CN110358704A - A kind of manioc waste fermentation special bacteria agent and preparation method thereof - Google Patents
A kind of manioc waste fermentation special bacteria agent and preparation method thereof Download PDFInfo
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- CN110358704A CN110358704A CN201910560673.5A CN201910560673A CN110358704A CN 110358704 A CN110358704 A CN 110358704A CN 201910560673 A CN201910560673 A CN 201910560673A CN 110358704 A CN110358704 A CN 110358704A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
- C05F5/006—Waste from chemical processing of material, e.g. diestillation, roasting, cooking
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
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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
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The present invention discloses a kind of manioc waste fermentation special bacteria agent and preparation method thereof, and of the present invention includes three kinds of bacterium, respectively highland bacillus, bacillus licheniformis, bacillus subtilis；Two kinds of fungi, respectively Trichoderma viride and candida tropicalis；Actinomyces are a kind of, for ash slightly red streptomyces.It can be very good symbiosis between bacterial strain in manioc waste fermentation special bacteria agent formula of the present invention, without obvious antagonism；It can accelerate the heating of manioc waste fermentation heap body; it can accelerate the fermenting speed of manioc waste compost material, mitigate the foul smell in heap body; improve manioc waste fermented quality; all indicators are better than the cassava slag muck body for being not added with microbial inoculum, the present invention is that manioc waste solid fermentation microbial inoculum quickly breeds and provides technical support in manioc waste matrix large-scale production.
Description
Technical field
The invention belongs to agricultural substrate technical field of microbial fermentation, and in particular to a kind of manioc waste fermentation special bacteria agent and
Preparation method.
Background technique
Cassava is also known as the wooden sweet potato or cassava, and root tuber is rich in starch, there is the good reputation of " king of starch ", " energy crop ".Cassava
Slag is that remaining residue after starch or industrial alcohol is produced and processed using cassava, these organic wastes are often dropped, not only
Land occupation resource, but also will cause serious environmental pollution.Manioc waste rich in nutrition content, containing up to 78.7% it is non-
Nitrogen compound, main component are soluble starch compound (such as monosaccharide, starch) and fiber substance, and content of starch is 40%
~50%, it is good carbon source.Currently, the research in terms of in relation to manioc waste resource utilization emerges one after another, manioc waste is as one
It is significant that kind of renewable resource in substitution turf does the Application effect in matrix, has many advantages, such as stable in physicochemical property, cheap.
Manioc waste is matrixing using fermentation as the processing method of the sustainable management of agriculture and forestry organic waste material and recycling, not only can be improved
Economic benefit, moreover it is possible to mitigate the problem of environmental pollution generated due to manioc waste air storage, but still lack manioc waste fermentation at present
Special microorganism microbial inoculum and products thereof.The microbial bacterial agent for developing and using highly effective and safe has carried out the matrixing fermentation of agriculture and forestry organic waste material
Trend as current industry development.
Summary of the invention
The object of the present invention is to provide a kind of manioc waste fermentation special bacteria agents and preparation method thereof, and the present invention is solid for manioc waste
Body fermenting agent is quickly bred and manioc waste scale matrix is produced and laid the foundation.
To achieve the goals above, technical solution provided by the invention are as follows:
A kind of manioc waste fermentation special bacteria agent, the manioc waste fermentation special bacteria agent includes three kinds of bacterium, respectively high
Ground bacillus (Bacillus altitudinis), bacillus licheniformis (Bacillus licheniformis), withered grass bud
Spore bacillus (Bacillus subtilis)；Two kinds of fungi, respectively Trichoderma viride (Trichoderma viride) and the torrid zone is false
Silk yeast (Candida tropicalis (Castell.) Berkhout)；Actinomyces one kind is ash slightly red streptomyces
(Streptomyces griseorubens)。
Highland bacillus (Bacillus altitudinis) of the present invention, bacillus licheniformis (Bacillus
Licheniformis), bacillus subtilis (Bacillus subtilis), Trichoderma viride (Trichoderma viride),
Candida tropicalis (Candida tropicalis (Castell.) Berkhout), ash slightly red streptomyces (Streptomyces
Griseorubens it) is known in the art bacterial strain, purchase acquisition can be carried out by commercially available approach, inventor is more by screening
A bacterial strain carries out manioc waste fermentation test research, it can be achieved that the contents of the present invention.
