CN103981107B - A kind of multifunctional agricultural microbiobacterial agent and using method thereof and effect - Google Patents
A kind of multifunctional agricultural microbiobacterial agent and using method thereof and effect Download PDFInfo
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Abstract
The invention discloses a kind of multifunctional agricultural microbiobacterial agent and using method thereof and effect.Described microbial inoculum is composited by subtilis 1 ~ 3 part, Bacillus licheniformis 0.5 ~ 2 part, sporotrichum thermophile 1 ~ 2 part, aspergillus oryzae 0.5 ~ 1 part and viride 0.5 ~ 3 part by weight, living bacteria count 5 × 10
8individual/g.This microbial inoculum has several functions, can humus organic material material and deodorizing, is applied to proportion of crop planting preventable disease and volume increase.Described organic materials refers to feces of livestock and poultry, straw crop stalk, mud, can pile the organic waste of the rich cellulose such as rotten domestic refuse and mushroom residue and protein.Apply described microbial inoculum process organic materials, the contrast with microbial inoculum is all better than than effect from bacterium number, available nutrient, C/N ratio, smell and fermentation period, applying the described bacteria fermentation cycle is 15 days, contrast 32 days, apply described microbial inoculum in green cucumber, diseases control reaches 60.20%, mu volume increase 15.6%.
Description
Technical field
The invention belongs to the Application Areas of agriculture microorganism in arviculture and organic waste treatment, relate to a kind of multi-functional agricultural microbial agent and using method thereof.
Background technology
Agricultural microbial agent, it is the active bacteria formulation be processed into after industrialization expansion is numerous by target microorganism or probiotics, be widely used in the plantation of farm crop, the becoming thoroughly decomposed and the improvement of environment of organic materials, 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. can be divided into by the microbe species included 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 can not get well process and application due to major part, cause serious harm to water body, air, soil, HUMAN HEALTH and the ecosystem.Solving 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 process organic waste produces bio-organic fertilizer, and be the Main overcome the above problems both at home and abroad, this technology is at home and abroad widely used.Along with the growth of 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 most 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 microbial agent function is mainly single, if any being used as organic matter decomposing inoculant, having as bacterial manure, also having and carry out disease-resistant as soil-repairing agent, function singleness, production cost is high, and added value is just low, and income is agriculturally few, affect the production of enterprise and the enthusiasm of peasant household's use, thus 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 of agricultural microbial agent, deodoriging properties, agricultural microbial agent and humus organic material material finished product thereof, be embodied in becoming thoroughly decomposed and deodorization functions of this agricultural microbial agent set pair organic materials, and be applied to the diseases prevention of proportion of crop planting and volume increase function in integrally.
Summary of the invention
The object of this invention is to provide a kind of agricultural microbial agent, it is characterized in that it is composited by decomposable asymmetric choice net Mierocrystalline cellulose, protein, deodorizing, multiple bacterial strain that is disease-resistant and bacterial manure several functions, it is made up of each single strain fermented product of following portions by weight: subtilis 1 ~ 3 part, Bacillus licheniformis 0.5 ~ 2 part, sporotrichum thermophile 1 ~ 2 part, aspergillus oryzae 0.5 ~ 1 part and viride 0.5 ~ 3 part; Organic material composting microbial inoculum living bacteria count is 5 × 10
8individual/g.
