CN102690763A - Enterobacter agglomerans with phosphate-solubilizing capacity, and application thereof - Google Patents
Enterobacter agglomerans with phosphate-solubilizing capacity, and application thereof Download PDFInfo
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Abstract
The invention discloses a strain of phosphate-solubilizing bacteria which is preserved in China general microbiological culture collection center (CGMCC) and is named as enterobacter agglomerans RKMC-7 in a classification way with the preservation number of CGMCC No. 5005. After a bactericide containing the enterobacter agglomerans is inoculated into the soil polluted by lead, the exchangeable form lead can be promoted to be fixed, and the fixing efficiency is 77-91.4%. The bactericide is simple in preparation method, rich in material source and easy in implementation of large-scale production, can realize the effect of carrying out agricultural production while repairing, and has important significance for guaranteeing grain safety and environmental protection.
Description
Technical field
The present invention relates to a kind of enterobacter agglomerans, belong to technical field of microbe application with phosphorus decomposing ability.
Background technology
Along with quick growth, industriallization and the fast development of urbanization of global population, heavy metal contamination has become the serious problems of China and even world's soil pollution.At present, about 5,000,000 t lead is discharged in the whole world every year on average.In past 50 years, the lead that is discharged in the global environment has 7.83 * 10 approximately
5T. wherein most of soil that gets into causes countries in the world soil Lead contamination in various degree to occur.The data presentation that China State Environmental Protection Administration in 2003 provides; The cultivated area of the nearly l/5 of China receives Lead contamination, and exceeding standard rate plumbous in suburb, China big and medium-sized cities vegetables, grain, fruit, meat and the livestock product is respectively 38.6%, 28.0%, 27.6%, 41.9% and 71.1%.It is thus clear that the reparation of heavy metal-polluted soil Lead contamination is extremely urgent.
In the heavy metal toxicity table, lead comes common the 3rd with genotoxic potential element.Excessive lead not only can block crop growth, reduces crop yield and quality, and nerve, digestion, immunity and reproductive system, the especially intelligence growth to children that can also damage the people through the enrichment of biologic chain cause serious hindrance.
At present, the technology of improvement soil Lead contamination mainly contains physico-chemical processes and bioremediation technology etc.The physico-chemical processes instant effect, but bigger to environmental perturbation, spend very high.At present; More for the research of the Pb in the using soluble phosphoric acid salt curing soil both at home and abroad; But use the phosphoric acid salt curing technology, need apply a large amount of phosphoric acid salt, therefore spend higher; Phosphoric acid salt except with soil in lead form the indissoluble material such as pyromorphite, also can form multiple mixture with other material in the soil such as calcium, iron, aluminium etc.; In addition, the use of solidifying agent can change Soil structure, also possibly produce certain influence to soil microorganisms; Also have some researchs to think and the enrichment that can cause phosphorus in the soil of using of phosphorus solidifying agent make the leaching property increase of plumbous and phosphorus, thereby possibly cause that phosphorus and lead content increase in underground water and the surface water body.
The biological prosthetic method is a kind of very promising restorative procedure, and studying more at present is the super enriching plant recovery technique, has been found that multiple super enriching plant with strong heavy metal accumulation ability so far.But because the Pb super enriching plant kind of having found is still few, living weight is less, and the speed of growth is slow, and repairing efficiency is long, still is difficult to use in the reparation of actual Pb contaminated soil at present.In addition, China is populous, has found out that farmland area contaminated or that pollution condition is not clear is very big, and reality is to have to carry out farm crop in the farmland that failed to understand by heavy metal contamination or pollution condition produce.But, utilize the super enriching plant recovery technique to last long and take a large amount of farmlands, do not suit to apply at some low polluted farmland soils.Therefore; Under the prerequisite that does not influence agriculture prodn; The seed selection heavy metal is refused the plant improved seeds that (low) absorbs; The heavy metal migration is a kind of novel, effective heavy metals in farmland Pollution abatement technology in measure in-situ passivation, the impedance soil-botanical systems such as utilization biology, chemistry, has become the focus of domestic and international polluted agricultural land recovery technique research at present.
