CN102598978A - Method for improving chromium resistance and cadmium resistance of Festuca arundinacea with compost fermentation liquid - Google Patents
Method for improving chromium resistance and cadmium resistance of Festuca arundinacea with compost fermentation liquid Download PDFInfo
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- CN102598978A CN102598978A CN2012100519813A CN201210051981A CN102598978A CN 102598978 A CN102598978 A CN 102598978A CN 2012100519813 A CN2012100519813 A CN 2012100519813A CN 201210051981 A CN201210051981 A CN 201210051981A CN 102598978 A CN102598978 A CN 102598978A
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Abstract
The invention discloses a method for improving chromium resistance and cadmium resistance of Festuca arundinace with compost fermentation liquid, which comprises the following steps: adding 200g of oven-dried soil into disposable plastic cups of a diameter 7cm and seeding 0.8g of Festuca arundinace seeds, respectively adding microbial inoculums to lawn plants after seeding for 10 days, respectively adding cadmium chloride solution and potassium dichromate solution to soil, repeating the experiment for three times (60 samples in all), continuing culturing at room temperature while feeding water at a fixed amount every day during the experiment to ensure uniform environment among treatment chambers, and measuring indicators after growing for 30 days. The experimental results shows that after the Bacillus thuringiensis preparation is added, the underground dry weight and the root-top ratio of Festuca arundinace are increased by 137.5% and 102.9% respectively compared with the underground dry weight and the root-top ratio of the control, and the lawn plant growth is promoted, and the chromium resistance and cadmium resistance of Festuca arundinace are remarkably improved.
Description
Technical field
The invention belongs to environmental protection technical field, relate to application with the probiotics that extracts in the producing fertilizer from refuse in daily life.A kind of bacillus thuringiensis zymotic fluid that adopts of saying so more specifically improves the method for Festuca Arundinacea to Cr and Cd resistance.
Background technology
Composted (Composting) is meant to be had under the condition of control, makes organic waste under microorganism (being mainly bacterium) effect, degrade, and the process that organic matter is transformed to stable humus direction.Product after the composted is referred to as compost (Compost).Composting process can reduce 40%-50% effectively with the volume of mixture, and can rely on the heat that metabolism in the hot stage produces and kill the cause of disease material in the rubbish.Compost is not a new technology, but handles urban solid garbage in this way, meets the interests of environment, because in composting process, remove or reduced the toxicity in the urban solid garbage and made that final product can be used to utilize again.The program of compost comprises preliminary treatment, fermenting raw materials and the post processing of raw material, and composting process receives the influence of factors such as moisture, oxygen content, C/N ratio, temperature, pH and ventilation situation.
The essence of composted process is microorganism metabolism under optimum conditions; In the composted process; Solubilized organic substance in the house refuse sees through cells of microorganisms wall and cell membrane and is absorbed by Institute of Micro-biology; And microorganism changes into inorganic matter to the organic matter that absorbs through the vital movement process of self.Microorganism is playing the part of important role in composting process, closely related with compost cycle and compost quality.Therefore, the research of microorganism is quickened composting process to exploitation compost microbe resource in the compost, shortens the compost cycle, improves compost quality and all has great importance.
Microbe species is various, and occurring in nature only has the microorganism of only a few to obtain identifying that the kind of can survive, cultivating is few especially, is at most the l% of microorganism total amount.At present, carried out the research of series of theories and practice both at home and abroad for the microorganism in the compost.The environmental problem that possibly occur for the application compost has also caused people's attention; But how to address these problems and also lack careful deep research; At present; How to improve the quality of producing fertilizer from refuse in daily life, issuable environmental problem is the focus that the related scientific research personnel study concern always in the solution compost application process.The research that changes for the microorganism in composition of the microorganism in the compost and the composting process has many reports, but for the rare report of research that microorganism in the compost is separated, is applied to other matrix.
