TW201139681A - Napier grass and uses thereof - Google Patents

Abstract

Disclosed are culture media, compositions and methods for culturing cellulolytic microbes for cellulase production.

Description

201139681 VI. Description of the Invention: [Technical Field] The present application relates to a medium, a composition and a method for cultivating a cellulose-decomposing microorganism for producing a cellulase. [Prior Art] Most of the research on the '(iv) veganase system in the past decades has focused on the decomposition of cellulose-decomposed biomass for the production of enriched (en)dch materials (such as ethanol, nitrogen and methyl ketone). . Therefore, many related technologies have been developed (Sheehan and Himme丨, 1999). The key to these biotechnologies is the practical goal of achieving microbial and enzymatic cellulose hydrolysis (Lynd et al.), but the biotechnologies that can be used to convert cellulose to biofuels are relatively promising but relatively lacking in-depth research. In addition, the use of naturally occurring lignocellulose (from wood, grass, forestry waste, waste paper, municipal waste and agricultural residues such as corn stalks and straws) to produce cellulase trends has become significant worldwide. increase. In all raw materials, agricultural biomass (consisting of more than 5% cellulose and hemicellulose) is an excellent carbon source for the production of microbial enzymes due to the extremely low operating costs. Reducing the cost of microbial enzyme production has become an important research in the current research and development of biofuels - the goal. Due to the wide application potential of cellulase in industrial processes (such as animal feed, powder processing, koji and wine making, grain alcohol germination, fruit and vegetable processing, pulp and paper, and textiles) (Bhat, 2_), A large number of studies have focused on cellulases. Many microorganisms that produce various cellulolytic enzymes have been studied for decades (Lynd et al., reading methods for increasing cellulase production 155455.doc 201139681 have been widely reported, including immersion and solid fermentation (Grajek, 1987). ), different types and concentrations of substrates (pavarina and Durrant, 2002), using carbon sources and nitrogen sources for regulation (L〇ckingt〇n et al., 2002), co-cultivation to produce cellulase (|&% Et al., 2006) and strain improvement (Adsul et al., 2007). Based on the need to use cellulase for saccharification of cellulose-based materials, screening has high cellulase activity and leaves no residue in the hydrolysate. Microorganisms such as cellobiose and having a thermo-pH stability enzyme will be beneficial. Cost issues are critical in the development and production of cellulases with applicability, high activity and high productivity. The substance is a major expense for growing cellulolytic microorganisms for producing cellulase. A cost-effective medium is required. [Invention] The present invention relates to a Effect of growing medium and microbial cellulose decomposition method for generating cellulase Accordingly, one feature of the aspect of the present invention is characterized by containing a wolf tail;. The NP) grass (Pennisetum purpureum pwrpwrewm)) of the medium. In one example, the culture medium contains about 0.11⁄4 to 5 〇/〇 (W/v) NP grass debris. The medium may contain an N source. In one example, the medium is sterilized. In another example, the medium further contains an antibiotic. In a first aspect, the invention features a method of culturing cellulolytic microorganisms. The method comprises the steps of: obtaining the above medium; and cultivating the cells in the medium under conditions permitting growth of cells of the cellulolytic microorganism. Examples of cellulolytic microorganisms include, but are not limited to, 155455.doc 201139681 Fusarium (FMsar/ww spp.), Aspergillus spp.) and Pseudomonas sp. (·Λ^ΜΓ〇·;?〇Γα spp. ). Preferred cellulolytic microorganisms to be used may be fungi such as Aspergillus niger (ApeazV/w·?wz'ger), Candida utilis {Candida /?e"icw/〇ia), Kluyveromyces cerevisiae (French / wyveromyces fragilis), Phanerochaete chrysosporium, Schizophyllum (5^/π·ζ〇ρ/7/ζ>>//μ/« cowiWM«e) and Richter Wood Mildew (TWc/zoaferwa reese/); bacteria, such as Agrobacterium (_4rongro0<3Cier/Mm) ATCC 21400, Bacillus licheniformis (Sctci/Zws am^y/o/kwe/aciews), ring bacillus (Bacillus circulans), Bacillus subtilis, Bacillus subtilis SMCciwogewe·?), CeHumonas fimi, Cellulomonas uda, Clostridium thermocellum C7〇iirM/ww thermocellum) 'Escherichia adecar boxy lata', Ewinrich c/zrysawiemi, MicroSsporfl and high temperature single bacteria FZ (772erwo«c »v〇ra ΓΧ); or obtained from, for example, the yellow-breasted termites (AeiicMh'iermes //<3v//?e·?), the wooden boat sister «y/c>_p/7ag^), the Australian ridge home Ants (Copioiermes /aciews), Black-breasted termites (TVaswmermes walkeri), Termites (Leucotermes (Reticulitermes), 1 Helix pomatia, Cryptocercus punctulates, 食本蹲{Panesthia cribrata), Mulberry serrata (Ca/o/awpra e/e) and other microbes of the six-spotted scorpion ((e〇icap/7ewi