CN102626034A - Method by utilizing microorganisms and ryegrasses to collectively repair garbage compost heavy-metal seepage system - Google Patents
Method by utilizing microorganisms and ryegrasses to collectively repair garbage compost heavy-metal seepage system Download PDFInfo
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Abstract
The invention discloses a method by utilizing microorganisms and ryegrasses to collectively repair a garbage compost heavy-metal seepage system, which comprises the steps of filling 500g of air-dried garden soil into a polyvinyl chloride (PVC) pipe with diameter of 7.5cm and length of 25cm, filling 60g of a horizontal partition layer into the PVC pipe, and then filling 280g of compost into the PVC pipe; seeding about 0.6g of seeds of ryegrasses inside the PVC pipe, applying microorganism agents with different concentrations onto the surface of the compost after 30 days of the seeding, cutting grasses after 10 days, and measuring variation of content of each one of heavy metals such as cadium (Cd), chromium (Cr), copper (Cu), lead (Pb) and zinc (Zn). An experimental result shows that when the horizontal partition layer is paved above the soil and below the compost, migration of harmful substances and heavy metals from the garbage compost into the soil can be reduced, so that the time for lawn plants to absorb the heavy metals in the compost and the concentration of the heavy metals inside the lawn plants can be increased under the induction of chemical chelon or the synergy of the microorganisms, and the migration of the heavy metals into deep soil can be reduced.
Description
Technical field
The invention belongs to garbage compost in urban lawn build and plant technology Application for Field, relate to and adopt microorganism and perennial ryegrass to unite the method for reparation garbage compost heavy metal seepage system.
Background technology
In recent years, the problem of environmental pollution that is caused by house refuse has become one of focus of social extensive concern.Along with the raising day by day of China's living standards of the people, the domestic waste generation also increases thereupon.Consumer garbage compost as lawn matrix, not only can be avoided food chain, solved the problem of outlet of rubbish simultaneously again.Garbage compost has good physicochemical property, abundant nutrient, is to substitute the more satisfactory material of soil as lawn matrix.Populous in China, land resources is poor relatively, has more realistic meaning with garbage compost as lawn matrix.Some lawn plants can grow in the heavier matrix of heavy metal pollution, and fast growth, regeneration capacity are strong, have bigger using value in chemical method and collaborative the reparation in the garbage compost heavy metal of biological method.Therefore when garbage compost was as lawn matrix, its heavy metal can obtain the reparation of lawn plant again, had so not only increased the application potential of garbage compost but also had improved the safety of its use.
At present, China has tentatively launched consumer garbage compost is used for the research work of greenery patches, gardens matrix or fertilizer, and garbage compost adds soil with the form of fertilizer usually, can improve soil property, improves turfgrass biomass, chlorophyll content etc.But the heavy metal that contains high concentration in the garbage compost when it improves soil, edaphophyte and microorganism, also produces harm to them.So, propose garbage compost to be applied to the actual idea that must remove heavy metal wherein if want.In the garbage compost application, how to prevent or reduce heavy metal to get into soil and underground water generation, become a big problem that will solve.
Discover, add EDTA and can significantly improve the enriching quantity of heavy metal in supplying the examination lawn plant.When utilizing EDTA to work in coordination with phytoremediation; Because the compound that EDTA and EDTA and heavy metal chelating form has very strong dissolubility and bio-toxicity; And in soil, be difficult to, so cause environmental problems such as polluting deep soil and groundwater contamination possibly by light, chemical substance and biological degradation.But NTA (Nitrilotriacetic acid) was applied to scale remover in 50 years in the past always, in analytical chemistry, was used as complexometric titration agent and screening agent, Chang Zuowei activator in catalytic kinetics is analyzed.Yet unite the method for repairing garbage compost heavy metal seepage system about microorganism and perennial ryegrass, do not see bibliographical information.
The microorganism recovery technique remedied method costs such as the collaborative phytoremediation of phytoremediation method, chemical extraction method, chemical lixiviate high, used shortcoming such as limitation.Experiment proof Frankia is not strong to certain density Cu, Co, Ni, Cd and Hg susceptibility.Zhou etc. (2007) show that the Cu that mycorhiza can be subdued by maize root system absorption transports to overground part.The Cu, Hg, Pb, Cd and Cr 5 heavy metal species that in microbiological culture media, have added variable concentrations; Escherichia coli, bacillus subtilis are coerced cultivation; The result finds that Hg, Cd toxicity are stronger, and the propagation of 2 kinds of bacteriums is suppressed when high concentration, and bacillus subtilis is more responsive; When heavy metal concentration>behind 50 mg/L, 5 heavy metal species are Hg to the toxicity of two kinds of bacteriums in proper order>Cd>Cu>Cr ≈ Pb.The key property that microorganism is repaired is the heavy metal ion that can tolerate high concentration, and is common like Cu, Zn, Ni etc.To these characteristics, there is the researcher to begin to attempt adopting composite flora to unite at present and repairs.Complex microorganism is made up of by the proper ratio co-incubation two or more microorganism; Can give full play to the synergy advantage of colony; Experiment shows that its composted efficient of garbage compost of inoculating complex microorganism significantly improves, and can improve the germination rate of seed.
