CN102634465B - Acinetobacter with authigenic nitrogen fixation capacity and application thereof - Google Patents
Acinetobacter with authigenic nitrogen fixation capacity and application thereof Download PDFInfo
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Abstract
The invention discloses an acinetobacter with authigenic nitrogen fixation capacity, which is classified and named as Ymu7-3 and is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No. 5006. The free living nitrogen fixing bacteria has high nitrogen fixing capacity, is capable of resisting the growth of Cd with higher concentration, and can secrete heteroauxin so as to generate iron atom; and the prepared microbial bacterium agent can remarkably promote the growth of hyper-accumulating plants, increase the biomass liveweight of the hyper-accumulating plants, greatly improve the heavy metal enrichment factor and transfer coefficient of the hyper-accumulating plants, and improve the ecological environment of the soil, thus being environment-friendly and low in cost.
Description
Technical field
The present invention relates to the acinetobacter calcoaceticus that a strain has spontaneous nitrogen fixing capacity, this ultraviolet ray for acinetobacter calcoaceticus-plasma body complex mutation obtains, and has the ability of preventing from heavy metal growth, can be used for heavy metal pollution of soil environmental improvement, belongs to technical field of microbe application.
Background technology
Heavy metal contamination refers to the environmental pollution being caused by heavy metal or its compound, mainly by due to the human factors such as mining, exhaust gas emission, sewage irrigation.In recent years, along with the growth of population, a large amount of uses of fast development, chemical fertilizer and the agricultural chemicals etc. of industry increasingly sharpen heavy metal pollution of soil.
Cd is the heavy metal that a kind of toxicity is very large, and world-shaking Japan " itai-itai " causes because of cadmium pollution.Cadmium can replace the calcium in bone, makes skeleton softening, and bone is broken into pieces; Cadmium also can cause gastrointestinal dysfunction, disturbs the enzyme system of zinc in human body and organism, causes hypertension.Cadmium is many-sided to the murder by poisoning of tissue and organ, and treatment is very difficult.Therefore, various countries have all made extremely strict regulation to the cadmium in industrial discharge " three wastes ".Japan's regulation rice surpasses 1 mg/kg containing cadmium and is " cadmium rice ", forbids eating.Japanese Environment Room regulation 0.3ppm is the highest normal contents of cadmium concentration in rice.
At present, the technology for heavy metal pollution of soil reparation mainly contains: physics reparation, chemistry are repaired and biological restoration.Physical restoration treatment effect is better, but quantities is large, and cost is high.Chemical restoration mainly comprises the methods such as chemical modifying, surfactant washing and organic improvement.Its advantage is simple to operate, and cost is lower, and shortcoming is fundamentally not remove heavy metal, and the modifying agent adding has the risk of secondary pollution.Biological restoration comprises animal reparation, phytoremediation, microorganism reparation and microorganism and plant combined reparation etc.Plant extract is the restorative procedure of current most study, this method is to utilize the plant of super accumulation heavy metal to absorb the heavy metal ion in soil, heavy metal ion is enriched in a large number to the over-ground part of plant, by gathering in or pulling out, the over-ground part of plant reduces and removal heavy metal-polluted soil.The super accumulation plant of having reported has penny cress genus, Herba avenae fatuae, Indian mustard, rape, alfalfa, false indigo etc., is mainly used at present removing Pb, Cd etc. in contaminated soil.The advantage of this method is to repair thoroughly, there is no secondary pollution, and shortcoming is the shortcomings such as most of super accumulation plant exists poor growth, biomass is little, Heavy Metal Accumulation amount is not high, and repair time is long.Plant-microorganism united restoration method is to study in recent years more a kind of new restorative procedure both at home and abroad, and the method is to utilize the efficiency of the super accumulation of microbial augmentation phytoremediation, thoroughly removes the heavy metal in contaminated soil.In soil-microorganism-plant symbiosis environment, microorganism can be converted into the secretory product of soil with organic matter and root system of plant small molecules and utilize for self, simultaneously, the heavy metal of the organic acids such as the siderophore that microorganism produces in self metabolic process, indolylacetic acid (IAA), oxalic acid in can activating soil, the available state of heavy metal is increased, and microorganism also has very strong redox ability, can reduce to iron and manganese oxides, discharge heavy metal, thereby improve super accumulation plant to the enrichment of heavy metal and transhipment.In addition, microorganism also can be synthesized IAA, siderophore, and the effect such as acc deaminase and fixed nitrogen, phosphorus decomposing and potassium decomposing Promoting plant growth, increases plant-growth amount, thereby accelerates the removal of heavy metal.As the people such as Whiting access containing in the soil of heavy metal Zn of sterilizing by blue colza of holding back of surface sterilization, access has the bacterium of Zn resistance again, it is 2 times of plant that do not connect bacterium that the plant shoot of result access bacterium divides the amount of accumulating heavy metal Zn, the total Zn amount of accumulating in over-ground part and root, access bacterium for not accessing 4 times of schizomycete semi-invariant.