ί/ζ'/αίαίΜ·?) In a third aspect, the invention features a method of producing a cellulase. The method comprises the steps of: obtaining the above medium and culturing in the culture 155455.doc 201139681 in a condition permitting the expression of cellulase a cellulolytic microorganism containing a nucleic acid encoding a cellulase (such as a fungus selected from the group consisting of a sickle (four), a genus of the genus Trichophyton, and a genus Neurospora); "the cultured cell or medium is purified by a cellulase. In the fourth aspect, the present invention is characterized by a composition comprising the above medium and cells of a cellulolytic microorganism such as a fungus selected from the group consisting of Fusarium, Quercus and Fusarium. The above methods and compositions are useful for producing saccharable sugars and energy from lignocellulosic materials. Thus, in the fifth aspect, the present invention is characterized by a method for producing acid-smelling sugar from wood fiber and alizarin substances. The method comprises providing a composition having cells of the above medium and cellulolytic microorganisms; and contacting the composition with a lignocellulosic material to produce a fermentable sugar. In an example, . Nutrient 3 has about 11% to 5%, such as 〇%% (w/v qingcao pieces. The soluble sugar can be glucose, xylose, arabinose, galactose, mannose, rat = sugar, recommended sugar , fructose, lactose, maltose, trehalose, cellobiose: oligosaccharides (such as fiber oligomers or xylose oligomers) or any combination thereof. Examples of 2 substances include cellulosic animal waste, municipal solid waste red Stand, waste paper, yard waste, agricultural residues, forestry residues and mergings. The method may further comprise converting the sugar sugar into vinegar production. 4. The transformation step may be carried out by microorganisms or Enzymatic treatment to produce hydrazine: a sample stalk is characterized by a method of self-lignin cellulosic material. The method comprises: providing a composition of the above composition in contact with a lignocellulosic mulch, a priming substance to produce The sugar is converted; the yeast sugar is converted to produce a combustible yeast product; and the combustible yeast product is burned or the solid waste or residue is hydrolyzed to generate energy. In a seventh aspect, the features of the invention In a feed or food addition A method of processing, biopulping or complex treatment, the method comprising providing the above composition comprising a medium and a cell of a cellulolytic microorganism; and contacting the composition with a substance to be processed or treated. The invention is characterized by a bioreactor comprising a lignocellulosic material and the above composition. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the present invention will be apparent from the scope of the invention. The present invention is based, at least in part, on the surprisingly finding that Pennisetum can be used as an efficient and inexpensive method for growing cellulolytic enzymes that produce cellulase. Major carbon sources. The wolftail (Np) grass (grass) found in tropical and subtropical regions has been widely used as a feed for anti-animals and its composition, nature and some genetic information have been fully studied; however, No report shows the use of grass in the preparation of microbial cellulose. The present invention provides Np grass for growth from straw or sweet A new application of an effective and inexpensive c source for cellulolytic fungi isolated from bagasse composting (ie, Fusarium, Aspergillus, and Neurospora). The results show that when using l% (w/v) NP grass as When the main c source of the three fungi, the total cellulase activity (FPase) increased significantly (for Fusarium, Aspergillus and Neurospora from 0.008 to 0.085, from 〇85 to 〇〇85, from 0.002 increased to 0.124 U/ml) (Figure 丨). After optimizing all individual culture conditions to 155455.doc 201139681, the optimal cellulase yields of Fusarium and Vibrio were 018 to 0.22 U/ml, respectively. And 0.14 to 〇_16 u/m b in the studied C source sugar, CMC and NP grass), containing 1% called medium not only = good cellulose decomposition activity 'and tested at all. The source is the cheapest combination. This has made N P grass a major C source for the improvement of biofuels that must be produced by cellulolytic microorganisms to produce cellulase, the textile industry and other industries. For example, cellulases produce acid-sweet sugar and energy from lignocellulosic matter. The term "wood f-cellulosic material" as used herein refers to a substance containing cellulose and/or hemicellulose. In general, substances such as & also contain xylan, lignin, protein and carbohydrates such as starch and sugar. For example, 'in the stems, leaves, shells, skins and cobs of plants or leaves, branches and xylem of trees. The process of converting complex carbohydrates, such as starch, cellulose or hemicellulose, into saccharable sugars is also referred to herein as "saccharification." A saccharable sugar as used herein refers