CN105272621A - Preparation and application of composite bioactive material taking attapulgite to load bacillus - Google Patents
Preparation and application of composite bioactive material taking attapulgite to load bacillus Download PDFInfo
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- CN105272621A CN105272621A CN201510725541.5A CN201510725541A CN105272621A CN 105272621 A CN105272621 A CN 105272621A CN 201510725541 A CN201510725541 A CN 201510725541A CN 105272621 A CN105272621 A CN 105272621A
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Abstract
The invention relates to a method for preparing a composite material which takes modified or unmodified attapulgite as a carrier to immobilize bacillus, belonging to the technical fields of microbial fertilizers, biological control of plant diseases, soil remediation and pollution abatement. A microorganism-mineral immobilization technology is realized by means of preparing a proper amount of attapulgite with a proper amount of bacillus viable bacteria preparation according to a certain technology. The composite material prepared according to the method disclosed by the invention has the advantages that the plant growth can be promoted, the soil-borne diseases, such as root rot, blight, soft rot and the like, of various crops can be inhibited, and the effect of preventing threadworm from spawning is achieved; the composite material further has the capabilities of keeping the local moisture of plant root systems, and enhancing the plant resistance to drought, saline and alkaline as well as oxidization; moreover, the composite material can be further used for purifying organic pollutants in water, absorbing soil heavy metals and relieving soil hardening, so that the effects of soil moisture conservation and fertilizer conservation are achieved. The composite material prepared according to the method disclosed by the invention is an environment-friendly composite material, which has the advantages that environment harm and pollution can be prevented through correct use of the composite material, and a wide application prospect is obtained.
Description
Technical field the invention belongs to microorganism field.Relate generally to preparation and the application thereof of the mineral composite of attapulgite Immobilized Bacillus Sp.
Background technology
Along with people are for the attention of ecotope and the continuous increase to green safety, organic pollution-free food demand, environmentally friendly agricultural chemicals and chemical fertilizer become the study hotspot in Agriculture Production problem increasingly.Since over half a century, although chemical pesticide control disease and pest plays an important role to agriculture production, but long-term unreasonable use chemical pesticide creates problems, the height poison of such as chemical pesticide, high residue not only work the mischief to the health of people, animal, and severe contamination is caused to soil, water body, air, destroy the eubiosis.Meanwhile, due to the continuous enhancing of germ and pest resistance to insecticide, usage quantity and the usage frequency of agricultural chemicals continue to increase, and have occurred the vicious cycle that dosage and disease increase progressively mutually.Secondly, due to the long-term unreasonable a large amount of use inorganic fertilizer of China, destroy the structure of soil, soil compaction is serious, Soil structure overbalance, causes Fertilizer application amount to increase and without positive effect.Address these problems the substitute of the traditional agriculture chemical finding environmentally friendly and ecological balance type in the urgent need to us.
Biological control based on biological pesticide is at present most environmental protection, ecological prevention and controls, utilizes this method, can be come pest control, disease and weeds by the direct or indirect mode of action, and nontoxic to people and animals, free from environmental pollution; High specificity is killed and wounded to disease and pest, does not injure natural enemy and beneficial organism, can keep ecological balance.At present, biological pesticide based on genus bacillus has been widely used in field and spread uses, particularly bacillus amyloliquefaciens can be grown at plant root rapidly surely, by a large amount of Fast-propagation, compete nutrition and living space, secretion antibacterial substance and proteolytic enzyme with pathogenic bacteria, excite the comprehensive action such as resistance immunologic mechanism, Promoting plant growth of plant materials to form prevention to soil-borne disease and prevention effect.
Attapulgite (palygorskite) also known as polygorskite, because of character such as its tool layer, chain, fibrous crystalline structure and nano level hole passage microtexture and tool adsorptivity, slow-releasing, dispersiveness, suspension and the strong exchangeabilities of ion.Attapulgite is a kind of Mineral resources, and it contains 12 kinds of trace elements needed for life entity, and the attapulgite ore deposit iodine that wherein Gansu is domestic, selenium, Zn content are higher.Attapulgite, owing to having larger specific surface area and certain cation exchange capacity, makes it can change the materialization property of soil, coordinates the liquid manure gas heat of soil, increases the preserve moisture and fertility ability of soil.The porous crystal structure of attapulgite, can improve the transparent performance to moisture, can strengthen its water-retentivity to sand, can strengthen its water-permeable, be not only desirable water-holding agent, and effectively can prevent nitrogen volatilization loss the sticky soil that heavily hardens.Attapulgite is not only good soil improvement material, and itself is exactly fertilizer, attapulgite breeze is joined in soil go separately, just can reach the effect making increasing crop yield 5% ~ 10%.