Consumer garbage compost as lawn matrix, not only can be avoided food chain, solved the problem of outlet of rubbish simultaneously again.But contain materials such as heavy metal in the garbage compost and possibly cause adverse influence environment; This is the major issue during compost is used always, from garbage compost, separates and extracts useful microorganism fungus kind, is mixed with different microbial bacterial agents and is inoculated in the turf establishment system; Both can improve the utilization ratio of compost; Solve simultaneously the environmental threat that compost heavy metal etc. possibly constitute again, embodied the superiority of biologic product, met the requirement of sustainable development; Through studying of the influence of different compost microbe microbial inoculums to lawn plant preventing from heavy metal ability; Purpose is in order to screen suitable microbial bacterial agent, improves quality and the resistance of lawn plant, and the quality of improving lawn soil matrix provides theoretical foundation.
Bacillus thuringiensis is present maximum of output in the world and the microorganism insecticide that is successfully used to prevent and treat farming, woods, pest of stored grain and some sanitary insect pests; Bacillus thuringiensis is when forming gemma; Can form a kind of parasporal crystal of being made up of insecticidal crystal protein, its main active insecticidal components is a parasporal crystal protein.Discover that this albumen has the specific killing effect to multiple agriculture and forestry injurious insect.Behind the insect's food-taking gemma mixed crystal; Be degraded under the effect of midgut alkaline environment and the protease of crystal in insect bodies and form insecticidal activity albumen; Result of study shows that synergy has toxic action in various degree to insect between independent effect of these toxalbumin or different albumen; Further strengthen the research of bacillus thuringiensis aspect control and Pest Control harm, realize that for the protection environment for human survival sustainable development is significant.Bacillus thuringiensis,Bt (Bacillus thuringiensis is called for short Bt) is the biological insecticides that current production rate is maximum, use is the widest.About adopt the bacillus thuringiensis ferment filtrate to be used for to improve Festuca Arundinacea to the method for Cr and Cd resistance capacity still for seeing bibliographical information.
The heavy metal pollution of soil problem is to influence agricultural product quality and safety always; The serious environmental problems that is detrimental to health; The biologically active of utilizing microorganism is to the affine absorption of heavy metal or be translated into the low toxicity product; Thereby reducing the heavy metal pollution degree, is the important channel of biological restoration heavy metal pollution.Microorganism can secrete metabolism products such as organic acid in process of growth, have dissolving heavy metal and the ability that contains the mineral of heavy metal.This research joins the external source heavy metal solution in the lawn soil matrix that contains different compost microbe microbial inoculums; Inquire into different compost microbes to alleviating of the harmful effect of external source heavy metal to lawn plant; For utilizing the microorganism Adsorption of Heavy Metal Ions, the reduction heavy metal provides reference to the infringement of lawn plant.
Summary of the invention
The bacillus thuringiensis that the present invention adopts (latin name:
Bacillus thuringiensis) culture presevation number: CFCC10206, depositary institution: China Forest microorganism fungus kind preservation administrative center externally can provide.
Physio-biochemical characteristics: cell is Gram-positive, and is shaft-like, and size is 1.0-1.2 * 3-5 μ m.Form half spore crystal, arabinose mannitol is not produced acid produce amylase, nitrate reduction is to nitrite.
Bacillus thuringiensis toxicity: to the people, animal low toxicity, the oral acute LD of rat
50852.7-856.7 milligram/kilogram is to low toxicities such as poultry, birds, fish, poultrys.
The bacillus thuringiensis ferment filtrate that the present invention will be mixed with variable concentrations is inoculated in the turf establishment system.Improve the method for Festuca Arundinacea through research compost zymotic fluid, for the planting of lawn plant provides foundation to Cr and Cd resistance capacity.
