KR101043443B1 - Method for Producing Bio-Ethanol Using Simultaneous Saccharification and Fermentation with the Supernatant of the Waste of Culture Broth - Google Patents

Abstract

The present invention relates to a method for producing bioethanol by the co-saccharification fermentation process of beer fermentation waste supernatant. The method of producing bioethanol by the co-glycosification fermentation process of the beer fermentation waste supernatant comprises the steps of: (a) separating the supernatant from the beer fermentation waste liquor generated as a by-product during beer production; (b) fermenting the separated supernatant at a temperature of 30-70 ° C. to produce bioethanol; (c) separating the yeast from the fermentation broth containing the bioethanol; And (d) fractionating the fermentation broth from which the yeast is separated to obtain bioethanol. In the case of using the method of producing bioethanol by the co-saccharification fermentation process of the beer fermentation waste supernatant according to the present invention, it is possible to produce bioethanol economically without adding the microbial saccharification enzyme and starch cellulose biomass for saccharification. Can produce.

Beer Fermentation, Lung Supernatant, Co-Saccharification, Fermentation, Bio Ethanol

Description

Method for Producing Bio-Ethanol Using Simultaneous Saccharification and Fermentation with the Supernatant of the Waste of Culture Broth}

The present invention relates to a method for producing bioethanol by a co-glycosylation fermentation process of beer fermentation waste supernatant, using nutrients such as starch, protein, and traces of beer waste yeast present in beer fermentation waste supernatant discarded after beer fermentation. The present invention relates to a method for producing bioethanol simply and economically by co-glycosifying fermentation without additional input of sugar and additional input of yeast.

Fossil fuels such as coal, petroleum, and natural gas are currently widely used, but they are expected to be depleted within a few decades, and have problems causing environmental problems such as greenhouse gases. In order to solve this problem, methods using nuclear power, solar heat, wind power, etc. have been studied, but the energy produced from them is power energy, and is not suitable as a fuel for transportation. Thus, biofuels have been discussed as the most realistic alternative energy that can be used as a transportation fuel.

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. Starting with corn ethanol for fuel from the United States, it now produces large amounts of ethanol for fuel in Brazil and around the world. Bioethanol is an energy that is compatible with existing energy systems. Bioethanol is recognized for its value as clean energy because it is renewable energy, reduces greenhouse gas emissions due to low amount of carbon emitted, and reduces air pollution.It can be used in combination with commercially available gasoline. The advantage is that you can use the gas station as it is.

The mixed fuel of bioethanol and gasoline is divided into E10, E20, and E100 according to the mixing ratio.E10 gasoline containing 10% of bioethanol is widely used around the world.In Brazil, where biofuel is developed, 22% ~ Some 25% of the mix is mandatory.

Substrates used for bioethanol production can be classified into starch-based systems such as corn, wheat, barley, potatoes, sweet potatoes, and sugar-based systems such as sugar cane and sugar beets.They are limited in their quantity to replace fossil fuels. As it uses sugar- or starch-based raw materials that can be used for this food, not only the use of food as an energy source but also the supply and demand problem may arise if food demand increases in the future. It is a problem in terms of cost.

In addition, the wood-based system 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. In addition to the increase in the process cost due to the process, due to the crystalline structure made 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 glycosylation yield. In other words, when wood-based biomass is used as a biomass, the raw material cost is about 30%, but it costs more than 60% for the pretreatment and saccharification process and cellulase enzyme production.

To reduce the cost of bioethanol, sulfurous acid pulp liquor and whey with carbohydrates as a by-product after pulp or food processing are currently used for industrial production of ethanol. However, even if ethanol is produced using food by-products or waste resources, there is still a problem of undergoing complex pretreatment and saccharification and fermentation.

In order to solve the above problems, Korean Patent No. 866032 discloses a method for producing bioethanol using beer fermentation by-products, that is, direct fractionation of beer fermentation waste yeast liquid to extract ethanol, or use beer fermentation waste yeast liquid as fermentation strains. Start a process for producing ethanol from biomass such as corn, sugar cane or the like, or use the supernatant of beer fermented waste yeast as a culture medium and inoculate microbial cellulose producing strain to produce microbial cellulose, and then use it as biomass. Although the method of manufacturing is disclosed, when the beer fermentation waste yeast liquor is fractionated directly without a separate process, the sugar in the waste yeast liquor cannot be used as a raw material of ethanol and the distillation efficiency is reduced due to the presence of many impurities during the distillation separation. And also fermented bacteria mainly using fermented yeast brewer's fermentation In the case of producing bioethanol from biomass, there is still a problem in that the biomass pretreatment process is required, and additional glycosylation enzyme is added.