Attapulgite is owing to having higher cation exchange capacity and specific surface area, can adsorb and complexation heavy metal ion, thus reduce Heavy Metals in Soils and reduce Pesticide-Polluted Soil, but research shows, the power of polygorskite heavy metal cation adsorption is relevant with atomic radius with adion electronegativity, has certain selectivity; In addition, attapulgite also can be used as organic pollutants in water body and administers material, but some researchs show, attapulgite often and other non-biological material compound use, although result can improve adsorption efficiency, some non-biological material such as gac etc. easily causes secondary pollution of water.
Microorganism is also widely used in heavy-metal contaminated soil reparation and Organic pollutants wastewater treatment, wherein, especially give prominence to such pollution effects of genus bacillus process, there is the very thick netted peptidoglycan structure of one deck in the cell wall of Bacillus strain, in addition the bacterial strain such as such as bacillus amyloliquefaciens can secrete the secondary metabolite that exocellular polysaccharide, class collagen protein etc. have adhesion effect, make it have powerful Adsorption of Heavy Metals and the ability of Organic pollutants, in addition, they are also by the Adsorption of Heavy Metals such as ion-exchange, complexing action; In water body scrubbing, genus bacillus mainly through heavy metal ion redox, methylate and demethylation be used for repair water body, but because microorganism active is larger by the impact of environmental factors, independent applying microorganism carries out restoration of the ecosystem not easily gathering in the environment, survival rate is low, and then affects its repairing effect.
The two performance comprehensive above, the shortcoming applying single material not only can be made up with the composite bio-active material of attapulgite loaded genus bacillus, the effect of the two can also be strengthened, fix and the matrix material of load genus bacillus through attapulgite, as bio-feritlizer and agricultural chemicals: can not only Promoting plant growth, suppress soil-borne disease, also there is efficiency slow release effect; As ecological restoration materials: long-acting and ecology can concentrate rehabilitating soil and water pollution with open arms, therefore, this microorganism-mineral composite has a good application prospect.
Summary of the invention
The object of the invention is for overcome chemical pesticide and the hazard residue of chemical fertilizer in farmland, single-material to provide as fertilizer or the low present situation of soil sewage modifying agent validity one can not only Promoting plant growth, prevent and treat soil-borne disease, the residual quantity of can also alleviate soil compaction, strengthen the organic content of soil, improve heavy metal in preserving soil moisture fertilizer conservation performance, degraded soil and objectionable impurities also improves the novel environmental friendly microorganism-mineral composite of quality of agricultural product.
For achieving the above object, the present invention propose a kind of with modification and non-modified attapulgite for carrier fixes preparation method and the application thereof of the matrix material of genus bacillus.
Technical scheme of the present invention is:
One, the present invention is by following material and percentage weight ratio thereof: attapulgite 100%, genus bacillus 2% ~ 8%.Containing genus bacillus living spores number 5.0 × 10 in every gram of this compound manure
9~ 2.0 × 10
10individual.
Two, preparation process of the present invention:
Attapulgite mud acid (alkali) modification method for preparing:
(1), take purification attapulgite 10g and put into 250mL Erlenmeyer flask, add properties-correcting agent 1mol/LHCI (2mol/LNaOH), purification attapulgite and HCI (NaOH) mix with the ratio of 1:10.
(2), the sample mixed is put under 25 DEG C of conditions in 150rpm shaking table vibration modification 2h.
(3), take out sample and carry out centrifugation at 4000rpm whizzer, discard supernatant liquid, then with distilled water wash and centrifugation.
(4), with the nitre alkali silver solution of 0.1mol/L detect without after chlorion, dry under the condition of 105 DEG C, obtained attapulgite modified.
Fermentation of bacillus preparation method:
(1), first order seed is cultivated
-80 DEG C of frozen former bacterial classifications to be rule on plate culture medium activation culture, 36 ~ 40 DEG C of incubated overnight.With the mono-clonal colony inoculation that transfering loop picking flat board grows in 50mL sterilizing seed liquor substratum (peptone 10g/L, yeast extract 5g/L, NaCI10g/L are housed, pH7.0) 150mL triangular flask, 37 DEG C, 200rpm shaking table shaking culture 8-10h, obtains bacteria suspension; All be inoculated in by the 50mL seed bacteria suspension of cultivation in the large tank of 6L that 2.7L sterilizing seed liquor substratum is housed, tank mouth 16 layers of sterile gauze seal to ensure dissolved oxygen amount, 37 DEG C, 200rpm shaking table shaking culture 12-16h.
