CN103981107A - Multifunctional agricultural microbial agent as well as application method and effects thereof - Google Patents
Multifunctional agricultural microbial agent as well as application method and effects thereof Download PDFInfo
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- CN103981107A CN103981107A CN201410242397.5A CN201410242397A CN103981107A CN 103981107 A CN103981107 A CN 103981107A CN 201410242397 A CN201410242397 A CN 201410242397A CN 103981107 A CN103981107 A CN 103981107A
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Classifications
-
- 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 invention discloses a multifunctional agricultural microbial agent as well as an application method and effects thereof. The microbial agent is compounded from 1-3 parts by weight of bacillus subtilis, 0.5-2 parts by weight of Bacillus licheniformis,1-2 parts by weight of Sporotrichum thermophile, 0.5-1 part by weight of aspergillus oryzae and 0.5-3 parts by weight of trichoderma viride, and has the effective viable count of 5*10<8> per g. The microbial agent has a plurality of functions, is capable of decomposing organic materials and deodorizing, and can be applied to preventing diseases and increasing production in crop planting. The organic materials refer to organic waste materials rich in cellulose and protein such as excrements of livestock, crop straws, sludge, compostable household refuse and spent mushroom compost. The microbial agent is applied to treating the organic materials, and has better effects than the control without the microbial agent in the aspects of microbial content, available nutrient, C/N ratio, odor and fermentation period; the period of fermentation by using the microbial agent is 15 days, while the period of fermentation of the control is 32 days; and if the microbial agent is applied to cucumber planting, the disease control efficiency can be 60.20% and the production increase per mu can be 15.6%.
Description
Technical field
The invention belongs to the Application Areas of agriculture microorganism on arviculture and organic waste treatment, relate to a kind of multi-functional agricultural microorganism microbial inoculum and using method thereof.
Background technology
Agricultural microorganism microbial inoculum, through industrialization, to expand numerous active bacteria formulation of being processed into afterwards by target microorganism or probiotics, the becoming thoroughly decomposed and the improvement of environment of plantation, organic materials that is widely used in farm crop, can be divided into Rhizobium Inoculant, vinelandii microbial inoculum, phosphorus decomposing quasi-microorganism microbial inoculum, silicate microbiobacterial agent, photosynthetic bacterium microbial inoculum, organic matter decomposing inoculant, growth promoting bacteria agent and bush mycorrhiza agent etc. by the microbe species including or functional performance.
The total organic waste quantity discharged of China is 41.3～43.4 * 10
8t, wherein Amount of poultry excrements has reached 18.84 hundred million t, stalk generation reaches 6.4 hundred million t, sludge discharge is about 6,670,000 t, along with China's expanding economy, the generation of mushroom slag, Chinese medicine slag, gardens cutting object waste also rapidly occurs, and these wastes, because major part can not get well processing and application, have caused serious harm to water body, atmosphere, soil, HUMAN HEALTH and the ecosystem.Solve the outlet of these organic wastes, protection of the environment, is current problem anxious to be resolved.Application organic material composting microbial inoculum fast decoupled is processed organic waste and is produced bio-organic fertilizer, is the Main overcoming the above problems both at home and abroad, and this technology is at home and abroad widely used.Growth along with the economic fast and stable of China, living standards of the people improve constantly, the green agricultural product of the few laxative of consumption and Shaoshi chemical fertilizer, become the basic demand of China's food consumption, and the application of microbial fertilizer and bio-organic fertilizer is the inevitable requirement of green agriculture production.
At present, various agricultural microorganism microbial inoculum functions are mainly single, if any as organic matter decomposing inoculant, have as bacterial manure, also have and carry out disease-resistant as soil-repairing agent, function singleness, production cost is high, and added value is just low, and the income in agricultural is few, affect the production of enterprise and the enthusiasm that peasant household is used, thereby affect the quick solution of above problem.
Based on this, special proposition the present invention.This invention considers disease-resistant performance and the bacterial manure performance of the performance of becoming thoroughly decomposed, deodoriging properties, agricultural microorganism microbial inoculum and the humus organic material material finished product thereof of agricultural microorganism microbial inoculum, be embodied in becoming thoroughly decomposed and deodorization functions of this agricultural microorganism microbial inoculum set pair organic materials, and be applied to the diseases prevention of proportion of crop planting and volume increase function in one.
Summary of the invention
The object of this invention is to provide a kind of agricultural microorganism microbial inoculum, it is characterized in that it by can decomposition of cellulose, protein, deodorizing, multiple bacterial strain disease-resistant and bacterial manure several functions is composited, it is comprised of each single strain fermented product of following portions by weight: 0.5～3 part of 1～3 part of subtilis, 0.5～2 part of Bacillus licheniformis, 1～2 part of sporotrichum thermophile, 0.5～1 part of aspergillus oryzae and viride; Organic material composting microbial inoculum living bacteria count is 5 * 10
8individual/g.