Therefore, the present inventors have made diligent efforts to solve the above problems, as a result of separating the supernatant from the beer fermentation waste liquid generated as a by-product in the beer production process, and fermented under optimum conditions to produce ethanol and fractional distillation thereof, Performing the saccharification and fermentation at the same time without a separate pre-treatment process, confirming the fact that ethanol can be prepared simply without supplying additional strains and nutritional medium, and completed the present invention.

It is an object of the present invention to omit the pretreatment process by using the fermentation waste liquid of the discarded beer and the yield of bioethanol can be improved without adding the microethanol and yeast required for the bioethanol fermentation process and saccharification and fermentation performed simultaneously. The development of an ethanol fermentation process provides a way to reduce the production and separation costs of bioethanol.

Another object of the present invention is to provide a method of reducing waste treatment costs and reducing the production cost of bioethanol from the fermentation industry by producing bioethanol using the fermentation broth supernatant of the discarded beer as a medium substitute.

In order to achieve the above object, the present invention comprises the steps of (a) separating the supernatant from the beer fermentation waste liquor generated as a by-product in the beer manufacturing process; (b) fermenting the separated supernatant at a temperature of 30-70 ° C. to produce bioethanol; (c) separating the yeast from the fermentation broth containing the bioethanol; And (d) fractionating and distilling the fermentation broth from which the yeast is separated to obtain bioethanol.

In the present invention, the supernatant of the beer fermentation waste liquid may be obtained by centrifugation for about 20 minutes at 2,000 ~ 6,000 rpm.

In the present invention, the supernatant separated from the beer fermentation waste liquid may be characterized by comprising yeast, starch, protein, alpha amylase, beta amylase, hemicellulose, phosphatese, protease and glucanase. .

In the present invention, the fermentation may be characterized by stirring or stationary fermentation for 1 to 14 days.

In the case of using the method of producing bioethanol by the co-saccharification fermentation process of beer fermentation waste supernatant according to the present invention, it is possible to produce bioethanol economically without adding microbial saccharifying enzyme for starch and starch cellulose biomass. Can produce In addition, by utilizing the beer fermentation waste liquid, not only can reduce the waste treatment cost but also can reduce the cost of bioethanol production can be widely used in the commercial production of bioethanol.

In the present invention, when fermentation of beer fermentation waste supernatant under optimal fermentation conditions, it was confirmed that bioethanol can be produced without the addition of a separate strain, glycosylase and nutrient medium.

In the present invention, after separating the supernatant by centrifuging the beer fermentation waste liquid discarded in the beer factory, ethanol was produced by varying the fermentation temperature, fermentation time, fermentation method and the like. As a result, 10% or more bioethanol was produced when the beer fermented supernatant was stirred or stirred for 1 to 14 days at a temperature of 30 to 70 ° C. without the addition of a separate strain, glycosylation enzyme and nutrient medium.

Accordingly, the present invention in one aspect, (a) separating the supernatant from the beer fermentation waste liquid generated as a by-product in the beer manufacturing process; (b) fermenting the separated supernatant at a temperature of 30-70 ° C. to produce bioethanol; (c) separating the yeast from the fermentation broth containing the bioethanol; And (d) fractionating and distilling the fermentation broth from which the yeast is separated to obtain bioethanol.

In the present invention, the supernatant of the beer fermentation waste liquid may be obtained by centrifugation for about 20 minutes at 2,000 ~ 6,000 rpm. If the centrifugation is less than 2,000rpm centrifugation is not performed properly or centrifugation for a long time, there is a fear that the yeast bacteria do not exist in the supernatant when centrifugation exceeds 6,000rpm.

In the present invention, the supernatant separated from the beer fermentation waste liquid is yeast, starch, protein, alpha-amylase, beta-amylase, hemicellulase, phosphotase ), Protease and glucanase.