Nitrogen is one of macroelement essential in crop growth process, to the contribution of crop ultimate capacity, is 40%-50%.The heavy metal of high density can make plant lose root nodule tubercle ability, thereby suppresses the symbiotic nitrogen fixation process in soil completely, easily causes plant nitrogen stress.If rely on merely nitrogen fertilizer application, not only uneconomical, and easily cause soil compaction, excessive the using of nitrogenous fertilizer can be caused the pollution of underground water nitrite and the eutrophication of rivers and lakes.The nitrogen of azotobacter agent in can fixed air, is transformed into ammonium nitrogen by nitrogen, and to compare use range wider with root nodule bacterium, produces and use all more for convenience.But all there is poor growth, preventing from heavy metal growth and the lower shortcoming of nitrogen fixing capacity in most of wild vinelandii, can not meet the needs that contaminated soil is repaired.For this reason, adopt various methodologies to break the eubolism of bacterial classification, make it to produce needed target meta-bolites (as proteolytic enzyme), improve its resistance capacity etc., reach this object, major measure is exactly to carry out the seed selection of bacterial classification, as carries out physics and chemistry mutagenesis etc.
Summary of the invention
One of object of the present invention is to obtain by ultraviolet ray-plasma body complex mutation the acinetobacter calcoaceticus that a strain can be used for the spontaneous nitrogen fixing capacity of having of heavy metal pollution of soil environmental improvement and preventing from heavy metal energy for growth.
Two of object of the present invention is to provide microbiobacterial agent containing above-mentioned acinetobacter calcoaceticus and preparation method thereof;
Three of object of the present invention is to provide the application of above-mentioned acinetobacter calcoaceticus in heavy metal pollution of soil environmental improvement and soil ecology reparation.
Implementation procedure of the present invention is as follows:
An acinetobacter calcoaceticus with spontaneous nitrogen fixing capacity, its Classification And Nomenclature be acinetobacter calcoaceticus (
acinetobacter sp.) Ymu7-3, on June 29th, 2011, be deposited in (address:, deposit number is CGMCC No.5006, China Committee for Culture Collection of Microorganisms's common micro-organisms center No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica).
It is yellow that YMU7-3 bacterium colony in culture medium A is, circular protrusion, and neat in edge, periphery of bacterial colonies has transparent circle, G
-, aerobic or amphimicrobian, is elongated rod shape, 28 ~ 32 ℃ of its suitableeest growth temperatures, and the suitableeest pH value is 6.0 ~ 8.0; Its nitrogen fixing capacity is at 0.43 ~ 0.68nmol10
7cfu
-1h
-1, separate inorganic phosphorus ability and be respectively 236mg/l; Its ability of producing indolylacetic acid (IAA) is 33.25 ~ 45.62 μ g/ml; The ability (A/Ar) of producing siderophore is 0.206 ~ 0.63; The active size of its acc deaminase is 0.468 ~ 0.712 μ mol/ mg/h.
Described culture medium A consists of: yeast powder 5-10g, NaCl 2-5g, peptone 5-10g, FeSO
47H
2o 0.005-0.01g, Na
2moO
42H
2o 0.0025-0.005 g, distilled water 1000ml, p H 7.0 ~ 7.2.