Summary of the invention
The present invention systematically inquires into the potential risk of heavy metal leaching migration when microorganism, NTA fortification of plants are repaired through the research to heavy metal accumulation, leaching etc. in the composting substrate seepage system of lawn, and proposes the method for preventing and treating accordingly.
For realizing that above-mentioned purpose the invention discloses following technology contents:
A kind of microorganism and perennial ryegrass are united the method for repairing garbage compost heavy metal seepage system, it is characterized in that being undertaken by following step:
(1) experiment material is prepared
The experiment selected English ryegrass (
Lolium perenneL.) be experiment material; Consumer garbage compost is taken from Tianjin Xiao Dian composting plant; Supply examination soil to get the dark topsoil of 0-20 cm; 2 mm sieve is subsequent use excessively respectively after air-dry for compost and garden mould; Interlayer material is served as reasons down and supremely is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively; Bacillus subtilis, actinomycetes separate the consumer garbage compost of the Xiao Dian garbage compost treatment plant from Tianjin; Leaching liquor: with (NH
4)
2SO
4, Na
2SO
4, K
2SO
4, MgSO
4, Ca (NO
3)
2, Mg (NO
3)
2, H
2SO
4Make SO
4 2-, NO
3 -, Cl
-, NH
+, Mg
2+, Ca
2+, K
+, Na
+Concentration is respectively 14.96,6.54,1.68,3.71,0.82,1.38,0.64 and 0.78 mg/L, and uses HCl allotment pH subsequent use as the leaching liquor of 5.5-6;
(2) experimental technique
1) the lawn matrix compost utilizes and the interlayer setting
Selecting the pvc pipe diameter for use is 7.5cm, the long tube of 25cm, pipe bottom be fixed with diameter be the nylon wire of 0.2 mm with soil in the stay pipe and compost, pipe is sling, the below of pipe is furnished with taper bottle graft percolate;
2) the air-dry garden mould of 500g of in pvc pipe, packing into is paved, and reinstalls horizontal interlayer and is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively from the bottom to top, reinstalls the 280g compost, paves on interlayer;
3) foundation of microbial bacterial agent and lawn matrix interlayer system
0.5 g perennial ryegrass is sowed in the pvc pipe, behind the sowing 10d,
Add 1.4 mL distilled water in the I;
Add 0.7 mL beef-protein medium and 0.7 mL Gause I medium in the II;
Add 1.4 mL bacillus subtilises in the III;
Add 1.4 mL actinomycetes in the IV;
Add 0.7 mL liquid bacillus subtilis and the actinomycetic complexing agent of 0.7 mL liquid in the V;
After connecing bacterium, lawn plant regrowth 30d cradles, and places 80 ℃ baking oven to dry to weight the lawn plant overground part under cradling, the note ground biomass;
4) cradle second day with the artificial leaching liquor of 150 mL, be equivalent to the precipitation of 34.0 mm, carry out the drip washing first time; Per afterwards 7 d drip washing once, each 150mL drenches 600 mL totally 4 times; Weigh respectively for 4 times, and be collected in the same conical flask, mix to be measured; Wherein perennial ryegrass was all watered with running water between planting season, and keeping its whole field capacity is 60-65%.Temperature and relative moisture indoor between planting season are respectively 14-25 ℃ and 30-55%, and intensity of illumination is 350-800
μMol m
-2s
-1
5) utilize the content of heavy metal Cd, Cr, Cu, Pb and Ni in the TAS-990 atomic absorption spectrophotometer digestive juice.
Zeolite of the present invention is a natural zeolite, and sieve is got particle diameter less than 4mm's; Vermiculite is taken from Japan great foreign garden company, gets particle diameter less than 4mm's; Sawdust is taken from the discarded offcuts of processing factory, and above material is dried to constant weight for equal 80 ℃, and is subsequent use.
Bacillus subtilis of the present invention, actinomycetes all have commercially available.Also can adopt the consumer garbage compost of Tianjin Xiao Dian garbage compost treatment plant to separate.The method of its separation is: isolated bacterial classification purifying was cultivated for 2 generations; Get activated spawn, insert and be equipped with in the 250 mL triangular flasks of 50 mL liquid nutrient mediums, 180 r/min thermophilics are cultivated; Bacillus subtilis strain with the activation of transfer needle picking; Access is equipped with in the 250 mL triangular flasks of the liquid beef-protein medium of 50 mL, and 180 r/min thermophilics are cultivated 12 h, obtain this experiment and use bacillus subtilis; With the actinomycetes bacterial classification of transfer needle picking activation, insert in the 250 mL triangular flasks that the liquid Gause I medium of 50 mL is housed, 180 r/min thermophilics are cultivated 96h, obtain this experiment and use actinomycetes.
