CN108913616A - A method of bio-bacterial manure is prepared using tobacco leaf residue - Google Patents
A method of bio-bacterial manure is prepared using tobacco leaf residue Download PDFInfo
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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
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Abstract
The invention discloses a kind of methods using the preparation bio-bacterial manure of tobacco leaf residue.The seed liquor of bacillus subtilis, colloid bacillus cereus and Paecilomyces lilacinus is added as culture medium in residue after extracting active constituent using dry tobacco leaf or fresh tobacco leaf, and solid state rheology 7~15 days after mixing can obtain corresponding bio-bacterial manure, and wherein gemma number is more than 109cfu/g.The present invention, without adding other nutriments, has saved production cost using the tobacco leaf residue after effective component extracting as solid medium；Microorganism in product exists with spore form, can long-time stable save, ensure that the microbial activity of product.Product is rich in beneficial microbe and organic matter, and soil organic matter content can be improved, and improves soil microenvironment, reduces fertilizer amount, reduces pest and disease damage, improves crop quality and yield.
Description
Technical field
The invention belongs to biological husbantry technical fields, are related to a kind of preparation of composite biological fertilizer, and are related to discarded cigarette
The comprehensive utilization of leaf residue.
Background technique
China's leaf tobacco production is throughout the whole nation, and annual yield of tobacco is 400~5,000,000 tons, the annual amount of tobacco waste
There are 900,000~1,000,000 tons, accounts for about the 18%~25% of total output.In addition, being abandoned because of weather, pest and disease damage or mismanagement or not
Field fresh tobacco leaf quantity that is qualified and abandoning is also very big, therefore the comprehensive utilization of tobacco waste has become domestic tobacco business
One of important research direction.For tobacco leaf rich in protein, carbohydrate, lignin, cellulose, organic acid, pectin etc., N-P-K content is aobvious
It writes and is higher than cereal crops stalk, especially total nitrogen up to 1.5%~3%, total reducing sugar is up to 10%~20%, and total potassium is up to 2%~5%.It mentions
Tobacco leaf residue after taking effective component (such as nicotine, Salanesol) is still rich in organic matter and other microelements, is that microorganism is solid
The good matrix of state culture is badly in need of developing simple and easy microculture technique.
Solid state rheology refers to the process of that microorganism grows and is metabolized on the culture medium almost without free water, has equipment
Simply, low energy consumption, easy to operate, post-processing is simple, pollution less, non-wastewater discharge, the advantages that culture medium is simple substantially.With novel
The discovery of strain excellent and the application of technique of gene detection, the condition of solid state rheology are gradually improved, from single culture culture toward
In hybrid bacterial strain culture.Bio-bacterial manure generally includes multiple-microorganism, and common strain has bacillus subtilis, colloid gemma bar
Bacterium, bacillus licheniformis, bacillus megaterium, side chain bacillus brevis, bacillus amyloliquefaciens, Paecilomyces lilacinus etc..
From the document published in recent years it can be found that with agriculture and forestry organic waste material (such as stalk, edible fungi residue, corncob
Deng), industrial waste biomass (such as sugaring distillery residue) and all kinds of cheap biomass resources of animal wastes be that raw material production is given birth to
The method of object bacterial manure mainly there are several types of：1) biomass and microbial inoculum are simply mixed, such as CN 107673936 A, CN
107721713 A etc..2) first compost is decomposed by biomass, then adds the mixing of several functions microorganism, such as CN
107746356 106,336,269 106187590 A etc. of A, CN of A, CN.3) two-part solid state rheology：First compost maturity biology
Then matter adds functional microorganism solid state rheology, such as 106007824 A of CN.4) solid-state training is carried out by matrix of biomass
It supports, such as 107,337,517 107417457 A of A, CN of CN.
There is the report of some tobacco leaf bio-bacterial manures in the prior art, the tobacco leaf utilized is mainly that waste/hypo-tobacco leaf or field are discarded
Fresh tobacco leaves, such as 105,175,193 103274784 A of A, CN of CN103274785A, CN.Wherein, the active material of tobacco leaf
It is not extracted by, greatly causes the wasting of resources.The production technology of use is mainly compost, and formula is complicated, needs to add many
Auxiliary material, it is time-consuming very long, it usually needs 1~2 month.Common issue existing for this compost production method is：Microorganism was producing
The situation of change of interaction, type and strain quantity is unintelligible in journey, it is difficult to effectively control final strain quantity and ratio
Example.Therefore, in the field of comprehensive utilization of tobacco leaf, it is also necessary to which further widening roadbed, exploitation can ground and existing Tobacco Leaf Industry skills
The chimeric technology of art frame, to deepen the application of raw material.
