CN105750311B - A kind of lead cadmium combined pollution soil comprehensive organism repairing method - Google Patents
A kind of lead cadmium combined pollution soil comprehensive organism repairing method Download PDFInfo
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- CN105750311B CN105750311B CN201610167393.4A CN201610167393A CN105750311B CN 105750311 B CN105750311 B CN 105750311B CN 201610167393 A CN201610167393 A CN 201610167393A CN 105750311 B CN105750311 B CN 105750311B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
Abstract
The invention discloses a kind of lead cadmium combined pollution soil comprehensive organism repairing methods, for the soil of lead cadmium combined pollution, based on being repaired with over-accumulated plant, the microorganism remediation and animal reparation of effect of plant restoration can be improved by being aided with, enhance lead in soil, the biological effectiveness of cadmium and the growth and absorption that thus promote plant, improves the overall efficiency of contaminated soil remediation.The present invention smoothly realizes the biology in situ reparation to soil, has a significant effect, and does not generate secondary pollution to environment, is the developing direction that following heavy-metal contaminated soil repairs field.
Description
Technical field
The present invention relates to the biological prosthetic of soil pollution, especially a kind of comprehensive organism reparation of lead cadmium combined pollution soil
Technology.
Background technology
Soil is one of most basic, most important natural resources that the mankind depend on for existence and development and human ecology ring
The important component and filter in border, however the situation is tense for the soil pollution in China.With China's industrialization and urbanization into
The quickening of journey, a variety of pollutants containing heavy metal enter soil, cause heavy metal pollution of soil getting worse by all means.
The national irrigating region investigation carried out according to the Ministry of Agriculture of China, in about 1,400,000 hm2Irrigating region in, by the soil of heavy metal pollution
Ground area accounts for the 64.8% of the irrigating region gross area, and wherein slight pollution accounts for 46.7%, and intermediate pollution accounts for 9.7%, serious to pollute
Account for 8.4%（Chen Zhiliang, 2002）.Currently, the cultivated area of national heavy metal pollution is up to 20,000,000 hm2, account for about the total face in arable land
Long-pending 1/5 leads to have the content of beary metal of 12,000,000 t grains exceeded every year, and leads to 10,000,000 t of grain drop in production, and the two is straight
Economic loss is connect up to more than 200 hundred million yuan (paddy Zhao, 2008).In addition, China also has a large amount of industrial and mining area soils and urban land etc. are non-to plough
It is polluted by heavy metal on ground（Wang Zhilou, 2010）.
In The Soils of Hebei heavy metal pollution situation allows of no optimist, lead, cadmium pollution outstanding problem.According to Hebei province's environment in 2004
Situation bulletin reports that the whole province's sewage irrigation area is 53277 hectares, focuses on the water-based Hutuohe River of Ziyahe River, the rivers Xiao, ox
7 rivers such as tail river, vast sea ditch, Fu Yanghe, azure water canal, north row river, relate generally to Shijiazhuang, Xingtai, Handan, Hengshui, Cangzhou 5
City.Wang Yuanzhong etc. 2005 is to 15 county of Hebei province, 20.53 ten thousand hm2Wheat, main maize area farmland 1535 pedotheques in
The survey showed that for the pollution situation of heavy metal：8 heavy metal species of Hebei province main maize area are in Hebei province main maize area
There is different degrees of pollution accumulation phenomenon;The whole province's gross contamination area has accounted for the 12.0% of the monitoring gross area.Meng Fanqiao etc.
2004 to the rivers the Xiao irrigating region in Hebei province and mansion river irrigating region studies have shown that the dirt by 20 years fills, the river Fu He, Xiao two is big
Cd is more than China in the soil in blowdown area《Standard of soil environment quality》Secondary standard, have apparent pollution.Luan Wenlou etc.
Farmland topsoil contents of heavy metal elements, the pollution shape Shijiazhuang City irrigating region upper, middle and lower swum in 3 sections for 2009
Condition, form etc. have carried out investigation and research.As a result, it has been found that：Study the heavy metal in area's topsoil（In addition to As）In irrigating region
Upper, middle and lower trip has different degrees of enrichment, and wherein Hg, Cd content is higher, heavy metal average content in the topsoil of downstream
It is above, middle reaches, downstream Cd average contents are 8.51 times of In The Soils of Hebei background value, for national soil background
8.25 again.
