KR101856849B1 - Method of producing glucose from chinese cabbage wastes and algae culture media containing glucose - Google Patents

Abstract

The present invention relates to a method for producing glucose from a Chinese cabbage waste and to a culture solution for producing biodiesel of a microalgae containing an enzyme hydrolyzate obtained by treating a cellulase obtained from Trichoderma harzianum to a sulfuric acid treated Chinese cabbage waste, Enzymatic hydrolysis of cabbage waste using cellulase derived from Trichoderma harzianum produces glucose, which can greatly reduce the production cost of biodiesel production using microalgae.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing glucose from cabbage wastes and a method for producing glucose from a cabbage waste,

The present invention relates to a method for producing glucose from a Chinese cabbage waste and a microalgae culture liquid for producing biodiesel containing glucose produced by the method, and more particularly, to a method for producing a Chinese cabbage waste by using a cellulase derived from Trichoderma harzianum To produce glucose by enzymatic hydrolysis.

Petroleum fuels have long been warned of depletion. It is biofuels that can replace petroleum fuels that are depleted. In particular, biofuels produced through microalgae can reduce CO2 emissions, which are greenhouse gases emitted by fuel use. This can prevent the acceleration of global warming and reduce the cost of production and distribution. have.

Biofuels are produced by sugar fermentation of microorganisms. Because sugar production from nature can reduce the production cost of biofuels, a method for producing sugars through enzymatic degradation of cellulose of lignocellulosic biomass is widely used worldwide. However, most of the lignocellulosic biomass is composed of lignin, hemicellulose, and cellulose, which are difficult to degrade enzymes. Therefore, it is difficult to produce biofuels efficiently by a simple method. Accordingly, the biomass requires a pretreatment process, an enzyme treatment, and a sugar fermentation process.

There are various methods of pretreatment of biomass such as dilute acid, alkali and hydrogen peroxide. Among them, dilute acid is used because a small amount of acid is used, which is inexpensive in process and effective for pretreatment. In particular, H ₂ SO ₄ has been used commercially in a variety of biomass by pretreatment with dilute acid.

In the enzymatic treatment, three cellulases such as β-glucosidase, endo-1,4-β-glucanase and exo-1,4-β-glucanase are necessary to decompose cellulose. Endo-1,4-β-glucanase hydrolyzes cellulosic β-1,4-linkage indiscriminately into cellobiose units. Exo-1,4-β-glucanase is similar to endo but recognizes the non-reducing end of cellulose and hydrolyzes to cellobiose unit. The cellobiose and other water-soluble cello-oligosaccarides thus produced are degraded into glucose by β-glucosidase. These enzymes are secreted from microorganisms such as fungi and bacteria.

Microalgae are the most efficient in biofuel production. Most microalgae can synthesize fats through inorganic carbon and organic carbon sources. The composition of the fat depends on the species of microalgae. The fat of microalgae is divided into neutral fat and polar fat. Among them, triglycerides, which are triglycerides, are the most important fats for producing biodiesel. Microalgae require acetyl coenzyme A to produce this triglyceride. It is formed from inorganic or organic carbon sources. In particular, Chlorella protothecoides is known to store high biomass content and fat in cells under heterotrophic conditions in which organic sources such as glucose, acetate or glucose are provided. However, since the addition of glucose to the culture medium accounts for 80% of the total cost of the culture, it is necessary to industrially lower the cost of biodiesel production from the microalgae culture.

Currently, biofuels, which are widely used in the world, are produced by microbial fermentation through saccharification process from lignocellulosic biomass such as corn, wheat, rice and sugar cane. However, since they are all crops, they are used for fuel production and cause food shortages. However, the use of waste agricultural products not only can escape from such problems but also has an environmental advantage. In the present invention, the sugar production process to be added to the culture of C. protothecoides for biodiesel production has been developed through saccharification process of Chinese cabbage, which is a waste agricultural product, with a focus on biodiesel production cost reduction.

1. Korean Patent Publication No. 2012-0036883 2. Korean Patent No. 1554874

It is an object of the present invention to provide a process for producing glucose from cabbage wastes.

Yet another object of the present invention is to provide a microalgae culture medium for producing biodiesel containing enzyme hydrolyzate obtained by treating cellulase obtained from Trichoderma harzianum in sulfuric acid treated Chinese cabbage wastes and a method for producing biodiesel using the same.

In order to accomplish the above object, the present invention provides a method for treating a Chinese cabbage waste, And treating the sulfuric acid-treated Chinese cabbage waste with a cellulase derived from Trichoderma harzianum to perform enzymatic hydrolysis.

