CN105733963A - Composite fermentation straw degradation agent, preparation method and application - Google Patents
Composite fermentation straw degradation agent, preparation method and application Download PDFInfo
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Abstract
The invention relates to the technical field of mixed culture of two different microorganisms, in particular to a composite fermentation straw degradation agent, a preparation method and application. The straw degradation agent is compound strains obtained by confront culture of Trichoderma reesei and Trichoderma harzianum obtained after physical mutagenesis is performed respectively. The preparation method and application comprise the steps of performing physical mutagenesis to obtain Trichoderma reesei mutant strains and Trichoderma harzianum mutant strains, high-activity compound strains are obtained through screening, purification and enlarged culture, and then two bacterial strains are inoculated according to crop varieties with different function advantages to be subjected to composite culture fermentation. According to the technical scheme, the advantages of the Trichoderma reesei and the Trichoderma harzianum are supplemented and combined, the compound bacterial strains can generate high-content and multi-activity cellulase, crop straw can be effectively degraded within 10-13 d, crops can resist soil disease, growth is promoted, secondary pollution of emission generated by incineration of straw can be reduced, and application of microbial fermentation in agricultural production is improved.
Description
Technical field
The present invention relates to two kinds of different cultivars microorganism mixed culture technique fields, be specifically related to a kind of composite fermentation
Straw degradative agent, preparation method and application.
Background technology
Lignocellulose is plant cell wall important composition composition, thus raw material sources is extremely extensive, such as standing grain originally
The straw (such as Oryza sativa L., Semen Maydis, Semen Tritici aestivi, Semen Fagopyri Esculenti, Caulis Sacchari sinensis, Semen setariae and Sorghum vulgare Pers. etc.) of section crop, Chun Xinghua section
Class (such as fresh kidney beans, Semen Glycines, Semen phaseoli radiati, Semen Phaseoli etc.) and the vegetables straw of Cruciferae, be fermentable synthesis
Suppression parasitic disease, the natural biomass materials of infectivity cause of disease fungus growth.But in view of single kind fungus
Because of the architectural difference of enzyme cellulolytic in various extracellular microbials, enzyme specificity is the most different, adapts to raw material
Property different, there is the conversion coefficient decomposited low, the deficiencies such as cycle length, vigor are low, it is necessary to take two or more
The combination of microorganism fungus kind, studies high efficient technology, and just can be suitable for the exploitation to a large amount of baroque straws should
With.
Have appreciated that reesei spores preparation facture restriction farmland straw returns the bottleneck of field rehabilitating soil structure and is at present
Weak effect, time length, cost are high, underlying reason is that the hydrolysis of lignocellulose class hydrolytic enzyme in microbial body relatively
Less, vigor is low, content is low.Although it is the most a large amount of to the Trichoderma harzianum granule of straw hydrolysis on soil on market
Occurring, academy of agricultural sciences's clay fertilizer institute of many provinces, agricultural universities and colleges and minority key university all develop Trichoderma spp. hydrolyzing straw enzyme
Preparation, but practice shows, and owing to using, strain is dull, vigor is low, and in thalline, the kind of enzyme is few, and straw
Stalk kind is many, and lignocellulose chemical constitution is complicated, and hydrolase of the same race is different because of kind bacterium source, i.e.
Making enzyme activity high, but the enzyme activity shown is different, effect is the most different, it is impossible to adapt to peasant to different seasons
The needs that joint institute harvesting and stalk processes in time, hydrolyzing straw cycle short person about 2～3 weeks, elder needs more than January even
Longer.The fungus of hydrolyzing straw, different fungus combined crosswise and solid-liquid fermentation technique is how selected to become biological
Worker faces a difficult problem for urgent solution.
In recent years, Jiang Like, Cheng Beijiu, Wei the resisting at Trichoderma harzianum plant growth regulator such as gentle Yu Mei are practiced
Find when disease, growth promotion and the application of increase yield that the polydirectional mutation strain of same bacterial strain Trichoderma spp. is basis set by adjusting cultivation
Close technique, the polynary fermentation (ZL.201310058602.8) that a bacterium is multiplex can be realized, both reached institute's tunning
Can effective disease resistance, can increase production again, moreover it is possible to make crop degeneration-resistant (harm as arid, saline and alkaline).