The present invention further discloses the using method of agricultural microorganism microbial inoculum, it is characterized in that being undertaken by following step:
1, as the using method of organic matter decomposing inoculant and reodorant:
(1) adjust carbon-nitrogen ratio (25～30:1) and 55～60% (w/w) moisture of material, every 5 side's organic materialss add 1 kilogram of decomposing agent, turn over and smash evenly;
(2) the bank growth mixing is trapezoidal, 1 meter of heap top width, 2 meters of heap bottom width, pile 0.8～1 meter of high summer and autumn, 1.2～1.4 meters of winter-spring season, and heap is long to be determined according to place, and every heap volume is no less than 5 sides;
(3) after fermentation starts, when 20～30 lis of material rice temperature rise to 55 ℃, keep three days, then turn over and smash once;
(4) when heap temperature rises to after 55 ℃ again, keep three days, then turn over and smash once; According to heap tender feeling condition, turn over and smash afterwards, temperature upward period does not turn over and smashes, and temperature stops rising and turns over and smash, and repeat this step, until heap temperature is identical with external temperature, moisture is down to 30% when following, completed fermenting process, whole fermentation period is generally 15～25 days;
2, as disease-resistant microbial inoculum and agricultural be the using method of bacteria agent:
(1) seed dressing: after seed is squirted, mix thoroughly with microbial inoculum, with every seed-coat, be all stained with microbial inoculum and be advisable, after seed dressing, dry in the shade, sowing immediately, 3 kilograms of every mu of consumptions, dress seed and are applicable to the crop of direct program request;
(2) seed soaking: in the ratio of 1:50, microbial inoculum is immersed in water through fully stirring and place 4 hours, gets supernatant liquor and soak seed 12-24 hour, then through drying in the shade, can sow, 3 kilograms of mu consumptions;
(3) be stained with root: microbial inoculum and water, in the ratio furnishing pasty state of 1:10, and are sticked on seedling root to field planting immediately, 5 kilograms of mu consumptions equably;
(4) fill with root: in the ratio of 1:25, microbial inoculum is mixed and stirred evenly with water, then pour into crop root, 5 kilograms of mu consumptions;
(5) spread manuer in holes: after being mixed, microbial inoculum and fine earth put into every cave, and then transplanting or dibbling, 5 kilograms of mu fertilizer application amounts.
Agricultural microorganism microbial inoculum provided by the invention has several functions, comprises the becoming thoroughly decomposed and deodorization functions of organic materials, is applied to diseases prevention and the volume increase function of proportion of crop planting.The quick composting that specifically can be used for the rich cellulose such as feces of livestock and poultry (ox, pig, chicken, sheep, horse, duck excrement), stalk, mud, rubbish and mushroom residue and protein organic materials, can be used as the fermentative deodorizing that reodorant carries out organic materials, can be used as the control that disease-resistant microbial inoculum carries out corps diseases, can also as bacterial manure, be applied to the cultivation of farm crop, play the effect of volume increase.
Agricultural microorganism microbial inoculum provided by the invention, its activeconstituents comprises subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), sporotrichum thermophile (Sporotrichum thermophile Apinis), aspergillus oryzae (Aspergillus oryzae) and viride (Trichoderma viride).
The weight ratio of described subtilis, described Bacillus licheniformis, described sporotrichum thermophile, described aspergillus oryzae and described viride is 1～3:0.5～2:1～2:0.5～1:0.5～3.
It is the bacterial strain of ACCC 11089 that described subtilis specifically can be Chinese agriculture microbial strains preservation administrative center deposit number.It is the bacterial strain of ACCC 04249 that described Bacillus licheniformis specifically can be Chinese agriculture microbial strains preservation administrative center deposit number.It is the bacterial strain of ACCC 30346 that described sporotrichum thermophile specifically can be Chinese agriculture microbial strains preservation administrative center deposit number.It is the bacterial strain of ACCC 30155 that described aspergillus oryzae specifically can be Chinese agriculture microbial strains preservation administrative center deposit number.It is the bacterial strain of ACCC 30166 that described viride specifically can be Chinese agriculture microbial strains preservation administrative center deposit number.
Rich in proteins and the cellulosic organic wastes such as agricultural microorganism microbial inoculum energy quick composting feces of livestock and poultry provided by the invention, stalk, there is good deodoriging properties, and then output high-quality can be disease-resistant bio-organic fertilizer, the disadvantage such as can avoid because organic waste becomes thoroughly decomposed that thorough do not brought disease and pest is high, seedling rate is low, burns seedling, dead seedling, weeds are many.By the become thoroughly decomposed bio-organic fertilizer product produced of described organic material composting microbial inoculum, can significantly increase the content of the nutritive elements such as the soil organism and phosphorus, potassium, improve quantity of useful microbe in Nutrient Transformation rate and product, promote soil remediation, culture fertility, can reduce fertilizer application amount, strengthens the disease resistance of crop simultaneously, improve crop quality, reduce and drop into, improve output, promote to increase income.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
Following bacterial strain is all available from Chinese agriculture microbial strains preservation administrative center (Agricultural Culture Collection of China is called for short ACCC):
Subtilis (Bacillus subtilis): ACCC deposit number is 11089, is called for short ACCC11089;
Bacillus licheniformis (Bacillus licheniformis): ACCC deposit number is 04249, is called for short ACCC 04249;
Sporotrichum thermophile (Sporotrichum thermophile Apinis): ACCC deposit number is 30346, is called for short ACCC30346;
Aspergillus oryzae (Aspergillus oryzae): ACCC deposit number is 30155, is called for short ACCC30155;
Viride (Trichoderma viride): ACCC deposit number is 30166, is called for short ACCC30166.
embodiment 1:
the preparation of substratum and bacterial classification
One, the preparation of substratum
1, potato, glucose agar medium (PDA)
200 grams of potatos removing skin, are cut into small pieces, and add water 1000ml and boil 30 minutes, elimination potato ball, filtrate is complemented to 1000ml, add 20 grams of glucose, 15 grams, agar, dissolves rear packing, 15 pounds of sterilizings 30 minutes, pH nature, does not add agar during obtaining liq substratum.
2, nutrition nutrient agar
By peptone 5g, sodium-chlor 5g, extractum carnis 3g and distilled water 1000ml, formed.PH7.0(solid medium adds agar 15-20g).
3, fungus solids fermention medium
Wheat bran 80g, dregs of beans 10g, (NH
4)
2sO
41.0g, KH
2pO
40.2g, MgSO
47H
200.05g, 80mL distilled water.
