KR101147503B1 - Mutant strain of brettanomyces sp. and producing method of biofuel using the same - Google Patents

Abstract

PURPOSE: A Brettanomyces sp. mutant strain and a method for preparing biofuel using the same are provided to ferment monosaccharides of red algae without reduction of fermentation speed and to enhance production. CONSTITUTION: A Brettanomyces sp. mutant strain(deposit number: KCCM11216P) ferments carbon source and converts into ethanol. The carbon source includes monosaccharides, polysaccharides, disaccharides, or combination thereof. The Brettanomyces sp. mutant strain is prepared by mutation such as UV ray, ethyl methane sulfonate(EMS), N-methyl-N'-nitro-N-nitrosoguanidine(NTG) or combination thereof.

Description

Bretanomyces genus mutant strain and a method for producing a biofuel using the same {MUTANT STRAIN OF BRETTANOMYCES SP. AND PRODUCING METHOD OF BIOFUEL USING THE SAME}

The present application relates to a strain of the genus Bretanomyces and a method for producing a biofuel using the same.

Since the Industrial Revolution, excessive use of fossil fuels such as petroleum has caused serious problems such as high oil prices, environmental pollution, and climate change due to exhaustion of the main stream. At the time when new alternative energy (alternative fuel, nuclear power, solar, geothermal, wind, hydropower, etc.) is urgently needed to solve all of these problems, bioenergy does not emit any harmful substances, unlike fossil fuels. It is getting attention.

Biofuel is a generic term for energy obtained by using biomass as a raw material, and is obtained through direct combustion, alcohol fermentation, methane fermentation, and the like. Biomass, a raw material of biofuel, is divided into sugar-based (sugar cane, sugar beet, etc.), starch-based (corn, potato, sweet potato, etc.), and wood-based (wood, rice straw, waste paper, etc.). In the case of raw materials, biofuel can be directly converted into biofuel through a fermentation process following a relatively simple pretreatment process. However, in the case of starch and wood, biofuel is manufactured through fermentation process using saccharified liquid after proper pretreatment and saccharification process. can do. Wood-based materials can use waste wood in the form of urban waste or forest by-products scattered throughout the forest as raw materials, and there is no useful value as food, so the stability of supply and demand of raw materials can be secured, but the lignin removal pretreatment process must be accompanied in the process. Due to the increase in the process cost, due to the crystalline structure consisting of hydrogen bonds, which is a characteristic of the wood-based cellulose substrate, there is a disadvantage that the economic efficiency is low due to low saccharification yield.

As of 2006, bioethanol is produced at around 50.1 billion liters worldwide. The world production of biofuels using saccharides, in particular bioethanol, is about 17.7 billion liters (as of 2006), and the main producers are Brazil, India and Taiwan. (Global Bio Energy Partnership (GBEP), 2006). Brazil is actively producing bioethanol for transportation using sugarcane, which is an abundant resource, and various types of ethanol-mixed gasoline (gasohol) are being spread. In 2003, FFV (Flexible Fuel Vehicle), which can operate even if the ethanol and gasoline contents change, began to be sold. As of May 2005, it occupied about 50% of the total sales of passenger cars. In recent years, it has exceeded 80 billion liters in 2011 due to increased demand and opacity of oil energy supply.

On the other hand, algae are largely divided into macroalgae and microalgae, and large algae include red algae, brown algae, green algae, microalgae, chlorella and spirulina. Seaweed production is estimated at around 14 million tonnes per year worldwide and is expected to increase to more than 22 million tonnes by 2020. These production amounts to about 23% of the total aquaculture production, more than 90% of which consists of brown seaweed such as seaweed and kelp, and red algae such as laver, seaweed, and stalks. The production of algae farming in Korea is now about 500,000 tons, which is slightly lower than about 700,000 tons in the mid-90s, but the total area of the farms is about 70,000 ha, which is higher than about 60,000 ha in the mid-90s. Among these algae, red algae are particularly marine-derived biomass that has excellent growth potential, wide available cultivation area, can be harvested four times a year in the waters of Korea, and can be harvested up to six times a year using the subtropical climate of Southeast Asia. In addition, the use of high cost resources such as freshwater, land, and fertilizers is low, and in the case of wood, there is no lignin component that must be removed, so the manufacturing process is simple and ethanol is expected to be produced at the starch level. For example, Gelidium, Gelidium , a Moroccan loot amansii , Morocco) 70-80% of carbohydrates (milk and fiber) and 20-30% of carbohydrates (protein, lipids, and other) are the most efficient as ethanol-producing raw materials. It can be used as. In particular, galactose accounts for 26% of total dry weight and 34% of carbohydrates, and glucose is known to form 17% of total dry weight and 22% of carbohydrates in the form of cellulose.

