KR20230060720A - Lignocellulosic biomass pretreatment method using nanoparticles - Google Patents

Abstract

The present invention relates to a lignocellulosic biomass pretreatment method using nanoparticles, and specifically, to a method for decomposing lignin from lignocellulosic biomass under conditions of room temperature and pressure by using at least one of cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP) under conditions with or without the addition of hydrogen peroxide (H_2O_2) in pretreating lignocellulosic biomass.

Description

Lignocellulosic biomass pretreatment method using nanoparticles}

The present invention relates to a method for pre-treating lignocellulosic biomass using nanoparticles, and more particularly, in pre-treating lignocellulosic biomass, cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles ( The present invention relates to a method for decomposing lignin from lignocellulosic biomass at room temperature and pressure under conditions of no addition or addition of hydrogen peroxide by utilizing laccase and peroxidase-mimicking properties of Ce-FeONP).

Biofuels, such as bioethanol, which is a renewable fuel, are one of the growing alternative energies worldwide considering environmental, economic and energy security issues. Overall food supply and land sustainability have raised concerns about producing biofuels from edible resource biomass (first generation biomass) derived from food crops such as corn, sugar cane and sugar beets.

This led to the emergence of non-edible resource biomass (second generation biomass) for the production of biofuels and biochemicals. Accordingly, demand for biomass degradation enzymes such as cellulase, hemicellulase, and lignin peroxidase is expected to increase for the production of second-generation biomass-derived bioethanol.

Lignocellulosic biomass (LCB), the most abundantly available resource on earth, is a lignocellulosic biomass, and it does not compete with food supply like edible biomass in that it is a non-edible resource cellulose biomass. It is receiving great attention as a resource.

The lignocellulosic biomass as described above has a problem in that cellulose (35 to 60%) and hemicellulose (15 to 30%) polymer components are entangled with lignin (15 to 20%) to form a network, hindering hydrolysis. For the same reason, in order to extract fermented sugar from lignocellulosic biomass, a pretreatment process to decompose lignin is required.

To date, numerous chemical, physical, biological and physicochemical pretreatment methods have been used as pretreatment methods for lignocellulosic biomass. However, most of the conventional pretreatment processes as described above require excessive costs and generate toxic compounds during the reaction.

More specifically, there is a problem of significantly increasing the total production cost of the bioconversion process and reducing the yield of biofuel due to the inhibitor compound generated in the pretreatment process of lignocellulosic biomass.

In addition, the biofuel production process includes a process of fermenting the separated cellulose and hemicellulose after the pretreatment process for decomposition of lignin as described above. Under harsh conditions, toxic substances such as furfural and hydroxymethyl furfural are produced, and the toxic substances produced in this way inhibit decomposition and fermentation by cellulolytic enzymes and fermentation yeast. There was a problem.

In addition, in the case of biomass pretreated using acid and alkali, washing is required, and thus there is a problem that land or water area is directly or indirectly contaminated by the washing water flowing out.

As the above conventional physical, chemical and physicochemical pretreatment methods have various problems, a pretreatment method using enzymes and microorganisms that degrade lignin has been proposed as an alternative, but there is a problem of low stability and excessive production cost. .

On the other hand, current nanomaterials (nanoparticles, nanotubes, nanoporous matrices, nanofibers, etc.) have various industrial applications due to their unique properties such as large surface area, low mass transfer limitation, reusability, and easy selective separation of reaction mixtures under magnetic field. is receiving a lot of attention from

Nanoparticles with unique enzyme-mimicking properties, commonly referred to as nanozymes, are being explored today as replacements for natural enzymes in several biomedical, industrial and energy-related applications. In particular, iron oxide nanoparticles have been reported to have unique peroxidase-mimicking properties. The main feature of nanozymes is that their properties can be controlled by changing their shape and size or doping them with other nanoparticles.

As such, as a technology related to lignocellulosic biomass pretreatment using nanoparticles, Koo et al. (H Koo, BK Salunke, B Iskandarani, WG Oh, BS Kim, Improved degradation of lignocellulosic biomass pretreated by Fenton-like reaction using Fe3O4 magnetic nanoparticles, Biotechnology and Bioprocess Engineering 22 (5), 597-603, 2017) reported that the production of glucose by hydrolysis enzyme increased after pretreatment of lignocellulosic biomass using magnetic iron oxide (Fe ₃ O ₄ ) nanoparticles. did

Accordingly, in the present invention, by using cerium oxide nanoparticles (CeONP) and cerium-doped magnetic iron oxide nanoparticles (Ce-FeONP) to pre-treat lignocellulosic biomass, lignin decomposition efficiency and sugar production by hydrolase It is intended to propose a new lignocellulosic biomass pretreatment method that can further increase the

On the other hand, as prior patent documents in the technical field such as the present invention, Korean Patent Registration No. 10-2102063 B1 (2020.04.10.), Korean Patent Publication No. 10-2017-0044778 A (2017.04.26.) and Korea Patent Publication Publication No. 10-2013-0046205 A (2013.05.07.) discloses a method for producing bioethanol from biomass using nanoparticles such as magnetic iron oxide (Fe ₃ O ₄ ), but the prior patent documents are biomass As using nanoparticles in the saccharification process, a technique using cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP) in a pretreatment process to decompose lignin in lignocellulosic biomass has not been proposed.