The present invention further discloses the using method of agricultural microbial agent, 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) grow up trapezoidal by the bank of mixing, heap top width 1 meter, heap bottom width 2 meters, piles high summer and autumn 0.8 ~ 1 meter, winter-spring season 1.2 ~ 1.4 meters, and heap is long to be determined according to place, often piles volume and is no less than 5 sides;
(3), after fermentation starts, keep three days when 20 ~ 30 lis of material rice temperature rise to 55 DEG C, then turn over and smash once;
(4) after heap temperature rises to 55 DEG C again, keep three days, then turn over and smash once; Carry out turning over smashing according to heap tender feeling condition afterwards, temperature upward period does not turn over and smashes, and temperature stops rising then to turn over smashing, and repeat this step, until when heap temperature is identical with external temperature, moisture is down to below 30%, namely complete 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) dress seed: mix thoroughly with microbial inoculum after being squirted by seed, be all stained with microbial inoculum and be advisable, after seed dressing, dry in the shade, sow immediately with every seed-coat, every mu of consumption 3 kilograms, dresses seed and is applicable to the crop of direct program request;
(2) soak seed: in the ratio of 1:50, microbial inoculum is immersed in warp in water and fully stirs and place 4 hours, getting supernatant liquor and soak seed 12-24 hour, then through drying in the shade, can sow, mu consumption 3 kilograms;
(3) root is stained with: microbial inoculum and the water ratio furnishing pasty state in 1:10, and stick on seedling root equably, field planting immediately, mu consumption 5 kilograms;
(4) fill with root: in the ratio of 1:25, microbial inoculum is mixed with water and stirs evenly, then pour into crop root, mu consumption 5 kilograms;
(5) spread manuer in holes: after microbial inoculum and fine earth being mixed, put into every cave, and then transplant or dibbling, mu fertilizer application amount 5 kilograms.
Agricultural microbial agent provided by the invention has several functions, comprises becoming thoroughly decomposed and deodorization functions to organic materials, is applied to diseases prevention and the volume increase function of proportion of crop planting.Specifically can be used for the quick composting of 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, the cultivation of farm crop can also be applied to as bacterial manure, play the effect of volume increase.
Agricultural microbial agent provided by the invention, its activeconstituents comprises subtilis (Bacillussubtilis), Bacillus licheniformis (Bacilluslicheniformis), sporotrichum thermophile (SporotrichumthermophileApinis), aspergillus oryzae (Aspergillusoryzae) and viride (Trichodermaviride).
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 ACCC11089 that described subtilis specifically can be Chinese agriculture Microbiological Culture Collection administrative center deposit number.It is the bacterial strain of ACCC04249 that described Bacillus licheniformis specifically can be Chinese agriculture Microbiological Culture Collection administrative center deposit number.It is the bacterial strain of ACCC30346 that described sporotrichum thermophile specifically can be Chinese agriculture Microbiological Culture Collection administrative center deposit number.It is the bacterial strain of ACCC30155 that described aspergillus oryzae specifically can be Chinese agriculture Microbiological Culture Collection administrative center deposit number.It is the bacterial strain of ACCC30166 that described viride specifically can be Chinese agriculture Microbiological Culture Collection administrative center deposit number.
Rich in proteins and the cellulosic organic wastes such as agricultural microbial agent 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, can avoid because of organic waste become thoroughly decomposed that thorough do not brought disease and pest is high, seedling rate is low, burn seedling, the disadvantage such as dead seedling, weeds are many.The content of the soil organism and the nutritive element such as phosphorus, potassium significantly can be increased by the described organic material composting microbial inoculum bio-organic fertilizer product produced that becomes thoroughly decomposed, improve quantity of useful microbe in nutrient conversion efficiency 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 the present invention better, 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 for three times, results averaged.
Following bacterial strain is all available from Chinese agriculture Microbiological Culture Collection administrative center (AgriculturalCultureCollectionofChina is called for short ACCC):
Subtilis (Bacillussubtilis): ACCC deposit number is 11089, is called for short ACCC11089;
Bacillus licheniformis (Bacilluslicheniformis): ACCC deposit number is 04249, is called for short ACCC04249;
Sporotrichum thermophile (SporotrichumthermophileApinis): ACCC deposit number is 30346, is called for short ACCC30346;
Aspergillus oryzae (Aspergillusoryzae): ACCC deposit number is 30155, is called for short ACCC30155;
Viride (Trichodermaviride): 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)
Remove the potato 200 grams of skin, be cut into small pieces, the 1000ml that adds water boils 30 minutes, elimination potato ball, filtrate is complemented to 1000ml, adds glucose 20 grams, 15 grams, agar, dissolves rear packing, 15 pounds of sterilizings 30 minutes, pH nature, does not add agar during obtaining liq substratum.