The repairing and treating of heavy-metal contaminated soil is that the weight solved is currently badly in need of in current international research hotspot and China
Want problem.Soil is once contaminated by heavy metals, just has the characteristics that the seriousness of irreversibility, concealment and consequence, not only may be used
Lead to crop yield and the decline of quality, more can bring serious harm to human health by the way that food chain enters human body
（Nabulo, G., 2006；Ma Zhihong, 2007）.It is significant that the heavy metal of contaminated soil environment is primarily referred to as bio-toxicity
Cadmium, mercury, lead and metalloid arsenic.Cadmium is classified as first kind human carcinogen by International Agency for Research on Cancer (IARC)（Li Zhaohui,
2010）.Lead is mainly the nervous centralis by poisoning human body, the damage of the multiple organs such as system, liver and spleen is caused to body, especially
It is to endanger more very the next-generation youngsters and children of motherland（Luo Ya, 2012；Wu Qianqian, 2014）.Therefore, heavy-metal contaminated soil
Repairing and treating be research hotspot outside Now Domestic.Especially after China joined WTO, asked by the heavy metals exceeding standard of agricultural product
Trade barrier caused by topic will generate large impact more to China's agricultural, and the improvement of heavy metal pollution of soil is that China is currently badly in need of
The major issue of solution.
Heavy metal repairing research field development process is developed into biological prosthetic with initial reason, change restorative procedure both at home and abroad,
And to comprehensive repair trend development.Domestic and international expert has carried out numerous studies in succession, wherein early stage with it is traditional chemically and physically
Based on administering method（Renella .G, 2002）, many developed countries in Europe and North America open in 80 mid-nineties 90 of 20th century
The Primary Study work of bioremediation technology is opened up（Nabulo, G., 2006）, and complete some practical processing engineerings.
(bioremediation) technology biological prosthetic in recent years is increasingly becoming an emerging environmental organism hot spot of countries in the world concern
Technology, the concept of broad sense refer to reducing the concentration of poisonous and harmful substances in environment using the vital metabolic activity of biology or making it
It is completely innoxious, to enable the environment that polluted partially or even wholly to be restored to the process of reset condition（Zhou Qixing,
Wei Shu and 2007）.Biological prosthetic, including microorganism remediation and the phytoremediation of narrow sense, it is primarily referred to as utilizing biotechnology
The hard-degraded substance such as macromolecular organic pollution, heavy metal etc. entered in soil environment is administered（Yan Xiaoming, He Jin
Column, 2002）.
In conclusion the reparation of heavy-metal contaminated soil is a system engineering, and in natural ecosystems, heavy metal
Pollution mainly exists with combined pollution（Han Guiqi, Xu Weihong, 2010）, single recovery technique is extremely difficult to desired effect（Cui
De Jie, Zhang Yulong, 2004）.
Invention content
The technical problem to be solved by the present invention is to overcome various deficiencies of the prior art, provide a kind of lead efficiently, economic
Cadmium combined pollution soil comprehensive organism repairing method.
In order to solve the above technical problems, the technical solution used in the present invention is as follows.
A kind of lead cadmium combined pollution soil comprehensive organism repairing method, for the soil of lead cadmium combined pollution, with super accumulation
Based on phytoremediation, it is aided with the microorganism remediation and animal reparation that can improve effect of plant restoration, enhances lead in soil, cadmium
Biological effectiveness and the growth and absorption for thus promoting plant, improve the overall efficiency of contaminated soil remediation.
As a preferred technical solution of the present invention, this method adds animal reparation to add microorganism remediation using phytoremediation
Combined remediation method.
As a preferred technical solution of the present invention, the phytoremediation uses sudangrass, the animal reparation to use
Earthworm, the microorganism remediation use the composite bacteria agent containing sulfur bacteria, nitrifier, amonifying bacteria and nitrogen-fixing bacteria；The plant,
Animal, microorganism, joint repair operating procedure be：Before sudangrass is sowed, keep soil moisture 50%-70% by composite bacteria agent
Shallow 3-5cm mixings of turning over are manured into soil；Then after sudangrass seedling all sprouts, temperature is when 18-25 DEG C and soil moisture are in 50%-70%
Launch earthworm.