Total phosphorous is approximately 0.04-0.1% in the soil, but has only the 1-2.5% wherein can be by the plant absorbing utilization, mostly accumulates in soil for the indissoluble attitude.The whole world is annual uses about 3,000 ten thousand tons of phosphorus fertilizers, but wherein 80% because absorption, deposition or fixed action become the invalid phosphorus that plant is difficult to absorb.Phosphate solubilizing bacteria can be converted into the form that can absorb with the stationary state phosphorus that plant in the soil is difficult to absorb, and when having Lead contamination in the soil, the lead in free state phosphorus and the soil forms the pyromorphite of indissoluble etc., reduces plumbous bioavailability.Phosphate solubilizing bacteria has very strong rhizosphere effect, and the phosphoric acid salt that the phosphate solubilizing bacteria of rhizosphere can make the lead in the soil form insoluble is enriched in plant root, suppresses Pb from the transfer of plant root to over-ground part, improves the security of food.In addition, the part phosphate solubilizing bacteria also can produce siderophore, indolylacetic acid, have ACC desaminase activity etc. and promote plant-growth except that having the phosphorus decomposing ability.A little less than but the wild phosphate solubilizing bacteria of nature all exists growth alleviation phosphorus ability, and in contaminate environment, the wild strain growth is suppressed, and can not satisfy and pollute the needs of repairing.For this reason, need to adopt various methodologies to break the homergy of bacterial classification, improve its energy for growth, phosphorus decomposing ability and resistance capacity etc., reach this purpose, major measure is exactly to carry out the seed selection of bacterial classification, as carries out physics and chemomorphosis etc.
Summary of the invention
One of the object of the invention is to obtain the enterobacter agglomerans that a strain can be used for efficient phosphorus decomposing ability of having of heavy metal pollution of soil environmental improvement and preventing from heavy metal Pb energy for growth through ultraviolet ray-plasma body complex mutation.
Two of the object of the invention provides microbiobacterial agent that contains above-mentioned enterobacter agglomerans and preparation method thereof.
Three of the object of the invention provides the application of above-mentioned enterobacter agglomerans in improvement of heavy metal-polluted soil Pb contaminate environment and restoration of the ecosystem.
Implementation procedure of the present invention is following:
A kind of enterobacter agglomerans with efficient phosphorus decomposing ability, its classification called after group enterobacteria (
Enterobacter agglomerans) RKMC-7, being preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (BeiChen West Road, Chaoyang District, BeiJing City No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute) on June 29th, 2011, deposit number is CGMCC No.5005.
RKMC-7 colony colour on solid medium A is creamy white circular protrusions, neat in edge; Bacterium colony is rounded on solid medium B, and pale has transparent circle on every side; G
-, aerobic or amphimicrobian is elongated rod shape; 28 ~ 32 ℃ of its righttest growth temperatures, the righttest pH value is 6.8 ~ 7.5; Separating the inorganic phosphorus ability among the liquid medium within C is 635.4 ± 15.6 mg/L; Can produce oxalic acid, lactic acid, Hydrocerol A and succsinic acid; Make the pH of nutrient solution B reduce to 4.13-3.54 from initial 6.8-7.5, the ability of producing indolylacetic acid is 64.7 ± 5.2mg/l, can produce siderophore; Can resist penicillium mould, cephalosporin, penbritin, Oxacyclotetradecane,erythromycin deriv growth; Can tolerate the Pb of 1200mg/L at solid medium A
2+Grow, in plumbiferous liquid nutrient medium A, can tolerate the Pb of 1000mg/l
2+Growth; In edatope, has certain growth vigor.
Said solid medium A consists of: Tryptones 5-10 g, yeast powder 3-5 g, NaCl 5-10 g, zero(ppm) water 1000 mL, agar powder 15-20g, zero(ppm) water 1000ml, pH value 6.8-7.5.
Described substratum B consists of: glucose 10 g, Ca
3(PO
4)
25-13 g, MgCl
26H
2O 5 g, MgSO
47H
2O 0.25 g, KCl 0.2-0.5 g, (NH
4)
2SO
40.1-0.2g, agar powder 15 g, zero(ppm) water 1000 mL, pH 6.8-7.5.