2, nutrient agar
Be made up of peptone 5g, sodium-chlor 5g, extractum carnis 3g and distilled water 1000ml.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 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 inclined-plane switching respectively, 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 medium are all nutrient agar, 15 grams of agar are added during preparation solid medium, 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, through 121 DEG C of real tank sterilizings after 30 minutes, cultivate 48 hours, when in every ml fermented liquid, bacterium number reaches 6 × 10 at 28 DEG C ~ 30 DEG C
8individual/g.Then on the vermiculite being adsorbed in 1 part by the fermented liquid of weight ratio 3 parts respectively, normal temperature dries and namely 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 strain.Inclined-plane and Shake flask medium 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, cultivate 48 hours for 28 DEG C ~ 30 DEG C, when occurring in shaking flask that density is large, 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 DEG C of real tank sterilizings after 30 minutes, cultivate 48 hours at 28 DEG C ~ 30 DEG C, after the mycelium pellet that density is large appears in fermented liquid, be inoculated on fungus solids fermention medium, inoculum size 10%, 121 DEG C, 0.1MPa autoclaving 30min, 25 DEG C of solid fermentations cultivate 48h.In solid-state fermentation tank, the loading amount of substratum is 70% of volume, and sporotrichum thermophile, aspergillus oryzae and viride spore count reach 6 × 10 respectively
8individual/g, 5 × 10
8individual/g and 5 × 10
8individual/g.
Four, the preparation of agricultural microbial agent (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 that rear obtained composite fungus agent is mixed in 1 ~ 3:0.5 ~ 2:1 ~ 2:0.5 ~ 1:0.5 ~ 3 mixing thoroughly, and composite fungus agent living bacteria count reaches 5 × 10
8individual/g.
embodiment 2:
mutual antagonistic experiment between bacterial strain
Subtilis after activation, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae and viride are carried out the mutual antagonistic experiment between bacterial strain, and combination of two, is divided into into 20 kinds of array modes, each combination three repetition.
Adopt steel ring method to carry out bacteriostatic test, bacterial strain is cultivated in corresponding substratum 2-3 days (190r/min, 28 DEG C) and make fermented liquid.The fermented liquid 0.1mL getting wherein a kind of bacterium (A) is applied on PDA flat board, then on the solid medium of each flat board, put the steel ring of two prior sterilizings, the fermented liquid of any one bacterium (B) outside the A bacterium adding 0.25mL again in each steel ring, each process in triplicate, with the plate only connecing A bacterium for contrast.Cultivate and the antibacterial situation of routine observation for 28 DEG C.
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 that Antagonistic reaction is negative, "+" represents that Antagonistic reaction is positive
Test-results shows subtilis, Bacillus licheniformis, sporotrichum thermophile, unrestraint effect between aspergillus oryzae and viride 5 kinds of bacterium, and Antagonistic reaction is feminine gender.
embodiment 3:
the mensuration of single bacterial strain and composite fungus agent Mierocrystalline cellulose and breaks down proteins ability
Subtilis thalline can the enzyme such as self synthetic proteins enzyme, cellulase, α-amylase and lipase, can the organic substance such as Mierocrystalline cellulose effectively 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 DEG C, energy a large amount of eccrine fiber element enzyme, becomes organic by quick for the organism such as stalk, feces of livestock and poultry, peat composed of rotten mosses decomposition; Aspergillus oryzae is that a class produces the bacterial strain of prozyme, except product proteolytic enzyme, and also can cellulase-producing, amylase, saccharifying enzyme, phytase etc.; Viride is one of institute's cellulase-producing the highest active bacterial strain, and the cellulase produced has Degradation to organic substance, and effect is very good.
One, cellulase activity measuring method
1, reagent and solution
Substrate solution: accurately take 0.625g sodium carboxymethyl-cellulose, is dissolved in 100.0mL sodium phosphate buffer (0.2M, pH6.0), and heated and stirred makes it to dissolve.
