CN101591679B - Method for improving utilization rate in native grass-based fuel ethanol production by using mixed strains - Google Patents
Method for improving utilization rate in native grass-based fuel ethanol production by using mixed strains Download PDFInfo
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- CN101591679B CN101591679B CN2009100320210A CN200910032021A CN101591679B CN 101591679 B CN101591679 B CN 101591679B CN 2009100320210 A CN2009100320210 A CN 2009100320210A CN 200910032021 A CN200910032021 A CN 200910032021A CN 101591679 B CN101591679 B CN 101591679B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of perennial grass-based green renewable biomass fuel ethanol production and particularly relates to a method for improving the utilization rate in native perennial grass and mixed grass thereof-based green renewable biomass fuel ethanol production by using self-screened, mixed and optimized strains. The method comprises the following steps: firstly, selecting lignin degrading bacteria and cellulose degrading bacteria, wherein the lignin degrading bacteria and the cellulose degrading bacteria can grow together; and secondly mixing coriolus versiclor, laetiporus sulphureus, trichoderma viride, aspergillus niger and hairy mould two by two by an engineering preparation mode of solid state fermentation rescreening, strain combination optimization and mixed strain optimization, wherein solid state fermentation preferably selects mixed strain combinations of coriolus versiclor and trichoderma viride as well as coriolus versiclor and hairy mould, and the solid state fermentation of native perennial mixed grass improves lignin degradation rate by 46.25 percent, cellulose degradation rate by 10.09 percent and hemicelluloses by 11 percent compared with single-strain fermentation. The method is simple and easy in operation, effectively improves the lignin degradation rate and the utilization rate and conversion rate of wood fiber substance, greatly reduces production cost, improves the utilization rate and conversion rate in the perennial nativegrass-based fuel ethanol production and provides scientific support for the development of energy grass industry.
Description
One, technical field
The invention belongs to and utilize natural perennial grass to produce green renewable biomass alcohol fuel technical field; Be specifically related to utilize the hybrid optimization bacterial classification of autonomous screening, improve the method that natural perennial grass and miscellany thereof grass is produced green renewable biomass alcohol fuel utilization ratio.
Two, background technology
The green bio alcohol fuel is not add denaturing agent, the absolute ethyl alcohol that can directly act as a fuel and use, and this fuel in use can reduce the consumption to Nonrenewable energy resources; The amount of hydrocarbon gas of emitting after the burning and carbon oxide gas obviously is less than the amount of this type of gas that regular price gasoline burning produced, thereby can effectively reduce Greenhouse effect; The alcohol fuel storing is convenient, and raw material sources is green low-carbon gasoline in the renewable material of green.Therefore, the use of alcohol fuel meets the needs of the energy, environment, agricultural development, is for solving a kind of novel, green, the reproducible substitute energy that a series of problems such as energy dilemma, environmental degradation, food shortage grow up.
See that from feedstock property two stages have been experienced in the development of alcohol fuel.Fs is to be the developmental stage of main production raw material with carbohydrate, starch based: this stage production technique is simple; But raw materials cost accounts for more than 40% of production cost; And the increase of output has certain limit; Cost also is difficult to reduce, and is example with american corn raw material production alcohol fuel: be used for 2,500,000 tons of the every increases of corn of fuel ethanol production, the corn price 1.20-2.00 dollar that just goes up; It is more limited that China can be used for the grain raw material of Production of Fuel Ethanol, forms the awkward present situation that fuel ethanol production " is striven ground with grain, striven grain with the people "; Subordinate phase is to be master's development stages to utilize the lignocellulose material: this type substance classes is various, is renewable resources the abundantest on the earth; Year living weight is huge, is containing huge biomass energy, can furnish ample material for fuel ethanol production.Natural perennial grass monoid is wherein a kind of as the lignocellulose material, in the production of fuel ethanol process, have raw materials cost inexpensive, be prone to obtain, the raw material year-round supply; Do not occupy cultivated land with the farmland, do not strive feed with livestock; The ecological benefits outstanding feature is a kind of raw material that has using value in the fuel ethanol production process.
