WO2013032212A2 - RabG3bCA를 이용한 형질전환된 나무 및 이의 용도 - Google Patents
RabG3bCA를 이용한 형질전환된 나무 및 이의 용도 Download PDFInfo
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Definitions
- the present invention relates to a tree transformed with RabG3bCA and its use.
- Carbon-based plants on Earth are reported to be ten times the world's oil consumption and five times the energy consumption of all kinds.
- the availability of global biomass crops is in the order of rice straw, wheat straw, corn stover, bagasse, etc.
- the total amount is 1549 trillion kg.
- the raw materials of alternative energy bioethanol which is the biggest issue at present, are largely divided into sugar-based (sugar cane, sugar beet, etc.), starch-based (corn, potato, sweet potato, etc.), and wood-based (thinning, waste wood, rice straw, etc.).
- bioethanol production technology uses both saccharide and starch biomass, which are food resources, it is closely related to the food supply and demand situation of civilization.
- second-generation biomass which is cheaper and less supply-demanded
- Representative wood-based biomass crops include poplar and eucalyptus, and these trees are attracting attention as important raw materials for bioethanol production and various products such as paper, pulp and fiber. Because of this economic importance, researches have been conducted to increase the quantity and quality of wood, and therefore, the study of the synthesis pathway and the synthesis control mechanism of wood is very important.
- the wood layer of the tree is composed of secondary xylems and is mainly composed of vessel elements and fibers.
- Wood, or secondary water ducts are made in the vascular cambium of the inner seed plants, such as poplar and eucalyptus, and are completed through the development of secondary cell wall formation and programmed cell death.
- poplar has been an important crop for research as a model tree for research purposes and as a biomass tree.
- various molecular biological databases and systems such as deciphering Poplar's genome sequence, EST, accumulation of microarray data, developing molecular markers, and establishing transformation technologies, are essential for the activation of Poplar research. It is an important foundation.
- this perennial tree is rich in cellulose and hemicellulose, the major constituents of the sugar conversion, and does not require large chemical inputs, requiring less investment in cultivation [Baucher, M., Halpin, C., Petit- Conil, M., Boerjan, W. 2003. Lignin: genetic engineering and 166 impact on pulping. Crit Rev Biochem Mol Biol , 38 (4), 305-50.].
- Lignin modification improves fermentable sugar yields for 168 biofuel production.
- the present inventors are conducting a study on the catheter differentiation process, which is a representative cell layer of the water pipe.
- RabG3b a small GTP-binding protein found through the analysis of secretory proteins in Arabidopsis.
- the Arabidopsis RabG3b is involved in autophagy and catheter differentiation, and autophagy may be involved in catheter differentiation. I thought about the possibilities.
- Various molecular biological and cytological studies have found that RabG3b is involved in catheter differentiation of plants, ie, water tube development, which is a mechanism through which RabG3b regulates autophagy.
- autophagy was found to be an important process for catheter cell differentiation.
- the RabG3bCA-transformed tree of the present invention is expected to be very useful industrially because it is possible to produce paper, pulp, and bioethanol due to an increase in cellulose and / or glucose content due to an increase in fiber cell length compared to wild-type trees.
- an object of the present invention to provide a tree in which an active mutant (RabG3bCA) gene of Arabidopsis-derived RabG3b is transformed.
- Another object of the present invention is to provide a paper based on a tree transformed with RabG3bCA and a pulp produced from the transformed tree.
- Another object of the present invention is to provide a biomass produced from trees transformed with RabG3bCA.
- Another object of the present invention is a method for producing biomass using wood
- the tree is to provide a method for producing a biomass is a tree transformed RabG3bCA.
- the present invention provides a tree transformed with an active mutant of RabG3b (RabG3bCA).
- the present invention also provides papers based on trees transformed with RabG3bCA and pulp produced from the transformed trees.
- the present invention also provides a biomass produced from trees transformed with RabG3bCA.
- the present invention also provides a method for producing biomass using wood
- the tree provides a method for producing biomass, which is a tree transformed with RabG3bCA.
- the cellulose and / or glucose content as a raw material of the bioethanol was increased by 10% while the hemicellulose content which hindered cellulose degradation was decreased by 25%, so that the trees transformed with RabG3bCA according to the present invention were industrialized. It can be used as a valuable biomass / bioenergy tree.
