CN102827801B - Napier grass (pennisetum purpureum) and application thereof - Google Patents

Abstract

The invention relates to napier grass (pennisetum purpureum) and an application thereof. The invention discloses a culture medium, a composition, and a method used for culturing cellulose-decomposing microbes for producing cellulase.

Description

Herba penniseti and uses thereof

Technical field

Subject application relates to for cultivating cellulose decomposition microorganism to produce the substratum of cellulase, composition and method.

Background technology

In many decades in the past, the major part research for cellulase system mainly concentrates on cellulose decomposition Wood Adhesives from Biomass to produce rich energy (energy-rich) material (such as ethanol, hydrogen and methane).Therefore, many correlation techniques (Xia Erhan (Sheehan) and extra large Gadamer (Himmel), 1999) have been developed.The key of these biotechnologys is the practical target (people such as Li De (Lynd), 2002) realizing microorganism and enzymatic cellulose hydrolysis example.But, although can be used at present cellulose conversion being become the biotechnological means of biofuel to have much prospect, relatively lack further investigation.In addition, the trend of the lignocellulose of natural formation (being derived from timber, grass, forest trimmings, waste paper, municipal waste, and agricultural residue, such as corn stalk and dogstail) production of cellulose enzyme is used worldwide obviously to increase.In all raw materials, agricultural biomass (forming by more than 50% Mierocrystalline cellulose and hemicellulose) forms the splendid carbon source of producing microbial enzyme of making a living because operational cost is extremely low.Reduce microbial enzyme production cost and become biofuel research and development instantly and the important research target of producing.

Due to the application potential huge (Bo Aite (Bhat) of cellulase in numerous industrial technology (such as animal-feed, starch processing, koji and wine brewing, grain alcohol fermentation, fruits and vegetables processing, paper pulp papermaking industry and textile industry), 2000), large quantity research concentrates on cellulase.The microorganism of the various cellulolytic enzyme of many generations have studied many decades (people such as Li De (Lynd), 2002).Some cultural methods for increasing yield of cellulase are widely reported, comprise submerged fermentation and solid state fermentation (Gai Jiake (Grajek), 1987), substrate (Pa Walinna (Pavarina) and the Durant (Durrant) of dissimilar and concentration, 2002) Carbon and nitrogen sources, is utilized to carry out regulating (the people such as Luo Kejindun (Lockington), 2002) Dual culture, is carried out to produce the cellulase (people such as king (Wang), 2006) and strain improvement (people such as Ai De Sol (Adsul), 2007).Consider the saccharification needing to use cellulase to carry out based on cellulosic material, therefore screening has in high-cellulose enzymic activity, hydrolysate the microorganism that stays without resistates (such as cellobiose) and have heat-pH stability enzyme by for useful.

In the research and development of cellulase with suitability, high reactivity and high productivity and production process, Cost Problems is a critical problem.The chemical substance preparing substratum is that growth cellulose decomposition microorganism is to produce typical case's cost of cellulase.One is needed to have cost-benefit substratum.

Summary of the invention

The present invention relates to, for growing cellulose decomposition microorganism with what produce cellulase, there is cost-benefit substratum and method.

Therefore, the feature of one aspect of the present invention is a kind of containing wolf tail (Napier; NP) substratum of grass (napier grass (Pennisetum purpureum)).In one example, substratum comprises about 0.1% to 5% (w/v) NP grass fragment.Substratum can contain nitrogen (N) source.In one example, substratum is through sterilizing.In another example, substratum is further containing microbiotic.

