KR101108875B1 - Processes for the pretreatment of rice straw using TiO2, and processes for the production of saccharides and bioethanol using the same - Google Patents

Abstract

The present invention relates to a pretreatment method of lignocellulosic biomass using a photocatalyst, and more particularly, to a pretreatment method of lignocellulosic biomass using titanium oxide and the production of sugar compounds and bioethanol from the pretreated biomass. Regarding the method, the pretreatment method according to the present invention has the advantage of being suitable for large-capacity processes because it is possible to reduce the environmental pollution, input time and cost due to chemical treatment as well as the production of efficient bioethanol.

Photocatalyst, lignocellulosic, sugar compound, bioethanol

Description

Processes for the pretreatment of rice straw using TiO2, and processes for the production of saccharides and bioethanol using the same}

The present invention relates to a pretreatment method of lignocellulosic biomass using a photocatalyst, and more particularly, to a pretreatment method of lignocellulosic biomass using titanium oxide and the production of sugar compounds and bioethanol from the pretreated biomass. It is about a method.

Due to the depletion of fossil fuels and the global warming caused by the generation of gases such as carbon dioxide produced during use, and concerns about national security due to the lack of inherent energy resources, corn, sugar cane, sugar beet, etc. The technology for producing ethanol from vegetable biomass resources, which are agricultural wastes, is receiving attention again. The plant biomass is a sustainable resource that can be produced indefinitely on the earth as long as the sun exists. At its core is lignocellulose, which contains cellulose that can be converted to glucose.

Vegetable biomass refers to a plant that is biosynthesized through photosynthesis process including the magnetization of sunlight and carbon dioxide. The main component is lignocellulosic, a complex composed of cellulose, hemicellulose, and lignin.

Therefore, in order to produce ethanol or various compounds using lignocellulosic material, lignocellulosic cellulose and carbohydrates must be converted into fermentable sugars capable of ethanol fermentation.

To convert cellulose, a key substrate in lignocellulosic, to glucose, hydrolysis must be done in which one water molecule is added per two glucose units in cellulose. Such hydrolysis includes enzymatic saccharification by cellulase derived from fungi or bacteria that degrade wood, and chemical saccharification by catalysts such as acids and alkalis. Since chemical glycosylation is very slow compared to enzyme reactions, it must be carried out at high temperature, and there is no specificity. Therefore, enzymatic hydrolysis using cellulase is preferable because a large number of byproducts are produced at high temperatures.

In general, hydrolysis of cellulose by enzymes must be preceded by pretreatment due to the durability of cellulose itself and the hard degradability of lignocellulosic.

In general, pretreatment has been accomplished by physical and chemical methods. The physical method is slow and the energy consumption is high, the economic efficiency is low, and the chemical method uses a strong acid or a strong alkaline compound, the process cost is high, it is unsuitable for high-volume process, high toxicity, adversely affect the environment, There is an urgent need to develop effective pretreatment methods that can reduce the time and cost required for the process without causing environmental problems.

Therefore, the present invention overcomes the industrially difficult limitations of the physical and chemical methods presented so far, such as high temperature, high pressure, and pretreatment methods using an acid or a base alone or in a mixture, as a method of using a photocatalyst for pretreatment. Lee, et al. Pretreatment using existing compound mixtures [Yong-Jae Lee, Chang-Ho Chung, Donal F. Day, Sugarcane bassage oxidation using a combination of hypochlorite and peroxidase, Bioresource Technology (2009) 100: 935-941 It is also confirmed that the differentiation and completed the present invention.

An object of the present invention is to provide a method for pretreatment of lignocellulosic biomass using hydroxide radical (OH radical) having a strong oxidation in the presence of a photocatalyst.

It is also an object of the present invention to provide a method for producing a sugar compound from the lignocellulosic biomass pretreated according to the pretreatment method of the lignocellulosic biomass.

It is also an object of the present invention to provide a method for producing bioethanol by fermenting a sugar compound generated from lignocellulosic biomass pretreated according to the pretreatment method of the lignocellulosic biomass.

It is also an object of the present invention to provide a bioethanol produced according to the production method of the bioethanol.

The present invention provides a pre-treatment method for lignocellulosic biomass characterized by decomposing lignin by irradiating light to lignocellulosic biomass in the presence of a photocatalyst.