Red algae bioethanol production consists of saccharification, fermentation, separation and purification. Glycation is a process of converting carbohydrates (polysaccharides) in the form of polymers into monosaccharides, and there are physical glycosylation techniques using chemicals such as acids and biological glycosylation techniques using enzymes. The monomer obtained in the saccharification process is fermented with ethanol through the fermentation process by microbial metabolism, and the fermentation broth is separated and purified to produce ethanol for fuel.

 The saccharification process of red algae bioethanol production involves the hydrolysis of glucoside bonds using gallic acid agar, which is present in red algae, with acids or enzymes, resulting in D-galactose, 3,6-anhydro-L-galactose (3). , 6-anhydro-L-galactose (AHG).

Most ethanol-producing strains are known to convert glucose to ethanol in most cases. However, in red algae, galactose, which is a saccharified product of galactan, is the main monosaccharide for fermentation, and thus, a strain having excellent galactose utilization needs to be developed. In the production of red algae bioethanol, fermentation is inhibited by high galactose concentration and high ethanol concentration in ethanol fermentation, and the representative fermentation produced by pyrolysis of C ₆ monosaccharides produced in saccharification process at high pressure. Inhibition products such as hydroxymethylfurfural (Hydroxymethylfurfural; 5-HMF) such as inhibit the growth of microorganisms can reduce the fermentation rate or ethanol production yield can be very low. Fermentation inhibitors There are active carbon and ion resin treatment methods to remove HMF from fermentation broth, but the process becomes complicated and the production cost increases when designing the process. Therefore, during the ethanol fermentation process, it is urgent to develop a strain capable of producing fermentation without being affected by the inhibitor 5-HMF, having strong resistance and converting galactose to ethanol with high efficiency.

Accordingly, there is a need for the development of new strains for effectively converting biomass with a high galactose content such as red algae into bioethanol. Korean Patent No. 10-1075602 discloses a method for preparing bioethanol by degrading red algae using a mutated strain of Bretanomyces custersii.

Therefore, the present application is to provide a strain strain of Bretanomyces genus and a method for producing a biofuel using the same.

However, the problems to be solved by the present invention are not limited to the problems described above, and other problems not described can be clearly understood by those skilled in the art from the following description.

A first aspect of the present application provides a Bretanomyces brookselensis mutant strain (Accession Number: KCCM11216P), which ferments a carbon source and converts it to ethanol.

The second aspect of the present application, the production of ethanol comprising contacting the Bretanomyces brookselensis variant strain (Accession Number: KCCM11216P) of the first aspect of the present application with a carbon source to produce ethanol by fermentation of the carbon source Provide a method.

In conventional red algae bioethanol production, fermentation is inhibited by high galactose concentration and ethanol concentration produced in fermentation, and growth of microorganisms is inhibited by substances such as 5-HMF, a representative fermentation inhibitory product present in saccharified solution. There was a problem that the fermentation rate is reduced or ethanol production is lowered, but the Bretanomyces brookselensis mutant strain prepared by mutating brethanomyces, a yeast isolated from beer, is effectively a monosaccharide of red algae without decreasing the fermentation rate. Can be fermented with ethanol and production of ethanol can be increased even in the presence of 5-HMF.

Figure 1 is a schematic diagram showing the entire process of the variation, isolation, and ethanol production capacity of the strain according to an embodiment of the present application.
Figure 2 is a graph showing the ethanol production ability results of the strain according to an embodiment of the present application.

Hereinafter, with reference to the accompanying drawings will be described in detail the embodiments and embodiments of the present application to be easily carried out by those of ordinary skill in the art.

