CN103667069A - Agricultural straw degrading fungus penicillium oxalicum NJGZ-2 and fungal agent thereof - Google Patents
Agricultural straw degrading fungus penicillium oxalicum NJGZ-2 and fungal agent thereof Download PDFInfo
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Abstract
The invention relates to a normal-temperature fungus NJGZ-2 capable of degrading agricultural waste straw, belonging to the agricultural waste resource utilization technology. The class of the strain NJGZ-2 is named penicillium oxalicum, and the culture is collected in the China General Microbiological Culture Collection Center (CGMCC), with collection number CGMCC No.7527. Tests indicate that after the rice straw is inoculated with a spore suspension (1% 10<7> spores/mL), the weight loss ratio of the rice straw is 59.7% after 7 days of 170r/min liquid fermentation culture at 30 DEG C. In the fermentation crude enzyme extraction liquid, the activities of incision enzyme, beta-glucosidase, xylanase, beta-xylosidase and total cellulose are 113.3U/gds, 11.6U/gds, 1,439.7U/gds, 2.0U/gds and 13.8U/gds respectively. The crude enzyme liquid can hydrolyze low-concentration alkali-treated corncob to generate high-concentration reducing sugar (962.2mg of reducing sugar/gram of substrate). According to the invention, the agricultural waste straw is degraded by the microbial fermentation technology and is converted into organic fertilizer, therefore, the waste can be turned into wealth to protect the environment, and application prospects are broad.
Description
One, technical field
The present invention relates to strain agricultural stalk degradative fungi and a microbial inoculum thereof, belong to intensive agriculture production technology, be exclusively used in the discarded stalk of degraded and produce organic fertilizer, realize reclaiming organic waste.
Two, background technology
China Shi Yige large agricultural country, how processing agricultural crop straw is the difficult problem that most of agricultures district faces, if a large amount of discarded stalks is processed not in time, not only affects normal farming, and has become the place of disease and pest breeding; As it is committed to the flames, serious environment pollution not only, has also wasted useful resources and the energy contained in stalk simultaneously.Utilize straw degradative bacterium to process discarded stalk, there is environmental protection, the feature such as energy-conservation, efficient, meet the requirement of Sustainable development.Agriculture waste stalk is a kind of most valuable agricultural resource, contains mineral nutrient element and the trace elements such as abundant organic carbon and a large amount of nitrogen, phosphorus, potassium, silicon, and rationally utilizing this resource is a vital task of agricultural sustainable development.Yet, in stalk, contain a large amount of xylogen and Mierocrystalline cellulose, not perishable, therefore should not go back on the spot field and process, be not suitable for yet and make feed.So most stalks are all dropped as waste or are burned, in various degree ground contamination local life production environment, also caused the waste of precious resources.
A large amount of researchs show, straw-returning can effectively increase soil organic matter content, improvement soil, and culture fertility, is of great significance for alleviating Chinese soil nitrogen, phosphorus, potash fertilizer contradiction tool out of proportion.But it is few because Chinese population, to plough, and arable land multiple crop index is high, and reverse cropping time is short, and stalk carbon-nitrogen ratio is high in addition, to straw-returning, brings difficulty, and effect is poor.Therefore by stalk, being processed and be made into fertilizer, is the technology of an energy environment protection doulbe-sides' victory.The recycling treatment of stalk includes Physical, chemical method and biological process, and front two class methods processing costs are high, and easily causes secondary pollution, therefore mostly adopts at present microorganism treatment.Microorganism treatment is exactly that stalk is decomposed to microbial inoculum by adding specific stalk, under the condition of field normal temperature, accelerates decomposition and the conversion of stalk.Stalk resource is by the oligosaccharides that resolves into glucose, wood sugar, cellobiose, solubility of the cell wall degradation relevant enzyme of microorganism secretion, is the preferential carbon source nutritive substance utilizing of most of biologies and by direct trans-utilization efficiently.Therefore, utilize cellulose decomposition microbial inoculum to set up the novel biological straw important channel that also field technology will become solves a large amount of stalk resources.
By inoculation straw degradative microbial inoculum, accelerate the decomposition of lignocellulose material in substrate, mycetogenetic born of the same parents' outer fiber element lytic enzyme activity is far away higher than bacteriogenic cellulose decomposition enzymic activity.If can screen corresponding efficient function stem, use it for degraded and the fertiliser production of discarded stalk, will have good application prospect.