Experimental result of the present invention shows: horizontal interlayer shop is supreme with soil, under the compost; The harmful substance and the heavy metal that can reduce in the garbage compost move in soil; Perhaps microorganism is down collaborative under chelating agent is induced to make lawn plant; Increase time and the enrichment of heavy metal in its body that it absorbs the heavy metal in the compost, and reduce the migration of heavy metal to deep soil.
Bacillus subtilis (the Latin formal name used at school that the present invention adopts
Bacillus subtilis:) (preserving number CGMCC N0 2548), middle northern bio tech ltd externally can provide.Chinese patent number is: ZL200710062503.1.
Physio-biochemical characteristics: the bacillus subtilis somatic cells is a rod-short, and gemma is arranged, and the size of examining under a microscope thalline is 0.4～0.8 μ m * 2.3～3.7 μ m.It is more sturdy shaft-like, misaligned that somatic cells is.Gram is positive.At the solid culture primary surface, thalline forms translucent light yellow bacterium colony, the bacterium colony thickness, and the edge is irregular.
Soil actinomycetes fermenting liquid disclosed by the invention is disclosed identical with application number 200610122277.7.
Bacillus subtilis, actinomycetes that the present invention adopts the consumer garbage compost of Tianjin Xiao Dian garbage compost treatment plant to separate and draws are identical with the characteristic of existing bibliographical information, (preserving number CGMCC N0 2548; CCTCC N0 M206067 bacterial strain) solid not preservation.
Bacillus subtilis is one of bacterial classification that has in the bacillus application potential.Increasing for the research of bacillus application facet both at home and abroad in recent years.Actinomycetes can promote plant metabolism, improve plant resistance to environment stress.Fermentation of bacillus subtilis liquid, actinomycete fermentation filter that the present invention will be mixed with variable concentrations are inoculated in the turf establishment system.Through the fermentation of bacillus subtilis filtrating of research variable concentrations, the influence that actinomycete fermentation filtrating is sprouted ryegrass seed, for the ecological planting of lawn plant provides foundation.
The present invention further discloses said method and increasing heavy metal enrichment obstruction Cd in plant corpus, Cr, the application of the vertical migration aspect of Cu and Ni; Its method is to adopt 0.7 mL liquid bacillus subtilis and the actinomycetic complexing agent of 0.7 mL liquid and horizontal interlayer to form interlayer by 20g zeolite, 20g vermiculite and 20g sawdust respectively from the bottom to top.
This experimental studies results shows, in general complex microorganism to increase heavy metal in plant corpus enrichment and to hindering Cd, Cr, the vertical migration of Cu and Ni is all remarkable.Wherein bacillus subtilis is the most remarkable to the vertical migration that hinders Pb.
The more detailed child of the present invention is following by method:
1 experiment material and method
1.1 soil, compost and lawn plant
(1) plant is used in experiment
The more common English ryegrass of this experiment selected northern China (
Lolium perenneL.) be experiment material.
(2) experiment is with compost and soil and physics and chemistry thereof
Consumer garbage compost is taken from Tianjin Xiao Dian composting plant.Supply examination soil to take from the dark topsoil of the sample plot 0-20 cm of Tianjin Normal University.2 mm sieve is subsequent use excessively respectively after air-dry for compost and garden mould.
The compost physicochemical property is: pH 7.62, the content of organic matter 12.12%, full nitrogen 5.18%, available phosphorus 77.92 mg/Kg, saturation moisture content 0.76 mL/g.Its heavy metal Cd, Cr, Cu, Pb, the Ni background value is respectively 1.97,67.00, and 238.73,172.11,33.42
μG/g.Soil property is: pH 8.30, the content of organic matter 4.68%, full nitrogen 0.21%, available phosphorus 22.03 mg/kg, saturation moisture content 0.58 mL/g.Its heavy metal Cd, Cr, Cu, Pb, the background value of Ni is respectively 0.18,2.13, and 9.33,5.75,2.64
μG/g.
1.2 interlayer material is selected
This experiment interlayer material is zeolite, vermiculite, sawdust.Zeolite is a natural zeolite, and sieve is got particle diameter less than 4mm's; Vermiculite is taken from Japan great foreign garden company, gets particle diameter less than 4mm's.Sawdust is taken from the discarded offcuts of processing factory, and above material is dried to constant weight for equal 80 ℃, and is subsequent use.
This experiment interlayer is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively from the bottom to top, is total to high 4cm in the pvc pipe of packing into.