It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and like reference numerals designate like parts throughout the specification.

Throughout this specification, when a portion is "connected" to another portion, this includes not only "directly connected" but also "electrically connected" with another element in between. do.

Throughout this specification, when a member is located “on” another member, this includes not only when one member is in contact with another member but also when another member is present between the two members.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

As used herein, the terms "about", "substantially", and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are provided to aid the understanding herein. In order to prevent the unfair use of unscrupulous infringers. In addition, throughout this specification, "step to" or "step of" does not mean "step for."

Throughout this specification, the term “combinations of these” included in the expression of the mark of the form refers to one or more mixtures or combinations selected from the group consisting of the elements described in the mark of the form of the mark. It means to include one or more selected from the group consisting of elements.

Hereinafter, with reference to the embodiments and examples and drawings with respect to Bretanomyces genus strains of the present application and a method for producing a biofuel using the same. However, the present application is not limited to these embodiments, examples and drawings.

A first aspect of the present disclosure may provide a Bretanomyces brookselensis mutant strain (Accession Number: KCCM11216P) that ferments a carbon source to convert to ethanol. The Bretanomyces Brookselens mutant strain was named Bretanomyces Brookselenssis BSM0016, and was deposited with the Korea Microorganism Conservation Center on November 4, 2011 under accession number KCCM11216P. The Bretanomyces Brookeslensis BSM0016 is a fermentation growth inhibitory substance (5-HMF) resistant strain and exhibits aerobicity.

According to one embodiment of the present application, the Bretanomyces brookelensis mutant strain may be mutated by mutagenesis, for example, the parent strain Brettanomyces Brookselenssis ( Brettanomyces bruxellensis ; BB) may be modified by treating the strain selected from the group consisting of ultraviolet light, ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and combinations thereof. However, the present invention is not limited thereto.

Referring to Figure 1, it will be described in detail an embodiment of the method for producing the Bretanomyces Brookelenosis mutant strain.

The parent strain Bretanomyces brookelensis is cultured in a culture medium, such as, for example, YPG broth at about 20 ° C. to about 40 ° C., for example at about 30 ° C., from about 50 rpm to about 300 rpm, for example about 150 after preincubation for an appropriate time at rpm, for example about 24 hours, ultraviolet light, ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and combinations thereof Selected from the group consisting of, such as, for example, NTG treatment, suitable for at about 20 ℃ to about 40 ℃, for example about 30 ℃ while shaking at about 50 rpm to about 300 rpm, for example about 150 rpm Mutations can be caused by incubation for a period of time, for example about 20 minutes. Then, the culture solution is plated on the YPG plate and incubated, the parent strain and the number of colonies are compared and 50% survival time is measured, only excellent colonies are isolated and passaged to isolate the variant strains of the present application.

The Brettanomyces bruxellensis (BB) is a yeast isolated from beer and has been traditionally used for wine fermentation for a long time and is a strain capable of producing bioethanol using galactose. However, growth is inhibited by 5-HMF, a representative fermentation inhibition product present in the saccharified solution, and the fermentation rate is decreased. Thus, by mutating the Bretanomyces Bruxelensis by mutation method, for example, the Bretanomyces Brookselensis high ethanol fermentation efficiency even at high 5-HMF conditions of about 5 g / L concentration or more Mutant strains can be prepared.

According to the exemplary embodiment of the present application, the carbon source may include, but is not limited to, one selected from the group consisting of monosaccharides, disaccharides, polysaccharides, and combinations thereof. The monosaccharide may comprise one selected from the group consisting of galactose, glucose, fructose and combinations thereof, the disaccharide may comprise one selected from the group consisting of sucrose, maltose, lactose and combinations thereof, The polysaccharide may include, but is not limited to, one selected from the group consisting of radish, starch, fibrin, carrageenan, alginic acid, and combinations thereof.