Three, summary of the invention
1. technical problem
The object of the invention is to screen the normal temperature fungi penicillium oxalicum that agriculture waste stalk can be efficiently decomposed in a strain, it can efficiently decompose agriculture waste stalk, makes the discarded stalk resource in farmland, guarantees shaping up of sustainable agriculture.
2. technical scheme
The normal temperature fungi of one strain energy degrading straw, this fungi NJGZ-2 belongs to penicillium oxalicum (Penicillium oxalicum), be deposited at China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 27th, 2013, depositary institution address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, culture presevation number is CGMCC NO.7527; Its biological characteristics is, on PDA flat board, is shallow white, profile rule in the middle of initial stage bacterium colony, and outward appearance is fine hair shape.Hyphal cell is thread to interweave, tool tabula; Conidiophore is not had a podocyte, and penicillus is symmetrical twin type, and the elongated gradual change of bottle stalk is sharp-pointed; Conidium is oval, is blue-greenish colour.Adopt fungi universal primer ITS1 and ITS4 to carry out pcr amplification, will after PCR product connection pMD-19T carrier, check order, institute's calling sequence is compared through NCBI, has 99% similarity with Penicillium oxalicum strain a1s2_d38.Choose some bacterial strain drawing systems close with it and grow evolutionary tree, and in conjunction with bacterium colony and mycelia morphological feature, identify that this bacterial strain is penicillium oxalicum.
NJGZ-2 can grow and produce transparent hydrolysis circle on the Congo red substratum of CMC-.The Congo red culture medium prescription of CMC-is: Xylo-Mucine (CMC-Na) 10g, (NH
4)
2sO
42g, KH
2pO
41g, MgSO
47H
2o0.5g, Congo red 0.2g, agar 20g, water 1000mL, 121 ℃ of autoclaving 20min.
The preparation method of straw degradative bacterium NJGZ-2 spore suspension (microbial inoculum) is:
The preparation of liquid nutrient medium: dry the rice straw powder 20g after pulverizing, (NH
4)
2sO
41.4g, KH
2pO
42g, MgSO
47H
2o0.3g, CaCl
20.4g, Tryptones 1g, FeSO
47H
2o0.0075g; MnSO
4h
2o0.0025g; ZnSO
40.002g, CoCl
20.003g, water 1000mL, 121 ℃ of autoclaving 20min;
Rice straw powder culture medium prescription is: dry the rice straw powder 10g after pulverizing, KH
2p
43.0g; NaNO
33.0g; CaCl
20.5g; MgSO
47H
2o0.5g, FeSO
47H
2o0.0075g, MnSO
4h
2o0.0025g, ZnSO
40.002g, CoCl
20.003g, agar 20g, water 1000mL, 121 ℃ of autoclaving 20min;
The preparation of liquid nutrient medium: dry the rice straw powder 20g after pulverizing, (NH
4)
2sO
41.4g, KH
2pO
42g, MgSO
47H
2o0.3g, CaCl
20.4g, Tryptones 1g, FeSO
47H
2o0.0075g, MnSO
4h
2o0.0025g, ZnSO
40.002g, CoCl
20.003g, water 1000mL, 121 ℃ of autoclaving 20min;
Bacterial strain NJGZ-2 is seeded in the triangular flask that 50mL straw powder substratum is housed, under 30 ℃ of conditions, static cultivation 7d, adds 30mL sterilized water and goes in triangular flask, is placed on shaking table and shakes 30min, with being spore suspension microbial inoculum after filtered through gauze culture.Inoculum size by 1% is inoculated in liquid nutrient medium, under 30 ℃ of conditions, with 170r/min, cultivates 6d, and thalline and impurity are removed in centrifuging, obtain crude enzyme liquid.
Enzymolysis stalk reaction system: 0.5g stalk powder, 20mL crude enzyme liquid (pH5.0), in the centrifuge tube with screw socket cap of 50mL, 50 ℃, 200r/min, is hydrolyzed 72 hours.
Stalk powder used is crossed 40 mesh sieves after referring to and pulverizing, and water content is below 10%.
The above-mentioned spore suspension microbial inoculum agriculture waste stalk that can be used for degrading.