Korean Registered Patent Publication No. 10-2102063 B1 (2020.04.10.) Korean Patent Publication No. 10-2017-0044778 A (2017.04.26.) Korean Patent Publication No. 10-2013-0046205 A (2013.05.07.)

H Koo, BK Salunke, B Iskandarani, WG Oh, BS Kim, Improved degradation of lignocellulosic biomass pretreated by Fenton-like reaction using Fe3O4 magnetic nanoparticles, Biotechnology and Bioprocess Engineering 22 (5), 597-603, 2017

The present invention was created to solve the above problems, and in pre-treating lignocellulosic biomass, by using cerium oxide nanoparticles (CeONP) and cerium-doped magnetic iron oxide nanoparticles (Ce-FeONP), It increases the efficiency of lignin degradation in biomass and prevents the production of toxic substances that inhibit decomposition and fermentation by cellulolytic enzymes and fermentation yeast after the pretreatment process, further increasing sugar production by hydrolytic enzymes, and is environmentally friendly and pretreatment The purpose is to provide a lignocellulosic biomass pretreatment method using novel nanoparticles that can reduce the process cost.

In order to solve the above problems, the present invention provides a catalyst for pretreatment of lignocellulosic biomass, characterized in that it includes at least one of cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP). to provide. The cerium content of the cerium-doped nanoparticles may range from 1 to 10 wt%.

In one embodiment of the present invention, the nanoparticles may be nanoparticles not supported on a support, or the nanoparticles may be supported on a support.

In addition, the present invention mixes and reacts at least one of cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP) in lignocellulosic biomass with or without hydrogen peroxide (H ₂ O ₂ ). , It provides a lignocellulosic biomass pretreatment method using nanoparticles, characterized in that the lignin in the lignocellulosic biomass is subjected to a decomposition reaction.

In the pretreatment method, the decomposition reaction may be carried out at 20 to 90 ° C. and 1 to 5 bar, the concentration of hydrogen peroxide (H ₂ O ₂ ) is 1 M or less, and the concentration of the nanoparticles is 1 to 10 g/L It may be, and the loading of biomass may be 5 to 30% (w / v).

In addition, the present invention enzymatically hydrolyzes lignocellulosic biomass pretreated by the pretreatment method according to the present invention to produce fermentable sugars including glucose and xylose, lignocellulose using nanoparticles A method for producing fermentable sugars from biomass is provided.

The present invention produces fermentable sugars in biomass by using at least one of cerium oxide nanoparticles (CeONP) and cerium-doped magnetic iron oxide nanoparticles (Ce-FeONP) in pretreatment of lignocellulosic biomass It has the effect of selectively decomposing the lignin part without decomposing the cellulose component that can be used for ethanol production.

In addition, the present invention can pre-treat biomass under normal temperature and pressure conditions, so that toxic substances such as furfural and hydroxymethyl furfural generated under high temperature and high pressure conditions are not generated. Therefore, there is an effect of preventing the decomposition and fermentation of cellulose and hemicellulose by cellulose hydrolase and fermentation yeast from being inhibited by toxic substances in the subsequent process.

In addition, the present invention has an effect of improving sugar production by improving the decomposition and fermentation effect of cellulose and hemicellulose by hydrolytic enzymes and fermenting yeasts by increasing the pore surface area, pore size and pore volume of biomass after pretreatment. there is.

In addition, in the present invention, in pre-treating lignocellulosic biomass using at least one of cerium oxide nanoparticles (CeONP) and cerium-doped magnetic iron oxide nanoparticles (Ce-FeONP), by optimizing pre-treatment conditions, There is an effect of further increasing the lignin decomposition efficiency and sugar production in the subsequent process.

In addition, in the pretreatment of lignocellulosic biomass as described above, the present invention uses at least one of cerium oxide nanoparticles (CeONP) and cerium-doped magnetic iron oxide nanoparticles (Ce-FeONP) to prevent washing contamination. It is eco-friendly because no water is generated, has high stability, and the nanoparticles used in the pretreatment can be recovered and reused, thereby reducing the cost of the pretreatment process.

1 is a schematic diagram showing lignin decomposition using cerium-doped iron oxide nanoparticles according to an embodiment of the present invention.
2 shows (a) EDS, (b) SEM, and (c) TEM results of cerium-doped iron oxide nanoparticles (Ce-FeONP) synthesized according to an embodiment of the present invention.
3 is a result showing the peroxidase mimetic activity of cerium-doped iron oxide nanoparticles (Ce-FeONP) synthesized according to an embodiment of the present invention.
Figure 4 is an experimental result of measuring the lignin decomposition rate using the pretreatment time, nanoparticle concentration, solid load and H ₂ O ₂ concentration as process parameters in one embodiment of the present invention.
5 is data showing the saccharification results of 50 g/L of pretreated corncob biomass and untreated samples (control group) according to an embodiment of the present invention.
6 is a view for explaining recovery and reuse of nanoparticles according to an embodiment of the present invention. (a) is an image of biomass after nanoparticle treatment and washing, and (b) is data showing the reuse of the recovered catalyst (Ce-FeO-H ₂ O ₂ ) and its effect on lignin decomposition.