The acinetobacter calcoaceticus with spontaneous nitrogen fixing capacity provided by the invention obtains by mutagenic breeding method, comprises the following steps:
1) using laboratory from the azotobacter acinetobacter calcoaceticus YSGD07 of plant rhizosphere screening as starting strain;
2) mutagenic and breeding
(1) prepare the single cell suspension of starting strain YSGD07
Starting strain YSGD07 is inoculated in liquid nutrient medium A, 28-32 ℃, 120-150rpm cultivates 12-16hrs, centrifugal, with stroke-physiological saline solution washing, is placed 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: yeast powder 5-10g, NaCl 2-5g, peptone 5-10g, FeSO
47H
2o 0.005-0.01g, Na
2moO
42H
2o 0.0025-0.005 g, distilled water 1000ml, p H 7.0 ~ 7.2;
(2) ultraviolet mutagenesis
The bacteria suspension of step (1) gained is regulated respectively to concentration to 10
5-10
7cFU/ml, gets 0.1ml and coats the Cd containing 400-800mg/L
2+upper without nitrogen solid medium B, carry out ultraviolet mutagenesis, the frequency of ultraviolet mutagenesis is 10-18W, irradiation distance is 25-50cm, irradiation time 5-10min; 28-32 ℃ of standing cultivation 5-7days, select 25-30 larger single bacterium colony, carrying out shaking flask sieves again, by Acetylene Reduction method, measure the nitrogenase activity of each bacterial strain, select the highest bacterial strain of 5-8 strain nitrogenase activity, make respectively again shaking flask and sieve again, select the bacterial strain YSGD07-Z of the high and good stability of a strain nitrogenase activity, make bacteria suspension for the mutagenesis of next step plasma body;
Described without nitrogen solid medium B component is: glucose 10-20g, KH
2pO
40.2g, MgSO
47H
2o 0.1-0.2g, NaCl 0.2g, CaSO
42H
2o 0.2g, CaCO
35g, CdCl
20.080-0.32g, agar 15-18g, distilled water 1000ml, p H 7.0 ~ 7.2;
The step that described shake flask fermentation sieves is again: a 25-30 first above-mentioned separation being obtained is inoculated in the above-mentioned liquid nutrient medium A of 100ml, cultivates 8-12hrs.Get in the Erlenmeyer flask that 5ml bacterium liquid is inoculated into the 250ml that 100ml liquid nutrient medium C is housed, 28 ℃, after 150rpm shaking table shaking culture 48hrs, change tampon into rubber plug, sealed membrane drips wax sealing, and syringe extracts the air of 10-15%, injects the acetylene gas of 10-15%, continue reaction, by Acetylene Reduction method, measure nitrogenase activity;
Described liquid nutrient medium C component is: glucose 10-20 g, KH
2pO
40.2-0.41 g, K
2hPO
40.3-0.52 g, CaCl
20.1-0.2 g, MgSO
47H
2o 0.1-0.2 g, FeSO
47H
2o 0.005-0.01g, Na
2moO
42H
2o 0.0025-0.005 g, distilled water 1000ml, p H 7.0 ~ 7.2;
(3) plasma body mutagenesis
By the YSGD07-Z bacterial strain of step (2) gained, make 10
5-10
7the bacteria suspension of CFU/ml, getting 0.1-0.2ml evenly coats in sterile petri dish, culture dish is put on the electrode below plasma, regulate the position of top electrode, make the distance between upper/lower electrode be controlled at 3-8mm left and right, 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.After mutagenesis, immediately with stroke-physiological saline solution or phosphoric acid salt wash-out, coat the Cd containing 400-800mg/L
2+upper without nitrogen solid medium B, then carry out shaking flask and sieve again, choose the strain bacterium YSGD07-Z3 that a strain nitrogenase activity is the highest, make bacteria suspension for next step mutagenesis; The same step of method (2) that described shaking flask is sieved again;
(4) the YSGD07-Z3 bacteria suspension viable count of step (3) gained is adjusted to 10
5-10
7cFU/ml, is cycled to repeat ultraviolet mutagenesis → plasma body mutagenesis 1-2 time, finally obtains strain tolerance heavy metal Cd and the highest bacterial strain Ymu7-3 of nitrogenase activity.