To sum up, production of the present invention for the growth characteristic and microbial-bacterial fertilizer using status and tobacco leaf of waste tobacco leaf
Status develops a kind of tobacco leaf bio-bacterial manure, and production process is simple, and the number and ratio of every kind of bacterium are clear, and the invention is for cigarette
The development and utilization of utilization and the bio-bacterial manure of grassland industry waste tobacco leaf all have significance.
Summary of the invention
It is an object of that present invention to provide a kind of technical solutions for making full use of the tobacco leaf residue after extracting active material.It is benefit
Mixed bacterium solid state rheology, the method for preparing bio-bacterial manure are carried out with the tobacco leaf residue that activated product industrial chain is discarded.
To achieve the above object, the present invention discloses a kind of method using the preparation bio-bacterial manure of tobacco leaf residue, the method
Including the step of using tobacco leaf residue as solid medium, inoculation mixed bacteria carries out solid state rheology；The wherein mixed bacteria
It is bacillus subtilis (Bacillus subtilis), colloid bacillus cereus (Bacillus mucilaginosus) and pale purple
The mixture of paecilomycerol (Paecilomyces lilacinus).
The present invention is using waste tobacco leaf residue as raw material, inoculation bacillus subtilis, colloid bacillus cereus and Paecilomyces lilacinus
The seed liquor of bacterium, is mixed, and is improved the composition ratio of the substances such as N, P, organic matter, is obtained composite microbiological fertilizer, real
The high-valued comprehensive utilization of tobacco waste is showed.
Compared with prior art, the invention has the advantages that：
One, raw material sources are cheap extensively.The present invention uses waste tobacco leaf and water as culture raw material completely, and source is wide
It is general, it is cheap, it is translated into a kind of important biomass resource, effectively using strain fermentation without complicated pretreatment
Be utilized starch in waste tobacco leaf, organic acid, protein isoreactivity substance, and be rich in the micro member such as C, N, P and K, Ca, Mg
Element.
Two, production cost is low.The solid state rheology technology that the present invention uses, have equipment is simple, low energy consumption, it is easy to operate, after
Processing is simple, pollution less, the simple advantage of non-wastewater discharge, culture medium substantially；Selected strain can in 10 DEG C or more of room temperature
Normal growth greatlies simplify equipment investment without stringent temperature control and humid control.
Three, sense organ smell is good.Raw material of the present invention is waste tobacco leaf, fragrant with strong tobacco, meanwhile, incubation
In, the fragrance materials such as raw 3-hydroxy-2-butanone of producing bacillus subtilis have been mixed into a light butter aroma in strong tobacco perfume；
Paecilomyces lilacinus can produce lavender villiform mycelium, keep product color sense organ more preferable.
Four, multi-cultur es are mixed, and simplify culture process.Three kinds of bacterium access tobacco leaf culture medium simultaneously, mix in tobacco leaf
Culture is closed, interaction forms a relatively stable micro-ecological environment, is conducive to the existence growth of three kinds of bacterium in the soil.
By adjusting the inoculative proportion of different bacterium, the product of different strain ratio finally can be obtained, that realizes strain quantity and ratio has
Effect control.Moreover, N content dramatically increases in bacterial manure after fermentation, the quality of bacterial manure is further improved.
Detailed description of the invention
Fig. 1 is viable count change curve when three kinds of bacterium are mixed under the conditions of temperature is 15 DEG C~25 DEG C indoors.
Fig. 2 is gemma number change curve when three kinds of bacterium are mixed under the conditions of temperature is 15 DEG C~25 DEG C indoors.
Fig. 3 is viable count change curve when three kinds of bacterium are mixed in 30 DEG C of insulating boxs.
Fig. 4 is gemma number change curve when three kinds of bacterium are mixed in 30 DEG C of insulating boxs.