For realizing above-mentioned purpose, the present invention provides following technical scheme:
A kind of compost fermentation liquid improves the method for anti-Cr of Festuca Arundinacea and Cd ability, it is characterized in that being undertaken by following step:
(1)The separation of bacillus thuringiensis: bacillus thuringiensis itself has commercially available; The consumer garbage compost that also can separate Xiao Dian garbage compost treatment plant from Tianjin; The present invention is identical with commercially available bacillus thuringiensis from the resulting bacillus thuringiensis Physiology and biochemistry of consumer garbage compost character, and the method for separation is following:
1) fresh compost sample is taken by weighing 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration; Make in the sample microorganism fully and be uniformly distributed in the liquid; In triangular flask, get the lml mother liquor with liquid-transfering gun, add abundant mixing in the Boiling tube that fills the 9ml sterile water, this is l0
-1The dilute solution of concentration according to said method is diluted to l0 respectively
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several kinds of concentration; Use liquid-transfering gun to draw 0.1 ml concn respectively and be l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on the beef extract-peptone solid plate medium, and each dilution factor triplicate, sterile water are blank; With aseptic glass slicker bacteria suspension is evenly spread upon on the whole beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat board that will contain the beef extract-peptone solid culture medium is inverted in 37 ℃ of constant incubators to be cultivated 1 day;
2) bacillus thuringiensis preparation of fermentation liquid:
A. isolated bacillus thuringiensis bacterial classification purifying was cultivated for 2 generations; Insert then 180 r/min37 ℃ cultivation is housed in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa, draws the microbial growth curve; According to the growth curve acquisition time of growth of microorganism to stationary phase, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid;
B. get purifying bacillus thuringiensis bacterial classification; Access is equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 8～12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation; The clump count of bacillus thuringiensis (individual) wherein
Conclusion: the clump count situation through the bacillus thuringiensis that on beef-protein medium, grows can find out that the bacillus thuringiensis clump count in the compost is higher than the soil contrast, because l0
-4In the soil under times concentration and l0
-5, l0
-6Concentration under bacterium colony number average in compost and the soil be less than 30, therefore can't carry out statistical counting.
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
Compost fermentation liquid improves the method for Festuca Arundinacea to Cr and Cd resistance capacity:
A cup body that with diameter is the disposal plastic cup of 7cm encases lucifuge with newspaper, in each plastic cup, adds 250g soil, sowing Festuca Arundinacea grass seeds 0.8g, and experiment is to repeat for 3 times; When treating lawn plant growth 5cm, Xiang Tuzhong adds the 1ml ferment filtrate, does not avoid the nutrition in the liquid nutrient medium to disturb; Control group adopts aseptic liquid nutrient medium (the bacillus thuringiensis medium is beef-protein medium, and Gu is established contrast and compared respectively), room temperature plantation; Cultivated after 60 days on the lawn, measures the growth indexes of turfgrass: ground biomass, underground biomass; Plant height, chlorophyll, and lawn plant content of beary metal;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
The prepared bacillus thuringiensis of the present invention with from consumer garbage compost prepared bacillus thuringiensis Physiology and biochemistry character and commercially available identical, so not preservation.
Qualification result about bacillus thuringiensis:
The picking bacterium colony similar with bacillus thuringiensis on beef-protein medium finds that the bacterium colony that on medium, forms is circle, white; The edge is smooth, and is opaque, and Gram is positive; It is shaft-like that thalline is examined under a microscope ovalize, and gemma is oval, carries out parasporal crystal dyeing with bromophenol blue and sarranine dye liquor; Observed red thalline under the mirror, colourless gemma and blue parasporal crystal, it is bacillus thuringiensis (with commercially available identical) for a Preliminary Identification.
The Physiology and biochemistry experimental result of bacterium
With Preliminary Identification is that the bacterial strain of bacillus thuringiensis is decided to be bacterial strain to be measured, to its detection of carrying out the Physiology and biochemistry experiment, obtains result such as following table:
The Physiology and biochemistry experimental result of bacterium
Bacterial strain to be measured has been carried out the Physiology and biochemistry test, shown in the result as above shows.Bacterial strain to be measured is a Gram-positive, the hydrogen peroxide enzyme positive, and the V.P reacting positive, glucose fermentation produces acid, and hydrolyzed starch, gelatin can both utilize citrate.
The distinguishing feature of bacterial strain to be measured is: bacterial strain to be measured produces acid to the azymic of D-wood sugar, and acid, not aerogenesis are produced in the azymic of D-mannitol.According to above experimental result, identify that further bacterial strain to be measured is bacillus thuringiensis (with commercially available identical).With the bacterial strain purification storage, use in order to subsequent experimental.
The more detailed test method of the present invention is following:
1. materials and methods
1.1 experiment material
Experiment separates the consumer garbage compost of the Xiao Dian garbage compost treatment plant from Tianjin with bacillus thuringiensis, and lawn plant is selected Festuca Arundinacea (Tall fescue), supplies examination soil to take from that the degree of depth is the 5-15cm topsoil in the Tianjin Normal University campus; The soil texture is a dauk; Its character is: pH 7.44, the content of organic matter 4.68%, full nitrogen 0.21%; Available phosphorus 22.03mgkg-1, saturation moisture content 0.58mlg-1.