Hereinafter, with reference to the accompanying drawings, a lignocellulosic biomass pretreatment method using nanoparticles will be described in detail so that those skilled in the art can easily practice the present invention.

In describing the principles of preferred embodiments of the present invention in detail, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

In addition, since the embodiments described in this specification and the configurations shown in the drawings are only one of the most preferred embodiments of the present invention and do not represent all of the technical ideas of the present invention, various alternatives may be used at the time of this application. It should be understood that there may be equivalents and variations.

The present invention is a catalyst for pre-treating lignocellulose, and includes at least one of cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP).

The iron oxide nanoparticles (CeONP) have laccase mimicking properties, and the cerium-doped iron oxide nanoparticles (Ce-FeONP) have laccase or peroxidase mimicking properties, respectively, without addition of hydrogen peroxide at room temperature and normal pressure or has a catalytic activity to decompose lignin from lignocellulosic biomass under addition conditions.

The size of the cerium oxide nanoparticles (CeONP) may range from 30 to 60 nm, and may take various shapes, but is preferably spherical.

In addition, the present invention is a method for preparing the cerium oxide nanoparticles (CeONP), comprising the steps of (a) adding chitosan to an acetic acid solution to prepare a chitosan-acetic acid solution; (b) mixing a Ce precursor with the chitosan-acetic acid solution to prepare a Ce precursor mixed solution; (c) mixing and reacting hydrogen peroxide (H ₂ O ₂ ) with the Ce precursor mixture; (d) separating cerium oxide nanoparticles (CeONP) from the reaction product and washing/drying;

The Ce precursor is cerium acetate hydrate, cerium acetylacetonate hydrate, cerium bromide, cerium carbonate hydrate, cerium chloride, cerium chloride hepta Hydrate (cerium chloride heptahydrate), cerium 2-ethylhexanoate, cerium fluoride, cerium fluoride, cerium hydroxide, cerium iodide (cerium iodide), cerium nitrate hexahydrate, cerium oxalate hydrate, cerium sulfate, cerium sulfate hydrate and cerium sulfate Any one selected from the group is used.

The cerium-doped iron oxide nanoparticles (Ce-FeONP) are obtained by doping/mixing iron oxide with cerium, wherein the iron oxide is at least one selected from FeO, Fe ₂ O ₃ , and Fe ₃ O ₄ , and the content of cerium is 1 to 10. It may be 10 wt%, preferably 5 wt%.

The cerium nanoparticles (CeONP) and the cerium-doped iron oxide nanoparticles (Ce-FeONP) may be used in the form of nanoparticles without a support, or the nanoparticles may be used in the form of being supported on a support.

As the support, commonly used supports may be used without limitation, but are not limited thereto, but are exemplified by alumina, ceria, zirconia, silica, titania, magnesia, activated carbon, graphite, carbon nanotubes, graphene, fullerene, Graphene oxide, zeolite, metal organic framework (MOF), spinel, perovskite, hydrotalcite, tantalum oxide, molybdenum oxide, tungsten oxide, diatomaceous earth, bauxite, clay, magnesium silicate, silicon carbide, ceramic, It may be at least one selected from carborundum, quartz, thoria, chromite, rutile, ilmenite zircon, and the like. In addition, the shape of the support may be particles, extrudates, monoliths, fibers, meshes, and nets.

The size of the cerium-doped iron oxide nanoparticles (Ce-FeONP) may range from 6 to 13 nm, and may take various shapes, but is preferably spherical.

In addition, the present invention is a method for producing the cerium-doped iron oxide particles (Ce-FeONP), comprising the steps of (a) preparing a Fe precursor solution by dissolving the Fe precursor in distilled water; (b) preparing an ammonia mixture solution by adding an ammonia solution to the Fe precursor solution; (c) mixing and aging a Ce precursor solution with the ammonia mixture solution; (d) separating cerium-doped iron oxide nanoparticles (Ce-FeONP) from the aged solution and washing/drying;

Step (a) is a step of preparing an Fe precursor solution, and the Fe precursor includes iron (II) chloride FeCl ₂ , iron (III) chloride (III) FeCl ₃ , ferric acetate, iron cupferronate, iron At least one selected from iron pentacarbonyl and the like is used.

The step (b) is a step of adding and mixing an ammonia solution to the Fe precursor solution, and the amount of ammonia to be added is 50 ml of 28% ammonia water (density 0.9) based on 50 to 150 ml of the Fe precursor solution. .

The temperature at the time of mixing the aqueous ammonia solution in step (b) may be in the range of 20 to 90 °C. Preferably it is 60 degreeC. When the temperature is less than 20 ° C, nucleation is low and incomplete growth of nanoparticles may occur, and when the temperature exceeds 90 ° C, ammonia water may volatilize.