1.3 the preparation of microbial inoculum
Experiment separates the consumer garbage compost of the Xiao Dian garbage compost treatment plant from Tianjin with bacillus subtilis, actinomycetes.Get the fresh compost of 10 g and be added in the aqua sterilisa of 90 mL, the compost suspension of configuration suitable concentration is got the suspension 100 that mixes
μL joins on the corresponding solid plate medium; Thermophilic is cultivated; According to colonial morphology, picking carries out Gram's stain (identifying bacterium), spore staining method (identifying bacillus subtilis) and printingout method (identifying actinomycetes) with the known bacillus subtilis bacterium colony more similar with actinomycetes to be identified.According to qualification result, isolated bacterial classification purifying was cultivated for 2 generations, get activated spawn; Access is equipped with in the 250 mL triangular flasks of 50 mL liquid nutrient mediums; 180 r/min thermophilics are cultivated, and select for use 600 nm wavelength to carry out turbidimetric assay, are ordinate with the OD value of bacteria suspension; Incubation time is an abscissa, draws the microbial growth curve.
According to the growth curve acquisition time of growth of microorganism to stationary phase, be 8-18h the stationary phase that draws bacillus subtilis, and be 84-108h actinomycetic stationary phase.With the Bacillus subtilis strain of transfer needle picking activation, insert in the 250 mL triangular flasks that the liquid beef-protein medium of 50 mL is housed, 180 r/min thermophilics are cultivated 12 h, obtain this experiment and use bacillus subtilis; With the actinomycetes bacterial classification of transfer needle picking activation, insert in the 250 mL triangular flasks that the liquid Gause I medium of 50 mL is housed, 180 r/min thermophilics are cultivated 96h, obtain this experiment and use actinomycetes.
1.4 leaching liquor preparation
According to Han Yu (2008) investigation whole day Jinshi City Precipitation pH scope is 4.00-8.24, and a year average is 5.59, belongs to acid precipitation.This experiment is with (NH
4)
2SO
4, Na
2SO
4, K
2SO
4, MgSO
4, Ca (NO
3)
2, Mg (NO
3)
2, H
2SO
4Make SO
4 2-, NO
3 -, Cl
-, NH
+, Mg
2+, Ca
2+, K
+, Na
+Concentration is respectively 14.96,6.54,1.68,3.71,0.82,1.38,0.64 and 0.78 mg/L, and use HCl allotment pH is 5.59 leaching liquor.
1.5 experimental technique
(1) the lawn matrix compost utilizes and the interlayer setting
The pvc pipe diameter is 7.5cm, and it is cut into 6 of the short tubes of 21cm, 24 of the long tubes of 25cm respectively.All pipe bottoms all be fixed with diameter be the nylon wire of 0.2 mm with soil in the stay pipe and compost, pipe is sling, the below of pipe is furnished with taper bottle graft percolate.
Soil plough horizon is the topsoil of 0-20cm.This experimental simulation topsoil is provided with the high 12cm of garden mould, the high 8cm of compost.The preparation height is 6 of the short tubes of 20cm, and height is 24 of the long tubes of 24cm.The preparation of short tube: the air-dry garden mould of 500g of packing into earlier, pave, reinstall the 280g compost, pave on garden mould; The preparation of long tube: the garden mould that the 500g that packs into is air-dry, pave, reinstall horizontal interlayer, reinstall the 280g compost, pave on interlayer.
(2) foundation of microbial bacterial agent and lawn matrix interlayer system
Lawn plant is selected English ryegrass for use.0.5 g perennial ryegrass is sowed in the pvc pipe, and every group of 3 repetition handled and seen table 1.After sowing 10 d, add 1.4 mL distilled water in the I; Add 0.7 mL beef-protein medium and 0.7 mL Gause I medium in the II; Add 1.4 mL bacillus subtilises in the III; Add 1.4 mL actinomycetes in the IV; Adding 0.7 mL liquid bacillus subtilis and 0.7 mL liquid are actinomycetic compound in the V.After connecing bacterium, lawn plant regrowth 30 d cradle (long 40 d of perennial ryegrass symbiosis), place 80 ℃ baking oven to dry to weight the lawn plant overground part under cradling, the note ground biomass.Cradle second day and carry out drip washing first time with the artificial leaching liquors of 150 mL (precipitation that is equivalent to 34.0 mm), per afterwards 7 d drip washing once, each 150 mL, 600 mL are gone in totally 4 pouring.Weigh respectively for 4 times, and be collected in the same conical flask, mix to be measured.
All with the running water pouring, keep its whole field capacity is 60% to lawn plant between planting season.Temperature and relative moisture indoor between planting season are respectively 14-25 ℃ and 30-55%, and intensity of illumination is 350-800
μMol m
-2s
-1
Inoculation of table 1 microbial bacterial agent and interlayer processing scheme
(4) content of beary metal analysis
Rye carelessly appearance all takes by weighing 0.2 g, with 10 mL HNO
3With 2 mL HClO
4120-140 ℃ of following electric hot plate digestion, treat the cancellation of pale brown look cigarette, gained solution with 1% nitric acid constant volume to 25 mL.Percolate is got 20 mL, with 8 mL HNO
3With 2 mL HClO
4120-140 ℃ of following electric hot plate digestion, treat the cancellation of pale brown look cigarette, gained solution with 1% nitric acid constant volume to 50 mL.Utilize heavy metal in the TAS-990 atomic absorption spectrophotometer digestive juice (Cd, Cr, Cu, Pb and Ni) content at last.