3. beneficial effect
This bacterial strain can produce hydrolysis circle (Fig. 1) on the Congo red substratum of CMC-, can on the substratum for sole carbon source, grow at agricultural wastes straw stalks such as () paddy rice, wheat, corns.This bacterium piece is inoculated in after rice straw powder substratum, and static cultivation 7d under 30 ℃ of conditions adds after sterilized water filters mycelia and can obtain spore suspension.Test shows, rice straw inoculating spores (1% 10
7spore/mL) after suspension, solid fermentation 14d under 170r/min liquid fermentation and culture 7d and quiescent conditions under 30 ℃ of conditions, the rate of weight loss of rice straw is respectively 59.7% and 32.5%.After liquid fermenting completes, filter mycelia and solid impurity and obtain thick zyme extract, its restriction endonuclease, beta-glucosidase, zytase, xylobiase is alive and the plain enzymic activity of total fiber is respectively 113.3U/gds, 11.6U/gds, 1439.7U/gds, 2.0U/gds and 13.8U/gds.Crude enzyme liquid can be hydrolyzed the reducing sugar (962.2mg reducing sugar/g substrate) of the corn cob generation high density of low concentration alkali processing; the present invention utilizes the method degraded agriculture waste stalk of microorganism fermentation to make it be converted into organic fertilizer; can turn waste into wealth, protection of the environment, have broad application prospects.
Straw degradative fungi NJGZ-2 can make organic materials by discarding after agricultural stalk fermentation is decomposed, and accelerates straw-returning and becomes thoroughly decomposed, and not only can turn waste into wealth, protection of the environment, can also promote sustainable development, and has broad application prospects.
Accompanying drawing explanation
Fig. 1 identifies the phylogenetic tree of NJGZ-2
The hydrolysis circle that Fig. 2 NJGZ-2 produces on the Congo red flat board of CMC-
Fig. 3 NJGZ-2ITS rRNA nucleotide sequence
Contrast effect and Electronic Speculum figure before and after the degraded of Fig. 4 rice straw
The thick enzymic hydrolysis stalk of Fig. 5 produces reducing sugar
Four, embodiment
1) efficient straw degradation bacteria strains is separated: this degradation bacteria is to screen and obtain from Pattern of Zijinshan rots leaf soil.Utilize gradient dilution coating method that the suspension of the pedotheque of stepwise dilution is applied to the Congo red flat board of CMC-, cultivate screen fibre element degradation bacteria for 30 ℃.The Congo red culture medium prescription of above-mentioned CMC-is: Xylo-Mucine (CMC-Na) 10g, (NH
4)
2sO
42g, KH
2pO
41g, MgSO
47H
2o0.5g, Congo red 0.2g, agar 20g, water 1000mL, 121 ℃ of autoclaving 20min.
Select and on the Congo red flat board of CMC-, produce transparent circle greatly and the bacterial strain purifies and separates faster of growing, then, they are seeded to take on the rice straw powder substratum that rice straw is sole carbon source and cultivate, relatively each strain growth speed and growing state, finally obtain can efficient degradation tabacco straw high temperature fungi NJGZ-2.On PDA flat board, in the middle of initial stage bacterium colony, be shallow white, profile rule, outward appearance is fine hair shape.Hyphal cell is thread to interweave, tool tabula; Conidiophore is not had a podocyte, and penicillus is symmetrical twin type, and the elongated gradual change of bottle stalk is sharp-pointed; Conidium is oval, is blue-greenish colour.Adopt fungi universal primer ITS1 and ITS4 to carry out pcr amplification, will after PCR product connection pMD-19T carrier, check order, institute's calling sequence is compared through NCBI, has 99% similarity with Penicillium oxalicum strain a1s2_d38.Choose some bacterial strain drawing systems close with it and grow evolutionary tree, and in conjunction with bacterium colony and mycelia morphological feature, identify that this bacterial strain is penicillium oxalicum (Penicillium oxalicum).
2) straw degradative bacterium NJGZ-2 spore suspension microbial inoculum
Bacterial strain NJGZ-2 is seeded in the triangular flask that 50mL straw powder substratum is housed, under 30 ℃ of conditions, static cultivation 7d, adds 30mL sterilized water and goes in triangular flask, is placed on shaking table and shakes 30min, with being spore suspension microbial inoculum after filtered through gauze culture.