As a preferred technical solution of the present invention, the pollution level of soil is measured first, and by soil contamination grade
Delimit is slight pollution, intermediate pollution or serious pollution；If soil belongs to slight pollution, using phytoremediation mode or
Add the joint repair mode of microorganism remediation using phytoremediation plus animal reparation；If soil belongs to intermediate pollution, use
Phytoremediation mode or the joint repair mode for using phytoremediation that animal reparation is added to add microorganism remediation；If soil belongs to
Serious pollution, then the joint repair mode for using phytoremediation that animal reparation is added to add microorganism remediation.
As a preferred technical solution of the present invention, the demarcation method of the soil contamination grade is as follows：To in soil
Lead content be measured, if lead content be no more than 300mg/kg, delimit be slight pollution, if lead content is more than
300mg/kg and be no more than 600mg/kg, then delimit be intermediate pollution, if lead content be more than 600mg/kg, delimit be severe
Pollution.
As a preferred technical solution of the present invention, the cadmium content in soil is measured, if cadmium content does not surpass
1.5mg/kg is crossed, then it is slight pollution to delimit；If cadmium content is more than 1.5mg/kg and is no more than 6mg/kg, it is moderate to delimit
Pollution；If cadmium content is more than 6mg/kg, it is serious pollution to delimit.
As a preferred technical solution of the present invention, the phytoremediation uses sudangrass, the animal reparation to use
Earthworm, the microorganism remediation use the composite bacteria agent containing sulfur bacteria, nitrifier, amonifying bacteria and nitrogen-fixing bacteria；The plant,
Animal, microorganism, joint repair operating procedure be：Before sudangrass is sowed, keep soil moisture 50%-70% by composite bacteria agent
Shallow 3-5cm mixings of turning over are manured into soil；Then after sudangrass seedling all sprouts, temperature is when 18-25 DEG C and soil moisture are in 50%-70%
Launch earthworm.
As a preferred technical solution of the present invention, the phytoremediation is using common over-accumulated plant kind or adopts
With transgenosis Sudan. grass Varieties.
As a preferred technical solution of the present invention, the preparation process of the transgenosis Sudan. grass Varieties includes：
A, the preparation of receptor：Full neat the Sudan's grass seed is chosen, is rinsed well with tap water, 1% NaCLO disinfections
Seed is placed in by high pressure sterilization and in the 90 mm culture dishes of diameter with two layers of filter paper, adds 7ml sterile waters by 10 min,
Light culture 2-3d under the conditions of 25 DEG C；When the young shoot length on stem reaches 0.5cm, plumule and part are pruned with knife blade
Blade is put into after exposing growing point in the plate for being covered with dry filter paper, is placed at room temperature for 5min and is siphoned away stem with sterilized filter paper item
After the moisture that point overflows, room temperature places 20min again；
B, receptor is infected and is co-cultured：Takara companies are synthesizedTCMT3Gene DNA fragment has passed through intermediate carrier
PAHC25 adds ubi-1 promoters and no terminators at TCMT3 genes both ends, is then implemented in the plasmid containing T-sequence again
In Pcambia0390, finally it is transformed into agrobacterium strains EHA105；, design examine foreign geneTCMT3Special primer pair
Transformant is detected, and finally " Micro trauma bud growing point Agrobacterium-mediated Transformation technology " is utilized to import external source to sudangrass bud growing point
GeneTCMT3；The Agrobacterium of single bacterium colony is taken with sterile Tip choicests, is accessed in the LB liquid medium containing antibiotic, in 28
DEG C, culture to bacterial concentration is OD600=0.6 under the conditions of 220 rpm, 4,000rpm, thalline were collected by centrifugation by 5min, suspend again
3 times in original bacteria liquid volume are infected in liquid, this infects liquid and contains 100 μm of ol/L AS, 100mg/L F68,1/10MS salt, 1% grape
Sugar, 4% maltose, PH=5.6；It is dipped with transgenosis brush and infects liquid alignment growth pricking method brush two to after three times, be placed in diameter 90
In the sterile petri dish of mm, this sterile petri dish is dark under the conditions of being placed in 24-25 DEG C added with 2 layers of sterilizing filter paper, sterile water 0.5ml
Cultivate 2-3d；
C, the culture and management of transformation seedlings：After co-cultivation, receptor is transferred in the culturing pot equipped with vermiculite soil, in 20
DEG C, intermediate house after culture 7d in the illumination box of 16h/d, it is ripe after single plant harvest, only harvest main fringe；
D, the screening of transformation generation：By the material of harvest, each main fringe randomly selects 30 kinds and enters in culturing pot respectively, puts
Enter greenhouse；When seedling grows to a leaf wholeheartedly, blade is smeared with 20mg/LPPT, after 7 d, non-resistance seedling is pulled out, resistance seedling is moved
It plants in flowerpot, Molecular Detection further is carried out to determine transgenic seedling to foreign gene.