In addition, after mixing the aqueous ammonia solution, stirring is continued for 1 to 2 hours. If the time is less than 1 hour, particle generation may not be sufficient, and if the time exceeds 2 hours, there may be a problem in that the manufacturing time is prolonged.

The step (c) is a step of adding the Ce precursor to the aqueous ammonia solution in the step (b) and aging it. In this case, as the Ce precursor, at least one selected from cerium nitrate and cerium hydrochloride may be used. The addition of the Ce precursor may be added in the form of a powder of the Ce precursor, or may be added in a form dissolved in an aqueous solution in advance. The addition amount of the Ce precursor is added so that the mass converted to Ce is 1 to 10 wt% based on the mass of Fe present in the solution. Preferably it is added so that it becomes 5 wt%. At this time, the temperature of the aqueous ammonia solution may be in the range of 20 to 90 ° C, and preferably maintains the same temperature as in step (b).

Aging in step (c) may be for 12 to 36 hours. If the time is less than 12 hours, particle generation may not be sufficient, and if the time exceeds 36 hours, there may be a problem as long as the manufacturing time becomes long.

The aging is generally performed in the presence of an inert gas or air, preferably in an air atmosphere.

Step (d) is a step of separating, washing, and drying the generated cerium-doped iron oxide nanoparticles (Ce-FeONP).

The separation may be performed using a general separation method such as separation membrane, filtration after precipitation, centrifugation, or separation using a magnet, and centrifugation is preferable.

Washing is to remove excess ammonia and unreacted Fe and Ce precursors, and can be washed using water, alcohol, or a mixture thereof, until excess ammonia and unreacted Fe, Ce precursors are gone. It can be washed repeatedly several times. It is common to perform washing at room temperature.

Drying is sufficiently dried at -80 to 30 ° C. The drying may be performed under normal pressure or reduced pressure, and may be preferably freeze-dried.

In addition, as shown in the schematic diagram of FIG. 1, the present invention provides a method for pre-treating lignocellulosic biomass using the cerium-doped iron oxide nanoparticles (Ce-FeONP). In FIG. 1, corn cob was used as a feedstock for lignocellulosic biomass, but the type of lignocellulosic is not limited thereto.

The method of the present invention mixes and reacts cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP) with or without the addition of hydrogen peroxide (H ₂ O ₂ ) to lignocellulosic biomass, It is characterized by decomposing lignin in lignocellulosic biomass.

At this time, the added hydrogen peroxide may be in the form of an aqueous solution, and the concentration of hydrogen peroxide is preferably 1 M or less. When hydrogen peroxide is greater than 1 M, there may be a problem that the lignin removal efficiency does not significantly increase.

In addition, the amount of the biomass in the solution, may be 5 to 20% (w / v). If the amount of biomass is less than 5% (w / v), the concentration of sugars produced may be too low, and if it is more than 20% (w / v), the viscosity of the reaction mixture may be too high.

In the lignocellulosic biomass pretreatment method according to the present invention, the content of the nanoparticles (Ce-FeONP) may be 1 to 10 g/L in terms of the weight of the nanoparticles. When the amount of the nanoparticles is less than 1 g/L, the lignin removal efficiency may not be sufficient, and when the amount exceeds 10 g/L, the lignin removal efficiency may not increase any more.

In addition, in the lignocellulosic biomass pretreatment method according to the present invention, the temperature range of the pretreatment reaction may be 20 to 90 ° C, the reaction pressure may be 1 to 5 bar, and the reaction time may be 1 to 48 hours.

In addition, in the lignocellulosic biomass pretreatment method according to the present invention, the load of biomass is preferably 5 to 30% (w / v).

Hereinafter, the excellence of the present invention will be described through Examples and Comparative Examples.

[Example]

Microbial strains, culture media and enzyme production

A fungal strain of Fusarium verticillioides ( GenBank accession number PRJNA664836) isolated from corn cob (CC) biomass was used for cellulase production.

Fungal strains were propagated on PDA gradient medium at 28 °C. For inoculation, 2 ml of spore solution containing 1.8 × 10 ⁶ spores was used in 50 ml culture medium. RSM optimized basal medium (RSM-BM) was used for cellulase enzyme production. The composition of RSM-BM medium is as follows (g/L). CaCl ₂ 0.15; KH ₂ PO ₄ 1.0; (NH ₄ ) ₂ SO ₄ 0.50; factor 0.60; peptone 0.45; yeast extract 0.15; MgSO _4. 7H ₂ O 0.15; FeSO ₄ .7H ₂ O 0.0075; MnSO ₄ .7H ₂ O 0.0024; ZnSO ₄ .7H ₂ O 0.0021; CoCl ₂ 0.003; Tween 80 1.0 ml.

Cellulase production was performed under liquid fermentation conditions using Fusarium verticillioides fungal strains. Liquid fermentation was carried out in a 250 ml Erlenmeyer flask containing 70 ml of fermentation medium with cellulose 1% (w/v) and corncob 2.5% (w/v) substrates. The flask was inoculated with spores (approximately 10 ⁷ ) derived from a 15-day-old culture grown on a PDA slope and incubated at 28 °C with agitation at 150 rpm. Samples were taken every 2 days and centrifuged at 7,500 rpm for 10 minutes to collect the supernatant. All supernatant samples were assayed for extracellular cellulase activity and protein content.

