CN101486988B - Method for degrading ligno-cellulose and special cellulose decomposing bacteria therefor - Google Patents
Method for degrading ligno-cellulose and special cellulose decomposing bacteria therefor Download PDFInfo
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Abstract
The invention discloses a lignocellulose decomposing method and a specific cellulose decomposing fungus strain thereof. The invention provides a cellulose decomposing fungus strain L-A with CCTCC NO. of M208163, and further provides a culture medium that is used for cultivating the cellulose decomposing fungus strain L-A with CCTCC NO. of M208163, as well as applications of the cellulose decomposing fungus strain L-A with CCTCC NO. of M208163 in lignocellulose decomposition. The lignocellulose decomposing method is favorable for solving the current situation that lignocellulose resource cannot reach complete utilization. The lignocellulose that is preliminarily decomposed by the cellulose decomposing fungus strain L-A can be used as fertilizer for improving and reserving soil fertility, as poultry feedstuff for promoting the development of animal husbandry, and as raw materials of marsh gas fermentation and ethanol production for obtaining clean energies. The lignocellulose decomposing method and the specific cellulose decomposing fungus strain transform cheap regenerative plant cellulose resources into environment-friendly products, thus changing waste into valuables and having great economic value and social significance.
Description
Technical field
The method and the special cellulose thereof that the present invention relates to a kind of lignocellulose degradation decompose fungus strain.
Background technology
Lignocellulose is mainly become to be grouped into for three kinds by Mierocrystalline cellulose, hemicellulose and xylogen, and they mutually combine and form complicated supramolecular complex.Lignocellulose is the abundantest biomass resource of China, comprises a large amount of agricultural crop straws, industry and forestry waste and municipal solid wastes etc.Lignocellulose resource major part fails to be fully utilized at present.Especially agricultural crop straw, it has big, the characteristics wide and wide in variety that distribute of output, and application potential is huge.After lignocellulose is broken down into small organic molecule, can be used as fertilizer, can be used as animal and fowl fodder, can be used as the raw material of biogas fermentation and alcohol production and obtain clean energy to promote animal husbandry development to improve and preservation of fertility.Therefore, separation and Culture has the microorganism of cellulose-decomposing ability, with the renewable plant cellulose resource conversion of cheapness is environment amenable product, turns waste into wealth, and is one of the effective way that solves China's energy and environment problem, realizes the Sustainable development of China's agricultural.
The researchist finds when separation screening has the bacterium of decomposition of cellulose ability, the bacterium with cellulose-decomposing ability that is separated in the experiment all is the composite microbial system that two or more bacterium is formed, and independent bacterium is difficult to or can only faint decomposition of cellulose.That is to say that bacterium is finished by two or more bacterium mixed fermentives cellulosic efficient decomposition.In order to study the cellulosic mechanism of bacterial degradation better, the population composition of analyzing bacterium in the composite microbial system is necessary and important.
Past is adopted traditional isolation cultivation method mostly to the research of microbial diversity and microbial population composition, but the microorganism of some kind is difficult to realize pure culture, thereby is difficult to utilize traditional microorganism separation and Culture technology to obtain complete microbial population information in the sample.The research that does not rely on traditional cultured microorganism population composition that develops into of Protocols in Molecular Biology provides strong support.The Protocols in Molecular Biology that is applied to microbial population structural research mainly comprises denaturing gradient gel electrophoresis (Denaturing Gradient GelElectrophoresis, DGGE) technology, single strand conformation polymorphism (Single-Strand ConformationPolymorphism, SSCP) technology, fluorescence in situ hybridization (Fluorescent In SituHybridization, FISH) technology and 16S rDNA clone library technology etc.Wherein, 16S rDNA clone library technology has been widely used in the multifarious research of environmental microorganism, it both can the analytic sample microorganism kind, can reflect the relative proportion of various microorganisms in the sample in relative quantification ground again, the most important thing is to detect the microorganism that can not under laboratory condition, obtain pure culture by this technology.The structure of 16S rDNA clone library and analysis comprise following a few step: at first extract sample total DNA, by round pcr amplification 16S rRNA gene; 16S rDNA fragment after the amplification is linked to each other with carrier, over to the intestinal bacteria of changing then; Set up clone library by selecting positive colony; Insertion sequencing fragment with positive colony, utilize Blast software in 16S rDNA sequence that order-checking obtains and NCBI, known 16S rDNA sequence to compare, and utilize other software to analyze, just know that the bacterial classification population in the library is formed and relative populations.