The bacillus thuringiensis preparation of fermentation liquid:
A. isolated bacillus thuringiensis bacterial classification purifying was cultivated for 2 generations; Insert then and be equipped with in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; 180 r/min37 ℃ cultivation; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa; Draw the microbial growth curve, obtain the time that bacillus thuringiensis grows to stationary phase according to growth curve;
B. get purifying bacillus thuringiensis bacterial classification; Insert respectively and be equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 8～12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
This is tested used external source heavy metal and comes from caddy and potassium bichromate solution, and formulated by caddy and potassium bichromate solid respectively, its concentration is respectively, and cadmium concentration is 2mg/kg (soil), and chromium concn is 5mg/kg (soil).
1.2 experimental technique:
Compost fermentation liquid improves the method for Festuca Arundinacea to Cr and Cd resistance capacity:
A cup body that with diameter is the disposal plastic cup of 7cm encases lucifuge with newspaper, in each plastic cup, adds 250g soil, sowing Festuca Arundinacea grass seeds 0.8g, and experiment is to repeat for 3 times; When treating lawn plant growth 5cm, Xiang Tuzhong adds the 1ml ferment filtrate, does not avoid the nutrition in the liquid nutrient medium to disturb; Control group adopts aseptic liquid nutrient medium (the bacillus thuringiensis medium is beef-protein medium, and Gu is established contrast and compared respectively), room temperature plantation; Cultivated after 60 days on the lawn, measures the growth indexes of turfgrass: ground biomass, underground biomass; Plant height, chlorophyll, and measure the lawn plant content of beary metal.
At diameter is to add 200g oven dry soil in the disposal plastic cup of 7cm, and sowing tall fescue seed 0.8g after waiting to sow 10d, adds the various microbial inoculums of 1ml respectively on lawn plant; Control group adds aseptic liquid nutrient medium, behind microbial inoculum growth 5d, in soil, adds caddy and potassium bichromate solution respectively; Experiment is to repeat for 3 times, totally 60 samples, and indoor thermophilic continues to cultivate; Experimental session quantitative water supply every day guarantees that environment is consistent between each processing, treats to measure each item index after it was grown 30 days.
1.3 each item index determining
1.3.1 the mensuration of lawn plant content of beary metal
Lawn plant acrial part after cradling and the under ground portion of cleaning are used oven for drying; The lawn plant acrial part and the under ground portion sample that take by weighing respectively after 0.2g is dried are put into the small beaker through the pernitric acid acidifying, add 10ml nitric acid, are placed on the electric hot plate and heat; When the liquid in the beaker takes off beaker during near evaporate to dryness; Nitric acid with 1% is with the resolution of precipitate in the beaker and shift, constant volume, the acrial part constant volume is to 10 ml volumetric flasks, the under ground portion constant volume is to 50 ml volumetric flasks; Adopt heavy metal (Cr, Cd) content in the atomic absorption spectrometry solution.
1.3.2 the mensuration of lawn plant growth indexes
The back 15d that germinates promptly inoculates the plant height that began to measure lawn plant after the microbial inoculum in 5 days, whenever later on measures 1 time at a distance from 10d, cradles behind the sowing 45d, measures on the ground and underground biomass, chlorophyll content.
1.3.3 statistical analysis
Data analysis adopts Excel 2003 and SPSS 17.0 statistical analysis softwares to analyze.
2 development results
2.1 compost microbe is to the influence of lawn plant absorption external source heavy metal
The different compost microbe microbial inoculums of table 1 are to the influence (microgram/milligram) of lawn plant absorption external source chromium concn
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Different compost microbe microbial inoculums are as shown in table 1 to the influence of lawn plant absorption external source chromium; Add three kinds of microbial inoculums of bacillus thuringiensis lawn plant absorption external source chromium is had inhibitory action significantly; After adding the compost microbe microbial inoculum; Compare with contrast, the Festuca Arundinacea under ground portion has reduced by 67.3% respectively to the absorption of external source chromium after the adding bacillus thuringiensis microbial inoculum.