to a monosaccharide such as glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose, fructose, lactose, maltose, trehalose or cellobiose. Lignocellulosic materials may include virgin plant biomass and/or non-primitive plant biomass, such as agricultural biomass, commercial organisms, constructs and destructive debris, municipal solid waste, waste paper, and yard waste. Common forms of lignocellulosic material include trees, shrubs and grasses, wheat, wheat straw, bagasse, corn, corn husks, corn kernels, including stalks of corn, rice, wheat, and barley (including wet Products and by-product fibers of municipal and dry grinding, as well as municipal solid waste, waste paper and yard waste. The lignocellulosic material may also be 155455.doc 201139681 (but not limited to) herbal matter, agricultural residues, forestry residues, and paper pulp residues. "Agricultural biomass" includes branches, bushes, stems, jade and corn husks, energy crops, forests, fruits, flowers, scorpions, grasses, grasses, crops, leaves, bark, needles, Logs, roots, seedlings, short-rotation woody crops, shrubs, switchgrass, trees, vegetables, peels, vines, beet cypress, wheat noodles, oatmeal, hardwood and = (excluding wood containing harmful substances) ), organic substances produced by agricultural processes (including farming and forestry activities, including forestry wood waste in particular) or mixtures thereof. = Examples include (but are not limited to) branches, bushes, factory waste, municipal wood waste, cities: waste, felled waste, forest (four) thinning, short-rotation wood, industrial waste, wheat, Wheat, oats, straw, big, rye, flax, stalk, soybean, rice husk, oat husk, king: rice, corn stalk, corn stalk, corn gluten feed, corn husk , corn grain 1 fiber, big netch grass, duck-like rubbing fiber 2: ΓΓ °, foxtail grass, beet stalk, mandarin quince fruit pulp, seed shell, shrub 1 waste, lawn trim, cotton, consumption , trees, products, Chenggu slag, grain wet or dry grinding products and sub-agricultural residues solid waste, waste paper, yard waste, herbal matter, industrial residues, forestry residues, urban clusters, Papermaking hinders residues, branches, bushes, stalks,:: Ershi paper energy crops, forests, fruits, flowers/kayuskins, bark, needle-like leaves, roots, seedlings, shrubs, switchgrass, trees, 155,455. Doc 201139681 Vegetables, peels , m plants, beet noodles, wheat #, oat hulls, hardwoods and softwoods, organic waste from agricultural processes, forestry wood waste or combinations thereof. In this study, Fusarium, Qufu, and Rhizoctonia were cultured in a medium containing Wolftail (NP) grass (Ceramic), CMC or glucose as the main carbon source to produce extracellular fibers. Enzyme. The results showed that when the three isolated fungi were cultured with P/0 (W/V) NP grass, the total cellulase activity (based on the .μ measurement) increased significantly for Aspergillus, Fusarium and Pulmonary The genus increased from 〇(no NP) to 〇〇85 u/ml〇% Np), increased from 〇嶋 to 0.085 U/ml, and increased from 〇·002 to 〇124 u/ml; Wv) about i to 2 lings of total cellulase activity during CMC culture. The optimum cellulase activity was achieved under optimized culture conditions (〇18 to 22 u/ml and 〇u to 〇Μ U/ml for Aspergillus and Neurospora, respectively). In addition, the cellulase collected from the 1% Np batch has a good positive temperature and temperature operating range' and is stable at a wide pH value. In all C sources tested, the medium containing (4) not only achieves optimal cellulolytics. Active' and the cheapest combination. This discovery makes NP grass a biofuel that is required to improve cellulase production by cellulolytic fungi. The textile industry and other major carbon sources for the industry. Thus, the media, compositions, and methods disclosed herein can be used in feed or food additive processing, biopulping, or compounding in the manner described in U.S. Patent Application Nos. 20,100,047, 869 and 2008, 013, 958, and U.S. Patent Nos. 5,232, 851, 5, 677, 151, 6, 061, 063, 6, 602, and 7, 272, 677. In the context of processing, the contents of each case are incorporated herein by reference. The following specific examples are to be construed as illustrative only and not limiting the remainder of the invention in any way. There is no need to clarify that the skilled artisan can make the most of the present invention based on the description herein. All publications cited herein are hereby incorporated by reference in their entirety. In addition, any mechanism proposed below does not limit the scope of the claimed invention in any way. 1. INTRODUCTION In this study, three cellulase-producing strains, Fusarium, Aspergillus, and Neurospora were used. The culture conditions of the three fungal strains were optimized to achieve optimal cellulase activity. Three different natural or artificial carbon sources (i.e., Pennisetum, CMC or Glucose) were supplemented to the medium to assess their potential to increase cellulase production at lower cost. 2. Method U quality NP grass was obtained from Taiwan Animal Production Laboratory (Taiwan Uvestock Researeh)