Specific embodiment
It include using tobacco leaf residue as solid medium in technical solution of the present invention, inoculation mixed bacteria carries out solid state rheology
The step of；Wherein the mixed bacteria is bacillus subtilis (Bacillus subtilis), colloid bacillus cereus
The mixture of (Bacillus mucilaginosus) and Paecilomyces lilacinus (Paecilomyces lilacinus).
Specifically, it in the mixed bacteria, is calculated according to viable count, it is colloid bacillus cereus, bacillus subtilis, pale purple
The ratio of paecilomycerol is 1~10: 1~10: 1.
It further specifically describes, mixed bacteria described above can pass through the seed culture fluid of three kinds of microorganisms of mixing
It obtains；The preparation method of the seed culture fluid is：Three kinds of microorganisms are respectively connected to its seed culture medium, 150~200r/
Min, 30~37 DEG C of shaking table cultures to OD620It is 4~6, wherein：
The seed culture medium of colloid bacillus cereus is 5~50 times of dilutions of molasses of the ammonium phosphate containing 0.2~1g/100ml, excellent
Select 10~30 times of dilutions；
Bacillus subtilis seed culture medium is 5~50 times of dilutions of molasses, preferably 10~30 times of dilutions；
Paecilomyces lilacinus seed culture medium is 5~50 times of dilutions of molasses, preferably 10~30 times of dilutions.
On the other hand, in technical solution of the present invention, microbial fermentation characteristically is carried out using tobacco leaf residue.Described
Tobacco leaf residue, which is tobacco leaf, eliminates nicotine, Salanesol, chlorogenic acid isoreactivity substance and surplus after removing organic solvent by extraction
Remaining part point.In specific embodiment, the tobacco leaf residue is residue of the tobacco leaf after active material extracts, residual according to drying
Slag poidometer, in tobacco leaf residue, nicotine content is no more than 0.5mg/g, and Salanesol content is no more than 0.2mg/g, chlorogenic acid content
No more than 0.5mg/g.The removal of these active materials can be carried out by the technical solution that this field has been described, such as
Method documented by CN104086425A.The present invention provides a kind of specific extractive technique scheme, by the active material in tobacco leaf
It extracts, remaining tobacco leaf residue is to be used for bio-bacterial manure fermentation of the invention, and the mode is：By smashed dry tobacco leaf with
The ethanol water of 90% (volumetric concentration) is according to feed liquid mass ratio 1:5~10 mixing, it is small in 50-80 DEG C of hot reflow treatment 0.5
When, it repeats 1-5 times, residue removing ethyl alcohol and drying after extraction.More specifically, the preparation method of the tobacco leaf residue is：It takes
100g dry tobacco leaf, is added 1L90% ethyl alcohol, 50 DEG C of heat reflux 30min, and filtering separates tobacco leaf and extracting solution, tobacco leaf continues to use
It flows back in heat, heat together flows back 3 times, each 30min.Then tobacco leaf residue is dried, as used in the embodiment of the present invention
Tobacco leaf residue can be used for preparing tobacco leaf solid medium.Residual activity substance is surveyed by following methods in gained dry tobacco leaf residue
It is fixed：2g dry tobacco leaf residue is taken, is added 2mL ethanol solution (70%), ultrasonic 30min, centrifuging and taking supernatant is used for liquid chromatographic detection
Nicotine and chlorogenic acid.2g dry tobacco leaf residue is taken, 5mL dehydrated alcohol, ultrasonic 30min is added, centrifuging and taking supernatant is used for liquid chromatogram
Detect Salanesol.It can be calculated by HPLC data, handled by said extracted active material, contain cigarette in every gram of dry tobacco leaf residue
164.71 μ g of alkali, 41.25 μ g of Salanesol, 173.22 μ g of chlorogenic acid.
In another specific embodiment of the invention, the solid medium is tobacco leaf residue and water according to mass ratio
1: 1.2~2.0 mixture.It is preferred that 1: 1.4~1.6.The mixed bacteria inoculum concentration be solid medium gross mass 1~
15%.
In another specific embodiment, the incubation time of the solid state rheology is 7~15 days, is turned over every 24~48h
Material is primary, and cultivation temperature is 10~37 DEG C, preferably 20~30 DEG C.
The contents of the present invention are described further below in conjunction with non-limiting embodiment, these embodiments can make this
The present invention, the content of but do not limit the invention in any way is more fully understood in the those of ordinary skill in field.