(2), secondary propagation enlarged culturing
Use RTY-MZ100L-1000L liquid fermentation tank to carry out propagation and expand fermentation.Fermenter volume 1000L.Fermentation volume is 800L, first add water after tank body sky goes out 400L, add seed liquor substratum, add again edible soybean oil as defoamer and start stir, stirring velocity is 300rpm, finally add water to 800L and mix fermention medium, start real going out, temperature of going out in fact is 126 DEG C of sterilizing 1h, when temperature drops to 85 DEG C, open stirrer after fermentor tank sterilizing to 300rpm, continue to add when being cooled to 42 DEG C the seed liquor that step (1) is cultivated, leavening temperature 42 DEG C cultivation about 12h ~ 16h, dissolved oxygen amount remains on about 60%, and being cultured to bacterium colony concentration is 1.0 × 10
10~ 9.9 × 10
11cFU.
(3), solid state fermentation microbial inoculum preparation
The genus bacillus nutrient solution that step (2) is fermented centrifugal 40min under the condition of normal temperature, 5000r/min; Semi-lactosi protective material is put in thalline centrifugation, more aseptically vacuum lyophilization, pulverize and sieve rear for subsequent use.Wherein in step (3), thalline centrifugal sediment and protectant mass ratio are 1:1.
Three, the composite mineral fertilizer material of attapulgite loaded bacillus amyloliquefaciens is to the field control effect of soil-borne disease of crop.
From Lanzhou, peaceful with finding lily blight (Fusariumoxysporum) morbidity ground, boxthorn root maize ear rot (Fusariumsolani) morbidity ground and capsicum epidemic disease (PhytophthoracapsiciLeonian) morbidity, the morbidity finally determined by incidence, evenly be divided into three blocks, lily and each district of capsicum add up 300 strains, the each district of matrimony vine adds up 100 strains, and the incidence before investigation fertilising, shown in table 1 specific as follows.
This efficiency test has on intercurrent disease crop in land for growing field crops to carry out, dispenser Qian Mei slightly falls ill community, if check plot not dispenser, therefore, sickness rate is added up to during peak period after administration until control group morbidity, and dispenser group is lighter than the control group sickness rate of not dispenser, namely this light partial difference is the prevention effect of this fertilizer.Field efficacy calculates based on existing bibliographical information:
In formula, EF is prevention effect; T is the final state of an illness in control district, and T0 is its initial state of an illness; Ck is not for prevent and treat district (check plot) the final state of an illness, and ck0 is its initial state of an illness.
The incidence of blight, root rot and epidemic disease before table 1 field test is fertile
Fertilization mode adopts to turn over and buries and foliage-spray, and use 20kg or every strain spray 50-100ml liquid for every mu, treat that check plot plant is to onset peak period investigation diseased plant number and statistics, result is as shown in following table 2-3.
The composite mineral fertilizer material of table 2 attapulgite loaded bacillus amyloliquefaciens is to the field efficacy of lily wilt and boxthorn root maize ear rot
The composite mineral fertilizer material of table 3 attapulgite loaded bacillus amyloliquefaciens is to the field efficacy of capsicum epidemic disease
Four, the composite mineral fertilizer material of attapulgite loaded bacillus amyloliquefaciens is on the impact of plant growth.
Use the composite mineral fertilizer material of attapulgite loaded bacillus amyloliquefaciens with demonstrating in Yongning Ningxia county silage corn, use 20kg for every mu, record output during results, result is as shown in table 4.
The composite mineral fertilizer material of table 4 attapulgite loaded bacillus amyloliquefaciens is on the impact of silage corn output
Benefit of the present invention is: the field test results from table 1 to table 3, the mineral composite of attapulgite loaded bacillus amyloliquefaciens of the present invention is ploughed and is executed and the soil-borne disease of foliage-spray to crop, as all good disease control effects of tool such as lily blight (Fusariumoxysporum), boxthorn root maize ear rot (Fusariumsolani) and capsicum epidemic diseases (PhytophthoracapsiciLeonian), more than 58% is reached to the field efficacy of each disease; In addition, also can be found out by table 4, this microorganism-mineral composite also has the effect of Promoting plant growth, silage corn output can be made comparatively to contrast and to improve nearly 30%, can Promoting plant growth the residual quantity of the heavy metal had in degraded soil and objectionable impurities, alleviate soil compaction, strengthen the organic content of soil, improve preserving soil moisture fertilizer conservation performance, and improve the effect of quality of agricultural product.Therefore, this product, as novel environment-friendly and bio agriculture chemical material, has wide market application foreground.
