KR20160008000A - Rice straw saccharification method using combined pretreatment including radiation and alkali, and microalgal lipid production method using same - Google Patents

Abstract

The present invention relates to a rice straw saccharification method using pre-treatment both radiation and alkali, and to a lipid production method of microalgae using the same. The rice straw saccharification method using pre-treatment both radiation and alkali can be helpfully used in producing lipid using the microalgae, by increasing a cellulose content when treating both a large amount of gamma rays and the alkali, and increasing decomposable properties of rice straw which removes a large amount of lignin and hemicellulose, increasing a glucose and a xylose yield in enzyme hydrolysis, and confirming that Chlorella protothecoides, which are ipid-producing microalgae, produce lipid when culturing a rice straw hydrolyzate as a carbon source.

Description

[0001] The present invention relates to a method of producing a rice straw saccharification method using a pretreatment method using radiation and alkali, and a microalgae lipid production method using the same,

The present invention relates to a method for saccharification of rice straw using pretreatment for combination of radiation and alkali, and a method for producing microalgae using the same.

Lignocellulosic biomass is composed of cellulose, hemicellulose and lignin, which is the most abundant and renewable source on the planet. Lignocellulosic biomass includes agricultural residues, forestry residues, animal and human wastes, which are biofuels and value-added biomass. biomaterials (Himmel et al., 2007). In addition, impacts on the environment due to collisions between food and energy resources, the need for rapid growth for energy, the reduction of stored oil, and the use of fossil fuels have been highlighted in lignocellulosic biomass to produce biofuels (Cassman and Liska, 2007). However, lignocellulose requires pretreatment to overcome the natural biomass recalcitrance of natural biomass due to chemical interactions between components (Himmel et al., 2007) , By inhibiting the enzymatic digestion of lignocellulosic biomass, which allows many physicochemical structures and constituent components to liver the sugars necessary for fermentation, thereby reducing lignin destruction and degradation of lignin for enzymatic degradation of lignocellulosic biomass, It is well known in the art that pretreatment for exposure of hemicellulose is an essential step.

Various pretreatment methods, including physical, chemical and biological pretreatment, have been developed to increase the effectiveness of enzymatic hydrolysis and reduce costs in the production of biofuels using lignocellulosic biomass (Sun and Cheng, 2002; Moiser, et al., 2005; Kumar, et al., 2009; Menon and Rao, 2012). Combined pretreatment, in which two or more pretreatment techniques are combined to increase the degradability of the enzyme, is also generally performed, but there are options for many types of pretreatment methods, and each pretreatment method has its own advantages and disadvantages (disadvantage), it is difficult to make an ideal choice. In addition, the optimal pretreatment method depends on the type of biomass, its economics and its environmental impact. Thus, there is a greater scope to reduce the operating costs of the pretreatment and subsequent enzymatic hydrolysis steps to obtain fermentation sugars as compared to other processes.

Ionizing radiation such as gamma rays and electron beams can modify or interfere with the structure of the lignocellulose by penetration into the lignocellulose-based structure and generation of free radicals. For this reason, radiation pretreatment has long been studied as a physical pretreatment method to increase the enzymatic hydrolysis of lignocellulosic biomass. Preliminary results indicate that degradation of lignocellulosic biomass enzymes is increased after radiation pretreatment (Kamakura and Kaetsu, 1978; Khan, 1986; Chodsu et al., 1993; Xin and Kumakura, 1993). Recently, the effect of ionizing radiation pretreatment on lignocellulosic biomass has been investigated in detail (Chunping et al., 2008; Yang, et al., 2008; Bak et al., 2009; Driscoll, et al., 2009 , 2012; Karthika et al., 2012a), the effect of irradiation on the crystallinity of lignocellulosic biomass ), Inducing a decrease in degree of polymerization, and increasing the surface area for increasing enzyme accessibility and enzyme degradability. Generally, the radiation pretreatment of lignocellulosic biomass is performed in combination with other physical or chemical pretreatments such as mechanical milling and acid or alkaline dilution pretreatment, which reduces the amount of radiation required, Promotes the enzymatic degradation of cellulosic biomass. However, the theoretical yield of glucose when combined pretreatment in previous studies was generally lower than that obtained from lignocellulose treated physico-chemical or chemically (Bak et al., 2009). Other pretreatment methods are used in enzymatic hydrolysis due to differences in the nature of the biomass, such as absorbed energy, source, enzyme loading, and efficiency (Karthika et al ., 2012). Therefore, a pretreatment method for treating lignocellulosic biomass using radiation pretreatment and other pretreatment methods and industrially useful industrial enzymes should be designed to be economical.

Among the various types of lignocellulosic biomass, rice straw is known as a potential candidate for biofuel production since it is known as the world's most abundant and renewable raw material (Binod et al., 2010; Sarkar et al., 2012). In the past few years, extensive research has been undertaken to convert rice straw into pretreatment methods and convert straw straw into biofuels, particularly bioethanol (Sun and Cheng, 2002; Kumar et al., 2009; Binod et al., 2010 ). However, production of biodiesel using lignocellulosic biomass is less well known than extensive and useful information on the production of bioethanol from leathers of cellulosic biomass. In addition, they are used in combination with sugar, such as acetic acid, furfural, HMF, heavy metal and water soluble regnin, which are known to inhibit microbial metabolism, ) (Silva et al., 2013). Therefore, the effect of lignocellulose hydrolysates on cell growth and oil production must be evaluated before the hydrolyzate is applied as a carbon source to a large scale fermentation process.