Unless otherwise specified, strain used in the present invention is as follows：
Bacillus subtilis：By Bacillus subtilis strain (CGMCC13141 and the respectively biological, mountain purchased from Qingdao root
The green Gansu Province in east is biological, the bacillus subtilis microbial agent of the strong emerging biology in the North Sea, Jiangxi Shun Quan biology, moral Johnson & Johnson object) access tobacco leaf solid-state
Domestication is cultivated in culture medium, filters out eugonic bacterial strain, by the discovery of plate antagonistic experiment without obvious antagonism, LB training
Guarantor bacterium is spare after supporting base mixed culture.
Colloid bacillus cereus：It will be cultivated in colloid bacillus cereus strain (being purchased from open-minded biology) access tobacco leaf solid medium
Domestication, filters out eugonic bacterial strain, and it is spare that bacterium is protected after LB culture medium culture.
Paecilomyces lilacinus：It will be cultivated in colloid bacillus cereus strain (being purchased from open-minded biology) access tobacco leaf solid medium
Domestication, filters out eugonic bacterial strain, and it is spare that bacterium is protected after PDA culture medium culture.
The composition of culture medium used in this specification is as follows：
LB culture medium (1L)：Peptone 10g, yeast powder 5g, NaCl 10g.PH is adjusted to 7.0,121 DEG C of high pressures with NaOH
Sterilize 20min.
PDA culture medium：Potato 200g adds boiling rotten, with eight layers of filtered through gauze, glucose 20g is added, adds water after cooling
Complement to 1L, 115 DEG C of sterilizing 20min.
Bacillus subtilis seed culture medium (1L)：Molasses (sugar content 42%~50%) 33.3g, adds water to 1L, and 115 DEG C
Sterilize 20min.
Colloid bacillus cereus seed culture medium (1L)：Molasses (sugar content 42%~50%) 33.3g, ammonium phosphate 4g adds water
To 1L, 115 DEG C of sterilizing 20min.
Paecilomyces lilacinus seed culture medium (1L)：Molasses (sugar content 42%~50%) 33.3g, adds water to 1L, and 115 DEG C
Sterilize 20min.
The activation method of strain is as follows in this specification：
The activation of bacillus subtilis：It goes bail on superclean bench and is stored in the strain 1mL of -70 DEG C of refrigerators and is inoculated in
In 100mLLB culture medium, 200r/min, 37 DEG C of shaking table cultures 20h, OD620Reach 4~6.
The activation of colloid bacillus cereus：It goes bail on superclean bench and is stored in the strain 1mL of -70 DEG C of refrigerators and is inoculated in
In 100mLLB culture medium, 200r/min, 30 DEG C of shaking table cultures 20h, OD620Reach 4~6.
The activation of Paecilomyces lilacinus：It goes bail on superclean bench and is stored in the strain 1mL of -70 DEG C of refrigerators and is inoculated in
In 100mLPDA culture medium, 200r/min, 30 DEG C of shaking table cultures 20h, OD620Reach 4~6.
Analysis method used in this specification is as follows：
Measurement (the OD of cell density620)：When liquid culture, it is control to be centrifuged the clear liquid of removal thallus, is surveyed in 620nm
Determine absorbance.
Dilution spread flat band method measures the viable count in sample：According to《Microbial manure examination and test of products regulation (NY2321-
2013)》In living bacteria count detection method (5.1 living bacteria count) carry out.
The measurement of gemma (spore) number：Weighing solid state fermentation sample adds water to mix, 80 DEG C of heating water bath 20min, then stands
20min, subsequent operation according to《Microbial manure examination and test of products regulation (NY2321-2013)》In living bacteria count detection side
Method (5.1 living bacteria count) carries out.
Embodiment 1
Prepare microorganism seed liquid
(1) 1mL bacillus subtilis bacterium solution of activation culture 20h in LB culture medium is taken to be inoculated in 100mL withered grass gemma
Bacillus seed culture medium expands culture, 200r/min, 37 DEG C of shaking table cultures 20h, OD620Reach 4~6, as withered grass gemma
Bacillus seed liquor.