Summary of the invention
The method and the special cellulose thereof that the purpose of this invention is to provide a kind of lignocellulose degradation decompose fungus strain.
Cellulose-decomposing bacterium provided by the present invention system, name is called L-A, and (be called for short CCTCC, the address is: Wuhan University), preserving number is CCTCC NO:M208163 to be preserved in Chinese typical culture collection center on October 17th, 2008.
This cellulose-decomposing bacterium is that L-A has capacity of decomposition efficiently to lignocellulose.
The present invention also provides a kind of being used to cultivate the substratum that cellulose-decomposing bacterium is L-A CCTCC NO:M208163, and every liter of substratum is composed of the following components: yeast powder, 1-10g; Casein peptone, 1-2g; K
2HPO
4, 0.1-3g; MgSO
47H
2O, 0.1-1g; CaCl
2H
2O, 0.5-1.5g; NaHCO
3, 0.2-1.5g; NaCl, 0.1-1g; NH
4Cl, 0.1-1g; FeCl
3, 0.001-0.01g; All the other are water.
The pH of described substratum can be 6.8-8.0.
It is the application of L-A CCTCC NO:M208163 in lignocellulose degradation that the present invention also protects cellulose-decomposing bacterium.
Lignocellulose is the abundantest biomass resource of China, comprises a large amount of agricultural crop straws, industry and forestry waste and municipal solid wastes etc.Cellulose-decomposing bacterium of the present invention is the degraded that L-A CCTCC NO:M208163 can be applicable to various lignocelluloses, as agricultural crop straw, straw, bagasse etc.
In the described application, the temperature of lignocellulose degradation can be 30-60 ℃, is preferably 40-50 ℃.
The present invention also protects a kind of method of lignocellulose degradation, is that the described cellulose-decomposing bacterium of cultivation is L-A in containing the substratum of lignocellulose.
The described substratum that contains lignocellulose is that to be used for cultivating cellulose-decomposing bacterium be that the substratum of L-A CCTCCNO:M208163 adds lignocellulose and obtains described.
In the method for described lignocellulose degradation, the culture temperature of cultivating described cellulose-decomposing bacterium and be L-A is 30-60 ℃, is preferably 40-50 ℃.
Cultivate described cellulose-decomposing bacterium and be L-A or use described cellulose-decomposing bacterium when being L-A, can adopt the rotating speed of 150-200rpm.
The present invention helps to solve the present situation that the lignocellulose resource can not be fully utilized.Adopting cellulose-decomposing bacterium of the present invention is after L-A tentatively degrades lignocellulose, can be used as fertilizer to improve and preservation of fertility, can be used as animal and fowl fodder to promote animal husbandry development, can be used as the raw material of biogas fermentation and alcohol production and obtain clean energy.The present invention is environment amenable product with the renewable plant cellulose resource conversion of cheapness, turns waste into wealth, and has great economic worth and social effect.
Description of drawings
Fig. 1 is the analysis process figure that bacterial population is formed among the L-A for cellulose-decomposing bacterium.
Fig. 2 is the gel electrophoresis figure of 16S rDNA amplified production.
Fig. 3 is the gel electrophoresis figure of the 16S rDNA amplified production of purifying recovery.
Fig. 4 is the gel electrophoresis figure of colony PCR amplification product.
Fig. 5 carries out restriction analysis for the colony PCR amplification product to 30 positive colonies.
Fig. 6 shows that cellulose-decomposing bacterium is the decomposition of L-A to filter paper.
Fig. 7 shows that cellulose-decomposing bacterium is the decomposition of L-A to straw.
Fig. 8 shows that cellulose-decomposing bacterium is the decomposition of L-A to bagasse.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
Every liter of substratum contains following component: yeast powder, 5g; Casein peptone, 1.5g; K
2HPO
4, 1.5g; MgSO
47H
2O, 0.5g; CaCl
2H
2O, 1g; NaHCO
3, 0.9g; NaCl, 0.6g; NH
4Cl, 0.6g; FeCl
3, 0.006g; PH7.2.