With respect to carbohydrate and starch based crop, be that substrate production alcohol fuel process exists certain bottleneck with the lignocellulose material, this is because the complex structure of lignocellulose material is difficult to degraded.The lignocellulose material mainly comprises three kinds of staples: Mierocrystalline cellulose, semicellulose and xylogen, and wherein Mierocrystalline cellulose is the bone material, xylogen and semicellulose are dispersed among the Mierocrystalline cellulose with the form of container.Xylogen is formed by lubanol, sinapyl alcohol with to the dehydrogenated polymers and the P-coumaric acid of tonquinol; Because the copolymerizationization of these three types of alcohol has produced inhomogeneous, unglazed revolving property, intersection bonding, high dispersing and the biodegradable fragrant superpolymer of high resistance; Attack to most mikrobes has resistance; Thereby the decomposition of xylogen is slow especially, and also can't form fermentable saccharide behind the lignin degradation.In complete lignum, xylogen exists with a kind of form of physical barriers, is enclosed in the Mierocrystalline cellulose periphery, and the protection Mierocrystalline cellulose exempts from mikrobe and attacks and the degrading enzyme attack, and its degraded must be decomposed prior to Mierocrystalline cellulose.Therefore, the utilization of lignocellulose material must be carried out suitable pre-treatment earlier, to remove delignification or to destroy the xylogen layer, reduces cellulosic percent crystallinity, improves cellulosic accessibility, thereby reaches the purpose of effectively utilizing fibrous matter.Conventional pretreatment mode comprises physics, chemistry, biology or hybrid system.Wherein physics pre-treatment cost is high, and condition is harsh; Chemical Pretreatment reagent reclaims difficulty, cost is high, the loss of effective components rate is big; Physical chemistry pre-treatment cost is complicated, and environmental pollution is serious, reagent reclaims problems such as difficulty.Biological Pretreatment has the treatment condition gentleness, equipment requirements is low, and therefore the advantage that processing cost is cheap, environmental pollution is little, is often used in process of production.Biological Pretreatment mainly directly acts on the lignocellulose material through the ligocellulose degradation's enzyme that is decomposed in the microorganism growth process, thereby reaches the principle of effective utilization.It is less that occurring in nature can carry out the microbe species of lignocellulose mass degradation, and exist the production by biological enzyme activity low, and long grades of degradation cycle needs the further problem of solution.Thereby effectively improving the degradation capability of mikrobe, the degradation cycle that shortens mikrobe will effectively improve the effect of Biological Pretreatment, and then improve the utilization ratio of lignocellulose material production alcohol fuel.
Occurring in nature, the degraded fully of lignocellulose material is accomplished under fungi, bacterium and the acting in conjunction of corresponding microorganism group, and wherein fungi plays a leading role.The kind of the enzyme that the different microorganisms process of growth is produced and the different amts of enzyme; The character of enzyme is also different accordingly with the action effect of enzyme; Therefore different bacterial classifications is mixed, can produce certain synergistic effect, thereby effectively improve degraded the lignocellulose material.The fungi that can carry out lignin degradation comprises whiterot fungi, brown rot fungus, soft-rot bacterium, and wherein the former two's lignin degradation ability is stronger, mainly produces lignin-degrading enzymes; The latter's cellulose degradation ability is stronger, mainly produces the cellulose degradation enzyme, and they are also corresponding different to the degradation effect of lignocellulose material.The lignocellulose degradation bacteria is mixed, utilize mutual synergistic effect between bacterial classification, the ligocellulose degradation's enzyme in conjunction with different can improve the degradation rate to the lignocellulose material, improves the usability of its fuel ethanol production accordingly.The cardinal principle of the present invention research is to utilize perennial grass effect fuel ethanol production raw material reproducible, that can provide in the anniversary, green, environmental protection; Lignin-degrading bacteria is mixed with cellulose-degrading bacteria; Utilize the difference of different strain to produce the enzyme characteristic, thereby effectively improve the method that perennial native grass produces the alcohol fuel utilization ratio.
Owing to utilize perennial native grass to produce alcohol fuel, and utilize stalk and grain in storage for years to produce alcohol fuel to compare, have raw materials cost cheap, be easy to get, renewable, not with the people strive grain, not with grain strive, the characteristics of not striving feed with livestock; And the associate strain synergy process of utilizing our independently to screen; Conditional request is easy, equipment is simple, processing cost is cheap; Can effectively improve perennial native grass production fuel ethanol production efficient; Therefore, the present invention has important and practical meanings and Scientific Application and is worth for improving utilization ratio and the transformation efficiency that natural perennial grass produces alcohol fuel.