Enterobacter agglomerans with phosphorus decomposing ability provided by the invention obtains through mutagenic breeding method, may further comprise the steps:
1) with the laboratory from the enterobacter agglomerans KMC with higher phosphorus decomposing ability of plant rhizosphere screening as starting strain;
2) mutagenic and breeding
(1) single cell suspension of preparation starting strain KMC
Starting strain KMC is inoculated among the liquid nutrient medium A, and 28-32 ℃, 150-180rpm cultivates 8-12hrs, and is centrifugal, with the SPSS washing, places in the triangular flask that granulated glass sphere is housed, and vibration makes it be dispersed into single celled bacteria suspension;
Described culture medium A consists of: Tryptones 5-10 g, yeast powder 3-5 g, NaCl 5-10 g, zero(ppm) water 1000ml, pH value 6.8-7.5;
(2) ultraviolet mutagenesis
The bacteria suspension of step (1) gained is regulated concentration to 10 respectively
5-10
7CFU/ml gets 0.1ml and coats and contain 600-1500mg/L Pb
2+On the solid medium B, carry out ultraviolet mutagenesis, the frequency of ultraviolet mutagenesis is 10-18W, and irradiation distance is 25-50cm, irradiation time 5-10min; 28-30 ℃ leaves standstill cultivation 5-7days; Select 20-30 single bacterium colony that transparent circle is bigger, shake the multiple sieve of bottle, measure the phosphorus decomposing ability of each bacterial strain with molybdenum blue colorimetric method; Select the highest bacterial strain of 5-8 strain phosphorus decomposing activity; Shake the multiple sieve of bottle more respectively, select the bacterial strain RKMC of active height of a strain phosphorus decomposing and good stability, process the mutagenesis that bacteria suspension is used for next step plasma body;
The step that described shake flask fermentation sieves again is: the higher inoculation of 20-30 transparent circle diameter D that at first above-mentioned separation is obtained and colony diameter d ratio is cultivated 8-12hrs in the above-mentioned liquid nutrient medium A of 100ml.Get in the Erlenmeyer flask that 5ml bacterium liquid is inoculated into the 250ml that 100ml liquid nutrient medium B is housed, 28-32 ℃, 150rpm shaking table shaking culture 5-7days;
(3) plasma body mutagenesis
With the RKMC bacterial strain of step (2) gained, process 10
5-10
7The bacteria suspension of CFU/ml is got 0.1-0.2ml and is evenly coated in the sterile petri dish, and petridish is put on the electrode below the plasma; Regulate the position of top electrode, make that the distance between the upper/lower electrode is controlled at about 3-8mm, regulating voltage is 3-5V; Electric current is 0.5-0.8A; Make air or argon gas discharging, obtain uniform air or argon medium barrier discharge plasma, be 2-7min discharge time.Immediately with SPSS or phosphoric acid salt wash-out, coat and contain 600-1000mg/L Pb after the mutagenesis
2+On the solid medium B, shake the multiple sieve of bottle again, choose the highest strain bacterium RKMC-P of a strain phosphorus decomposing activity, process bacteria suspension and be used for next step mutagenesis; The described same step of method (2) of shaking the multiple sieve of bottle;
(4) the bacteria suspension viable count with step (3) gained transfers to 10
5-10
7CFU/ml, cycle repeats ultraviolet mutagenesis → plasma body mutagenesis 1-2 time obtains strain tolerance heavy metal Pb and the highest bacterial strain RKMC-7 of phosphorus decomposing activity at last.
RKMC-7 identifies with enterobacter agglomerans to have high homology through 16SrDNA, compares with parent KMC, and the base likelihood is 95.79%.
The present invention also provides a kind of efficient phosphorus decomposing microbial inoculum, can be liquid bacterial agent or solid fungicide based on nutrients carrier difference.
The preparation of microbial inoculum may further comprise the steps:
1) strain fermentation
1. with frozen RKMC-7 quick-thawing under 37 ℃ of conditions, be equipped with in the test tube of 10-15ml liquid nutrient medium A according to the inoculum size access of 0.5-1%, 28-32 ℃, leave standstill and cultivate 8-12hrs, obtain the primary seed solution of RKMC-7;
2. secondary triangular flask liquid culture
Primary seed solution is equipped with in the triangular flask of 50-100ml liquid nutrient medium A according to the inoculum size access of 3-5%, and 28-32 ℃, 150-200rpm cultivates 8-12hrs;
3. ferment tank
The one-level nutrient solution of the RKMC-7 inoculum size according to 5-10% is inserted respectively in the fermentor tank that fermention medium C is housed, carry out fermentation culture, jar warm 28-32 ℃, cultivate pH6.8-7.5, aerobic culture 30-48 hrs;
Described culture medium C consists of: wheat bran 5-10g, dregs of beans 30-50g, Semen Maydis powder 3-5g, KH
2PO
45-10g/ml, K
2HPO
40.1-0.2g/ml, Ca
2(PO
4)
35-13 g, MgSO
47H
2O 5-10 g, water 1000ml, p H 6.8 ~ 7.5;
2) straw powder, turfy soil, wheat bran and dregs of beans being pulverized with high speed disintegrator, crossed 60 mesh sieves, is the ratio uniform mixing of 20-30:20-30:5-10:30-50 according to mass percent;
3) fermented liquid that obtains in the step 1) is squeezed into container for storing liquid and promptly get liquid phosphorus decomposing microbial inoculum, living bacteria count can reach 5-10 * 10
9CFU/ml is with dosage and the step 2 of fermented liquid according to 50-100ml/kg) in the solid state substrate mixing of gained, 28-32 ℃ of fermentation 5-7days promptly obtains solid fungicide, cell density reaches 1-2 * 10
9CFU/g.
Phosphorus decomposing microbial inoculum liquid bacterial agent provided by the invention soaks seed or plant root 2-5hrs with 100-200 liquid bacterial agent diluent doubly when plant is soaked seed or soaks root; Irritate root in nursery stage or vegetative period and adopt 300-600 liquid bacterial agent diluent 10-40ml/Kg soil dosage pouring diluent doubly, the whole growth phase is irritated root 1-3 time; Solid-state microbial inoculum is as the base manure and the use of topdressing, and using dosage is a 5-20g/Kg soil.
