CN102550809A - Method for degrading compound strain silage cotton stalks by using white rot fungi and lignocellulose - Google Patents
Method for degrading compound strain silage cotton stalks by using white rot fungi and lignocellulose Download PDFInfo
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Abstract
The invention discloses a method for degrading compound strain silage cotton stalks by using white rot fungi and lignocellulose. The method comprises the following steps of: sampling fresh cow manure, dunghill and cotton land plow layer soil (0 to 40cm) and rumen liquor, adding 5g of samples respectively into 100mL of PCS culture medium, and performing closed culture at the temperature of 37 DEG C; and after celluloses are disintegrated, transferring 10 percent (v/v) of samples to a fresh PCS culture medium, making 23 generations of transfer, getting rid of cultures which are unstable and lose the capacity of decomposition, leaving the cultures which are high in capacity of decomposition, and after the disintegration of the celluloses is stable, performing primary screening, and thus obtaining mixed strains with disintegrated celluloses. The method has the advantages of low energy consumption, simplicity in operation and no environmental pollution.
Description
Technical field
The invention belongs to agricultural technology field, relate to a kind of method of utilizing the cotton stalk of the collaborative ligocellulose degradation of whiterot fungi composite microbial system ensiling.
Background technology
Lignocellulosic extensively is present in the plant cell; It is the maximum organic renewable resource of occurring in nature content; In plant; Lignin is connected with hemicellulose through chemical bond and is wrapped in then outside the cellulose, forms lignocellulosic, and lignin constitutes the main component of plant skeleton with cellulose.Lignin is owing to the complicated of bonding with various bio-stables is difficult for being degraded by microorganisms.Solve and utilize this problem of lignocellulosic how efficiently, key is how to degrade and is wrapped in the lignin and the hemicellulose of cellulose crystal outside, amasss, makes cellulose to be easy to degraded and utilization thereby increase cellulose surface.
Traditional physico-chemical method probably can remove 50% lignin; And make fiber become amorphous; But cost is higher, needs expensive professional equipment and consumes a large amount of energy, and cause secondary pollution easily; And Biological Pretreatment has advantages such as energy consumption is low, simple to operate and free from environmental pollution, more and more receives people's attention.
If it is found that can lignin degrading the microbial host fungi, can lignin degrading about fungi, think that mostly it can produce some lignin degrading enzymes.In the prior art research more, the enzyme that degradation capability is stronger mainly contains lignin peroxidase, manganese-dependent peroxidase and laccase.In above-mentioned 3 the important enzyme, other all participate in or the degraded of lignin had certain influence like glucose oxidase, catalase and some reductases and protease etc.
The bacterial species of lignocellulose degradation is a lot of in the prior art, and actinomyces are one type of stronger trichobacterias of degradation capability of generally acknowledging, comprise streptomycete, arthrobacterium, small single-cell bacteria, Nocard's bacillus etc.
In sum, people mainly concentrate on the screening of single bacterial strain at present, or the research lignin-degrading bacteria, or the research cellulose-degrading bacteria, do not find effect composite flora preferably as yet.
Summary of the invention
The objective of the invention is to overcome above-mentioned technological deficiency, a kind of method of utilizing the cotton stalk of the collaborative ligocellulose degradation of whiterot fungi composite microbial system ensiling is provided.Its technical scheme is:
A kind of method of utilizing the cotton stalk of the collaborative ligocellulose degradation of whiterot fungi composite microbial system ensiling, the present invention has screened composite flora, also collaborative in addition whiterot fungi; Also screened the ensiling flora, to realize cotton stalk ensiling, the roughage that solves the South Sinkiang is not enough; Turn waste into wealth, realize the pattern that breeding combines.
May further comprise the steps:
(1) screening of the cotton stalk of degraded ligocellulose degradation composite microbial system
1) will be that MG0, compost are that MG1, cotton field topsoil 0～40cm are that the sample of fetching MG2 and the rumen fluid is MG3 from fresh cow dung, and respectively get 5g/mL and join in the 100ml PCS culture medium, 37 ℃ of cultivations, static, CO
2Cultivate in the incubator.