Summary of the invention
Present invention aim to address above-mentioned the deficiencies in the prior art, it is provided that a kind of fungus group producing dissimilar enzyme
Close polynary fermentation system, to decomposing with separate sources straw, realize straw-returning with getting twice the result with half the effort, change
Good Soil structure.
The present invention is achieved by the following technical solutions:
A kind of composite fermentation straw degradative agent, described straw degradative agent be trichoderma reesei with Trichoderma harzianum respectively through thing
The compound fungus of opposite culture gained after reason mutation.
The preparation method of a kind of composite fermentation straw degradative agent, comprises the following steps:
(1) buy from market or nature separates wild trichoderma reesei and Trichoderma harzianum；
(2) N is used+Or Ar+Or CO2 +Trichoderma reesei and Trichoderma harzianum are carried out respectively the injection of ion beam, and
Compare with matched group, separate trichoderma reesei mutant and Trichoderma harzianum mutant；
(3) trichoderma reesei mutant and Trichoderma harzianum mutant are cultivated respectively in PDA culture medium, screening
Go out bacterium colony shinny, eugonic, then trichoderma reesei mutant is inoculated into cellulose screening culture medium, Kazakhstan
Thatch Trichoderma spp. mutant is inoculated in chitin screening culture medium, and screening is purified into high dynamic strain；
(4) by the high vigor trichoderma reesei mutant filtered out and Trichoderma harzianum mutant point on same flat board
Opposite culture, is used for making two kinds of mutants to complement each other the other side's nutrient, the other side that mutually promotes growth；Repeat three
Secondary, mix amplification culture, measure its functional enzyme activity.
The preparation method of a kind of composite fermentation straw degradative agent of the present invention, step (2) described ion beam
Implantation dosage be 10kev.
The preparation method of a kind of composite fermentation straw degradative agent of the present invention, step (3) described PDA trains
Support base to be prepared by following substances in percentage by weight concentration: Rhizoma Solani tuber osi 2～3%；Fish protein powder 0.5～3.3%；
FeSO40.01～0.03%；KH2PO40.02～0.20%；MgSO40.05～0.35%；Na2B2O7·10H2O
0.025～0.05%；Na2SeO30.025～0.05%；ZnSO40.01～0.02%；Surplus is water.
The application of a kind of composite fermentation straw degradative agent, according to different crops kind, preparation solid fermentation is cultivated
Base, adds and treats degrading straw, and inoculation composite fermentation straw degradative agent carries out fermentation culture.
The application of a kind of composite fermentation straw degradative agent of the present invention, described solid fermentation culture medium includes
Ferment base material, buffer, nitrogen-containing compound and Tween-20, and the weight of dry accounts for the 30～50% of total amount.
The application of a kind of composite fermentation straw degradative agent of the present invention, described composite fermentation straw degradative agent
Inoculum concentration is 0.5 × 108～1.5 × 108Individual/mL.
The application of a kind of composite fermentation straw degradative agent of the present invention, the condition of described fermentation culture is:
PH6～7, temperature 27～29 DEG C, pressure 0.05～0.1MPa, ferments 10～13 days.
The beneficial effects of the present invention is:
The present invention uses two kind Trichoderma spp.: trichoderma reesei can produce the degrading activity of high-load and many identification types
Cellulase, but it is not good enough to promote plant growth ability；Trichoderma harzianum can make crop anti-soil disease can preferably promote again to make
Thing grows, and lignocellulose degradation ability；By two kinds of Trichoderma spp. compound criteria, it is compound that composition is had complementary advantages
Fermentation system, its product can effectively degrade crop material, and it is sick and promote plant growth to be resistant to soil, improves
Microbial fermenters application in agricultural production.
