The method according to [4], wherein the saccharifying microorganism is a microorganism that presents at least one saccharifying enzyme on its cell surface.

[0017] [6] The method according to any one of [3] to [5], wherein a reaction solution is prepared containing the above-mentioned dried macroalgae and at least one saccharifying enzyme, and step (a) is performed.

[0018] [7] The method according to any one of [2] to [6], wherein the saccharifying enzyme comprises cellulase and hemicellulase.

[0019] [8] A method according to any one of [3] to [7], wherein step (a) is performed, and then step (b) is performed.

[0020] [9] The method according to any one of [3] to [7], wherein steps (a) and (b) are carried out simultaneously or consecutively in the same reaction solution.

[0021]

[10] The method according to any one of [1] to [9], wherein the alcohol-fermenting microorganism is an alcohol-fermenting yeast.

[0022]

[11] The method according to any one of [1] to

[10] , wherein the alcohol-fermenting microorganism is an ethanol-fermenting yeast.

[0023]

[12] The method according to any one of [1] to

[11] , wherein the alcohol-fermenting microorganism is a yeast having the ability to utilize alginic acid and / or mannitol.

[0024]

[13] The method according to any one of [3] to

[12] , wherein the alcohol-fermenting microorganism is a microorganism that has the ability to saccharify the dried macroalgae, and step (a) is performed using the alcohol-fermenting microorganism in addition to the saccharifying enzyme.

[0025]

[14] The method according to any one of [1] to

[13] , wherein the alcohol-fermenting microorganism expresses endo-type alginate lyase, which decomposes alginate into oligosaccharides, and exo-type alginate lyase, which decomposes oligosaccharides into monosaccharides, in a manner that is presented on the cell surface, and is a microorganism that has the ability to assimilate mannitol.

[0026]

[15] The method according to any one of [1] to

[14] , wherein the dried macroalgae is a dried brown algae.

[0027] According to embodiments of the present invention, alcohol can be produced efficiently and in high yield by directly using dried macroalgae as a substrate without complicated processing or operations.

[0028] Figure 1A is a flowchart showing an example of one embodiment of the present invention. Figure 1B is a diagram showing the results of the example (test example 1). Figure 2A is a flowchart showing another embodiment of the present invention. Figure 2B is a diagram showing the results of the example (test example 2). Figure 3A is a diagram showing the results of the example (test example 3). Figure 3B is a diagram showing the results of the example (test example 3). Figure 3C is a diagram showing the results of the example (test example 3). Figure 3D is a diagram showing the results of the example (test example 3). Figure 4A is a diagram showing the results of the example (test example 4). Figure 4B is a diagram showing the results of the example (test example 4). Figure 5A is a diagram showing the results of the example (test example 5). Figure 5B is a diagram showing the results of the example (test example 5). Figure 5C is a diagram showing the results of the example (test example 5). Figure 5D is a diagram showing the results of the example (test example 5).

[0029] According to an aspect of the present invention, a method for producing alcohol is provided, comprising (b) carrying out alcoholic fermentation using alcohol-fermenting microorganisms in a culture medium containing saccharified dried macroalgae, wherein the water content of the dried macroalgae is 20 wt% or less.

[0030] In some embodiments, the "saccharified products of dried macroalgae" described above are obtained by performing a saccharification treatment on dried macroalgae using a saccharifying enzyme.

[0031] Therefore, in certain embodiments, the method according to the present invention may further include, as a step performed before or simultaneously with step (b), (a) performing a saccharification treatment on the dried macroalgae using a saccharifying enzyme to obtain the saccharified product.

[0032] The method according to the present invention will be described below by showing examples of various embodiments that may be adopted in the present invention, along with optional steps (a) and essential steps (b) adopted in the method according to the present invention, as well as other optional steps. However, the method according to the present invention is not limited to the embodiments shown below.

[0033] (Dried Macroalgae) In this invention, "macroalgae" should be interpreted literally and refers to algae that are distinguished from microalgae and are relatively larger than microalgae. Examples of macroalgae include the following: Brown algae: Ecklonia genus such as Ecklonia cava subsp. Kurome, Ecklonia cava, and Ecklonia stolonifera; Eisenia genus such as Eisenia bicyclos and Eisenia nipponica; Undaria pinnatifida, Laminaria spp., and Sargassum fusiforme. Red algae: Neopyropia tenera, Gelidium elegans Kuetzing, Gelidium pacificum Okamura, Meristotheca papulosa, Nemacystus decipiens. Yellow-green algae: Pseudodichotomosiphon constricta, Vaucheria longicaulis. Green algae: Ulva species (Ulvaceae), Glycine, Glycine, Glycine, and Glycine. Conjugating algae: Spirogyra and Spirogyra. Charophytes (algae belonging to the order Charales, class Charophyceae): Chara braunii.

[0034] In some embodiments, the macroalgae is at least one selected from the group consisting of brown algae, red algae, yellow-green algae, green algae, conjugated algae, and charophytes, and is preferably at least one selected from the group consisting of brown algae, red algae, and green algae. In a more preferred embodiment, the macroalgae includes brown algae. This is because brown algae contains relatively abundant mannitol and alginic acid that can serve as substrates for alcohol-fermenting microorganisms and is particularly abundant among macroalgae in the marine ecosystem. Therefore, it is suitable as a raw material substrate and can be expected to improve the alcohol yield.

[0035] In the embodiments according to the present invention, as the "dried macroalgae", depending on the life cycle of the macroalgae used, for example, thalli such as sporophytes, female or male gametophytes can be used. However, it is not limited thereto, and any living body of macroalgae that can serve as a substrate for alcohol-fermenting microorganisms can be used without particular limitation.

[0036] In the present invention, "drying" in "dried macroalgae" means that as a material to be subjected to saccharification treatment or alcohol fermentation, dehydration drying treatment is performed so that its water content is 20 wt% or less.

[0037] In addition, as shown in the examples, the above water content (wt%) can be determined by the following method. First, place a sample of dried macroalgae, whose weight (g) has been accurately measured in advance, on a sufficiently dried sample dish, and put this into a constant temperature bath at, for example, 105°C to evaporate the water contained in each sample until the measured weight reaches a substantially constant value. After the measured weight reaches a substantially constant value, measure the weight (g) of the sample again. Then, calculate the weight loss corresponding to the water contained in the sample as the water content, and the water content rate (%) can be calculated as the percentage of the water content (g) to the total weight (g) of the sample used for the measurement. The value obtained by subtracting the water content (g) from the total weight (g) of the sample used for the measurement is referred to as the "solid content weight" (g).

[0038] The specific method for dehydration and drying to obtain the above-mentioned "dried macroalgae" is not particularly limited, and known methods can be used. For example, in addition to drying by forced air, reduced pressure drying, freeze-drying, and drying using silica gel or various desiccants, methods such as leaving the water-retaining algae on the ground for a predetermined time to dehydrate and dry them, and sun-drying can be used. Within a range that does not cause any particular contradiction, the dehydration and drying treatment may be carried out at room temperature or with heating, either simultaneously or sequentially, using one or more combinations of these methods.

[0039] In a particular embodiment, the method according to the present invention further includes, prior to steps (a) and (b), subjecting the macroalgae used as raw material to the dehydration and drying treatment described above (p).

[0040] Furthermore, “dried macroalgae” may be flaked or pulverized macroalgae. In some embodiments, the process further includes (q) flaking or pulverizing the macroalgae prior to steps (a) and (b). In certain embodiments, step (q) is performed before and / or simultaneously with and / or after step (p). For example, the thallus of raw macroalgae collected from a body of water may be cut into pieces (e.g., chips) of a predetermined shape / size, then the obtained raw pieces may be dried to obtain dried thallus fragments, and then subjected to a pulverizing process to obtain dried macroalgae powder or granules. Alternatively, the thallus of raw macroalgae collected from a body of water may be dried without cutting to obtain dried macroalgae, which may then be subjected to a coarse crushing process to obtain dried chips or flaks, and then further subjected to a fine pulverizing process to obtain dried seaweed powder. Furthermore, the dried macroalgae do not necessarily have to be in the form of fine powder or granules; relatively large dried materials such as chips or fragments may be used as raw materials, as long as saccharification and alcoholic fermentation can be achieved.