Over the last decade, microalgae have been used for sustainable, biodegradable biodiesel production due to their high oil content, strong environmental adaptability, shorter life cycles than energy crops used for biodiesel production, and uncultivated areas. Has been considered as one of the most potent raw materials (Chisti, 2007; Li et al., 2008; Meng et al., 2009). However, algae-based biodiesel production is limited due to the high cost of the fermentation substrate, which accounts for 80% of the total media cost (see Li et al., 2007). Chlorella protothecoides, which grow rapidly among various groups for microalgae, high biomass and lipid production under high heterotrophic cultivation and exhibit high oil content, are used for organic carbon input, And that it uses a variety of carbon sources that can reduce costs for the environment. These include, for example, sugar cane juice (Cheng et al., 2009b), waste molasses (Yan et al., 2011), crude glycerol (O'Grady and Morgan, 2011), starch hydrolysates from the jerusalem artichoke (Cheng et al., 2009a), sweet sorghum (Gao et al., 2010), corn powder (Xu et al., 2006) and cassava (Wei et al., 2009; Lu et al., 2010) were used to produce oil for the production of biodiesel as a carbon source of chlorella proto- Respectively. These results indicate that these carbon sources can be used for oil production through the subculture of chlorella proto-choide with high oil content and for the production of high quality biodiesel. However, the long-term viability of grain-based biofuel production is controversial (Cassman and Liska, 2007; Groom et al., 2008). In addition, cereal-based biofuel production has a moral dilemma for the use of crops and land for fuel-to-food (Sun and Cheng 2002; Cassman and Liska, 2007). Nonetheless, only a few studies have confirmed that hydrolysates from lignocellulosic biomass can be replaced by glucose or grain-based feedstocks for microalgae oil production (Li et al. , 2007; EL-Sheekh et al., 2012). Therefore, further and deeper studies for the production of microalgae oil using lignocellulose hydrolyzate are needed for sustainable and economical biodiesel production.

Therefore, when investigating the oil production method using microalgae, the present inventors increased the cellulose content by removing the large amount of lignin and hemicellulose when the gamma ray and alkali were used together to improve the degradability of the rice straw, It was confirmed that when the rice straw hydrolyzate was cultured with a carbon source, chlorella protocheeed, which is a lipid production microalgae, produced lipid significantly, and thus the pretreatment of rice straw and alkaline rice straw The present inventors completed the present invention by confirming that the rice straw hydrolyzate produced using the saccharification method can be used for the production method of microalgae lipid by establishing optimal conditions for the production of microbial lipid using the rice straw saccharification method of the present invention .

It is an object of the present invention to provide a rice straw saccharification method using pretreatment for combination of radiation and alkali and a microalgae production method using the same.

In order to achieve the above object, the present invention provides a pretreatment method of lignocellulosic biomass, which comprises lignocellulosic biomass in combination with radiation and alkali.

The present invention also provides a method for saccharification of lignocellulosic biomass comprising the steps of:

1) treating lignocellulosic biomass with gamma irradiation and alkali treatment; And

2) treating the pretreated lignocellulosic biomass of step 1) above with a saccharifying enzyme.

The present invention also provides a saccharified liquid of lignocellulosic biomass prepared by the method of the present invention.

In addition, the present invention provides a microalgae culture medium containing glycosylated lignocellulosic biomass of the present invention.

The present invention also provides a method for producing microalgae lipids comprising the steps of:

1) culturing microalgae in the microalgae culture medium of the present invention; And

2) obtaining a lipid from the cultured medium of step 1) above.

The method of the present invention for improving the degradability of rice straw by increasing the cellulose content and removing a large amount of lignin and hemicellulose when the gamma ray and alkali are used in combination, And chylella protothecide, which is a lipid production microalgae, was found to produce lipid when the rice straw hydrolyzate was cultivated as a carbon source, thereby providing an optimum method for producing lipid using microalgae Respectively.

FIG. 1 is a graph showing changes in glucose and xylose yields by pretreatment of rice straw with gamma ray and alkali.
FIG. 2 is a graph showing a change in glucose yield due to loading of Cellic CTec2 enzyme in the hydrolysis of pretreated rice straw.
FIG. 3A is a graph showing changes in concentration of glucose and biomass of chlorella protothewide when a pure glucose or rice straw hydrolyzate is subcultured with a carbon source; FIG.
?: Concentration of glucose residue when pure glucose is used as a carbon source;
●: cell concentration when pure glucose is used as a carbon source;
?: Concentration of glucose residue when rice straw hydrolyzate was used as a carbon source; And
▲: Cell concentration when rice straw hydrolyzate was used as a carbon source.
FIG. 3B is a graph showing changes in the concentrations of glucose and biomass of chlorella protocide when a nutrient mixture of pure glucose or rice straw hydrolyzate is mixed with carbon source:
?: Concentration of glucose residue when pure glucose is used as a carbon source;
●: cell concentration when pure glucose is used as a carbon source;
?: Concentration of glucose residue when rice straw hydrolyzate was used as a carbon source; And
▲: Cell concentration when rice straw hydrolyzate was used as a carbon source.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for pretreatment of lignocellulosic biomass comprising combining lignocellulosic biomass with radiation and alkali.

The lignocellulosic biomass may be selected from the group consisting of rice straw, hardwood, softwood, herbaceous, recycled paper, waste paper, wood chips, pulp and paper wastes, waste wood, liverwood, cornstalks, , Straw, sugar cane, bargain, agricultural products, agricultural waste, and animal manure. In a preferred embodiment of the present invention, rice straw is the most preferred.