Beer manufacturing process is largely divided into malt manufacturing process, malt manufacturing process, fermentation process, and product process. Malt can be produced by germinating beer barley. During beer barley germination, enzymes such as amylase, hemicellulase, protease, and phosphotase, which are necessary enzymes for wort such as glycosylase and proteolytic enzymes, are activated or biosynthesized in barley particles. And insoluble substances such as proteins can be solubilized. Malt has its own enzymes that break down starch and protein into fermentable sugars and amino acids, so that the beer fermentation waste liquid is rich in enzymes, starches, sugars, proteins, etc. necessary for saccharification.

In general, the fermentation process is performed by adding beer yeast to the cooled wort and alcohol fermentation to make the fermentable sugar in the wort into ethyl alcohol. It is divided into post-fermentation, which is made into finished products. The fermentation process of wort is carried out under adiabatic cooling conditions to maintain 5 ~ 10 ℃, the beer is not suitable for drinking, since the main fermentation finished beer is fermented by gradual fermentation of the remaining substrate while maintaining a low temperature in the post-fermentation tank and at the same time carbon dioxide gas It is contained.

In beer brewer's yeast, starch is glycosylated by enzymes native to malt formation in the beer fermentation process, and fermented at low temperatures. In the beer fermentation process, unlike other liquor manufacturing processes, only simple yeast is used without yeast fungi.

Therefore, in the present invention, bioethanol fermentation can be performed without a pretreatment process, which is essential for the process of producing bioethanol from cellulose-based biomass, and bioethanol fermentation can be performed without addition of microbial strain or enzyme required for saccharification. It can be carried out, there is an advantage that can be performed simultaneously with the saccharification and ethanol fermentation without the addition of nutrients such as yeast, starch, protein in the medium.

In the present invention, the fermentation may be characterized by stirring or static fermentation for 1 to 14 days.

The fermentation temperature, fermentation time and fermentation method may have a complementary effect on ethanol production. For example, when shaken fermentation at 30 ℃ 10 days, fermentation at 50 ℃ shake fermentation 5 days, fermentation at 60 ℃ shake fermentation at 3 ~ 5 days more than 10% ethanol can be produced. In addition, when left to ferment at 60 ℃, can be produced more than 10% ethanol on the fifth day.

In order to prevent the disappearance of the yeast in the fermentation process, it is preferable to perform the fermentation at a temperature of less than 70 ℃.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Experimental Example 1 Measurement of CFU of Yeast Bacteria in Fermented Lung Supernatant

Beer fermentation waste yeast liquid was centrifuged at 3000 rpm for 20 minutes to obtain a supernatant, 0.1 ml of the initial supernatant was plated on a solid medium of the composition shown in Table 1 and incubated at 30 ° C. for 3 days. Colonies formed on the solid medium were observed under a microscope, and the number of colonies (CFU) of the yeast was measured. As a result, the yeast was found to be 70 / ml.

ingredient Volume (/ L) Glucose (SIGMA, USA)
Yeast Extract (DIFCO, USA)
Peptone (DIFCO, USA)
Succinic acid (WACO PURE CHEMICAL, USA)
Acetic acid (Dongyang Chemical, Korea) 10 g
10 g
7 g
0.2 g
1.5 ml

Experimental Example 2: Bioethanol production by shaking fermentation by adding yeast after sterilizing beer fermentation waste supernatant

500 ml of supernatant obtained by centrifuging beer fermentation waste liquid (waste yeast solution) at 3000 rpm for 20 minutes was sterilized at 121 ^o C for 15 minutes in a 1000 ml culture tank, and 50 g of wet waste yeast obtained by centrifugation was put together in the culture tank. The culture vessel was sealed and shaken for 60 hours at 150 rpm while maintaining the temperature at 40 and 55 ^° C., respectively. As a result of measuring the ethanol concentration of the fermentation broth, the concentration of ethanol reached 10.2% at 40 ^° C and 13.1% at 55 ^° C. As a result, beer fermentation waste supernatant was found to contain sufficient nutrients for ethanol fermentation.

Example 1 Bioethanol Production by Static Fermentation

500 ml of the supernatant obtained by centrifuging the beer fermentation waste liquid at 3000 rpm for 20 minutes was placed in a 1000 ml culture vessel and sealed, and then left to ferment for 21 days at 30 ^° C. without adding nutrients and yeast. Ethanol concentration in the fermentation broth during the fermentation was measured by date, and the results are shown in Table 2 below.