Strain used in manioc waste microbial bacterial agent of the present invention: highland bacillus, bacillus licheniformis, Trichoderma viride and
Ash slightly red streptomyces, have stronger capacity of decomposition to lignin, cellulose and hemicellulose in cassava slag muck body etc., are conducive to
Accelerate heap temperature to increase, extend the time of megathermal period, promote conversion of the fermentation process ammonium nitrogen to nitrate nitrogen, reduction was fermented
Nitrogen loss in journey, it is final to improve the matrixing product quality of manioc waste, without obvious antagonism between bacterial strain.
In a preferred embodiment, manioc waste fermentation special bacteria agent of the present invention, highland bacillus: ground
Clothing bacillus: bacillus subtilis: Trichoderma viride: candida tropicalis: the volume ratio of ash slightly red streptomyces is (1~5): (1
~5): (1~5): (1~5): (1~5): (1~5)；Further preferably, highland bacillus: bacillus licheniformis: withered grass
Bacillus: Trichoderma viride: candida tropicalis: the volume ratio of ash slightly red streptomyces is (1~3): (1~3): (1~3): (1
~3): (1~3): (1~3)；It is further preferred that highland bacillus, bacillus licheniformis, bacillus subtilis, green wood
Mould, candida tropicalis and ash slightly red streptomyces mix in equal volume.
The present invention also provides the preparation methods of the manioc waste fermenting agent, which is characterized in that
(1) highland bacillus, bacillus licheniformis, bacillus subtilis, Trichoderma viride, the false silk ferment in the torrid zone are cultivated respectively
The suspension of female and ash slightly 6 kinds of microorganisms of red streptomyces；
(2) suspension of 6 kinds of microorganisms is uniformly mixed, obtains manioc waste liquid composite fermentation microbial inoculum；
(3) manioc waste liquid composite fermentation microbial inoculum obtained by step (2) is inoculated in the fermenter equipped with wheat bran base-material
Manioc waste fermentation special bacteria agent is made in middle culture after sieving.
The preparation of the suspension of 6 kinds of microorganisms can be prepared using the method for the prior art in step (1) of the present invention,
In a kind of preferred embodiment, the suspension that step (1) culture obtains, wherein highland bacillus suspension, bacillus licheniformis
The OD of suspension, bacillus subtilis bacteria suspension600It is 0.5~1.5；Trichoderma viride suspension, candida tropicalis suspension and ash are slightly red
Strepto- bacteria suspension miospore concentration is (0.5~1.5) * 107A/ml.
In a preferred embodiment, step (2) highland bacillus suspension: bacillus licheniformis suspension: withered grass
Bacillus suspension: Trichoderma viride suspension: candida tropicalis suspension: the volume ratio of ash slightly red streptomyces suspension is (1~5):
(1~5): (1~5): (1~5): (1~5): (1~5), preferably (1~3): (1~3): (1~3): (1~3): (1~3):
(1~3), more preferably isometric mixing.
In a preferred embodiment, the water content of step (3) wheat bran base-material is within 10%, it is preferred that cassava
Slag liquid state composite bacteria agent is inoculated in equipped with moisture content after wheat bran base-material within 30%, and preferably 15~30%.
In a preferred embodiment, the mass ratio of manioc waste liquid composite bacteria agent and wheat bran base-material be 1:(3~
5), preferably 1:4；The temperature cultivated in preferred fermenter is 25~30 DEG C, cultivates 6~7d.
Application of the manioc waste fermentation special bacteria agent of the present invention in manioc waste fermentation.
The present invention carries out the culture of bacterium solution shaking flask after activating each bacterial strain, by carrying out antagonism verifying to addition external source strain,
It was found that being inoculated in the fermenter equipped with base-materials such as wheat brans without obvious antagonism and carrying out fermented and cultured, it is made after drying sieving
Manioc waste fermentation special solid microbial inoculum.