The radiation may be any one selected from the group consisting of gamma rays, electron beams, UV, and X rays. According to a preferred embodiment of the present invention, gamma rays are most preferable. The gamma ray is preferably irradiated using a gamma ray emitted from a radioisotope such as cobalt (Co) -60, krypton (Kr) -85, strontium (Sr) -90 or cesium (Cs) -137, Co) -60 radioisotope, but it is not limited thereto. The radiation dose is preferably 10 to 300 kGy, more preferably 10 to 200 kGy, and most preferably 25 to 100 kGy. When the irradiation dose is less than 10 kGy, the saccharification efficiency is lowered. When the irradiation dose is more than 300 kGy, there is no change in the saccharification efficiency compared to the irradiation dose, which is inefficient in terms of energy.

It is preferable that the alkali is any one selected from the group consisting of ammonia water, NaOH, Ca (OH) ₂ , and Na ₂ S, but it is most preferred that the alkali is NaOH according to a specific embodiment of the present invention. The concentration of NaOH is preferably 0.5 to 3.0% (w / v), more preferably 1.0 to 2.0% (w / v) and most preferably 1.0% (w / v). When the alkali concentration is less than 0.5% (w / v), there is a problem that the saccharification efficiency is lowered, and when it exceeds 3.0% (w / v), there is a problem that the pH of the saccharide is changed. The alkali treatment is preferably performed at 5 to 20 ml per 1 g of lignocellulosic biomass, more preferably at 9 ml per 1 g of lignocellulosic biomass, In the case of less than 5 ml per 1 g of nocellulose biomass, there is a problem that the saccharification efficiency is lowered, and when it exceeds 20 ml, there is no change in the saccharification efficiency compared with the usage amount, which is inefficient in terms of cost.

In addition, it is most preferable that the combination treatment is performed sequentially by irradiation with gamma rays and alkali, but the present invention is not limited thereto.

In a specific embodiment of the present invention, the rice straw saccharification method using the pretreatment for combination of radiation and alkali improves the degradability of rice straw by increasing the cellulose content and removing a large amount of lignin and hemicellulose (see Table 1) (See Fig. 1), it can be used for the pretreatment of lignocellulosic biomass.

The present invention also provides a method for saccharification of lignocellulosic biomass comprising the steps of:

1) treating lignocellulosic biomass with gamma irradiation and alkali treatment; And

2) treating the pretreated lignocellulosic biomass of step 1) above with a saccharifying enzyme.

In addition, the present invention provides a saccharified liquid of lignocellulosic biomass prepared by the above method and a microalgae culture medium containing the same.

The lignocellulosic biomass may be selected from the group consisting of rice straw, hardwood, softwood, herbaceous, recycled paper, waste paper, wood chips, pulp and paper wastes, waste wood, liverwood, cornstalks, , Straw, sugar cane, bargain, agricultural products, agricultural waste, and animal manure. In a preferred embodiment of the present invention, rice straw is the most preferred.

The radiation may be any one selected from the group consisting of gamma rays, electron beams, UV, and X rays. According to a preferred embodiment of the present invention, gamma rays are most preferable. The gamma ray is preferably irradiated using a gamma ray emitted from a radioisotope such as cobalt (Co) -60, krypton (Kr) -85, strontium (Sr) -90 or cesium (Cs) -137, Co) -60 radioisotope, but it is not limited thereto. The radiation dose is preferably 10 to 300 kGy, more preferably 10 to 200 kGy, and most preferably 25 to 100 kGy. When the irradiation dose is less than 10 kGy, the saccharification efficiency is lowered. When the irradiation dose is more than 300 kGy, there is no change in the saccharification efficiency compared to the irradiation dose, which is inefficient in terms of energy.

It is preferable that the alkali is any one selected from the group consisting of ammonia water, NaOH, Ca (OH) ₂ , and Na ₂ S, but it is most preferred that the alkali is NaOH according to a specific embodiment of the present invention. The concentration of NaOH is preferably 0.5 to 3.0% (w / v), more preferably 1.0 to 2.0% (w / v) and most preferably 1.0% (w / v). When the alkali concentration is less than 0.5% (w / v), there is a problem that the saccharification efficiency is lowered, and when it exceeds 3.0% (w / v), there is a problem that the pH of the saccharide is changed. The alkali treatment is preferably performed at 5 to 20 ml per 1 g of lignocellulosic biomass, more preferably at 9 ml per 1 g of lignocellulosic biomass, In the case of less than 5 ml per 1 g of nocellulose biomass, there is a problem that the saccharification efficiency is lowered, and when it exceeds 20 ml, there is no change in the saccharification efficiency compared with the usage amount, which is inefficient in terms of cost.

It is most preferable that the combination treatment is performed sequentially by irradiation with gamma rays and alkali, but it is not limited thereto.

In addition, the microalgae may be selected from the group consisting of Nannochloropsis, Chlorella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, ), Spirulina (Spirulina), Amfora (Amphora), and oak preferably composed of any one selected from the group consisting of Pseudomonas (Ochromonas), and chlorella minu tea Shima (chlorella minutissima), chlorella Pierre D'Dosa (chlorella pyrenoidosa), Chlorella Varia Billy's (Chlorella variabilis , Chlorella vulgaris) and protocol Chlorella Teco id (and more preferably one selected from the group consisting of Chlorella protothecoides), in accordance with a preferred embodiment of the present invention is most preferably Chlorella Teco protocol id (Chlorella protothecoides).

In a specific example of the present invention, the rice straw saccharification method using the pretreatment for combination of radiation and alkali improves the degradability of rice straw by removing a large amount of lignin and hemicellulose, and increases the content of cellulose (see Table 1) It has been confirmed that it can be used for pretreatment of lignocellulosic biomass by directly reducing the residue recovery (see Table 2) and directly confirming that the concentration of minerals is changed (see Table 3).