Synthesis of Cerium Oxide Nanoparticles (CeONP)

Chitosan (0.5 g) was added to a 3% (w/v) acetic acid solution and completely dissolved. Then, 1 g of cerium nitrate hexahydrate was mixed with the chitosan-acetic acid solution for 1 hour. A stoichiometric amount of hydrogen peroxide was added dropwise to the solution and mixed thoroughly until the color changed from colorless to orange. Thereafter, the final solution was centrifuged at 15000 rpm for 30 minutes and washed three times with water and ethanol. Finally, the recovered nanoparticles were lyophilized and stored for further use.

Synthesis of iron oxide nanoparticles (FeONP)

Ammonia was added to distilled water along with 1M FeCl ₂ and 2M FeCl ₃ in a ratio of 1:1 (v/v), followed by stirring at 60 °C overnight. The formed nanoparticles were washed with distilled water and lyophilized.

Synthesis of cerium-doped iron oxide nanoparticles (Ce-FeONP)

FeCl ₂ and FeCl ₃ were taken in distilled water at a molar ratio of 1:2 and mixed with an ammonia solution (1:1, v/v) at 60 °C for 1 hour. Then, 5 wt% of cerium nitrate hexahydrate was added to the solution and mixed overnight. The formed nanoparticles were centrifuged at 15000 rpm for 30 minutes and washed three times with water and ethanol. Finally, the recovered nanoparticles were lyophilized and stored for further use.

[Test Example 1]

Characterization and results of synthesized cerium-doped iron oxide nanoparticles (Ce-FeONP)

The synthesis of Ce-FeONP was confirmed by characterization with EDS, SEM, TEM, etc. 2 shows (a) EDS, (b) SEM, and (c) TEM results of cerium-doped iron oxide nanoparticles (Ce-FeONP) synthesized according to an embodiment of the present invention.

As shown therein, the cerium-doped iron oxide nanoparticles (Ce-FeONP) according to the present invention have a structure in which iron oxide particles are doped with nano-cerium particles.

[Test Example 2]

Measurement of laccase and peroxidase mimetic activity of nanoparticles

The laccase and peroxidase mimicking activities of the prepared nanoparticles were confirmed by the following method. 330 μL acetate buffer (0.1 M, pH 4.16), 300 μL 3,3′,5,5′-tetramethylbenzidine (TMB) (6 mM), 100 μL hydrogen peroxide (H ₂ O ₂ ) (0.75 M) and 50 μL nano The particle solution (4 mg/mL) was added to the 24-well plate, mixed for 30 seconds, and stirred at room temperature for 30 minutes. Then, the absorbance of the mixture was measured at 652 nm. In this way, the absorbance of TMB+H ₂ O ₂ , TMB+CeONP, TMB+FeONP, and TMB+Ce-FeONP was also measured, respectively. Lacase mimetic activity is the activity shown without the addition of hydrogen peroxide, and peroxidase mimetic activity is the activity shown when hydrogen peroxide is added.

Measurement of laccase and peroxidase mimetic activity of nanoparticles

The laccase and peroxidase mimicking activities of the synthesized nanoparticles are shown in FIG. 3 . The absorbance peak at 652 nm and color change (from colorless to blue) can indicate laccase and peroxidase mimetic activity (presence of oxidized TMB). Almost no color change of TMB was observed in the absence of a catalyst, indicating that only H ₂ O ₂ did not oxidize TMB. When H ₂ O ₂ was not added, CeONP turned blue in the presence of TMB, indicating that it had laccase mimetic activity, but FeONP and Ce-FeONP showed a low change. The addition of H ₂ O ₂ changed the color of the solution from colorless to blue, indicating that the synthesized iron oxide nanoparticles have peroxidase mimetic activity. Ce-FeONP showed higher peroxidase activity than FeONP. The combination of the two catalytic metals appears to enhance hydroxyl radical generation as in the Fenton reaction. These laccase and peroxidase mimicking activities of the synthesized nanoparticles were utilized for lignin degradation.

[Test Example 3]

Biochemical analysis before and after biomass pretreatment using nanoparticles (Ce-FeONP)

The ash and moisture contents of the raw biomass were determined through the AOAC protocol. The lignin content of the biomass was evaluated through iodine measurement. 0.1 N potassium permanganate (7.5 mL) was used to oxidize the lignin present in the biomass in an acidic environment (4 N sulfuric acid, 7.5 mL) at room temperature. After stirring at room temperature for 10 min, potassium iodide (1 N, 1.5 mL) was added to the reaction mixture to form free iodine. The formed free iodine was titrated with 0.1 N sodium thiosulfate solution using 0.2% starch as an indicator. A blank titration was performed without biomass using equal volumes of water and reagents. The lignin content of the biomass was determined by equations (1) and (2).