- Figure 2 shows the morphological comparison between RabG3bCA transformed poplar tree (OX) and wild type tree (WT) [(a) Transgenic poplar tree (OX) and wild type tree (WT) grown in soil for 10 weeks. Comparison pictures between; (b) stem length; (c) stem diameter; Stem growth of transformed poplar trees (OX) and wild type trees (WT) was measured for 10 weeks at weekly intervals (b, c); (d) comparison of tree rings numbers of transformed poplar trees (OX) and wild type trees (WT) grown for 10 weeks; (e) comparison of the weight of transformed poplar trees (OX) and wild type trees (WT) 10 weeks prior to drying; (f) comparison of the weight of transformed poplar trees (OX) and wild type trees (WT) 10 weeks after drying; 5 repetitions of mean ⁇ standard deviation, t-test; * P ⁇ 0.05 ** P ⁇ 0.01]
- FIG. 5 shows changes in related elements and fibroblasts in the poplar tree (OX) stems transformed with wild type tree (WT) and RabG3bCA.
- Figure 6 compares the change in cell size of the woodwind in RabG3bCA transformed wood (OX) [(a) fibroblast length; (b) fibroblast diameter; (c) associated element length; (d) associated element diameter; Obtained by separating cells by taking 10-week-old WT and lower and middle stems of OX-8 stem from soil; values are mean ⁇ standard deviation of 25 cells measured; t-test; * P ⁇ 0.05 ** P ⁇ 0.01].
- Figure 9 shows the expression analysis of genes associated with the development of woodwinds in the trunks of young poplar trees [(a) Comparative analysis of genes involved in the formation of secondary cell walls; (b) comparative analysis of genes associated with cell death; (c) Comparative analysis of genes related to autophagy, the results of which were repeated three times and expressed as mean ⁇ standard deviation.
- Figure 10 shows the glucose yield of three poplar trees (WT, OX-3, OX-8) before and after pretreatment with catalysts of sulfuric acid, sodium hydroxide and aqueous ammonia, respectively.
- the present invention relates to trees to which the active mutant of RabG3b (RabG3bCA) gene has been transformed.
- the tree may include any tree made of paper pulp, including softwoods such as poplar, eucalyptus, birch, beech, aspen and oak, and conifers such as pine, fir and larch.
- RabG3b is derived from Arabidopsis , but RabG3b genes or similar genes from other species of interest may also be used.
- the RabG3bCA gene is represented by the nucleotide sequence of SEQ ID NO: 1.
- the transformed tree according to the invention is characterized in that the cellulose and / or Glucose content was increased. In addition, fibroblast length was increased. This is a result of the increased trait of the water canal.
- the present invention may include a pulp produced from the transformed tree or a paper based on the transformed tree.
- the present invention may include biomass produced from the transformed tree.
- the present invention may include a method for producing biomass using the transformed tree. It is carried out in the same way as the general biomass production method using wood.
- the biomass includes all the biomass produced from the wood-based system, and may specifically include sugars such as cellulose and glucose.
- the transformed tree may be pretreated with sodium hydroxide and then hydrolyzed with an enzyme to produce biomass.
- the present invention may include a method for producing pulp using the transformed tree. It is carried out in the same way as the pulp production method using general wood.
- the present invention overexpresses the active mutant (RabG3bCA) gene of RabG3b, thereby causing cellulose and / or Methods of making transformed trees with increased glucose.
- Poplar trees have environmental and economic value and are also one of the important trees in the timber industry [Kaneda et al. (2010) Journal of Integrative Plant Biology 52, 234-243].
- the most important biological resource in wood is the wood or secondary woodwind part, which is used for the production of recyclable paper, pulp and biofuels [Demura et al. (2010) Current Opinion in Plant Biology 13, 299-304.
- Recent genetic and genetic studies have revealed some important key genes in tree production and broadened our understanding of tree production.
- RabG3bCA has not been described in the development process of woodwind at Arabidopsis.