Two, the activation of bacterial classification
Subtilis and Bacillus licheniformis are seeded on nutrition nutrient agar substratum; Sporotrichum thermophile, aspergillus oryzae and viride are seeded on PDA substratum.
Subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae and viride are carried out respectively to inclined-plane switching, access respective ramp substratum, activation culture 2-3 days.
Three, single bacterial classification preparation
1, the cultivation of bacteria culture and fermentation
Wherein 2 strains are bacterial isolateses, they are subtilis and Bacillus licheniformis, slant medium and shake-flask culture base are all nutrition nutrient agar, during preparation solid medium, add 15 grams of agar, seeding tank and fermentation tank culture medium are 1000kg water, the soya-bean milk of 8.75kg soybean, the starch of 6.5kg, 1kg glucose, CaCO
3100g, FeCl
310g, pH7.0,, cultivates 48 hours at 28 ℃～30 ℃ after 30 minutes through 121 ℃ of real tank sterilizings, and in every ml fermented liquid, bacterium number reaches 6 * 10
8individual/g.Then by the fermented liquid of 3 parts of weight ratios respectively, being adsorbed on the vermiculite of 1 part normal temperature dries and obtains subtilis and Bacillus licheniformis single strain microbial inoculum.
2, the cultivation of fungi strain and fermentation
Fungal bacterial strain comprises sporotrichum thermophile, aspergillus oryzae and viride 3 fungal strains.Inclined-plane and shake-flask culture base are potato, glucose agar medium, and slant medium adds agar.Liquid nutrient medium is sub-packed in 500ml triangular flask after sterilizing, accesses each fungi slant strains, shaking table speed 180r/min, cultivates 48 hours for 28 ℃～30 ℃, occurs that density is large in shaking flask, and mycelium pellet is obviously not muddy.
Fungal bacterial strain seeding tank and fermentation cylinder for fermentation substratum are the cooking liquor of 1000kg water, the soya-bean milk of 8.75kg soybean, the starch of 6.5kg, 1kg glucose and 2kg potato, pH7.0, through 121 ℃ of real tank sterilizings after 30 minutes, at 28 ℃～30 ℃, cultivate 48 hours, after mycelium pellet that density is large appears in fermented liquid, be inoculated on fungus solids fermention medium, inoculum size 10%, 121 ℃, 0.1MPa autoclaving 30min, 25 ℃ of solid fermentations are cultivated 48h.In solid-state fermentation tank, the loading amount of substratum is volume 70%, and sporotrichum thermophile, aspergillus oryzae and viride spore count reach respectively 6 * 10
8individual/g, 5 * 10
8individual/g and 5 * 10
8individual/g.
Four, the preparation of agricultural microorganism microbial inoculum (representing with composite fungus agent below)
The described subtilis of producing as stated above, described Bacillus licheniformis, described sporotrichum thermophile, described aspergillus oryzae and described viride list bacterial classification weight ratio are to make composite fungus agent after 1～3:0.5～2:1～2:0.5～1:0.5～3 mixing is mixed thoroughly, and composite fungus agent living bacteria count reaches 5 * 10
8individual/g.
embodiment 2:
mutual antagonistic experiment between bacterial strain
Subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae and viride after activation are carried out to the mutual antagonistic experiment between bacterial strain, and combination of two, is divided into into 20 kinds of array modes, and each combines three repetitions.
Adopt steel ring method to carry out bacteriostatic test, bacterial strain is cultivated in corresponding substratum to 2-3 days (190r/min, 28 ℃) and made fermented liquid.Getting the wherein fermented liquid 0.1mL of a kind of bacterium (A) is applied on PDA flat board, then on each dull and stereotyped solid medium, put the steel ring of two prior sterilizings, the fermented liquid that adds again any bacterium (B) outside the A bacterium of 0.25mL in each steel ring, each is processed in triplicate, and the plate that only connects A bacterium of take is contrast.Cultivate and the antibacterial situation of routine observation for 28 ℃.
The results are shown in Table 1.
Antagonistic effect between table 1 bacterial classification
| ? | Subtilis | Bacillus licheniformis | Sporotrichum thermophile | Aspergillus oryzae | Viride |
| Subtilis | ? | - | - | - | - |
| Bacillus licheniformis | - | ? | - | - | - |
| Sporotrichum thermophile | - | - | ? | - | - |
| Aspergillus oryzae | - | - | - | ? | - |
| Viride | - | - | - | - | ? |
Note: "-" represents antagonism reaction negative, "+" represents antagonism reacting positive
Test-results shows unrestraint effect between subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae and 5 kinds of bacterium of viride, and antagonism reaction is all negative.
embodiment 3:
the mensuration of single bacterial strain and composite fungus agent Mierocrystalline cellulose and protein capacity of decomposition
The enzymes such as subtilis thalline can self synthetic proteins enzyme, cellulase, α-amylase and lipase, the effectively organic substance such as the Mierocrystalline cellulose in degradation of organic substances material and protein; Bacillus licheniformis can produce various active enzyme, as proteolytic enzyme, cellulase, amylase, lipase, polygalacturonase, dextranase etc., can also produce the multiple enzymatic factor simultaneously; Sporotrichum thermophile is typical thermophilic cellulose decomposition fungi, and at 40～50 ℃, a large amount of eccrine fibers element of energy enzymes, become organic by the quick decomposition of the organism such as stalk, feces of livestock and poultry, the peat composed of rotten mosses; Aspergillus oryzae is the bacterial strain that a class is produced prozyme, except producing proteolytic enzyme, and also can cellulase-producing, amylase, saccharifying enzyme, phytase etc.; Viride is one of bacterial strain that institute's cellulase-producing activity is the highest, and the cellulase producing has Degradation to organic substance, and effect is very good.
