CN102598909A - Method for regulating activity of soil matrix enzyme with Bacillus thuringiensis fermentation liquid - Google Patents
Method for regulating activity of soil matrix enzyme with Bacillus thuringiensis fermentation liquid Download PDFInfo
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Abstract
The invention discloses a method for regulating activity of a soil matrix enzyme with Bacillus thuringiensis fermentation liquid, which comprises the following steps: adding 30g of lawn soil into 6.5cm culture dishes, respectively adding 150mul compost microbial fermentation filtrates of different concentrations into the soil, adding an aseptic liquid culture medium into a control group, uniformly spreading 50 full Festuca arundinacea seeds in each culture dish, planting at room temperature, and measuring indicators after seed germination. Experimental results show that after the Bacillus thuringiensis fermentation liquid is added into the lawn soil matrix, the activity of the soil matrix enzyme can be significantly improved, meanwhile, the stress resistance of lawn plants is enhanced, and the growth of lawn plants is promoted.
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 method that adopts the bacillus thuringiensis zymotic fluid to regulate the soil matrix enzymic activity of saying so more specifically.
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 the influence that different compost microbe microbial inoculums is lived to the lawn soil enzyme; 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 adopting method that the bacillus thuringiensis ferment filtrate is used for regulating the tall fescue turf soil enzyme still for seeing bibliographical information.
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.Adopt the bacillus thuringiensis zymotic fluid to regulate the method for soil matrix enzymic activity through research, for the planting of lawn plant provides foundation.
For realizing above-mentioned purpose, the present invention provides following technical scheme:
A kind of method that adopts the bacillus thuringiensis zymotic fluid to regulate the soil matrix enzymic activity 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; 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; 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;
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.
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
Actinomycetes are used in experiment, bacillus subtilis, and bacillus thuringiensis separates the consumer garbage compost of the Xiao Dian garbage compost treatment plant from Tianjin; 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, and 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.
1.2 experimental technique:
Isolated bacterial classification purifying was cultivated for 2 generations; Get activated spawn, insert and be equipped with in the 250 mL triangular flasks of 50mL liquid nutrient medium, 180 r/min thermophilics are cultivated; Select for use the 600nm wavelength to carry out turbidimetric assay; OD value with bacteria suspension is an ordinate, and incubation time is an abscissa, draws the microbial growth curve.Obtain microbial inoculum according to growth curve cultured strain under suitable temperature and time.
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 the about 5cm of lawn plant growth, in soil, add 1ml different microorganisms microbial inoculum respectively, do not avoid the nutrition interference in the liquid nutrient medium; Control group adopts aseptic liquid nutrient medium (the actinomycetes medium is the Gause I medium, and bacillus subtilis and bacillus thuringiensis medium are beef-protein medium, and Gu is established two groups of contrasts and compared respectively); Room temperature plantation, lawn were cultivated after 60 days, measured the growth indexes (ground biomass of turfgrass; Underground biomass; Plant height, chlorophyll), and measure soil enzyme activities.
1.2.1 the assay method of lawn plant growth indexes
The every 10d in back that germinates measures 1 plant height, cradles behind the sowing 70d, measures fresh weight, dry weight, root dry weight and chlorophyll on the ground.Method is: use the dissection scissors that lawn plant is cut at root, earlier it is carried out the mensuration of fresh weight with electronic balance, then the lawn plant acrial part is placed the 1h that completes under 105 ℃ of conditions of baking oven earlier, dry to constant weight for 80 ℃, measure the dry weight of overground part.Clean the soil in each plastic cup, then root is placed the 1h that completes under 105 ℃ of conditions of baking oven, dry to constant weight for 80 ℃, measure the dry weight of root.