[0041] Fragmentation or pulverization of such large algae can be carried out by various known methods. For example, cutting tools such as knives and scissors, mixers, homogenizers, rotary crushers, French presses, stone mortars, pestles, glass beads, dry bead mills, media stirring mills such as ball mills (rolling type, vibration type, etc.), jet mills, cutter mills, high-speed rotating impact mills (pin mills, etc.), roll mills, hammer mills, and ultrasonic treatment. It is only necessary to fragment or pulverize using one or a combination of two or more of various instruments and devices until it reaches the desired size.

[0042] In some embodiments, by performing a screen treatment to remove fragments or particles larger than a certain size from the fragmented or pulverized large algae, a dried large algae fragment or pulverized product with fragments or particles larger than a certain size removed may be obtained. The dried large algae fragment or pulverized product thus screen-treated may be used as the dried large algae in step (a), or a medium containing the saccharified product of the fragment or pulverized product may be used in step (b). In a specific embodiment, the method according to the present invention may further include (r) subjecting the fragmented or pulverized large algae to a screen treatment after step (q).

[0043] The screen treatment can be carried out using one or a combination of two or more of various instruments and devices such as various sieves, vibrating screen devices (for example, grizzly type, finger type, mesh type), rotary screen devices (for example, disk type, trommel type, rotary type).

[0044] In certain embodiments, dried macroalgae are specified in the Japanese Industrial Standard "JIS The nominal mesh openings according to "Z8801-1" are 26.5 mm, 22.4 mm, 19 mm, 16 mm, 13.2 mm, 11.2 mm, 9.5 mm, 8 mm, 6.7 mm, 5.6 mm, 4.75 mm, 4 mm, 3.35 mm, 2.8 mm, 2.36 mm, 2 mm, 1.7 mm, 1.4 mm, 1.18 mm, 1 mm, 850 μm, 710 μm, 600 μm, 500 μm, 425 μm, 355 μm, 300 μm, 250 μm, 212 μm, 180 μm, 150 μm, 125 μm, 106 μm, 90 μm, 75 μm, 63 μm, 53 μm, 45 μm, 38 μm, 32 μm, and 25 μm. It is preferable that the material contains fragments or pulverized material that pass through a standard sieve of a value selected from the group consisting of 20 μm (preferably 180 μm, 150 μm, 125 μm, 106 μm, 90 μm, and 75 μm) (more preferably 150 μm), the content of which fragments or pulverized material is preferably 50 wt% or more, more preferably 60 wt% or more, even more preferably 70 wt% or more, even more preferably 90 wt% or more, and even more preferably 95 wt% or more relative to the total amount of dried macroalgae, and it is most preferable that the dried macroalgae being tested substantially consists of such fragments or pulverized material.

[0045] In another embodiment, two of the above nominal opening values ​​are combined as upper and lower limits in a way that does not cause contradiction, and the resulting particle size (maximum diameter) range of dried macroalgae is used as the dried macroalgae in step (a), or the saccharified product of the said particle size or pulverized product (e.g., the saccharified product obtained in step (a)) is used as the "saccharified product of dried macroalgae" in step (b). The particle size (maximum diameter) of dried macroalgae is obtained by combining the above-mentioned fragmentation / pulverization equipment or apparatus that can produce a particle size (maximum particle size) within the above-mentioned numerical range from among the various equipment or apparatus for fragmentation or pulverization described above, and the above-mentioned equipment or apparatus that can filter-separate the particle size (maximum particle size) within the above-mentioned numerical range from among the various equipment or apparatus for screen processing described above. In a more specific embodiment, approximately 15 μm to approximately 19 mm, approximately 15 μm to approximately 18 mm, approximately 15 μm to approximately 14 mm, approximately 15 μm to approximately 10 mm, approximately 15 μm to approximately 9 mm, approximately 15 μm to approximately 7 mm, approximately 15 μm to approximately 6 mm, approximately 15 μm to approximately 7 mm, approximately 15 μm to approximately 4.5 mm, approximately 50 μm to approximately 4.5 mm, approximately 60 μm to approximately 4.5 mm, approximately 70 μm to approximately 4.5 mm, approximately 85 μm to approximately 4.5 mm, approximately 100 μm to approximately 4.5 mm, approximately Finely chopped or pulverized dried macroalgae belonging to the particle size (maximum diameter) range of 120 μm to approximately 4.5 mm, approximately 140 μm to approximately 4.5 mm, approximately 148 μm to approximately 4.2 mm, approximately 148 μm to approximately 3.4 mm, approximately 148 μm to approximately 3 mm, approximately 148 μm to approximately 2.9 mm, approximately 148 μm to approximately 2.4 mm, or approximately 148 μm to approximately 2.2 mm, or saccharified products thereof, may also be used in step (a) or step (b) as described above.

[0046] Furthermore, the identification of dried macroalgae using the standard sieve described above is significant for identifying the structure or physical properties of the dried macroalgae fragments or pulverized material that can be used in the method according to the present invention, and does not necessarily indicate the existence of a screen processing step. However, the existence of a screen processing step (r) using a standard sieve or a sieve equivalent to the one specified in the above Japanese Industrial Standards is not excluded in the method according to the present invention.

[0047] (Saccharifying enzymes) In the present invention, any saccharifying enzyme can be used without particular limitation, as long as it has the enzymatic activity to decompose at least one selected from the group consisting of cellulose, hemicellulose, laminarin (laminaran), alginic acid, λ-carrageenan, and pectin, which are components contained in dried large seaweed, and to produce carbohydrates (various hexoses, various pentoses, oligosaccharides, etc.) that serve as substrates for microbial reactions and microbial growth. Specifically, any saccharifying enzyme can be used that has at least one enzymatic activity selected from the group consisting of cellulase activity, hemicellulase activity, endo-type / exo-type alginate lyase activity, λ-carrageenase activity, and pectinase activity (for example, polygalacturonase, pectin lyase, pectin esterase, pectin methylesterase, etc.).

[0048] In some embodiments, the saccharifying enzyme can be an enzyme having at least one of the following enzymatic activities: endoglucanase activity (EC 3.2.1.4) which cleaves the cellulose molecule from within; exoglucanase (cellobiohydrolase) activity (e.g., reducing end decomposition type: EC 3.2.1.176, non-reducing end decomposition type: EC 3.2.1.91) which decomposes the reducing or non-reducing end of cellulose and releases cellobiose, etc.; β-glucosidase (EC 3.2.1.21, etc.); alginate lyase activity (EC 4.2.2.3 or EC 4.2.2.11); λ-carraginase activity (EC 3.2.1.162); or pectinase activity.

[0049] In certain embodiments, hemicellulases that break down hemicellulose into monosaccharides or oligosaccharides such as xylose may also be used as saccharifying enzymes. Typical examples of hemicellulose include mannan, β-1,4-glucan, xylan, and xyloglucan, and in certain embodiments, hemicellulases that break down these hemicelluloses can be used. In more specific embodiments, at least one hemicellulase exhibiting the activity of at least one enzyme selected from the group consisting of xylanase, xylosidase, mannanase, pectinase, galactosidase, glucuronidase, and arabinofuranosidase can be used.

[0050] Furthermore, it is preferable to use an enzyme mixture comprising multiple cellulases and hemicellulases as the saccharifying enzyme, from the viewpoint of exhibiting sufficient enzymatic activity.

[0051] Furthermore, the origin of the saccharifying enzyme is not particularly limited; for example, enzymes derived from microorganisms such as filamentous fungi, basidiomycetes, and bacteria can be used, and they may be wild-type enzymes or genetically modified enzymes with enhanced specific functions. Examples of microorganisms possessing various saccharifying enzymes include the genera Trichoderma, Acremonium, Aspergillus, Bacillus, Pseudomonas, Penicillium, Aeromonus, Irpex, Sporotrichum, and Humicola. Examples include filamentous fungi, basidiomycetes, and bacteria belonging to genera such as Flavobacterium, Pseudoalteromonas, Vibrio (including Vibrio splendidus, Non-Patent Literature 3), and Saccharophagus (including Saccharophagus degradans, Non-Patent Literature 1). One or more of the above-mentioned saccharifying enzymes derived from these microorganisms may be used.