One, cellulase activity measuring method
1, reagent and solution
Substrate solution: accurately take 0.625 g sodium carboxymethyl-cellulose, be dissolved in 100.0 mL sodium phosphate buffers (0.2 M, pH 6.0), heated and stirred makes it to dissolve.
DNS developer: take 10.0 g 3,5-dinitrosalicylic acid is dissolved in distilled water, add 20.0 g sodium hydroxide, 200.0 g Seignette salts and 500.0 ml water, after heating for dissolving, add again re-distilled phenol 2.0 g, sodium sulphite anhydrous 99.3 0.5 g cooling after all dissolving, be dissolved to 1000.0 mL.
2, plant and instrument
Spectrophotometer, electronic balance, vibrator incubator.
3, Specification Curve of Increasing
Standard glucose solution: dissolve 25.0mg glucose with distilled water and be settled to 25.0mL.
Get 5 with the test tube of 20.0 ml scales, by upper table, measure reagent.In every pipe, add 1.0 ml 2.0 M sodium hydroxide solutions and 2.0 ml DNS nitrite ions, shaking up to be placed in boiling water bath accurately heats after 5.0 min, flowing water is cooling, distilled water is settled to 20.0 ml, after shaking up, in 490 nm places, measure the OD value of each pipe, the μ g number of glucose of take is X-coordinate, and OD value is ordinate zou, drawing standard curve.
Table 2 Specification Curve of Increasing table
| Test tube number | Standard glucose solution (ml) | Sodium phosphate buffer (0.2M pH 6.0) (ml) | Glucose amount in test tube (μ g) |
| 1 | 0 | 5 | 0 |
| 2 | 0.4 | 4.6 | 400 |
| 3 | 0.7 | 4.2 | 800 |
| 4 | 1.6 | 3.4 | 1600 |
| 5 | 3.2 | 1.8 | 3200 |
4, former state enzyme liquid
Take 10 times of distilled water dilutings for 5.0g sample, fully vibration, static 30min, filters, and its filtrate is former state enzyme liquid.
5, determination step
Get 3 with the test tube of 20.0ml scale, 1 as blank, and all the other 2 as parallel sample pipe.In sample hose, add 1.0ml former state enzyme liquid, then in 3 test tubes, add respectively 4.0ml to be preheated to the substrate solution of 60 ℃, in the water-bath of 60 ℃, reacting 20min takes out, every pipe adds 2.0M sodium hydroxide solution and the 2.0ml DNS nitrite ion of 1.0ml immediately, adds 1.0ml former state enzyme liquid after shaking up in control tube again.3 test tubes are put into boiling water bath, after colour developing 5min, take out immediately, flowing water is cooling, is dissolved to 20.0ml, surveys its OD value with spectrophotometer in 490nm place.
6, cellulase activity calculates:
According to typical curve, be converted into glucose μ g number, U=(M
1-M
0)/20
In formula: U: the enzyme activity of sample (μ g/min); M
0: contrast glucose amount (μ g)
M
1: example weight (μ g) after decomposing; 20: the enzyme-to-substrate reaction times (min)
1ml former state enzyme liquid, per minute produces 1 μ g glucose and is defined as 1 enzyme activity unit (u).
Two, proteinase activity measuring method
1, reagent: according to the regulation in QB/T 1803.
2, the preparation of former state enzyme liquid
Take sample 10.0g(or 10.0ml), add in the triangular flask (150ml) that granulated glass sphere is housed, then add the phosphoric acid buffer (pH7.5) of certain volume to dissolve, 200 r/min shaking table vibration 30min, then four layers of filtered through gauze, filtrate is again in the centrifugal 20min of 3000r/min.Supernatant liquor after centrifugal is diluted to proper concn according to damping fluid for enzyme activity (pH7.5), for test.
3, determination step: other just has two places to change according to the regulation of QB/T1803 in concrete mensuration: the one, the reaction times (adding after casein) changes 30min into by 10min; The 2nd, former static filtration changes centrifugal 5min into and filters.
Three, enzyme assay result
To prepare subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, viride and composite fungus agent and carry out as stated above cellulase and amylase activity mensuration, measurement result sees the following form.
Table 3 enzymic activity situation
| Process | Cellulase activity (u/ml) | Protease activity (u/ml) |
| Subtilis | 68.00 | 27.20 |
| Bacillus licheniformis | 56.20 | 35.20 |
| Sporotrichum thermophile | 71.58 | - |
| Aspergillus oryzae | 76.32 | 27.23 |
| Viride | 80.62 | - |
| Composite fungus agent | 78.70 | 31.58 |
Detected result shows, the composite fungus agent being comprised of subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, viride several bacterial strains has very strong Mierocrystalline cellulose and protein capacity of decomposition, higher than the requirement of cellulase activity 30 (u/ml) and the protease activity 15 (u/ml) of organic matter decomposing inoculant national Specification, composite fungus agent can be used as organic matter decomposing inoculant and uses.
embodiment 4:
the mensuration of single bacterial strain and composite bacteria fermentation deodorizing capability
Subtilis can secrete the function of a large amount of chitinases, there are stronger proteolytic enzyme, lipase, diastatic activity, can carry out the stink of livestock-raising animal waste, rubbish, manure pit, manure pit and process, reduce ight soil stink and noxious gas emission, thereby reduce environmental pollution; Bacillus licheniformis can be decomposed hazardous and noxious substances; Sporotrichum thermophile, aspergillus oryzae and viride can play certain deodorizing effect by secreting corresponding enzymic activity material.
One, agents useful for same and fermented substrate
1, sulphuric acid soln: sulfuric acid 0.005mol, is settled to 1L with distilled water.