1.6 data analysis
Data analysis is accomplished with EXCEL2003 and the analysis of SPSS14.0 statistical software.
Heavy metal accumulation amount=heavy metal concentration * biomass dry weight.
Heavy metal diafiltration ratio=(total metals in heavy metal drip washing total amount/compost) * 100%.
2 results and analysis
2.1 different microorganisms microbial inoculum and interlayer are provided with the influence to the lawn plant biomass
Can find out that from table 2 interlayer can increase the biomass of perennial ryegrass, but to the raising of perennial ryegrass not significantly (
p>0.05).The inoculation microbial inoculum to the biomass of lawn plant increase significantly (
p<0.05).It is the most remarkable to the influence that improves the perennial ryegrass biomass that composite bacteria agent capable is worked in coordination with horizontal interlayer (
), and
handled in comparison has respectively improved 18.8% and 34.2%.
Table 2 different disposal is to the influence (g/PVC) of lawn plant biomass
| Different disposal | Perennial ryegrass |
 |
0.733±0.025b |
 |
0.768±0.018b |
 |
0.853±0.012a |
 |
0.836±0.009a |
 |
0.871±0.023a |
Annotate:
: no interlayer;
: inoculum medium only;
: the inoculation bacillus subtilis;
: the inoculation actinomycetes;
: inoculation bacillus and actinomycetic compound bacteria (
-
Interlayer all is set), down together.
2.2 different microorganisms microbial inoculum and interlayer are provided with the influence to the lawn plant plant height
Different disposal is seen table 3 to the influence of perennial ryegrass plant height, connects the processing that the processing perennial ryegrass plant height that interlayer is arranged before the bacterium is higher than no interlayer, but each handle between the difference of perennial ryegrass plant height not obvious.After connecing bacterium; After perennial ryegrass grows to 21d; The increase of plant height is slow; Behind the 30d,
,
,
and
have improved 14.0%, 23.9%, 22.0% and 29.9% than
respectively.
3 different disposal are to the influence (cm) of perennial ryegrass plant height
2.3 different microorganisms microbial inoculum and interlayer are provided with the influence to the lawn plant enriching heavy metal
Concentration and the enriching quantity of heavy metal in the lawn plant body.From table, can find out that interlayer is set can increase the concentration of heavy metal in plant corpus.Interlayer only is set but does not have under the microbial inoculum, perennial ryegrass is the most remarkable to the enrichment of Cr.Adding different microbial inoculums all can be significantly (
p<0.05) enrichment of increase heavy metal in the body of plant lawn.Wherein under the processing of composite bacteria agent capable (
); Best to the enrichment of heavy metal in plant corpus, perennial ryegrass to Cd, Cr, Cu, Pb and Ni respectively than interlayer only is set but do not have microbial inoculum and handle (
) 2.69,1.25,1.42,2.04 and 1.79 times; The processing of the collaborative horizontal interlayer of microbial bacterial agent is perhaps only added single microbial inoculum to the accumulation ability of heavy metal in plant corpus greater than horizontal interlayer only is set; Can find out also that from table composite bacteria agent capable increases more remarkable to the enrichment in the lawn plant body of heavy metal.
The enrichment of table 4Cd in the lawn plant body
The enrichment of table 5Cr in the lawn plant body
The enrichment of table 6 Cu in the lawn plant body
The enrichment of table 7Pb in the lawn plant body
The enrichment of table 8Ni in the lawn plant body
2.4 utilization and interlayer are provided with the influence to heavy metal diafiltration behavior and general layout
Table 9 has been listed Cd in each pvc pipe percolate of planting perennial ryegrass, Cr, Cu, the total content of Pb and Ni.The result shows that microorganism has suppressed the stripping property and the mobility of heavy metal.Simultaneously, experimental data also is illustrated under the rainfall, lawn plant behind the microbial action and horizontal separation layer to the leaching of heavy metal migration have certain inhibitory action (
p>0.05).Bacillus subtilis effect is the most remarkable to the stripping that reduces Pb down, the Pb content in the filtrating than the reduction of not adding microbial inoculum 2 times.Cr under the compound bacteria effect, Cu, the animal migration of Pb and Ni is lower than actinomycetes.
The total amount and the percentage of heavy metal in the pvc pipe percolate of the different microbial inoculums processing of table 9 perennial ryegrass
3 conclusions
This experiment is spread interlayer with discarded object between compost and soil, purpose is the heavy-metal contaminated soil that hinders in the compost.Experimental result shows, horizontal interlayer interlayer helps the growth of lawn plant can increase biomass and the plant height of lawn plant, and interlayer has also increased the content of plant heavy metal of body simultaneously, and hinder know clearly heavy metal with vertical migration.