- Cellulose is an important secondary cell wall component used in biofuel production and is broken down by cellulose degrading enzymes to be glycosylated and converted to glucose [Abramson et al. (2010) Plant Science 178, 61-72. Lignin and hemicellulose act as physical barriers to the enzymatic degradation of cellulose, and geneticists and chemists are developing methods to reduce and further degrade the biosynthesis of lignin and xylan [Pilate et al. (2002) Nature Biotechnology 20, 607-612 .; Yang et al. (2004) Biotechnology & Bioengineering 86, 88-95 .; Weng et al. (2008) Current Opinion in Biotechnology 19, 166-172.
- the present invention shows that RabG3bCA has been altered in order to improve the quality of trees as biofuels. (10% increase in cellulose and glucose, 25% decrease in xylan but no significant change in lignin content (Table 3))
- the increase in the ratio of cellulose to xylan makes secondary cell wall degradation easier and therefore bio Ethanol production can be further increased [Sticklen M. (2006) Current Opinion in Biotechnology 17, 315-319 .; Pauly et al. (2010) Current Opinion in Plant Biology 13, 305-312.
- Direct movement of the cells of the vesicles to the tip is very important for the nutrient supply to the extended zones and this redistribution process can be controlled by autophagy.
- the length of the fibrous cells is a very important factor, so it is possible to manufacture paper and pulp using wood transformed with RabG3bCA.
- Poplar trees transformed with RabG3bCA showed a glucose yield of at least 20% from 100 g biomass despite the amount of lignin similar to wild-type trees. This may be due in particular to the effect of the RabG3bCA gene, which results in faster growth in OX8, loosening the binding of cellulose with other wood components such as hemicellulose and lignin and subsequently facilitating cellulase access to cellulose with less steric hindrance. Also, considering the total biomass weight, possible glucose production will be significantly expanded since OX8 exhibits a two-fold increase in weight within the same time period (see Figures 2 (e), (f)).
- glucose yield after catalyst pretreatment differed depending on the catalyst. Hemicellulose removal yield was found to be higher in sulfuric acid immersion than in other catalysts, showing the lowest digestibility. This may be due to the reduced accessibility of enzymes to cellulose molecules due to low cellulose recovery and lignin removal. Sodium hydroxide has proven to be the most effective catalyst for poplar in terms of digestibility and glucose yield.
- RabG3bCA The inventors of the study of overexpression of RabG3bCA have led to morphological changes in the development of woodwind in both the Arabidopsis edodes and perennial poplar trees. It has also been found that there is a physiological action leading to apoptosis by RabG3b autophagy and promoting secondary cell wall production during tree formation. Based on the present invention, the continuous increase of RabG3b and autophagy elements can increase the quality of the tree and can be applied to forestry. Moreover, known factors associated with RabG3b have provided new information to the complex network of elements of the woodwind development process and will contribute to a higher physiological understanding of the tree.
- BH1 P. alba x P. tremula var. Glandulosa BH1
- the RabG3bCA structure was previously cloned into the pBI121 plant expression vector (Kwon et al (2009) Journal of Plant Biology 52, 79-87.) And by Agrobacterium-mediated transformation of poplar tree cells. Choi et al (2005) Journal of Plant Biology 48, 351-355 .; Kim YH, Kim et al (2011) Plant Biotechnology Journal 9, 334-347.].
- Transformed cells were callus-induced medium containing 500 mg / L Cefotaxime and 50 mg / L kanamycin (Murashige and Skoog [MS] medium, pH 5.8, 3% sucrose, 0.8% agar, 1 mg / l 2,4). -dichlorophenoxyacetic acid, 0.01 mg / l benzylaminopurine, and 0.1 mg / l 1-naphthylacetic acid [NAA]). The roots were transferred to tree medium and regenerated. During the transformation process, the medium was maintained at 25 ° C. in the culture chamber, exposed to white light (30 ⁇ mol / m 2 ⁇ s) for 16 hours and left under dark conditions for 8 hours.
- Rooted plants had an acclimatization period in pollen and were transferred to greenhouse soils for further study. Plants were cultured and tested in a greenhouse, 28-30 ° C., 12 h light / 12 h dark conditions.
- Plant growth was determined by measuring the length of the stem from the root growth point to the base of the stem, the diameter of the base of the stem, and the number and length of the cuts. To make the initial plant the same length, when the plant grew to less than 20 cm in the soil, the base was cut and measured at the same time at the same time, and the measurement was repeated 3 to 5 times.