The present invention also provides a kind of preparation method of more specifically manioc waste fermenting agent,
(a) bacterium used in microbial inoculum the preparation of test tube slant strain: is inoculated in beef extract test tube slant culture medium, institute respectively
It is inoculated in the test tube slant PDA culture medium with fungi, actinomyces used are inoculated in Gause I test tube slant culture medium, after culture
Each microorganism test tube slant strain of microbial inoculum；
(b) the antagonism verifying between two plants of external source strains: by the false silk ferment of the bacillus subtilis of step (1) activation and the torrid zone
Female slant strains are crossed culture in PDA culture medium, 28 DEG C in biochemical cultivation case culture 5 days, if two plants of bacterium growing ways of intersection
Well, show two plants of bacterium without obvious antagonism；
(c) preparation of each microbial bacteria suspension: by test tube slant strain transposing prepared by step (1) in fluid nutrient medium
In, shaking flask culture is carried out, by bacterium bacteria suspension culture to OD600About 1, the spore concentration of fungi and actinomyces is about 1*107
When a/ml, each microbial bacteria suspension is obtained；
(d) antagonism of the original strain of manioc waste and external source strain is verified: by the original strain bacteria suspension of manioc waste in step (3)
It is added to PDA plate even spread with 0.1/ ware, the sterile former filter paper of 3 diameter 6mm is affixed on thereon in triangle, in filter paper
Upper addition 20ul bacillus subtilis bacteria suspension (Candida tropicalis suspension) is placed in 28 DEG C of biochemical cultivation case and cultivates seven days,
If bacterium colony grows fine, no antibacterial ring size occurs, and shows between original strain and external source strain without obvious antagonism.
(e) preparation of manioc waste liquid composite fermentation microbial inoculum: the bacterium of all microorganism fungus kinds prepared by step (3) is outstanding
Liquid is mixed in same volume ratio, obtains manioc waste liquid composite bacteria agent；
(f) preparation of manioc waste solid fungicide: manioc waste liquid composite fermentation microbial inoculum obtained by step (5) is inoculated in
It is cultivated in fermenter equipped with wheat bran base-material, manioc waste fermenting agent is made after sieving.
Further, in the step (a), test tube used uses 18*180mm, silica gel plug 15-19mm, beef extract inclined-plane
The proportion of culture medium is that beef extract 1.5g, peptone 5.0g, NaCl2.5g, agar 9.0g are added in every 500ml aqua sterilisa, adjusts pH
7.4-7.6；The proportion of PDA slant medium is that potato 100g, glucose 10g, agar powder are added in every 500ml aqua sterilisa
9g, natural pH；The proportion of Gause I slant medium is that KNO is added in every 500ml aqua sterilisa30.50g, soluble starch
10.0g K2HPO40.25g, MgSO4.7H2O 0.25g, NaCl 0.25g, FeSO40.005g, agar 9g adjust pH 7.4-
7.6。
Further, it in the step (a), bacterium, fungi is inoculated in slant medium is placed under the conditions of 28 DEG C and train
It supports 2~3 days, actinomyces is inoculated under the conditions of Gause I slant medium is placed on 28 DEG C and are cultivated 5~7 days, after length is good
It is placed in 4 DEG C of refrigerators and saves.
Further, in the step (c), each strain is inoculated in the 150ml conical flask equipped with fluid nutrient medium, is shaken
Bottle culture parameters are set as 200r/min, and shake culture is for 24 hours.
Further, in the step (f), wheat bran is handled by ultraviolet-sterilization, and fermenter is surface in disinfection plastic barrel
Lid sterile gauze is cultivated 6~7 days in 21 DEG C~28 DEG C environment, is turned over even 1 time manually daily to get manioc waste fermenting agent, is used
Method of dilution butteron on plate detects living bacteria count in microbial inoculum, and bacterium, fungi and actinomyces living bacteria count reach micro- life in solid fungicide
Object microbial inoculum national standard (>=0.5 × 108cfu/g)。
The invention has the following beneficial effects:
Manioc waste fermenting agent provided by the invention, can be very good symbiosis between bacterial strain, without short of money between microorganism fungus kind
Anti- effect makes manioc waste solid fermentation microbial inoculum, easy to operation, practicability is stronger, is produced into using Barrel-type fermentation method
The batch production of substrate fermentation microbial inoculum product may be implemented in this reduction.