It is using advantageous effect caused by above-mentioned technical proposal：
Presently found hyperaccumulative plant only extracts certain metal mostly, and the heavy metal pollution of soil is often several heavy
The combined pollution of metal, and forefathers study that most hyperaccumulative plant biomass in the nature found are low, growth is slow
Slowly, plant is short and small, limits its removal efficiency to Heavy Metals in Soil Contaminated, is also unfavorable for the mechanized operation of large area.This
Invention is for the purpose of the overall efficiency for improving phytoremediation, through ours the study found that TcMT3 is the super accumulation of penny cress platymiscium
The key-gene of heavy metal imports super accumulation heavy metal characteristic gene by transgenic technology（TcMT3）Has certain weight to itself
In the Sudan's cursive script that metal enrichment ability, biomass are big, the speed of growth is fast, excess of export accumulation type genetically modified plants are cultivated, and herein
On the basis of, it proposes based on phytoremediation, is aided with the means such as animal, microorganism and Agro-ecology measure, enhance lead, cadmium in soil
Biological effectiveness, promote the growth and absorption of plant, to improve over-accumulated plant comprehensive repair efficiency effective measures；
This is the innovative technique for being different from previous single repair mode.
The present invention be in conjunction with plant, animal, microorganism and Agro-ecology measure in one biological synthesis recovery technique, it is suitable
Profit realizes the biology in situ reparation to soil, has a significant effect, and does not generate secondary pollution to environment, is following
Heavy-metal contaminated soil repairs the developing direction in field.The present invention is better than the comprehensive repair of single reparation means on repairing effect
Technology：Such as：Jia Chan（Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology；Agro-environment science journal, 6 phases in 2014）Sudangrass can be multiple from cadmium-pyrene
It closes and absorbs cadmium in contaminated soil, the plant removal rate of cadmium is between 4.23%-17.88% in soil, and the reparation of this patent is imitated
For fruit from slightly to serious pollution, the removal rate of cadmium is both greater than 60%.The present invention is at environmental-friendly aspect better than easy bands such as physical chemistry
Carry out the recovery technique of secondary pollution；The physics such as surface restoration agents and chelating agent such as comparison addition hydroxyapatite, potassium chloride, EDTA
Chemical repair method（Wang Li, 2015；Dong Shanyan, 2002）, this hair will not bring soil because of artificial addition chemicals
Secondary pollution, with based on plant extract, the substance secreted by earthworm, microorganism etc. itself biology beneficial to soil is to promote
Into, promoted effect of plant restoration supplemented by, be environmentally friendly reparation approach.
In addition, classification and the differentiation treatment technology of the present invention, have both efficient and economic two-fold advantage, are this fields
Pioneering creation.Specific efficacy parameter of the invention test example and embodiment as detailed below.
Description of the drawings
Fig. 1 is the scene photograph of development test of the present invention.
Fig. 2 be 2 slight lead cadmium combined pollution geobiont repair of embodiment column design sketch and sudangrass to Pb, Cd
Absorb total amount block diagram.
Fig. 3 be 3 moderate lead cadmium combined pollution geobiont repair of embodiment column design sketch and sudangrass to Pb, Cd
Absorb total amount block diagram.
Fig. 4 be 4 severe lead cadmium combined pollution geobiont repair of embodiment column design sketch and sudangrass to Pb, Cd
Absorb total amount block diagram.