Bacillus subtilis is one of bacterial classification that has in the bacillus application potential.Actinomycetes can promote plant metabolism, improve plant resistance to environment stress.This experimental studies results in general complex microorganism to increase heavy metal in plant corpus enrichment and to hindering Cd, Cr, the vertical migration of Cu and Ni is all remarkable.Bacillus subtilis is the most remarkable to the vertical migration that hinders Pb.
Description of drawings:
Fig. 1 is the growth curve of bacillus subtilis;
Fig. 2 is actinomycetic growth curve.
Embodiment
Below in conjunction with embodiment the present invention is described; The scheme of embodiment described here; Do not limit the present invention; One of skill in the art can make improvements and change according to spirit of the present invention, and described these improvement and variation all should be regarded as within the scope of the invention, and scope of the present invention and essence are limited claim.Bacillus subtilis, actinomycetes and existing document (preserving number CGMCC N0 2548 wherein; CCTCC N0 M206067 bacterial strain) characteristic of report is identical, has commercially availablely, also can obtain according to the method for embodiment 1, from the resulting bacillus subtilis of consumer garbage compost, actinomycetes Physiology and biochemistry 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.
The preparation of microbial inoculum
Experiment separates the consumer garbage compost of the Xiao Dian garbage compost treatment plant from Tianjin with bacillus subtilis, actinomycetes.Get the fresh compost of 10 g and be added in the aqua sterilisa of 90 mL, the compost suspension of configuration suitable concentration is got the suspension 100 that mixes
μL joins on the corresponding solid plate medium; Thermophilic is cultivated; According to colonial morphology, picking carries out Gram's stain (identifying bacterium), spore staining method (identifying bacillus subtilis) and printingout method (identifying actinomycetes) with the known bacillus subtilis bacterium colony more similar with actinomycetes to be identified.According to qualification result, isolated bacterial classification purifying was cultivated for 2 generations, get activated spawn; Access is equipped with in the 250 mL triangular flasks of 50 mL liquid nutrient mediums; 180 r/min thermophilics are cultivated, and select for use 600 nm wavelength to carry out turbidimetric assay, are ordinate with the OD value of bacteria suspension; Incubation time is an abscissa, draws the microbial growth curve.
According to the growth curve acquisition time of growth of microorganism to stationary phase, be 8-18h the stationary phase that draws bacillus subtilis, and be 84-108h actinomycetic stationary phase.With the Bacillus subtilis strain of transfer needle picking activation, insert in the 250 mL triangular flasks that the liquid beef-protein medium of 50 mL is housed, 180 r/min thermophilics are cultivated 12 h, obtain this experiment and use bacillus subtilis; With the actinomycetes bacterial classification of transfer needle picking activation, insert in the 250 mL triangular flasks that the liquid Gause I medium of 50 mL is housed, 180 r/min thermophilics are cultivated 96h, obtain this experiment and use actinomycetes.
Bacillus subtilis (preserving number CGMCC N0 2548) is inserted in the 250 mL triangular flasks that the liquid beef-protein medium of 50 mL is housed, and 180 r/min thermophilics are cultivated 12 h, obtain this experiment and use bacillus subtilis;
Actinomycetes (preserving number CCTCC N0 M206067) are inserted in the 250 mL triangular flasks that the liquid Gause I medium of 50 mL is housed, and 180 r/min thermophilics are cultivated 96h, obtain this experiment and use actinomycetes.
Embodiment 3
(1) experiment material is prepared
The experiment selected English ryegrass (
Lolium perenneL.) be experiment material; Consumer garbage compost is taken from Tianjin Xiao Dian composting plant; Supply examination soil to get the dark topsoil of 0-20 cm; 2 mm sieve is subsequent use excessively respectively after air-dry for compost and garden mould; Interlayer material is served as reasons down and supremely is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively; Bacillus subtilis, actinomycetes separate the consumer garbage compost of the Xiao Dian garbage compost treatment plant from Tianjin; Leaching liquor: with (NH
4)
2SO
4, Na
2SO
4, K
2SO
4, MgSO
4, Ca (NO
3)
2, Mg (NO
3)
2, H
2SO
4Make SO
4 2-, NO
3 -, Cl
-, NH
+, Mg
2+, Ca
2+, K
+, Na
+Concentration is respectively 14.96,6.54,1.68,3.71,0.82,1.38,0.64 and 0.78 mg/L, and to use HCl allotment pH be that 5.5 leaching liquor is subsequent use;
(2) experimental technique (bacterial strain is selected from embodiment 1)
1) the lawn matrix compost utilizes and the interlayer setting
Selecting the pvc pipe diameter for use is 7.5cm, the long tube of 25cm, pipe bottom be fixed with diameter be the nylon wire of 0.2 mm with soil in the stay pipe and compost, pipe is sling, the below of pipe is furnished with taper bottle graft percolate;