The present invention also provides a method for producing microalgae lipids comprising the steps of:

1) culturing microalgae in the microalgae culture medium of the present invention; And

2) obtaining a lipid from the cultured medium of step 1) above.

The lignocellulosic biomass may be selected from the group consisting of rice straw, hardwood, softwood, herbaceous, recycled paper, waste paper, wood chips, pulp and paper wastes, waste wood, liverwood, cornstalks, , Straw, sugar cane, bargain, agricultural products, agricultural waste, and animal manure. In a preferred embodiment of the present invention, rice straw is the most preferred.

The radiation may be any one selected from the group consisting of gamma rays, electron beams, UV, and X rays. According to a preferred embodiment of the present invention, gamma rays are most preferable. The gamma ray is preferably irradiated using a gamma ray emitted from a radioisotope such as cobalt (Co) -60, krypton (Kr) -85, strontium (Sr) -90 or cesium (Cs) -137, Co) -60 radioisotope, but it is not limited thereto. The radiation dose is preferably 10 to 300 kGy, more preferably 10 to 200 kGy, and most preferably 25 to 100 kGy. When the irradiation dose is less than 10 kGy, the saccharification efficiency is lowered. When the irradiation dose is more than 300 kGy, there is no change in the saccharification efficiency compared to the irradiation dose, which is inefficient in terms of energy.

It is preferable that the alkali is any one selected from the group consisting of ammonia water, NaOH, Ca (OH) ₂ , and Na ₂ S, but it is most preferred that the alkali is NaOH according to a specific embodiment of the present invention. The concentration of NaOH is preferably 0.5 to 3.0% (w / v), more preferably 1.0 to 2.0% (w / v) and most preferably 1.0% (w / v). When the alkali concentration is less than 0.5% (w / v), there is a problem that the saccharification efficiency is lowered, and when it exceeds 3.0% (w / v), there is a problem that the pH of the saccharide is changed. The alkali treatment is preferably performed at 5 to 20 ml per 1 g of lignocellulosic biomass, more preferably at 9 ml per 1 g of lignocellulosic biomass, In the case of less than 5 ml per 1 g of nocellulose biomass, there is a problem that the saccharification efficiency is lowered, and when it exceeds 20 ml, there is no change in the saccharification efficiency compared with the usage amount, which is inefficient in terms of cost.

It is most preferable that the combination treatment is performed sequentially by irradiation with gamma rays and alkali, but it is not limited thereto.

In addition, the microalgae may be selected from the group consisting of Nannochloropsis, Chlorella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, ), Spirulina (Spirulina), Amfora (Amphora), and oak preferably composed of any one selected from the group consisting of Pseudomonas (Ochromonas), and chlorella minu tea Shima (chlorella minutissima), chlorella Pierre D'Dosa (chlorella pyrenoidosa), Chlorella Varia Billy's (Chlorella variabilis , Chlorella vulgaris) and protocol Chlorella Teco id (and more preferably one selected from the group consisting of Chlorella protothecoides), in accordance with a preferred embodiment of the present invention is most preferably Chlorella Teco protocol id (Chlorella protothecoides).

In a specific example of the present invention, when a microalgae, chlorella prototheoid, which produces lipids, is cultured in a medium using a rice straw hydrolyzate prepared by the saccharification method of the present invention as a carbon source, chlorella protococoide grows, By producing lipids (see FIG. 3), it was confirmed that the rice straw hydrolyzate produced by the saccharification method of the present invention can be used for producing lipids using microalgae.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

It should be noted, however, that the following examples and experimental examples are illustrative of the present invention, but the present invention is not limited by the following examples and experimental examples.

< Example  1> Confirmation of composition change of rice straw by pretreatment

Weight reduction and compositional changes in biomass are important indicators of the effectiveness of the pretreatment method. In addition, chemical composition of rice straw was significantly changed by various components such as farm, used variety, harvesting season, and storage environment, and composition and weight change between untreated or pretreated rice straw biomass decreased the effect of pretreatment method It is first analyzed for comparison.

<1-1> Preparation of rice straw

Rice straw was grown in Jeongeup (Korea), cut to a maximum of 5 cm before pretreatment, washed with tap water to remove extraneous matter, and dried in an oven at 70 ° C. until constant weight was reached . It was then stored in a polystyrene bag until it was used.

<1-2> Pretreatment of rice straw

In order to increase glucose recovery from rice straw, gamma irradiation and alkaline (0.1% NaOH) pretreatment methods were performed.

Specifically, for the purpose of gamma irradiation and alkali pretreatment, the rice straw was finely cut into a glass tube and irradiated with a dose rate of 10 kGy / h using a Cobalt-60 machine of the Korea Atomic Energy Research Institute (Jeongeup, Korea) To an optimal absorbed dose of gamma rays. For alkaline pretreatment, 10 g of rice straw was mixed with 90 ml of 1.0% NaOH (w / w) solution and allowed to stand at room temperature (25 to 30 ° C) for 5 days. After pretreatment, wet rice straw was collected by filtration and washed with deionized water until neutral pH was reached. All pretreated rice straws were dried at 70 ° C until constant weight was reached. All the rice straw samples were prepared by grinding through a 420 nm sieve using a pulverizer (Korea Medi, Korea) prior to the following steps.

<1-3> Preprocessed  Analysis of composition of rice straw

In order to confirm the compositional change of the rice straw pretreated by the method of Example <1-2>, a two stage acid hydrolysis protocol designed by the National Renewable Energy Laboratory was performed .