The photocatalyst is titanium oxide of a material to the self-cleaning (self-cleaning) and the second hydrophilic action to the light as a catalyst to the titanium oxide particles in the nano-scale (TiO ₂₎ will receive the ultraviolet rays of sunlight or ultraviolet light electron (e ^-) And holes (H ⁺ ), and especially holes (H ⁺ ) form a hydroxide radical (OH radical) having a strong oxidation effect and have a sterilizing effect [Klaus P. K, Iris F. Chaberny, Karl Massholder, Manfred Stickler, Volker W. Benz, Hans-GSonntag, Lothar Erdinger, Chemosphere (2003) 53: 71-77]. In addition, the former generates oxygen adsorbed on the photocatalyst as oxygen ions (O ₂ ⁻ ), which is a reactive active species, which is added to the oxidation reaction intermediate, which is odorous or decomposes organic matters. As a solution to the problems of volatile organic compounds (VOCs) occurring after finishing materials and constructions [Hodgson AT, Destaillats H, Sullivan DP, Fisk WJ, Performance of ultraviolet photocatalytic oxidation for indoor] air cleaning applications. (2007) Indoor Air. 17: 305-16.

Hereinafter, the configuration of the present invention with reference to the accompanying drawings will be described in detail.

The present invention comprises the steps of: a) immersing lignocellulosic biomass in a photocatalyst dispersion; And b) irradiating the immersed lignocellulosic biomass with ultraviolet rays. It provides a method for pretreatment of lignocellulosic biomass comprising a.

The photocatalyst dispersion according to the present invention is characterized in that the titanium oxide (TiO ₂ ) liquid containing 0.05 to 0.5% by weight of titanium oxide (TiO ₂ ), the ultraviolet irradiation is to irradiate ultraviolet rays of 230nm to 400nm wavelength 60 to 240 minutes To be characterized.

The photocatalyst is a hydrophilic photocatalyst material such as titanium oxide (TiO ₂ ), zinc oxide (ZnO), tin oxide (SnO ₂ ), strontium titanate (SrTiO ₃ ), tungsten oxide (WO ₃ ), bismuth oxide (Bi ₂ O ₃ ), and At least one photocatalyst selected from iron oxide (Fe ₂ O ₃ ) is used, more preferably titanium oxide (TiO ₂ ). As a more specific example, it is preferable to soak the straw remaining in the autumn harvested with lignocellulosic biomass in 0.1 wt% titanium oxide (TiO ₂ ) dispersion to completely absorb the dispersion, and then irradiate ultraviolet rays with 254 nm wavelength for 120 minutes. . The biomass is not limited to rice straw. After pretreatment using titanium oxide (TiO ₂ ) according to the present invention, rice straw was found to have a large physical and morphological change. See FIG. 1.

The present invention provides a method for producing a sugar compound from the lignocellulosic biomass pretreated according to the pretreatment method of the lignocellulosic biomass.

The sugar compound according to the present invention is produced through a saccharification process and may be performed by acid saccharification or enzyme saccharification according to a conventional method, but more preferably by enzymatic saccharification.

The enzyme glycosylation uses glycosylase, glycoenzyme having the activity of endoglucanase, carboxymethylcellulose, β-glucosidase and pNP-glucosidase or mixtures thereof, and 100 parts by weight of biomes. It is characterized by using in an amount of 1 to 30 parts by weight.

In addition, the present invention provides a method for producing bioethanol by fermenting a sugar compound generated from the lignocellulosic biomass pretreated according to the pretreatment method of the lignocellulosic biomass.

In the present invention, to produce bioethanol, the fermentation process is performed after the saccharification process. The fermentation is performed by using a fermentation strain, the fermentation strain Pichia steffitis ( Pichia stipitis ) Saccharomyces using KCCM 12009, Saccharomyces cerevisiae ) and Pichia steffitis stipitis ) bacteria can be used at the same time, so the use of bacteria is not limited.

The fermentation strain is characterized by incubating the fermentation strain in a volume ratio of 1: 0.5 to 1.5 in the ethanol fermentation medium containing a sugar compound according to the present invention, the temperature 28 to 32 ℃, characterized in that the culture for 24 to 96 hours.

In addition, the present invention provides a bioethanol produced according to the production method of the bioethanol.

More specifically, according to the pre-treatment method according to the present invention, the saccharification process is performed by adding 0.25 ml per 1 g of biomass to Axelase 1000 as a saccharase, and after saccharification, glucose and xylose in the degradation product. The amount was measured. As a result, in untreated rice straw, the hydrolysis rate was only 0.00-0.02 g / L by saccharase, whereas in pre-processed rice straw, up to 10.5 g / L for glucose and 3.21 g / L for xylose were produced. The hydrolysis rate was confirmed. See FIGS. 2, 3, 4 and 6.