Acinetobacter calcoaceticus (
acinetobacter sp.) Ymu7-3 and acinetobacter calcoaceticus have high homology, the result of strain identification is as follows:
GGGGGGGGGGGGCTTACCATGCAGTCGACGCCCCGCAAGGGGAGTGGCAGACGGGTGAGTAACGCGTGGGAACATACCCTTTCCTGCGGAATAGCTCCGGGAAACTGGAATTAATACCGCATACGCCCTACGGGGGAAAGATTTATCGGGGAAGGATTGGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACCGGAGAAGATAATGACGGTATCCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGATATTTAAGTCAGGGGTGAAATCCCAGAGCTCAACTCTGGAACTGCCTTTGATACTGGGTATCTTGAGTATGGAAGAGGTAAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCTCTTGACATTCGGGGTTTGGGCAGTGGAGACATTGTCCTTCAGTTAGGCTGGCCCAGAACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGTTTAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCAGCATTTAGTTTGGCACTCTAGGGGACTGCCGTGATAGCCGGAGAGGAAGGTGGGGATGACGTTCAGTCTCATGGCCTTTACCGGCCTGGGCTACCACGTGCTACAATGGTGGTGAACGTGGCAGTAAACAGCGATTGTCAGCTATTCTCAGCATTCAGTCGATGTACTTTGCATCTGAGTGG。
The present invention also provides a kind of high-efficiency nitrogen-fixing microbial inoculum, according to nutrition carrier difference, can be liquid bacterial agent or solid fungicide.
The preparation of microbial inoculum comprises the following steps:
1) strain fermentation
1. one-level triangular flask liquid culture
By frozen Ymu7-3 quick-thawing under 37 ℃ of conditions, 28-32 ℃, shaking culture 15-20hrs are housed in the triangular flask of liquid nutrient medium A according to the inoculum size access of 0.5-1%;
Described culture medium A consists of: yeast powder 5-10g, NaCl 2-5g, peptone 5-10g, FeSO
47H
2o 0.005-0.01g, Na
2moO
42H
2o 0.0025-0.005 g, distilled water 1000ml, p H 7.0 ~ 7.2;
2. second order fermentation tank fermentation
1) the one-level nutrient solution of Ymu7-3 is accessed respectively according to the inoculum size of 5-10% in the fermentor tank that fermention medium E is housed, carry out fermentation culture, tank temperature 28-32 ℃, cultivates pH7-8, aerated culture 20-30 hrs;
Described substratum E consists of: sucrose 5-10g, starch 2-3g, dregs of beans 20-30g, KH
2pO
40.2-0.41 g, K
2hPO
40.3-0.52 g, CaCl
20.1-0.2 g, MgSO
47H
2o 0.1-0.2 g, Na
2moO
42H
2o 0.0025-0.005 g, water 1000ml, p H 7.0 ~ 7.2;
2) straw powder, turfy soil, rice chaff are pulverized with high speed disintegrator, cross 60 mesh sieves, the ratio that is 50-70:20-30:10-20 according to mass percent is evenly mixed, add again certain density Sodium orthomolybdate and ferrous sulfate, wherein Sodium orthomolybdate is 0.2 ~ 0.5g/Kg, and ferrous sulfate is 0.05 ~ 0.08g/Kg;
3) the Ymu7-3 fermented liquid obtaining in step 1) is squeezed into container for storing liquid and obtains liquid high-efficiency nitrogen-fixing microbial inoculum, by fermented liquid according to dosage and the step 2 of 50-100ml/kg) in the solid state substrate of gained mix, 28-32 ℃ of fermentation 5-7days, obtains solid fungicide.
Efficient azotobacter agent liquid bacterial agent provided by the invention soaks while root, nursery stage or vegetative period filling with root and adds in proportion while can be used for the seed soaking of super accumulation plant, transplanting, when soaking seed, plant soaks seed or plant root 2-5hrs with 50-200 diluent doubly, fill with root in nursery stage or vegetative period and adopt the dosage of 20-40ml/Kg soil to water diluent, fill with root 1-3 whole vegetative period; Solid-state microbial inoculum can be used as base manure and the use of topdressing, and using dosage is 5-20g/Kg soil, and in soil, the clearance of Cd has increased 50.4-178.7% than control group.
Advantage of the present invention and beneficial effect:
1) using plasma mutagenesis and the ultraviolet mutagenesis method of circular treatment repeatedly, has guaranteed the stability of mutant strain;
2) azotobacter providing has high nitrogen fixing capacity, can resist the Cd growth of higher concentration, can secrete indolylacetic acid, produce iron atom, has ACC desaminase activity etc., and stabilization characteristics of genetics goes down to posterity 14 times.Can promote the growth of super accumulation plant, improve coefficient of concentration and the transhipment coefficient of super accumulation plant;
3) microbial inoculum nutritional requirement of the present invention simple, easily cultivate, growth cycle is short, the large production that can carry out mass-producing;
4) microbial inoculum of the present invention can obviously promote the growth of super accumulation plant, increases the biomass of super tired massive planting thing, greatly improves heavy metal accumulation coefficient and the transhipment coefficient of super accumulation plant, can improve ecological environment of soil again, environmental friendliness and with low cost;
5) for developing the quick removal of heavy metal in plant growth-promoting rhizobacteria reinforced soil, provide new way, the improvement of Heavy-metal Polluted Environment and restoration of the ecosystem have been had great importance.