Table 2 compost microbe is to the influence (microgram/milligram) of lawn plant absorption external source cadmium concentration
Can know by table 2, add the bacillus thuringiensis microbial inoculum to lawn plant absorption external source cadmium all have minimizing effect significantly (
p<0.05); After adding the bacillus thuringiensis microbial inoculum; Compare with contrast, the Festuca Arundinacea under ground portion has reduced by 101.8% to the absorption of external source cadmium after the adding bacillus thuringiensis microbial inoculum, and the Festuca Arundinacea acrial part has reduced by 46.5% to the absorption of external source cadmium after the adding bacillus thuringiensis microbial inoculum.
2.2 the influence of lawn plant plant height under the compost microbe external source heavy metal condition
The influence of lawn plant plant height under the different compost microbe external source of the table 3 chromium condition (centimetre)
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Can know by table 3; Under external source chromium condition; Adding compost microbe microbial inoculum then is that according to having improved 8.8%, compost actinomycetes microbial inoculum does not have influence significantly to the Festuca Arundinacea plant height in the Festuca Arundinacea plant height comparison that adds the bacillus thuringiensis microbial inoculum for the influence of Festuca Arundinacea plant height.
The influence of lawn plant plant height under the different compost microbe external source of the table 4 cadmium condition (centimetre)
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
It is as shown in table 4 to the plant height influence of lawn plant under external source cadmium condition, to add different compost microbe microbial inoculums; Adding compost microbe microbial inoculum for the influence of Festuca Arundinacea plant height then is; 5d after adding the bacillus thuringiensis microbial inoculum, experimental group plant height all be significantly higher than control group (
p<0.05), after meeting bacterium 5d, 15d, 25d, 35d, the Festuca Arundinacea plant height comparison of inoculation bacillus thuringiensis microbial inoculum is shone and has been improved 9.8%, 13.7%, 11.9%, 5.6%,
2.3 the influence of lawn plant chlorophyll content under the compost microbe external source heavy metal condition
The influence (milligram/gram fresh weight) of lawn plant chlorophyll content under the different compost microbe external source of the table 5 chromium condition
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Under external source chromium condition; Different compost microbe microbial inoculums to the influence of lawn plant chlorophyll content shown in table .5; Add the bacillus thuringiensis microbial inoculum chlorophyll a of lawn plant Festuca Arundinacea and total chlorophyll content are had significantly facilitation (P < 0.05); Comparison is according to having improved 28.4%, 24.4%, and different compost microbe microbial inoculums then do not improve Festuca Arundinacea chlorophyll b content significantly.
The influence (milligram/gram fresh weight) of lawn plant chlorophyll content under the different compost microbe external source of the table 6 cadmium condition
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Can know that by table 6 under external source cadmium condition, Festuca Arundinacea chlorophyll a, chlorophyll b are compared with contrast also with total chlorophyll content after the adding compost microbe microbial inoculum increases to some extent, but does not reach degree significantly.
2.4 the influence of lawn plant biomass under the compost microbe external source heavy metal condition
The influence (gram/basin) of lawn plant biomass under the different compost microbe external source of the table 7 chromium condition
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Under the external source chromium condition, the underground dry weight comparison that adds Festuca Arundinacea behind the bacillus thuringiensis has been according to having improved 63.6%, and ground biomass and fresh and dried ratio and root/shoot ratio are not had obvious facilitation.
The influence (gram/basin) of lawn plant biomass under the different compost microbe external source of the table 8 cadmium condition
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Can know that by table 8 under external source cadmium condition, the underground dry weight of Festuca Arundinacea is compared with contrast with root/shoot ratio and improved 102.9% respectively after the adding bacillus thuringiensis microbial inoculum.
3 development conclusions
In this research, the compost microbe microbial inoculum has reduced the absorption of plant to heavy metal, has promoted the growth of plant, provides with reference to description of drawings for utilizing the removal of microorganisms heavy metal in soil:
Fig. 1 is the growth curve of bacillus thuringiensis;
Fig. 2 is a bacillus thuringiensis bacterium colony photo.