2, starch indicating liquid: 1g starch adding distil water 10ml, injects the distilled water that 200ml boils, micro-2min that boils.
3, tetrabromo-mcresolsulfonphthalein ethanolic soln: 0.10g tetrabromo-mcresolsulfonphthalein, is settled to 100ml with ethanol.
4, methyl red-tetrabromo-mcresolsulfonphthalein indicator: 0.1% tetrabromo-mcresolsulfonphthalein ethanolic soln of 3 volumes mixes with 0.2% methyl red ethanolic soln of 1 volume.
5, acetic acid zinc solution: take 50g zinc acetate and be dissolved in a small amount of distilled water, add 2 Glacial acetic acid, stir to solution and become limpid, add 2 ethanol, be settled to 1L with distilled water.
6, methyl red ethanolic soln preparation: 0.20g methyl red, is settled to 100ml with ethanol.
7, boric acid solution: 20.41g boric acid and 1000ml distilled water.
8, iodine standard solution: 13g iodine and 35g potassiumiodide are dissolved in 100mL distilled water, are settled to 1000mL with distilled water, shake up and are stored in brown bottle.
9, hypo solution: 13g five water Sulfothiorine and 0.5g sodium carbonate, be settled to 1L with distilled water, slowly boils 10min, is contained in brown bottle.
10, hydrochloric acid (1+1) solution: by 1 volume water and 1 volume 37% hydrochloric acid.
11, fermentative deodorizing matrix: be comprised of dried poultrymanure 60g, straw powder 10g and water, water content is 50%.
Two, measuring method
The main component of the foul smell that compost produces is ammonia and hydrogen sulfide, very large to people and animals' harm, and in embodiment, main ammonia detection, sulfurated hydrogen detection and the organoleptic detection of adopting evaluated the deodorizing capability of microbiobacterial agent.
1, ammonia detection method
By 2% boric acid solution, absorb the ammonia in gas, then use the titration of 0.005mol/L sulphuric acid soln, during titration, first dripping methyl red-tetrabromo-mcresolsulfonphthalein mixture indicator 5-6 drips in having absorbed the boric acid solution of ammonia, use 0.005mol/L sulphuric acid soln titration again until the pink terminal that arrives calculates ammonia level according to the consumption of sulphuric acid soln (volume).
Reaction equation is: (1) NH
3+ H
3bO
3→ NH
4h
2bO
3; (2) NH
4h
2bO
3+ H
2sO
4→ (NH
4)
2sO
4+ 2H
3bO
3.
The calculation formula of the amount of substance of ammonia: n
1=0.005*(V
1-V
01) * 10
-3; n
1the amount of substance of-ammonia (mol), the amount of substance concentration (mol/L) of 0.005-sulfuric acid, V
1the consumption of sulphuric acid soln (ml) during the titration of-test solution, V
01the consumption of sulphuric acid soln (ml) during-blank titration.
2, sulfurated hydrogen detection method
Iodimetry,iodometry is to absorb the hydrogen sulfide in unstripped gas with zinc acetate, adds excess iodine standard titration solution S in slightly acidic solution
2-be oxidized to simple substance S, remaining iodine sodium thiosulfate solution titrated, according to hypo solution consumption, calculates hydrogen sulfide content; 5mL iodine standard solution, 5mL hydrochloric acid (1+1) solution are added in the 50g/L zinc acetate solution that has absorbed hydrogen sulfide, confined reaction 5 minutes, use 0.05mol/L sodium thiosulfate solution titrated, during nearly terminal, drip 0.5% Starch Indicator, to becoming colorless, record the volume of hypo solution used, can calculate the content of hydrogen sulfide.
The reaction formula of iodometric titrationiodimetry titration: (1) H
2s+Zn
2+→ ZnS ↓+2H
+; (2) ZnS+I
2→ Zn
2++ 2I
-+ S ↓; (3) I
2+ 2S
2o
3 2-→ 2I
-+ S
4o
6 2-.
The calculation formula of the amount of substance of hydrogen sulfide: n
2=[0.5-0.05*(V
02-V
2)/2] * 10
-3; n
2the amount of substance of-hydrogen sulfide (mol), the amount of substance of 0.5-standard iodine solution (mol), the amount of substance concentration (mol/L) of 0.05-hypo solution, V
02the consumption of hypo solution (ml) during-blank titration, V
2the consumption of hypo solution (ml) during the titration of-test solution.
3, experimental technique
Subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae and viride after activation are cultivated 2 days with liquid nutrient medium respectively, obtained the bacterium liquid of each bacterial strain.
Bacterium liquid and the composite fungus agent of each bacterial strain are tested respectively as follows: bacterium liquid is connected in fermentative deodorizing matrix, in 500mL beaker, mix (every part of fermentative deodorizing matrix inoculation 10mL bacterium liquid), as experiment sample, fermentative deodorizing matrix sample is in contrast set, kraft paper and the sealing of one deck plastic paper for each sample.Each sample (six kinds of experiment samples and a kind of check sample) arranges three parallel processing, and each parallel processing arranges three repetitions, and result is got the mean value of three repetitions.The ammonia of the small beaker of 2% boric acid solution of containing 20mL in first parallel middle placement in order to produce in absorbing fermenting process, measures with acidimetry on the 7th day; The hydrogen sulfide of the 50g/L acetic acid zinc solution of containing 20mL in second parallel middle placement in order to produce in absorbing fermenting process, the 7th day with iodometric determination; The 3rd parallel, within the 7th day, carries out sense organ mensuration.
4, experimental result
The results are shown in Table 4.