Specific implementation mode
The present invention is described in detail in following embodiment.Various raw materials used in the present invention and items of equipment are conventional city
Product is sold, can be bought and be directly obtained by market.Wherein, sudangrass grows kind using summer, it is preferred to use the present invention
Transformed variety " holding back indigo plant-the Sudan ".Microorganism is formed using composite bacteria agent：By sulfur bacteria, nitrifier, amonifying bacteria, fixed nitrogen
The multiple-microorganisms flora such as bacterium forms, and purchases from high standard bioengineering Co., Ltd, product type：Unigrow microbial bacterial agents,
Using density：30 ~ 50 kg/hm of solid fungicide2Or 2 ~ 3.5 kg/ mus, application method：Before sowing, soil moisture is kept
60% or so, it is shallow to turn over 3-5cm mixings.Earthworm is conventional commercial kind, throw-in density：6.7×105Item/mu larva（Every cubic metre
It about charges seed 1000）, the release time is temperature control 2-3 days after emergence：18-25 DEG C, soil moisture control：60% or so.It is rotten
Phytic acid（Mass ratio with soil is 10%）：Shijiazhuang humic acid plant production.
The biological prosthetic experimental design of 1. lead cadmium combined pollution soil of embodiment
It is as shown in the table for the basic condition of examination soil：
Table 1 is for examination physiochemical properties of soil and content of beary metal
Soil designs 4 and adds level, respectively： CK、C1（Lead 200mg/kg, cadmium 1mg/kg）、C2（Lead 400mg/kg,
5 mg/kg of cadmium）C3（Lead 800mg/kg, 10 mg/kg of cadmium）.The Pb of corresponding amount（NO3）2、Cd（NO3）2It is made into various concentration
Solution is uniformly mixed with 2mm sieve soil is crossed, soil 8kg is filled per basin, and apply base fertilizer repeatedly（Each processing sowing amount is identical, N, P
30kg/ha is closed, K closes 15 kg/ha）, the plant variety of plantation is：South Africa hybrid Sudan grass seedSudangrass（Latin name
(Sorghum×Sudan,Sorghum bicolor×S.sudanense)）.
Experiment sets 96 basins altogether, and four heavy metals add level, repeat three times, eight processing, respectively：
Processing 1（T1）：Plant（It is equivalent to CK）；
Processing 2（T2）：Plant+microorganism；
Processing 3（T3）：Plant+animal；
Processing 4（T4）：Plant+humic acid；
Processing 5（T5）：Plant+microorganism+animal；
Processing 6（T6）：Plant+microorganism+humic acid；
Processing 7（T7）：Plant+animal+humic acid；
Processing 8（T8）：Plant+microorganism+animal+humic acid.
Plant the Sudan. grass Varieties of summer growth.After planting, it grows 10-15 days, after seedling all sprouts, launches earthworm, plant
Growth period is 95 days, harvests its overground part, while washing out root system, is dried under 70 degree, weighs the dry weight of overground part and root.Plant
Sample HNO3-HCLO4Then graphite furnace-aas determination cadmium content is used in digestion；Soil total P b, Cd HCl-
HNO3-HClO4- HF clear up, then high-content with flame-atomic absorption spectrography (AAS), low content use graphite furnace-Atomic absorption
Lead, cadmium content in spectrographic determination soil compare it and repair difference.
The biological prosthetic experiment of 2. slight lead cadmium combined pollution soil of embodiment
Test result is shown in attached drawing 2.As seen from Figure 2, total in soil slight pollution processing by 95 days repair processes
The content of Pb, Cd decline more notable（When Alpha=0.05）Be C1T1, C1T2, C1T3, C1T4, C1T5, this 5 kinds processing
Lower Pb removal rates are respectively 34.21%, 31.02%, 27.89%, 32.12%, 30.78%, Cd removal rates be respectively 92.06%,
92.46%, 91.83%, 91.90%, 92.54%, Pb, Cd content difference is not notable in soil after the lower reparation of this 5 kinds processing（Alpha=
When 0.05）.Total amount is absorbed to Pb, Cd in conjunction with Fig. 2 sudangrasses as it can be seen that the lower sudangrass of C1T5 processing is to Pb absorptions total amount
1.14mg is significantly higher than other 7 kinds processing（When Alpha=0.05）, and be 0.15% to the contribution rate of Pb removal amounts in soil；
Sudangrass is significantly higher than other processing to Cd absorption total amounts under C1T1, C1T2, C1T3, C1T4, C1T5 processing, and at C1T5
The lower Cd of reason absorbs total amount and reaches 0.21mg, is the maximum value in each processing, accounts for Cd in soil and removes the 0.22% of total amount.Therefore
C1T1 handles economical and effective, C1T5 processing（I.e.：Plant+microorganism+animal）For the best repair of heavy metal slight pollution soil
Combination.