2) the air-dry garden mould of 500g of in pvc pipe, packing into is paved, and reinstalls horizontal interlayer and is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively from the bottom to top, reinstalls the 280g compost, paves on interlayer;
3) foundation of microbial bacterial agent and lawn matrix interlayer system
0.5 g perennial ryegrass is sowed in the pvc pipe, behind the sowing 10d,
Add 1.4 mL distilled water in the I;
Add 0.7 mL beef-protein medium and 0.7 mL Gause I medium in the II;
Add 1.4 mL bacillus subtilises in the III;
Add 1.4 mL actinomycetes in the IV;
Add 0.7 mL liquid bacillus subtilis and the actinomycetic complexing agent of 0.7 mL liquid in the V;
After connecing bacterium, lawn plant regrowth 30d cradles, and places 80 ℃ baking oven to dry to weight the lawn plant overground part under cradling, the note ground biomass;
4) cradle second day with the artificial leaching liquor of 150 mL, be equivalent to the precipitation of 34.0 mm, carry out the drip washing first time; Per afterwards 7 d drip washing once, each 150mL drenches 600 mL totally 4 times; Weigh respectively for 4 times, and be collected in the same conical flask, mix to be measured; Wherein perennial ryegrass was all watered with running water between planting season, and keeping its whole field capacity is 60%.Temperature and relative moisture indoor between planting season are respectively 16 ℃ and 40%, and intensity of illumination is 400
μMol m
-2s
-1
5) utilize the content of heavy metal Cd, Cr, Cu, Pb and Ni in the TAS-990 atomic absorption spectrophotometer digestive juice.Its mesolite is a natural zeolite, and sieve is got particle diameter less than 4mm's; Vermiculite is taken from Japan great foreign garden company, gets particle diameter less than 4mm's; Sawdust is taken from the discarded offcuts of processing factory, and above material is dried to constant weight for equal 80 ℃, and is subsequent use.
(1) experiment material is prepared
The experiment selected English ryegrass (
Lolium perenneL.) be experiment material; Consumer garbage compost is taken from Tianjin Xiao Dian composting plant; Supply examination soil to get the dark topsoil of 0-20 cm; 2 mm sieve is subsequent use excessively respectively after air-dry for compost and garden mould; Interlayer material is served as reasons down and supremely is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively; Bacillus subtilis, actinomycetes separate the consumer garbage compost of the Xiao Dian garbage compost treatment plant from Tianjin; Leaching liquor: with (NH
4)
2SO
4, Na
2SO
4, K
2SO
4, MgSO
4, Ca (NO
3)
2, Mg (NO
3)
2, H
2SO
4Make SO
4 2-, NO
3 -, Cl
-, NH
+, Mg
2+, Ca
2+, K
+, Na
+Concentration is respectively 14.96,6.54,1.68,3.71,0.82,1.38,0.64 and 0.78 mg/L, and to use HCl allotment pH be that 5.5 leaching liquor is subsequent use;
(2) experimental technique (bacterial strain is selected from embodiment 2)
1) the lawn matrix compost utilizes and the interlayer setting
Selecting the pvc pipe diameter for use is 7.5cm, the long tube of 25cm, pipe bottom be fixed with diameter be the nylon wire of 0.2 mm with soil in the stay pipe and compost, pipe is sling, the below of pipe is furnished with taper bottle graft percolate;
2) the air-dry garden mould of 500g of in pvc pipe, packing into is paved, and reinstalls horizontal interlayer and is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively from the bottom to top, reinstalls the 280g compost, paves on interlayer;
3) foundation of microbial bacterial agent and lawn matrix interlayer system
0.5 g perennial ryegrass is sowed in the pvc pipe, behind the sowing 10d,
Add 1.4 mL distilled water in the I;
Add 0.7 mL beef-protein medium and 0.7 mL Gause I medium in the II;
Add 1.4 mL bacillus subtilises in the III;
Add 1.4 mL actinomycetes in the IV;
Add 0.7 mL liquid bacillus subtilis and the actinomycetic complexing agent of 0.7 mL liquid in the V;
After connecing bacterium, lawn plant regrowth 30d cradles, and places 80 ℃ baking oven to dry to weight the lawn plant overground part under cradling, the note ground biomass;
4) cradle second day with the artificial leaching liquor of 150 mL, be equivalent to the precipitation of 34.0 mm, carry out the drip washing first time; Per afterwards 7 d drip washing once, each 150mL drenches 600 mL totally 4 times; Weigh respectively for 4 times, and be collected in the same conical flask, mix to be measured; Wherein perennial ryegrass was all watered with running water between planting season, and keeping its whole field capacity is 65%.Temperature and relative moisture indoor between planting season are respectively 25 ℃ and 55%, and intensity of illumination is 800
μMol m
-2s
-1
5) utilize the content of heavy metal Cd, Cr, Cu, Pb and Ni in the TAS-990 atomic absorption spectrophotometer digestive juice.Its mesolite is a natural zeolite, and sieve is got particle diameter less than 4mm's; Vermiculite is taken from Japan great foreign garden company, gets particle diameter less than 4mm's; Sawdust is taken from the discarded offcuts of processing factory, and above material is dried to constant weight for equal 80 ℃, and is subsequent use.