- the stems of the poplar tree were fixed in 0.1 M phosphate buffer (pH 7.4) with a solution containing 2.5% glutaraldehyde and 4% p-formaldehyde at 4 ° C. for 4 hours, and 0.1 M phosphate buffer (pH 7.4). Rinsed and fixed at room temperature for 2 hours with 1% OsO 4 . After rinsing with 0.1 M phosphate solution, the samples were dehydrated and filled in LR White Resin (London Resin). Cross-sections (1 ⁇ m) were prepared using Ultramicrotome (RMC MT X) and then stained and observed under a light microscope. After cutting to 60-70 nm and briefly stained with filtered 1% toluidine blue (toluidine blue).
- RMC MT X Ultramicrotome
- LTG LysoTracker Green
- stem sections (1 cm) away from the bottom and middle of wild-type and transformed poplar trees grown in soil for 10 weeks were cut into 2 mm x 1 mm x 2 mm pieces. .
- Pieces of wood were immediately immersed in Schulze's reagent containing 6% KClO 3 in 50% nitric acid and stored at room temperature for 1 week. These samples were then shaken vigorously at 60 ° C. for 30 minutes to separate into individual cells. The called individual cells were washed three times with distilled water, placed on a microscope slide and covered with a cover glass. The length and width of the cells were measured by observing with an optical microscope (Olympus, BX51TRF).
- the stem (2 g) was ground to 40-60 mesh in liquid nitrogen, extracted with 20 ml acetone (99.9%, reagent grade) for 8 hours at room temperature, and Whatman No. It filtered with 2 filter paper. The acetone-extracted sample was then extracted in boiling water for 2 hours, and Whatman No. It filtered with 2 filter paper and air-dried.
- sample (200 mg) from which the unnecessary elements were removed was dissolved in 72% sulfuric acid (1.5 ml) for 2 hours at 20 ° C. It was then diluted in boiling water containing 3% sulfuric acid and the sample solution was boiled for 3 hours. The boiled sample solution was then cooled to room temperature overnight to precipitate Klason lignin and filtered through a 1G4 magnetic crucible filter.
- the monosaccharide composition on the cell wall was estimated based on the interpretation of the 1 H-NMR spectra at the anomeric hydrogen peak integration as described (Shin et al (2008) Cellulose 15, 255-260.).
- Crystallinity analysis of cellulose was performed by X-RAY diffractometer (Bruker D5005) (40 Kv, 40 mA) on samples from which previously unnecessary elements were removed.
- RNAs 1.5 ⁇ g were used for first strand cDNAs synthesis using the Power cDNA Synthesis Kit (iNtRON) according to the manufacturer's protocol.
- Quantitative real-time PCR was performed using a KAPA SYBR FAST qPCR master mix in the Light Cycler 480 system (Roche).
- Tested gene expression levels were normalized to constitutive expression levels of 18S rRNA and calculated using the 2- ⁇ method [Schmittgen et al. (2008) Nature Protocols 3, 1101-1108.
- the experiment was repeated three times with biologically independent samples.
- Proteins were separated on 15% SDS-polyacrylamide gels, transferred to nitrocellulose membranes and then incubated with anti-RabG3b antibody overnight at 4 ° C.
- Antibody binding proteins were detected by using an ECL system (Amersham Biosciences) by incubating with conjugated secondary antibodies.
- Genomic DNA was extracted from poplar leaves by using DNAzol solution (MRC) [Chomczynski et al. (1997) BioTechniques 22, 550-553.], Restriction enzyme Eco RI overnight.
- the cut DNA was then separated on a 0.7% agarose gel and transferred to a Hybond-XL membrane (Amersham Bioscience).
- the blots were hybridized with the [ ⁇ - 32 P] dCTP-labeled RabG3b cDNA probe in the hybridization buffer at 60 ° C. for 24 hours.
- Hybridized blots were washed continuously with 2 ⁇ SSC and 1 ⁇ SSC at 50 ° C. for 30 minutes and exposed to X-ray film.
- the pretreated biomass was filtered using a filtration cloth (22-25 ⁇ m, Calbiochem, La Jolla, Calif.) And a sufficient amount of liquid was obtained until the pH reached 6-7. After washing with water it was dried in an oven for 3 days.