According to the exemplary embodiment of the present application, the carbon source may include, but is not limited to, one selected from the group consisting of a saccharide system, a starch system, a wood system, algae, and combinations thereof. The sugar system may include, for example, sugar cane, sugar beet, and the like, but is not limited thereto. The starch system may include, for example, corn, potatoes, sweet potatoes, etc., but is not limited thereto. The wood system may include, for example, wood, rice straw, waste paper, etc., but is not limited thereto. The algae may include one selected from the group consisting of red algae, brown algae, green algae, and combinations thereof, for example, but may not be limited to red algae. The red alga is, for example, wood starfish, laver, kotoni, dog gambling, rounded seaweed, ox radish, buckwheat, green starfish, pods, jindubal, sesame gambling, spiny radish, silk grass, edible leaf, stone star, stone and Jinu It may be selected from the group consisting of Ari, but is not limited thereto. The brown algae, for example, from the group consisting of brown seaweed, kelp, barn horse, folk eggplant, shellfish, gorimae, seaweed, Ecklonia cava, gompi, rhubarb, iron seaweed cousin, mabanban, hoe sang mabanban, jichung and 톳 It may be selected, but is not limited thereto. The green algae may be selected from the group consisting of Cheongtae, Hakkham, green, hearing, bead hearing, jade and salt juice, but is not limited thereto.

The second aspect of the present application, ethanol comprising producing ethanol by culturing the fermented Bretanomyces brookselensis variant strain (Accession Number: KCCM11216P) of the first aspect of the present application in a culture medium containing a carbon source and the like and fermentation It is possible to provide a method for producing.

According to one embodiment of the present application, the culture medium may include 5-HMF, but is not limited thereto.

Referring to Figure 1, it will be described in detail the ethanol production method according to an embodiment of the present application.

Bretanomyces brookselensis mutant strain (Accession No .: KCCM11216P) was inoculated in a culture medium, for example, YPG medium, followed by incubation for about 24 hours at about 20 ° C. to about 40 ° C. with shaking at about 150 rpm. , Transfer to YPG medium containing 5-HMF and measure the ethanol production while incubating for about 108 hours.

According to the exemplary embodiment of the present application, the carbon source may include, but is not limited to, one selected from the group consisting of monosaccharides, disaccharides, polysaccharides, and combinations thereof. The monosaccharide may comprise one selected from the group consisting of galactose, glucose, fructose and combinations thereof, the disaccharide may comprise one selected from the group consisting of sucrose, maltose, lactose and combinations thereof, The polysaccharide may include, but is not limited to, one selected from the group consisting of radish, starch, fibrin, carrageenan, alginic acid, and combinations thereof.

According to the exemplary embodiment of the present application, the carbon source may include, but is not limited to, one selected from the group consisting of saccharide, starch, wood, algae, and combinations thereof. The sugar system may include, for example, sugar cane, sugar beet, and the like, but is not limited thereto. The starch system may include, for example, corn, potatoes, sweet potatoes, etc., but is not limited thereto. The wood system may include, for example, wood, rice straw, waste paper, etc., but is not limited thereto. The algae may include one selected from the group consisting of red algae, brown algae, green algae, and combinations thereof, for example, but may not be limited to red algae. The red alga is, for example, wood starfish, laver, kotoni, dog gambling, rounded seaweed, ox radish, buckwheat, green starfish, pods, jindubal, sesame gambling, spiny radish, silk grass, edible leaf, stone star, stone and Jinu It may be selected from the group consisting of Ari, but is not limited thereto. The brown algae, for example, from the group consisting of brown seaweed, kelp, barn horse, folk eggplant, shellfish, gorimae, seaweed, Ecklonia cava, gompi, rhubarb, iron seaweed cousin, mabanban, hoe sang mabanban, jichung and 톳 It may be selected, but is not limited thereto. The green algae may be selected from the group consisting of Cheongtae, Hakkham, green, hearing, bead hearing, jade and salt juice, but is not limited thereto.