Measuring chlorophyll content (Zhang Zhiliang etc., 2003; )
Get the 0.2g plant leaf blade in mortar, add a small amount of quartz sand and a little 80% acetone, fully grind, homogenate is changed in the 15ml centrifuge tube; And with an amount of 80% washing with acetone alms bowl body, change in the lump in the centrifuge tube, be settled to 10mL with 80% acetone; Centrifugal 4000r/min, 10min abandons deposition after centrifugal; Get supernatant and be transferred in the test tube, as contrast, be determined at the absorbance of 663nm, 645nm wavelength respectively with ultraviolet specrophotometer with 80% acetone.And according to formula calculating chlorophyll content.
Chl?a=12.7×A
663-2.59×A
645 Chl?b=22.9×A
645-4.67×A
663
1.2.2 soil enzyme activities assay method
The mensuration of invertase adopts sodium thiosulfate titration (Guan Songyin, 1986)
Step: get the 10g soil specimen in the 100ml triangular flask, with 1.5ml O for toluene 15min.Add 10ml20% sucrose solution and 10mlpH5.5 acetate-phosphate buffer, be placed on after shaking up in 37 ℃ of insulating boxs and cultivate 24h.After cultivating end, add 50ml distilled water, sway the back and filter.Get 20ml and filtrate in the 100ml triangular flask, add 10ml film solution, boiling water bath 10min adds 3ml33% liquor kalii iodide and 4ml sulfuric acid after being cooled to room temperature again.Use the sodium thiosulfate titration then, before add starch indicator to terminal, titration disappears to blue again.Replacing soil matrix with water is contrast.Sucrase active is represented with the contrast and the difference of the sodium thiosulfate titration milliliter number of experiment.
The mensuration of urase adopts colorimetric method
Step: get the air-dry soil sample of 5g, place the 50ml triangular flask, add 1ml toluene.Add 10ml 10% urea liquid and 20mlpH6.7 citrate buffer behind the 15min.Shake up the back and in 37 ℃ of insulating boxs, cultivate 24h.Get 3ml filtrating after the filtration and inject the 50ml volumetric flask, adding distil water adds 4ml sodium phenate solution and 3ml liquor natrii hypochloritis again to 20ml then,, develops the color constant volume behind the 20min with adding with shaking up.On the inherent spectrophotometer of 1h in wavelength 578nm place colorimetric.Urease activity with 24h after NH in the 1g soil
3The milligram numerical table of-N shows.
The NH that a-checks in from calibration curve in the formula
3-N milligram is counted the coefficient that 2-is converted into 1g soil
Catalase adopts permanganimetric method
Step: get the air-dry soil of 2g, place the 100ml triangular flask, and inject 40ml distilled water and 5ml 0.3% hydrogenperoxide steam generator.Vibration 20min.Add 5ml sulfuric acid, again suspension in the bottle is filtered.Draw 25ml filtrating then, with permanganate titration to rose pink terminal point.
Be used for the potassium permanganate amount (milliliter number) that titration soil filtrating consumed and be B, be used for the original potassium permanganate amount that the hydrogen peroxide mixed liquor consumed of titration 25ml (milliliter number) and be A.
The * T of catalase activity=(A-B).
T is the corrected value of permanganate titration degree in the formula.
The polyphenol oxidase enzymatic determination adopts the pyrogallol colorimetric method
Step: get the air-dry soil sample of 1g, place the 50ml triangular flask, add 10ml 1% pyrogallol solution, be placed on after swaying in 30 ℃ of insulating boxs and cultivate 2h.Add 4ml pH4.5 citric acid-phosphate buffer after the taking-up, add the 35ml ether again, firmly sway for several times, extraction 30min.The plain painted ether of purple nutgall that will contain dissolving carries out colorimetric under 430nm.Polyphenol oxidase activity: the milligram numerical table plain with purple nutgall in the 1g soil behind the 2h shows.
What peroxidase adopted is the pyrogallol colorimetric method
Step: get 1g soil and place the 50ml triangular flask, inject 10ml 1% pyrogallol solution and 2ml 0.5% hydrogenperoxide steam generator then.The vibration back is by measuring polyphenol oxidase step extraction colorimetric.