Manioc waste fermenting agent of the present invention can accelerate the heating of manioc waste fermentation heap body, can accelerate cassava slag muck
The fermenting speed of rotten material mitigates foul smell in heap body, improves manioc waste fermented quality, all indicators are better than be not added with bacterium
The cassava slag muck body of agent, the present invention are that manioc waste solid fermentation microbial inoculum is quickly bred and in manioc waste matrix large-scale production
Technical support is provided.
Detailed description of the invention
Fig. 1 is shown as a kind of Technology Roadmap of manioc waste fermentation special bacteria agent and preparation method thereof.
Fig. 2 is shown as the antagonistic effect picture between two plants of external source strains.
Fig. 3 is shown as original strain and bacillus subtilis antagonistic effect picture.
Fig. 4 is shown as original strain and candida tropicalis antagonism verifies picture.
Fig. 5 is shown as manioc waste fermentation special solid microbial inoculum finished product.
Fig. 6 is shown as addition manioc waste fermentation special solid microbial inoculum (T3) and is not added with microbial inoculum spontaneous fermentation (CK) and addition
The temperature change of commercially available microbial inoculum compares.
Fig. 7 be shown as addition addition manioc waste fermentation special solid microbial inoculum (T3) and be not added with microbial inoculum spontaneous fermentation (CK) and
Add the water-cut variation comparison of commercially available microbial inoculum.
Specific embodiment
Below with reference to embodiment, the present invention will be further described, it should be understood that specific embodiment described herein is only
To explain the present invention, it is not intended to limit the present invention, all letters under concept thereof of the invention to preparation method of the present invention
Single improve belongs within protection scope of the present invention.Following example test method without specific conditions, usually according to
The known approaches of this field.Test material as used in the following examples is unless otherwise specified from conventional biochemical reagent quotient
What shop was commercially available.
Embodiment 1:
A kind of manioc waste fermentation special bacteria agent as shown in Fig. 1 and preparation method thereof, specific steps are as follows:
(1) preparation of test tube slant strain: by bacterium used in microbial inoculum: highland bacillus, bacillus licheniformis and withered grass
Bacillus (be purchased from store north receive biology) be inoculated in respectively pH 7.4 beef extract test tube slant culture medium (culture medium at
Be divided into: beef extract 1.5g, peptone 5.0g, NaCl 2.5g, agar 9.0g, aqua sterilisa 500ml), fungi Trichoderma viride and
Candida tropicalis (being purchased from store Bei Na biotech firm) is inoculated in the test tube slant the PDA culture medium (culture medium of nature pH
Ingredient: potato 100g, glucose 10g, agar powder 9g, aqua sterilisa 500ml), actinomyces ash used omits red streptomyces (can also be by
Store Bei Na biotech firm buys) it is inoculated in the Gause I test tube slant culture medium (medium component are as follows: KNO that pH is 7.43
0.50g, soluble starch 10.0g, K2HPO40.25g, MgSO4.7H2O 0.25g, NaCl 0.25g, FeSO40.005g, fine jade
Rouge 9g, aqua sterilisa 500ml), test tube specification uses 18*180mm, and silica gel plug uses 15~19mm, in 28 DEG C of biochemical trainings after inoculation
Feeding case carries out inversion culture, after 3 days bacterium, fungi test tube slant strain are placed in 4 DEG C of refrigerators and save backup, after 6 days unwrapping wire
Bacterium test tube slant strain is placed in 4 DEG C of refrigerators and saves backup.