In one embodiment of the present invention, the sulfuric acid can treat the Chinese cabbage waste at a concentration of 0.2 to 0.6%.

In one embodiment of the present invention, the cellulase can be obtained by culturing Trichoderma harzianum at 25 to 30 DEG C, 100 to 150 rpm and pH 6 to 7. The activity of the cellulase may be 0.3-0.5 FPU / ml and the cellulase may be treated with sulfuric acid-treated cabbage waste at a dose of 0.15-0.4 FPU / ml.

In one embodiment of the present invention, the enzyme hydrolysis can be carried out at a pH of 5 and a temperature range of 40-50 ° C.

The present invention also provides a microalgae culture medium for producing biodiesel comprising an enzyme hydrolyzate obtained by treating cellulase obtained from Trichoderma harzianum in a sulfuric acid treated Chinese cabbage waste.

In one embodiment of the present invention, the microalgae can be used without limitation as long as they are used for producing biodiesel. The microalgae are Chlorella spp., Scenedesmus spp., Stigoclonium spp. Or Dunaliella spp., For example, but not limited to, Chlorella protothecoides.

In one embodiment of the present invention, the enzyme hydrolyzate may be a grape sugar, and the glucose may be contained in the culture solution at a concentration of 20% (v / v) or less.

The present invention also provides a method for producing biodiesel, comprising culturing microalgae in a culture solution for producing biodiesel according to the present invention, and then extracting and separating lipids and fatty acids from the culture solution.

In one embodiment of the present invention, the microalgae can be used without limitation as long as they are used for producing biodiesel. The microalgae are Chlorella spp., Scenedesmus spp., Stigoclonium spp. Or Dunaliella spp., For example, but not limited to, Chlorella protothecoides.

The present invention can produce glucose to be supplied for the production of biodiesel using the Chinese cabbage waste discharged in large quantities in the domestic market and can greatly reduce the production cost of biodiesel to about 1/2, The amount of glucose produced can be increased. In addition, according to the present invention, by producing and using a cellulase from Trichoderma harzianum, the process cost can be reduced to almost 1/10 as compared with a commercially available mixed enzyme.

FIG. 1 shows the glucose production yields of cellulase derived from three Trichoderma harzianum strains over time according to one embodiment of the present invention.
Figure 2 shows the yield of glucose production with time of cellulase derived from Trichoderma harzianum cultured under optimal conditions in one embodiment of the present invention.
FIG. 3 shows (A) the production yield of cellulase from Trichoderma harzianum and (B) the yield of glucose production of the cellulase according to pH in one embodiment of the present invention.
FIG. 4 shows the yield of glucose production according to various pretreatments of Chinese cabbage according to an embodiment of the present invention.
FIG. 5 shows the yield of glucose production according to the concentration of sulfuric acid pretreated in Chinese cabbage according to an embodiment of the present invention.
6 shows the growth rate of Chlorella protothecoides according to the concentration of sulfuric acid pretreated in Chinese cabbage in an embodiment of the present invention.
FIG. 7 shows the production yield of glucose according to temperature during the sulfuric acid pretreatment of Chinese cabbage according to an embodiment of the present invention.
FIG. 8 shows the production yield of glucose according to pH during the sulfuric acid pretreatment of Chinese cabbage according to an embodiment of the present invention.
9 is a graph showing the production yield of glucose according to whether or not washed after pretreatment of sulfuric acid in Chinese cabbage according to an embodiment of the present invention, wherein (A) conventional hydrolytic enzyme cocktail and (B) cellulose after treatment according to the present invention Glucose production yield.
FIG. 10 shows the yield of glucose production according to temperature during the culture of Chlorella protothecoides in an embodiment of the present invention.
11 shows the yield of glucose production according to the amount of cellulase added to the culture of Chlorella protothecoides according to an embodiment of the present invention.
12 shows the yield of glucose production according to the content of Chinese cabbage in an embodiment of the present invention.
FIG. 13 shows the total glucose production amount and (B) glucose production yield in (A) culture medium of commercial cellulase and cellulase of the present invention in one embodiment of the present invention.
FIG. 14 shows growth rates of Chlorella protothecoides according to glucose content in one embodiment of the present invention.
15 shows the yield of glucose production according to cellulose from Chinese cabbage and other biomass in one embodiment of the present invention.

The present invention relates to a method for treating a Chinese cabbage waste, And treating the sulfuric acid treated Chinese cabbage waste with a cellulase derived from Trichoderma harzianum to perform enzymatic hydrolysis.