Kappa number = (P × f) / W (One)

where P represents the mL of 0.1 N potassium permanganate solution consumed by the sample, f represents the correction factor for 50% permanganate consumption (f = 1), and W represents the weight of the dry sample (0.05 g).

Lignin content (%) = (Kappa number × 0.155) × 100 (2)

Quantification of cellulose was performed according to the semi-micro measurement method, and hemicellulose was quantified by the Antron method.

Biochemical analysis results before and after biomass pretreatment using nanoparticles (Ce-FeONP)

Table 1 shows the composition of lignin, cellulose, and hemicellulose in biomass before and after pretreatment. For reference, the corncob biomass used in the present invention contained a large amount of cellulose (38.69%, w/w) and hemicellulose (32.14%, w/w). The lignin content of the biomass was 12.40%, w/w. The lignin content was 14.28%, w/w, as determined by the NREL method. Moisture (6.9%, w/w) and ash content (10%, w/w) were also obtained.

The lignin content of biomass decreased significantly (1.78 times) after pretreatment with nanoparticles, whereas the hemicellulose content slightly decreased. Cellulose content was 1.07 times higher than before pretreatment. That is, the nanoparticles (Ce-FeONP) indicate that they generally act to decompose only lignin without significantly affecting cellulose and hemicellulose in biomass. For reference, the fact that the reducing sugar concentration of the pre-treatment liquid in Table 1 below is 23.71 g/L seems to be due to the small amount of decomposition of hemicellulose during the pre-treatment process.

ingredient before pretreatment after pretreatment Lignin (%, w/w) 12.40 6.94 Cellulose (%, w/w) 38.69 41.47 Hemicellulose (%, w/w) 32.14 28.20 Reducing sugar (g/L) - 23.71

[Test Example 4]

Determination of lignin degradation for various process parameters

Three types of nanoparticles (FeONP, CeONP and Ce-FeONP) were used to study the effect on lignin degradation of corncob biomass. The initial process parameters are: Pretreatment time (1 to 30 h), nanoparticle concentration (1 to 10 g/L), solid loading (5 to 30%, w/v) and H ₂ O ₂ concentration (0.1 to 1 M). A control experiment using only H ₂ O ₂ was also performed (FIG. 4). For reference, the test conditions in FIG. 4 are as follows. (a) Effect of pretreatment time (H ₂ O ₂ concentration: 0.1 M, solid loading: 15% (w/v), Ce-FeONP concentration: 2 g/L), (b) Effect of H ₂ O ₂ concentration ( Pretreatment time: 24 hours, solid loading: 15% (w/v), Ce-FeONP concentration: 2 g/L), (c) Effect of solid loading (pretreatment time: 24 hours, H ₂ O ₂ concentration: 0.75 M , Ce-FeONP concentration: 2 g/L), (d) Effect of nanoparticle (NP) concentration (pretreatment time: 24 hours, H ₂ O ₂ concentration: 0.75 M, solid loading: 20% (w/v)

The method for measuring the percentage of lignin degradation is as follows. First, lignin degradation (pretreatment) of corncobs was performed in an Erlenmeyer conical flask (50 mL) containing nanoparticles and lignocellulosic biomass. Before adding the biomass, the nanoparticles were sonicated in distilled water to obtain a homogeneous solution. After adding biomass and H ₂ O ₂ , the mixture was stirred at 25 °C and 200 rpm. Samples were taken at fixed time intervals and samples were recovered after solid-liquid separation. After solid-liquid separation, the solid biomass was dried overnight at 60 °C. The dried biomass was further used for estimation of residual lignin through iodine measurement. The percentage of lignin degradation in the pretreated biomass was calculated according to equation (3).

% delignification = (initial lignin - final lignin)/(initial lignin) × 100 (3)

Lignin degradation measurement results for various process parameters

Measurement results of lignin degradation by pretreatment time (1 to 30 hours), nanoparticle concentration (1 to 10 g/L), solid load (5 to 30%, w/v), and H ₂ O ₂ concentration (0.1 to 1 M) is shown in Figure 4.

As shown, the optimal lignin decomposition conditions were 24 hours of pretreatment, H ₂ O ₂ concentration of 0.75 M, solid loading of 20% (w/v), and nanoparticle concentration of 4 g/L. As a result of performing a control experiment using only H ₂ O ₂ without nanoparticles under optimal conditions, no lignin degradation was observed, indicating the role of nanoparticles in the process. Under optimal conditions, the lignin degradation efficiency was 44%. It is known that a lignin degradation efficiency of 20 to 65% is sufficient to improve the release of fermentable sugars from pretreated biomass.

Meanwhile, lignin decomposition experiments were performed using cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP) in the absence of H ₂ O ₂ conditions. Pretreatment time: 24 hours, nanoparticle concentration: 2 g/L, solid loading: 20.8 ± 2.01% for cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce -FeONP) showed 15.3 ± 2.14% of the lignin degradation efficiency in both nanoparticles without H ₂ O ₂ addition. This is thought to be due to the laccase mimetic activity of cerium oxide nanoparticles.