Antagonism verifying between (2) two plants of external source strains: by the false silk ferment of the bacillus subtilis of step (1) activation and the torrid zone
Female slant strains are crossed culture in PDA culture medium, 28 DEG C in biochemical cultivation case culture 5 days, as shown in Fig. 2, intersection two
Strain bacterium grows fine, and two plants of bacterium are without obvious antagonism.
(3) preparation of each microbial bacteria suspension: by bacterium prepared by step (1), fungi, actinomyces test tube slant strain
Transposing is in the 20ml conical flask that beef extract fluid nutrient medium, PDA liquid medium, Gause I fluid nutrient medium are housed respectively
In, under the conditions of 28 DEG C, 160 turns are shaken bacterium culture for 24 hours, by bacterium bacteria suspension culture to OD600About 1, the spore of fungi and actinomyces
Concentration is about 1*107When a/ml, each microbial bacteria suspension is obtained.
(4) antagonism of the original strain of manioc waste and external source strain is verified: by the original strain bacteria suspension of manioc waste in step (3)
It is added to PDA plate even spread with 0.1ml/ ware, the sterile former filter paper of 3 diameter 6mm is affixed on thereon in triangle, in filter paper
10ul bacillus subtilis bacteria suspension (Candida tropicalis suspension) is added on piece, is placed in 28 DEG C of biochemical cultivation case and cultivates 6 days,
As shown in Figure 3, Figure 4, bacterium colony grows fine, and no antibacterial ring size occurs, then without antagonism between original strain and external source strain.
(5) preparation of manioc waste liquid composite fermentation microbial inoculum: the bacterium of all microorganism fungus kinds prepared by step (3) is outstanding
Liquid is mixed in same volume ratio, obtains manioc waste liquid composite fermentation microbial inoculum.
(6) preparation of manioc waste solid fungicide: manioc waste liquid composite fermentation microbial inoculum obtained by step (5) is inoculated in
It is cultivated in fermenter equipped with wheat bran base-material, as shown in figure 5, manioc waste solid fungicide is made after sieving.
Solid state fermentation base-material used is wheat bran (being handled using preceding ultraviolet sterilization), by moisture control within 10%, so
Afterwards according to manioc waste liquid microbial inoculum: base-material=2:8 (mass ratio) is uniformly mixed, and is controlled moisture content within 30%, is placed in disinfection
It in fermenter, stirs, after material moistening 1h, surface cover sterile gauze, 28 DEG C~33 DEG C culture 7d manually turn over even 1 time daily, obtain cassava
Slag special solid microbial inoculum, using living bacteria count in method of dilution butteron on plate detection microbial inoculum.Bacterium, fungi and actinomyces in solid fungicide
Living bacteria count reaches microbial bacterial agent national standard (>=0.5 × 108cfu/g)。
Embodiment 2:
A kind of manioc waste fermentation special bacteria agent as shown in Fig. 1 and preparation method thereof, specific steps are as follows:
(1) preparation of test tube slant strain: by bacterium used in microbial inoculum: highland bacillus, bacillus licheniformis and withered grass
Bacillus (be purchased from store north receive biology) be inoculated in respectively pH 7.6 beef extract test tube slant culture medium (culture medium at
Be divided into: beef extract 1.5g, peptone 5.0g, NaCl 2.5g, agar 9.0g, aqua sterilisa 500ml), fungi Trichoderma viride and
Candida tropicalis (be purchased from store north receive biology) be inoculated in nature pH the test tube slant PDA culture medium (medium component:
Potato 100g, glucose 10g, agar powder 9g, aqua sterilisa 500ml), actinomyces ash used omits red streptomyces (can also be by store
Bei Na biotech firm buys) it is inoculated in the Gause I test tube slant culture medium (medium component are as follows: KNO that pH is 7.63
0.50g, soluble starch 10.0g, K2HPO4 0.25g, MgSO4.7H2O 0.25g, NaCl 0.25g, FeSO40.005g, fine jade
Rouge 9g, aqua sterilisa 500ml), test tube specification uses 18*180mm, and silica gel plug uses 15~19mm, in 30 DEG C of biochemical trainings after inoculation
Feeding case carries out inversion culture, after 2 days bacterium, fungi test tube slant strain are placed in 4 DEG C of refrigerators and save backup, after 5 days unwrapping wire
Bacterium test tube slant strain is placed in 4 DEG C of refrigerators and saves backup.