Effective Days (day) Ethanol Concentration in Fermentation Broth (%) 0 4.0 4 6.0 7 9.5 14 7.4 21 0

As shown in Table 2, when the stationary fermentation was performed at 30 ℃, it showed the highest ethanol concentration (9.5%) on the 7th day, after which the concentration gradually decreased to confirm that the ethanol concentration is 0% on the 21st day.

Example 2: Yeast Growth in Fermentation Broth by Static Fermentation

The beer fermentation waste liquid was centrifuged at 3000 rpm for 20 minutes in 500 ml of the supernatant, which was sealed in a 1000 ml culture tank, and then fermented at 30 ^° C. During the fermentation, 0.1 ml of the culture medium was optionally taken, diluted 10,000-fold, inoculated into the solid medium of Table 1, and incubated for 3 days. Colonies formed on the solid medium (colony) was observed under a microscope, and the results of measuring the colony count (CFU) of the yeast is shown in Table 3.

Effective Days (day) Culture Yeast Count (CFU / mL) 0 7.0 × 10 ¹ 5 4.0 × 10 ² 7 2.0 × 10 ⁵

As shown in Table 3, the number of yeast was confirmed to increase with the culture day.

Example 3: Increase of Various Bacteria in Fermentation Broth by Static Fermentation

The beer fermentation waste liquid was centrifuged at 3000 rpm for 20 minutes in 500 ml of the supernatant, which was sealed in a 1000 ml culture tank, and then fermented at 30 ^° C. During the fermentation, 0.1 ml of the culture medium was optionally taken, diluted 10,000-fold, inoculated into the solid medium of Table 1, and incubated for 3 days. Colonies formed on the solid medium (colony) was observed under a microscope, and the results of measuring the colony number (CFU) of the bacteria is shown in Table 4.

Days of Culture (day) Culture bacterial count (CFU / mL) 0 7.1 × 10 ² 3 2.2 × 10 ⁴ 5 2.1 × 10 ⁵ 14 4.2 × 10 ⁶

As shown in Table 4, the number of bacteria increased with the day of culture and reached 2.1 × 10 ⁵ / mL on the 5th day of culture.

Experimental Example 3: Bioethanol Production by Static Fermentation of Fermented Lung Supernatant with Glucose

In order to check whether there is enough yeast and other nutrients necessary for ethanol fermentation in beer fermentation waste supernatant, 500 ml of supernatant obtained by centrifuging beer fermentation waste liquid at 3000 rpm for 20 minutes was sterilized at 121 ^o C for 15 minutes in a 1000 ml culture tank. After adding 100 g of glucose, the culture vessel was sealed and left to ferment for 7 days while maintaining the temperature at 30 ^° C.

After fermentation, the concentration of ethanol in the fermentation broth was measured and found to be 16%. Therefore, it can be confirmed that beer fermentation waste supernatant contains yeast sufficient for ethanol fermentation and other nutrients necessary for fermentation.

Example 4 Bioethanol Production by Shaking Fermentation

500 ml of the supernatant obtained by centrifuging the beer fermentation waste liquid at 3000 rpm for 20 minutes was placed in a 1000 ml culture tank and sealed, followed by shaking fermentation at 150 rpm at 30 ^° C. for 21 days. Ethanol concentration in the fermentation broth during the fermentation was measured by date, and the results are shown in Table 5 below.

Effective Days (day) Ethanol Concentration in Fermentation Broth (%) 0 4.0 4 6.7 14 5.3 21 0

As shown in Table 5, when shaking fermentation was performed at 30 ° C., the highest ethanol concentration (6.7%) was shown on day 4, and then the concentration was gradually decreased to confirm that ethanol concentration was 0% on day 21. .

Example 5: Bioethanol Production by Shaking Fermentation According to Culture Temperature

500 ml of the supernatant obtained by centrifuging the beer fermentation waste liquid at 3000 rpm for 20 minutes were put in three 1000 ml culture tanks, and the culture tank was sealed, and then shaken and fermented at 150 rpm while maintaining the temperature at 30, 50, and 60 ^o C, respectively. . The ethanol concentration of the fermentation broth was measured according to the fermentation time, and the results are shown in Table 6.