Institute) (Hsin-hua, Tainan, Taiwan). Carboxymethylcellulose (CMC), glucose, β_p-germanyl-phenyl-sugar (β_ρΝρ〇), Whatman paper No. 1 and all the chemicals used in this study were from Sigma. . CMC, Waterman No. 1 paper and β-pNPG were the substrates for CMCase, β-glucosidase and FPase measurements, respectively. Among the three fungi, CMC, glucose and NP grass were used as a carbon source at a concentration of i% (w/v) to study the production of cellulase. The NP grass was air-dried using a chopper to cut into small pieces and ground to a smaller size. The particles 'and then finally separated by passing through a 0.45 mm screen. The medium is prepared using a portion that passes through the screen and a commercially available chemical. 155455.doc 201139681 u Inoculation and culture conditions Maintain Fp and RS_N strains on pDA loam on 5 to 7 natural and RS-lanthanide at 35 °C at 2 rc, in 丁 丁 v v ', here At the time, the sporulation was in good condition: fresh hyphae from this culture was used as the inoculum. The vegetative suspension was prepared by washing the slant culture with $ ml sterilized 。 2 。. Spore suspension using a hemocytometer The liquid was counted up to 1 8 spores/ml. 2 ml of spore suspension of Fusarium, Aspergillus and Neurospora was found in 25 ml containing 100 ml of MR medium (excluding 〇1% soy peptone) Growth in the mi flask, cellulase production was studied using 1 〇 / NP grass, 1% CMC 41% glucose as carbon source. Cellulase activity of three fungi grown in different carbon sources was recorded during 5 days of culture. The optimal concentration of MNp grass is to use different percentages of NP and cultured in MR medium (excluding 0.1% soy peptone) under the optimal growth conditions. 2.3 The crude fiber enzyme preparation of 3 fungi will be used 11⁄2 Fusarium, Aspergillus and veins cultured in NP grass The culture medium of the genus Neurospora was centrifuged at 8,0〇〇xg for 20 minutes to remove the hyphae and the unused material. The supernatant was filtered through the Waterman No. 1 paper and the 〇45 μηι membrane, and then borrowed. It was concentrated by filtration using an Amicon concentrator with a 3 kDa membrane (Vivaspin 20). The FPase activity loss during the filtration process was less than 3%. » Add 0.01% (v/w) sodium azide (NaN3) to the supernatant. Prevents microbial growth. Concentrated crude enzyme preparation containing 0.01% NaN3 and 〇·〇ΐ〇/0 phenyl decane sulfonium fluoride (pmSF, a serine protease inhibitor) can be stored at 4 ° C for 1 week. After still retaining 80% activity. Enzyme characteristics were studied using crude enzyme preparations, including optimal pH, pH stability, optimal temperature and thermal stability. 155455.doc -12- 201139681 2.4 Cellulase (CMCase, FPase and β-glucosidase activity assays for the slave activity test, using the corresponding 1% substrate as described in the method, measuring the CMCase, β-Portuguese extract of the crude enzyme extract of Fusarium at 40 ° C The activity of budase and FPase, but for Aspergillus and Neurospora, the measurement was carried out at 50 C. The reaction at 200 μΐ The compound (j 〇〇y crude enzyme and 丄〇〇μΐ in 2 mM of 100 mM NaOAC, 1 〇〇 mM NaCl (pH 5.0)) was measured for reduction from 1% CMC release in 10 minutes. Sugar was used to determine CMCase. Fpase activity was determined using 3 mg No. 1 (Wartman) crepe paper as the substrate in a 2 〇〇 reaction mixture (as above) for 1 hour. After the addition of 2 μl of mononitrosalicylic acid (DNS) and heating at 1 ° C for 1 min, the total release reducing sugar was determined using the modified DNS method (wood et al., 1988). One unit (IU) CMCase and FPase activity is defined as the amount of enzyme that releases 1 μηιοί glucose (as reducing sugar equivalent) per minute under assay conditions. About ρ-glucosidase activity by using β-pNPG (final concentration: 1 mM) with a 10 μΐ enzyme solution prepared in sodium acetate buffer (100 mM NaOAC, 1 mM Nacn, pH 5.0) Three fungi assays were performed by mixing the resulting 〇5〇μ1 working volume. After 1 minute of incubation, the reaction was terminated by the addition of 5 〇 μ1 2 Μ sodium carbonate. The absorbance of the reaction mixtures of the three fungi was measured at 400 nm using a spectrophotometer. The β-glucosidase activity was calculated based on the 4-nitrophenol phenol calibration curve. The enzyme activity of one unit is defined as the release of 丨μιη〇1 4-nitrobenzene equivalent per minute. 