In this method, the yield of glucose production can be increased by optimizing the conditions of the sulfuric acid treatment step of the Chinese cabbage. Specifically, the sulfuric acid can treat the cabbage waste at a concentration of 0.2 to 0.6%, preferably 0.25 to 0.5%, most preferably 0.5%.

The cellulase may be obtained by culturing Trichoderma harzianum under the conditions of 25 to 30 DEG C, 100 to 150 rpm and pH 6 to 7. When the cellulase is cultured under the conditions of 28 DEG C, 130 rpm, pH 6, about 0.3 to 0.5 FPU / ml, preferably about 0.4 FPU / ml, can be obtained. The cellulase can be produced at a cost of almost 1/10 the level of a commercially available mixed enzyme. The cellulase may be treated in a sulfuric acid treated cabbage waste at an input of 0.15 to 0.4 FPU / ml, and preferably at an input of 0.24 FPU / ml.

In one embodiment of the present invention, the optimum conditions for the enzymatic hydrolysis can be carried out at a pH of 5 and a temperature range of 40 to 50 ° C, preferably at a temperature of 45 ° C.

The present invention also provides a culture medium for the production of biodiesel of microalgae, for example, Chlorella protothecoides, which comprises an enzyme hydrolyzate obtained by treating cellulase obtained from Trichoderma harzianum in a sulfuric acid treated Chinese cabbage waste.

In one embodiment of the present invention, the enzyme hydrolyzate may be a grape sugar. The glucose may be contained in the culture solution at a concentration of 20% (v / v) or less, and when the concentration exceeds 20%, the growth of the microalgae may be inhibited.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that the following examples are merely illustrative of the present invention and that the scope of the present invention is not limited to these examples.

≪ Example 1 >

Cellulase production from T. harzianum

T. harzianum (KACC 40549, 40784, 40793) was distributed from the National Agricultural Genetic Resources Information Center. Stock culture was subcultured at 28 ° C in MYG medium (0.2% malt extract, 0.2% yeast extract, 2% glucose and 2% agar). T. harzianum cultured on MYG agar plates for 7 days was collected with 0.02% tween 80 spores. Collected spores were cell counted. For the production of fungal cellulase, Mandel's medium (Carbon 10 g L ^-1 , Peptone 1.0 g L ^-1 , Urea 0.3 g L ^-1 , Tween 80 2.0 mL, Antifoam 2.0 mL, (NH ₄ ) ₂ SO ₄ 1.4 g L ^-1 , KH ₂ PO ₄ 2.0 g L ^-1 CaCl 0.3 g L ^-1 MgSO ₄ .7H ₂ O 0.3 g L ^-1 , Fe ₂ SO ₄ .7H ₂ O 0.005 g L ^-1 , MnSO ₄ .7H ₂ O 0.0016 g · L ^-1 ZnSO ₄ · 7H ₂ O 0.0014 g · L ^-1 , CoCl ₂ · 7H ₂ O 0.002 g · L ^-1 ) was used. The collected spores were inoculated into Mandel's medium at 3 * 10 ⁵ spores / ml. Each strain was incubated at 28 ° C and 130 rpm. After incubation, the cells were stored at 4 ° C and used.

Cellulase activity from T. harzianum was measured by Filter paper assay. 1 x 6 cm paper (wattman no. 1) was placed in a 1.5 ml tube, and then 1 ml of 50 mM sodium acetate at pH 5.0 and 0.5 ml of fungal cellulase were added. Thereafter, the reaction was carried out in a water bath at 50 DEG C for 1 hour. Next, it was boiled for 15 minutes and then cooled for 5 minutes. Glucose from paper was analyzed by glucose kit (Sigma GAGO 20) and absorbance was measured at 540 nm.

The ATCC no. 40549, 40784, and 40793 of T. harzianum. The strains were cultured for 6 days and the enzyme activities of the cultures were measured daily. 40784 strain was the highest (Fig. 1).

≪ Example 2 >

Determination of conditions for optimal activity of cellulase

T. harzianum ATCC no. The optimum cell culture time and pH of cellulase activity in 40784 strain were investigated. The highest activity was observed at 5 days after incubation in Mandel's medium at 28 ° C and 130 rpm for 6 days (FIG. 2). Also, when cultured under the same conditions, the enzyme concentration was high at pH 6 (FIG. 3A) and the activity was highest at pH 7 (FIG. 3B). At this time, the activity of the enzyme secreted from T. harzianum averaged about 0.4 FPU / ml.

≪ Example 3 >

Determination of pretreatment conditions of Chinese cabbage

Chinese cabbage was purchased at a general store. The Chinese cabbage was washed and the soil was removed, and the moisture of the leaf surface was dried at room temperature for about 2 hours. Dried Chinese cabbage was changed through a mixer. The ground cabbage was stored at 20 ° C until use.