Advantage of the present invention and beneficial effect:
1) using plasma mutagenesis and the ultraviolet mutagenesis method of circular treatment has repeatedly guaranteed the stability of mutant strain;
2) phosphate solubilizing bacteria that provides has high phosphorus decomposing ability, can resist the Pb growth of higher concentration, can secrete indolylacetic acid, produce iron atom, and stabilization characteristics of genetics goes down to posterity 14 times.Can promote plant-growth, promote plumbous enrichment, suppress the ability of plumbous migration from underground part toward over-ground part at plant root;
3) the big production of mass-producing is lacked, can be carried out to simple, the easy cultivation of microbial inoculum nutritional requirement of the present invention, growth cycle;
4) microbial inoculum of the present invention can obviously promote the growth of plant; Promote plumbous enrichment at plant root; Suppress plumbous from underground ability of moving toward over-ground part; Through selectivity in contaminated soil plantation farm crop, collect plant root etc. and focus on, reduce or finally remove the Lead contamination in the soil.Environmental friendliness and with low cost;
5) for the lead in short the livings bacterium fixing soil of development and use plant rhizosphere provides new way, can carry out the reparation of agriculture prodn limit in the limit, the improvement of food safety and heavy-metal contaminated soil is had great importance.
Description of drawings
Dull and stereotyped photo and stereoscan photograph that Fig. 1 grows in solid medium A, solid medium B respectively for RKMC-7;
Fig. 2 produces siderophore qualitative experiment photo for RKMC-7;
Fig. 3 produces the organic acid high-efficient liquid phase chromatogram for RKMC-7;
Fig. 4 is that RKMC-7 is to antibiotic susceptibility;
Fig. 5 is the anti-Pb of RKMC-7
2+Growth figure;
Fig. 6 is a R KMC-7 mitotic stability;
Fig. 7 is that R KMC-7 liquid bacterial agent is to fixed action plumbous in the soil;
The corn pot experiment of Fig. 8 in leaded soil, carrying out, and in soil, inoculate the RKMC-7 solid fungicide: wherein Fig. 8 A is the content of corn root Pb; Fig. 8 B is the content of the Pb of corn stem; Fig. 8 C is the content of leaf of Semen Maydis Pb; Fig. 8 D is the content of Pb in the maize kernel; Fig. 8 E is a maize kernel output; Fig. 8 F is the plumbous content of exchangeable species in the soil behind the harvest corn.
Embodiment
Embodiment 1:According to the mutagenic breeding method with enterobacter agglomerans of phosphorus decomposing ability provided by the invention, mutagenesis screening can tolerate the strong mutagenic strain of phosphorus decomposing ability of heavy metal lead, may further comprise the steps:
1) with the laboratory from the enterobacter agglomerans KMC with higher phosphorus decomposing ability of plant rhizosphere screening as starting strain;
2) mutagenic and breeding
(1) single cell suspension of preparation starting strain KMC
Starting strain KMC is inoculated among the liquid nutrient medium A, and 28 ℃, 150rpm cultivates 12hrs, and is centrifugal, with the SPSS washing, places in the triangular flask that granulated glass sphere is housed, and vibration makes it be dispersed into single celled bacteria suspension;
Described culture medium A consists of: Tryptones 5 g, yeast powder 5 g, NaCl 10 g, zero(ppm) water 1000ml, pH value 7.2;
(2) ultraviolet mutagenesis
The bacteria suspension of step (1) gained is regulated concentration to 10 respectively
7CFU/ml gets 0.1ml and coats and contain 1200mg/L Pb
2+On the solid medium B, carry out ultraviolet mutagenesis, the frequency of ultraviolet mutagenesis is 15W, and irradiation distance is 30cm, irradiation time 5min; 28 ℃ leave standstill cultivation 7days; Select 20 single bacterium colonies that transparent circle is bigger, shake the multiple sieve of bottle, measure the phosphorus decomposing ability of each bacterial strain with molybdenum blue colorimetric method; Select the highest bacterial strain of 5 strain phosphorus decomposing activity; Shake the multiple sieve of bottle more respectively, select the bacterial strain RKMC of active height of a strain phosphorus decomposing and good stability, process the mutagenesis that bacteria suspension is used for next step plasma body;
The step that described shake flask fermentation sieves again is: the higher inoculation of 20 transparent circle diameter D that at first above-mentioned separation obtained and colony diameter d ratio is cultivated 12hrs in the above-mentioned liquid nutrient medium A of 100ml.Get in the Erlenmeyer flask that 5ml bacterium liquid is inoculated into the 250ml that 100ml liquid nutrient medium B is housed, 28 ℃, 150rpm shaking table shaking culture 7days;
(3) plasma body mutagenesis
With the RKMC bacterial strain of step (2) gained, process 10