The most popular species of red algae are the most diverse species and excellent growth. On the basis of dry weight, about 15-25% of cellulose is composed of cellulose and about 50-70% of radish is mainly composed of galactan. It consists of less than 15% protein and less than 7% lipid. As the brown algae, seaweed, kelp, barn horse, folk eggplant, shellfish, hooked seaweed, seaweed, Ecklonia cava, gompi, rhubarb, iron seaweed cousin, mabanban, hoesan mabanban, jichungyi, 톳 and the like may be used. It doesn't happen. Brown algae are multicellular bodies and are best differentiated among algae. The green algae may be used, but are not limited to Cheongtae, Hakkham, blue, auditory, bead hearing, jade, salt-jumping, and the like. Green algae have chlorophyll and make starch by photosynthesis. Looking at the components of brown algae and green algae, brown algae contain about 30-40% of alginic acid and about 5-6% of fibrin, and green algae contain about 40-50% of starch, the main component of carbohydrate, and 5% of fibrin. It contains less than.

The method for extracting polysaccharide materials such as radish, cellulose, starch, carrageenan, alginic acid and the like from the biomass is not particularly limited, and any method known in the art may be used. According to one preferred embodiment, the biomass, for example, seaweeds are immersed in an aqueous alkali solution for a predetermined time and then washed with water, and the washed seaweeds are immersed in an extraction solvent made of an acidic chemical for a predetermined time to extract the radish, carrageenan, and alginic acid components. The step of collecting residual fibrin and starch may extract the radish, carrageenan, alginic acid and starch or fiber. At this time, the extraction temperature is not particularly limited, but is preferably in the range of about 80 to about 150 ℃. The acidic chemicals include H ₂ SO ₄ , HCl, HBr, HNO ₃ , CH ₃ COOH, HCOOH, HClO ₄ (perchloric acid), H ₃ PO ₄ (phosphoric acid), PTSA (paratoluene sulfonic acid) or commercial solid acids. However, the present invention is not limited thereto, but the alkaline aqueous solution may include potassium hydroxide, sodium hydroxide, calcium hydroxide, aqueous ammonia solution, and the like, but is not necessarily limited thereto. Monosaccharides can be obtained by treating and extracting a polysaccharide substance such as radish, starch, fibrin, carrageenan, alginic acid and the like by treating with an appropriate degrading enzyme and / or a hydrolysis catalyst.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present application is not limited thereto.

[ Example ]

< Example  1> EMS Using Bretanomyces Brookselensis  Preparation of Mutant Strains

Brettanomyces mother strain Brettanomyces bruxellensis ) (Korea Microorganism Conservation Center, Accession No .: KCCM 11490) was added to 100 ml of YPG (Yeast extract 10 g / L, Peptone 20 g / L, Galactose 200 g / L) medium at 30 ° C, pH 7 at 150 rpm. After incubation for 24 hours, mutations were made using EMS. Variation method is as follows (Mobini-Dehkordi et al (2008 ) Isolation of a Novel Mutant Strains of Saccharomyces cerevisiae by an Ethyl Methane Sulfonate-Induced Mutagenesis Approach as a High Producer of Bioethanol J. Biosci Bioeng, 105....: 403-408).

The shake cultured 10 ml of the parent strain was washed with 10 ml of sterile water and then again with 0.1 M sodium phosphate buffer. 1.7 ml of 0.1 M sodium phosphate buffer was added and vortexed followed by 50 μL of EMS (3% of final volume). Mutations were stopped by adding 8 ml of 5% sodium thiosulphate every 15 minutes with shaking culture at 150 ° C. at 30 ° C., pH 7 for 60 minutes. 9 mL of sterile water was added to each culture solution, vortexed, and then plated on YPG plates to compare the number of colonies with no EMS added colonies, and screened for excellent strains using 50% survival time.

< Example  2> NTG Using Bretanomyces Brookselensis  Preparation of Mutant Strains

Brettanomyces mother strain Brettanomyces bruxellensis ) (Korea Microorganism Conservation Center, Accession No .: KCCM 11490) was added to 100 ml YPG (Yeast extract 10 g / L, Peptone 20 g / L, Galactose 200 g / L) broth at 30 ° C, pH 7 at 150 rpm. After incubation for 24 hours, mutations were made using NTG. Variation method is as follows (Kim Soo-ki (2007) Increased Carotenoid Production in Xanthophyllomyces dendrorhous G276 Using Plant Extracts. Journal of Microbiology ., Vol. 45, No. 2. p128-132).