The biological prosthetic experiment of 3. moderate lead cadmium combined pollution soil of embodiment
Test result is shown in attached drawing 3.As seen from Figure 3, by 95 days repair processes, Pb repairing effects exist in soil
There are significant differences under C2T4, C2T5 processing（When Alpha=0.05）, P in soil b contents are without significance difference between remaining is handled after reparation
It is different；And Cd content differences are not notable in the lower soil of each processing（When Alpha=0.05）.However, Pb, Cd exist in soil after repairing
The lower content of C2T5 processing is minimum, and removal rate is respectively 26.28%, 77.89%.As seen from Figure 3, between different disposal sudangrass to Pb,
The absorption total amount of Cd is without significant difference（When Alpha=0.05）, lower Pb, Cd absorption total amount of wherein C2T5 processing is maximum, respectively
0.70mg, 0.49mg account for Pb, Cd in soil and remove 0.07%, the 0.48% of total amount.Therefore, C2T1 handles economical and effective, at C2T5
Reason（I.e.：Plant+microorganism+animal）It is combined for the best repair of heavy metal intermediate pollution soil.
The biological prosthetic experiment of 4. severe lead cadmium combined pollution soil of embodiment
Test result is shown in attached drawing 4.From fig. 4, it can be seen that by 95 days repair processes, soil between respectively being handled under soil serious pollution
What Pb contents were minimum in earth is C3T5 processing, and is substantially less than other processing, and repairing effect is best, removal rate 39.18%；Weight
What Cd contents were minimum in degree contaminated soil is C3T1 processing and C1T5 processing, removal rate are respectively 65.97% and 64.51%, and two
Without significant difference between processing（When Alpha=0.05）.From fig. 4, it can be seen that sudangrass is poor different disposal between Pb, Cd absorption total amount
It is different notable（When Alpha=0.05）, maximum absorption differs 67% with minimal absorption amount, and the lower sudangrass of wherein C3T1 processing is to Pb
Uptake is significantly higher than other processing；And sudangrass is apparently higher than other processing to the absorption total amount C3T1 processing of Cd, is
1.01mg, maximum absorption differ 66% with minimal absorption amount.As it can be seen that C3T5 processing can preferably reduce in serious pollution soil
Pb, Cd concentration, while improving assimilation effect of the sudangrass to Pb, Cd.
Foregoing description is only proposed as the enforceable technical solution of the present invention, not as to the single of its technical solution itself
Restrictive condition.
Claims (1)
1. a kind of lead cadmium combined pollution soil comprehensive organism repairing method, it is characterised in that：For the soil of lead cadmium combined pollution,
Based on repairing with over-accumulated plant, it is aided with the microorganism remediation and animal reparation that can improve effect of plant restoration, enhances soil
Middle lead, the biological effectiveness of cadmium and the growth and absorption that thus promote plant, improve the overall efficiency of contaminated soil remediation；
The pollution level of soil is measured first, and it is slight pollution, intermediate pollution or serious pollution that soil contamination grade, which delimited,；
If soil belongs to slight pollution, animal reparation is added to add microorganism remediation using phytoremediation mode or using phytoremediation
Joint repair mode；If soil belongs to intermediate pollution, add animal using phytoremediation mode or using phytoremediation
Repair the joint repair mode for adding microorganism remediation；If soil belongs to serious pollution, animal reparation is added using phytoremediation
Add the joint repair mode of microorganism remediation；
The demarcation method of the soil contamination grade is as follows：Lead content in soil is measured, if lead content is no more than
300mg/kg, then it is slight pollution to delimit, if lead content is more than 300mg/kg and is no more than 600mg/kg, it is moderate to delimit
Pollution, if lead content is more than 600mg/kg, it is serious pollution to delimit；
The phytoremediation use sudangrass, the animal reparation use earthworm, the microorganism remediation use containing sulfur bacteria,
The composite bacteria agent of nitrifier, amonifying bacteria and nitrogen-fixing bacteria；The plant, animal, microbial association reparation operating procedure be：