2) treat after the cellulose disintegration that be transferred in the fresh PCS nutrient solution, inoculum concentration is 10% (v/v) again, the number generation of so transferring, eliminate and lose capacity of decomposition and unsettled culture, stay the strong culture of capacity of decomposition,
Regularly change propagating method: in per 5 generations, get 10mL, centrifugal 3500rmin
-1, abandon supernatant, add the 10mL SPSS, suspend, be inoculated on the fresh PCS culture medium.After treating that the cellulose disintegration time is stable, promptly Preliminary screening is decomposed mixed bacterial to cellulose.Obtaining at last degrading, cotton stalk lignocellulosic effect is strong, stable composite fungus strain MG0, MG1, MG2 and MG3.
Peptone cellulose nutrient solution (PCS) improved culture medium composition: peptone, cellulose (cotton stalk), yeast extract, each 5gL of NaCL
-1, CaCO
32gL
-1, K
2HPO
4, MgSO
4Each 0.5gL
-1, trace element solution 0.5mLL
-1, soil extraction (soil and deionized water mix with mass ratio at 1: 1, filter, and the sterilization back is subsequent use) 100mLL
-1Trace element solution composition (gL
-1): zinc sulfate 0.29, calcium chloride 0.24, copper sulphate 0.25, magnesium sulfate 0.17
(2) screening of ensiling composite microbial system
Utilize the MRS culture medium that mixed ensiling (mass ratio 1: 1) of South Sinkiang ensilage, cotton stalk and maize straw and cotton stalk are carried out ensiling, normal temperature, ensiling 40d.Take by weighing the 5g ensilage then, join in the 95mL MRS culture medium and cultivate, 37 ℃, static, CO
2Cultivate 24h in the incubator, screening pH drops to the compound system of ensiling below 4.0 fast.Then pH is dropped to the compound system below 4 fast, continue commentaries on classics and be commissioned to train foster.Drop to the compound system of stable ensiling below 4 fast up to filtering out pH.
MRS fluid nutrient medium (g/l): glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g; Dipotassium hydrogen phosphate 2g, sodium acetate 5g, magnesium sulfate 0.58g, manganese sulfate 0.25g; Tween-80 1mL, distilled water 1000mL, 121 ℃ of sterilization 20min, pH 6.2-6.4.
(3) cultivation of whiterot fungi
Whiterot fungi is that Phanerochaete chrysosporium (phanerochaete chrysosporium) utilizes key lab's donations by Production and Construction Corps of Xinjiang's Tarim Basin living resources conservation.
37 ℃ of condition of culture are cultivated in the dull and stereotyped electro-heating standing-temperature cultivator of PDA, and 6d is made into spore suspension before the utilization.
The PDA culture medium is formed. potato 200g, glucose 20g, agar 15～20g, running water 1000mL, pH nature.
Compost described in the step 1) of the present invention is the mixing of cow dung, pig manure, chicken manure, ratio 2: 1: 1,37 ℃, fermentation 15d.
Compared with prior art, beneficial effect of the present invention is:
1, the present invention has energy consumption advantage low, simple to operate and free from environmental pollution.
2, technical scheme of the present invention is the synergy of multiple microorganism.
3, use the inventive method good degrading effect, cotton stalk microbial degradation starts very fast, just can reach enzyme peak alive at 5d, explains that the cotton stalk of handling through dilute acid pretreatment helps the degraded of microorganism.
