KR101075602B1 - Mutant Strain of Brettanomyces custersii and Method of Ethanol Production Using the Same - Google Patents

Abstract

The present invention relates to a Bretanomyces custersii mutant strain having excellent fermentation ability using galactose and a method for producing ethanol using the same, and the microbial strain according to the present invention has a very high galactose use efficiency, and has a high content of galactose. For example, there is an advantage that can significantly improve the time and cost of producing ethanol from algae, algae.

Description

Mutant Strain of Brettanomyces custersii and Method of Ethanol Production Using the Same}

The present invention is Brettanomyces custersii ) The present invention relates to a mutant strain and a method for producing ethanol using the same, and more particularly, to a novel Bretanomyces custersii mutant strain having excellent fermentation ability using galactose and a method for producing ethanol using the same.

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.

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 somewhat 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 (Table 1). 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.

Chemical Composition of Glydium Amansi Cellulose (fiber)
(%) Baby (Galactan) Other (protein, lipids, ash)
(%) Galactose (%) 3,6-AHG (%) 16.6 25.6 33.0 24.8

Currently, Saccharomyces cerevisiae and Bretanomyces custersii, microorganisms mainly used for ethanol fermentation, have been reported to have lower fermentation capacity using galactose than glucose (Keating et al. (2004). ), Characterization of a unique ethanologenic yeast capable of fermenting galactose, Enzyme and Microbioal Technology, Vol. 35, pp. 242-253). Therefore, in order to effectively convert biomass with a high content of galactose, such as red algae, to ethanol, it is necessary to discover a new strain having excellent galactose availability.

The present invention has been made to improve the problems of the conventional ethanol fermentation strain and the biofuel manufacturing method using the same, a novel strain having excellent galactose use fermentation ability by improving the low galactose utilization which was a problem in the conventional fermentation and It is an object to provide a method for producing ethanol using the same.

In order to achieve the above object, the present invention provides a novel microbial strain belonging to Bretanomyces custersii species that can ferment ethanol using a carbon source.

The microbial strain of the present invention was obtained by mutating Bretanomyces custersii yeast strains discovered from nature by ultraviolet irradiation, and the microbial strains were transmitted to the Korea Biotechnology Center of Korea Biotechnology Research Institute on March 5, 2009. Deposited (Accession Number: KCTC18154P). Bretanomyces custersii KCTC18154P microbial strain of the present invention has the following microbial properties.

end. Morphological characteristics

-Yeast ovate and elliptical, 3-10 × 3-20 ㎛.

I. Characteristics of incubation period

-It shows spherical shape, ovoid shape, cylinder shape and oval shape in liquid culture and breeds by germination method.

All. Physiological properties

-Breathable

-Growth temperature: 20-40 ℃

-Optimum growth temperature: 25-33 ℃

-Growth pH: 4.0-7.0

Optimum growth pH: 4.8-5.5

-Optimum temperature for fermentation: 27-30 ℃

la. Carbon Sources Used: Glucose, Sugar, Maltose, Galactose

In addition, any composition including monosaccharides, disaccharides, and polysaccharides may be used as the carbon source. The monosaccharides include galactose, glucose, fructose, and the like, and the disaccharides include sucrose, maltose, lactose, and the like, and the polysaccharides include daikon radish, starch, fiber, carrageenan, alginic acid, and the like. However, other carbon sources may be used without limitation. Preferably these carbon sources can be extracted from biomass such as sugar based (sugar cane, sugar beets, etc.), starch based (corn, potatoes, sweet potatoes, etc.), wood based (wood, rice straw, waste paper, etc.), or seaweeds.

The present invention also provides a method for producing ethanol using the Bretanomyces custersii mutant strain.

Ethanol production method of the present invention

(1) preculturing by inoculating Bretanomyces custersii mutant strain (Accession Number: KCTC18154P) into the culture medium; And

(2) adding the preculture to a fermenter containing a carbon source to ethanol fermentation.

In step (1), the preculture of the Bretanomyces custersii variant strain is aerobic at 50-300 rpm, preferably about 150 rpm, at a culture temperature of 20-40 ° C., preferably about 30 ° C. It is preferable to culture. In addition, the culture medium is preferably YEPD (yeast extract 10 g / l, peptone 20 g / l, dextrose 20 g / l) medium, but is not limited to this, can be used without limitation microbial culture medium have.