The present invention by the two kinds of microbial fungis combinations of trichoderma reesei and Trichoderma harzianum to crop material (as corn cob,
Caulis et Folium Oryzae, straw and broomcorn straw powder) fermentation, the most anti-soil-borne disease, can quickly make again straw Organic substance return field,
Improvement Soil structure, increases soil fertility, the promotion less Semen Maydis of comprehensive utilization of resources, particularly industrial use,
Sorghum vulgare Pers., beans and wheat stalk.
Trichoderma reesei and Trichoderma harzianum have been carried out orienting (employing N by the present invention+Or Ar+Or CO2 +Radiation) prominent
Become, filter out energy fast degradation content the most respectively high, in constituent structure complexity cellulose energy efficient degradation
Family name's Trichoderma spp. mutant, this fungus compensate for energy high anti-soil disease and promotes plant growth, and decomposition of cellulose vigor
The shortcoming of weak Trichoderma harzianum, effectively raises the microorganism utilization to farmland straw, promotes soil remediation
The marketization.
The present invention is used to be combined into by two kinds of performance differences of formation deficiency fungus and is degraded into separation structure complexity
Straw system, make straw decompose entrance soil as early as possible, change conventional incineration and solve straw and go back to field, can not only make
Organic time field, increases soil fertility, and reduces crop pest, and significantly reduces and release because burning generation smog
Put CO2、NO2、SO2Deng the pollutant producing greenhouse effect.Meanwhile avoid murdering in soil having in a large number
Prebiotic thing, as decomposed the microorganism of organic matter in Lumbricus and soil, destroys Soil structure, causes soil
Increase production ability glides, and excites peasant the most potentially and strengthens minimizing to harm soil, the fertilizer and pesticide of grain security
The consciousness used.The modern agricultural production that advances that this method can be strong is used to make the reform of agricultural tillage system.
The inventive method can realize straw-returning with getting twice the result with half the effort, and improves Soil structure, makes soil carbon sequestration reach
More than 3%, phosphorus potassium content improved 0.5%～1.0% more originally.Improve soil fertilizer water-holding capacity, it is achieved stable yields,
High yield, reduces the wasting of resources and secondary pollution produced because crop straw burning processes, reduces the soil disease to crop
Evil, and reach the effect achieved many things at one stroke.
Accompanying drawing explanation
Fig. 1 is trichoderma reesei of the present invention and Trichoderma harzianum complementation growing principle schematic diagram.
Wherein, I is Trichoderma harzianum mutant, and II is trichoderma reesei mutant, and A is the excellent of each mutant
Gesture enzyme, B is the inferior position enzyme of each mutant.
Fig. 2 is the technique for applying flow chart of composite fermentation straw degradative agent of the present invention.
Detailed description of the invention
For being best understood from the present invention, below in conjunction with embodiment and accompanying drawing, the invention will be further described, real below
Executing example is only that the present invention will be described rather than is limited it.
Embodiment one
Bacterial screening
(1) activation of strain: will from rotten timber isolated wild trichoderma reesei and trichoderma harzianum strain
It is inoculated in PDA culture medium cultivation 5-7d respectively, carries out activated spawn；
(2) spore suspension is prepared: the spore after activating with 10mL aseptic water washing pours the nothing of 150mL into
In bacterium triangular flask, inside there is bead, then on constant-temperature table, shake 20min with 120r/min, make spore ball fill
It scatter.
(3) ion beam mutation: take the spore being grown in PDA culture medium and make spore suspension, make spore
Amount is 106/ ml, takes 0.1mL and is uniformly applied in sterilized blank culture dish, be placed on super-clean bench, with aseptic
Mild wind dries.Use N respectively+(or Ar+Or CO2 +) restraint the injection carrying out ion beam with the dosage of 10kev,
Two wares of each strain, separately make a vacuum comparison.
(4) by spore under the aseptic washing of flat board 1mL of ion beam mutation, then by spore liquid dilute after with
Every plate 100uL is coated with in PDA culture medium, cultivates under the conditions of being then placed in 27-28 DEG C, treats on flat board long
When going out single bacterium colony, choose single bacterium colony with toothpick and use the method for photocopy to be inoculated into cellulose screening culture medium respectively
In (trichoderma reesei) or chitin screening culture medium (Trichoderma harzianum), after three days, measure the transparent of each bacterium colony
Loop diameter, and select the bigger bacterial strain of transparent circle and carry out postsearch screening；Original strain takes same method to survey thoroughly
Bright circle size.