Embodiment:
Embodiment 1:
The present invention is by following material and percentage weight ratio thereof: attapulgite 100%, bacillus amyloliquefaciens (Bacillusamyloliquefaciens) 5%, containing bacillus amyloliquefaciens living spores number 1.0 ~ 2.0 × 10 in every gram of this compound manure
11individual.
Preparation process of the present invention:
Attapulgite mud acid modification method for preparing:
(1), take purification attapulgite 10g and put into 250mL Erlenmeyer flask, add properties-correcting agent 1mol/LHCI, purification attapulgite and HCI mix with the ratio of 1:10.
(2), the sample mixed is put under 25 DEG C of conditions in 150rpm shaking table vibration modification 2h.
(3), take out sample and carry out centrifugation at 4000rpm whizzer, discard supernatant liquid, then with distilled water wash and centrifugation.
(4), with the nitre alkali silver solution of 0.1mol/L detect without after chlorion, dry under the condition of 105 DEG C, obtained attapulgite modified.
Bacillus amyloliquefaciens fermentation preparation:
(1), first order seed is cultivated
-80 DEG C of frozen former bacterial classifications to be rule on plate culture medium activation culture, 36 ~ 40 DEG C of incubated overnight.With the mono-clonal colony inoculation that transfering loop picking flat board grows in 50mL sterilizing seed liquor substratum (peptone 10g/L, yeast extract 5g/L, NaCI10g/L are housed, pH7.0) 150mL triangular flask, 37 DEG C, 200rpm shaking table shaking culture 8-10h, obtains bacteria suspension; All be inoculated in by the 50mL seed bacteria suspension of cultivation in the large tank of 6L that 2.7L sterilizing seed liquor substratum is housed, tank mouth 16 layers of sterile gauze seal to ensure dissolved oxygen amount, 37 DEG C, 200rpm shaking table shaking culture 12-16h.
(2), secondary propagation enlarged culturing
Use RTY-MZ100L-1000L liquid fermentation tank to carry out propagation and expand fermentation.Fermenter volume 1000L.Fermentation volume is 800L, first add water after tank body sky goes out 400L, add seed liquor substratum, add again edible soybean oil as defoamer and start stir, stirring velocity is 300rpm, finally add water to 800L and mix fermention medium, start real going out, temperature of going out in fact is 126 DEG C of sterilizing 1h, when temperature drops to 85 DEG C, open stirrer after fermentor tank sterilizing to 300rpm, continue to add when being cooled to 42 DEG C the seed liquor that step (1) is cultivated, leavening temperature 42 DEG C cultivation about 12h ~ 16h, dissolved oxygen amount remains on about 60%, and being cultured to bacterium colony concentration is 1.0 × 10
10~ 9.9 × 10
11cFU.
(3), solid state fermentation microbial inoculum preparation
The genus bacillus nutrient solution that step (2) is fermented centrifugal 40min under the condition of normal temperature, 5000r/min; Semi-lactosi protective material is put in thalline centrifugation, more aseptically vacuum lyophilization.Wherein in this step (3), thalline centrifugal sediment and protectant mass ratio are 1:1.
The preparation of the composite mineral fertilizer material of the attapulgite modified load bacillus amyloliquefaciens of acid
Take sour attapulgite modified and bacillus amyloliquefaciens agent by above-mentioned weight part, with pulverizer, attapulgite and bacillus amyloliquefaciens agent are pulverized rear mistake 800 mesh sieve respectively, make particulate material by certain technique.
Application process: should base fertilizer be made, namely evenly spread fertilizer over the fields before turning over or after ploughing, turn over (rake) buries, and also can do foliage-spray with 1000 times of diluents; Also can topdress, should early unsuitable evening, execute rear earthing, water good.General crop every mu uses 20kg, please according to the growth needs of all kinds of crop when user uses, increases in proportion and joins other nutrient.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. an attapulgite loaded genus bacillus composite bio-active material, is characterized in that, bacillus viable bacteria and gemma thereof is fixed in attapulgite material.
2. a kind of attapulgite loaded genus bacillus composite bio-active material according to claim 1, is characterized in that, material and percentage weight ratio thereof: attapulgite 90-99.9%, the high dense preparation 0.1% ~ 10% of genus bacillus.Containing genus bacillus living spores number 2.0 × 10 in every gram of this matrix material
8~ 2.0 × 10
10individual.