Antagonism verifying between (2) two plants of external source strains: by the false silk ferment of the bacillus subtilis of step (1) activation and the torrid zone
Female slant strains are crossed culture in PDA culture medium, 33 DEG C in biochemical cultivation case culture 7 days, two plants of bacterium growing ways of intersection are good
Good, then two plants of bacterium are without obvious antagonism.
(3) preparation of each microbial bacteria suspension: by bacterium prepared by step (1), fungi, actinomyces test tube slant strain
Transposing is in the 2000ml conical flask that beef extract fluid nutrient medium, PDA liquid medium, Gause I fluid nutrient medium are housed respectively
In, under the conditions of 33 DEG C, 250 turns are shaken bacterium culture for 24 hours, by bacterium bacteria suspension culture to OD600About 1, the spore of fungi and actinomyces
Concentration is about 1*107When a/ml, each microbial bacteria suspension is obtained.
(4) antagonism of the original strain of manioc waste and external source strain is verified: by the original strain bacteria suspension of manioc waste in step (3)
It is added to PDA plate even spread with 0.2ml/ ware, the sterile former filter paper of 3 diameter 6mm is affixed on thereon in triangle, in filter paper
20ul bacillus subtilis bacteria suspension (Candida tropicalis suspension) is added on piece, is placed in 33 DEG C of biochemical cultivation case and cultivates 7 days,
Bacterium colony grows fine, and no antibacterial ring size occurs, then without antagonism between original strain and external source strain.
(5) preparation of manioc waste liquid composite fermentation microbial inoculum: the bacterium of all microorganism fungus kinds prepared by step (3) is outstanding
Liquid is mixed in same volume ratio, obtains manioc waste liquid composite fermentation microbial inoculum.
(6) preparation of manioc waste solid fungicide: manioc waste liquid composite fermentation microbial inoculum obtained by step (5) is inoculated in
It is cultivated in fermenter equipped with wheat bran base-material, manioc waste solid fungicide is made after sieving.
Solid state fermentation base-material used is wheat bran (being handled using preceding ultraviolet sterilization), by moisture control within 10%, so
Afterwards according to manioc waste liquid microbial inoculum: base-material=2:8 (mass ratio) is uniformly mixed, and is controlled moisture content within 30%, is placed in disinfection
Fermenter stirs, and after material moistening 2h, surface cover sterile gauze, 28 DEG C of culture 7d manually turn over even 1 time daily, it is dedicated solid to obtain manioc waste
Body microbial inoculum, using living bacteria count in method of dilution butteron on plate detection microbial inoculum.Bacterium, fungi and the effective viable bacteria of actinomyces in solid fungicide
Number reaches microbial bacterial agent national standard (>=0.5 × 108cfu/g)。
Embodiment 3:
A kind of manioc waste fermentation special bacteria agent as shown in Fig. 1 and preparation method thereof, specific steps are as follows:
(1) preparation of test tube slant strain: by bacterium used in microbial inoculum: highland bacillus, bacillus licheniformis and withered grass
Bacillus (be purchased from store north receive biology) is inoculated in the beef extract test tube slant culture medium (training of pH 7.4~7.6 respectively
Support based component are as follows: beef extract 1.5g, peptone 5.0g, NaCl 2.5g, agar 9.0g, aqua sterilisa 500ml), fungi green
Trichoderma and candida tropicalis (be purchased from store north receive biology) are inoculated in the test tube slant the PDA culture medium (culture of nature pH
Based component: potato 100g, glucose 10g, agar powder 9g, aqua sterilisa 500ml), actinomyces ash used omits red streptomyces (can also
By store, Bei Na biotech firm is bought) it is inoculated in the Gause I test tube slant culture medium (medium component that pH is 7.4~7.6
Are as follows: KNO30.50g, soluble starch 10.0g, K2HPO40.25g, MgSO4.7H2O 0.25g, NaCl 0.25g, FeSO4
0.005g, agar 9g, aqua sterilisa 500ml), test tube specification uses 18*180mm, and silica gel plug uses 15~19mm, in 28 after inoculation
DEG C~30 DEG C of biochemical cultivation cases carry out inversion culture, after 2 days bacterium, fungi test tube slant strain be placed in 4 DEG C of refrigerators save it is standby
With, after 5 days actinomyces test tube slant strain is placed in 4 DEG C of refrigerators and saves backup.