Table 4 fermentative deodorizing ability
| Process | Foul smell grade | Ammonia level (10 -3mol） | Hydrogen sulfide content (10 -3mol） |
| Subtilis | + | 0.00260 | 0.32321 |
| Bacillus licheniformis | ++ | 0.00390 | 0.45913 |
| Sporotrichum thermophile | +++ | 0.00425 | 0.47342 |
| Aspergillus oryzae | ++ | 0.00410 | 0.43617 |
| Viride | ++ | 0.00385 | 0.45912 |
| Composite fungus agent | + | 0.00210 | 0.31628 |
| Contrast | ++++ | 0.00713 | 0.65432 |
Note: how many expression sense organs of "+" are measured foul smell grade, "+" more multilist shows that sense organ measures more smelly.
Detected result in conjunction with organoleptic detection and ammonia and hydrogen sulfide content shows, the composite fungus agent deodorizing effect being comprised of subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae and viride is significantly better than single bacterial strain and contrast.
embodiment 5:
composite fungus agent organic materials become thoroughly decomposed and deodorizing on application
1, materials and methods
Composting material: chicken manure, cow dung, maize straw, mud, can pile rotten domestic refuse and mushroom residue, weight ratio is 25:25:15:15:10:10.C/N ratio is 38.
If two processing, process: add described invention organic material composting microbial inoculum; Contrast: with the blank of microbial inoculum, each processes 2T raw material, repeats for three times, averages, and buttress type fermentation reactor system.Fermentation period calculates gained till being down to room temperature with leavening temperature, ferment and two processing were carried out to sampling analysis in the 15th day, comprise available N, P, K total amount (%), C/N ratio, bacterium number and intestinal bacteria, average, and the physical condition of observing fermentation, result is as shown in the table:
2, results and analysis
Table 5 composite fungus agent becomes thoroughly decomposed and deodorizing effect
As shown in Table 5, from indices, process ferment effect and be better than contrast, wherein process leavening temperature and in 2 days, can reach 55 ℃, contrast needs 4 days; The Bearing performance of fermentation is active for processing microbe population growth and breeding, and bacterium number and available N, P, K content are as shown in table 5 all higher than contrast, and fermentation period also shortens greatly, wherein be treated to 15 days, contrast 32 days, process fermentation color chocolate, odorless, and contrast is light color, frowziness.The aftertreatment in 15 days of fermenting is respectively 15.3,17.60 with contrast C/N ratio, and C/N is lower than, and microorganism is better to cellulose degradation effect in compost organic materials.Therefore,, from bacterium number, available nutrient, C/N ratio, smell and fermentation period effect, illustrate and add described composite fungus agent all to play facilitation effect to becoming thoroughly decomposed of organic materials with deodorizing.
embodiment 6:
the application on crop disease control of single bacterial strain and composite fungus agent
Subtilis thalline can produce subtilyne, polymyxin, nystatin, linear gramicidins isoreactivity material in process of growth, these active substances have obvious restraining effect to pathogenic bacterium, be mixed with multiple bacterial classification, in agriculture production, there is vital role; Bacillus licheniformis can produce activity resistent material, and has unique biology and take oxygen mechanism of action by force, can suppress the growth and breeding of pathogenic bacterium; Energy Fast-propagation after sporotrichum thermophile buries, can grow nonparasitically upon another plant at root table, can " invade " again Gen Nei and crop and form " VA Mycorrhizal Fungi ", not only can regulate the ecotope of crop root soil microorganisms, increase soil beneficial microorganism population and quantity, and can also reduce the occurrence frequency of crop root disease, receive the effect of controlling bacterium with bacterium; Aspergillus oryzae can produce useful metabolite, suppresses and kills harmful bacteria; Viride is again a kind of resourceful antagonistic microbe, has important effect in plant pathology biological control, has protection and treatment double effects, can effectively prevent and treat soil-borne disease.
One, bacteriostatic test
Adopt steel ring method to carry out bacteriostatic test, subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, viride and composite fungus agent after activation are carried out to bacteriostatic test with cucumber fusarium axysporum respectively, each combines three repetitions.
Each bacterial strain is cultivated in corresponding substratum to 2-3 days (190r/min, 28 ℃) and made fermented liquid, it is 10 that cultured cucumber fusarium axysporum is made to spore concentration
6the bacteria suspension of individual/mL.Getting cucumber fusarium axysporum bacteria suspension 0.1mL is applied on PDA flat board, then on each dull and stereotyped solid medium, put the steel ring of two prior sterilizings, above-mentioned each fermented liquid and the composite bacteria liquid that in each steel ring, add again 0.25mL, each is processed in triplicate, and the plate that only connects cucumber fusarium axysporum bacteria suspension of take is contrast.Cultivate and the antibacterial situation of routine observation for 28 ℃.
Each bacterial strain of table 6 and the bacteriostasis of composite fungus agent to cucumber fusarium axysporum
Note: "+" represents antagonism reacting positive, and "+" more multilist shows that inhibition is larger.
Test-results shows, subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, viride and composite fungus agent all have fungistatic effect to cucumber fusarium axysporum, and wherein composite fungus agent is the strongest.
Two, field test
1, test site and varieties of plant
Test vegetables are cucumber.
2, field design
Totally 7 processing, each processes 3 repetitions, community area 10m
2, each repeats survival after transplant 50 strain cucumber seedlings, and 7 processing are by usual conventional fertilizer application.
Process 1: subtilis; Process 2: Bacillus licheniformis; Process 3: sporotrichum thermophile; Process 4: aspergillus oryzae; Process 5: viride; Process 6: composite fungus agent; Process 7: contrast.