Description of drawings
Fig. 1 is the flow chart of the inventive method;
Fig. 2 is the cellulose composite microbial system screening process figure of the cotton stalk of degraded;
Fig. 3 is the screening process figure of ensiling composite microbial system;
Fig. 4 is a whiterot fungi, the compound and ensiling effect assessment figure of wood fibre degraded composite microbial system and ensiling composite microbial system;
Fig. 5 is the plain enzyme activity determination of compound series fiber figure as a result;
Fig. 6 decomposes three kinds of enzymatic activitys of the cotton compound MG1 of being of stalk lignocellulosic of sour preliminary treatment dynamically to scheme;
Fig. 7 is OD and pH dynamic change figure;
Fig. 8 is the influence figure of different nitrogen sources to cellulase activity;
Fig. 9 is that different nitrogen sources is to the cellulose degraded design sketch;
Figure 10 is the influence figure of different temperatures to cellulase activity;
Figure 11 is that different temperatures is to the cellulose degraded design sketch;
Figure 12 is the influence figure of PH to cellulase activity;
Figure 13 is that PH is to the cellulose degraded design sketch;
Figure 14 is the influence figure of inoculum concentration to cellulase activity;
Figure 15 is that inoculum concentration is to the cellulose degraded design sketch;
Figure 16 is the different DGGE that cultivate ligocellulose degradation's composite microbial system of algebraically;
Figure 17 is the different DGGE that cultivate the ensiling composite microbial system in generation.
The specific embodiment
Below in conjunction with the accompanying drawing and the specific embodiment the present invention is done explanation in further detail.
Referring to Fig. 1-Fig. 3, a kind of method of utilizing the cotton stalk of the collaborative ligocellulose degradation of whiterot fungi composite microbial system ensiling may further comprise the steps:
(1) screening of the cotton stalk of degraded ligocellulose degradation composite microbial system
1) will be MG0, the compost (mixing of cow dung, pig manure, chicken manure from fresh cow dung; Ratio 2: 1: 1,37 ℃, fermentation 15d) be that MG1, cotton field topsoil 0～40cm are that the sample of fetching in MG2 and the rumen fluid is MG3; Respectively getting 5g/mL joins in the 100ml PCS culture medium; 37 ℃ of cultivations, static, CO
2Cultivate in the incubator.
2) treat after the cellulose disintegration that be transferred in the fresh PCS nutrient solution, inoculum concentration is 10% (v/v) again, the number generation of so transferring, eliminate and lose capacity of decomposition and unsettled culture, stay the strong culture of capacity of decomposition,
Regularly change propagating method: in per 5 generations, get 10mL, centrifugal 3500rmin
-1, abandon supernatant, add the 10mL SPSS, suspend, be inoculated on the fresh PCS culture medium.After treating that the cellulose disintegration time is stable, promptly Preliminary screening is decomposed mixed bacterial to cellulose.Obtaining at last degrading, cotton stalk lignocellulosic effect is strong, stable composite fungus strain MG0, MG1, MG2 and MG3.
PCS medium component: peptone cellulose nutrient solution (PCS) improved culture medium composition: peptone, cellulose (cotton stalk), yeast extract, each 5gL of NaCL
-1, CaCO
32gL
-1, K
2HPO
4, MgSO
4Each 0.5gL
-1, trace element solution 0.5mLL
-1, soil extraction (soil and deionized water mix with mass ratio at 1: 1, filter, and the sterilization back is subsequent use) 100mLL
-1Trace element solution composition (gL
-1): zinc sulfate 0.29, calcium chloride 0.24, copper sulphate 0.25, magnesium sulfate 0.17
(2) screening of ensiling composite microbial system
Utilize the MRS culture medium that mixed ensiling (mass ratio 1: 1) of South Sinkiang ensilage, cotton stalk and maize straw and cotton stalk are carried out ensiling, normal temperature, ensiling 40d.Take by weighing the 5g ensilage then, join in the 95mL MRS culture medium and cultivate, 37 ℃, static, CO
2Cultivate 24h in the incubator, screening pH drops to the compound system of ensiling below 4.0 fast.Then pH is dropped to the compound system below 4 fast, continue commentaries on classics and be commissioned to train foster.Drop to the compound system of stable ensiling below 4 fast up to filtering out pH.
MRS fluid nutrient medium (g/l): glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g; Dipotassium hydrogen phosphate 2g, sodium acetate 5g, magnesium sulfate 0.58g, manganese sulfate 0.25g; Tween-80 1mL, distilled water 1000mL, 121 ℃ of sterilization 20min, pH 6.2-6.4.