In addition, inoculating the fermentation broth containing the sugar compound in order to produce bio ethanol Pichia steffitis KCCM 12009 was subjected to a fermentation process at a temperature of 30 ℃ 3 days to produce bio ethanol, according to the present invention The pretreatment method did not adversely affect the activity of glycation enzymes and fermentation microorganisms, so that enzymes and yeasts could be reacted under optimized conditions in the saccharification process and fermentation process, respectively, to efficiently produce bioethanol.

The pretreatment method according to the present invention differs from the conventional pretreatment method, which has a complex process for removing residues affecting enzyme and fermentation yeast activity. There is an advantage that can easily pre-treat lignocellulosic vise mass in a reduction and eco-friendly way.

The sugar compound obtained by the pretreatment method according to the present invention can be used very efficiently in biotechnological processes using enzymes, which not only reduces environmental pollution caused by chemical treatment to produce bioethanol, but also adds time and cost. It can reduce the cost and has the advantage of being suitable for high volume process.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and it is apparent to those skilled in the art that various changes or modifications can be made within the idea and scope of the present invention.

At this time, if there is no other definition in the technical and scientific terms used, it has a meaning generally understood by those of ordinary skill in the art.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

[ Example  1] titanium oxide TiO ₂ Pretreatment using

(1) straw preparation

Rice straw left after harvesting in Damyang, Jeollanam-do was used as a sample. The rice straw was left to dry and cut into 2-3 cm. Rice straw was produced as a 50 mesh powder and used in the experiment. Rice straw consists of about 35% cellulose, 25% hemicellulose and 12% lignin in dry weight.

(2) titanium oxide ( TiO ₂ Pretreatment using

1 wt% titanium oxide (TiO ₂ ) dispersion was prepared as a stock solution and used for rice straw treatment, and freshly prepared immediately before treatment. Titanium oxide (TiO ₂ ) stock solution was diluted to the desired concentration by adding distilled water was used for pretreatment. 20 g of rice straw was immersed in 200 ml of an appropriate concentration of titanium oxide (TiO ₂ ) dispersion and spread evenly in a pyrex container so as to be sufficiently wet. This vessel was irradiated with a UV lamp for a suitable time. After the irradiation, the titanium oxide (TiO ₂ ) dispersion was removed and washed with distilled water to remove the titanium oxide (TiO ₂ ) component. After washing, water was removed and the remaining pretreated rice straw was used for enzyme saccharification. The straw straw appearance before and after the pretreatment was photographed and shown in FIG. 1.

[ Example  2] by enzyme Saccharification

20 g of rice straw pretreated in Example 1 was treated with Axelase 1000 (accellerase 1000, Genencor, USA) to perform saccharification. Axelase 1000 is a mixed enzyme, which has multiple enzymatic activity, and the main enzymes are endoglucanize, exoglucanize, hemicellulase and beta-glucanase. After adding 200 ml of water to 20 g of pretreated rice straw, enzyme was added and the reaction was carried out at 60 ° C. for 24 hours. At this time, the enzyme was 0.25 ml per gram of rice straw (g) with respect to dry weight before pretreatment (endoglucanase activity: 2,500 carboxymethylcellulose U / g, beta-glucosidase activity: 400 pNP-glucoside U / g) ) Was added. After glycosylation, the amount of glucose and xylose products of rice straw was measured by thin layer chromatography (TLC) method and electrolyte analyzer Multiparameter Bioanalytical System YSI 7100 MBS (YSI Life Science, USA).

[ Example  3] titanium oxide ( TiO ₂ ) Check the optimum condition of pretreatment according to the concentration of dispersion

To prepare various concentrations of titanium oxide (TiO ₂₎ dispersion of the pre-processing to determine the optimum conditions according to the concentration of the titanium oxide (TiO ₂₎ dispersion liquid was confirmed the effect. Concentrations used are 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5% and 1.0% by weight. Experimental method was the same as the pretreatment method of Example 1, the wavelength of the UV lamp used was 254 nm, UV irradiation time was confirmed by fixing to 3 hours. The results are shown in FIG.