In second aspect, the invention is characterized in a kind of cultivation cellulose decomposition method of microorganism.Said method comprising the steps of: obtain above-mentioned substratum; With in described substratum, under the condition allowing the growth of cellulose decomposition microbial cell, cultivate described cell.The example of cellulose decomposition microorganism includes, but is not limited to Fusarium (Fusarium spp.), Eurotium (Aspergillus spp.) and Neurospora (Neurospora spp.).The cellulose decomposition microorganism of preferred use can be fungi, such as black-koji mould (Aspergillus niger), candida pelliculosa (Candida pelliculosa), Kluyveromyces fragilis (Kluyveromyces fragilis), Phanerochaete chrysosporium (Phanerochaete chrysosporium), Split-gill (Schizopphyllum commune) and Trichodermareesei (Trichoderma reesei), bacterium, such as Agrobacterium (Agrobacterium) ATCC 21400, bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus circulans (Bacillus circulans), subtilis (Bacillus subtilis), Bacteroides succinogenes (Bacteroides succinogenes), Cellulomonase fimi (Cellumonas fimi), moist cellulomonas cartae (Cellulomonas uda), Clostridium thermocellum (Clostridium thermocellum), Escherichia adecarboxylata (Escherichia adecarboxylata), Erwinia chrysanthemi (Erwinia chrysantemi), microbispora bispora (Microbispora bispora) and thermomonospora fusca YX (Thermonospora YX), or from such as eastern subterranean termite (Reticulitermes flavipes), food wooden boat maggot (Xylophaga), Australia rib Coptotermes formosanus Shtrari． (Coptotermes lacteus), black chest resembles termite (Nasutitermes walkeri), large and termite (Leucotermes speratus) (Reticulitermes (Reticulitermes)), housing helix (Helixpomatia), hidden tail Lian (Cryptocercus punctulates), eat wooden cockroach (Panesthia cribrata), other microorganism that the animals such as mulberry Apriona germari (Calolampra elegans) and the cockroach that lives in caves (Geoscapheus dilatatus) obtain.

In a third aspect, the invention is characterized in a kind of method producing cellulase.Said method comprising the steps of: obtain above-mentioned substratum, and in described substratum, allowing the cell of the cellulose decomposition microorganism (being such as selected from the fungi of Fusarium, Eurotium and Neurospora) cultivating the nucleic acid containing encoding cellulase under the condition expressing cellulase; With from institute's cultured cells or substratum purifying cellulose enzyme.

In fourth aspect, the invention is characterized in that one has the composition of the cell of above-mentioned substratum and cellulose decomposition microorganism (being such as selected from the fungi of Fusarium, Eurotium and Neurospora).Aforesaid method and composition can be used, produce fermentable sugars and energy by lignocellulosic materials.

Thus, in the 5th, the invention is characterized in a kind of method being produced fermentable sugars by lignocellulosic materials.Described method comprises the composition providing and have above-mentioned substratum and cellulose decomposition microbial cell; Contact to produce fermentable sugars with making described composition with lignocellulosic materials.In one example, substratum containing having an appointment 0.1% to 5%, such as 1.0% (w/v) NP grass fragment.Fermentable sugars can be glucose, wood sugar, pectinose, semi-lactosi, seminose, rhamnosyl, sucrose, fructose, lactose, maltose, trehalose, cellobiose, oligosaccharides (such as Mierocrystalline cellulose oligopolymer or xylo-oligomers) or its arbitrary combination.The example of lignocellulosic materials comprises cellulosic animal waste, municipal solid waste, waste paper, garden waste material, agricultural residue, forestry residue and arbitrary combination thereof.Described method can comprise step fermentable sugars being changed into tunning further.This step of converting is undertaken by fermentable or ferment treatment.

In in the 6th, the invention is characterized in a kind of by the energy-producing method of lignocellulosic materials.Described method comprises: provide above-mentioned composition; Described composition is made to contact to produce fermentable sugars with lignocellulosic materials; Transform described fermentable sugars to produce combustible fermentation product; With burn described combustible fermentation product or water-disintegrable solid waste or resistates with generate energy.

In in the 7th, the invention is characterized in a kind of method for feed or foodstuff additive processing, bio-pulping or mixture process.Described method comprises provides above-mentioned composition, and it comprises substratum and cellulose decomposition microbial cell; Contact with material that is to be processed or that process with making described composition.

In eighth aspect, the invention is characterized in a kind of bio-reactor, it contains lignocellulosic materials and above-mentioned composition.

The details of one or more embodiments of the present invention will be set forth in institute's accompanying drawings and following description.Describe and graphic and claims according to these, will be easy to understand further feature of the present invention, object and advantage.

