CN102598977B - Method for improving chromium resistance and cadmium resistance of ryegrass with compost fermentation liquid - Google Patents
Method for improving chromium resistance and cadmium resistance of ryegrass with compost fermentation liquid Download PDFInfo
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- CN102598977B CN102598977B CN 201210051928 CN201210051928A CN102598977B CN 102598977 B CN102598977 B CN 102598977B CN 201210051928 CN201210051928 CN 201210051928 CN 201210051928 A CN201210051928 A CN 201210051928A CN 102598977 B CN102598977 B CN 102598977B
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Abstract
The invention discloses a method for improving chromium resistance and cadmium resistance of ryegrass 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 0.5g of ryegrass 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 lawn plant growth is promoted, and the chromium resistance and cadmium resistance of ryegrass are improved.
Description
Technical field
The invention belongs to environmental protection technical field, relate to the 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 perennial ryegrass to the method for Cr and Cd resistance capacity.
Background technology
Compostization (Composting) refers to having 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 compostization is referred to as compost (Compost).Composting process can reduce 40%-50% effectively with the volume of mixture, and can rely on heat that metabolism in the hot stage produces and kill 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 remove in composting process or reduced the toxicity in the urban solid garbage and made final product can be used to carry out recycling.The program of compost comprises preliminary treatment, fermenting raw materials and the post processing of raw material, and composting process is subjected to the influence of factors such as moisture, oxygen content, C/N ratio, temperature, pH and ventilation situation.
The essence of compost process is microorganism metabolism under optimum conditions, in the compost 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 by 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 accelerated 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 may 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 the 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 may cause adverse influence to environment, this is the major issue during compost is used always, from garbage compost, separate and extract useful microorganism fungus kind, being mixed with different microbial bacterial agents is inoculated in the turf establishment system, both can improve the utilization ratio of compost, simultaneously solved the environmental threat that compost heavy metal etc. may constitute again, embody the superiority of biologic product, meet the requirement of sustainable development, improve perennial ryegrass to the method for Cr and Cd resistance capacity by research compost microbial bacterial agent, purpose is in order to screen suitable microbial bacterial agent, and the quality and the resistance that improve lawn plant provide theoretical foundation.
Bacillus thuringiensis is the 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 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; crystal in insect bodies the midgut alkaline environment and the effect of protease under be degraded and form insecticidal activity albumen; result of study show these toxalbumin separately between effect or different albumen synergy insect is had in various degree toxic action; further strengthen the research of bacillus thuringiensis aspect control and control pest damage, realize that for the protection environment for human survival sustainable development is significant.Bacillus thuringiensis,Bt (Bacillus thuringiensis is called for short Bt) is current production rate maximum, the widest biological insecticides of use.About adopting the bacillus thuringiensis ferment filtrate to be used for improving the method for perennial ryegrass preventing from heavy metal ability 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 the external source heavy metal to the harmful effect of 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 perennial ryegrass to the method for Cr and Cd resistance capacity by research compost zymotic fluid, for the planting of the anti-heavy metal of lawn plant provides foundation.
For achieving the above object, the invention provides following technical scheme:
A kind of compost fermentation liquid improves the method for the anti-Cr of perennial ryegrass 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; Also can separate the consumer garbage compost from Tianjin Xiao Dian garbage compost treatment plant; 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 as follows:
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, get the lml mother liquor with liquid-transfering gun in triangular flask, 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 concentration; Drawing 0.1 ml concn with liquid-transfering gun respectively is 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 abscissa, draws the microbial growth curve; Obtain growth of microorganism to time of stationary phase according to growth curve, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate 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 viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um with bacterium liquid by 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 by the bacillus thuringiensis that grows at beef-protein medium as can be seen, 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 filtrates and 8 times of filtrates respectively, standby;
4) compost fermentation liquid improves perennial ryegrass to the method for Cr and Cd resistance capacity:
Be to add 200g oven dry soil in the disposal plastic cup of 7cm at diameter, and sowing ryegrass seed 0.5g, after waiting to sow 10d, on lawn plant, add 1ml compost fermentation liquid respectively, control group adds aseptic liquid nutrient medium, behind microbial inoculum growth 5d, in soil, add caddy and potassium bichromate solution respectively, experiment is 3 repetitions, totally 60 samples, indoor thermophilic continue to cultivate experimental session quantitative water supply every day, guarantee environment unanimity between each processing, treat to measure every index after it was grown 30 days;
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, from the bacterial classification of a purifying generation picking colony repeat again this process be two generations of purifying purifying wherein cultivate 2 and be commissioned to train to support and refer 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 the prepared bacillus thuringiensis Physiology and biochemistry character of consumer garbage compost and commercially available identical, so not preservation.
Qualification result about bacillus thuringiensis:
The picking bacterium colony similar to bacillus thuringiensis on beef-protein medium, discovery is circle at the bacterium colony that medium forms, white, and the edge is smooth, opaque, Gram is positive, and it is shaft-like that thalline is examined under a microscope ovalize, and gemma is oval, carry 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 Preliminary Identification.