3. a kind of attapulgite loaded genus bacillus composite bio-active material according to claim 1 or 2, is characterized in that, the optimal load viable count of attapulgite modified load genus bacillus is 2.0 × 10
9/ gram.
4. a kind of attapulgite loaded genus bacillus composite bio-active material preparation method according to claim 1 or 2, is characterized in that, acid is attapulgite modified to be prepared according to the following steps:
(1) take purification attapulgite 10g and put into 250mL Erlenmeyer flask, add properties-correcting agent 1mol/LHCI, purification attapulgite and HCI mix with the ratio of 1:10.
(2) sample mixed is put under 25 DEG C of conditions in 150rpm shaking table vibration modification 2h.
(3) take out sample and carry out centrifugation at 4000rpm whizzer, discard supernatant liquid, then with distilled water wash and centrifugation.
(4) detect without after chlorion with the nitre alkali silver solution of 0.1mol/L, dry under the condition of 105 DEG C, obtained attapulgite modified.
5. a kind of attapulgite loaded genus bacillus composite bio-active material preparation method according to claim 1 or 2, it is characterized in that, alkali modification attapulgite is prepared according to the following steps: (1) takes purification attapulgite 10g and puts into 250mL Erlenmeyer flask, add properties-correcting agent 2mol/LNaOH, purification attapulgite and NaOH mix with the ratio of 1:10.
(2) sample mixed is put under 25 DEG C of conditions in 150rpm shaking table vibration modification 2h.
(3) take out sample and carry out centrifugation at 4000rpm whizzer, discard supernatant liquid, then with distilled water wash and centrifugation.
(4) centrifugation is dried under the condition of 105 DEG C, obtained attapulgite modified.
6. a kind of attapulgite loaded genus bacillus composite bio-active material preparation method according to claim 1 or 2, it is characterized in that, genus bacillus used is prepared this matrix material according to the following steps:
(1) first order seed is cultivated
-80 DEG C of frozen former bacterial classifications to be rule on plate culture medium activation culture, 37 DEG C of incubated overnight.With the mono-clonal colony inoculation that transfering loop picking flat board grows in 50mL sterilizing seed liquor substratum (peptone 10g/L, yeast extract 5g/L, NaCI10g/L are housed, pH7.0) 150mL triangular flask, 37 DEG C, 200rpm shaking table shaking culture 8-10h, obtains bacteria suspension; All be inoculated in by the 50mL seed bacteria suspension of cultivation in the large tank of 6L that 2.7L sterilizing seed liquor substratum is housed, tank mouth 16 layers of sterile gauze seal to ensure dissolved oxygen amount, 37 DEG C, 200rpm shaking table shaking culture 12-16h.
(2) secondary propagation enlarged culturing
Use RTY-MZ100L-1000L liquid fermentation tank to carry out propagation and expand fermentation.Fermenter volume 1000L.Fermentation volume is 800L, first add water after tank body sky goes out 400L, add seed liquor substratum, add again edible soybean oil as defoamer and start stir, stirring velocity is 300rpm, finally add water to 800L and mix fermention medium, start real going out, temperature of going out in fact is 126 DEG C of sterilizing 1h, when temperature drops to 85 DEG C, open stirrer after fermentor tank sterilizing to 300rpm, continue to add when being cooled to 42 DEG C the seed liquor that step (1) is cultivated, leavening temperature 42 DEG C cultivation about 15h, dissolved oxygen amount remains on about 60%, and being cultured to bacterium colony concentration is 1.0 × 10
10~ 9.9 × 10
11cFU.
(3) solid state fermentation microbial inoculum preparation
The genus bacillus nutrient solution that step (2) is fermented centrifugal 40min under the condition of normal temperature, 5000r/min; Semi-lactosi protective material is put in thalline centrifugation, more aseptically vacuum lyophilization, pulverize and sieve rear for subsequent use.Wherein in step (3), thalline centrifugal sediment and protectant mass ratio are 1:1.
7. a kind of application of attapulgite loaded genus bacillus composite bio-active material according to claim 1 or 2, is characterized in that, mainly prevent and treat wilt, pine root fungus, ralstonia solanacearum, epidemic disease or rhizoctonia solani.
8. a kind of application of attapulgite loaded genus bacillus composite bio-active material according to claim 1 or 2, is characterized in that, for the effect of adsorbing heavy metal in soil and other objectionable impuritiess, alleviate soil compaction, improve preserving soil moisture and fertilizer conservation.
9. a kind of application of attapulgite loaded genus bacillus composite bio-active material according to claim 1 or 2, is characterized in that, for adsorption treatment water pollution organism.
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