Really, through we the national patent net (
Www.sipo.gov.cn/sipo/default.htm) go up with pertinent literature internet retrieval result and show that the research of " utilizing the hybrid optimization bacterial classification to improve perennial native grass production alcohol fuel utilization ratio " does not appear in the newspapers as yet, for we declare patent of invention foundation is provided.
Three, summary of the invention
1, the technical issues that need to address of the present invention
Key point of the present invention is to select suitable biological inoculum type, carries out the problem that the different microorganisms bacterial classification cooperatively interacts.The present invention utilizes the combined hybrid cultivation in twos of several kinds of different perennial lignin degradation bacterial classifications and cellulose degradation bacterial classification; Through associate strain primary dcreening operation, multiple sieve, optimization and engineering preparation; Searching can symplastic growth, efficiently produce the hybrid optimization flora of ligocellulose degradation's enzyme, thereby realizes regulation and control and the optimization to perennial native grass degradation process, and degradability is high; Decomposing force is strong; Can be effective utilization of perennial native grass, effective biological degradation flora is provided, thereby solve the gordian technique difficult problem of lignocellulose material production alcohol fuel.
2, technology contents main points of the present invention
(1) primary election of associate strain: through a large amount of bacterial classification tests, two kinds of lignin-degrading bacterias of initial option: variegated rainbow conk (Coriolus versicolor), Laetiporus sulphureus (Bull. Ex Fr.) Bond. Et Singer. (Laetiporus sulphureus); The bacterial classification of two kinds of efficient cellulase-producings: viride (Trichoderma viride), revolve spore bacterium (Cochliobolus.sp); The efficient bacterial classification that produces beta-glucosidase enzyme: black mold (Aspergillus niger) bacterial classification; With the bacterial classification of the bacterial classification of selecting as mix and match; Two two mix, and are basic medium with potato culture (PDA), and slant strains is inoculated on the PDA flat board; Observe the phenomenon of mixed growth between bacterial classification, observe the speed of growth of associate strain, the interactional size between outward appearance, bacterial classification of growing;
(2) solid state fermentation final election: further utilizing solid-state fermentation culture medium, is the solid state fermentation raw material with perennial native grass, carries out solid-state degradation experiment.Utilize inorganic salt solution required nutritive element to be provided for associate strain; The fixedly culture temperature of solid state fermentation and incubation time; And measure the variation of three cellulose contents in lignin degradation enzymic activity that the solid ferment process associate strain produces, cellulose degradation enzymic activity, the natural perennial grass, the associate strain type that screening can the coordinated solid state fermentation;
(3) bacterial classification optimum combination: the kind of the degrading enzyme that associate strain and single culture produced and the height of degrading enzymatic activity are compared; The preferred degrading enzymatic activity that is produced is strong; The hybrid optimization combination that lignocellulose mass degradation rate is high, the phenomenon of further observing its mixed growth.
(4) the engineering preparation of optimization associate strain: under certain conditions with the optimization associate strain that is filtered out; Become production bacterial classification through enlarged culturing with some amount and quality; With the usefulness as further expansion biomass and synthetic product, its engineering preparation comprises spore preparation and mycelium preparation.
3, beneficial effect of the present invention
(1) operating process is simple: bacterial classification mixes different with single culture Degradation principle, but processing mode is identical, utilizes the enzyme of microorganisms that perennial grass is directly acted on, so the present invention is simple to operate, treatment condition are gentle, accessibility is strong.
(2) low production cost: owing in treating processes, need not use the instrument and equipment high temperature resistant, high pressure resistant, that erosion resistance is strong; And do not need too much to use materials such as extra water, electricity, chemical reagent, thereby it is cheap to analyze input cost.
(3) can effectively improve the lignin degradation rate: because after optimizing the bacterial classification mixing; Can improve the collaborative mutually short effect of bacterial classification; Inulinase-producing activity improves greatly, the vigor of the favourable enzyme of lignin degradation is strengthened greatly, thereby decompose corresponding being enhanced of degradation capability of xylogen.Experiment based on us is visible, and after the mixed bacteria cooperation was optimized in utilization, the lignin degradation rate had improved 46.25%, and cellulose degradation rate has improved 10.09%, and the hemicellulose degradation rate has improved 11%.