[0052] Furthermore, various commercially available saccharifying enzymes are also available and can be used. For example, product names such as Novozym® 613, Novozym® 476, Celluzyme®, Celluclast®, Carezyme®, FiberCare®, Cellic® CTec / CTec2 / CTec3 (Novozymes Inc.); Optimase CX, Multifect® A40, Pergalase®, Optimase®, Accellerase. ＴＭ 1000, Accellerase ＴＭ 1500, AccelleraseＴＭ Product lines such as TRIO (DuPont, Danisco); product lines such as Cellulase Onozuka (registered trademark), Macerozyme (registered trademark) (Yakult Pharmaceutical Co., Ltd.); Spartec (trademark) CEL100, Pyrolase (registered trademark) Cellulase, Pyrolase (registered trademark) 200cellulase, Pyrolase (registered trademark) HT Cellulase (BASF); Acremo Examples include CellulaseKM, CellulaseTP5-KYOWA (Kyowa Chemical Co., Ltd.); GODO-TCL, Besselex (Godo Shusei Co., Ltd.); Cellulizer ACE (Nagase ChemteX Corporation); Alginate Lyase S (Nagase Vita Co., Ltd.); Pectinase XP-534NEO (Nagase Vita Co., Ltd.).

[0053] In certain embodiments, the saccharifying enzyme may optionally include, in addition to the above-mentioned enzyme, one or more other enzymes useful for liquefying or saccharifying dried large seaweed. Examples of such other enzymes include agarase, xylanase, mannanase, protease, fucoidanase, and glucanase.

[0054] In the method according to the present invention, when obtaining saccharified products of dried macroalgae, the saccharification treatment of dried macroalgae with at least one saccharifying enzyme (i.e., step (a)) may be carried out by embodiments i) and / or ii) below, or the saccharified products of dried macroalgae obtained thereby may be used in step (b). i) The reaction solution or culture medium containing dried macroalgae contains at least one saccharifying enzyme, ii) The reaction solution or culture medium containing dried macroalgae contains an alcohol-fermenting microorganism or other microorganism that secretes or expresses at least one saccharifying enzyme in the form of cell surface presentation. Here, embodiment i) can be carried out, for example, by adding at least one extracted or purified enzyme (e.g., in the form of an enzyme solution, powder, or other solid) to the reaction solution or culture medium together with the dried macroalgae.

[0055] (Alcohol-fermenting microorganisms) In the present invention, the type of alcohol-fermenting microorganism is not particularly limited, and any microorganism having the ability to ferment alcohol such as ethanol or butanol can be used without particular restriction. In addition, in the present invention, the microorganism may be a prokaryote or a eukaryote, and may be a wild species, a mutant or a genetically modified organism.

[0056] In some embodiments, the microorganism is at least one selected from the group consisting of archaea, bacteria, cyanobacteria, microalgae, and fungi. Furthermore, in some embodiments, the microorganism is a microorganism capable of producing a specific substance.

[0057] In certain embodiments, the microorganisms may be one or more alcohol-fermenting microorganisms capable of producing alcohols, such as methanol, ethanol, propanol, and butanol. In more preferred embodiments, the microorganisms used are alcohol-fermenting yeasts (particularly preferably ethanol-fermenting yeasts).

[0058] Examples of alcohol-producing microorganisms include the genera Saccharomyces, such as Saccharomyces cerevisiae; the genera Schizosaccharomyces, such as Schizosaccharomyces pombe; the genera Pichia, such as Pichia stipitis and Pichia kudriavzevii (e.g., RZ8-1); the genera Pachysolen, such as Pachysolen tannophilus; Kluyveromyces lactis and Kluyveromyces marsianus. Examples include yeasts belonging to the genus Kluyveromyces, such as marxianus (e.g., DMKU3-1042), and the genus Candida, such as Candida shehatae, Candida shehatae, and Candida glabrata NF (e.g., RI3163), as well as some bacteria such as Clostridium thermocellum, Clostridium thermohydrosulfuricum, Thermoanaerobacter ethanolicus, and Zymomonas mobilis.

[0059] Furthermore, in certain embodiments, alcohol-fermenting microorganisms having pentose assimilation ability (e.g., microorganisms having xylose and / or arabinose assimilation ability) may be used. More specifically, as alcohol-fermenting microorganisms having xylose and / or arabinose assimilation ability, genetically modified yeast for ethanol fermentation to which xylose and / or arabinose assimilation ability has been conferred may be used. Since ethanol-fermenting yeasts to which xylose and / or arabinose assimilation ability has been conferred by the introduction of various heterologous genes are also known, these may be used in the present invention [e.g., Appl. Biochem. Biotechnol., 105-108:277-286 (2003); Appl. Microbiol. Biotechnol., 73:1039-1046 (2007); J. Biosci. Bioeng.]. , 106:306-309 (2008). ;Appl. Microbiol. Biotechnol. , 82, 1037-1047 (2009); Appl. Environ. Microbiol. , 69, 4144-4150 (2003); Appl. Environ. Microbiol. , 73, 4881-4891 (2007); Microb. Cell Fact. , 8, 40 (2009); Appl. Environ. Microbiol. , 75, 907-914 (2009)].

[0060] Furthermore, in another embodiment, an alcohol-fermenting microorganism possessing cellobiose assimilation ability may be employed. For example, ethanol-fermenting yeasts to which cellobiose assimilation ability has been conferred by the introduction of various heterologous genes are known, and these may also be employed in the present invention [e.g. (Science, 330, 84-86 (2010); Proc. Natl. Acad. Sci. U.S.A., 108, 504-509 (2011))]. Furthermore, in another embodiment, heat-resistant yeast capable of fermentation at temperatures above 37°C, around 40-50°C, may be used (for example, J. Biosci. Bioeng. 2010; 110: 176-179; Braz. J. Microbiol. 49(2): 378-391(2018); World J Microbiol Biotechnol. 1992; 8: 259-263).

[0061] Furthermore, in another preferred embodiment, an alcohol-fermenting microorganism having the ability to assimilate alginate and / or mannitol is employed. Examples of such alcohol-fermenting microorganisms having assimilation ability include microorganisms that satisfy at least one selected from the group consisting of (A), (B), and (C) below: (A) Having one or more genes that can be expressed in a manner of cell surface presentation that encode at least one or both of the following: endo-type alginate lyases that decompose alginate into oligosaccharides (e.g., Alg7A, Alg7D, Alg18J from the alginate-assimilated marine bacterium Saccharophagus degradans; Non-Patent Literature 1, and Appl Microbiol Biotechnol. 2016 Feb; 100(4) pp1723-1732) and exo-type alginate lyases that further decompose the above oligosaccharides into monosaccharides (e.g., Alg7K from the above marine bacterium; the above prior art literature). Furthermore, in the reaction solution, the monosaccharide produced from the oligosaccharide by the action of the exo-type alginate lyase is spontaneously converted to 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH).

[0062] (B) DEH transporters that take DEH from outside the cell into the cell (cytoplasm) (e.g., DHT1 from the algal fungus Asteromyces cluciatus; WO / 2013 / 115959), DEH reductases that convert DEH taken inside the cell (cytoplasm) to 2-keto-3-deoxy-D-gluconic acid (KDG) (e.g., dehR from the mollusk Vibrio splendidas; WO / 2013 / 115959), KD The organism is capable of expressing one or more genes encoding the following proteins: a KDG kinase that converts G to 2-keto-3-deoxy-6-phosphogluconate (KDPG) (e.g., kdgK from Escherichia coli; WO / 2013 / 115959), and a KDPG aldolase that decomposes KDPG into pyruvate and glyceraldehyde phosphate (e.g., kdgpA from Vibrio splendidas; WO / 2013 / 115959). (C) The cell has the ability to express one or more genes encoding proteins for mannitol transporters that take mannitol from extracellular to intracellular (cytoplasm) (e.g., HXT13, HXT15, HXT16, HXT17 from the budding yeast Saccharomyces cerevisiae; Non-Patent Literature 2; P. Jordan, J.Y. Choe, E. Boles, M. Oreb: Sci. Rep., 6, 23502 (2016)), and mannitol-2-dehydrogenase (MDH) [e.g., the MDH gene (YNR073C) from budding yeast, and the paralog DSF1 (YEL070W) of the same gene; the above references]. In the cell, the fructose converted from mannitol by MDH is metabolized to fructose-6-phosphate and used as an intermediate in glycolysis.

[0063] The satisfaction of (A), (B), and (C) above may be achieved by genetic manipulation of microorganisms, and / or by mutagen treatment or the application of specific selective pressures to microorganisms (for example, culture breeding in a nutrient-deficient medium in which the microorganism cannot normally grow), and / or by properties inherently possessed by wild-type microbial species.