Accompanying drawing explanation
Fig. 1 is the dull and stereotyped photo of Ymu7-3 and stereoscan photograph;
Fig. 2 is that Ymu7-3 produces iron ion qualitative experiment photo;
Fig. 3 is Ymu7-3 mitotic stability;
Fig. 4 is that Ymu7-3 is containing Cd
2+growing state in liquid nutrient medium A;
Fig. 5 is that Indian mustard is at the biomass (dry weight) containing in Cd soil;
Fig. 6 is the content of Indian mustard over-ground part accumulation Cd;
Fig. 7 is the clearance of Indian mustard to Cd;
Fig. 8 is that alfalfa is at the biomass containing in Cd soil;
Fig. 9 is the content of alfalfa over-ground part accumulation Cd;
Figure 10 is the clearance of alfalfa to Cd;
Figure 11 is that rape is at the biomass containing in Cd soil;
Figure 12 is the content of rape over-ground part accumulation Cd;
Figure 13 is the clearance of rape to Cd.
Embodiment
According to the screening method of azotobacter strain provided by the invention, seed selection can tolerate the high azotobacter strain of nitrogenase activity of heavy metal cadmium, and the step of preparation is as follows:
1) using laboratory from the azotobacter acinetobacter calcoaceticus YSGD07 of plant rhizosphere screening as starting strain;
2) mutagenic and breeding
(1) prepare the single cell suspension of starting strain YSGD07
Starting strain YSGD07 is inoculated in liquid nutrient medium A, and 28 ℃, 150rpm cultivates 16hrs, centrifugal, with stroke-physiological saline solution washing, is placed 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: yeast powder 10g, NaCl 5g, peptone 10g, FeSO
47H
2o 0.005g, Na
2moO
42H
2o 0.0025g, distilled water 1000ml, p H 7.0 ~ 7.2.
(2) ultraviolet mutagenesis
The bacteria suspension of step (1) gained is regulated to concentration to 10
5between CFU/ml, get 0.1ml and coat the Cd containing 600mg/L
2+upper without nitrogen solid medium B, carry out ultraviolet mutagenesis, the frequency of ultraviolet mutagenesis is 18W, irradiation distance is 25cm, irradiation time 5min; 28 ℃ of standing cultivation 5days, select 25 larger bacterium colonies, carry out shaking flask and sieve again, by Acetylene Reduction method, measure the nitrogenase activity of each bacterial strain, select altogether the bacterial strain of the high and good stability of 8 strain nitrogenase activities, make bacteria suspension for the mutagenesis of next step plasma body;
Described without the contained component of nitrogen solid medium B is: glucose 20g, KH
2pO
40.41g, MgSO
47H
2o 0.2g, NaCl 0.2g, CaSO
42H
2o 0.2g, CaCO
35g, CdCl
20.32g, agar 18g, distilled water 1000ml, p H 7.0 ~ 7.2
The step that described shake flask fermentation sieves is again: the 50 strain azotobacters that first above-mentioned separation obtained are inoculated in the above-mentioned liquid nutrient medium A of 100ml, cultivates 12hrs.Respectively get in the Erlenmeyer flask that 5ml bacterium liquid is inoculated into the 250ml that 100ml liquid nutrient medium C is housed, 28 ℃, after 150rpm shaking table shaking culture 48hrs, change tampon into rubber plug, sealed membrane drips wax sealing, and syringe extracts 10 air, injects 10 acetylene gas, continue reaction, by Acetylene Reduction method, measure nitrogenase activity;
The described contained component of liquid nutrient medium C is: glucose 20 g, KH
2pO
40.41 g, K
2hPO
40.52g, CaCl
20.2 g, MgSO
47H
2o 0.1 g, FeSO
47H
2o 0.005, Na
2moO
42H
2o 0.0025 g, distilled water 1000ml, p H 7.0 ~ 7.2.