Accompanying drawing explanation

Fig. 1 a to Fig. 1 c shows compared with use 1%CMC, the figure that use 1% (w/v) NP grass makes the plain production of enzyme of the total fiber of (a) Fusarium, (b) Eurotium and (c) Neurospora 3 kinds of fungies (FPase activity) increase.There is not soy peptone and cultivating in MR substratum using 1%NP or 1%CMC as primary carbon source in all bacterial strains.The bacterial strain cultivated is appointed as control experiment (namely without NP) in not containing the same medium of above-mentioned carbon source.General condition is pH 7.0,25 DEG C to 35 DEG C and 150rpm, tests in contrast with 1%NP grass.

Fig. 2 a to Fig. 2 c is the figure that the NP grass of display optimum concn makes total fiber element production of enzyme (FPase) of (a) Fusarium, (b) Eurotium and (c) Neurospora 3 kinds of fungies obviously increase.There is not soy peptone and cultivating under corresponding optimum growth conditions in MR substratum using the NP of different weight percentage as primary carbon source in all bacterial strains.General condition is pH 7.0,25 DEG C to 35 DEG C and 150rpm, tests in contrast with 1%NP grass.

Embodiment

The present invention at least partly based on being surprised to find that Herba penniseti can be used as effective and cheap primary carbon source for growth cellulose decomposition fungi, thus produces cellulase.

Wolf tail (NP) grass (napier grass), is common in subtropical and tropical zones, has been widely used as the feed of ruminating animal, and its composition, character and portion gene group information are well studied; But, there is no report display and NP grass can be utilized to produce microbial cellulase.The invention provides NP grass as effective and cheap carbon source to grow the new opplication of the cellulose decomposition fungi (i.e. Fusarium, Eurotium and Neurospora) be separated from straw or bagasse compost at first.Result shows, when the primary carbon source of use 1% (w/v) NP grass as these 3 kinds of fungies, total fiber element enzymic activity (FPase) significantly increases (Fusarium, Eurotium and Neurospora respectively from 0.008 increasing to 0.085, from 0 increasing to 0.085, increasing to 0.124U/ml from 0.002) (Fig. 1).After all indivedual culture condition optimizings, the best yield of cellulase of Fusarium and Neurospora is respectively 0.18 to 0.22U/ml and 0.14 to 0.16U/ml.In studied carbon source (i.e. glucose, CMC and NP grass), the substratum containing 1%NP grass not only reaches best cellulolytic activity, and is combination the most cheap in the carbon source of all tests.This discovery makes NP grass become the primary carbon source that can additionally select improved and produced the required cellulase of biofuel, textile industry and other industry by cellulose decomposition microorganism.

For example, when producing fermentable sugars and energy by lignocellulosic materials, cellulase can be used.As used herein term " lignocellulosic materials " refers to the material containing Mierocrystalline cellulose and/or hemicellulose.In general, these materials are also containing xylan, xylogen, protein, and carbohydrate, such as starch and sugar.For example, at the stem of plant, leaf, shell, crust and cob (cob), or lignocellulose can be found in the leaf of trees, branch and xylem.Complex carbohydrates (such as starch, Mierocrystalline cellulose or hemicellulose) changes into the process of fermentable sugars in this article also referred to as " saccharification ".As used herein fermentable sugars refers to monose, such as glucose, wood sugar, pectinose, semi-lactosi, seminose, rhamnosyl, sucrose, fructose, lactose, maltose, trehalose or cellobiose.Lignocellulosic materials can comprise primordial plant biomass and/or non-primary plant biomass, such as agricultural biomass, business organism, buildings and ruins chip, municipal solid waste, waste paper and garden waste material.The common form of lignocellulosic materials comprises tree, shrub and grass, wheat, wheat stalk, bagasse, corn, maize peel, corn grain (comprising the fiber from grain), the product obtained by cereal (comprising wet grinding and dry grinding) such as grinding such as corn, rice, Wheat and barley and by product, and municipal solid waste, waste paper and garden waste material.Lignocellulosic materials also can be (but being not limited to) herbaceous material, agricultural residue, forestry residue and paper mill residue." agricultural biomass " comprises branch, thicket, rattan, corn and maize peel, energy crop, forest, fruit, flower, cereal, grass, herbaceous crops, leaf, bark, acicular leaf (needle), log, root, nursery stock, short circulation phase tree crop (short rotation woody crop), shrub, switchgrass (switch grass), trees, vegetables, pericarp, vine, beet pulp, Wheat bran, oat shell, hardwood and cork (not comprising the timber contained hazardous substance), the organic waste materials that agricultural procedure produces (comprises and cultivating and forestry activities, especially forestry Wood waste is comprised) or its mixture.