- RabG3b is highly expressed under wood differentiation conditions such as brassinolide / boric acid administration and programmed apoptosis [Kwon et al. (2009) Journal of Plant Biology 52, 79-87 .; Kwon et al. (2010) Plant Journal 64, 151-164.
- RabG3b has been shown to regulate the differentiation of woodwind elements through its own autophagy in the Arabidopsis [Kwon et al. (2010) Plant Journal 64, 151-164.
- Poplar trees transformed with RabG3bCA were prepared through the cauliflower mosaic virus 35S promoter. Fourteen lines were found in gene expression transformed using RT-PCR and western blotting, which showed good agreement in transcription and protein levels (FIG. 1).
- FIG. 2A Stem growth of poplar trees transformed with RabG3bCA was first measured (FIG. 2A).
- Transformed poplar trees P. alba x P. tremula var. Glandulosa ) (OX) grew faster than control (WT) in both medium and soil.
- WT control
- the control group grew 5 cm and the transformed trees grew about 7-8 cm for 5 weeks.
- trees transformed with RabG3bCA grew 13-20% more in stem length and 10-12% thicker than in the control [Fig. 2 (b), (c)].
- Interstitial numbers also increased about 10% in the transformed poplar trees (FIG. 2 (d)).
- the trees transformed with RabG3bCA weighed about twice as much as those of the control group (Fig. 2 (e), (f)).
- the tree grown for 5 weeks in the medium was cut off the lower part of the stem to observe the pattern of the ducts [Fig. 3].
- the overall vascular morphology was similar in the transformed tree or in the control group, the deformation of the wood canal was markedly increased in the transformed tree (Fig. 3 (a)-(c)).
- Fluorescence analysis of lignin showed that lignin formation and secondary cell wall formation were further increased in RabG3bCA-transformed trees compared to the control group (Fig. 3 (d)-(f)).
- the development of the ducts was further observed in trees grown in soil.
- cellulose and lignin Two major structural components, cellulose and lignin, were observed.
- the lignin did not change in the transformed poplar tree compared to the control, but the cellulose increased approximately 10% (OX-8).
- the total polysaccharide content was similar between the control and the transformed poplar trees.
- the amount of glucose, the primary component of cellulose was about 6-11% higher in transformed trees.
- the hemicellulose, xylan and xylose the major component of xylan
- the crystallinity of cellulose did not differ between control and transformed trees.
- FIG. 8 In order to observe whether the result obtained by this stain was applied directly to the poplar tree, the sample obtained in the second section was analyzed by scanning electron microscopy [FIG. 8]. Differentiation of primary woodwind cell lines was observed equally in both wild-type and transformed trees (Figs. 8 (a), (g)). In the woodwind cells of wild-type trees, many parts were filled with intact cell contents and some cells had secondary cell walls deposited (Fig. 8 (b)). This shows that these cells are in the early stages of differentiation. Notably, autophagy structures in the wild-type tree cell line were surrounding and disintegrating organelles and structures (Figs. 8 (c)-(f)).
- the present inventors have identified two groups (monolignol synthetic genes ( 4CL3 , C3H3 , CCoAOMT1 , COMT2 , CAD1 , CAD4 , CAD10 ) and cell carbohydrate synthetic genes ( CesA4 , CesA7 , CesA8 , CesA18 , IRX8 , IRX9 , FRA8 ) that are involved in secondary cell wall formation). ) Expression levels were compared (Fig. 9 (a)). Most cell wall synthesis genes except for a small number (eg CesA4 ) were increased in RabG3bCA transformed trees.
- the inventors also expressed the expression level of two groups of genes that degrade cell contents: genes related to programmed apoptosis ( peroxidase e, VPE , and MC9 ) and autophagy related genes ( ATG8d1 , ATG8f2 , ATG8i ). Analyzes [Courtois-Moreau et al. (2009) Plant Journal 58, 260-274 (FIG. 9 (b), (c)). As with phenotypic features in which cell walls in transformed trees have increased degradation and increased formation of autophagy structures, these degraded genes have been further increased in transformed trees. These results demonstrate that the transgenic phenotype and the degree of differentiation of woodwind in RabG3bCA transfected trees are associated with the expression of these genes.