(3) plasma body mutagenesis
The bacterial strain of 8 strain nitrogenase activities of above-mentioned selection are high and good stability, makes 10
5the bacteria suspension of CFU/ml, getting 0.1ml evenly coats in sterile petri dish, culture dish is put on the electrode below plasma, regulating the distance between upper/lower electrode is 3mm, voltage is 3V, and electric current is 0.5A, utilizes atmospherical discharges, obtain uniform air dielectric barrier discharge plasma, be 3min discharge time.After mutagenesis, immediately with stroke-physiological saline solution or phosphoric acid salt wash-out, coat the Cd containing 600mg/L
2+upper without nitrogen solid medium B, then carry out shaking flask and sieve again, obtain the bacterial strain of the high and good stability of 5 strain nitrogenase activities, make bacteria suspension for next step mutagenesis.
(4) be cycled to repeat ultraviolet mutagenesis and plasma body mutagenesis 1-2 time, finally obtain strain tolerance heavy metal Cd and the high bacterial strain YMU7-3 of nitrogenase activity.
Fig. 1 is the dull and stereotyped photo of Ymu7-3 and stereoscan photograph, and Fig. 2 is that Ymu7-3 produces iron ion qualitative experiment photo, and Fig. 3 is Ymu7-3 mitotic stability, and table 1 is wild azotobacter and the contrast of mutagenic strain nitrogenase activity.
According to the method for preparing azotogen provided by the invention, preparation can tolerate the high azotogen of nitrogenase activity of heavy metal cadmium, and the step of preparation is as follows:
The preparation of microbial inoculum comprises the following steps:
1, strain fermentation
1. one-level triangular flask liquid culture
By frozen Ymu7-3 quick-thawing under 37 ℃ of conditions, according to 0.5% inoculum size access, be equipped with in the triangular flask of liquid nutrient medium A, 28 ℃, 150rpm shaking culture 18hrs;
Described liquid nutrient medium A consists of: described culture medium A consists of: yeast powder 10g, NaCl 5g, peptone 10g, FeSO
47H
2o 0.005g, Na
2moO
42H
2o 0.0025g, distilled water 1000ml, p H 7.0 ~ 7.2
2. second order fermentation tank fermentation
1) the one-level nutrient solution of Ymu7-3 is equipped with in the fermentor tank of liquid nutrient medium E according to 5% inoculum size access, carries out fermentation culture, 28 ℃ of tank temperature, cultivate pH7-8, aerated culture 28 hrs;
Described liquid nutrient medium E consists of: sucrose 10g, starch 3g, dregs of beans 30g, KH
2pO
40.41 g, K
2hPO
40.52 g, CaCl
20.2 g, MgSO
47H
2o 0.2 g, Na
2moO
42H
2o 0.005 g, water 1000ml, p H 7.0 ~ 7.2;
2) straw powder, turfy soil, rice chaff are pulverized with high speed disintegrator, cross 60 mesh sieves, the ratio that is 50:30:20 according to mass percent is evenly mixed, and after Sodium orthomolybdate and ferrous sulfate are dissolved, according to Sodium orthomolybdate, is that 0.3g/Kg ferrous sulfate is that the dosage of 0.05g/Kg adds and fully mixes;
3) the Ymu7-3 fermented liquid obtaining in step 1) is directly squeezed into container for storing liquid and obtains liquid high-efficiency nitrogen-fixing microbial inoculum, by the fermented liquid obtaining according to dosage and the step 2 of 100ml/kg) in the solid state substrate of gained mix, 28 ℃ of fermentation 5days.Colony counting method is counted YMU7-3 quantity in solid-state microbial inoculum can reach 6.2 * 10
9-1.1 * 10
11cFU/g.
Use embodiment 2 microbial inoculums at strengthening Indian mustard cadmium pollution soil repair, its concrete steps are:
Potted plant soil picks up from Shaanxi Province's Chang'an county agricultural land soil, for the total Cd content of examination soil, is 0.25mg/kg, and soil is established 4 and added Cd
2+level, is respectively 0,20,40,80mg/kg, the CdCl of respective amount
2wiring solution-forming, makes repeatedly to mix with heavy metal for examination soil standing balance 30days.Experiment is established respectively blank group, is added urea group and with microbial inoculum group.After emerging, every basin retains 3 strains., the liquid bacterial agent of above-mentioned preparation is diluted according to the dilution proportion of 1:100, the every basin of method that adopts root to fill with waters the bacterium liquid of 50ml dilution, every 3 weeks, water once, the group that does not add bacterium is watered the dead thalline of Isodose, and the concentration that adds urea group urea is 0.12g/L.Plant strain growth is gathered in the crops for 50 days afterwards, measures respectively overground part dry weight, HNO
3-HClO
4digestion, Cd concentration in each sample of aas determination, the overground part that calculates different treatment is inhaled the soil sanitation rate of Cd amount and each processing.