In this method, the example of lignocellulosic materials used includes, but is not limited to orchard beta pruning, grove, factory waste, city Wood waste, municipal waste, felling waste material, forest thinning (forest thinning), short circulation phase tree crop, industrial waste, wheat, wheat stalk, oat straw, straw, Barley straw, rye stalk, flax stalk, soybean peel, rice husk, oat shell, sugarcane, corn, corn stalk, maize straw, corn gluten feed, mealie, maize peel, corn grain, grain fiber, large net cogongrass (prairie grass), orchardgrass shape friction standing grain (gamagrass), foxtail, beet pulp, citrus pulp, seed hulls, cellulosic animal waste, turf-mown thing, cotton, marine alga, trees, shrub, grass, bagasse, the product of cereal wet grinding or dry grinding and by product, municipal solid waste, waste paper, garden waste material, herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper, paper pulp, paper mill residue, branch, thicket, rattan, corn, maize peel, energy crop, forest, fruit, flower, cereal, grass, herbaceous crops, leaf, bark, acicular leaf, log, root, nursery stock, shrub, switchgrass, trees, vegetables, pericarp, vine, beet pulp, Wheat bran, oat shell, hardwood and cork, the organic waste materials that agricultural procedure produces, forestry Wood waste or its combination.

In this research, with containing wolf tail (NP) grass (napier grass), CMC or glucose these Fusariums of culture medium culturing as primary carbon source, Eurotium and Neurospora fungal bacterial strain, to produce cellulase in extracellular.Result shows, when the fungi of these 3 kinds separation cultivated by use 1% (w/v) NP grass, total fiber element enzymic activity (based on FPase observed value) of Eurotium, Fusarium and Neurospora significantly increases, and increases to 0.085U/ml (1%NP), increases to 0.085U/ml with from 0.002 increasing to 0.124U/ml from 0.008 respectively from 0 (without NP); About 1 to 23 times of the plain enzymic activity of total fiber when it is for cultivating with 1% (w/v) CMC.Best cellulase activity (for Eurotium and Neurospora, being respectively 0.18 to 0.22U/ml and 0.14 to 0.16U/ml) is issued at optimized culture condition.In addition, from the 1%NP batch of cellulase collected, there is good pH value and temperature working range, and stable under extensive pH value.In the carbon source of all tests, the substratum containing NP grass not only reaches best cellulolytic activity, but also is the most cheap combination.This discovery provides NP grass to select as the extra utilization of primary carbon source, produces the required cellulase of biofuel, textile industry and other industry to improve by cellulose decomposition fungi.

Therefore, substratum disclosed herein, composition and method can be used in feed or foodstuff additive processing, bio-pulping or mixture process by the mode described in U. S. application case 20100047869 and 20080131958 and United States Patent (USP) 5232851,5677151,6451063,6602700 and 7226773, and the content of described each case is incorporated herein by reference.

Following specific examples only should be interpreted as that tool is illustrative, and not so that no matter any mode limits rest part of the present invention.Without the need to illustrating further, believe that one of ordinary skill in the art can maximally utilise the present invention according to description herein.The mode that all publications quoted herein are quoted all is in full incorporated herein.In addition, any mechanism hereafter proposed limits advocated scope of invention never in any form.