[0064] In certain embodiments, step (b) may include, as alcohol-fermenting microorganisms: 1) Saccharomyces cerevisiae (e.g., BY4742, BY4741, AH109, ​​DBY877, EBY100, SEY6210, T8-1D, YPH500), 2) Saccharomyces paradoxus strain NBRC0259 (Journal of Bioscience and Bioengineering, Vol. 116, No. 3, September 2013, pp. 327-332), 3) Alginic acid and mannitol-assimilating Saccharomyces cerevisiae strain AM1 (Non-Patent Literature 1). 4) Saccharomyces cerevisiae BAL2759 strain [MATα; LYS2-ENO1t:pFBA1-Ec_kdgK(optSc); LEU2-ENO1t:pFBA1-Vs_kdgpA(optSc); ADE2-ENO1t:pTDH3-Vs_dehR(optSc); HIS3-ENO1t:pTDH3-iM3; TRP1; URA3-ENO1t:TDH3-Ac_DHT1(optSc); suc2-Δ9; GAL], Saccharomyces cerevisiae BAL2772 ​​strain [MATα; LYS2-ENO1t:pFBA1-Ec_kdgK(optSc); LEU2-ENO1t:pFBA1-Vs_kdgpA(optSc); ADE2-ENO1t:pTDH3-At_dehR; HIS3-ENO1t:pTDH3-iM3; TRP1; URA3-ENO1t:pTDH3-Ac_DTH1(optSc); suc2-Δ9; GAL], Saccharomyces cerevisiae BAL2956 strain [MATα; LYS2-ENO1t:pFBA1-Ec_kdgK(optSc); LEU2-ENO1t:pFBA1-Vs_kdgpA(optSc); ADE2-ENO1t:pTDH3-Vh_dehR(optSc); HIS3-ENO1t:pTDH3-iM3; TRP1; URA3-ENO1t:pTDH3-Ac_DTH1(optSc); suc2-Δ9;[GAL] (Non-Patent Literature 2), 5) At least one selected from the group consisting of recombinant Escherichia coli BAL1366 strain and BAL1611 strain (Non-Patent Literature 3, Supporting Online Material: https: / / www.science.org / doi / 10.1126 / science.1214547), which have been conferred with alginate assimilation ability by introducing genes encoding alginate lyase derived from Vibrio splendidus, etc., and whose heterologous ethanol production pathway has been constructed by introducing genes encoding pyruvate decarboxylase (Pdc) and alcohol dehydrogenase B (AdhB) derived from Zymomonas movilis.

[0065] Furthermore, by subjecting various yeasts (e.g., budding yeasts such as BY4741, BY4742, AH109, ​​DBY877, EBY100, SEY6210, T8-1D, YPH500, etc.) to breeding methods using a medium containing only mannitol as a carbon source (e.g., SM medium, YPM medium, etc.), strains that have acquired mannitol assimilation ability can be selected and obtained (Appl Environ Microbiol. 2014 Dec 11;81(1):9-16). That is, in another embodiment, in step (b), a mannitol-assimilating strain obtained by the breeding method described above can also be used as the alcohol-fermenting microorganism. Furthermore, in another specific embodiment, step (b) may also use a mannitol-assimilating bacterial strain obtained by the breeding method described above that, in addition to mannitol assimilation ability, has salt tolerance (for example, the ability to survive in the presence of 1 M NaCl and produce alcohol from mannitol) and exhibits non-aggregating or agglutinating properties in a predetermined culture medium (for example, SM medium or SC medium). More specifically, the above-mentioned literature describes how, by subjecting the budding yeast strain BY4742 to the above breeding method, various strains (mutants) exhibiting properties such as salt tolerance, non-aggregating or agglutinating properties in addition to mannitol assimilation, and possessing various mutations in the TUP1 gene and / or CYC8 (Ssn6) gene (TABLE 1 of the same literature). As also described in the second paragraph of the "RESULTS" section in the right-hand column on page 10 of the same literature, generally, by subjecting various fission yeast strains to the above breeding method, mutants exhibiting mannitol assimilation ability and the properties described above can be obtained with high frequency. Therefore, it is possible to easily design embodiments using any of these mutants, and it can be said that the method according to the present invention can be implemented by such embodiments.

[0066] In some embodiments, the method according to the present invention may further include, as shown in Figures 1A and 2A, (s) pre-culturing an alcohol-fermenting microorganism in a predetermined medium prior to step (b) to obtain a pre-culture of the alcohol-fermenting microorganism (i.e., a culture in which the microorganism has grown to a predetermined cell density). In certain embodiments, the pre-culture obtained in step (s) may be used as is as the alcohol-fermenting microorganism in step (b) without any processing such as concentration (for example, as shown in the process flow in Figure 2A). Furthermore, in another particular embodiment, the cells (bacterial cells) grown from the medium in the pre-culture obtained in step (s) may be separated by operations such as centrifugation or decantation, the separated cells (bacterial cells) may be concentrated by processing such as suspending them in fresh medium or reaction medium, and the concentrated cells (bacterial cells) may be used as the alcohol-fermenting microorganism in step (b) (for example, as shown in the process flow in Figure 1A). According to these embodiments, it is possible to obtain a pre-culture in which alcohol-fermenting microorganisms have already grown to a predetermined cell density and are in a logarithmic growth phase, which can be said to be a more active state of microbial metabolism, or in an active state that has not yet reached the stationary phase or death phase. Furthermore, since the cell density of the microorganisms at the start of the alcohol fermentation reaction in step (b) can be controlled to a predetermined value or range, it is possible to easily control the alcohol fermentation in step (b) and achieve highly reproducible and efficient alcohol production. Therefore, embodiments employing step (s) are preferably adopted.

[0067] (Order of steps (a) and (b) and basic composition of saccharification / fermentation reaction solution) If the method according to the present invention further includes step (a) in addition to step (b), either of the following order of steps (i) and (ii) can be adopted as the order of steps (a) and (b). (i) Step (a) is performed, and then step (b) is performed. (ii) Steps (a) and (b) are performed simultaneously or sequentially in the same system.

[0068] In the method according to the present invention, the preparation methods for the saccharification reaction solution in step (a) and the fermentation reaction solution in step (b), as well as the timing and method of adding each component to constitute each reaction solution, can be selected as appropriate within the scope that does not cause any technical inconsistencies or problems, and are not particularly limited.

[0069] Specifically, the main components constituting the above saccharification / fermentation reaction solution include the following (v), (w), (x-1), (x-2), (y), and (z): (v) a predetermined amount of at least one saccharifying enzyme; (w) a predetermined amount of alcohol-fermenting microorganisms (e.g., a pre-culture of alcohol-fermenting microorganisms grown in a predetermined medium or a cell concentrate prepared from said pre-culture); (x-1) a predetermined amount of dried macroalgae; (x-2) a predetermined amount of saccharified products of dried macroalgae; (y) water as a solvent; (z) culture medium components.

[0070] In some embodiments, in the essential step (b), a fermentation reaction solution containing the above-mentioned components (w), (x-2), and (y), and optionally (z), is prepared, and alcoholic fermentation is carried out. However, the persistence of active saccharifying enzymes in the saccharified product of dried macroalgae related to component (x-2), and the resulting progress of the saccharification reaction in step (b), are not excluded from embodiments of the present invention.

[0071] In another embodiment, a process sequence (i) is adopted that includes step (a) in addition to step (b), in which a fermentation reaction solution containing the above-mentioned components (v), (x-1) and (y) is prepared, and saccharification of dried macroalgae is carried out to obtain saccharified products of dried macroalgae, which are then subjected to step (b). That is, in this embodiment, in step (b), a fermentation reaction solution is prepared that contains a predetermined amount of the saccharified products of dried macroalgae obtained via step (a) as the above-mentioned component (x-2), and also contains the above-mentioned components (w) and (y), as well as optionally (z), and alcoholic fermentation is carried out.

[0072] In yet another embodiment, a process sequence (ii) is adopted that includes step (a) in addition to step (b), and in steps (a) and (b) which are performed simultaneously in the same system, a saccharification and fermentation reaction solution containing the above components (v), (w), (x-1) and (y), and optionally (z), is prepared, and the saccharification reaction and the alcohol fermentation reaction are carried out simultaneously or sequentially in the same system.