The culture medium preparation method is: FeCl
3Be mixed with the storage liquid of 1g/L.Take by weighing the corresponding medicine of residue in proportion and pour in the beaker that part of running water is housed in advance, medicine fully dissolves the back and is settled to 990ml with tap water, adds the FeCl of 6ml
3Storage liquid is transferred pH to 7.2, and last constant volume is to 1000ml.
Every 10ml substratum branch is installed in the common test tube (placing the filter paper bar of 12 * 100mm in advance) of 18 * 180mm.
One, cellulose-decomposing bacterium is the acquisition of L-A
The anaerobic activated sludge sample that 0.5g is picked up from Guangxi cassava alcohol waste liquid UASB-TLP treatment system is inoculated in the test tube that substratum is housed.The enrichment screening is carried out in test tube speed oscillation cultivation with 180rpm under 40-50 ℃ temperature condition.After 72 hours, the inoculum size of the nutrient solution in the test tube with 5% (volume percent) is inoculated in the new test tube that substratum is housed.Shaking culture is carried out until the complete disintegration of filter paper (15-20 hour) in the inoculation back under 40-50 ℃ of temperature.Repeat to transfer 3 times by same procedure, obtain cellulose-decomposing bacterium system.
Two, cellulose-decomposing bacterium is the analysis that bacterial population is formed among the L-A
Analysis process figure sees Fig. 1.
1, the extraction of total DNA
The cellulose-decomposing bacterium system that step 1 is obtained is seeded in the substratum, and inoculum size is 5% (volume percent), cultivates 20 hours for 40-50 ℃.
The 1ml nutrient solution is put into 1.5ml Eppendorf pipe, 12000rpm, 4 ℃ of centrifugal 5 minutes collection thalline wash thalline 2-3 time with 1 * TES.Thalline is suspended among the 435 μ l TES again breaks up thalline, add N,O-Diacetylmuramidase (20mg/ml) 125 μ l, Proteinase K (10mg/ml) 2.5 μ l, 37 ℃ of water-baths are gently shaken and are incubated 4 hours, add 50 μ l 20%SDS, gently shaking in 37 ℃ of water-baths is incubated 30 minutes, adds 5M NaClO
4125 μ l do not have protein film to the interface 3-4 time with the extracting of equal-volume (560 μ l) phenol-chloroform-primary isoamyl alcohol (25: 24: 1) and occur.Drawing the upper strata water places ice bath to add the 3M NaAc-EDTA-Na of 1/10 volume
2, add 2 times of cold dehydrated alcohols of volume, upset mixing, ice bath 5 minutes.Centrifugal 20 minutes deposit D NA of 12000rpm.Add 70% ethanol desalination, 4 ℃ of placements of spending the night, centrifugal 10 minutes of 7000rpm, sucking-off ethanol.Air-dry, add the second distillation water dissolution.Transferring DNA concentration after the electrophoresis detection is 25 μ g/ml.
2, pcr amplification 16S rDNA partial sequence
Universal primer: 530F (5 '-GTG CCA GCA/G GCC GCG G-3 ')
1490R(5′-GTG?CCA?GCA/G?GCC?GCG?G-3′)
PCR reaction system (50 μ l):
5 * damping fluid, 10.0 μ l
Mg
2+(25mM) 3.0μl
dNTP(10mM) 1.0μl
530F(50μM) 0.5μl
1490R(50μM) 0.5μl
Template DNA 1.0 μ l
GoTaq archaeal dna polymerase 0.25 μ l
ddH
2O 33.75μl
The PCR reaction conditions:
Pre-sex change: 95 ℃ of 5min;
Circulation: 30 circulations of 95 ℃ of 72 ℃ of 1min of 1min 55 2min;
Extend: 72 ℃ of 5min.
Through electrophoresis detection, prove the PCR product that obtains about 1000bp.The electrophorogram of PCR product is seen Fig. 2.
3, PCR product purification
Adopt the Wizard SV Gel and PCR Clean-up System (catalog number: A9281) purifying of Promega company.Concrete operations are: after gel electrophoresis the 1.5ml centrifuge tube is put in required band cutting-out.The weight of weighing adhesive tape, and add the film binding soln with the amount of 1 μ l/mg, 50-65 ℃ of insulation dissolving.Pouring the mixture after the dissolving into the pillar room temperature placed after 1 minute with 16000 rotating speeds centrifugal 1 minute.Adding 700 μ l washes coating solution and adds 500 μ l after centrifugal 1 minute again with same rotating speed and washed coating solution centrifugal 5 minutes.Pillar is transferred to adding 50 μ l nuclease free water in the clean centrifuge tube gently, rearmounted 4 ℃ of preservations in centrifugal 1 minute.