3. Results and discussion The influence of carbon (C) sources such as the complexity of the culture medium and the complexity of the structure of the substrate may be affected. 155455.doc 201139681 The filamentous fungus secretes hydrolase (Kurchenko et al., 2001). In this study, it was estimated that different C sources (i.e., NP grass and CMC) improved the cellulase secretion potential of Fusarium, Aspergillus, and Neurospora. The results showed that the total cellulase activity (FPase) of the three fungi grown in the medium without the C source supplement was extremely low. Once in the presence of 1% (w/v) NP grass (but in the absence of organic N) (Table 1 and Figure 1), FPase activity increased significantly by about 6 to 1.5 times (ie for Fusarium) For Aspergillus and Neurospora, increased from 0.062 U/ml, from 0.008 to 0.085 U/ml and from 0.002 to 0.124 U/ml, respectively, indicating that NP can induce cellulose in large quantities. Enzyme production. The CMCase and β-glucosidase activities also showed a similar increase (Table), and the activity was increased to a greater extent than FPase. In addition, the FPase activity of the batch cultured with i% Νρ grass was cultured with 1% CMC. Approximately double the batch. These results indicate that NP grass can be used as an ideal c source to improve the production of cellulase in the cellulolytic fungi inoculated in this study. In addition, the optimal Np grass for cellulase production is further evaluated. Concentration. Generally, for Fusarium and Neurospora, Fpase activity increases with the increase of Np grass 'except for 5% Np (Fig. 2b and Fusarium, Aspergillus and Pseudomonas) The maximum FPase appeared after 4 to 5 days of 2%, 5%, and 3% of the grass, respectively; this value is even more than the value of ι〇/〇, and the Np-based medium developed by the Institute Qing. The cost of the medium' and the cost of the C source is usually more than one-half of the total cost of the medium (Table 2). It is concluded that NP stem sputum ra μ, a soap for the production of economic cellulase is more J55455.doc - 14- 201139681 Table 1. Enhancing the cellulose of three fungi using l% (v/w) NP grass as the main C source (FPase, CMCase and β-glucosidase) yield. C* source fungal strain enzyme (mU/ml) FPase CMCase β-glucosidase without C source Aspergillus 0.8 7.2 19 1% Pennisetum Aspergillus 85 1024 217 1% CMC Aspergillus 3.7 132 46 No C-derived Fusarium 0 0 0 Γ/. Fusarium oxysporum 62.1 676.8 349.2 1% CMC Recording genus 13.3 513.6 125.5 No C-source genus 1.9 12.8 1.2 1°/. Pennisetum genus 124 866 214 1% CMC genus 112 928 237 * After 5 days of incubation under the optimal growth conditions, test each in MR medium without soy peptone Fungal strains. 155455.doc -15· 201139681 4哒砩#进./OOSA^^Sil^要(窭诹尔喵UM3)£3”^脊03朱密:>嫦^姝^瞎阳^”&lt ; 155455.doc (古<<#) Drinking·Considerable Savings Efficiency (%) 43-53 (1 τTMΗ Ο Total Cost (NT$/L) [C Source + Medium] 8.71-10.71 16.71 18.71 i 1 j Cost (NT$/L) 6.71 Medium P? ifttV ^ Ο 〜 ~ w ΊΙΪ 1 ^ g磡s General cost (NT$/L) (N Ο C source 1% Pennisetum 1% CMC 玑 Move g •16- 201139681 Conclusion When using NP grass as the main carbon When three kinds of fungi reached FPase activity using 1% CMC as the main carbon source activity when FPase 1.1 to 23 times; and 60 to 105 times the grass without NP FPase activity. Among the C sources studied (i.e., glucose, CMC, and NP grass), the best NP grass concentration of the medium was used to achieve not only optimal cellulolytic activity, but also the cheapest combination among all C sources tested. The above properties indicate that cellulases derived from RS-A, Fp and RS-N can be used as key enzymes for the preparation of bioethanol from cellulose. Other Embodiments All of the features disclosed in this specification can be combined in any combination. Features disclosed in this specification may be replaced by alternative features for the same, equivalent or similar purpose. Accordingly, unless expressly stated otherwise, the features disclosed are only one example of a series of generally equivalent or similar features. A number of embodiments of the invention have been described. It will be understood, however, that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. references.