7The bacteria suspension of CFU/ml is got 0.1ml and is evenly coated in the sterile petri dish, and petridish is put on the electrode below the plasma; Regulate the position of top electrode, make that the distance between the upper/lower electrode is controlled at about 3mm, regulating voltage is 3V; Electric current is 0.5A; Make atmospherical discharges, obtain uniform air dielectric barrier discharge plasma, be 3min discharge time.Immediately with SPSS or phosphoric acid salt wash-out, coat and contain 1200mg/L Pb after the mutagenesis
2+On the solid medium B, shake the multiple sieve of bottle again, choose the highest strain bacterium RKMC-P of a strain phosphorus decomposing activity, process bacteria suspension and be used for next step mutagenesis; The described same step of method (2) of shaking the multiple sieve of bottle;
(4) the bacteria suspension viable count with step (3) gained transfers to 10
7CFU/ml, cycle repeats ultraviolet mutagenesis → plasma body mutagenesis 1 time obtains strain tolerance heavy metal Pb and the highest bacterial strain RKMC-7 of phosphorus decomposing activity at last.The character of KMC and RKMC-7 is more as shown in table 1, can find out from table 1: the phosphorus decomposing of bacterial strain can increase 98.5%. before the force rate mutagenesis after the mutagenesis
The character of table 1 KMC and RKMC-7 relatively
RKMC-7 sees accompanying drawing 1 at dull and stereotyped photo of in solid medium A, solid medium B, growing respectively and stereoscan photograph, and as can be seen from the figure: RKMC-7 can produce the obvious transparent circle on solid medium B, has higher phosphorus decomposing ability; Accompanying drawing 2 produces siderophore qualitative experiment photo for RKMC-7, and as can be seen from the figure: RKMC-7 can produce a certain amount of siderophore; Accompanying drawing 3 produces the organic acid high-efficient liquid phase chromatogram for RKMC-7, and as can be seen from the figure, RKMC-7 can produce at least 4 kinds of organic acids, comprises oxalic acid, lactic acid, Hydrocerol A and succsinic acid, and wherein the output with Hydrocerol A is the highest; Accompanying drawing 4 is that RKMC-7 is to antibiotic susceptibility; Adopt the Kirby Bauer scraps of paper to detect RKMC-7 to antibiotic susceptibility, what wherein produce transparent circle has Amikacin Sulphate, penbritin, Oxacyclotetradecane,erythromycin deriv, trimethoprim-sulfamethoxazole, norfloxicin, qingfengmeisu qiong and a CIPROFLOXACIN USP 24; No transparent circle penicillium mould, paraxin and cephazolin arranged.As can be seen from the figure: RKMC-7 has the ability of anti-penicillium mould, paraxin and cephazolin growth, in edatope, has certain growth vigor.
Embodiment 2:RKMC-7 is to Pb
2+Resistance, determination step is following:
RKMC-7 is inoculated in enrichment culture among the liquid nutrient medium A, grows to exponential phase, behind centrifugal 10 min of 8000 rpm, the aseptic NaCl solution washing with 0.85% three times, using 0.85% aseptic NaCl solution adjustment bacterial concentration again is 10
7CFU/ ml draws 1 mL adding and contains 80 mL interpolation different concns Pb
2+(0,400,800,1000,1200mg Pb
2+In/l) the 250 mL triangular flasks of liquid nutrient medium D, Pb
2+With Pb (NO
3)
2Form add, 28 ° of C cultivated after 5 days, 600 nm measure the absorption photometric value of nutrient solution, the result shows: at Pb
2+When concentration reached 1000mg/L, RKMC-7 still can grow, and explained that RKMC-7 can tolerate the Pb of higher concentration
2+, in polluting comparatively serious Pb contaminated soil, can grow, have the potentiality of repairing the Pb contaminated soil.The anti-Pb of RKMC-7
2+Growth figure is shown in accompanying drawing 5, as can be seen from the figure: in the liquid medium within, RKMC-7 can resist the Pb of 1000mg/L
2+Growth.
Embodiment 3:RKMC-7 stability is measured, and concrete steps are following:
With frozen RKMC-7 quick-thawing under 37 ℃ of conditions; Inoculum size according to 1% inserts and is equipped with in the test tube of 10ml liquid nutrient medium A, 28 ℃, leaves standstill and cultivates 12hrs; Centrifugal; Behind the aseptic NaCl solution washing with 0.85% three times, suspend with 0.85% aseptic NaCl solution, the adjustment cell concentration is 1 * 10 again
7CFU/ml, the inoculum size according to 2% inserts among the liquid nutrient medium B, and 150rpm cultivated 7 days, measured the content of supernatant titanium pigment with molybdenum blue colorimetric method; Nutrient solution among the above-mentioned liquid medium within A is changed among the fresh liquid nutrient medium A according to 1% inoculum size again, repeat above-mentioned steps, go down to posterity altogether 14 times; Measure the phosphorus decomposing ability of RKMC-7; The result sees accompanying drawing 6, as can be seen from the figure goes down to posterity the phosphorus decomposing ability kept stable of RKMC-7 14 times; This shows that dissolving phosphorus mutant strain characteristic is not the adaptation to environment, but due to the stable transgenation.