(H ₂ SO ₄ ), hydrogen peroxide (PAA), hydrogen peroxide and sulfuric acid (PAA + H ₂ SO ₄ ), phosphoric acid and sulfuric acid (PO ₄ + H ₂ SO ₄ ) Respectively. As a result, glucose yield was the highest when treated with sulfuric acid (FIG. 4). In addition to the acid treatment, the yield of glucose was lower than that of sulfuric acid when the alkali method was applied.

<Example 4>

Determination of treatment concentration of sulfuric acid

The concentration of sulfuric acid treatment for the removal of lignin and expansion of tissue of Chinese cabbage was investigated.

Sulfuric acid (catalog B3010-1250, Junsei) was used for the pretreatment of Chinese cabbage. Sulfuric acid was added to the gallinel cabbage, which had about 5% dry cabbage. 0.1%, 0.25%, 0.5%, and 1% sulfuric acid were added to the Chinese cabbage to select the addition concentration, and the mixture was subjected to high temperature and high pressure treatment at 50 ° C for 40 minutes. To investigate the treatment efficiency, industrially marketed enzymes (sigma C2730 1 FPU / ml, sigma C6105 3.5 FPU / ml) were treated with sulfuric acid in Chinese cabbage at 45 ° C and 130 rpm for 2 days. Glucose output was then measured using a glucose kit (Sigma GAGO 20). As a result, the yield of glucose through the enzyme was 0.5%, the glucose content was the highest, and the 0.25% glucose content was the second highest (FIG. 5).

C. protothecoides was affected by sulfuric acid pretreatment. The absorbance was measured by culturing C. protothecoides with 0%, 0.25%, and 0.5% concentrations of sulfuric acid in the medium, respectively. As a result, it was confirmed that the difference between the control group and the group treated with sulfuric acid was not large (FIG.

The optimum temperature in the sulfuric acid pretreatment was investigated. Enzymes secreted from T. harzianum were added 0.16 FPU / ml and hydrolyzed at 130 rpm for 2 days. As a result, the reaction of the Chinese cabbage at 50 ° C after sulfuric acid treatment showed the greatest effect on glucose yield (FIG. 7).

< Example  5>

Evaluation of Glucose Production by Cabbage Washing

The effect of acid and acid by-products of cabbage juice on enzyme hydrolysis was investigated before acid hydrolysis of acid treated Chinese cabbage. The 0.25% acid treated Chinese cabbage was washed with 0, 1.5, and 3 times distilled water, respectively, of the extracted chinese cabbage (g). The Sigma cocktail enzyme and fungal enzyme were then treated at 50 ° C and 130 rpm for 2 days. As a result, both groups showed lower glucose yields compared to the untreated group when washed with distilled water (FIG. 9). It was expected that there would be a loss of glucose in the process of washing and purifying the cabbage.

&Lt; Example 6 >

Determination of glucose production conditions

ATCC no. 5, grown in Manderl's medium at pH 7, 28 ° C, 130 rpm for 5 days. The optimum pH, temperature and enzyme loading of 40784 strain of cellulase were investigated. As a result, glucose yield was the highest at pH 5 (FIG. 8). The enzyme activity was measured at 40, 45 and 50 ℃ for 2 days. The results were similar to those of 45 ℃ and 50 ℃. For this reason, 45 캜 was set to an enzyme-compatible temperature (Fig. 10). Treatment of 0.24 FPU / ml with 0.4 FPU / ml of the enzyme secreted from T. harziaum at 0.08, 0.16, 0.24 and 0.32 FPU / ml was the most efficient (FIG. 11).

After optimizing the activity of the enzyme, the glucose yield according to the content of Chinese cabbage was investigated. The dry weight of Chinese cabbage was about 5%, which was diluted to 1.7%, 2.8%, 3.9% and 5.0%. As a result, glucose yield was the highest in 5.0% of Chinese cabbage (FIG. 12).

&Lt; Example 7 >

Production of glucose

The acid treated Chinese cabbage was adjusted to pH 5.0, and adjusted to a total concentration of 50 mM acetate. After the sterilization, the acid treated cabbage products were treated with the enzyme secreted from T. harziaum. One milliliter of sample was collected before enzyme reaction and stored at -20 ° C. The enzyme reaction was carried out at 50 ° C and 130 rpm for 3 days. After the reaction, 1 ml of sample was collected and glucose yield was measured with a glucose kit (Sigma GAGO 20) together with the sample before enzyme reaction.