Bhat, M.K., 2000. Cellulases and related enzymes in biotechnology. 18, 355-83 0

Grajek, W., 1987. Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid-state fermentation. MicroMo/26, 126-129.

Kurchenko, IM, Zhdanova, NM, Sokolova, OV, 2001. Study 155455.doc •17- 201139681 of availability of some hydrolytic and redox enzymes in strains of Fusarium oxysporum (Schlecht.) Snyd. and Hans, isolated from different habitats. , 63, 34-43.

Lee, M., Hwang, S., Chiou, P.W. 2000. Metabolizable energy of roughage in Taiwan. iSwcj// 36(3), 251-259 0

Lockington, R.A., Rodbourn, L., Barnett, S., Carter, C.J., Kelly, J.M., 2002. Regulation by carbon and nitrogen sources of a family of cellulases in Aspergillus nidulans. Fungal Genet Biol, 37, 190-6.

Lynd, L.R., Weimer, P.J., van Zyl, W.H., Pretorius, I.S., 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev, 66, 506-ΊΊ, color record.

Pavarina, E.C., Durrant, L.R., 2002. Growth of lignocellulosic-fermenting fungi on different substrates under low oxygenation conditions. 98-100, 663-77 0

Prakash, B., Dhali, A., Mondal, M., Sangtam, M., Khate, K., Rathore, SS, Rajkhowa, C. 2008. Effect of feeding Lagerstroemia speciosa and conventional fodder based rations on nutrient utilization, ruminal Metabolites and body weight gain in mithun (Bos frontalis). J Anim Physiol Anim Nutr (Berl), 92(5), 591-6.

Sheehan, J., Himmel, M., 1999. Enzymes, Energy, and the Environment: A Strategic Perspective on the U.S. Department of Energy's Research and Development Activities for Bioethanol. 155455.doc •18· 201139681

Biotecknol Pr〇g, S17-S27.