DNS developer: take 10.0g3,5-dinitrosalicylic acid is dissolved in distilled water, add 20.0g sodium hydroxide, 200.0g Seignette salt and 500.0ml water, add re-distilled phenol 2.0g, sodium sulphite anhydrous 99.3 0.5g cooling after all dissolving after heating for dissolving again, be dissolved to 1000.0mL.
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 test tubes with 20.0ml scale, measure reagent by upper table.Often add 1.0ml2.0M sodium hydroxide solution and 2.0mlDNS nitrite ion in pipe, shake up and to be placed in boiling water bath accurately after heating 5.0min, flowing water cools, distilled water is settled to 20.0ml, measure the OD value of each pipe in 490nm place after shaking up, with the μ g number of glucose for X-coordinate, 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 (μ g) in test tube |
| 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 5.0g sample distilled water diluting 10 times, fully vibrate, static 30min, filter, its filtrate is former state enzyme liquid.
5, determination step
Get 3 test tubes with 20.0ml scale, 1 as blank, all the other 2 as parallel sample pipe.1.0ml former state enzyme liquid is added in sample hose, then the substrate solution that 4.0ml has been preheated to 60 DEG C is added respectively in 3 test tubes, in the water-bath of 60 DEG C, react 20min take out, often manage the 2.0M sodium hydroxide solution and 2.0mlDNS nitrite ion that add 1.0ml immediately, in control tube, after shaking up, add 1.0ml former state enzyme liquid again.3 test tubes are put into boiling water bath, and take out immediately after colour developing 5min, flowing water cools, and is dissolved to 20.0ml, surveys its OD value with spectrophotometer in 490nm place.
6, cellulase activity calculates:
Glucose μ g number is converted into, U=(M according to typical curve
1-M
0)/20
In formula: U: the enzyme activity (μ g/min) of sample; 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/T1803.
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 the phosphoric acid buffer (pH7.5) adding certain volume dissolves, 200r/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 enzyme activity damping fluid (pH7.5), for test.
3, determination step: other regulation according to QB/T1803 just has two places to change in concrete mensuration: is the reaction times (after adding casein) change 30min into by 10min; Two is that 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 cellulase and amylase activity mensuration as stated above, 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 be made up of subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, viride several bacterial strains has very strong Mierocrystalline cellulose and breaks down proteins ability, higher than the cellulase activity 30 (u/ml) of organic matter decomposing inoculant national Specification and the requirement of protease activity 15 (u/ml), composite fungus agent can use as organic matter decomposing inoculant.
embodiment 4:
the mensuration of single bacterial strain and composite bacteria fermentation deodorizing capability
Subtilis can secrete the function of a large amount of chitinase, there are stronger proteolytic enzyme, lipase, diastatic activity, the stink process of livestock-raising animal waste, rubbish, manure pit, manure pit can be carried out, reduce stool odor and noxious gas emission, thus reduce environmental pollution; Bacillus licheniformis decomposable asymmetric choice net hazardous and noxious substances; Sporotrichum thermophile, aspergillus oryzae and viride can play certain deodorizing effect by the corresponding enzymic activity material of secretion.
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, adds 2 Glacial acetic acid, stirs and becomes limpid to solution, 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, be settled to 1000mL, shake up and be stored in brown bottle with distilled water.
9, hypo solution: 13g five water Sulfothiorine and 0.5g sodium carbonate, be settled to 1L with distilled water, slowly boil 10min, be 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 made up 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 the harm of people and animals, and in embodiment, main ammonia detection, sulfurated hydrogen detection and the deodorizing capability of organoleptic detection to microbiobacterial agent of adopting is evaluated.
1, ammonia detection method
The ammonia in gas is absorbed by 2% boric acid solution, then the titration of 0.005mol/L sulphuric acid soln is used, first drip during titration that methyl red-tetrabromo-mcresolsulfonphthalein mixture indicator 5-6 drips in the boric acid solution absorbing ammonia, again with 0.005mol/L sulphuric acid soln titration until pinkly namely arrive terminal, the consumption (volume) according to sulphuric acid soln calculates ammonia level.