(3) cultivation of whiterot fungi
Whiterot fungi is that Phanerochaete chrysosporium (phanerochaete chrysosporium) utilizes key lab's donations by Production and Construction Corps of Xinjiang's Tarim Basin living resources conservation.
37 ℃ of condition of culture are cultivated in the dull and stereotyped electro-heating standing-temperature cultivator of PDA, and 6d is made into spore suspension before the utilization.
The PDA culture medium is formed. potato 200g, glucose 20g, agar 15～20g, running water 1000mL, pH nature.
Fig. 4 is a whiterot fungi, the compound and ensiling effect assessment figure of wood fibre degraded composite microbial system and ensiling composite microbial system.
Carry out compound and the ensiling effect assessment through ligocellulose degradation's composite microbial system of having obtained and ensiling lactic acid fungus strain and whiterot fungi; At first carrying out antagonism measures; Confirm its no antagonism; Carry out mixed culture fermentation then, measure relevant index (enzymatic activity, degradation rate, external fermentation, cud degradation rate etc.) then, confirm best combination.
The plain enzyme activity determination result of compound series fiber sees Fig. 5, and the CMC enzyme of soil is lived and is that it is 6.74U/mL that 18.23U/mL, FPA enzyme live, beta-glucosidase 12.67U/mL; The CMC enzyme of compost 13.91U/mL alive, FPA enzyme 6.47U/mL alive, beta-glucosidase 11.34U/mL; The CMC enzyme of cow dung 11.95U/mL alive, FPA enzyme 8.29U/mL alive, beta-glucosidase 11.03U/mL; The CMC enzyme of rumen fluid 12.26U/mL alive, FPA enzyme 5.95U/mL alive, beta-glucosidase enzyme 11.39U/mL alive.The compound FPA of the being enzyme of the cellulose work that wherein is derived from cow dung is higher than other composite microbial systems, and the cellulase-producing ability is the strongest.
Decompose three kinds of enzymatic activitys of the cotton compound MG1 of being of stalk lignocellulosic of sour preliminary treatment, OD and pH dynamic change, specifically see Fig. 6, Fig. 7.
From the 2d that ferments, measure CMC, FPA and β-G enzymatic activity every day, the enzyme activity attitude of measuring the cotton stalk of microbial degradation acid preliminary treatment in the 8d changes, and is as shown in Figure 1.Ligocellulose degradation is compound to be that MG1 is very fast to the decomposition startup of the pretreated cotton stalk of acid, and the enzymatic activity amplitude of variation is little, and keeps higher enzymatic activity; Possibly be the result of composite microbial system MG1 by multiple microorganism compound action, CMC and FPA enzyme are lived and all peak value occurred in 5d, and enzyme is lived afterwards has decline slightly; All maintain state more stably; β-G enzymatic activity is higher in the 5d enzymatic activity, possibly be more sugar of growth of microorganism consumption, has reduced the inhibitory action of sugar to β-G enzyme; From the enzyme angle changing of living, can be about 5d with the microbial degradation periodic Control of the cotton stalk of sour preliminary treatment.So quick startup produces sugar, for lactic acid bacteria carries out hay silage sufficient carbon source is provided.
In addition, also measured OD and the variation of pH value in cotton stalk sweat, seen Fig. 7, at 5d, it is maximum that the OD value reaches, and pH descends fast, helps the activity performance of cellulase, also favourable cotton stalk hay silage.