Liquid nutrient medium D moity is: sucrose 10 g, NH
4NO
30.5 g, MgCl
26H
2O 0.5 g, K
2HPO
40.1 g, NaCl 0.1 g, yeast powder 0.25 g, zero(ppm) water 1000 mL, pH 7.2.
Embodiment 4:The preparation method of RKMC-7 microbial inoculum may further comprise the steps:
1) strain fermentation
1. with frozen RKMC-7 quick-thawing under 37 ℃ of conditions, the inoculum size according to 1% inserts and is equipped with in the test tube of 15ml liquid nutrient medium A, and 28 ℃, leave standstill and cultivate 12hrs, obtain the primary seed solution of RKMC-7;
2. secondary triangular flask liquid culture
Primary seed solution is equipped with in the triangular flask of 100ml liquid nutrient medium A according to 5% inoculum size access, and 28 ℃, 150rpm cultivates 12hrs;
3. ferment tank
The one-level nutrient solution of RKMC-7 is inserted respectively in the fermentor tank that fermention medium C is housed according to 5% inoculum size, carry out fermentation culture, 28 ℃ of jar temperature are cultivated pH 7.2, aerobic culture 48 hrs;
Described culture medium C consists of: wheat bran 10g, dregs of beans 50g, Semen Maydis powder 5g, KH
2PO
410g/ml, K
2HPO
40.1g/ml, Ca
2(PO
4)
313 g, MgSO
47H
2O 5 g, water 1000ml, p H 7.0.
2) straw powder, turfy soil, wheat bran and dregs of beans being pulverized with high speed disintegrator, crossed 60 mesh sieves, is the ratio uniform mixing of 20:30:10:40 according to mass percent;
3) fermented liquid that obtains in the step 1) is squeezed into container for storing liquid and promptly get liquid phosphorus decomposing microbial inoculum, living bacteria count reaches 5 * 10
9CFU/ml; With dosage and the step 2 of fermented liquid according to 50-100ml/kg) in the solid state substrate mixing of gained, 28 ℃ of fermentation 7days promptly obtain solid fungicide, living bacteria count reaches 10
9CFU/g.
Embodiment 5:The RKMC-7 inoculation may further comprise the steps the solvency action of invalid phosphorus in the soil:
Soil is packed in the triangular flask, and every bottled 100g that goes into is according to 0,200, the dosage of 800mg P/kg soil adds calcium phosphate; Mix the back sterilization; Add among the 20ml embodiment two liquid bacterial agent of preparation respectively, control group adds the dead thalline of Isodose respectively, every group all establish three parallel; Cultivated 21 days for 25 ℃ behind the abundant mixing, use 0.5M NaHCO
3(transferring pH to 8.5 with concentrated NaOH solution) solution lixiviate is according to the ratio adding NaHCO of 20ml solution/g soil
3Solution, 25 ℃ of vibration 30min, molybdenum antimony resistance colorimetric method is measured the content of phosphorus in the supernatant.
Experimental result shows: soil quick-effective phosphor content is respectively 8.35,11.25,15.49 mg/kg in the control group, and the experimental group available phosphorus contents is respectively 24.68,52.01,80.02 mg/kg, is respectively doubly 2.96,4.62 and 5.17 times of control group; Explain that this microbial inoculum can effectively dissolve the insoluble phosphorus in the soil, improve the content of rapid available phosphorus in the soil.
Embodiment 6:The RKMC-7 inoculation is to fixed action plumbous in the soil, and concrete steps are following:
1) supplying examination soil lead content is 22.34 mg/kg, is the flowerpot of 21 cm, high 30 cm with the soil diameter of packing into, and every basin is adorned native 5 kg.Respectively according to 0,400,800,1200mg Pb
2+The dosage of/kg soil adds Pb (NO
3)
2Solution, the plumbous actual content of each experimental group soil is respectively: 22.34,422.34,822.34 and 1222.34mg/kg, each dose groups establish 6 parallel, abundant every deionized water, balance 30 days of watering a time at a distance from 5 days behind the mixing;
2) the every basin soil with above-mentioned preparation takes out respectively in the 100g adding 250ml triangular flask; The liquid bacterial agent that adds preparation among the 10ml embodiment 2; Dead thalline to add Isodose is a control group; Each experimental group establish three parallel, fully behind the mixing 25 ℃ cultivated 28 days, adding 8 ml concentration according to every gram dry ground is the MgCl of 1M
2(pH 7.0) solution, (25 ± 1) ℃, 1h is extracted in the 150rpm vibration, and centrifugal 15 minutes of 8000rpm gets supernatant is measured exchangeable species Pb in the soil with flame atomic absorption spectrometry content.The result shows: add the live bacteria agent group and compare the content that can obviously reduce dissoluble lead in the soil with control group, 0,400,800,1200mg Pb
2+In the dose groups of/kg soil, the content comparison that adds interchangeability Pb in the live bacteria agent group soil has reduced by 4.28,11.67,4.88 and 5.86 times respectively according to group, and up to 77%-91.4%, the result sees accompanying drawing 7 to microbial inoculum to the fixed efficiency of interchangeability Pb.