For reference, it was observed that the phenol content representing lignin degradation gradually increased during the lignin degradation process using corncob biomass nanoparticles as the pretreatment time increased (Table 2).

Preprocessing time (h) Phenol content (mg/mL) One 0.095 6 0.278 12 0.285 18 0.301 24 0.412 30 0.420

[Test Example 5]

Enzymatic hydrolysis of biomass after pretreatment and measurement of reducing sugar concentration

Hydrolysis of Ce-FeONP-treated corncob biomass and untreated corncob biomass (control) samples (50 g/L) was carried out in 50 ml citrate buffer (50 mM, pH 5.5) and two concentrations of enzyme (10 FPU/g substrate, 20 FPU/g substrate) in 250 ml flasks. The hydrolysis reaction was carried out at 50 °C with stirring at 200 rpm. Samples were taken at regular time intervals and analyzed to determine glucose, xylose and total reducing sugar concentrations after hydrolysis up to 48 hours.

The concentrations of glucose and xylose released after hydrolysis were measured using high-performance liquid chromatography (HPLC, YL 9100, Young-Lin, Inc., Korea). Samples were centrifuged and filtered through a 0.2 μm filter. Each sample was injected onto a Bio-Rad Aminex hpx-87h column at 65 °C using a refractive index detector and eluted from the column with a mobile phase of 5 mM H ₂ SO ₄ at a flow rate of 0.6 ml/min. Total reducing sugar concentration was analyzed using the DNS method.

Enzymatic hydrolysis of biomass after pretreatment and measurement of reducing sugar concentration

Cellulase and hemicellulase enzyme preparations from isolated fungal strains were used to evaluate saccharification of 50 g/L of pretreated corncob biomass and untreated samples (control). For both the 10 and 20 FPU enzyme (per g substrate) concentrations used for hydrolysis, optimal hydrolysis was obtained after 24 h of saccharification at 50 °C. Hydrolysis of untreated corncob biomass showed negligible sugar release.

As shown in Figure 5 (a), when 10 FPU enzyme/g substrate was used, 12.5 g/L glucose and 6.99 g/L xylose were produced after 24 hours of saccharification. When pretreated corn cob biomass was hydrolyzed with 20 FPU enzyme/g substrate, it released up to 18.1 g/L glucose and 9.12 g/L xylose after 24 h (Fig. 5(b)). The resulting reducing sugars were 34.5 g/L for 10 FPU enzyme/g substrate and 46.6 g/L for 20 FPU enzyme/g substrate (Fig. 5(a) and 5(b)).

[Test Example 6]

Identification of lignin degradation intermediates using GC-MS

GC-MS was used to analyze low molecular weight lignin-degrading compounds produced during the pretreatment process (Table 3).

As a result of lignin degradation by Ce-FeONP, various compounds such as 4-methylquinoline (RT 5.72), 2(3H)-furanone (RT 9.186), xylitol (RT 13.53), etc. A low molecular weight compound was identified.

These aromatic compounds identified after decomposition of lignin are related to the oxidation of sinapyl, coniferyl, and p -coumaroyl alcohol, which are considered the basic units constituting the lignin molecule. In addition to aromatic alcohols in their various forms, cinnamic acid (RT 8.442), propanoic acid (RT 4.695), benzoic acid (RT 7.993), maleic acid (RT 10.690) and succinic acid (RT 16.46) ) was also confirmed. In addition, among aldehydes, benzaldehyde (RT 16.837) and 3-bromo-5-ethoxy-4-hydroxybenzaldehyde (RT 21.470) were also identified.

ingredient 0 h ^1st h 3 ^rd h ^5th h ^6th h ^7th h Retention time
(min) Relative composition by area (%) Propanoic acid - + + + + + 4.695 0.41 Lepidine (4-methylquinolin) - - - + - - 5.720 0.24 Butanoic acid - + + + + + 5.860 1.18 Acetic acid - + + + + + 5.974 0.07 4-Methylesculetin - + + + + + 7.592 0.25 Benzoic acid - + + + + + 7.993 0.10 Cinnamic acid - - - + + + 8.442 0.07 2(3H)-Furanone - + + + + + 9.186 0.04 Malic acid - + + + + + 10.690 1.76 Butane - + + + + + 11.00 1.18 D-Glucitol - + + + + + 12.97 0.14 Xylitol - + + + + + 13.53 3.03 β-D-Galactofuranose - + + + + + 14.84 3.07 D-Xylose - + + + + + 15.34 9.64 D-Glucose - + + + + + 16.22 9.33 Succinic acid - + + + + + 16.46 0.66 Benzaldehyde - + - - - - 16.837 0.08 Octadecanoic acid - + + + + + 18.283 1.17 3-Bromo-5-ethoxy-4-hydroxybenzaldehyde - + + + + + 21.470 0.88 Bis(trimethylsily)estradiol - + - - - - 21.800 0.14 Scopolin - + - - - - 21.900 3.68 D-Arabinose - + + + + + 22.545 0.09 D-Ribose - + + + + + 22.597 0.82

Due to its polyphenol aromatic structure and high energy density, lignin is considered a high value-added renewable raw material that can be used in biorefineries for the production of fine chemicals, fuels and functional materials. Decomposition into low molecular weight compounds such as aromatic alcohols, acidic compounds and aldehydes has high commercial prospects due to applications in the perfume industry. In addition, 1,2-benzenedicarboxylic acid, 2-butenedioic acid, and stearic acid (octadecanoic acid) are major components of fuel diesel (fatty acid methyl ester) produced from various raw materials. act as a component.