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 (mol) of-ammonia, the substance withdrawl syndrome (mol/L) of 0.005-sulfuric acid, V
1the consumption (ml) of sulphuric acid soln during the titration of-test solution, V
01the consumption (ml) of sulphuric acid soln during-blank titration.
2, sulfurated hydrogen detection method
Iodimetry,iodometry is the hydrogen sulfide absorbed with zinc acetate in unstripped gas, 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, calculate hydrogen sulfide content; 5mL iodine standard solution, 5mL hydrochloric acid (1+1) solution being added absorbs in the 50g/L zinc acetate solution of hydrogen sulfide, confined reaction 5 minutes, use 0.05mol/L sodium thiosulfate solution titrated, 0.5% Starch Indicator is dripped during nearly terminal, to becoming colorless, record the volume of hypo solution used, the content of hydrogen sulfide can be calculated.
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 (mol) of-hydrogen sulfide, the amount of substance (mol) of 0.5-standard iodine solution, the substance withdrawl syndrome (mol/L) of 0.05-hypo solution, V
02the consumption (ml) of hypo solution during-blank titration, V
2the consumption (ml) of hypo solution during the titration of-test solution.
3, experimental technique
Subtilis after activation, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae and viride are cultivated 2 days with liquid nutrient medium respectively, obtains the bacterium liquid of each bacterial strain.
The bacterium liquid of each bacterial strain and composite fungus agent are tested respectively as follows: bacterium liquid is connected on fermentative deodorizing Medium Culture, mix in 500mL beaker (every part of fermentative deodorizing matrix inoculation 10mL bacterium liquid), as experiment sample, fermentative deodorizing matrix sample is in contrast set, each sample kraft paper and the sealing of one deck plastic paper.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 gets three mean values repeated.In first parallel middle placement, contain the small beaker of 2% boric acid solution of 20mL in order to absorb the ammonia produced in fermenting process, within the 7th day, measure with acidimetry; In second parallel middle placement, contain the 50g/L acetic acid zinc solution of 20mL in order to absorb the hydrogen sulfide produced in fermenting process, the 7th day with iodometric determination; 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 "+" measure foul smell grade, "+" more multilist to show 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 be made up of subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae and viride is significantly better than single bacterial strain and contrast.
embodiment 5:
composite fungus agent becoming thoroughly decomposed and the application in deodorizing at organic materials
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 process, process: add described invention organic material composting microbial inoculum; Contrast: with the blank of microbial inoculum, each process 2T raw material, repeats, averages, buttress type fermentation reactor system for three times.Fermentation period calculates gained till being down to room temperature with leavening temperature, ferment and sampling analysis was carried out to two process in the 15th day, comprise available N, P, K total amount (%), C/N ratio, bacterium number and intestinal bacteria, average, and observe the physical condition of 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 is better than contrast, wherein processes leavening temperature and can reach 55 DEG C in 2 days, contrast needs 4 days; The result of fermentation shows as process microbe population growth and breeding and enlivens, 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.Ferment aftertreatment in 15 days and contrast C/N ratio be respectively 15.3,17.60, C/N lower than, 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 add described composite fungus agent to organic materials become thoroughly decomposed and deodorizing all serves facilitation effect.
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, with multiple bacterial classification mixture, in agriculture production, there is vital role; Bacillus licheniformis can produce activity resistent material, and the biology with uniqueness takes 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, Gen Nei and crop formation " VA Mycorrhizal Fungi " " can be invaded " again, not only can regulate the ecotope of crop root soil microorganisms, increase soil beneficial microorganism population and quantity, and the occurrence frequency of crop root disease can also be reduced, receive the effect of fungus treatment; Aspergillus oryzae can produce beneficial metabolites, suppresses and kills harmful bacteria; Viride is again a kind of resourceful antagonistic microbe, in plant pathology biological control, have important effect, has protection and treatment double effects, effectively can prevent and treat soil-borne disease.