3) preparation of crude enzyme liquid
In the liquid nutrient medium that volume inoculating spores suspension microbial inoculum according to 1% is carbon source in rice straw, under 30 ℃ of 170r/min conditions, cultivate 6d, thalline and impurity are removed in centrifuging, obtain crude enzyme liquid.Measure restriction endonuclease, beta-glucosidase, zytase in crude enzyme liquid, xylobiase is alive and the plain enzymic activity of total fiber.
Gds represents every gram of dry weight substrate.It can be seen from the table, this bacterium can produce the enzyme of a large amount of relevant Mierocrystalline celluloses and hemicellulose degraded, for its different stalk substrates of efficiently degrading fast provide the foundation.
Enzymolysis stalk reaction system: 0.5g stalk powder, 20mL crude enzyme liquid (pH5.0), in the centrifuge tube with screw socket cap of 50mL, 50 ℃, 200r/min, is hydrolyzed 72 hours.Different time points sampling utilizes DNS method to measure the reducing sugar content of its release.
Result shows, the crude enzyme liquid that NJGZ-2 produces, can promptly be hydrolyzed different agriculture waste stalks, produces utilizable reducing sugar, can in 24 hours, be hydrolyzed into reducing sugar by the pretreated stalk of 2%NaOH 90.
Mensuration and definition that enzyme is lived: the mensuration of enzymic activity is with reference to International Standards Method.Under corresponding condition, at 50 ℃, the enzyme amount that in 1min, catalytic substrate hydrolysis generates 1 μ mol resultant is defined as Yi Ge unit of enzyme activity (U).Adopt DNS method to measure reducing sugar content.
The compound method of DNS reagent: 6.3g3,5-dinitrosalicylic acid and 21g NaOH, be added to 500mL containing in the hydrothermal solution of 185g Seignette salt, then add 5g crystalline phenol and 5g S-WAT, stirring and dissolving.Cooling rear adding distil water is settled to 1000mL, stores in brown bottle standby after one week.
3) rice straw degradation effect:
Adopt the method for triangular flask liquid fermenting to study the degradation effect of this bacterium to rice straw.50 ℃ of oven dry of each 500mL triangular flask packing, shred the long rice straw 4g to 2-3cm, liquid nutrient medium 200mL.Each bottle graft kind 2mL10
7the spore suspension microbial inoculum of spore/mL concentration, mixes and is placed on 30 ℃ of constant temperature 170r/min cultivations, to inoculate equivalent sterilized water as contrast.The metamorphosis of routine observation rice straw, after 7 days, washes away the mycelia on stalk, dries to constant weight, measures its rate of weight loss, by electron microscopic observation straw degradative situation.
Result shows, after inoculation, in 24h nutrient solution, starts to occur mycelia floss, and now, rice straw starts deliquescing, and disintegration disperses.During to 48h, grown a large amount of white hypha groups.Blank process in stalk still less than what large variation, the still complete decomposition not having clearly.The stalk degradation of substrates rate of inoculation NJGZ-2 is 59.6%, and the degradation rate of contrast substrate is 15.9%.
Inoculate as can be seen from the above data degradation bacteria NJGZ-2 and can significantly accelerate the decomposition of stalk and the wherein conversion of composition, accelerate decomposition and the conversion of substrate.
Claims (5)
1. the fungi NJGZ-2 of a strain degraded agricultural stalk, this fungi belongs to penicillium oxalicum (Penicillium oxalicum), be deposited at Chinese microorganism strain on April 27th, 2013 and preserve management committee's common micro-organisms center, culture presevation number is CGMCC NO.7527; Its biological characteristics is, on PDA flat board, is shallow white, profile rule in the middle of initial stage bacterium colony, and outward appearance is fine hair shape; Hyphal cell is thread to interweave, tool tabula; Conidiophore is not had a podocyte, and penicillus is symmetrical twin type, and the elongated gradual change of bottle stalk is sharp-pointed; Conidium is oval, is blue-greenish colour.
2. the application of agricultural stalk degradative fungi aspect straw degradative as claimed in claim 1.