The electrophorogram of the PCR product that purifying reclaims is seen Fig. 3.Among Fig. 3,1,2: the PCR product that purifying reclaims.
4, the PCR product is connected with carrier
Connection carrier select for use Promega company pGEM T-easy carrier (catalog number: A1360), linked system such as following table:
4 ℃ of placements of spending the night behind the mixing.
Provide in the pGEM T-easy carrier of Control DNA for Promega company.
5, transform
Connection product transformed into escherichia coli competent cell DH5 α (Beijing thing technology limited liability company of vast safe Hang Seng) with step 4.Get 50 μ l respectively, 100 μ l, 200 μ l are coated with flat board, cultivate 12-16 hour in 37 ℃ of incubators.According to the principle picking white colony dibbling of blue hickie screening on flat board, 50 bacterium colonies of each dull and stereotyped dibbling, the position mark number (1-50) of each bacterium colony was cultivated 12 hours for 37 ℃.
6, the screening of positive colony
Adopt the bacterium colony round pcr.Thalline with a little different bacterium colony of toothpick picking (totally 60 bacterium colonies) adds respectively in the PCR pipe that 50 μ l sterilized waters are housed in advance, carries out mark (identical with the lable number on the flat board).With the PCR pipe in 95 ℃ the heating 5 minutes, be chilled to room temperature after, centrifugal 5 minutes of 15000rpm.Getting 10 μ l supernatants is that template is carried out PCR, and primer is SP6 (5 '-CAT ACG ATT TAG GTG ACA CTA TAG-3 ') and T7 (5 '-TAATAC GAC TCA CTA TAG GG-3 ').Bacterium colony PCR system composition and reaction conditions are identical with above-mentioned 16S rDNA amplification condition.Get 10 μ l PCR products and determine positive colony, determined the positive bacterium colony of 43 strains altogether through gel detection.The gel detection of part bacterium colony the results are shown in Figure 4.Among Fig. 4,1-15: the amplified production of bacterium colony PCR.
7, with restriction enzyme Msp I the colony PCR amplification product of 30 positive colonies is carried out restriction analysis
It is as follows that enzyme is cut system (25 μ l):
Deionized water 9.5 μ l
10 * damping fluid, 5 μ l
Msp?I(10U/μl) 0.5μl
Mix rearmounted 37 ℃ water bath heat preservation 2-5 hour.
Enzyme cuts the back to place 30 minutes termination enzymes at 60-70 ℃ and cuts.Get 10 μ l enzymes and cut product and carry out electrophoresis detection proof and have 5 kinds of different enzymes to cut banding pattern, 5 OTU (Operational Taxonomic Unit) are promptly arranged, see Fig. 5.Among Fig. 5, swimming lane 1,6,7,8,9 is first kind of banding pattern; Swimming lane 3,10,11 is second kind of banding pattern; Swimming lane 4,5 is the third banding pattern; Swimming lane 2 is the 4th a kind of banding pattern; Swimming lane 12 is the 5th a kind of banding pattern.
8, positive colony order-checking and Phylogenetic Analysis
Picking represents the positive colony colony inoculation of 5 different banding patterns on the LB flat board, send order-checking company to check order after 37 ℃ of incubated overnight.Institute calling sequence input NCBI website is compared with existing sequence among Blast program and the Genbank.The result shows: the bacterial population of cellulose-decomposing bacterium system is made up of 5 kinds of bacteriums (bacterium A, bacterium B, bacterium C, bacterium D and bacterium E).Bacterium A belongs to bacillus pumilus and belongs to (Brevibacillus), reaches 99-100% with the homology of Brevibacillus brevis, and bacterium A accounts for 35% of total plate count.Bacterium B, bacterium C, bacterium D and bacterium E all belong to series bacillus and belong to (Paenibacillus).The homology of bacterium B and Paenibacillus illinoisensis reaches 96%, the homology of bacterium C and Paenibacillusdaejeonensis reaches 96%, the homology of bacterium D and Paenibacillus lentimorbus reaches 95%, and the homology of bacterium E and Paenibacillus sp. reaches 94%.Bacterium B accounts for 5% of total plate count.Bacterium C accounts for 50% of total plate count.Bacterium D accounts for 5% of total plate count.Bacterium E accounts for 5% of total plate count.