[0073] According to embodiments of the present invention that include steps (a) and (b) performed simultaneously or sequentially within the same system, the production of saccharified products that serve as substrates for alcohol-fermenting microorganisms by the saccharification reaction of dried macroalgae in step (a) and the alcohol fermentation using the saccharified products as a substrate in step (b) can be performed simultaneously and sequentially within the same system. Therefore, efficient production of alcohol from dried macroalgae becomes possible, and such embodiments are preferably adopted.

[0074] In the embodiments of the present invention, there are microbial species that can grow and produce alcoholic fermentation as long as the saccharified dried macroalgae related to (x-2), which serve as the substrate, are present in the reaction solution. Therefore, the culture medium components related to (z) above are not necessarily essential components of the fermentation reaction solution in step (b). Whether or not to add culture medium components should be decided according to the type and properties of the alcohol-fermenting microorganisms used, and if culture medium components are added, the specific contents of the culture medium components should also be decided as appropriate.

[0075] In addition, in steps (a) and / or (b), there are no particular restrictions on the mixing method or the order in which the above components (w), (x-1), (x-2), (y), and (z) are added when preparing the reaction solution. Suitable conditions can be appropriately selected depending on the device used, the structure of the reactor, etc.

[0076] In the saccharification reaction solution prepared in step (a), the predetermined amount of saccharifying enzyme [element (v)] depends on conditions such as the enzyme activity unit of the various saccharifying enzymes, but in some embodiments, it is set so that, with respect to 100 parts by weight of dried macroalgae [element (x-1)] to be saccharified in the reaction system, it falls within a range that adopts one of the following lower limits, a range that adopts one of the following upper limits, or a numerical range that combines one of the lower and upper limits in a non-contradictory manner. Lower limit: approximately 0.005 parts by weight or more, approximately 0.01 parts by weight or more, approximately 0.5 parts by weight or more, approximately 1 part by weight or more, or approximately 5 parts by weight or more; Upper limit: approximately 1000 parts by weight or less, approximately 900 parts by weight or less, approximately 600 parts by weight or less, approximately 500 parts by weight or less, approximately 200 parts by weight or less, approximately 150 parts by weight or less, approximately 100 parts by weight or less, approximately 90 parts by weight or less, approximately 60 parts by weight or less, approximately 50 parts by weight or less, approximately 40 parts by weight or less, approximately 30 parts by weight or less, approximately 20 parts by weight or less, or approximately 15 parts by weight or less. Note that the above-mentioned "100 parts by weight of dried macroalgae [element (x-1)]" may be calculated including the water content, or it may be calculated as the solid content weight after subtracting the water content.

[0077] In a particular embodiment, the predetermined amount of saccharifying enzyme [element (v)] can be set to fall within the following numerical ranges relative to 100 parts by weight of dried macroalgae [element (x-1)]: for example, about 0.005 parts by weight to about 100 parts by weight, about 0.01 parts by weight or more to about 60 parts by weight, about 0.5 parts by weight to about 50 parts by weight, about 0.5 parts by weight to about 40 parts by weight, about 0.5 parts by weight to about 30 parts by weight, about 0.5 parts by weight to about 20 parts by weight, or about 0.5 parts by weight to about 15 parts by weight.

[0078] Furthermore, in certain embodiments, the predetermined amount of alcohol-fermenting microorganisms [element (w)] (amount of microorganisms at the start of the alcohol production reaction) may be set as wet cell weight in the range of, for example, about 0.01 to about 100 parts by weight, preferably about 0.5 to about 80 parts by weight, more preferably about 1 to about 60 parts by weight, or about 1 to about 50 parts by weight, per 100 parts by weight of dried macroalgae and / or their saccharified products [components (x-1) and / or (x-2)].

[0079] Furthermore, in some embodiments, the concentration of alcohol-fermenting microorganisms in the reaction solution at the start of alcohol production is not particularly limited, but can be adjusted to, for example, about 2 to about 60, preferably about 5 to about 50, more preferably about 10 to about 45, and even more preferably about 15 to about 40, in terms of turbidity OD600. In another embodiment, the concentration of alcohol-fermenting microorganisms in the reaction solution at the start of alcohol production can be adjusted to, for example, about 2 to about 60, preferably about 5 to about 125 g / L, more preferably about 10 to about 113 g / L, and even more preferably about 15 to about 100 g / L, in terms of wet cell weight.

[0080] The relationship between turbidity (OD600) in a suspension containing microorganisms (e.g., culture medium, reaction solution, etc.), dry cell weight, and wet cell weight is as follows: • Dry cell weight when OD600 = 1 ≈ 0.2–0.5 g / L, • 1 g dry cell weight ≈ 5 g wet cell weight, • Wet cell weight when OD600 = 1 ≈ 1–2.5 g / L.

[0081] Therefore, taking these relationships into consideration and referring to the various numerical ranges described above, the amount or initial concentration of dried macroalgae and / or their saccharified products [components (x-1) and / or (x-2)], saccharifying enzymes [element (v)], alcohol-fermenting microorganisms, etc., added to the reaction solution can be appropriately set.

[0082] Furthermore, Novozymes, the manufacturer of the Cellic® CTec3 and other products mentioned above, has proposed an evaluation method using an enzyme activity unit called "BHU(2)HS" (basically, an enzyme activity unit based on hydrolysis activity for cellulose in crushed corn stover) as an index for evaluating the performance of a multi-saccharifying enzyme mix suitable for the saccharification reaction of biomass materials and estimating the required enzyme activity unit (Novozymes Technical Information "BHU(2)HS, Biomass hydrolysis activity by FCD"). Therefore, the required amount of "at least one saccharifying enzyme" used in the present invention may be appropriately determined using this enzyme activity unit as an index, or by utilizing a fluorescent in vitro enzyme assay system (Methods Enzymol. 2012, 510, pp. 19-36) applied to the measurement of the said enzyme activity unit. For example, the saccharifying enzyme mix Cellic® CTec3 HS provided by Novozymes has an enzyme activity of approximately 1000 to 2000 BHU(2)HS / g according to the above enzyme activity unit. In a particular embodiment, at least one saccharifying enzyme [element (v)] exhibiting an enzyme activity equivalent to approximately 1000 to 2000 BHU(2)HS / g may be prepared in advance and added in predetermined amounts (g) belonging to each of the above numerical ranges to 100 parts by weight of dried macroalgae [element (x-1)], or an amount of at least one saccharifying enzyme [element (v)] equivalent to the amount of enzyme activity unit (BHU(2)HS) corresponding to the total predetermined amount (g) may be added.

[0083] Furthermore, in another embodiment, at least one of the above-mentioned saccharifying enzymes [element (v)] may be added to 100 parts by weight of dried macroalgae [element (x-1)] in the range of approximately 5.0 to approximately 2,000,000, approximately 10 to approximately 1,800,000, approximately 500 to approximately 1,200,000, approximately 1,000 to approximately 200,000, approximately 5,000 to approximately 400,000, or approximately 5,000 to approximately 300,000, most preferably approximately 5,000 to approximately 200,000, using enzyme activity units with BHU(2)HS.

[0084] In the present invention, the saccharification / fermentation reaction solution in step (a) and / or (b) is not necessarily essential, but the types of components it contains can be appropriately selected from the viewpoint of favorably expressing saccharifying enzyme activity or alcohol fermentation reaction.

[0085] For example, in some embodiments, each reaction solution may contain at least one of the following (P) to (T): (P) at least one selected from the group consisting of molasses (e.g., derived from sugarcane, sugar beet, or corn), malt extract, and whey as a culture medium component; (Q) at least one selected from the group consisting of corn steep liquor, yeast extract, soybean meal, and peptone (protein hydrolysate) as a culture medium component; (R) at least one nitrogen source as a culture medium component [e.g., an inorganic nitrogen source including ammonium salts (e.g., ammonium sulfate, ammonium carbonate, ammonium bicarbonate, ammonium phosphate), sodium nitrate, potassium nitrate, and ammonia; an organic nitrogen source including urea]; (S) a buffer as a component of the culture medium and / or saccharifying enzyme reaction solution (e.g., at least one inorganic buffer (e.g., K ２ HPO ４ 7KH ２ PO ４ CaCO ３ ); (T)LB medium, TB medium, YEP (yeast extract peptone) medium, YPD (yeast extract peptone dextrose) medium, YPM (yeast extract peptone mannitol) medium, YPG (yeast extract peptone glycerol) medium, YNB medium, DOB (Drop Out Base) medium, CSM (Complete Supplement Mixture) medium, SCSM (Hopkins Mixture, Synthetic Complete Supplement Mixture) medium, SC (Kaiser Mixture, Synthetic Complete Drop-Out) Various typical culture media such as (U) Mixture medium, SD (Synthetic Defined) medium, BSM (Brent Supplement Mixture) medium, HSM (Hollenberg Supplement Mixture) medium, etc.; (U) Antifoaming agent.