Peroxidase activity, behind 2h, the plain milligram of the purple nutgall that generates in 1g soil numerical table shows.
1.3 statistical analysis
Data analysis adopts Excel 2003 and SPSS 17.0 statistical analysis softwares to analyze.
1.4 development results
1.4.1 the compost microbe microbial inoculum is to the influence of Festuca Arundinacea plant height
Table 1 compost microbe microbial inoculum to the influence of Festuca Arundinacea plant height (centimetre)
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
The compost microbe microbial inoculum all has facilitation significantly to the plant height of lawn plant Festuca Arundinacea; At the seed germination initial stage; Different compost microbe microbial inoculums are to the not significantly influence (1) of plant height of Festuca Arundinacea, and at after planting 30 days, microbial bacterial agent began to manifest to the facilitation of Festuca Arundinacea plant height; Festuca Arundinacea plant height with the bacillus thuringiensis microbial inoculum is handled is compared according to having increased by 29.1%; Cultivated when cradling in 60 days to the lawn, the experimental group of handling with the compost microbe microbial inoculum is still remarkable to the facilitation of Festuca Arundinacea plant height, and comparison is according to having increased by 31.8% respectively.
1.4.2 the compost microbe microbial inoculum is to the influence of Festuca Arundinacea biomass
The different compost microbe microbial inoculums of table 2 are to the influence (gram/basin) of Festuca Arundinacea biomass
Can know that by table 2 the underground dry weight of experimental group that different compost microbe microbial inoculums are handled, ground fresh weight are compared with contrast all to have significantly with the ground dry weight to be increased.The underground dry weight of experimental group, ground dry weight with the bacillus thuringiensis microbial inoculum is handled are compared respectively according to having increased by 60.0%, 31.8%.In lawn matrix, add different compost microbe microbial inoculums for the root/shoot ratio of lawn plant Festuca Arundinacea, fresh and dried ratio not significantly facilitation (
p>0.05).
1.4.3 the compost microbe microbial inoculum is to the influence of soil urease liveness
The different compost microbe microbial inoculums of table 3 are to the influence of soil enzyme activities
Can be found out that by Fig. 3 with the experimental group that the compost microbe microbial inoculum is handled, urease activity is significantly higher than control group, especially adds the experimental group of bacillus thuringiensis, urease activity is compared with contrast also has remarkable increase, is respectively 2.77 times and 2.45 times of contrast.
1.4.4 the compost microbe microbial inoculum is to the active influence of soil saccharase
Invertase is present in all soil; Can resolve into the glucose and the fructose that can be absorbed by plant and edaphon to HMW sucrose molecule in the soil; For the soil organisms body provides the abundant energy; The invertase activity of soil, with the humus in the soil, the content of water-soluble organic matter and clay and microbial numbers and activity thereof are proportionate.In neutral calcium soil, the activity of invertase is the highest, is that neutral mold sandy soil and acid soil are lower secondly.The sucrase active of soil has reflected the rule of soil organic carbon accumulation with decomposition and inversion.Can find out that therefrom different compost microbe microbial inoculums all has facilitation significantly to the activity of soil saccharase, has added the experimental group of bacillus thuringiensis, sucrase active is compared with control group has increased by 08.7% respectively.
1.4.5 the compost microbe microbial inoculum is to the influence of soil polyphenol oxidase activity
Polyphenol oxidase is participated in is the process that aromatic organic compounds in the soil is converted into the humus component, polyphenol oxidase can enzymatic one, two and trihydric phenol be oxidized to corresponding quinone.Research shows that certain micro-organisms forms the ability of dark-coloured humic substance, depends on whether they contain phenol type oxidase, and in general, the polyphenol oxidase activity of peat soil is higher than mineral soil significantly.The activity of polyphenol oxidase and the humification degree of soil humic substances are to be negative correlation.Therefore, the activity of soil polyphenol oxidase can be explained the humification process of soil to a certain extent.In this research, the experimental group polyphenol oxidase activity that adds the compost microbe microbial inoculum is significantly higher than control group, is respectively 20.6 times, 7.7 times and 8.4 times of contrast, and this explanation adds compost microbe and quickened soil detritus degree to a certain extent.