- Sulfuric acid, sodium hydroxide, and ammonia were used as catalysts for the pretreatment of lignocellulosic.
- Pretreatment conditions for each catalyst were determined based on our previous studies.
- insoluble solids recovery with 60 FPU cellulase in sulfuric acid, sodium hydroxide, and ammonia pretreatment were 49.0%, 58.7%, 67.2%, respectively, and the enzymatic digestibility was 54.4%, 58.7%, 44.9%, respectively. Due to mild delignification at 23.8%, 37.3% and 36.4% levels in each treatment, the digestibility was 1.6-, 1.7- and 1.3-fold higher than untreated WT showing 34.8% digestion, respectively.
- insoluble solids recovery from OX8 treated with sulfuric acid, sodium hydroxide, and ammonia pretreatment were 44.1%, 60.0% and 63.7%, respectively, and the enzymatic digestibility was 61.9%, 69.4% and 61.3%, respectively.
- 1.4-, 1.6- and 1.4-fold improvements were developed as compared to the treated 43.0% digestibility when exhibiting 27.6%, 37.6%, and 38.4% delignification at each pretreatment condition.
- Experiments on ammonia recycle leaching hybrid poplar showed that, despite removal of more than 60% lignin, digestibility with 15 FPU cellulase was less than 50% due to non-productive binding of the enzyme to residual lignin and lignin.
- the transgenic poplars OX3 and OX8 in this study may be lignocellulosic suitable for the production of high value products such as ethanol.
- SEQ ID NO: 1 Arabidopsis RabG3b constitutively active gene
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Claims (15)
- RabG3b의 활성형 돌연변이체(RabG3bCA) 유전자가 형질전환된 나무.
- 제 1 항에 있어서,상기 RabG3bCA 유전자는 서열번호 1의 염기서열인 형질전환된 나무.
- 제 1 항에 있어서,상기 RabG3b는 애기장대 유래인 형질전환된 나무.
- 제 1 항에 있어서,상응하는 야생형 식물과 비교하여 셀룰로오스 및/또는 글루코오스 함량이 증가된 형질전환된 나무.
- 제 1 항에 있어서,상응하는 야생형 식물과 비교하여 섬유세포 길이가 증가된 형질전환된 나무.
- 제 1 항에 있어서,상응하는 야생형 식물과 비교하여 물관부가 증가된 형질전환된 나무.
- 제 1 항에 있어서,상기 나무는 포플러, 유칼립투스, 자작나무, 너도밤나무, 미루나무, 참나무, 소나무, 전나무 및 낙엽송으로 이루어진 군에서 선택된 하나 이상인 형질전환된 나무.
- 청구항 1의 형질전환된 나무를 원료로 하는 종이.
- 청구항 1의 형질전환된 나무로부터 생산된 펄프.
- 청구항 1의 형질전환된 나무로부터 생산된 바이오매스.
- 나무를 이용한 바이오매스의 생산방법에 있어서,상기 나무는 청구항 1의 형질전환된 나무인 바이오매스의 생산방법.
- 제 11 항에 있어서,상기 나무를 수산화나트륨으로 전처리한 후, 효소로 가수분해하여 바이오매스를 생산하는 바이오매스의 생산방법.
- 나무를 이용한 펄프의 생산방법에 있어서,상기 나무는 청구항 1의 형질전환된 나무인 펄프의 생산방법.
- RabG3b의 활성형 돌연변이체(RabG3bCA) 유전자를 과발현시켜 바이오매스가 증가된 형질전환된 나무를 제조하는 방법.
- 제 14 항에 있어서,상기 바이오매스가 셀룰로오스 및/또는 글루코오스인 방법.
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US14/241,278 US9551004B2 (en) | 2011-08-30 | 2012-08-29 | Transgenic tree induced by RABG3BCA and use thereof |
JP2014528272A JP5967674B2 (ja) | 2011-08-30 | 2012-08-29 | RabG3bCAを利用した形質転換された樹木及びその用途 |
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KR10-2012-0094285 | 2012-08-28 |
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JPH08503126A (ja) * | 1992-08-06 | 1996-04-09 | ザ・テキサス・エイ・アンド・エム・ユニバーシティ・システム | バイオマスの前処理方法 |
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