Embodiment 7:RKMC-7 inoculation corn is to the repair of Lead contamination in the soil
Each plumbous dose groups of potted plant soil of embodiment 6 step 1) preparation is divided into microbial inoculum group and control group; Every group establish three parallel; With the solid fungicide of microbial inoculum group according to dosage adding embodiment three preparations of 5g solid fungicide/kg soil, control group adds the dead thalline of Isodose, fully behind the mixing; 8 corn seeds of every basin sowing, every basin keeps 3 strains during final singling.
Behind the harvest corn root, stem, leaf, seed are handled respectively, with after nitric acid-Perchloric Acid Digestion with NITRATE BY FLAME ATOMIC spectrophotometric determination lead content separately.It is the MgCl of 1M that the potted plant soil of every basin adds 8 ml concentration according to every gram dry ground
2(pH 7.0) solution, 25 ± 1 ℃, 1h is extracted in the 150rpm vibration, and centrifugal 15 minutes of 8000rpm gets supernatant is measured exchangeable species Pb in the soil with flame atomic absorption spectrometry content.
The result shows: can significantly increase the lead content of corn root with microbial inoculum, and plumbous content is compared with control group and declined to a great extent in stem, leaf and the seed.The corn root lead content is measured the result shown in accompanying drawing 8A: the lead content that adds live bacteria agent group corn root is compared obvious increase with control group, 0,400,800,1200mgPb
2+In the dose groups of/kg soil, the lead content that adds live bacteria agent group corn root is respectively 2.04,2.04,2.13 and 1.85 times of corresponding control group; Corn stem lead content is measured the result shown in accompanying drawing 8B: the lead content that adds live bacteria agent group corn stem is compared obvious reduction with control group, the lead content that adds live bacteria agent group corn stem has reduced by 1.19,3.15,1.88 and 2.38 times than corresponding control group respectively; Leaf of Semen Maydis portion lead content is measured the result shown in accompanying drawing 8C: the lead content that adds live bacteria agent group leaf of Semen Maydis portion is compared obvious reduction with control group, the lead content that adds live bacteria agent group leaf of Semen Maydis portion has reduced by 1.47,1.52,1.47 and 3.44 times than corresponding control group respectively; The maize kernel lead content is measured the result shown in accompanying drawing 8D: the lead content that adds live bacteria agent group maize kernel is compared obvious reduction with control group; The lead content that adds live bacteria agent group maize kernel has reduced by 1.0,4.33,7.43 and 5.83 times than corresponding control group respectively; The seed lead content all meet national regulation the grain hygienic standard (≤0.2mg/kg, GB2715-2005); The yield result of maize kernel is shown in accompanying drawing 8E: add live bacteria agent group corn yield and be greatly improved than corresponding control group, the output that adds live bacteria agent group maize kernel has increased by 25.6%, 10%, 46.3% and 51.4% than corresponding control group respectively; The potted plant content results of accomplishing exchangeable species Pb in the soil of back is shown in accompanying drawing 8F: the content that adds exchangeable species Pb in the live bacteria agent group soil is than the obvious reduction of control group, and the content comparison that adds exchangeable species Pb in the live bacteria agent group soil has reduced by 1.30,24.3,5.05 and 6.56 times respectively according to organizing.Therefore, this microbial inoculum has good repairing effect to the soil of Lead contamination, and the reparation of the output, security and the soil that improve grain is had great importance.
Claims (8)
1. enterobacter agglomerans with phosphorus decomposing ability, its classification called after enterobacter agglomerans (
Enterobacter agglomerans) RKMC-7, being preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.5005.