Antagonism verifying between (2) two plants of external source strains: by the false silk ferment of the bacillus subtilis of step (1) activation and the torrid zone
Female slant strains are crossed culture in PDA culture medium, 28 DEG C in biochemical cultivation case culture 3~5 days, two plants of bacterium growing ways of intersection
Well, then two plants of bacterium without antagonism.
(3) preparation of each microbial bacteria suspension: by bacterium prepared by step (1), fungi, actinomyces test tube slant strain
Transposing is in the conical flask that beef extract fluid nutrient medium, PDA liquid medium, Gause I fluid nutrient medium are housed respectively, and 28
DEG C~33 DEG C under the conditions of, 160~250 turns shake bacterium culture for 24 hours, by bacterium bacteria suspension culture to OD600About 1, fungi and actinomyces
Spore concentration be about 1*107When a/ml, each microbial bacteria suspension is obtained.
(4) antagonism of the original strain of manioc waste and external source strain is verified: by the original strain bacteria suspension of manioc waste in step (3)
It is added to PDA plate even spread with 0.1ml~0.2ml/ ware, the sterile former filter paper of 3 diameter 6mm is affixed on it in triangle
On, 10ul~20ul bacillus subtilis bacteria suspension (Candida tropicalis suspension) is added on filter paper, is placed in biochemical training
It supports 28 DEG C of case to cultivate 5~7 days, bacterium colony grows fine, and no antibacterial ring size occurs, then without obvious short of money between original strain and external source strain
Anti- effect.
(5) preparation of manioc waste liquid composite fermentation microbial inoculum: the bacterium of all microorganism fungus kinds prepared by step (3) is outstanding
Liquid is mixed in same volume ratio, obtains manioc waste liquid composite fermentation microbial inoculum.
(6) preparation of manioc waste solid fungicide: manioc waste liquid composite fermentation microbial inoculum obtained by step (5) is inoculated in
It is cultivated in fermenter equipped with wheat bran base-material, manioc waste solid fungicide is made after sieving.
Solid state fermentation base-material used is wheat bran (being handled using preceding ultraviolet sterilization), by moisture control within 10%, so
Afterwards according to manioc waste liquid microbial inoculum: base-material=2:8 (mass ratio) is uniformly mixed, and is controlled moisture content within 30%, is placed in disinfection
It in fermenter, stirs, after material moistening l~2h, surface cover sterile gauze, 28 DEG C of 6~7d of culture manually turn over even 1 time daily, obtain cassava
Slag special solid microbial inoculum, using living bacteria count in method of dilution butteron on plate detection microbial inoculum.Bacterium, fungi and actinomyces in solid fungicide
Living bacteria count reaches microbial bacterial agent national standard (>=0.5 × 108cfu/g)。
Embodiment 4:
Manioc waste fermentation special solid microbial inoculum compost Contrast on effect, as shown in Figure 6, Figure 7:
The compost comparative test of manioc waste fermentation special solid microbial inoculum, it is main using manioc waste solid obtained by the present invention
Microbial inoculum is added microbial inoculum by 1 ‰ mass ratio, adjusts manioc waste moisture content to 60%~70%, build up 1.9m wide, 1.2m high, 5m long
A heap fermented (T3), to be not added with the heap body of microbial inoculum spontaneous fermentation as control.Main compost effect has: addition manioc waste
The heap body of special solid microbial inoculum can accelerated warming phase temperature raising, extend megathermal period fermentation time, be conducive to manioc waste material
The killing of middle worm's ovum and pathogen, at the 10th day of compost, temperature reached 55.93 DEG C, than being not added with microbial inoculum (CK) and addition city
The heap body for selling microbial inoculum enters the megathermal period in advance；In composting process, what the heap body that addition solid fungicide carries out decomposed fermentation generated
Unhappy smell is significantly less than not plus the heap body of bacteria fermentation, the use of manioc waste solid fungicide can reduce stink to a certain extent
Generation；In addition, the heap body moisture content decrease speed of addition manioc waste special solid microbial inoculum to be faster than CK and addition is general commercially available
The heap body of microbial inoculum.