In step (2), the ethanol fermentation is preferably carried out in an anaerobic state at a culture temperature of 20-40 ℃, preferably about 30 ℃. In addition, the initial pH is preferably 5.0 to 5.5, the inoculation amount is 20 to 30% by weight, but is not necessarily limited thereto. In addition, any composition including monosaccharides, disaccharides, and polysaccharides may be used as the carbon source. The monosaccharides include galactose, glucose, fructose, and the like, and the disaccharides include sucrose, maltose, lactose, and the like, and the polysaccharides include daikon radish, starch, fiber, carrageenan, alginic acid, and the like. However, other carbon sources may be used without limitation. Preferably these carbon sources can be extracted from biomass such as sugar based (sugar cane, sugar beets, etc.), starch based (corn, potatoes, sweet potatoes, etc.), wood based (wood, rice straw, waste paper, etc.), or seaweeds. As the algae, large algae such as red algae, brown algae and green algae may be used without limitation. As the red algae, wood starfish, laver, kotoni, dog gambling, round stone laver, ox radish, buckwheat, green grass, walnut, jindubal, sesame gourd, spiny radish, silk grass, vulgaris, stone star, stone tree, jinari But it is not limited thereto, and among them, it is preferable to use a stump. The most popular species of red algae are the most diverse species, and the growth is excellent. The dry weight accounts for about 15-25% of cellulose, cellulose, and about 50-70% 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 80 to 150 ° C. The acidic agent may be H ₂ SO ₄ , HCl, HBr, HNO ₃ , CH ₃ COOH, HCOOH, HClO ₄ (perchloric acid), H ₃ PO ₄ (phosphoric acid), PTSA (para-toluene sulfonic acid) or a commercial solid. Acids and the like, but is not necessarily limited thereto, and the alkali aqueous solution may include potassium hydroxide, sodium hydroxide, calcium hydroxide, aqueous ammonia solution, but is not necessarily limited thereto. The monosaccharide can be obtained by treating the polysaccharide substance extracts such as radish, starch, fibrin, carrageenan, alginic acid, and the like with appropriate enzymes and / or hydrolysis catalysts.

Since the microbial strain according to the present invention has excellent galactose utilization efficiency, it is possible to drastically improve the time and cost of producing ethanol from biomass having a high galactose content, such as algae red algae.

1 is a graph showing a fermentation result of a high concentration of galactose / glucose using the Bretanomyces custersii mutant strain of the present invention.
Figure 2 is a graph showing the results of fermentation of high concentration of galactose / glucose using the conventional Bretanomyces custersii strain.
Figure 3 is a graph showing the results of fermentation of the native sugar saccharin of Bretanomyces custersii mutant strain of the present invention.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only illustrative of the present invention in detail, and the content of the present invention is not limited by the examples.

Example 1 Preparation of Bretanomyces Custersii Mutant Strains

The mother strain Bretanomyces custersii cultured in the saccharified liquid-agar medium obtained by steam-exposure of fibrin-based biomass was irradiated with energy of 1,200 erg / mm 2 using ultraviolet rays having a wavelength of 245 nm to induce artificial mutation. The culture solution thus treated was inoculated onto a plate medium (galactose 2%, yeast extract 1%, peptone 2%, agar 1.5%), and then cultured at 30 ° C. for 3 days to separate and grow only colonies having good growth. Bretanomyces custersii mutant strains and parent strains isolated therefrom were fermented for 3 days under optimal fermentation conditions using galactose 2, 3 and 4 wt% sugar solution. The fermentation yield was higher than 25%. In addition, as a result of confirming the fermentation ability of galactose for each generation while continuing subculture until reaching 20 generations in order to confirm genetic stability, the mutant strain has a fermentation performance with the primary cultured mutant strain until reaching 20 generations. It appeared to be at the same level. The present inventors deposited the Bretanomyces custersii mutant strain at the Korea Biotechnology Center, Korea Research Institute of Bioscience and Biotechnology on March 5, 2009 (Accession Number: KCTC18154P). The Brentanomyses custersii KCTC18154P strain was shown to have the following bacterial characteristics.

end. Morphological characteristics

-Yeast ovate and elliptical, 3-10 × 3-20 ㎛.

I. Characteristics of incubation period

-It shows spherical shape, ovoid shape, cylinder shape and oval shape in liquid culture and breeds by germination method.

All. Physiological properties

-Breathable

-Growth temperature: 20-40 ℃

-Optimum growth temperature: 25-33 ℃

-Growth pH: 4.0-7.0

Optimum growth pH: 4.8-5.5

-Optimum temperature for fermentation: 27-30 ℃

la. Carbon Sources Used: Glucose, Sugar, Maltose, Galactose

Example  2. Bretanomyces Custerssee  Mutant strains and conventional Bretanomyces Ku Galactose / Glucose of Sterciy Strains Mixed sugar  Fermentation comparison

Comparative experiments of the Bretanomyces custersii mutant strain of the present invention and the conventional Bretanomyces custerishi KCCM11490 strain were carried out under the same high concentration of mixed sugars (initial galactose 75 g / l, glucose 25 g / l). . To this end, the strain preserved in a solid medium was inoculated with platinum in YEPD (yeast extract 10 g / l, peptone 20 g / l, dextrose 20 g / l) medium, and stirred at 30 rpm at 150 ° C. for 24 hours. Preculture in aerobic state. Fermentation experiments were performed in 150 ml medium (yeast extract 10 g / l, peptone 20 g / l, galactose 50, 75, 100, 120 g / l) inoculated with 25% preculture at an initial pH of 5.0-5.5 and a temperature of 30 ° C. Progressed in anaerobic state for 48 hours.