3. a microbial inoculum for the degrading straw that prepared by employing fungi claimed in claim 1, it is characterized in that being prepared from by following methods:
1) preparation of rice straw powder substratum: dry the rice straw powder 10g after pulverizing, KH
2pO
43.0g, NaNO
33.0g, CaCl
20.5g, MgSO
47H
2o0.5g, FeSO
47H
2o0.0075g, MnSO
4h
2o0.0025g, ZnSO
40.002g, CoCl
20.003g, agar 20g, water 1000mL, 121 ℃ of autoclaving 20min;
2) preparation of liquid nutrient medium: dry the rice straw powder 20g after pulverizing, (NH
4)
2sO
41.4g, KH
2pO
42g, MgSO
47H
2o0.3g, CaCl
20.4g, Tryptones 1g, FeSO
47H
2o0.0075g, MnSO
4h
2o0.0025g, ZnSO
40.002g, CoCl
20.003g, water 1000mL, 121 ℃ of autoclaving 20min;
3) bacterial strain NJGZ-2 described in claim 1 is seeded in the triangular flask that 50mL straw powder substratum is housed, static cultivation 7d under 30 ℃ of conditions, adding 30mL sterilized water is placed on spore on shaking table, to shake 30min, with being spore suspension after filtered through gauze culture, with can concentrating or dilute to reach working concentration with sterilized water after blood counting chamber counting, be generally 10
7spore/mL, makes straw degradative microbial inoculum; Inoculum size by 1% is inoculated in straw powder liquid nutrient medium, under 30 ℃ of conditions, with 170r/min, cultivates 6d, and thalline and impurity are removed in centrifuging, obtain crude enzyme liquid.
4. microbial inoculum as claimed in claim 3, is characterized in that straw powder is to cross 40 mesh sieves after pulverizing, and water content is below 10%.
5. the application of microbial inoculum aspect degrading straw described in claim 3 or 4.
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Cited By (8)
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| CN104003772A (en) * | 2014-05-29 | 2014-08-27 | 南京农业大学 | Method of accelerating decomposition of maize straw retention |
| CN106348798A (en) * | 2016-08-29 | 2017-01-25 | 宿州市埇桥区杨园种植专业合作社 | Method for returning crop stalks to field by microbial fermentation |
| CN106434386A (en) * | 2016-11-24 | 2017-02-22 | 郑州大学 | Microorganism and microbial inoculum for degrading straws as well as preparation method and application of microbial inoculum |
| CN108976042A (en) * | 2018-09-14 | 2018-12-11 | 苏州格瑞格登新材料科技有限公司 | A kind of organic fertilizer and preparation method of agricultural wastes degradation |
| CN109400352A (en) * | 2018-11-12 | 2019-03-01 | 吉林建筑大学 | A method of it is fermented and is recycled using stalk microbe |
| CN110295118A (en) * | 2019-08-05 | 2019-10-01 | 湖南泰谷生态工程有限公司 | A kind of degraded cellulose, the Tarlaromyces flavus bacterial strain for adsorbing cadmium and its application |
| CN110343623A (en) * | 2019-08-05 | 2019-10-18 | 湖南泰谷生态工程有限公司 | A kind of degraded cellulose, the penicillium oxalicum bacterial strain for adsorbing cadmium and its application |
| CN110564667A (en) * | 2019-09-25 | 2019-12-13 | 云南省烟草农业科学研究院 | method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
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| CN104003772A (en) * | 2014-05-29 | 2014-08-27 | 南京农业大学 | Method of accelerating decomposition of maize straw retention |
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| CN106434386B (en) * | 2016-11-24 | 2019-06-21 | 郑州大学 | For the microorganism of degrading straw, microbial inoculum and its preparation method and application |
| CN108976042A (en) * | 2018-09-14 | 2018-12-11 | 苏州格瑞格登新材料科技有限公司 | A kind of organic fertilizer and preparation method of agricultural wastes degradation |
| CN109400352A (en) * | 2018-11-12 | 2019-03-01 | 吉林建筑大学 | A method of it is fermented and is recycled using stalk microbe |
| CN110295118A (en) * | 2019-08-05 | 2019-10-01 | 湖南泰谷生态工程有限公司 | A kind of degraded cellulose, the Tarlaromyces flavus bacterial strain for adsorbing cadmium and its application |
| CN110343623A (en) * | 2019-08-05 | 2019-10-18 | 湖南泰谷生态工程有限公司 | A kind of degraded cellulose, the penicillium oxalicum bacterial strain for adsorbing cadmium and its application |
| CN110564667A (en) * | 2019-09-25 | 2019-12-13 | 云南省烟草农业科学研究院 | method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
| CN110564667B (en) * | 2019-09-25 | 2023-02-28 | 云南省烟草农业科学研究院 | Method for efficiently suspending and concentrating zoospores of plant pathogenic oomycetes and application of zoospores |
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