Specifically, each sample was hydrolyzed with 72% sulfuric acid at 30 ° C for 1 hour and then hydrolyzed with 3% sulfuric acid at 121 ° C for 1 hour. The sterilized hydrolyzate solution was neutralized to pH 6.0 by the addition of calcium carbonate and filtered under reduced pressure. The sugars released by hydrolysis are then transferred to a high-pressure liquid chromatography system (Agilent 1200, Agilent Technologies, USA) equipped with a refractive index detector (Agilent 1260, (Bio-Rad Laboratories, USA) at 65 ° C with a flow rate of 0.6 ml / min. All samples were filtered with a 0.2 μm filter and diluted with eluent before being analyzed on HPLC. It was performed using various concentrations of pure monomeric sugar and used as a standard. The theoretical glucose and xylose contents were calculated by multiplying the glucose and xylose contents by conversion factors 0.9 and 0.88, respectively, which was the mass obtained during the hydrolysis of glycan and xylan mass. The moisture content was measured by the weight loss of a 1 g sample of rice straw dried at 105 ° C and the content of acid insoluble lignin (Klason lignin) -free and oven-dried filter cake were weighed, and the residue recovery, delignification, and cellulosic recovery of the gamma-irradiated or alkali-treated rice straw samples, The loss of cellulose and xylan were determined by weighing with untreated rice straw samples as control. The elemental composition was determined by inductively coupled plasma (ICP) from Korea Basic Science Institute.

As a result, as shown in Table 1, when the composition was compared with the composition of the untreated rice straw, the cellulose content was increased up to 40.5% when pretreated with alkali alone, and the contents of xylenes, lignin, extractives and water were 42.9% 23.4%, 10.8%, and 44.3%, respectively. However, when the gamma ray and alkali were pretreated in combination, the cellulose content was further increased up to 60%, and the increase amount was dependent on the gamma ray dose. On the other hand, the level of other components decreased in dependence on the gamma dose. The combined pretreatment increased the removal rates of xylan, lignin, extract, and water to 63.8%, 40.2%, 32.3%, and 58.7%, respectively. In addition, sugar degradation products such as furfural and hydroxymethyl-furfural, which are known to inhibit cell growth, were not identified (Table 1). Lignin is known to limit access to carbohydrates by physical barriers or by unproductive binding of enzymes (Palonen, 2004; Berlin et al., 2007). Thus, an increase in cellulose content, and a decrease in xylans and lignin, will facilitate the process of enzyme hydrolysis. It was clearly confirmed that the combination pretreatment could increase the enzyme digestibility of the rice straw after a large amount of lignin and hemicellulose content was removed by the combined pretreatment.

Identification of major components of rice straw by pretreatment Pretreatment condition Composition analysis (w / w,%) ^a Cellulose Xylian Clason lignin Water-soluble extract Water content Untreated 37.5 10.5 18.4 9.3 9.7 25 kGy 37.1 10.8 18.2 9.2 9.6 50 kGy 37.4 10.6 18.3 9.5 9.3 75 kGy 37.3 10.3 18.2 9.4 9.1 100 kGy 37.9 10.6 18.5 9.4 9.1 NaOH 52.7 6.0 14.1 8.3 5.4 25 kGy + NaOH 52.7 5.9 13.6 8.1 5.5 50 kGy + NaOH 54.2 5.8 12.5 8.1 4.3 75 kGy + NaOH 56.9 4.4 11.5 7.2 4.3 100 kGy + NaOH 60.0 3.8 11.0 6.3 4.0

^a Results are expressed as the average percentage of components obtained by three runs.

It is known in the art that alkaline pretreatment methods can cause effective delignification as well as swelling of biomass by promoting hydrolysis and improving glucose recovery from biomass (Kumar et al ., 2009). In addition, because it can be used at lower temperatures and pressures than other pretreatment methods, it can be performed in an ambient condition that results in lower sugar degradation as compared to acid pretreatment. Thus, alkaline pretreatment has been identified as the most effective method for destroying the structure of lignocellulosic biomass compared to acid or an oxidizing reagent (Gaspar et al., 2007; Binod et al., 2010). However, as subsequent biodegradation of the biomass is required for the subsequent enzymatic hydrolysis process, additional washing steps are required, and the water-soluble hemicellulose and lignin can be removed in the washing step, but the content of the different constituents, including cellulose, Lt; / RTI &gt;

As shown in Table 2, based on the residue recovery after pretreatment, the combined pretreatment can significantly reduce not only the content of xylan and lignin but also the total biomass recovery (Table 2). Although alkali treatment is known to cause less loss of cellulose content than acid or hydrothermal treatment (Carvalheiro et al., 2008), it is known that the cellulose content in the range of 11-15.4% based on the original weight of rice straw Investigation-dependent losses also occur.

Solid recovery of pre-treated rice straw and loss of major components of gamma-ray and alkali-treated rice straw Pretreatment condition Residue recovery
(w / w,%) ^a Component loss (w / w,%) ^b Cellulose Xylian Lignin NaOH 65.1 8.5 62.8 50.2 25 kGy + NaOH 63.2 11.0 64.5 52.8 50 kGy + NaOH 60.5 12.5 66.5 58.6 75 kGy + NaOH 56.8 13.9 76.4 64.2 100 kGy + NaOH 52.9 15.4 80.9 68.5

^a 100 g of rice straw were pretreated and the results were expressed as the average percentage of the oven-dried rice straw performed three times.

^b . Residue recovery and component analysis in Table 1 were determined based on percentage.