In addition, the lignin remaining in the pretreated biomass can be used for liquid and gaseous fuel production. Remaining lignin can be extracted from the pretreated biomass with an appropriate solvent. The extracted lignin can be further used to produce syngas through gasification, which in turn can produce methanol and other chemicals. In addition, bio-oil can be produced by pyrolysis of solvent-treated lignin at high temperature and high pressure. Most of the extracted lignin can be used as low-grade boiler fuel to transfer heat and power.

[Test Example 7]

Measurement of changes in surface area, pore volume and pore size before and after pretreatment

The surface area, pore size and pore volume of corncob biomass before and after pretreatment with nanoparticles were estimated by BET and BJH methods and are shown in Table 4.

Surface area, pore volume and pore size change measurement results before and after pretreatment

The pore size of the pretreated biomass increased from 18.97 nm before pretreatment to 30.90 nm. In general, small pores are not easily accessible to hydrolytic enzymes, whereas enzyme diffusion through large pores greatly increases the rate of enzymatic hydrolysis of cellulose and hemicellulose. The pretreated substrate recorded an increase in surface area (1.48 m ² /g) compared to untreated biomass (0.59 m ² /g). The total pore volume of the pretreated biomass increased from 0.0028 mL/g before pretreatment to 0.0049 mL/g. An increase in biomass porosity in terms of surface area, pore size and pore volume can further improve the accessibility of hydrolases for production of fermentable sugars.

Surface area (m²/g) Pore volume (mL/g) Pore size (nm) before pretreatment 0.59 0.0028 18.97 after pretreatment 1.48 0.0049 30.90

[Test Example 8]

Nanoparticle Recovery and Reuse

Besides the peroxidase-mimicking activity of Ce-FeONPs to degrade lignin in biomass, the magnetic properties of the nanoparticles have another advantage, such that they are easily separated from the reaction mixture.

The recovery and reuse of Ce-FeONPs were investigated through three sequential runs with the recovered nanoparticles. 6 (a) is an image of biomass after nanoparticle treatment and washing, and (b) is data showing the reuse of the recovered catalyst (Ce-FeO-H ₂ O ₂ ) and its effect on lignin decomposition.

It can be seen that the nanoparticles synthesized in (a) of FIG. 6 are attached to the surface of the biomass and can be easily recovered through washing after sonication. Also, as shown in (b) of Fig. 6, the lignin degradation rates of corncobs pretreated with recovered Ce-FeONP-H ₂ O ₂ were 37.1% in the second run and 30.8% in the third run. The recovery of nanoparticles was about 50% of the initial concentration.

As described above, the present invention developed a new eco-friendly biomass pretreatment method by utilizing the enzyme-mimicking properties of cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP).

Although the present invention has been described above with reference to the accompanying drawings, this is merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the technical protection scope of the present invention should be determined by the claims below.

Claims (8)

As a catalyst for decomposing lignin in lignocellulosic biomass,
The catalyst is a catalyst for pretreatment of lignocellulosic biomass, characterized in that it comprises at least one or more of cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP). According to claim 1,
The cerium-doped nanoparticles,
The content of cerium is characterized in that the range of 1 to 10 wt%, lignocellulosic biomass pretreatment catalyst. According to claim 1,
The nanoparticles are nanoparticles not supported on a support, or a catalyst for pretreatment of lignocellulosic biomass, characterized in that the nanoparticles are supported on the support. By mixing and reacting at least one of cerium oxide nanoparticles (CeONP) and cerium-doped iron oxide nanoparticles (Ce-FeONP) in lignocellulosic biomass with or without the addition of hydrogen peroxide (H ₂ O ₂ ), lignocellulosic A method for pre-treating lignocellulosic biomass using nanoparticles, characterized in that lignin in biomass is subjected to a decomposition reaction. According to claim 4,
The decomposition reaction is characterized in that carried out at 20 to 90 ℃, 1 to 5 bar, lignocellulosic biomass pretreatment method using nanoparticles. According to claim 4,
The hydrogen peroxide (H ₂ O ₂ ) concentration is 1 M or less, and the concentration of the nanoparticles is 1 to 10 g / L, characterized in that, lignocellulosic biomass pretreatment method using nanoparticles. According to claim 4,
Characterized in that the loading of biomass is 5 to 30% (w / v), lignocellulosic biomass pretreatment method using nanoparticles. Lignocellulosic biomass using nanoparticles, characterized in that fermentable sugars including glucose and xylose are produced by enzymatic hydrolysis of the lignocellulosic biomass pretreated by the method of any one of claims 4 to 7. A method for producing fermentable sugars from cellulosic biomass.

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