The cellulose-decomposing bacterium that step 1 is obtained is called after L-A, on October 17th, 2008 it is stored in Chinese typical culture collection center (address: in the Wuhan University), and preservation registration number is CCTCC NO:M208163.
Every liter of substratum contains following component: yeast powder, 5g; Casein peptone, 1.5g; K
2HPO
4, 1.5g; MgSO
47H
2O, 0.5g; CaCl
2H
2O, 1g; NaHCO
3, 0.9g; NaCl, 0.6g; NH
4Cl, 0.6g; FeCl
3, 0.006g; PH7.2.
The culture medium preparation method is: FeCl
3Be mixed with the storage liquid of 1g/L.Take by weighing the corresponding medicine of residue in proportion and pour in the beaker that part of running water is housed in advance, medicine fully dissolves the back and is settled to 990ml with tap water, adds the FeCl of 6ml
3Storage liquid is transferred pH to 7.2, and last constant volume is to 1000ml.
Every 10ml substratum branch is installed in the common test tube (placing the filter paper bar of 12 * 100mm in advance) of 18 * 180mm.
The cellulose-decomposing bacterium that the step 1 of embodiment 1 is obtained is that L-A is seeded in the test tube that contains substratum, and inoculum size is 5% (volume percent), inoculates 40 test tubes.40 samples are divided into four groups, every group of 10 samples.Four groups of samples are cultivated with the speed oscillation of 180rpm under 30-40 ℃, 40-50 ℃, 50-60 ℃, 70 ℃ temperature condition respectively.
At first grow yellow bacterium colony on the filter paper bar in the test tube of under 40-50 ℃ of condition, cultivating and also clear up gradually, except that 70 ℃, under other temperature condition, also successively grow yellow bacterium colony on the filter paper bar and also clear up gradually.Show that cellulose-decomposing bacterium is that L-A all can grow at 30-60 ℃, optimum growth temperature is 40-50 ℃.
Every liter of substratum contains following component: yeast powder, 1g; Casein peptone, 1g; K
2HPO
4, 0.1g; MgSO
47H
2O, 0.1g; CaCl
2H
2O, 0.5g; NaHCO
3, 0.2g; NaCl, 0.1g; NH
4Cl, 0.1g; FeCl
3, 0.001g; PH6.8.
The culture medium preparation method is: FeCl
3Be mixed with the storage liquid of 1g/L.Take by weighing the corresponding medicine of residue in proportion and pour in the beaker that part of running water is housed in advance, medicine fully dissolves the back and is settled to 990ml with tap water, adds the FeCl of 1ml
3Storage liquid is transferred pH to 6.8, and last constant volume is to 1000ml.
Every 10ml substratum branch is installed in the common test tube of 18 * 180mm.
Get 30 test tubes that substratum is housed, be divided into three groups, 10 every group.In first group of test tube, add oven dry straw (adding 0.1g oven dry straw in each test tube), in second group of test tube, add oven dry bagasse (adding 0.3g oven dry bagasse in each test tube).In the 3rd group of test tube, place the filter paper bar of 12 * 100mm.
The cellulose-decomposing bacterium that the step 1 of embodiment 1 is obtained is that L-A is seeded in each test tube, inoculum size is 5% (volume percent), 40 ℃, 150rpm shaking culture, and the result shows, filter paper is disintegration fully in 24 hours, and straw and bagasse are in the two most of decomposition in week back.
Fig. 6 is the decomposition of L-A to filter paper for cellulose-decomposing bacterium; A: the inoculation cellulose-decomposing bacterium is the filter paper before the L-A; B: the inoculation cellulose-decomposing bacterium is 24 hours a filter paper behind the L-A.
Fig. 7 is the decomposition of L-A to straw for cellulose-decomposing bacterium; A: the inoculation cellulose-decomposing bacterium is the straw before the L-A; B: the inoculation cellulose-decomposing bacterium is the straw of L-A after two weeks.