The Physiology and biochemistry experimental result of bacterium
Be that the bacterial strain of bacillus thuringiensis is decided to be bacterial strain to be measured with Preliminary Identification, to its detection of carrying out the Physiology and biochemistry experiment, obtain 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 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 as follows:
1. materials and methods
1.1 experiment material
Experiment separates consumer garbage compost from Tianjin Xiao Dian garbage compost treatment plant with bacillus thuringiensis, lawn plant is selected perennial ryegrass, take from for examination soil that the degree of depth is the 5-15cm topsoil in the Tianjin Normal University campus, the soil texture is 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, select for use the 600nm wavelength to carry out turbidimetric assay, OD value with bacteria suspension is ordinate, incubation time is 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 viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um with bacterium liquid by 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 filtrates and 8 times of filtrates respectively, standby;
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), chromium concn is 5mg/kg(soil).
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.
1.2 experimental technique:
Be to add 200g oven dry soil in the disposal plastic cup of 7cm at diameter, sowing ryegrass seed 0.5g, after waiting to sow 10d, on lawn plant, add 1ml bacillus thuringiensis microbial inoculum respectively, control group adds aseptic liquid nutrient medium, behind microbial inoculum growth 5d, in soil, add caddy and potassium bichromate solution respectively, experiment is 3 repetitions, totally 60 samples, indoor thermophilic continue to cultivate experimental session quantitative water supply every day, guarantee environment unanimity between each processing, treat to measure every index after it was grown 30 days.
1.3 every index determining
1.3.1 the mensuration of lawn plant content of beary metal
With the lawn plant acrial part after cradling and clean under ground portion oven for drying, take by weighing lawn plant acrial part after the 0.2g oven dry and under ground portion sample respectively and put into small beaker through the pernitric acid acidifying, add 10ml nitric acid, be 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 precipitation dissolving in the beaker and transfer, 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 namely inoculates the plant height that began to measure lawn plant after the microbial inoculum in 5 days, measures 1 time every 10d later on, cradles behind the sowing 45d, measures on the ground and underground biomass, chlorophyll content concrete grammar (same 3.1.2.1).
1.3.3 statistical analysis
Data analysis adopts Excel 2003 and SPSS 17.0 statistical analysis softwares to analyze.
2 results and analysis
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: * represents in the table
p＜0.05, * * represents
p＜0.01, down together
The influence of lawn plant plant height under the 2 compost microbe external source heavy metal conditions
The influence of lawn plant plant height under the different compost microbe external source of the table 2 chromium condition (centimetre)
Annotate: * represents in the table
p＜0.05, * * represents
p＜0.01, down together
As shown in Table 2, under external source chromium condition, adding compost microbe microbial inoculum has facilitation significantly for the plant height growth of perennial ryegrass, after inserting microbial inoculum 15d, As time goes on, the plant height of the experimental group of adding bacillus thuringiensis microbial inoculum is compared with control group and has been increased by 9.9%.
The influence of lawn plant plant height under the different compost microbe external source of the table 3 cadmium condition (centimetre)
Annotate: * represents in the table
p＜0.05, * * represents
p＜0.01, down together
It is as shown in table 3 to the plant height influence of lawn plant to add different compost microbe microbial inoculums under external source cadmium condition, facilitation to the perennial ryegrass plant height namely appears in adding bacillus thuringiensis microbial inoculum after meeting the 5d of bacterium, As time goes on increased and 5.4% than contrast.
The influence of lawn plant chlorophyll content under the 3 compost microbe external source heavy metal conditions
The influence of lawn plant chlorophyll content under the different compost microbe external source of the table 4 chromium condition (milligram/gram fresh weight)
Annotate: * represents in the table
p＜0.05, * * represents
p＜0.01, down together
Under external source chromium condition, different compost microbe microbial inoculums are as shown in table 4 to the influence of lawn plant chlorophyll content, adding bacillus thuringiensis has facilitation for perennial ryegrass chlorophyll content a and total chlorophyll content, has increased by 15.2% and 13.7% than contrast respectively.
The influence of lawn plant biomass under the 4 compost microbe external source heavy metal conditions
The influence (gram/basin) of lawn plant biomass under the different compost microbe external source of the table 5 cadmium condition
Annotate: * represents in the table
p＜0.05, * * represents
p＜0.01, down together
As shown in Table 5, under external source cadmium condition, the underground dry weight of perennial ryegrass has improved 72.7% than contrast behind the adding bacillus thuringiensis, and fresh weight, ground dry weight, fresh and dried ratio and root/shoot ratio do not have significantly effect on the ground to perennial ryegrass.
3 development conclusions
In this research, different compost microbe microbial inoculums all shows inhibitory action significantly to the external source content of beary metal of lawn plant absorption.The compost microbe microbial inoculum also has certain facilitation for plant height, chlorophyll content, ground and the underground biomass etc. of lawn plant perennial ryegrass, utilize microorganism can improve plant to the resistance of heavy metal, reduce the infringement of heavy metal on plants, promote the growth of plant.This research provides explanation with reference to the accompanying drawings for utilizing the removal of microorganisms heavy metal in soil:
Fig. 1 is the growth curve of bacillus thuringiensis;
Fig. 2 is bacillus thuringiensis bacterium colony photo.