As visible in Fig. 4-7 result, with microbial inoculum with add urea and all can promote the growth of Indian mustard, with the dry weight of microbial inoculum group Indian mustard, than control group, increased 28.4-71.9%, and add urea group, than control group, increased 2.99-14.6%; With microbial inoculum with add urea and all can promote Indian mustard over-ground part to Cd
2+enrichment, with the Cd of microbial inoculum group Indian mustard accumulation
2+amount has increased 48.4-66.8% than control group, and add urea group, than control group, has only increased 3.7-13.6%; With microbial inoculum group Indian mustard to Cd in soil
2+clearance than control group, increased 98-178.7%, and add urea group, only than control group, increased 8.06-30.2%.Therefore, this microbial inoculum can significantly improve the biomass of Indian mustard and to Cd
2+enrichment, accelerate Cd in soil
2+removal.
Use solid fungicide provided by the invention for strengthening alfalfa cadmium pollution soil repair, its concrete steps are:
Potted plant soil is identical with embodiment 3, and soil is established 3 and added Cd
2+level, is respectively 0,20,50mg/kg, and blank group and with microbial inoculum group is established respectively in experiment.Solid fungicide and soil are added to soil and fully mix according to the consumption of 15g/kg soil, plantation alfalfa, after emerging, every basin retains 10 strains.Plant strain growth is gathered in the crops for 45 days afterwards, measures respectively overground part dry weight, HNO
3-HClO
4digestion, Cd concentration in each sample of aas determination, the suction Cd amount of the overground part of calculating different treatment and the soil sanitation rate of each processing.
As visible in Fig. 8-10 result, with microbial inoculum, can promote the growth of alfalfa, with the dry weight of microbial inoculum group alfalfa, than control group, increased 24.4-31.3%; Microbial inoculum can promote Indian mustard over-ground part to Cd
2+enrichment, with the Cd of microbial inoculum group alfalfa accumulation
2+amount has increased 49.2.4-57.8% than control group; With microbial inoculum group alfalfa to Cd in soil
2+purification rate than control group, increased 88.9-107%.
embodiment 5
Use solid fungicide provided by the invention for strengthening rape cadmium pollution soil repair, its concrete steps are:
Potted plant soil is identical with embodiment 3, and soil is established 3 and added Cd
2+level, is respectively 0,20,50mg/kg, and experiment is established respectively blank group and with microbial inoculum group.Rape is first grown seedlings in without Cd soil, select the Brassica campestris L seedling that growing way is identical and dilute immersion root with the liquid bacterial agent of above-mentioned 1:50, the dead bacterium immersion root of same dose for control group, every basin is transplanted 3 strains.Growth after three weeks again with the liquid bacterial agent dilution liquid irrigating root of 1:200, control group adds the dead microbial inoculum of same dosage, all the other conditions are identical with embodiment tri-, plant strain growth 45 days is results afterwards, Cd concentration in the dry weight of measuring respectively overground part and each sample.
Figure 11-13 result is visible, can obviously promote the growth of rape with microbial inoculum, with the dry weight of microbial inoculum rape, than control group, has increased 15.1-29.4%; With microbial inoculum, can promote rape over-ground part to Cd
2+enrichment, with the Cd of microbial inoculum group rape accumulation
2+amount has increased 27.5-34.5% than control group; With microbial inoculum group rape to Cd in soil
2+purification rate than control group, increased 50.4-72.8%.
Claims (5)
1. an acinetobacter calcoaceticus with spontaneous nitrogen fixing capacity, its Classification And Nomenclature be acinetobacter calcoaceticus (
acinetobacter sp.) Ymu7-3, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.5006.
2. the microbiobacterial agent that contains acinetobacter calcoaceticus described in claim 1.