[0086] Here, the content of components (P) and / or (Q) in the reaction solution can be appropriately determined according to the properties of the saccharifying enzyme and microorganism used, as well as the desired saccharification and microbial reaction, and is not particularly limited, but is, for example, about 0.5 to about 60 parts by weight, about 0.5 to about 50 parts by weight, about 0.5 to about 40 parts by weight, preferably about 1.0 to about 40 parts by weight, about 1.0 to about 30 parts by weight, about 1.0 to about 20 parts by weight, more preferably about 1.5 to about 20 parts by weight, and even more preferably about 2.0 to about 10 parts by weight, relative to about 100 parts by weight of water as the main solvent. In addition, the content of (R) in the reaction solution is not limited in the same way as that of components (P) and / or (Q), but is, for example, about 0.1 to about 5 parts by weight, about 0.1 to about 4 parts by weight, preferably about 0.5 to about 3 parts by weight, about 0.5 to about 4 parts by weight, more preferably about 1.0 to about 4 parts by weight, or about 1.0 to about 3 parts by weight, per about 100 parts by weight of water as the main solvent.

[0087] Regarding component (T), the saccharification / fermentation reaction solution may be constructed based on the composition of various typical culture media, or component (T) may be contained in the saccharification / fermentation reaction solution as a result of mixing a culture of alcohol-fermenting microorganisms pre-cultured in the various typical culture media with dried macroalgae or their saccharified products and other components.

[0088] In addition, the composition of the culture medium used in the above-described pre-culture step (s) is not particularly limited and can be appropriately designed based on the type and characteristics of the alcohol-fermenting microorganisms used. For example, a culture medium containing at least one of the above (P) to (T) may be used, and in particular, from the viewpoint of suitable and efficient growth, it is preferable to use a culture medium containing the artificial culture medium related to (T), or to consist of said artificial culture medium.

[0089] (Obtaining Alcohol) As described above, the method according to the present invention, in step (b), uses saccharified dried macroalgae as a substrate to carry out an alcohol fermentation reaction by alcohol-fermenting microorganisms to produce alcohol, and obtains the alcohol as a target substance.

[0090] The type of alcohol obtained as the target substance is naturally determined by the specific alcohol species production ability of the microorganisms used in process (b), and examples include methanol, ethanol, propanol, and butanol.

[0091] In some embodiments, the method according to the present invention includes, after step (b), extracting and / or separating and / or concentrating and / or purifying the alcohol produced as the target substance.

[0092] Methods for extracting, separating, concentrating, and purifying various alcohols can be any known technique, such as distillation, organic solvent extraction, salting out, filtration, ultrafiltration, ion exchange chromatography, and affinity chromatography. One of these techniques may be used, or a combination of several may be used.

[0093] The uses of the alcohol produced in this invention are not limited in any way, but examples include pharmaceutical / medical uses, industrial uses, fuel uses, cosmetic uses, etc. In addition, the alcohol produced in this invention may be consumed for various purposes, or it may be an intermediate raw material used in the production of the final product.

[0094] Although specific embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications, alterations, and combinations of each configuration, element, and feature can be adopted without departing from the spirit of the present invention.

[0095] In this application, unless otherwise specified, the terms "contains," "constitutes," "includes," "possesses," and "constitutes" do not exclude the existence of elements other than those explicitly stated with these terms, and these terms are often used interchangeably. In addition, in this application, the meaning of the term "substantial" is that the element corresponding to the subject is composed mainly of the constituent elements referred to as the object, while not excluding the inclusion of impurities as constituent elements. Embodiments in which the terms "contains," "constitutes," "includes," "possesses," and "constitutes" are replaced with "substantial" to the extent that no particular contradiction arises are also explicitly stated herein.

[0096] Furthermore, the contents of Japanese Patent Application No. 2024-226292, which forms the basis of the priority claim in this application, and each of the prior art documents referenced herein are incorporated herein by reference for all purposes and constitute a part of this specification, and are expressly provided herein as elements that may constitute a part of the above embodiments, in particular to the extent that they do not contradict each other.

[0097] First, the following shows the various materials common to all of the test examples 1 to 5.

[0098] 1) The thallus of *Cymbidium goeringii*, collected from a dry, large-scale algal area, was dried in the sun and then roughly broken into pieces of about 1 cm to 3 cm to obtain dried thallus chips. Next, these dried thallus chips were crushed into a powder using a mixer, and the resulting powder was passed through a 150 μm mesh sieve. The resulting powder sample (hereinafter referred to as "dried *Cymbidium goeringii* powder") was used as a raw material in the ethanol production tests shown in the following Test Examples 1 to 3. In Test Example 4, dried *Cymbidium goeringii* samples prepared separately as described later were used, and the above-mentioned dried *Cymbidium goeringii* powder was used as a comparative control. The moisture content (%) of the dried *Cymbidium goeringii* powder was calculated to be 9.8%. The method for measuring the moisture content is as follows. First, each chrome sample, whose weight (g) had been accurately measured beforehand, was placed on a thoroughly dried aluminum dish. This dish was then placed in a 105°C constant temperature bath to evaporate the water contained in each sample until the weight reached a constant mass, and the weight (g) of each sample was measured again. The weight loss corresponding to the water contained in each sample was then calculated as the water content, and the water content (%) was calculated as the percentage of the water content (g) relative to the total weight (g) of the samples used for measurement.

[0099] 2) Saccharifying enzyme solution: Commercial product "CTec3" [manufactured by Novozymes] (hereinafter simply referred to as "CTec3").

[0100] 3) In alcohol-fermenting microorganism test examples 1 and 2, Saccharomyces cerevisiae strain AM1 (Non-Patent Literature 1, hereinafter simply referred to as "strain AM1") was used as the alcohol-fermenting microorganism, and in test example 3, strain AM1 and Saccharomyces cerevisiae strain SEY6210 (ATCC96099) were used as the alcohol-fermenting microorganisms.

[0101] In the following test examples 1 to 4, the amount of each alcohol-fermenting microorganism [element (w)] at the start of ethanol production culture was adjusted to a turbidity OD600 of 15 to 40, as shown in each test example. That is, considering the relationship between turbidity (OD600) and the dry cell weight and wet cell weight described in the above embodiment, it is estimated that the wet cell weight will be in the range of 15 to 100 g / L in the OD600 range of 15 to 40.

[0102] [Test Example 1] In this test example, ethanol was produced by generally using the following materials and performing saccharification treatment and ethanol fermentation of dried macroalgae in this order according to the procedure shown in FIG. 1A.

[0103] 11.1 g (solid content weight: 10.0 g) of dried Clome powder was weighed, and a suspension obtained by suspending it in 87.9 g of water was put into a 250 mL culture tank. After adjusting the pH of the suspension put into the culture tank to 4.5 by using 6M HCl, 1 g of Ctec3 was added to the suspension, and saccharification treatment was performed while stirring at 37 ° C. for 24 hours to obtain a saccharification treatment solution (saccharide) of dried Clome powder.

[0104] In addition, simultaneously with the enzymatic saccharification treatment of seaweed, separately, cell growth culture (pre-culture) of AM1 strain, which is ethanol-fermenting yeast, was carried out. The glycerol stock of AM1 strain was inoculated onto YPD agar medium under aseptic conditions and cultured at 30 ° C. The grown bacteria were suspended in an appropriate amount of YPD medium, and inoculated into 5 L of YPD medium prepared in advance in a 10 L culture tank so that the initial turbidity OD600 became 0.02. The cell growth culture was carried out by setting the culture temperature at 30 ° C., the rotation speed of the stirring blade at 250 rpm, and the aeration rate at 1 L / min. During the culture, the pH was maintained at 6.0 by NH ３ (5N). After 18 hours, consumption of glucose was confirmed, and the turbidity OD600 at that time was 18.5. 162 mL of the culture solution was collected and centrifuged (5000 xg, 5 minutes), and the supernatant was discarded. The obtained cell pellet was resuspended in 162 mL of PBS buffer and then centrifuged (5000 xg, 5 minutes), and the supernatant was discarded. 20 mL of 5×YP (50 mM MgCl ２ addition) was added to the obtained washed cell pellet to obtain a cell suspension (estimated to be about OD600 = 150).