One, bacteriostatic test
Adopt steel ring method to carry out bacteriostatic test, the subtilis after activation, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, viride and composite fungus agent are carried out bacteriostatic test with cucumber fusarium axysporum, each combination three repetition respectively.
Each bacterial strain is cultivated in corresponding substratum 2-3 days (190r/min, 28 DEG C) and make fermented liquid, cultured cucumber fusarium axysporum being made spore concentration is 10
6the bacteria suspension of individual/mL.Getting cucumber fusarium axysporum bacteria suspension 0.1mL is applied on PDA flat board, then on the solid medium of each flat board, put the steel ring of two prior sterilizings, above-mentioned each fermented liquid and the composite bacteria liquid of 0.25mL is added again in each steel ring, each process in triplicate, with the plate only connecing cucumber fusarium axysporum bacteria suspension for contrast.Cultivate and the antibacterial situation of routine observation for 28 DEG C.
The each bacterial strain of table 6 and composite fungus agent are to the bacteriostasis of cucumber fusarium axysporum
Note: "+" represents that Antagonistic reaction is 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 process, each process 3 repetition, plot area 10m
2, each repetition survival after transplant 50 strain cucumber seedlings, 7 process 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
The 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 comparatively contrast seedling rate and protection effect is all good, and wherein composite fungus agent is best, and preventive effect reaches 60.20%.
embodiment 7:
single bacterial strain and the application of composite fungus agent on arviculture is increased production
Subtilis can produce multiple enzyme, amino acid, VITAMIN, and minimizing breaks down proteins is ammonia waste, for playing the function increasing nutrient in agricultural; 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 ", crop utilization is supplied after the Nutrient Transformation that root system can not directly can be absorbed, 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; Organic matter contained by aspergillus oryzae can make in stalk and the element such as phosphorus, potassium become the nutrition needed for plant-growth, and produce a large amount of beneficial microorganism, stimulate crop production, improve the soil organism, improve 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 process, each process 3 repetition, plot area 10m
2, each repetition survival after transplant 50 strain cucumber seedlings, 7 process 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
The 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 use as bacterial manure in farmland.
embodiment 8:
the proportioning of composite fungus agent
Agricultural microbial agent 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, illustrate by subtilis, Bacillus licheniformis, sporotrichum thermophile, aspergillus oryzae, the composite fungus agent of viride 5 kinds of bacterium compositions, 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, the cultivation of farm crop can also be applied to as agricultural microbial agent, 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, grow up trapezoidal by the bank of mixing, heap top width 1 meter, heap bottom width 2 meters, piles high summer and autumn 0.8 ~ 1 meter, winter-spring season 1.2 ~ 1.4 meters, and heap is long to be determined according to place, often piles volume and is no less than 5 sides.
3, after fermentation starts, keep three days when temperature of charge (20 ~ 30 centimetres of depths) rises to 55 DEG C, then turn over and smash once.If fermentation initial stage temperature do not rise, then adjust moisture to recommendation ratio and turn over every day smash once until pile temperature rising.
4, after heap temperature rises to 55 DEG C again, keep three days, then turn over and smash once;
5, carry out turning over smashing according to heap tender feeling condition afterwards, temperature upward period does not turn over and smashes, and temperature stops rising then to turn over smashing, and repeat this step, until when heap temperature is identical with external temperature, moisture is down to below 30%, namely complete 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 adjustment microbial inoculum consumption.
1, dress seed
After seed is squirted and microbial inoculum mix thoroughly, be all stained with microbial inoculum with every seed-coat and be advisable, after seed dressing, dry in the shade (avoiding direct sunlight), sows immediately, every mu of consumption about 3 kilograms.Seed dressing is applicable to the crop of direct program request, as cotton, peanut, Chinese cabbage, radish, watermelon, muskmelon etc.
2, soak seed
Some seed coats are thicker or plant the harder seed of shell, can adopt the method for seed soaking.Specific practice in the ratio of 1:50, microbial inoculum is immersed in warp in water fully stir and place 4 hours, getting supernatant liquor and soak seed 12-24 hour, then through drying in the shade, can sow, mu consumption about 3 kilograms.