Embodiment
In order to explain enforcement of the present invention more fully, provide following preparation method's embodiment.These embodiments only are to explain rather than limit the scope of the invention.Wherein bacillus thuringiensis (
Bacillusthuringiensis) have commercially availablely, also can be obtained 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 adopts 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, get the lml mother liquor with liquid-transfering gun in triangular flask, 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 concentration; Drawing 0.1 ml concn with liquid-transfering gun respectively is 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, select for use the 600nm wavelength to carry out turbidimetric assay, OD value with bacteria suspension is ordinate, incubation time is 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 by the bacillus thuringiensis that grows at beef-protein medium as can be seen, 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 viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um with bacterium liquid by 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 filtrates and 8 times of filtrates respectively, standby;
Get a part of filtered fluid and be diluted to 4 times of filtrates and 8 times of filtrates respectively, standby;
4) compost fermentation liquid improves perennial ryegrass to the method for Cr and Cd resistance capacity:
Be to add 200g oven dry soil in the disposal plastic cup of 7cm at diameter, and sow ryegrass seed 0.5g respectively, after waiting to sow 10d, on lawn plant, add 1ml compost fermentation liquid respectively, control group adds aseptic liquid nutrient medium, behind microbial inoculum growth 5d, in soil, add caddy and potassium bichromate solution respectively, experiment is 3 repetitions, totally 60 samples, indoor thermophilic continue to cultivate experimental session quantitative water supply every day, guarantee environment unanimity between each processing, treat to measure every index after it was grown 30 days;
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, select for use the 600nm wavelength to carry out turbidimetric assay, OD value with bacteria suspension is ordinate, incubation time is 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 viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um with bacterium liquid by 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 filtrates and 8 times of filtrates respectively, standby;
Get a part of filtered fluid and be diluted to 4 times of filtrates and 8 times of filtrates respectively, standby;
4) compost fermentation liquid improves perennial ryegrass to the method for Cr and Cd resistance capacity:
Be to add 200g oven dry soil in the disposal plastic cup of 7cm at diameter, and sowing ryegrass seed 0.5g, after waiting to sow 10d, on lawn plant, add 1ml compost fermentation liquid respectively, control group adds aseptic liquid nutrient medium, behind microbial inoculum growth 5d, in soil, add caddy and potassium bichromate solution respectively, experiment is 3 repetitions, totally 60 samples, indoor thermophilic continue to cultivate experimental session quantitative water supply every day, guarantee environment unanimity between each processing, treat to measure every index after it was grown 30 days;
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 describes in detail, being familiar with this technology personage can be well understood to, can carry out various variations and modification not breaking away under above-mentioned claim and the spirit, all foundations technical spirit of the present invention all belongs to the scope of technical solution of the present invention to any simple modification, equivalent variations and modification that above embodiment does.And the present invention also is not subject to the embodiment that gives an actual example in the specification.
Claims (1)
1. a compost fermentation liquid improves the method for the anti-Cr of perennial ryegrass and Cd ability, it is characterized in that being undertaken by following step:
1) separation of bacillus thuringiensis:
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, get the lml mother liquor with liquid-transfering gun in triangular flask, 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 concentration; Drawing 0.1 ml concn with liquid-transfering gun respectively is 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, select for use the 600nm wavelength to carry out turbidimetric assay, OD value with bacteria suspension is ordinate, incubation time is 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 viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um with bacterium liquid by 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 filtrates and 8 times of filtrates respectively, standby;
4) compost fermentation liquid improves perennial ryegrass to the method for Cr and Cd resistance capacity:
Be to add 200g oven dry soil in the disposal plastic cup of 7cm at diameter, and sowing ryegrass seed 0.5g, after waiting to sow 10d, on lawn plant, add 1ml compost fermentation liquid respectively, control group adds aseptic liquid nutrient medium, behind microbial inoculum growth 5d, in soil, add caddy and potassium bichromate solution respectively, experiment is 3 repetitions, totally 60 samples, indoor thermophilic continue to cultivate experimental session quantitative water supply every day, guarantee environment unanimity between each processing, treat to measure every index after it was grown 30 days;
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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| CN108690813A (en) * | 2018-05-24 | 2018-10-23 | 鲁东大学 | A kind of preparation method and applications of silicate-dissolving microbe microbial inoculum |
| CN109329015A (en) * | 2018-09-17 | 2019-02-15 | 南京农业大学 | A kind of method and its application using Pseudomonas fluorescence category bacterial strain Ca02 water planting rye grass |
| CN109315278A (en) * | 2018-09-17 | 2019-02-12 | 南京农业大学 | A kind of method and its application using Enterobacter bacterial strain Oj06 water planting rye grass |
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