1. introduction:

In this research, use 3 kinds of bacterial strains producing cellulase: Fusarium, Eurotium and Neurospora.The culture condition of these 3 kinds of fungal bacterial strains through optimizing to reach best cellulase activity.3 kinds of different natural or artificial carbon sources (i.e. Herba penniseti, CMC or glucose) are added in substratum to assess the potential that it increases yield of cellulase at lower cost.

2. method

2.1 substrate

NP grass is from Taiwan livestock products test institute (Taiwan Livestock Research Institute) (letter China (Hsin-hua), Taiwan south (Tainan, Taiwan)).Carboxymethyl cellulose (CMC), glucose, β-in nitro-phenyl-glucoside (β-pNPG), Whatman (Whatman) No. 1 paper and this research use all chemical substances all purchased from Sigma (Sigma).CMC, Whatman No. 1 paper and β-pNPG are respectively the substrate that CMCase, beta-glucosidase enzyme and FPase measure.Be that the CMC of 1% (w/v), glucose and NP grass is used as carbon source to check the output of 3 kinds of fungi cellulases by concentration.By air-dry for NP grass, use knife mill to cut into pieces, grind to form comparatively small-particle, be then finally separated through 0.45mm screen cloth.The preparation of substratum will be used for by the part of screen cloth and available commodity chemicals.

2.2 inoculation and culture condition

Fp and RS-N bacterial strain to be maintained at 28 DEG C on PDA agar 5 to 7 days, at RS-A being maintained at 35 DEG C 5 to 7 days simultaneously, now, sporulation all right.Use the fresh mycelia of culture since then as inoculum.By the water washing slant culture with 5ml sterilizing, obtained conidial suspension.Use hemocytometer to count spore suspension, obtain 10 ⁸individual spore/milliliter.2 of Fusarium, Eurotium and Neurospora milliliters of spore suspensions are grown in the 250ml flask containing 100ml MR substratum (not containing 0.1% soy peptone), wherein uses 1%NP grass, 1%CMC or 1% glucose to check that cellulase produces as carbon source respectively.Between 5 days incubation periods, be recorded in the cellulase activity of the 3 kinds of fungies grown in different carbon source.For the optimum concn of NP grass, adopt the NP of different weight percentage and cultivate with the MR substratum not containing 0.1% soy peptone under corresponding optimum growth conditions.

The 2.3 robust fibre zymins obtained by 3 kinds of fungies

To stem from cultivate with 1%NP grass Fusarium, Eurotium and Neurospora nutrient solution 8, under 000 × g centrifugal 20 minutes with the substrate removing mycelia and do not utilize.Supernatant liquor filters via Whatman No. 1 paper and 0.45 μm of film, and then concentrated by using and having Amy health (Amicon) concentrating unit of 3kDa film (Vivaspin 20) to filter, wherein in filtration procedure, FPase loss of activity is less than 3%.0.01% (v/w) sodiumazide (NaN is added in supernatant liquor ₃) to prevent microorganism growth.Containing 0.01%NaN ₃can store at 4 DEG C with the concentrated crude zyme preparation of 0.01% phenylmethanesulfonyl fluoride (PMSF, a kind of serpin), and still can retain the activity of 80% after 1 week.Use crude zyme preparation studying enzyme feature, comprise optimal ph, pH stability, optimum temps and thermostability.