Fig. 8 is the decomposition of L-A to bagasse for cellulose-decomposing bacterium; A: the inoculation cellulose-decomposing bacterium is the bagasse before the L-A; B: the inoculation cellulose-decomposing bacterium is the bagasse of L-A after two weeks.
The result shows: cellulose-decomposing bacterium is that L-A decomposes obviously filter paper, straw and bagasse.
The straw weight that adds is deducted the dry weight of the dregs that obtain after filtration after the decomposition, be the reduction (bagasse with) of straw weight.
After two weeks, the weight of straw reduces 71%, and the weight of bagasse reduces 64%.
Every liter of substratum contains following component: yeast powder, 10g; Casein peptone, 2g; K
2HPO
4, 3g; MgSO
47H
2O, 1g; CaCl
2H
2O, 1.5g; NaHCO
3, 1.5g; NaCl, 1g; NH
4Cl, 1g; FeCl
3, 0.01g; PH8.0.
The culture medium preparation method is: FeCl
3Be mixed with the storage liquid of 1g/L.Take by weighing the corresponding medicine of residue in proportion and pour in the beaker that part of running water is housed in advance, medicine fully dissolves the back and is settled to 990ml with tap water, adds the FeCl of 10ml
3Storage liquid is transferred pH to 8.0, and last constant volume is to 1000ml.
Every 10ml substratum branch is installed in the common test tube of 18 * 180mm.
Get 30 test tubes that substratum is housed, be divided into three groups, 10 every group.In first group of test tube, add oven dry straw (adding 0.1g oven dry straw in each test tube), in second group of test tube, add oven dry bagasse (adding 0.3g oven dry bagasse in each test tube).In the 3rd group of test tube, place the filter paper bar of 12 * 100mm.
The cellulose-decomposing bacterium that the step 1 of embodiment 1 is obtained is that L-A is seeded in each test tube, inoculum size is 5% (volume percent), 50 ℃, 200rpm shaking culture result show that filter paper is disintegration fully in 24 hours, and straw and bagasse are in two week backs most of decomposition.。
The result shows: cellulose-decomposing bacterium is that L-A decomposes obviously filter paper, straw and bagasse.
The straw weight that adds is deducted the dry weight of the dregs that obtain after filtration after the decomposition, be the reduction (bagasse with) of straw weight.
After two weeks, the weight of straw reduces 68%, and the weight of bagasse reduces 70%.
Claims (9)
1. cellulose-decomposing bacterium is L-A CCTCC NO:M208163.
2. cellulose-decomposing bacterium is the application of L-A CCTCC NO:M208163 in lignocellulose degradation.
3. application as claimed in claim 2 is characterized in that: described lignocellulose is straw or bagasse.
4. as claim 2 or 3 described application, it is characterized in that: in the described application, the temperature of lignocellulose degradation is 30-60 ℃.
5. application as claimed in claim 4 is characterized in that: in the described application, the temperature of lignocellulose degradation is 40-50 ℃.
6. the method for a lignocellulose degradation is that the described cellulose-decomposing bacterium of cultivation claim 1 is L-A in containing the substratum of lignocellulose.
7. method as claimed in claim 6 is characterized in that: the described substratum that contains lignocellulose is that to cultivate cellulose-decomposing bacterium in as described below being used for be that the substratum of L-A CCTCC NO:M208163 adds lignocellulose and obtains;
It is described that to be used to cultivate cellulose-decomposing bacterium be that every liter of substratum of substratum of L-A CCTCC NO:M208163 is composed of the following components: yeast powder, 1-10g; Casein peptone, 1-2g; K
2HPO
4, 0.1-3g; MgSO
47H
2O, 0.1-1g; CaCl
2H
2O, 0.5-1.5g; NaHCO
3, 0.2-1.5g; NaCl, 0.1-1g; NH
4Cl, 0.1-1g; FeCl
3, 0.001-0.01g; All the other are water.
8. as claim 6 or 7 described methods, it is characterized in that: in the described method, the culture temperature of cultivating described cellulose-decomposing bacterium and be L-A is 30-60 ℃.
9. method as claimed in claim 8 is characterized in that: in the described method, the culture temperature of cultivating described cellulose-decomposing bacterium and be L-A is 40-50 ℃.
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