(2) 1mL colloid bacillus cereus bacterium solution of activation culture 20h in LB culture medium is taken to be inoculated in 100mL colloid gemma
Bacillus seed culture medium expands culture, 200r/min, 30 DEG C of shaking table cultures 20h, OD620Reach 4~6, as colloid gemma
Bacillus seed liquor.
(3) 1mL Paecilomyces lilacinus bacterium solution of activation culture 20h in PDA culture medium is taken to be inoculated in the pale purple quasi- blueness of 100mL
Mould seed culture medium expands culture, 200r/min, 30 DEG C of shaking table cultures 20h, OD620Reach 4~6, as pale purple quasi- blueness
Mould seed liquor.
(4) three kinds of seed liquors of above-mentioned preparation are mixed in a certain ratio, as mixed bacterium seed liquor.
Embodiment 2
Influence experiment of the raw tobacco material to thalli growth
(1) the dry tobacco leaf residue of 60g is taken, according to the solid-liquid ratio of residue and water is 1 in 500mL triangular flask:1.8 (quality
Than) mixing, tobacco leaf culture medium is made.The bacillus subtilis seed liquor of gross mass 10% is accessed, stands training in 37 DEG C of insulating boxs
It supports 4 days, it is primary every 48h stirring.Plate culture meter viable count and gemma number.Viable count and gemma number be respectively 2.5 ×
1013Cfu/g and 5 × 1010cfu/g。
(2) will untreated drying tobacco leaf crush after be used as tobacco leaf culture medium raw material, according to above-mentioned (1) method into
The fermentation of row bacillus subtilis.Incubation time is 5 days and 7 days, and obtained viable count is respectively 2.15 × 108Cfu/g and
6.37×108Cfu/g, gemma number are respectively 4.68 × 108Cfu/g and 8.45 × 108cfu/g。
As it can be seen that using by pretreated tobacco leaf residue, as fermentation raw material, viable count is higher, is also easier in shorter week
Phase reaches expected culture effect.
Embodiment 3
The comparison of single culture culture and composite bacteria culture
(1) dry tobacco leaf residue 35g and water 49mL are placed in the thin mouth conical flask of 250mL, 115 DEG C of sterilizing 20min.Access is total
The bacillus subtilis seed liquor of quality 10%, the stationary culture in 30 DEG C of insulating boxs are primary every 48h stirring.Viable count and
Gemma number reached maximum value at the 10th day, and wherein viable count is 7 × 1013Cfu/g, gemma number are 1.5 × 1013cfu/g。
(2) dry tobacco leaf residue 35g and water 49mL are placed in the thin mouth conical flask of 250mL, 115 DEG C of sterilizing 20min.Access is total
The colloid bacillus cereus seed liquor of quality 10%, the stationary culture in 30 DEG C of insulating boxs are primary every 48h stirring.Viable count and
Gemma number reached maximum value at the 12nd day, and wherein viable count is 2 × 1013Cfu/g, gemma number are 1.6 × 1013cfu/g。
(3) dry tobacco leaf residue 35g and water 49mL are placed in the thin mouth conical flask of 250mL, 115 DEG C of sterilizing 20min.Access is total
The Paecilomyces lilacinus seed liquor of quality 10%, the stationary culture in 30 DEG C of insulating boxs are primary every 48h stirring.Viable count and
Gemma number reached maximum value at the 10th day, and wherein viable count is 1 × 1014Cfu/g, gemma number are 2 × 1013cfu/g。
(4) dry tobacco leaf residue 35g and water 49mL are placed in the thin mouth conical flask of 250mL, 115 DEG C of sterilizing 20min.Access is total
(ratio of three kinds of bacterium is 1 to the mixed bacterium seed liquor of quality 10%:1:1), the stationary culture in 30 DEG C of insulating boxs, every 48h stirring
Once.The sum of viable bacteria reached maximum value at the 8th day, added up to 1.5 × 1015Cfu/g, total spore content reached 2.5 at the 10th day
×1011Cfu/g, incubation time extend, and gemma number continues growing.