(4) can effectively improve the utilization ratio of lignocellulose material: owing to utilize associate strain the degradation rate of lignocellulose is improved than single culture; Make effectively breaking that three plain crosslinking structures obtain; Thereby can effectively improve the utilization ratio of lignocellulose material; For the lignocellulose material provides competent decomposition bacterial classification, improved the production of fuel ethanol utilization ratio of raw materials.
Four, description of drawings
1, Fig. 1: show that variegated rainbow conk and viride are blended in the upgrowth situation on the flat board.
2, Fig. 2: show viride and the upgrowth situation of Laetiporus sulphureus (Bull. Ex Fr.) Bond. Et Singer. SJ on flat board.
3, Fig. 3: show variegated rainbow conk and the upgrowth situation that becomes mildewed on flat board.
4, Fig. 4: show and become mildewed and the upgrowth situation of black mold on flat board.
Five, specific embodiments
1, medium preparation and spawn culture condition:
(1) PDA substratum (gl
-1) preparation: remove skin fresh potato 200g and be cut into small pieces and put into pot, add water 1000ml, boil 20min after, use two-layer filtered through gauze.Add glucose 20g, agar 17g in the filtered liq, tap water is settled to 1000ml, 121 ℃ of sterilization 20min.Do not add corresponding agar in the PDA liquid nutrient medium.
(2) solid fermentation medium preparation: 5g cogongrass powder, add the 30ml inorganic salt solution, put into the triangular flask of 300ml, 120 ℃ of sterilization 20min.
(3) inorganic salt solution (gl
-1) preparation: the Massadeh prescription is done change a little, wherein contains NaCl 0.5, MgSO
40.5, (NH
4) SO
40.5, KH
2PO
40.5, Ca (NO
3)
20.3, FeSO
4.7H
2O 0.01, MnSO
40.015, ZnSO
4.7H
2O0.015, Co (NO
3)
20.01.
(4) spawn culture condition: bacterial classification primary election is dull and stereotyped in the PDA solid culture with multiple sieve, 28 ℃ of conditions are cultivated, associate strain in the distance of the inoculation on the flat board at a distance of about 2cm.Three kinds of bacterial classifications are respectively inoculated 3 points on flat board, form two dels; In the solid ferment process bacterial classification two two is joined mutually, three point method is inoculated in the solid fermentation substratum, and 28 ℃ of solid state fermentations are cultivated 15d.
(5) preparation of enzyme activity determination liquid: accurately take by weighing solid state fermentation appearance 1g and put into the 20ml centrifuge tube, add deionized water, be put in 150rpm.min in 1: 10 ratio
-1Behind the vibration lixiviate 1h, again centrifuge tube is put into 5000rpm.min in the shaking table
-1Centrifugal 15min in the whizzer of rotating speed, supernatant are enzyme activity determination liquid.
2, composition measurement employing method:
(1) measures composition: laccase (Lac), manganese-dependent peroxidase (MnP), LIP (LiP), carboxymethylcelluloenzyme enzyme (CMC), filter paper enzyme activity, xylogen, Mierocrystalline cellulose, semicellulose.
(2) measure the employing method: the mensuration of Lac, MnP, LiP, CMC, filter paper enzyme, beta-glucosidase activity adopts spectrophotometry; Measuring substrate is respectively methyl catechol, veratryl alcohol, manganous sulfate, sodium cellulose glycolate, filter paper and salicin, and enzymic activity is shown 1U with the mmole numerical table that PM generates substrate.The mensuration of xylogen, Mierocrystalline cellulose and semicellulose adopts improvement Wang Yuwan method, after handling with neutral and acid detergent, uses acetone and washing again, after the residue oven dry, calculates with minusing.
3, growth, product enzyme and three plain observations of decomposing variation:
(1) the upgrowth situation observation of associate strain on flat board:
The upgrowth situation of associate strain sees accompanying drawing 1-4 for details, and is visible from Fig. 1-4, after the bacterial classification combination, according to the difference of associate strain, different growth phenomenons occurs.Specifically with the speed of growth of bacterium colony, the propagation rate of bacterium colony, the covering intensity of bacterium colony is the index of observation.It is relatively slow to show as strain growth, and the bacterial strain that is constantly enlarged by another strain gradually covers; The growth of two strain bacterium is identical with expansion, in the time of being investigated, occupies the certain culture space separately, and does not have mutual intersection contact between the bacterial strain; Strain growth is very fast, and bacterium colony is expanded rapidly, but mutual contact phenomena do not occur between the bacterium colony, and tangible space is arranged between bacterium colony; The growth of two kinds of bacterial strains is all better, and the mycelia elongation is very fast, and in the contact position of bacterium colony, mycelia is interlaced.