In addition, as shown in Table 3, significant changes were observed in the concentrations of minerals in the neutralized rice straw after the combined pretreatment. Potassium, calcium, and magnesium were the major elements of untreated rice straw in the minerals, but the potassium content was significantly reduced (22 folds) when combined pretreatment, and calcium and magnesium The content increased. The sodium content also increased after pretreatment, probably due to alkaline pretreatment. Traces of non-essential heavy metals such as arsenic, cadmium, and lead, which can inhibit cell growth during fermentation, have also been identified (Table 3). This is necessary for in-depth analysis of the composition of biomass pretreated after pre-treatment to apply it in the fermentation process.

Comparison of element composition before and after pre-combination treatment Element (mg / kg) Untreated 100 kGy + NaOH Al 156.83 8.12 58.29 ± 5.65 As 1.21 ± 0.03 ND ^a Ca 1605.93 + - 21.30 2805.35 + - 123.53 CD 0.06 ± 0.01 0.07 ± 0.01 Cr 30.44 + 1.43 14.30 ± 0.36 Cu 2.71 ± 0.06 6.46 0.41 Fe 185.21 + - 6.36 215.27 ± 15.74 K 3743.71 + - 0.32 169.11 + - 12.96 Mg 629.97 + - 7.06 1016.06 + - 46.19 Mn 304.73 ± 12.51 515.47 ± 48.62 Mo 0.33 ± 0.01 ND ^a Na 98.70 + - 0.95 383.19 ± 17.76 Ni 2.84 ± 0.19 2.01 ± 0.1 Phosphate 356.86 ± 5.83 73.55 + - 4.95 Pb 3.22 + 0.02 5.12 ± 1.48 Sulfate 304.12 ± 1.09 277.20 ± 12.93 Zn 38.06 + - 0.43 55.49 + - 3.51

Data are presented as the mean of three measurements of standard deviation.

Below ^a detection limit

< Example  2> Confirmation of effect by pretreatment in enzyme hydrolysis

In general, the efficiency of enzymatic hydrolysis of biomass is increased when the combination of enzymes such as cellulase, xylanase and glucosidase is used alone as a cellulase. Enzymatic hydrolysis was carried out with Cellic CTec2 (Novozymes, Denmark) and Cellic HTec2 (Novozymes, Denmark), which are currently used as industrial enzymes, and Cellic HTec2 was used for efficient enzymatic hydrolysis of cellulose in rice straw. which was used to decompose the barrier.

Specifically, the FPU (filter paper unit) of Cellic CTec2 (120 FPU / ml) was measured according to the method of Ghose, a well-known method, and the FXU (fungal) of Cellic HTec2 (2500 FXU / xylanase unit) was used to calculate the loading. Enzyme hydrolysis was performed with 50 mM sodium citrate buffer containing meropenem (Sigma-Aldrich, USA) at a concentration of 12.5 mg / l and substrate consistency was 20% (w / v). The rice straw sample was immersed in the buffer and heated 30 minutes before the addition of the enzyme. The reaction was carried out in a shaking water bath (JEIO TECH, Korea) at 200 rpm at 50 DEG C, . The progress of the enzymatic hydrolysis was measured at regular intervals by estimating the reduced sugar in the rice straw hydrolyzate using HPLC following the protocol described above. All experiments were carried out three times, and the mean value of the values was shown.

As a result, the theoretical yield of glucose and xylose after enzyme hydrolysis (based on the weight of the rice straw after pretreatment) is as shown in Fig. The initial hydrolysis rate for all rice straw samples was significantly higher than the subsequent hydrolysis rate, which was the same as other studies. Selective initial hydrolysis of amorphous cellulose or subsequent insufficiency of the new catalytic site may explain this phenomenon (Eriksson et al., 2002; Zhong et al., 2009). Glucose and xylose yields of gamma - irradiated rice straw increased in a dose - dependent manner, and glucose was a major component of enzymatic rice straw hydrolyzate. When compared to the untreated control, the yield of glucose and glial was increased by 2-fold by gamma-ray pretreatment, but the conversion of cellulose and xylen to monomers was still less than 50%, which was consistent with previous reports Bak et al., 2009). This means that the pretreatment method of radiation is very simple, but using it alone shows that it is insufficient for pretreatment of rice straw. Alkali pretreatment showed higher glucose and xylose yields than gamma - ray pretreatment, and conversion rates of cellulose and xylenes to monomers were 74.2% and 85.8% after 72 hours, respectively. In order to increase the conversion rate, pretreatment with alkaline pretreatment was performed after gamma irradiation. As a result, the pretreatment for the pretreatment of rice straw was the most effective method, and the maximum conversion rates of cellulose and xylan after 72 hours were 92.3% and 98.9% (Xin and Kumakura, 1993; Zhang and Cai, 2008; Zhong et al., 2009; Waeonukul et al., 2007), which shows the maximum conversion rate of rice straw to monomeric sugar among the reported ones (Fig. , 2012; Yang et al., 2012; Hou et al., 2013).

In many studies of the combined use of one or two pretreatments, the addition of? -Glucosidases and hemicellulose, and accessory enzymes such as ionic or non-ionic additives, (Waeonukul et al., 2012).