Embodiment
In order to explain enforcement of the present invention more fully, following preparation method's embodiment is provided.These embodiments only are to explain rather than limit scope of the present invention.Wherein bacillus thuringiensis has commercially availablely, also can obtain according to the method for embodiment 1, from the resulting bacillus thuringiensis Physiology and biochemistry of consumer garbage compost character with commercially available identical.The beef extract-peptone solid culture medium that is adopted has commercially available.Plating medium is: the medium of Gause I also has commercially available.
(1)The separation of bacillus thuringiensis:
1) fresh compost sample is taken by weighing 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration; Make in the sample microorganism fully and be uniformly distributed in the liquid; In triangular flask, get the lml mother liquor with liquid-transfering gun, add abundant mixing in the Boiling tube that fills the 9ml sterile water, this is l0
-1The dilute solution of concentration according to said method is diluted to l0 respectively
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several kinds of concentration; Use liquid-transfering gun to draw 0.1 ml concn respectively and be l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on the beef extract-peptone solid plate medium, and each dilution factor triplicate, sterile water are blank; With aseptic glass slicker bacteria suspension is evenly spread upon on the whole beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat board that will contain the beef extract-peptone solid culture medium is inverted in 37 ℃ of constant incubators to be cultivated 1 day;
2) bacillus thuringiensis preparation of fermentation liquid:
A. isolated bacillus thuringiensis bacterial classification purifying was cultivated for 2 generations; Insert then and be equipped with in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; 180 r/min37 ℃ cultivation; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa; Draw the microbial growth curve, obtain the time that bacillus thuringiensis grows to stationary phase according to growth curve;
The clump count of bacillus thuringiensis (individual) wherein
Conclusion: the clump count situation through the bacillus thuringiensis that on beef-protein medium, grows can find out that the bacillus thuringiensis clump count in the compost is higher than the soil contrast, because l0
-4In the soil under times concentration and l0
-5, l0
-6Concentration under bacterium colony number average in compost and the soil be less than 30, therefore can't carry out statistical counting.
B. get purifying bacillus thuringiensis bacterial classification; Access is equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 8～12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
4) compost fermentation liquid improves the method for Festuca Arundinacea to Cr and Cd resistance capacity:
A cup body that with diameter is the disposal plastic cup of 7cm encases lucifuge with newspaper, in each plastic cup, adds 250g soil, sows Festuca Arundinacea grass seeds 0.5g respectively, and experiment is to repeat for 3 times; When treating lawn plant growth 5cm, Xiang Tuzhong adds the 1ml ferment filtrate, does not avoid the nutrition in the liquid nutrient medium to disturb; Control group adopts aseptic liquid nutrient medium (the bacillus thuringiensis medium is beef-protein medium, and Gu is established contrast and compared respectively), room temperature plantation; Cultivated after 60 days on the lawn, measures the growth indexes of turfgrass: ground biomass, underground biomass; Plant height, chlorophyll, and measure the plant content of beary metal;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
1) bacillus thuringiensis preparation of fermentation liquid:
A. the bacillus thuringiensis bacterial classification purifying of buying was cultivated for 2 generations; Insert then and be equipped with in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; 180 r/min37 ℃ cultivation; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa; Draw the microbial growth curve, obtain the time that bacillus thuringiensis grows to stationary phase according to growth curve; Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
B. get purifying bacillus thuringiensis bacterial classification; Access is equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
4) compost fermentation liquid improves the method for Festuca Arundinacea to Cr and Cd resistance capacity:
A cup body that with diameter is the disposal plastic cup of 7cm encases lucifuge with newspaper, in each plastic cup, adds 250g soil, sows Festuca Arundinacea grass seeds 0.5g respectively, and experiment is to repeat for 3 times; When treating lawn plant growth 5cm, Xiang Tuzhong adds the 1ml ferment filtrate, does not avoid the nutrition in the liquid nutrient medium to disturb; Control group adopts aseptic liquid nutrient medium (the bacillus thuringiensis medium is beef-protein medium, and Gu is established contrast and compared respectively), room temperature plantation; Cultivated after 60 days on the lawn, measures the growth indexes of turfgrass: ground biomass, underground biomass; Plant height, chlorophyll, and measure soil enzyme activities.Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
The present invention is after the preferred embodiment that specifies; Being familiar with this technological personage can be well understood to; Do not break away from above-mentioned claim with spirit under can carry out various variations and modification; All foundations technical spirit of the present invention all belongs to the scope of technical scheme of the present invention to any simple modification, equivalent variations and modification that above embodiment did.And the embodiment that the present invention also is not subject in the specification to be given an actual example.