2.4 cellulases (CMCase, FPase and beta-glucosidase enzyme) activation analysis

For carbon source active testing, it is active that corresponding 1% substrate described in using method measures the CMCase of the thick enzyme extract of Fusarium, beta-glucosidase enzyme and FPase at 40 DEG C, but for Eurotium and Neurospora, then measure at 50 DEG C.CMCase is measured by measuring the reducing sugar discharged by 1%CMC in 10 minutes in 200 μ l reaction mixtures (the thick enzyme of 100 μ l and 2% substrate of 100 μ l in 100mM NaOAC, 100mM NaCl (pH 5.0)).Use 3mg No. 1 (Whatman) filter paper as substrate, in 200 μ l reaction mixtures (as above), measure FPase through 1 hour active.Use DNS method people such as (, 1988) Woods (Wood) improved, to heat at 100 DEG C after 10 minutes at interpolation 200 μ l dinitrosalicylic acid (DNS), measure the total reducing sugars of release.Unit (IU) CMCase and FPase activity is defined as, and under analysis condition, per minute discharges the enzyme amount of 1 μm of ol glucose (as reducing sugar equivalent).For beta-glucosidase activity, by β-pNPG (ultimate density: 1mM) and the 10 μ l enzyme solution prepared in sodium acetate buffer (100mM NaOAC, 100mM NaCl, pH5.0) are mixed to get the analysis that 150 μ l working volumes carry out 3 kinds of fungies.Cultivate after 10 minutes, by adding 50 μ l 2M sodium carbonate termination reactions.Under 400nm, the absorbancy of the reaction mixture of 3 kinds of fungies is measured with spectrophotometer.Beta-glucosidase activity is calculated based on 4-nitrophenols working curve.The enzymic activity of a unit is defined as the equivalents that per minute discharges 1 μm of ol 4-nitrophenols.

3. result and discussion

The impact in carbon (C) source

The such as cultivation such as substratum complicacy and substrate structure complicacy factor may affect filamentous fungus secretion lytic enzyme people such as (, 2001) Ku Erqinke (Kurchenko).In this research, estimate that different carbon sources (i.e. NP grass and CMC) improves the potential of the Cellulase secretion of Fusarium, Eurotium and Neurospora.Result shows, and 3 kinds of rear total fibers element enzymic activity (FPase) of fungi growth in the substratum supplemented without carbon source are extremely low.Once cultivate (table 1 and Fig. 1) under at existence 1% (w/v) NP grass but without organonitrogen condition, namely FPase activity significantly increases about 60 to 105 times (namely for Fusarium, Eurotium and Neurospora, increase to 0.062U/ml, from 0.008 increasing to 0.085U/ml with from 0.002 increasing to 0.124U/ml from 0 respectively), this represents a large amount of cellulase of NP grass induction generation.CMCase and beta-glucosidase activity also show similar improvement (table 1), and compared with FPase, the active degree improved is larger.In addition, with 1%NP grass cultivate batch FPase activity be with 1%CMC cultivation batch about 1 to 23 times.These results show, NP grass can be used as desirable carbon source and produces with the cellulase improveing the cellulose decomposition fungi be separated in this research.

In addition, assessment produces the optimum N P grass concentration of cellulase further.Usually, for Fusarium and Neurospora, the active amount with NP grass of FPase raises and strengthens, but except 5%NP (Fig. 2 a and Fig. 2 c).FPase maximum value is there is in Fusarium, Eurotium and Neurospora after cultivating 4 to 5 days respectively under 2%, 5% and 3%NP grass exist; This value is even higher than the value of 1%NP.Substratum based on the NP grass expection of researching and developing in this research can save the culture medium cost of 50%, and carbon source cost is generally (table 2) over half of total culture medium cost.Infer accordingly, NP grass can be the comparatively Jia Diwuyuan for economic cellulase production.

Table 1. uses 1% (v/w) NP grass to strengthen Mierocrystalline cellulose (FPase, CMCase and the beta-glucosidase enzyme) output of 3 kinds of fungies as primary carbon source.

^*cultivate after 5 days under corresponding optimum growth conditions, in the MR substratum of not soybean protein-containing peptone, test each fungal bacterial strain.

Table 2. uses Herba penniseti as carbon source saving at least 50% culture medium cost compared with the carbon source determined (CMC or glucose)

^*trace element is added to the MR substratum supply nitrogenous source of not soybean protein-containing peptone.

Conclusion

When using NP grass as primary carbon source, FPase activity that 3 kinds of fungies reach is use 1%CMC as 1.1 to 23 times of FPase activity during primary carbon source; And be 60 to 105 times without FPase activity during NP grass.In studied carbon source (i.e. glucose, CMC and NP grass), use the substratum of optimum N P grass concentration not only to reach best cellulolytic activity, but also be combination the most cheap in the carbon source of all tests.Above character shows, the cellulase deriving from RS-A, Fp and RS-N can be used as the key enzyme being prepared bio-ethanol by Mierocrystalline cellulose.