1.4.6 the compost microbe microbial inoculum is to the influence of soil peroxidase activity
Peroxidase has important function in the forming process of soil humus; Because the activity of microorganism has formed hydrogen peroxide with some oxidasic effect, the peroxidase in the soil utilizes the oxygen in these oxides to come organic matter such as phenol, amine and some heterocyclic compound etc. in the oxidation soil in soil.The power of peroxidase activity and humus forming process is proportionate.Different compost microbe microbial inoculums have obvious facilitation to the increase of soil peroxidase activity, and the experimental group peroxidase activity that adds the bacillus thuringiensis microbial inoculum is 4.3 times of contrast.
2 development conclusions
Soil enzyme is the important composition of estimating soil quality; Main live body or residual body from edaphon, plant and animal; Participation comprises the natural world material circulation of soil biochemistry process, in the forming process of generation, growth and the soil fertility of soil, plays an important role.This research is that matrix is set up the turf establishment system with soil; Respectively to wherein adding the bacillus thuringiensis microbial bacterial agent; The result shows that adding the compost microbe microbial inoculum all has humidification significantly for soil urease, invertase, catalase, polyphenol oxidase and peroxidase activity.Adding compost microbe impels urease activity to strengthen; Satisfied the needed ammonia nitrogen nutritive element of plant itself; Help promoting the utilization of plant to urea and nitrogen; The compost microbe microbial inoculum can make the soil saccharase increased activity, possibly be because the carbon in the microbial metabolic products is that invertase provides more enzymatic matrix, causes thereby accelerated the organic conversion of macromolecule.In soil, add the compost microbe microbial inoculum, help improving the soil microbiology activity, promote the conversion of soil nutrient, thereby improve catalatic activity.In addition; The adding compost microbe also has obvious facilitation for the growth indexes of lawn plant Festuca Arundinacea; The plant height of Festuca Arundinacea, biomass and chlorophyll content have been compared with contrast significantly to be increased; This has explained that also adding compost microbe microbial inoculum has improved soil enzyme activities, has changed soil quality to a certain extent, thereby has promoted the growth of lawn plant.
Description of drawings:
Fig. 1 is the growth curve of bacillus thuringiensis;
Fig. 2 is a bacillus thuringiensis bacterium colony photo;
Fig. 3 is the urase calibration curve;
The different compost microbe microbial inoculums of Fig. 4 are to the influence of soil urease liveness;
The different compost microbe microbial inoculums of Fig. 5 are to the active influence of soil saccharase;
The different compost microbe microbial inoculums of Fig. 6 are to the influence of soil catalase activity;
Fig. 7 polyphenol oxidase calibration curve;
The different compost microbe microbial inoculums of Fig. 8 are to the influence of soil peroxidase activity.
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; 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;
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.
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; Insert respectively and be 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;
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.
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 bacillus thuringiensis zymotic fluid is regulated the method for soil matrix enzymic activity, 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; 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; 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;
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.
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| CN115868506A (en) * | 2022-11-18 | 2023-03-31 | 山东农业大学 | Application of bacillus thuringiensis in soil phosphorus dissolution, plant growth promotion and soil enzyme system metabolism regulation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105543331A (en) * | 2016-01-21 | 2016-05-04 | 苏州科铭生物技术有限公司 | Soil peroxidase activity test kit and method thereof |
| CN115868506A (en) * | 2022-11-18 | 2023-03-31 | 山东农业大学 | Application of bacillus thuringiensis in soil phosphorus dissolution, plant growth promotion and soil enzyme system metabolism regulation |
| CN115868506B (en) * | 2022-11-18 | 2024-01-02 | 山东农业大学 | Application of bacillus thuringiensis in dissolving phosphorus in soil, promoting plant growth and regulating metabolism of soil enzyme system |
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