(2) bacterial classification produces the observation of enzyme situation on solid medium:
Visible from table 2, different strain is handled the product enzyme situation on solid medium, and is visible by table 1, black mold high yield beta-glucosidase enzyme, and variegated rainbow conk high yield Lac also produces higher CMC simultaneously; Viride high yield CMC; SJ produces three kinds of cellulases and MnP simultaneously, but does not produce Lac; The bacterium that becomes mildewed produces certain CMC, filter paper enzyme activity and beta-glucosidase enzyme.
The height of the enzymic activity that the bacterial classification mixed culture is produced different during with kind and bacterial classification single culture, can be found out by table 1 result: variegated rainbow conk and viride mixed culture, the activity of lignin-degrading enzymes and cellulase improve during than single culture; Variegated rainbow conk is mixed with becoming mildewed, and the activity of various enzymes is high during than the bacterial classification single culture, and the increase of its cellulase activity is than rainbow conk and wood height during mould the mixing; Variegated rainbow conk is mixed the back cellulase activity and is enhanced with black mold, and the activity of lignin-degrading enzymes and beta-glucosidase enzyme descends; Black mold is with after viride mixes, and cellulase activity strengthens greatly, but the lignin degradation enzymic activity descends during than the bacterial classification single culture; Do not produce Lac when black mold and SJ mixed culture, MnP high when producing than the SJ single culture is active; SJ and wooden mould mixed culture, its cellulase activity raises, but low during the wooden mould single culture of MnP activity; Its beta-glucosidase activity significantly increases when becoming mildewed with the black mold mixed culture, but filter paper enzyme and the work of CMC enzyme are low during than the black mold single culture; Low when becoming mildewed with wooden mould activity of mixing several kinds of enzymes that produced all than the bacterial classification single culture.Variegated rainbow conk is mixed with trichoderma viride, viride mixes with black mold, variegated rainbow conk is mixed the generation of enzyme and the raising of enzymic activity with the bacterium that becomes mildewed, and effect positive, that help each other and tangible facilitation effect are arranged.
Table 1. different strain is handled the product enzyme situation on solid medium
(3) three plain changing conditions observations in the native grass cogongrass:
Visible from table 2, single culture and associate strain are to the influence of cogongrass composition; Visible by table 3, single culture solid state fermentation 15d, the content of three kinds of staples has taken place to change in various degree in the cogongrass.Several kinds of bacterial classifications all have certain degradation capability to lignocellulose.Take temperature from the variation of total fiber element, the total fiber cellulose content of inoculation trichoderma viride processing reduces maximum, and next is for variegated rainbow conk and become mildewed the reduction amount minimum that the total fiber of SJ and black mold is plain.See that from the changing conditions of semicellulose the different treatment hemicellulose level reduces all not obvious, the hemicellulose level that SJ handles does not almost change.Take temperature from cellulosic variation, the ability of viride degraded cellulose is the strongest, and secondly for becoming mildewed and variegated rainbow conk, the resolving power of black mold is minimum.Take temperature from the variation of xylogen, the ability of variegated rainbow conk lignin degrading is the strongest, is SJ and viride secondly.Optimize the bacterial classification mixed culture, significant variation has taken place in three cellulose contents in the cogongrass, has several kinds of bacterial classifications combinations that three plain degradeds in the cogongrass are had tangible facilitation effect, and they are that viride mixes with variegated rainbow conk respectively, variegated rainbow conk and becoming mildewed.Trichoderma viride mixes with variegated rainbow conk, and is best to the degradation effect of xylogen in the cogongrass; SJ mixes the degradation effect of xylogen also better with black mold, SJ with trichoderma viride; Black mold mixes with viride, can effectively promote the degraded to Mierocrystalline cellulose and semicellulose, and hemicellulose level has descended 4.2%.