(5) inoculation of postsearch screening is cultivated in PDA culture medium preservation, the bacterium that will preserve after five days
Strain spore under aseptic washing, is inoculated in fermentation cellulose screening culture medium or the training of chitin liquid by spore liquid
Supporting base, (being centrifuged under the conditions of 12000r/min by fermentation liquid, supernatant is slightly to take crude enzyme liquid after four days
Enzyme liquid) carry out enzyme activity determination, and the transparent circle result of result with each bacterial strain is compared.Original strain is also
Enzyme activity size is compared after doing same process.Being determined as by the bacterial strain that enzyme activity is maximum is High Cellulase Production or several
The bacterial strain of fourth matter enzyme.
Embodiment two
Produce the preparation of Spore cultivation base
PDA culture medium is prepared (with weight percent concentration proportioning) by following material: Rhizoma Solani tuber osi 2～3%；Fish
Egg albumen powder 0.5～3.3%；FeSO40.01～0.03%；KH2PO40.02～0.20%；MgSO40.05～0.35%；
Na2B2O7·10H2O 0.025～0.05%；Na2SeO30.025～0.05%；ZnSO40.01～0.02%；Surplus
For water.It is the certain base leaving out some gene according to used bacterial strain, produces auxotrophic strain, at this
Culture medium is added Mel 3%, is allowed to improve spore amount (using after sterilizing).
Embodiment three
The amplification culture of compound fungus fermentation system
Preparation PDA culture medium kind, will be obtained two bacterial strain mutant points on same flat board, at a distance of 4cm
Make opposite culture, in triplicate, separately set trichoderma reesei mutant and Trichoderma harzianum mutant list is cultivated and compared,
Being inverted in 28 DEG C of warm and humid constant casees, every day, timing was observed, such as Fig. 1.
Trichoderma reesei mutant and Trichoderma harzianum opposite culture on identical PDA solid medium, trichoderma reesei
Mutant i.e. contacts in 3d with Trichoderma harzianum mutant, and 4d grows in respective point sample district, and bacterium colony is equal
The most uniform, shinny, alternate in green white, occur without the growth of a kind of fungus and suppress what another fungus grew to show
As, show and be complementary to one another the other side's nutrient, the growth of the other side that mutually promotes.Fig. 1 shows that I group of sudden change group is produced
(gene sequencing has six subversivenesses sudden changes for the cellulolytic enzyme activity of raw disease resistance and chitinase activity decline
Caused), but II group of B substance can be provided so that it is super normal growth；II. gene sequencing is that growth promotion gibberellins closes
Becoming enzyme gene to have frameshift mutation at 20, lose growth promotion function, scarce growth promotion functional materials, I then
Somatotrophic gibberellins gene is without damage, it is possible to provide the material that trichoderma reesei growing power is weak, and it is right to make to adapt to as early as possible
The hydrolysis of lignocellulose-containing hexose needed for providing metabolism, strengthen the degraded to straw.
Embodiment four
Solid fermentation culture medium is prepared
Depending on plantation chief crop breediness, other crops can increase and decrease as one sees fit, and total amount is to mix it with water
Front dry substance mixture amplitude is 30～50%.
1. rice straw powder class (granularity 120 mesh is containing Oryza sativa L. var. glutinosa Matsum., celestial rice or the raw rice of drought): 45.0%, milled powders of cornstalk:
20%, Testa Tritici: 20%.
2. corn stalk powder class: corn stalk powder 45%, Testa Tritici 15%, rice straw powder 25%.
3. Caulis Sacchari sinensis bar powder class: Caulis Sacchari sinensis bar powder 45%, rice straw powder 25%, other powder of straw 15% are (such as beans straw
Stalk and other powder of straw), additive.