In the present invention, the influence of loading of Cellic CTec2 in the hydrolysis of rice straw was confirmed, and specifically, conversion of cellulose to glucose was confirmed. As shown in Fig. 2, the initial conversion of cellulose increases when Cellic CTec2 loading is high Respectively. In addition, the effect of enzyme concentration on cellulolytic conversion was not significant in untreated rice straw. When 2 FPU was used in untreated rice straw, the conversion of cellulose was 22%, and it increased to 28% when 12 FPU was used. In addition, the effect of enzyme concentration was more significant in alkaline pretreated rice straw than in rice straw pretreated with gamma irradiation. When the gamma - irradiated rice straw was decomposed, conversion rate increased from 32% to 66% when using enzyme 2 FPU, and from 56% to 12% when 2 FPU was used for alkaline pretreatment And 84%, respectively. When the enzyme 2 FPU was added to the hydrolysis reaction of gamma and alkaline pretreated rice straw, the conversion of cellulose was 47% after 24 hours of decomposition. However, when 8 FPU was added, the theoretical glucose yield of 85.2% . In addition, the hydrolysis reaction only increased the theoretical glucose yields of 5.4% and 2.7% after 48 and 96 hours, respectively, and increased the enzyme concentration to 12 FPU, Compared to only 2% (Fig. 2).

< Experimental Example  1> Rice straw singer resolvent  Chlorella proto-choide ( Chlorella  protothecoides ) Growth Confirmation

<1-1> Chlorella Prototheoid  Ready

Chlorella prototyoid strain 25 was obtained from the UTEX algae culture collection (Texas Univ. Of Austin, USA) and was cultivated in a medium containing 3 g / l yeast extract as a base medium for culture, medium) was used (Bischoff and Bolds, 1963).

<1-2> Rice straw singer resolvent  Chlorella in the medium Prototheoid  Growth confirmation

To confirm the possibility of rice straw for producing biodiesel, rice straw hydrolyzate prepared from rice straw pretreated with alkali and gamma ray (100 kGy) was added to a carbon source in chlorella prototype growth medium, Respectively.

Specifically, batch cultivation was performed by adding 10 g of carbon source (equal volume of glucose with the appropriate volume of pure glucose or rice straw hydrolyzate added) and 3 g / l yeast extract with a nitrogen source instead of NaNO ₃ Was carried out in a 500 ml baffled flask containing 150 ml BBM and the pH of the medium was adjusted to 6.8. Algae (~ 5% V / V) were inoculated and cultured in a rotary shaker at 25 ° C with stirring at 120 rpm. Mixotrophic cultures were exposed to light for 12 hours, and heterotrophic cultivation was maintained in the dark.

As a result, when the rice straw hydrolyzate was used as the sole carbon source (subculture), the glucose of the rice straw hydrolyzate was almost used, and the cell concentration reached 6.5 g / L after 8 days of culture. The results were similar to those when using purified glucose, and when 10 g / l glucose was added to the medium, the maximum cell concentration was 8.7 g / l. The difference in yield when using rice straw hydrolyzate and purified glucose is expected to be due to impurities including other sugars or organic acids in the rice straw hydrolyzate which inhibits the growth of chlorella proto-trychoids. Growth of chlorella protothecide in rice straw hydrolyzate medium was performed by mixed nutrition culture, in which the carbon consumption rate for cell mass was 92%, which was higher than 65% in the dependent culture. However, the final cell concentration in the mixed nutrient culture was 4 g / ℓ, which was lower than the final cell concentration of 6.5 g / ℓ in the subculture culture. In addition, the cell concentration was higher when using the purified glucose than in the case of rice straw hydrolyzate in the mixed nutrition culture (Fig. 3).

It is known that the effect of glucose concentration on microalgae growth can be confirmed by lipid production by Chlorella vulgaris (Liang, et al., 2009). Under mixed nutrient culture, the addition of 5% and 10% glucose showed a growth inhibitory effect, which was also confirmed in chlorella protothecoids supplemented with glucose in the 1.5 and 6% range (Xiong et al., 2008). However, the influence of glucose on microalgae growth was not clearly confirmed.

Thus, this result indicates that rice straw can be used as an alternative to expensive carbon sources for the cultivation of microalgae producing biodiesel without changing the culture conditions.

< Experimental Example  2> Glucose  Or rice straw singer resolvent  Algae lipid between media lipid ) Production comparison

The following experiments were conducted to analyze the total lipid content and fatty acid analysis of algae cultured in a medium using glucose or rice straw hydrolyzate as a carbon source.

Specifically, for the lipid content analysis, the micro-algae growth was measured by measuring the absorbance of the culture broth at 540 nm using a UV-Vis spectrophotometer (Biochrome, UK) as a control. Respectively. The algal biomass was calculated using the following equation, where y (g / l) is the dry cell weight and x is the absorbance of the resuspension suspended at 540 nm.

For the dry weighing of the microalgae biomass, the microalgal culture was filtered using a GF / C glass fiber filter (GE Healthcare, UK) and the filter containing the biomass was placed in a 70 ° C convection oven convection oven).

After incubation, algae cells were obtained by centrifugation at 4,000 rpm for 15 minutes, and the cell pellet was lyophilized in high vaccum for 2 days, and 0.2 g of dried algae was suspended in 0.5 ml of distilled water and The mixture was mixed with 3 ml of chloroform / methanol (2: 1, v / v), and the mixed mixture was stirred for 20 minutes and then centrifuged at 10,000 rpm for 10 minutes to obtain a chloroform layer phase) was transferred to a weighted tube. The sample was repeatedly extracted five times and dried by adding a nitrogen gas at 60 DEG C in a dry block bath (MG-2000, EYELA, Japan). Lipid content was expressed as a percentage (lipid weight / biomass weight x%).