Embodiment 4
Inoculative proportion tests the influence that compound bacteria is grown
Dry tobacco leaf residue 35g and water 49mL are placed in the thin mouth conical flask of 250mL, 115 DEG C of sterilizing 20min.Access 8.4mL
Mixed bacterium seed liquor, wherein viable count ratio colloid bacillus cereus: bacillus subtilis in solid medium: Paecilomyces lilacinus
It is 1:1:1,3:1:2 and 6: 1: 3.Stationary culture in 30 DEG C of insulating boxs, it is primary every 48h stirring.Plate culture meter viable count and
Gemma number, three kinds of bacterium are distinguished with morphological differences.The sum of viable bacteria reached maximum value, respectively 1.5 × 10 at the 8th day15cfu/g、
4.5×1015Cfu/g and 5.2 × 1014The ratio of cfu/g, three kinds of bacterium therein are respectively 2.7:1:6.1,6.8:1:6.8 and
3.0:1:1.3.13rd day total spore content is respectively 3.42 × 1011cfu/g、1.8×1012Cfu/g and 6.9 × 1012Cfu/g,
The ratio of three kinds of bacterium therein is respectively 2.7:1:2.4,1.4:1:1.1 and 1.6:1:1.1.
Embodiment 5
The preparation of bacterial manure is mixed under the conditions of room temperature
(1) dry tobacco leaf residue 35g and water 49mL are placed in the thin mouth conical flask of 250mL, 115 DEG C of sterilizing 20min.Access
The mixed bacterium seed liquor of 8.4mL, wherein viable count ratio colloid bacillus cereus: bacillus subtilis in solid medium: pale purple quasi-
Penicillium notatum is 6: 3: 1.Room temperature is stationary culture under the conditions of 15 DEG C~25 DEG C, primary every 48h stirring.Plate culture meter is living
Bacterium number and gemma number, three kinds of bacterium are distinguished with morphological differences.Viable count and gemma number growth curve difference are as depicted in figs. 1 and 2.?
0-8 days, the biomass of microorganism gradually increased, and gradually decreased after viable count reaches maximum value within the 8th day, maximum value can reach
1014cfu/g.Generation gemma is less within first 8 days, increases rapidly later, wherein colloid bacillus cereus Number of spores is most.To the 11st
It, the gemma number of every kind of bacterium reaches 1011cfu/g.The gemma ratio of 13rd day three kinds of bacterium is colloid bacillus cereus: withered grass gemma
Bacillus: Paecilomyces lilacinus=1.7: 0.7: 1.Dry pack can obtain bio-bacterial manure.
(2) dry tobacco leaf residue 80g and water 112mL are placed in the thin mouth conical flask of 500mL, 115 DEG C of sterilizing 20min.Access
The mixed bacterium seed liquor of 19.2mL, wherein viable count ratio is colloid bacillus cereus：Bacillus subtilis：Paecilomyces lilacinus=6:
3:1.Room temperature is stationary culture under the conditions of 15 DEG C~20 DEG C, primary every 48h stirring.Plate culture meter viable count and gemma
Number, three kinds of bacterium are distinguished with morphological differences.Viable count and gemma number growth curve difference are as shown in Figure 3 and Figure 4.Culture 10 days, three
Kind bacterium gemma number reaches 109Cfu/g, ratio are colloid bacillus cereus：Bacillus subtilis：Paecilomyces lilacinus=3: 1.9:
1.Dry pack can obtain this microbial compound bacterial fertilizer.
Embodiment 6
Regulation of the Hybrid NC machine tool to bacterial manure pH
Dry tobacco leaf residue 35g and water 49mL are placed in the thin mouth conical flask of 250mL, 115 DEG C of sterilizing 20min.It is respectively connected to
(ratio of three kinds of bacterium is 1 to the bacillus subtilis seed liquor and seed mixture liquid of gross mass 10%:1:1), in 30 DEG C of insulating boxs
Interior stationary culture, it is primary every 48h stirring and sample 0.3g measurement pH, according to 1：10 (samples：Water) mass ratio be added it is pure
Water (pH 6.48), stirs evenly, and places 30min, is measured with pH meter, recorded after stable reading, three groups of each sample is parallel.
In thallus Initial stage of culture, solid state rheology condition is faintly acid, and with the progress that thalli growth is metabolized, pH is gradually increased, wherein withered
Careless bacillus single bacterium culture substrate pH increases significantly, and 72h reaches maximum value, about 9 or so, later in 8.5~9.5 waves
It is dynamic；Gradually increase within 7 days before Hybrid NC machine tool substrate pH, in the 8th day beginning held stationary, 8~8.5 or so.In no additional substance
Under conditions of, Hybrid NC machine tool more effectively controls the pH value of product.