4. additive 20%: (containing fish flour, domestic animals and fowls slaughter rear blood powder to compound protein powder, and silkworm pupa albumen (contains
Amount 8%, chitin 7%)).
5. Tween-20: according to requiring configuration.
Embodiment five
The degraded of straw
(1) constituent in straw
Straw refers to the herbaceous plant stem after defoliation, flower, fruit and root.Be mainly composed of cellulose, hemicellulose,
Protein, pectic substance and a small amount of lignin；The monomeric species constituting these compositions is various, the straw of separate sources
In composition and constitute kinds of ingredients monomer the most different.Therefore, if straw is effectively degraded, absolutely not one
Kind microorganism, but the compound system of two or more microorganism compositions.
(2) grinding and processing of component
1. the processing principle of one pack system
In view of final goods areSpheroidal particle, is outward component and the inclusion agents such as peat,
Fungus is comprised and protects in pouch, be then sprinkling upon in soil sprouting with planting seed, rely on straw institute in soil
Grow containing nutritional labeling and other trace element, and Straw decomposing is become Organic substance, suppress or kill non-
Group's fungus, reaches the target of anti-crop pest.For ease of fermentation, both need to improve microorganism and reactant is connect
Contacting surface, keeps there is a fixed gap between granule again, is beneficial to ventilation.Need to make granularity is 0.01～1.0mm
, it is simple to pelletize.
2. ferment base material
Corn stalk powder
Rice straw powder
Straw powder
Caulis Sacchari sinensis powder of straw
Other herbaceous plant straw powder (such as beans and the crop material of Cruciferae)
3. additive (itrogenous organic substance)
A fish flour
B blood powder
C animal (shellfish, Limax, spiral shell) residuum
D ammonium sulfate
4. fermentation pH buffer system
0.05mol/L pH5.0 ± 0.2 citrate buffer solution (citric acid-sodium citrate)
5. 1.0%Tween-20
(3) solid fermentation system is set up
1. bacterial strain prepares: take screened two mutant strain trichoderma reesei mutant, Trichoderma harzianum mutant divides
It is not seeded in two culture medium of PDA, activates 5-7d respectively.
2. seed amplification culture: institute's activated strains 10mL sterilized water is washed down respectively and is transferred to aseptic
In 150mL conical flask, then with 120r/min vibration 20min on constant-temperature table, make spore ball abundant
After scattering, count with blood cell calculator, with 1 × 108The inoculum concentration of individual/mL is inoculated in fermentation medium.
(4) fermentation
1. fermentation medium is canned: make culture medium material and proportion of composing is prepared, then follow fermentation tank
After capacity tinning, addition pH5.0 sodium citrate buffer mixes and stirs regulation pH to 6.5, i.e. uses steam
High pressure (113 DEG C, pressure 1.050Pa) is sterilized, and is cooled to room temperature.
2. inoculation: by made seed fungus suspension by 1 × 108Individual/mL inoculum concentration, inoculates, stirs.
3. fermentation starting inoculation after, on request in 28 ± 0.5 DEG C, pressure 0.08MPa carry out ferment 11d.
4. the termination fermented: in above-mentioned sweat, through device window, after observing overall process change 1d,
Culture medium starts a small amount of white colony occur；Start caking on 2-3d culture base-material, and have a large amount of hair fleece；
There is light green color spore in the base material surface of fermentation 4d；After 5-6d, whole culture medium presents green；7-8d base material
Color gradually turns yellow, and shows that spore is the most ripe.Sampling detection, the result that spore increases shows 1-10d bacterial strain
Being constantly in growth stage, wherein 9-10d strain growth is the fastest, and during 10-12d, the speed of growth keeps constant, place
In resting state.Therefore the suitable cycle of solid fermentation is 10d and liquid fermentation cycle 5d, and the cycle is the longest.
This is because fermentation base ingredients is complicated, the conduction of heat is relatively slow and moisture scatter and disappear caused.Its fermentation period reaches
11d can opening discharging.Concrete technology flow process is as shown in Figure 2.