Other embodiment

The all features disclosed in this specification sheets may be combined with into any combination.Each feature disclosed in this specification sheets all can by for identical, quite or similar object alternative characteristics replace.Thus, unless separately clearly defined, otherwise each feature disclosed just general quite or similar characteristics series example.

Many embodiments of the present invention have been described.But should be understood that and can carry out various amendment when not deviating from the spirit and scope of the present invention.Therefore, other embodiment is in the scope of following claims.

Reference:

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Claims (14)

1. cultivate a cellulose decomposition method of microorganism, it comprises

Acquisition comprises 0.1% to 5% (w/v) wolf tail (Napier; NP) substratum of grass (napier grass (Pennisetum purpureum)) fragment; With

In described substratum, under the condition allowing the growth of cellulose decomposition microbial cell, cultivate described cell;

Wherein said cellulose decomposition microorganism is selected from the fungi of Fusarium (Fusarium spp.), Eurotium (Aspergillus spp.) and Neurospora (Neurospora spp.) for one or more.

2. method according to claim 1, wherein said substratum comprises 1% to 3% (w/v) wolf tail (NP) grass (napier grass) fragment.

3. method according to claim 1 and 2, wherein said substratum is through sterilizing.

4. method according to claim 1 and 2, wherein said substratum comprises microbiotic further.

5. produce a method for cellulase, it comprises

Obtain the substratum defined in the method according to claim arbitrary in Claims 1-4; With

Allowing the cellulose decomposition microbial cell of the nucleic acid cultivated under the condition expressing cellulase containing the described cellulase of coding; With

From cellulase described in described cultured cells or described substratum purifying;

Wherein said cellulose decomposition microorganism is selected from the fungi of Fusarium (Fusarium spp.), Eurotium (Aspergillus spp.) and Neurospora (Neurospora spp.) for one or more.

6. a composition, it comprises the substratum and cellulose decomposition microbial cell that define in the method according to claim arbitrary in Claims 1-4, and wherein said cellulose decomposition microorganism is selected from the fungi of Fusarium (Fusarium spp.), Eurotium (Aspergillus spp.) and Neurospora (Neurospora spp.) for one or more.

7. produced a method for fermentable sugars by lignocellulosic materials, it comprises

Providing package is containing composition according to claim 6;

Described composition is made to contact to produce fermentable sugars with lignocellulosic materials;

Wherein said cellulose decomposition microorganism is selected from the fungi of Fusarium (Fusarium spp.), Eurotium (Aspergillus spp.) and Neurospora (Neurospora spp.) for one or more.

8. method according to claim 7, wherein said fermentable sugars is selected from the group be made up of glucose, wood sugar, pectinose, semi-lactosi, seminose, rhamnosyl, sucrose, fructose, lactose, maltose, trehalose, cellobiose, oligosaccharides and any combination thereof.

9. method according to claim 7, wherein said lignocellulosic materials is selected from the group be made up of cellulosic animal waste, municipal solid waste, waste paper, garden waste material, agricultural residue, forestry residue and any combination thereof.

10. the method according to claim arbitrary in claim 7 to 9, it comprises further described fermentable sugars is changed into tunning.

11. methods according to claim 10, wherein said step of converting is undertaken by fermentable or ferment treatment.

12. 1 kinds by the energy-producing method of lignocellulosic materials, it comprises

Composition according to claim 6 is provided;

Described composition is made to contact to produce fermentable sugars with described lignocellulosic materials;

Transform described fermentable sugars to produce combustible fermentation product; With

Burn described combustible fermentation product or water-disintegrable solid waste or resistates with generate energy.

13. 1 kinds of bio-reactors, it contains lignocellulosic materials and composition according to claim 6.

14. 1 kinds of methods for feed or foodstuff additive processing, bio-pulping or mixture process, it comprises

Providing package is containing composition according to claim 6; With

Described composition is contacted with material that is to be processed or that process;

Wherein said cellulose decomposition microorganism is selected from the fungi of Fusarium (Fusarium spp.), Eurotium (Aspergillus spp.) and Neurospora (Neurospora spp.) for one or more.