Before sudangrass sowing, keep soil moisture 50%-70% that the shallow 3-5cm mixings of turning over of composite bacteria agent are manured into soil；Then sudangrass is waited for
After seedling all sprouts, temperature launches earthworm when 18-25 DEG C and soil moisture are in 50%-70%；
The phytoremediation uses transgenosis Sudan. grass Varieties；
The preparation process of the transgenosis Sudan. grass Varieties includes：
A, the preparation of receptor：Full neat the Sudan's grass seed is chosen, is rinsed well with tap water, 1% NaCLO disinfections 10
Seed is placed in by high pressure sterilization and in the 90 mm culture dishes of diameter with two layers of filter paper, adds 7ml sterile waters by min,
Light culture 2-3d under the conditions of 25 DEG C；When the young shoot length on stem reaches 0.5cm, plumule and part leaf are pruned with knife blade
Piece is put into after exposing growing point in the plate for being covered with dry filter paper, is placed at room temperature for 5min and is siphoned away stem apex with sterilized filter paper item
After the moisture of spilling, room temperature places 20min again；
B, receptor is infected and is co-cultured：Takara companies are synthesizedTCMT3Gene DNA fragment has passed through intermediate carrier
PAHC25 adds ubi-1 promoters and no terminators at TCMT3 genes both ends, is then implemented in the plasmid containing T-sequence again
In Pcambia0390, finally it is transformed into agrobacterium strains EHA105；Foreign gene is examined in designTCMT3Special primer pair
Transformant is detected, and finally " Micro trauma bud growing point Agrobacterium-mediated Transformation technology " is utilized to import external source to sudangrass bud growing point
GeneTCMT3；The Agrobacterium of single bacterium colony is taken with sterile Tip choicests, is accessed in the LB liquid medium containing antibiotic, in 28
DEG C, culture to bacterial concentration is OD600=0.6 under the conditions of 220 rpm, 4,000rpm, thalline were collected by centrifugation by 5min, suspend again
3 times in original bacteria liquid volume are infected in liquid, this infects liquid and contains 100 μm of ol/L AS, 100mg/L F68,1/10MS salt, 1% grape
Sugar, 4% maltose, PH=5.6；It is dipped with transgenosis brush and infects liquid alignment growth pricking method brush two to after three times, be placed in diameter 90
In the sterile petri dish of mm, this sterile petri dish is dark under the conditions of being placed in 24-25 DEG C added with 2 layers of sterilizing filter paper, sterile water 0.5ml
Cultivate 2-3d；
C, the culture and management of transformation seedlings：After co-cultivation, receptor is transferred in the culturing pot equipped with vermiculite soil, in 20 DEG C,
Cultivate intermediate house after 7d in the illumination box of 16h/d, it is ripe after single plant harvest, only harvest main fringe；
D, the screening of transformation generation：By the material of harvest, each main fringe randomly selects 30 kinds and enters in culturing pot respectively, is put into temperature
Room；When seedling grows to a leaf wholeheartedly, blade is smeared with 20mg/LPPT, after 7 d, non-resistance seedling is pulled out, resistance transplantation of seedlings is entered
In flowerpot, Molecular Detection further is carried out to determine transgenic seedling to foreign gene.
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| CN109894461B (en) * | 2019-03-14 | 2021-10-26 | 四川师范大学 | Bioremediation method of waste drilling mud |
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| CN110283845B (en) * | 2019-07-29 | 2021-08-13 | 中国科学院地理科学与资源研究所 | Phytoremediation method for applying transgenic plant composite system to polluted soil |
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| CN110877048B (en) * | 2019-12-05 | 2023-12-01 | 河南省科学院生物研究所有限责任公司 | Method for repairing lead-chromium composite severely-polluted soil by utilizing humic acid and sudan grass |
| CN113369296A (en) * | 2021-06-15 | 2021-09-10 | 广州市金龙峰环保设备工程股份有限公司 | Method for improving farmland polluted soil with slight heavy metal pollution |
| CN113732053A (en) * | 2021-10-13 | 2021-12-03 | 江苏景然生态建设集团有限公司 | Heavy metal soil remediation agent |
| CN114752499A (en) * | 2022-04-02 | 2022-07-15 | 河南中烟工业有限责任公司 | Microorganism for repairing cadmium/lead polluted soil and repairing method |
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