The above embodiment is only to be described the preferred embodiment of the present invention, not to the present invention
Scope be defined, on the premise of designing spirit without departing from the present invention, those of ordinary skill in the art to this
The various deformation made of technical scheme of invention and improvement, all should fall into the guarantor that claims of the present invention determines
In the range of protecting.
Claims (8)
1. a composite fermentation straw degradative agent, it is characterised in that: described straw degradative agent be trichoderma reesei with
The Trichoderma harzianum compound fungus of opposite culture gained after physical mutagenesis respectively.
2. the preparation method of the composite fermentation straw degradative agent described in a claim 1, it is characterised in that
Comprise the following steps:
(1) buy from market or nature separates wild trichoderma reesei and Trichoderma harzianum；
(2) N is used+Or Ar+Or CO2 +Trichoderma reesei and Trichoderma harzianum are carried out respectively the injection of ion beam, and
Compare with matched group, separate trichoderma reesei mutant and Trichoderma harzianum mutant；
(3) trichoderma reesei mutant and Trichoderma harzianum mutant are cultivated respectively in PDA culture medium, screening
Go out bacterium colony shinny, eugonic, then trichoderma reesei mutant is inoculated into cellulose screening culture medium, Kazakhstan
Thatch Trichoderma spp. mutant is inoculated in chitin screening culture medium, and screening is purified into high dynamic strain；
(4) by the high vigor trichoderma reesei mutant filtered out and Trichoderma harzianum mutant point on same flat board
Opposite culture, is used for making two kinds of mutants to complement each other the other side's nutrient, the other side that mutually promotes growth；Repeat three
Secondary, mix amplification culture, measure its functional enzyme activity.
The preparation method of a kind of composite fermentation straw degradative agent the most according to claim 2, its feature exists
In: the implantation dosage of step (2) described ion beam is 10kev.
The preparation method of a kind of composite fermentation straw degradative agent the most according to claim 2, its feature exists
In: step (3) described PDA culture medium is prepared by following substances in percentage by weight concentration: Rhizoma Solani tuber osi 2～3%；
Fish protein powder 0.5～3.3%；FeSO40.01～0.03%；KH2PO40.02～0.20%；MgSO40.05～0.35%；
Na2B2O7·10H2O 0.025～0.05%；Na2SeO30.025～0.05%；ZnSO40.01～0.02%；Surplus
For water.
5. the application of composite fermentation straw degradative agent described in a claim 1, it is characterised in that: according to not
Same variety of crops, prepares solid fermentation culture medium, adds and treats degrading straw, inoculates composite fermentation straw degradative
Agent carries out fermentation culture.
The application of a kind of composite fermentation straw degradative agent the most according to claim 5, it is characterised in that:
Described solid fermentation culture medium includes ferment base material, buffer, nitrogen-containing compound and Tween-20, and dry
The weight of material accounts for the 30～50% of total amount.
The application of a kind of composite fermentation straw degradative agent the most according to claim 5, it is characterised in that:
The inoculum concentration of described composite fermentation straw degradative agent is 0.5 × 108～1.5 × 108Individual/mL.
The application of a kind of composite fermentation straw degradative agent the most according to claim 5, it is characterised in that
The condition of described fermentation culture is: pH6～7, temperature 27～29 DEG C, pressure 0.05～0.1MPa, fermentation 10～13
My god.
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| CN114032227A (en) * | 2021-09-16 | 2022-02-11 | 厦门大学 | Composite cellulase for saccharification of lignocellulose and preparation method thereof |
| CN114260283A (en) * | 2021-11-19 | 2022-04-01 | 南开大学 | Method for converting soluble organic matters based on delignification-based disposable tableware |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110476969A (en) * | 2019-08-29 | 2019-11-22 | 段青文 | Accelerate the composition and its preparation method and application of degradation of pesticide |
| CN114032227A (en) * | 2021-09-16 | 2022-02-11 | 厦门大学 | Composite cellulase for saccharification of lignocellulose and preparation method thereof |
| CN114260283A (en) * | 2021-11-19 | 2022-04-01 | 南开大学 | Method for converting soluble organic matters based on delignification-based disposable tableware |
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