After washing 1 ml of the shaken mother strain with 0.1 M sodium citrate (pH 5.5), 5 ml of 0.1 M sodium citrate buffer was added, and NTG solution (1 μg / ml) was added to 10 μg / ml and 30 μg / ml. , 50 μg / ml, and 100 μg / ml were added. Shake incubation at 150 ° C. at 30 ° C., pH 7 for 20 minutes, followed by washing with 1 ml of 0.1 M potassium phosphate buffer. When 300 μl of 0.1 M potassium phosphate buffer was added and plated on YPG plate to compare the number of colonies of parent strain with NTG solution, the survival rate was 69% and 30 μg when the concentration of NTG solution was 10 μg / ml. 78% for / ml and 17% for 50 ㎍ / ml, but the survival rate was 49% when the concentration of NTG solution was 100 ㎍ / ml. Therefore, excellent strains were selected by screening with 100 ㎍ / ml NTG solution. .

The Bretanomyces brookselensis mutant strain prepared as above was named Bretanomyces brookselensis BSM0016, and deposited with the Korea Microorganism Conservation Center as of November 4, 2011 with accession number KCCM11216P.

< Experimental Example  1> Bretanomyces Brookselensis  Ethanol from Mutant Strains Generation  evaluation

Bretanomyces brookselensis mutant strain and parent strain Bretanomyces brookselensis prepared as in Example 2, high concentration galactose culture medium (Yeast extract 10 g / L to which 5 g / L 5-HMF was added) , Peptone 20 g / L, Galactose 200 g / L) and then incubated for 30 hours at 30 ℃, pH 7 for 150 hours, ethanol production was analyzed by HPLC. In addition, after culturing the parent strain Bretanomyces Brookelensis in a culture medium without 5-HMF for comparison, ethanol production ability was analyzed by HPLC.

As can be seen in Figure 2, the parent strain exhibits about 58 g / L ethanol production capacity, whereas the Bretanomyces Brookelensis mutant strain of the present application shows an ethanol production capacity of about 87 g / L, Bretanomyces brookselensis mutant of the ethanol production was confirmed that 1.5-times higher than the conventional microbial strain under 5-HMF conditions with high galactose content.

The foregoing description of the application is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present application is shown by the claims below rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. .

Korea Microbial Conservation Center KCCM11216P 20111104

Claims (12)

Bretanomyces brookselensis mutant strain (Accession No .: KCCM11216P), which ferments the carbon source to ethanol.
The method of claim 1,
Wherein the carbon source comprises one selected from the group consisting of monosaccharides, disaccharides, polysaccharides and combinations thereof, Bretanomyces Brookselens mutant strain.
The method of claim 1,
The Bretanomyces brookselensis variant strain is mutated by mutation, Bretanomyces brookselensis variant strain.
The method of claim 3, wherein
Wherein said mutagenesis comprises treating one selected from the group consisting of ultraviolet light, ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and combinations thereof. Bretanomyces brookselensis mutant strain. Ethanol comprising producing a ethanol by culturing the Bretanomyces brookselensis variant strain (Accession Number: KCCM11216P) according to any one of claims 1 to 4 in a culture medium containing a carbon source to ferment the carbon source. Method of preparation.
The method of claim 5, wherein
The culture medium is one containing 5-HMF, ethanol production method.
The method of claim 5, wherein
Wherein the carbon source comprises one selected from the group consisting of monosaccharides, disaccharides, polysaccharides, and combinations thereof.
The method of claim 7, wherein
Wherein said monosaccharide comprises one selected from the group consisting of galactose, glucose, fructose and combinations thereof.
The method of claim 7, wherein
Wherein the disaccharide is sucrose, maltose, lactose and a combination of those selected from the group consisting of, ethanol production method.
The method of claim 7, wherein
Wherein said polysaccharide comprises one selected from the group consisting of daikon, starch, fibrin, carrageenan, alginic acid, and combinations thereof.
The method of claim 5, wherein
Wherein the carbon source comprises a one selected from the group consisting of sugar-based, starch-based, wood-based, seaweed and combinations thereof, ethanol production method.
The method of claim 11,
The algae will include red algae, brown algae, green algae and those selected from the group consisting of, ethanol production method.

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