The result was 9.18 g / L for glucose and 0.99 to 1.30 g / L for glucose. It can be seen that the concentration of the titanium oxide (TiO ₂ ) dispersion having the highest glucose and xylose glycosylation is 0.1% by weight. Therefore, the optimum conditions for the next UV irradiation time and UV wavelength types were fixed at this concentration.

[ Example  4] Check the optimum condition of pretreatment according to the type of UV wavelength

In order to determine the optimum pretreatment conditions according to the type of UV wavelength, it was immersed in the titanium oxide (TiO ₂ ) dispersion using UV (ultraviolet, UV) and UV (UV) with 254 nm and 365 nm wavelengths. Rice straw was examined, and the experimental method was the same as the pretreatment method of Example 1 above. At this time, the concentration of the titanium oxide (TiO ₂ ) dispersion is 0.1% by weight, and the irradiation time was fixed at 3 hours to react. The results are shown in FIG.

As a result, the glucose produced by 254 nm UV irradiation was 8.84 g / L, the xylose was 1.25 g / L. The glucose produced by medium-wave UV irradiation was 8.75 g / L, the xylose was 1.24 g / L, and 365 nm. The glucose produced upon irradiation with ultraviolet rays was 8.38 g / L and xylose was 1.10 g / L.

The pretreatment conditions according to the ultraviolet wavelength are almost similar, but it can be seen that it has the maximum value of enzyme glycosylation when irradiated with 254 nm ultraviolet rays with a slight difference. In addition, 254 nm UV is the most commonly used lamp in general research, making it easy to experiment.

[ Example  5]: Confirm the optimum condition of pretreatment according to UV irradiation time

In order to confirm the optimum conditions for pretreatment according to the ultraviolet irradiation time, ultraviolet rays were irradiated at intervals of 30 minutes for 0 to 240 minutes, and the degree of glycation was confirmed by treating the enzyme. The experimental method was the same as the pretreatment method of Example 1, and the concentration of the titanium oxide (TiO ₂ ) dispersion was 0.1 wt%, and the experiment was performed after fixing at 254 nm ultraviolet wavelength. The results are shown in FIG.

As a result, the production of glucose and xylose increased up to 120 minutes. The peak of 120 minutes was found to be nearly constant or slightly decreased. At 120 minutes glucose was 9.19 g / L and xylose was 1.70 g / L glycosylated product.

Therefore, when the results are summarized in Examples 2 to 5, the optimum conditions for pretreatment using titanium oxide (TiO ₂ ) and ultraviolet light are 0.1 wt% of the concentration of the titanium oxide (TiO ₂ ) dispersion and the wavelength of ultraviolet light is 254 nm. , UV treatment time was confirmed to have the maximum value of enzyme saccharification when 120 minutes treatment.

[ Example  6] titanium oxide ( TiO ₂ Dispersion Axelize  Effect on activity

In general, the pretreatment method using acid for saccharification confirms that the titanium oxide (TiO ₂ ) dispersion has a negative effect on the activity of axelase because an acid residue that affects the enzyme remains upon saccharification. Carboxymethylcellulose (CMC) was prepared in a 0.1 wt% titanium oxide (TiO ₂ ) dispersion to 1.0 wt%. At this time, the control was used to dissolve carboxymethyl cellulose in water. Axelase 1000 was added to 1.0 wt% carboxymethyl cellulose to 0.25 mg / g, and the reaction was carried out at 60 ° C. for 24 hours. Enzyme activity was confirmed by thin layer chromatography (Thin layer chromatography). The results are shown in FIG.

As a result, Axelase 1000 can be confirmed that even in the presence of titanium oxide (TiO ₂ ) to decompose the carboxymethyl cellulose to produce oligosaccharides, the most of the glucose is generated is not affected by the enzyme activity.

The results also clearly show that a biotechnological process is possible using enzymes to saccharify UV treated biomass after immersion in titanium oxide (TiO ₂ ) dispersions.

[ Example  7] Production of Bioethanol

After pretreatment of 20 g of rice straw under the optimum pretreatment conditions identified from the above example, that is, the concentration of the titanium oxide (TiO ₂ ) dispersion was 0.1 wt%, the wavelength of ultraviolet ray was 254 nm, and the treatment time of ultraviolet ray was 120 minutes. Fermentation medium was produced by adding 0.17 g of yeast nitrogen base (without amino acid with ammonium sulfate, Difco), 0.227 g of urea, and 0.656 g of peptone as a nitrogen source to 100 ml of the sugar compound obtained by treating Axelase 1000. The sugar compound obtained after the optimal pretreatment had glucose of 10.5 g / L and xylose of 3.21 g / L. Also, the glycosylated product was identified by thin layer chromatography (TLC) and is shown in FIG. 6. .