For fatty acid analysis, fatty acid was extracted from 1 ㎖ NaOH-CH ₃ OH from 50 ㎎ dried algae and 10 g / ℓ tridecanoic acid (C13: 0) was added as an internal standard for quantification of fatty acids. The mixture was stirred in a constant temperature water bath at 75 DEG C for 10 minutes and cooled at room temperature. Then, boron trifluoride-methanol solution (1: 2, v / v) was added, stirred at 75 ° C for 10 minutes, and cooled at room temperature. Then 0.3 ml of saturated salt solution was added to form a solution layer, followed by 2 ml of hexane, and the mixture was centrifuged. The upper fatty acid layer was used to perform GC-linked mass spectrometry analysis on an Agilent HP 6890N machine equipped with a 5975 inert mass selective detector (5975 inert) capillary column) DB-5MS (length 30 m, ID 0.25 mm, thickness 0.25 탆, Agilent Technologies) was used. At this time, the size of the sample was 1 μl, the carrier gas was helium at a flow rate of 1.0 ml / min, the solvent delay time was 5 minutes, and the injection temperature was 200 ° C. The initial oven temperature was 130 [deg.] C for 1 minute, which increased by 5 [deg.] C per minute to 200 [deg.] C and maintained at this temperature for 5 minutes. Fatty acids were identified by direct comparison of their mass spectral database and retention index with the NIST 05 mass spectral database (NIST 05 mass spectral database). Data were expressed as mean values measured three times.

As a result, as shown in Table 4, the total lipid content and fatty acid composition in the dependent cultures did not show any significant difference between the chlorella proto-promoted in the glucose and rice straw hydrolyzate medium. Based on cell dry weight, total lipid content was about 55% and total fatty acid content was 45%. The major fatty acids in chlorella protothecide were 9-octadecenoic acid and the major fatty acids were hexadecanoic acid, 9,12-octadecadienoic acid, Z , Z) and octadecatrienoic acid. The total lipid content and fatty acid composition in the mixed nutrition culture showed no difference between chlorella prototyoids grown in the glucose and rice straw hydrolyzate media. In the subculture and mixed nutrient culture of chlorella prototype, the total lipid content was the same, and the total fatty acid content decreased slightly in the mixed nutrition culture, but this was not statistically significant. As to the fatty acid composition, the content of 9-octadecanoic acid was decreased, but the content of 9,12,15-octadecatrienoic acid was increased (Table 4).

Other On carbon  Chlorella according to Prototheoid  Identification of fatty acid composition of lipid extract Component Relevant FAME content (%) Dependent culture Mixed nutrition culture Glucose Rice straw
Hydrolyzate Glucose Rice straw
Hydrolyzate 7,10-Hexadecadienoic acid, mothyl ester 2.13 2.30 9.02 1.64 7,10,13-Hexadecatrienoic acid, methyl ester 3.11 3.91 4.17 5.90 Hexadecanoic acid, methyl ester 15.38 16.26 20.40 20.56 Heptadecanoic acid, methyl ester 0.47 0.60 0.59 0.48 9,12-Octadecadienoic acid (Z, Z) -, methyl ester 16.91 17.10 10.52 11.59 9-Octadecenoic acid, methyl ester 51.37 47.57 31.81 34.77 9,12,15-Octadecatrienoic acid methyl ester 9.57 10.93 21.76 23.14 Octadecanoic acid, methyl ester 1.05 1.32 1.74 1.92 TL / CDW (%) b 55.77 54.97 56.37 55.10 TFA / CDW (%) c 45.62 45.37 40.95 40.04 TFA / TL (%) d 81.80 82.54 72.65 72.66 TFA yield (g / l) 4.00 2.95 2.63 1.57

FAME composition is expressed as the weight percentage of all FAME.

Data were measured three times and expressed as mean with standard deviation.

It is known that the lipid content of chlorella bulgarians was also the same in the subculture and mixed nutrient culture, thus the results indicate that the rice straw hydrolyzate can be used as a glucose replacement carbon source which does not cause any change in fatty acid content and composition.

Claims (13)

A method of pretreatment of lignocellulosic biomass comprising combining lignocellulosic biomass with radiation and alkali.
The pretreatment method according to claim 1, wherein the lignocellulosic biomass is rice straw.
The pretreatment method according to claim 1, wherein the radiation is any one selected from the group consisting of gamma rays, electron beams, UV and X rays.
4. The pretreatment method according to claim 3, wherein the gamma ray is irradiated at a dose of 10 to 150 kGy.
The pretreatment method according to claim 1, wherein the alkali is NaOH.
6. The pretreatment method according to claim 5, wherein the concentration of NaOH is 1.0 to 3.0% (w / v).
The pretreatment method according to claim 1, wherein the alkali is treated with 5 to 20 ml per gram of lignocellulosic biomass.
1) treating lignocellulosic biomass with gamma irradiation and alkali treatment; And
2) treating the pretreated lignocellulosic biomass of step 1) with a saccharifying enzyme.
9. The saccharification method according to claim 8, wherein the combination treatment is an alkali treatment after irradiation with gamma rays.
A saccharified liquid of lignocellulosic biomass prepared by the method of claim 8.
10. A microalgae culture medium containing the glycosylated lignocellulosic biomass of claim 10.
The method of claim 11 wherein the microalgae Chlorella minu tea Shima (Chlorella minutissima), Chlorella Pierre D'Dosa (Chlorella pyrenoidosa), Chlorella Varia Billy's (Chlorella variabilis , Chlorella wherein the microalgae culture medium is any one selected from the group consisting of Clostridium vulgaris and Chlorella protothecoides .
1) culturing the microalgae in the microalgae culture medium of claim 10; And
2) obtaining a lipid from the cultured medium of step 1).

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