Claims (10)
1. a kind of manioc waste fermenting agent, which is characterized in that the fermenting agent includes three kinds of bacterium, respectively highland gemma
Bacillus, bacillus licheniformis, bacillus subtilis；Two kinds of fungi, respectively Trichoderma viride and candida tropicalis；Actinomyces one
Kind, for ash slightly red streptomyces.
2. a kind of manioc waste fermenting agent according to claim 1, which is characterized in that highland bacillus: lichens gemma
Bacillus: bacillus subtilis: Trichoderma viride: candida tropicalis: the volume ratio of ash slightly red streptomyces is (1~5): (1~5):
(1~5): (1~5): (1~5): (1~5).
3. a kind of manioc waste fermenting agent according to claim 2, which is characterized in that highland bacillus: lichens gemma
Bacillus: bacillus subtilis: Trichoderma viride: candida tropicalis: the volume ratio of ash slightly red streptomyces is (1~3): (1~3):
(1~3): (1~3): (1~3): (1~3).
4. a kind of manioc waste fermenting agent according to claim 3, which is characterized in that highland bacillus, lichens gemma
Bacillus, bacillus subtilis, Trichoderma viride, candida tropicalis and ash slightly red streptomyces mix in equal volume.
5. the preparation method of manioc waste fermenting agent described in claim 1, which is characterized in that
(1) respectively cultivate highland bacillus, bacillus licheniformis, bacillus subtilis, Trichoderma viride, candida tropicalis and
The suspension of ash slightly 6 kinds of microorganisms of red streptomyces；
(2) suspension of 6 kinds of microorganisms is uniformly mixed, obtains manioc waste liquid composite fermentation microbial inoculum；
(3) manioc waste liquid composite fermentation microbial inoculum obtained by step (2) is inoculated in the fermenter equipped with wheat bran base-material and is trained
It supports, manioc waste fermentation special bacteria agent is made after sieving.
6. the preparation method of manioc waste fermenting agent according to claim 5, which is characterized in that step (1) culture obtains
Suspension, the wherein OD of highland bacillus suspension, bacillus licheniformis suspension, bacillus subtilis bacteria suspension600Be 0.5~
1.5；Trichoderma viride suspension, candida tropicalis suspension and ash slightly red streptomyces suspension miospore concentration are (0.5~1.5) * 107
A/ml.
7. the preparation method of manioc waste fermenting agent according to claim 5, which is characterized in that step (2) highland gemma
Bacillus suspension: bacillus licheniformis suspension: bacillus subtilis bacteria suspension: Trichoderma viride suspension: candida tropicalis suspension: ash is slightly
The volume ratio of red streptomyces suspension is (1~5): (1~5): (1~5): (1~5): (1~5): (1~5), preferably (1~3):
(1~3): (1~3): (1~3): (1~3): (1~3), more preferably isometric mixing.
8. the preparation method of manioc waste fermenting agent according to claim 5, which is characterized in that step (3) wheat bran base-material
Water content within 10%, it is preferred that manioc waste liquid composite bacteria agent is inoculated in equipped with moisture content after wheat bran base-material 30%
Within, preferably 15~30%.
9. the preparation method of manioc waste fermenting agent according to claim 5, which is characterized in that manioc waste liquid compound bacteria
The mass ratio of agent and wheat bran base-material is 1:(3~5), preferably 1:4；The temperature cultivated in preferred fermenter is 25~30 DEG C,
Cultivate 6~7d.
10. application of the manioc waste fermentation special bacteria agent described in claim 1 in manioc waste fermentation.
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