Wood, TM·, Willis, AW.aSTK” 1988· Preparation of crystalline, amorphous, and dyed cellulase substrates. Method Less Mo/. Academic Press, pp. 19-25. [Simplified Schematic] Figure la to Figure lc Shows that for 3 fungi (a) Fusarium, (b) Aspergillus, and (c) Neurospora, 1% (w/v) NP grass is used to enhance total fiber compared to 1% CMC. Map of prime enzyme production (Fpase activity). All strains were cultured in MR medium in the absence of soy protein glands and supplemented with 1% NP*1% CMC as the main carbon source. Cultured in the same medium without the above c source. The strain was designated as a control experiment (ie, no Np) ^ General conditions were pH 7.0, 25 C to 35 ° C, and 150 rpm, with 1.0 NP grass as a control experiment. Figure 2a to Figure 2c show for 3 species For the fungi Fusarium, Aspergillus and (c) Neurospora, Np grass significantly enhances the optimal concentration of total cellulase production (FPase). All strains are in MR medium in the absence of soy protein glands. Different percentages of Np were used as the main carbon source to culture under the optimal growth conditions. The general condition was pH 7 〇, but it was to "it, and 15 rpm, with 1% NP grass as a control experiment. 155455.doc •19·

Claims (1)

201139681 VII. Patent application scope · 1. A culture medium containing wolftail (Napier; NP) grass (Pennhetum pUrpureum, 2. The medium of μ1, wherein the medium contains about 0.1% to 5% (w/v) wolftail (ΝΡ) grass (grass) fragments. 3. The medium of spray 2, wherein the medium contains about 丨% to 3% (w/v) wolftail (NP) grass The medium according to any one of items 1 to 3, wherein the medium is sterilized. The medium according to any one of the items 3 to 3, wherein the medium further comprises an antibiotic 6. A method of cultivating a cellulolytic microorganism, the method comprising: obtaining a medium according to any one of claims 5 to 5; and cultivating the medium under conditions allowing cell growth of the cellulolytic microorganism 7. The method of claim 6, wherein the cellulolytic microorganism is one or more selected from the group consisting of Fusarium spp.), Aspergillus (Liu 6 Mail SPP), and Neurospora spp) Fungus. A method for producing a cellulase, the method comprising: obtaining a medium according to any one of claims 5 to 5; and cultivating a cellulose containing a nucleic acid encoding the cellulase under conditions permitting expression of a cellulase Decomposing the cells of the microorganism; and purifying the cellulase from the cells cultured in the colon or the medium. 9. The method according to claim 8, wherein the cellulolytic microorganism is one or more 155455.doc 201139681 is a fungus selected from the group consisting of Fusarium, Aspergillus and Neurospora. A composition comprising the medium of any one of the items (1) and the cell of the cellulolytic microorganism. 11. As requested! ! The composition, wherein the cellulolytic microorganism is one or more fungi selected from the group consisting of Fusarium, Aspergillus and Neurospora. A method for producing a sucrose-derived sugar from a lignocellulosic material f, comprising: a composition comprising a medium according to any one of claims 1 to 5 and a cell of a cellulolytic microorganism; The composition is contacted with a lignocellulosic material to produce a fermentable sugar. 13. The method of claim 12, wherein the saccharide is selected from the group consisting of glucose, sugar: arabinose, galactose, mannose, rhamnose, sucrose, fruit sugar, (iv) sugar, sea uranium, fiber s The group consisting of sugar combinations 乔 次,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, a group of objects and any combination thereof. 15. In the case of the method of 14th, the saccharide is converted into a fermented product. 3 will be smashed 16. =:: method 'one step by micro-17. The hai composition is contacted with the lignocellulosic material to produce sugar 155455.doc 201139681; transforming the fermentable sugar to Producing a combustible fermentation product; and burning the combustible vinegar product or hydrolyzing solid waste or residue to generate energy. & 18. 19. A bioreactor' which contains a lignocellulosic material and a composition as claimed in claim 1 or 11. a method for processing a feed or food additive, a biopulping or a composite treatment, providing a composition comprising a medium according to any one of the claims m and a cell of a cellulolytic microorganism; and allowing the composition to be treated Contact with processed or treated materials. 155455.doc