Table 3 has been represented the degradation rate of different strain processing to heterogeneity in the cogongrass; Visible by table 3, it is better that single culture is handled cellulosic degradation effect, and degradation rate can reach 30%-49%; And relatively poor to the degradation effect of xylogen and semicellulose, particularly the degradation rate of semicellulose is lower.Associate strain is handled; Degraded has in various degree taken place in three cellulose contents in the cogongrass; Wherein cellulosic degradation rate has improved 10.09%, becomes mildewed and mixes cellulosic degradation rate maximum with trichoderma viride, and variegated rainbow conk is mixed cellulosic degradation rate minimum with SJ; The degradation rate of xylogen is degraded than single culture, and degradation rate has improved 46.25%,
Table 2. different strain is handled the changing conditions of three elements in the cogongrass
Table 3. different strain is handled the degradation rate to three elements in the cogongrass
Wherein variegated rainbow conk is mixed the degradation rate of xylogen the highest with trichoderma viride, secondly be the variegated rainbow conk and the bacterium mixed culture that becomes mildewed, and it is the poorest that the bacterium that becomes mildewed mixes the degradation effect of xylogen with trichoderma viride; It is less that different strain is handled double cellulosic degradation rate, the single culture degraded, and the degradation rate of the semicellulose of cogongrass is 4.05-16.0%, and bacterial classification mixes double cellulosic degraded has obvious facilitation, and degradation rate is 20.86-33.45%; Bacterial classification mixes the plain degradation rate of back total fiber and improves, and degradation rate is 41.01-46.70.The strains solid fermented process of hybrid optimization has obvious facilitation to three of native grass plain degradeds, has improved the utilization ratio that native grass produces alcohol fuel.
4. the engineering of hybrid optimization bacterial classification preparation:
With the optimization associate strain that is filtered out under certain conditions; Become production bacterial classification through enlarged culturing with some amount and quality; With the usefulness as further expansion biomass and synthetic product, its engineering preparation comprises serial process (detailed process: slightly) such as spore preparation and mycelium preparation.
Claims (2)
1. one kind is improved natural perennial grass and the careless method of producing green renewable biomass alcohol fuel utilising efficiency of miscellany thereof; It is characterized in that mixing with cellulose-degrading bacteria after primary dcreening operation, multiple sieve, optimization and engineering are prepared from, specifically constitute by following steps perennial lignin-degrading bacteria:
(1) mixed bacteria primary election: on the PDA solid medium; Through a large amount of tests; Perennial lignin-degrading bacteria that initial option can mix and cellulose-degrading bacteria Mixed Microbes group; With the PDA culture medium is basal medium; Slant strains was cultivated 7 days; Observe the speed of growth of bacterial classification on the PDA culture medium, the performance of growth outward appearance, bacterial classification interphase interaction and collaborative; Select suitability, variegated rainbow conk and Trichoderma viride bacterial classification, Trichoderma viride and two kinds of combinations of aspergillus niger that concertedness is strong at last, prepare material as optimizing suitable mixed bacteria engineering bacteria;
(2) solid state fermentation final election: utilize solid-state fermentation culture medium; Optimize mixed bacteria for two groups that can mix; Solid state fermentation is measured the variation of three cellulose contents in lignin degradation enzymatic activity, cellulose degradation enzymatic activity, the native grass; Screening can the coordinated solid state fermentation mixed bacteria; Wherein variegated rainbow conk is with after Trichoderma viride mixes, and the lignin degradation enzymatic activity improves, and the lignin degradation rate has great raising; Trichoderma viride is with after aspergillus niger mixes, and cellulase activity obviously improves;
(3) solid state fermentation culture optimization: the kind of the degrading enzyme that associate strain and single culture produced and the height of degrading enzymatic activity are compared; The preferred degrading enzymatic activity that is produced is strong; Its mixed growth and the collaborative degradation effect of imitating are mutually further observed in two combinations of hybrid optimization that perennial lignocellulose mass degradation rate is high;
(4) optimize the preparation of bacterial classification integration engineering: optimize suitable associate strain with two that are filtered out and become production bacterial classification with some amount and quality through enlarged culturing; With the usefulness as further expansion biomass and synthetic product, its engineering preparation comprises spore preparation and mycelium preparation.
2. produce the application in the alcohol fuel according to the said a kind of method that improves natural perennial grass and the green renewable biomass alcohol fuel utilising efficiency of miscellany grass production thereof of claim 1 native grass.
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