[0105] The cell suspension thus obtained was added to the saccharification treatment solution of the above-mentioned dried Clome (estimated to be about 25 as cell OD). Ethanol production culture (ethanol fermentation) was carried out by setting the culture temperature at 37 ° C., the rotation speed of the stirring blade at 200 rpm, and the aeration rate at 0 L / min. During the culture, the pH of the culture solution was maintained at 6.0 by using 10N NaOH.

[0106] At 0, 2, 4, 24, 96, 120, 144, 192, and 264 hours after the start of cultivation, a portion of the culture medium was sampled, and the concentrations of glucose, mannitol (fermentation substrate), and ethanol at each time point were measured. The results are shown in Table 1 and Figure 1B.

[0107]

[0108] As can be seen from Table 1 and Figure 1B, the ethanol concentration increased as the mannitol concentration, which is the substrate, decreased with the progression of the culture time. As shown in Table 1, the ethanol concentration at 264 hours after the start of culture was 7.05 g / L. In other words, in this test example, since the culture volume was 0.12 L, 0.85 g of ethanol could be produced from 10 g of dried chrome solids.

[0109] [Test Example 2] In this test example, the same materials as in Test Example 1 were used, but ethanol was produced by simultaneously carrying out saccharification treatment and ethanol fermentation of dried macroalgae according to the procedure shown in Figure 2A.

[0110] The glycerol stock of strain AM1 was inoculated onto YPD agar under sterile conditions and cultured at 30°C. Next, the grown cells were suspended in an appropriate amount of YPD medium and inoculated into 100 mL of pre-prepared YPD medium in a 250 mL culture vessel so that the initial turbidity OD600 was 0.02. Cell growth culture was carried out at a culture temperature of 30°C, a stirring blade rotation speed of 250 rpm, and aeration rate of 20 mL / min. During culture, NH ３ The pH was maintained at 6.0 using (5N). Glucose consumption was observed after 24.5 hours, and the OD600 at that time was 18.9.

[0111] Next, the culture temperature was changed to 33°C, the rotation speed of the stirring blade to 200 rpm, and the aeration rate to 0 mL / min. Then, 11.1 g of dried chrome powder (moisture content 9.8%) (solid weight 10.0 g) and 1 g of saccharifying enzyme solution (CTec3) were added to the culture medium, and ethanol production culture was carried out. During the culture, the pH of the culture medium was maintained at 6.0 using NaOH (10N).

[0112] At 0, 2, 4, 24, 120, 144, 168, 192, 264, and 288 hours after the start of the ethanol production culture described above, a portion of the culture medium was sampled, and the concentrations of glucose, mannitol (fermentation substrate), and ethanol at each time point were measured. The results are shown in Table 2 and Figure 2B.

[0113] As can be seen from Table 2 and Graph 2B, the ethanol concentration increased as the mannitol concentration, which is the substrate, decreased with the progression of culture time. The increase in ethanol concentration from the start of ethanol production culture to 288 hours later was 9.74 g / L. In other words, in this test example, since the culture volume was 0.10 L, 0.97 g of ethanol could be produced from 10 g of dried chrome solids.

[0114] [Test Example 3] In this test example, as shown in Figure 2A, the bacterial cells were grown by pre-culture, similar to Test Example 2. Then, dried seaweed and saccharifying enzymes were added to the culture medium, and ethanol production was carried out by simultaneously promoting saccharification and fermentation. In addition to the ethanol production test series of strain AM1 (with and without saccharifying enzymes), the ethanol production test series of Saccharomyces cerevisiae SEY6210 strain (with and without saccharifying enzymes) was also included as an ethanol-fermenting microorganism. The procedure is shown below.

[0115] Glycerol stocks of strains AM1 and SEY6210 were inoculated onto YPD agar plates under sterile conditions and cultured at 30°C. The grown bacteria were suspended in an appropriate amount of YPD medium and inoculated into 100 mL of pre-prepared YPD medium in a 250 mL culture vessel so that the initial turbidity OD600 was 0.005. Cell growth culture was carried out with the culture temperature set to 30°C, the rotation speed of the stirring blade set to 550 rpm, and the aeration rate set to 20 mL / min. During culture, NH ３The pH of the culture medium was maintained at 6.0 using (5N). In the AM1 strain test series (with and without saccharifying enzyme), glucose consumption was confirmed 21 hours after the start of culture, and the OD600 at that time was 33.7 and 33.6, respectively. In the SEY6210 strain test series (with and without saccharifying enzyme), glucose consumption was confirmed 41 hours after the start of culture, and the OD600 at that time was 19.1 and 18.4, respectively.

[0116] Next, for each test series, the culture temperature was changed to 33°C, the rotation speed of the stirring blade to 200 rpm, and the aeration rate to 0 mL / min. Then, 11.1 g of dried chrome powder (moisture content 9.8%) (solid weight 10.0 g) was added. For the test series of AM1 strain and SEY6210 strain used for saccharification enzyme addition, 1 g of saccharification enzyme solution (CTec3) was added, and ethanol production culture was carried out. During culture, the pH of each culture medium was maintained at 6.0 using NaOH (10N).

[0117] At each time point from the start of the ethanol production culture as described above to the start of the reaction as shown in Table 3, a portion of the culture medium was sampled for each test series, and the concentrations of glucose, mannitol (fermentation substrate), and ethanol at each time point were measured. The results are shown in Table 3, Figure 3A, Figure 3B, Figure 3C, and Figure 3B.

[0118]

[0119] As can be seen from Table 3 and Figure 3, in both strains AM1 and SEY621, in the samples to which saccharifying enzymes were added ("strain AM1, enzyme added" and "strain SEY6210, enzyme added"), an increase in ethanol concentration was observed as the mannitol concentration, which is the substrate, decreased with the progression of culture time. Looking at it in more detail, the increase in ethanol concentration from the start of ethanol production culture to 168 hours was 6.50 g / L for "strain AM1, enzyme added," whereas no increase in ethanol concentration was detected for "strain AM1, no enzyme added." Furthermore, in "strain SEY6210, enzyme added," the increase in ethanol concentration from the start of ethanol production culture to 168 hours was 2.67 g / L, while no increase in ethanol concentration was detected for "strain SEY6210, no enzyme added."

[0120] The results of this test showed that both strain AM1 and strain SEY6210 could produce ethanol when saccharifying enzymes were added. However, strain AM1, which has alginate assimilation ability and mannitol assimilation ability, showed a higher ethanol yield compared to strain SEY6210.

[0121] Furthermore, the results of this test show that when using dried materials of macroalgae such as *Crocidolomia* directly as substrate material and producing substances through a microbial process, treating the substrate material with saccharifying enzymes significantly improves the ethanol yield. Additionally, using microorganisms that have the ability to assimilate alginate, alginate deoxysodium (4-deoxy-L-threo-5-hexosloth-uronic acid), and mannitol, such as strain AM1, is even more effective in improving the yield of the target substance.

[0122] [Test Example 4] In this test example, ethanol production was carried out using dried Chromium algae fragments or pulverized materials prepared at several particle sizes. The details of the procedure are shown below.

[0123] Using a Power Mill P-04S (manufactured by Dalton Co., Ltd.) equipped with the following screens (1) to (3), three types of dried Chromium algae samples with different particle sizes were obtained from dried Chromium algae chips. In addition to the test series using these three types of samples, a test series using the dried Chromium algae powder samples used in Test Examples 1 to 3 was also added as a comparative control. (1) Stainless steel round hole screen φ4.0 mm, (2) Stainless steel round hole screen φ2.0 mm, (3) Mesh screen with a mesh opening of 1.0 mm (JIS Z 8801). In addition, in Table 4 and Figures 4A and 4B described later, the test series for the dried chrome samples obtained using the screens described in (1) to (3) above (hereinafter referred to as "Sample (1)", "Sample (2)", "Sample (3)", etc.) and the dried chrome powder samples (hereinafter referred to as "Sample (4)", etc.) are respectively labeled as "φ4.0 mm", "φ2.0 mm", "1.0 mm mesh", and "powder".