3, test-results
Each bacterial strain of table 7 and composite fungus agent field disease-preventing effect
| Process | Average number of seedling | Average diseased plant number | Average diseased plant rate (%) | Preventive effect (%) |
| Process 1 | 47.67 | 3.33 | 6.99 | 49.60 |
| Process 2 | 48.00 | 5.33 | 11.11 | 19.90 |
| Process 3 | 46.33 | 5.33 | 11.50 | 17.09 |
| Process 4 | 46.00 | 4.00 | 8.70 | 37.27 |
| Process 5 | 47.67 | 3.00 | 6.29 | 54.65 |
| Process 6 | 48.33 | 2.67 | 5.52 | 60.20 |
| Process 7 | 45.67 | 6.33 | 13.87 | - |
Test-results shows, subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, viride and composite fungus agent contrast seedling rate and protection effect is all good, and wherein composite fungus agent is best, and preventive effect reaches 60.20%.
embodiment 7:
the application in arviculture volume increase of single bacterial strain and composite fungus agent
Subtilis can produce multiple enzyme, amino acid, VITAMIN, reduces protein and is decomposed into ammonia waste, for agricultural, can play the function that increases nutrient; Bacillus licheniformis is applied in the production of soil remediation and bio-fertilizer, P and K decomposing successful; Energy Fast-propagation after sporotrichum thermophile buries, energy and crop form " VA Mycorrhizal Fungi ", after the Nutrient Transformation that root system can not directly can be absorbed, supply with crop utilization, also can secrete the inorganic nutriment in a fertilizer such as organic nutrient and N, P, K, and multiple amino acids, VITAMIN, phytokinin and plant hormone, make crop obtain higher yield and fine quality; Aspergillus oryzae can make the elements such as organic matter contained in stalk and phosphorus, potassium become the required nutrition of plant-growth, and produces a large amount of beneficial microorganisms, stimulates crop production, improves the soil organism, improves Soil structure.
1, test site and varieties of plant
Test site is facility plastic greenhouse ground, great Meng village town, Wuqing, Tianjin, and planting vegetable is cucumber.
2, field design
Totally 7 processing, each processes 3 repetitions, community area 10m
2, each repeats survival after transplant 50 strain cucumber seedlings, and 7 processing are by usual conventional fertilizer application.
Process 1: subtilis; Process 2: Bacillus licheniformis; Process 3: sporotrichum thermophile; Process 4: aspergillus oryzae; Process 5: viride; Process 6: composite fungus agent; Process 7: contrast.
3, test-results
Each bacterial strain of table 8 and composite fungus agent field effect of increasing production
| Process | Community mean yield (kg) | Per mu yield (kg) | Volume increase (%) |
| Process 1 | 103.5 | 6900 | 13.1 |
| Process 2 | 97.3 | 6485 | 6.3 |
| Process 3 | 101.7 | 6780 | 11.2 |
| Process 4 | 93.8 | 6250 | 2.5 |
| Process 5 | 97.1 | 6475 | 6.2 |
| Process 6 | 105.7 | 7050 | 15.6 |
| Process 7 | 91.5 | 6100 | - |
Test-results shows, subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, viride and composite fungus agent all have effect of increasing production than contrast on green cucumber, wherein composite fungus agent is best, better than single bacterial strain, can be used as bacterial manure and uses in farmland.
embodiment 8:
the proportioning of composite fungus agent
Agricultural microorganism microbial inoculum is formulated as follows (by weight): the weight ratio of described subtilis, described Bacillus licheniformis, described sporotrichum thermophile, described aspergillus oryzae and described viride is 1～3:0.5～2:1～2:0.5～1:0.5～3.
The composition of table 9 multifunctional agricultural microbiobacterial agent
| Title | Subtilis | Bacillus licheniformis | Sporotrichum thermophile | Aspergillus oryzae | Viride | Composite fungus agent living bacteria count |
| （1） | 1.0 | 2.0 | 1.0 | 0.5 | 2.0 | 5×10 8Individual/g |
| （2） | 2.0 | 1.5 | 1.5 | 1.0 | 1.5 | 5×10 8Individual/g |
| （3） | 2.5 | 1.0 | 2.0 | 1.0 | 1.0 | 5×10 8Individual/g |
| （4） | 3.0 | 0.5 | 1.0 | 0.5 | 3.0 | 5×10 8Individual/g |
embodiment 9:
the using method of composite fungus agent
By above embodiment, explanation is by subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, the composite fungus agent that 5 kinds of bacterium of viride form, there is the effect of several functions, can be used as organic matter decomposing inoculant and carry out feces of livestock and poultry (ox, pig, chicken, sheep, horse, duck excrement), stalk, mud, the quick composting of the rich cellulose such as rubbish and mushroom residue and protein organic materials, can be used as the deodorizing that reodorant carries out organic materials, can be used as the control that disease-resistant microbial inoculum carries out crop pest, can also be applied to as agricultural microorganism microbial inoculum the cultivation of farm crop, play the effect of volume increase.
One, as the using method of organic matter decomposing inoculant and reodorant
1, adjust the carbon-nitrogen ratio (25～30:1) of material and moisture (55～60%, with hand one hold hand seam drip be advisable), every 5 side's organic materialss add 1 kilogram of decomposing agent, turn over and smash evenly.
2, the bank growth mixing is trapezoidal, 1 meter of heap top width, 2 meters of heap bottom width, pile 0.8～1 meter of high summer and autumn, 1.2～1.4 meters of winter-spring season, and heap is long to be determined according to place, and every heap volume is no less than 5 sides.
3, after fermentation starts, when temperature of charge (20～30 centimetres of depths) rises to 55 ℃, keep three days, then turn over and smash once.If fermentation initial stage temperature does not rise, adjust moisture to recommendation ratio and turn over every day and smash once until pile temperature rising.