As a result, the galactose consumption rate and the maximum ethanol concentration of the Bretanomyces custersii mutant strains of the present invention were 90.6% and 40.3 g / l, respectively (Fig. 1). In contrast, the conventional Bretanomyces costerie The galactose consumption rate and the maximum ethanol concentration of Shi KKCM11490 strain were 60.1% and 10.0 g / l, respectively (FIG. 2). From the above results, it can be seen that the mutant strain of the present invention is significantly higher in ethanol fermentation efficiency than the conventional microbial strain, through which the novel Bretanomyces custersii mutant strain of the present invention has high galactose content. It was found to be useful for ethanol fermentation in. On the other hand, based on the maximum value (0.51 g ethanol / g hex charcoal) is converted to ethanol (see formula below), the ethanol yield confirmed in this example was 79.0%.

C ₆ H ₁₂ O ₆ (galactose or glucose) → 2CH ₃ CH ₂ OH (ethanol) + 2CO ₂

Example  3. Bretanomyces Custersi  Root of the Mutant Strain Saccharification  Investigation of efficiency

Ethanol fermentation was carried out using Bretanomyces custersii mutant strains from the root saccharin of red algae, a representative biomass with a high galactose content. To this end, the preculture of the Bretanomyces custersii mutant strain was prepared in the same manner as described in Example 2, and the fermentation experiment was carried out with 150 ml of saponin saccharified solution inoculated with 25% of the preculture solution at a temperature of 30 ° C. It proceeded in anaerobic state for hours. The root sugar saccharification liquid was prepared by acidifying the Moroccan root grass native. Acid glycosylation conditions were 15% solids / liquid ratio, 150 ° C., 15 minutes, and the catalyst used H ₂ SO ₄ 1%. Initial galactose and glucose concentrations were 34.8 and 9.5 g / l, respectively.

As a result, after 48 hours of fermentation, galactose and glucose were consumed 89.6% and 100%, respectively, and the final ethanol concentration was 18.2 g / l (FIG. 3). Ethanol yield was 80.4%, it was confirmed that the variant strain and ethanol production method provided in the present invention can be useful for ethanol fermentation of biomass containing galactose.

Korea Research Institute of Bioscience and Biotechnology KCTC18154P March 5, 2009

Claims (17)

Bretanomyces custersii mutant strain (accession number: KCTC18154P) that ferments ethanol using a carbon source. The method according to claim 1,
The carbon source is a Bretanomyces custersii variant strain selected from the group consisting of monosaccharides, disaccharides and polysaccharides. (1) preincubation of the Bretanomyces custersy mutant strain (Accession Number: KCTC18154P) of claim 1 in a culture medium; And
(2) ethanol production method comprising the step of adding the pre-culture solution to the fermentation tank containing a carbon source to ethanol fermentation. The method according to claim 3,
The pre-culture of step (1) is ethanol production method of culturing in aerobic state at 50-300 rpm at a culture temperature of 20-40 ℃. The method according to claim 3,
The culture medium of step (1) is ethanol production method of YEPD (yeast extract 10 g / l, peptone 20 g / l, dextrose 20 g / l) medium. The method according to claim 3,
The ethanol fermentation is ethanol production method carried out in an anaerobic state at a culture temperature of 20-40 ℃. The method according to claim 3,
The carbon source is ethanol production method selected from the group consisting of monosaccharides, disaccharides and polysaccharides. The method according to claim 7,
Wherein said monosaccharide is selected from the group consisting of galactose, glucose and fructose. The method according to claim 7,
The disaccharide is ethanol production method selected from the group consisting of sucrose, maltose and lactose. The method according to claim 7,
The polysaccharide is ethanol production method selected from the group consisting of radish, starch, fibrin, carrageenan and alginic acid. The method according to any one of claims 7 to 10,
The carbon source is an ethanol production method is extracted from a raw material selected from the group consisting of sugar, starch, wood and algae. The method of claim 11,
The seaweed is ethanol production method selected from the group consisting of red algae, brown algae and green algae. The method of claim 12,
The red algae are from the group consisting of woodworm, laver, kotoney, dog gambling, boulder, ox radish, buckwheat, buckwheat fern, pansy, jindubal, gambling gourd, spiny radish, silk grass, vulgaris, stone star, stone tree and zinnia Ethanol manufacturing method selected. The method of claim 12,
The brown algae is prepared from ethanol selected from the group consisting of brown seaweed, seaweed, sea bream, walnut malt, shellfish, falcon, seaweed, Ecklonia cava, gompi, rhubarb, sesame seaweed cousin, mabanban, hoesan mabanban, worms Way. The method of claim 12,
The green alga is ethanol production method selected from the group consisting of Cheongtae, Hakkham, green, hearing, bead hearing, jade and salt jujube. The method of claim 11,
Extraction of the carbon source from the algae is the step of immersing the algae in an aqueous alkali solution and washing with water; And ethanol production step of immersing the washed algae in an extraction solvent for a predetermined time. The method according to claim 16,
The extractant is H ₂ SO ₄ , HCl, HBr, HNO ₃ , CH ₃ COOH, HCOOH, HClO ₄ , H ₃ PO ₄ , PTSA and a commercial solid acid is selected from the group consisting of solid acids.

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