3. the preparation method of microbiobacterial agent described in claim 2, comprises the following steps:
1) strain fermentation
1. one-level triangular flask liquid culture
By frozen Ymu7-3 quick-thawing under 37 ℃ of conditions, 28-32 ℃, shaking culture 15-20 hour are housed in the triangular flask of liquid nutrient medium A according to the inoculum size access of 0.5-1%;
Described liquid nutrient medium A consists of: yeast powder 5-10g, NaCl 2-5g, peptone 5-10g, FeSO
47H
2o 0.005-0.01g, Na
2moO
42H
2o 0.0025-0.005 g, distilled water 1000ml, p H 7.0 ~ 7.2;
2. second order fermentation tank fermentation
1) the one-level nutrient solution of Ymu7-3 is accessed respectively according to the inoculum size of 5-10% in the fermentor tank that fermention medium E is housed, carry out fermentation culture, tank temperature 28-32 ℃, pH 7-8, aerated culture 20-30 hour;
Described substratum E consists of: sucrose 5-10g, starch 2-3g, dregs of beans 20-30g, KH
2pO
40.2-0.41 g, K
2hPO
40.3-0.52 g, CaCl
20.1-0.2 g, MgSO
47H
2o 0.1-0.2 g, Na
2moO
42H
2o 0.0025-0.005 g, water 1000ml, pH 7.0 ~ 7.2;
2) straw powder, turfy soil, rice chaff were pulverized to 60 mesh sieves, the ratio that is 50-70:20-30:10-20 according to mass percent is evenly mixed, then adds Sodium orthomolybdate and ferrous sulfate, and wherein Sodium orthomolybdate is 0.2 ~ 0.5g/Kg, and ferrous sulfate is 0.05 ~ 0.08g/Kg;
3) the Ymu7-3 fermented liquid obtaining in step 1) is squeezed into container for storing liquid and obtains liquid high-efficiency nitrogen-fixing microbial inoculum, by fermented liquid according to dosage and the step 2 of 50-100ml/kg) in the solid state substrate of gained mix, 28-32 ℃ of fermentation 5-7 days, obtains solid fungicide.
4. the application of acinetobacter calcoaceticus in the environmental improvement of soil heavy metal cadmium ionic soil described in claim 1.
5. the application of acinetobacter calcoaceticus in the restoration of the ecosystem of soil heavy metal cadmium ionic soil described in claim 1.
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| CN109423489B (en) * | 2017-08-28 | 2021-11-23 | 江苏食品药品职业技术学院 | Continuous microorganism physical mutation breeding method |
| CN108774625B (en) * | 2017-12-29 | 2021-02-23 | 浙江双良商达环保有限公司 | Acinetobacter CL04 and application thereof in village and town sewage dephosphorization treatment |
| CN110343634B (en) * | 2019-06-04 | 2020-06-26 | 青岛农业大学 | Acinetobacter 3P29 culture medium and fermentation method |
| CN113403224B (en) * | 2021-05-28 | 2023-05-26 | 华南农业大学 | Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof |
| CN113337445B (en) * | 2021-07-06 | 2022-07-05 | 福建省农业科学院农业生物资源研究所 | Gemonad RF4 and application thereof |
| CN114250177B (en) * | 2021-12-21 | 2022-05-24 | 东北林业大学 | Acinetobacter and application thereof in improving stress resistance of plants |
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| CN101709282A (en) * | 2009-12-23 | 2010-05-19 | 河北科技大学 | Organophosphorus pesticide degrading bacterium and method for producing microbial inoculum thereof |
| CN101921710A (en) * | 2010-08-24 | 2010-12-22 | 河北省环境科学研究院 | Repairing agent for microbes in water bodies of excessive culture zones |
| CN101974448A (en) * | 2010-09-07 | 2011-02-16 | 新疆农业科学院微生物应用研究所 | Acinetobacter and application thereof to biological treatment of heavy metal ions |
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| CN101709282A (en) * | 2009-12-23 | 2010-05-19 | 河北科技大学 | Organophosphorus pesticide degrading bacterium and method for producing microbial inoculum thereof |
| CN101921710A (en) * | 2010-08-24 | 2010-12-22 | 河北省环境科学研究院 | Repairing agent for microbes in water bodies of excessive culture zones |
| CN101974448A (en) * | 2010-09-07 | 2011-02-16 | 新疆农业科学院微生物应用研究所 | Acinetobacter and application thereof to biological treatment of heavy metal ions |
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