4, when heap temperature rises to after 55 ℃ again, keep three days, then turn over and smash once;
5, according to heap tender feeling condition, turn over and smash afterwards, temperature upward period does not turn over and smashes, and temperature stops rising and turns over and smash, and repeat this step, until heap temperature is identical with external temperature, moisture is down to 30% when following, completed fermenting process, whole fermentation period is generally 15～25 days.
Two, as the using method of disease-resistant microbial inoculum and bio-bacterial manure
According to different fertilizing methods, soil fertility condition, crop yield and fertilizing time, suitably adjust microbial inoculum consumption.
1, seed dressing
After seed is squirted, mix thoroughly with microbial inoculum, be all stained with microbial inoculum be advisable with every seed-coat, after seed dressing, dry in the shade (avoiding direct sunlight), sows 3 kilograms of left and right of every mu of consumption immediately.Seed dressing is applicable to the crop of direct program request, as cotton, peanut, Chinese cabbage, radish, watermelon, muskmelon etc.
2, seed soaking
Some kind skins are thicker or plant the harder seed of shell, can adopt the method for seed soaking.Specific practice is in the ratio of 1:50, microbial inoculum to be immersed in water through fully stirring and place 4 hours, gets supernatant liquor and soaks seed 12-24 hour, then through drying in the shade, can sow mu 3 kilograms of left and right of consumption.
3, be stained with root
When the crops such as vegetables are transplanted seedlings field planting, being stained with root is a kind of important application process.Specific practice be microbial inoculum and water in the ratio furnishing pasty state of 1:10, and stick in equably on seedling root field planting immediately, mu 5 kilograms of left and right of consumption.
4, fill with root
In the ratio of 1:25, microbial inoculum is mixed and stirred evenly with water, then pour into crop root, mu 5 kilograms of left and right of consumption.Fill with root and be applicable to fruit tree, vegetables and flowers etc.
5, spread manuer in holes
When dibbling or transplanting, all can take the method for spreading manuer in holes.For ease of using, after microbial inoculum and fine earth can being mixed, put into every cave, and then transplanting or dibbling, mu 5 kilograms of left and right of fertilizer application amount.
Claims (8)
1. a multifunctional agricultural microbiobacterial agent, it is characterized in that it by can decomposition of cellulose, protein, deodorizing, multiple bacterial strain disease-resistant and bacterial manure several functions is composited, it is comprised of each single strain fermented product of following portions by weight: 1～3 part of subtilis, 0.5～2 part of Bacillus licheniformis, 1～2 part of sporotrichum thermophile, 0.5～1 part of aspergillus oryzae, 0.5～3 part of viride; Agricultural microorganism microbial inoculum living bacteria count is 5 * 10
8individual/g.
2. the using method of agricultural microorganism microbial inoculum described in claim 1, is characterized in that being undertaken by following step:
One, as the using method of organic matter decomposing inoculant and reodorant:
(1) adjust carbon-nitrogen ratio (25～30:1) and 55～60% (w/w) moisture of material, every 5 side's organic materialss add 1 kilogram of decomposing agent, turn over and smash evenly;
(2) the bank growth mixing is trapezoidal, 1 meter of heap top width, 2 meters of heap bottom width, pile 0.8～1 meter of high summer and autumn, 1.2～1.4 meters of winter-spring season, and heap is long to be determined according to place, and every heap volume is no less than 5 sides;
(3) after fermentation starts, when 20～30 lis of material rice temperature rise to 55 ℃, keep three days, then turn over and smash once
(4) when heap temperature rises to after 55 ℃ again, keep three days, then turn over and smash once; According to heap tender feeling condition, turn over and smash afterwards, temperature upward period does not turn over and smashes, and temperature stops rising and turns over and smash, and repeat this step, until heap temperature is identical with external temperature, moisture is down to 30% when following, completed fermenting process, whole fermentation period is generally 15～25 days;
Two, as the using method of disease-resistant microbial inoculum and bio-bacterial manure:
(1) seed dressing: after seed is squirted, mix thoroughly with microbial inoculum, with every seed-coat, be all stained with microbial inoculum and be advisable, after seed dressing, dry in the shade, sowing immediately, 3 kilograms of every mu of consumptions, dress seed and are applicable to the crop of direct program request;
(2) seed soaking: in the ratio of 1:50, microbial inoculum is immersed in water through fully stirring and place 4 hours, gets supernatant liquor and soak seed 12-24 hour, then through drying in the shade, can sow, 3 kilograms of mu consumptions;
(3) be stained with root: microbial inoculum and water, in the ratio furnishing pasty state of 1:10, and are sticked on seedling root to field planting immediately, 5 kilograms of mu consumptions equably;
(4) fill with root: in the ratio of 1:25, microbial inoculum is mixed and stirred evenly with water, then pour into crop root, 5 kilograms of mu consumptions;
(5) spread manuer in holes: after being mixed, microbial inoculum and fine earth put into every cave, and then transplanting or dibbling, 5 kilograms of mu fertilizer application amounts.
3. described in claim 1, agricultural microorganism microbial inoculum has several functions, comprises the becoming thoroughly decomposed and deodorization functions of organic materials, is applied to diseases prevention and the volume increase function of proportion of crop planting.
Described in claim 1 agricultural microorganism microbial inoculum in the application of the capacity of decomposition of Mierocrystalline cellulose and protein.
Described in claim 1 agricultural microorganism microbial inoculum organic materials become thoroughly decomposed and deodorizing on application.
6. the application of agricultural microorganism microbial inoculum on crop disease control described in claim 1.
7. the application of agricultural microorganism microbial inoculum in arviculture volume increase described in claim 1.
8. application claimed in claim 5, wherein said organic materials refers to feces of livestock and poultry (ox, pig, chicken, sheep, horse, duck excrement), straw crop stalk, mud, can pile the organic waste of the rich cellulose such as rotten domestic refuse and mushroom residue and protein.
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