The single factor experiment result
Four kinds of nitrogenous sources are to the influence of the cotton stalk lignocellulosic of dilute acid pretreatment MG1 degradation effect
Ammonium sulfate, ammonium nitrate, urea and the peptone of four kinds of different nitrogen sources of employing is compound to the cotton stalk of sour preliminary treatment to be the influence of MG1 degradation effect, and the result is like Fig. 8, shown in 9.As nitrogenous source, can keep the highest CMC, FPA and β-G enzymatic activity with urea, and conversion coefficient and cellulose degradation rate all be higher than other nitrogenous source, explain that urea can promote the compound MG1 of being to produce enzyme, the conversion coefficient that has improved cotton stalk and cotton stalk weight-loss ratio effectively.But, also can tentatively draw from Fig. 3, FPA enzymatic activity effect for nitrogen all is better than organic nitrogen, and possible inorganic nitrogen helps the dissolving of lignin, for the effect of cellulase provides more space.From Fig. 9, can draw the processing that urea is nitrogenous source, conversion coefficient and weight-loss ratio are apparently higher than other nitrogenous source, but the treatment effect of ammonium nitrate is the poorest, and possible nitrate nitrogen is unfavorable for the utilization of the compound MG1 of being.
Different temperatures is compound to the cotton stalk of dilute acid pretreatment to be the influence of MG1 degradation effect
Can find out from Figure 10,11; Along with temperature raises, FPA enzyme, β-G enzyme are lived and are remained unchanged basically, maintain higher enzymatic activity; Heat-resisting ability is strong; The activity of CMC enzyme keeps higher activity at 32 ℃, 37 ℃, 42 ℃, and just temperature is bigger to the influence of CMC enzymatic activity, influences little to FPA enzyme, β-G enzyme.Conversion coefficient and cellulose degradation rate reach peak for 32 ℃, and 42 ℃ of work of CMC enzyme, conversion coefficient and cellulose degradation rates all have raising in various degree.It seems that comprehensively 32～37 ℃ is more excellent fermentation temperature.Though enzymatic activity changes little, conversion coefficient changes greatly with weight-loss ratio, and this maybe be owing to temperature has bigger influence to the composition of the compound MG1 of being.
Initial pH value is seen Figure 12, Figure 13 to the influence of the compound MG1 of the being degradation effect of the cotton stalk of dilute acid pretreatment.
Can find out from Figure 12, pH is between 4.8～6.8, and the compound MG1 of being can keep higher CMC, FPA, β-G enzymatic activity; PH is about 4.8, and conversion coefficient is the highest, and pH is maximum at 5.8 left and right sides weight-loss ratios; And make cotton stalk that higher conversion coefficient and weight-loss ratio arranged, initial pH value is adjusted between the 4.8-5.8.In general, so select initial pH value 5.8 to be advisable.The adaptability to pH of three kinds of cellulases is bigger, but the variation of conversion coefficient and weight-loss ratio is bigger, maybe be because the compound MG1 of being changes relatively more responsive causing to pH.PH is 5.8 o'clock, helps microbial growth and produces enzyme.
Inoculum concentration is compound to the cotton stalk of dilute acid pretreatment to be the influence of MG1 degradation effect: from Figure 14,15 curvilinear motions, the activity change of FPA enzyme is little, and CMC and β-G enzyme activity when 1% and 10% inoculum concentration is the highest.Conversion coefficient is increasing with the increase of inoculum concentration always, 10% inoculum concentration, and conversion coefficient is the highest, and the cotton stalk weight-loss ratio of 5% inoculum concentration reaches maximum.This possibly be that 10% inoculum concentration has been brought more nutrient into, and the ability of inducing the compound MG1 of being to produce cellulase weakens.Can obtain the highest cotton stalk weight-loss ratio and higher conversion coefficient with 5.0% inoculum concentration, with inoculum concentration 5.0% for more preferably selecting.
The cotton stalk of dilute acid pretreatment is compound to be the orthogonal experiments of MG1 degraded
Table 1 is compound to be the MG1 orthogonal experiments
Each processing factor of table 2 is compound to the cotton stalk of dilute acid pretreatment to be the F value table of MG1 degradation effect influence
Annotate: * * representes that influence reaches the utmost point level of signifiance (P＜0.01), and * representes that influence reaches the level of signifiance (P＜0.05).
F value result sees table 2; CMC, FPA, β-G enzyme are lived for pH, nitrogenous source kind and inoculum concentration and the influence of conversion coefficient and weight-loss ratio reaches the level of signifiance (P＜0.01); Temperature reaches the level of signifiance (P＜0.01) to the influence of FPA enzyme and weight-loss ratio; Influence to conversion coefficient reaches the level of signifiance (P＜0.05), and the influence that CMC, β-G enzyme are lived does not reach the level of signifiance.