Fermentation time
(Day) Ethanol Concentration in Fermentation Broth (%) Fermentation temperature 30 ^o C 50 ^o C 60 ^o C 0 4.5 4.5 4.5 3 7.1 7.5 10.2 5 7.4 10.7 12.9 7 10.4 5.6 5.3 10 11.5 3.4 5.3

As shown in Table 6, the concentration of ethanol varies depending on the fermentation time and the fermentation temperature, and the higher the fermentation temperature increases with the fermentation temperature until the 5th day of fermentation, but at 30 ° C on the 7th and 10th days after the 5th day. Ethanol concentration continued to increase when fermented, but it was confirmed that the ethanol concentration gradually decreased when fermented at 50 ℃ and 60 ℃.

Example 6: Bioethanol Production by Static Fermentation According to Culture Temperature

500 ml of the supernatant obtained by centrifuging the beer fermentation waste liquid at 3000 rpm for 20 minutes was placed in three 1000 ml culture tanks, and the culture tanks were sealed, and the fermentation was performed while maintaining the temperature at 30, 50, and 60 ^° C., respectively. The ethanol concentration of the fermentation broth was measured according to the fermentation time, and the results are shown in Table 7.

Fermentation time
(Day) Ethanol Concentration in Fermentation Broth (%) Fermentation temperature 30 ^o C 50 ^o C 60 ^o C 0 4.5 4.5 4.5 3 6.7 7.5 9.6 5 7.4 9.0 11.8 7 9.2 5.4 5.3 10 7.0 4.6 5.3

As shown in Table 7, the concentration of ethanol varies depending on the fermentation time and fermentation temperature, and when fermented at 30 ° C., the 7th day (9.2%), when fermented at 50 ° C., the 5th day (9.0%), at 60 ° C. When the fermentation was confirmed that the highest ethanol concentration on the 5th day (11.8%).

Example 7: Yeast Bacteria Increase by Static Fermentation According to Culture Temperature

500 ml of the supernatant obtained by centrifuging the beer fermentation waste liquid at 3000 rpm for 20 minutes were put in three 1000 ml culture tanks, and the culture tanks were sealed, and the fermentation was performed while maintaining the temperature at 30 and 80 ^° C., respectively. During the fermentation, 0.1 ml of the culture medium was optionally taken, diluted 10,000-fold, inoculated into the solid medium of Table 1, and incubated for 3 days. Colonies formed on the solid medium (colony) was observed under a microscope, the results of measuring the colony number (CFU) of the yeast is shown in Table 8.

Fermentation time
(Day) Yeast Bacteria in Fermentation Broth (CFU / mL) Fermentation temperature 30 ^o C 60 ^o C 80 ^o C 0 7.0 × 10 ¹ 7.0 × 10 ¹ 7.0 × 10 ¹ One - - - 2 - 2.0 × 10 ⁴ 0 3 - 0 5 4.0 x 10 ² 0 7 2.0 × 10 ⁵ -

As shown in Table 8, the number of yeast is increased as the fermentation (culture) temperature is increased, but when the fermentation at 80 ℃ or more was confirmed that the yeast disappeared after 3 days.

As described above in detail the specific parts of the present invention, it is apparent to those skilled in the art that such specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a flow chart illustrating a method for producing bioethanol by the co-glycosylation fermentation process of beer fermentation waste supernatant according to an embodiment of the present invention.

Claims (4)

Bioethanol production method by the co-glycosylation of beer fermentation waste supernatant comprising the following steps: (a) separating the supernatant from the beer fermentation waste liquor generated as a by-product during beer production; (b) fermenting the separated supernatant at a temperature of 30-70 ° C. to produce bioethanol; (c) separating the yeast from the fermentation broth containing the bioethanol; And (d) fractionating the fermentation broth from which the yeast is separated to obtain bioethanol. The method of claim 1, wherein the supernatant of the beer fermentation waste liquid is obtained by centrifugation for about 20 minutes at 2,000 to 6,000 rpm.  The method of claim 1, wherein the supernatant separated from the beer fermentation waste liquor comprises yeast, starch, protein, alpha amylase, beta amylase, hemicellulose, phosphotese, protease and glucanase How to. The method of claim 1, wherein the fermentation is characterized in that shaking or stationary fermentation for 1 to 14 days.

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