In contrast, the control medium was produced by adding 11.85 g / L glucose and 3.3 g / L xylose instead of the sugar compound. For the production of bioethanol was used as a fermentation strain Pichia steffitis KCCM 12009 purchased from the Korea Microorganism Conservation Center. After inoculating Pichia steffitis colonies on YPX medium (1 g yeast extract per 100 ml, 2 g peptone, 2 g xylose) for 18 hours, the absorbance (OD600) of the strain at 600 nm wavelength was 10. When inoculated into fermentation broth at a volume ratio of 1/100 by performing a fermentation process for 3 days at a temperature of 30 ℃ to produce bioethanol.

As a result, the amount of bioethanol produced after the fermentation using the sugar compound was measured using a multiparameter bioanalytical system YSI 7100 MBS (YSI Life Science, USA), which is an electrolyte analyzer, and is shown in FIG. 7.

That is, glucose was all consumed after 1 day (FIG. 7A), and xylose was consumed after 2 days (FIG. 7B). In addition, in the case of bioethanol production, the control group was 2.442 g / L ethanol production, ethanol production was 2.715 g / L when using the pre-glycosylated rice straw according to the present invention (Fig. 7c).

As can be seen from the above results, the pretreatment method of lignocellulosic biomass according to the present invention can be said to be an efficient pretreatment process that can produce bioethanol from rice straw which is a waste product obtained after pretreatment-glycosylation in a very simple manner. .

1 is a view showing the form of rice straw after pretreatment according to the present invention.

2 is a graph measuring the yield of the sugar compound according to the concentration of the titanium oxide (TiO ₂ ) dispersion in the pre-treatment conditions,

(■: glucose, □: xylose)

3 is a graph measuring the rate of hydrolysis of rice straw according to the type of ultraviolet wavelength as a pretreatment condition,

(■: glucose, □: xylose)

4 is a graph confirming the yield of the sugar compound according to the ultraviolet irradiation time in the pre-treatment conditions,

(■: glucose, □: xylose)

5 is a result of confirming the effect of titanium oxide (TiO ₂ ) dispersion on the activity of axelase by a thin membrane chromatography method,

(1: carboxymethyl cellulose, 2: titanium oxide dispersion, 3: carboxymethyl cellulose + titanium oxide dispersion)

6 is a result of confirming the obtained sugar compound by a thin membrane chromatography method after the pretreatment according to the present invention,

(1: glucose, 2: xylose, 3: pretreatment conditions)

7 is a graph confirming the obtained sugar compound and the yield of bioethanol using the same after the pretreatment according to the present invention.

(a: glucose, b: xylose, c: bioethanol)

Claims (12)

delete delete delete delete a) pretreatment by irradiating UV light of 250 nm to 380 nm to lignocellulosic biomass immersed in a titanium oxide (TiO ₂ ) dispersion; b) preparing glucose and xylose at the same time by treating the pretreated lignocellulosic biomass with glycosylation enzyme; And c) inoculating the prepared glucose and xylose with Pichia stipitis KCCM 12009 strain; A method of producing bioethanol from lignocellulosic biomass comprising a. The method of claim 5, The method of producing bioethanol from lignocellulosic biomass, characterized in that the titanium oxide (TiO ₂ ) dispersion of step a) comprises 0.05 to 0.5% by weight of titanium oxide (TiO ₂ ). The method of claim 5, UV irradiation of step a) is a method for producing bioethanol from lignocellulosic biomass, characterized in that for 60 to 240 minutes irradiation. The method of claim 5, The glucose and xylose of step b) are added to 1 to 30 parts by weight of endoglucanase, carboxymethylcellulose, β-glucosidase, pNP-glucosidase or a mixture thereof based on 100 parts by weight of the pretreated biomass. A method for producing bioethanol from lignocellulosic biomass, which is obtained by saccharification. delete delete The method of claim 5, Culture step c) was inoculated with Pichia stipitis KCCM 12009 strain in a volume ratio of 1: 0.5 to 1.5 in ethanol fermentation medium containing glucose and xylose, temperature 28 to 32 ℃, time 24 Method for producing bioethanol from lignocellulosic biomass, characterized in that for culturing for 96 hours. delete

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