Claims (4)
1. microorganism and perennial ryegrass are united the method for repairing garbage compost heavy metal seepage system, it is characterized in that being undertaken by following step:
(1) experiment material is prepared
The experiment selected English ryegrass (
Lolium perenneL.) be experiment material; Consumer garbage compost is taken from Tianjin Xiao Dian composting plant; Supply examination soil to get the dark topsoil of 0-20 cm; 2 mm sieve is subsequent use excessively respectively after air-dry for compost and garden mould; Interlayer material is served as reasons down and supremely is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively; Bacillus subtilis, actinomycetes separate the consumer garbage compost of the Xiao Dian garbage compost treatment plant from Tianjin; Leaching liquor: with (NH
4)
2SO
4, Na
2SO
4, K
2SO
4, MgSO
4, Ca (NO
3)
2, Mg (NO
3)
2, H
2SO
4Make SO
4 2-, NO
3 -, Cl
-, NH
+, Mg
2+, Ca
2+, K
+, Na
+Concentration is respectively 14.96,6.54,1.68,3.71,0.82,1.38,0.64 and 0.78 mg/L, and uses HCl allotment pH subsequent use as the leaching liquor of 5.5-6;
(2) experimental technique
1) the lawn matrix compost utilizes and the interlayer setting
Selecting the pvc pipe diameter for use is 7.5cm, the long tube of 25cm, pipe bottom be fixed with diameter be the nylon wire of 0.2 mm with soil in the stay pipe and compost, pipe is sling, the below of pipe is furnished with taper bottle graft percolate;
2) the air-dry garden mould of 500g of in pvc pipe, packing into is paved, and reinstalls horizontal interlayer and is made up of 20g zeolite, 20g vermiculite and 20g sawdust respectively from the bottom to top, reinstalls the 280g compost, paves on interlayer;
3) foundation of microbial bacterial agent and lawn matrix interlayer system
0.5 g perennial ryegrass is sowed in the pvc pipe, behind the sowing 10d,
Add 1.4 mL distilled water in the I;
Add 0.7 mL beef-protein medium and 0.7 mL Gause I medium in the II;
Add 1.4 mL bacillus subtilises in the III;
Add 1.4 mL actinomycetes in the IV;
Add 0.7 mL liquid bacillus subtilis and the actinomycetic complexing agent of 0.7 mL liquid in the V;
After connecing bacterium, lawn plant regrowth 30d cradles, and places 80 ℃ baking oven to dry to weight the lawn plant overground part under cradling, the note ground biomass;
4) cradle second day with the artificial leaching liquor of 150 mL, be equivalent to the precipitation of 34.0 mm, carry out the drip washing first time; Per afterwards 7 d drip washing once, each 150mL drenches 600 mL totally 4 times; Weigh respectively for 4 times, and be collected in the same conical flask, mix to be measured; Wherein perennial ryegrass was all watered with running water between planting season, and keeping its whole field capacity is 60-65%; Temperature and relative moisture indoor between planting season are respectively 14-25 ℃ and 30-55%, and intensity of illumination is 350-800
μMol m
-2s
-1
5) utilize the content of heavy metal Cd, Cr, Cu, Pb and Ni in the TAS-990 atomic absorption spectrophotometer digestive juice.
2. the described method of claim 1, its mesolite is a natural zeolite, sieve is got particle diameter less than 4mm's; Vermiculite is taken from Japan great foreign garden company, gets particle diameter less than 4mm's; Sawdust is taken from the discarded offcuts of processing factory, and above material is dried to constant weight for equal 80 ℃, and is subsequent use.
3. the described method of claim 1, wherein bacillus subtilis, the actinomycetes method of separating is: isolated bacterial classification purifying was cultivated for 2 generations, get activated spawn; Access is equipped with in the 250 mL triangular flasks of 50 mL liquid nutrient mediums; 180 r/min thermophilics are cultivated, and with the Bacillus subtilis strain of transfer needle picking activation, insert in the 250 mL triangular flasks that the liquid beef-protein medium of 50 mL is housed; 180 r/min thermophilics are cultivated 12 h, obtain this experiment and use bacillus subtilis; With the actinomycetes bacterial classification of transfer needle picking activation, insert in the 250 mL triangular flasks that the liquid Gause I medium of 50 mL is housed, 180 r/min thermophilics are cultivated 96h, obtain this experiment and use actinomycetes.
4. the said method of claim 1 is increasing heavy metal enrichment obstruction Cd in plant corpus, Cr, the application of the vertical migration aspect of Cu and Ni; Its method is to adopt 0.7 mL liquid bacillus subtilis and the actinomycetic complexing agent of 0.7 mL liquid and horizontal interlayer to form interlayer by 20g zeolite, 20g vermiculite and 20g sawdust respectively from the bottom to top.
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