The influence that each factor is lived to CMC, FPA, β-G enzyme
Table 3 is compound to be that the MG1CMC enzyme is lived, the FPA enzyme is lived and β-the G activity ratio
Annotate: 1,2,3 be respectively the mean value of each factor on level 1,2,3; R is the extreme difference value; Letter representation mean value multiple ratio otherness after the numeral, different lowercases represent that each horizontal differences of mean value is remarkable, and different capitalizations represent that each horizontal differences of mean value is extremely remarkable, down together.
Compound three kinds of enzyme range analysis of the MG1 of being result sees table; The influence degree that each factor is lived to the cotton stalk microbial degradation of sour preliminary treatment CMC enzyme is followed successively by: the initial pH value of inoculum concentration＞fermentation＞nitrogenous source kind＞fermentation temperature; The influence degree that the FPA enzyme is lived is followed successively by: the initial pH value＞inoculum concentration of nitrogenous source kind＞fermentation temperature＞fermentation, the influence degree that β-G enzyme is lived is followed successively by: the initial pH value＞inoculum concentration of nitrogenous source kind＞fermentation temperature＞fermentation.
It is thus clear that the nitrogenous source kind has the greatest impact to what the cotton stalk microbial degradation of sour preliminary treatment enzyme was lived, explain that the nitrogenous source that is fit to possibly be the key that growth of microorganism produces enzyme.See table through mean value intuitive analysis and The result of multiple comparisons; Can draw CMC enzyme combination of process parameters alive and FPA enzyme optimum alive is A3B3C2D1; That is: nitrogenous source is a peptone, 37 ℃ of fermentation temperatures, initial pH value 7.0; Inoculum concentration 1.0%, the cotton stalk of microbial degradation acid preliminary treatment can be kept the highest CMC enzyme and live; β-G enzyme optimum combination of process parameters alive is A2B1-3C3D2, that is: nitrogenous source is a urea, and three levels of fermentation temperature choose one wantonly, initial pH value 9.0, inoculum concentration 5.0%.
Each factor is to the influence of cotton stalk conversion coefficient and cellulose degradation rate
Table 4 conversion coefficient and comparison of weight-loss ratio mean value and range analysis
Can know that through range analysis table 4 each factor is followed successively by respectively the cotton stalk conversion coefficient of sour preliminary treatment influence degree: initial pH value＞inoculum concentration＞nitrogenous source kind＞fermentation temperature ferments; Each factor is followed successively by respectively the influence degree of cellulose degradation rate: initial pH value＞nitrogenous source kind＞inoculum concentration＞fermentation temperature ferments.Fermentation temperature is most important to the influence of conversion coefficient, and as shown in table 4, and nitrogenous source, fermentation initial pH and inoculum concentration are to the influence of conversion coefficient extremely significantly (P＜0.01), and visible regulation and control fermentation pH is very important to effective saccharification of cotton stalk; Inoculum concentration is the principal element that influences cellulose degradation rate, suitably selects nitrogenous source can promote cellulosic degraded effectively in the research, and the saccharification of producing enzyme and cotton stalk for microbial growth lays the foundation.
Can draw conversion coefficient optimum process parameter combinations through mean value intuitive analysis and The result of multiple comparisons (table 4) is A3B2C2D1, that is: nitrogenous source is a peptone, 37 ℃ of fermentation temperatures, and initial pH value 6.8, inoculum concentration 1.0%, the cotton stalk conversion coefficient of dilute acid pretreatment is the highest; Cotton stalk weight-loss ratio optimum process parameter combinations is A1B3C2D1, that is: inoculum concentration 1.0%, and nitrogenous source is (NH
4)
2SO
4, 42 ℃ of fermentation temperatures, the initial pH value of fermenting is 6.8.