[0124] Glycerol stock of strain AM1 was inoculated onto YPD agar under sterile conditions and cultured at 30°C. The grown bacteria were suspended in an appropriate amount of YPD medium and inoculated into 100 mL of YPD medium prepared in a 250 mL culture vessel so that the initial turbidity OD600 was 0.005. Cell growth culture was carried out with the culture temperature set to 30°C, the rotation speed of the stirring blade set to 550 rpm, and the aeration rate set to 20 mL / min. During culture, NH ３ The pH of the culture medium was maintained at 6.0 using (5N). Glucose consumption was confirmed 21 hours after the start of incubation, and the OD600 values ​​for samples (1) to (4) at that time were 35.1, 34.5, 35.2, and 35.3, respectively.

[0125] For the pre-culture solutions of each test series obtained from the above bacterial growth culture, the culture temperature was changed to 33°C, the rotation speed of the stirring blade to 200 rpm, and the aeration rate to 0 mL / min. Then, 11.1 g of each of the above dried chromium samples was added to each, followed by 1 g of the enzyme solution CTec3, and ethanol production culture was carried out. During the culture, the pH of each culture solution was maintained at 6.0 with NaOH (10N).

[0126] At each time point from the start of the ethanol production culture as described above to the start of the reaction as shown in Table 4, sampling was performed as appropriate for each test series, and the concentrations of glucose, mannitol (fermentation substrate), and ethanol at each time point were measured. The results are shown in Table 4 and Figures 4A and 4B.

[0127]

[0128] As can be seen from Table 4 and Figures 4A and 4B, the above-mentioned dried chrome samples (1) to (3) obtained using various screens showed an unexplained decrease in ethanol concentration in the early stages of ethanol production. However, compared to the ethanol concentration at the start of the ethanol production reaction (when dried chrome was added), an increase in ethanol concentration was confirmed in each sample: 4.64 g / L by 288 hours for sample (1), 5.15 g / L by 288 hours for sample (2), and 4.76 g / L by 336 hours for sample (3). Compared with the series of chrome powder samples (4) included as a comparison, although the rate of increase in ethanol was small for samples (1) to (3), by extending the reaction time, it was possible to achieve an ethanol yield equivalent to that of the chrome powder sample (4), and the results were comparable when considering actual ethanol production.

[0129] This test example demonstrates that ethanol production is possible according to the embodiments of the present invention, even when using relatively large particle size dried macroalgae as raw materials (fine fragments or pulverized material).

[0130] [Test Example 5] In this test example, a new strain of Saccharomyces cerevisiae that acquired the ability to utilize mannitol was created through breeding, and this strain was used as an alcohol-fermenting microorganism to produce ethanol. The details of the procedure are shown below.

[0131] First, following the breeding method described in Apple Environ Microbiol. 2014 Dec 11;81(1):9-16., strain SEY6210 was subcultured in a medium containing mannitol as the sole carbon source, and four strains (SEY-TI01, SEY-TI02, SEY-TI03, and SEY-TI04) that acquired mannitol assimilation ability were obtained.

[0132] Glycerol stocks of these strains were inoculated onto YPD agar under sterile conditions and cultured at 30°C. The grown bacteria were suspended in an appropriate amount of YPD medium, and this was inoculated into 100 mL of YPD medium prepared in a 250 mL culture vessel so that the initial turbidity was 0.005. Then, bacterial growth culture was carried out with the culture temperature set to 30°C, the rotation speed of the stirring blade set to 550 rpm, and the aeration rate set to 20 mL / min. During culture, 5N NH40 was used. ３ The pH of the culture medium was maintained at 6.0. For SEY-TI01, SEY-TI02, and SEY-TI03, glucose consumption was confirmed after 47 hours, and the OD600 values ​​at that time were 17.8, 14.2, and 17.5, respectively. For SEY-TI04, cultivation was continued until 65 hours later, and the next procedure was performed when the remaining glucose concentration reached 18.0 g / L and the OD600 value reached 16.6.

[0133] Next, the culture temperature was changed to 33°C, the rotation speed of the stirring blade to 200 rpm, and the aeration rate to 0 mL / min. Then, 11.1 g of dried chrome powder (moisture content 9.8%) (solid weight 10.0 g) and 1 g of enzyme solution CTec3 were added to the culture medium, and ethanol production culture was carried out. During the culture, the pH of the culture medium was maintained at 6.0 with 10N NaOH.

[0134] The culture medium was sampled at each predetermined time point after the start of ethanol production culture, and the concentrations of glucose, mannitol, and ethanol were measured at each point in time. The results are shown in Table 5 and Figures 5A, 5B, 5C, and 5D.

[0135]

[0136] As can be seen from Table 5 and Figures 5A to 5D, in all strains of SEY-TI01, SEY-TI02, SEY-TI03, and SEY-TI04, an increase in ethanol production was observed as mannitol was consumed over time. Furthermore, for each strain of SEY-TI01, SEY-TI02, SEY-TI03, and SEY-TI04, the increase in ethanol concentration (ΔEtOH) from the start of ethanol production culture to 192 hours later was confirmed to be 6.0 g / L, 5.6 g / L, 9.2 g / L, and 9.5 g / L, respectively. This confirmed that all mannitol-assimilating strains produced in this test were able to produce a substantial amount of ethanol from mannitol derived from dried krom powder used as a raw material.

[0137] (Summary) As described above, the results of Test Examples 1 to 5 show that, according to the embodiments of the present invention, dried material of macroalgae such as *Crocidolomia* (which has been turned into a powder by a relatively simple grinding process) can be sufficiently saccharified by a saccharifying enzyme without undergoing complicated chemical treatments such as extraction of organic solvents such as hexane and alcohol, and alkaline extraction. Furthermore, the resulting saccharified liquid can be immediately used as a substrate for alcohol fermentation by alcohol-fermenting microorganisms, thus demonstrating that alcohol can be produced simply and efficiently.

[0138] This invention has high industrial applicability in fields such as biotechnology, fine chemicals, and material production.

Claims

1. A method for producing alcohol, comprising (b) carrying out alcohol fermentation using alcohol-fermenting microorganisms in a culture medium containing saccharified products of dried macroalgae, wherein the moisture content of the dried macroalgae is 20 wt% or less.

2. The method according to claim 1, wherein the saccharified product is obtained by performing a saccharification treatment on the dried macroalga using at least one saccharifying enzyme.

3. The method according to claim 1, further comprising: (a) performing a saccharification treatment on the dried macroalgae using at least one saccharifying enzyme to obtain the saccharified product, as a step performed before or simultaneously with step (b).

4. The method according to claim 3, wherein a solution containing the above-mentioned dried macroalgae and saccharifying microorganisms is prepared, and step (a) is performed.

5. The method according to claim 4, wherein the saccharifying microorganism is a microorganism that presents at least one saccharifying enzyme on its cell surface.

6. The method according to claim 3, wherein a reaction solution containing the above-mentioned dried macroalgae and at least one saccharifying enzyme is prepared, and step (a) is carried out.

7. The method according to claim 5 or 6, wherein the at least one saccharifying enzyme comprises cellulase and hemicellulase.

8. The method according to claim 3, wherein step (a) is performed, and then step (b) is performed.

9. The method according to claim 3, wherein steps (a) and (b) are carried out simultaneously or consecutively in the same reaction solution.

10. The method according to claim 1, wherein the alcohol-fermenting microorganism is an alcohol-fermenting yeast.

11. The method according to claim 1, wherein the alcohol-fermenting microorganism is an ethanol-fermenting yeast.

12. The method according to claim 1, wherein the alcohol-fermenting microorganism is a yeast having the ability to utilize alginic acid and / or mannitol.

13. The method according to claim 3, wherein the alcohol-fermenting microorganism is a microorganism that has the ability to saccharify the dried macroalgae, and step (a) is carried out using the alcohol-fermenting microorganism in addition to the saccharifying enzyme.

14. The method according to claim 13, wherein the alcohol-fermenting microorganism expresses endo-type alginate lyase, which decomposes alginate into oligosaccharides, and exo-type alginate lyase, which decomposes oligosaccharides into monosaccharides, in a manner that is presented on the cell surface, and is a microorganism that has the ability to assimilate mannitol.

15. The method according to claim 1, wherein the dried macroalgae is a dried brown algae.

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