3, root is stained with
When the crops such as vegetables transplant seedlings field planting, being stained with root is a kind of important application process.Specific practice is microbial inoculum and the water ratio furnishing pasty state in 1:10, and sticks on seedling root equably, field planting immediately, mu consumption about 5 kilograms.
4, root is filled with
In the ratio of 1:25, microbial inoculum is mixed with water and stirs evenly, then pour into crop root, mu consumption about 5 kilograms.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 transplant or dibbling, mu fertilizer application amount about 5 kilograms.
Claims (7)
1. a multifunctional agricultural microbiobacterial agent, it is characterized in that it is composited by decomposable asymmetric choice net Mierocrystalline cellulose, protein, deodorizing, multiple bacterial strain that is disease-resistant and bacterial manure several functions, it is made up of each single strain fermented product of following portions by weight: subtilis ACCC110891 ~ 3 part, Bacillus licheniformis ACCC042490.5 ~ 2 part, sporotrichum thermophile ACCC303461 ~ 2 part, aspergillus oryzae ACCC301550.5 ~ 1 part, viride ACCC301660.5 ~ 3 part; Agricultural microbial agent living bacteria count is 5 × 10
8individual/g.
2. the using method of agricultural microbial agent 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) grow up trapezoidal by the bank of mixing, heap top width 1 meter, heap bottom width 2 meters, piles high summer and autumn 0.8 ~ 1 meter, winter-spring season 1.2 ~ 1.4 meters, and heap is long to be determined according to place, often piles volume and is no less than 5 sides;
(3), after fermentation starts, keep three days when 20 ~ 30 lis of material rice temperature rise to 55 DEG C, then turn over and smash once;
(4) after heap temperature rises to 55 DEG C again, keep three days, then turn over and smash once; Carry out turning over smashing according to heap tender feeling condition afterwards, temperature upward period does not turn over and smashes, and temperature stops rising then to turn over smashing, and repeat this step, until when heap temperature is identical with external temperature, moisture is down to below 30%, namely complete fermenting process, whole fermentation period is 15 ~ 25 days;
Two, as the using method of disease-resistant microbial inoculum and bio-bacterial manure:
(1) dress seed: mix thoroughly with microbial inoculum after being squirted by seed, be all stained with microbial inoculum and be advisable, after seed dressing, dry in the shade, sow immediately with every seed-coat, every mu of consumption 3 kilograms, dresses seed and is applicable to the crop of direct program request;
(2) soak seed: in the ratio of 1:50, microbial inoculum is immersed in warp in water and fully stirs and place 4 hours, getting supernatant liquor and soak seed 12-24 hour, then through drying in the shade, can sow, mu consumption 3 kilograms;
(3) root is stained with: microbial inoculum and the water ratio furnishing pasty state in 1:10, and stick on seedling root equably, field planting immediately, mu consumption 5 kilograms;
(4) fill with root: in the ratio of 1:25, microbial inoculum is mixed with water and stirs evenly, then pour into crop root, mu consumption 5 kilograms;
(5) spread manuer in holes: after microbial inoculum and fine earth being mixed, put into every cave, and then transplant or dibbling, mu fertilizer application amount 5 kilograms.
3. the application of agricultural microbial agent described in claim 1 in the decomposition of Mierocrystalline cellulose and protein.
4. the becoming thoroughly decomposed and the application in deodorizing at organic materials of agricultural microbial agent described in claim 1.
5. the application of agricultural microbial agent described in claim 1 on crop disease control.
6. the application of agricultural microbial agent described in claim 1 in arviculture volume increase.
7. application according to claim 4, wherein organic materials refers to feces of livestock and poultry, crop material, mud, can pile the organic waste of rotten domestic refuse and mushroom residue rich cellulose and protein; Livestock and poultry wherein refer to: ox, pig, chicken, sheep, horse, duck.
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