Claims (1)
1. a compost fermentation liquid improves the method for anti-Cr of Festuca Arundinacea and Cd ability, it is characterized in that being undertaken by following step:
(1)The separation of bacillus thuringiensis:
1) fresh compost sample is taken by weighing 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration; Make in the sample microorganism fully and be uniformly distributed in the liquid; In triangular flask, get the lml mother liquor with liquid-transfering gun, add abundant mixing in the Boiling tube that fills the 9ml sterile water, this is l0
-1The dilute solution of concentration according to said method is diluted to l0 respectively
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several kinds of concentration; Use liquid-transfering gun to draw 0.1 ml concn respectively and be l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on the beef extract-peptone solid plate medium, and each dilution factor triplicate, sterile water are blank; With aseptic glass slicker bacteria suspension is evenly spread upon on the whole beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat board that will contain the beef extract-peptone solid culture medium is inverted in 37 ℃ of constant incubators to be cultivated 1 day;
2) bacillus thuringiensis preparation of fermentation liquid:
A. isolated bacillus thuringiensis bacterial classification purifying was cultivated for 2 generations; Insert then and be equipped with in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; 180 r/min37 ℃ cultivation; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa; Draw the microbial growth curve, obtain the time that bacillus thuringiensis grows to stationary phase according to growth curve;
B. get purifying bacillus thuringiensis bacterial classification; Access is equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 8～12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid: get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
4) compost fermentation liquid improves the method for anti-Cr of Festuca Arundinacea and Cd ability: the cup body that with diameter is the disposal plastic cup of 7cm encases lucifuge with newspaper, in each plastic cup, adds 250g soil, sowing Festuca Arundinacea grass seeds 0.8g, and experiment is 3 repetitions; When treating lawn plant growth 5cm, Xiang Tuzhong adds the 1ml ferment filtrate, does not avoid the nutrition in the liquid nutrient medium to disturb; Control group adopts aseptic liquid nutrient medium (the bacillus thuringiensis medium is beef-protein medium, and Gu is established contrast and compared respectively), room temperature plantation; Cultivated after 60 days on the lawn, measures the growth indexes of turfgrass: ground biomass, underground biomass; Plant height, chlorophyll, lawn plant content of beary metal;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
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| CN105315998A (en) * | 2015-10-14 | 2016-02-10 | 济南益邦生物科技有限公司 | Passivating agent for reducing activity of Pb and Cr in alkaline soil and application thereof |
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| CN110408562A (en) * | 2019-07-12 | 2019-11-05 | 南京农业大学 | A kind of preparation method and application of cadmium pollution soil repair and the complex micro organism fungicide for promoting plant growth |
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| CN104892291A (en) * | 2015-05-07 | 2015-09-09 | 天津师范大学 | Application of chlorpyrifos for reinforcing turfgrass to extract house refuse compost heavy metal |
| CN105315998A (en) * | 2015-10-14 | 2016-02-10 | 济南益邦生物科技有限公司 | Passivating agent for reducing activity of Pb and Cr in alkaline soil and application thereof |
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| CN110144226A (en) * | 2019-06-13 | 2019-08-20 | 生态环境部南京环境科学研究所 | A kind of cogongrass growth-promoting media and preparation method thereof applied to mining soil reparation |
| CN110144226B (en) * | 2019-06-13 | 2021-04-09 | 生态环境部南京环境科学研究所 | Festuca arundinacea growth solution applied to mining area soil remediation and preparation method thereof |
| CN110408562A (en) * | 2019-07-12 | 2019-11-05 | 南京农业大学 | A kind of preparation method and application of cadmium pollution soil repair and the complex micro organism fungicide for promoting plant growth |
| CN110408562B (en) * | 2019-07-12 | 2021-05-04 | 南京农业大学 | Preparation method and application of compound microbial agent for repairing cadmium-polluted soil and promoting plant growth |
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