Embodiment 7
Influence of the residual ethanol to mixed bacterium solid state rheology in dry tobacco leaf
Dry tobacco leaf residue 35g and water 49mL (ethyl alcohol for containing 1%, 2%, 3% respectively according to percent by volume) are placed in
In the thin mouth conical flask of 250mL, 115 DEG C of sterilizing 20min.The mixed bacterium seed liquor of 8.4mL is accessed, wherein viable count ratio is colloid bud
Spore bacillus：Bacillus subtilis：Paecilomyces lilacinus=6: 3: 1.Temperature is 8 DEG C~15 DEG C stationary cultures indoors, every 48h
Stirring is primary.Plate culture meter viable count and gemma number, three kinds of bacterium are distinguished with morphological differences.Culture 8 days, the gemma number of three kinds of bacterium
It can reach 106cfu/g。
Embodiment 8
Fresh tobacco leaf residue is raw material preparation mixing bacterial manure
Fresh tobacco leaf residue 20g and water 40mL are placed in the thin mouth conical flask of 500mL, 115 DEG C of sterilizing 20min.Access 6mL's
Mixed bacterium seed liquor, wherein viable count ratio is colloid bacillus cereus：Bacillus subtilis：Paecilomyces lilacinus=6: 3: 1.In room
Interior temperature (8 DEG C~15 DEG C) stationary culture, it is primary every 48h stirring.Plate culture meter viable count and gemma number, three kinds of bacterium are with shape
State difference is distinguished.Culture 7 days, only the gemma number of bacillus subtilis reaches 109cfu/g。
Claims (8)
1. a kind of method using the preparation bio-bacterial manure of tobacco leaf residue, which is characterized in that including using tobacco leaf residue as solid state rheology
The step of base, inoculation mixed bacteria carries out solid state rheology；Wherein the mixed bacteria is bacillus subtilis, colloid gemma bar
The mixture of bacterium and Paecilomyces lilacinus.
2. the method according to claim 1, wherein being calculated in the mixed bacteria according to viable count, colloid
Bacillus, bacillus subtilis, Paecilomyces lilacinus ratio be 1~10: 1~10: 1.
3. according to the method described in claim 2, it is characterized in that, the kind that the mixed bacteria passes through three kinds of microorganisms of mixing
Sub- culture solution obtains；
The preparation method of the seed culture fluid is：Three kinds of microorganisms are respectively connected to its seed culture medium, 150~200r/
Min, 30~37 DEG C of shaking table cultures to OD620It is 4~6, wherein：
The seed culture medium of colloid bacillus cereus is 5~50 times of dilutions of molasses of the ammonium phosphate containing 0.2~1g/100ml；
Bacillus subtilis seed culture medium is 5~50 times of dilutions of molasses；
Paecilomyces lilacinus seed culture medium is 5~50 times of dilutions of molasses.
4. the method according to claim 1, wherein the solid medium is tobacco leaf residue and water according to matter
Amount compares 1: 1.2~2.0 mixture.
5. according to the method described in claim 4, it is characterized in that, the mixed bacteria inoculum concentration is the total matter of solid medium
The 1~15% of amount.
6. according to the method described in claim 5, it is characterized in that, the incubation time of the solid state rheology is 7~15 days, often
Primary every 24~48h stirring, cultivation temperature is 10~37 DEG C.
7. according to the method described in claim 4, it is characterized in that, the tobacco leaf residue is tobacco leaf after active material extracts
Residue, according to dry residue weight meter, in tobacco leaf residue, nicotine content is no more than 0.5mg/g, and Salanesol content is no more than
0.2mg/g, chlorogenic acid content are no more than 0.5mg/g.
8. the method according to the description of claim 7 is characterized in that the tobacco leaf residue is prepared by following methods：By powder
Drying tobacco leaf after broken and 90% ethanol water according to feed liquid mass ratio 1:5~10 mixing, in 50-80 DEG C of hot reflow treatment
It 0.5 hour, repeats 1-5 times, residue removing ethyl alcohol and drying after extraction.
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| CN201810683858.0A CN108913616B (en) | 2018-06-28 | 2018-06-28 | Method for preparing biological bacterial fertilizer by utilizing tobacco leaf residues |
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