&Lt; Example 8 >

T. to harziaum from  Comparison of activities of secreted enzymes and commercial enzymes

ATCC no. (Cellulase 0.4 FPU / ml) and commercial mixed enzyme (sigma C2730 0.013 FPU / ml, sigma C6105 0.046 FPU / ml) directly obtained from the culture of 40784 strain. Each enzyme was treated with 0.15 FPU / ml in 0.25% sulfuric acid-treated Chinese cabbage, and the amount of glucose was measured at 45 ° C and 130 rpm for 4 days. As a result of the glucose yield, the activity of the commercial mixed enzyme was about twice as high (Fig. 13). However, considering the cost of the process, the commercial mixed enzyme cost about 800 won to produce 1 g of glucose, while the enzyme secreted from T. harziaum costs only about 90 won (Table 1). This suggests that the use of enzymes secreted from T. harziaum is appropriate when considering activity and process costs.

< Example  9>

Culture of C. protothecoides

C. protothecoides is PB5 medium (NaNO ₃ 2.94 mM, K ₂ HPO ₄ 0.43 mM, MgSO ₄ .7H ₂ O 0.3 mM, CaCl ₂ .2H ₂ O 0.17 mM, KH ₂ PO ₄ 1.29 mM, NaCl 0.43 mM. Yeast extract 0.50%, HEPES 10 mM, Vitamin B12 0.1 μM, Biotin 0.1 μM, Thiamine 6.5 μM) and subcultured at 26 ° C.

As in Example 7, the saccharide produced from Chinese cabbage was added to PB5 culture medium at 20, 40, 60, and 80% (V / V). The equivalent was adjusted to 2% and algae were inoculated at 3 × 10 ⁶ algaes / ml. The inoculated microalgae were cultured at 26 ° C and 130 rpm, and samples were collected every 24 hours for the growth and residual glucose was measured by glucose kit (Sigma GAGO 20).

The sugar produced from Chinese cabbage was added to the microalgae culture at 0, 20, 40, 60 and 80% (V / V). C. protothecoides was examined for growth of microalgae at 26 ° C and 130 rpm for 5 days. When samples were taken every day and added with 20% of Chinese cabbage, growth was not affected, but it was observed that there was a serious inhibition of growth of C. protothecoides at higher concentrations (Fig. 14).

< Example  10>

Production of glucose using various biomes

Sugar production was investigated through a process developed using different types of biomass (residue after palm oil extraction, rice straw, tea leaves). After standard grind the rice straw, tea leaves, farm residues in grinder, were pretreated biomass of 5% to 0.5% H ₂ SO _4. Then, the enzyme secreted from T. harzianum was added at 0.24 FPU / ml and reacted at 45 ° C and 130 rpm. The glucose concentration was measured by glucose kit (Sigma GAGO 20) at 24 ℃.

As a result, it can be seen that rice straw has a sugar production as high as about 4 mg / ml (Fig. 15). However, since the amount of sugar contained in the cabbage itself is large, the total amount of the total sugar produced is twice as high as that of rice straw (Fig. 15).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

Crushing the Chinese cabbage waste and then treating the sulfuric acid; And
Treating the sulfuric acid treated Chinese cabbage waste with the cellulase obtained from Trichoderma harzianum (KACC No. 40784) to perform enzyme hydrolysis
&Lt; / RTI &gt; The method according to claim 1,
Wherein the sulfuric acid is treated at a concentration of 0.2 to 0.6%. The method according to claim 1,
Wherein said cellulase is obtained by culturing Trichoderma harzianum (KACC No. 40784) at 25 to 30 DEG C, 100 to 150 rpm and pH 6 to 7. The method according to claim 1,
Wherein the activity of the cellulase is 0.3 to 0.5 FPU / ml. The method according to claim 1,
Wherein the cellulase is treated at a dosage of 0.15 to 0.4 FPU / ml. The method according to claim 1,
Wherein the enzyme hydrolysis is carried out at a pH of 5 and a temperature range of 40 to 50 占 폚. A microalgae culture for producing biodiesel comprising an enzyme hydrolyzate obtained by treating cellulase obtained from Trichoderma harzianum (KACC No. 40784) in a sulfuric acid treated Chinese cabbage waste. 8. The method of claim 7,
Wherein the enzyme hydrolyzate comprises glucose. 9. The method of claim 8,
Wherein the culture medium contains the glucose at a concentration of 20% (v / v) or less. A method for producing biodiesel comprising culturing microalgae in a culture solution for producing biodiesel according to claim 7, and extracting and separating lipids and fatty acids from the culture solution.

Images