2. the enterobacter agglomerans with phosphorus decomposing ability according to claim 1, it is characterized in that this bacterial strain has following characteristic: colony colour is creamy white on solid medium A, circular protrusions, neat in edge; Bacterium colony is rounded on solid medium B, and pale has transparent circle on every side; G
-, aerobic or amphimicrobian is elongated rod shape; 28 ~ 32 ℃ of optimum growth temperatures, optimum pH are 6.8 ~ 7.5; Separating the inorganic phosphorus ability among the liquid medium within C is 635.4 ± 15.6 mg/L; Can produce oxalic acid, lactic acid, Hydrocerol A and succsinic acid; Make the pH of nutrient solution A reduce to 4.13-3.54 from initial 6.8-7.5, the ability of producing indolylacetic acid is 64.7 ± 5.2mg/l, can produce siderophore; Can resist penicillium mould, cephalosporin, penbritin, Oxacyclotetradecane,erythromycin deriv growth; Can tolerate the Pb of 1200mg/L at solid medium A
2+Grow, in plumbiferous liquid nutrient medium A, can tolerate the Pb of 1000mg/L
2+Growth;
Said solid medium A consists of: Tryptones 5-10 g, yeast powder 3-5 g, NaCl 5-10 g, zero(ppm) water 1000 mL, agar powder 15-20g, zero(ppm) water 1000ml, pH value 6.8-7.5;
Described substratum B consists of: glucose 10 g, Ca
3(PO
4)
25-13 g, MgCl
26H
2O 5 g, MgSO
47H
2O 0.25 g, KCl 0.2-0.5 g, (NH
4)
2SO
40.1-0.2g, agar powder 15 g, zero(ppm) water 1000 mL, pH 6.8-7.5.
3. the microbiobacterial agent that contains the said enterobacter agglomerans of claim 1.
4. the preparation method of the said microbiobacterial agent of claim 3 may further comprise the steps:
1) strain fermentation
1. with the frozen enterobacteria RKMC-7 of group quick-thawing under 37 ℃ of conditions, be equipped with in the test tube of 10-15ml liquid nutrient medium A according to the inoculum size access of 0.5-1%, 28-32 ℃, leave standstill and cultivate 8-12hrs, obtain the primary seed solution of RKMC-7;
2. secondary triangular flask liquid culture
Primary seed solution is equipped with in the triangular flask of 50-100ml liquid nutrient medium A according to the inoculum size access of 3-5%, and 28-32 ℃, 150-200rpm cultivates 8-12hrs;
3. ferment tank
The one-level nutrient solution of the RKMC-7 inoculum size according to 5-10% is inserted respectively in the fermentor tank that fermention medium C is housed, carry out fermentation culture, jar warm 28-32 ℃, cultivate pH6.8-7.5, aerobic culture 30-48 hrs;
Described culture medium C consists of: wheat bran 5-10g, dregs of beans 30-50g, Semen Maydis powder 3-5g, KH
2PO
45-10g/ml, K
2HPO
40.1-0.2g/ml, Ca
2(PO
4)
35-13 g, MgSO
47H
2O 5-10 g, water 1000ml, pH 6.8 ~ 7.5;
2) straw powder, turfy soil, wheat bran and dregs of beans being pulverized with high speed disintegrator, crossed 60 mesh sieves, is the ratio uniform mixing of 20-30:20-30:5-10:30-50 according to mass percent;
3) fermented liquid that obtains in the step 1) is squeezed into container for storing liquid and promptly get liquid phosphorus decomposing microbial inoculum, living bacteria count reaches 5-10 * 10
9CFU/ml; With dosage and the step 2 of fermented liquid according to 50-100ml/kg) in the solid state substrate mixing of gained, 28-32 ℃ of fermentation 5-7 days promptly obtains solid fungicide, cell density reaches 1-2 * 10
9CFU/g.
5. the application of the said enterobacter agglomerans of claim 1 in the heavy metal pollution of soil environmental improvement.
6. according to the said application of claim 5, it is characterized in that heavy metal is a lead ion.
7. according to the said application of claim 5; It is characterized in that method of use is: when plant is soaked seed or soaks root, soak seed or plant root 2-5hrs with 100-200 liquid bacterial agent diluent doubly; Irritate root in nursery stage or vegetative period and adopt 300-600 liquid bacterial agent diluent 10-40ml/Kg soil dosage pouring diluent doubly, the whole growth phase is irritated root 1-3 time; Solid-state microbial inoculum is as the base manure and the use of topdressing, and using dosage is a 5-20g/Kg soil.
8. the application of the said enterobacter agglomerans of claim 1 in soil ecology is repaired.
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| CN104212747A (en) * | 2014-09-17 | 2014-12-17 | 黑龙江省科学院微生物研究所 | Enterobacter cloacae having phosphorus solubilizing function and used for degrading imazethapyr |
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| CN103718690A (en) * | 2013-12-27 | 2014-04-16 | 中国矿业大学(北京) | Phosphate solubilizing bacterium inoculation method for promoting growth of plants in poor soil |
| CN103718690B (en) * | 2013-12-27 | 2015-10-28 | 中国矿业大学(北京) | A kind ofly promote that in lean soil, the phosphate solubilizing bacteria of plant growth connects bacterium method |
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