Through single factor and orthogonal test, optimized the technology of the cotton stalk microbial degradation of sour preliminary treatment, drawn the optimum process parameter and be: 42 ℃ of fermentation temperatures, nitrogenous source are (NH4) 2SO4, inoculum concentration 1.0%, the initial pH value of fermenting is 6.8.Under optimal conditions, the CMC enzyme is lived, the FPA enzyme is lived ,-G enzyme, conversion coefficient and weight-loss ratio can reach 47.12U/mL, 24.35U/mL, 13.05U/mL, 37.21% and 26.38% respectively.
Like Figure 16, the cellulose degradation composite microbial system is made up of microorganism in 6.
Whiterot fungi (white rot fungi); Can lignin degrading, hemicellulose and cellulosic characteristic; Experiment selected representative strain Phanerochaete chrysosporium (Phanerochaete chrysosporium) has the ability of stronger lignin degrading, is widely used at present.
Test utilizes the cultivation of continuously fermenting of MRS fluid nutrient medium from the ensilage of South Sinkiang, obtained stable composite ensiling fungus strain, 24h, and pH drops to below 4.0 fast.
Referring to Figure 17, find that through DGGE the ensiling composite microbial system is made up of bacterial strain in 4.
The above; Be merely the preferable specific embodiment of the present invention; Protection scope of the present invention is not limited thereto; Any technical staff who is familiar with the present technique field is in the technical scope that the present invention discloses, and the simple change of the technical scheme that obtains or equivalence replacement all fall in protection scope of the present invention with may be obvious that.
Claims (2)
1. a method of utilizing the cotton stalk of the collaborative ligocellulose degradation of whiterot fungi composite microbial system ensiling is characterized in that, may further comprise the steps:
1) will be MG0, compost from fresh cow dung, 37 ℃, fermentation 15d for MG1, cotton field topsoil 0～40cm are that the sample of fetching in MG2 and the rumen fluid is MG3, respectively get 5g/mL and joins in the 100ml PCS culture medium, and 37 ℃ of cultivations are static, CO
2Cultivate in the incubator;
2) treat after the cellulose disintegration that be transferred in the fresh PCS nutrient solution, inoculum concentration is 10% (v/v) again, the number generation of so transferring, eliminate and lose capacity of decomposition and unsettled culture, stay the strong culture of capacity of decomposition,
Wherein regularly change propagating method: in per 5 generations, get 10mL, centrifugal 3500rmin
-1, abandon supernatant, add the 10mL SPSS, suspend; Be inoculated on the fresh PCS culture medium, treat that the cellulose disintegration time is stable after, promptly Preliminary screening is decomposed mixed bacterial to cellulose, tentatively obtaining degrading, cotton stalk lignocellulosic effect is strong, stable composite fungus strain MG0; MG1, MG2 and MG3 utilize MG0, MG1 then; MG2 and MG3 carry out solid fermentation to cotton stalk, and obtaining the best flora of effect is MG0, then MG1 is carried out enzymatic productivity and is optimized, and obtains condition of enzyme production preferably.
2. the method for utilizing the cotton stalk of the collaborative ligocellulose degradation of whiterot fungi composite microbial system ensiling according to claim 1 is characterized in that, the mixing of the compost cow dung described in the step 1), pig manure, chicken manure, and its mixed proportion is 2: 1: 1.
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| CN105166509A (en) * | 2015-09-14 | 2015-12-23 | 安徽三兴饲料有限公司 | Production method of feed for growing and fattening pigs |
| CN105918648A (en) * | 2016-04-07 | 2016-09-07 | 延边大学 | Composite biological preparation used for fattening cattle and preparation method thereof and application method thereof |
| CN106495847A (en) * | 2016-10-14 | 2017-03-15 | 安徽格义循环经济产业园有限公司 | Lignin slow release Water soluble fertilizer and preparation method thereof |
| CN115537349A (en) * | 2022-06-20 | 2022-12-30 | 天津科技大学 | Straw degrading bacterium and application thereof |
| CN115537349B (en) * | 2022-06-20 | 2023-11-07 | 天津科技大学 | Straw degrading bacterium and application thereof |
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